COMBINATION OF MARKERS AT THE ESTROGEN- AND VITAMIN D-RECEPTOR GENES OR EQUIVALENTS THEREOF TO PROGNOSE A RESPONSE TO OSTEOPOROSIS THERAPY

Abstract: The present invention relates to a method for determining an individual's predisposition to osteoporosis, development of osteoporosis and/or responsiveness 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's predisposition to osteoporosis, development of osteoporosis and/or responsiveness to therapy for osteoporosis.

Description: TITLE OF THE INVENTION COMBINATION OF MARKERS AT THE ESTROGEN- AND VITAMIN D- RECEPTOR GENES OR EQUIVALENTS THEREOF TO PROGNOSE A RESPONSE TO OSTEOPOROSIS THERAPY FIELD OF THE INVENTION 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 As well, 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 BACKGROUND OF THE INVENTION Osteoporosis is a multifactoπal 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 One current model of osteoporosis defines two distinct entities, namely type I (postmenopausal) osteoporosis and type II (age-related) osteoporosis 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. Genetic factors have always been considered an important component to individual risk for osteoporosis, but its role is incompletely understood. Indeed, studies of monozygotic (MZ) and dizygotic (DZ) twins have shown that BMD was more strongly correlated in MZ than DZ twin pairs, suggesting that BMD has a genetic component and that genetic factors could account for up to 80 to 90% of the variability in BMD. Although the molecular basis of osteoporosis is undetermined, segregation analyses suggest that its genetic control very likely includes many susceptibility genes. Apart from rare genetic mutations that significantly alter the function of a gene, the hypothesis underlying allelic association studies in osteoporosis is that genetic polymorphisms with small functional differences may increase or decrease risk for osteoporosis, resulting in significant differences in BD during perimenopause and/or thereafter A few candidate genes for osteoporosis have been studied so far. One thing remains, the molecular basis of osteoporosis and/or low bone density remains incompletely understood. Segregation analyses suggest that its genetic control very likely includes many succeptibility genes Polymorphisms in the vitamin D receptor (VDR) gene located on chromosome 12 have been reported to be correlated with the levels of osteocalcin (BGP), a marker of bone turnover. Another candidate gene with a potential role on individual variability in BD is the estrogen receptor gene (ESR1), located on chromosome 6 (U.S. P. 5,834,200) Also, recent reports on association between COLIAI1 variants and BMD in two different populations make this locus a strong candidate as one of the genes involved in genetic determinant of BMD, and possibly the risk of osteoporosis. In addition, interleukin 1 receptor antagonist, collagen type Iα1 and Iα2, transforming growth factor β1 , α 2 HS-glycoprotein, interleukin-6, and apoiipoprotein E have been related to osteoporosis as well. Recently, the polymorphic (CAG)n site in the androgen receptor gene has been associated with bone mineral density of the femoral neck and lumbar spine (Sowers et al. 1999, J. Bone Mineral Res. 14(8):1411-1419). However, the stratistical relationship between BMD and AR genotype was not very significant in terms of p-value (Sowers et al. supra). It has been suggested that common polymorphisms of (VDR) could significantly contribute to the variance of BMD in postmenopausal women It is now considered that the effect of VDR polymorphism alone on BMD is probably modest, as opposed to what was originally thought (Morrison, N.A., et al., Nature, 367:284-287, 1994 and USP 5,593,033). Moreover, it was recently found that common ESR polymorphisms could also be associated with BMD but others failed to observe such an effect. Analysis of the publications relating to the association between VDR or ESR and BMD and/or osteoporosis are contradictory (USP 5,834,200 and references therein). It would be highly desirable to be provided with a genetic assay for determining the predisposition to osteoporosis and/or response to osteoporosis treatment. While some markers have been identified as genetic determinants for BMD and/or as risk factors in osteoporosis, there remains a need to identify new markers therefor. More specifically, there remains a need to provide means to determine a predisposition to osteoporosis and/or low bone density and/or responsiveness to therapy to osteoporosis or to the prevention of low bone density, by analyzing allelic variations in genes associated with osteoporosis and/or low bone density. There also remains a need to identify the molecular determinants at the genetic level enabling women to significantly benefit from a hormone replacement therapy (HRT). In addition there remains a need to identify the patients who are likely to benefit from the HRT and those that will not show as good a level of osteoporosis protection Further, there remains a need to provide assays to screen for compounds (i e hormones, molecules acting on hormone receptors or other agents) that could be beneficial to the patients not showing as good a level of osteoporosis protection upon HRT The present invention seeks to meet these and other needs The present description refers to a number of documents, the content of which is herein incorporated by reference, in their entirety SUMMARY OF THE INVENTION 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 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, prognosis or treatment) 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 of ESR and VDR which can predict a much more significant response to HRT thus provides the means to better predict and adapt preventive and therapeutic measures for osteoporosis Having identified a combination of VDR and ESR (i e bb-PP) that predicts a significant response to HRT provides the means to identify agents which could enable the same type of HRT response in non bb-PP women In a particular