WO2001038576A2 - Polymorphismes humains a nucleotide unique - Google Patents

Polymorphismes humains a nucleotide unique Download PDF

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WO2001038576A2
WO2001038576A2 PCT/US2000/031639 US0031639W WO0138576A2 WO 2001038576 A2 WO2001038576 A2 WO 2001038576A2 US 0031639 W US0031639 W US 0031639W WO 0138576 A2 WO0138576 A2 WO 0138576A2
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nucleic acid
ofthe
polymoφhic
allele
nucleotide
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PCT/US2000/031639
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WO2001038576A3 (fr
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Michele Cargill
James S. Ireland
Eric S. Lander
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Whitehead Institute For Biomedical Research
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the variant form may confer an evolutionary advantage or disadvantage relative to a progenitor form, or may be neutral.
  • a variant form confers a lethal disadvantage and is not transmitted to subsequent generations of the organism.
  • a variant form confers an evolutionary advantage to the species and is eventually incorporated into the DNA of many or most members of the species and effectively becomes the progenitor form.
  • a restriction fragment length polymorphism is a variation in DNA sequence that alters the length of a restriction fragment (Botstein et al., Am. J. Hum. Genet. 32, 314-331 (1980)). The restriction fragment length polymorphism may create or delete a restriction site, thus changing the length of the restriction fragment.
  • RFLPs have been widely used in human and animal genetic analyses (see WO 90/13668; WO 90/11369; Donis-Keller, Cell 51, 319-337 (1987); Lander et al, Genetics 121, 85-99 (1989)). When a heritable trait can be linked to a particular RFLP, the presence of the RFLP in an individual can be used to predict the likelihood that the animal will also exhibit the trait.
  • VNTR variable number tandem repeat
  • polymo ⁇ hisms are far more frequent than RFLPs, STRs and VNTRs.
  • Some single nucleotide polymo ⁇ hisms occur in protein-coding nucleic acid sequences (coding sequence SNP (cSNP)), in which case one of the polymo ⁇ hic forms may give rise to the expression of a defective or otherwise variant protein and, potentially, a genetic disease.
  • genes in which polymo ⁇ hisms within coding sequences give rise to genetic disease include ⁇ -globin (sickle cell anemia), apoE4 (Alzheimer's Disease), Factor V Leiden (thrombosis), and CFTR (cystic fibrosis).
  • cSNPs can alter the codon sequence of the gene and therefore specify an alternative amino acid. Such changes are called “missense” when another amino acid is substituted, and “nonsense” when the alternative codon specifies a stop signal in protein translation. When the cSNP does not alter the amino acid specified the cSNP is called “silent”.
  • Single nucleotide polymo ⁇ hisms occur in noncoding regions. Some of these polymo ⁇ hisms may also result in defective protein expression (e.g., as a result of defective splicing). Other single nucleotide polymo ⁇ hisms have no phenotypic effects. Single nucleotide polymo ⁇ hisms can be used in the same manner as RFLPs and VNTRs, but offer several advantages. Single nucleotide polymo ⁇ hisms occur with greater frequency and are spaced more uniformly throughout the genome than other forms of polymo ⁇ hism.
  • the greater frequency and uniformity of single nucleotide polymo ⁇ hisms means that there is a greater probability that such a polymo ⁇ hism will be found in close proximity to a genetic locus of interest than would be the case for other polymo ⁇ hisms.
  • the different forms of characterized single nucleotide polymo ⁇ hisms are often easier to distinguish than other types of polymo ⁇ hism (e.g., by use of assays employing allele-specific hybridization probes or primers). Only a small percentage of the total repository of polymo ⁇ hisms in humans and other organisms has been identified. The limited number of polymo ⁇ hisms identified to date is due to the large amount of work required for their detection by conventional methods.
  • a conventional approach to identifying polymo ⁇ hisms might be to sequence the same stretch of DNA in a population of individuals by dideoxy sequencing.
  • the amount of work increases in proportion to both the length of sequence and the number of individuals in a population and becomes impractical for large stretches of DNA or large numbers of persons.
  • SUMMARY OF THE INVENTION Work described herein pertains to the identification of polymo ⁇ hisms which can predispose individuals to disease by resequencing large numbers of genes in a large number of individuals.
  • SNPs SNPs in these genes have been discovered (see the Table).
  • Some of these SNPs are cSNPs which specify a different amino acid sequence (shown as mutation type "M” in the Table), some of the SNPs are silent cSNPs (shown as mutation type "S” in the Table), and some of these cSNPs specify a stop signal in protein translation (shown as mutation type "N" in the Table).
  • the invention relates to a nucleic acid molecule which comprises a single nucleotide polymo ⁇ hism at a specific location.
  • the invention relates to the variant allele of a gene having a single nucleotide polymo ⁇ hism, which variant allele differs from a reference allele by one nucleotide at the site(s) identified in the Table.
  • Complements of these nucleic acid segments are also included in the invention.
  • the segments can be DNA or RNA, and can be double- or single-stranded. Segments can be, for example, 5-10, 5-15, 10-20, 5-25, 10-30, 10-50 or 10-100 bases long.
  • the invention further provides allele-specific oligonucleotides that hybridize to a nucleic acid molecule comprising a single nucleotide polymo ⁇ hism or to the complement of the nucleic acid molecule. These oligonucleotides can be probes or primers.
