WO2003025225A2

WO2003025225A2 - Pthr1 gene polymorphisms

Info

Publication number: WO2003025225A2
Application number: PCT/GB2002/004287
Authority: WO
Inventors: Emma L. Duncan; Matthew Brown; John A H. Wass; Peng Y. Woon
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2001-09-20
Filing date: 2002-09-20
Publication date: 2003-03-27
Anticipated expiration: 2004-03-20
Also published as: WO2003025225A3; AU2002324234A1; GB0122730D0

Abstract

A method of diagnosing the existence of or susceptibility to a bone disorder, which method comprises determining whether the subject has a polymorphism associated with low bone mineral density (BMD) in the parathyroid hormone receptor-1 (PTHR1) gene, thereby determining the susceptibility of the subject to a bone disorder.

Description

GENE LINKAGE Field of the Invention

The invention relates to methods for identifying individuals who are susceptible to bone disorders, in particular to osteoporosis. The invention also relates to methods for treating individuals who have been identified as being susceptible to bone disorders.

Background to the Invention

Evidence from twin and family studies suggest that osteoporosis has a substantial genetic component and that it is likely that several genes are involved. Estimates of the genetic contribution to peak bone mass range up to 85% of the population periods.Α similar magnitude of genetic control over post menopausal bone loss has also been reported. Several genes have been suggested to be associated with osteoporosis including the vitamin D receptor, type 1 collagen Al, interleukin- 6, transforming growth factor-βl, estrogen receptor 1 and parathyroid hormone receptor 1 but the data did not reach the generally regarded threshold for significant linkage and the genetic influence of a given candidate gene could not be distinguished from that of nearby genes. In addition, contradictory studies exist for each of these findings. Therefore, clearly a large component of the genetic variants for osteoporosis remains unexplained.

The present inventors have previously demonstrated possible evidence of linkage between the PTHR1 locus and bone density in a family based study, with LOD scores ranging between 2.7 and 3.5 at femoral neck and 1.5 to 2.2 at lumbar spine. However, this linkage is of borderline significance because for sib-pair based analysis the thresholds for significant linkage vary between 3.6 and 4.2 (Nyholt, AM J Hum Genet 2000; 67: 282-288). Further, the PTHR1 locus has not shown^' up in • other genome scans in humans (see for example Devoto et al Eur J Hum Genet 1998; 6: 151-157, Roller et al J Clin Endocrine! Metab 2000; 85: 3116-3120). Although the present inventors have previously reported linkage to the PTHR1 locus, they have not specifically shown that the PTHR1 gene is responsible. From the limited evidence available it is equally likely that another gene could be responsible.

Summary of the Invention

The present inventors have demonstrated conclusively for the first time that there is association between the PTHR1 gene and bone mineral density (BMD).

BMD is a major determinant of bone fragility in later life. People with low BMD are said to have osteoporosis.

In particular, the present inventors have shown that a polymorphism at position 311 in exon M7 of the PTHR1 gene (C61407T) is associated with BMD both within families and in collections of unrelated individuals. This is a novel finding and means that at least one of the variants of the PTHR1 gene is involved in determining BMD. The polymorphism at position 311 in exon M7 is a C/T base pair change. The polymorphism itself does not change the sequence of PTHR1 and is therefore unlikely to be involved directly. Rather, it is most likely inherited together with another variant on the same haplotype, the other variant being involved in osteoporosis.

The present inventors have also discovered a novel variant of the PTHR1 gene within the U3 region of the promoter of the gene. The present inventors have discovered that the reported sequence of the U3 region is very inaccurate and have determined the correct sequence of the U3 promoter region. The effect of the errors in the published sequence (from the http: //genome.ucsc.edu) is that PCR amplification of the U3 region is made very difficult and the identification of minor variations in sequence between individuals is made problematic because the base line sequence is wrong. There is now disclosed a novel variant of the PTHR1 gene U3 promoter region having a C/T base pair change at position 89 (C40850T). It is believed that this variant is implicated in BMD control as it may interfere with binding of nuclear activating factors which induce transcription of the PTHR1 gene.

Additionally there is now disclosed a polymorphism in the U4 promoter region of the PTHR1 gene (U₄-(AAAG)₅.₇), which is associated with BMD and is therefore proposed as a polymorphism indicative of susceptibility to a bone disorder, especially osteoporosis. This polymorphism is a tetranucleotide repeat (AAAG)_n, for example as indicated in SEQ ID NO: 7.

