WO2006091092A1

WO2006091092A1 - Polymorphism analysis of the estrogen receptor and vitamin d receptor to predict therapy responsiveness in the treatment of osteoporosis

Info

Publication number: WO2006091092A1
Application number: PCT/NL2006/000106
Authority: WO
Inventors: Chris J. Mugglestone
Original assignee: Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno
Priority date: 2005-02-28
Filing date: 2006-02-28
Publication date: 2006-08-31

Abstract

The invention relates to the treatment of osteoporosis. More specifically, it relates to genetic markers that allow to predict the response to calcium/vitamin D therapy in an osteoporotic subject. Provided is a method for predicting whether an osteoporotic subject will respond to calcium / vitamin D therapy, comprising a) isolating genomic DNA from said subject; b) determining the presence or absence of at least one polymorphism in the genomic DNA, wherein said polymorphism is selected from the group of polymorphisms consisting of the T/C estrogen receptor-a codon 10 (ER10) polymorphism, the C/G estrogen receptor-a codon 325 (ER325) polymorphism, the G/A vitamin D receptor Bsm1 (VDR BsmI) polymorphism, the T/C vitamin D receptor Fok1 (VDR FokI) polymorphism and the T/C vitamin D receptor TaqI (VDR TaqI) polymorphism; and c) correlating the presence or absence of said polymorphism with the responsiveness to calcium /vitamin D therapy.

Description

POLYMORPHISM ANALYSIS OF THE ESTROGEN RECEPTOR AND VITAMIN D RECEPTOR TO PREDICT THERAPY RESPONSIVENESS IN THE TREATMENT OF OSTEOPOROSIS

The invention relates to the treatment of osteoporosis. More specifically, it relates to genetic markers that allow to predict the response to calcium/vitamin D therapy in an osteoporotic subject.

Post-menopausal osteoporosis is a major healthcare problem that results in significant morbidity and mortality (Genant et al. 1999). The disease affects 75 million people in the USA, Europe and Japan combined. It is characterized by low bone mass and micro-architectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fractures, most frequently of the vertebrae, the proximal femur and the distal forearm. In 1994 a study group from the WHO established a more operational definition of osteoporosis that is based on the measurement of bone mineral density (BMD): a BMD more than 2.5 SD below the mean value of peak bone mass in healthy young adults. Several recent studies have confirmed that the use of calcaneal ultrasound to diagnose osteoporosis is comparable to the use of axial BMD (Frost et al. 2001; Greenspan et al., 2001; Langton et al, 2000).

The genetic basis for osteoporosis is not fully conclusive, and no single gene has been shown to reliably predict osteoporosis risk. This could be because osteoporosis is a polygenic disorder, determined by the effects of several genes, each with modest effect on bone mass and other determinants of fracture risk (Stewart et al, 2000). Population based studies and case controlled studies have identified polymorphisms in several genes, notably vitamin D receptor (VDR)(Sheehan et al 2001, Uitterlinden et al 2001, Eissman 2001, Owada et al, 1996), collagen (COLlAl) (Uitterlinden et al 2001, Efstathiadou et al 2001, Mcgurgan et al 2001, Mann V 2001, Uitterlinden et al, 1998), oestrogen receptor (ER) (Albagha et al 2001, Salmen et al 2000, Ongphiphadhanakul et al, 2000) and calcitonin/parathyroid hormone receptors (Hosoi et al, 1999, Masi et al, 1998, Taboulet et al, 1998). Interestingly, these polymorphisms are associated with the putative targets of the four classes of drugs most commonly being used. Other genetic variations implicated include the interleukin-6 locus (Ota et al, 1999), the interleukin 1 receptor antagonist (IL- IRA) gene (Keen et al, 1998), the matrix GIa protein gene (MGP) (Tsukamoto et al, 2000), the calcium-sensing receptor (CASR) gene (Tsukamoto et al, 1998), tumor necrosis factor receptor 2 (TNFR2) gene (Spotila et al, 2000) and the transforming growth factor-betal (TGF-betal) gene (Bertoldo et al, 2000). At present, four broad classes of drug treatment are commonly used to treat osteoporosis, namely calcium/vitamin D supplement, bisphosphonates, hormone replacement therapy (HRT) and calcitonin. For every class of treatment there are a significant number of treated patients who are considered 'non-responders'. Although no definitive explanation is available, a genetic basis for a subject being either a "responder" or a 'non-responder" to a particular therapy is being widely investigated. Ca/vit D therapy is regarded as a basic requirement for all therapies for osteoporosis and is, of the four classes, the most often prescribed for the treatment of osteoporosis.

