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  • 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
  • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
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  • 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
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  • 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
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
  • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/172—Haplotypes

Definitions

• the present invention relates to osteoporosis and/or bone mineral density
• the invention further relates to a combination of markers at the estrogen receptor and vitamin D receptor or equivalents thereof to prognose a response to osteoporosis therapy
• the invention relates to a method for determining osteoporosis susceptibility, prognosis and response to therapy based on a determination of a combination of genotype at the estrogen receptor and the vitamin D receptor loci or at markers in linkage disequilibrium with these loci
• the invention further relates to screening assays to identify and select agents which can be used in the treatment of osteoporosis
• Osteoporosis is a multifacto ⁇ al disease that involves a reduction in bone mineral density (BMD) leading to an increased risk of fracture It is a major public health concern, especially in postmenopausal women Many determinants of BMD such as age, sex, race, body weight, state of hypoestrogenism, calcium and vitamin D intake are well known, but the genetic factors underlying individual variation of bone density (BD) remain obscure
• Type I osteoporosis which typically affects trabecular bones becomes apparent in women within 5 to 10 years after menopause (as detected by bone densitometry) and is the consequence of increased bone remodelling secondary to estrogen deficiency
• Type II osteoporosis affects both sexes but is twice as common in women than in men and occurs mainly in individuals aged over 70 years.
• Type II osteoporosis affects both cancellous and cortical bones and is generally considered to be the consequence of significant changes that occur after age 65 in vitamin D/calcium/parathyroid hormone metabolic pathway.
• the type l/type II model of involutional osteoporosis is currently supported by several kinds of evidence, mainly differences in fracture patterns, pattern of bone loss and parathyroid function, as well as hormonal mechanisms of bone loss.
• ESR1 estrogen receptor gene
• HRT hormone replacement therapy
• One aim of the present invention is to provide a genetic assay for determining the predisposition to osteoporosis and/or response to osteoporosis treatment
• Another aim of the present invention is to use a combination of at least one polymorphism of the estrogen receptor (ESR) or an equivalent thereof and a polymorphism of the VDR or an equivalent thereof as a marker for osteoporosis susceptibility and/or response to hormone replacement therapy
• ESR estrogen receptor
• One of a polymorphism of the ESR gene, or any polymorphism in linkage disequilibrium therewith when combined with one of a polymorphism of the VDR gene, or any polymorphism in linkage disequilibrium therewith, can also be used as a test for osteoporosis susceptibility or for low bone mass, for responsiveness to treatment for osteoporosis, for osteoporosis prognosis or severity, or as a means to classify patients in clinical trial for osteoporosis (screening, diagnosis, pro
• One of a polymorphism of the ESR gene, or any polymorphism in linkage disequilibrium therewith, when combined with one of a polymorphism of the VDR gene, or any polymorphism in linkage disequilibrium therewith, can further be used as a test for screening drugs for osteoporosis or for determining the best treatment for osteoporosis
• Another aim of the present invention is to provide a method of assessing the outcome of HRT of a patient, which comprises determining one of a polymorphism of the ESR gene, or any polymorphism in linkage disequilibrium therewith, and one of a polymorphism of the VDR gene, or any polymorphism in linkage disequilibrium therewith, in a biological sample of the patient, wherein a specific genotype combination is associated with a significantly higher HRT response While it is known that HRT is responsible for an increase of bone density in women, the response is usually modest and a small proportion of women do not respond The identification of the combination
• the genotype combination prognosing a significant response to HRT is VDR-bb/ESR-PP
• the present invention also relates to vectors, including expression vectors harboring at least one of a VDR gene (or fragment or fusion thereof) and an ESR gene (or fragment or fusion thereof) having genotypes and combination of genotypes in accordance with the present invention (i e a prognosing a significant response to HRT, VDR-bb/ESR-PP, or other genotypes isolated from patients or genetically engineered), cells harboring such vectors, and non-human animals harboring such vectors or cells
• Another aim of the present invention is to provide means of identifying young women that will be at risk of osteoporosis after their menopause and to categorize those that are likely to respond significantly to HRT by displaying a significant increase their BMD to reach a higher peak bone mass