  • the invention further provides a method of analyzing a nucleic acid from an individual.
  • the method allows the determination of whether the reference or variant base is present at any one of the polymo ⁇ hic sites shown in the Table.
  • a set of bases occupying a set of the polymo ⁇ hic sites shown in the Table is determined.
  • This type of analysis can be performed on a number of individuals, who are also tested (previously, concurrently or subsequently) for the presence of a disease phenotype. The presence or absence of disease phenotype is then correlated with a base or set of bases present at the polymo ⁇ hic site or sites in the individuals tested.
  • the invention further relates to a method of predicting the presence, absence, likelihood of the presence or absence, or severity of a particular phenotype or disorder associated with a particular genotype.
  • the method comprises obtaining a nucleic acid sample from an individual and determining the identity of one or more bases (nucleotides) at specific (e.g., polymo ⁇ hic) sites of nucleic acid molecules described herein, wherein the presence of a particular base at that site is correlated with a specified phenotype or disorder, thereby predicting the presence, absence, likelihood of the presence or absence, or severity of the phenotype or disorder in the individual.
  • the present invention relates to a nucleic acid molecule which comprises a single nucleotide polymo ⁇ hism (SNP) at a specific location.
  • the nucleic acid molecule e.g., a gene, which includes the SNP has at least two alleles, referred to herein as the reference allele and the variant allele.
  • the reference allele (prototypical or wild type allele) has been designated arbitrarily and typically corresponds to the nucleotide sequence of the nucleic acid molecule which has been deposited with GenBank or TIGR under a given Accession number.
  • the variant allele differs from the reference allele by one nucleotide at the site(s) identified in the Table.
  • the present invention also relates to variant alleles of the described genes and to complements of the variant alleles.
  • the invention further relates to portions of the variant alleles and portions of complements of the variant alleles which comprise (encompass) the site of the SNP and are at least 5 nucleotides in length. Portions can be, for example, 5-10, 5-15, 10-20, 5-25, 10-30, 10-50 or 10-100 bases long.
  • a portion of a variant allele which is 21 nucleotides in length includes the single nucleotide polymo ⁇ hism (the nucleotide which differs from the reference allele at that site) and twenty additional nucleotides which flank the site in the variant allele.
  • polymo ⁇ hisms which are the subject of this invention are defined in the Table with respect to the reference sequence deposited in GenBank or TIGR under the Accession number indicated.
  • the invention relates to a portion of a gene (e.g., astrotactin (ASTN)) having a nucleotide sequence as deposited in GenBank (e.g., AB006627) comprising a single nucleotide polymo ⁇ hism at a specific position (e.g., nucleotide 587).
  • GenBank e.g., AB006627
  • the reference nucleotide (G) for this polymo ⁇ hic form of ASTN is shown in column 8
  • the variant nucleotide (C) is shown in column 9 of the Table.
  • the nucleic acid molecule of the invention comprises the variant nucleotide at the polymo ⁇ hic position.
  • the invention relates to a nucleic acid molecule which comprises the nucleic acid sequence shown in row 1, column 6, of the Table having a "c" at nucleotide position 587.
  • the nucleotide sequences of the invention can be double- or single-stranded.
  • the invention further provides allele-specific oligonucleotides that hybridize to a gene comprising a single nucleotide polymo ⁇ hism or to the complement of the gene. Such oligonucleotides will hybridize to one polymo ⁇ hic form of the nucleic acid molecules described herein but not to the other polymo ⁇ hic form(s) of the sequence. Thus, such oligonucleotides can be used to determine the presence or absence of particular alleles of the polymo ⁇ hic sequences described herein. These oligonucleotides can be probes or primers.
  • the invention further provides a method of analyzing a nucleic acid sample from an individual.
  • the method determines which base is present at any one of the polymo ⁇ hic sites shown in the Table.
  • a set of bases occupying a set of the polymo ⁇ hic sites shown in the Table is determined.
  • This type of analysis can be performed on a number of individuals, who are also tested (previously, concurrently or subsequently) for the presence of a disease phenotype.
  • the presence or absence of disease phenotype is then correlated with a base or set of bases present at the polymo ⁇ hic site or sites in the individuals tested.
  • the invention further relates to a method of predicting the presence, absence, likelihood of the presence or absence, or severity of a particular phenotype or disorder associated with a particular genotype.
  • the method comprises obtaining a nucleic acid sample from an individual and determining the identity of one or more bases (nucleotides) at polymo ⁇ hic sites of nucleic acid molecules described herein, wherein the presence of a particular base is correlated with a specified phenotype or disorder, thereby predicting the presence, absence, likelihood of the presence or absence, or severity of the phenotype or disorder in the individual.
  • the correlation between a particular polymo ⁇ hic form of a gene and a phenotype can thus be used in methods of diagnosis of that phenotype, as well as in the development of treatments for the phenotype.
  • An oligonucleotide can be DNA or RNA, and single- or double-stranded. Oligonucleotides can be naturally occurring or synthetic, but are typically prepared by synthetic means. Preferred oligonucleotides of the invention include segments of DNA, or their complements, which include any one of the polymo ⁇ hic sites shown in the Table. The segments can be between 5 and 250 bases, and, in specific embodiments, are between 5-10, 5-20, 10-20, 10-50, 20-50 or 10-100 bases. For example, the segment can be 21 bases. The polymo ⁇ hic site can occur within any position of the segment. The segments can be from any of the allelic forms of DNA shown in the Table.