Thus, there is a clear clinical need for a diagnostic test or assay to identify patients who are likely not to benefit from Ca/vit D supplementation, for example vitamin D sufficient subjects, and thus should be considered for alternative therapy. Furthermore, it would be desirable to predict the therapy responsiveness of a subject who would theoretically benefit from Ca/vit D therapy, i.e. a vitamin D deficient subject. This would enable a medical practitioner to distinguish between "responders" and "non-responders" prior to treatment, aiding in selecting the appropriate therapy for that individual patient.

In search for genetic markers that aid in identifying an osteoporotic subject that would benefit from Ca/vitD therapy before the onset of therapy, the present inventors set out to investigate the occurrence of known single nucleotide polymorphisms (SNPs) of the vitamin D receptor (VDR) and the human estrogen receptor-α (ER) in osteoporotic patients. The design of the study is described in detail below. Briefly, non-osteoporotic subjects served as a control group. Osteoporotic subjects received oral calcium/vit D supplementation therapy during 3 months. Before and after the treatment, biochemical analysis was performed to determine whether or not a subject had responded to the therapy. Furthermore, all subjects were genotyped for the following single nucleotide polymorphisms: ERlO, ER325, VDR Bsml, VDR Fokl and VDRTaql. Genomic analysis was performed by PCR using pairs of dual labeled probes. It was found that subjects who responded to Ca/vitamin D treatment (responders) and those who did not (non-responders) segregated towards specific genotypes. Herewith, the invention provides a method for predicting whether an osteoporotic subject, preferably a human subject, will respond to calcium / vitamin D therapy. A method of the invention comprises the steps of a) isolating genomic DNA from said subject; b) determining the presence or absence of at least one (germline) polymorphism in the genomic DNA, wherein said polymorphism is selected from the group of polymorphisms consisting of the T/C estrogen receptor-α codon 10 (ERlO) polymorphism, the C/G estrogen receptor-α codon 325 (EK325) polymorphism, the G/A vitamin D receptor Bsnil (VDR Bsml) polymorphism, the T/C vitamin D receptor Fokl (VDR Fold) polymorphism and the T/C vitamin D receptor Taql (VDR Taql) polymorphism; and c) correlating the presence or absence of said at least one polymorphism with the responsiveness to calcium /Vitamin D therapy.

The VDR and ER genes were selected based on the hypothesis that calcium metabolism (systemically or locally) in a subject, and therewith also responsiveness to calcium therapy, is genetically regulated. For example, it had been demonstrated that Ca metabolism is dependent on certain VDR genotypes, and consequently higher doses of calcium/vitamin D may be required (Dawson-Hughes, et al, 1995). The relationship between VDR genotype and estrogen had also been suggested from the results of a study examining the effects of Ca intake on BMD in VDR-genotyped peri- menopausal women (MacDonald, et al, 2000).

EP-A-I 054 066 and US 5,593,833 do mention some correlation between the absence or presence of a polymorphism and therapy responsiveness. EP-A-I 054 066 provides a method for anticipating the sensitivity to a plurality of therapies for osteoporosis by determining polymorphisms of VDR, ER and ApoE. Yet, they did not address responsiveness to the combined calcium/vitamin D therapy and they used different polymorphisms in the ER gene. Further, they conclude that the VDR genotype B(-) having no wild-type allele, i.e. homozygous for the VDR Bsml polymorphism has a high sensitivity to vitamin D. This is in contrast with the date presented here.

Also US 5,593,833 describes a correlation between VDR Bsml and therapy responsiveness. The overall conclusion with respect to calcitriol treatment are, however, not equivocal; the publication fails to clearly teach on basis of which genotype a subject should be eligible for calcitriol treatment.

