WO2010073266A1 - Sequence variants predictive of risk of kidney stones and bone mineral density - Google Patents

Sequence variants predictive of risk of kidney stones and bone mineral density Download PDF

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WO2010073266A1
WO2010073266A1 PCT/IS2009/000017 IS2009000017W WO2010073266A1 WO 2010073266 A1 WO2010073266 A1 WO 2010073266A1 IS 2009000017 W IS2009000017 W IS 2009000017W WO 2010073266 A1 WO2010073266 A1 WO 2010073266A1
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allele
condition
risk
markers
marker
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Hilma Holm
Gudmar Thorleifsson
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Decode Genetics Ehf
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    • A61K31/549—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame having two or more nitrogen atoms in the same ring, e.g. hydrochlorothiazide
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • kidney stone disease kidney stones; renal stones
  • the lifetime risk of stone formation exceeds 12% in men and 5% in women in the United States and recurrence rates are high at 30% to 40% within 5 years of the initial event.
  • the vast majority of kidney stones ( ⁇ 80%) contain calcium while other forms, including uric acid stones, struvite and cystine stones, are far less common (Coe FL, Evan A, Worcester E. J Clin Invest 2005;115: 2598- 608).
  • the formation of kidney stones is generally associated with a metabolic abnormahtv , most commonly primary hypercalciu ⁇ a, found in 5-10% of the general population and approximately 60% of adult patients with stones.
  • Bartters syndrome caused by mutations of the bumetamde-sensitive Na-K-Cl (NKCC2) cotransporter, the renal outer-medullary potassium channel (ROMK), the voltage-gated chloride channel, CLC-Kb, or of its subunit Barttin, or the calcium-sensing receptor, CaSR; Dent ' s disease, due to mutations of the chloride/proton antiporter, CLC-5; and familial hypomagnesemia with hypercalciu ⁇ a and nephrocalcinosis (FHHNC) resulting from mutations in claudins (CLDN16 and CLDN19), major structural constituents of the intercellular tight junction barrier.
  • NKCC2 bumetamde-sensitive Na-K-Cl
  • ROMK renal outer-medullary potassium channel
  • CaSR calcium-sensing receptor
  • Kidney stones and hypercalciurea are known to be associated with bone mineral density.
  • the association of hypercalciu ⁇ a with bone mineral density has been extensively explored and the overall data appears to suggest a risk for bone loss in individuals with kidney stones, primarily those with hypercalciu ⁇ a, and possibly associated with enhanced bone turnover (S ⁇ vastava T, Alon US., Pediatr Nephrol 2007;22: 1659-73, Caudarella R, et al. , Urol Int 2004,72 Suppl 1.17- 9).
  • Metabolic acidosis is also known to induce bone resorption although bone involvement is rarely present in dRTA (Rodriguez Soriano J., J Am Soc Nephrol 2002; 13:2160-70).
  • Genetic risk is in general conferred by subtle differences in the genome among individuals in a population. Genomic differences between individuals most frequently due to single nucleotide polymorphisms (SNP), although other variations are also important. SNP are located on average every 1000 base pairs in the human genome. Accordingly, a typical human gene containing 250,000 base pairs may contain 250 different SNPs. Only a minor number of SNPs are located in exons and alter the amino acid sequence of the protein encoded by the gene. Most SNPs may have little or no effect on gene function, while others may alter transcription, splicing, translation, or stability of the mRNA encoded by the gene. Additional genetic polymorphism in the human genome is caused by insertion, deletion, translocation, or inversion of either short or long stretches of DNA. Genetic polymorphisms conferring kidney stones risk may therefore directly alter the amino acid sequence of proteins, may increase the amount of protein produced from the gene, or may decrease the amount of protein produced by the gene.
  • SNP single nucleotide polymorphisms
  • genetic testing for such risk factors is becoming important for clinical medicine. Examples are apolipoprotein E testing to identify genetic carriers of the apoE4 polymorphism in dementia patients for the differential diagnosis of Alzheimer's kidney stones, and of Factor V Leiden testing for predisposition to deep venous thrombosis. More importantly, in the treatment of cancer, diagnosis of genetic variants in tumor cells is used for the selection of the most appropriate treatment regime for the individual patient.
  • the present invention is based on the association of the human CLDN 14 gene with conditions that include kidney stones, serum parathyroid hormone levels, serum bicarbonate levels, bone mineral density and osteoporosis. Variants associated with the CLDN 14 gene have been shown to be associated with risk of these conditions.
  • the invention provides a method of determining a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in e human individual, the method comprising obtaining nucleic acid sequence data about a human individual identifying at least one allele of at least one polymorphic marker associated with the human CLDN14 gene, wherein different alleles of the at least one polymorphic marker are associated with different susceptibilities to the at least one condition in humans, and determining a susceptibility to the at least one condition from the nucleic acid sequence data.
  • the invention also provides a method of determining a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, the method comprising : obtaining nucleic acid sequence data about a human individual identifying both alleles of at least two polymorphic markers associated with the human CLDN14 gene, determine the identity of at least one haplotype based on the sequence data, and determining a susceptibility to the at least one condition from the haplotype data
  • the invention also provides a method of diagnosing a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, the method comprising, obtaining CLDN14 amino acid sequence data about at least one encoded CLDN14 protein of a human individual, identifying at least one polymorphic site associated with the CLDN14 amino acid sequence, wherein different amino acids of the at least one polymorphic site are associated with different susceptibilities to the at least one condition in humans, and diagnosing susceptibility to the at least one condition from the amino acid sequence data.
  • the invention provides a method of determining a susceptibility of at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, the method comprising determining the identity of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one marker is selected from the group of markers located within the LD block C21, wherein susceptibility of the at least one condition is correlated with the identity of the at least one allele.
  • the invention also provides a method of identification of a marker for use in assessing susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, the method comprising
  • the invention can also be used to assess individuals for probability of response to a therapeutic agent for preventing and/or ameliorating symptoms associated with at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, comprising: determining the identity of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one polymorphic marker is selected from polymorphic markers associated with the human CLDN 14 gene, wherein determination of the identity of the at least one allele of the at least one marker is indicative of a probability of a positive response to the therapeutic agent.
  • Prognosis of an individual experiencing symptoms associated with, or an individual diagnosed with, at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis is also provided, the method comprising : obtaining nucleic acid sequence data about a human individual identifying at least one allele of at least one polymorphic marker associated with the human CLDN14 gene, wherein different alleles of the at least one polymorphic marker are associated with different susceptibilities to the at least one condition in humans, andpredicti ⁇ g prognosis of the individual from the nucleic acid sequence data.
  • Treatment outcome of an individual undergoing treatment for at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis is also provided by the invention, the method comprising obtaining nucleic acid sequence data about a human individual identifying at least one allele of at least one polymorphic marker associated with the human CLDN 14 gene, wherein different alleles of the at least one polymorphic marker are associated with different susceptibilities to the at least one condition in humans, and predicting treatment outcome of the individual from the nucleic acid sequence data.
  • kits for assessing susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, the kit comprising ( ⁇ ) reagents for selectively detecting at least one allele of at least one polymorphic marker in the genome of the individual, wherein the at least one polymorphic marker is selected from the group consisting of rsl90068, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773,
  • the invention also provides in another aspect the use of an oligonucleotide probe in the manufacture of a diagnostic reagent for diagnosing and/or assessing susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, wherein the probe is capable of selectively hybridizing to a segment of a nucleic acid whose nucleotide sequence is given by SEQ ID NO: 1 that comprises at least one polymorphic site, wherein the segment is 15-500 nucleotides in length.
  • the invention provides a computer-readable medium having computer executable instructions for determining susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, the computer readable medium compr ⁇ s ⁇ ng:data indicative of at least one polymorphic markerja routine stored on the computer readable medium and adapted to be executed by a processor to determine risk of developing the at least one condition for the at least one polymorphic marker; wherein the at least one polymorphic marker is associated with the human CLDN14 gene.
  • Another computer-implemented aspect provides an apparatus for determining a genetic indicator in a human individual for at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, comprising. a processor; a computer readable memory having computer executable instructions adapted to be executed on the processor to analyze marker and/or haplotype information for at least one human individual with respect to at least one polymorphic marker associated with the human CLDN14 gene, and generate an output based on the marker or haplotype information, wherein the output comprises a risk measure of the at least one marker or haplotype as a genetic indicator of the at least one condition for the human individual.
  • the invention also provides pharmaceutical compositions comprising polypeptides encoded by a human CLDN14 gene or fragments thereof, methods of prophylaxis therapy of conditions selected from the group consisting of kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis using pharmaceutical compositions that include agents that regulate expression, activity or physical state of a CLDN14 gene, its encoding RNA or protein in an individual, human CLDN14 polypeptides and oligonucleotides for uses as medicaments, and methods of therapy , that select individuals for treatment based on the genetic status of at least one CLDN14 variant that correlates with increased risk of the condition.
  • the invention in general relates to markers associated with the CLDN14 gene, in particular markers within, or in linkage disequilibrium with, the LD block C21 as defined herein.
  • ExemplaiV markers are provide e.g. in Table 1 herein. Certain embodiments relate to one or more of the markers rs219778, rs219779, rs219780 and rs219781. Further embodiments of the invention are presented in the following description of various embodiments of the invention.
  • Figure 1 shows a Q-Q plot of the 303,120 adjusted Chi 2 -statistics from a GWA analysis of kidney stones.
  • the equiangular line (black line) is included in the plot for reference purpose.
  • the dashed horizontal line indicates the threshold for genome-wide significance assuming a Bonferroni correction for the 303,120 SNPs tested.
  • the red dots correspond to results for all the 303,120 SNPs, while for the blue dots, the 166 SNPs in a 1 Mb region on chromosome 21 centered on the CLDN14 gene have been excluded.
  • Figure 2 describes the 21q22 ⁇ ocus;
  • A presents the pair-wise correlation structure in a 200 kb interval (36.45 - 36.95 Mb, NCBI B36) on Chromosome 21.
  • the upper plot shows pair-wise D' for 454 common SNPs (with MAF > 5%) from the HapMap (v22) CEU dataset.
  • the lower plot shows the corresponding r 2 values.
  • B shows the estimated recombination rates (saRR) in cM/Mb from the HapMap Phase II data(Frazer KA, et al., Nature 2007;449:851-61).
  • C displays the location of known genes in the region and
  • D reveals the schematic view of the association with kidney stones for all the markers tested in the genome-wide scan. All panels use the same horizontal scale shown in panel D).
  • Figure 3 provides a diagram illustrating a computer-implemented system utilizing risk variants. , as described herein.
  • nucleic acid sequences are written left to right in a 5' to 3' orientation.
  • Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range.
  • all technical and scientific terms used herein have the same meaning as commonly understood by the ordinary person skilled in the art to which the invention pertains.
  • the marker can comprise any allele of any variant type found in the genome, including SNPs, mini- or microsatellites, translocations and copy number variations (insertions, deletions, duplications).
  • Polymorphic markers can be of any measurable frequency in the population. For mapping of kidney stone genes, polymorphic markers with population frequency higher than 5-10% are in general most useful. However, polymorphic markers may also have lower population frequencies, such as 1-5% frequency, or even lower frequency, in particular copy number variations (CNVs). The term shall, in the present context, be taken to include polymorphic markers with any population frequency.
  • an “allele” refers to the nucleotide sequence of a given locus (position) on a chromosome.
  • a polymorphic marker allele thus refers to the composition (i.e., sequence) of the marker on a chromosome.
  • CEPH sample Centre d'Etudes du Polymorphisme Humain, genomics repository, CEPH sample 1347-02
  • the shorter allele of each microsatellite in this sample is set as 0 and all other alleles in other samples are numbered in relation to this reference.
  • allele 1 is 1 bp longer than the shorter allele in the CEPH sample
  • allele 2 is 2 bp longer than the shorter allele in the CEPH sample
  • allele 3 is 3 bp longer than the lower allele in the CEPH sample, etc.
  • allele - 1 is 1 bp shorter than the shorter allele in the CEPH sample
  • allele -2 is 2 bp shorter than the shorter allele in the CEPH sample, ' etc.
  • Sequence conucleotide ambiguity as described herein is as proposed by IUPAC-IUB. These codes are compatible with the codes used by the EMBL, GenBank, and PIR databases.
  • a nucleotide position at which more than one sequence is possible in a population is referred to herein as a "polymorphic site”.
  • a "Single Nucleotide Polymorphism” or "SNP” is a DNA sequence variation occurring when a single nucleotide at a specific location in the genome differs between members of a species or between paired chromosomes in an individual. Most SNP polymorphisms have two alleles. Each individual is in this instance either homozygous for one allele of the polymorphism (i.e. both chromosomal copies of the individual have the same nucleotide at the SNP location), or the ⁇ individual is heterozygous (i.e. the two sister chromosomes of the individual contain different nucleotides).
  • the SNP nomenclature as reported herein refers to the official Reference SNP (rs) ID identification tag as assigned to each unique SNP by the National Center for Biotechnological Information (NCBI).
  • a “variant”, as described herein, refers to a segment of DNA that differs from the reference DNA.
  • a “marker” or a “polymorphic marker”, as defined herein, is a variant. Alleles that differ from the reference are referred to as “variant” alleles.
  • a “microsatellite” is a polymorphic marker that has multiple small repeats of bases that are 2-8 - nucleotides in length (such as CA repeats) at a particular site, in which the number of repeat lengths varies in the general population.
  • An “indel” is a common form of polymorphism, comprising a small insertion or deletion that is typically only a few nucleotides long.
  • haplotype refers to a segment of genomic DNA that is characterized by a specific combination of alleles arranged along the segment.
  • a haplotype comprises one member of the pair of alleles for each polymorphic marker or locus along the segment.
  • the haplotype can comprise two or more alleles, three or more alleles, four or more alleles, or five or more alleles.
  • Haplotypes are described herein in the context of the marker name and the allele of the marker in that haplotype, e.g., "2 rs219781" refers to the 2 allele of marker rs219781 being in the haplotype, and is equivalent to "rs219781 allele 2".
  • susceptibility refers to the proneness of an individual towards the development of a certain state (e.g., a certain trait or phenotype), or towards being less able to resist a particular state than the average individual.
  • the term encompasses both increased susceptibility and decreased susceptibility.
  • particular alleles at polymorphic markers and/or haplotypes of the invention as described herein may for example be characteristic of increased susceptibility (i.e., increased risk) of kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, as characterized by a relative risk (RR) or odds ratio (OR) of greater than one for the particular allele or haplotype.
  • the markers and/or haplotypes of the invention are characteristic of decreased susceptibility (i.e., decreased risk) of one or more of these conditions, as characterized by a relative risk of less than one.
  • look-up table is a table that correlates one form of data to another form, or one or more forms of data to a predicted outcome to which the data is relevant, such as phenotype or trait.
  • a look-up table can comprise a correlation between allelic data for at least one polymorphic marker and a particular trait or phenotype, such as a particular kidney stones diagnosis, that an individual who comprises the particular allelic data is likely to display, or is more likely to display than individuals who do not comprise the particular allelic data.
  • Look-up tables can be multidimensional, i.e.
  • a "computer-readable medium” is an information storage medium that can be accessed by a computer using a commercially available or custom-made interface.
  • Exemplary compute- readable media include memory (e.g., RAM, ROM, flash memory, etc.), optical storage media (e.g., CD-ROM), magnetic storage media (e.g., computer hard drives, floppy disks, etc.), punch cards, or other commercially available media.
  • Information may be transferred between a system of interest and a medium, between computers, or between computers and the computer- readable medium for storage or acess of stored information. Such transmission can be electrical, or by other available methods, such as IR links, wireless connections, etc.
  • nucleic acid sample refers to a sample obtained from an individual that contains nucleic acid (DNA or RNA).
  • the nucleic acid sample comprises genomic DNA.
  • a nucleic acid sample can be obtained from any source that contains genomic DNA, including a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs.
  • Kidney stones refers to a disease state in which crystal aggregations of dissolved minerals, also called renal calculi, form in the kidneys, urinary tract or bladder. Kidney stones are sometimes also referred to as “nephrolithiasis”, “kidney stone disease”, or “renal stones”.
  • therapeutic agent for kidney stones refers to an agent that can be used to ameliorate or prevent symptoms associated with kidney stones.
  • kidney stones-associated nucleic acid refers to a nucleic acid that has been found to be associated to kidney stones. This includes, but is not limited to, the markers and haplotypes described herein and markers and haplotypes in strong linkage disequilibrium (LD) therewith.
  • a kidney stone-associated nucleic acid refers to an LD-block found to be associated with Type 2 diabetes through at least one polymorphic marker located within the LD block.
  • LD block C21 refers to the Linkage Disequilibrium (LD) block on Chromosome 21 corresponding to position 36,697,419 - 36,770,504 of NCBI (National Center for Biotechnology Information) Build 36 (SEQ ID NO: 1).
  • variants associated with the human CLDN14 gene are associated with risk of kidney stones, hypercalciurea, bone mineral density, reduced serum bicarbonate and increased urinary calcium concentration. Certain variants in the CLDN14 gene have been found to confer risk of these conditions, such that the presence of particular alleles at these variants confer increased risk of the condition, and certain other alleles confer decreased risk of the condition. These variants, and other variants that are correlated to these variants, are useful for certain diagnostic applications.
  • the present invention provides methods of determining a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual.
  • the method comprises obtaining nucleic acid sequence data about a human individual identifying at least one allele of at least one polymorphic marker associated with the human CLDN14 gene, wherein different alleles of the at least one : polymorphic marker are associated with different susceptibilities to the at least one condition in '1 humans, and determining a susceptibility to the at least one condition from the nucleic acid sequence data.
  • nucleic acid marker changes the codon of a polypeptide encoded by the nucleic acid, then the marker will also result in alternate sequence at the amino acid level of the encoded polypeptide (polypeptide markers).
  • Determination of the identity of particular alleles at polymorphic markers in a nucleic acid or particular alleles at polypeptide markers comprises whether particular alleles are present at a certain position in the sequence. Sequence data identifying a particular allele at a marker comprises sufficient sequence to detect the particular allele.
  • sequence data can ⁇ comprise sequence at a single position, i.e. the identity of a nucleotide or amino acid at a single position within a sequence.
  • the allele can be the allele of the complementary strand of DNA, such that the nucleic acid data includes the identification of at least one allele which is complementary to the allele at the opposite strand.
  • nucleic acid sequence for at least two polymorphic markers it may be useful to determine the nucleic acid sequence for at least two polymorphic markers. In other embodiments, the nucleic acid sequence for at least three, at least four or at least five or more polymorphic markers is determined.
  • Haplotype information can be derived from an analysis of two or more polymorphic markers. Thus, in certain : embodiments, a further step is performed, whereby haplotype information is derived based on sequence data for at least two polymorphic markers.
  • sequence data about both alleles of polymorphic markers associated , with the CLDN 14 gene are obtained, and the identity of at least one haplotype based on the sequence data is determined, and a susceptibility to the condition is determined from the haplotype data.
  • determination of a susceptibility comprises comparing the nucleic acid sequence data to a database containing correlation data between the at least one polymorphic marker and susceptibility to the at least one condition.
  • the database comprises at least one risk measure of susceptibility to at least one condition for the at least one marker.
  • the sequence database can for example be provided as a look-up table that contains data that indicates the susceptibility of at least one condition for any one, or a plurality of, particular polymorphisms.
  • the database may also contain data that indicates the susceptibility for a particular haplotype that comprises at least two polymorphic markers.
  • Obtaining nucleic acid sequence data can in certain embodiments comprise obtaining a biological sample from the human individual and analyzing sequence of the at least one polymorphic marker in nucleic acid in the sample. Analyzing sequence can comprise determining the presence or absence of at least one allele of the at least one polymorphic marker. Determination of the presence of a particular susceptibility allele (e.g. , an at-risk allele) is indicative of susceptibility to the condition in the human individual. Determination of the absence of a particular suscepbility allele is indicative that the particular susceptibility due to the at least one, polymorphic marker is not present in the individual.
  • a particular susceptibility allele e.g. , an at-risk allele
  • obtaining nucleic acid sequence data comprises obtaining nucleic acid • sequence information from a preexisting record.
  • the preexisting record can for example be a ⁇ • computer file or database containing sequence data, such as genotype data, for the human individual, for the at least one polymorphic marker.
  • Susceptibility determined by the diagnostic methods of the invention can be reported to a particular entity.
  • the at least one entity is selected from the group consisting of the individual, a guardian of the individual, a genetic service provider, a physician, a medical organization, and a medical insurer.
  • determination of a susceptibility comprises comparing the nucleic acid sequence data to a database containing correlation data between the at least one polymorphic marker and susceptibility to the condition.
  • the database comprises at least one risk measure of susceptibility to the condition for the at least one polymorphic marker.
  • the database comprises a look-up table containing at least one risk measure of the at least one condition for the at least one polymorphic marker.
  • obtaining nucleic acid sequence data comprises obtaining a biological . sample from the human individual and analyzing sequence of the at least one polymorphic marker in nucleic acid in the sample. Analyzing sequence of the at least one polymorphic marker can comprise determining the presence or absence of at least one allele of the at least one polymorphic marker. Obtaining nucleic acid sequence data can also comprise obtaining nucleic acid sequence information from a preexisting record. Certain embodiments of the invention relate to obtaining nucleic acid sequence data about at least two polymorphic markers associated with the CLDN14 gene. Other embodiments may relate to obtaining sequence data about more than two polymorphic markers, including three, four, five, six, seven, eight, nine or ten or more polymorphic markers.
  • the markers associated with the CLDN 14 gene are in certain embodiments markers within LD block C21 as set forth in SEQ ID NO: 1 herein.
  • Obtaining sequence data may in certain embodiments relate to determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, or in a genotype dataset from the individual. Obtaining information about the absence or presence of particular marker alleles represents sequence information for the marker, identifying particular marker alleles.
  • the at least one polymorphic marker is selected from ⁇ polymorphic markers associated with the human CLDN14 gene. Certain embodiments relate to markers within LD block C21. Certain preferred embodiments relate to markers selected from ' the group consisting of rsll ⁇ .88346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, ⁇ • rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076
  • One preferred embodiment relates to markers selected from the group consisting of rsl 1088346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364 rsl70183, rsl3051987, rsl70182, rs218638, rs218641, rs218642,
  • anchor markers such as rs219781, rs219780, rs219779, • ⁇ ' rs219778 can be considered to be in linkage disequilibrium with themselves.
  • markers in linkage disequilibrium with the anchor markers include the foregoing surrogate marker list (as also presented in Table 1), excluding the surrogate markers rs219781, rs219780, rs219779, rs219778.
  • Another preferred embodiment relates to markers selected from the group consisting of rsllO88346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs21978l, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364 and rsl70183.
  • Particularly preferred embodiments relate to markers selected from the group consisting of rs219778, rs219779, rs219780 and rs219781. Certain preferred embodiments relate to marker rs219778. Certain other embodiments relate to markers rs219779. Certain other embodiments relate to markers rs219780. Certain other embodiments relate to markers rs219781. Certain embodiments further relate to surrogate markers of any one of the markers rs219778, rs219779, rs219780 and rs219781, which can suitably be selected from the markers presented , in Table 1 (Ia and/or Ib) herein.
  • a further step of assessing the frequency of at least one haplotype in the individual is performed.
  • two or more markers including three, four, five, six, seven, eight, nine or ten or more markers can be included in the haplotype.
  • the at least one haplotype comprises markers that are all in LD with at least one of rs219778, rs219779, rs219780 and rs219781.
  • determination of the presence of rs219778 allele T, rs219779 allele C, rs219780 allele C and/or rs219781 allele C, or marker alleles or haplotypes in linkage disequilibrium therewith is indicative of increased susceptibility to the condition.
  • the opposite (alternate) allele of particular SNPs are in such embodiments indicative of a decreased susceptibility to the conditions.
  • the presence of rs219778 allele C, rs219779 allele T, rs219780 allele A and/or rs219781 allele A is indicative of decreased susceptibility to the at least one condition.
  • Certain embodiments of the invention thus relate to individuals who are homozygous for at-risk alleles for at least one polymorphic marker selected from the group consisting of rsl 1088346, rsl90068, rs2835342, ;_ rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364, rsl70183, rsl3051987,
  • Preferred embodiments relate to individuals who are homozygous for any one, or a combination of, rs219778 allele C, rs219779 allele T, rs219780 allele A and/or rs219781 allele A, or marker alleles or haplotypes in linkage disequilibrium therewith.
  • the markers conferring risk of at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, as described herein, can be combined with other genetic markers for one or more of these conditions.
  • Such markers are typically not in linkage disequilibrium with any one of the markers described herein, in particular markers rs219778, rs219779, rs219780 and rs219781. Any of the mehods described herein can be practiced by combining the genetic risk factors described herein with such additional genetic risk factors for the condition.
  • a further step comprising determining whether at least one at-risk allele of at least one at-risk variant for at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum " bicarbonate levels, reduced bone mineral density and osteoporosis not in linkage disequilibrium with any one of the markers rs219778, rs219779, rs219780 and rs219781 is present in a sample comprising genomic DNA from a human individual or a genotype dataset derived from a human individual. Determination of the presence of the marker allele may be determined by obtaining - nucleic acid sequence data identifying the particular allele.
  • markers in other locations in the genome can be useful in combination with the markers of the present invention, so as to determine overall risk of the condition based on multiple genetic factors.
  • Selection of , markers that are not in linkage disequilibrium (not in LD) can be based on a suitable measure for linkage disequilibrium, as described further herein.
  • markers that are not in linkage disequilibrium have values for the LD measure r 2 between the markers of less than 0.2.
  • markers that are not in LD have values for r 2 between the markers of less than 0.15, including less than 0.10, less than 0.05, less than 0.02 and less than 0.01.
  • Other suitable cutoff values for establishing that markers are not in LD are contemplated, including values bridging any of these values.
  • the genetic markers of the invention can also be combined with non-genetic information to establish overall risk for an individual.
  • a further step is included, comprising analyzing non-genetic information to make risk assessment, diagnosis, or prognosis: of the individual.
  • the non-genetic information can be any information pertaining to the disease' status of the indiviudal or other information that can influence the estimate of overall risk of the condition for the individual.
  • the non-genetic information is selected from the group consisting of low water consumption, hypercalciurea, high-protein diet, high-sodium . diet, low-calcium diet, obesity, high blood pressure, lack of physical activity, family history of kidney stones, previous kidney stones, vitamin A deficiency, hyperparathyroidism, kidney infection and history of gastric bypass surgery, inflammatory bowel disease or chronic diarrhea.
  • Certain aspects of the invention relate to a method of identification of a marker for use in assessing susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, the method comprising : identifying at least one polymorphic marker in linkage disequilibrium with at least one of rs219778, rs219779, rs219780, and rs219781 (e.g., one or more of the markers set forth in Table 1 herein); determining the genotype status of a sample of individuals diagnosed with, or having a susceptibility to, the condition; and determining the genotype status of a sample of control individuals; wherein a significant difference in frequency of at least one allele in at least one polymorphism in individuals diagnosed with, or having a susceptibility to, the condition, as compared with the frequency of the at least one allele in the control sample is indicative of the at least one polymorphism being useful for
  • a significant difference can be estimated on statistical analysis of allelic counts at certain polymorphic markers in patients and controls.
  • a significant difference is based on a calculated P- value between patients and controls of less than 0.05.
  • a significant difference is based on a lower value of the calculated P-value, such as less than 0.005, 0.0005, or 0.00005.
  • an increase in frequency of the at least one allele in the at least one polymorphism in individuals diagnosed with, or having a susceptibility to, the condition, as compared with the frequency of the at least one allele in the control sample is indicative of the at least one polymorphism being useful for assessing increased susceptibility to the condition.
  • a decrease in frequency of the at least one allele in the at least one polymorphism in individuals diagnosed with, or having a susceptibility to, the condition, as compared with the frequency of the at least one allele in the control sample is indicative of the at least one polymorphism being useful for assessing decreased susceptibility to, or protection against, the condition.
  • the invention also relates to a method of genotyping a nucleic acid sample obtained from a human individual comprising determining whether at least one allele of at least one polymorphic marker is present in a nucleic acid sample from the individual sample, wherein the at least one marker is selected from rsllO88346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs72
  • genotyping comprises amplifying a segment of a nucleic acid that comprises the at least one polymorphic marker by Polymerase Chain Reaction (PCR), using a nucleotide primer pair flanking the at least one polymorphic marker.
  • PCR Polymerase Chain Reaction
  • genotyping is performed using a process selected from allele-specific probe hybridization, allele-specific primer extension, allele-specific amplification, nucleic acid sequencing, 5'-exonuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformation analysis and microarray technology.
  • the microarray technology is Molecular Inversion Probe array technology or BeadArray Technologies.
  • the process comprises allele-specific probe hybridization.
  • the process comprises microrray technology.
  • One preferred embodiment comprises the steps of ( 1) contacting copies of the nucleic acid with a detection oligonucleotide probe and an enhancer oligonucleotide probe under conditions for specific hybridization of the oligonucleotide probe with the nucleic acid; wherein (a) the detection oligonucleotide probe is from 5-100 nucleotides in length and specifically hybridizes to a first segment of a nucleic acid whose nucleotide sequence is given by SEQ ID NO: 1; (b) the detection oligonucleotide probe comprises a detectable label at its 3' terminus and a quenching moiety at its 5' terminus; (c) the enhancer oligonucleotide is from 5-100 nucleotides in length and is complementary to a second segment of the nucleotide sequence that is 5' relative to the,.
  • oligonucleotide probe such that the enhancer oligonucleotide is located 3' relative to the detection oligonucleotide probe when both oligonucleotides are hybridized to the nucleic acid; and (d) a single base gap exists between the first segment and the second segment, such that when the oligonucleotide probe and the enhancer oligonucleotide probe are both hybridized to the nucleic acid, a single base gap exists between the oligonucleotides; (2) treating the nucleic acid with an endonuclease that will cleave the detectable label from the 3 1 terminus of the detection probe to release free detectable label when the detection probe is hybridized to the nucleic acid; and (3) measuring free detectable label, wherein the presence of the free detectable label indicates that the detection probe specifically hybridizes to the first segment of the nucleic acid, and indicates the sequence of the polymorphic site as the complement of the detection probe.
  • the invention also relates to the use of an oligonucleotide probe in the manufacture of a reagent for diagnosing and/or assessing susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, wherein the probe hybridizes to a segment of a nucleic acid with nucleotide sequence as set forth in SEQ ID NO: 1, wherein the probe is 15-500 nucleotides in length.
  • the probe is about 16 to about 100 nucleotides in length.
  • the probe is about 20 to about 50 nucleotides in length.
  • the probe is about 20 to about 30 nucleotides in length.
  • markers that are described herein to be associated with risk of conditions selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis may be used, alone or in any particular combination, in all various aspects of the invention, including the methods, kits, uses, apparatus, procedures described herein.
  • This relates in particular to markers associated with the human CLDN 14 gene, and in particular embodiments markers within LD block C21, and markers as set forth in Table 1, or markers in linkage disequilibrium therewith.
  • linkage disequilbrium is determined using the linkage disequilibrium measures r 2 and
  • linkage disequilibrium between marker is defined as r 2 > 0.1.
  • linkage disequilibrium is defined as r 2 > 0.2.
  • linkage disequilibrium such as r 2 > 0.25, r 2 > 0.3, r 2 > 0.35, r 2 > 0.4, r 2 > 0.45, r 2 > 0.5, r 2 > 0.55, r 2 > 0.6, r 2 > 0.65, r 2 > 0.7, r 2 > 0.75, r 2 > 0.8, r 2 > 0.85, r 2 > 0.9, r 2 > 0.95, r 2 > 0.96, r 2 > 0.97, r 2 > 0.98, or r 2 > 0.99.
  • Linkage disequilibrium can in certain embodiments also be defined as
  • linkage disequilibrium is defined as fulfilling two criteria of r 2 and
  • are also possible and within scope of the present invention, including but not limited to the values for these parameters set forth in the above. Assessment for markers and haplotypes
  • the genomic sequence within populations is not identical when individuals are compared. Rather, the genome exhibits sequence variability between individuals at many locations in the genome. Such variations in sequence are commonly referred to as polymorphisms, and there are many such sites within each genome
  • the human genome exhibits sequence , ⁇ variations which occur on average every 500 base pairs.
  • the most common sequence variant consists of base variations at a single base position in the genome, and such sequence variants, or polymorphisms, are commonly called Single Nucleotide Polymorphisms ("SNPs"). These SNPs are believed to have occurred in a single mutational event, and therefore there are usually two possible alleles possible at each SNPsite; the original allele and the mutated allele.
  • a polymorphic microsatellite has multiple small repeats of bases (such as CA repeats, TG on the complimentary strand) at a particular site in which the number of repeat lengths varies in the general population.
  • each version of the sequence with respect to the polymorphic site represents a specific allele of the polymorphic site.
  • These sequence variants can all be referred to as polymorphisms, occurring at specific polymorphic sites characteristic of the sequence variant in question.
  • polymorphisms can comprise any number of specific alleles.
  • the polymorphism is characterized by the presence of two or more alleles in any given population.
  • the polymorphism is characterized by the presence of three or more alleles.
  • the polymorphism is characterized by four or more alleles, fiva or more alleles, six or more alleles, seven or more alleles, nine or more alleles, or ten or more alleles. All such polymorphisms can be utilized in the methods and kits of the present invention, and are thus within the scope of the invention.
  • SNPs Due to their abundance, SNPs account for a majority of sequence variation in the human genome. Over 6 million SNPs have been validated to date
  • CNVs are known to affect gene expression, phenotypic variation and adaptation by disrupting gene dosage, and are also known to cause kidney stones (microdeletion and microduplication disorders) and confer risk of common complex kidney stones, including HIV-I infection and glomerulonephritis (Redon, R., et al. Nature 23:444-454 (2006)). It is thus possible that either previously described or unknown CNVs represent causative variants in linkage disequilibrium with the markers described herein.
  • Methods for detecting CNVs include comparative genomic hybridization (CGH) and genotyping, including use of genotyping arrays, as described by Carter (Nature Genetics 39:S16-S21 (2007)).
  • the Database of Genomic Variants http://projects.tcag.ca/variation/) contains updated information about the location, type and ,. size of described CNVs. The database currently contains data for over 15,000 CNVs.
  • reference is made to different alleles at a polymorphic site without choosing a reference allele.
  • a reference sequence can be referred to for a particular polymorphic site.
  • the reference allele is sometimes referred to as the "wild-type” allele and it usually is chosen as either the first sequenced allele or as the allele from a "non -affected" individual ⁇ e.g., an individual that does not display a trait or disease phenotype).
  • Alleles for SNP markers as referred to herein refer to the bases A, C, G or T as they occur at the polymorphic site in the SNP assay employed.
  • the assay employed may be designed to specifically detect the presence of one or both of the two bases possible, i.e. A and G.
  • variant sequence refers to a sequence that differs from the reference sequence but is otherwise substantially similar. Alleles at the polymorphic genetic markers described herein are variants. Variants can include changes that affect a polypeptide.
  • Sequence differences when compared to a reference nucleotide sequence, can include the insertion or deletion of a single nucleotide, or of more than one nucleotide, resulting in a frame shift; the change of at least one nucleotide, resulting in a change in the encoded amino acid; the change of at least one nucleotide, resulting in the , generation of a premature stop codon; the deletion of several nucleotides, resulting in a deletion of one or more amino acids encoded by the nucleotides; the insertion of one or several nucleotides, such as by unequal recombination or gene conversion, resulting in an interruption of the coding sequence of a reading frame; duplication of all or a part of a sequence; transposition; or a rearrangement of a nucleotide sequence,.
  • sequence changes can alter the polypeptide encoded by the nucleic acid.
  • the change in the nucleic acid sequence causes a frame shift
  • the frame shift can result in a change in the encoded amino acids, and/or cen result in the generation of a premature stop codon, causing generation of a truncated polypeptide.
  • a polymorphism associated with a kidney stones or trait can be a synonymous change in one or more nucleotides (i.e., a change that does not result in a change in the amino acid sequence).
  • Such a polymorphism can, for example, alter splice sites, affect the stability or' transport of mRNA, or otherwise affect the transcription or translation of an encoded polypeptide.
  • polypeptide encoded by the reference nucleotide sequence is the "reference” polypeptide with a particular reference amino acid sequence
  • polypeptides encoded by variant alleles are referred to as "variant" polypeptides with variant amino acid sequences.
  • a haplotype refers to a segment of DNA that is characterized by a specific combination of alleles arranged along the segment.
  • a haplotype comprises one member of the pair of alleles for each polymorphic marker or locus .
  • the haplotype can comprise two or more alleles, three or more alleles, four or more alleles, or five or more alleles, each allele corresponding to a specific polymorphic marker along the segment.
  • Haplotypes can comprise a combination of various polymorphic markers, e.g., SNPs and microsatellites, having particular alleles at the polymorphic sites. The haplotypes thus comprise a combination of alleles at various genetic markers.
  • Detecting specific polymorphic markers and/or haplotypes can be accomplished by methods known in the art for detecting sequences at polymorphic sites.
  • standard techniques for genotyping for the presence of SNPs and/or microsatellite markers can be used, such as fluorescence-based techniques (e.g., Chen, X. et a/., Genome Res. 9(5): 492-98 ( 1999); Kutyavin et al., Nucleic Acid Res. 34:el28 (2006)), utilizing PCR, LCR, Nested PCR and other techniques for nucleic acid amplification.
  • Specific commercial methodologies available for SNP genotyping include, but are not limited to, TaqMan genotyping assays and SNPIex platforms (Applied Biosystems), gel electrophoresis (Applied Biosystems), mass spectrometry (e.g.,
  • MassARRAY system from Sequenom minisequencing methods, real-time PCR, Bio-Plex system (BioRad), CEQ and SNPstream systems (Beckman), array hybridization technology(e.g., .;
  • one or more alleles at polymorphic markers including microsatellites, SNPs or other types of polymorphic markers, can be identified.
  • genotypes of un-genotyped relatives For every un-genotyped case, it is possible to calculate the probability of the genotypes of its relatives given its four possible phased genotypes. In practice it may be preferable to include only the genotypes of the case's parents, children, siblings, half-siblings (and the half-sibling's . parents), grand-parents, grand-children (and the grand-children's parents) and spouses. It will be assumed that the individuals in the small sub-pedigrees created around each case are not related through any path not included in the pedigree. It is also assumed that alleles that are not transmitted to the case have the same frequency - the population allele frequency. The probability of the genotypes of the case's relatives can then be computed by:
  • ⁇ denotes the A allele's frequency in the cases. Assuming the genotypes of each set of relatives are independent, this allows us to write down a likelihood function for ⁇ :
  • the likelihood function in (*) may be thought of as a pseudolikelihood approximation of the full likelihood function for ⁇ which properly accounts for all dependencies.
  • genotyped cases and controls in a case-control association study are not independent and applying the case-control method to related cases and controls is an analogous approximation.
  • the method of genomic control (Devlin, B. et al., Nat Genet 36, 1129-30; author reply 1131 (2004)) has proven to be successful at adjusting case-control test statistics for relatedness. The method of genomic control to account for the dependence between the terms in the pseudolikelihood is therefore typically used to produce a valid test statistic.
  • individual who is at an increased susceptibility (i.e., increased risk)' for forming kidney stones is an individual in whom at least one specific allele at one or more ⁇ ⁇ polymorphic marker or haplotype conferring increased susceptibility (increased risk) for kidney stones is identified (i.e., at-risk marker alleles or haplotypes).
  • the at-risk marker or haplotype is one that confers an increased risk (increased susceptibility) of kidney stones.
  • significance associated with a marker or haplotype is measured by a relative risk (RR).
  • significance associated with a marker or haplotye is measured by an odds ratio (OR).
  • the significance is measured by a percentage.
  • a significant increased risk is measured as a risk (relative risk and/or odds ratio) of at least 1.10, including but not limited to: at least 1.11, at least 1.12, at least 1.13, at least 1.14, at least 1.15, at least 1.16, at least 1.17, at least 1.18, at least 1.19, atleast 1.20, at least 1.21, at least 1.22, at least 1.23, at least 1.24, at least 1.25, at least 1.26, at least 1.27, at least 1.28, at least 1.29, at least 1.30, at least 1.40, at least 1.50, at least 1.60, at least 1.70, at least 1.80, at least 1.90, and at least 2.0.
  • a risk relative risk and/or odds ratio
  • a risk (relative risk and/or odds ratio) of at least 1.20 is significant.
  • a risk of at least 1.21 is significant.
  • a risk of at least 1.22 is significant.
  • a relative risk of at least 1.23 is significant.
  • a significant increase in risk is at least 1.24.
  • a significant increase in risk is at least about 10%, including but not limited to at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, or at least " 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • a significant increase in risk is at least 20%.
  • a significant increase in risk is at least 21%, at least 22%, at least 23%, at least 24%, or at least 25%.
  • a significant increase in risk is characterized-. ⁇ by a p-value, such as a p-value of less than 0.05, less than 0.01, less than 0.001, less than 0.0001, less than 0.00001, less than 0.000001, less than 0.0000001, less than 0.00000001, or t less than 0.000000001.
  • An at-risk polymorphic marker or haplotype of the present invention is one where at least one ' allele of at least one marker or haplotype is more frequently present in an individual at risk for the disease or trait (affected), compared to the frequency of its presence in a comparison group (control), and wherein the presence of the at-risk allele of the marker or the at-risk haplotype is indicative of susceptibility to the disease or trait.
  • the control group may in one embodiment be a population sample, i.e. a random sample from the general population.
  • the control group is represented by a group of individuals who are disease-free. Such disease-free controls may in one embodiment be characterized by the absence of one or more specific disease-associated symptoms.
  • the disease-free control group is characterized by the absence of one or more disease-specific risk factors.
  • risk ⁇ factors are in one embodiment at least one environmental risk factor.
  • Representative environmental factors are natural products, minerals or other chemicals which are known to affect, or contemplated to affect, the risk of developing the specific disease or trait.
  • Other environmental risk factors are risk factors related to lifestyle, including but not limited to food ; and drink habits, geographical location of mam habitat, and occupational risk factors.
  • the risk factors comprise at least one additional genetic risk factor.
  • protective effect ⁇ of certain alleles or haplotypes are generally multiplicative.
  • certain embodiments of the invention relate to individuals who are homozygous for at least one at-risk or protective variant (marker allele or haplotype), as described herein.
  • the effect of homozygous individuals is higher than would be expected based on the multiplicative model. This is indicative of a recessive component to the effect, which means that two copies of the variant, when brought together, have a greater overall effect than would be expected if the effect of each variant is added assuming no interaction between the two copies.
  • a simple test for correlation would be a Fisher-exact test on a two by two table.
  • the two by two table is constructed out of the number of chromosomes that include both of the markers or haplotypes, one of the markers or haplotypes but not the other and neither of the markers or haplotypes.
  • Other statistical tests of association known to the skilled person are also contemplated and are also within scope of the invention.
  • an individual who is at a decreased susceptibility (i.e., at a decreased risk) for a disease or trait is an individual in whom at least one specific allele at one or more polymorphic marker or haplotype conferring decreased susceptibility for the disease or trait is identified.
  • the marker alleles and/or haplotypes conferring decreased risk are also said to be protective.
  • the protective marker or haplotype is one that confers a significant decreased risk (or susceptibility) of the disease or trait.
  • significant decreased risk is measured as a relative risk (or odds ratio) of less than 0.9, including but not limited to less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2 and less than 0.1. In one particular embodiment, significant decreased risk is less than 0.7. In another embodiment, significant decreased risk is less than 0.5. In yet another embodiment, significant decreased risk is less than 0.3.
  • the decrease in risk is at least 20%, including but not limited to at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%', at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and at least 98%.
  • a significant decrease in risk is at least about 30%.
  • a significant decrease in risk is at least about 50%.
  • the decrease in risk is at least about 70%.
  • Other cutoffs or ranges as deemed suitable by the person skilled in the art to characterize the invention are however also contemplated, and those are also within scope of the present invention. 5
  • markers with two alleles present in the population being studied such as SNPs
  • the other allele of the marker will be found in decreased frequency in the group of individuals with the trait or disease, compared with controls.
  • one allele of the marker (the one found in increased frequency in individuals with the trait or disease) will be the at-risk allele, while the other allele will be a protective allele.
  • a genetic variant associated with a disease or a trait can be used alone to predict the risk of the disease for a given genotype.
  • a biallelic marker such as a SNP
  • the risk associated with each genotype can be estimated from the genotype of the individual. Risk associated with variants at multiple loci can be used to estimate overall risk.
  • Overall risk assessment calculations for a plurality of risk variants usually assume that the relative risks of different genetic variants multiply, i.e. the overall risk (e.g. , RR or OR) associated with a particular genotype combination is the product of the risk values for the genotype at each locus. If the risk presented is the relative risk for a person, or a specific genotype for a person, compared to a reference population with matched gender and ethnicity, then the combined risk - is the product of the locus specific risk values - and which also corresponds to an overall risk estimate compared with the population.
  • the overall risk e.g. , RR or OR
  • the combined risk corresponds to an estimate that compares the person with a given combination of genotypes at all loci to a group of individuals who do not carry risk variants at any of those loci.
  • the group of non-carriers of any at risk variant has the lowest estimated risk and has a combined risk , compared with itself (i.e., non-carriers) of 1.0, but has an overall risk, compare with the population, of less than 1.0. It should be noted that the group of non-carriers can potentially be very small, especially for large number of loci, and in that case, its relevance is correspondingly small.
  • the multiplicative model is a parsimonious model that usually fits the data of complex traits reasonably well. Deviations from multiplicity have been rarely described in the context of common variants for common diseases, and if reported are usually only suggestive since very large sample sizes are usually required to be able to demonstrate statistical interactions between loci.
  • the combined or overall risk associated with a plurality of variants described herein may be assessed. Furthermore the overall risk associated with at least one marker described herein in combination with any other markers associated with risk of kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis can be assessed.
  • Linkage Disequilibrium refers to a non-random assortment of two genetic elements. For example, if a particular genetic element (e.g., an allele of a polymorphic marker, or a haplotype) occurs in a population at a frequency of 0.50 (50%) and another element occurs at a frequency of 0.50 (50%), then the predicted occurrance of a person's having both elements is 0.25 (25%), assuming a random distribution of the elements.
  • a particular genetic element e.g., an allele of a polymorphic marker, or a haplotype
  • Allele or haplotype frequencies can be determined in a population by genotyping individuals in a population and determining the frequency of the occurence of each ' allele or haplotype in the population. For populations of diploids, e.g., human populations, individuals will typically have two alleles or allelic combinations for each genetic element (e.g., a marker, haplotype or gene).
  • is defined in such a way that it is equal to 1 if just two or three of the possible haplotypes are ' - present, and it is ⁇ 1 if all four possible haplotypes are present. Therefore, a value of
  • the measure r 2 represents the statistical correlation between two sites, and takes the value of 1 if only two haplotypes are 1 J present.
  • the r 2 measure is arguably the most relevant measure for association mapping, because there is a simple inverse relationship between r 2 and the sample size required to detect association between susceptibility loci and SNPs. These measures are defined for pairs of sites, but for some applications a determination of how strong LD is across an entire region that contains many polymorphic sites might be desirable (e.g., testing whether the strength of LD differs significantly among loci or across populations, or whether there is more or less LD in a region than predicted under a particular model). Measuring LD across a region is not straightforward, but one approach is to use the measure r, which was developed in population genetics.
  • r measures how much recombination would be required under a particular population, model to generate the LD that is seen in the data. This type of method can potentially also ; ' provide a statistically rigorous approach to the problem of determining whether LD data provide evidence for the presence of recombination hotspots.
  • a significant r 2 value can be at least 0.1 such as at least 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or at lesat 0.99.
  • the significant r 2 value can be at least 0.2.
  • linkage disequilibrium as described herein refers to linkage disequilibrium characterized by values of
  • linkage disequilibrium represents a correlation :' between alleles of distinct markers, It is measured by correlation coefficient or
  • linkage disequilibrium is defined in terms of values for both the r 2 and
  • a significant linkage disequilibrium is defined as r 2 > 0.1 and
  • a significant linkage j disequilibrium is defined as r 2 > 0.2 and
  • r 2 > 0.2 is , « significant linkage disequilibrium..
  • r 2 > 0.5 is significant linkage disequilibrium.
  • r 2 > 0.7 is significant linkage disequilibrium.
  • r 2 > 0.8 is significant linkage disequilibrium.
  • Linkage disequilibrium can be determined in a single human population, as defined herein, or it can be . determined in a collection of samples comprising individuals from more than one human population.
  • LD is determined in a sample from one or more of the HapMap populations (Caucasian, african, Japanese, Chinese), as defined
  • LD is determined in the CEU population of the HapMap samples. In another embodiment, LD is determined in the YRI population. In yet ' another embodiment, LD is determined in samples from the Icelandic population.
  • Genomic LD maps have been generated across the genome, and such LD maps have been , proposed to serve as framework for mapping disease-genes (Risen, N. & Merkiangas, K, Science 273: 1516-1517 (1996); Maniatis, N., et al., Proc Natl Acad Sci USA 99:2228-2233 (2002); Reich, DE et al, Nature 411 : 199-204 (2001)).
  • blocks can be defined as regions of DNA that have limited haplotype diversity (see, e.g., Daly, M. et al., Nature Genet. 29:229-232 (2001); Patil, N. et ai, Science 294: 1719-1723 (2001); Dawson, E. et al., Nature ⁇ 418: 544-548 (2002); Zhang, K. et al., Proc. Natl. Acad. Sci. USA 99: 7335-7339 (2002)), or as regions between transition zones having extensive historical recombination, identified using linkage disequilibrium (see, e.g., Gabriel, S. B.
  • the map reveals the enormous variation in recombination across the genome, with recombination rates as high as 10-60 cM/Mb in hotspots, while closer to 0 in intervening regions, which thus represent regions of limited haplotype diversity and high LD.
  • the map can therefore be used to define haplotype blocks/LD blocks as regions flanked by ; recombination hotspots.
  • haplotype block or "LD block” includes blocks defined by any of the above described characteristics, or other alternative methods used by the person skilled in the art to define such regions.
  • Haplotype blocks (LD blocks) can be used to map associations between phenotype and haplotype status, using single markers or haplotypes comprising a plurality of markers.
  • the main haplotypes can be identified in each haplotype block, and then a set of "tagging" SNPs or markers (the smallest set of SNPs or markers needed to distinguish among the haplotypes) can then be identified. These tagging SNPs or markers can then be used in assessment of samples from groups of individuals, in order to identify association between phenotype and haplotype. if desired, neighboring haplotype blocks can be assessed concurrently, as there may also exist linkage disequilibrium among the haplotype blocks.
  • markers used to detect association thus in a sense represent "tags" for a genomic region (i.e., a haplotype block or LD block) that is associating with a given disease or trait, and as such are useful for use in the methods and kits of the present invention.
  • Table 1 provides exemplary surrogate markers in linkage disequilibrium with the markers shown herein to be associated with risk of kidney stones and related conditions. Any one of these surrogate markers could suitably be chosen to detect the association to kidney stones described herein.
  • markers which are more tightly correlated [i.e., in greater LD) with the anchor markers are better surrogate markers than those which are less tightly correlated.
  • One or more causative (functional) variants or mutations may reside within the region found to ' be associating to the disease or trait.
  • the functional variant may be another SNP, a tandem ⁇ repeat polymorphism (such as a minisatellite or a microsatelhte), a transposable element, or a ' copy number variation, such as an inversion, deletion or insertion.
  • Such variants in LD with the ; variants described herein may confer a higher relative risk (RR) or odds ratio (OR) than observ ⁇ 1 for the tagging markers used to detect the association.
  • the present invention thus refers to the ⁇ markers used for detecting association to the disease, as described herein, as well as markers iri linkage disequilibrium with the markers.
  • markers that are in LD with the markers and/or haplotypes of the invention, as described herein may be used as surrogate markers.
  • the surrogate markers have in one embodiment relative risk (RR) and/or odds ratio (OR) values smaller than for the markers or haplotypes initially found to be J 5 associating with the disease, as described herein.