• An aim of the present invention is thus to provide means of identification of target sub-groups of women for osteoporosis prevention measures/programs
• Another aim of the present invention is to provide means to determine which sub-group of post menopausal women will most
• Another aim of the present invention is to provide an assay to screen for drugs for the treatment and/or prevention of osteoporosis Having identified a combination of markers which predict a very positive outcome to HRT and markers which predict a negative or moderate outcome to HRT, assays can be set-up to screen agents and select drugs which could be used in the treatment or prevention of osteoporosis, thereby increasing the proportion of women which can be treated in order to increase very significantly their bone density, bone mass and protect against osteoporosis
• such assays can be designed using cells from patients having a known genotype at the loci of the present invention, these cells harboring recombinant vectors enabling an assessment of the functionality of the ESR and VDR
• assays that could be used in accordance with the present invention include cis-trans assays similar to those described in USP 4,981 ,784
• the cell line expressing the HRT-positive response genotype combination can be used as a positive control for the functionality of the estrogen and vitamin D receptors
• the determination of the combinations of allelic variations in the ESR and VDR genes can be combined to the determination of allelic variations in other genes/markers linked to the predisposition to osteoporosis and/or low bone density and/or responsiveness to therapy to osteoporosis or to the prevention of low bone density
• This combination of genotype analyses could lead to better diagnoses programs and/or treatment of osteoporosis and low bone density related diseases
• Non- limiting examples of such markers could lead to better diagnoses programs and/or treatment of osteo
• osteoporosis is significantly more preponderant in women, it can also be a morbidity-inducing disease in men
• the present invention is meant to also cover men
• a method of determining an individual's predisposition to osteoporosis and/or low or high bone density, development of osteoporosis and/or responsiveness to therapy for osteoporosis or for a prevention of low bone density which comprises determining a combination of estrogen receptor polymorphism (directly or indirectly by linkage disequilibrium) and vitamin D receptor polymorphism (directly or indirectly by linkage disequilibrium) in a biological sample of the individual and analyzing allelic variation in the estrogen receptor and vitamin D receptor genes of the individual, thereby determining an individual's predisposition to osteoporosis, development of osteoporosis and/or responsiveness to therapy for osteoporosis
• a method for determining susceptibility to osteoporosis, and/or response to therapy for osteoporosis The method comprises the step of determining the estrogen receptor genotype and the vitamin D receptor genotype of the individual, thereby determining an individual
• the step of determining the estrogen receptor genotype and the vitamin D receptor genotype preferably comprises restriction endonuclease digestion.
• the step of determining the estrogen receptor genotype and the vitamin D receptor genotype could also comprise hybridizing with allele specific oligonucleotides.
• the method of the present invention preferably further comprises a step prior to determining the estrogen receptor genotype and the vitamin D receptor genotype, of amplifying a segment of the estrogen receptor gene or of the vitamin D receptor gene.
• the amplification is carried out using polymerase chain reaction.
• a pair of primers is designed to specifically amplify a segment of the estrogen receptor gene or of the vitamin D receptor gene.
• This pair of primers is preferably derived from a nucleic acid sequence of the estrogen receptor gene or flanking the gene to amplify a segment of the estrogen receptor gene, or derived from a nucleic acid sequence of the vitamin D receptor gene or flanking the gene to amplify a segment of the vitamin D receptor gene.
• the pair of primers used for amplifying the segment of the estrogen receptor gene is defined as follows: ⁇ '-TGCCACCCTA TCTGTATCTT TTCC-3' SEQ ID NO:1 and 5'-TCTTTCTCTG CCACCCTGGC GTC-3' SEQ ID NO:2.
• the pair of primers used for amplifying the segment of the vitamin D receptor gene is selected from the group consisting of: ⁇ '-CAACCAAGAC TACAAGTACC GCGTCAGTGA-3' (SEQ ID NO:3) and ⁇ '-TATCGTGAGT AAGGCAGGAG AGGGAGACC-3' (SEQ ID NO:4); and 5'-AGCTGGCCCT GGCACTGACT CTGCTCT-3' (SEQ ID NO:5) and ⁇ '-ATGGAAACAC CTTGCTTCTT CTCCCTC-3' (SEQ ID NO:6).
• the ESR and VDR genes can be designed, based on the known sequences of the ESR and VDR genes, as commonly known in the art.
• Restriction fragment length polymorphism can be used to determine the polymorphisms at the ESR and VDR loci (and equivalent loci).
• Pvull or isoschizomers thereof is preferably used for determining the estrogen receptor genotype.