  • nucleotide As used herein, the terms “nucleotide”, “base” and “nucleic acid” are intended to be equivalent.
  • nucleotide sequence As used herein, the terms “nucleotide sequence”, “nucleic acid sequence”, “nucleic acid molecule” and “segment” are intended to be equivalent.
  • Hybridization probes are oligonucleotides which bind in a base-specific manner to a complementary strand of nucleic acid. Such probes include peptide nucleic acids, as described in Nielsen et al., Science 254, 1497-1500 (1991). Probes can be any length suitable for specific hybridization to the target nucleic acid sequence.
  • the most appropriate length of the probe may vary depending upon the hybridization method in which it is being used; for example, particular lengths may be more appropriate for use in micro fabricated arrays, while other lengths may be more suitable for use in classical hybridization methods. Such optimizations are known to the skilled artisan.
  • Suitable probes and primers can range from about 5 nucleotides to about 30 nucleotides in length.
  • probes and primers can be 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28 or 30 nucleotides in length.
  • the probe or primer preferably overlaps at least one polymo ⁇ hic site occupied by any of the possible variant nucleotides.
  • the nucleotide sequence can correspond to the coding sequence of the allele or to the complement of the coding sequence of the allele.
  • the term "primer” refers to a single-stranded oligonucleotide which acts as a point of initiation of template-directed DNA synthesis under appropriate conditions (e.g., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
  • the appropriate length of a primer depends on the intended use of the primer, but typically ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.
  • a primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with a template.
  • primer site refers to the area of the target DNA to which a primer hybridizes.
  • primer pair refers to a set of primers including a 5' (upstream) primer that hybridizes with the 5' end of the DNA sequence to be amplified and a 3' (downstream) primer that hybridizes with the complement of the 3' end of the sequence to be amplified.
  • linkage describes the tendency of genes, alleles, loci or genetic markers to be inherited together as a result of their location on the same chromosome. It can be measured by percent recombination between the two genes, alleles, loci or genetic markers.
  • polymo ⁇ hism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population.
  • a polymo ⁇ hic marker or site is the locus at which divergence occurs.
  • Preferred markers have at least two alleles, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population.
  • a polymo ⁇ hic locus may be as small as one base pair.
  • Polymo ⁇ hic markers include restriction fragment length polymo ⁇ hisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu.
  • the first identified allelic form is arbitrarily designated as the reference form and other allelic forms are designated as alternative or variant alleles.
  • the allelic form occurring most frequently in a selected population is sometimes referred to as the wildtype form. Diploid organisms may be homozygous or heterozygous for allelic forms.
  • a diallelic or biallelic polymo ⁇ hism has two forms.
  • a triallelic polymo ⁇ hism has three forms.
  • SNPs may alter the function of the encoded proteins.
  • the discovery of the SNP facilitates biochemical analysis of the variants and the development of assays to characterize the variants and to screen for pharmaceutical that would interact directly with on or another form of the protein.
  • SNPs may also alter the regulation of the gene at the transcriptional or post-transcriptional level.
  • SNPs include silent SNPs
  • SNPs also enable the development of specific DNA, RNA, or protein-based diagnostics that detect the presence or absence of the polymo ⁇ hism in particular conditions.
  • a single nucleotide polymo ⁇ hism occurs at a polymo ⁇ hic site occupied by a single nucleotide, which is the site of variation between allelic sequences.
  • the site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations).
  • a single nucleotide polymo ⁇ hism usually arises due to substitution of one nucleotide for another at the polymo ⁇ hic site.
  • a transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine.
  • a transversion is the replacement of a purine by a pyrimidine or vice versa.
  • Single nucleotide polymo ⁇ hisms can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele.
  • the polymo ⁇ hic site is occupied by a base other than the reference base. For example, where the reference allele contains the base "T" at the polymo ⁇ hic site, the altered allele can contain a "C", "G" or "A" at the polymo ⁇ hic site.
  • Hybridizations are usually performed under stringent conditions, for example, at a salt concentration of no more than 1 M and a temperature of at least 25°C.
  • stringent conditions for example, at a salt concentration of no more than 1 M and a temperature of at least 25°C.
  • 5X SSPE 750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4
  • a temperature of 25-30°C, or equivalent conditions are suitable for allele-specific probe hybridizations.
  • Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between the target nucleotide sequence and the primer or probe used.
  • isolated is used herein to indicate that the material in question exists in a physical milieu distinct from that in which it occurs in nature.
  • an isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs.
  • the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix.
  • the material may be purified to essential homogeneity, for example as determined by PAGE or column chromatography such as HPLC.
  • an isolated nucleic acid comprises at least about 50, 80 or 90 percent (on a molar basis) of all macromolecular species present.
  • Novel Polymo ⁇ hisms of the Invention The novel polymo ⁇ hisms ofthe invention are shown in the Table. Columns one and two show designations for the indicated polymo ⁇ hism. Column three shows the Genbank or TIGR Accession number for the wild type (or reference) allele. Column four shows the location ofthe polymo ⁇ hic site in the nucleic acid sequence with reference to the Genbank or TIGR sequence shown in column three. Column five shows common names for the gene in which the polymo ⁇ hism is located. Column six shows the polymo ⁇ hism and a portion ofthe 3' and 5' flanking sequence ofthe gene. Column seven shows the type of mutation; N, non-sense, S, silent, M, missense. Columns eight and nine show the reference and alternate nucleotides, respectively, at the polymo ⁇ hic site. Columns ten and eleven show the reference and alternate amino acids, respectively, encoded by the reference and variant, respectively, alleles.