  • the surrogate markers have RR or OR values greater than those initially determined for the markers initially found to be associating with the disease, as described herein.
  • An example of such an embodiment would be a rare, or relatively rare (such as ⁇ 10% allelic population frequency) variant in LD with a more * , common variant (> 10% population frequency) initially found to be associating with the disease', such as the variants described herein. Identifying and using such markers for detecting the j association discovered by the inventors as described herein can be performed by routine methods well known to the person skilled in the art, and are therefore within the scope of the present invention.
  • the frequencies of haplotypes in patient and control groups can be estimated using an ⁇ ; expectation-maximization algorithm (Dempster A. et al., J. R. Stat. Soc. B 1 39: 1-38 (1977)). An implementation of this algorithm that can handle missing genotypes and uncertainty with the • phase can be used. Under the null hypothesis, the patients and the controls are assumed to * have identical frequencies. Using a likelihood approach, an alternative hypothesis is tested, where a candidate at-nsk-haplotype, which can include the markers described herein, is allowed to have a higher frequency in patients than controls, while the ratios of the frequencies of other haplotypes are assumed to be the same in both groups. Likelihoods are maximized separately s . under both hypotheses and a corresponding 1-df likelihood ratio statistic is used to evaluate the statistical significance.
  • At- ⁇ sk and protective markers and haplotypes within a susceptibility region for example within an LD block, association of all possible combinations of genotyped markers within the region is studied.
  • the combined patient and control groups can be randomly divided into two sets, equal in size to the original group of patients and controls.
  • the marker and haplotype analysis is then repeated and the most significant p-value registered is determined.
  • This i randomization scheme can be repeated, for example, over 100 times to construct an empirical ,' distribution of p-values.
  • a p-value of ⁇ 0.05 is indicative of a significant marker and/or haplotype association. ., '
  • haplotype analysis involves using likelihood-based inference applied to] NEsted MOdels (Gretarsdottir S., et al., Nat. Genet. 35: 131-38 (2003)).
  • the method is [ implemented in the program NEMO, which allows for many polymorphic markers, SNPs and microsatellites.
  • the method and software are specifically designed for case-control studies where the purpose is to identify haplotype groups that confer different risks. It is also a tool for c. studying LD structures.
  • maximum likelihood estimates, likelihood ratios and p-values are calculated directly, with the aid of the EM algorithm, for the observed data treating it as a missing-data problem.
  • the Fisher exact test can be used to calculate two- sided p-values for each individual allele. Usually, all p-values are presented unadjusted for multiple comparisons unless specifically indicated.
  • the presented frequencies are allelic frequencies as opposed to earner frequencies.
  • first and second-degree relatives can be eliminated from the patient list.
  • the test can be repeated for association correcting for any remaining relatedness among the patients, by extending a variance adjustment procedure previously described (Risch, N. & Teng, J.
  • the method of genomic controls (Devlin, B. & Roeder, K. Biometrics 55:997 (1999)) can also be used to adjust for the relatedness of the individuals and possible stratification. The differences are in general very small as expected. To assess the significance of single-marker association corrected for multiple testing we can carry out a randomization test using the same genotype data.
  • Cohorts of patients and controls can be randomized and the association analysis redone multiple times (e.g., up to 500,000 times) and the p-value is the fraction of replications that produced a p-value for some ⁇ marker allele that is lower than or equal to the p-value we observed using the original patient and control cohorts.
  • relative risk and the population attributable risk (PAR) can be calculated assuming a multiplicative model (haplotype relative risk model) (Terwilliger, J. D. & Ott, J., Hum. Hered. 42:337-46 (1992) and FaIk, CT. & Rubinstein, P, Ann. Hum. Genet. 51 (Pt j;:227-33 (1987)), i.e., that the risks of the two alleles/haplotypes a person carries multiply.
  • a multiplicative model haplotype relative risk model
  • haplotypes are independent, i.e., in Hardy-Weinberg equilibrium, within the affected population as well as within the control population. As a consequence, haplotype counts of the affecteds and controls each have multinomial distributions, but with different haplotype frequencies under the alternative hypothesis.
  • risk(/?,)/risk(fy) (fJ Pi)Kf ] / P j )
  • f and p denote, respectively, frequencies in the affected population and in ' ' the control population. While there is some power loss if the true model is not multiplicative, the loss tends to be mild except for extreme cases. Most importantly, p-values are always valid since they are computed with respect to null hypothesis.
  • An association signal detected in one association study may be replicated in a second cohort, .. ideally from a different population (e.g., different region of same country, or a different country) of the same or different ethnicity.
  • the advantage of replication studies is that the number of tests performed in the replication study, and hence the less stringent the statistical measure that is applied. For example, for a genome-wide search for susceptibility variants for a particular disease or trait using 300,000 SNPs, a correction for the 300,000 tests performed (one for each SNP) can be performed. Since many SNPs on the arrays typically used are correlated ⁇ i.e., in LD), they are not independent. Thus, the correction is conservative.
  • the results from several case-control cohorts can also be combined to provide an overall assessment of the underlying effect.
  • the methodology commonly used to combine results from multiple genetic association studies is the Mantel-Haenszel model (Mantel and Haenszel, J Natl ⁇ , Cancer Inst 22:719-48 (1959)).
  • the model is designed to deal with the situation where association results from different populations, with each possibly having a different population frequency of the genetic variant, are combined.
  • the model combines the results assuming that the effect of the variant on the risk of the disease , a measured by the OR or RR, is the same in all populations, while the frequency of the variant may differ between the poplations.
  • an absolute risk of developing a disease or trait defined as the chance of a person developing the specific kidney stones or trait over a specified time-period.
  • a woman's lifetime absolute risk of breast cancer is one in nine. That is to say, one woman in every nine will develop breast cancer at some point in their lives.
  • Risk is typically measured by looking at very large numbers of people, rather than at a particular individual. Risk is often presented in terms of Absolute Risk (AR) and Relative Risk (RR).
  • AR Absolute Risk
  • RR Relative Risk
  • Relative Risk is used to compare risks associating with two variants or the risks of two different groups of people. For ⁇ example, it can be used to compare a group of people with a certain genotype with another group having a different genotype.
  • a relative risk of 2 means that one group has, twice the chance of developing a disease as the other group.
  • Certain polymorphic markers have been found to be predictive of risk of kidney stones, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral • ' density and osteoporosis in humans. Risk assessment based on this finding may involve the use of the markers for determining a susceptibility to any one of these conditions.
  • Particular alleles of polymorphic markers e.g., SNPs
  • SNPs polymorphic markers
  • these marker alleles have predictive value " for detecting a risk or a susceptibility to any one of these conditions in an individual.
  • Tagging markers in linkage disequilibrium with at-risk variants (or protective variants) described herein . can also be used as surrogates for these markers (and/or haplotypes). Such surrogate markers can be located within a particular haplotype block or LD block. Such surrogate markers can also sometimes be located outside the physical boundaries of such a haplotype block or LD block, either in close vicinity of the LD block/haplotype block, but possibly also located in a more distant genomic location.
  • Long-distance LD can for example arise if particular genomic regions (e.g., genes) are in a functional relationship. For example, if two genes encode proteins that play a role in a shared metabolic pathway, then particular variants in one gene may have a direct impact on observed variants for the other gene. Let us consider the case where a variant in one gene leads to increased expression of the gene product. To counteract this effect and preserve overall flux of the particular pathway, this variant may have led to selection of one (or more) variants at a " ; second gene that conferes decreased expression levels of that gene. These two genes may be .
  • genomic regions e.g., genes
  • Markers with smaller values of r 2 than 1 can also be surrogates for the at-risk variant, or alternatively represent variants with relative ⁇ , risk values as high as or possibly even higher than the at-risk variant.
  • the at-risk variant identified may not be the functional variant itself, but is in this instance in linkage disequilibrium with the true functional variant.
  • the functional variant may for example be a tandem repeat, such as a minisatellite or a microsatellite, a transposable element (e.g., an AIu element), or a structural alteration, such as a deletion, insertion or inversion (sometimes also called copy number variations, or CNVs).
  • the present invention encompasses the assessment of such surrogate markers for the markers as disclosed herein.
  • markers are annotated, mapped , and listed in public databases, as well known to the skilled person ⁇ e.g. , as illustrated by the markers in Table 1), or can alternatively be readily identified by sequencing the region or a part of the region identified by the markers of the present invention in a group of individuals, and identify polymorphisms in the resulting group of sequences.
  • the person skilled in the art can readily and without undue experimentation genotype surrogate markers in linkage disequilibrium with the markers and/or haplotypes as described herein.
  • the tagging or surrogate markers in LD with the at-risk variants detected also have predictive value for detecting association to the condition, or a susceptibility to the condition, in an individual. ;:
  • the present invention can in certain embodiments be practiced by assessing a sample comprising genomic DNA from an individual for the presence of variants described herein to be , associated with kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis.
  • Such assessment typically steps that detect the presence or absence of at least one allele of at least : one polymorphic marker in the genome of the individual, using methods well known to the skilled person and further described herein, and based on the outcome of such assessment, determine whether the individual from whom the sample is derived is at increased or decreased risk (increased or decreased susceptibility) of kidney stones, hypercalciurea, elevated serum ., parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density' and/or osteoporosis. Detecting particular alleles of polymorphic markers can in certain embodiments be done by obtaining nucleic acid sequence data about a particular human individual, that identifies at least one allele of at least one polymorphic marker.
  • nucleic acid sequence data can comprise nucleic acid sequence at a single nucleotide position, which is sufficient to identify alleles at SNPs.
  • the nucleic acid sequence data can also comprise sequence at any other number of nucleotide positions, in particular for genetic markers that comprise multiple nuclotide positions, and can be anywhere from two to hundreds • of thousands, possibly even millions, of nucleotides (in particular, in the case of copy number ' variations (CNVs)).
  • the invention can be practiced utilizing a dataset comprising information about the genotype status of particular polylmorphic markers, such as any one of the markers shown herein to be associated with risk of kidney stones, hypercal ⁇ urea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis (or markers in linkage disequilibrium with any one of those markers).
  • polylmorphic markers such as any one of the markers shown herein to be associated with risk of kidney stones, hypercal ⁇ urea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis (or markers in linkage disequilibrium with any one of those markers).
  • a dataset containing information about such genetic status for example in the form of genotype counts at a certain polymorphic marker, or a plurality of markers (e.g., an indication of the presence or absence of certain at-risk alleles), or actual genotypes for one or more markers, can be queried for the presence or absence of certain at-risk alleles at certain polymorphic markers associated with any one of kidney stones, hypercalciurea, elevated serum parathyroid ' hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis .
  • a positive result for the presence of the variant e.g., marker allele or haplotype
  • Determination of the absence of the variant allele is indicative of the individual not being at increased suscepbility of the condition caused by the particular variant.
  • a polymorphic marker is correlated to a a condition or disease (such as kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis) by , referencing genotype data for the polymorphic marker to a look-up table that comprises correlations between at least one allele of the polymorphism and the condition.
  • the table comprises a correlation for one polymorhpism.
  • the table comprises a correlation for a plurality of polymorhpisms.
  • a risk for the condition can be identified in the individual from whom the sample is derived.
  • the correlation is reported as a statistical measure.
  • the statistical measure may be reported as a risk measure, such as a relative risk (RR), an absolute risk (AR) or an odds ratio (OR).
  • the markers described herein may be useful for risk assessment and diagnostic purposes, either alone or in combination.
  • Results of risk of kidney stones or related conditions based on genotype results for the markers described herein can also be combined with data for other genetic markers or risk factors for the kidney stones or the related conditions, to establish overall risk.
  • the association may have significant implications.
  • relatively common variants may have significant contribution to the overall risk (Population Attributable Risk is high), or combination of markers can be used to define groups of individual who, based on the combined risk of the markers, are at even greater , overall risk.
  • a plurality of variants is used for overall risk assessment.
  • variants are in one embodiment selected from the variants as disclosed herein.
  • Other embodiments include the use of the variants of the present invention in combination with other variants known to be useful for diagnosing a susceptibility to kidney stones or related conditions, including hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone,-, mineral density and osteoporosis.
  • the genotype status of a plurality of markers and/or haplotypes is determined in an individual, and the status of the individual compared with the population frequency of the associated variants, or the frequency of the variants in clinically healthy subjects, such as age-matched and sex-matched subjects.
  • Methods known in the art such as multivariate analyses or joint risk analyses or other methods known to the skilled person, may subsequently be used to determine the overall risk conferred based on the genotype status at the multiple loci. Assessment of risk based on such analysis may subsequently be used in the methods, uses and kits of the invention, as described herein.
  • the haplotype block structure of the human genome has the effect that a large number of variants (markers and/or haplotypes) in linkage disequilibrium with the . variant originally associated with a kidney stones or trait may be used as surrogate markers for' assessing association to the kidney stones or trait.
  • the number of such surrogate markers will , depend on factors such as the historical recombination rate in the region, the mutational frequency in the region (i.e., the number of polymorphic sites or markers in the region), and the extent of LD (size of the LD block) in the region.
  • markers are usually located within the physical boundaries of the LD block or haplotype block in question as defined using the methods described herein, or by other methods known to the person skilled in the art. However, sometimes marker and haplotype association is found to extend beyond the physical boundaries' of the haplotype block as defined, as discussed in the above. Such markers and/or haplotypes . may in those cases be also used as surrogate markers and/or haplotypes for the markers and/or haplotypes physically residing within the haplotype block as defined.
  • markers and haplotypes in LD are also within the scope of the invention, even if they are physically located beyond the boundaries of the haplotype block as defined.
  • the opposite allele to the allele found to be in excess in , individuals with kidney stones or related conditions is found in decreased frequency in those individuals.
  • Such marker alleles and/or haplotypes in LD are thus protective ' for the condition, i.e. they confer a decreased risk or susceptibility of individuals carrying these ⁇ markers and/or haplotypes developing the condition.
  • haplotypes comprise, in some cases, " a combination of various genetic markers, e.g., SNPs and microsatellites. Detecting haplotypes ' can be accomplished by methods known in the art and/or described herein for detecting sequences at polymorphic sites. Furthermore, correlation between certain haplotypes or sets of markers and kidney stones or related conditions can be verified using standard techniques. A representative example of a simple test for correlation would be a Fisher-exact test on a two by two table.
  • a marker allele or haplotype found to be associated with kidney stones' or related conditions is one in > which the marker allele or haplotype is more frequently present in an individual at risk for kidney stones or the related condition (affecteds), compared to the frequency of its presence in a healthy individual (control), or in randombly selected individual from the population, wherein the presence of the marker allele or haplotype is indicative of a susceptibility of kidney stones or the related condition.
  • At-risk markers in linkage disequilibrium with one or - more markers shown herein to be associated with kidney stones and related conditions are tagging markers that are also predictive of risk for kidney stones or related conditions.
  • the methods and kits of the invention can be utilized from samples containing nucleic acid material (DNA or RNA) from any source and from any individual, or from genotype data derived from such samples.
  • the individual is a human individual.
  • the individual can be an adult, child, or fetus.
  • the nucleic acid source may be any sample comprising nucleic acid material, including biological samples, or a sample comprising nucleic acid material derived therefrom.
  • the present invention also provides for assessing markers and/or haplotypes in individuals who are members of a target population.
  • a target population is in one embodiment a population or group of individuals at risk of developing the , kidney stones, based on other genetic factors, biomarkers, biophysical parameters (e.g. , serum bicarbonate levels, urinary calcium levels, serum parathyroid hormone levels, bone mineral ; density measurements), or general health and/or lifestyle parameters (e.g. , history of kidney stones, previous diagnosis of kidney stones, family history of kidney stones).
  • the invention provides for embodiments that include individuals from specific age subgroups, such as those over the age of 40, over age of 45, or over age of 50, 55, 60, 65, 70, 75, 80, or 85.
  • Other embodiments of the Invention pertain to other age groups, such as individuals aged less than 85, such as less than age 80, less than age 75, or less than age 70, 65, 60, 55, 50, 45, 40, 35, or age 30.
  • Other embodiments relate to individuals with previous history of kidney stones in any of the age ranges described in the above. It is also contemplated that a range of ' , ages may be relevant in certain embodiments, such as age at onset at more than age 45 but less than age 60. Other age ranges are however also contemplated, including all age ranges bracketed by the age values listed in the above.
  • the invention furthermore relates to individuals of either gender, males or females.
  • the Icelandic population is a Caucasian population of Northern European ancestry.
  • a large number of studies reporting results of genetic linkage and association in the Icelandic population have been published in the last few years. Many of those studies show replication of variants, originally identified in the Icelandic population as being associating with a particular kidney stones, in other populations (Styrkarsdottir, U., et a/. N Engl J Med Apr 29 2008 (Epub ahead of ; print); Thorgeirsson, T., et al. Nature 452:638-42 (2008); Gudmundsson, J., et al. Nat Genet. 40: 281-3 (2008); Stacey, S. N., et al., Nat Genet.
  • Such embodiments relate to human subjects that are from one or more human population including, but not limited to, Caucasian populations, European ; . populations, American populations, Eurasian populations, Asian populations, Central/South Asian populations, East Asian populations, Middle Eastern populations, African populations, Hispanic populations, and Oceanian populations.
  • European populations include, but are not limited to, Swedish, Norwegian, Finnish, Russian, Danish, Icelandic, Irish, Kelt, English, Scottish, Dutch, Belgian, French, German, Spanish, Portugues, Italian, Polish, Bulgarian, Slavic, Serbian, Laun, Czech, Greek and Vietnamese populations.
  • the invention furthermore in other embodiments can be * practiced in specific human populations that include Bantu, Mandenk, Yoruba, San, Mbuti Pygmy, Orcadian, Adygei, Russian, Sardinian, Tuscan, Mozabite, Bedouin, Druze, Vietnamese, Balochi, .; Brahui, Makrani, Sindhi, Pathan, Burusho, Hazara, Uygur, Kalash, Han, Dai, Daur, Hezhen, Lahu, Miao, Oroqen, She, Tujia, Tu, Xibo, Yi, Mongolan, Naxi, Cambodian, Japanese, Yakut, Melanesian, Papuan, Karitianan, Surui, Colmbian, Maya and Pima. '
  • the invention relates to populations that include black African ancestry such as populations comprising persons of African descent or lineage.
  • Black African ancestry may be determined by self reporting as African-Americans, Afro-Americans, Black Americans, being a member of the black race or being a member of the negro race.
  • African Americans or Black Americans are those persons living in North America and having origins in any of the black racial groups of Africa.
  • self-reported persons of black African ancestry may have at least one parent of black African ancestry or at least one grandparent of black African ancestry.
  • the invention relates to individuals of Caucasian origin. The racial contribution in individual subjects may also be determined by genetic analysis. Genetic analysis of ancestry may be carried out using unlinked microsatellite markers such as those set out in Smith et al. (Am J Hum Genet 74, 1001-13 (2004)).
  • the invention relates to markers and/or haplotypes identified in specific populations, as described in the above.
  • measures of linkage disequilibrium (LD) may give different results when applied to different populations. This is due to different population history of different human populations as well as differential selective pressures that may have led to differences in LD in specific genomic regions.
  • certain markers e.g. SNP markers, have different population frequncy in different populations, or are polymorphic in one population but not in another. The person skilled in the art will however apply the methods available and as , thought herein to practice the present invention in any given human population.
  • This may include assessment of polymorphic markers in the LD region of the present invention, so as to .. identify those markers that give strongest association within the specific population.
  • the at-risk variants of the present invention may reside on different haplotype background and in different frequencies in various human populations.
  • the invention can be practiced in any given human population.
  • variants described herein in general do not, by themselves, provide an absolute identification of individuals who will develop a particular disease.
  • the variants described herein do however indicate increased and/or decreased likelihood that individuals carrying the at-risk or protective variants of the invention will develop one or more of kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis. It has been discovered that certain variants confer increase risk of developing these disorders, as ' supported by the results presented herein.
  • This information is extremely valuable in itself, as outlined in more detail in the below, as it can be used to, for example, initiate preventive measures at an early stage, perform regular physical exams to monitor the progress and/or appearance of symptoms, or to schedule exams at a regular interval to identify early symptoms ' , so as to be able to apply treatment at an early stage.
  • the knowledge about a genetic variant that confers a risk of developing a disease or condition * offers the opportunity to apply a genetic test to distinguish between individuals with increased risk of developing the condition (i.e. carriers of the at-risk variant) and those with decreased risk of developing the condition (i.e. carriers of the protective variant).
  • the core values of genetic '; testing, for individuals belonging to both of the above mentioned groups, are the possibilities of 1 being able to diagnose the condition, or a predisposition to the condition, at an early stage and ' provide information to the clinician about prognosis/aggressiveness of the condition in order to " be able to apply the most appropriate treatment.
  • kidney stones go unnoticed until they cause acute symptoms - specifically, pain associated with the stone going through the ureter, excluding other possible sources of the pain.
  • kidney stones are discovered during course of looking for the cause of urinary tract infections or blood in the urine. Suspicion of kidney stones is usually followed by . blood analysis, to look for excess calcium or uric acid, as well as a 24-hour collection of urine to check for stone-forming minerals. Imaging tests may also be performed, the most common being a helical CT (computer tomography) scan without contrast material. Pregnant women, or others who should avoid radiation exposure, may undergo ultrasound examination to establish diagnosis. Ultrasound may however miss small stones, especially if located in the ureter or : bladder.
  • Kidney stones may become recurrent, especially in those with more than one kidney stone and in those with a family history of kidney stones.
  • Preventive measures than may be taken to minimize the risk of kidney stones include (i) increased volume of fluids consumed; and (ii) altered diet, for example by increasing fiber consumption, eating less meat, consumption of moderate or high amounts of calcium-rich foods, avoiding food that is high in oxalate (e.g., dark green vegetables, nuts, chocolate), and minimizing salt in your diet.
  • Preventive medication may also be taken, which - depending on the type of stones you have or are likely to have - may include, for calcium stones: thiazides ⁇ e.g., hydrochlorothiazide, chlorthalidone), potassium citrate, orthophosphate and chlestyramine; for uric acid stones: potassium citrate, sodium bicarbonate, and allopurinol; for cystine stones: potassium citrate, pinicillamine, tipronin and captopril; for struvite stones: urease inhibitors (e.g., lithostat).
  • thiazides ⁇ e.g., hydrochlorothiazide, chlorthalidone
  • potassium citrate orthophosphate and chlestyramine
  • uric acid stones potassium citrate, sodium bicarbonate, and allopurinol
  • cystine stones potassium citrate, pinicillamine, tipronin and captopril
  • struvite stones urease inhibitors (e.g., lit
  • the most common metabolic risk factor for kidney stones in adults and children is hypercalciurea. Furthermore, about 20% of patients with hypercalciurea have a family history of kidney stones, showing that the disorders are intertwined.
  • Determination of the presence of a genetic risk factor for kidney stones or traits that predispose to kidney stones, such as hypercalciurea may be used to guide individuals towards a diet that minimizes risk of kidney stones. This may be done in conjunction with blood and urine measurements to determine whether the individual has hypercalciurea, decreased serum bicarbonate levels or decreased serum bicarbonate levels. Individuals testing positive for one or more of those risk factors and who also are carrying at least one genetic risk factor for kidney stones, as described herein, may also especially benefit from preventive measures for kidney ' stones. A family history of kidney stones may also be used in combination with the genetic test , and biomarker evaluation(s) to assess the appropriate course of action for the individual.
  • Individuals determined to have an increased genetic risk of kidney stones may be monitered more closely. For example, regular determination of urinary calcium levels may be performed. As urinary calcium levels are increased, recommendation of a calcium-reducing diet may be recommended, to minimize the risk of stone formation.
  • Individual disease risk management ' may also take into account other factors, including family history of kidney stones, family history of hypercalciurea or other metabolic risk factors of kidney stones, and a history of other related disorders.
  • a personal risk management profile may thus include genetic testing in combination with a medical history of the individual, and his/her immediate family, and may also include additional biochemical tests. This way, an individualized disease risk management program may be developed, based on a combination of genetic risk factors, biochemical measurements, medical history and family history.
  • the genetic profiling may be key in deciding which individuals should be recommended for extensive follow-up and/or monitoring, including monitoring of calcium levels in urine, oxalate levels in urine, uric acid levels in urine, cystine levels in urine and citrate levels in urine, for early prevention and detection of stones.
  • DNA-genotyping of markers associated with decreased bone mineral density could be of value in identifying those individuals that could benefit from earlier BMD measurements in order to target preventive start of anti-fracture therapy.
  • BMD measurements or DNA-genotyping of markers associated with decreased BMD
  • DNA-genotyping of markers associated with decreased BMD could be especially of value in first-degree relatives' of individuals with low bone mass. This should especially be applied to individuals likely to go into a phase of bone loss such as women at the menopause or individuals receiving certain medications known to affect the bone metabolism such as glucocorticoids. It is also possible that bone density measurements and DNA-genotyping could be of value in children of families with strikingly low bone mass indicating genetic influence.
  • low peak bone mass might be related to insufficient response to mechanical or physical loading at ⁇ [ young age when the bones are fast growing. It is possible that those individuals diagnosed by DXA or by DNA testing need extra physical exercise to gain optimal peak bone mass. It is also possible that safe drug therapy could be of use in individuals with strikingly low bone mass at young age if the drug is anabolic on the osteoblasts.
  • Variants associated with decreased BMD might also be used as diagnostics markers for those that would benefit the most from treatment options that target osteoporosis. For example, denosumbab is currently being tested in a phase III clinical trial aimed at preventing bone loss , with fracture as an end point. Such markers might also be used for identifying those individuals that are/will be receiving certain medications known to induce bone loss, such as glucocorticoids, prior to treatment in order to identify those that need contra-treatment for preventing the bone loss, i.e. individuals at risk of low bone mass for whom such additional bone loss will be detrimental and cause low trauma fractures.
  • certain medications known to induce bone loss such as glucocorticoids
  • the invention also encompasses methods of assessing an individual for probability of response to a therapeutic agents, methods for predicting the effectiveness of a therapeutic agent, nucleic acids, polypeptides and antibodies and computer-implemented functions. Kits for use in the various methods presented herein are also encompassed by the invention. Diagnostic and screening methods
  • the present invention pertains to methods of diagnosing, or aiding in : the diagnosis of, at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis or a susceptibility to the condition, by detecting particular alleles at genetic markers that appear more frequently in subjects with these conditions or subjects who are susceptible to the conditions.
  • the invention is a method of determining a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, by detecting, or determining the identity of, at least one allele of at least one polymorphic marker (e.g., the markers described herein).
  • the present invention describes methods whereby detection of particular alleles of particular markers or haplotypes is indicative of a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis.
  • the present invention pertains in some embodiments to methods of clinical applications of diagnosis, e.g., diagnosis performed by a medical professional.
  • the invention pertains to methods of diagnosis or determination of a susceptibility performed by a layman.
  • the layman can be the customer of a genotyping service.
  • the layman may also be a genotype or sequence data service provider, who performs genotype analysis on a DNA sample from an individual, in order to provide service related to genetic risk factors for particular traits or kidney stoness, based on the genotype/sequence status of the individual (i.e., the customer).
  • genotyping technologies including high-throughput ganotyping of SNP markers, such as Molecular Inversion Probe array technology (e.g., Affymetrix ..
  • genotype data can be generated by determination of the identity of certain marker alleles by direct sequencing of genomic DNA.
  • the resulting genotype information which can be made available to the individual, can be compared' to information about kidney stones or trait risk associated with various SNPs, including information from public litterature and scientific publications.
  • kidney stones(or related conditions)-associated alleles as described herein can thus for example be performed by the individual, through analysis of his/her genotype data, by a health professional based on results of a clinical test, or by a third party, including the genotype service provider.
  • the third party may also be service provider who interprets genotype information from the customer to provide service related to specific genetic risk factors, including the genetic markers described herein.
  • the diagnosis or determination of a susceptibility of genetic risk can be made by health professionals, genetic counselors, third parties providing genotyping service, third parties providing risk assessment service or by the layman (e.g., the * _ individual), based on information about the genotype status of an individual and knowledge about the risk conferred by particular genetic risk factors (e.g., particular SNPs).
  • diagnosis can be made by health professionals, genetic counselors, third parties providing genotyping service, third parties providing risk assessment service or by the layman (e.g., the * _ individual), based on information about the genotype status of an individual and knowledge about the risk conferred by particular genetic risk factors (e.g., particular SNPs).
  • the term “diagnosing”, “diagnose a susceptibility” and “determine a susceptibility” is ⁇ -, meant to refer to any available diagnostic method, including those mentioned above. ⁇ ' ⁇
  • a sample containing genomic DNA from an individual is collected.
  • sample can for example be a buccal swab, a saliva sample, a blood sample, or other suitable samples containing genomic DNA, as described further herein.
  • the genomic DNA is then analyzed using any common technique available to the skilled person, such as high-throughput , ' array technologies. Results from such genotyping are stored in a convenient data storage unit, such as a data carrier, including computer databases, data storage disks, or by other convenient data storage means.
  • the computer database is an object database, a relational database or a post-relational database.
  • genotype data is subsequently analyzed for the presence of certain variants known to be susceptibility variants for a particular human conditions, such as the genetic variants described herein.
  • Genotype data can be retrieved from . the data storage unit using any convenient data query method.
  • Calculating risk conferred by a particular genotype for the individual can be based on comparing the genotype of the individual to previously determined risk (expressed as a relative risk (RR) or and odds ratio (OR), for example) for the genotype, for example for an heterozygous carrier of an at-risk variant. In other embodiments, risk for homozygous carriers of particular at-risk variants is calculated.
  • RR relative risk
  • OR odds ratio
  • the calculated risk for the individual can be the relative risk for a person, or for a specific genotype of a person, compared to the average population with matched gender and ethnicity.
  • the average population risk can be expressed as a weighted average of the risks of different genotypes, using results from a reference population, and the appropriate calculations to calculate the risk of a genotype group relative to the population can then be performed.
  • the risk for an individual is based on a comparison of particular genotypes, for example heterozygous carriers of an at-risk allele of a marker compared with non-carriers of the at-risk allele.
  • population average may in certain embodiments be more convenient, since it provides a measure which is easy to interpret for the user, i.e. a measure that gives the risk for the individual, based on his/her genotype, compared with the average in the population.
  • the calculated risk estimated can be made available to the customer via a website, preferably a secure website.
  • a service provider will include in the provided service all of the steps of isolating genomic DNA from a sample provided by the customer, performing genotyping of the isolated DNA, calculating genetic risk based on the genotype data, and report the risk to the customer.
  • the service provider will include in the service the interpretation of genotype data for the individual, i.e. , risk estimates for particular genetic variants based on the genotype data for the individual .
  • the service provider may include service that includes genotyping service and interpretation of the genotype data, starting from a sample of isolated DNA from the individual (the customer).
  • Overall risk for multiple risk variants can be performed using standard methodology. For example, assuming a multiplicative model, i.e. assuming that the risk of individual risk variants ⁇ multiply to establish the overall effect, allows for a straight-forward calculation of the overall risk for multiple markers.
  • significance of association of particular marker alleles or haplotypes is 1 characterized by a p value ⁇ 0.05.
  • the significance of association is characterized by smaller p-values, such as ⁇ 0.01, ⁇ 0.001, ⁇ 0.0001, ⁇ 0.00001, ⁇ 0.000001, ; . ⁇ 0.0000001, ⁇ 0.00000001 or ⁇ 0.000000001.
  • the significant risk is characterized by particular values of Relative Risk (RR) or Absolute Risk (AR), or lifetime risk.
  • the presence of particular marker alleles or haplotypes is indicative of a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis.
  • Diagnostic methods involve determining whether particular alleles or " haplotypes that are associated with risk of kidney stones or related conditions are present in particular individuals.
  • the haplotypes described herein include combinations of alleles at various genetic markers ⁇ e.g., SNPs, microsatellites or other genetic variants).
  • the detection of the particular genetic marker alleles that make up particular haplotypes can be performed by a variety of methods described herein and/or known in the art.
  • genetic markers can be detected at the nucleic acid level (e.g., by direct nucleotide sequencing, or by other genotyping means known to the skilled in the art) or at the amino acid level if the genetic marker affects the coding sequence of an encocded protein (e.g., CLDN14; e.g., by protein sequencing or by immunoassays using antibodies that recognize such a protein).
  • an encocded protein e.g., CLDN14; e.g., by protein sequencing or by immunoassays using antibodies that recognize such a protein.
  • the marker alleles or haplotypes of the present invention correspond to fragments of genomic segments associated with risk of kidney stones and related conditions. These segments in certain embodiments comprise a portion of the human CLDN14 gene.
  • Such fragments encompass the " DNA sequence of the polymorphic marker or haplotype in question, but may also include DNA segments in strong LD (linkage disequilibrium) with the marker or haplotype.
  • such segments comprises segments in LD with the marker or haplotype as determined by a value of r 2 greater than 0.2 and/or
  • determination of a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis is accomplished using hybridization methods, (see Current Protocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley & Sons, including all supplements).
  • the presence of a specific marker allele can be indicated by sequence-specific hybridization of a nucleic acid probe specific for the particular allele.
  • the presence of more than one specific marker allele or a specific haplotype can be indicated by using several sequence-specific nucleic acid probes, each being specific for a particular allele.
  • a sequence-specific probe can be directed to hybridize to genomic DNA, RNA, or cDNA.
  • a "nucleic acid probe”, as used herein, can be a DNA probe or an RNA probe that hybridizes to a complementary sequence.
  • One of skill in the art would know how to design such a probe so that sequence specific hybridization will occur only if a particular allele is present in a genomic sequence from a test sample.
  • the invention can also be reduced to practice using any convenient genotyping method, including commercially available technologies and methods for genotyping particular polymorphic markers.
  • a hybridization sample can be formed by contacting the test sample containing a nucleic acid, such as a genomic DNA sample, with at least one nucleic acid probe.
  • a probe for detecting mRIMA or genomic DNA is a labeled nucleic acid probe that is capable of hybridizing to mRNA or genomic DNA sequences described herein.
  • the nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof,' such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length that is , sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA.
  • the nucleic acid probe can comprise all or a portion of the nucleotide sequence of the LD block C21, as described herein, optionally comprising at least one allele of a marker described herein, or at least one haplotype described herein, or the probe can be the complementary sequence of such a sequence.
  • the nucleic acid probe is a portion of the nucleotide sequence of LD Block C21, as described herein, optionally comprising at least one allele of a marker described herein, or at least one allele of one polymorphic marker or haplotype comprising at least one polymorphic marker described herein, or the probe can be the complementary sequence of such a sequence.
  • Other suitable probes for use in the diagnostic assays of the invention are described herein.
  • Hybridization can be performed by methods well known to the person skilled in the art (see, e.g., Current Protocols in Molecular Biology, Ausubel, F. et al. , eds., John Wiley & Sons, including all supplements).
  • hybridization refers to specific hybridization, i.e., hybridization with no mismatches (exact hybridization).
  • the hybridization conditions for specific hybridization are high stringency.
  • Specific hybridization if present, is detected using standard methods. If specific hybridization occurs between the nucleic acid probe and the nucleic acid in the test sample, then the sample • contains the allele that is complementary to the nucleotide that is present in the nucleic acid probe.
  • the process can be repeated for any markers of the present invention, or markers that . make up a haplotype of the present invention, or multiple probes can be used concurrently to detect more than one marker alleles at a time. It is also possible to design a single probe containing more than one marker alleles of a particular haplotype (e.g., a probe containing alleles complementary to 2, 3, 4, 5 or all of the markers that make up a particular haplotype).
  • Detection of the particular markers of the haplotype in the sample is indicative that the source of the sample has the particular haplotype (e.g., an at-risk haplotype).
  • a method utilizing a detection oligonucleotide probe comprising a fluorescent moiety or group at its 3' terminus and a quencher at its 5' terminus, and an enhancer oligonucleotide, is employed, as described by Kutyavin et al. (Nucleic Acid Res. 34:el28 (2006)).
  • the fluorescent moiety can be Gig Harbor Green or Yakima Yellow, or other suitable fluorescent moieties.
  • the detection probe is designed to hybridize to a short nucleotide sequence that includes the SNP polymorphism to be detected.
  • the SNP is anywhere from the terminal residue to -6 residues from the 3' end of the detection probe.
  • the enhancer is a short oligonucleotide probe which hybridizes to the DNA template 3' relative to the detection probe.
  • the probes are designed such that a single nucleotide gap exists between the detection probe and the enhancer nucleotide probe when both are bound to the template.
  • the gap creates a synthetic abasic site that is recognized by an endonuclease, such as Endonuclease IV.
  • the ⁇ enzyme cleaves the dye off the fully complementary detection probe, but cannot cleave a detection probe containing a mismatch.
  • assessment of the presence of a particular allele defined by nucleotide sequence of the detection probe can be performed.
  • the detection probe can be of any suitable size, although preferably the probe is relatively short. In one embodiment, the probe is from 5-100 nucleotides in length. In another embodiment, the probe is from 10-50 nucleotides in length, and in another embodiment, the probe is from 12-30 nucleotides in length. Other lengths of the probe are possible and within scope of the skill of the average person skilled in the art.
  • the DNA template containing the SNP polymorphism is amplified by Polymerase Chain Reaction (PCR) prior to detection.
  • PCR Polymerase Chain Reaction
  • the amplified DNA serves as the template for the detection probe and the enhancer probe.
  • Certain embodiments of the detection probe, the enhancer probe, and/or the primers used for amplification of the template by PCR include the use of modified bases, including modified A and modified G,
  • modified bases can be useful for adjusting the melting temperature of ⁇ , the nucleotide molecule (probe and/or primer) to the template DNA, for example for increasing the melting temperature in regions containing a low percentage of G or C bases, in which modified A with the capability of forming three hydrogen bonds to its complementary T can be ' used, or for decreasing the melting temperature in regions containing a high percentage of G or C bases, for example by using modified G bases that form only two hydrogen bonds to their complementary C base in a double stranded DNA molecule.
  • modified bases are used in the design of the detection nucleotide probe. Any modified base known to the skilled person can be selected in these methods, and the selection of suitable bases is well within the scope of the skilled person based on the teachings herein and known bases available from commercial sources as known to the skilled person.
  • a peptide nucleic acid (PNA) probe can be used in addition to, or instead of, a nucleic acid probe in the hybridization methods described herein.
  • a PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, Nielsen, P., et a/., Bioconjug. Chem. 5:3-7 (1994)).
  • the PNA probe can be designed to specifically hybridize to a molecule in a sample suspected of containing one or more of the marker alleles or haplotypes that are associated with risk of at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis. Hybridization of the PNA probe is thus diagnostic risk of these conditions.
  • a test sample containing genomic DNA obtained from the ' subject is collected and the polymerase chain reaction (PCR) is used to amplify a fragment comprising one ore more markers or haplotypes of the present invention.
  • PCR polymerase chain reaction
  • identification of a particular marker allele or haplotype can be accomplished using a variety of methods (e.g., sequence analysis, analysis by restriction digestion, specific hybridization, single stranded conformation polymorphism assays (SSCP), electrophoretic analysis, etc.).
  • diagnosis is accomplished by expression analysis, for example by using quantitative PCR (kinetic thermal cycling).
  • This technique can, for example, utilize commercially available technologies, such as TaqMan ® (Applied Biosystems, Foster City, CA).
  • the technique - can assess the presence of an alteration in the expression or composition of a polypeptide or splicing variant(s), for example of the CLDN14 gene. Further, the expression of the variant(s) can be quantified as physically or functionally different.
  • restriction digestion in another embodiment, analysis by restriction digestion can be' used to detect a particular allele if the allele results in the creation or elimination of a restriction' site relative to a reference sequence.
  • Restriction fragment length polymorphism (RFLP) analysis can be conducted, e.g., as described in Current Protocols in Molecular Biology, supra. The digestion pattern of the relevant DNA fragment indicates the presence or absence of the particular allele in the sample.
  • Sequence analysis can also be used to detect specific alleles or haplotypes. Therefore, in one embodiment, determination of the presence or absence of a particular marker alleles or haplotypes comprises sequence analysis of a test sample of DNA or RNA obtained from a subject or individual. PCR or other appropriate methods can be used to amplify a portion of a nucleic acid that contains a particular polymorphic marker or haplotype, and the presence of specific alleles can then be detected directly by sequencing the polymorphic site (or multiple polymorphic sites in a haplotype) of the genomic DNA in the sample.
  • arrays of oligonucleotide probes that are complementary to target : . nucleic acid sequence segments from a subject can be used to identify particular alleles at polymorphic sites.
  • an oligonucleotide array can be used.
  • Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. These arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods that incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods, or by other methods known to the person skilled in the art (see, e.g., Bier, F. F., ef al.
  • nucleic acid analysis that are available to those skilled in the art can be used ; : to detect a particular allele at a polymorphic site.
  • Representative methods include, for example, direct manual sequencing (Church and Gilbert, Proc. Natl. Acad. Sci. USA, 81 : 1991-1995 ( 1988); Sanger, F., et al., Proc. Natl. Acad. Sci. USA, 74: 5463-5467 (1977); Beavis, et al., U.S. Patent No.
  • CMC chemical mismatch cleavage
  • RNase ⁇ protection assays Myers, R., et al. , Science, 230: 1242-1246 ( 1985); use of polypeptides that recognize nucleotide mismatches, such as E. coli mutS protein; and allele-specific PCR.
  • diagnosis of, or a determination of, a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and ; ⁇ osteoporosis can be made by examining expression and/or composition of a polypeptide encoded by a nucleic acid associated with the condition, in those instances where the genetic marker(s) or haplotype(s) of the present invention result in a change in the composition or expression of ' the polypeptide.
  • the polypeptide is a CLDN 14 polypeptide encoded by the human CLDN14 gene.
  • determination of a susceptibility to the condition can be made by examining expression and/or composition of such a polypeptide, in those instances where the genetic marker or haplotype of the present invention results in a change in the composition or . expression of the polypeptide.
  • the haplotypes and markers of the present invention that are associaed to kidney stones and related conditions may play a role through their effect on particular genes, such as the CLDN14 gene. Possible mechanisms affecting these genes include, e.g. , effects on transcription, effects on RNA splicing, alterations in relative amounts of alternative splice forms of mRNA, effects on RNA stability, effects on transport from the nucleus to cytoplasm, and effects on the efficiency and accuracy of translation.
  • regulatory element affecting gene expression may be located far away, even as far as tenths or hundreds of kilobases away, from the promoter region of a gene.
  • assaying for the presence or absence of at least one allele of at least one polymorphic marker of the present invention it is thus possible to assess the expression level of nearby genes. It is thus contemplated that the detection of the markers or haplotypes of the present invention can be used for assessing expression for one or more of such genes, including but not limited to the human CLDN14 gene.
  • a variety of methods can be used for detecting protein expression levels, including enzyme linked immunosorbent assays (ELISA), Western blots, immunoprecipitations and immunofluorescence.
  • ELISA enzyme linked immunosorbent assays
  • a test sample from a subject is assessed for the presence of an alteration in the expression and/or an alteration in composition of the polypeptide encoded by a particular nucleic acid.
  • An alteration in expression of a polypeptide can be, for example, an alteration in the quantitative polypeptide expression (i.e., the amount of polypeptide produced).
  • An alteration in the composition of a polypeptide may be an alteration in the qualitative polypeptide expression (e.g., expression of a mutant polypeptide or of a different splicing variant).
  • determination of a susceptibility to kidney stones or related conditions is made by detecting a particular splicing variant of a gene associated with the condition (e.g., the CLDN14 gene), or a particular pattern of splicing
  • an "alteration" in the polypeptide expression or composition refers to an alteration in expression or composition in a test sample, as compared to the expression or composition of the polypeptide in a control sample.
  • a control sample is a sample that corresponds to the test sample (e.g., is from the same type of cells), and is from a subject who is not affected by, and/or who does not have, a susceptibility to, the condition.
  • the control sample is from a subject that does not possess a particular marker allele or haplotype associated with kidney stones or related conditions, as described herein.
  • the presence of one or more different splicing variants in the test sample, or the presence of significantly different amounts of different splicing variants in the test sample, as compared with the control sample can be indicative of a susceptibility to , kidney stones or related conditions.
  • An alteration in the expression or composition of the polypeptide in the test sample, as compared with the control sample can be indicative of a specific allele in the instance where the allele alters a splice site relative to the reference in the control sample.
  • an antibody e.g., an antibody with a detectable label
  • a polypeptide encoded by a nucleic acid associated with at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis can,- be used.
  • the polypeptide is a CLDN 14 polypeptide.
  • Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., Fv, Fab, Fab', F(ab') 2 ) can be used.
  • labeled with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • indirect labeling include detection of a primary antibody using a labeled secondary antibody (e.g., a fluorescently-labeled secondary antibody) and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.
  • the level or amount of a polypeptide in a test sample is compared with the level or amount of the polypeptide in a control sample.
  • a level or amount of the polypeptide in the test sample that is higher or lower than the level or amount of the polypeptide in the control sample, such that the difference is statistically significant is indicative of an alteration in the expression of the polypeptide encoded by the nucleic acid, and is diagnostic for a particular allele or haplotype responsible for causing the difference in expression.
  • the composition of the polypeptide in a test sample is compared with the composition of the polypeptide in a control sample.
  • both the level or amount and the composition of the polypeptide can be assessed in the test sample and in the control sample.
  • determination of a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum •', bicarbonate levels, reduced bone mineral density and osteoporosis is made by detecting at least one marker or haplotype as described herein in combination with an additional protein-based, RNA-based or DNA-based assay.
  • Kits useful in the methods of the invention comprise components useful in any of the methods ⁇ ; described herein, including for example, primers for nucleic acid amplification, hybridization probes, restriction enzymes (e.g., for RFLP analysis), allele-specific oligonucleotides, antibodies that bind to an altered polypeptide encoded by a nucleic acid of the invention as described herein (e.g., a genomic segment comprising at least one polymorphic marker and/or haplotype of the • present invention) or to a non-altered (native) polypeptide encoded by a nucleic acid of the invention as described herein, means for amplification of nucleic acids as described herein, means for analyzing the nucleic acid sequence of nucleic acids as described herein, means for analyzing the amino acid sequence of a polypeptide encoded by a nucleic acid described herein, ; etc.
  • kits can for example include necessary buffers, nucleic acid primers for amplifying -: nucleic acids of the invention (e.g. , a nucleic acid segment comprising one or more of the polymorphic markers as described herein), and reagents for allele-specific detection of the fragments amplified using such primers and necessary enzymes (e.g., DNA polymerase). Additionally, kits can provide reagents for assays to be used in combination with the methods of the present invention, e.g., reagents for use with other diagnostic assays for kidney stones or related conditions.
  • nucleic acid primers for amplifying -: nucleic acids of the invention e.g. , a nucleic acid segment comprising one or more of the polymorphic markers as described herein
  • reagents for allele-specific detection of the fragments amplified using such primers and necessary enzymes e.g., DNA polymerase.
  • kits can provide reagents for assays to be used in combination
  • the invention pertains to a kit for assaying a sample from a subject to detect a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone 7 , mineral density and osteoporosis in a subject, wherein the kit comprises reagents necessary for selectively detecting at least one allele of at least one polymorphism of the present invention in the genome of the individual.
  • the reagents comprise at least one . contiguous oligonucleotide that hybridizes to a fragment of the genome of the individual comprising at least one polymorphism of the present invention.
  • the reagents comprise at least one pair of oligonucleotides that hybridize to opposite strands of a genomic segment obtained from a subject, wherein each oligonucleotide primer pair is designed to selectively amplify a fragment of the genome of the individual that includes at least one polymorphism associated with risk of the condition.
  • the polymorphism is selected from the group consisting of the polymorphisms as listed in Table 1, • and polymorphic markers in linkage disequilibrium therewith.
  • the fragment is at least 20 base pairs in size.
  • oligonucleotides or nucleic acids can be designed using portions of the nucleic acid sequence flanking polymorphisms (e.g., SNPs or microsatellites) that are associated with risk of the at least one . condition.
  • the kit comprises one or more labeled nucleic acids capable . of allele-specific detection of one or more specific polymorphic markers or haplotypes, and reagents for detection of the label.
  • Suitable labels include, e.g. , a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.
  • the polymorphic marker or haplotype to be detected by the reagents of the kit comprises one or more markers, two or more markers, three or more markers, four or more markers or five or more markers.
  • the polymorphic marker or , haplotype comprises at least one marker selected from the group of markers set forth in Table 1.
  • the marker or haplotype to be detected comprises at least one marker from the group of markers in strong linkage disequilibrium, as defined by values of r 2 greater than 0.2, to at least one of the group of markers listed in Table 1.
  • the marker or haplotype to be detected is selected from rs219778, rs219779, rs219780 and rs219781.
  • the kit for detecting the markers of the invention comprises a detection oligonucleotide probe, that hybridizes to a segment of template DNA containing a SNP polymorphisms to be detected, an enhancer oligonucleotide probe and an endonuclease.
  • the detection oligonucleotide probe comprises a fluorescent moiety or • group at its 3' terminus and a quencher at its 5' terminus, and an enhancer oligonucleotide, is , employed, as described by Kutyavin et al. (Nucleic Acid Res. 34:el28 (2006)).
  • the fluorescent, moiety can be Gig Harbor Green or Yakima Yellow, or other suitable fluorescent moieties.
  • the , detection probe is designed to hybridize to a short nucleotide sequence that includes the SNP polymorphism to be detected.
  • the SNP is anywhere from the terminal residue to -6 residues from the 3' end of the detection probe.
  • the enhancer is a short oligonucleotide probe which hybridizes to the DNA template 3' relative to the detection probe.
  • the probes are designed such that a single nucleotide gap exists between the detection probe and the enhancer nucleotide probe when both are bound to the template. The gap creates a synthetic abasic site ' that is recognized by an endonuclease, such as Endonuclease IV.
  • the enzyme cleaves the dye off the fully complementary detection probe, but cannot cleave a detection probe containing a mismatch.
  • assessment of the presence of a particular allele defined by nucleotide sequence of the detection probe can be performed.
  • the detection probe can be of any suitable size, although preferably the probe is relatively short. In one embodiment, the probe is from 5-100 nucleotides in length. In another embodiment, the i probe is from 10-50 nucleotides in length, and in another embodiment, the probe is from 12-30 nucleotides in length. Other lengths of the probe are possible and within scope of the skill of the average person skilled in the art.
  • the DNA template containing the SNP polymorphism is amplified by Polymerase Chain Reaction (PCR) prior to detection, and primers for such amplification are included in the reagent kit.
  • PCR Polymerase Chain Reaction
  • the amplified DNA serves as the template for the detection probe and the enhancer probe.
  • the DNA template is amplified by means of Whole Genome Amplification (WGA) methods, prior to assessment for the presence of specific polymorphic markers as described herein. Standard methods well known to the skilled person for performing WGA may be utilized, and are within scope of the invention.
  • reagents for performing WGA are included in the reagent kit.
  • modified bases including modified A and modified G.
  • modified bases can be useful for adjusting the melting temperature of the nucleotide molecule (probe and/or primer) to the template DNA, for example for increasing the melting temperature in regions containing a low percentage of G or C bases, in which modified A with the capability of forming three hydrogen bonds to its complementary T can be used, or for decreasing the melting temperature in regions containing a high percentage of G or C bases, for example by using modified G bases that form only two hydrogen bonds to their complementary C base in a double stranded DNA molecule.
  • modified bases are used in the design of the detection nucleotide probe. Any modified base known to the skilled person can be selected in these methods, and the selection of suitable bases is well within the scope of the skilled person based on the teachings herein and known bases available from commercial sources as known to the skilled person.
  • determination of the presence of the marker or haplotype is indicative of a susceptibility (increased susceptibility or decreased susceptibility) to at least one condition • selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis.
  • determination of the presence of the marker or haplotype is indicative of response to a therapeutic agent for at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis.
  • the presence of the marker or haplotype is indicative of prognosis of at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis.
  • the presence of the marker or haplotype is indicative of progress of treatment of the at elast one condition.