• Bsml, Taql, Apal or Fokl, or isoschizomers thereof is preferably used for determining the vitamin D receptor genotype.
• the estrogen receptor genotype is determined using a polymorphic variant site in linkage disequilibrium with at least one allelic variant as detected with Pvull in a restriction endonuclease digestion.
• the vitamin D receptor genotype is determined using a polymorphic variant site in linkage disequilibrium with at least one allelic variant as detected with Bsml, Taql, Apal or Fokl in the restriction endonuclease digestion. More preferably, the vitamin D receptor genotype is determined using Bsml.
• the Taql, Apal or Fokl polymorphisms of the VDR gene are non-limiting examples of polymorphisms (or equivalents) which are in linkage disequilibrium with the Bsml polymorphism and can be used in accordance with the present invention.
• RFLP restriction fragment length polymorphism
• polymorphism refers to any sequence in the human genome which exists in more than one version or variant in the population.
• estrogen hormone- and vitamin D-related medical conditions refers to, without limitation, any estrogen and vitamin D-dependent diseases
• linkage disequilibrium refers to any degree of non- ⁇ random genetic association between one or more allele(s) of two different polymorphic DNA sequences, that is due to the physical proximity of the two loci Linkage disequilibrium is present when two DNA segments that are very close to each other on a given chromosome will tend to remain unseparated for several generations with the consequence that alleles of a DNA polymorphism 0 (or marker) in one segment will show a non-random association with the alleles of a different DNA polymorphism (or marker) located in the other DNA segment nearby Hence, testing of one of a marker in linkage desiquilibnum with the polymorphisms of the present invention at the ESR or VDR genes (indirect testing), will give almost the same information as testing for the ESR and VDR ⁇ polymorphisms directly This situation is encountered throughout all the human genome when two DNA polymorphisms that are very close to each other are studied Such a linkage disequilibrium with several
• estrogen receptor polymorphism or "genetic marker” are intended to include, without limitation, Pvull (GDB (Genome Data Base) #G00-155-446), Pssl (GDB #G00-155-447), SACI (GDB #G00- 155-448), Xbal (GDB # G00-155-440) as well as the following ESR non-RFLP 5 polymorphisms GDB #G00-162-450, #G00-162-541 , and any other allelic variant of the estrogen receptor gene that show some degree of linkage disequilibrium in any population sub-group with at least one of the above- mentioned estrogen receptor polymorphisms
• vitamin D receptor polymorphism or “genetic 0 marker” are intended to include, without limitation, Bsml, Taql, Apal or Fokl and any other allelic variant of the vitamin D receptor gene that show some degree of linkage disequilibrium in any population sub-group with at least one of the above-mentioned vitamin D receptor polymorphisms.
• the estrogen receptor gene and vitamin D receptor gene polymorphism sites in accordance with the present invention can be located within the estrogen receptor gene, or on each side thereof, or within the vitamin D receptor gene, or on each side thereof provided that they are on the same chromosome and in linkage disequilibrium with the ESR and VDR polymorphisms of the present invention.
• Distances between markers in linkage disequilibrium can vary widely (below 50 kb to more than 1 mega base) depending on the genetic structure of the population and is ascertainable by a statistically significant association between the markers.
• the findings described herein may have important repercussions on the screening for low BMD in post-menopausal women but also for the implementation of screening programs aimed at preventing low BMD in women and for identification of women which would benefit from hormone replacement therapy at their menopause. Also, it may be useful for other applications in the prevention and treatment of osteoporosis and perhaps on other important diseases or conditions showing an estrogen- and vitamin D- hormone related medical condition.
• the present invention should not be limited to the identification of the polymorphisms at the DNA level (whether on genomic DNA, amplified DNA, cDNA or the like) Indeed, the herein-identified polymorphisms could be detected at the imRNA or protein level. Such detections of polymorphism identification on mRNA or protein are known in the art.