The present inventors also have evidence to suggest that the following novel polymorphisms are associated with BMD: a G/A base pair change at position 317 of SEQ ID NO: 6, a G/A base pair change at position 86 of SEQ ID NO: 8 (G36506A), a G/A base pair change at position 88 of SEQ ID NO: 9 (G36808A), a C/G base pair change at position 108 of SEQ ID NO: 10 (C40428G), a C/G base pair change at position 86 of SEQ ID NO: 11 (C41426G), a G/T base pair change at position 99 of SEQ ID NO: 12 (G52659T), a C/T base pair change at position 73 of SEQ ID NO: 13 (C52813T), an A/C base pair change at position 126 of SEQ ID NO: 13 (A52866C), a deletion of GACA from positions 101 to 104 of SEQ ID NO: 14 (54821-54824- GACA-DEL) and a C/A base pair change at position 68 of SEQ ID NO: 16 (C56708A).

Accordingly, the present invention provides a method of diagnosing the existence of or susceptibility to a bone disorder, especially osteoporosis, which method comprises determining whether the subject has a polymorphism associated with low bone mineral density (BMD) in the parathyroid hormone receptor- 1 (PTHR1) gene, thereby determining the susceptibility of the subject to a bone disorder. The present invention further provides the use of a substance or composition in the manufacture of a medicament for use in the treatment or prevention of a bone disorder in a subject, which subject has been identified as being susceptible to a bone disorder using a diagnostic method of the invention. In a preferred aspect, the substance is parathyroid hormone (PTH), parathyroid hormone related peptide (PTHrP) or osteoprotegrin (OPG), or wherein the composition comprises PTH, PTHrP, OPG and/or a related compound.

Alternatively, the invention provides a method of treating or preventing a bone disorder in a subject, which method comprises:

(i) determining whether the subj ect has a polymorphism associated with low BMD in the PTHR1 gene, thereby determining whether the , subject is susceptible to a bone disorder; and (ii) administering to a subject identified in (i) as susceptible to a bone disorder a therapeutically effective amount of an agent which reduces the risk of a bone disorder. The present invention also provides a probe or primer for use in a diagnostic method of the invention, which probe or primer is capable of selectively detecting a polymorphism in the PTHRl gene associated with low BMD, and a kit suitable for use in a method for determining the susceptibility of a subject to a bone disorder, which kit comprises such a probe or primer. ^" The invention further provides a method for identifying an agent which modulates the expression of the PTHRl gene in a subject having a polymoφhism in a promoter region of the PTHRl gene, which method comprises:

(i) providing a cell comprising the promoter region of the PTHRl gene having said polymoφhism, wherein said promoter region is operably linked to the coding sequence of a gene;

(ii) contacting said cell with a test agent;

(iii) monitoring the expression of said gene, thereby determining whether the test agent modulates the expression of PTHRl gene in said subject. Also provided is an agent identified by such a method which modulates expression of the PTHRl gene in a subject having a polymoφhism in a promoter region of the PTHRl gene. An agent identified by such a method may be synthesised, or may be incoφorated into a pharmaceutical composition. The invention also provides the use of such an agent in the manufacture of a medicament for use in a method of treating a bone disorder, and a method of treating a bone disorder, which method comprises:

(i) identifying an agent that modulates the expression of the

PTHRl gene in a subject having a polymoφhism associated with low BMD in a promoter region of the PTHRl gene; and (ii) ₍ administering a therapeutically effective amount of an agent identified in (i) to a subject as defined in (i). The present invention further provides a method of treating a bone disorder, comprising providing an agent that complements a polymoφhism in the PTHRl gene that is associated with low BMD. Also provided is a method of screening for an agent that will complement such a polymoφhism, an agent identified by such a screening method and a method of screening which further comprises synthesising an agent and/or incorporating it into a pharmaceutical composition.

Also provided is a method for determining the efficacy of an agent useful in the treatment of a bone disorder in a subj ect having a polymoφhism associated with low BMD in the PTHRl gene, which method comprises:

(i) providing a PTHRl gene having said polymoφhism, or a protein encoded thereby; (ii) contacting said protein or gene with said agent; and (iii) determining whether said protein or gene interacts with said agent.

Brief description of the Figures

Figure 1 shows the structure of the PTHRl 5' UTR.