Jurada S. et al. (J. Steroid Biochem. MoI. Biol. 78:15-20, 2001), Hoshino S. et al. (J. Bone Miner. Metab. 18:153-157, 2000), Rapuri P.B. et al. (J. Steroid Biochem. MoI. Biol. 89:503-506, 2004) and Thakkinstain A. et al. (Osteopor. Int. 15:729-734, 2004)' all disclose the association of various polymorphisms with clinical parameters related to osteoporosis, such as bone mineral density, urinary calcium excretion and rate of bone loss. However, they do not mention any relationship between the polymorphisms and therapy responsiveness, which is the focus of the present invention.

Tables IA, IB and 1C show the genetic and biochemical data of, respectively, the responders to calcium/vitamin D therapy, non-responders and controls. Tables 2A through 2E list the segregation of each genotype (either homozygous for the wild-type allele, heterozygous for the wild-type and polymorphic allele or homozygous for the polymorphic allele) within each of the groups. Table 2A shows that, of the total number of responders analyzed, 41% has the non-polymorphic EElO T/T genotype; 36% is heterozygous and 23% is homozygous for the ERlO polymorphism. Accordingly, the frequency of the wild-type T-allele among the responders is 59% and that of polymorphic C-allele 41%. The numbers between brackets in Tables 2A-E indicate the probability that a subject with that genotype will have the particular phenotype. For example, a subject with the ERlO T/T genotype has a 63% chance of being a responder, 22% chance of being a non-responder and 15% chance of being a control.

The present findings can be used in a diagnostic test to identify patients who are less likely to respond to calcium and vitamin D treatment and should therefore be considered for an alternative therapy, such as bisphosphonates, hormone replacement therapy (HRT) and/or calcitonin. Such a test may comprise the analysis of a single (e.g. ERlO) or multiple genomic polymorphisms. For example, the allele frequency of the VDR Fokl polymorphism is detected in combination with the HER325 and/or the VDR Taql allele frequency.

The term "osteoporotic subject" as used herein refers to a person who is diagnosed with osteoporosis based on standard clinical criteria, for instance quantitative heel (calcaneal) ultrasound (QUS), blood analysis for standard biochemistry, vitamin D, parathyroid hormone (PTH) and calcium, and urine analysis for deoxypyrodinoline (DPD) and calcium. The osteoporotic subject is often a female human subject of post-menopausal age, e.g. 50 years or older. Ca/vit D supplementation therapy is typically prescribed only to those patients who are known to be deficient in vitamin D, as these subjects are most likely to benefit from Ca/vit D therapy. Vitamin D sufficient osteoporotic patients should be considered for an alternative therapy. Thus, predicting the responsiveness to a Ca/Vit D supplementation therapy is particularly relevant for those patients who, in theory, are most likely to benefit from such a therapy. The term "responsive" indicates that a significant change in one or more relevant parameters can be observed in an subject upon exposure to calcium/vitamin D supplement therapy. Examples of relevant parameters include vitamin D levels and PTH levels in the blood. Preferably, the PTH level in a responsive subject is increased with more than 10 pmol/1 following a 3 months therapy with oral calcium/vitamin D. "Calcium/vitamin D supplement therapy" refers to conventional combined calcium/vitamin D therapies prescribed for osteoporotic patients, typically the daily oral intake of calcium (e.g. 1,200 mg) and vitamin D (e.g. 400-800 units). Various types of commercially available drugs are suitably used in such oral supplementation therapies, for instance Adcal-D3®. Table 2A shows that the ERlO genotype of responders associated with a high percentage (41%) of homozygosity for the wt allele (T/T genotype) compared with non- responders (14%) and controls (10%). In other words, an osteoporotic person without the ERlO polymorphism has a 63% chance (41/(41+14+10)) of being a responder to Ca/VitD therapy versus a 22% change of being a non-responder. The allelic frequency of polymorphic C allele is 41% in responders versus 61% and 50% in non-responders and controls, respectively. Genotyping an osteoporotic subject for the presence of the ERlO polymorphism can thus be used to predict whether the subject will benefit from vitD/ca therapy. If the subject is homozygous for the wild-type T-allele (i.e. the ERlO polymorphism being absent), , the subject has an increased chance of being responsive. If a heterozygous T/C or a homozyhous C/C genotype is observed, the subject is more likely to be unresponsive. Thus, the presence of the polymorphic C allele in a subject is indicative of a reduced responsiveness to VitD/ca therapy. Such subjects could be given either an increased dosage (in case of vitamin D deficiency) or alternative therapy.