embodiment, 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 benefit from osteoporosis treatment(s) and eventually predict their response to therapy or choose the optimal preventive pharmacotherapy Another aim of the present invention is to identify means of prediction and management of BMD as well as biological parameters for the establishment of population-based osteoporosis prevention and intervention programs In addition, it is an aim of the present invention to provide a method of selecting a combination of alleles which is suitable for designing an assay to screen compounds which can promote BMD and increase bone mass, thereby protecting against osteoporosis 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 In a particular embodiment, 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 Non-limiting examples of assays that could be used in accordance with the present invention include cis-trans assays similar to those described in USP 4,981 ,784 Of course, it will be understood that 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 It shall be understood that 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 include, COLIAI1, mterleuktn 1 receptor antagonist, collagen type Iα1 and Iα2, transforming growth β1 , α 2 HS- glycoprotein, ιnterleukιn-6, and apolipoprotem E (WO 94/03633) It shall also be understood that although osteoporosis is significantly more preponderant in women, it can also be a morbidity-inducing disease in men Thus, the present invention is meant to also cover men In accordance with the present invention, there is therefore provided 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 In accordance with the present invention there is provided 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's susceptibility to osteoporosis and/or response to therapy for osteoporosis Numerous methods for determining a genotype are known and available to the skilled artisan. All these genotype determination methods are within the scope of the present invention. In a preferred embodiment of the present invention, 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. In one particularly preferred embodiment, the amplification is carried out using polymerase chain reaction. In a particular embodiment, 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. In accordance with a preferred embodiment of the present invention, the pair of primers used for amplifying the segment of the estrogen receptor gene is defined as follows: δ'-TGCCACCCTA TCTGTATCTT TTCC-3' SEQ ID NO:1 and 5'-TCTTTCTCTG CCACCCTGGC GTC-3' SEQ ID NO:2. In accordance with a further preferred embodiment of the present invention, the pair of primers used for amplifying the segment of the vitamin D receptor gene is selected from the group consisting of: δ'-CAACCAAGAC TACAAGTACC GCGTCAGTGA-3' (SEQ ID NO:3) and δ'-TATCGTGAGT AAGGCAGGAG AGGGAGACC-3' (SEQ ID NO:4); and 5'-AGCTGGCCCT GGCACTGACT CTGCTCT-3' (SEQ ID NO:5) and δ'-ATGGAAACAC CTTGCTTCTT CTCCCTC-3' (SEQ ID NO:6). Of course, other primer pairs 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. In accordance with another embodiment of the present invention, 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. Similarly, 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. In order to provide a clear and consistent understanding of terms used in the present description, a number of definitions are provided hereinbelow. As used herein the term "RFLP" refers to restriction fragment length polymorphism. The terms "polymorphism", "DNA polymorphism" and the like, refer to any sequence in the human genome which exists in more than one version or variant in the population. The term "estrogen hormone- and vitamin D-related medical conditions" refers to, without limitation, any estrogen and vitamin D-dependent diseases The term "linkage disequilibrium" refers to any degree of non- δ 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 δ 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 polymorphisms in the vitamin D receptor gene are reported in Morπsson et al , 1994 (Nature 367 284-287) Various degrees of linkage disequilibrium can be encountered between two 0 genetic markers so that some are more closely associated than others The terms "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 The terms "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. It shall be recognized by the person skilled in the art to which the present invention pertains, that since some of the polymorphisms herein identified in the ESR and VDR genes can be within the coding region of the gene and therefore expressed, that 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 δ 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 δ' to 3' direction, from left to right, using the one letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission Unless defined otherwise, the scientific and technological δ terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains Generally, the procedures for cell cultures, infection, molecular biology methods and the like are common methods used in the art Such standard techniques can be found in reference manuals such as for example Sambrook et al (1989, 0 Molecular Cloning -A Laboratory Manual, Cold Spring Harbor Laboratories) and Ausubel et al (1994, Current Protocols in Molecular Biology, Wiley, New York) The present description refers to a number of routinely used recombinant DNA (rDNA) technology terms Nevertheless, definitions of selected examples of such rDNA terms are provided for clarity and consistency 5 As used herein, "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 synthesized DNA can be double-stranded or single-stranded (coding strand or non-coding strand [antisense]) The term "recombinant DNA" as known in the art refers to a DNA molecule resulting from the joining of DNA segments This is often referred to as genetic engineering The term "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 The terminology "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 The nucleic acid (i e DNA or RNA) 