Figure 2 is a comparison of the published sequence of the U3 region of the PTHRl gene (from http: //genome.ucsc.edu) and the sequence of the U3 region determined by the present inventors. The published sequence is labelled "query" and the new sequence is labelled "sbjct".

Figure 3 shows multipoint Graphs of Additional Markers for PTHRl region.

Brief Description of the Sequence Listing

SEQ ID NO: 1 is the publicly available sequence of the U3 region of the PTHRl gene from http://genome.ucsc.edu.

SEQ ID NO: 2 is the sequence of the U3 region of the PTHRl gene as determined by the present inventors. SEQ ID NO: 3 is the wild type sequence of a part of the U3 promoter region of the PTHR_jl gene as determined by the present inventors.

SEQ ID NO: 4 is the sequence of a part of the U3 promoter region of the PTHRl gene which is a variant of the wild type sequence including a polymoφhism at position 89 (C40850T). SEQ ID NO: 5 is the sequence of the M6-M7 exon region of the PTHRl gene which is a variant of the wild type sequence including a polymoφhism at position 311 (C61407T).

SEQ ID NO: 6 is the sequence of the Tl region of the PTHRl gene which is a variant of the wild type sequence including a polymoφhism at position 317 (G62133A).

SEQ ID NO: 7 is a sequence from the U4 region of the PTHRl gene which includes a tetranucleotide repeat at positions 440 to 467 (U4-(AAAG)_5-7-φts).

SEQ ID NO: 8 is a sequence from between the Ul and U2 regions of the PTHRl gene from positions 36421 to 36600 of the PTHRl gene according to the publically available sequence of clone AC094020 and including a G/A polymoφhism at position 86 (G36506A).

SEQ ID NO: 9 is a sequence from between the Ul and U2 regions of the PTHRl gene from positions 36721 to 36900 of the PTHRl gene according to the publically available sequence of clone AC094020 and including a G/A polymoφhism at position 88 (G36808A).

SEQ ID NO: 10 is a sequence from between the U2 and U3 regions of the PTHRl gene from positions 40321 to 40500 of the PTHRl gene according to the publically available sequence of clone AC094020 and including a C/G polymoφhism at position 108 (C40428G). SEQ ID NO: 11 is a sequence from between the U3 and U4 regions of the

PTHRl gene from positions 41341 to 41520 of the PTHRl gene according to the publically available sequence of clone AC094020 and including a C/G polymoφhism at position 86 (C41426G).

SEQ ID NO: 12 is an intronic sequence of the PTHRl gene from positions 52561 to 52740 of the PTHRl gene according to the publically available sequence of clone AC094₎020 and including a G/T polymoφhism at position 99 (G52659T).

SEQ ID NO: 13 is an intronic sequence of the PTHRl gene from positions 52741 to 52980 of the PTHRl gene according to the publically available sequence of clone AC094020 and including a C/T polymoφhism at position 73 (C52813T) and an A/C polymoφhism at position 126 (A52866C).

SEQ ID NO: 14 is the wild type sequence of the PTHRl gene from positions 54721 to 54900 of the PTHRl gene according to the publically available sequence of clone AC094020 and including a GACA insertion polymoφhism at positions 101 to 104. SEQ ID NO: 15 is the mutant sequence of the PTHRl gene from positions

54721 to 54900 of the PTHRl gene according to the publically available sequence of clone AC09402θ^"aήd including a GACA deletion polymoφhism (54821-54824- GACA-DEL).

SEQ ID NO: 16 is a potion of the coding sequence of the PTHRl gene from positions 56641 to 56820 of the PTHRl gene according to the publically available sequence of clone AC094020 and including a silent C/A polymoφhism at position 68 (C56708A).

Detailed Description of The Invention The present inventors have shown conclusively for the first time that polymoφhisms within the parathyroid hormone receptor- 1 gene are associated with bone disorders. More specifically, the present inventors have shown that a polymoφhism in the M6-M7 exon at position 311 as defined with reference to SEQ ID NO: 5 is associated with reduced bone mineral density. The polymoφhism at this position results in the replacement of a cytosine (C) residue by a thymidine (T) residue. This polymoφhism does not result in a codon change and must therefore be in linkage disequilibrium with another polymoφhism which has a functional effect. The present inventors have also obtained an accurate sequence of the U3 promoter region of the PTHRl gene and have identified a novel polymoφhism at nucleotide position 89 of the U3 promoter region as defined by reference to SEQ ID NO: 3. The polymoφhisjn at this position results in the replacement of a cytosine (C) residue by a thymidine (T) residue. Other polymoφhisms that the inventors have identified as being associated with BMD include those shown as positions 317 of SEQ ID NO: 6, 86 of SEQ ID NO: 3, 88 of SEQ ID NO: 9, 108 of SEQ ID NO: 10, 86 of SEQ ID NO: l l, 99 of SEQ ID NO: 12, 73 and 126 of SEQ ID NO: 13 and 68 of SEQ ID NO: 16. The polymoφhisms shown in SEQ ID NOs: 8, 9, 10, 11, 12, 13, 15 and 16 are novel polymoφhisms. A polymoφhism in a promoter region may affect expression of the PTHRl gene and thus affect bone mineral density.