  • the kit further comprises a set of instructions for using the reagents comprising the kit.
  • a pharmaceutical pack comprising a therapeutic agent and a set of instructions for administration of the therapeutic agent to humans diagnostically tested for one or more variants of the present invention, as disclosed herein.
  • the therapeutic agent can be a small molecule drug, an antibody, a peptide, an antisense or RNAi molecule, or other therapeutic molecules.
  • an individual identified as a carrier of at least one variant of the present invention is instructed to take a prescribed dose of the therapeutic agent.
  • an individual identified as a homozygous carrier of at least one variant of the present invention is instructed to take a prescribed dose of the therapeutic agent.
  • an individual identified as a non-carrier of at least one variant of the present invention is instructed to take a prescribed dose of the therapeutic agent.
  • the variants (markers and/or haplotypes) disclosed herein to confer increased risk of at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis can ' also be used to identify novel therapeutic targets for the condition.
  • genes containing, or in linkage disequilibrium with, one or more of these variants, or their products, as well as genes or their products that are directly or indirectly regulated by or interact with these variant genes or their products can be targeted for the development of therapeutic agents.
  • Therapeutic agents may comprise one or more of, for example, small non-protein and non- nucleic acid molecules, proteins, peptides, protein fragments, nucleic acids (DNA, RNA), PNA - (peptide nucleic acids), or their derivatives or mimetics which can modulate the function and/or levels of the target genes or their gene products.
  • the therapeutic agent targets the CLDN14 gene.
  • the therapeutic agent is a human CLDN 14 polypeptide or a fragment thereof.
  • the CLDN14 polypeptide may be developed as a medicament using any suitable method of preparation, formulation and delivery, which are all well known to the skilled person.
  • the therapeutic agent is an oligonucleotide.
  • an oligonucleotide probe which is capable of hybridizing to a portion or fragment of the humand CLDN14 gene, may be prepared as a medicament for delivery to ameliorate symptoms associated with any one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis.
  • nucleic acids and/or variants described herein, or nucleic acids comprising their complementary sequence may be used as antisense constructs to control gene expression in cells, tissues or organs.
  • the methodology associated with antisense techniques is well known to ' the skilled artisan, and is for example described and reviewed in Antis ⁇ ns ⁇ Drug Technology: Principles, Strategies, and Applications, Crooke, ed., Marcel Dekker Inc., New York (2001).
  • antisense agents are comprised of single stranded oligonucleotides that are capable of binding to a complimentary nucleotide segment. By binding the appropriate target sequence, an RNA-RNA, DNA-DNA or RNA-DNA duplex is formed.
  • the antisense oligonucleotides are complementary to the sense or coding strand of a gene. It is also possible to form a triple helix, where the antisense oligonucleotide binds to duplex DNA.
  • antisense oligonucleotide Several classes of antisense oligonucleotide are known to those skilled in the art, including cleavers and blockers.
  • the former bind to target RNA sites, activate intracellular nucleases ⁇ e.g., RnaseH or Rnase L), that cleave the target RNA.
  • Blockers bind to target RNA, inhibit protein translation by steric hindrance of the ribosomes. Examples of blockers include nucleic acids, morpholino compounds, locked nucleic acids and methylphosphonates (Thompson, Drug Discovery Today, 7:912-917 (2002)).
  • Antisense oligonucleotides are useful directly as therapeutic agents, and are also useful for determining and validating gene function, for example by gene knock-out or gene knock-down experiments. Antisense technology is further described in Lavery er a/. , Curr. Opin. Drug Discov. Devel. 6:561-569 (2003), Stephens er a/., Curr. Opin. MoI. Then 5: 118-122 (2003), Kurreck, Eur. J. Biochem. 270: 1628-44 (2003), Dias er a/., MoI. ' Cancer Ter. 1:347-55 (2002), Chen, Methods MoI. Med. 75:621-636 (2003), Wang er a/., Curr.
  • the antisense agent is an oligonucleotide that is capable of binding to a nucleotide segment of the CLDN14 gene.
  • Antisense nucleotides can be from 5-500 nucleotides . in length, including 5-200 nucleotides, 5-100 nucleotides, 8-50 nucleotides, and 8-30 nucleotides.
  • the antisense nucleotides is from 14-50 nucleotides in length, includign 14-40 nucleotides and 14-30 nucleotides.
  • the antisense nucleotide is capable of binding to a nucleotide segment of the CLDN14 as set forth in SEQ ID NO: 1.
  • the variants described herein can be used for the selection and design of antisense reagents that are specific for particular variants. Using information about the variants described herein, antisense oligonucleotides or other antisense molecules that specifically target mRNA molecules that contain one or more variants of the invention can be designed. In this manner, expression of mRNA molecules that contain one or more variant of the present invention (markers and/or haplotypes) can be inhibited or blocked.
  • the antisense molecules are designed to specifically bind a particular allelic form (i.e., one or several variants (alleles and/or haplotypes)) of the target nucleic acid, thereby inhibiting translation of a product originating from this specific allele or haplotype, but which do not bind other or alternate variants at the specific polymorphic sites of the target nucleic acid molecule.
  • allelic form i.e., one or several variants (alleles and/or haplotypes)
  • antisense molecules can be used to inactivate mRNA so as to inhibit gene expression, and thus protein expression, the molecules can be used for development of treatment of kidney stones or related conditions.
  • the methodology can involve cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated.
  • mRNA regions include, for example, . • protein-coding regions, in particular protein-coding regions corresponding to catalytic activity, . substrate and/or ligand binding sites, or other functional domains of a protein.
  • RNA interference also called gene silencing, is based on using double-stranded RNA molecules (dsRNA) to turn off specific genes.
  • dsRNA double-stranded RNA molecules
  • siRNA small interfering RNA
  • the siRNA molecules are typically about 20, 21, 22 or 23 nucleotides in length.
  • one aspect of the invention relates to isolated nucleic acid molecules, and the use of those molecules for RNA interference, i.e. as small interfering RNA molecules (siRNA).
  • the isolated nucleic acid molecules are 18-26 nucleotides in length, preferably 19-25 nucleotides in length, more preferably 20-24 nucleotides in length, and more preferably 21, 22 or 23 nucleotides in length.
  • RNAi-mediated gene silencing originates in endogenously encoded primary' microRNA (pri-miRNA) transcripts, which are processed in the cell to generate precursor miRNA (pre-miRNA). These miRNA molecules are exported from the nucleus to the cytoplasm, where they undergo processing to generate mature miRNA molecules (miRNA), which direct translational inhibition by recognizing target sites in the 3' untranslated regions of mRNAs, and subsequent mRNA degradation by processing P-bodies (reviewed in Kim & Rossi, Nature Rev. . Genet. 8: 173-204 (2007)).
  • RNAi Clinical applications of RNAi include the incorporation of synthetic siRNA duplexes, which preferably are approximately 20-23 nucleotides in size, and preferably have 3' overlaps of 2 nucleotides. Knockdown of gene expression is established by sequence-specific design for the target mRNA. Several commercial sites for optimal design and synthesis of such molecules are; known to those skilled in the art.
  • siRNA molecules typically 25-30 nucleotides in length, preferably about 27 nucleotides
  • shRNAs small hairpin RNAs
  • the latter are naturally expressed, as described in Amarzguioui et al. ⁇ [FEBS Lett. 579: 5974-81 (2005)).
  • Chemically synthetic siRNAs and shRNAs are substrates for in vivo processing, and in some cases provide more potent gene-silencing than shorter designs (Kim et al.. Nature Biotechnol. 23:222-226 (2005); Siolas et al., Nature Biotechnol. 23 :227-231 (2005)).
  • siRNAs provide for transient silencing of gene expression, because their intracellular concentration is diluted by subsequent cell divisions.
  • expressed shRNAs mediate long-term, stable knockdown of target transcripts, for as long as transcription of the shRNA takes place (Marques et al., Nature Biotechnol. 23 :559-565 (2006); Brummelkamp et al., Science 296: 550-553 (2002)).
  • RNAi molecules including siRNA, miRNA and shRNA, act in a sequence-dependent manner
  • the variants presented herein ⁇ e.g., the markers set forth in Table 1) can be used to design RNAi reagents that recognize specific nucleic acid molecules comprising specific alleles and/or haplotypes (e.g., the alleles and/or haplotypes of the present invention), while not recognizing nucleic acid molecules comprising other alleles or haplotypes.
  • RNAi reagents can thus recognize and destroy the target nucleic acid molecules.
  • RNAi reagents can be useful as therapeutic agents (i.e., for turning off kidney stones-associated genes or kidney stones-associated gene variants), but may also be useful for characterizing and . validating gene function (e.g., by gene knock-out or gene knock-down experiments).
  • RNAi may be performed by a range of methodologies known to those skilled in the art. Methods utilizing non-viral delivery include cholesterol, stable nucleic acid-lipid particle (SNALP), heavy-chain antibody fragment (Fab), aptamers and nanoparticles. Viral delivery methods include use of lentivirus, adenovirus and adeno-associated virus.
  • the siRNA molecules are in some embodiments chemically modified to increase their stability. This can include modifications at the 2' position of the ribose, including 2'-O-methylpurines and 2'- fluoropyrimidines, which provide resistance to Rnase activity. Other chemical modifications are possible and known to those skilled in the art.
  • a genetic defect leading to increased predisposition or risk for development of a condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, or a defect causing the kidney stones may be corrected permanently by administering to a subject carrying the defect a nucleic acid fragment that incorporates a repair sequence that supplies the normal/wild-type nucleotide(s) at the site of the genetic defect.
  • site-specific repair sequence may co ⁇ compass an RIMA/DNA oligonucleotide that operates to promote endogenous repair of a subject's genomic DNA.
  • the administration of the repair sequence may be performed by an appropriate vehicle, such as a complex with polyethelenimine, encapsulated in anionic liposomes, a viral vector such as an adenovirus vector, or other pharmaceutical compositions suitable for promoting intracellular uptake of the adminstered nucleic acid.
  • an appropriate vehicle such as a complex with polyethelenimine, encapsulated in anionic liposomes, a viral vector such as an adenovirus vector, or other pharmaceutical compositions suitable for promoting intracellular uptake of the adminstered nucleic acid.
  • the genetic: defect may then be overcome, since the chimeric oligonucleotides induce the incorporation of the normal sequence into the genome of the subject, leading to expression of the normal/wild-type gene product.
  • the replacement is propagated, thus rendering a permanent repair and alleviation of the symptoms associated with the kidney stones or condition.
  • the present invention provides methods for identifying compounds or agents that can be used to treat conditions such as kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis.
  • the variants of the invention are useful as targets for the identification and/or development of therapeutic agents.
  • such methods include assaying the ability of an agent or compound to modulate the activity and/or expression of a nucleic acid that includes at least one of the variants (markers and/or haplotypes) of the present invention, or the encoded product of the nucleic acid ⁇ e.g., CLDN14). This in turn can be used to identify agents or compounds that inhibit or alter the undesired activity or expression of the encoded nucleic acid product.
  • Cell-based systems include cells naturally expressing the nucleic acid molecules of interest, or recombinant cells that have been genetically modified so as to express a certain desired nucleic acid molecule.
  • Variant gene expression in a patient can be assessed by expression of a variant-containing nucleic acid sequence (for example, a gene containing at least one variant of the present invention, which can be transcribed into RNA containing the at least one variant, and in turn translated into protein), or by altered expression of a normal/wild-type nucleic acid sequence -' due to variants affecting the level or pattern of expression of the normal transcripts, for example variants in the regulatory or control region of the gene.
  • Assays for gene expression include direct nucleic acid assays (mRNA), assays for expressed protein levels, or assays of collateral compounds involved in a pathway, for example a signal pathway.
  • mRNA direct nucleic acid assays
  • assays for expressed protein levels or assays of collateral compounds involved in a pathway, for example a signal pathway.
  • the expression of genes that are up- or down-regulated in response to the signal pathway can also be assayed.,.
  • One embodiment includes operably linking a reporter gene, such as
  • Modulators of gene expression can in one embodiment be identified when a cell is contacted with a candidate compound or agent, and the expression of mRNA is determined. The expression level of mRNA in the presence of the candidate compound or agent is compared to the expression level in the absence of the compound or agent. Based on this comparison, candidate compounds or agents can be identified as those modulating the gene expression of the variant gene. When expression of mRNA or the encoded protein is statistically significantly greater in the presence of the candidate compound or agent than in its absence, then the candidate compound or agent is identified as a stimulator or up-regulator of expression of the nucleic acid. When nucleic acid expression or protein level is statistically significantly less in the presence of . nurse the candidate compound or agent than in its absence, then the candidate compound is identified, as an inhibitor or down-regulator of the nucleic acid expression.
  • the invention further provides methods of treatment using a compound identified through drug (compound and/or agent) screening as a gene modulator (i.e. stimulator and/or inhibitor of gene expression).
  • a gene modulator i.e. stimulator and/or inhibitor of gene expression
  • kidney stones pass spontaneously - i.e., those 4mm or less ( ⁇ 90% of kidney stones); on the other hand, stones larger than 6mm require some sort of intervention. Stone passage may be encouraged by increased hydration, medication for treating infection and reducing pain, and , diuretics to encourage urine flow and prevent further stone formation.
  • narcotics such as morphine, ] codeine and derivatives
  • alpha adrenergic blocking agents such as Flomax (active compound (R)-5-(2-(2-(2- ethoxyphenoxy)ethylamino)propyl)-2-methoxybenzenesulfonamide), Uroxatral (Alfuzosin, active compound /V-[3-[(4-amino-6,7-dimethoxy-quinazolin-2-yl)- methyl-amino]propyl] tetrahydrofuran- 2-carboxamide), Terazosin (Hytrin, active compound [4-(4-amino-6,7- dimethoxy-quinazolin-2-yl) piperazin-1-yl]- tetrahydrofuran-2-yl-methanone) or Doxazosin (brand names Cardura/Carduran, active compound [4-(4-amino-6,7-dimethoxy- quinazolin-2-yl) piperazin
  • Flomax
  • Urologic intervention may be required when pain is persistent and severe, in renal failure and when there is kidney infection. Most common intervention is Extracorporeal Shock Wave Lithotripsy (ESWL).
  • ESWL Extracorporeal Shock Wave Lithotripsy
  • invasive procedures including ureteroscopic fragmentation, using laser, ultrasonic or mechanical (shock wave, pneumatic) energy to fragment larger stones.
  • Percutaneous nephrolithotomy or open surgery may be required for large or complicated stones or stones which fail other less invasive treatments.
  • the variants of the present invention may determine the manner in which a therapeutic agent and/or method acts on the body, or the way in which the body metabolizes the therapeutic agent.
  • the presence of a particular allele at a polymorphic site as described herein is indicative of a different response, e.g. a different response rate, to a particular treatment modality for kidney stones.
  • a patient who presents with symptoms indicative of kidney stones, and carrying a certain at-risk markers for kidney stones as described herein would respond better to, or worse to, a specific therapeutic, drug and/or other therapy used to treat the kidney stones. Therefore, the presence or absence of the marker allele (or haplotype) could aid in deciding what treatment is appropriate for the patient. For example, for a newly diagnosed patient, the presence of a marker or haplotype of the present invention may be assessed (e.g.
  • the patient's carrier status could be used to help determine which particular treatment modality should be administered.
  • the value lies within the possibilities of being able to determine or predict the likely severity of the kidney stones in the individual at an early stage, to select the most appropriate treatment, and provide information to the clinician about prognosis/aggressiveness of the kidney stones in order to be able to apply the most appropriate treatment.
  • Appropriate medication may also be administered for at-risk variant carriers.
  • the present invention also relates to methods of monitoring progress or effectiveness of a treatment for kidney stones. This can be done based on the genotype and/or haplotype status of the markers and haplotypes of the present invention, i.e., by assessing the absence or ', presence of at least one allele of at least one polymorphic marker as disclosed herein, or by monitoring expression of genes that are associated with the variants (markers and haplotypes) of the present invention (e.g., the CLDN14 gene).
  • the risk gene mRNA or the encoded polypeptide can be measured in a tissue sample (e.g. , a peripheral blood sample, or a biopsy sample). Expression levels and/or mRNA levels can thus be determined before and during treatment to monitor its effectiveness.
  • biological networks or metabolic pathways related to the markers and haplotypes of the present invention can be monitored by determining mRNA and/or polypeptide levels. This, can be done for example, by monitoring expression levels or polypeptides for several genes belonging to the network and/or pathway, in samples taken before and during treatment. Alternatively, metabolites belonging to the biological network or metabolic pathway can be determined before and during treatment. Effectiveness of the treatment is determined by comparing observed changes in expression levels/metabolite levels during treatment to corresponding data from healthy subjects.
  • the variants described herein may be useful for such a purpose.
  • a prognosis of the individual may be determined.
  • at-risk variants for one or more of the above-mentioned conditions is predictive of a worse prognosis of the condition.
  • the markers of the present invention can be used to increase power and effectiveness of clinical trials.
  • individuals who are carriers of at least one at-risk variant ⁇ for kidney stones or related conditions may be more likely to respond favorably to a particular treatment modality.
  • individuals who carry at-risk variants for gene(s) in a pathway and/or metabolic network (e.g., CLDNl 4-associated genes) for which a particular treatment (e.g., small molecule drug) is targeting are more likely to be responders to the treatment.
  • individuals who carry at-risk variants for a gene, which ; expression and/or function is altered by the at-risk variant (e.g., CLDN 14), are more likely to be responders to a treatment modality targeting that gene, its expression or its gene product.
  • This application can improve the safety of clinical trials, but can also enhance the chance that a clinical trial will demonstrate statistically significant efficacy, which may be limited to a certain sub-group of the population.
  • one possible outcome of such a trial is that carriers of certain genetic variants, e.g., the markers and haplotypes of the present invention, are statistically significantly likely to show positive response to the therapeutic agent, i.e.
  • kidney stones experience alleviation of symptoms, or prevention of symptoms, associated with kidney stones, or experience less severe symptoms (e.g., small stones) or no symptoms (prophylactic therapy, preventln ⁇ i or ! minimizing kidney stone formation) when taking the therapeutic agent or drug as prescribed.
  • the markers and haplotypes of the present invention can be used for targeting the selection of pharmaceutical agents for specific individuals.
  • Personalized selection of treatment modalities, lifestyle changes, e.g., dietary changes, or combination of lifestyle changes and administration of particular treatment can be realized by the utilization of the at-risk variants of the present invention.
  • Dietary changes or modifications may include one or more of- increased water consumption, low protein, nitrogen and sodium diet, low oxalate diet ⁇ e.g., low ' chocolate, nuts, soybean, rhubarb, spinach consumption), reduced cola beverage diet, avoidance of large doses of vitamin C, and supplements that include thiazides, potassium citrate, magnesium citrate, and/or allopurinol.
  • the knowledge of an individual's status for particular markers of the present invention can be useful for selection of treatment options, which may also be combined with dietary changes or modifications.
  • Certain combinations of variants may be suitable for one selection of treatment options (e.g, prophylactic therapy or dietary changes), while other gene variant combinations may target other treatment options.
  • combinations of genetic variants may be assessed, including one variant,' two variants, three variants, or four or more variants, as needed to determine with clinically reliable accuracy the selection of treatment module.
  • the methods and information described hereib may be implemented, in all or in part, as computer executable instructions on known computer ' readable media.
  • the methods described herein may be implemented in hardware/
  • the method may be implemented in software stored in, for example, one or more memories or other computer readable medium and implemented on one or more processors.
  • the processors may be associated with one or more controllers, calculation units and/or other units of a computer system, or implanted in firmware as desired.
  • the routines may be stored in any computer readable memory such as in RAM, P.OM, flash memory, a magnetic disk, a laser disk, or other storage medium, as is also known.
  • this software may be delivered to a computing device via any known delivery method including, for example, over a communication channel such as a telephone line, the Internet, a - wireless connection, etc., or via a transportable medium, such as a computer readable disk, flash drive, etc.
  • a communication channel such as a telephone line, the Internet, a - wireless connection, etc.
  • a transportable medium such as a computer readable disk, flash drive, etc.
  • the various steps described above may be implemented as various blocks, operations, tools, modules and techniques which, in turn, may be implemented in hardware, firmware, software, or any combination of hardware, firmware, and/or software.
  • some or all of the blocks, operations, techniques, etc. may be implemented in, for example, a custom integrated circuit (IC), an application specific integrated circuit (ASIC), a field programmable logic array (FPGA), a programmable logic array (PLA), etc. :
  • the software When implemented in software, the software may be stored in any known computer readable ' medium such as on a magnetic disk, an optical disk, or other storage medium, in a RAM or ROM or flash memory of a computer, processor, hard disk drive, optical disk drive, tape drive, etc.
  • the software may be delivered to a user or a computing system via any known delivery method including, for example, on a computer readable disk or other transportable computer storage mechanism.
  • Fig. 3 illustrates an example of a suitable computing system environment 100 on which a system for the steps of the claimed method and apparatus may be implemented.
  • the computing system environment 100 is only one example of a suitable computing environment and is not intended to suggest any limitation as to the scope of use or functionality of the method or apparatus of '! the claims. Neither should the computing environment 100 be interpreted as having any dependency or requirement relating to any one or combination of components illustrated in the exemplary operating environment 100.
  • the steps of the claimed method and system are operational with numerous other general purpose or special purpose computing system environments or configurations.
  • Examples of well known computing systems, environments, and/or configurations that may be suitable for use with the methods or system of the claims include, but are not limited to, personal computers, server computers, hand-held or laptop devices, multiprocessor systems, microprocessor-based systems, set top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, distributed computing environments that include any of the above systems or devices, and the like.
  • program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types.
  • the methods and apparatus may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network.
  • program modules may be located in both local and remote computer storage media including memory storage devices.
  • an exemplary system for implementing the steps of the claimed method and system includes a general purpose computing device in the form of a computer 110.
  • Components of computer 110 may include, but are not limited to, a processing unit 120, a system memory 130, and a system bus 121 that couples various system components including the system memory to the processing unit 120.
  • the system bus 121 may be any of several types of bus structures including a memory bus or memory controller, a peripheral bus, and a ,, local bus using any of a variety of bus architectures.
  • such architectures include Industry Standard Architecture (ISA) bus, Micro Channel Architecture (MCA) bus, Enhanced ISA (EISA) bus, Video Electronics Standards Association (VESA) local bus, and Peripheral Component Interconnect (PCI) bus also known as Mezzanine bus.
  • ISA Industry Standard Architecture
  • MCA Micro Channel Architecture
  • EISA Enhanced ISA
  • VESA Video Electronics Standards Association
  • PCI Peripheral Component Interconnect
  • Computer 110 typically includes a variety of computer readable media.
  • Computer readable media can be any available media that can be accessed by computer 110 and includes both volatile and nonvolatile media, removable and non-removable media.
  • Computer readable media may comprise computer storage media and communication media.
  • Computer storage media includes both volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data.
  • Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage - devices, or any other medium which can be used to store the desired information and which can accessed by computer 110.
  • Communication media typically embodies computer readable instructions, data structures, program modules or other data in a modulated data signal such as a carrier wave or other transport mechanism and includes any information delivery media.
  • modulated data signal means a signal that has one or more of its characteristics set or ' changed in such a manner as to encode information in the signal.
  • communication media includes wired media such as a wired network or direct-wired ' connection, and wireless media such as acoustic, RF, infrared and other wireless media. Combinations of the any of the above should also be included within the scope of computer readable media.
  • the system memory 130 includes computer storage media in the form of volatile and/or nonvolatile memory such as read only memory (ROM) 131 and random access memory (RAM) 132.
  • ROM read only memory
  • RAM random access memory
  • BIOS basic input/output system
  • RAM 132 typically contains data and/or program modules that are immediately accessible to and/or presently being operated on by processing unit 120.
  • Fig. 3 illustrates operating system 134, application programs 135, other program modules 136, and program data 137.
  • the computer 110 may also include other removable/non-removable, volatile/nonvolatile computer storage media.
  • Fig. 3 illustrates a hard disk drive 140 that ' reads from or writes to non-removable, nonvolatile magnetic media, a magnetic disk drive 151 that reads from or writes to a removable, nonvolatile magnetic disk 152, and an optical disk drive 155 that reads from or writes to a removable, nonvolatile optical disk 156 such as a CD ROM or other optical media.
  • removable/non-removable, volatile/nonvolatile computer storage media that can be used in the exemplary operating environment include, but are not limited to, magnetic tape cassettes, flash memory cards, digital versatile disks, digital video tape, solid state RAM, solid state ROM, and the like.
  • the hard disk drive 141 is typically connected to the system bus 121 through a non-removable memory interface such as interface 140, and magnetic disk drive 151 and optical disk drive 155 are typically connected to the system bus 121 by a removable memory interface, such as interface 150.
  • hard disk drive 141 is illustrated as storing operating system 144, application programs 145, other program modules 146, and program data 147. Note that these components can either be the same as or different from operating system 134, application programs 135, other program modules 136, and program data 137. Operating system 144, application programs 145, other program modules 146, and program data 147 are given different numbers here to illustrate that, at a minimum, they are different copies.
  • a user may enter commands and information into the computer 20 through input devices such as a keyboard 162 and pointing device 161, commonly referred to as a mouse, trackball or touch pad.
  • Other input devices may include a microphone, joystick, game pad, satellite dish, scanner, or the like.
  • These and other input devices are often connected to the processing unit 120 through a user input interface 160 that is coupled to the system bus, but may be connected by other interface and bus structures, such as a parallel port, game port or a universal serial bus (USB).
  • a monitor 191 or other type of display device is also connected to the system bus 121 via an interface, such as a video interface 190.
  • computers may also include other peripheral output devices such as speakers 197 and printer 196, which may be connected through an output peripheral interface 190.
  • the computer 110 may operate in a networked environment using logical connections to one or more remote computers, such as a remote computer 180.
  • the remote computer 180 may be a personal computer, a server, a router, a network PC, a peer device or other common network f . node, and typically includes many or all of the elements described above relative to the computer 110, although only a memory storage device 181 has been illustrated in Fig. 3.
  • the logical connections depicted in Fig. 3 include a local area network (LAN) 171 and a wide area network (WAN) 173, but may also include other networks.
  • LAN local area network
  • WAN wide area network
  • Such networking environments are commonplace in offices, enterprise-wide computer networks, intranets and the Internet.
  • the computer 110 When used in a LAN networking environment, the computer 110 is connected to the LAN 171 through a network interface or adapter 170. When used in a WAN networking environment, the computer 110 typically includes a modem 172 or other means for establishing communications over the WAN 173, such as the Internet.
  • the modem 172 which may be internal or external, may be connected to the system bus 121 via the user input interface 160, or other appropriate mechanism.
  • program modules depicted relative to the computer 110, or portions thereof may be stored in the remote memory storage device.
  • Fig. 3 illustrates remote application programs 185 as residing on memory device 181. It will be appreciated that the network connections shown are exemplary and other means of establishing a communications link between the computers may be used.
  • the risk evaluation system and method, and other elements have been described as preferably being implemented in software, they may be implemented in hardware, firmware, etc., and may be implemented by any other processor.
  • the elements described herein may be implemented in a standard multi-purpose CPU or on specifically designed hardware or ; firmware such as an application-specific integrated circuit (ASIC) or other hard-wired device as desired, including, but not limited to, the computer 110 of Fig. 3.
  • the software routine may be stored in any computer readable memory such as on a magnetic disk, a laser disk, or other storage medium, in a RAM or ROM of a computer or processor, in any database, etc.
  • this software may be delivered to a user or a diagnostic system via any known or desired delivery method including, for example, on a computer readable disk or other transportable computer storage mechanism or over a communication channel such as a telephone line, the internet, wireless communication, etc. (which are viewed as being the same as or interchangeable with providing such software via a transportable storage medium).
  • the invention relates to computer-implemented applications using the polymorphic markers and haplotypes described herein, and genotype and/or kidney stones-association data (or association data for related conditions of kidney stones) derived therefrom.
  • Such applications can be useful for storing, manipulating or otherwise analyzing genotype data that is useful in the methods of the invention.
  • One example pertains to storing genotype information derived from an individual on readable media, so as to be able to provide the genotype information to a third party (e.g., the individual, a guardian of the individual, a health care provider or genetic analysis service provider), or for deriving information from the genotype data, e.g. , by comparing the genotype data to information about genetic risk factors contributing to increased susceptibility to the kidney stones and related conditions, and reporting results based on such comparison.
  • a third party e.g., the individual, a guardian of the individual, a health care provider or genetic analysis service provider
  • computer-readable media has capabilities of storing ( ⁇ ) identifer information for at least one polymorphic marker or a haplotype, as described herein; (ii) an indicator of the frequency of at least one allele of said at least one marker, or the frequency of a haplotype, in individuals with the condition; and an indicator of the frequency of at least one allele of said at least one marker, or the frequency of a haplotype, in a reference population.
  • the reference population can be a kidney stones-free population of individuals. Alternatively, the reference population is a random sample from the general population, and is thus representative of the population at large.
  • the frequency indicator may be a calculated frequency, a count of alleles , and/or haplotype copies, or normalized or otherwise manipulated values of the actual frequencies that are suitable for the particular medium.
  • markers and haplotypes described herein to be associated with increased susceptibility (e.g. , increased risk) of kidney stones and related conditions are in certain embodiments useful for interpretation and/or analysis of genotype data.
  • an identification of an at-risk allele for at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, as shown herein, or an allele at a polymorphic marker in LD , with any one of such markers, is indicative of the individual from whom the genotype data originates is at increased risk of the condition.
  • genotype data may be generated for at least one polymorphic marker shown herein to be associated with the condition, or a marker in linkage disequilibrium therewith.
  • the genotype data is subsequently made available to a third party, such as the individual from whom the data originates, his/her guardian or representative, a physician or health care worker, genetic counselor, or insurance agent, for example via a user interface accessable over the internet, together with an interpretation of the genotype data, e.g., in the form of a risk measure (such as an absolute risk (AR), risk ratio (RR) or odds ratio (OR)) for the kidney stones.
  • a risk measure such as an absolute risk (AR), risk ratio (RR) or odds ratio (OR)
  • At-risk markers identified in a genotype dataset derived from an individual are assessed and results from the assessment of the risk conferred by the presence of such at-risk varians in the dataset are made available to . ; the third party, for example via a secure web interface, or by other communication means.
  • the results of such risk assessment can be reported in numeric form (e.g., by risk values, such as absolute risk, relative risk, and/or an odds ratio, or by a percentage increase in risk compared with a reference), by graphical means, or by other means suitable to illustrate the risk to the individual from whom the genotype data is derived.
  • nucleic acids and polypeptides described herein can be used in methods and kits of the present invention.
  • An "isolated" nucleic acid molecule is one that is separated from nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA library).
  • an isolated nucleic acid of the invention can be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix.
  • the material can be purified to essential homogeneity, for example as determined by polyacrylamide gel electrophoresis (PAGE) or column chromatography (e.g., HPLC).
  • An isolated nucleic acid molecule of the invention can comprise at least about 50%, at least about 80% or at least about 90% (on a molar basis) of all macromolecular species present.
  • genomic DNA the term "isolated" also can refer to nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated.
  • the isolated nucleic acid molecule can contain less than about 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of the nucleotides that flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived.
  • nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.
  • recombinant DNA contained in a vector is included in the definition of "isolated” as used herein.
  • isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells or heterologous organisms, as well as partially or substantially purified DNA molecules in solution.
  • isolated nucleic acid molecules also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the present invention.
  • An isolated nucleic acid molecule or nucleotide sequence can include a nucleic acid molecule or . nucleotide sequence that is synthesized chemically or by recombinant means.
  • Such isolated nucleotide sequences are useful, for example, in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g. , from other mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the gene in tissue (e.g., human tissue), such as by Northern blot analysis or other hybridization techniques.
  • homologous sequences e.g. , from other mammalian species
  • gene mapping e.g., by in situ hybridization with chromosomes
  • tissue e.g., human tissue
  • the invention also pertains to nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein ⁇ e.g. , nucleic acid molecules that specifically hybridize to a nucleotide sequence containing a polymorphic site associated with a marker or haplotype described herein).
  • nucleic acid molecules can be detected and/or isolated by allele- or sequence-specific hybridization (e.g., under high stringency conditions).
  • Stringency conditions and methods for nucleic acid hybridizations are well known to the skilled person (see, e.g. , Current Protocols in Molecular Biology, Ausubel, F. et al, John Wiley & Sons, (1998), and Kraus, M. and Aaronson, S'., Methods Enzymol., 200:546-556 (1991), the entire teachings of which are incorporated by reference herein.
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, of the length of the reference sequence.
  • Another example of an algorithm is BLAT (Kent, WJ. Genome Res. 12:656-64 (2002)).
  • the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software package (Accelrys, Cambridge, UK). .,
  • the present invention also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleic acid that comprises, or consists of, the nucleotide sequence of LD Block C21, or a nucleotide sequence comprising, or consisting of, the complement of the nucleotide sequence of LD Block C21, wherein the nucleotide sequence comprises at least one polymorphic allele contained in the markers and haplotypes described herein.
  • the nucleic acid fragments of the invention are at least about 15, , at least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200, 500, 1000, 10,000 or more nucleotides in length.
  • probes or primers are oligonucleotides that hybridize in a base- specific manner to a complementary strand of a nucleic acid molecule.
  • probes and primers include polypeptide nucleic acids (PNA), as described in Nielsen, P. et al., Science 254: 1497-1500 (1991).
  • PNA polypeptide nucleic acids
  • a probe or primer comprises a region of nucleotide sequence that hybridizes to at least about 15, typically about 20-25, and in certain embodiments about 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule.
  • the probe or primer comprises at least one allele of at least one polymorphic marker or at least one haplotype described herein, or the complement thereof.
  • a probe or primer can comprise 100 or fewer nucleotides; for example, in certain embodiments from 6 to 50 nucleotides, or, for example, from 12 to 30 nucleotides.
  • the probe or primer is at least 70% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical, to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence.
  • the probe or primer is capable, of selectively hybridizing to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence.
  • the probe or primer further comprises a label, e.g., a . radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.
  • the nucleic acid molecules of the invention can be identified and isolated using standard molecular biology techniques well known to the skilled person.
  • the amplified DNA can be labeled (e.g. , radiolabeled, fluorescently labeled) and used as a probe for screening a cDNA library derived from human cells.
  • the cDNA can be derived from mRNA and contained in a suitable vector.
  • Corresponding clones can be isolated, DNA obtained following in vivo excision, and the cloned insert can be sequenced in either or both orientations by art- recognized methods to identify the correct reading frame encoding a polypeptide of the (i appropriate molecular weight. Using these or similar methods, the polypeptide and the DNA encoding the polypeptide can be isolated, sequenced and further characterized.
  • the invention also provides antibodies which bind to an epitope comprising either a variant amino acid sequence (e.g., comprising an amino acid substitution) encoded by a variant allele or the reference amino acid sequence encoded by the corresponding non-variant or wild-type allele.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. , molecules that contain antigen-binding sites ' that specifically bind an antigen.
  • a molecule that specifically binds to a polypeptide of the invention is a molecule that binds to that polypeptide or a fragment thereof, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind to a polypeptide of the invention.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.
  • Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a desired immunogen, e.g., polypeptide of the invention or a fragment thereof.
  • a desired immunogen e.g., polypeptide of the invention or a fragment thereof.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • the' antibody molecules directed against the polypeptide can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein, Nature 256:495-497 ( 1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72 (1983)), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,1985, Inc., pp. . 77-96) or trioma techniques.
  • hybridomas The technology for producing hybridomas is well known (see generally Current Protocols in Immunology (1994) Coligan et al., (eds.) John Wiley & Sons, Inc., New York, NY). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a polypeptide of the invention.
  • lymphocytes typically splenocytes
  • a monoclonal antibody to a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g. , an antibody phage display library) with the polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene . Su/fZAPTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can , be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al., Bio/Technology 9: 1370-1372 (1991); Hay et al. , Hum. Antibod. Hybridomas 3:81-85 ( 1992); Huse et al., Science 246: 1275-1281 (1989); and Griffiths et al., EMBO J. 12:725-734 (1993).
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies, . comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.
  • antibodies of the invention can be used to isolate a polypeptide of the invention by standard techniques, such as affinity chromatography or immunoprecipitation.
  • a polypeptide-specific antibody can facilitate the purification of natural polypeptide from cells and of recombinantly produced polypeptide expressed in host cells.
  • an antibody specific for a polypeptide of the invention can be used to detect the polypeptide (e.g., in a cellular lysate, cell supernatant, or tissue sample) in order to evaluate the abundance and pattern of expression of the polypeptide.
  • Antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, - determine the efficacy of a given treatment regimen.
  • the antibody can be coupled to a detectable substance to facilitate its detection. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.
  • Antibodies may also be useful in pharmacogenomic analysis.
  • antibodies against variant proteins encoded by nucleic acids according to the invention such as variant proteins that are encoded by nucleic acids that contain at least one polymorpic marker of the invention, can be used to identify individuals that require modified treatment modalities.
  • Antibodies can furthermore be useful for assessing expression of variant proteins in in an individual with a predisposition to condition related to the function of the protein, in particular a condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis.
  • the protein is a human CLDN 14 protein.
  • Antibodies specific for a variant protein that is encoded by a nucleic acid that comprises at least one polymorphic marker or haplotype as described herein can be used to screen for the presence of the variant protein, for example to screen for a predisposition to the condition as indicated by the presence of the variant protein.
  • Antibodies can be used in other methods. Thus, antibodies are useful as diagnostic tools for J evaluating proteins, such as variant proteins of the invention, in conjunction with analysis by electrophoretic mobility, isoelectric point, tryptic or other protease digest, or for use in other physical assays known to those skilled in the art. Antibodies may also be used in tissue typing. In one such embodiment, a specific variant protein has been correlated with expression in a specific tissue type, and antibodies specific for the variant protein can then be used to identify the specific tissue type.
  • Subcellular localization of proteins can also be determined using antibodies, and can be applied to assess aberrant subcellular localization of the protein in cells in various tissues. Such use can be applied in genetic testing, but also in monitoring a particular treatment modality. In the case where treatment is aimed at correcting the expression level or presence of the variant protein or aberrant tissue distribution or developmental expression of the variant protein, antibodies specific for the variant protein or fragments thereof can be used to monitor therapeutic efficacy.
  • Antibodies are further useful for inhibiting variant protein function, for example by blocking the binding of a variant protein to a binding molecule or partner. Such uses can also be applied in a therapeutic context in which treatment involves inhibiting a variant protein's function.
  • An antibody can be for example be used to block or competitively inhibit binding, thereby modulating (i.e., agonizing or antagonizing) the activity of the protein.
  • Antibodies can be prepared against specific protein fragments containing sites required for specific function or against an intact protein that is associated with a cell or cell membrane. For administration in .
  • an antibody may be linked with an additional therapeutic payload, such as radionuclide, an enzyme, an immunogenic epitope, or a cytotoxic agent, including bacterial toxins (diphtheria or plant toxins, such as ricin).
  • an additional therapeutic payload such as radionuclide, an enzyme, an immunogenic epitope, or a cytotoxic agent, including bacterial toxins (diphtheria or plant toxins, such as ricin).
  • the in vivo half-life of an antibody or a fragment thereof may be ⁇ increased by pegylation through conjugation to polyethylene glycol.
  • kits for using antibodies in the methods described herein includes, but is not limited to, kits for detecting the presence of a variant protein in a test sample.
  • kits for detecting the presence of a variant protein in a test sample comprises antibodies such as a labelled or labelable antibody and a compound or agent for detecting variant proteins in a biological sample, means for determining the amount or the presence and/or absence of variant protein in the sample, and means for comparing the amount of variant protein in the sample with a standard, as well as instructions for use of the kit.
  • kits for detecting the presence of a variant protein in a test sample comprises antibodies such as a labelled or labelable antibody and a compound or agent for detecting variant proteins in a biological sample, means for determining the amount or the presence and/or absence of variant protein in the sample, and means for comparing the amount of variant protein in the sample with a standard, as well as instructions for use of the kit.
  • the present invention will now be exemplified by the following non-limiting example.
  • the Icelandic kidney stone cases consisted of patients with confirmed radiopaque kidney stones, diagnosed in the years 1983-2003, from the Icelandic Kidney Stone Registry at Landspitali University Hospital, Reykjavik, Iceland. The study was approved by the Icelandic Data Protection Authority and the National Bioethics Committee of Iceland.
  • the Icelandic bone density sample-set comprises individuals who had bone density measurements (DEXA, Hologic QDR4500A) at the lumbar spine (L2-L4) and the hip (total hip) and participated in the osteoporosis genetics program or other genetic programs at deCODE genetics. All participants gave written informed consent and the study was approved by the Icelandic Data Protection Authority and the National Bioethics Committee of Iceland.
  • the Dutch patients with kidney stones were recruited from two sources: The outpatient clinics of the Radboud University Nijmegen Medical Center (RUNMC) and The Nijmegen Biomedical Study. All patients who present to the outpatient clinics of the RUNMC are invited to participate in a study on the effects of genes and lifestyle on the development of urological diseases. In case of consent, the patients fill out a lifestyle questionnaire and donate a blood sample for DNA isolation.
  • RUNMC Radboud University Nijmegen Medical Center
  • Nijmegen Biomedical Study All patients who present to the outpatient clinics of the RUNMC are invited to participate in a study on the effects of genes and lifestyle on the development of urological diseases. In case of consent, the patients fill out a lifestyle questionnaire and donate a blood sample for DNA isolation.
  • PROF Study Prospective Epidemiological Risk Factor
  • allele and haplotype frequencies are estimated by maximum likelihood and tests of differences between cases and controls are performed using a generalized likelihood ratio test. This method is particularly useful in situations where there are some missing genotypes for the ⁇ marker of interest and genotypes of another marker, which is in strong LD with the marker of interest, are used to provide some partial information. This was used in the association tests, to ensure that the comparison of the highly correlated markers was done using the same number of individuals.
  • maximum likelihood estimates, likelihood ratios and P values are computed directly for the observed data, and hence the loss of information due to uncertainty in phase and missing genotypes is automatically . captured by the likelihood ratios.
  • results from multiple case-control groups were combined using a Mantel-Haenszel model (Mantel, N. Haenszel, W., J Natl Cancer Inst 22: 719 (1959)) in which the groups were allowed • to have different population frequencies for alleles, haplotypes and genotypes but were assumed to have common relative risks.
  • the correlation between the genetic variants and the various biochemical traits was done by regressing the individual trait values on the number of copies of the at-risk variant an individual carries.
  • both the effect estimate and the P values are calculated by regressing standardized age, sex and weight adjusted trait values on the number of copies of the risk variant an individual carries.
  • the adjustment factors for relatedness for the various biochemical traits were estimated by simulations as described above and ranged from 1 (no adjustment) to 1.030 depending on the trait in question.
  • the adjustment factors for correlation with BMD were 1.165 (1.188) for hip (spine) for females, 1 (1.023) for hip (spine) for males, and 1.168 (1.198) of hip (spine) for the sexes combined.
  • the 5 ⁇ l PCR amplification reactions were set up on the Zymark SciClone ALH 300 robotic workstation in a 384 well PCR plate and amplified on a MJR Tetrad (Trademark of MJ. Research, Inc., Watertown, MA). Ampure (Agencourt) 384 PCR filters were used to remove unincorporated PCR primers and mononucleotides from the PCR reaction. l
  • Serum ionized calcium and serum bicarbonate were measured with an ion selective electrode (ABL 700, Radiometer Denmark); serum alkaline phosphatase (ALP) and urinary calcium and creatinine with a dry chemistry autoanalyzer (Vitros, Rochester, MN, USA); intact serum parathyroid hormone and serum CTx were measured using ECLIA (ElectroChemiLuminscence Immuno Assay, Elecsys 2010, Roche Diagnostics, Germany); Serum 25(OH)D (RIA; DiaSorin, USA) and serum cystatin C with PEIA (Particle-Enhanced Immunoturbidometric Assay; DAKO, Denmark).
  • ABL 700 ion selective electrode
  • ALP serum alkaline phosphatase
  • urinary calcium and creatinine with a dry chemistry autoanalyzer
  • intact serum parathyroid hormone and serum CTx were measured using ECLIA (ElectroChemiLuminscence Immuno Assay, Elecsys 2010, Roche Diagnostics,
  • the two SNPs are located in the same linkage disequilibrium (LD) block, 2,020 bases apart on chromosome 21q22.13, on either side of the last exon of claudin 14 (CLDN14) ( Figure 2), a gene involved in regulation of paracellular permeability at epithelial tight junctions ((Angelow S, er a/., Am J Physiol Renal Physiol 2008;295: F867-76)).
  • LD linkage disequilibrium
  • Figure 2 a gene involved in regulation of paracellular permeability at epithelial tight junctions
  • the CLDN14 gene is one of 24 members of the claudin family of membrane proteins that , regulate paracellular passage of ions and small solutes at epithelial tight junctions (Krause G, et al., Biochim Biophys Acta 2008; 1778: 631-45).
  • the specific distribution and function of the various claudins is considered an important determinant of the paracellular transport properties. , of different epithelia (Krause G, et al., Biochim Biophys Acta 2008;1778:631-45).
  • a renal disorder, autosomal recessive familial hypomagnesemia with hypercalciuria and nephrocalcinosis is caused by mutations in the claudin 16 (CLDN16) and claudin 19 (CLDN19) genes, ; , both expressed in the loop of Henle ((Angelow S, et al., Am J Physiol Renal Physiol 2008;295: F867-76);Simon, D. B., et al., Science 285: 103 (1999); Konrad, M., et al. Am J Hum: - Genet 79:949 (2006)). Kidney stone formation is a common finding in FHHNC.
  • the gene M associating with kidney stones in our study, CLDN14 is also expressed in the kidney, both in the loop of Henle and the proximal convoluted tubule, as well as in the epithelia of several other .; organs, and has been observed to selectively decrease cation paracellular permeability (Elkouby,- Naor, L, et al., Cell Tissue Res 333:427 (2008); Ben-Yosef, T., et al., Hum MoI Genet 79:949 (2006)).
  • CLDN8 which functions as a negative regulator of paracellular transport of cations, similar to CLDN14, also appears to act specifically as a negative regulator of protons, bicarbonate and ammonium ions in the distal nephron (Angelow, S. et al., J Physiol 571 : 1,5 (2006)).
  • the association between CLDN14 and serum bicarbonate level in our study suggestsia role for CLDN14 in acid-base balance, possibly directly through regulation of paracellular ion transport.
  • CLDN14 has been found to be expressed in the proximal tubule of the kidney where the reclamation of the bulk of filtered bicarbonate occurs as well as synthesis of ammonium, a major component of renal acid excretion. ⁇ ;.
  • Serum Albumin 0.10 (0.15) 0.42 0.12 (0.16) 0.48
  • Serum Alkaline phosphotase (Total) (U/L) 2.81 (2.31 ) 0.098 4.10 (2.45) 0.038
  • Serum Bicarbonate (mEq/L) -0.24 (0.1 1) 0.013 -0.25 (0.12) 0.0087
  • Serum Creatinine (micromol/L) -0.55 (0.87) 0.49 -0.51 (0.93) 0.39
  • Serum CTx (U/L) 0.017 (0.01 1) 0.098 0.016 (0.012) 0.038
  • Serum Cystacin C (mg/L) 0.0044 (0.0131) 0.49 0.0034 (0.0139) 0.51
  • Serum Ionized Calcium (mmol/L) 0.00061 (0.00205) 0.79 0.0018 (0.0022) 0.44
  • Serum Parathyroid Hormone - ELECS (pg/ml) 2.46 (0.96) 0.005 2.48 ( 1.03) 0.0072
  • Table 4 Correlation between the two CLDN14 exonic variants that associate with kidney stones, rs219780-C and rs219779-C, and BMD. The correlation is tested in a set of 8,450 Icelandic individuals of both sexes and 3,601 Danish females. The table shows the correlation with sex, age, and weight adjusted values of BMD for hip and spine separately, and for the Icelandic sample set for males, females and both sexes combined. Shown are the estimated effects expressed as standardized values per copy of the SNP allele, the corresponding standard errors of the mean (s.e.m.) and P values. For the Icelandic sample set, both the standard errors of the mean and P values have been adjusted for relatedness of the study individuals using simulations. Also shown is the result for the two sample sets combined.
  • the table includes the location (in IMCBI Build 36) of the identified variant, the corresponding rs-name if known, the minor allele and its frequency in controls and cases, the allelic OR and P value for association with kidney stone formation, the location of the variant within the gene region and the amino acid change where applicable. All P values have been adjusted for the relatedness of the cases and control by dividing the corresponding chi-square statistic by 1.023 (determined by simulations as described in Methods).
  • the association is calculated for the combined Icelandic and Dutch sample sets and for each SNP the association is tested conditional on the observed association to each of the other SNPs.
  • the table includes the SNP, the tested allele and the corresponding OR and P values and the adjusted P values corresponding to an association test conditional on each of the other SNPs.
  • Table 8 Genotype Specific Odds Ratios for the Risk Alleles of rs219780 and rs219779. Shown is the risk for heterozygous carriers (CT) and homozygous carriers (CC) of rs219780 and rs219779 compared to the risk for non-carriers (TT), together with 95% confidence intervals (CI), both for the combined Icelandic discovery and replication sample set and the sample set from the Netherlands, and for the sample sets combined using a Mantel-Haenzel model. For the Icelandic samples set the P values are adjusted for relatedness using simulations.
  • CT heterozygous carriers
  • CC homozygous carriers
  • TT risk for non-carriers
  • CI 95% confidence intervals
  • NCIN A Genotype specific Odds Ratio
  • Table 10 The association with kidney stones of 12 variants, at six distinct loci, that have previously been shown to association with variation in BMD. Results are shown for the Icelandic discovery data set of 1,507 individuals with kidney stones and 34,033 controls. Shown is the loci, the variant, the position of the variant, the tested allele, the frequency in cases and controls, the OR and the P values. For all variants the tested allele is the allele that correlates with lower bone mass density. All P values were adjusted for relatedness as described in the method section.
  • Table 11 Correlation between 12 variants at six distinct loci, previously associated with variation in BMD, and urinary calcium, serum bicarbonate and parathyroid hormone levels. The correlation is calculated for as subset of 706 of the 1,026 individuals in the Study on Bone and Mineral Health Cohort. Shown are the loci, the variant, the position of the variant, the tested allele, and the effect (s.e.m) and P values for each of the three biochemical traits. The effect and the corresponding standard error of the mean are obtained by regressing the unadjusted trait values on the number of risk allele an individual carries. The P value was calculated by regressing the sex and age adjusted and inverse normal transformed trait values on the number of risk allele carried. For all variants the tested allele is the allele that correlates with lower bone mass density. All P values were adjusted for relatedness as described in the method section.
  • Urine Calcium Serum Bicarbonate Hormone - ELECS (mmol/L) (mEq/L)

Abstract

Variants for assessing risk of conditions such as kidney stones, hypercalciurea reduced serum bicarbonate levels, reduced bone mineral density and osteroporosis are described. Such variants are useful in diagnostic applications for these conditions. Also described are kits, apparatus and media for use in such applications.