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 In general, 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 As commonly known in the art, the oligonucleotide probes and primers can be designed by taking into consideration the melting point of hydπzidation 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 adenine (A), guanine (G), thymine (T) and/or cytosine (C), in a double-stranded form, and comprises or includes a "regulatory element" according to the present invention, as the term is defined herein. The term "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 δ' 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. In the case of a hybridization to a nitrocellulose filter, as for example in the well known Southern blotting procedure, a nitrocellulose filter can be incubated overnight at 6δ°C with a labeled probe in a solution containing 50% formamide, high salt (δ x SSC or δ x SSPE), δ x Denhardt's solution, 1 % SDS, and 100 μg/ml denatured carrier DNA (i.e. salmon sperm DNA) 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 6δ°C (high stringency). The selected temperature is based on the melting temperature (Tm) of the DNA hybrid. Of course, RNA-DNA hybrids can also be formed and detected. In such cases, 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 α-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. The types of detection methods in which 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. Although the present invention is not specifically dependent on the use of a label for the detection of a particular nucleic acid sequence, such a label might be beneficial, by increasing the sensitivity of the detection. Furthermore, it enables automation. 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. As commonly known, 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 As used herein, "o gonucleotides" or "oligos" define a molecule having two or more nucleotides (πbo 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 As used herein, 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 Qβ 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 Polymerase chain reaction (PCR) is carried out in accordance with known techniques See, e g , U S Pat Nos 4,683,195, 4,683,202, 4,800,159, and 4,965,188 (the disclosures of all three U S Patent are incorporated herein by reference) In general, PCR involves, a treatment of a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) under hybridizing conditions, with one oligonucleotide primer for each strand of the specific sequence to be detected. 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. Following a sufficient number of rounds of synthesis of extension products, 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. For a review on PCR techniques (see PCR Protocols, A Guide to Methods and Amplifications, Michael et al. Eds, Acad. Press, 1990). Ligase chain reaction (LCR) is carried out in accordance with known techniques (Weiss, 1991 , Science 254:1292). Adaptation of the protocol to meet the desired needs can be carried out by a person of ordinary skill. Strand displacement amplification (SDA) is also carried out in accordance with known techniques or adaptations thereof to meet the particular needs (Walker et al., 1992, Proc. Natl. Acad. Sci. USA 89:392-396; and ibid., 1992, Nucleic Acids Res. 20:1691-1696). As used herein, 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. a 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 The term "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, β-galactosidase, and the like which can be juxtaposed or joined to heterologous control regions or to heterologous polypeptides. The term "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. The term "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. The terminology "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. 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 Thus, 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. 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 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, centπfugation, ion exchange chromatography ) In addition, 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 For purposes of the present invention, 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 Within the promoter will be found 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-Dalgarno sequences in addition to the -10 and -35 consensus sequences In accordance with one embodiment of the present invention, an expression vector can be constructed to assess the functionality of specific alleles of the ESR and VDR genes and of the interaction of such alleles Non- limiting examples of such expression vectors include a vector comprising the estrogen responsive element and/or vitamin D responsive element (the cis sequences [i.e. 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. When such a vector is tranfected in a cell expressing ESR and VDR, the modulating effect of the promoter activity can be assessed by determining the level of expression of the reporter gene. In one embodiment, 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. These cells can serve to screen for compounds that modulate the promoter activity, in order to identify compounds that could be used to treat especially, patients predicted to be significantly less responsive to HRT. Of course, it will be understood that the ESR and VDR genes expressed by these cells can be modified at will (i.e. by in vitro mutagenesis or the like). Similarly, numerous combinations of genotypes can be tested in such assay to dissect the functional relationship between the ESR and VDR genotypes. It will also be clear to the skilled artisan, that such 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. As used herein, 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 The same applies to derivatives of 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 When relating to a protein sequence, 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 Thus, the term "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 As used herein, "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 The term "allele" defines an alternative form of a gene which occupies a given locus on a chromosome As commonly known, a "mutation" is a detectable change in the genetic material which can be transmitted to a daughter cell As well known, a mutation can be, for example, a detectable change in one or more deoxynbonucleotide For example, 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. As used herein, the term "purified" refers to a molecule having been separated from a cellular component. Thus, for example, 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. 