  • Polymo ⁇ hisms are detected in a target nucleic acid from an individual being analyzed.
  • genomic DNA virtually any biological sample (other than pure red blood cells) is suitable.
  • tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair.
  • tissue sample must be obtained from an organ in which the target nucleic acid is expressed.
  • the target nucleic acid is a cytochrome P450
  • the liver is a suitable source.
  • PCR DNA Amplification
  • PCR Protocols A Guide to Methods and Applications (eds. Innis, et al., Academic Press, San Diego, CA, 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Patent 4,683,202.
  • LCR ligase chain reaction
  • NASBA nucleic acid based sequence amplification
  • the latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • the first type of analysis is carried out to identify polymo ⁇ hic sites not previously characterized (i.e., to identify new polymo ⁇ hisms). This analysis compares target sequences in different individuals to identify points of variation, i.e., polymo ⁇ hic sites.
  • de novo characterization is carried out to identify polymo ⁇ hic sites not previously characterized (i.e., to identify new polymo ⁇ hisms).
  • This analysis compares target sequences in different individuals to identify points of variation, i.e., polymo ⁇ hic sites.
  • groups of individuals representing the greatest ethnic diversity among humans and greatest breed and species variety in plants and animals patterns characteristic ofthe most common alleles/haplotypes ofthe locus can be identified, and the frequencies of such alleles/haplotypes in the population can be determined. Additional allelic frequencies can be determined for subpopulations characterized by criteria such as geography, race, or gender.
  • the de novo identification of polymo ⁇ hisms ofthe invention is described in the Examples section.
  • the second type of analysis determines which form(s) of a characterized (known) polymo ⁇ hism are present in individuals under test. There are a variety of suitable procedures, some of which are discussed in turn below.
  • Allele-specific probes for analyzing polymo ⁇ hisms is described by e.g., Saiki et al., Nature 324, 163-166 (1986); Dattagupta, EP 235,726, Saiki, WO 89/11548. Allele-specific probes can be designed that hybridize to a segment of target DNA from one individual but do not hybridize to the corresponding segment from another individual due to the presence of different polymo ⁇ hic forms in the respective segments from the two individuals. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one ofthe alleles.
  • Some probes are designed to hybridize to a segment of target DNA such that the polymo ⁇ hic site aligns with a central position (e.g., in a 15-mer at the 7 position; in a 16-mer, at either the 8 or 9 position) ofthe probe.
  • This design of probe achieves good discrimination in hybridization between different allelic forms.
  • Allele-specific probes are often used in pairs, one member of a pair showing a perfect match to a reference form of a target sequence and the other member showing a perfect match to a variant form. Several pairs of probes can then be immobilized on the same support for simultaneous analysis of multiple polymo ⁇ hisms within the same target sequence.
  • the polymo ⁇ hisms can also be identified by hybridization to nucleic acid arrays, some examples of which are described in WO 95/11995. The same arrays or different arrays can be used for analysis of characterized polymo ⁇ hisms.
  • WO 95/11995 also describes subarrays that are optimized for detection of a variant form of a precharacterized polymo ⁇ hism. Such a subarray contains probes designed to be complementary to a second reference sequence, which is an allelic variant ofthe first reference sequence. The second group of probes is designed by the same principles as described, except that the probes exhibit complementarity to the second reference sequence.
  • a second group can be particularly useful for analyzing short subsequences ofthe primary reference sequence in which multiple mutations are expected to occur within a short distance commensurate with the length ofthe probes (e.g., two or more mutations within 9 to 21 bases).
  • An allele-specific primer hybridizes to a site on target DNA overlapping a polymo ⁇ hism and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. See Gibbs, Nucleic Acid Res. 17, 2427-2448 (1989). This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers, resulting in a detectable product which indicates the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymo ⁇ hic site and the other of which exhibits perfect complementarity to a distal site. The single-base mismatch prevents amplification and no detectable product is formed.
  • the method works best when the mismatch is included in the 3'-most position ofthe oligonucleotide aligned with the polymo ⁇ hism because this position is most destabilizing to elongation from the primer (see, e.g., WO 93/22456).
  • Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. Erlich, ed., PCR Technology,
  • Alleles of target sequences can be differentiated using single-strand conformation polymo ⁇ hism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al., Proc. Nat. Acad. Sci. 86, 2766-2770 (1989).
  • Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single stranded amplification products.
  • Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence.
  • the different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence differences between alleles of target sequences.
  • An alternative method for identifying and analyzing polymo ⁇ hisms is based on single-base extension (SBE) of a fluorescently-labeled primer coupled with fluorescence resonance energy transfer (FRET) between the label ofthe added base and the label ofthe primer.
  • SBE single-base extension
  • FRET fluorescence resonance energy transfer
  • the method such as that described by Chen et al., (PNAS 94:10756-61 (1997)), uses a locus-specific oligonucleotide primer labeled on the 5' terminus with 5-carboxyfluorescein (FAM). This labeled primer is designed so that the 3' end is immediately adjacent to the polymo ⁇ hic site of interest.