Description

Sequence variants predictive of risk of kidney stones and bone mineral density
INTRODUCTION
Nephrolithiasis (kidney stone disease, kidney stones; renal stones) is a common condition. The lifetime risk of stone formation exceeds 12% in men and 5% in women in the United States and recurrence rates are high at 30% to 40% within 5 years of the initial event. The vast majority of kidney stones (~80%) contain calcium while other forms, including uric acid stones, struvite and cystine stones, are far less common (Coe FL, Evan A, Worcester E. J Clin Invest 2005;115: 2598- 608). The formation of kidney stones is generally associated with a metabolic abnormahtv , most commonly primary hypercalciuπa, found in 5-10% of the general population and approximately 60% of adult patients with stones. Other less common abnormalities include hyperuπcosuria, primary hyperparathyroidism, hyperoxaluria and urinary acid-base abnormalities (Coe FL, Evan A, Worcester E. J CIm Invest 2005, 115:2598-608). Both nephrolithiasis and primary hypercalciuπa have a well recognized heritable contribution Between 35% and 65% of stone formers and up to 70% of subjects with hypercalciuπa have been found to have relatives with nephrolithiasis ( Polito C, et al. Pediatr Nephrol 2000; 14.1102- 4). Calcium stones have also been associated with several monogenic mendelian traits, many of which affect ion channels and transporters (Stechman MJ, et al. , Ann N Y Acad Sci 2007; 1116:461-84). Examples include Bartters syndrome, caused by mutations of the bumetamde-sensitive Na-K-Cl (NKCC2) cotransporter, the renal outer-medullary potassium channel (ROMK), the voltage-gated chloride channel, CLC-Kb, or of its subunit Barttin, or the calcium-sensing receptor, CaSR; Dent 's disease, due to mutations of the chloride/proton antiporter, CLC-5; and familial hypomagnesemia with hypercalciuπa and nephrocalcinosis (FHHNC) resulting from mutations in claudins (CLDN16 and CLDN19), major structural constituents of the intercellular tight junction barrier.
Kidney stones and hypercalciurea are known to be associated with bone mineral density. The association of hypercalciuπa with bone mineral density has been extensively explored and the overall data appears to suggest a risk for bone loss in individuals with kidney stones, primarily those with hypercalciuπa, and possibly associated with enhanced bone turnover (Sπvastava T, Alon US., Pediatr Nephrol 2007;22: 1659-73, Caudarella R, et al. , Urol Int 2004,72 Suppl 1.17- 9). Metabolic acidosis is also known to induce bone resorption although bone involvement is rarely present in dRTA (Rodriguez Soriano J., J Am Soc Nephrol 2002; 13:2160-70).
Genetic risk is in general conferred by subtle differences in the genome among individuals in a population. Genomic differences between individuals most frequently due to single nucleotide polymorphisms (SNP), although other variations are also important. SNP are located on average every 1000 base pairs in the human genome. Accordingly, a typical human gene containing 250,000 base pairs may contain 250 different SNPs. Only a minor number of SNPs are located in exons and alter the amino acid sequence of the protein encoded by the gene. Most SNPs may have little or no effect on gene function, while others may alter transcription, splicing, translation, or stability of the mRNA encoded by the gene. Additional genetic polymorphism in the human genome is caused by insertion, deletion, translocation, or inversion of either short or long stretches of DNA. Genetic polymorphisms conferring kidney stones risk may therefore directly alter the amino acid sequence of proteins, may increase the amount of protein produced from the gene, or may decrease the amount of protein produced by the gene.
As genetic polymorphisms conferring risk of the common diseases are uncovered, genetic testing for such risk factors is becoming important for clinical medicine. Examples are apolipoprotein E testing to identify genetic carriers of the apoE4 polymorphism in dementia patients for the differential diagnosis of Alzheimer's kidney stones, and of Factor V Leiden testing for predisposition to deep venous thrombosis. More importantly, in the treatment of cancer, diagnosis of genetic variants in tumor cells is used for the selection of the most appropriate treatment regime for the individual patient. In breast cancer, genetic variation in estrogen receptor expression or heregulin type 2 (Her2) receptor tyrosine kinase expression determine if anti-estrogenic drugs (tamoxifen) or anti-Her2 antibody (Herceptin) will be incorporated into the treatment plan. In chronic myeloid leukemia (CML) diagnosis of the Philadelphia chromosome genetic translocation fusing the genes encoding the Bcr and AbI receptor tyrosine kinases indicates that Gleevec (STI571), a specific inhibitor of the Bcr-Abl kinase should be used for treatment of the cancer. For CML patients with such a genetic alteration, inhibition of the Bcr- Abl kinase leads to rapid elimination of the tumor cells and remission from leukemia. Furthermore, genetic testing based on the identification of common SNP variants that confer increased risk of common diseases such as Breast Cancer, Prostate Cancer, Myocardial Infarction, Type 2 Diabetes and Atrial Fibrillation is now available.
There is an unmet need for genetic variants that confer susceptibility of kidney stones and related conditions in the general population. Identification of such variants may have important diagnostic applications, as they can be used to identify those at particularly high risk of these ' conditions, prompting those individuals to take available precautions, either by pharmacological means or by dietary adjustments. The present invention provides such variants.
SUMMARY OF THE INVENTION
The present invention is based on the association of the human CLDN 14 gene with conditions that include kidney stones, serum parathyroid hormone levels, serum bicarbonate levels, bone mineral density and osteoporosis. Variants associated with the CLDN 14 gene have been shown to be associated with risk of these conditions. In a first aspect, the invention provides a method of determining a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in e human individual, the method comprising obtaining nucleic acid sequence data about a human individual identifying at least one allele of at least one polymorphic marker associated with the human CLDN14 gene, wherein different alleles of the at least one polymorphic marker are associated with different susceptibilities to the at least one condition in humans, and determining a susceptibility to the at least one condition from the nucleic acid sequence data.
The invention also provides a method of determining a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, the method comprising : obtaining nucleic acid sequence data about a human individual identifying both alleles of at least two polymorphic markers associated with the human CLDN14 gene, determine the identity of at least one haplotype based on the sequence data, and determining a susceptibility to the at least one condition from the haplotype data
Also provided is a method of diagnosing a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, the method comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, or in a genotype dataset from the individual, wherein the at least one polymorphic marker is associated with the CLDN14 gene, and wherein the presence of the at least one allele is indicative of a susceptibility to the at least one condition.
The invention also provides a method of diagnosing a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, the method comprising, obtaining CLDN14 amino acid sequence data about at least one encoded CLDN14 protein of a human individual, identifying at least one polymorphic site associated with the CLDN14 amino acid sequence, wherein different amino acids of the at least one polymorphic site are associated with different susceptibilities to the at least one condition in humans, and diagnosing susceptibility to the at least one condition from the amino acid sequence data.
Furthermore, the invention provides a method of determining a susceptibility of at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, the method comprising determining the identity of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one marker is selected from the group of markers located within the LD block C21, wherein susceptibility of the at least one condition is correlated with the identity of the at least one allele.
The invention also provides a method of identification of a marker for use in assessing susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, the method comprising
a. identifying at least one polymorphism associated with the CLDN14 gene;
b. determining the genotype status of a sample of individuals diagnosed with, or having a susceptibility to, the condition; and
c. determining the genotype status of a control sample;
wherein determination of a significant difference in frequency of at least one allele in the at least one polymorphism in individuals diagnosed with, or having a susceptibility to, the condition, as compared with the frequency of the at least one allele in the control sample is indicative of the at least one polymorphism being useful for assessing susceptibility to the condition.
The invention can also be used to assess individuals for probability of response to a therapeutic agent for preventing and/or ameliorating symptoms associated with at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, comprising: determining the identity of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one polymorphic marker is selected from polymorphic markers associated with the human CLDN 14 gene, wherein determination of the identity of the at least one allele of the at least one marker is indicative of a probability of a positive response to the therapeutic agent.
Prognosis of an individual experiencing symptoms associated with, or an individual diagnosed with, at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis is also provided, the method comprising : obtaining nucleic acid sequence data about a human individual identifying at least one allele of at least one polymorphic marker associated with the human CLDN14 gene, wherein different alleles of the at least one polymorphic marker are associated with different susceptibilities to the at least one condition in humans, andpredictiπg prognosis of the individual from the nucleic acid sequence data.
Treatment outcome of an individual undergoing treatment for at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis is also provided by the invention, the method comprising obtaining nucleic acid sequence data about a human individual identifying at least one allele of at least one polymorphic marker associated with the human CLDN 14 gene, wherein different alleles of the at least one polymorphic marker are associated with different susceptibilities to the at least one condition in humans, and predicting treatment outcome of the individual from the nucleic acid sequence data.
The invention also provides kits. In one such aspect the invention provides a kit for assessing susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, the kit comprising (ι) reagents for selectively detecting at least one allele of at least one polymorphic marker in the genome of the individual, wherein the at least one polymorphic marker is selected from the group consisting of rsl90068, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364 and rsl70183, and markers in linkage disequilibrium therewith, and (ιι) a collection of data comprising correlation data between the polymorphic markers assessed by the kit and susceptibility to the at least one condition.
The invention also provides in another aspect the use of an oligonucleotide probe in the manufacture of a diagnostic reagent for diagnosing and/or assessing susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, wherein the probe is capable of selectively hybridizing to a segment of a nucleic acid whose nucleotide sequence is given by SEQ ID NO: 1 that comprises at least one polymorphic site, wherein the segment is 15-500 nucleotides in length.
Computer-implemented aspects are also provided. In one such aspect, the invention provides a computer-readable medium having computer executable instructions for determining susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, the computer readable medium comprιsιng:data indicative of at least one polymorphic markerja routine stored on the computer readable medium and adapted to be executed by a processor to determine risk of developing the at least one condition for the at least one polymorphic marker; wherein the at least one polymorphic marker is associated with the human CLDN14 gene.
Another computer-implemented aspect provides an apparatus for determining a genetic indicator in a human individual for at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, comprising. a processor; a computer readable memory having computer executable instructions adapted to be executed on the processor to analyze marker and/or haplotype information for at least one human individual with respect to at least one polymorphic marker associated with the human CLDN14 gene, and generate an output based on the marker or haplotype information, wherein the output comprises a risk measure of the at least one marker or haplotype as a genetic indicator of the at least one condition for the human individual.
The invention also provides pharmaceutical compositions comprising polypeptides encoded by a human CLDN14 gene or fragments thereof, methods of prophylaxis therapy of conditions selected from the group consisting of kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis using pharmaceutical compositions that include agents that regulate expression, activity or physical state of a CLDN14 gene, its encoding RNA or protein in an individual, human CLDN14 polypeptides and oligonucleotides for uses as medicaments, and methods of therapy , that select individuals for treatment based on the genetic status of at least one CLDN14 variant that correlates with increased risk of the condition.
The invention in general relates to markers associated with the CLDN14 gene, in particular markers within, or in linkage disequilibrium with, the LD block C21 as defined herein. ExemplaiV markers are provide e.g. in Table 1 herein. Certain embodiments relate to one or more of the markers rs219778, rs219779, rs219780 and rs219781. Further embodiments of the invention are presented in the following description of various embodiments of the invention.
It should be understood that all combinations of features described herein are contemplated, even if the combination of feature is not specifically found in the same sentence or paragraph herein. This includes in particular the use of all markers disclosed herein, alone or in combination, for analysis individually or in haplotypes, in all aspects of the invention as described herein.
BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention.
Figure 1 shows a Q-Q plot of the 303,120 adjusted Chi2-statistics from a GWA analysis of kidney stones. The equiangular line (black line) is included in the plot for reference purpose. The dashed horizontal line indicates the threshold for genome-wide significance assuming a Bonferroni correction for the 303,120 SNPs tested. The red dots correspond to results for all the 303,120 SNPs, while for the blue dots, the 166 SNPs in a 1 Mb region on chromosome 21 centered on the CLDN14 gene have been excluded.
Figure 2 describes the 21q22 \ocus;(A) presents the pair-wise correlation structure in a 200 kb interval (36.45 - 36.95 Mb, NCBI B36) on Chromosome 21. The upper plot shows pair-wise D' for 454 common SNPs (with MAF > 5%) from the HapMap (v22) CEU dataset. The lower plot shows the corresponding r2 values. (B) shows the estimated recombination rates (saRR) in cM/Mb from the HapMap Phase II data(Frazer KA, et al., Nature 2007;449:851-61). (C) displays the location of known genes in the region and (D) reveals the schematic view of the association with kidney stones for all the markers tested in the genome-wide scan. All panels use the same horizontal scale shown in panel D).
Figure 3 provides a diagram illustrating a computer-implemented system utilizing risk variants., as described herein.
DETAILED DESCRIPTION
Definitions
Unless otherwise indicated, nucleic acid sequences are written left to right in a 5' to 3' orientation. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by the ordinary person skilled in the art to which the invention pertains.
The following terms shall, in the present context, have the meaning as indicated:
A "polymorphic marker", sometime referred to as a "marker", as described herein, refers to a genomic polymorphic site. Each polymorphic marker has at least two sequence variations characteristic of particular alleles at the polymorphic site. Thus, genetic association to a polymorphic marker implies that there is association to at least one specific allele of that particular polymorphic marker. The marker can comprise any allele of any variant type found in the genome, including SNPs, mini- or microsatellites, translocations and copy number variations (insertions, deletions, duplications). Polymorphic markers can be of any measurable frequency in the population. For mapping of kidney stone genes, polymorphic markers with population frequency higher than 5-10% are in general most useful. However, polymorphic markers may also have lower population frequencies, such as 1-5% frequency, or even lower frequency, in particular copy number variations (CNVs). The term shall, in the present context, be taken to include polymorphic markers with any population frequency.
An "allele" refers to the nucleotide sequence of a given locus (position) on a chromosome. A polymorphic marker allele thus refers to the composition (i.e., sequence) of the marker on a chromosome. Genomic DNA from an individual contains two alleles (e.g., allele-specific sequences) for any given polymorphic marker, representative of each copy of the marker on each chromosome. Sequence codes for nucleotides used herein are: A = 1, C = 2, G = 3, T = , 4. For microsatellite alleles, the CEPH sample (Centre d'Etudes du Polymorphisme Humain, genomics repository, CEPH sample 1347-02) is used as a reference, the shorter allele of each microsatellite in this sample is set as 0 and all other alleles in other samples are numbered in relation to this reference. Thus, e.g. , allele 1 is 1 bp longer than the shorter allele in the CEPH sample, allele 2 is 2 bp longer than the shorter allele in the CEPH sample, allele 3 is 3 bp longer than the lower allele in the CEPH sample, etc., and allele - 1 is 1 bp shorter than the shorter allele in the CEPH sample, allele -2 is 2 bp shorter than the shorter allele in the CEPH sample, ' etc.
Sequence conucleotide ambiguity as described herein is as proposed by IUPAC-IUB. These codes are compatible with the codes used by the EMBL, GenBank, and PIR databases.
Figure imgf000009_0001
A nucleotide position at which more than one sequence is possible in a population (either a natural population or a synthetic population, e.g. , a library of synthetic molecules) is referred to herein as a "polymorphic site".
A "Single Nucleotide Polymorphism" or "SNP" is a DNA sequence variation occurring when a single nucleotide at a specific location in the genome differs between members of a species or between paired chromosomes in an individual. Most SNP polymorphisms have two alleles. Each individual is in this instance either homozygous for one allele of the polymorphism (i.e. both chromosomal copies of the individual have the same nucleotide at the SNP location), or the \ individual is heterozygous (i.e. the two sister chromosomes of the individual contain different nucleotides). The SNP nomenclature as reported herein refers to the official Reference SNP (rs) ID identification tag as assigned to each unique SNP by the National Center for Biotechnological Information (NCBI).
A "variant", as described herein, refers to a segment of DNA that differs from the reference DNA. A "marker" or a "polymorphic marker", as defined herein, is a variant. Alleles that differ from the reference are referred to as "variant" alleles. A "microsatellite" is a polymorphic marker that has multiple small repeats of bases that are 2-8 - nucleotides in length (such as CA repeats) at a particular site, in which the number of repeat lengths varies in the general population. An "indel" is a common form of polymorphism, comprising a small insertion or deletion that is typically only a few nucleotides long.
A "haplotype," as described herein, refers to a segment of genomic DNA that is characterized by a specific combination of alleles arranged along the segment. For diploid organisms such as humans, a haplotype comprises one member of the pair of alleles for each polymorphic marker or locus along the segment. In a certain embodiment, the haplotype can comprise two or more alleles, three or more alleles, four or more alleles, or five or more alleles. Haplotypes are described herein in the context of the marker name and the allele of the marker in that haplotype, e.g., "2 rs219781" refers to the 2 allele of marker rs219781 being in the haplotype, and is equivalent to "rs219781 allele 2". Furthermore, allelic codes in haplotypes are as for individual markers, i.e. I = A, 2 = C, 3 = G and 4 = T.
The term "susceptibility", as described herein, refers to the proneness of an individual towards the development of a certain state (e.g., a certain trait or phenotype), or towards being less able to resist a particular state than the average individual. The term encompasses both increased susceptibility and decreased susceptibility. Thus, particular alleles at polymorphic markers and/or haplotypes of the invention as described herein may for example be characteristic of increased susceptibility (i.e., increased risk) of kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, as characterized by a relative risk (RR) or odds ratio (OR) of greater than one for the particular allele or haplotype. Alternatively, the markers and/or haplotypes of the invention are characteristic of decreased susceptibility (i.e., decreased risk) of one or more of these conditions, as characterized by a relative risk of less than one.
The term "and/or" shall in the present context be understood to indicate that either or both of the items connected by it are involved. In other words, the term herein shall be taken to mean "one or the other or both".
The term "look-up table", as described herein, is a table that correlates one form of data to another form, or one or more forms of data to a predicted outcome to which the data is relevant, such as phenotype or trait. For example, a look-up table can comprise a correlation between allelic data for at least one polymorphic marker and a particular trait or phenotype, such as a particular kidney stones diagnosis, that an individual who comprises the particular allelic data is likely to display, or is more likely to display than individuals who do not comprise the particular allelic data. Look-up tables can be multidimensional, i.e. they can contain information about multiple alleles for single markers simultaneously, or the can contain information about multiple markers, and they may also comprise other factors, such as particulars about kidney stonesdiagnoses, racial information, biomarkers, biochemical measurements, therapeutic methods or drugs, etc. A "computer-readable medium", is an information storage medium that can be accessed by a computer using a commercially available or custom-made interface. Exemplary compute- readable media include memory (e.g., RAM, ROM, flash memory, etc.), optical storage media (e.g., CD-ROM), magnetic storage media (e.g., computer hard drives, floppy disks, etc.), punch cards, or other commercially available media. Information may be transferred between a system of interest and a medium, between computers, or between computers and the computer- readable medium for storage or acess of stored information. Such transmission can be electrical, or by other available methods, such as IR links, wireless connections, etc.
A "nucleic acid sample" as described herein, refers to a sample obtained from an individual that contains nucleic acid (DNA or RNA). In certain embodiments, i.e. the detection of specific polymorphic markers and/or haplotypes, the nucleic acid sample comprises genomic DNA. Such a nucleic acid sample can be obtained from any source that contains genomic DNA, including a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs.
"Kidney stones", as described herein, refers to a disease state in which crystal aggregations of dissolved minerals, also called renal calculi, form in the kidneys, urinary tract or bladder. Kidney stones are sometimes also referred to as "nephrolithiasis", "kidney stone disease", or "renal stones".
The term " therapeutic agent for kidney stones" refers to an agent that can be used to ameliorate or prevent symptoms associated with kidney stones.
The term "kidney stones-associated nucleic acid", as described herein, refers to a nucleic acid that has been found to be associated to kidney stones. This includes, but is not limited to, the markers and haplotypes described herein and markers and haplotypes in strong linkage disequilibrium (LD) therewith. In one embodiment, a kidney stone-associated nucleic acid refers to an LD-block found to be associated with Type 2 diabetes through at least one polymorphic marker located within the LD block.
The term "LD block C21", as described herein, refers to the Linkage Disequilibrium (LD) block on Chromosome 21 corresponding to position 36,697,419 - 36,770,504 of NCBI (National Center for Biotechnology Information) Build 36 (SEQ ID NO: 1).
Identification of variants on chromosome 21 associated with risk of kidney stones
The present inventors have discovered that variants associated with the human CLDN14 gene ,. are associated with risk of kidney stones, hypercalciurea, bone mineral density, reduced serum bicarbonate and increased urinary calcium concentration. Certain variants in the CLDN14 gene have been found to confer risk of these conditions, such that the presence of particular alleles at these variants confer increased risk of the condition, and certain other alleles confer decreased risk of the condition. These variants, and other variants that are correlated to these variants, are useful for certain diagnostic applications.
Methods of determining susceptibility to kidney stones
Accordingly, the present invention provides methods of determining a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual. In one aspect, the method comprises obtaining nucleic acid sequence data about a human individual identifying at least one allele of at least one polymorphic marker associated with the human CLDN14 gene, wherein different alleles of the at least one : polymorphic marker are associated with different susceptibilities to the at least one condition in'1 humans, and determining a susceptibility to the at least one condition from the nucleic acid sequence data.
In a general sense, genetic markers lead to alternate sequences at the nucleic acid level. If the nucleic acid marker changes the codon of a polypeptide encoded by the nucleic acid, then the marker will also result in alternate sequence at the amino acid level of the encoded polypeptide (polypeptide markers). Determination of the identity of particular alleles at polymorphic markers in a nucleic acid or particular alleles at polypeptide markers comprises whether particular alleles are present at a certain position in the sequence. Sequence data identifying a particular allele at a marker comprises sufficient sequence to detect the particular allele. For single nucleotide polymorphisms (SNPs) or amino acid polymorphisms described herein, sequence data can ά comprise sequence at a single position, i.e. the identity of a nucleotide or amino acid at a single position within a sequence. Alternatively, the allele can be the allele of the complementary strand of DNA, such that the nucleic acid data includes the identification of at least one allele which is complementary to the allele at the opposite strand.
In certain embodiments, it may be useful to determine the nucleic acid sequence for at least two polymorphic markers. In other embodiments, the nucleic acid sequence for at least three, at least four or at least five or more polymorphic markers is determined. Haplotype information can be derived from an analysis of two or more polymorphic markers. Thus, in certain : embodiments, a further step is performed, whereby haplotype information is derived based on sequence data for at least two polymorphic markers.
In certain embodiments, sequence data about both alleles of polymorphic markers associated , with the CLDN 14 gene are obtained, and the identity of at least one haplotype based on the sequence data is determined, and a susceptibility to the condition is determined from the haplotype data. In certain embodiments, determination of a susceptibility comprises comparing the nucleic acid sequence data to a database containing correlation data between the at least one polymorphic marker and susceptibility to the at least one condition. In some embodiments, the database comprises at least one risk measure of susceptibility to at least one condition for the at least one marker. The sequence database can for example be provided as a look-up table that contains data that indicates the susceptibility of at least one condition for any one, or a plurality of, particular polymorphisms. The database may also contain data that indicates the susceptibility for a particular haplotype that comprises at least two polymorphic markers.
Obtaining nucleic acid sequence data can in certain embodiments comprise obtaining a biological sample from the human individual and analyzing sequence of the at least one polymorphic marker in nucleic acid in the sample. Analyzing sequence can comprise determining the presence or absence of at least one allele of the at least one polymorphic marker. Determination of the presence of a particular susceptibility allele (e.g. , an at-risk allele) is indicative of susceptibility to the condition in the human individual. Determination of the absence of a particular suscepbility allele is indicative that the particular susceptibility due to the at least one, polymorphic marker is not present in the individual.
In some embodiments, obtaining nucleic acid sequence data comprises obtaining nucleic acid • sequence information from a preexisting record. The preexisting record can for example be a ■ • computer file or database containing sequence data, such as genotype data, for the human individual, for the at least one polymorphic marker.
Susceptibility determined by the diagnostic methods of the invention can be reported to a particular entity. In some embodiments, the at least one entity is selected from the group consisting of the individual, a guardian of the individual, a genetic service provider, a physician, a medical organization, and a medical insurer.
In certain embodiments of the invention, determination of a susceptibility comprises comparing the nucleic acid sequence data to a database containing correlation data between the at least one polymorphic marker and susceptibility to the condition. In one such embodiment, the database comprises at least one risk measure of susceptibility to the condition for the at least one polymorphic marker. In another embodiment, the database comprises a look-up table containing at least one risk measure of the at least one condition for the at least one polymorphic marker.
In certain embodiments, obtaining nucleic acid sequence data comprises obtaining a biological . sample from the human individual and analyzing sequence of the at least one polymorphic marker in nucleic acid in the sample. Analyzing sequence of the at least one polymorphic marker can comprise determining the presence or absence of at least one allele of the at least one polymorphic marker. Obtaining nucleic acid sequence data can also comprise obtaining nucleic acid sequence information from a preexisting record. Certain embodiments of the invention relate to obtaining nucleic acid sequence data about at least two polymorphic markers associated with the CLDN14 gene. Other embodiments may relate to obtaining sequence data about more than two polymorphic markers, including three, four, five, six, seven, eight, nine or ten or more polymorphic markers.
The markers associated with the CLDN 14 gene are in certain embodiments markers within LD block C21 as set forth in SEQ ID NO: 1 herein.
Obtaining sequence data may in certain embodiments relate to determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, or in a genotype dataset from the individual. Obtaining information about the absence or presence of particular marker alleles represents sequence information for the marker, identifying particular marker alleles.
In certain embodiments of the invention, the at least one polymorphic marker is selected from ■ polymorphic markers associated with the human CLDN14 gene. Certain embodiments relate to markers within LD block C21. Certain preferred embodiments relate to markers selected from ' the group consisting of rsllθ.88346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, ■• rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364 and rsl70183, which are the. markers listed in Table Ia, and markers in linkage disequililbπum therewith.
One preferred embodiment relates to markers selected from the group consisting of rsl 1088346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364 rsl70183, rsl3051987, rsl70182, rs218638, rs218641, rs218642, rs218643, rs219759, rs219761, rs219765, rs219766, rs219767, rs219768, rs219772, rs219774, rs219782, rs219783, rs219785, rs219790, rs2244014, rs2249115, rs2633329, rs2633331, rs2633334, rs2776288, rs2835304, rs2835305, rs2835309, rs2835318, rs2835319, rs2835348, rs2835349, rs2845762, rs2845764, rs2845766, rs2845769, rs2850087, rs2850102, rs2850108, rs2850110, rs56183874, rs9975445, S.36470266, S.36472078, S.36478541, s.36487357, S.36489391, S.36492368, s.36571651, s.36598656, s.36623095, s.36644230, S.36690993, s.36691722, S.36693496, s.36694516, S.36696258, S.36696674, s.36708810, s.36722244, s.36724712, s.36730418, S.36730731, S.36732831, S.36733004, s.36734680, s.36738144, S.36738223, S.36738919, s.36739280, s.36739821, s.36749630, s.36749760, S.36751378, S.36757110, S.36757852, S.36776041, S.36777630, S.36777724, s.36777871, S.36778050, S.36780792, S.36781080, s.37075430, s.37158027, which are the markers listed in Table Ia and Ib herein (i.e., markers rs219781, rs219780, rs219779, rs219778 and markers in linkage disequilibrium therewith). Obviously anchor markers such as rs219781, rs219780, rs219779, •■ ' rs219778 can be considered to be in linkage disequilibrium with themselves. As a consequence; markers in linkage disequilibrium with the anchor markers include the foregoing surrogate marker list (as also presented in Table 1), excluding the surrogate markers rs219781, rs219780, rs219779, rs219778. Another preferred embodiment relates to markers selected from the group consisting of rsllO88346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs21978l, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364 and rsl70183.
Particularly preferred embodiments relate to markers selected from the group consisting of rs219778, rs219779, rs219780 and rs219781. Certain preferred embodiments relate to marker rs219778. Certain other embodiments relate to markers rs219779. Certain other embodiments relate to markers rs219780. Certain other embodiments relate to markers rs219781. Certain embodiments further relate to surrogate markers of any one of the markers rs219778, rs219779, rs219780 and rs219781, which can suitably be selected from the markers presented , in Table 1 (Ia and/or Ib) herein.
In certain embodiments of the invention, a further step of assessing the frequency of at least one haplotype in the individual is performed. In such embodiments, two or more markers, including three, four, five, six, seven, eight, nine or ten or more markers can be included in the haplotype. In certain embodiments, the at least one haplotype comprises markers that are all in LD with at least one of rs219778, rs219779, rs219780 and rs219781.
In certain embodiments, determination of the presence of rs219778 allele T, rs219779 allele C, rs219780 allele C and/or rs219781 allele C, or marker alleles or haplotypes in linkage disequilibrium therewith, is indicative of increased susceptibility to the condition. The opposite (alternate) allele of particular SNPs are in such embodiments indicative of a decreased susceptibility to the conditions. Thus, in certain embodiments, the presence of rs219778 allele C, rs219779 allele T, rs219780 allele A and/or rs219781 allele A is indicative of decreased susceptibility to the at least one condition.
The skilled person will appreciate that homozygous individuals for particular at-risk or protective marker alleles or haplotypes are at particularly high and/or low risk. Since the effects of each .. copy of a particular risk or protective allele are usually additive, individuals who carry two copies of an at-risk allele or haplotype are at particularly high risk. Certain embodiments of the invention thus relate to individuals who are homozygous for at-risk alleles for at least one polymorphic marker selected from the group consisting of rsl 1088346, rsl90068, rs2835342, ;_ rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364, rsl70183, rsl3051987, rsl70182, rs218638, rs218641, rs218642, rs218643, rs219759, rs219761, rs219765, rs219766, rs219767, rs219768, rs219772, rs219774, rs219782, rs219783, rs219785, rs219790, rs2244014, rs2249115, rs2633329, rs2633331, rs2633334, rs2776288, rs2835304, rs2835305, rs2835309, rs2835318, rs2835319, rs2835348, rs2835349, rs2845762, rs2845764, rs2845766, rs2845769, rs2850087, rs2850102, rs2850108, rs2850110, rs56183874, rs9975445, S.36470266, s.36472078, s.36478541, s.36487357, s.36489391, s.36492368, S.36571651, S.36598656, s.36623095, s.36644230, s.36690993, s.36691722, s.36693496, S.36694516, s.36696258, s.36696674, s.36708810, s.36722244, s.36724712, s.36730418, S.36730731, s.36732831, S.36733004, s.36734680, s.36738144, s.36738223, S.36738919, S.36739280, s.36739821, s.36749630, s.36749760, s.36751378, s.36757110, s.36757852, s.36776041, s.36777630, s.36777724, s.36777871, s.36778050, s.36780792, s.36781080, \ s.37075430, and s.37158027. Preferred embodiments relate to individuals who are homozygous for any one, or a combination of, rs219778 allele C, rs219779 allele T, rs219780 allele A and/or rs219781 allele A, or marker alleles or haplotypes in linkage disequilibrium therewith.
The markers conferring risk of at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, as described herein, can be combined with other genetic markers for one or more of these conditions. Such markers are typically not in linkage disequilibrium with any one of the markers described herein, in particular markers rs219778, rs219779, rs219780 and rs219781. Any of the mehods described herein can be practiced by combining the genetic risk factors described herein with such additional genetic risk factors for the condition.
Thus, in certain embodiments, a further step is included, comprising determining whether at least one at-risk allele of at least one at-risk variant for at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum " bicarbonate levels, reduced bone mineral density and osteoporosis not in linkage disequilibrium with any one of the markers rs219778, rs219779, rs219780 and rs219781 is present in a sample comprising genomic DNA from a human individual or a genotype dataset derived from a human individual. Determination of the presence of the marker allele may be determined by obtaining - nucleic acid sequence data identifying the particular allele. Thus, genetic markers in other locations in the genome can be useful in combination with the markers of the present invention, so as to determine overall risk of the condition based on multiple genetic factors. Selection of , markers that are not in linkage disequilibrium (not in LD) can be based on a suitable measure for linkage disequilibrium, as described further herein. In certain embodiments, markers that are not in linkage disequilibrium have values for the LD measure r2 between the markers of less than 0.2. In certain other embodiments, markers that are not in LD have values for r2 between the markers of less than 0.15, including less than 0.10, less than 0.05, less than 0.02 and less than 0.01. Other suitable cutoff values for establishing that markers are not in LD are contemplated, including values bridging any of these values.
The genetic markers of the invention can also be combined with non-genetic information to establish overall risk for an individual. Thus, in certain embodiments, a further step is included, comprising analyzing non-genetic information to make risk assessment, diagnosis, or prognosis: of the individual. The non-genetic information can be any information pertaining to the disease' status of the indiviudal or other information that can influence the estimate of overall risk of the condition for the individual. In one embodiment, the non-genetic information is selected from the group consisting of low water consumption, hypercalciurea, high-protein diet, high-sodium . diet, low-calcium diet, obesity, high blood pressure, lack of physical activity, family history of kidney stones, previous kidney stones, vitamin A deficiency, hyperparathyroidism, kidney infection and history of gastric bypass surgery, inflammatory bowel disease or chronic diarrhea.
Certain aspects of the invention relate to a method of identification of a marker for use in assessing susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, the method comprising : identifying at least one polymorphic marker in linkage disequilibrium with at least one of rs219778, rs219779, rs219780, and rs219781 (e.g., one or more of the markers set forth in Table 1 herein); determining the genotype status of a sample of individuals diagnosed with, or having a susceptibility to, the condition; and determining the genotype status of a sample of control individuals; wherein a significant difference in frequency of at least one allele in at least one polymorphism in individuals diagnosed with, or having a susceptibility to, the condition, as compared with the frequency of the at least one allele in the control sample is indicative of the at least one polymorphism being useful for assessing susceptibility to the condition. Significant difference can be estimated on statistical analysis of allelic counts at certain polymorphic markers in patients and controls. In one embodiment, a significant difference is based on a calculated P- value between patients and controls of less than 0.05. In other embodiments, a significant difference is based on a lower value of the calculated P-value, such as less than 0.005, 0.0005, or 0.00005. In one embodiment, an increase in frequency of the at least one allele in the at least one polymorphism in individuals diagnosed with, or having a susceptibility to, the condition, as compared with the frequency of the at least one allele in the control sample is indicative of the at least one polymorphism being useful for assessing increased susceptibility to the condition. In another embodiment, a decrease in frequency of the at least one allele in the at least one polymorphism in individuals diagnosed with, or having a susceptibility to, the condition, as compared with the frequency of the at least one allele in the control sample is indicative of the at least one polymorphism being useful for assessing decreased susceptibility to, or protection against, the condition.
The invention also relates to a method of genotyping a nucleic acid sample obtained from a human individual comprising determining whether at least one allele of at least one polymorphic marker is present in a nucleic acid sample from the individual sample, wherein the at least one marker is selected from rsllO88346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364, rsl70183, rsl3051987, rsl70182, rs218638, rs218641, rs218642, rs218643, rs219759, rs219761, rs219765, rs219766, rs219767, rs219768, rs219772, rs219774, rs219782, rs219783, rs219785, rs219790, rs2244014, rs2249115, rs2633329f rs2633331, rs2633334, rs2776288, rs2835304, rs2835305,, rs2835309, rs2835318, rs2835319, rs2835348, rs2835349, rs2845762, rs2845764, rs2845766) rs2845769, rs2850087, rs2850102, rs2850108, rs2850110, rs56183874, rs9975445, s.36470266, s.36472078, s.36478541, s.36487357, S.36489391, s.36492368, s.36571651, S.36598656, s.36623095, s.36644230, s.36690993, S.36691722, s.36693496, s.36694516, S.36696258, s.36696674, S.36708810, S.36722244, s.36724712, S.36730418, s.36730731, ' s.36732831, s.36733004, s.36734680, s.36738144, s.36738223, s.36738919, s.36739280, s.36739821, s.36749630, s.36749760, s.36751378, s.36757110, s.36757852, s.36776041, s.36777630, s.36777724, S.36777871, s.36778050, s.36780792, s.36781080, S.37075430, s.37158027, and markers in linkage disequilibrium therewith, and wherein determination of the presence of the at least one allele in the sample is indicative of a susceptibility to a condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in the individual. In one embodiment, determination of the presence of rs219778 allele T, rs219779 allele C, rs219780 allele C and rs219781 allele C is indicative of increased suscepbtibility of the condition in the individual. In one embodiment, genotyping comprises amplifying a segment of a nucleic acid that comprises the at least one polymorphic marker by Polymerase Chain Reaction (PCR), using a nucleotide primer pair flanking the at least one polymorphic marker. In another embodiment, genotyping is performed using a process selected from allele-specific probe hybridization, allele-specific primer extension, allele-specific amplification, nucleic acid sequencing, 5'-exonuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformation analysis and microarray technology. In one embodiment, the microarray technology is Molecular Inversion Probe array technology or BeadArray Technologies. In one embodiment, the process comprises allele-specific probe hybridization. In another embodiment, the process comprises microrray technology. One preferred embodiment comprises the steps of ( 1) contacting copies of the nucleic acid with a detection oligonucleotide probe and an enhancer oligonucleotide probe under conditions for specific hybridization of the oligonucleotide probe with the nucleic acid; wherein (a) the detection oligonucleotide probe is from 5-100 nucleotides in length and specifically hybridizes to a first segment of a nucleic acid whose nucleotide sequence is given by SEQ ID NO: 1; (b) the detection oligonucleotide probe comprises a detectable label at its 3' terminus and a quenching moiety at its 5' terminus; (c) the enhancer oligonucleotide is from 5-100 nucleotides in length and is complementary to a second segment of the nucleotide sequence that is 5' relative to the,. oligonucleotide probe, such that the enhancer oligonucleotide is located 3' relative to the detection oligonucleotide probe when both oligonucleotides are hybridized to the nucleic acid; and (d) a single base gap exists between the first segment and the second segment, such that when the oligonucleotide probe and the enhancer oligonucleotide probe are both hybridized to the nucleic acid, a single base gap exists between the oligonucleotides; (2) treating the nucleic acid with an endonuclease that will cleave the detectable label from the 31 terminus of the detection probe to release free detectable label when the detection probe is hybridized to the nucleic acid; and (3) measuring free detectable label, wherein the presence of the free detectable label indicates that the detection probe specifically hybridizes to the first segment of the nucleic acid, and indicates the sequence of the polymorphic site as the complement of the detection probe.
The invention also relates to the use of an oligonucleotide probe in the manufacture of a reagent for diagnosing and/or assessing susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, wherein the probe hybridizes to a segment of a nucleic acid with nucleotide sequence as set forth in SEQ ID NO: 1, wherein the probe is 15-500 nucleotides in length. In certain embodiments, the probe is about 16 to about 100 nucleotides in length. In certain other embodiments, the probe is about 20 to about 50 nucleotides in length. In certain other embodiments, the probe is about 20 to about 30 nucleotides in length.
The skilled person will appreciate that the markers that are described herein to be associated with risk of conditions selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis may be used, alone or in any particular combination, in all various aspects of the invention, including the methods, kits, uses, apparatus, procedures described herein. This relates in particular to markers associated with the human CLDN 14 gene, and in particular embodiments markers within LD block C21, and markers as set forth in Table 1, or markers in linkage disequilibrium therewith.
In certain embodiments of the invention, linkage disequilbrium is determined using the linkage disequilibrium measures r2 and |D'|, which give a quantitative measure of the extent of linkage disequilibrium (LD) between two genetic element (e.g., polymorphic markers). Certain numerical values of these measures for particular markers are indicative of the markers being in linkage disequilibrium, as described further herein. In one embodiment of the invention, linkage disequilibrium between marker (Ae., LD values indicative of the markers being in linkage disequilibrium) is defined as r2 > 0.1. In another embodiment, linkage disequilibrium is defined as r2 > 0.2. Other embodiments can include other definitions of linkage disequilibrium, such as r2 > 0.25, r2 > 0.3, r2 > 0.35, r2 > 0.4, r2 > 0.45, r2 > 0.5, r2 > 0.55, r2 > 0.6, r2 > 0.65, r2 > 0.7, r2 > 0.75, r2 > 0.8, r2 > 0.85, r2 > 0.9, r2 > 0.95, r2 > 0.96, r2 > 0.97, r2 > 0.98, or r2 > 0.99. Linkage disequilibrium can in certain embodiments also be defined as |D'| > 0.2, or as * | D'| > 0.3, | D'| > 0.4, |D'| > 0.5, | D'| > 0.6, | D'| > 0.7, |D'| > 0.8, |D'| > 0.9, | D'| > 0.95, | D'| > 0.98 or | D'[ > 0.99. In certain embodiments, linkage disequilibrium is defined as fulfilling two criteria of r2 and |D'|, such as r2 > 0.2 and | D'| > 0.8. Other combinations of values for r2 and |D'| are also possible and within scope of the present invention, including but not limited to the values for these parameters set forth in the above. Assessment for markers and haplotypes
The genomic sequence within populations is not identical when individuals are compared. Rather, the genome exhibits sequence variability between individuals at many locations in the genome. Such variations in sequence are commonly referred to as polymorphisms, and there are many such sites within each genome For example, the human genome exhibits sequence ,■ variations which occur on average every 500 base pairs. The most common sequence variant consists of base variations at a single base position in the genome, and such sequence variants, or polymorphisms, are commonly called Single Nucleotide Polymorphisms ("SNPs"). These SNPs are believed to have occurred in a single mutational event, and therefore there are usually two possible alleles possible at each SNPsite; the original allele and the mutated allele. Due to natural genetic drift and possibly also selective pressure, the original mutation has resulted in a polymorphism characterized by a particular frequency of its alleles in any given population. Many other types of sequence variants are found in the human genome, including mini - and microsatellites, and insertions, deletions andinversions (also called copy number variations
(CNVs)). A polymorphic microsatellite has multiple small repeats of bases (such as CA repeats, TG on the complimentary strand) at a particular site in which the number of repeat lengths varies in the general population. In general terms, each version of the sequence with respect to the polymorphic site represents a specific allele of the polymorphic site. These sequence variants can all be referred to as polymorphisms, occurring at specific polymorphic sites characteristic of the sequence variant in question. In general terms, polymorphisms can comprise any number of specific alleles. Thus in one embodiment of the invention, the polymorphism is characterized by the presence of two or more alleles in any given population. In another embodiment, the polymorphism is characterized by the presence of three or more alleles. In other embodiments, the polymorphism is characterized by four or more alleles, fiva or more alleles, six or more alleles, seven or more alleles, nine or more alleles, or ten or more alleles. All such polymorphisms can be utilized in the methods and kits of the present invention, and are thus within the scope of the invention.
Due to their abundance, SNPs account for a majority of sequence variation in the human genome. Over 6 million SNPs have been validated to date
(http://www.ncbi. nlm.nih.gov/projects/SNP/snp_summarγ.cgι). However, CNVs are receiving ' increased attention. These large-scale polymorphisms (typically lkb or larger) account for polymorphic variation affecting a substantial proportion of the assembled human genome; known CNVs covery over 15% of the human genome sequence (Estivill, X Armengol; L., PIoS Genetics 3: 1787-99 (2007). A http://projects.tcag. ca/variation/). Most of these polymorphisms are however very rare, and on average affect only a fraction of the genomic sequence of each individual. CNVs are known to affect gene expression, phenotypic variation and adaptation by disrupting gene dosage, and are also known to cause kidney stones (microdeletion and microduplication disorders) and confer risk of common complex kidney stones, including HIV-I infection and glomerulonephritis (Redon, R., et al. Nature 23:444-454 (2006)). It is thus possible that either previously described or unknown CNVs represent causative variants in linkage disequilibrium with the markers described herein. Methods for detecting CNVs include comparative genomic hybridization (CGH) and genotyping, including use of genotyping arrays, as described by Carter (Nature Genetics 39:S16-S21 (2007)). The Database of Genomic Variants (http://projects.tcag.ca/variation/) contains updated information about the location, type and ,. size of described CNVs. The database currently contains data for over 15,000 CNVs.
In some instances, reference is made to different alleles at a polymorphic site without choosing a reference allele. Alternatively, a reference sequence can be referred to for a particular polymorphic site. The reference allele is sometimes referred to as the "wild-type" allele and it usually is chosen as either the first sequenced allele or as the allele from a "non -affected" individual {e.g., an individual that does not display a trait or disease phenotype).
Alleles for SNP markers as referred to herein refer to the bases A, C, G or T as they occur at the polymorphic site in the SNP assay employed. The allele codes for SNPs used herein are as follows: 1= A, 2=C, 3=G, 4=T. The person skilled in the art will however realise that by assaying or reading the opposite DNA strand, the complementary allele can in each case be measured. Thus, for a polymorphic site (polymorphic marker) characterized by an A/G polymorphism, the assay employed may be designed to specifically detect the presence of one or both of the two bases possible, i.e. A and G. Alternatively, by designing an assay that is designed to detect the complimentary strand on the DNA template, the presence of the complementary bases T and C can be measured. Quantitatively (for example, in terms of risk estimates), identical results would be obtained from measurement of either DNA strand ( + strand or - strand).
Typically, a reference sequence is referred to for a particular sequence. Alleles that differ from' the reference are sometimes referred to as "variant" alleles. A variant sequence, as used herein, refers to a sequence that differs from the reference sequence but is otherwise substantially similar. Alleles at the polymorphic genetic markers described herein are variants. Variants can include changes that affect a polypeptide. Sequence differences, when compared to a reference nucleotide sequence, can include the insertion or deletion of a single nucleotide, or of more than one nucleotide, resulting in a frame shift; the change of at least one nucleotide, resulting in a change in the encoded amino acid; the change of at least one nucleotide, resulting in the , generation of a premature stop codon; the deletion of several nucleotides, resulting in a deletion of one or more amino acids encoded by the nucleotides; the insertion of one or several nucleotides, such as by unequal recombination or gene conversion, resulting in an interruption of the coding sequence of a reading frame; duplication of all or a part of a sequence; transposition; or a rearrangement of a nucleotide sequence,. Such sequence changes can alter the polypeptide encoded by the nucleic acid. For example, if the change in the nucleic acid sequence causes a frame shift, the frame shift can result in a change in the encoded amino acids, and/or cen result in the generation of a premature stop codon, causing generation of a truncated polypeptide. Alternatively, a polymorphism associated with a kidney stones or trait can be a synonymous change in one or more nucleotides (i.e., a change that does not result in a change in the amino acid sequence). Such a polymorphism can, for example, alter splice sites, affect the stability or' transport of mRNA, or otherwise affect the transcription or translation of an encoded polypeptide. It can also alter DNA to increase the possibility that structural changes, such as amplifications or deletions, occur at the somatic level. The polypeptide encoded by the reference nucleotide sequence is the "reference" polypeptide with a particular reference amino acid sequence, and polypeptides encoded by variant alleles are referred to as "variant" polypeptides with variant amino acid sequences.
A haplotype refers to a segment of DNA that is characterized by a specific combination of alleles arranged along the segment. For diploid organisms such as humans, a haplotype comprises one member of the pair of alleles for each polymorphic marker or locus . In a certain embodiment," the haplotype can comprise two or more alleles, three or more alleles, four or more alleles, or five or more alleles, each allele corresponding to a specific polymorphic marker along the segment. Haplotypes can comprise a combination of various polymorphic markers, e.g., SNPs and microsatellites, having particular alleles at the polymorphic sites. The haplotypes thus comprise a combination of alleles at various genetic markers.
Detecting specific polymorphic markers and/or haplotypes can be accomplished by methods known in the art for detecting sequences at polymorphic sites. For example, standard techniques for genotyping for the presence of SNPs and/or microsatellite markers can be used, such as fluorescence-based techniques (e.g., Chen, X. et a/., Genome Res. 9(5): 492-98 ( 1999); Kutyavin et al., Nucleic Acid Res. 34:el28 (2006)), utilizing PCR, LCR, Nested PCR and other techniques for nucleic acid amplification. Specific commercial methodologies available for SNP genotyping include, but are not limited to, TaqMan genotyping assays and SNPIex platforms (Applied Biosystems), gel electrophoresis (Applied Biosystems), mass spectrometry (e.g.,
MassARRAY system from Sequenom), minisequencing methods, real-time PCR, Bio-Plex system (BioRad), CEQ and SNPstream systems (Beckman), array hybridization technology(e.g., .;
Affymetrix GeneChip; Perlegen), BeadArray Technologies (e.g. , Illumina GoldenGate and Infinium assays), array tag technology (e.g., Parallele), and endonuclease-based fluorescence hybridization technology (Invader; Third Wave). Some of the available array platforms, including Affymetrix SNP Array 6.0 and Illumina CNV370-Duo and IM BeadChips, include SNPs that tag certain CNVs. This allows detection of CNVs via surrogate SNPs included in these platforms. Thus, by use of these or other methods available to the person skilled in the art, one or more alleles at polymorphic markers, including microsatellites, SNPs or other types of polymorphic markers, can be identified.
It is possible to impute or predict genotypes for un-genotyped relatives of genotyped individuals. For every un-genotyped case, it is possible to calculate the probability of the genotypes of its relatives given its four possible phased genotypes. In practice it may be preferable to include only the genotypes of the case's parents, children, siblings, half-siblings (and the half-sibling's . parents), grand-parents, grand-children (and the grand-children's parents) and spouses. It will be assumed that the individuals in the small sub-pedigrees created around each case are not related through any path not included in the pedigree. It is also assumed that alleles that are not transmitted to the case have the same frequency - the population allele frequency. The probability of the genotypes of the case's relatives can then be computed by:
Pr(genotypes of relatives; #) = of relatives | h) ,
Figure imgf000023_0001
where θ denotes the A allele's frequency in the cases. Assuming the genotypes of each set of relatives are independent, this allows us to write down a likelihood function for θ:
L(θ) = ]~JPr(genotypesof relativesof casez;#) . (*)
This assumption of independence is usually not correct. Accounting for the dependence between individuals is a difficult and potentially prohibitively expensive computational task. The likelihood function in (*) may be thought of as a pseudolikelihood approximation of the full likelihood function for θ which properly accounts for all dependencies. In general, the genotyped cases and controls in a case-control association study are not independent and applying the case-control method to related cases and controls is an analogous approximation. The method of genomic control (Devlin, B. et al., Nat Genet 36, 1129-30; author reply 1131 (2004)) has proven to be successful at adjusting case-control test statistics for relatedness. The method of genomic control to account for the dependence between the terms in the pseudolikelihood is therefore typically used to produce a valid test statistic.
Fisher's information can be used to estimate the effective sample size of the part of the pseudolikelihood due to un-genotyped cases. Breaking the total Fisher information, /, into the part due to genotyped cases, J9, and the part due to ungenotyped cases, Iu, I = I0 + Iuι and denoting the number of genotyped cases with N, the effective sample size due to the ungenotyped cases is estimated by — N . g
In the present context, and individual who is at an increased susceptibility (i.e., increased risk)' for forming kidney stones, is an individual in whom at least one specific allele at one or more ■ ■ polymorphic marker or haplotype conferring increased susceptibility (increased risk) for kidney stones is identified (i.e., at-risk marker alleles or haplotypes). The at-risk marker or haplotype is one that confers an increased risk (increased susceptibility) of kidney stones. In one embodiment, significance associated with a marker or haplotype is measured by a relative risk (RR). In another embodiment, significance associated with a marker or haplotye is measured by an odds ratio (OR). In a further embodiment, the significance is measured by a percentage. In one embodiment, a significant increased risk is measured as a risk (relative risk and/or odds ratio) of at least 1.10, including but not limited to: at least 1.11, at least 1.12, at least 1.13, at least 1.14, at least 1.15, at least 1.16, at least 1.17, at least 1.18, at least 1.19, atleast 1.20, at least 1.21, at least 1.22, at least 1.23, at least 1.24, at least 1.25, at least 1.26, at least 1.27, at least 1.28, at least 1.29, at least 1.30, at least 1.40, at least 1.50, at least 1.60, at least 1.70, at least 1.80, at least 1.90, and at least 2.0. In a particular embodiment, a risk (relative risk and/or odds ratio) of at least 1.20 is significant. In another particular embodiment, a risk of at least 1.21 is significant. In yet another embodiment, a risk of at least 1.22 is significant. In a further embodiment, a relative risk of at least 1.23 is significant. In another further embodiment, a significant increase in risk is at least 1.24. In other embodiments, a significant increase in risk is at least about 10%, including but not limited to at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, or at least" 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In one particular embodiment, a significant increase in risk is at least 20%. In other embodiments, a significant increase in risk is at least 21%, at least 22%, at least 23%, at least 24%, or at least 25%. Other cutoffs or ranges as deemed suitable by the person skilled in the \ art to characterize the invention are however also contemplated, and those are also within scope of the present invention. In certain embodiments, a significant increase in risk is characterized-.^ by a p-value, such as a p-value of less than 0.05, less than 0.01, less than 0.001, less than 0.0001, less than 0.00001, less than 0.000001, less than 0.0000001, less than 0.00000001, ort less than 0.000000001.