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. The terms "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 As will be understood by the person of ordinary skill, macromolecules having non-naturally occurring modifications are also within the scope of the term "molecule". For example, peptidomimetics, well known in the pharmaceutical industry and generally referred to as peptide analogs can be generated by modelling as mentioned above. Similarly, in a preferred embodiment, 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 Alternatively, 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). As used herein, agonists and antagonists also include potentiators of known compounds with such agonist or antagonist properties. In one embodiment, 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. In certain embodiments, 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 For example, 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. Of course, 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). It shall be understood that the "in vivo" experimental model can also be used to carry out an "in vitro" assay For example, cellular extracts from the indicator cells can be prepared and used in an "in vitro" test. A non- limiting example thereof include binding assays As used herein the recitation "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. In another embodiment, 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. In a particular embodiment, the reporter gene is luciferase or β-Gal. In some embodiments, it might be beneficial to express a fusion protein. The design of constructs therefor and the expression and production of fusion proteins and are well known in the art (Sambrook et al., 1989, supra; and Ausubel et al., 1994, supra). Non limiting examples of such fusion proteins include a hemagiutinin fusions and Gluthione-S-transferase (GST) fusions and Maltose binding protein (MBP) fusions. In certain embodiments, 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 In certain embodiments, 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. In certain embodiments, it might also be beneficial to introduce a linker (commonly known) between the interaction domain and the heterologous polypeptide portion. Such fusion protein find utility in the assays of the present invention as well as for purification purposes, detection purposes and the like. For certainty, the sequences and polypeptides useful to practice the invention include without being limited thereto mutants, homologs, subtypes, alleles and the like. It shall be understood that generally, the 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. It should be understood that the 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. However, 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. In addition, 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. In prokaryotes, yeast, and mammalian cells for example, the transfecting DNA may be maintained on a episomal element such as a plasmid. With respect to eukaryotic cells, 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. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transfecting DNA. Transfection methods are well known in the art (Sambrook et al., 1989, supra; Ausubel et al., 1994 supra). The use of a mammalian cell as indicator can provide the advantage of furnishing an intermediate factor, which permits for example the interaction of two polypeptides which are tested, that might not be present in lower eukaryotes or prokaryotes. It will be understood that extracts from mammalian cells for example could be used in certain embodiments, to compensate for the lack of certain factors. In general, techniques for preparing antibodies (including monoclonal antibodies and hybridomas) and for detecting antigens using antibodies are well known in the art (Campbell, 1984, In "Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology", Elsevier Science Publisher, Amsterdam, The Netherlands) and in Harlow et al., 1988 (in: Antibody-A Laboratory Manual, CSH Laboratories) The present invention also provides polyclonal, monoclonal antibodies, or humanized versions thereof, chimeric antibodies and the like which inhibit or neutralize their respective interaction domains and/or are specific thereto. From the specification and appended claims, the term therapeutic agent should be taken in a broad sense so as to also include a combination of at least two such therapeutic agents. Further, the DNA segments or proteins according to the present invention could be introduced into individuals in a number of ways. For example, 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. Alternatively, 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 For administration to humans, 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. Typically, 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. Pharmaceutically acceptable preparations 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.) For the administration of polypeptides, antagonists, agonists and the like, 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. HRT) comprising an assessment of a combination of genotypes at the ESR and VDR loci (or loci in linkage desiquilibrium therewith) using a nucleic acid fragment, a protein or a ligand, or a restriction enzyme in accordance with the present invention. For example, a compartmentalized kit 