  • the labeled primer is hybridized to the locus, and single base extension of the labeled primer is performed with fluorescently-labeled dideoxyribonucleotides (ddNTPs) in dye-terminator sequencing fashion.
  • ddNTPs fluorescently-labeled dideoxyribonucleotides
  • An increase in fluorescence ofthe added ddNTP in response to excitation at the wavelength ofthe labeled primer is used to infer the identity ofthe added nucleotide.
  • this information can be used in a number of methods.
  • polymo ⁇ hisms ofthe invention are often used in conjunction with polymo ⁇ hisms in distal genes.
  • Preferred polymo ⁇ hisms for use in forensics are biallelic because the population frequencies of two polymo ⁇ hic forms can usually be determined with greater accuracy than those of multiple polymo ⁇ hic forms at multi-allelic loci.
  • the capacity to identify a distinguishing or unique set of forensic markers in an individual is useful for forensic analysis. For example, one can determine whether a blood sample from a suspect matches a blood or other tissue sample from a crime scene by determining whether the set of polymo ⁇ hic forms occupying selected polymo ⁇ hic sites is the same in the suspect and the sample. If the set of polymo ⁇ hic markers does not match between a suspect and a sample, it can be concluded (barring experimental error) that the suspect was not the source ofthe sample. If the set of markers does match, one can conclude that the DNA from the suspect is consistent with that found at the crime scene.
  • p(ID) is the probability that two random individuals have the same polymo ⁇ hic or allelic form at a given polymo ⁇ hic site. In biallelic loci, four genotypes are possible: AA, AB, BA, and BB.
  • the object of paternity testing is usually to determine whether a male is the father of a child. In most cases, the mother of the child is known and thus, the mother's contribution to the child's genotype can be traced. Paternity testing investigates whether the part ofthe child's genotype not attributable to the mother is consistent with that ofthe putative father. Paternity testing can be performed by analyzing sets of polymo ⁇ hisms in the putative father and the child. If the set of polymo ⁇ hisms in the child attributable to the father does not match the set of polymo ⁇ hisms of the putative father, it can be concluded, barring experimental error, that the putative father is not the real father. If the set of polymo ⁇ hisms in the child attributable to the father does match the set of polymo ⁇ hisms ofthe putative father, a statistical calculation can be performed to determine the probability of coincidental match.
  • cum p(exc) 1 - cum p(non-exc).
  • the cumulative probability of exclusion of a random male is very high. This probability can be taken into account in assessing the liability of a putative father whose polymo ⁇ hic marker set matches the child's polymo ⁇ hic marker set attributable to his/her father.
  • the polymo ⁇ hisms ofthe invention may contribute to the phenotype of an organism in different ways. Some polymo ⁇ hisms occur within a protein coding sequence and contribute to phenotype by affecting protein structure. The effect may be neutral, beneficial or detrimental, or both beneficial and detrimental, depending on the circumstances. For example, a heterozygous sickle cell mutation confers resistance to malaria, but a homozygous sickle cell mutation is usually lethal. Other polymo ⁇ hisms occur in noncoding regions but may exert phenotypic effects indirectly via influence on replication, transcription, and translation. A single polymo ⁇ hism may affect more than one phenotypic trait. Likewise, a single phenotypic trait may be affected by polymo ⁇ hisms in different genes. Further, some polymo ⁇ hisms predispose an individual to a distinct mutation that is causally related to a certain phenotype.
  • Phenotypic traits include diseases that have known but hitherto unmapped genetic components (e.g., agammaglobulimenia, diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy, Wiskott-Aldrich syndrome, Fabry's disease, familial hypercholesterolemia, polycystic kidney disease, hereditary spherocytosis, von Willebrand's disease, tuberous sclerosis, hereditary hemorrhagic telangiectasia, familial colonic polyposis, Ehlers-Danlos syndrome, osteogenesis imperfecta, and acute intermittent po ⁇ hyria).
  • agammaglobulimenia e.g., diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy, Wiskott-Aldrich syndrome, Fabry's disease, familial hypercholesterolemia, polycystic kidney disease, hereditary spherocytosis, von Willebrand's disease
  • Phenotypic traits also include symptoms of, or susceptibility to, multifactorial diseases of which a component is or may be genetic, such as autoimmune diseases, inflammation, cancer, diseases ofthe nervous system, and infection by pathogenic microorganisms.
  • autoimmune diseases include rheumatoid arthritis, multiple sclerosis, diabetes (insulin-dependent and non-independent), systemic lupus erythematosus and Graves disease.
  • Some examples of cancers include cancers ofthe bladder, brain, breast, colon, esophagus, kidney, leukemia, liver, lung, oral cavity, ovary, pancreas, prostate, skin, stomach and uterus.
  • Phenotypic traits also include characteristics such as longevity, appearance (e.g., baldness, obesity), strength, speed, endurance, fertility, and susceptibility or receptivity to particular drugs or therapeutic treatments.
  • the correlation of one or more polymo ⁇ hisms with phenotypic traits can be facilitated by knowledge ofthe gene product ofthe wild type (reference) gene.