An at-risk polymorphic marker or haplotype of the present invention is one where at least one ' allele of at least one marker or haplotype is more frequently present in an individual at risk for the disease or trait (affected), compared to the frequency of its presence in a comparison group (control), and wherein the presence of the at-risk allele of the marker or the at-risk haplotype is indicative of susceptibility to the disease or trait. The control group may in one embodiment be a population sample, i.e. a random sample from the general population. In another embodiment, the control group is represented by a group of individuals who are disease-free. Such disease-free controls may in one embodiment be characterized by the absence of one or more specific disease-associated symptoms. In another embodiment, the disease-free control group is characterized by the absence of one or more disease-specific risk factors. Such risk ■ factors are in one embodiment at least one environmental risk factor. Representative environmental factors are natural products, minerals or other chemicals which are known to affect, or contemplated to affect, the risk of developing the specific disease or trait. Other environmental risk factors are risk factors related to lifestyle, including but not limited to food ; and drink habits, geographical location of mam habitat, and occupational risk factors. In another embodiment, the risk factors comprise at least one additional genetic risk factor.
Individuals who are homozygous for an at-risk allele of a polymorphic marker (i.e., individuals carrying two copies of the at-risk allele in their genome), or individuals who are homozygous for an at-risk haplotype are at particularly high risk. The contribution of each allelic copy is usually independent, i.e. the overall effect multiplies, such that homozygous individuals for an at-πsk . variant (allele or haplotype) with a risk of 1.2 in heterozygous carriers are expected to have a risk of 1.2x1.2 = 1.44. Likewise, the protective effect ^of certain alleles or haplotypes are generally multiplicative. Thus, certain embodiments of the invention relate to individuals who are homozygous for at least one at-risk or protective variant (marker allele or haplotype), as described herein. Sometimes, the effect of homozygous individuals is higher than would be expected based on the multiplicative model. This is indicative of a recessive component to the effect, which means that two copies of the variant, when brought together, have a greater overall effect than would be expected if the effect of each variant is added assuming no interaction between the two copies.
As an example of a simple test for correlation would be a Fisher-exact test on a two by two table. Given a cohort of chromosomes, the two by two table is constructed out of the number of chromosomes that include both of the markers or haplotypes, one of the markers or haplotypes but not the other and neither of the markers or haplotypes. Other statistical tests of association known to the skilled person are also contemplated and are also within scope of the invention.
In other embodiments of the invention, an individual who is at a decreased susceptibility (i.e., at a decreased risk) for a disease or trait is an individual in whom at least one specific allele at one or more polymorphic marker or haplotype conferring decreased susceptibility for the disease or trait is identified. The marker alleles and/or haplotypes conferring decreased risk are also said to be protective. In one aspect, the protective marker or haplotype is one that confers a significant decreased risk (or susceptibility) of the disease or trait. In one embodiment, significant decreased risk is measured as a relative risk (or odds ratio) of less than 0.9, including but not limited to less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2 and less than 0.1. In one particular embodiment, significant decreased risk is less than 0.7. In another embodiment, significant decreased risk is less than 0.5. In yet another embodiment, significant decreased risk is less than 0.3. In another embodiment, the decrease in risk (or susceptibility) is at least 20%, including but not limited to at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%', at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and at least 98%. In one particular embodiment, a significant decrease in risk is at least about 30%. In another embodiment, a significant decrease in risk is at least about 50%. In another embodiment, the decrease in risk is at least about 70%. Other cutoffs or ranges as deemed suitable by the person skilled in the art to characterize the invention are however also contemplated, and those are also within scope of the present invention. 5
The person skilled in the art will appreciate that for markers with two alleles present in the population being studied (such as SNPs), and wherein one allele is found in increased frequency in a group of individuals with a trait or disease in the population, compared with controls, the other allele of the marker will be found in decreased frequency in the group of individuals with the trait or disease, compared with controls. In such a case, one allele of the marker (the one found in increased frequency in individuals with the trait or disease) will be the at-risk allele, while the other allele will be a protective allele. 'A
A genetic variant associated with a disease or a trait can be used alone to predict the risk of the disease for a given genotype. For a biallelic marker, such as a SNP, there are 3 possible genotypes: homozygote for the at risk variant, heterozygote, and non carrier of the at risk '. variant. The risk associated with each genotype can be estimated from the genotype of the individual. Risk associated with variants at multiple loci can be used to estimate overall risk. For multiple SNP variants, there are k possible genotypes k = 3" χ 2"; where n is the number autosomal loci and p the number of gonosomal (sex chromosomal) loci. Overall risk assessment calculations for a plurality of risk variants usually assume that the relative risks of different genetic variants multiply, i.e. the overall risk (e.g. , RR or OR) associated with a particular genotype combination is the product of the risk values for the genotype at each locus. If the risk presented is the relative risk for a person, or a specific genotype for a person, compared to a reference population with matched gender and ethnicity, then the combined risk - is the product of the locus specific risk values - and which also corresponds to an overall risk estimate compared with the population. If the risk for a person is based on a comparison to non-carriers of the at risk allele, then the combined risk corresponds to an estimate that compares the person with a given combination of genotypes at all loci to a group of individuals who do not carry risk variants at any of those loci. The group of non-carriers of any at risk variant has the lowest estimated risk and has a combined risk, compared with itself (i.e., non-carriers) of 1.0, but has an overall risk, compare with the population, of less than 1.0. It should be noted that the group of non-carriers can potentially be very small, especially for large number of loci, and in that case, its relevance is correspondingly small.
The multiplicative model is a parsimonious model that usually fits the data of complex traits reasonably well. Deviations from multiplicity have been rarely described in the context of common variants for common diseases, and if reported are usually only suggestive since very large sample sizes are usually required to be able to demonstrate statistical interactions between loci.
By way of an example, let us consider a total of eight variants that have been described to associate with prostate cancer (Gudmundsson, J., et al., Nat Genet 39:631-7 (2007),
Gudmundsson, J., et al., Nat Genet 39:977-83 (2007); Yeager, M., et al, Nat Genet 39:645-49 (2007), Amundadottir, L, el al., Nat Genet 38:652-8 (2006); Haiman, C. A., et al., Nat Genet 39:638-44 (2007)). Seven of these loci are on autosomes, and the remaining locus is on chromosome X. The total number of theoretical genotypic combinations is then 37 x 21 = 4374. Some of those genotypic classes are very rare, but are still possible, and should be considered for overall risk assessment. It is likely that the multiplicative model applied in the case of [[ multiple genetic variant will also be valid in conjugation with non-genetic risk variants assuming that the genetic variant does not clearly correlate with the "environmental" factor. In other words, genetic and non-genetic at-risk variants can be assessed under the multiplicative model to estimate combined risk, assuming that the non-genetic and genetic risk factors do not interact.
Using the same quantitative approach, the combined or overall risk associated with a plurality of variants described herein may be assessed. Furthermore the overall risk associated with at least one marker described herein in combination with any other markers associated with risk of kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis can be assessed.
Linkage Disequilibrium
The natural phenomenon of recombination, which occurs on average once for each chromosomal pair during each meiotic event, represents one way in which nature provides variations in sequence (and biological function by consequence). It has been discovered that recombination ' does not occur randomly in the genome; rather, there are large variations in the frequency of recombination rates, resulting in small regions of high recombination frequency (also called recombination hotspots) and larger regions of low recombination frequency, which are commonly referred to as Linkage Disequilibrium (LD) blocks (Myers, S. et al., Biochem Soc Trans 34: 526- 530 (2006); Jeffreys, A.]., et al., Nature Genet 29: 217-222 (2001); May, C. A., et al., Nature ' Genet 31: 272-275(2002)). )
Linkage Disequilibrium (LD) refers to a non-random assortment of two genetic elements. For example, if a particular genetic element (e.g., an allele of a polymorphic marker, or a haplotype) occurs in a population at a frequency of 0.50 (50%) and another element occurs at a frequency of 0.50 (50%), then the predicted occurrance of a person's having both elements is 0.25 (25%), assuming a random distribution of the elements. However, if it is discovered that the two elements occur together at a frequency higher than 0.25, then the elements are said to be in ; linkage disequilibrium, since they tend to be inherited together at a higher rate than what their independent frequencies of occurrence (e.g., allele or haplotype frequencies) would predict. Roughly speaking, LD is generally correlated with the frequency of recombination events between the two elements. Allele or haplotype frequencies can be determined in a population by genotyping individuals in a population and determining the frequency of the occurence of each ' allele or haplotype in the population. For populations of diploids, e.g., human populations, individuals will typically have two alleles or allelic combinations for each genetic element (e.g., a marker, haplotype or gene).
Many different measures have been proposed for assessing the strength of linkage disequilibrium (LD; reviewed in Devlin, B. & Risen, N., Genomics 29:311-22 (1995))). Most capture the ; strength of association between pairs of biallelic sites. Two important pairwise measures of LD 1 are r2 (sometimes denoted Δ2) and | D'| (Lewontin, R., Genetics 49:49-67 ( 1964); Hill, W. G. & , Robertson, A. Theor. Appl. Genet. 22:226-231 (1968)). Both measures range from 0 (no disequilibrium) to 1 ('complete' disequilibrium), but their interpretation is slightly different. | D'| is defined in such a way that it is equal to 1 if just two or three of the possible haplotypes are '- present, and it is <1 if all four possible haplotypes are present. Therefore, a value of | D'| that is < 1 indicates that historical recombination may have occurred between two sites (recurrent mutation can also cause | D'| to be < 1, but for single nucleotide polymorphisms (SNPs) this is , usually regarded as being less likely than recombination). The measure r2 represents the statistical correlation between two sites, and takes the value of 1 if only two haplotypes are 1J present.
The r2 measure is arguably the most relevant measure for association mapping, because there is a simple inverse relationship between r2 and the sample size required to detect association between susceptibility loci and SNPs. These measures are defined for pairs of sites, but for some applications a determination of how strong LD is across an entire region that contains many polymorphic sites might be desirable (e.g., testing whether the strength of LD differs significantly among loci or across populations, or whether there is more or less LD in a region than predicted under a particular model). Measuring LD across a region is not straightforward, but one approach is to use the measure r, which was developed in population genetics. Roughl/ speaking, r measures how much recombination would be required under a particular population, model to generate the LD that is seen in the data. This type of method can potentially also ;' provide a statistically rigorous approach to the problem of determining whether LD data provide evidence for the presence of recombination hotspots. For the methods described herein, a significant r2 value can be at least 0.1 such as at least 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or at lesat 0.99. In one preferred embodiment, the significant r2 value can be at least 0.2. Alternatively, linkage disequilibrium as described herein, refers to linkage disequilibrium characterized by values of | D'| of at least 0.2, such as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.85, 0.9, ,? 0.95, 0.96, 0.97, 0.98, or at least 0.99. Thus, linkage disequilibrium represents a correlation :' between alleles of distinct markers, It is measured by correlation coefficient or | D'| (r2 up to 1.0 and |D'| up to 1.0). In certain embodiments, linkage disequilibrium is defined in terms of values for both the r2 and |D'| measures. In one such embodiment, a significant linkage disequilibrium is defined as r2 > 0.1 and | D'| >0.8. In another embodiment, a significant linkage j disequilibrium is defined as r2 > 0.2 and |D'| >0.9. In one preferred embodiment, r2 > 0.2 is ,« significant linkage disequilibrium.. In another preferred embodiment, r2 > 0.5 is significant linkage disequilibrium. In yet another preferred embodiment, r2 > 0.7 is significant linkage disequilibrium. In a further preferred embodiment, r2 > 0.8 is significant linkage disequilibrium. Other combinations and permutations of values of r2 and | D'|for determining linkage disequilibrium are also contemplated, and are also within the scope of the invention. Linkage disequilibrium can be determined in a single human population, as defined herein, or it can be . determined in a collection of samples comprising individuals from more than one human population. In one embodiment of the invention, LD is determined in a sample from one or more of the HapMap populations (Caucasian, african, Japanese, Chinese), as defined
(http://www.hapmap.org). In one such embodiment, LD is determined in the CEU population of the HapMap samples. In another embodiment, LD is determined in the YRI population. In yet ' another embodiment, LD is determined in samples from the Icelandic population.
If all polymorphisms in the genome were independent at the population level (i.e., no LD), then every single one of them would need to be investigated in association studies, to assess all the different polymorphic states. However, due to linkage disequilibrium between polymorphisms, tightly linked polymorphisms are strongly correlated, which reduces the number of polymorphisms that need to be investigated in an association study to observe a significant association. Another consequence of LD is that many polymorphisms may give an association signal due to the fact that these polymorphisms are strongly correlated.
Genomic LD maps have been generated across the genome, and such LD maps have been , proposed to serve as framework for mapping disease-genes (Risen, N. & Merkiangas, K, Science 273: 1516-1517 (1996); Maniatis, N., et al., Proc Natl Acad Sci USA 99:2228-2233 (2002); Reich, DE et al, Nature 411 : 199-204 (2001)).
It is now established that many portions of the human genome can be broken into series of discrete haplotype blocks containing a few common haplotypes; for these blocks, linkage disequilibrium data provides little evidence indicating recombination (see, e.g., Wall., J. D. and Pritchard, J. K., Nature Reviews Genetics 4:587-597 (2003); Daly, M. et al., Nature Genet. 29:229-232 (2001); Gabriel, S. B. et al., Science 296:2225-2229 (2002); Patil, N. et al., Science 294: 1719-1723 (2001); Dawson, E. et ah, Nature 418: 544-548 (2002); Phillips, M.S. et al., Nature Genet. 33:382-387 (2003)).
There are two main methods for defining these haplotype blocks: blocks can be defined as regions of DNA that have limited haplotype diversity (see, e.g., Daly, M. et al., Nature Genet. 29:229-232 (2001); Patil, N. et ai, Science 294: 1719-1723 (2001); Dawson, E. et al., Nature ■ 418: 544-548 (2002); Zhang, K. et al., Proc. Natl. Acad. Sci. USA 99: 7335-7339 (2002)), or as regions between transition zones having extensive historical recombination, identified using linkage disequilibrium (see, e.g., Gabriel, S. B. et al., Science 296: 2225-2229 (2002); Phillips, M.S. et al., Nature Genet. 33:382-387 (2003); Wang, N. et al.r Am. J. Hum. Genet. 71 : 1227- 1234 (2002); Stumpf, M. P., and Goldstein, D. B., Curr. Biol. 13: 1-8 (2003)). More recently, a fine-scale map of recombination rates and corresponding hotspots across the human genome has been generated (Myers, S., et al., Science 310:321-32324 (2005); Myers, S. et al., Biochem' Soc Trans 34:526530 (2006)). The map reveals the enormous variation in recombination across the genome, with recombination rates as high as 10-60 cM/Mb in hotspots, while closer to 0 in intervening regions, which thus represent regions of limited haplotype diversity and high LD. The map can therefore be used to define haplotype blocks/LD blocks as regions flanked by ; recombination hotspots. As used herein, the terms "haplotype block" or "LD block" includes blocks defined by any of the above described characteristics, or other alternative methods used by the person skilled in the art to define such regions. Haplotype blocks (LD blocks) can be used to map associations between phenotype and haplotype status, using single markers or haplotypes comprising a plurality of markers. The main haplotypes can be identified in each haplotype block, and then a set of "tagging" SNPs or markers (the smallest set of SNPs or markers needed to distinguish among the haplotypes) can then be identified. These tagging SNPs or markers can then be used in assessment of samples from groups of individuals, in order to identify association between phenotype and haplotype. if desired, neighboring haplotype blocks can be assessed concurrently, as there may also exist linkage disequilibrium among the haplotype blocks.
It has thus become apparent that for any given observed association to a polymorphic marker in the genome, it is likely that additional markers in the genome also show association. This is a natural consequence of the uneven distribution of LD across the genome, as observed by the large variation in recombination rates. The markers used to detect association thus in a sense represent "tags" for a genomic region (i.e., a haplotype block or LD block) that is associating with a given disease or trait, and as such are useful for use in the methods and kits of the present invention. Table 1 provides exemplary surrogate markers in linkage disequilibrium with the markers shown herein to be associated with risk of kidney stones and related conditions. Any one of these surrogate markers could suitably be chosen to detect the association to kidney stones described herein. The skilled person will appreciate that markers which are more tightly correlated [i.e., in greater LD) with the anchor markers are better surrogate markers than those which are less tightly correlated.
Table 1. (A) SNPs in the interval 36.25 Mb to 37.25 Mb (NCBI Build 36) on chromosome 21, that have correlation coefficient (r2) > 0.2 with markers rs219778, rs219779, rs219780 and rs219781. The correlation is calculated based on the HapMap CEU dataset (version 22). For each SNP the table includes two measures of correlation, D-prime (D') and correlation coefficient squared (r2), between the SNP and the two exonic variants, and the corresponding P-values for the significance of the correlation.
O
Figure imgf000031_0001
Table 1. (B) Surrogate markers of anchor markers rs219778, rs219779, rs219780 and rs219781. Markers were selected using data for Caucasian samples from the publically available 1000 Genomes project (http://www.1000genomes.org). Markers that have not been assigned rs names are identified by their position in NCBI Build 36 of the human genome assembly. Shown are risk alleles for the surrogate markers, i.e. alleles that are correlated with the corresponding allele of the anchor markers, rs219778-T, rs219779-C, rs219780-C and rs219781-C. Linkage disequilibrium measures D' and R2, and corresponding p-value, are also shown, and a reference to the sequence listing identifying the >- particular SNP and furthermore, position of the markers within the Chr21 LD block, (Seq ID No 1) where applicable. j
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
One or more causative (functional) variants or mutations may reside within the region found to ' be associating to the disease or trait. The functional variant may be another SNP, a tandem ■ repeat polymorphism (such as a minisatellite or a microsatelhte), a transposable element, or a ' copy number variation, such as an inversion, deletion or insertion. Such variants in LD with the; variants described herein may confer a higher relative risk (RR) or odds ratio (OR) than observέα1 for the tagging markers used to detect the association. The present invention thus refers to theϊ markers used for detecting association to the disease, as described herein, as well as markers iri linkage disequilibrium with the markers. Thus, in certain embodiments of the invention, markers that are in LD with the markers and/or haplotypes of the invention, as described herein, may be used as surrogate markers. The surrogate markers have in one embodiment relative risk (RR) and/or odds ratio (OR) values smaller than for the markers or haplotypes initially found to be J5 associating with the disease, as described herein. In other embodiments, the surrogate markers have RR or OR values greater than those initially determined for the markers initially found to be associating with the disease, as described herein. An example of such an embodiment would be a rare, or relatively rare (such as < 10% allelic population frequency) variant in LD with a more*, common variant (> 10% population frequency) initially found to be associating with the disease', such as the variants described herein. Identifying and using such markers for detecting the j association discovered by the inventors as described herein can be performed by routine methods well known to the person skilled in the art, and are therefore within the scope of the present invention.
Determination of haplotype frequency
The frequencies of haplotypes in patient and control groups can be estimated using an <<; expectation-maximization algorithm (Dempster A. et al., J. R. Stat. Soc. B1 39: 1-38 (1977)). An implementation of this algorithm that can handle missing genotypes and uncertainty with the • phase can be used. Under the null hypothesis, the patients and the controls are assumed to * have identical frequencies. Using a likelihood approach, an alternative hypothesis is tested, where a candidate at-nsk-haplotype, which can include the markers described herein, is allowed to have a higher frequency in patients than controls, while the ratios of the frequencies of other haplotypes are assumed to be the same in both groups. Likelihoods are maximized separately s. under both hypotheses and a corresponding 1-df likelihood ratio statistic is used to evaluate the statistical significance.
To look for at-πsk and protective markers and haplotypes within a susceptibility region, for example within an LD block, association of all possible combinations of genotyped markers within the region is studied. The combined patient and control groups can be randomly divided into two sets, equal in size to the original group of patients and controls. The marker and haplotype analysis is then repeated and the most significant p-value registered is determined. This i randomization scheme can be repeated, for example, over 100 times to construct an empirical ,' distribution of p-values. In a preferred embodiment, a p-value of <0.05 is indicative of a significant marker and/or haplotype association. .,'
Haplotype Analysis
One general approach to haplotype analysis involves using likelihood-based inference applied to] NEsted MOdels (Gretarsdottir S., et al., Nat. Genet. 35: 131-38 (2003)). The method is [ implemented in the program NEMO, which allows for many polymorphic markers, SNPs and microsatellites. The method and software are specifically designed for case-control studies where the purpose is to identify haplotype groups that confer different risks. It is also a tool for c. studying LD structures. In NEMO, maximum likelihood estimates, likelihood ratios and p-values are calculated directly, with the aid of the EM algorithm, for the observed data treating it as a missing-data problem.
Even though likelihood ratio tests based on likelihoods computed directly for the observed data, which have captured the information loss due to uncertainty in phase and missing genotypes, Ii can be relied on to give valid p-values, it would still be of interest to know how much information had been lost due to the information being incomplete. The information measure for haplotype ' analysis is described in Nicolae and Kong (Technical Report 537, Department of Statistics, University of Statistics, University of Chicago; Biometrics, 60(2) :368-75 (2004)) as a natural ; extension of information measures defined for linkage analysis, and is implemented in NEMO. Y
For single marker association to a disease, the Fisher exact test can be used to calculate two- sided p-values for each individual allele. Usually, all p-values are presented unadjusted for multiple comparisons unless specifically indicated. The presented frequencies (for microsatellites, SNPs and haplotypes) are allelic frequencies as opposed to earner frequencies. To minimize any bias due the relatedness of the patients who were recruited as families to the study, first and second-degree relatives can be eliminated from the patient list. Furthermore, the test can be repeated for association correcting for any remaining relatedness among the patients, by extending a variance adjustment procedure previously described (Risch, N. & Teng, J. Genome Res., 8: 1273-1288 (1998)) for sibships so that it can be applied to general familial relationships, and present both adjusted and unadjusted p-values for comparison. The method of genomic controls (Devlin, B. & Roeder, K. Biometrics 55:997 (1999)) can also be used to adjust for the relatedness of the individuals and possible stratification. The differences are in general very small as expected. To assess the significance of single-marker association corrected for multiple testing we can carry out a randomization test using the same genotype data. Cohorts of patients and controls can be randomized and the association analysis redone multiple times (e.g., up to 500,000 times) and the p-value is the fraction of replications that produced a p-value for some \ marker allele that is lower than or equal to the p-value we observed using the original patient and control cohorts.
For both single-marker and haplotype analyses, relative risk (RR) and the population attributable risk (PAR) can be calculated assuming a multiplicative model (haplotype relative risk model) (Terwilliger, J. D. & Ott, J., Hum. Hered. 42:337-46 (1992) and FaIk, CT. & Rubinstein, P, Ann. Hum. Genet. 51 (Pt j;:227-33 (1987)), i.e., that the risks of the two alleles/haplotypes a person carries multiply. For example, if RR is the risk of A relative to a, then the risk of a person homozygote AA will be RR times that of a heterozygote Aa and RR2 times that of a homozygote aa. The multiplicative model has a nice property that simplifies analysis and computations — haplotypes are independent, i.e., in Hardy-Weinberg equilibrium, within the affected population as well as within the control population. As a consequence, haplotype counts of the affecteds and controls each have multinomial distributions, but with different haplotype frequencies under the alternative hypothesis. Specifically, for two haplotypes, h, and h]t risk(/?,)/risk(fy) = (fJ Pi)Kf]/ Pj) , where f and p denote, respectively, frequencies in the affected population and in '' the control population. While there is some power loss if the true model is not multiplicative, the loss tends to be mild except for extreme cases. Most importantly, p-values are always valid since they are computed with respect to null hypothesis.
An association signal detected in one association study may be replicated in a second cohort, .. ideally from a different population (e.g., different region of same country, or a different country) of the same or different ethnicity. The advantage of replication studies is that the number of tests performed in the replication study, and hence the less stringent the statistical measure that is applied. For example, for a genome-wide search for susceptibility variants for a particular disease or trait using 300,000 SNPs, a correction for the 300,000 tests performed (one for each SNP) can be performed. Since many SNPs on the arrays typically used are correlated {i.e., in LD), they are not independent. Thus, the correction is conservative. Nevertheless, applying this correction factor requires an observed P-value of less than 0.05/300,000 = 1.7 x 10"7 for the ', signal to be considered significant applying this conservative test on results from a single study cohort. Obviously, signals found in a genome-wide association study with P-values less than this conservative threshold are a measure of a true genetic effect, and replication in additional cohorts is not necessarily from a statistical point of view. However, since the correction factor - depends on the number of statistical tests performed, if one signal (one SNP) from an initial study is replicated in a second case-control cohort, the appropriate statistical test for significance is that for a single statistical test, i.e., P-value less than 0.05. Replication studies in one or even several additional case-control cohorts have the added advantage of providing assessment of the association signal in additional populations, thus simultaneously confirming the initial finding and providing an assessment of the overall significance of the genetic variant(s) being tested in human populations in general.
The results from several case-control cohorts can also be combined to provide an overall assessment of the underlying effect. The methodology commonly used to combine results from multiple genetic association studies is the Mantel-Haenszel model (Mantel and Haenszel, J Natl ■, Cancer Inst 22:719-48 (1959)). The model is designed to deal with the situation where association results from different populations, with each possibly having a different population frequency of the genetic variant, are combined. The model combines the results assuming that the effect of the variant on the risk of the disease , a measured by the OR or RR, is the same in all populations, while the frequency of the variant may differ between the poplations. Combining the results from several populations has the added advantage that the overall power to detect a real underlying association signal is increased, due to the increased statistical power provided by the combined cohorts. Furthermore, any deficiencies in individual studies, for example due to f. unequal matching of cases and controls or population stratification will tend to balance out wheη results from multiple cohorts are combined, again providing a better estimate of the true underlying genetic effect.
Risk assessment and Diagnostics
Within any given population, there is an absolute risk of developing a disease or trait, defined as the chance of a person developing the specific kidney stones or trait over a specified time-period. For example, a woman's lifetime absolute risk of breast cancer is one in nine. That is to say, one woman in every nine will develop breast cancer at some point in their lives. Risk is typically measured by looking at very large numbers of people, rather than at a particular individual. Risk is often presented in terms of Absolute Risk (AR) and Relative Risk (RR). Relative Risk is used to compare risks associating with two variants or the risks of two different groups of people. For { example, it can be used to compare a group of people with a certain genotype with another group having a different genotype. For a disease, a relative risk of 2 means that one group has, twice the chance of developing a disease as the other group. The risk presented is usually the relative risk for a person, or a specific genotype of a person, compared to the population with >' matched gender and ethnicity. Risks of two individuals of the same gender and ethnicity could be compared in a simple manner. For example, if, compared to the population, the first individual has relative risk 1.5 and the second has relative risk 0.5, then the risk of the first individual compared to the second individual is 1.5/0.5 = 3. - '
Certain polymorphic markers have been found to be predictive of risk of kidney stones, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral • ' density and osteoporosis in humans. Risk assessment based on this finding may involve the use of the markers for determining a susceptibility to any one of these conditions. Particular alleles of polymorphic markers (e.g., SNPs) are found more frequently in individuals with kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum i, bicarbonate levels, reduced bone mineral density and/or osteoporosis, than in individuals without diagnosis of any one of these conditions. Therefore, these marker alleles have predictive value " for detecting a risk or a susceptibility to any one of these conditions in an individual. Tagging markers in linkage disequilibrium with at-risk variants (or protective variants) described herein . can also be used as surrogates for these markers (and/or haplotypes). Such surrogate markers can be located within a particular haplotype block or LD block. Such surrogate markers can also sometimes be located outside the physical boundaries of such a haplotype block or LD block, either in close vicinity of the LD block/haplotype block, but possibly also located in a more distant genomic location.
Long-distance LD can for example arise if particular genomic regions (e.g., genes) are in a functional relationship. For example, if two genes encode proteins that play a role in a shared metabolic pathway, then particular variants in one gene may have a direct impact on observed variants for the other gene. Let us consider the case where a variant in one gene leads to increased expression of the gene product. To counteract this effect and preserve overall flux of the particular pathway, this variant may have led to selection of one (or more) variants at a "; second gene that conferes decreased expression levels of that gene. These two genes may be .• located in different genomic locations, possibly on different chromosomes, but variants within the genes are in apparent LD, not because of their shared physical location within a region of high 1 LD, but rather due to evolutionary forces. Such LD is also contemplated and within scope of the present invention. The skilled person will appreciate that many other scenarios of functional gene-gene interaction are possible, and the particular example discussed here represents only '\ one such possible scenario. Markers with values of r2 equal to 1 are perfect surrogates for the at-risk variants, i.e. genotypes for one marker perfectly predicts genotypes for the other. Markers with smaller values of r2 than 1 can also be surrogates for the at-risk variant, or alternatively represent variants with relative ^, risk values as high as or possibly even higher than the at-risk variant. The at-risk variant identified may not be the functional variant itself, but is in this instance in linkage disequilibrium with the true functional variant. The functional variant may for example be a tandem repeat, such as a minisatellite or a microsatellite, a transposable element (e.g., an AIu element), or a structural alteration, such as a deletion, insertion or inversion (sometimes also called copy number variations, or CNVs). The present invention encompasses the assessment of such surrogate markers for the markers as disclosed herein. Such markers are annotated, mapped , and listed in public databases, as well known to the skilled person {e.g. , as illustrated by the markers in Table 1), or can alternatively be readily identified by sequencing the region or a part of the region identified by the markers of the present invention in a group of individuals, and identify polymorphisms in the resulting group of sequences. As a consequence, the person skilled in the art can readily and without undue experimentation genotype surrogate markers in linkage disequilibrium with the markers and/or haplotypes as described herein. The tagging or surrogate markers in LD with the at-risk variants detected, also have predictive value for detecting association to the condition, or a susceptibility to the condition, in an individual. ;:
The present invention can in certain embodiments be practiced by assessing a sample comprising genomic DNA from an individual for the presence of variants described herein to be , associated with kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis. Such assessment typically steps that detect the presence or absence of at least one allele of at least : one polymorphic marker in the genome of the individual, using methods well known to the skilled person and further described herein, and based on the outcome of such assessment, determine whether the individual from whom the sample is derived is at increased or decreased risk (increased or decreased susceptibility) of kidney stones, hypercalciurea, elevated serum ., parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density' and/or osteoporosis. Detecting particular alleles of polymorphic markers can in certain embodiments be done by obtaining nucleic acid sequence data about a particular human individual, that identifies at least one allele of at least one polymorphic marker. Different alleles of the at least one marker are associated with different susceptibility to the kidney stones in humans. Obtaining nucleic acid sequence data can comprise nucleic acid sequence at a single nucleotide position, which is sufficient to identify alleles at SNPs. The nucleic acid sequence data can also comprise sequence at any other number of nucleotide positions, in particular for genetic markers that comprise multiple nuclotide positions, and can be anywhere from two to hundreds • of thousands, possibly even millions, of nucleotides (in particular, in the case of copy number ' variations (CNVs)).
In certain embodiments, the invention can be practiced utilizing a dataset comprising information about the genotype status of particular polylmorphic markers, such as any one of the markers shown herein to be associated with risk of kidney stones, hypercalαurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis (or markers in linkage disequilibrium with any one of those markers). In other words, a dataset containing information about such genetic status, for example in the form of genotype counts at a certain polymorphic marker, or a plurality of markers (e.g., an indication of the presence or absence of certain at-risk alleles), or actual genotypes for one or more markers, can be queried for the presence or absence of certain at-risk alleles at certain polymorphic markers associated with any one of kidney stones, hypercalciurea, elevated serum parathyroid ' hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis . A positive result for the presence of the variant (e.g., marker allele or haplotype) is indicative of the individual from which the dataset is derived is at increased susceptibility (increased risk) of the condition. Determination of the absence of the variant allele is indicative of the individual not being at increased suscepbility of the condition caused by the particular variant.
In certain embodiments of the invention, a polymorphic marker is correlated to a a condition or disease (such as kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis) by , referencing genotype data for the polymorphic marker to a look-up table that comprises correlations between at least one allele of the polymorphism and the condition. In some embodiments, the table comprises a correlation for one polymorhpism. In other embodiments, the table comprises a correlation for a plurality of polymorhpisms. In both scenarios, by referencing to a look-up table that gives an indication of a correlation between a marker and the condition, a risk for the condition, or a susceptibility to the condition, can be identified in the individual from whom the sample is derived. In some embodiments, the correlation is reported as a statistical measure. The statistical measure may be reported as a risk measure, such as a relative risk (RR), an absolute risk (AR) or an odds ratio (OR).
The markers described herein, e.g., the markers presented in Table 1, may be useful for risk assessment and diagnostic purposes, either alone or in combination. Results of risk of kidney stones or related conditions based on genotype results for the markers described herein can also be combined with data for other genetic markers or risk factors for the kidney stones or the related conditions, to establish overall risk. Thus, even in cases where the increase in risk by individual markers is relatively modest, e.g. on the order of 10-30%, the association may have significant implications. Thus, relatively common variants may have significant contribution to the overall risk (Population Attributable Risk is high), or combination of markers can be used to define groups of individual who, based on the combined risk of the markers, are at even greater, overall risk.
Thus, in certain embodiments of the invention, a plurality of variants (genetic markers, biomarkers and/or haplotypes) is used for overall risk assessment. These variants are in one embodiment selected from the variants as disclosed herein. Other embodiments include the use of the variants of the present invention in combination with other variants known to be useful for diagnosing a susceptibility to kidney stones or related conditions, including hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone,-, mineral density and osteoporosis. In such embodiments, the genotype status of a plurality of markers and/or haplotypes is determined in an individual, and the status of the individual compared with the population frequency of the associated variants, or the frequency of the variants in clinically healthy subjects, such as age-matched and sex-matched subjects. Methods known in the art, such as multivariate analyses or joint risk analyses or other methods known to the skilled person, may subsequently be used to determine the overall risk conferred based on the genotype status at the multiple loci. Assessment of risk based on such analysis may subsequently be used in the methods, uses and kits of the invention, as described herein.
As described in the above, the haplotype block structure of the human genome has the effect that a large number of variants (markers and/or haplotypes) in linkage disequilibrium with the . variant originally associated with a kidney stones or trait may be used as surrogate markers for' assessing association to the kidney stones or trait. The number of such surrogate markers will , depend on factors such as the historical recombination rate in the region, the mutational frequency in the region (i.e., the number of polymorphic sites or markers in the region), and the extent of LD (size of the LD block) in the region. These markers are usually located within the physical boundaries of the LD block or haplotype block in question as defined using the methods described herein, or by other methods known to the person skilled in the art. However, sometimes marker and haplotype association is found to extend beyond the physical boundaries' of the haplotype block as defined, as discussed in the above. Such markers and/or haplotypes . may in those cases be also used as surrogate markers and/or haplotypes for the markers and/or haplotypes physically residing within the haplotype block as defined. As a consequence, markers and haplotypes in LD (typically characterized by inter-marker r2 values of greater than 0.1, such as r2 greater than 0.2, including r2 greater than 0.3, also including markers correlated by values for r2 greater than 0.4) with the markers and haplotypes of the present invention are also within the scope of the invention, even if they are physically located beyond the boundaries of the haplotype block as defined. This includes markers that are described herein (e.g., Table 1), but may also include other markers that are in strong LD (e.g., characterized by r2 greater than 0.1. or 0.2 and/or | D'| > 0.8) with one or more of the markers listed in Table 1.
For the SNP markers described herein, the opposite allele to the allele found to be in excess in , individuals with kidney stones or related conditions (at-πsk allele) is found in decreased frequency in those individuals. Such marker alleles and/or haplotypes in LD are thus protective' for the condition, i.e. they confer a decreased risk or susceptibility of individuals carrying these ■ markers and/or haplotypes developing the condition.
Certain variants of the present invention, including certain haplotypes comprise, in some cases," a combination of various genetic markers, e.g., SNPs and microsatellites. Detecting haplotypes ' can be accomplished by methods known in the art and/or described herein for detecting sequences at polymorphic sites. Furthermore, correlation between certain haplotypes or sets of markers and kidney stones or related conditions can be verified using standard techniques. A representative example of a simple test for correlation would be a Fisher-exact test on a two by two table.
In specific embodiments, a marker allele or haplotype found to be associated with kidney stones' or related conditions (e.g., hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis) is one in > which the marker allele or haplotype is more frequently present in an individual at risk for kidney stones or the related condition (affecteds), compared to the frequency of its presence in a healthy individual (control), or in randombly selected individual from the population, wherein the presence of the marker allele or haplotype is indicative of a susceptibility of kidney stones or the related condition. In other embodiments, at-risk markers in linkage disequilibrium with one or - more markers shown herein to be associated with kidney stones and related conditions (e.g., marker alleles as listed in Table 1) are tagging markers that are also predictive of risk for kidney stones or related conditions.
Study population
In a general sense, the methods and kits of the invention can be utilized from samples containing nucleic acid material (DNA or RNA) from any source and from any individual, or from genotype data derived from such samples. In preferred embodiments, the individual is a human individual. The individual can be an adult, child, or fetus. The nucleic acid source may be any sample comprising nucleic acid material, including biological samples, or a sample comprising nucleic acid material derived therefrom. The present invention also provides for assessing markers and/or haplotypes in individuals who are members of a target population. Such a target population is in one embodiment a population or group of individuals at risk of developing the , kidney stones, based on other genetic factors, biomarkers, biophysical parameters (e.g. , serum bicarbonate levels, urinary calcium levels, serum parathyroid hormone levels, bone mineral ; density measurements), or general health and/or lifestyle parameters (e.g. , history of kidney stones, previous diagnosis of kidney stones, family history of kidney stones).
The invention provides for embodiments that include individuals from specific age subgroups, such as those over the age of 40, over age of 45, or over age of 50, 55, 60, 65, 70, 75, 80, or 85. Other embodiments of the Invention pertain to other age groups, such as individuals aged less than 85, such as less than age 80, less than age 75, or less than age 70, 65, 60, 55, 50, 45, 40, 35, or age 30. Other embodiments relate to individuals with previous history of kidney stones in any of the age ranges described in the above. It is also contemplated that a range of ' , ages may be relevant in certain embodiments, such as age at onset at more than age 45 but less than age 60. Other age ranges are however also contemplated, including all age ranges bracketed by the age values listed in the above. The invention furthermore relates to individuals of either gender, males or females.
The Icelandic population is a Caucasian population of Northern European ancestry. A large number of studies reporting results of genetic linkage and association in the Icelandic population have been published in the last few years. Many of those studies show replication of variants, originally identified in the Icelandic population as being associating with a particular kidney stones, in other populations (Styrkarsdottir, U., et a/. N Engl J Med Apr 29 2008 (Epub ahead of; print); Thorgeirsson, T., et al. Nature 452:638-42 (2008); Gudmundsson, J., et al. Nat Genet. 40: 281-3 (2008); Stacey, S. N., et al., Nat Genet. 39:865-69 (2007); Helgadottir, A., et al., Science 316: 1491-93 (2007); Steinthorsdottir, V., et al., Nat Genet. 39:770-75 (2007); Gudmundsson, J., et al., Nat Genet. 39:631-37 (2007); Frayling, TM, Nature Reviews Genet 8:657-662 (2007); Amundadottir, L.T., et al., Nat Genet. 38:652-58 (2006); Grant, S. F., et al./ Nat Genet. 38:320-23 (2006)). Thus, genetic findings in the Icelandic population have in general been replicated in other populations, including populations from Africa and Asia.
It is thus believed that the markers of the present invention found to be associated with kidney , stones and related conditions will show similar association in other human populations. Particular embodiments comprising individual human populations are thus also contemplated and within the scope of the invention. Such embodiments relate to human subjects that are from one or more human population including, but not limited to, Caucasian populations, European ;. populations, American populations, Eurasian populations, Asian populations, Central/South Asian populations, East Asian populations, Middle Eastern populations, African populations, Hispanic populations, and Oceanian populations. European populations include, but are not limited to, Swedish, Norwegian, Finnish, Russian, Danish, Icelandic, Irish, Kelt, English, Scottish, Dutch, Belgian, French, German, Spanish, Portugues, Italian, Polish, Bulgarian, Slavic, Serbian, Bosnian, Czech, Greek and Turkish populations. The invention furthermore in other embodiments can be* practiced in specific human populations that include Bantu, Mandenk, Yoruba, San, Mbuti Pygmy, Orcadian, Adygei, Russian, Sardinian, Tuscan, Mozabite, Bedouin, Druze, Palestinian, Balochi, .; Brahui, Makrani, Sindhi, Pathan, Burusho, Hazara, Uygur, Kalash, Han, Dai, Daur, Hezhen, Lahu, Miao, Oroqen, She, Tujia, Tu, Xibo, Yi, Mongolan, Naxi, Cambodian, Japanese, Yakut, Melanesian, Papuan, Karitianan, Surui, Colmbian, Maya and Pima. '
In certain embodiments, the invention relates to populations that include black African ancestry such as populations comprising persons of African descent or lineage. Black African ancestry ., may be determined by self reporting as African-Americans, Afro-Americans, Black Americans, being a member of the black race or being a member of the negro race. For example, African Americans or Black Americans are those persons living in North America and having origins in any of the black racial groups of Africa. In another example, self-reported persons of black African ancestry may have at least one parent of black African ancestry or at least one grandparent of black African ancestry. In another embodiment, the invention relates to individuals of Caucasian origin. The racial contribution in individual subjects may also be determined by genetic analysis. Genetic analysis of ancestry may be carried out using unlinked microsatellite markers such as those set out in Smith et al. (Am J Hum Genet 74, 1001-13 (2004)).
In certain embodiments, the invention relates to markers and/or haplotypes identified in specific populations, as described in the above. The person skilled in the art will appreciate that measures of linkage disequilibrium (LD) may give different results when applied to different populations. This is due to different population history of different human populations as well as differential selective pressures that may have led to differences in LD in specific genomic regions. It is also well known to the person skilled in the art that certain markers, e.g. SNP markers, have different population frequncy in different populations, or are polymorphic in one population but not in another. The person skilled in the art will however apply the methods available and as , thought herein to practice the present invention in any given human population. This may include assessment of polymorphic markers in the LD region of the present invention, so as to .. identify those markers that give strongest association within the specific population. Thus, the at-risk variants of the present invention may reside on different haplotype background and in different frequencies in various human populations. However, utilizing methods known in the art and the markers of the present invention, the invention can be practiced in any given human population.
Utility of Genetic Testing
The person skilled in the art will appreciate and understand that the variants described herein in general do not, by themselves, provide an absolute identification of individuals who will develop a particular disease. The variants described herein do however indicate increased and/or decreased likelihood that individuals carrying the at-risk or protective variants of the invention will develop one or more of kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis. It has been discovered that certain variants confer increase risk of developing these disorders, as ' supported by the results presented herein. This information is extremely valuable in itself, as outlined in more detail in the below, as it can be used to, for example, initiate preventive measures at an early stage, perform regular physical exams to monitor the progress and/or appearance of symptoms, or to schedule exams at a regular interval to identify early symptoms', so as to be able to apply treatment at an early stage.
The knowledge about a genetic variant that confers a risk of developing a disease or condition * offers the opportunity to apply a genetic test to distinguish between individuals with increased risk of developing the condition (i.e. carriers of the at-risk variant) and those with decreased risk of developing the condition (i.e. carriers of the protective variant). The core values of genetic '; testing, for individuals belonging to both of the above mentioned groups, are the possibilities of1 being able to diagnose the condition, or a predisposition to the condition, at an early stage and ' provide information to the clinician about prognosis/aggressiveness of the condition in order to " be able to apply the most appropriate treatment.
Many kidney stones go unnoticed until they cause acute symptoms - specifically, pain associated with the stone going through the ureter, excluding other possible sources of the pain. Sometimes, however, kidney stones are discovered during course of looking for the cause of urinary tract infections or blood in the urine. Suspicion of kidney stones is usually followed by . blood analysis, to look for excess calcium or uric acid, as well as a 24-hour collection of urine to check for stone-forming minerals. Imaging tests may also be performed, the most common being a helical CT (computer tomography) scan without contrast material. Pregnant women, or others who should avoid radiation exposure, may undergo ultrasound examination to establish diagnosis. Ultrasound may however miss small stones, especially if located in the ureter or : bladder.
Kidney stones may become recurrent, especially in those with more than one kidney stone and in those with a family history of kidney stones. Preventive measures than may be taken to minimize the risk of kidney stones include (i) increased volume of fluids consumed; and (ii) altered diet, for example by increasing fiber consumption, eating less meat, consumption of moderate or high amounts of calcium-rich foods, avoiding food that is high in oxalate (e.g., dark green vegetables, nuts, chocolate), and minimizing salt in your diet. Preventive medication may also be taken, which - depending on the type of stones you have or are likely to have - may include, for calcium stones: thiazides {e.g., hydrochlorothiazide, chlorthalidone), potassium citrate, orthophosphate and chlestyramine; for uric acid stones: potassium citrate, sodium bicarbonate, and allopurinol; for cystine stones: potassium citrate, pinicillamine, tipronin and captopril; for struvite stones: urease inhibitors (e.g., lithostat).
The most common metabolic risk factor for kidney stones in adults and children is hypercalciurea. Furthermore, about 20% of patients with hypercalciurea have a family history of kidney stones, showing that the disorders are intertwined.
Determination of the presence of a genetic risk factor for kidney stones or traits that predispose to kidney stones, such as hypercalciurea, may be used to guide individuals towards a diet that minimizes risk of kidney stones. This may be done in conjunction with blood and urine measurements to determine whether the individual has hypercalciurea, decreased serum bicarbonate levels or decreased serum bicarbonate levels. Individuals testing positive for one or more of those risk factors and who also are carrying at least one genetic risk factor for kidney stones, as described herein, may also especially benefit from preventive measures for kidney ' stones. A family history of kidney stones may also be used in combination with the genetic test , and biomarker evaluation(s) to assess the appropriate course of action for the individual.
Individuals determined to have an increased genetic risk of kidney stones may be monitered more closely. For example, regular determination of urinary calcium levels may be performed. As urinary calcium levels are increased, recommendation of a calcium-reducing diet may be recommended, to minimize the risk of stone formation. Individual disease risk management ' may also take into account other factors, including family history of kidney stones, family history of hypercalciurea or other metabolic risk factors of kidney stones, and a history of other related disorders. A personal risk management profile may thus include genetic testing in combination with a medical history of the individual, and his/her immediate family, and may also include additional biochemical tests. This way, an individualized disease risk management program may be developed, based on a combination of genetic risk factors, biochemical measurements, medical history and family history. In a practical medical setting, the genetic profiling may be key in deciding which individuals should be recommended for extensive follow-up and/or monitoring, including monitoring of calcium levels in urine, oxalate levels in urine, uric acid levels in urine, cystine levels in urine and citrate levels in urine, for early prevention and detection of stones.
WHO has recommended that diagnosis of osteoporosis should be made when T-score on bone mineral density measurement by dual energy X-ray absorptiometry (DXA) is -2.5 or lower, compared to the bone mineral density of young women. They also suggested that the term osteopenia or low bone mass be applied when T-scores are from -1.0 to -2.5. Because osteopenia is much more common than osteoporosis, approximately half of fragility fractures : occur in the osteopenic group, although the relative risk of fracture is higher in the osteoporotic population. The current practice is to perform DXA of the lumbar vertebrae and the hip including the femoral neck. Z-score (the bone density in the patient as compared to other people of the same age and size expressed as number of SD above or below the mean) is also used in clinical, practice as risk of fractures increases about two-fold for each SD below the mean.
Other approaches for measuring bone mass such as ultrasound of the heel can be used to assess whether DXA is indicated but not for diagnosis or assessment of response to therapy. Quantitative computed tomography can also be used but is more costly and results in greater exposure to radiation and DXA.
Laboratory assessment is not used to screen for the presence of osteoporosis but is routinely used to exclude secondary causes of osteoporosis. Biochemical markers of increased bone resorption have been shown to be associated with an increasing fracture risk. However, these markers show substantial variability and there are insufficient data to support their use in deciding for or against bone densitometry or pharmacotherapy.
Guidelines have been issued from several professional societies. Most of them have recommended that all women should have a measurement of bone mineral density at the age of 65 because of the sharp increase in the incidence of fracture that occurs in association with low bone mass after that age as well as clinical trials showing the reduction in the risk of fracture when these women are treated. Some of these guidelines have recommended DXA measurements below the age of 65 for postmenopausal women with multiple risk factors such as prior fragility fracture and family history of fractures. The guidelines do not, however, specify how risk factors should be assisted or weighted. Some of these societies have also provided additional guidelines for testing in men, pre-menopausal women and children.
DNA-genotyping of markers associated with decreased bone mineral density could be of value in identifying those individuals that could benefit from earlier BMD measurements in order to target preventive start of anti-fracture therapy. For example, BMD measurements (or DNA-genotyping of markers associated with decreased BMD) could be especially of value in first-degree relatives' of individuals with low bone mass. This should especially be applied to individuals likely to go into a phase of bone loss such as women at the menopause or individuals receiving certain medications known to affect the bone metabolism such as glucocorticoids. It is also possible that bone density measurements and DNA-genotyping could be of value in children of families with strikingly low bone mass indicating genetic influence. The possibility has been raised that low peak bone mass might be related to insufficient response to mechanical or physical loading at }[ young age when the bones are fast growing. It is possible that those individuals diagnosed by DXA or by DNA testing need extra physical exercise to gain optimal peak bone mass. It is also possible that safe drug therapy could be of use in individuals with strikingly low bone mass at young age if the drug is anabolic on the osteoblasts.
Variants associated with decreased BMD might also be used as diagnostics markers for those that would benefit the most from treatment options that target osteoporosis. For example, denosumbab is currently being tested in a phase III clinical trial aimed at preventing bone loss , with fracture as an end point. Such markers might also be used for identifying those individuals that are/will be receiving certain medications known to induce bone loss, such as glucocorticoids, prior to treatment in order to identify those that need contra-treatment for preventing the bone loss, i.e. individuals at risk of low bone mass for whom such additional bone loss will be detrimental and cause low trauma fractures.
METHODS
Methods for risk assessment and risk management are described herein and are encompassed by the invention. The invention also encompasses methods of assessing an individual for probability of response to a therapeutic agents, methods for predicting the effectiveness of a therapeutic agent, nucleic acids, polypeptides and antibodies and computer-implemented functions. Kits for use in the various methods presented herein are also encompassed by the invention. Diagnostic and screening methods
In certain embodiments, the present invention pertains to methods of diagnosing, or aiding in : the diagnosis of, at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis or a susceptibility to the condition, by detecting particular alleles at genetic markers that appear more frequently in subjects with these conditions or subjects who are susceptible to the conditions. In particular embodiments, the invention is a method of determining a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, by detecting, or determining the identity of, at least one allele of at least one polymorphic marker (e.g., the markers described herein). The present invention describes methods whereby detection of particular alleles of particular markers or haplotypes is indicative of a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis.
The present invention pertains in some embodiments to methods of clinical applications of diagnosis, e.g., diagnosis performed by a medical professional. In other embodiments, the invention pertains to methods of diagnosis or determination of a susceptibility performed by a layman. The layman can be the customer of a genotyping service. The layman may also be a genotype or sequence data service provider, who performs genotype analysis on a DNA sample from an individual, in order to provide service related to genetic risk factors for particular traits or kidney stoness, based on the genotype/sequence status of the individual (i.e., the customer). Recent technological advances in genotyping technologies, including high-throughput ganotyping of SNP markers, such as Molecular Inversion Probe array technology (e.g., Affymetrix ..