The position of the single nucleotide polymoφhisms (SNP) are defined above by reference to the sequences set out in SEQ ID NOs: 3, 5, 6, 8, 9, 10, 11, 12, 13 and 16. If the subject carries an allelic variant of the sequence set out in SEQ ID NO: 3, 5, 6, 8, 9, 10, 11, 12, 13 or 16 the nucleotide of interest is the one in the allelic variant which corresponds to position 89 in SEQ ID NO: 3, 311 in SEQ ID NO: 5, 317 in SEQ ID NO: 6, 86 in SEQ ID NO: 8, 88 in SEQ ID NO: 9, 108 in SEQ ID NO: 10, 86 in SEQ ID NO: 11, 99 in SEQ ID NO: 12, 73 or 126 in SEQ ID NO: 13 or 68 of SEQ ID NO: 16. Those skilled in the art will be able to determine the appropriate nucleotide. Comparison of an allelic variant with the sequence set out in SEQ ID NO: 3, 5, 6, 8, 9, 10, 11, 12 , 13 or.16 using for example the PILEUP program referred to below, will allow the nucleotide corresponding to that at position 89, 311, 317, 86, 88, 108, 99, 73, 126 or 68 to be identified.

The present inventors have identified a polymoφhism in the U4 promoter region of the PTHRl gene. This polymoφhism takes the form of a tetranucleotide repeat, (AAAG)_n. Preferably, n is from 3 to 5. This is a polymoφhism indicative of susceptibility to a bone disorder, especially osteoporosis. The location of the tetranucleotide repeat begins at position 440 as defined by reference to SEQ ID NO: 7. If the subject carries an allelic variant of the sequence set out in SEQ ID NO: 7, the location of interest is the one in the variant which corresponds to the region starting at position 440 in SEQ ID NO: 7. Those skilled in the art will be able to determine the appropriate location. Comparison of an allelic variant with the sequence set out in SEQ ID NO: 7, using for example the PILEUP program referred to below, will allow the location of the tetranucleotide repeat corresponding to that starting at position 440 of SEQ ID NO: 7 to be identified.

The inventors have also identified a insertion/deletion polymoφhism as shown in SEQ ID NOs: 14 and 15. The GACA sequence at positions 101 to 104 of SEQ ID NO : 14 is absent from the polymoφhic allele shown in SEQ ID NO : 15. This intronic polymoφhism is also associated with BMD.

Other polymoφhisms of the PTHRl gene are known in the art. For Example, variants in the U4 promoter region and within the coding region of the gene, including in exons G, M4, M7 and Tl and in the introns between E2 and E3 and between G and Ml.

Preferred polymoφhisms include those shown in SEQ ID NO: 8 (G36506A), SEQ ID NO: 9 (G36808A), SEQ ID NO: 13 (G52813T and A52866C), SEQ ID NO: 7 (U4-(AAAG)n-φts), SEQ ID NO: 15 (54821-54824-GACA-DEL) and SEQ ID NO: 5 (C61407T). The invention provides methods for determining whether a subject is susceptible to a bone disorder, and in particular osteoporosis. The method comprises determining whether'the subject has a polymoφhism in the PTHRl gene, which polymoφhism is associated with reduced bone mineral density (BMD). BMD is a major determinant of bone fragility in later life. People with low BMD are said to have osteoporosis. Any BMD values below the normal range are said to be low. For example, the World Health Organisation defines osteoporosis as a bone density at any site with a T-score of less than -2.5 and osteopaenia as a T-score of -1.0 to -2.5 where a T-score is the number of standard deviations below the mean peak bone mass for that gender (WHO Technical Report Series 1994; 9: 1137-1141). Normal BMD is in the range of T-score > -1.0. The actual amount of bone present depends on numerous factors, including bone site, gender and age. A polymoφhism in the PTHRl gene associated with low BMD is preferably associated with a BMD of less than such as of less than -1.5, -2.0, -2.5, -3.0 or -4.0. The polymoφhism may be associated with a decrease in BMD which would be defined as osteoporosis or a decrease in BMD which would be defined as osteopaenia. Geneifally, the subject will be a human. The subject may be asymptomatic for osteoporosis or alternatively may show clinical symptoms of predisposition to osteoporosis, for example low bone mineral density (BMD).