Analysis of the ER325 genotype in the different groups of subjects revealed that homozygosity of the polymorphic G-allele tends to associate with the non- responsive phenotype (see Table 2B). Two of the 14 non-responsive subjects displayed the G/G genotype, whereas this genotype was observed in only 1 out of 22 responders and in none of the control subjects. The allelic frequency of the polymorphic G-allele is slightly increased in non-responders (29% versus 23% and 20% for responders and controls, respectively). Thus, in case a subject is homozygous for the ER325 polymorphism, there is an increased chance that the subject will not respond to Vit D/Ca therapy.

Tables 2C, 2D and 2E show the results of the three polymorphisms of the VDR that were investigated. The VDR Bsml polymorphism (Table 2C) involves a G to A transition. Some correlation was observed between the absence of the VDR Bsml polymorphism (i.e. homozygosity for the wild-type G-allele) and therapy responsiveness: 41% of the subjects with the G/G genotype was responsive, while 33% was a non-responsive subject and 26% belonged to the controls. The heterozygous G/A phenotype was relatively more frequent in the control group. Determining the presence ox absence of the polymorphic A-allele can thus be used to predict whether an osteoporotic subject will respond to vitD/ca therapy. If the VDR Bsml polymorphism is absent.

The VDR Fold polymorphism (Table 2D) involves a T to C transition. A homozygous wild-type genotype (T/T) was found in only 10% of the control subjects compared with nearly 70% in the osteoporotic groups of responders and non- responders. A subject with a heterozygous genotype has a 70% probability to belong to the control group, and a probability of 21% and 9% to be a responder or a non- responder, respectively. Thus, an osteoporotic subject who is heterozygous for VDRFokI has an increased chance of being responsive, whereas a homozygous polymorphic VDR Fold genotype is indicative of being unresponsive. The published allelic frequency of the VDR Fokl polymorphism in a healthy control population is 37%. The present study shows that the frequency of the polymorphic C-allele is reduced (23/27%) in osteoporotic subjects. This indicates that the VDR Fokl genotype of a female osteoporotic population differs from the normal population. Herewith, the invention also provides a method to diagnose or screen for osteoporosis in a subject, preferably a female aged 50 years or older, comprising isolating genomic DNA from said subject; determining the presence or absence of the T/C vitamin D receptor Fokl (VDR Fold) polymorphism in said genomic DNA; and correlating the presence or absence of said VDR Fold polymorphism with a risk of having or developing osteoporosis, wherein the absence of said polymorphism is indicative of an increased risk.

The VDR Taql polymorphism (Table 2E) also involves a T to C transition. A responsive phenotype is relatively often associated with a homozygous T/T genotype and less often with a heterozygous T/C genotype compared with non-responders or controls. The allelic frequency of the polymorphic C-allele is 50% in the non- responders versus 43 and 44% in the responders and controls, respectively. Thus, if an osteoporotic subject carries the polymorphic C-allele, she has an increased chance of being unresponsive to vit D/ca therapy. The method provided herein to predict a subject's responsiveness to calcium/vitD supplementation therapy only requires a determination of the subject's genotype with regard to one of the five germline polymorphisms mentioned above. To that end, genomic DNA can be isolated from the subject using standard techniques. Preferably, the genomic DNA is isolated from a blood sample obtained from the subject. Commercial ready-to-use kits for the isolation of genomic DNA are available from various suppliers, for example the GenomicPrep™ Blood DNA Isolation Kit for purification of high-quality genomic DNA from whole blood, bone marrow and nucleated blood can be obtained from Amersham Biosciences. The Wizard® Genomic DNA Purification Kit from Promega provides a versatile solution-based system for the manual isolation of DNA from blood or mammalian tissue.

Following preparation of the genomic DNA, the presence of one or more single- nucleotide polymorphisms (SNPs) according to the invention is detected using standard techniques. There are many techniques for SNP detection and genotyping known in the art, such as restriction fragment length polymorphism PCR (RFLP- PCR), single-strand conformation polymorphism (SSCP) analysis, allele specific hybridization, primer extension, allele specific oligonucleotide ligation, sequencing. In one embodiment, real time PCR analysis using two sets of specific primers and probes (one set for the wild-type allele and one set for the polymorphic allele) is used to detect the presence or the absence of a polymorphism.