  • the genes in which cSNPs ofthe present invention have been identified are genes which have been previously sequenced and characterized in one of their allelic forms. Correlation is performed for a population of individuals who have been tested for the presence or absence of a phenotypic trait of interest and for polymo ⁇ hic markers sets. To perform such analysis, the presence or absence of a set of polymo ⁇ hisms (i.e. a polymo ⁇ hic set) is determined for a set ofthe individuals, some of whom exhibit a particular trait, and some of which exhibit lack ofthe trait.
  • the alleles of each polymo ⁇ hism ofthe set are then reviewed to determine whether the presence or absence of a particular allele is associated with the trait of interest. Correlation can be performed by standard statistical methods such as a ⁇ -squared test and statistically significant correlations between polymo ⁇ hic form(s) and phenotypic characteristics are noted. For example, it might be found that the presence of allele Al at polymo ⁇ hism A correlates with heart disease. As a further example, it might be found that the combined presence of allele Al at polymo ⁇ hism A and allele Bl at polymo ⁇ hism B correlates with increased milk production of a farm animal.
  • Such correlations can be exploited in several ways.
  • detection ofthe polymo ⁇ hic form set in a human or animal patient may justify immediate administration of treatment, or at least the institution of regular monitoring ofthe patient.
  • Detection of a polymo ⁇ hic form correlated with serious disease in a couple contemplating a family may also be valuable to the couple in their reproductive decisions.
  • the female partner might elect to undergo in vitro fertilization to avoid the possibility of transmitting such a polymo ⁇ hism from her husband to her offspring.
  • bovine mitochondrial polymo ⁇ hisms in a breeding program to improve milk production in cows.
  • each cow was assigned a value of 1 if variant or 0 if wildtype with respect to a prototypical mitochondrial DNA sequence at each of 17 locations considered.
  • Each production trait was analyzed individually with the following animal model:
  • Y, jlcpn ⁇ + YS, + P j + X k + ⁇ , + ... ⁇ 17 + PE n + a profession +e p
  • Y ljb ⁇ p is the milk, fat, fat percentage, SNF, SNF percentage, energy concentration, or lactation energy record
  • ⁇ is an overall mean
  • YS is the effect common to all cows calving in year-season
  • X k is the effect common to cows in either the high or average selection line
  • ⁇ , to ⁇ 17 are the binomial regressions of production record on mtDNA D-loop sequence polymo ⁇ hisms
  • PE n is permanent environmental effect common to all records of cow n
  • a municipality is effect of animal n and is composed ofthe additive genetic contribution of sire and dam breeding values and a Mendelian sampling effect
  • e p is a random residual.
  • Such analysis is useful for mapping a genetic locus associated with a phenotypic trait to a chromosomal position, and thereby cloning gene(s) responsible for the trait.
  • Lander et al Proc. Natl. Acad. Sci. (USA) 83, 7353-7357 (1986); Lander et al, Proc. Natl. Acad. Sci. (USA) 84, 2363-2367 (1987); Donis-Keller et al, Cell 51, 319-337 (1987); Lander et al, Genetics 121, 185-199 (1989)).
  • Genes localized by linkage can be cloned by a process known as directional cloning. See Wainwright, Med. J.
  • LOD log ofthe odds
  • a lod value is the relative likelihood of obtaining observed segregation data for a marker and a genetic locus when the two are located at a recombination fraction ⁇ , versus the situation in which the two are not linked, and thus segregating independently (Thompson & Thompson, Genetics in Medicine (5th ed, W.B. Saunders Company, Philadelphia, 1991); Strachan, "Mapping the human genome” in The Human Genome (BIOS Scientific Publishers Ltd, Oxford), Chapter 4).
  • the likelihood at a given value of ⁇ is: probability of data if loci linked at ⁇ to probability of data if loci unlinked.
  • the computed likelihoods are usually expressed as the log 10 of this ratio (i.e., a lod score). For example, a lod score of 3 indicates 1000:1 odds against an apparent observed linkage being a coincidence.
  • the use of logarithms allows data collected from different families to be combined by simple addition. Computer programs are available for the calculation of lod scores for differing values of ⁇ (e.g., LIPED, MLINK (Lathrop, Proc. Nat. Acad. Sci. (USA) 81, 3443-3446 (1984)).
  • a recombination fraction may be determined from mathematical tables. See Smith et al, Mathematical tables for research workers in human genetics (Churchill, London, 1961); Smith, Ann. Hum. Genet. 32, 127-150 (1968). The value of ⁇ at which the lod score is the highest is considered to be the best estimate ofthe recombination fraction.
  • Positive lod score values suggest that the two loci are linked, whereas negative values suggest that linkage is less likely (at that value of ⁇ ) than the possibility that the two loci are unlinked.
  • a combined lod score of +3 or greater is considered definitive evidence that two loci are linked.
  • a negative lod score of - 2 or less is taken as definitive evidence against linkage ofthe two loci being compared.
  • Negative linkage data are useful in excluding a chromosome or a segment thereof from consideration. The search focuses on the remaining non- excluded chromosomal locations.
  • Chromosome mapping is the construction of a series of chromosome descriptions that depict the position and spacing of unique, identifiable biochemical landmarks, including genes, that occur on the DNA of chromosomes.
  • the nucleic acid molecules disclosed herein provide additional unique, identifiable biochemical landmarks for use in mapping chromosomes and for use as chromosomal markers, particularly where the chromosomal location ofthe reference allele is already known.