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 BRIEF DESCRIPTION OF THE DRAWINGS Having thus generally described the invention, reference will now be made to the accompanying drawings, showing by way of illustration a preferred embodiment thereof, and in which 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) Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments with reference to the accompanying drawing which is exemplary and should not be interpreted as limiting the scope of the present invention DESCRIPTION OF THE PREFERRED EMBODIMENT in accordance with the present invention, the effect of genotypes and genotype combinations on BD in women taking estrogen replacement therapy (HRT) compared with those not on HRT was investigated In this study 426 unrelated healthy postmenopausal women were both genotyped for common variants at the vitamin D (VDR) and estrogen (ESR) receptors and evaluated for their bone density (BD) as determined by age-adjusted right calcaneal bone ultradensitometry (heel-SI Z-score) Globally, HRT is highly associated with an increased heel-SI (p = 0 0005) Interestingly, in accordance with a preferred embodiment of the invention, it was found that a specific genotype combination (VDR-bb/ESR-PP) representing 9 5% of the cohort was responsible for 40-60% of the HRT effect on BD in the whole group Women bearing the VDR-bb/ESR-PP genotype combination who received HRT for 2 to δ years had a 20% higher heel-SI (SD difference = 1 3, p = 0 003) compared with those bearing the same genotypes combination whom had received HRT for < 5 years. This may represent a 2-3 times decrease in the risk of fracture. Our findings suggest that response to HRT may involve gene-gene interactions. In accordance with the present invention, the interaction between hormone replacement therapy and genotype on bone density was also investigated. Four hundreds and twenty five (425) healthy postmenopausal French-Canadian women were genotyped for VDR (Bsml) and ESR (Pvull) polymorphisms. These women had a previous BD measurement by a calcaneal bone ultrasound measurement (heel-SI), a technique that recently showed to be a good predictor of the risk of osteoporosis and which appears to discriminate between individuals with and without a risk of fracture as well as X-ray-based BMD measurements such as DXA. Furthermore, ultrasound densitometry is simple, portable, inexpensive and it represents an interesting alternative to BMD measurements as it evaluates different bone properties also related to susceptibility to bone fracture. The 425 women (mean age 64 ± 7 years) had an average of 15.6 years postmenopause; 48%(207/425) of the subjects had never received HRT while 32% (134/425) had received HRT for at least five years (12.8 + 6.7; mean + SD). When the whole cohort is considered, there is no association between either VDR or ESR polymorphisms and heel-SI. As a whole, women who received HRT for ≥ 5 years had a significantly higher mean heel-SI compared with women that received HRT for < 5 years (n = 425, SD difference = 0.4, p = 0.0005); this corresponds to a 6% difference in age-adjusted heel-SI. These findings show a highly significant difference in heel-SI between women who received HRT for ≥ 5 years compared with women who had received HRT for < 5 years in a relatively large scale cross-sectional study. Eventhough there has been very few long term placebo-control studies on the effect of hormone-replacement therapy (HRT) to prevent fracture risk in postmenopausal women, in a ten-year study, HRT was found to be a definite benefit. Many studies have demonstrated the benefits of HRT on BD, even after only two years of therapy. It was also found that response to HRT was heterogeneous, some women responding strongly to HRT while approximately 8% were considered as non-responders. This raises the possibility that genetic determinants as well as gene-environment interactions may modulate individual response to HRT. Therefore, in accordance with the present invention it was initially investigated for possible combined effects of HRT and either VDR or ESR polymorphisms separately on BD and found no particular interaction pattern other than an increased BD in women who received HRT (Figs. IA and IB). In Figs. 1A to 1C, Bone density is expressed as age-adjusted heel stiffness index (SI) Z-scores. Vitamin D receptor (VDR) Bsml and estrogen receptor (ESR) Pvull were genotyped by polymerase chain reaction as described below. For each genotype (1A, 1 B) or genotypes combination (1C), average heel-SI Z-score in women whom had received hormone replacement therapy (HRT) for at least five years (HORM ≥ 5 yrs) are shown as (♦) while those whom had received HRT for less than five years (HORM < 5 yrs) are shown as (D). The number of subjects in each genotype (1A, 1B) or genotype combination (1C) is shown in parentheses. Error bars represent standard errors When the whole cohort was analyzed irrespective of HRT, there was a significant statistical difference in the average age-adjusted heel-SI (Z-scores) between women who received HRT ≥ 5 years and women who received HRT < 5 years (p = 0.0005). The symbol "*" indicates p < 0.10, and the symbol " ** " indicates p > 0.06 for within VDR or ESR genotype comparisons (1 A, 1 B) between women in the HRT ≥ 5 years and women in the HRT < δ years subgroups. However, stratification of the sample by combined VDR and ESR genotypes showed that a specific genotype combination accounted for a major part of the global effect of HRT on BD Twenty one percent (21 %) of the variance in age-adjusted heel-SI could be attributed to HRT in women with the VDR-bb/ESR-PP genotype combination while only about 2% of the variance in age-adjusted heel-SI could be attributed to the influence of HRT when the other VDR/ESR genotypes combinations were considered (Table 1). In fact, this translates into a 1.3 SD (20%) difference (p = 0.003) in age-adjusted heel-SI between women bearing the VDR-bb/ESR-PP genotype combination who received HRT for ≥ δ years as compared to those who received HRT for < 5 five