• Non- limiting examples include detection based on oligos designed to hybridize to mRNA or ligands such as antibodies which are specific to the encoded polymorphism (i e specific to the protein fragment encoded by the distinct polymorphisms)
• a non-limiting example of such a polymorphism which could be detected at the mRNA or protein level is the Fokl polymorphism of the VDR gene which creates an initiator methionme upstream of the normal AUG ⁇ Since the polymorphisms of the present invention are expressed, one of the advantages of the present invention is to enable a determination of the polymorphisms in the ESR and VDR genes, in easily obtainable cells which express these genes
• a non-limiting example thereof is lymphocytes, thereby enabling a genotyping from a simple blood sample 0
• Nucleotide sequences are presented herein by single strand, in the ⁇ ' to 3' direction, from left to right, using the one letter nucleotide symbols as commonly used in the art and in
• nucleic acid molecule refers to a polymer of nucleotides Non-limiting examples thereof include DNA (i e genomic DNA, cDNA) and RNA molecules (i e mRNA) The nucleic acid molecule can be obtained by cloning techniques or synth
• DNA segment is used herein, to refer to a DNA molecule comprising a linear stretch or sequence of nucleotides This sequence when read in accordance with the genetic code, can encode a linear stretch or sequence of ammo acids which can be referred to as a polypeptide, protein, protein fragment and the like
• amplification pair refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction Other types of amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below As commonly known in the art, the oligos are designed to bind to a complementary sequence under selected conditions
• nucleic acid i e DNA or RNA
• the nucleic acid for practicing the present invention may be obtained according to well known methods
• Oligonucleotide probes or primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed
• the oligonucleotide probes or primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system
• the oligonucleotide probes and primers can be designed by taking into consideration the melting point of hyd ⁇ zidation thereof with its targeted sequence (see below and in Sambrook et al , 1989, Molecular Cloning -A Laboratory Manual, 2nd Edition, CSH Laboratories, Ausubel et al , 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc , N Y )
• the term "oligonucleotide" or "DNA" molecule or sequence refers to a molecule comprised of the deoxyribonucleotides
• oligonucleotide or "DNA” can be found in linear DNA molecules or fragments, viruses, plasmids, vectors, chromosomes or synthetically derived DNA. As used herein, particular double-stranded DNA sequences may be described according to the normal convention of giving only the sequence in the ⁇ ' to 3' direction.
• Nucleic acid hybridization refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure. Examples of hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al., 1989, supra and Ausubel et al., 1989, supra) and are commonly known in the art.
• a nitrocellulose filter can be incubated overnight at 6 ⁇ °C with a labeled probe in a solution containing 50% formamide, high salt ( ⁇ x SSC or ⁇ x SSPE), ⁇ x Denhardt's solution, 1 % SDS, and 100 ⁇ g/ml denatured carrier DNA (i.e.
• the non-specifically binding probe can then be washed off the filter by several washes in 0.2 x SSC/0.1% SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 42°C (moderate stringency) or 6 ⁇ °C (high stringency).
• the selected temperature is based on the melting temperature (Tm) of the DNA hybrid.
• Tm melting temperature
• RNA-DNA hybrids can also be formed and detected.
• the conditions of hybridization and washing can be adapted according to well known methods by the person of ordinary skill. Stringent conditions will be preferably used (Sambrook et al.,1989, supra).
• Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and ⁇ -nucleotides and the like. Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann. Reports Med. Chem. 23:296 and Moran et al , 1987, Nucleic acid molecule. Acids Res., 14:5019 Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.
• RNA ribonucleic acid
• DNA deoxyribonucleic acid
• probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection). Although less preferred, labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds. More recently, PNAs have been described (Nielsen et al. 1999, Current Opin. Biotechnol. 10:71-76). PNAs could also be used to detect the polymorphisms of the present invention. Other detection methods include kits containing probes on a dipstick setup and the like.
• Probes can be labeled according to numerous well known methods (Sambrook et al , 1989, supra). Non-limiting examples of labels include 3 H, 14 C, 32 P, and 35 S. Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radionucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.
• radioactive nucleotides can be incorporated into probes of the invention by several methods.