Whereas it may be sufficient to detect one SNP in a method of the invention in order to predict the therapy responsiveness of an osteoporotic subject, for example the ERlO polymorphism, detection of two or more different SNPs is preferred to increase the reliability of the outcome. In one embodiment, a method of the invention comprises detection of the ERlO, the VDR Bsml and the VDRTaql polymorphisms. Presence of the wild type allele (T/T) in ERlO; (G/G) in VDR Bsml; and (T/T) in VDR Taql are indicative of an osteoporotic subject being more likely to be responsive to Ca/vitamin D therapy.

EXPERIMENTAL SECTION

A single-centre, case controlled, clinical study was performed to investigate the relationship between the genotype and calcium/vitamin D metabolism in osteoporosis.

The study was conducted in two phases (see flowcharts of Fig. 1 and Fig. 2). Phase 1 included screening, routine diagnostic tests (biochemistry for renal function, hepatic function and calcium/vitamin D homeostasis) and treatment with calcium and vitamin D when necessary. Phase 2 consisted of further biochemical assessments and genotyping.

The population under study consisted of osteoporosis patients and controls. The subjects were female and they were aged 50 years or older. The subjects completed a witnessed written informed consent form for phase 2 of the study. Subjects had a satisfactorily documented medical history, and blood biochemistry (for renal and liver function) and urinalysis with no clinically significant abnormalities for their age. The following subjects were excluded from the study: subjects with any evidence or history of clinically significant renal or hepatic dysfunction, subjects with any history of metastatic disease, multiple myeloma, or any other condition resulting in hypercalcaemia, subjects who have participated in a clinical trial in the three months prior to study commencement and subjects who were on therapy known to affect calcium or bone metabolism. Number of patients: Recruited: 108; Completed: 54

Excluded: 54 (incomplete data, concomitant medication and medical conditions)

Concomitant Medication: The subjects' regular medication was documented at screening and follow-up. If medication other than the investigational product or the subjects' regular medication was taken at any time from study screening, details were recorded on the case report form. Subjects were asked at 3 months whether they had taken any medication other than that documented in their medication diary.

Study Medication: Oral calcium 1200 mg (2 Adcal D3 chewable tablets Oral vitamin D 800 units taken at night).

Dosing: Subjects who were osteoporotic and vitamin D deficient were prescribed 1200 mg oral calcium and 800U vitamin D, which they took for the duration of the trial. Calcium/vitamin D was not prescribed to those who acted as controls (i.e. non- osteoporotic patients).

Sampling: Approximately 15 ml of venous blood was drawn from a forearm vein at screening. This was used for biochemical analyses and genotyping. A further 15 ml sample was drawn at 3 months. Urine was analysed at screening and 3 months.

Data Analyses: The preliminary results were analysed at 3 months to see whether differences in calcium/vitamin D homeostasis segregate with different genotypes. Calcium/vitamin D homeostasis was assessed by the subjects' response to Ca/vitamin D treatment, i.e. >10 pmol/1 PTH suppression at 3 months. Genotype correlations were performed using allele frequency analysis.

Subjects were recruited from the King's College Hospital (KCH)osteoporosis clinic. A standard NHS osteoporosis work-up was performed at screening. This included: quantitative heel (calcaneal) ultrasound (QUS), blood analysis for standard biochemistry, vitamin D, PTH and calcium, and urine analysis for deoxypyrodinoline (DPD) and calcium. Subjects with osteoporosis levels 12 [Q: what does this mean?] were started on oral calcium and vitamin D supplements. Those with normal QUS did not receive any Ca/vitamin D supplementation, and acted as controls. After 3 months of treatment, urine and blood analyses were repeated. A sample was obtained for genotyping (either at baseline or 3 months). The difference in vitamin D levels and PTH levels were assessed to distinguish between subjects who responded well to Ca/vitamin D treatment and those who did not respond well to the treatment. Subjects who experienced any unresolved adverse events (i.e. fractures) were withdrawn from the study, but followed up by KCH at the osteoporosis clinic.

Bias Reduction

Subjects were identified by a unique code. Therefore when blood samples and results were analysed, the analysts were blinded to the identity of the subject, and whether they were on oral calcium and vitamin D treatment or not.