GeneChip), and BeadArray Technologies (e.g., Illumina GoldenGate and Infinium assays) have made it possible for individuals to have their own genome assessed for up to one million SNPs simultaneously, at relatively little cost. Furthermore, genotype data can be generated by determination of the identity of certain marker alleles by direct sequencing of genomic DNA. The resulting genotype information, which can be made available to the individual, can be compared' to information about kidney stones or trait risk associated with various SNPs, including information from public litterature and scientific publications. The diagnostic application of kidney stones(or related conditions)-associated alleles as described herein, can thus for example be performed by the individual, through analysis of his/her genotype data, by a health professional based on results of a clinical test, or by a third party, including the genotype service provider. The third party may also be service provider who interprets genotype information from the customer to provide service related to specific genetic risk factors, including the genetic markers described herein. In other words, the diagnosis or determination of a susceptibility of genetic risk can be made by health professionals, genetic counselors, third parties providing genotyping service, third parties providing risk assessment service or by the layman (e.g., the *_ individual), based on information about the genotype status of an individual and knowledge about the risk conferred by particular genetic risk factors (e.g., particular SNPs). In the present context, the term "diagnosing", "diagnose a susceptibility" and "determine a susceptibility" is ■-, meant to refer to any available diagnostic method, including those mentioned above. ■ '■
In certain embodiments, a sample containing genomic DNA from an individual is collected. Such sample can for example be a buccal swab, a saliva sample, a blood sample, or other suitable samples containing genomic DNA, as described further herein. The genomic DNA is then analyzed using any common technique available to the skilled person, such as high-throughput , ' array technologies. Results from such genotyping are stored in a convenient data storage unit, such as a data carrier, including computer databases, data storage disks, or by other convenient data storage means. In certain embodiments, the computer database is an object database, a relational database or a post-relational database. The genotype data is subsequently analyzed for the presence of certain variants known to be susceptibility variants for a particular human conditions, such as the genetic variants described herein. Genotype data can be retrieved from . the data storage unit using any convenient data query method. Calculating risk conferred by a particular genotype for the individual can be based on comparing the genotype of the individual to previously determined risk (expressed as a relative risk (RR) or and odds ratio (OR), for example) for the genotype, for example for an heterozygous carrier of an at-risk variant. In other embodiments, risk for homozygous carriers of particular at-risk variants is calculated. The calculated risk for the individual can be the relative risk for a person, or for a specific genotype of a person, compared to the average population with matched gender and ethnicity. The average population risk can be expressed as a weighted average of the risks of different genotypes, using results from a reference population, and the appropriate calculations to calculate the risk of a genotype group relative to the population can then be performed. Alternatively, the risk for an individual is based on a comparison of particular genotypes, for example heterozygous carriers of an at-risk allele of a marker compared with non-carriers of the at-risk allele. Using the , population average may in certain embodiments be more convenient, since it provides a measure which is easy to interpret for the user, i.e. a measure that gives the risk for the individual, based on his/her genotype, compared with the average in the population. The calculated risk estimated can be made available to the customer via a website, preferably a secure website.
In certain embodiments, a service provider will include in the provided service all of the steps of isolating genomic DNA from a sample provided by the customer, performing genotyping of the isolated DNA, calculating genetic risk based on the genotype data, and report the risk to the customer. In some other embodiments, the service provider will include in the service the interpretation of genotype data for the individual, i.e. , risk estimates for particular genetic variants based on the genotype data for the individual . In some other embodiments, the service provider may include service that includes genotyping service and interpretation of the genotype data, starting from a sample of isolated DNA from the individual (the customer). Overall risk for multiple risk variants can be performed using standard methodology. For example, assuming a multiplicative model, i.e. assuming that the risk of individual risk variants ■ multiply to establish the overall effect, allows for a straight-forward calculation of the overall risk for multiple markers.
In certain embodiments, significance of association of particular marker alleles or haplotypes is 1 characterized by a p value < 0.05. In other embodiments, the significance of association is characterized by smaller p-values, such as < 0.01, <0.001, <0.0001, <0.00001, <0.000001, ;. <0.0000001, <0.00000001 or <0.000000001. In certain embodiments, the significant risk is characterized by particular values of Relative Risk (RR) or Absolute Risk (AR), or lifetime risk.
In these embodiments, the presence of particular marker alleles or haplotypes is indicative of a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis. Diagnostic methods involve determining whether particular alleles or " haplotypes that are associated with risk of kidney stones or related conditions are present in particular individuals. The haplotypes described herein include combinations of alleles at various genetic markers {e.g., SNPs, microsatellites or other genetic variants). The detection of the particular genetic marker alleles that make up particular haplotypes can be performed by a variety of methods described herein and/or known in the art. For example, genetic markers can be detected at the nucleic acid level (e.g., by direct nucleotide sequencing, or by other genotyping means known to the skilled in the art) or at the amino acid level if the genetic marker affects the coding sequence of an encocded protein (e.g., CLDN14; e.g., by protein sequencing or by immunoassays using antibodies that recognize such a protein). The marker alleles or haplotypes of the present invention correspond to fragments of genomic segments associated with risk of kidney stones and related conditions. These segments in certain embodiments comprise a portion of the human CLDN14 gene. Such fragments encompass the " DNA sequence of the polymorphic marker or haplotype in question, but may also include DNA segments in strong LD (linkage disequilibrium) with the marker or haplotype. In one embodiment, such segments comprises segments in LD with the marker or haplotype as determined by a value of r2 greater than 0.2 and/or |D'| > 0.8).
In one embodiment, determination of a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis is accomplished using hybridization methods, (see Current Protocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley & Sons, including all supplements). The presence of a specific marker allele can be indicated by sequence-specific hybridization of a nucleic acid probe specific for the particular allele. The presence of more than one specific marker allele or a specific haplotype can be indicated by using several sequence-specific nucleic acid probes, each being specific for a particular allele. A sequence-specific probe can be directed to hybridize to genomic DNA, RNA, or cDNA. A "nucleic acid probe", as used herein, can be a DNA probe or an RNA probe that hybridizes to a complementary sequence. One of skill in the art would know how to design such a probe so that sequence specific hybridization will occur only if a particular allele is present in a genomic sequence from a test sample. The invention can also be reduced to practice using any convenient genotyping method, including commercially available technologies and methods for genotyping particular polymorphic markers.
To determine a susceptibility to a condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral -j density and osteoporosis, a hybridization sample can be formed by contacting the test sample containing a nucleic acid, such as a genomic DNA sample, with at least one nucleic acid probe. A non-limiting example of a probe for detecting mRIMA or genomic DNA is a labeled nucleic acid probe that is capable of hybridizing to mRNA or genomic DNA sequences described herein. The nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof,' such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length that is , sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA. For example, the nucleic acid probe can comprise all or a portion of the nucleotide sequence of the LD block C21, as described herein, optionally comprising at least one allele of a marker described herein, or at least one haplotype described herein, or the probe can be the complementary sequence of such a sequence. In a particular embodiment, the nucleic acid probe is a portion of the nucleotide sequence of LD Block C21, as described herein, optionally comprising at least one allele of a marker described herein, or at least one allele of one polymorphic marker or haplotype comprising at least one polymorphic marker described herein, or the probe can be the complementary sequence of such a sequence. Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization can be performed by methods well known to the person skilled in the art (see, e.g., Current Protocols in Molecular Biology, Ausubel, F. et al. , eds., John Wiley & Sons, including all supplements). In one embodiment, hybridization refers to specific hybridization, i.e., hybridization with no mismatches (exact hybridization). In one embodiment, the hybridization conditions for specific hybridization are high stringency.
Specific hybridization, if present, is detected using standard methods. If specific hybridization occurs between the nucleic acid probe and the nucleic acid in the test sample, then the sample • contains the allele that is complementary to the nucleotide that is present in the nucleic acid probe. The process can be repeated for any markers of the present invention, or markers that . make up a haplotype of the present invention, or multiple probes can be used concurrently to detect more than one marker alleles at a time. It is also possible to design a single probe containing more than one marker alleles of a particular haplotype (e.g., a probe containing alleles complementary to 2, 3, 4, 5 or all of the markers that make up a particular haplotype). Detection of the particular markers of the haplotype in the sample is indicative that the source of the sample has the particular haplotype (e.g., an at-risk haplotype). In one preferred embodiment, a method utilizing a detection oligonucleotide probe comprising a fluorescent moiety or group at its 3' terminus and a quencher at its 5' terminus, and an enhancer oligonucleotide, is employed, as described by Kutyavin et al. (Nucleic Acid Res. 34:el28 (2006)). The fluorescent moiety can be Gig Harbor Green or Yakima Yellow, or other suitable fluorescent moieties. The detection probe is designed to hybridize to a short nucleotide sequence that includes the SNP polymorphism to be detected. Preferably, the SNP is anywhere from the terminal residue to -6 residues from the 3' end of the detection probe. The enhancer is a short oligonucleotide probe which hybridizes to the DNA template 3' relative to the detection probe. The probes are designed such that a single nucleotide gap exists between the detection probe and the enhancer nucleotide probe when both are bound to the template. The gap creates a synthetic abasic site that is recognized by an endonuclease, such as Endonuclease IV. The ■ enzyme cleaves the dye off the fully complementary detection probe, but cannot cleave a detection probe containing a mismatch. Thus, by measuring the fluorescence of the released fluorescent moiety, assessment of the presence of a particular allele defined by nucleotide sequence of the detection probe can be performed.
The detection probe can be of any suitable size, although preferably the probe is relatively short. In one embodiment, the probe is from 5-100 nucleotides in length. In another embodiment, the probe is from 10-50 nucleotides in length, and in another embodiment, the probe is from 12-30 nucleotides in length. Other lengths of the probe are possible and within scope of the skill of the average person skilled in the art.
In a preferred embodiment, the DNA template containing the SNP polymorphism is amplified by Polymerase Chain Reaction (PCR) prior to detection. In such an embodiment, the amplified DNA serves as the template for the detection probe and the enhancer probe.
Certain embodiments of the detection probe, the enhancer probe, and/or the primers used for amplification of the template by PCR include the use of modified bases, including modified A and modified G, The use of modified bases can be useful for adjusting the melting temperature of ■, the nucleotide molecule (probe and/or primer) to the template DNA, for example for increasing the melting temperature in regions containing a low percentage of G or C bases, in which modified A with the capability of forming three hydrogen bonds to its complementary T can be ' used, or for decreasing the melting temperature in regions containing a high percentage of G or C bases, for example by using modified G bases that form only two hydrogen bonds to their complementary C base in a double stranded DNA molecule. In a preferred embodiment, modified bases are used in the design of the detection nucleotide probe. Any modified base known to the skilled person can be selected in these methods, and the selection of suitable bases is well within the scope of the skilled person based on the teachings herein and known bases available from commercial sources as known to the skilled person.
Alternatively, a peptide nucleic acid (PNA) probe can be used in addition to, or instead of, a nucleic acid probe in the hybridization methods described herein. A PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, Nielsen, P., et a/., Bioconjug. Chem. 5:3-7 (1994)). The PNA probe can be designed to specifically hybridize to a molecule in a sample suspected of containing one or more of the marker alleles or haplotypes that are associated with risk of at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis. Hybridization of the PNA probe is thus diagnostic risk of these conditions.
In one embodiment of the invention, a test sample containing genomic DNA obtained from the ' subject is collected and the polymerase chain reaction (PCR) is used to amplify a fragment comprising one ore more markers or haplotypes of the present invention. As described herein, •, identification of a particular marker allele or haplotype can be accomplished using a variety of methods (e.g., sequence analysis, analysis by restriction digestion, specific hybridization, single stranded conformation polymorphism assays (SSCP), electrophoretic analysis, etc.). In another embodiment, diagnosis is accomplished by expression analysis, for example by using quantitative PCR (kinetic thermal cycling). This technique can, for example, utilize commercially available technologies, such as TaqMan® (Applied Biosystems, Foster City, CA). The technique - can assess the presence of an alteration in the expression or composition of a polypeptide or splicing variant(s), for example of the CLDN14 gene. Further, the expression of the variant(s) can be quantified as physically or functionally different.
In another embodiment of the methods of the invention, analysis by restriction digestion can be' used to detect a particular allele if the allele results in the creation or elimination of a restriction' site relative to a reference sequence. Restriction fragment length polymorphism (RFLP) analysis can be conducted, e.g., as described in Current Protocols in Molecular Biology, supra. The digestion pattern of the relevant DNA fragment indicates the presence or absence of the particular allele in the sample.
Sequence analysis can also be used to detect specific alleles or haplotypes. Therefore, in one embodiment, determination of the presence or absence of a particular marker alleles or haplotypes comprises sequence analysis of a test sample of DNA or RNA obtained from a subject or individual. PCR or other appropriate methods can be used to amplify a portion of a nucleic acid that contains a particular polymorphic marker or haplotype, and the presence of specific alleles can then be detected directly by sequencing the polymorphic site (or multiple polymorphic sites in a haplotype) of the genomic DNA in the sample.
In another embodiment, arrays of oligonucleotide probes that are complementary to target :. nucleic acid sequence segments from a subject, can be used to identify particular alleles at polymorphic sites. For example, an oligonucleotide array can be used. Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. These arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods that incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods, or by other methods known to the person skilled in the art (see, e.g., Bier, F. F., ef al. Adv Biochem Eπg Biotechnol 109:433-53 (2008); Hoheisel, J. D., Nat Rev Genet 7: 200-10 (2006); Fan, J. B., et
Figure imgf000056_0001
Methods Enzymol 410: 57-73 (2006); Raqoussis, J. & Elvidge, G., Expert Rev MoI Diagn 6: 145-52 (2006); Mockler, T.C , et al Genomics 85: 1-15 (2005), and references cited therein, the entire teachings of each of which are incorporated by reference herein). Many additional descriptions ' of the preparation and use of oligonucleotide arrays for detection of polymorphisms can be found, for example, in US 6,858,394, US 6,429,027, US 5,445,934, US 5,700,637, US 5,744,305, US 5,945,334, US 6,054,270, US 6,300,063, US 6,733,977, US 7,364,858, EP 619 321, and EP 373 203, the entire teachings of which are incorporated by reference herein. <
Other methods of nucleic acid analysis that are available to those skilled in the art can be used ; : to detect a particular allele at a polymorphic site. Representative methods include, for example, direct manual sequencing (Church and Gilbert, Proc. Natl. Acad. Sci. USA, 81 : 1991-1995 ( 1988); Sanger, F., et al., Proc. Natl. Acad. Sci. USA, 74: 5463-5467 (1977); Beavis, et al., U.S. Patent No. 5,288,644); automated fluorescent sequencing; single-stranded conformation polymorphism assays (SSCP); clamped denaturing gel electrophoresis (CDGE) ; denaturing gradient gel electrophoresis (DGGE) (Sheffield, V., et al. , Proc. Natl. Acad. Sci. USA, 86: 232-236 ( 1989)), mobility shift analysis (Orita, M., et al. , Proc. Natl. Acad. Sci. USA, 86: 2766-2770 (1989)), restriction enzyme analysis (Flavell, R., et al., Cell, 15:25-41 (1978); Geever, R., et al., Proc. Natl. Acad. Sci. USA, 78: 5081-5085 (1981)); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton, R., et al. , Proc. Natl. Acad. Sci. USA, 85:4397-4401 ( 1985)); RNase ■ protection assays (Myers, R., et al. , Science, 230: 1242-1246 ( 1985); use of polypeptides that recognize nucleotide mismatches, such as E. coli mutS protein; and allele-specific PCR.
In another embodiment of the invention, diagnosis of, or a determination of, a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and ;■■ osteoporosis can be made by examining expression and/or composition of a polypeptide encoded by a nucleic acid associated with the condition, in those instances where the genetic marker(s) or haplotype(s) of the present invention result in a change in the composition or expression of ' the polypeptide. In certain embodiments, the polypeptide is a CLDN 14 polypeptide encoded by the human CLDN14 gene. Thus, determination of a susceptibility to the condition can be made by examining expression and/or composition of such a polypeptide, in those instances where the genetic marker or haplotype of the present invention results in a change in the composition or . expression of the polypeptide. The haplotypes and markers of the present invention that are associaed to kidney stones and related conditions may play a role through their effect on particular genes, such as the CLDN14 gene. Possible mechanisms affecting these genes include, e.g. , effects on transcription, effects on RNA splicing, alterations in relative amounts of alternative splice forms of mRNA, effects on RNA stability, effects on transport from the nucleus to cytoplasm, and effects on the efficiency and accuracy of translation. It is well known that regulatory element affecting gene expression may be located far away, even as far as tenths or hundreds of kilobases away, from the promoter region of a gene. By assaying for the presence or absence of at least one allele of at least one polymorphic marker of the present invention, it is thus possible to assess the expression level of nearby genes. It is thus contemplated that the detection of the markers or haplotypes of the present invention can be used for assessing expression for one or more of such genes, including but not limited to the human CLDN14 gene.
A variety of methods can be used for detecting protein expression levels, including enzyme linked immunosorbent assays (ELISA), Western blots, immunoprecipitations and immunofluorescence. A test sample from a subject is assessed for the presence of an alteration in the expression and/or an alteration in composition of the polypeptide encoded by a particular nucleic acid. An alteration in expression of a polypeptide can be, for example, an alteration in the quantitative polypeptide expression (i.e., the amount of polypeptide produced). An alteration in the composition of a polypeptide may be an alteration in the qualitative polypeptide expression (e.g., expression of a mutant polypeptide or of a different splicing variant). In one embodiment, determination of a susceptibility to kidney stones or related conditions is made by detecting a particular splicing variant of a gene associated with the condition (e.g., the CLDN14 gene), or a particular pattern of splicing variants.
Both such alterations (quantitative and qualitative) can also be present. An "alteration" in the polypeptide expression or composition, as used herein, refers to an alteration in expression or composition in a test sample, as compared to the expression or composition of the polypeptide in a control sample. A control sample is a sample that corresponds to the test sample (e.g., is from the same type of cells), and is from a subject who is not affected by, and/or who does not have, a susceptibility to, the condition. In one embodiment, the control sample is from a subject that does not possess a particular marker allele or haplotype associated with kidney stones or related conditions, as described herein. Similarly, the presence of one or more different splicing variants in the test sample, or the presence of significantly different amounts of different splicing variants in the test sample, as compared with the control sample, can be indicative of a susceptibility to , kidney stones or related conditions. An alteration in the expression or composition of the polypeptide in the test sample, as compared with the control sample, can be indicative of a specific allele in the instance where the allele alters a splice site relative to the reference in the control sample. Various means of examining expression or composition of a polypeptide encoded by a nucleic acid are known to the person skilled in the art and can be used, including - spectroscopy, colorimetry, electrophoresis, isoelectric focusing, and immunoassays (e.g., David er a/., U.S. Pat. No. 4,376,110) such as immunoblotting (see, e.g., Current Protocols in Molecular Biology, particularly chapter 10, supra).
For example, in one embodiment, an antibody (e.g., an antibody with a detectable label) that is capable of binding to a polypeptide encoded by a nucleic acid associated with at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis can,- be used. In one embodiment, the polypeptide is a CLDN 14 polypeptide. Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., Fv, Fab, Fab', F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a labeled secondary antibody (e.g., a fluorescently-labeled secondary antibody) and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.
In one embodiment of this method, the level or amount of a polypeptide in a test sample is compared with the level or amount of the polypeptide in a control sample. A level or amount of the polypeptide in the test sample that is higher or lower than the level or amount of the polypeptide in the control sample, such that the difference is statistically significant, is indicative of an alteration in the expression of the polypeptide encoded by the nucleic acid, and is diagnostic for a particular allele or haplotype responsible for causing the difference in expression. Alternatively, the composition of the polypeptide in a test sample is compared with the composition of the polypeptide in a control sample. In another embodiment, both the level or amount and the composition of the polypeptide can be assessed in the test sample and in the control sample.
In another embodiment, determination of a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum •', bicarbonate levels, reduced bone mineral density and osteoporosis is made by detecting at least one marker or haplotype as described herein in combination with an additional protein-based, RNA-based or DNA-based assay.
Kits
Kits useful in the methods of the invention comprise components useful in any of the methods ι; described herein, including for example, primers for nucleic acid amplification, hybridization probes, restriction enzymes (e.g., for RFLP analysis), allele-specific oligonucleotides, antibodies that bind to an altered polypeptide encoded by a nucleic acid of the invention as described herein (e.g., a genomic segment comprising at least one polymorphic marker and/or haplotype of the • present invention) or to a non-altered (native) polypeptide encoded by a nucleic acid of the invention as described herein, means for amplification of nucleic acids as described herein, means for analyzing the nucleic acid sequence of nucleic acids as described herein, means for analyzing the amino acid sequence of a polypeptide encoded by a nucleic acid described herein, ; etc. The kits can for example include necessary buffers, nucleic acid primers for amplifying -: nucleic acids of the invention (e.g. , a nucleic acid segment comprising one or more of the polymorphic markers as described herein), and reagents for allele-specific detection of the fragments amplified using such primers and necessary enzymes (e.g., DNA polymerase). Additionally, kits can provide reagents for assays to be used in combination with the methods of the present invention, e.g., reagents for use with other diagnostic assays for kidney stones or related conditions.
In one embodiment, the invention pertains to a kit for assaying a sample from a subject to detect a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone7 , mineral density and osteoporosis in a subject, wherein the kit comprises reagents necessary for selectively detecting at least one allele of at least one polymorphism of the present invention in the genome of the individual. In a particular embodiment, the reagents comprise at least one . contiguous oligonucleotide that hybridizes to a fragment of the genome of the individual comprising at least one polymorphism of the present invention. In another embodiment, the reagents comprise at least one pair of oligonucleotides that hybridize to opposite strands of a genomic segment obtained from a subject, wherein each oligonucleotide primer pair is designed to selectively amplify a fragment of the genome of the individual that includes at least one polymorphism associated with risk of the condition. In one such embodiment, the polymorphism is selected from the group consisting of the polymorphisms as listed in Table 1, • and polymorphic markers in linkage disequilibrium therewith. In yet another embodiment the fragment is at least 20 base pairs in size. Such oligonucleotides or nucleic acids {e.g., oligonucleotide primers) can be designed using portions of the nucleic acid sequence flanking polymorphisms (e.g., SNPs or microsatellites) that are associated with risk of the at least one . condition. In another embodiment, the kit comprises one or more labeled nucleic acids capable . of allele-specific detection of one or more specific polymorphic markers or haplotypes, and reagents for detection of the label. Suitable labels include, e.g. , a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.
In particular embodiments, the polymorphic marker or haplotype to be detected by the reagents of the kit comprises one or more markers, two or more markers, three or more markers, four or more markers or five or more markers. In particular embodiments, the polymorphic marker or, haplotype comprises at least one marker selected from the group of markers set forth in Table 1. In another embodiment, the marker or haplotype to be detected comprises at least one marker from the group of markers in strong linkage disequilibrium, as defined by values of r2 greater than 0.2, to at least one of the group of markers listed in Table 1. In another embodiment, the marker or haplotype to be detected is selected from rs219778, rs219779, rs219780 and rs219781.
In one preferred embodiment, the kit for detecting the markers of the invention comprises a detection oligonucleotide probe, that hybridizes to a segment of template DNA containing a SNP polymorphisms to be detected, an enhancer oligonucleotide probe and an endonuclease. As explained in the above, the detection oligonucleotide probe comprises a fluorescent moiety or • group at its 3' terminus and a quencher at its 5' terminus, and an enhancer oligonucleotide, is , employed, as described by Kutyavin et al. (Nucleic Acid Res. 34:el28 (2006)). The fluorescent, moiety can be Gig Harbor Green or Yakima Yellow, or other suitable fluorescent moieties. The , detection probe is designed to hybridize to a short nucleotide sequence that includes the SNP polymorphism to be detected. Preferably, the SNP is anywhere from the terminal residue to -6 residues from the 3' end of the detection probe. The enhancer is a short oligonucleotide probe which hybridizes to the DNA template 3' relative to the detection probe. The probes are designed such that a single nucleotide gap exists between the detection probe and the enhancer nucleotide probe when both are bound to the template. The gap creates a synthetic abasic site ' that is recognized by an endonuclease, such as Endonuclease IV. The enzyme cleaves the dye off the fully complementary detection probe, but cannot cleave a detection probe containing a mismatch. Thus, by measuring the fluorescence of the released fluorescent moiety, assessment of the presence of a particular allele defined by nucleotide sequence of the detection probe can be performed.
The detection probe can be of any suitable size, although preferably the probe is relatively short. In one embodiment, the probe is from 5-100 nucleotides in length. In another embodiment, the i probe is from 10-50 nucleotides in length, and in another embodiment, the probe is from 12-30 nucleotides in length. Other lengths of the probe are possible and within scope of the skill of the average person skilled in the art.
In a preferred embodiment, the DNA template containing the SNP polymorphism is amplified by Polymerase Chain Reaction (PCR) prior to detection, and primers for such amplification are included in the reagent kit. In such an embodiment, the amplified DNA serves as the template for the detection probe and the enhancer probe.
In one embodiment, the DNA template is amplified by means of Whole Genome Amplification (WGA) methods, prior to assessment for the presence of specific polymorphic markers as described herein. Standard methods well known to the skilled person for performing WGA may be utilized, and are within scope of the invention. In one such embodiment, reagents for performing WGA are included in the reagent kit.
Certain embodiments of the detection probe, the enhancer probe, and/or the primers used for amplification of the template by PCR include the use of modified bases, including modified A and modified G. The use of modified bases can be useful for adjusting the melting temperature of the nucleotide molecule (probe and/or primer) to the template DNA, for example for increasing the melting temperature in regions containing a low percentage of G or C bases, in which modified A with the capability of forming three hydrogen bonds to its complementary T can be used, or for decreasing the melting temperature in regions containing a high percentage of G or C bases, for example by using modified G bases that form only two hydrogen bonds to their complementary C base in a double stranded DNA molecule. In a preferred embodiment, modified bases are used in the design of the detection nucleotide probe. Any modified base known to the skilled person can be selected in these methods, and the selection of suitable bases is well within the scope of the skilled person based on the teachings herein and known bases available from commercial sources as known to the skilled person.
In one such embodiment, determination of the presence of the marker or haplotype is indicative of a susceptibility (increased susceptibility or decreased susceptibility) to at least one condition • selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis. In another embodiment, determination of the presence of the marker or haplotype is indicative of response to a therapeutic agent for at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis. In another embodiment, the presence of the marker or haplotype is indicative of prognosis of at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis. In yet another embodiment, the presence of the marker or haplotype is indicative of progress of treatment of the at elast one condition.
In certain embodiments, the kit further comprises a set of instructions for using the reagents comprising the kit.
In a further aspect of the present invention, a pharmaceutical pack (kit) is provided, the pack comprising a therapeutic agent and a set of instructions for administration of the therapeutic agent to humans diagnostically tested for one or more variants of the present invention, as disclosed herein. The therapeutic agent can be a small molecule drug, an antibody, a peptide, an antisense or RNAi molecule, or other therapeutic molecules. In one embodiment, an individual identified as a carrier of at least one variant of the present invention is instructed to take a prescribed dose of the therapeutic agent. In one such embodiment, an individual identified as a homozygous carrier of at least one variant of the present invention is instructed to take a prescribed dose of the therapeutic agent. In another embodiment, an individual identified as a non-carrier of at least one variant of the present invention is instructed to take a prescribed dose of the therapeutic agent.
Therapeutic agents
The variants (markers and/or haplotypes) disclosed herein to confer increased risk of at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis can' also be used to identify novel therapeutic targets for the condition. For example, genes containing, or in linkage disequilibrium with, one or more of these variants, or their products, as well as genes or their products that are directly or indirectly regulated by or interact with these variant genes or their products, can be targeted for the development of therapeutic agents. Therapeutic agents may comprise one or more of, for example, small non-protein and non- nucleic acid molecules, proteins, peptides, protein fragments, nucleic acids (DNA, RNA), PNA - (peptide nucleic acids), or their derivatives or mimetics which can modulate the function and/or levels of the target genes or their gene products. In certain embodiments, the therapeutic agent targets the CLDN14 gene.
In one aspect, the therapeutic agent is a human CLDN 14 polypeptide or a fragment thereof. The CLDN14 polypeptide may be developed as a medicament using any suitable method of preparation, formulation and delivery, which are all well known to the skilled person.
In another aspect, the therapeutic agent is an oligonucleotide. For example, an oligonucleotide probe which is capable of hybridizing to a portion or fragment of the humand CLDN14 gene, may be prepared as a medicament for delivery to ameliorate symptoms associated with any one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis.
The nucleic acids and/or variants described herein, or nucleic acids comprising their complementary sequence, may be used as antisense constructs to control gene expression in cells, tissues or organs. The methodology associated with antisense techniques is well known to' the skilled artisan, and is for example described and reviewed in AntisβnsβDrug Technology: Principles, Strategies, and Applications, Crooke, ed., Marcel Dekker Inc., New York (2001). In general, antisense agents (antisense oligonucleotides) are comprised of single stranded oligonucleotides that are capable of binding to a complimentary nucleotide segment. By binding the appropriate target sequence, an RNA-RNA, DNA-DNA or RNA-DNA duplex is formed. The antisense oligonucleotides are complementary to the sense or coding strand of a gene. It is also possible to form a triple helix, where the antisense oligonucleotide binds to duplex DNA.
Several classes of antisense oligonucleotide are known to those skilled in the art, including cleavers and blockers. The former bind to target RNA sites, activate intracellular nucleases {e.g., RnaseH or Rnase L), that cleave the target RNA. Blockers bind to target RNA, inhibit protein translation by steric hindrance of the ribosomes. Examples of blockers include nucleic acids, morpholino compounds, locked nucleic acids and methylphosphonates (Thompson, Drug Discovery Today, 7:912-917 (2002)). Antisense oligonucleotides are useful directly as therapeutic agents, and are also useful for determining and validating gene function, for example by gene knock-out or gene knock-down experiments. Antisense technology is further described in Lavery er a/. , Curr. Opin. Drug Discov. Devel. 6:561-569 (2003), Stephens er a/., Curr. Opin. MoI. Then 5: 118-122 (2003), Kurreck, Eur. J. Biochem. 270: 1628-44 (2003), Dias er a/., MoI. ' Cancer Ter. 1:347-55 (2002), Chen, Methods MoI. Med. 75:621-636 (2003), Wang er a/., Curr. ' Cancer Drug Targets 1 : 177-96 (2001), and Bennett, Antisense Nucleic Acid Drug.Dev. 12:215- . 24 (2002). In certain embodiments, the antisense agent is an oligonucleotide that is capable of binding to a nucleotide segment of the CLDN14 gene. Antisense nucleotides can be from 5-500 nucleotides . in length, including 5-200 nucleotides, 5-100 nucleotides, 8-50 nucleotides, and 8-30 nucleotides. In certain preferred embodiments, the antisense nucleotides is from 14-50 nucleotides in length, includign 14-40 nucleotides and 14-30 nucleotides. In certain such embodiments, the antisense nucleotide is capable of binding to a nucleotide segment of the CLDN14 as set forth in SEQ ID NO: 1. U
The variants described herein can be used for the selection and design of antisense reagents that are specific for particular variants. Using information about the variants described herein, antisense oligonucleotides or other antisense molecules that specifically target mRNA molecules that contain one or more variants of the invention can be designed. In this manner, expression of mRNA molecules that contain one or more variant of the present invention (markers and/or haplotypes) can be inhibited or blocked. In one embodiment, the antisense molecules are designed to specifically bind a particular allelic form (i.e., one or several variants (alleles and/or haplotypes)) of the target nucleic acid, thereby inhibiting translation of a product originating from this specific allele or haplotype, but which do not bind other or alternate variants at the specific polymorphic sites of the target nucleic acid molecule.
As antisense molecules can be used to inactivate mRNA so as to inhibit gene expression, and thus protein expression, the molecules can be used for development of treatment of kidney stones or related conditions. The methodology can involve cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Such mRNA regions include, for example, .• protein-coding regions, in particular protein-coding regions corresponding to catalytic activity, . substrate and/or ligand binding sites, or other functional domains of a protein.
The phenomenon of RNA interference (RNAi) has been actively studied for the last decade, since its original discovery in C. elβgans (Fire et al., Nature 391 :806-11 ( 1998)), and in recent years its potential use in treatment of human kidney stones has been actively pursued (reviewed in Kim & Rossi, Nature Rev. Genet. 8: 173-204 (2007)). RNA interference (RNAi), also called gene silencing, is based on using double-stranded RNA molecules (dsRNA) to turn off specific genes. In the cell, cytoplasmic double-stranded RNA molecules (dsRNA) are processed by cellular complexes into small interfering RNA (siRNA). The siRNA guide the targeting of a protein-RNA complex to specific sites on a target mRNA, leading to cleavage of the mRNA (Thompson, Drug Discovery Today, 7:912-917 (2002)). The siRNA molecules are typically about 20, 21, 22 or 23 nucleotides in length. Thus, one aspect of the invention relates to isolated nucleic acid molecules, and the use of those molecules for RNA interference, i.e. as small interfering RNA molecules (siRNA). In one embodiment, the isolated nucleic acid molecules are 18-26 nucleotides in length, preferably 19-25 nucleotides in length, more preferably 20-24 nucleotides in length, and more preferably 21, 22 or 23 nucleotides in length. ' Another pathway for RNAi-mediated gene silencing originates in endogenously encoded primary' microRNA (pri-miRNA) transcripts, which are processed in the cell to generate precursor miRNA (pre-miRNA). These miRNA molecules are exported from the nucleus to the cytoplasm, where they undergo processing to generate mature miRNA molecules (miRNA), which direct translational inhibition by recognizing target sites in the 3' untranslated regions of mRNAs, and subsequent mRNA degradation by processing P-bodies (reviewed in Kim & Rossi, Nature Rev. . Genet. 8: 173-204 (2007)).
Clinical applications of RNAi include the incorporation of synthetic siRNA duplexes, which preferably are approximately 20-23 nucleotides in size, and preferably have 3' overlaps of 2 nucleotides. Knockdown of gene expression is established by sequence-specific design for the target mRNA. Several commercial sites for optimal design and synthesis of such molecules are; known to those skilled in the art.
Other applications provide longer siRNA molecules (typically 25-30 nucleotides in length, preferably about 27 nucleotides), as well as small hairpin RNAs (shRNAs; typically about 29 nucleotides in length). The latter are naturally expressed, as described in Amarzguioui et al. ■ [FEBS Lett. 579: 5974-81 (2005)). Chemically synthetic siRNAs and shRNAs are substrates for in vivo processing, and in some cases provide more potent gene-silencing than shorter designs (Kim et al.. Nature Biotechnol. 23:222-226 (2005); Siolas et al., Nature Biotechnol. 23 :227-231 (2005)). In general siRNAs provide for transient silencing of gene expression, because their intracellular concentration is diluted by subsequent cell divisions. By contrast, expressed shRNAs mediate long-term, stable knockdown of target transcripts, for as long as transcription of the shRNA takes place (Marques et al., Nature Biotechnol. 23 :559-565 (2006); Brummelkamp et al., Science 296: 550-553 (2002)).
Since RNAi molecules, including siRNA, miRNA and shRNA, act in a sequence-dependent manner, the variants presented herein {e.g., the markers set forth in Table 1) can be used to design RNAi reagents that recognize specific nucleic acid molecules comprising specific alleles and/or haplotypes (e.g., the alleles and/or haplotypes of the present invention), while not recognizing nucleic acid molecules comprising other alleles or haplotypes. These RNAi reagents can thus recognize and destroy the target nucleic acid molecules. As with antisense reagents, RNAi reagents can be useful as therapeutic agents (i.e., for turning off kidney stones-associated genes or kidney stones-associated gene variants), but may also be useful for characterizing and . validating gene function (e.g., by gene knock-out or gene knock-down experiments).
Delivery of RNAi may be performed by a range of methodologies known to those skilled in the art. Methods utilizing non-viral delivery include cholesterol, stable nucleic acid-lipid particle (SNALP), heavy-chain antibody fragment (Fab), aptamers and nanoparticles. Viral delivery methods include use of lentivirus, adenovirus and adeno-associated virus. The siRNA molecules are in some embodiments chemically modified to increase their stability. This can include modifications at the 2' position of the ribose, including 2'-O-methylpurines and 2'- fluoropyrimidines, which provide resistance to Rnase activity. Other chemical modifications are possible and known to those skilled in the art.
The following references provide a further summary of RNAi, and possibilities for targeting specific genes using RNAi: Kim & Rossi, Nat. Rev. Genet. 8: 173-184 (2007), Chen & Rajewsky,. Nat. Rev. Genet 8: 93-103 (2007), Reynolds, et al., Nat. Biotechnol. 22: 326-330 (2004), Chi et al., Proc. Natl. Acad. Sci. USA 100:6343-6346 (2003), Vickers et al., J. Biol. Chem. 278:7108- . 7118 (2003), Agami, Curr. OpIn. Chem. Biol. 6:829-834 (2002), Lavery, et al., Curr. Opin. Drug Discov. Devel. 6: 561-569 (2003), Shi, Trends Genet. 19:9-12 (2003), Shuey et al., Drug Discov. Today 7: 1040-46 (2002), McManus et al., Nat. Rev. Genet. 3:737-747 (2002), Xia et al., Nat. ■ Biotechnol. 20: 1006-10 (2002), Plasterk et al., curr. Opin. Genet. Dev. 10:562-7 (2000), Bosher et al., Nat. Cell Biol. 2: E31-6 (2000), and Hunter, Curr. Biol. 9:R440-442 (1999).
A genetic defect leading to increased predisposition or risk for development of a condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, or a defect causing the kidney stones, may be corrected permanently by administering to a subject carrying the defect a nucleic acid fragment that incorporates a repair sequence that supplies the normal/wild-type nucleotide(s) at the site of the genetic defect. Such site-specific repair sequence may coπcompass an RIMA/DNA oligonucleotide that operates to promote endogenous repair of a subject's genomic DNA. The administration of the repair sequence may be performed by an appropriate vehicle, such as a complex with polyethelenimine, encapsulated in anionic liposomes, a viral vector such as an adenovirus vector, or other pharmaceutical compositions suitable for promoting intracellular uptake of the adminstered nucleic acid. The genetic: defect may then be overcome, since the chimeric oligonucleotides induce the incorporation of the normal sequence into the genome of the subject, leading to expression of the normal/wild-type gene product. The replacement is propagated, thus rendering a permanent repair and alleviation of the symptoms associated with the kidney stones or condition.
The present invention provides methods for identifying compounds or agents that can be used to treat conditions such as kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis. Thus, the variants of the invention are useful as targets for the identification and/or development of therapeutic agents. In certain embodiments, such methods include assaying the ability of an agent or compound to modulate the activity and/or expression of a nucleic acid that includes at least one of the variants (markers and/or haplotypes) of the present invention, or the encoded product of the nucleic acid {e.g., CLDN14). This in turn can be used to identify agents or compounds that inhibit or alter the undesired activity or expression of the encoded nucleic acid product. Assays for performing such experiments can be performed in cell-based systems or in ' cell-free systems, as known to the skilled person. Cell-based systems include cells naturally expressing the nucleic acid molecules of interest, or recombinant cells that have been genetically modified so as to express a certain desired nucleic acid molecule. Variant gene expression in a patient can be assessed by expression of a variant-containing nucleic acid sequence (for example, a gene containing at least one variant of the present invention, which can be transcribed into RNA containing the at least one variant, and in turn translated into protein), or by altered expression of a normal/wild-type nucleic acid sequence -' due to variants affecting the level or pattern of expression of the normal transcripts, for example variants in the regulatory or control region of the gene. Assays for gene expression include direct nucleic acid assays (mRNA), assays for expressed protein levels, or assays of collateral compounds involved in a pathway, for example a signal pathway. Furthermore, the expression of genes that are up- or down-regulated in response to the signal pathway can also be assayed.,. One embodiment includes operably linking a reporter gene, such as luciferase, to the regulatory region of the gene(s) of interest.
Modulators of gene expression can in one embodiment be identified when a cell is contacted with a candidate compound or agent, and the expression of mRNA is determined. The expression level of mRNA in the presence of the candidate compound or agent is compared to the expression level in the absence of the compound or agent. Based on this comparison, candidate compounds or agents can be identified as those modulating the gene expression of the variant gene. When expression of mRNA or the encoded protein is statistically significantly greater in the presence of the candidate compound or agent than in its absence, then the candidate compound or agent is identified as a stimulator or up-regulator of expression of the nucleic acid. When nucleic acid expression or protein level is statistically significantly less in the presence of . „ the candidate compound or agent than in its absence, then the candidate compound is identified, as an inhibitor or down-regulator of the nucleic acid expression.
The invention further provides methods of treatment using a compound identified through drug (compound and/or agent) screening as a gene modulator (i.e. stimulator and/or inhibitor of gene expression).
Methods of assessing probability of response to therapeutic agents, methods of monitoring progress of treatment and methods of treatment
Most kidney stones pass spontaneously - i.e., those 4mm or less (~90% of kidney stones); on the other hand, stones larger than 6mm require some sort of intervention. Stone passage may be encouraged by increased hydration, medication for treating infection and reducing pain, and , diuretics to encourage urine flow and prevent further stone formation.
Pain management usually requires intravenous administration of narcotics (such as morphine, ] codeine and derivatives) in emergency rooms for acute situations; similar classes of drugs may also be effective when adminstered orally for less sever pain. This includes nonsteroidal antiinflammatory agents and narcotics, such as codeine or hydrocodone. Sometimes, patients are given alpha adrenergic blocking agents, such as Flomax (active compound (R)-5-(2-(2-(2- ethoxyphenoxy)ethylamino)propyl)-2-methoxybenzenesulfonamide), Uroxatral (Alfuzosin, active compound /V-[3-[(4-amino-6,7-dimethoxy-quinazolin-2-yl)- methyl-amino]propyl] tetrahydrofuran- 2-carboxamide), Terazosin (Hytrin, active compound [4-(4-amino-6,7- dimethoxy-quinazolin-2-yl) piperazin-1-yl]- tetrahydrofuran-2-yl-methanone) or Doxazosin (brand names Cardura/Carduran, active compound [4-(4-amino-6,7-dimethoxy- quinazolin-2-yl) piperazin-1-yl]- (2,5-dioxabicyclo[4.4.0] deca-6,8, 10-trien-4-yl) methanone), which act to reduct the muscle tone of the ureter and facilitate stone passage. For smaller stones, especially those near the bladder, such medication can increase spontaneous passage rate.
Urologic intervention may be required when pain is persistent and severe, in renal failure and when there is kidney infection. Most common intervention is Extracorporeal Shock Wave Lithotripsy (ESWL). Sometimes, invasive procedures are required, including ureteroscopic fragmentation, using laser, ultrasonic or mechanical (shock wave, pneumatic) energy to fragment larger stones. Percutaneous nephrolithotomy or open surgery may be required for large or complicated stones or stones which fail other less invasive treatments.
As is known in the art, individuals can have differential responses to a particular therapy (e.g., a therapeutic agent or therapeutic method). Pharmacogenomics addresses the issue of how genetic variations (e.g., the variants (markers and/or haplotypes) of the present invention) affect drug response, due to altered drug disposition and/or abnormal or altered action of the drug. Thus, the basis of the differential response may be genetically determined in part. Clinical outcomes due to genetic variations affecting drug response may result in toxicity of the drug in certain individuals (e.g., carriers or non-carriers of the genetic variants of the present invention), or therapeutic failure of the drug. Therefore, the variants of the present invention may determine the manner in which a therapeutic agent and/or method acts on the body, or the way in which the body metabolizes the therapeutic agent.
Accordingly, in one embodiment, the presence of a particular allele at a polymorphic site as described herein is indicative of a different response, e.g. a different response rate, to a particular treatment modality for kidney stones. This means that a patient who presents with symptoms indicative of kidney stones, and carrying a certain at-risk markers for kidney stones as described herein would respond better to, or worse to, a specific therapeutic, drug and/or other therapy used to treat the kidney stones. Therefore, the presence or absence of the marker allele (or haplotype) could aid in deciding what treatment is appropriate for the patient. For example, for a newly diagnosed patient, the presence of a marker or haplotype of the present invention may be assessed (e.g. , through testing DNA derived from a biological sample, or from genotype data, including sequence data, from the individual). If the patient is positive for a marker allele or haplotype (that is, at least one specific allele of the marker, or haplotype, is present), then the physician recommends one particular therapy, while if the patient is negativeι for the at least one allele of a marker, or a haplotype, then a different course of therapy may be recommended. For example, it is contemplated that certain patients carrying at-risk markers for kidney stones as described herein are more likely to present with severe form of kidney stones. For such individuals, aggressive administration of appropriate therapeutic intervention, as described in the above, may be appropriate, rather than a more common wait-and-see approach. Thus, the patient's carrier status could be used to help determine which particular treatment modality should be administered. The value lies within the possibilities of being able to determine or predict the likely severity of the kidney stones in the individual at an early stage, to select the most appropriate treatment, and provide information to the clinician about prognosis/aggressiveness of the kidney stones in order to be able to apply the most appropriate treatment. Appropriate medication may also be administered for at-risk variant carriers.
The present invention also relates to methods of monitoring progress or effectiveness of a treatment for kidney stones. This can be done based on the genotype and/or haplotype status of the markers and haplotypes of the present invention, i.e., by assessing the absence or ', presence of at least one allele of at least one polymorphic marker as disclosed herein, or by monitoring expression of genes that are associated with the variants (markers and haplotypes) of the present invention (e.g., the CLDN14 gene). The risk gene mRNA or the encoded polypeptide can be measured in a tissue sample (e.g. , a peripheral blood sample, or a biopsy sample). Expression levels and/or mRNA levels can thus be determined before and during treatment to monitor its effectiveness.
Alternatively, biological networks or metabolic pathways related to the markers and haplotypes of the present invention can be monitored by determining mRNA and/or polypeptide levels. This, can be done for example, by monitoring expression levels or polypeptides for several genes belonging to the network and/or pathway, in samples taken before and during treatment. Alternatively, metabolites belonging to the biological network or metabolic pathway can be determined before and during treatment. Effectiveness of the treatment is determined by comparing observed changes in expression levels/metabolite levels during treatment to corresponding data from healthy subjects.
It may also be beπefitial to be able to predict prognosis of individuals presenting with symptoms associated with kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density, and/or osteoporosis. It is contemplated that the variants described herein may be useful for such a purpose. Thus, by assaying for one or more of these variants, using any of the methods described herein, a prognosis of the individual may be determined. In certain embodiments, at-risk variants for one or more of the above-mentioned conditions is predictive of a worse prognosis of the condition.
In a further aspect, the markers of the present invention can be used to increase power and effectiveness of clinical trials. Thus, individuals who are carriers of at least one at-risk variant ^ for kidney stones or related conditions may be more likely to respond favorably to a particular treatment modality. In one embodiment, individuals who carry at-risk variants for gene(s) in a pathway and/or metabolic network (e.g., CLDNl 4-associated genes) for which a particular treatment (e.g., small molecule drug) is targeting, are more likely to be responders to the treatment. In another embodiment, individuals who carry at-risk variants for a gene, which ; expression and/or function is altered by the at-risk variant (e.g., CLDN 14), are more likely to be responders to a treatment modality targeting that gene, its expression or its gene product. This application can improve the safety of clinical trials, but can also enhance the chance that a clinical trial will demonstrate statistically significant efficacy, which may be limited to a certain sub-group of the population. Thus, one possible outcome of such a trial is that carriers of certain genetic variants, e.g., the markers and haplotypes of the present invention, are statistically significantly likely to show positive response to the therapeutic agent, i.e. experience alleviation of symptoms, or prevention of symptoms, associated with kidney stones, or experience less severe symptoms (e.g., small stones) or no symptoms (prophylactic therapy, preventlnςi or ! minimizing kidney stone formation) when taking the therapeutic agent or drug as prescribed.
In a further aspect, the markers and haplotypes of the present invention can be used for targeting the selection of pharmaceutical agents for specific individuals. Personalized selection of treatment modalities, lifestyle changes, e.g., dietary changes, or combination of lifestyle changes and administration of particular treatment, can be realized by the utilization of the at-risk variants of the present invention. Dietary changes or modifications may include one or more of- increased water consumption, low protein, nitrogen and sodium diet, low oxalate diet {e.g., low' chocolate, nuts, soybean, rhubarb, spinach consumption), reduced cola beverage diet, avoidance of large doses of vitamin C, and supplements that include thiazides, potassium citrate, magnesium citrate, and/or allopurinol. Thus, the knowledge of an individual's status for particular markers of the present invention, can be useful for selection of treatment options, which may also be combined with dietary changes or modifications. Certain combinations of variants may be suitable for one selection of treatment options (e.g, prophylactic therapy or dietary changes), while other gene variant combinations may target other treatment options. In certain embodiments, combinations of genetic variants may be assessed, including one variant,' two variants, three variants, or four or more variants, as needed to determine with clinically reliable accuracy the selection of treatment module.
Computer-implemented aspects
As understood by those of ordinary skill in the art, the methods and information described hereib may be implemented, in all or in part, as computer executable instructions on known computer ' readable media. For example, the methods described herein may be implemented in hardware/ Alternatively, the method may be implemented in software stored in, for example, one or more memories or other computer readable medium and implemented on one or more processors. As is known, the processors may be associated with one or more controllers, calculation units and/or other units of a computer system, or implanted in firmware as desired. If implemented in software, the routines may be stored in any computer readable memory such as in RAM, P.OM, flash memory, a magnetic disk, a laser disk, or other storage medium, as is also known. Likewise, this software may be delivered to a computing device via any known delivery method including, for example, over a communication channel such as a telephone line, the Internet, a - wireless connection, etc., or via a transportable medium, such as a computer readable disk, flash drive, etc.
More generally, and as understood by those of ordinary skill in the art, the various steps described above may be implemented as various blocks, operations, tools, modules and techniques which, in turn, may be implemented in hardware, firmware, software, or any combination of hardware, firmware, and/or software. When implemented in hardware, some or all of the blocks, operations, techniques, etc. may be implemented in, for example, a custom integrated circuit (IC), an application specific integrated circuit (ASIC), a field programmable logic array (FPGA), a programmable logic array (PLA), etc. :
When implemented in software, the software may be stored in any known computer readable ' medium such as on a magnetic disk, an optical disk, or other storage medium, in a RAM or ROM or flash memory of a computer, processor, hard disk drive, optical disk drive, tape drive, etc. Likewise, the software may be delivered to a user or a computing system via any known delivery method including, for example, on a computer readable disk or other transportable computer storage mechanism.
Fig. 3 illustrates an example of a suitable computing system environment 100 on which a system for the steps of the claimed method and apparatus may be implemented. The computing system environment 100 is only one example of a suitable computing environment and is not intended to suggest any limitation as to the scope of use or functionality of the method or apparatus of '! the claims. Neither should the computing environment 100 be interpreted as having any dependency or requirement relating to any one or combination of components illustrated in the exemplary operating environment 100. < '■'
The steps of the claimed method and system are operational with numerous other general purpose or special purpose computing system environments or configurations. Examples of well known computing systems, environments, and/or configurations that may be suitable for use with the methods or system of the claims include, but are not limited to, personal computers, server computers, hand-held or laptop devices, multiprocessor systems, microprocessor-based systems, set top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, distributed computing environments that include any of the above systems or devices, and the like.
The steps of the claimed method and system may be described in the general context of computer-executable instructions, such as program modules, being executed by a computer. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. The methods and apparatus may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network. In both integrated and distributed computing environments, program modules may be located in both local and remote computer storage media including memory storage devices. '
With reference to Fig. 3, an exemplary system for implementing the steps of the claimed method and system includes a general purpose computing device in the form of a computer 110. Components of computer 110 may include, but are not limited to, a processing unit 120, a system memory 130, and a system bus 121 that couples various system components including the system memory to the processing unit 120. The system bus 121 may be any of several types of bus structures including a memory bus or memory controller, a peripheral bus, and a ,, local bus using any of a variety of bus architectures. By way of example, and not limitation, such architectures include Industry Standard Architecture (ISA) bus, Micro Channel Architecture (MCA) bus, Enhanced ISA (EISA) bus, Video Electronics Standards Association (VESA) local bus, and Peripheral Component Interconnect (PCI) bus also known as Mezzanine bus.
Computer 110 typically includes a variety of computer readable media. Computer readable media can be any available media that can be accessed by computer 110 and includes both volatile and nonvolatile media, removable and non-removable media. By way of example, and not limitation, computer readable media may comprise computer storage media and communication media. Computer storage media includes both volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data. Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage - devices, or any other medium which can be used to store the desired information and which can accessed by computer 110. Communication media typically embodies computer readable instructions, data structures, program modules or other data in a modulated data signal such as a carrier wave or other transport mechanism and includes any information delivery media. The - term "modulated data signal" means a signal that has one or more of its characteristics set or ' changed in such a manner as to encode information in the signal. By way of example, and not limitation, communication media includes wired media such as a wired network or direct-wired ' connection, and wireless media such as acoustic, RF, infrared and other wireless media. Combinations of the any of the above should also be included within the scope of computer readable media.