The polymoφhism associated with a reduction in bone mineral density (BMD) may cause a change in the amino acid sequence of the PTHRl protein and may thus have a functional effect on the PTHRl protein. Alternatively, the polymorphism may have no effect on the amino acid sequence of the PTHRl protein. Such a polymoφhism may be in inherited with a second polymoφhism on the same haplotype that causes a change in the amino acid sequence of the PTHRl protein. A polymorphism may also be in a regulatory region of the PTHRl gene. A polymorphism in a regulatory region of the PTHRl gene may affect expression of the PTHRl protein.

A polymoφhism suitable for use in diagnosing the existence of or susceptibility to a bone disorder may be identified by screening the PTHRl gene of one or more individuals. One of skill in the art may determine whether a candidate polymoφhism can be typed to determine whether an individual has, or is susceptible to, a bone disorder, by determining whether the candidate polymoφhism is associated with low bone mineral density or is in linkage disequilibrium with a polymoφhism which is associated with low bone mineral density. Polymoφhisms which are in linkage disequilibrium with each other in a population tend to be found together on the same chromosome. Typically one is found at least 30% of the times, for example at least 40%, 50%, 70% or 90%, of the time the other is found on a particular chromosome in individuals in the population. Thus polymoφhisms which are not functional susceptibility polymoφhisms, but are in linkage disequilibrium with the functional polymoφhisms, may act as a marker indicating the presence of the functional polymoφhism. Polymoφhisms which are in linkage disequilibrium with any of the polymoφhisms mentioned herein are typically within 500kb, preferably within 400kb, 200kb, 100 kb, 50kb, lOkb, 5kb or 1 kb of the polymoφhism. Similarly the term "gene region" generally encompasses any of these distances from 5' to the transcription start site and 3' to the transcription termination site of Hie gene.

The polymoφhism is typically an insertion, deletion or substitution with a length of at least 1, 2, 5 or more base pairs or amino acids. The polymoφhism is typically a substitution of 1 base pair, i.e. a single polynucleotide polymoφhism (SNP). The term "polymoφhic position" is used herein to refer to the nucleotide position(s) which differ between polymoφhic variants of the PTHRl gene. The polymoφhism may be 5' to the coding region, in the coding region, in an intron or 3' to the coding region. The polymoφhism may be a functional mutation which contributes to a bone disorder, but may be a polymoφhism which is inherited together with a functional mutation of the PTHRl gene on the same chromosome or may be a polymoφhism in a regulatory region.

Generally the polymoφhism will be associated with reduced bone mineral density. The polymoφhism will generally cause, or be in linkage disequilibrium with a polymoφhism that causes, a change in any of the characteristics of the PTHRl protein, such as expression, activity, expression variant, cellular localisation or the pattern of expression in different tissues. The polymoφhism may modulate any of the following activities of the PTHRl : parathyroid hormone (PTH) binding, parathyroid hormone related peptide (PTHrP) binding, G-protein dissociation, internalisation, re-cycling, interactions with regulatory proteins or interactions with signalling complexes. The polymoφlnsm typically has an agonistic or antagonistic effect on any of these characteristics of the receptor. Generally this will lead to a consequent increase or decrease in the activity of the pathway.

A polymoφhism which can be typed to determine susceptibility to a bone disorder may be identified by a method comprising determining whether a candidate polymoφhism in the PTHRl gene is associated reduced bone mineral density and thereby determining whether the polymoφhism can be typed to determine susceptibility to a bone disorder.

Subjects identified as at risk from osteoporosis (i.e. subjects identified as susceptible or predisposed thereto) may be treated so as to reduce the risk of osteoporosisj Such treatment may take the form of, for example, a change of lifestyle or a pharmaceutical treatment.