• Non-limiting examples thereof include kinasing the 5' ends of the probes using gamma 32 P ATP and polynucleotide kinase, using the Klenow fragment of Pol I of E coll in the presence of radioactive dNTP (i e uniformly labeled DNA probe using random oligonucleotide primers in low-melt gels), using the SP6T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and
• oligonucleotides or “oligos” define a molecule having two or more nucleotides ( ⁇ bo or deoxynbonucleotides) The size of the oligo will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly by the person of ordinary skill
• An oligonucleotide can be synthetised chemically or derived by cloning according to well known methods
• a "primer” defines an oligonucleotide which is capable of annealing to a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions
• Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods See generally Kwoh et al , 1990, Am Biotechnol Lab 8 14-25 Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill Non-limiting examples of amplification techniques include polymerase chain reaction (PCR), hgase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the Q ⁇ replicase system and NASBA (Kwoh et al , 1989, Proc Natl Acad Sci USA 86, 1173-1177, Lizardi et al , 1988, BioTechnology 6 1197-1202, Malek et al , 1994, Methods Mol Biol , 28 253-260, and Sambrook et al , 1989, supra) Preferably, amplification will be carried out using PCR
• PCR Polymerase chain reaction
• a nucleic acid sample e.g., in the presence of a heat stable DNA polymerase
• An extension product of each primer which is synthesized is complementary to each of the two nucleic acid strands, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith.
• the extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers.
• the sample is analysed to assess whether the sequence or sequences to be detected are present. Detection of the amplified sequence may be carried out by visualization following EtBr staining of the DNA following gel electrophores, or using a detectable label in accordance with known techniques, and the like.
• EtBr staining of the DNA following gel electrophores, or using a detectable label in accordance with known techniques, and the like.
• Ligase chain reaction LCR is carried out in accordance with known techniques (Weiss, 1991 , Science 254:1292).
• SDA Strand displacement amplification
• the term "gene” is well known in the art and relates to a nucleic acid sequence defining a single protein or polypeptide.
• a "structural gene” defines a DNA sequence which is transcribed into RNA and translated into a protein having a specific amino acid sequence thereby giving rise the a specific polypeptide or protein. It will be readily recognized by the person of ordinary skill, that the nucleic acid sequence of the present invention can be incorporated into anyone of numerous established kit formats which are well known in the art A “heterologous" (i.e.
• heterologous gene region of a DNA molecule is a subsegment segment of DNA within a larger segment that is not found in association therewith in nature
• heterologous can be similarly used to define two polypeptidic segments not joined together in nature.
• Non-limiting examples of heterologous genes include reporter genes such as luciferase, chloramphenicol acetyl transferase, ⁇ -galactosidase, and the like which can be juxtaposed or joined to heterologous control regions or to heterologous polypeptides.
• vector is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned. Numerous types of vectors exist and are well known in the art.
• expression defines the process by which a gene is transcribed into mRNA (transcription), the mRNA is then being translated (translation) into one polypeptide (or protein) or more.
• expression vector defines a vector or vehicle as described above but designed to enable the expression of an inserted sequence following transformation into a host
• the cloned gene (inserted sequence) is usually placed under the control of control element sequences such as promoter sequences.
• control element sequences such as promoter sequences.
• the placing of a cloned gene under such control sequences is often refered to as being operably linked to control elements or sequences.
• Operably linked sequences may also include two segments that are transcribed onto the same RNA transcript
• two sequences such as a promoter and a "reporter sequence” are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence.
• two sequences In order to be "operably linked” it is not necessary that two sequences be immediately adjacent to one another
• Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host or both (shuttle vectors) and can additionally contain transcnptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites
• transcnptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites
• Prokaryotic expressions are useful for the preparation of large quantities of the protein encoded by the DNA sequence of interest
• This protein can be purified according to standard protocols that take advantage of the intrinsic properties thereof, such as size and charge (i e SDS gel electrophoresis, gel filtration, cent ⁇ fugation, ion exchange chromatography )
• the protein of interest can be purified via affinity chromatography using polyclonal or monoclonal antibodies The purified protein can be used for therapeutic applications
• the DNA construct can be a vector comprising a promoter that is operably linked to an oligonucleotide sequence of the present invention, which is in turn, operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule
• Promoter refers to a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence
• the promoter is bound at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background
• a transcription initiation site (conveniently defined by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase
• Eukaryotic promoters will often, but not always, contain "TATA" boses and "CCAT” boxes Prokaryotic promoters contain Shine-
• DNA sequence to which a factor binds] enabling estrogen- and vitamin D-dependent modulating effects of promoter activity are known in the art) operably linked to a chosen promoter and modulating the activity thereof, the promoter driving the expression of a reporter gene.
• the modulating effect of the promoter activity can be assessed by determining the level of expression of the reporter gene.