years (Fig. IC). Thorough analysis for cofounders did not reveal any difference. Moreover, these analyses were validated by comparing the findings of the present invention (using the age-adjusted heel-SI Z-scores for Caucasians available from the manufacturer's software) with results after age and weight adjustment from the sample analyzed in the present invention and results were similar. Data were also analyzed by comparing women currently receiving HRT with women not receiving HRT at the time of recruitment and results were similar. Table 1 ANOVA characteristics of the cohort as stratified by VDR/ESR genotypes combinations SS, sum of squares of the regression between age-adjusted heel-SI (Z-scores) and the presence of hormone replacement therapy (HRT, nominal variable) for ≥ 5 years vs < 5 years, % 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 SD difference, difference (in standard deviation inits) in age-adjusted heel-SI (Z-score) between women whom received HTR > 5 years and those whom received HRT < 5 Years The SI results from a combination of speed of sound (SOS) and broadband ultrasound attenuation (BUA) measurements. When BD date were analyzed using either SOS or BUA alone, results were similar (as with SI), so that the specific VDR/ESR genotypes combination-HRT interaction is reflected into both SI components, SOS and BUA. There are controversies over the actual genetic effect of common variants of candidate genes such as VDR and ESR on bone density, partly because these effects may be dissimilar between different populations and that several studies were on small samples, but also because it is difficult to control for the multiplicity of bone density determinants. Moreover, response to HRT among postmenopausal women is heterogeneous and a significant proportion of women keep loosing bone eventhough they are treated with HRT. Bone homeostasis is a complex biological process involving hormonal, biochemical and local factors and the mechanisms of hormonal responsiveness/resistance involve complex interactions. In accordance with the present invention, it was found that heterogeneity of the effect of HRT in postmenopausal women might be partly predicted by a combination of common polymorphisms at the VDR and ESR loci in this population This suggests that individual response to HRT is, at least partly, under the control of both VDR and ESR variants. Since both receptors are major BD determinants and that their actions are intertwined, it is conceivable that subtle differences in function due to polymorphisms in these genes may translate, after several decades, into significant qualitative as well as quantitative differences in target organs such as bone. However, the mechanism underlying VDR-ESR interactions resulting in an increased sensitivity to HRT in the specific VDR-bb/ESR-PP genotypes combination is unknown In the sample analyzed in the present invention, eventhough the majority of women appeared to obtain a beneficial effect of HRT on BD (p = 0.0005), only a minority of them (women bearing the VDR- bb/ESR-PP combination. 9.5% of the cohort) show a dramatic increase in BD when they received HRT for > 5 five years In fact, in the subgroup of women whom received HRT for > δ years, those carrying the VDR-bb/ESR-PP genotype combination had a significantly higher age-adjusted heel-SI compared with the rest of their cohort (SD difference = 1 0, p = 0 001 ), eventhough there was no difference in the subgroup of women whom had been on HRT for < δ years (p = 0 57) The benefits of HRT strongly outweigh its associated risks in women bearing the VDR-bb/ESR-PP genotypes combination, as long as this specific genotype combination is not shown to carry increased susceptibility to HRT associated undesired effects such as breast and endometnal tumors It is not unexpected that the increased benefit of HRT on BD in women carrying the VDR-bb/ESR-PP genotypes combination could disappear in these women if they were to choose alternative bone preserving therapies, such as biphosphonates, that operate under another mechanism unrelated to HRT In accordance with another embodiment of the invention, the Combined Vitamin D receptor (VDR) gene and Estrogen receptor 1 (ESR1) gene polymorphisms can be used as markers of bone response to osteoporosis therapy In accordance with a further embodiment of the present invention, there is also provided the use of combined vitamin D receptor (VDR) gene and estrogen receptor 1 (ESR1 ) gene polymorphisms, or polymorphisms in linkage disequilibrium, as a test for predicted response to osteoporosis therapy, as a test to classify patients in clinical trials for osteoporosis therapy, as a test for predicted response to osteoporosis therapy, as a test to screen for new drugs for osteoporosis therapy, or as a test to determine the best treatment for osteoporosis The present invention will be more readily understood by referring to the following example which is given to illustrate the invention rather than to limit its scope EXAMPLE 1 Combined Polymorphisms of the Vitamin D Receptor Gene and of the Estrogen Receptor Gene as a Marker for Osteoporosis Recruitment was achieved through voluntary response to local newspaper advertisement for a study on genetic and environmental factors affecting BMD in healthy postmenopausal women After informed and written consent was obtained, the subjects answered a detailed questionnaire derived from the Mediterranean osteoporosis (MEDOS) study questionnaire All women subsequently had a right heel BD measurement and a blood sample drawn All recruited women were included unless they had a medical condition affecting bone homeostasis or had used medications known to influence bone metabolism other than regular HRT Four hundreds and twenty five (425) healthy unrelated postmenopausal women aged between 42 and 85 were included in the study They were considered postmenopaused if they did not have their menses for at least one year before recruitment They were all Caucasian French-Canadian women living in the Quebec city metropolitan area, a