Serum samples for biomarker analysis

Blood samples were collected in a vacααtainer tube. Immediately after collection, blood was mixed well and put in a cooled box. Within 15-30 minutes after collection samples were centrifuged for 10 minutes at ca. 2000 x g at ca. 4°C. After centrifugation, 1 aliquot of ca. 5 ml plasma/serum (minimum 2 ml) was stored directly at <-20°C until analysis.

Genotyping studies The analysis of blood samples for genotyping was undertaken by TNO BIBRA International Ltd. The genetic variations analysed were:

Oestrogen Receptor

T/C codon 10 C/G codon 325

Vitamin D receptor

C/T exon 2 (Fokl) G/A intron 8 (Bsml) T/C exon 9 (Taql)

Allelic Discrimination Blood in vacutainers was stored at 4⁰C for up to 2 weeks before DNA was isolated using a Promega Genomic DNA Wizard Isolation kit. The DNA was then aliquoted and stored at -2O⁰C until required. Genomic analysis for allelic discrimination was performed on an ABI PRISM 7700 Sequence Detector using pairs of dual labelled probes, one labelled with PAM and TAMRA to detect one allele, the other labelled with VIC and TAMRA to detect the other allele.

Artificial templates were constructed for both genotypes of each polymorphism to act as controls. A common forward primer which spanned the whole forward primer and the 5'part of the shared region of the probe sequence and the unique reverse primer for each genotype spanning from the shared region of the probe sequence to the 3' end of the reverse primer, creating an overlap area, was incubated with Klenow DNA polymerase. This produced a piece of double stranded DNA suitable for use as a control template. Sigma Genosys supplied these oligonucleotides.

The DNA or control artificial template was placed in wells of an optical 96-well reaction plate with Universal master mix, forward and reverse primers and both probes. After 35 cycles of amplification the results were analysed using the allelic discrimination protocol of the ABI PRISM 7700 Sequence Detector and scored as wild type (wt) heterozygote (het) or homozygous /alternate allele (horn). All probes, primers, Universal master mix, optical plates and caps were obtained from Applied Biosystems.

Probe/Primer Sequences

Vitamin D Receptor (VDR; capital letter denotes non-cleavage) Taql (exon 9)

Forward CCC CGT GCC CAC AGA TC

Reverse TGT ACG TCT GCA GTG TGT TGG A

FAM probe (f ) CGC GCT GAT CGA G (mgb) VIC Probe (F) CGC GCT GAT TGA G(mgb) wt

Bsm I (intron 8)

Forward GGG ATT CTG AGG AAC TAG ATA AGC A

Reverse CAA GAG CAG AGC CCT GAG TAT TGG FAM Probe (b) ACA GGC CTG CAC ATT (mgb)

VIC Probe (B) CAG GCC TGC GCA T (mgb) wt

Fok I (exon 2)

Forward ACC GTG GCC TGC TTG CT Reverse AGG GTC AGG CAG GGA AGT G

FAM Probe (F) CAG GGA CGG AGG C (mgb) wt

VIC Probe (f) ACA GGG ATG GAG GCA (mgb)

Oestrogen Receptor

HERlO (codon 10; exon 1)

Forward CCA CGG ACC ATG ACC ATG A

Reverse GTT CCC TTG GAT CTG ATG CAG TAG

FAM Probe ACC AAA GCA TCT GGG AT (mgb) wt VIC Probe CCA AAG CAT CCG GGA T (mgb)

HER 325 (codon 325; exon 4) Forward TGA CGG CCG ACC AGA TG

Reverse CAC TGA AGG GTC TGG TAG GAT CAT FAM Probe ATG CTG AGC CCC CGA TAC TCT ATT CC wt

VIC Probe CTG AGC CCC CCA TAC TCT ATT CCG A

(mgb) denotes the presence of a Minor Groove Binder as part of the probe. Results

Tables IA, IB and 1C show the results of the statistical analysis of the genotyping. All statistical tests were 2-sided and performed at the 5% level of significance.

Table IA shows the genotype and allele frequencies in those patients responding to Calcium/Vitamin D therapy, as indicated by the fall in PTH levels. These results indicate that compared to previously published allele frequencies, these patients exhibit a small decrease in the frequencies of HER 10 and VDR Fok 1. There were no differences seen in mean Calcium levels pre-treatment compared with post-treatment, but mean vitamin D levels increased.