• the vector is transfected into a cell of a patient having the combination of genotypes of the ESR and VDR genes shown herein to predict a positive outcome to HRT, or in a cell from a patient having the combination of genotypes of the ESR and VDR genes shown herein to predict a negative outcome to HRT.
• ESR and VDR genes expressed by these cells can be modified at will (i.e. by in vitro mutagenesis or the like).
• numerous combinations of genotypes can be tested in such assay to dissect the functional relationship between the ESR and VDR genotypes.
• indicator cells expressing at least one of ESR and VDR could also be engineered by choosing a cell line and transfecting thereinto, chosen genotypes of the ESR and VDR genes and one expression vector as described above.
• Non-human transgenic animals expressing chosen combinations of the ESR and VDR genotypes could also be prepared and used to screen compounds that affect bone mineral density.
• the designation "functional derivative” denotes, in the context of a functional derivative of a sequence whether an nucleic acid or amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence.
• This functional derivative or equivalent may be a natural derivative or may be prepared synthetically.
• Such derivatives include amino acid sequences having substitutions, deletions, or additions of one or more ammo acids, provided that the biological activity of the protein is conserved
• nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained
• the substituting am o acid as chemico-physicai properties which are similar to that of the substituted amino acid
• the similar chemico-physicai properties include, similarities in charge, bulk ess, hydrophobicity, hydrophylicity and the like
• the term “functional derivatives" is intended to include “fragments", “segments", “variants”, “analogs” or "chemical derivatives" of the subject matter of the present invention
• variant refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention
• the functional derivatives of the present invention can be synthesized chemically or produced through recombinant DNA technology all these methods are well known in the art
• chemical derivatives is meant to cover additional chemical moieties not normally part of the subject matter of the invention Such moieties could affect the physico-chemical characteristic of the derivative (i e solubility, absorption, half life and the like, decrease of toxicity) Such moieties are exemplified in Remington's Pharmaceutical Sciences (1980) Methods of coupling these chemical-physical moieties to a polypeptide are well known in the art
• allele defines an alternative form of a gene which occupies a given locus on a chromosome
• a “mutation” is a detectable change in the genetic material which can be transmitted to a daughter cell
• a mutation can be, for example, a detectable change in one or more deoxynbonucleotide
• nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position.
• Spontaneous mutations and experimentally induced mutations exist.
• the result of a mutations of nucleic acid molecule is a mutant nucleic acid molecule.
• a mutant polypeptide can be encoded from this mutant nucleic acid molecule.
• the term "purified” refers to a molecule having been separated from a cellular component.
• a “purified protein” has been purified to a level not found in nature.
• a “substantially pure” molecule is a molecule that is lacking in all other cellular components.
• molecule As used herein, the terms “molecule”, “compound”, or “agent” are used interchangeably and broadly to refer to natural, synthetic or semi-synthetic molecules or compounds.
• the term “molecule” therefore denotes for example chemicals, macromolecules, cell or tissue extracts (from plants or animals) and the like.
• Non limiting examples of molecules include nucleic acid molecules, peptides, ligands, including antibodies, carbohydrates and pharmaceutical agents.
• the agents can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modelling methods such as computer modelling.
• rationally selected or “rationally designed” are meant to define compounds which have been chosen based on the configuration of the interaction domains of the present invention
• macromolecules having non-naturally occurring modifications are also within the scope of the term "molecule”.
• peptidomimetics well known in the pharmaceutical industry and generally referred to as peptide analogs can be generated by modelling as mentioned above.
• the polypeptides of the present invention are modified to enhance their stability It should be understood that in most cases this modification should not alter the biological activity of the protein
• the molecules identified in accordance with the teachings of the present invention have a therapeutic value in diseases or conditions in which the bone density of the animal is compromised by a combination of genotypes identified in accordance with the present invention
• the molecules identified in accordance with the teachings of the present invention find utility in the development of compounds which can modulate the bone density in an animal (i.e. positively modulate bone density and hence protect and/or treat against osteoporosis).
• agonists and antagonists also include potentiators of known compounds with such agonist or antagonist properties.
• modulators of the level or the activity of the ESR and VDR can be identified and selected by contacting the indicator cell with a compound or mixture or library of molecules for a fixed period of time.
• the "positive-outcome-predicting combination of genotypes of the ESR and VDR genotypes" can be used as positive controls.