  • the invention further provides variant forms of nucleic acids and corresponding proteins.
  • the nucleic acids comprise one ofthe sequences described in the Table, column 5, in which the polymo ⁇ hic position is occupied by one ofthe alternative bases for that position.
  • Some nucleic acids encode full-length variant forms of proteins.
  • variant proteins have the prototypical amino acid sequences encoded by nucleic acid sequences shown in the Table, column 5, (read so as to be in- frame with the full-length coding sequence of which it is a component) except at an amino acid encoded by a codon including one ofthe polymo ⁇ hic positions shown in the Table. That position is occupied by the variant or alternative amino acid shown in the Table.
  • Variant genes can be expressed in an expression vector in which a variant gene is operably linked to a native or other promoter.
  • the promoter is a eukaryotic promoter for expression in a mammalian cell.
  • the transcription regulation sequences typically include a heterologous promoter and optionally an enhancer which is recognized by the host.
  • the selection of an appropriate promoter for example t ⁇ , lac, phage promoters, glycolytic enzyme promoters and tRNA promoters, depends on the host selected.
  • Commercially available expression vectors can be used.
  • Vectors can include host-recognized replication systems, amplifiable genes, selectable markers, host sequences useful for insertion into the host genome, and the like.
  • the means of introducing the expression construct into a host cell varies depending upon the particular construction and the target host. Suitable means include fusion, conjugation, transfection, transduction, electroporation or injection, as described in Sambrook, supra.
  • a wide variety of host cells can be employed for expression ofthe variant gene, both prokaryotic and eukaryotic. Suitable host cells include bacteria such as E. coli, yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e.g., mouse, CHO, human and monkey cell lines and derivatives thereof. Preferred host cells are able to process the variant gene product to produce an appropriate mature polypeptide.
  • Protein product includes mRNA, peptide and protein products.
  • the protein may be isolated by conventional means of protein biochemistry and purification to obtain a substantially pure product, i.e., 80, 95 or 99% free of cell component contaminants, as described in Jacoby, Methods in Enzymology Volume 104, Academic Press, New York (1984); Scopes, Protein Purification, Principles and Practice, 2nd Edition, Springer- Verlag, New York (1987); and Deutscher (ed), Guide to Protein Purification, Methods in Enzymology, Vol. 182 (1990).
  • the invention further provides transgenic nonhuman animals capable of expressing an exogenous variant gene and or having one or both alleles of an endogenous variant gene inactivated. Expression of an exogenous variant gene is usually achieved by operably linking the gene to a promoter and optionally an enhancer, and microinjecting the construct into a zygote. See Hogan et al, "Manipulating the Mouse Embryo, A Laboratory Manual," Cold Spring Harbor Laboratory.
  • Inactivation of endogenous variant genes can be achieved by forming a transgene in which a cloned variant gene is inactivated by insertion of a positive selection marker. See Capecchi, Science 244, 1288-1292 (1989). The transgene is then introduced into an embryonic stem cell, where it undergoes homologous recombination with an endogenous variant gene. Mice and other rodents are preferred animals. Such animals provide useful drug screening systems.
  • the present invention includes biologically active fragments ofthe polypeptides, or analogs thereof, including organic molecules which simulate the interactions ofthe peptides.
  • biologically active fragments include any portion ofthe full-length polypeptide which confers a biological function on the variant gene product, including ligand binding, and antibody binding.
  • Ligand binding includes binding by nucleic acids, proteins or polypeptides, small biologically active molecules, or large cellular structures.
  • Antibodies that specifically bind to variant gene products but not to corresponding prototypical gene products are also provided.
  • Antibodies can be made by injecting mice or other animals with the variant gene product or synthetic peptide fragments thereof. Monoclonal antibodies are screened as are described, for example, in Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988); Goding, Monoclonal antibodies, Principles and Practice (2d ed.) Academic Press, New York (1986). Monoclonal antibodies are tested for specific immunoreactivity with a variant gene product and lack of immunoreactivity to the corresponding prototypical gene product. These antibodies are useful in diagnostic assays for detection ofthe variant form, or as an active ingredient in a pharmaceutical composition.
  • kits comprising at least one allele-specific oligonucleotide as described herein. Often, the kits contain one or more pairs of allele-specific oligonucleotides hybridizing to different forms of a polymo ⁇ hism. In some kits, the allele-specific oligonucleotides are provided immobilized to a substrate. For example, the same substrate can comprise allele-specific oligonucleotide probes for detecting at least 10, 100 or all ofthe polymo ⁇ hisms shown in the Table.
  • kits include, for example, restriction enzymes, reverse-transcriptase or polymerase, the substrate nucleoside triphosphates, means used to label (for example, an avidin-enzyme conjugate and enzyme substrate and chromogen if the label is biotin), and the appropriate buffers for reverse transcription, PCR, or hybridization reactions.
  • the kit also contains instructions for carrying out the methods.
  • a typical probe array used in this analysis has two groups of four sets of probes that respectively tile both strands of a reference sequence.
  • a first probe set comprises a plurality of probes exhibiting perfect complementarily with one ofthe reference sequences. Each probe in the first probe set has an interrogation position that corresponds to a nucleotide in the reference sequence.
  • the interrogation position is aligned with the corresponding nucleotide in the reference sequence, when the probe and reference sequence are aligned to maximize complementarily between the two.