Table IB below shows the genotype and allele frequencies in the non-responding osteoporotic patients. The main findings are increases in allele frequencies seen with HER 10, HER325, and VDR Taql and a decrease in VDR Fokl compared with previously published allele frequencies. Mean Calcium levels showed no change from baseline, but mean Vitamin D levels increased by the end of the treatment period compared with baseline. Table 1C below lists the genotypes and allele frequencies seen in the non-osteoporotic control group. These show little variations when compared with published frequencies rates, except for an increase in the frequency of VDR Fokl. Mean Calcium levels showed little change and mean Vitamin D levels remained above the level of 20.

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Table IA: genotype and allele frequencies in those patients responding to Calcium/Vitamin D therapy, as indicated by the fall in PTH levels. All statistical tests were 2-sided and performed at the 5% level of significance.

Table IA

Genotype and allele frequencies; Calcium, Vitamin D& PTH levels in Responders

GENOTYPES BASELINE 3 MONTHS

Patient ID HER 10 HER 325 VDR Bsm 1 VDR Fok1 VDR Taq1 calcium vit D PTH calciumvit D PTH

13 Horn Wt het horn het 2 63 14.5 60 2.56 23 2 24

16 Wt Wt het horn het 2 34 21 5 36 2.31 14.5 17

18 Het Wt Wt Wt Wt 2 33 9.6 33 2.38 41 23

30 Het het het Wt het 2.38 7.8 31 2.52 20.6 9

37 Het Wt Wt Wt wt 2 53 16 54 2.46 26.2 34

39 Wt het Wt Wt Wt 2.43 142 44 2 39 33.6 13

43 Wt het Wt Wt Wt 2.34 21.1 43 2.41 24.7 17

47 Wt Wt NT Wt horn 2 32 13 7 46 2.52 23.2 21

48 Wt wt het Wt het 2.31 12.6 120 2.33 16.8 66

52 Het Wt horn het horn 2 36 28 9 41 2.33 33.2 20 -4

56 Wt het Wt het Wt 247 16 4 49 2.51 38.1 34

59 Het Wt het Wt het 2.34 9 9 39 2 33 34.4 24

60 Wt Wt het Wt NT 2.52 32.3 43 2.4 31.7 31

63 Horn het horn horn hom 2.27 15.1 42 2.36 26.9 26

77 Wt wt wt het horn 24 12 58 2.49 32.2 28

80 Het Wt Wt Wt Wt 2.22 13 104 2.25 15 70

83 Horn het horn Wt hom 2 38 26 56 2.35 18 40

84 Wt wt het wt het 243 15 51 2.37 29.2 24

86 Het het Wt Wt wt 2.43 27 30 2.39 39.9 16

88 Horn het horn het Wt 2.36 17 40 2.44 17.7 20

89 Het Wt Wt wt hom 2.43 26 26 2.46 15.3 16

92 Horn horn wt Wt Wt 244 17 79 2.42 27.9 43 number in population n=22 n=22 n=21 n=22 n=21 n=22 n=22

X 18 10 15 10 18 2 39 17 6 51.1 2.41 26.5 28 gene frequency 0.41 0.23 0.36 0.23 0.43 previously published 0.48 0.22 0.42 0.37 0.41 frequency

Table IB shows the genotype and allele frequencies in the group of non-responders.

Table IB

Genotype and allele frequencies; Calcium, Vitamin D& PTH levels in non-responders

GENOTYPES BASELINE 3 MONTHS

Patient ID HER 10 : HER 325 VDR Bsm 1 VDR Fok1 VDR Taq1 calcium vit D PTH calcium vit D PTH