region of about 600 000 inhabitants The project was approved by the hospital's ethics committee Bone density measurements 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 Achiles™ 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 μl 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 μg) was resuspended into 100 μl 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 The 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 The same subjects were genotyped for a bialle c Pvull restriction site polymorphism in the first mtron of the ESR Two hundreds (200) ng of genomic DNA was amplified in 100 μl containing 0 δ μM of both forward 5'-TGCCACCCTA TCTGTATCTT TTCC-3' (SEQ ID NO 1) and reverse 5'-TCTTTCTCTG CCACCCTGGC GTC-3' (SEQ ID NO 2) primers, 200 pM of the four deoxynbonucleotides and 2 5 U of Taq polymerase and buffer (Promega Corporation, Madison, Wl) in 1 6 mM MgCI 2 PCR amplification included the following steps initial denaturation at 96°C for 7 mm followed by 35 cycles of amplification with denaturation at 94°C for 60 sec, annealing at 60°C for 60 sec and polymerase extension at 72°C for 4 mm A final extension at 72°C for 10 mm was also performed About 500 ng of the PCR product were digested with Pvull ESR (New England biolabs, Beverly, Ma) overnight at 37°C and the polymorphisms were visualized by ethidium bromide staining after migration in a 2% agarose gel electrophoresis Absence of the site (P) resulted into a 1 3 Kb fragment whereas presence of the Pvull site (p) resulted into 850 bp and 450 bp fragments. Data validation and statistical analyses VDR and ESR genotypes were interpreted blindly by three independent individuals and results that were not concordant for all three individuals were rejected Average age-adjusted heel-SI (Z-score) between the three genotypes for each biallelic loci (VDR and ESR) was compared by factorial analysis of variance (ANOVA) while unpaired t-test was used for two-way comparisons. Whether the age-adjusted heel SI (Z-score) formula available for Caucasians from the manufacturer's softwares (Lunar corporation, Madison, Wisconsin) could be applied in the sample tested in the present invention Was validated SI Z-scores (derived from the manufacturer's formulas) were compared with Z-scores after adjustment for age and weight derived from the sample's data obtained between VDR or ESR genotypes and similar results were found. Thereafter, data using aged-adjusted Z-scores generated from the manufacturer's software were analyzed since comparisons with other populations as well as assessment of fracture risk can be more easily inferred from Z-scores than from adjustment specific from the sample. Analysis of possible confounding variables showed that the number of years postmenopause (p < 0.0001), weight (p < 0.0001), body mass index (p < 0.0001) and numbers of years on HRT (p = 0.02) were significantly correlated with age-adjusted heel-SI. However, there was no difference (p > 0.5), except for the number of years of HRT, in these cofounders between the HRT < 5 years and HRT > 5 years subgroups. Irrespective of HRT, there was no difference for these cofounders when the VDR-bb/ESR-PP subgroup was compared with the rest of their cohort. In both the HRT ≥ 5 years and HRT < 5 years subgroups, there was no difference when the VDR-bb/ESR-PP subgroup was compared with the rest of their cohort. Finally, there was no difference between the VDR-bb/ESR-PP on HRT < 5 Years and 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). Although the present invention has been described hereinabove by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims SEQUENCE LISTING: 5'-TGCCACCCTA TCTGTATCTT TTCC-3' SEQ ID NO: 1 5'-TCTTTCTCTG CCACCCTGGC GTC-3' SEQ ID NO:2 5'-CAACCAAGAC TACAAGTACC GCGTCAGTGA-3' SEQ ID NO:3 5'-TATCGTGAGT AAGGCAGGAG AGGGAGACC-3' SEQ ID NO:4 5'-AGCTGGCCCT GGCACTGACT CTGCTCT-3' SEQ ID NO:5 δ'-ATGGAAACAC CTTGCTTCTT CTCCCTC-3' SEQ ID NO:6

Claims: WHAT IS CLAIMED IS: 1 A method for determining susceptibility to osteoporosis, and/or response to therapy for osteoporosis of a human patient, said method comprising the step of determining a combination of an estrogen receptor polymorphism or a polymorphism in linkage disequilibrium therewith and a vitamin D receptor polymorphism or a polymorphism in linkage disequilibrium therewith, of an individual in a biological sample of said human patient, wherein said estrogen receptor polymorphism is selected from a Pvull polymorphism located in intron 1 of the estrogen receptor gene and a DNA variant, equivalent, or mutation which shows linkage disequilibrium with one of the allele of said Pvull polymorphism, and wherein said vitamin D receptor polymorphism is selected from a Bsml polymorphism located in intron 8 of the vitamin D receptor gene and a DNA variant, equivalent, or mutation which shows linkage disequilibrium with one of the allele of said Bsml polymorphism 2 The method of claim 1 , wherein at least one of the steps of determining the estrogen receptor genotype, the vitamin D receptor genotype or the genotypes in linkage disequilibrium therewith comprises restriction endonuclease digestion 3 The method of claim 1 , wherein at least one of the steps of determining the estrogen receptor genotype, the vitamin D receptor genotype or the genotypes in linkage disequilibrium therewith comprises hybridizing with an allele specific oligonucleotide 4 The method of claim 2, which further comprises a step, prior to determining the estrogen receptor genotype, the vitamin D receptor genotype or the genotypes in linkage disequilibrium therewith, of amplifying a segment of the estrogen receptor gene using polymerase chain reaction. 5 The method of claim 3, which further comprises a step, δ prior to determining the estrogen receptor genotype and the vitamin D receptor genotype or the genotypes in linkage disequilibrium therewith, of amplifying a segment of the estrogen receptor gene using polymerase chain reaction. 