The system memory 130 includes computer storage media in the form of volatile and/or nonvolatile memory such as read only memory (ROM) 131 and random access memory (RAM) 132. A basic input/output system 133 (BIOS), containing the basic routines that help to transfer information between elements within computer 110, such as during start-up, is typically stored ., in ROM 131. RAM 132 typically contains data and/or program modules that are immediately accessible to and/or presently being operated on by processing unit 120. By way of example, , and not limitation, Fig. 3 illustrates operating system 134, application programs 135, other program modules 136, and program data 137.
The computer 110 may also include other removable/non-removable, volatile/nonvolatile computer storage media. By way of example only, Fig. 3 illustrates a hard disk drive 140 that ' reads from or writes to non-removable, nonvolatile magnetic media, a magnetic disk drive 151 that reads from or writes to a removable, nonvolatile magnetic disk 152, and an optical disk drive 155 that reads from or writes to a removable, nonvolatile optical disk 156 such as a CD ROM or other optical media. Other removable/non-removable, volatile/nonvolatile computer storage media that can be used in the exemplary operating environment include, but are not limited to, magnetic tape cassettes, flash memory cards, digital versatile disks, digital video tape, solid state RAM, solid state ROM, and the like. The hard disk drive 141 is typically connected to the system bus 121 through a non-removable memory interface such as interface 140, and magnetic disk drive 151 and optical disk drive 155 are typically connected to the system bus 121 by a removable memory interface, such as interface 150.
The drives and their associated computer storage media discussed above and illustrated in Fig. 3, provide storage of computer readable instructions, data structures, program modules and other data for the computer 110. In Fig. 3, for example, hard disk drive 141 is illustrated as storing operating system 144, application programs 145, other program modules 146, and program data 147. Note that these components can either be the same as or different from operating system 134, application programs 135, other program modules 136, and program data 137. Operating system 144, application programs 145, other program modules 146, and program data 147 are given different numbers here to illustrate that, at a minimum, they are different copies. A user may enter commands and information into the computer 20 through input devices such as a keyboard 162 and pointing device 161, commonly referred to as a mouse, trackball or touch pad. Other input devices (not shown) may include a microphone, joystick, game pad, satellite dish, scanner, or the like. These and other input devices are often connected to the processing unit 120 through a user input interface 160 that is coupled to the system bus, but may be connected by other interface and bus structures, such as a parallel port, game port or a universal serial bus (USB). A monitor 191 or other type of display device is also connected to the system bus 121 via an interface, such as a video interface 190. In addition to" the monitor, computers may also include other peripheral output devices such as speakers 197 and printer 196, which may be connected through an output peripheral interface 190.
The computer 110 may operate in a networked environment using logical connections to one or more remote computers, such as a remote computer 180. The remote computer 180 may be a personal computer, a server, a router, a network PC, a peer device or other common network f. node, and typically includes many or all of the elements described above relative to the computer 110, although only a memory storage device 181 has been illustrated in Fig. 3. The logical connections depicted in Fig. 3 include a local area network (LAN) 171 and a wide area network (WAN) 173, but may also include other networks. Such networking environments are commonplace in offices, enterprise-wide computer networks, intranets and the Internet.
When used in a LAN networking environment, the computer 110 is connected to the LAN 171 through a network interface or adapter 170. When used in a WAN networking environment, the computer 110 typically includes a modem 172 or other means for establishing communications over the WAN 173, such as the Internet. The modem 172, which may be internal or external, may be connected to the system bus 121 via the user input interface 160, or other appropriate mechanism. In a networked environment, program modules depicted relative to the computer 110, or portions thereof, may be stored in the remote memory storage device. By way of example, and not limitation, Fig. 3 illustrates remote application programs 185 as residing on memory device 181. It will be appreciated that the network connections shown are exemplary and other means of establishing a communications link between the computers may be used.
Although the forgoing text sets forth a detailed description of numerous different embodiments of the invention, it should be understood that the scope of the invention is defined by the words of the claims set forth at the end of this patent. The detailed description is to be construed a;; exemplary only and does not describe every possibly embodiment of the invention because describing every possible embodiment would be impractical, if not impossible. Numerous alternative embodiments could be implemented, using either current technology or technology developed after the filing date of this patent, which would still fall within the scope of the claims defining the invention.
While the risk evaluation system and method, and other elements, have been described as preferably being implemented in software, they may be implemented in hardware, firmware, etc., and may be implemented by any other processor. Thus, the elements described herein may be implemented in a standard multi-purpose CPU or on specifically designed hardware or ; firmware such as an application-specific integrated circuit (ASIC) or other hard-wired device as desired, including, but not limited to, the computer 110 of Fig. 3. When implemented in software, the software routine may be stored in any computer readable memory such as on a magnetic disk, a laser disk, or other storage medium, in a RAM or ROM of a computer or processor, in any database, etc. Likewise, this software may be delivered to a user or a diagnostic system via any known or desired delivery method including, for example, on a computer readable disk or other transportable computer storage mechanism or over a communication channel such as a telephone line, the internet, wireless communication, etc. (which are viewed as being the same as or interchangeable with providing such software via a transportable storage medium).
Thus, many modifications and variations may be made in the techniques and structures described and illustrated herein without departing from the spirit and scope of the present invention. Thus, it should be understood that the methods and apparatus described herein are illustrative only and are not limiting upon the scope of the invention. Accordingly, the invention relates to computer-implemented applications using the polymorphic markers and haplotypes described herein, and genotype and/or kidney stones-association data (or association data for related conditions of kidney stones) derived therefrom. Such applications can be useful for storing, manipulating or otherwise analyzing genotype data that is useful in the methods of the invention. One example pertains to storing genotype information derived from an individual on readable media, so as to be able to provide the genotype information to a third party (e.g., the individual, a guardian of the individual, a health care provider or genetic analysis service provider), or for deriving information from the genotype data, e.g. , by comparing the genotype data to information about genetic risk factors contributing to increased susceptibility to the kidney stones and related conditions, and reporting results based on such comparison.
In general terms, computer-readable media has capabilities of storing (ι) identifer information for at least one polymorphic marker or a haplotype, as described herein; (ii) an indicator of the frequency of at least one allele of said at least one marker, or the frequency of a haplotype, in individuals with the condition; and an indicator of the frequency of at least one allele of said at least one marker, or the frequency of a haplotype, in a reference population. The reference population can be a kidney stones-free population of individuals. Alternatively, the reference population is a random sample from the general population, and is thus representative of the population at large. The frequency indicator may be a calculated frequency, a count of alleles , and/or haplotype copies, or normalized or otherwise manipulated values of the actual frequencies that are suitable for the particular medium.
The markers and haplotypes described herein to be associated with increased susceptibility (e.g. , increased risk) of kidney stones and related conditions are in certain embodiments useful for interpretation and/or analysis of genotype data. Thus in certain embodiments, an identification of an at-risk allele for at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, as shown herein, or an allele at a polymorphic marker in LD , with any one of such markers, is indicative of the individual from whom the genotype data originates is at increased risk of the condition. In one such embodiment, genotype data may be generated for at least one polymorphic marker shown herein to be associated with the condition, or a marker in linkage disequilibrium therewith. The genotype data is subsequently made available to a third party, such as the individual from whom the data originates, his/her guardian or representative, a physician or health care worker, genetic counselor, or insurance agent, for example via a user interface accessable over the internet, together with an interpretation of the genotype data, e.g., in the form of a risk measure (such as an absolute risk (AR), risk ratio (RR) or odds ratio (OR)) for the kidney stones. In another embodiment, at-risk markers identified in a genotype dataset derived from an individual are assessed and results from the assessment of the risk conferred by the presence of such at-risk varians in the dataset are made available to .; the third party, for example via a secure web interface, or by other communication means. The results of such risk assessment can be reported in numeric form (e.g., by risk values, such as absolute risk, relative risk, and/or an odds ratio, or by a percentage increase in risk compared with a reference), by graphical means, or by other means suitable to illustrate the risk to the individual from whom the genotype data is derived.
Nucleic acids and polypeptides
The nucleic acids and polypeptides described herein can be used in methods and kits of the present invention. An "isolated" nucleic acid molecule, as used herein, is one that is separated from nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA library). For example, an isolated nucleic acid of the invention can be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix. In other circumstances, the material can be purified to essential homogeneity, for example as determined by polyacrylamide gel electrophoresis (PAGE) or column chromatography (e.g., HPLC). An isolated nucleic acid molecule of the invention can comprise at least about 50%, at least about 80% or at least about 90% (on a molar basis) of all macromolecular species present. With regard to genomic DNA, the term "isolated" also can refer to nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated. For example, the isolated nucleic acid molecule can contain less than about 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of the nucleotides that flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived.
The nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated. Thus, recombinant DNA contained in a vector is included in the definition of "isolated" as used herein. Also, isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells or heterologous organisms, as well as partially or substantially purified DNA molecules in solution. "Isolated" nucleic acid molecules also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the present invention. An isolated nucleic acid molecule or nucleotide sequence can include a nucleic acid molecule or . nucleotide sequence that is synthesized chemically or by recombinant means. Such isolated nucleotide sequences are useful, for example, in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g. , from other mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the gene in tissue (e.g., human tissue), such as by Northern blot analysis or other hybridization techniques.
The invention also pertains to nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein {e.g. , nucleic acid molecules that specifically hybridize to a nucleotide sequence containing a polymorphic site associated with a marker or haplotype described herein). Such nucleic acid molecules can be detected and/or isolated by allele- or sequence-specific hybridization (e.g., under high stringency conditions). Stringency conditions and methods for nucleic acid hybridizations are well known to the skilled person (see, e.g. , Current Protocols in Molecular Biology, Ausubel, F. et al, John Wiley & Sons, (1998), and Kraus, M. and Aaronson, S'., Methods Enzymol., 200:546-556 (1991), the entire teachings of which are incorporated by reference herein.
The percent identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison purposes {e.g., gaps can be introduced in the sequence of a first sequence). The nucleotides or amino acids at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions x 100). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, of the length of the reference sequence. The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A non-limiting example of such a mathematical algorithm is described in Karlin, S. ■ and Altschul, S., Proc. Natl. Acad. Sci. USA, 90:5873-5877 ( 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0), as described in Altschul, S. et al., Nucleic Acids Res., 25: 3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g. , NBLAST) can be used. See the website on the world wide web at ncbi.nlm.nih.gov. In one embodiment, parameters for sequence comparison can be set at score= 100, wordlength = 12, or can be varied (e.g., W=5 or W=20). Another example of an algorithm is BLAT (Kent, WJ. Genome Res. 12:656-64 (2002)).
Other examples include the algorithm of Myers and Miller, CABIOS (1989), ADVANCE and ADAM, as described in Torellis, A. and Robotti, C, Comput Appl. Biosci. 10:3-5 (1994); and FASTA described in Pearson, W. and Lipman, D., Proc. Natl. Acad. Sci. USA, 85:2444-48 (1988).
In another embodiment, the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software package (Accelrys, Cambridge, UK). .,
The present invention also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleic acid that comprises, or consists of, the nucleotide sequence of LD Block C21, or a nucleotide sequence comprising, or consisting of, the complement of the nucleotide sequence of LD Block C21, wherein the nucleotide sequence comprises at least one polymorphic allele contained in the markers and haplotypes described herein. The nucleic acid fragments of the invention are at least about 15, , at least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200, 500, 1000, 10,000 or more nucleotides in length. The nucleic acid fragments of the invention are used as probes or primers in assays such as those described herein. "Probes" or "primers" are oligonucleotides that hybridize in a base- specific manner to a complementary strand of a nucleic acid molecule. In addition to DNA and . RNA, such probes and primers include polypeptide nucleic acids (PNA), as described in Nielsen, P. et al., Science 254: 1497-1500 (1991). A probe or primer comprises a region of nucleotide sequence that hybridizes to at least about 15, typically about 20-25, and in certain embodiments about 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule. In one embodiment, the probe or primer comprises at least one allele of at least one polymorphic marker or at least one haplotype described herein, or the complement thereof. In particular embodiments, a probe or primer can comprise 100 or fewer nucleotides; for example, in certain embodiments from 6 to 50 nucleotides, or, for example, from 12 to 30 nucleotides. In other embodiments, the probe or primer is at least 70% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical, to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence. In another embodiment, the probe or primer is capable, of selectively hybridizing to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence. Often, the probe or primer further comprises a label, e.g., a . radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.
The nucleic acid molecules of the invention, such as those described above, can be identified and isolated using standard molecular biology techniques well known to the skilled person. The amplified DNA can be labeled (e.g. , radiolabeled, fluorescently labeled) and used as a probe for screening a cDNA library derived from human cells. The cDNA can be derived from mRNA and contained in a suitable vector. Corresponding clones can be isolated, DNA obtained following in vivo excision, and the cloned insert can be sequenced in either or both orientations by art- recognized methods to identify the correct reading frame encoding a polypeptide of the (i appropriate molecular weight. Using these or similar methods, the polypeptide and the DNA encoding the polypeptide can be isolated, sequenced and further characterized.
Antibodies
The invention also provides antibodies which bind to an epitope comprising either a variant amino acid sequence (e.g., comprising an amino acid substitution) encoded by a variant allele or the reference amino acid sequence encoded by the corresponding non-variant or wild-type allele. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. , molecules that contain antigen-binding sites ' that specifically bind an antigen. A molecule that specifically binds to a polypeptide of the invention is a molecule that binds to that polypeptide or a fragment thereof, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind to a polypeptide of the invention. The term "monoclonal antibody" or "monoclonal antibody ' composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of a polypeptide of the invention. A monoclonal antibody composition thus typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.
Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a desired immunogen, e.g., polypeptide of the invention or a fragment thereof. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the' antibody molecules directed against the polypeptide can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein, Nature 256:495-497 ( 1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72 (1983)), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,1985, Inc., pp. . 77-96) or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology (1994) Coligan et al., (eds.) John Wiley & Sons, Inc., New York, NY). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a polypeptide of the invention.
Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating a monoclonal antibody to a polypeptide of the invention (see, e.g., Current Protocols in Immunology, supra; Galfre et al., Nature 266: 55052 (1977); R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); and Lerner, Yale J. Biol. Med. 54:387-402 (1981)). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods that also would be useful.
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody to a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g. , an antibody phage display library) with the polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene . Su/fZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can , be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al., Bio/Technology 9: 1370-1372 (1991); Hay et al. , Hum. Antibod. Hybridomas 3:81-85 ( 1992); Huse et al., Science 246: 1275-1281 (1989); and Griffiths et al., EMBO J. 12:725-734 (1993).
Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, . comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.
In general, antibodies of the invention (e.g., a monoclonal antibody) can be used to isolate a polypeptide of the invention by standard techniques, such as affinity chromatography or immunoprecipitation. A polypeptide-specific antibody can facilitate the purification of natural polypeptide from cells and of recombinantly produced polypeptide expressed in host cells. Moreover, an antibody specific for a polypeptide of the invention can be used to detect the polypeptide (e.g., in a cellular lysate, cell supernatant, or tissue sample) in order to evaluate the abundance and pattern of expression of the polypeptide. Antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, - determine the efficacy of a given treatment regimen. The antibody can be coupled to a detectable substance to facilitate its detection. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; , examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;' an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.
Antibodies may also be useful in pharmacogenomic analysis. In such embodiments, antibodies against variant proteins encoded by nucleic acids according to the invention, such as variant proteins that are encoded by nucleic acids that contain at least one polymorpic marker of the invention, can be used to identify individuals that require modified treatment modalities.
Antibodies can furthermore be useful for assessing expression of variant proteins in in an individual with a predisposition to condition related to the function of the protein, in particular a condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis. In one embodiment, the protein is a human CLDN 14 protein. Antibodies specific for a variant protein that is encoded by a nucleic acid that comprises at least one polymorphic marker or haplotype as described herein can be used to screen for the presence of the variant protein, for example to screen for a predisposition to the condition as indicated by the presence of the variant protein.
Antibodies can be used in other methods. Thus, antibodies are useful as diagnostic tools for J evaluating proteins, such as variant proteins of the invention, in conjunction with analysis by electrophoretic mobility, isoelectric point, tryptic or other protease digest, or for use in other physical assays known to those skilled in the art. Antibodies may also be used in tissue typing. In one such embodiment, a specific variant protein has been correlated with expression in a specific tissue type, and antibodies specific for the variant protein can then be used to identify the specific tissue type.
Subcellular localization of proteins, including variant proteins, can also be determined using antibodies, and can be applied to assess aberrant subcellular localization of the protein in cells in various tissues. Such use can be applied in genetic testing, but also in monitoring a particular treatment modality. In the case where treatment is aimed at correcting the expression level or presence of the variant protein or aberrant tissue distribution or developmental expression of the variant protein, antibodies specific for the variant protein or fragments thereof can be used to monitor therapeutic efficacy.
Antibodies are further useful for inhibiting variant protein function, for example by blocking the binding of a variant protein to a binding molecule or partner. Such uses can also be applied in a therapeutic context in which treatment involves inhibiting a variant protein's function. An antibody can be for example be used to block or competitively inhibit binding, thereby modulating (i.e., agonizing or antagonizing) the activity of the protein. Antibodies can be prepared against specific protein fragments containing sites required for specific function or against an intact protein that is associated with a cell or cell membrane. For administration in . vivo, an antibody may be linked with an additional therapeutic payload, such as radionuclide, an enzyme, an immunogenic epitope, or a cytotoxic agent, including bacterial toxins (diphtheria or plant toxins, such as ricin). The in vivo half-life of an antibody or a fragment thereof may be ■ increased by pegylation through conjugation to polyethylene glycol.
The present invention further relates to kits for using antibodies in the methods described herein. This includes, but is not limited to, kits for detecting the presence of a variant protein in a test sample. One preferred embodiment comprises antibodies such as a labelled or labelable antibody and a compound or agent for detecting variant proteins in a biological sample, means for determining the amount or the presence and/or absence of variant protein in the sample, and means for comparing the amount of variant protein in the sample with a standard, as well as instructions for use of the kit. The present invention will now be exemplified by the following non-limiting example.
EXEMPLIFICATION
Sequence variants in the CLDN14 gene associate with kidney stones, serum bicarbonate and osteoporosis
With the aim of identifying sequence variants that confer risk of formation of kidney stones, we conducted a genome-wide association study using the Illumina HumanHap300 and HumanHapCIW370 bead chips. After quality filtering, 303,120 SNPs were tested individually for association with radiopaque kidney stones in a sample of 1,507 Icelandic patients and 34,033 Icelandic population controls. The results were adjusted for relatedness between individuals and potential population stratification using the method of genomic control by dividing the chi-square statistic with 1.078.
Methods
Subjects from Iceland
Kidney Stone Cohort
The Icelandic kidney stone cases consisted of patients with confirmed radiopaque kidney stones, diagnosed in the years 1983-2003, from the Icelandic Kidney Stone Registry at Landspitali University Hospital, Reykjavik, Iceland. The study was approved by the Icelandic Data Protection Authority and the National Bioethics Committee of Iceland.
Controls
The 34,033 Icelandic controls used in the genome-wide association study and the 4,726 controls used in the replication study, were selected among individuals that had participated in various genetic programs at deCODE genetics. Individuals that reported a history of kidney stones were excluded from the control set. The breakdown of the controls into the various genetic programs was approximately as follows: tremor (360), preeclampsia (780), endometriosis (330), psoriasis (850), type 2 diabetes (1450), Atzheimers disease (700), osteoarthritis (3800), schizophrenia (550), peripheral artery disease ( 1450), abdominal aortic aneurysm (380), chronic obstructive pulmonary disease (860), stroke (1830), osteoporosis (3230), coronary artery disease (4040), hypertension (2500), asthma (1470), Parkinson 's disease (320), sleep apnea (650), age-related macular degeneration (620), polycystic ovary syndrome (1380), rheumatoid arthritis (760), lung cancer (280), longevity (1580), benign prostatic hyperplasia (860), enuresis (890), migraine ; (1280), glaucoma (620), attention deficit hyperactivity disorder (560), prostate cancer (1280), . infectious diseases (3420), anxiety ( 1180), expression studies (870), autism (160), dyslexia (870), melanoma (550), colorectal cancer (890), deep vein thrombosis (950), restless leg syndrome (560), studies on addiction (3300), population controls (1180). Note that some of the controls have participated in more than one genetic program in which case their genotypes are only included once.
Study on Bone and Mineral Health Cohort
These individuals participated in a cross-sectional study on bone and mineral health in a random' sample of community-dwelling adults aged 30 to 85 years, in the Reykjavik area in Iceland (Gudmundsdottir, S. L., er a/. J Clin Densitom 8:80 (2005)). All subjects had hip (total hip; combined values at the femoral neck, trochanter and intertrochanter region) and lumbar spine bone density measured by dual energy X-ray absorptiometry (DEXA, Hologic QDR4500A), gave blood and urine samples, and answered a thorough questionnaire on medications and medical history. Subjects with diseases or taking medication known to affect bone tissue were exluded from the analysis. The National Bioethics Committee of Iceland approved the study, and all participants provided a written consent form.
Bone Density Samples
The Icelandic bone density sample-set comprises individuals who had bone density measurements (DEXA, Hologic QDR4500A) at the lumbar spine (L2-L4) and the hip (total hip) and participated in the osteoporosis genetics program or other genetic programs at deCODE genetics. All participants gave written informed consent and the study was approved by the Icelandic Data Protection Authority and the National Bioethics Committee of Iceland.
Subjects from the Netherlands
The Dutch patients with kidney stones were recruited from two sources: The outpatient clinics of the Radboud University Nijmegen Medical Center (RUNMC) and The Nijmegen Biomedical Study. All patients who present to the outpatient clinics of the RUNMC are invited to participate in a study on the effects of genes and lifestyle on the development of urological diseases. In case of consent, the patients fill out a lifestyle questionnaire and donate a blood sample for DNA isolation.
The details of the IMijmegen Biomedical Study were reported previously (Wetzels, J. F, et al. Kidney Int 72:632 (2007)). Briefly, this is a population based survey conducted by the Department of Epidemiology and Biostatistics and the Department of Clinical Chemistry of the •' Radboud University Nijmegen Medical Center (RUNMC), in which 9,371 individuals participated from a total of 22,500 age and sex stratified, randomly selected inhabitants of Nijmegen. The invited subjects were invited to participate in a study on gene-environment interactions in multifactorial diseases. All participants filled out a questionnaire on lifestyle and medical history' at baseline and 6500 of them donated blood samples for DNA isolation and biochemical studies. ' The controls for the study were also taken from the biobaπk of the Nijmegen Biomedical Study. All patients and controls were of self-reported European descent and were fully informed about the goals and the procedures of the study.
The study protocols for the recruitment of patients from outpatient clinics and the recruitment of participants to the Nijmegen Biomedical Study were approved by the Institutional Review Board of the RUNMC. All study subjects gave written informed consent.
Subjects from Denmark
The Danish samples were derived from the Prospective Epidemiological Risk Factor (PERF Study) (Bagger , Y. Z., βt al. Osteoporosis Int 17:471 (2006)). These are postmenopausal women (n = 3,884), in the age group 55-86 years, who participated in a prospective epidemiological study and in various clinical trials for osteoporosis at the Center for Clinical and Basic Research, Copenhagen. Baseline DEXA bone density measurement (Hologic QDR2000) at the hip (total hip) and lumbar spine (L2-L4) was used. The study was approved by the Ethics Committee of Copenhagen County and was in accordance with the principles of the Helsinki Declaration.
Illumina Genome-Wide Genotyping
All Icelandic case and control samples were assayed with the Infinium HumanHap300 or HumanHap370 SNP chips (Illumina, SanDiego, CA, USA), containing 317,503 and 370,404 haplotype tagging SNPs derived from phase I of the International HapMap project. Only SNPs present on both chips were included in the analysis and SNPs were excluded if they had (a) yield lower than 95% in cases or controls, (b) minor allele frequency less than 1% in the population, ' or (c) showed significant deviation from Hardy-Weinberg equilibrium in the controls (P < 0.001). Any samples with a call rate below 98% were excluded from the analysis. The final analysis included 303,120 SNPs.
Single SNP Genotyping
Single SNP genotyping for all samples was carried out at deCODE genetics in Reykjavik, Iceland, applying the same platform to all populations studied. All single SNP genotyping was carried out using the Centaurus (Nanogen) platform (Kutyavin, I.V., et al. Nucleic Acids Res 34:el28 (2006)). The quality of each Centaurus SNP assay was evaluated by genotyping each assay on ' the CEU samples and comparing the results with the HapMap data, all assays had mismatch rate <0.5%. Additionally, all markers were re-genotyped on more than 10% of samples typed with the Illumina platform resulting in an observed mismatch in less than <0.5% of samples.
Association Analysis
For association analysis we utilized a standard likelihood ratio statistic, implemented in the NEMO software (Gretarsdottir, S., et al., Nat Genet 35: 131 (2003)) to calculate two-sided P values and odds ratios (ORs) for each individual allele, assuming a multiplicative model for risk, i.e. that the risk of the two alleles a person carries multiplies (Rice, J. A., Mathematical Statistics and Data Analysis, Wadsmorth Inc., Belmont CA (1995)). Allelic frequencies, rather than carrier frequencies are presented for the markers and P values are given after adjustment for the relatedness of the subjects. When estimating genotype specific OR, genotype frequencies in the population were estimated assuming Hardy-Wemberg equilibrium.
In general, allele and haplotype frequencies are estimated by maximum likelihood and tests of differences between cases and controls are performed using a generalized likelihood ratio test. This method is particularly useful in situations where there are some missing genotypes for the < marker of interest and genotypes of another marker, which is in strong LD with the marker of interest, are used to provide some partial information. This was used in the association tests, to ensure that the comparison of the highly correlated markers was done using the same number of individuals. To handle uncertainties with phase and missing genotypes, maximum likelihood estimates, likelihood ratios and P values are computed directly for the observed data, and hence the loss of information due to uncertainty in phase and missing genotypes is automatically . captured by the likelihood ratios.
Results from multiple case-control groups were combined using a Mantel-Haenszel model (Mantel, N. Haenszel, W., J Natl Cancer Inst 22: 719 (1959)) in which the groups were allowed • to have different population frequencies for alleles, haplotypes and genotypes but were assumed to have common relative risks. The correlation between the genetic variants and the various biochemical traits was done by regressing the individual trait values on the number of copies of the at-risk variant an individual carries. To obtain effect estimates the regression was done using unadjusted trait values as response variables, while to estimate the significance of the correlation the regression was done using sex and age adjusted and standardized (by an inverse normal transform) trait values as response values to eliminate the effect of non-normality of the distribution of trait values. For ■ the regression between bone mineral density and the genetic variants, both the effect estimate and the P values are calculated by regressing standardized age, sex and weight adjusted trait values on the number of copies of the risk variant an individual carries.
Correction for Related ness of the Subjects and Genomic Control
Some of the individuals in both the Icelandic patient and control groups are related to each other, causing the chi-square test statistic to have a mean >1 and median >0.675. We estimated the inflation factor for the genome-wide association by calculating the average of the, 303,120 chi-square statistics, which was a method of genomic control (Devlin, B. Roeder, K. Biometrics 55:997 (1999)) to adjust for both relatedness and potential population stratification. The inflation factor was estimated as 1.078 and the results presented from the genome-wide association and in Tables 2 and 5 are based on adjusting the chi-square statistics by dividing each of them by 1.078. To adjust the association results for the Icelandic replication sample set^ and the combined replication and discovery sample set, where association results for a genome- wide set of SNPs is not available, we used a previously described procedure where we simulated genotypes through the genealogy of 708,683 Icelanders to estimate the adjustment factor (Stefansson, H., et al. Nat Genet 37: 129 (2005)). The adjustment factors for the replication and combined set of kidney stone cases and controls were 1.043 and 1.069, respectively.
The adjustment factors for relatedness for the various biochemical traits were estimated by simulations as described above and ranged from 1 (no adjustment) to 1.030 depending on the trait in question. Likewise, for the adjustment factors for correlation with BMD were 1.165 (1.188) for hip (spine) for females, 1 (1.023) for hip (spine) for males, and 1.168 (1.198) of hip (spine) for the sexes combined.
Sequencing of CLDN14
We resequenced the exons of the two CLDN14 Refseqs (NM_012130 and NM_144492) as well as the predicted exons of two GenBank registered human mRNAs (AJ566765 and AJ566766) and the sequence flanking the exons in 371 Icelandic cases and 270 Icelandic controls. Sequencing assays (ranging in amplimer size from 305 to 550 bp) were designed for each of the exons using NCBI assembly build 36 and the Winseq program (developed at deCODE genetics based on the Primer3 software (Rozen, S. & Skaltesky, H. Methods MoI Biol 132:365 (2000)). The 5μl PCR amplification reactions were set up on the Zymark SciClone ALH 300 robotic workstation in a 384 well PCR plate and amplified on a MJR Tetrad (Trademark of MJ. Research, Inc., Watertown, MA). Ampure (Agencourt) 384 PCR filters were used to remove unincorporated PCR primers and mononucleotides from the PCR reaction. l
Only the last exon of CLDN14 is translated. We found several, mostly rare, non-coding and intronic sequence variants as well as some synonymous and nonsynonymous coding variants (see Table 6). Two rare nonsynonymous variants caused the amino acid changes T4M (Uyguner, O., et at. Clin Genet 64:65 (2003)) and A163V. Of the protein coding exonic variants only the synonymous rs219780 (T229T) and rs219779 (R81R) were sufficiently frequent to use in our analysis. s"
,ι
Accession Numbers. Exons for sequencing were based on the Genbank Accession IDs for CLDN14 and are NM_144492, NM_012130 and AJ566765 and AJ566766. The position of the amino acids within CLDN 14 protein was based on Genpept Accession ID NP_036262.
Biochemistry
Fasting morning blood and second morning void urine samples were collected. Serum ionized calcium and serum bicarbonate were measured with an ion selective electrode (ABL 700, Radiometer Denmark); serum alkaline phosphatase (ALP) and urinary calcium and creatinine with a dry chemistry autoanalyzer (Vitros, Rochester, MN, USA); intact serum parathyroid hormone and serum CTx were measured using ECLIA (ElectroChemiLuminscence Immuno Assay, Elecsys 2010, Roche Diagnostics, Germany); Serum 25(OH)D (RIA; DiaSorin, USA) and serum cystatin C with PEIA (Particle-Enhanced Immunoturbidometric Assay; DAKO, Denmark).
Overall, two highly correlated SNPs (^ = I based on the HapMap CEPH Utah data) achieved genome-wide significance in the Icelandic discovery samples (P < 1.6χ lO"7, Figure 1 and Table 5), allele C of rs219781 with odds ratio (OR) = 1.30 and P = 3.2χlO 8 and allele T of rs219778 with OR = 1.30 and P = 3.2χlO'8. The at-risk alleles of the two variants are very common with a population frequency of 75% in the Icelandic control set. After adjusting for the association with rs219781, neither rs219778 nor any other SNP in the region showed significant association with kidney stones (Table 5). The two SNPs are located in the same linkage disequilibrium (LD) block, 2,020 bases apart on chromosome 21q22.13, on either side of the last exon of claudin 14 (CLDN14) (Figure 2), a gene involved in regulation of paracellular permeability at epithelial tight junctions ((Angelow S, er a/., Am J Physiol Renal Physiol 2008;295: F867-76)). In an attempt to replicate this observation, we typed the two variants in additional 1,520 Icelandic kidney stone patients and 4,726 Icelandic controls, and in 746 kidney stone patients , and 3,751 controls from the Netherlands. The T allele of rs219778 was significantly associated with kidney stones in both the Icelandic (P = 1.2χlO"s and OR = 1.25) and the Dutch (P = 0.015 and OR = 1.18) replication sets. The combined effect of rs219778-T in the discovery and the two replication sets was OR = 1.23 (95% CI: 1.16-1.31) with a corresponding P value of 1.7x10 12. Similar results were obtained for rs219781-C (OR = 1.23 (95% CI: 1.16-1.30) and P = 4.0x10" 12) (Table 2).
To search for causal variants, we resequenced the exons of the CLDN14 gene, and their immediate flanking regions in 371 kidney stone cases and 270 controls (see Methods), and found several non-coding and intronic sequence variants as well as some synonymous and nonsynonymous coding variants (Table 6). Of these, two common synonymous SNPs, rs219779 (R81R) and rs219780 (T229T), both located in the last exon of CLDN14, showed significant association with kidney stones. To further evaluate the effect of these variants, their genotypes were imputed for the discovery set, and the two variants directly genotyped in all replication samples (Methods). Through this effort, the two two variants showed very significant association with kidney stones, OR = 1.23 (95% CI: 1.16-1.31) and P = 1.7x10 12 for rs219779 and OR = 1.25 (95% CI: 1.17-1.33) and P = 4. OxIO"12 for rs219780 in the combined analysis of all sample sets (Table 2). Both rs219779 and rs219780 are highly correlated with the associated variants rs219778 and rs219781 {r2 = 1 and 0.77 between rs219778 and rs219779, and rs219780, respectively). The strong correlation between the four common variants and their similar effect in both the Icelandic and the Dutch sample sets did not allow for any further dissection of the signal (See conditional analysis in Table 7). Relative to noncarriers, trie genotype-specific OR for kidney stones is 1.33 and 1.64 for heterozygous and homozygous carriers of rs219780, respectively, and 1.27 and 1.55 for heterozygous and homozygous carriers of rs219779 (Table 8). !, y
The CLDN14 gene is one of 24 members of the claudin family of membrane proteins that , regulate paracellular passage of ions and small solutes at epithelial tight junctions (Krause G, et al., Biochim Biophys Acta 2008; 1778: 631-45). The specific distribution and function of the various claudins is considered an important determinant of the paracellular transport properties., of different epithelia (Krause G, et al., Biochim Biophys Acta 2008;1778:631-45). A renal disorder, autosomal recessive familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), is caused by mutations in the claudin 16 (CLDN16) and claudin 19 (CLDN19) genes, ; , both expressed in the loop of Henle ((Angelow S, et al., Am J Physiol Renal Physiol 2008;295: F867-76);Simon, D. B., et al., Science 285: 103 (1999); Konrad, M., et al. Am J Hum: - Genet 79:949 (2006)). Kidney stone formation is a common finding in FHHNC. The gene M associating with kidney stones in our study, CLDN14, is also expressed in the kidney, both in the loop of Henle and the proximal convoluted tubule, as well as in the epithelia of several other .; organs, and has been observed to selectively decrease cation paracellular permeability (Elkouby,- Naor, L, et al., Cell Tissue Res 333:427 (2008); Ben-Yosef, T., et al., Hum MoI Genet 79:949 (2006)). Mutations in CLDN14 have been found in families with nonsyndromic autosomal recessive deafness but hitherto neither kidney dysfunction nor kidney stone formation has been described in humans or mice with such mutations (Wilcox, E. R., et al. Hum MoI Genet 12:2049 -, (2003). In claudin 11/claudin 14 doubly mutant mice a mild enhancement in urine excretion of magnesium and calcium was observed (Elkouby-Naor, L., et al., Cell Tissue Res 333:427 (2008)), suggesting a role for claudin 14 in renal calcium excretion in mice. f.
In order to identify the biochemical pathway(s) that the kidney stone associated variants could be involved in, we tested the correlation between rs219779-C and rs219780-C and several biochemical values related to calcium metabolism (Table 3). The correlation was tested in a set of 1,026 population based individuals who had participated in a study of bone and mineral health in Icelandic men and women (Gudmundsdottir, S. L, et al., J CHn Densitom 8:80 (2005)). Both SNPs showed significant association with serum levels of bicarbonate, urinary calcium concentration, and serum parathyroid hormone (PTH). Each copy of the risk variant rs219779-C corresponded to a decrease in serum bicarbonate of 0.24 mEq/L (P = 0.013), an increase in urinary calcium concentration of 0.17 mmol/L (P = 0.038), and to an increase in PTH of 2.46 pg/ml (P = 0.005). The corresponding values for rs219780-C were -0.25 mEq/L (P = 0.0087), 0.27 mmol/L (P = 0.005), and 2.48 pg/ml (P = 0.0072).
The association of kidney stones with bone mineral density has been extensively explored and the overall data suggests a risk for bone loss in individuals with kidney stones, primarily those with hypercalciuria, possibly associated with enhanced bone turnover (Asplin, J. R., et al., Kidney Int 63:662 (2003)). We therefore assessed the association between the two variants, rs219779- C and rs219780-C, and bone mineral density (BMD) at the hip and spine in a sample of 8,450 Icelandic individuals and 3,601 Danish females. Both SNPs associated significantly with reduced bone density at the hip (total hip) and lumbar spine (L2-L4) in the Icelandic females (Table 4). While neither was significant, the effect on spine BMD in men was similar to that observed in women, whereas, the effect on hip BMD in men, although small, was in the opposite direction. In the Danish female samples the association was in the same direction but non-significant. In the combined analysis of the two sample sets each copy of the risk variants corresponded to -'a decrease in hip BMD of an estimated 0.063 (P = 0.000039) and 0.060 (P = 0.00039) standard deviations (SD) for rs219779-C and rs219780-C, respectively, and a decrease in spine BMD of 0.052 SD (P = 0.001) and 0.057 SD (P = 0.0077) for rs219779-C and rs219780-C, respectively. We repeated the analysis for the 8,450 Icelanders with BMD measurements by comparing individuals with and without kidney stones. Interestingly, the effect of rs219779-C on BMD both at the hip and spine was greater among kidney stone formers (Table S5). This effect was also seen for rs219780-C at the hip but not at the spine. However, the association of rs219779-C and rs219780-C with BMD remained significant after excluding kidney stone cases, suggesting that the effect of the variants on BMD is not through enrichment of kidney stone cases among individuals with low BMD. ,: We and others have previously reported on a number of sequence variants that affect BMD ((Styrkarsdottir, U. et al., N Engl J Med 358:2355 (2008); Richards, J. B. et al., Lancet 371 : 1505 (2008)). To further examine the relationship between kidney stones and BMD observed in the current study, we assessed the correlation of previously reported BMD-related variants with kidney stones. As shown in Table 10, of the 12 BMD-related variants we tested, 10 showed ήb association with kidney stones while two variants showed nominally significant association. Furthermore, we did not observe consistent association between the 12 variants and urinary calcium concentration, serum bicarbonate, and parathyroid hormone levels (Table 11). These findings support the view that the observed effect on both kidney stones and BMD in our study, is specifically mediated through the variants in CLDN14 as opposed to reflecting a more general relationship between these two phenotypes. £
We have shown that common variants in the gene CLDN14 associate with kidney stone formation. As these variants are common in the population, their population attributable risk (PAR) is substantial, or 27-30%. Furthermore, the alleles that confer risk of kidney stones correlate with lower serum bicarbonate levels, higher urinary calcium concentration, and reduced BMD. We postulate that the primary abnormalities associating with the variants in CLDN14 are decreased serum bicarbonate and increased urinary calcium concentration, and that kidney stones and decreased BMD may be caused by these metabolic abnormalities. Metabolic acidoses is known to both increase the release of calcium from bone (Lemann, Jr., J. et al. Am J Physiol, Renal Physiol 285: F811 (2003)), and reduce their reabsorption in the kidney tubules, causing hypercalciuria (Moe, O. W. Lancet 367:333 (2006)), and thus contributing to both decreased BM.D and kidney stone formation. Additionally, metabolic acidosis causes hypocitraturia, another important risk factor for calcium stones (Moe, O. W. Lancet 367:333 (2006)). As previously discussed, hypercalciuria is recognized as the most common metabolic risk factor for kidney stone disease, and reduced BMD has been linked to both kidney stones and hypercalciuria.
Several previous studies have suggested a role for claudins in acid-base homeostasis. For example, CLDN8 which functions as a negative regulator of paracellular transport of cations, similar to CLDN14, also appears to act specifically as a negative regulator of protons, bicarbonate and ammonium ions in the distal nephron (Angelow, S. et al., J Physiol 571 : 1,5 (2006)). The association between CLDN14 and serum bicarbonate level in our study suggestsia role for CLDN14 in acid-base balance, possibly directly through regulation of paracellular ion transport. CLDN14 has been found to be expressed in the proximal tubule of the kidney where the reclamation of the bulk of filtered bicarbonate occurs as well as synthesis of ammonium, a major component of renal acid excretion. ■;.
Whether the association between the CLDN14 variants and urinary calcium concentration is direct or indirect is unclear. It is, however, well recognized that acid-base balance and electrolyte transport, including that of calcium, are intricately coupled in the kidney. In conclusion, variants in the gene CLDN14 associate with kidney stones and reduced BMD. Further research is required to clarify the biological pathways interconnecting these phenotypes, CLDN14, and the associated metabolic abnormalities.
Table 2. Association between kidney stones and the four variants, rs219778-T, rs219781-C, rs219780-C and rs219779-C, all located in or close to the CLDN14 gene on chromosome 21q22.. Results are shown for the Icelandic discovery data set, an Icelandic replication dataset, the two Icelandic datasets combined, a replication dataset from the Netherlands, and for all the datasets combined using a Mantel-Haenzel model. Shown are the number of controls (/Vc) and cases (NA) for each study group, the frequency in controls and in cases, the OR with 95% confidence intervals (CI), and P values assuming the multiplicative model. For the Icelandic study groups, , the P values and CI were adjusted for relatedness as described in the method section.
Cohort (NcINx) Frequency
Variant AT Controls Cases OR (95% CI) P
Iceland I (34033/1507) rs219778T 0.754 0.799 1296(1.183-1.421) 3.2xlO"8 rs219781 C 0.754 0.799 1.297(1.183-1.422) 3.2x10'8 rs219780C 0.791 0.834 1329(1.201-1.469) 3.4x10"8 rs219779C 0.754 0.799 1.301 (1.185-1.428) 3.3xlO"8
Iceland II (4726/1520) rs219778T 0.744 0784 1248(1.130-1.378) 12x105 rs219781 C 0.744 0.783 1244(1.127-1.373) 1.5xlO"5 rs219780C 0.786 0.821 1.251 (1.122-1.394) 5.5x1 O5 rs219779C 0.743 0.784 1.256(1.137-1.386) 6.6x10-*
Iceland Combined (38759/3027) rs219778T 0.752 0.791 1.248(1.169-1.331) 2.7x10"" rs219781 C 0.752 0.791 1244(1.166-1.328) 45x10"" rs219780C 0.790 0.827 1272(1.185-1365) 2.4x10"" rs219779C 0.752 0.791 1.251 (1.172-1.336) 1.7x10""
The Netherlands (3751 /746) rs219778T 0.750 0.779 1.178(1.033-1.344) 0.015 rs219781 C 0.751 0.779 1.170(1.025-1.336) 0.02 rs219780C 0.794 0.818 1.167(1.014-1.344) 0.032 rs219779C 0.749 0.777 1 168(1.024-1.331) 0.02
Combined (42510/3773) rs219778T 1.234(1.164-1.308) l.7xlθ"12 rs219781 C 1.230(1.160-1.303) 4.OxIO"12 rs219780C 1.250(1.174-1.332) 4.OxIO"12 rs219779C 1.234(1.164-1.308) 1.7xlO"12
Table 3. Correlation between the two CLDN14 exonic variants that associate with kidney stones, rs219780-C and rs219779-C, and various biochemical measurements related to calcium metabolism. Correlation was tested in a population based set of 1,026 Icelanders. The effect and the corresponding standard error of the mean (s.e.m.) are obtained by regressing the unadjusted trait values on the number of risk allele and individual carries. The l value was : calculated by regressing the sex and age adjusted and invers normal transformed trait values of the number or risk allele carried. All P values and standard errors of the mean were adjusted for. relatedness by simulations as described in the method section.
rs219779-C rs219780-C ,.
Trait Effect (s.e.m.) P Effect (s.e.m.) P
Serum Albumin (mg/1) 0.10 (0.15) 0.42 0.12 (0.16) 0.48
Serum Alkaline phosphotase (Total) (U/L) 2.81 (2.31 ) 0.098 4.10 (2.45) 0.038
Serum Bicarbonate (mEq/L) -0.24 (0.1 1) 0.013 -0.25 (0.12) 0.0087
Serum Calcium (mmol/L) 0.0046 (0.0047) 0.38 0.0058 (0.0050) 0.33
Serum Creatinine (micromol/L) -0.55 (0.87) 0.49 -0.51 (0.93) 0.39
Serum CTx (U/L) 0.017 (0.01 1) 0.098 0.016 (0.012) 0.038
Serum Cystacin C (mg/L) 0.0044 (0.0131) 0.49 0.0034 (0.0139) 0.51
Serum Ionized Calcium (mmol/L) 0.00061 (0.00205) 0.79 0.0018 (0.0022) 0.44
Serum Magnesium (mmol/L) -0.0060 (0.0036) 0.2 -0.0061 (0.0039) 0.16
Serum Parathyroid Hormone - ELECS (pg/ml) 2.46 (0.96) 0.005 2.48 ( 1.03) 0.0072
Serum pH value -0.00095 (0.00175) 0.55 0.00025 (0.00187) 0.89
Serum Phosphate (mmol/L) 0.0037 (0.0083) 0.5 -0.0010 (0.0088) 0.98
Serum 1.25-(OH)2-D VitaminD (mmol/L) 1.96 (2.92) 0.59 0.61 (3.1 1 ) 0.91 '
Serum 25-hydroxy vitamin D (nmol/L) -0.47 ( 1.00) 0.61 - 1.45 (1.06) 0.19
Urine Calcium (mmol/L) 0.17 (0.09) 0.038 0.27 (0.10) 0.005
Urine Creatinine (mmol/L) -0.027 (0.301 ) 0.66 0.20 (0.32) 0.8
Table 4. Correlation between the two CLDN14 exonic variants that associate with kidney stones, rs219780-C and rs219779-C, and BMD. The correlation is tested in a set of 8,450 Icelandic individuals of both sexes and 3,601 Danish females. The table shows the correlation with sex, age, and weight adjusted values of BMD for hip and spine separately, and for the Icelandic sample set for males, females and both sexes combined. Shown are the estimated effects expressed as standardized values per copy of the SNP allele, the corresponding standard errors of the mean (s.e.m.) and P values. For the Icelandic sample set, both the standard errors of the mean and P values have been adjusted for relatedness of the study individuals using simulations. Also shown is the result for the two sample sets combined.
rs219779-C rs219780-C
Dataset («) Effect (s.e.m.) P Effect (s.e.m.) P
Iceland
Hip
All (8219) -0.074 (0.019) 0.000083 -0.070 (0.021 ) 0.00071
Males (1418) 0.002 (0.046) 0.97 0.029 (0.049) 0.57
Females (6801) -0.091 (0.021) 0.000013 -0.091 (0.022) 0.000062
Spine
All (8446) -0.069 (0.020) 0.00043 -0 066 (0.021 ) 0.002
Males (1437) -0.062 (0.051 ) 0.23 -0 075 (0.055) 0.17
Females (7009) -0.071 (0.021 ) 0.00088 -0.064 (0.023) 0.0055
Denmark
Hip
Females (3562) -0.042 (0.027) 0.12 -0.041 (0.029) 0.17
Spine
Females (3599) -0.021 (0.027) 0.45 -0.009 (0.029) 0.78
Combined
Hip
AU (1 1781) -0.063 (0.015) 0.000039 -0.060 (0.017) 0.00039
Females (10363) -0.073 (0.016) 0.00001 1 -0.073 (0.018) 0.000058
Spine
AU (12045) -0.052 (0.016) 0.001 -0.057 (0.022) 0.0077
Females (10608) -0.052 (0.017) 0.002 -0.055 (0.023) 0.019
Table 5. Results for 34 SNPs on the Illumina HumanHap300 and HumanHapCNV370 bead chips tested for association to kidney stones in the interval 36.65 to 36.85 Mb on chromosome 21q22 (NCBI build 36) in the GWA study. The results are based on 1,507 kidney stone cases and 34,033 controls from the Icelandic discovery case-control set. Also included are the corresponding results after adjusting for the observed association with the SNP rs219781 (indicated in bold).
Frequency Adjusted for rs219781
SNP Allele Position Cases Controls OR P ORadj Pad, rs218626 T 36674428 0.394 0.390 1.02 0.65 1.04 0.38 rs218628 T 36683748 0.395 0.381 1.06 0.14 0.99 0.85 rs2835341 G 36686531 0.221 0.219 1.01 0.86 1.09 0.072 rs1906483 G 36689527 0.221 0.219 1.01 0.84 1.09 0.066 rs190068 T 36703322 0.933 0.915 1.29 0.00053 1.09 0.34 rs218631 A 36706400 0.849 0.849 1.00 0.99 0.89 0.052 rs2835342 G 36707073 0.908 0.898 1.12 0.10 0.92 0.27 rs8596 G 36709276 0.893 0.892 1.00 0.96 0.93 0.29 rs218634 C 36715317 0.900 0.870 135 14χ10"6 1.17 0.050 rs218637 T 36720196 0.530 0.517 1.05 0.17 1.04 0.34 rs2835346 A 36725999 0.332 0.309 1.11 0.011 1.03 0.49 rs9981659 A 36728248 0.177 0.167 1.07 0.16 1.02 0.68 rs2835358 A 36738049 0.479 0.457 1.09 0.020 1.00 0.91 rs4378885 T 36745307 0.316 0.309 1.03 0.43 0.96 0.34 rs219781 C 36753017 0.799 0.754 1.30 3.2X10"8 - - rs128494 T 36754654 0.271 0.260 1.06 0.18 0.98 0.61 rs219778 T 36755037 0.799 0.754 1.30 3.2X10"8 1.00 0.80 rs219761 C 36759806 0.841 0.810 1.24 0.000025 1.09 0.15 rs219756 T 36762864 0.719 0.711 1.04 0.34 1.01 0.78 rs170183 T 36768730 0.557 0.517 1.17 0.000043 1.08 0.070 rs2249115 A 36769635 0.860 0.838 1.19 0.0011 1.06 0.35 rs219746 C 36770423 0.735 0.722 1.07 0.14 1.04 0.32 rs219745 T 36770539 0.735 0.722 1.07 0.14 1.04 0.31 rs2835365 T 36785735 0.650 0.620 1.14 0.0013 1.09 0.050 rs2850114 C 36786486 0.788 0.786 1.01 0.79 1.05 0.30 rs2071049 T 36786876 0.670 0.668 1.01 0.81 1.05 0.28 rs730265 A 36792282 0.179 0.176 1.02 0.70 1.03 0.55 rs2000546 T 36792843 0.618 0.613 1.02 0.63 1.01 0.78 rs2835368 T 36804933 0.117 0.111 1.05 0.38 1.05 0.43 rs2835370 C 36806021 0.088 0.087 1.00 0.96 1.00 0.96 rs2835380 A 36814485 0.056 0.052 1.06 0.48 1.07 0.45 rs8134973 A 36820524 0.747 0.745 1.01 0.84 1.00 0.98 πs2835389 G 36833861 0.394 0.384 1.05 0.27 1.04 0.27 rs448849 C 36847822 0.114 0.103 1.12 0.072 1.09 0.18 Table 6. Variants identified by sequencing the exons of CLDN14 and their flanking regions in 371 kidney stone cases and 270 controls. The table includes the location (in IMCBI Build 36) of the identified variant, the corresponding rs-name if known, the minor allele and its frequency in controls and cases, the allelic OR and P value for association with kidney stone formation, the location of the variant within the gene region and the amino acid change where applicable. All P values have been adjusted for the relatedness of the cases and control by dividing the corresponding chi-square statistic by 1.023 (determined by simulations as described in Methods).