  • For each probe in the first set there are three corresponding probes from three additional p be sets. Thus, there are four probes corresponding to each nucleotide in the reference sequence.
  • the probes from the three additional probe sets are identical to the corresponding probe from the first probe set except at the interrogation position, which occurs in the same position in each ofthe four corresponding probes from the four probe sets, and is occupied by a different nucleotide in the four probe sets. In the present analysis, probes were 25 nucleotides long. Arrays tiled for multiple different references sequences were included on the same substrate.
  • Genomic DNA was amplified in at least 50 individuals using 2.5 pmol each primer, 1.5 mM MgCl 2 , 100 ⁇ M dNTPs, 0.75 ⁇ M AmpliTaq GOLD polymerase, and 19 ng DNA in a 15 ⁇ l reaction.
  • Reactions were assembled using a PACKARD MultiPROBE robotic pipetting station and then put in MJ 96-well tetrad thermocyclers (96°C for 10 minutes, followed by 35 cycles of 96°C for 30 seconds, 59°C for 2 minutes, and 72°C for 2 minutes). A subset of the PCR assays for each individual were run on 3% NuSieve gels in 0.5X TBE to confirm that the reaction worked.
  • Low-density DNA chips (Affymetrix, Santa Clara CA) were hybridized following the manufacturer's instructions. Briefly, the hybridization cocktail consisted of 3M TMAC1, 10 mM Tris pH 7.8, 0.01% Triton X-100, 100 mg/ml herring sperm DNA (Gibco BRL), 200 pM control biotin-labeled oligo. The processed PCR products were denatured for 7 minutes at 100°C and then added to prewarmed (37°C) hybridization solution. The chips were hybridized overnight at 44°C.
  • Chips were washed in IX SSPET and 6X SSPET followed by staining with 2 ⁇ g/ml SARPE and 0.5 mg/ml acetylated BSA in 200 ⁇ l of 6X SSPET for 8 minutes at room temperature. Chips were scanned using a Molecular Dynamics scanner. Chip image files were analyzed using Ulysses (Affymetrix) which uses four algorithms to identify potential polymo ⁇ hisms. Candidate polymo ⁇ hisms were visually inspected and assigned a confidence value: high confidence candidates
  • the invention includes a number of general uses that can be expressed concisely as follows.
  • the invention provides for the use of any of the nucleic acid segments described above in the diagnosis or monitoring of diseases, such as cancer, inflammation, heart disease, diseases ofthe cardiovascular system, and infection by microorganisms.
  • the invention further provides for the use of any ofthe nucleic acid segments in the manufacture of a medicament for the treatment or prophylaxis of such diseases.
  • the invention further provides for the use of any ofthe DNA segments as a pharmaceutical. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope ofthe invention encompassed by the appended claims.

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Abstract

L'invention concerne des segments d'acide nucléique du génome humain, notamment des segments d'acide nucléique des gènes, y compris des sites polymorphes. L'invention concerne également des amorces et des sondes spécifiques aux allèles, s'hybridant à des régions adjacentes à ces sites ou contenant ces sites. On utilise ces acides nucléiques, ces amorces et ces sondes dans des applications telles que les corrélations phénotypiques, la médecine légale, la recherche de paternité, la médecine et les analyses génétiques.
PCT/US2000/031639 1999-11-24 2000-11-17 Polymorphismes humains a nucleotide unique WO2001038576A2 (fr)

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WO2003054230A2 (fr) * 2001-12-11 2003-07-03 King's College London Detection de maladie due a des taux anormaux d'oestrogenes
US7348140B1 (en) * 2001-07-25 2008-03-25 Acadia Pharmaceuticals, Inc. Clinical indications for genotyping polymorphic variants of G-protein coupled receptors
US20140377261A1 (en) * 2012-01-19 2014-12-25 Duke University Vaccines against antigens involved in therapy resistance and methods of using same
US10487143B2 (en) 2016-10-05 2019-11-26 Duke University Vaccines against HER3 antigens and methods of using the same

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001098316A2 (fr) * 2000-06-20 2001-12-27 Genaissance Pharmaceuticals, Inc. Haplotypes du gene chrne
WO2001098316A3 (fr) * 2000-06-20 2002-04-04 Genaissance Pharmaceuticals Haplotypes du gene chrne
US7348140B1 (en) * 2001-07-25 2008-03-25 Acadia Pharmaceuticals, Inc. Clinical indications for genotyping polymorphic variants of G-protein coupled receptors
WO2003054230A2 (fr) * 2001-12-11 2003-07-03 King's College London Detection de maladie due a des taux anormaux d'oestrogenes
WO2003054230A3 (fr) * 2001-12-11 2007-11-01 King S College London Detection de maladie due a des taux anormaux d'oestrogenes
US20140377261A1 (en) * 2012-01-19 2014-12-25 Duke University Vaccines against antigens involved in therapy resistance and methods of using same
US9956276B2 (en) * 2012-01-19 2018-05-01 Duke University Vaccines against antigens involved in therapy resistance and methods of using same
US11235043B2 (en) 2012-01-19 2022-02-01 Duke University Vaccines against antigens involved in therapy resistance and methods of using same
US10487143B2 (en) 2016-10-05 2019-11-26 Duke University Vaccines against HER3 antigens and methods of using the same

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