6 The method of claim 2, which further comprises a step, 0 prior to determining the estrogen receptor genotype and the vitamin D receptor genotype or the genotypes in linkage disequilibrium therewith, of amplifying a segment of the vitamin D receptor gene using polymerase chain reaction. 7. The method of claim 3, which further comprises a step, 5 prior to determining the estrogen receptor genotype and the vitamin D receptor genotype or the genotypes in linkage disequilibrium therewith, of amplifying a segment of the vitamin D receptor gene using polymerase chain reaction 8 The method of claim 2, wherein endonuclease Pvull or 0 isoschizomers thereof is used for determining the estrogen receptor genotype. 9. The method of claim 2, wherein at least one of endonuclease Bsml, Taql, Apal or Fokl, or isoschizomers thereof is used for 6 determining the vitamin D receptor genotype 10. The method of claim 4, wherein a pair of primers derived from a nucleic acid sequence of the estrogen receptor gene or flanking said gene is used in the polymerase chain reaction δ 11. The method of claim δ, wherein a pair of primers derived from a nucleic acid sequence of the vitamin D receptor gene or flanking said gene is used in the polymerase chain reaction. 12. The method of claims 10, wherein the segment of the 0 estrogen receptor gene is amplified using a pair of primers as defined as follows: δ'-TGCCACCCTA TCTGTATCTT TTCC-3' SEQ ID NO:1 and δ'-TCTTTCTCTG CCACCCTGGC GTC-3' SEQ ID NO:2. 6 13. The method of claims 11 , wherein the segment of the estrogen receptor gene is amplified using a pair of primers as defined as follows: 5'-TGCCACCCTA TCTGTATCTT TTCC-3' SEQ ID NO:1 and 5'-TCTTTCTCTG CCACCCTGGC GTC-3' SEQ ID NO:2. 0 14. The method of claims 10, wherein the segment of the vitamin D receptor gene is amplified using a pair of primers selected from the group consisting of: 5'-CAACCAAGAC TACAAGTACC GCGTCAGTGA-3' (SEQ ID NO: 3) and 6 δ'-TATCGTGAGT AAGGCAGGAG AGGGAGACC-3' (SEQ ID NO:4); and 5'-AGCTGGCCCT GGCACTGACT CTGCTCT-3' (SEQ ID NO.5) and δ'-ATGGAAACAC CTTGCTTCTT CTCCCTC-3' (SEQ ID NO:6) 15 The method of claim 2, wherein 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 the restriction endonuclease digestion 16 The method of claim 2, wherein 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 17 The method of claim 1 , wherein said sample is a DNA sample 18 The method of claim 1 , wherein said sample is an RNA sample 19 The method of claim 1 , wherein said sample is a protein sample 20 The method of claim 1 , wherein said determining of polymorphism comprises a determination of the nucleic acid sequence of a resulting amplified fragment 21 The method of claim 1 wherein said combination of polymorphisms comprises at least one oligonucleotide specific to a normal or variant estrogen receptor gene allele, vitamin D receptor gene allele or a normal or variant marker in linkage disequilibrium with said estrogen receptor gene allele and/or said vitamin D receptor gene allele 22 The method of one of the preceding claims, wherein low bone density is indicative of a predisposition of said patient to suffer from osteoporosis and/or bone fracture during post-menopause, while high bone density is indicative of a predisposition of said patient to be resistant to osteoporosis and/or bone fracture during post-menopause 23 A prognosis kit for determining an outcome of a osteoporosis treatment or prevention program for a human patient comprising (a) at least one nucleic acid fragment specific for estrogen receptor (ESR), wherein said fragment enables an assessment of the ESR polymorphism in the first intron of the ESR gene, or specific for a variant, equivalent, or mutation which shows linkage disequilibrium with one of the alleles of said ESR polymorphism, (b) at least one nucleic acid fragment specific for vitamin D receptor (VDR), wherein said fragment enables an assessment of the VDR polymorphism in intron 8 of the VDR gene, or specific for a variant, equivalent, or mutation which shows linkage disequilibrium with one of the alleles of said VDR polymorphism, whereby a combination of polymorphisms at the ESR and VDR genes, or markers in linkage disequilibrium therewith enables said determination of the outcome of said treatment 24 The kit of claim 23, wherein said treatment is a hormone replacement treatment 25 The method of any of the preceding claims, which further comprises determining the genotype of at least one other marker associated with predisposition to osteoporosis and/or low bone density 26. The method of claim 25, wherein said at least one other marker is selected from the androgen receptor gene (AR), COLIAI1 variants, interleukin 1 receptor antagonist, collagen type Iα1 and Iα2, transforming growth factor β1 , α 2 HS-glycoprotein, interleukin-6, and apolipoprotein E. 27 A method of using specific combinations of estrogen receptor alleles and vitamin D receptor alleles, or a variant, equivalent, or mutation thereof which shows linkage disequilibrium therewith, to set-up a screening assay for agents destined to modulate estrogen receptor and/or vitamin D receptor function for the purpose of increasing bone density and/or potentiating an action of an osteoporosis treatment program. 28. An assay for screening and selecting an agent which modulates bone density comprising: a) an expression vector comprising a promoter operably linked to a reporter gene, said promoter comprising an estrogen receptor response element and a vitamin D response element, said response elements affecting the activity of said promoter upon binding thereto of estrogen and vitamin D; b) a cell expressing a chosen allele of an estrogen receptor and a chosen allele of a vitamin D receptor, and harboring said vector of a); c) submitting said cell to at least one agent; and d) assaying a level of said reporte gene, whereby an agent can be selected when the level of said reporter gene is significantly modulated by the presence of said agent.

Inventors: ROUSSEAU FRANCOIS, ROUSSEAU, FRANCOIS
Applicants: ROUSSEAU FRANCOIS, SIGNALGENE INC, ROUSSEAU, FRANCOIS, SIGNALGENE INC.
Application year: 1999
Main classification: C12Q1
IPC codes: C12Q1