Plus Strand Minus Strand Frequency
Position Minor Minor Position of Variant Amino Acid Change NCBI36 SNP " IUPAC Allele IUPAC Allele Controls Cases OR P within the Gene g (NP_036262)
36754598 novel Y T R A 0.0075 0.0148 1 99 0 22 3' downstream
36754652 novel R A Y T 0.0149 00123 0.82 0 7 3' downstream
36755072 novel M A K T exon 2 of c * 'and 3 of " - 3' UTR
36755134 novel S G S C 0.0056 0.0056 0.99 099 exon 2 of c " and 3 of " - 3' UTR
36755165 rs219780 Y T R A 0.2252 0.1423 0.57 000019 exon 2 of c " and 3 of " ACG(Thr)->ACA(Thr) - T229T
36755376 novel R A Y T 0.0019 0 0 0 19 exon 2 of c * ' and 3 of * GCC(Ala)->GTC(Val) - A163V
36755621 rs219779 R A Y T 0.2519 0.1883 0 69 00077 exon 2 of " * ' and 3 of " CGC(Arg)->CGT(Arg;ι ■ R81R
36755762 novel Y T R A 0.0019 0.0014 0.73 083 exon 2 of ' * ' and 3 of ' GCG(Ala)->GCA(Ala) - A34A
36755801 novel * Y T R A 0 0185 0.0337 1.85 0 1 exon 2 of c " and 3 of " ACG(ThI )->ACT(Thr) - T21 T
36755849 novel R A Y T 0 0.0014 lnf 03 exon 2 of ° " ' and 3 of * GCC(Ala>->GCT(Ala) - A5A
36755853 novel " R A Y T 0.0167 0 0148 0.88 079 exon 2 of " ' ' and 3 of " ACG(Thr)->ATG(Met) - T4M
36755921 novel R A Y T 0.0037 0 0 007 exon 2 of c " 'and 3 of d - 5' UTR
36755938 novel Y T R A 0 0018 0.0014 0.72 082 exon 2 of c " 'and 3 of " - 5' UTR
36760291 rs219763 R G Y C 0.2626 0.2799 1.09 055 intronic
36760588 novel R G Y C 0 0.0027 lnf 0 14 intronic/exon 1 of c - 5' UTR
36771436 novel Y T R A 0.0396 0.031 0 78 0.41 intronic
36771488 novel Y T R A 00279 0.0459 1.68 0.095 intronic
36771498 novel R A Y T 0.0288 0.0301 1.04 0.9 intronic
36771639 novel Y T R A 0.0259 0.027 1.04 0.91 exon 2 of " - 5' UTR
36771755 rs219747 R A Y T 0.2819 0.2788 0.99 0.91 intronic
36771756 novel S C S G 0 0.0014 0 3 intronic
36771819 rs11365554 C/- del Gl- del 0.2907 0.2645 0.88 0.33 intronic
36773529 novel R A Y T 0.0186 0.0151 0.81 0.63 intronic
36773632 novel M A K T 0 0037 0 0 0066 intronic
36773651 novel Y T R A 0 00027 lnf 0 14 intronic
36773822 novel Y T R A 0013 0.004 0.31 0.08 exon 1 of " - 5' UTR
36773845 rs188733 S G S C 0.2611 0.2655 1.02 086 exon 1 of " - 5' UTR
36773871 rs219742 Y C R G 0 2611 02649 1.02 0.88 exon 1 of " - 5' UTR
36773878 novel Y T R A 0.0019 0.0041 2.19 0.48 exon 1 of " - 5' UTR
36774314 novel S G S C 0.0115 0.0151 1.32 0 59 intronic
36774449 novel Y T R A 0.0019 0 0 0 19 intronic ;
36804527 rs16994182 S G S C 0.0019 0 0 0.19 intronic
36804569 novel K G M C 0.0038 0.0055 1.47 0.66 exon 2 of * ' - 5' UTR )
36836795 ts928839 S G S C 0.0712 0.0796 1.13 0.58 intronic alslovel refers to SNPs who do not have an rs identifier in dbSNPI 29. b SNP does not have an rs identifier but has been cited in O. Uyguner ei al., CIiT Genet 64, 65 (JuI, 2003). C Refseq NM 012130 d Refseq NM_144492 e GenBank mRNA AJ566765 f GenBank mRNA AJ566766. g No vaπants found in exons 1 of e orf. Table 7. Association between kidney stones and the four SNPs typed in CLDN14. The association is calculated for the combined Icelandic and Dutch sample sets and for each SNP the association is tested conditional on the observed association to each of the other SNPs. The table includes the SNP, the tested allele and the corresponding OR and P values and the adjusted P values corresponding to an association test conditional on each of the other SNPs.
Adjusted P values
SNP Allele Position OR rs219781 rs219778 rs219780 rs219779 rs219781 C 36753017 1.230 4.0x10~12 na 0.019 0.11 0.22 rs219778 T 36755037 1.234 1.7x1012 0.0075 na 0.064 0.91 rs219780 C 36755177 1.250 4.0x10 0.098 0.16 na 0.16 rs219779 C 36755621 1.234 1.7x10 ,-12 0.068 0.65 0.059 na
Table 8. Genotype Specific Odds Ratios for the Risk Alleles of rs219780 and rs219779. Shown is the risk for heterozygous carriers (CT) and homozygous carriers (CC) of rs219780 and rs219779 compared to the risk for non-carriers (TT), together with 95% confidence intervals (CI), both for the combined Icelandic discovery and replication sample set and the sample set from the Netherlands, and for the sample sets combined using a Mantel-Haenzel model. For the Icelandic samples set the P values are adjusted for relatedness using simulations.
Cohort (NCINA) Genotype specific Odds Ratio
Variant AT TT CT (95% Cl) CC (95% Cl)
Iceland Combined (38759/3027) rs219780 1 1.24 (0.99-1.54) 1.58 (1.29-1.95) rs219779 1 1.24 (1.04-1.50) 1.56 (1.31-1.86)
The Netherlands (3751/746) rs219780 1 1.81 (1.14-2.87) 1.96 (1.24-3.08) rs219779 1 1.36 (0.96-1.93) 1.52 (1.07-2.16)
Combined (42510/3773) rs219780 1 1.33 (1.09-1.62) 1.64 (1.36-1.98) rs219779 1 1.27 (1.08-1.49) 1.55 (1.33-1.82)
Table 9. Ccorrelation of rs219780-C and rs219779-C with sex, age, and weight adjusted values of BMD for hip and spine separately for 564 Icelanders that have been diagnosed with kidney stones and in a set of 7,886 individuals without known kidney stone disease. Shown are the estimated effects expressed as standardized values per copy of the SNP allele, the corresponding standard errors of the mean (s.e.m.) and P values. Both the standard errors of the mean and P values have been adjusted for relatedness of the study individuals using simulations.
rs219779-C rs219780-C
Dataset (n) Effect (s.e.m.) P Effect (s.e.m.) P
Hip with KS (552) -0.139(0.082) 0.088 -0.096(0.089) 0.28 excl. KS (7667) -0.070(0.021) 0.00084 -0.067(0.023) 0.0038
Spine with KS (565) -0.079(0.091) 0.38 -0.029(0.099) 0.77 excl. KS (7883) -0.068(0.021) 0.0021 -0.068(0.024) 0.0047
Table 10. The association with kidney stones of 12 variants, at six distinct loci, that have previously been shown to association with variation in BMD. Results are shown for the Icelandic discovery data set of 1,507 individuals with kidney stones and 34,033 controls. Shown is the loci, the variant, the position of the variant, the tested allele, the frequency in cases and controls, the OR and the P values. For all variants the tested allele is the allele that correlates with lower bone mass density. All P values were adjusted for relatedness as described in the method section.
Frequency
Loci SNP Position Allele Controls Cases OR P
1p36.12 rs7524102 22571034 A 0.816 0.833 1.12 0.021
» rs6696981 22575445 G 0.857 0.873 1.15 0.017
6p21.32 - MHC rs3130340 32352605 T 0.786 0.790 1.02 0.62
6q25.1 - ESR1 rs9479055 151889660 C 0.349 0.358 1.04 0.33 rs4870044 151943102 T 0.281 0.290 1.05 0.30 rs 1038304 151974868 G 0.468 0.469 1.00 0.92 rs6929137 151978370 A 0.298 0.302 1.02 0.71 rs 1999805 152110057 C 0.438 0.423 0.94 0.11
8q24.12 - OPG rs6469804 120114010 A 0.511 0.505 0.98 0.57 rs6993813 120121419 C 0.495 0.489 0.98 0.53
11q13.2 - i.RP5 rs3736228 67957871 T 0.159 0.169 1.07 0.20
13q14.11 - RANKL rs9594759 41930593 T 0.625 0.638 1.06 0.17
Table 11. Correlation between 12 variants at six distinct loci, previously associated with variation in BMD, and urinary calcium, serum bicarbonate and parathyroid hormone levels. The correlation is calculated for as subset of 706 of the 1,026 individuals in the Study on Bone and Mineral Health Cohort. Shown are the loci, the variant, the position of the variant, the tested allele, and the effect (s.e.m) and P values for each of the three biochemical traits. The effect and the corresponding standard error of the mean are obtained by regressing the unadjusted trait values on the number of risk allele an individual carries. The P value was calculated by regressing the sex and age adjusted and inverse normal transformed trait values on the number of risk allele carried. For all variants the tested allele is the allele that correlates with lower bone mass density. All P values were adjusted for relatedness as described in the method section.
Seum Parathyroid
Urine Calcium Serum Bicarbonate Hormone - ELECS (mmol/L) (mEq/L)
(pg/mi)
Loci SNP Allele Effect (s.e.m) P Effect (s.e.m) P Effect (s.e.m) P
1p36.12 rs7524102 A 0.067 (0.123) 0.54 0.29 (0.15) 0.095 0.13 (1.28) 1 rs6696981 G 0.12 (0.13) 0.24 0.24 (0.17) 0.28 0.32 ( 1.40) 1
6p21.32 - MHC rs3130340 T 0.14 (0.12) 0.27 -0.16 (0.14) 0.25 -0.59 (1.21 ) 0.36
6q25.1 - ESR7 rs9479055 C -0.11 (0.10) 0.56 -0.11 (0.12) 0.35 0.11 (1 03) 0.93
» rs4870044 T -0.075 (0.106) 0.68 0.0036 (0.1253) 0.99 -0.72 (1.10) 0.33 rs1038304 G 0.12 (0.09) 0.37 -0.12 (0.11 ) 0.27 0.39 (0.97) 0.95 rs6929137 A 0.20 (0.10) 0.11 -0.064 (0.119) 0.46 -0.32 (1.03) 0.78 rs 1999805 C 0.062 (0.097) 0.45 -0.060 (0.117) 0.64 0.32 (1.00) 0.87
8q24.12 - OPG rs6469804 A 0.049 (0.092) 0.73 0.021 (0.113) 0.75 -1.08 (0.94) 0.24 rs6993813 C 0.029 (0.093) 0.81 -0.014 (0.116) 0.6 -0.75 (0.98) ,0.5
11q13.2 - LRP5 rs3736228 T -0.30 (0.14) 0.081 0.20 (0.17) 0.27 2.01 (1.43) 0.36
13q14.11 - RANKL rs9594759 T -0.069 (0.098) 0.5 -0.13 (0.11 ) 0.25 -0.53 (1.00) 0.59

Claims

1. A method of determining a susceptibility to at least one condition selected from kidney ! stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum i bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, the • ' method comprising:
obtaining nucleic acid sequence data about a human individual identifying at least one ailele of at least one polymorphic marker associated with the human CLDN14 gene, wherein different alleles of the at least one polymorphic marker are associated with different susceptibilities to the at least one condition in humans, and i determining a susceptibility to the at least one condition from the nucleic acid sequence data.
2. The method of claim 1, comprising obtaining nucleic acid sequence data about at least : two polymorphic markers associated with the CLDN 14 gene.
' 3. The method of claim 1 or claim 2, wherein determination of a susceptibility comprises comparing the nucleic acid sequence data to a database containing correlation data between polymorphic markers associated with the human CLDN14 gene and susceptibility to the at least one condition.
4. The method of claim 3, wherein the database comprises at least one risk measure of susceptibility to the at least one condition for the polymorphic markers associated with the CLDN14 gene.
5. The method of claim 3, wherein the database comprises a look-up table containing at least one risk measure of the at least one condition for the polymorphic markers.
6. The method of any one of the preceding claims, wherein obtaining nucleic acid sequence data comprises obtaining a biological sample from the human individual and analyzing sequence of the at least one polymorphic marker in nucleic acid in the sample.
7. The method of claim 6, wherein analyzing sequence of the at least one polymorphic marker comprises determining the presence or absence of at least one allele of the at least one polymorphic marker. .
8. The method of any one of claims 1-5, wherein the obtaining nucleic acid sequence data comprises obtaining nucleic acid sequence information from a preexisting record.
9. The method of any one of the preceding claims, further comprising reporting the susceptibility to at least one entity selected from the group consisting of the individual, a guardian of the individual, a genetic service provider, a physician, a medical organization, and a medical insurer.
10. The method of any one of the preceding claims, wherein the at least one polymorphic marker is selected from the group consisting of the markers rs219780, rs219778, rs219779, and . rs219781, and markers in linkage disequilibrium therewith.
11. The method of claim 10, wherein linkage disequilibrium is defined by numerical values of r2 of at least 0.2 and/or values of | D'| of at least 0.8.
* 12. The method of claim 10 or claim 11, wherein the at least one polymorphic marker is selected from the group consisting of the markers rsllO88346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364, rsl70183, rsl3051987, rsl70182, rs218638, rs218641, rs218642, rs218643, rs219759, rs219761, rs219765, rs219766, rs219767, rs219768, rs219772, rs219774, rs219782, rs219783, rs219785, rs219790, rs2244014, rs2249115, rs2633329, rs2633331, rs2633334, rs2776288, rs2835304, rs2835305, rs2835309, rs2835318, rs2835319, rs2835348, rs2835349, rs2845762', rs2845764, rs2845766, rs2845769, rs2850087, rs2850102, rs2850108, rs2850110, rs56183874, rs9975445, S.36470266, S.36472078, S.36478541, S.36487357, s.36489391, S.36492368, S.36571651, s.36598656, s.36623095, s.36644230, S.36690993, s.36691722, s.36693496, > s.36694516, s.36696258, s.36696674, s.36708810, s.36722244, s.36724712, s.36730418,
S.36730731, S.36732831, S.36733004, S.36734680, S.36738144, S.36738223, S.36738919, > s.36739280, S.36739821, s.36749630, s.36749760, S.36751378, s.36757110, s.36757852, , S.36776041, s.36777630, S.36777724, S.36777871, s.36778050, S.36780792, S.36781080, 1 S.37075430, and s.37158027.
13. The method of claim 12, wherein the at least one polymorphic marker is selected from the group consisting of rsllO88346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364 and rsl70183..
14. The method of any one of the preceding claims, wherein the at least one polymorphic marker is selected from the group consisting of rs219778, rs219779, rs219780, and rs219781."
15. A method of determining a susceptibility to at least one condition selected from kidney '■ stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, the method comprising:
obtaining nucleic acid sequence data about a human individual identifying both alleles of at least two polymorphic markers associated with the human CLDN 14 gene, determine the identity of at least one haplotype based on the sequence data, and
determining a susceptibility to the at least one condition from the haplotype data.
16. The method according to any one of the preceding Claims, wherein the at least one allele or haplotype is indicative of increased susceptibility to the condition.
17. The method according to Claim 16, wherein the increased susceptibility is characterized by a relative risk or an odds ratio of at least 1.15, including at least 1.20, at least 1.21, at least 1.22, at least 1.23, at least 1.24 and at least 1.25.
18. The method according to Claim 16 or 17, wherein the at least one allele is selected from the group consisting of rs219778 allele T, rs219779 allele C, rs219780 allele C and rs219781 s allele C.
i 19. The method of claim 18, wherein determination of homozygosity for rs219778 allele T, rs219779 allele C, rs219780 allele C and/or rs219781 allele C, or a marker allele in linkage disequilibrium therewith, is indicative of increased risk of the condition,
19. The method of any one of the Claims 1-15, wherein the presence of rs219778 allele C, rs219779 allele T, rs219780 allele A and/or rs219781 allele A is indicative of decreased susceptibility to the at least one condition. :
20. A method of diagnosing a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, the method comprising determining the presence or absence of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, or in a genotype dataset from the individual, wherein the at least one polymorphic marker is associated with the; CLDN14 gene, and wherein the presence of the at least one allele is indicative of a susceptibility to the at least one condition.
21. The method of claim 20, comprising determining both alleles of the at least one polymorphic marker.
22. The method of claim 21, further comprising determining the identity of at least one haplotype in the individual.
23. The method of any one of the claims 20 - 22, wherein the at least one polymorphic marker is selected from the group consisting of the markers rs219780, rs219778, rs219779, and rs219781, and markers in linkage disequilibrium therewith.
24. The method of any one of the claims 20 - 23, wherein the at least one polymorphic marker is selected from the group consisting of the markers rsllO88346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364, rsl70183, rsl3051987, rsl70182, rs218638, rs218641, rs218642, rs218643, rs219759, rs219761, rs219765, rs219766, rs219767, rs219768, rs219772, rs219774, rs219782, rs219783, rs219785, rs219790, rs2244014, rs2249115, rs2633329, rs2633331, rs2633334, rs2776288, rs2835304, rs2835305, rs2835309, rs2835318, rs2835319, rs2835348, rs2835349, rs2845762, rs2845764, rs2845766, rs2845769, rs2850087, rs2850102, rs2850108, rs2850110, rsb"61S3874, rs9975445, s.36470266, s.36472078, s.36478541, s.36487357, s.36489391, s.36492368, S.36571651, s.36598656, s.36623095, s.36644230, s.36690993, s.36691722, s.36693496, s.36694516, s.36696258, s.36696674, s.36708810, s.36722244, s.36724712, s.36730418, S.36730731, S.36732831, S.36733004, s.36734680, S.36738144, s.36738223, S.36738919, S.36739280, S.36739821, s.36749630, s.36749760, s.36751378, s.36757110, s.36757852,
S.36776041, s.36777630, s.36777724, s.36777871, s.36778050, s.36780792, s.36781080, , S.37075430, and S.37158027.
25. The method of any one of the claims 20 -24, wherein the at least one polymorphic marker is selected from the group consisting of rs219778, rs219779, rs219780, and rs219781.,.,
26. The method of any one of the Claims 20 - 25, wherein the at least one allele or haplotype is indicative of increased susceptibility to the condition.
27. The method of Claim 26, wherein the increased susceptibility is characterized by a relative risk or an odds ratio of at least 1.15, including at least 1.20, at least 1.21, at least 1.22, at least 1.23, at least 1.24 and at least 1.25.
28. The method of Claim 26 or 27, wherein the at least one allele is selected from the group, consisting of rs219778 allele T, rs219779 allele C, rs219780 allele C and rs219781 allele C.
29. The method of any one of the Claims 20 - 25, wherein the presence of rs219778 allele C, , rs219779 allele T, rs219780 allele T and/or rs219781 allele A is indicative of decreased susceptibility to the at least one condition.
30. The method of any one of the preceding Claims, wherein the condition is kidney stones and wherein the human individual further has at least one risk factor for kidney stones selected from the group consisting of: male sex, Caucasian ethnicity, low water consumption, hypercalciurea, high-protein diet, high-sodium diet, low-calcium diet, obesity, high blood pressure, lack of physical activity, family history of kidney stones, previous kidney stones, v vitamin A deficiency, hyperparathyroidism, kidney infection and history of gastric bypass surgery, inflammatory bowel disease or chronic diarrhea.
31. A method of diagnosing a susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, the method comprising:
obtaining CLDN14 amino acid sequence data about at least one encoded CLDN14 protein of a human individual,
identifying at least one polymorphic site associated with the CLDN 14 amino acid sequence, wherein different amino acids of the at least one polymorphic site are associated with different susceptibilities to the at least one condition in humans, and
diagnosing susceptibility to the at least one condition from the amino acid sequence data.
32. A method of determining a susceptibility of at least one condition selected from kidney ' stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, the method comprising determining the identity of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one marker is selected from the group of markers located within the LD block C21, wherein susceptibility of the at least one condition is correlated with the identity of the at least one allele.
33. The method of claim 32, comprising determining both alleles of at least one polymorphic marker.
34. The method of claim 32, further comprising assessing the identity of at least one haplotype in the individual, wherein the presence of the at least one haplotype is indicative of a- susceptibility to the at least one condition.
35. The method of any one of Claims 32 - 34, wherein the at least one polymorphic marker is selected from the group of markers listed in Table 1, and markers in linkage disequilibrium therewith.
36. The method of any one of the Claims 32 - 35, wherein the at least one polymorphic marker is selected from the group consisting of rsllO88346, rsl90068, rs2835342, rs218634, rs218639, ΓS172639, ΓS2244034, ΓS2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364 rsl70183, rsl3051987, rsl70182, rs218638, rs218641, rs218642, rs218643, rs219759, rs219761, rs219765, rs219766, rs219767, rs219768, rs219772, rs219774, rs219782, rs219783, rs219785, rs219790, rs2244014, rs2249115, rs2633329, rs2633331, rs2633334, rs2776288, rs2835304, rs2835305, rs2835309, rs2835318, rs2835319, rs2835348, rs2835349, rs2845762, rs2845764, rs2845766, rs2845769, rs2850087, rs2850102, rs2850108, rs2850110, rs56183874, rs9975445, s.36470266, s.36472078, s.36478541, s.36487357, s.36489391, s.36492368, S.36571651, S.36598656, S.36623095, S.36644230, S.36690993, S.36691722, S.36693496, S.36694516, s.36696258, s.36696674, s.36708810, s.36722244, s.36724712, s.36730418, S.36730731, s.36732831, s.36733004, s.36734680, s.36738144, s.36738223, s.36738919, S.36739280, S.36739821, S.36749630, S.36749760, S.36751378, S.36757110, S.36757852, S.36776041, s.36777630, S.36777724, s.36777871, s.36778050, s.36780792, s.36781080, S.37075430, and s.37158027. ;
37. The method of any one of the Claims 32 - 36, wherein the at least one polymorphic marker is selected from the group consisting of the markers rs219778, rs219779, rs219780, and rs219781.
38. A method of identification of a marker for use in assessing susceptibility to at least one . condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, the method comprising
d. identifying at least one polymorphism associated with the CLDN 14 gene;
e. determining the genotype status of a sample of individuals diagnosed with, or having a susceptibility to, the condition; and
f. determining the genotype status of a control sample;
wherein determination of a significant difference in frequency of at least one allele in the at least one polymorphism in individuals diagnosed with, or having a susceptibility to, the condition, as compared with the frequency of the at least one allele in the control sample is indicative of the at least one polymorphism being useful for assessing susceptibility to the condition.
39. The method of Claim 38, wherein the at least one polymorphism is in linkage disequilibrium to at least one polymorphic marker selected from the group consisting of rs219780, rs219778, rs219779, and rs219781.
40. The method of Claim 38 or 39, wherein an increase in frequency of the at least one allele in the at least one polymorphism in individuals diagnosed with, or having a susceptibility to, the condition, as compared with the frequency of the at least one allele in the control sample is indicative of the at least one polymorphism being useful for assessing increased susceptibility to the condition.
41. The method of Claim 68 or 69, wherein a decrease in frequency of the at least one allele in the at least one polymorphism in individuals diagnosed with, or having a susceptibility to, the " condition, as compared with the frequency of the at least one allele in the control sample is indicative of the at least one polymorphism being useful for assessing decreased susceptibility to, or protection against, the condition.
42. The method of any one of the preceding Claims, further comprising assessing at least one biomarker in a sample from the individual.
43. The method of Claim 42, wherein the sample is a blood sample.
44. The method of any one of the Claims 41 - 43, wherein the biomarker is CLDN 14 protein, or a fragment thereof.
45. The method of any one of the preceding Claims, further comprising analyzing non-genetic information to make overall risk assessment, diagnosis, or prognosis of the individual.
46. The method of Claim 45, wherein the non-genetic information is selected from the group consisting of low water consumption, hypercalciurea, high-protein diet, high-sodium diet, low- calcium diet, obesity, high blood pressure, lack of physical activity, family history of kidney stones, previous kidney stones, vitamin A deficiency, hyperparathyroidism, kidney infection and history of gastric bypass surgery, inflammatory bowel disease or chronic diarrhea.
47. The method of any one of the preceding Claims, further comprising analyzing expression levels of CLDN14 in a sample from the individual.
48. The method according to Claim 47, wherein the sample is a blood sample.
49. The method of Claim 47, wherein the sample is a nucleic acid sample.
50. The method of Claim 47, wherein the sample contains mRNA.
51. The method of Claim 47, wherein expression is determined by measuring CLDN 14 protein levels in the sample.
52. The method of any one of Claims 47 - 50, wherein expression is determined by measuring CLDN 14 mRNA levels in the sample.
53. A method of assessing an individual for probability of response to a therapeutic agent for preventing and/or ameliorating symptoms associated with at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum H bicarbonate levels, reduced bone mineral density and osteoporosis in a human individual, comprising: determining the identity of at least one allele of at least one polymorphic marker in a nucleic acid sample obtained from the individual, wherein the at least one polymorphic marker is selected from polymorphic markers associated with the human CLDN 14 gene, wherein determination of the identity of the at least one allele of the at least one marker is indicative of a probability of a positive response to the therapeutic agent.
54. The method of claim 53, wherein the at least one polymorphic marker is selected from the group consisting of rsllO88346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364 rsl70183, rsl3051987, rsl70182, rs218638, rs218641, rs218642, rs218643, rs219759, rs219761, rs219765, rs219766, rs219767, rs219768, rs219772, rs219774, rs219782, rs219783, rs219785, rs219790, rs2244014, rs2249115, rs2633329, rs2633331, rs2633334, rs2776288, rs2835304, rs2835305, rs2835309, rs2835318, rs2835319, rs2835348, rs2835349, rs2845762, rs2845764, rs2845766, rs2845769, rs2850087, rs2850102, rs2850108, rs2850110, rs56183874, rs9975445,
S.36470266, S.36472078, s.36478541, s.36487357, s.36489391, s.36492368, S.36571651, S.36598656, S.36623095, S.36644230, S.36690993, S.36691722, S.36693496, S.36694516, - S.36696258, s.36696674, S.36708810, s.36722244, s.36724712, s.36730418, s.36730731, S.36732831, S.36733004, s.36734680, S.36738144, s.36738223, S.36738919, s.36739280, S.36739821, S.36749630, S.36749760, S.36751378, S.36757110, S.36757852, S.36776041,
S.36777630, s.36777724, s.36777871, s.36778050, s.36780792, s.36781080, s.37075430, and S.37158027.
55. The method of claim 53 or 54, wherein the therapeutic agent is selected from the group consisting of thiazides (e.g., chlorothiazide, hydrocholorthiazide, bendroflumethiazide), potassium citrate, magnesium citrate and allopurinol.
56. A method of predicting prognosis of an individual experiencing symptoms associated r with, or an individual diagnosed with, at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, the method comprising
obtaining nucleic acid sequence data about a human individual identifying at least one allele of at least one polymorphic marker associated with the human CLDN 14 gene, wherein different alleles of the at least one polymorphic marker are associated with different susceptibilities to the at least one condition in humans, and
predicting prognosis of the individual from the nucleic acid sequence data. -
57. The method of claim 56, comprising obtaining nucleic acid sequence data about at least, two polymorphic markers associated with the CLDN 14 gene.
58. The method of claims 56 or 57, wherein the at least one polymorphic marker is selected from the group consisting of rsllO88346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364 rsl70183, rsl3051987, \[ rsl70182, rs218638, rs218641, rs218642, rs218643, rs219759, rs219761, rs219765, rs219766, rs219767, rs219768, rs219772, rs219774, rs219782, rs219783, rs219785, rs219790, rs2244014, rs2249115, rs2633329, rs2633331, rs2633334, rs2776288, rs2835304, rs2835305, rs2835309; rs2835318, rs2835319, rs2835348, rs2835349, rs2845762, rs2845764, rs2845766, TS2845769, rs2850087, rs2850102, rs2850108, rs2850110, rs56183874, rs9975445, s.36470266, s.36472078, s.36478541, s.36487357, s.36489391, s.36492368, s.36571651, " s.36598656, s.36623095, s.36644230, s.36690993, s.36691722, s.36693496, s.36694516, S.36696258, S.36696674, S.36708810, S.36722244, S.36724712, S.36730418, S.36730731, S.36732831, s.36733004, s.36734680, s.36738144, s.36738223, s.36738919, s.36739280, S.36739821, s.36749630, S.36749760, S.36751378, S.36757110, s.36757852, s.36776041, S.36777630, s.36777724, s.36777871, s.36778050, s.36780792, s.36781080, s.37075430, and s.37158027. >
59. A method of predicting treatment outcome of an individual undergoing treatment for at . least one condition selected from kidney stones, hyperca lei urea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and ' osteoporosis, the method comprising
obtaining nucleic acid sequence data about a human individual identifying at least one allele of at least one polymorphic marker associated with the human CLDN14 gene, wherein different alleles of the at least one polymorphic marker are associated with different susceptibilities to the at least one condition in humans, and
predicting treatment outcome of the individual from the nucleic acid sequence data.
60. The method of claim 59, comprising obtaining nucleic acid sequence data about at least two polymorphic markers associated with the CLDN 14 gene.
I
61. The method of claim 59 or 60, wherein the at least one polymorphic marker is selected t from the group consisting of rsllO88346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, : rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364, rsl70183, rsl3051987, rsl70182, rs218638, rs218641, rs218642, rs218643, rs219759, rs219761, rs219765, rs219766, rs219767, rs219768, rs219772, rs219774, rs219782, rs219783, rs219785, rs219790, rs2244014, rs2249115, rs2633329, rs2633331, rs2633334, rs2776288, rs2835304, rs2835305> rs2835309, rs2835318, rs2835319, rs2835348, rs2835349, rs2845762, rs2845764, rs2845766, rs2845769, rs2850087, rs2850102, rs2850108, rs2850110, rs56183874, rs9975445, s.36470266, s.36472078, s.36478541, s.36487357, s.36489391, s.36492368, s.36571651, S.36598656, S.36623095, S.36644230, S.36690993, S.36691722, s.36693496, S.36694516, s.36696258, s.36696674, s.36708810, s.36722244, s.36724712, s.36730418, s.36730731, S.36732831, s.36733004, s.36734680, s.36738144, s.36738223, s.36738919, s.36739280, S.36739821, S.36749630, S.36749760, S.36751378, S.36757110, S.36757852, S.36776041, i S.36777630, s.36777724, s.36777871, s.36778050, s.36780792, s.36781080, s.37075430, and S.37158027.
62. A kit for assessing susceptibility to at least one condition selected from kidney stones,, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, the kit comprising
(i) reagents for selectively detecting at least one allele of at least one polymorphic marker in the genome of the individual, wherein the at least one polymorphic marker is selected from the group consisting of rsllO88346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364 and rsl70183, and markers in linkage disequilibrium therewith, and
(ii) a collection of data comprising correlation data between the polymorphic markers assessed by the kit and susceptibility to the at least one condition.
63. The kit of Claim 62, wherein the at least one polymorphic marker is selected from the • group consisting of the group consisting of rsllO88346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364, rsl70183, rsl3051987, rsl70182, rs218638, rs218641, rs218642, rs218643, rs219759, rs219761, rs219765, rs219766, rs219767, rs219768, rs219772, rs219774, rs219782, r<,219783, rs219785, rs219790, rs2244014, rs2249115, rs2633329, rs2633331, rs2633334, rs2776288, rs2835304, rs2835305, rs2835309, rs2835318, rs2835319, rs2835348, rs2835349, rs2845762, rs2845764, rs2845766, rs2845769, rs2850087, rs2850102, rs2850108, rs2850110, rs56183874, rs9975445, s.36470266, s.36472078, s.36478541, s.36487357, s.36489391, s.36492368, S.36571651, S.36598656, s.36623095, S.36644230, S.36690993, S.36691722, S.36693496, S.36694516, s.36696258, s.36696674, s.36708810, s.36722244, S.36724712, S.36730418, S.36730731, s.36732831, s.36733004, s.36734680, S.36738144, s.36738223, s.36738919, S.36739280, S.36739821, s.36749630, s.36749760, s.36751378, S.36757110, s.36757852, s.36776041, s.36777630, s.36777724, s.36777871, s.36778050, s.36780792, s.36781080, ' s.37075430, and s.37158027.
64. The kit of claim 62 or claim 63, wherein the at least one polymorphic marker is selected from the group consisting of marker rs219778, rs219779, rs219780 and rs219781. '",
65. The kit of any one of the Claims 62 - 64, wherein the at least one allele is selected from' the group consisting of rs219778 allele T, rs219779 allele C, rs219780 allele C and rs219781 •'" allele C.
66. The kit of any one of the Claims 62 - 65, wherein the reagents comprise at least one contiguous oligonucleotide that hybridizes to a fragment of the genome of the individual comprising the at least one polymorphic marker, a buffer and a detectable label.
67. The kit of any one of the Claims 62 - 66, wherein the reagents comprise at least one pair of oligonucleotides that hybridize to opposite strands of a genomic nucleic acid segment obtained from the subject, wherein each oligonucleotide primer pair is designed to selectively amplify a fragment of the genome of the individual that includes one polymorphic marker, and wherein the fragment is at least 30 base pairs in size. !
68. The kit of claim 66 or claim 67, wherein the at least one oligonucleotide is completely complementary to the genome of the individual.
69. The kit of any one of the Claims 66 - 68, wherein the oligonucleotide is about 18 to about 50 nucleotides in length.
70. The kit of any one of the Claims 66 - 69, wherein the oligonucleotide is 20-30 nucleotides in length.
71. The kit of any one of the Claims 62 - 70, wherein the kit comprises:
a. a detection oligonucleotide probe that is from 5-100 nucleotides in length;
b. an enhancer oligonucleotide probe that is from 5-100 nucleotides in length; and
c. an endonuclease enzyme;
wherein the detection oligonucleotide probe specifically hybridizes to a first segment of the nucleic acid whose nucleotide sequence is given by SEQ ID NO: 1 that comprises at least one polymorphic site; and
wherein the detection oligonucleotide probe comprises a detectable label at its 3' terminus and a quenching moiety at its 5' terminus; and
wherein the enhancer oligonucleotide is from 5-100 nucleotides in length and is complementary to a second segment of the nucleotide sequence that is 5' relative to the oligonucleotide probe, such that the enhancer oligonucleotide is located 3' relative to the detection oligonucleotide probe when both oligonucleotides are hybridized to the nucleic acid; and
wherein a single base gap exists between the first segment and the second segment, such that" when the oligonucleotide probe and the enhancer oligonucleotide probe are both hybridized to the nucleic acid, a single base gap exists between the oligonucleotides; and wherein treating the nucleic acid with the endonuclease will cleave the detectable label from the 3' terminus of the detection probe to release free detectable label when the detection probe is hybridized to the nucleic acid.
72. Use of an oligonucleotide probe in the manufacture of a diagnostic reagent for diagnosing and/or assessing susceptibility to at least one condition selected from kidney stones, ' hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, wherein the probe is capable of selectively hybridizing to a segment of a nucleic acid whose nucleotide sequence is given by SEQ ID NO: 1 that comprises at least one polymorphic site, wherein the segment is 15-500 nucleotides in length.
73. The use according to Claim 72, wherein the polymorphic site is selected from the group consisting of:
rsllO88346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364, rsl70183, rsl3051987, rsl70182, rs218638, rs218641, rs218642, rs218643, rs219759, rs219761, rs219765, rs219766, rs219767, rs219768, rs219772, rs219774, rs219782, rs219783, rs219785, rs219790, rs2244014, rs2249115, rs2633329, rs2633331, rs2633334, rs2776288, rs2835304, rs2835305, rs2835309, rs2835318, rs2835319, rs2835348, rs2835349, rs2845762, rs2845764, rs2845766, rs2845769, rs2850087, rs2850102, rs2850108, rs2850110, rs56183874, rs9975445, s.36470266, s.36472078, S.36478541, s.36487357, s.36489391, S.36492368, s.36571651, s.36598656, s.36623095, S.36644230, S.36690993, S.36691722, S.36693496, S.36694516, S.36696258, S.36696674, • S.36708810, s.36722244, s.36724712, s.36730418, S.36730731, S.36732831, S.36733004, s.36734680, S.36738144, s.36738223, s.36738919, s.36739280, S.36739821, s.36749630, S.36749760, S.36751378, S.36757110, S.36757852, S.36776041, S.36777630, S.36777724, s.36777871, s.36778050, s.36780792, s.36781080, s.37075430, and s.37158027. ;
74. The use according to Claim 72 or claim 73, wherein the polymorphic site is selected from the group consisting of rs219778, rs219779, rs219780 and rs219781.
75. A computer-readable medium having computer executable instructions for determining " susceptibility to at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, the computer readable medium comprising: v
data indicative of at least one polymorphic marker; '! a routine stored on the computer readable medium and adapted to be executed by a processor . to determine risk of developing the at least one condition for the at least one polymorphic marker;
wherein the at least one polymorphic marker is associated with the human CLDN 14 gene.
76. The computer readable medium of claim 75, wherein the computer readable medium contains data indicative of at least two polymorphic markers.
77. The computer readable medium of claim 75 or claim 76, wherein the at least one polymorphic marker is selected from the group consisting of rsl 1088346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364, rsl70183, rsl3051987, rsl70182, rs218638, rs218641, rs218642, rs218643, rs219759, rs219761, rs219765, rs219766, rs219767, rs219768, rs219772, rs219774, rs219782, rs219783, rs219785, rs219790, rs2244014, rs2249115, rs2633329, rs2633331, rs2633334, rs2776288. rs2835304, rs2835305, rs2835309, rs2835318, rs2835319, rs2835348, rs2835349, rs2845762, rs2845764, rs2845766, rs2845769, rs2850087, rs2850102, rs2850108, rs2850110, rs56183874, rs9975445, S.36470266, s.36472078, s.36478541, s.36487357, s.36489391, s.36492368, S.36571651, S.36598656, S.36623095, S.36644230, S.36690993, S.36691722, S.36693496, S.36694516, s.36696258, s.36696674, s.36708810, s.36722244, s.36724712, s.36730418, S.36730731, S.36732831, S.36733004, s.36734680, S.36738144, s.36738223, S.36738919, S.36739280, s.36739821, s.36749630, s.36749760, S.36751378, s.36757110, s.36757852, S.36776041, s.36777630, s.36777724, s.36777871, s.36778050, s.36780792, s.36781080, S.37075430, and s.37158027.
78. The computer-readable medium of any one of Claims 75 - 77, wherein the at least one polymorphic marker is selected from the group consisting of marker rs219778, rs219779, rs219780 and rs219781.
79. The computer readable medium of any one of claims 75 - 78 , further comprising data 5 indicative of at least one haplotype comprising two or more polymorphic markers.
80. An apparatus for determining a genetic indicator in a human individual for at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, comprising:
a processor
a computer readable memory having computer executable instructions adapted to be executed on the processor to analyze marker and/or haplotype information for at least one human individual with respect to at least one polymorphic marker associated with the human CLDN 14 gene, and
generate an output based on the marker or haplotype information, wherein the output comprises a risk measure of the at least one marker or haplotype as a genetic indicator of the at least one condition for the human individual.
81. The apparatus of Claim 80, wherein the computer readable memory further comprises data indicative of the frequency of at least one allele of at least one polymorphic marker or at least one haplotype in a plurality of individuals with the condition, and data indicative of the frequency of at the least one allele of at least one polymorphic marker or at least one haplotype in a plurality of reference individuals, and wherein a risk measure is based on a comparison of ;; the at least one marker and/or haplotype status for the human individual to the data indicative of the frequency of the at least one marker and/or haplotype information for the plurality of individuals with the condition.
82. The apparatus of Claim 80, wherein the computer readable memory further comprises data indicative of the risk of developing the condition associated with at least one allele of at least one polymorphic marker or at least one haplotype, and wherein a risk measure for the human individual is based on a comparison of the at least one marker and/or haplotype status J for the human individual to the risk of the condition associated with the at least one allele of the at least one polymorphic marker or the at least one haplotype.
83. The apparatus of Claim 80, wherein the computer readable memory further comprises data indicative.of the frequency of at least one allele of at least one polymorphic marker or at least one haplotype in a plurality of individuals with the condition, and data indicative of the frequency of at the least one allele of at least one polymorphic marker or at least one haplotype in a plurality of reference individuals, and wherein risk of developing the condition is based on a comparison of the frequency of the at least one allele or haplotype in individuals diagnosed with, or presenting symptoms associated with, the condition, and reference individuals.
84. The apparatus of any one of the claims 80 - 83, wherein the at least one marker or haplotype comprises markers selected from the group consisting of rsl 1088346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364, rsl70183, rsl3051987, rsl70182, rs218638, rs218641, rs218642, rs218643, rs219759, rs219761, rs219765, rs219766, rs219767, rs219768, rs219772, rs219774, rs219782, rs219783, rs219785, rs219790, rs2244014, rs2249115, rs2633329, rs2633331, rs2633334, ■ rs2776288, rs2835304, rs2835305, rs2835309, rs2835318, rs2835319, rs2835348, rs2835349, rs2845762, rs2845764, rs2845766, rs2845769, rs2850087, rs2850102, rs2850108, rs2850110, rs56183874, rs9975445, S.36470266, S.36472078, S.36478541, S.36487357, S.36489391, s.36492368, s.36571651, s.36598656, s.36623095, s.36644230, s.36690993, s.36691722, S.36693496, s.36694516, s.36696258, s.36696674, s.36708810, s.36722244, s.36724712, S.36730418, s.36730731, s.36732831, s.36733004, s.36734680, s.36738144, s.36738223, S.36738919, S.36739280, S.36739821, S.36749630, S.36749760, S.36751378, S.36757110, s.36757852, S.36776041, s.36777630, s.36777724, s.36777871, s.36778050, s.36780792, s.36781080, S.37075430, and S.37158027.
85. The apparatus of any of the claims 80 - 84, wherein the at least one polymorphic marker is selected from the group consisting of rs219778, rs219779, rs219780 and rs219781.
86. The apparatus of any one of the Claims 80 - 85, wherein the risk measure is characterized by an Odds Ratio (OR) or a Relative Risk (RR).
87. A pharmaceutical composition for the treatment of at least one condition selected from kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, in an individual in need thereof, comprising a polypeptide encoded by a human CLDN 14 gene, or fragments thereof, and pharmacologically acceptable carriers and/or excipients.
88. A method of prophylaxis therapy of a condition selected from the group consisting of kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, comprising administering to a person in need thereof a pharmaceutical composition comprising an agent that regulates the expression, activity or physical state of the CLDN14 gene or its encoding RNA or protein in the > individual.
89. The method according to Claim 88, wherein the agent is selected from a small molecule compound, an oligonucleotide, a peptide and an antibody.
90. The method according to Claim 89, wherein said agent is an antisense molecule or interfering RNA.
91. The method according to Claim 90, wherein the antisense molecule or interfering RNA is an oligonucleotide molecule capable of hybridizing to at least a portion of the human CLDN 14 gene.
92. The method according to Claim 88, wherein said agent is an expression modifier.
93. The method according to Claim 88, wherein said agent is an activator, '
94. The method according to Claim 88, wherein said agent is a repressor.
95. A human CLDN 14 polypeptide for use as a medicament.
96. A human CLDN 14 polypeptide for the treatment of a condition selected from the group consisting of kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis. 4 o
97. An oligonucleotide capable of hybridizing to a portion of the nucleotide sequence of the human CLDN14 gene for use as a medicament.
98. A method of preventing or ameliorating symptoms associated with a condition selected from the group consisting of kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, the method comprising administering to an individual in need thereof a composition comprising a •, CLDN 14 polypeptide in a therapeutically effective amount.
99. A method of prophylaxis therapy for a condition selected from the group consisting of '■ kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, comprising :
selecting a human subject at risk for the condition;
administering to the subject a therapeutically effective amount of a composition comprising a ; therapeutic agent for the condition,
wherein the selecting comprises determining a CLDN14 variant for the human subject, and selecting for prophylaxis therapy a human subject with a CLDN14 variant that correlates with an increased risk for the condition.
100. The method of claim 99, wherein the selecting comprises determining the presence or absence of a genotype or haplotype in the CLDN 14 gene that correlates with increased risk of the condition.
101. The method of claim 100, wherein the genotype comprises at least one marker selected from rsl 1088346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, rs219771, rs219770, rs219769, rs7277076, rs2835364, rsl70183, rsl3051987, rsl70182, rs218638, rs218641, rs218642, rs218643, rs219759, rs219761, rs219765, rs219766, rs219767, rs219768, rs219772, rs219774, rs219782, rs219783, rs219785, rs219790, rs2244014, rs2249115, rs2633329, rs2633331, rs2633334, rs2776288, rs2835304, rs2835305, rs2835309> rs2835318, rs2835319, rs2835348, rs2835349, rs2845762, rs2845764, rs2845766, rs2845769, rs2850087, rs2850102, rs2850108, rs2850110, rs56183874, rs9975445, s.36470266, i
S.36472078, S.36478541, S.36487357, S.36489391, S.36492368, S.36571651, S.36598656, s.36623095, s.36644230, s.36690993, s.36691722, s.36693496, s.36694516, S.36696258, ■ S.36696674, s.36708810, s.36722244, s.36724712, s.36730418, s.36730731, s.36732831, -. s.36733004, s.36734680, s.36738144, s.36738223, s.36738919, s.36739280, s.36739821, S.36749630, s.36749760, s.36751378, s.36757110, s.36757852, s.36776041, s.36777630, s.36777724, s.36777871, s.36778050, s.36780792, s.36781080, s.37075430, and s.37158027..
102. The method of claim 100 or claim 101, wherein the selecting comprises selecting a human subject having a genotype that comprises rs219778 allele T, rs219779 allele C, rs219780 allele C and/or rs219781 allele C. >
103. The method of any one of claims 100 - 102, wherein the therapeutic agent is selected from the group consisting of thiazides (e.g., chlorothiazide, hydrocholorthiazide, bendroflumethiazide), potassium citrate, magnesium citrate and allopurinol.
104, The method of any one of the claims 100 - 103, wherein the human individual is : presymptomatic for the condition.
105. A therapeutic agent for a condition selected from the group consisting of kidney stones, ' hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis for treating a human subject with a CLDN 14 variant that correlates with an increased risk for the condition.
106. Use of a therapeutic agent for a condition selected from the group consisting of kidney stones, hypercalciurea, elevated serum parathyroid hormone levels, decreased serum bicarbonate levels, reduced bone mineral density and osteoporosis, for the manufacture of a medicament for treating the condition in a human subject with a CLDN14 variant that correlates with an increased risk of the condition.
107. The use or agent of claim 105 or 106, wherein the human subject has at least one genotype or haplotype in the CLDN 14 gene that correlates with increased risk of the ocular condition.
108. The use or agent of claim 107, wherein the genotype comprises at least one marker selected from rsl 1088346, rsl90068, rs2835342, rs218634, rs218639, rsl72639, rs2244034, rs2244237, rsl547369, rs219793, rs219791, rs219789, rs219788, rs219784, rs219781, rs219780, rs219779, rs219778, rs219777, rs219776, rs2835363, rs219775, rs219773, ' rs219771, rs219770, rs219769, rs7277076, rs2835364, rsl70183, rsl3051987, rsl70182, rs218638, rs218641, rs218642, rs218643, rs219759, rs219761, rs219765, rs219766, rs219767, rs219768, rs219772, rs219774, rs219782, rs219783, rs219785, rs219790, rs2244014, rs2249115, rs2633329, rs2633331, rs2633334, rs2776288, rs2835304, rs2835305, rs2835309, rs2835318, rs2835319, rs2835348, rs2835349, rs2845762, rs2845764, rs2845766, rs2845769;: rs2850087, rs2850102, rs2850108, rs2850110, rs56183874, rs9975445, s.36470266, s.36472078, s.36478541, s.36487357, s.36489391, s.36492368, s.36571651, s.36598656, S.36623095, S.36644230, S.36690993, S.36691722, S.36693496, S.36694516, S.36696258, s.36696674, s.36708810, s.36722244, s.36724712, s.36730418, s.36730731, s.36732831, S.36733004, s.36734680, s.36738144, s.36738223, s.36738919, S.36739280, s.36739821, S.36749630, S.36749760, S.36751378, S.36757110, S.36757852, S.36776041, S.36777630, s.36777724, s.36777871, s.36778050, s.36780792, S.36781080, S.37075430, and s.37158027!
109. The use or agent of any one of claims 105 - 107, wherein the human subject has a genotype that comprises rs219778 allele T, rs219779 allele C, rs219780 allele C and/or rs219781 allele C.
110. The use or agent of any one of the claims 105 - 109, wherein the therapeutic agent is ■ selected from the group consisting of thiazides (e.g., chlorothiazide, hydrocholorthiazide, ." bendroflumethiazide), potassium citrate, magnesium citrate and allopurinol.
111. The use or agent of any one of claims 105 - 110, wherein the human individual is presymptomatic for the condition.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8718950B2 (en) 2011-07-08 2014-05-06 The Medical College Of Wisconsin, Inc. Methods and apparatus for identification of disease associated mutations
US20140242609A1 (en) * 2013-02-26 2014-08-28 Washington University Diagnosis and treatment of kidney stones, methods and compositions therefor
US10295527B2 (en) 2016-03-14 2019-05-21 Bruce Yacyshyn Process and system for predicting responders and non-responders to mesalamine treatment of ulcerative colitis
US11946927B2 (en) 2016-03-14 2024-04-02 Musidora Biotechnology Llc Process and system for identifying individuals having a high risk of inflammatory bowel disease and a method of treatment

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000032826A1 (en) * 1998-11-30 2000-06-08 Drexel University Methods and kits for identifying individuals at risk of developing osteoporosis
WO2002022871A2 (en) * 2000-09-14 2002-03-21 Decode Genetics Ehf Polymorphic bone morphogenetic protein 2
WO2004065938A2 (en) * 2000-09-14 2004-08-05 Decode Genetics Ehf. Human osteoporosis gene
WO2004097044A1 (en) * 2003-04-29 2004-11-11 Oxagen Limited Method of diagnosins a genetic susceptibility for bone damage

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000032826A1 (en) * 1998-11-30 2000-06-08 Drexel University Methods and kits for identifying individuals at risk of developing osteoporosis
WO2002022871A2 (en) * 2000-09-14 2002-03-21 Decode Genetics Ehf Polymorphic bone morphogenetic protein 2
WO2004065938A2 (en) * 2000-09-14 2004-08-05 Decode Genetics Ehf. Human osteoporosis gene
WO2004097044A1 (en) * 2003-04-29 2004-11-11 Oxagen Limited Method of diagnosins a genetic susceptibility for bone damage

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SHOOFS M.W.C.J. ET AL: "Thiazide diuretics and the risk for hip fracture", ANNALS OF INTERNAL MEDICINE, vol. 139, no. 6, September 2003 (2003-09-01), pages 476 - 483 *
THORLEIFSSON G. ET AL: "Sequence variants in the CLDN14 gene associate with kidney stones and bone mineral density", NATURE GENETICS, vol. 41, no. 8, August 2009 (2009-08-01), pages 926 - 930 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8718950B2 (en) 2011-07-08 2014-05-06 The Medical College Of Wisconsin, Inc. Methods and apparatus for identification of disease associated mutations
US20140242609A1 (en) * 2013-02-26 2014-08-28 Washington University Diagnosis and treatment of kidney stones, methods and compositions therefor
US10295527B2 (en) 2016-03-14 2019-05-21 Bruce Yacyshyn Process and system for predicting responders and non-responders to mesalamine treatment of ulcerative colitis
US11199534B2 (en) 2016-03-14 2021-12-14 Bruce Yacyshyn Process and system for predicting responders and non-responders to mesalamine treatment of ulcerative colitis
US11946927B2 (en) 2016-03-14 2024-04-02 Musidora Biotechnology Llc Process and system for identifying individuals having a high risk of inflammatory bowel disease and a method of treatment

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