15 Horn Wt hom Wt hom 2.36 19.9 25 2.35 27.2 38

19 Het hom Wt hom Wt 2.42 20.4 17 2.33 13.1 28

20 Horn Wt het hom het 2.27 14.2 14 2.31 15.1 14

24 Het hom hom het hom 2.21 12.7 30 2.17 19.5 40

32 Wt het NT NT hom 2.27 8.4 66 2.29 10.4 85

33 Horn het het wt het 2.45 56.5 21 2.42 39 18

49 Wt Wt het Wt het 2.44 17.2 23 2.45 32.4 19

OO

57 Horn wt Wt wt Wt 2.47 17.9 22 2.4 25.3 15

70 Het Wt het wt het 2.28 14.5 2.46 19.2 34

87 Het Wt het wt het 2.26 14 25 2.57 18 50

104 Het het hom Wt hom 2.36 25.5 17 2.29 25.8 18

111 Horn wt Wt Wt Wt 2.33 17.1 24 2.3 24.3

116 Het het Wt hom Wt 2.29 6.2 162 2.35 26

120 Het Wt Wt wt NT 2.35 23.9 25 2.36 19.6 number in population n=14 n=14 n=13 N=13 n=13 n=13 n=11

X 17 8 11 7 13 2.34 19.2 36.2 2.36 22.5 32.6 gene frequency 0.60 0.29 0.42 0.27 0.50 previously published 0.48 0.22 0.42 0.37 0.41 frequency

Table 1C lists the genotypes and allele frequencies seen in the non-osteoporotic control group.

Table 3 Genotype and allele frequency; Calcium, Vitamin D& PTH levels in the control group

GENOTYPES BASELINE 3 MONTHS

Patient ID HER 10 HER 325 VDR Bsm 1 VDR Fok1 VDR Taq1 calcium vit D PTH calcium vit D PTH

17 Wt Wt het hom het 2.63 23.6 30 2.73 22.4 40

34 Het het Wt het Wt 2.46 20.8 17 2.39 25 25

42 Het wt Wt het Wt 2.44 25.4 57 2.39 28 33

46 Het Wt het het het 2.32 24 30 2.31 37 16

50 Het Wt het het het 2.51 20.1 31 2.52 20.1 20

61 Het het het het NT 2.4 15.7 25 2.54 37.4 10 VO

72 Horn Wt het hom het 2.57 25 43 2.5 16.5 65

85 Het Wt horn hom hom 2.44 28 23 2.34 27.9 28

107 Het het wt het wt 2.37 31 2.37 15.1 31

113 Het het hom Wt hom 2.38 23 44 2.35 18.9 number in population n=10 n=10 n=10 n=10 n=9 n=10 n=9

X 10 4 9 12 8 2.45 22.8 33.1 2.44 24.8 29.8 gene frequency 0.50 0.20 0.45 0.60 0.44 previously published 0.48 0.22 0.42 0.37 0.41 frequency

IMT = Not Testec i

Claims

1. A method for predicting whether an osteoporotic subject will respond to calcium / vitamin D therapy, comprising: a) isolating genomic DNA from said subject b) determining the presence or absence of at least one polymorphism in the genomic DNA, wherein said polymorphism is selected from the group of polymorphisms consisting of the T/C estrogen receptor-α codon 10 (ERlO) polymorphism, the C/G estrogen receptor-α codon 325 (ER325) polymorphism, the G/A vitamin D receptor Bsml (VDR Bsml) polymorphism, the T/C vitamin D receptor Fokl (VDR Fold) polymorphism and the T/C vitamin D receptor Taql (VDR Taql) polymorphism; and c) correlating the presence or absence of said polymorphism with the responsiveness to calcium /vitamin D therapy.

2. A method according to claim 1, wherein absence of the ERlO polymorphism is indicative of an increased chance of being responsive to said therapy.

3. A method according to claim 1 or 2, wherein homozygosity for the ER235 polymorphism is indicative of being unresponsive to said therapy.

4. A method according to any one of claims 1 to 3, wherein absence of the

VDR Bsml polymorphism is indicative of an increased chance of being responsive.

5. A method according to any one of claims 1 to 4, wherein heterozygosity for the VDR Fokl polymorphism is indicative of being responsive.

6. A method according to any one of claims 1 to 5, wherein absence of the

VDR Taql polymorphism is indicative of an increased chance of being responsive.

7. A method to diagnose or screen for osteoporosis in a subject, comprising: a) Isolating genomic DNA from said subject b) determining the presence or absence of the T/C vitamin D receptor Fokl

(VDR Fokl) polymorphism in said genomic DNA; and c) correlating the presence or absence of said VDR Fold polymorphism with a risk of having or developing osteoporosis, wherein the absence of said polymorphism is indicative of an increased risk of having or developing osteoporosis.

8. A method according to any one of claims 1 to 7, wherein said subject is a human female, preferably a female aged 50 years or older.