• An indicator cell in accordance with the present invention can be used to identify antagonists
• the test molecule or molecules are incubated with the host cell in conjunction with one or more agonists held at a fixed concentration.
• An indication and relative strength of the antagonistic properties of the moiecule(s) can be provided by comparing the level of gene expression in the indicator cell in the presence of the agonist, in the absence of test molecules vs in the presence thereof.
• the antagonistic effect of a molecule can also be determined in the absence of agonist, simply by comparing the level of expression of the reporter gene product in the presence and absence of the test molecule(s).
• the "in vivo" experimental model can also be used to carry out an "in vitro” assay
• cellular extracts from the indicator cells can be prepared and used in an "in vitro” test.
• a non- limiting example thereof include binding assays
• indicator cells refers to cells that express a combination of genotypes of ESR and VDR according to the present invention. As alluded to above, such indicator cells can be used in the screening assays of the present invention. In certain embodiments, the indicator cells have been engineered so as to express a chosen derivative, fragment, homolog, or mutant of the combination of genotypes of the present invention.
• the cells can be yeast cells or higher eukaryotic cells such as mammalian cells. In one particular embodiment, the indicator cell would be a yeast cell harboring vectors enabling the use of the two hybrid system technology, as well known in the art (Ausubel et al., 1994, supra) and can be used to test a compound or a library thereof.
• the cis-trans assay as described in USP 4,981,784 can be adapted and used in accordance with the present invention.
• Such an indicator cell could be used to rapidly screen at high-throughput a vast array of test molecules.
• the reporter gene is luciferase or ⁇ -Gal.
• fusion protein it might be beneficial to express a fusion protein.
• Non limiting examples of such fusion proteins include a hemagiutinin fusions and Gluthione-S-transferase (GST) fusions and Maltose binding protein (MBP) fusions.
• GST Gluthione-S-transferase
• MBP Maltose binding protein
• it might be beneficial to introduce a protease cleavage site between the two polypeptide sequences which have been fused Such protease cleavage sites between two heterologously fused polypeptides are well known in the art
• the protein of the present invention it might also be beneficial to fuse the protein of the present invention to signal peptide sequences enabling a secretion of the fusion protein from the host cell.
• Signal peptides from diverse organisms are well known in the art.
• Bacterial OmpA and yeast Suc2 are two non limiting examples of proteins containing signal sequences.
• Such fusion protein find utility in the assays of the present invention as well as for purification purposes, detection purposes and the like.
• sequences and polypeptides useful to practice the invention include without being limited thereto mutants, homologs, subtypes, alleles and the like.
• sequences of the present invention should encode a functional (albeit defective) ESR or VDR. It will be clear to the person of ordinary skill that whether the ESR or VDR sequence of the present invention, variant, derivative, or fragment thereof retains its function, can be determined by using the teachings and assays of the present invention and the general teachings of the art.
• ESR and VDR protein of the present invention can be modified, for example by in vitro mutagenesis, to dissect the structure-function relationship thereof and permit a better design and identification of modulating compounds.
• some derivative or analogs having lost their biological function may still find utility, for example for raising antibodies.
• These antibodies could be used for detection or purification purposes.
• these antibodies could also act as competitive or non-competitive inhibitor and be found to be modulators of the activity of the ESR or VDR protein of the present invention
• a host cell or indicator cell has been "transfected" by exogenous or heterologous DNA (e.g. a DNA construct) when such DNA has been introduced inside the cell
• the transfecting DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
• the transfecting DNA may be maintained on a episomal element such as a plasmid.
• a stably transfected cell is one in which the transfecting DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication.
• DNA segments or proteins according to the present invention could be introduced into individuals in a number of ways.
• cells can be isolated from the afflicted individual, transformed with a DNA construct according to the invention and reintroduced to the afflicted individual in a number of ways.
• the DNA construct can be administered directly to the afflicted individual.
• the DNA construct can also be delivered through a vehicle such as a liposome, which can be designed to be targeted to a specific cell type, and engineered to be administered through different routes
• the prescribing medical professional will ultimately determine the appropriate form and dosage for a given patient, and this can be expected to vary according to the chosen therapeutic regimen (i.e. DNA construct, protein, cells), the response and condition of the patient as well as the severity of the disease.
• Composition within the scope of the present invention should contain the active agent (i.e. molecule, hormone) in an amount effective to achieve the desired therapeutic effect while avoiding adverse side effects.
• the nucleic acids in accordance with the present invention can be administered to mammals (i.e. humans) in doses ranging from 0.006 to 1 mg per kg of body weight per day of the mammal which is treated.
• compositions and salts of the active agent are within the scope of the present invention and are well known in the art (Remington's Pharmaceutical Science, 16th Ed., Mack Ed.)
• the amount administered should be chosen so as to avoid adverse side effects.
• the dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0.001 to 50 mg/kg/day will be administered to the mammal.
• the present invention relates to a kit for predicting the outcome of a treatment aimed at preventing and/or treating osteoporosis (i.e.
• kits in accordance with the present invention includes any kit in which reagents are contained in separate containers.
• Such containers include small glass containers, plastic containers or strips of plastic or paper Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
• Such containers will include in one particular embodiment a container which will accept the test sample (DNA protein or cells), a container which contains the primers used in the assay, containers which contain enzymes, containers which contain wash reagents, and containers which contain the reagents used to detect the extension products
• Figs 1A to 1C illustrate the effect of hormone replacement therapy on bone density by VDR genotype (A), ESR genotype (B) and VDR * ESR genotypes combinations (C)
• HRT hormone-replacement therapy
• % variance HRT represents the age-adjusted heel-SI variance attributable to the presence of HRT for ⁇ 5 years within each group, all women, VDR-bb/ESR-PP genotypes combination subgroup, rest of the cohort (after the exclusion of women bearing the
• VDR-bb/ESR-PP genotypes combination It was calculated as the ratio of the sum of squares due to the regression (for each of the three groups) between age-adjusted heel-SI and the presence of HRT ⁇ 5 years vs HRT ⁇ 5 years over the total sum of square, and
• SI results from a combination of speed of sound (SOS) and broadband ultrasound attenuation (BUA) measurements.
• SOS speed of sound
• BUA broadband ultrasound attenuation
• VDR Vitamin D receptor
• ESR1 Estrogen receptor 1
• VDR vitamin D receptor
• ESR1 estrogen receptor 1
• Bone density of the right calcaneal bone was determined in 425 subjects by broadband ultrasound attenuation (BUA) and the speed of sound (SOS) as measured using the AchilesTM ultrasound bone densitometer (Lunar corporation, Madison, Wisconsin, USA)
• the stiffness index (SI) a combination of BUA and SOS, was calculated from the manufacturer's equation and expressed as a percentage (t-score) of young adults
• the mean coefficient of variation for the SI (which includes errors of both the BUA and SOS) was below 1 % Acoustic phantoms provided by the manufacturer were scanned daily and showed no drift VDR-Bsml Genotype analysis
• Genomic DNA was isolated from peripheral blood leukocytes by a mini-method necessitating only 200 ⁇ l of whole blood where all steps are processed in a single 1 5 ml tube (Rousseau, F , et al , Hum Mut , 4 61-54, 1994) Isolated DNA (5-7 ⁇ g) was resuspended into 100 ⁇ l TE 20 5 buffer (20 mM Tns, 5 mM EDTA), heated at 65°C for 4 hours and stored at 4°C until PCR was performed
• VDR-Bsml polymorphism was amplified by PCR and digested as described in Morrison, N A , et al (Morrison, N A , et al , Nature, 367 284-287, 1994) After Bsml digestion, genotypes were visualized by ethidium bromide after migration in a 2% agarose gel electrophoresis Absence of the polymorphic site (B) resulted in a 850 bp fragment while presence of the polymorphic site (b) resulted in 700 bp and 150 bp fragments (Morrison, N A , et al , Nature, 367 284-287, 1994) ESR-Pvull genotype analysis
• VDR and ESR genotypes were interpreted blindly by three independent individuals and results that were not concordant for all three individuals were rejected
• VDR-bb/ESR-PP VDR-bb/ESR-PP on HRT ⁇ 5 Years
• VDR-bb/ESR-PP on HRT ⁇ 5 years subgroups for these confounding variables Height, total calcium intake, physical activity level, smoking and alcohol intake were not significantly correlated (p > 0.05) with age-adjusted heel-SI Statistical analyses were performed using the JMP 3.0 statistical package (SAS Institute, Cary, North Carolina) and Statview 4.5 (Abacus concepts, Berkely, California).

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