The identity of the nucleotide at a polymoφhic position of the PTHRl gene is determined for at least one of the alleles carried by a subject. Typically the identity of the nucleotide at the polymoφhic position is determined for both alleles carried by a subject. If the subject has an allelic variant sequence of that shown in SEQ ID NO: 3, the identity of the nucleotide corresponding to that shown at position 89 as defined by reference to SEQ ID NO: 3 will typically be identified. The identity of that nucleotide may be determined for one or both alleles. If the subject has an allelic variant sequence of that shown in SEQ ID NO: 5, the identity of the nucleotide corresponding to that shown at position 311 as defined by reference to SEQ ID NO: 5 will typically be identified. Similarly, the identity of the nucleotides at any one or more of the following positions may be identified: 317 of SEQ ID NO: 6, 86 of SEQ ID NO: 8, 88 of SEQ ID NO: 9, 108 of SEQ ID NO: 10, 86 of SEQ ID NO: 11, 99 of SEQ ID NO: 12, 73 and/or 126 of SEQ ID NO: 13 and/or 68 of SEQ ID NO: 16. The identity of any one of the specified nucleotides may be determined for one or both alleles.

The identity of the nucleotide at a polymoφhic position of the PTHRl gene of the subject may be determined by any convenient method. Clearly, there are a large number of analytical techniques available to those skilled in the art for determining the identity of the a nucleotide at a given position. In general, however, a number of steps are common to whichever technique is used. Typically, a sample is obtained from the subject. Generally a nucleic acid preparation is prepared from the sample; and then the sequence of the nucleotide at a polymoφhic position may be determined.

The test sample is conveniently, a sample of blood, bronchoalveolar lavage fluid, sputum or any other body fluid or tissue obtained from an individual. Generally, nucleic acid, is prepared from the sample according techniques well- known to those skilled in the art. Typically, a preparation of total nuclear DNA is prepared. The nucleic acid preparation may then be used to determine the sequence at a polymoφhic position of the PTHRl gene. Alternatively, it may not be necessary to include a nucleic acid preparation step and a polymoφhism discrimination technique may be carried out directly on the sample.

In general the detection of allelic variation at a polymoφhic position requires a polymoφhism discrimination technique and a signal generation system. In addition, an optional amplification step is sometimes used.

Suitable polymoφhism discrimination techniques, some of which may include PCR, include:

General techniques, for example, DNA sequencing or sequencing by hybridisation;

Scanning techniques, for example protein truncation test (PTT) [not useful for the analysis of intron sequence polymoφhism], single-strand conformation polymoφhism analysis (SSCP), denaturing gradient gel eletrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), cleavase, heteroduplex analysis, chemical mismatch analysis (CMC) or enzymatic mismatch cleavage;

Hybridisation techniques, for example solid phase hybridisation (for example, dot blots, multiple allele specific diagnostic assay [MASDA], reverse dot blots, oligonucleotide arrays [DNA chips]) or solution phase hybridisation (for example, Taqman™ [US-A-5210015 and US-A-5487972], molecular beacons [Tyagi et al, 1996, Nature Biotechnology, 14, 303 and WO-A-95/13399]);

Extension based techniques, for example amplification refractory mutation system (ARMS™), amplification refractory mutation system linear extension (ALEX™) [EP-B-332435] or competitive oligonucleotide priming system (COPS) (Gibbs et al, Nucleic Acids Research 1989; 17: 2347); Incoφoration based techniques, for example mini-sequencing or arrayed primer extension (APEX);

Restriction enzyme based techniques, for example restriction fragment length polymoφhism (RFLP) or restriction site generating PCR;

Oligonucleotide based techniques: oligonucleotide ligation assay (OLA); and other types of assay, for example invader assay. The above list provides examples of suitable polymoφhism discrimination techniques and should not be construed as limiting.

Suitable signal generation or detection systems include:

Fluorescence based techniques, for example fluorescence resonance energy transfer, fluorescence quenching or fluorescence polarisation (GB-B-2228998); and

Other techniques, for example chemiluminescence, eletrochemiluminescence, raman, radioactivity, colorimetric, hybridisation protection assay, mass spectrometry.

Again, this list of signal generation or detection systems is merely illustrative and should not be construed as limiting. The oligonucleotides for use in determining whether a particular variant of the PTHRl gene is present in a subject may be any suitable oligonucleotides. The design of suitable oligonucleotides will be apparent to the person skilled in the art. Suitable oligonucleotides will be of any convenient length, for example up to about 50 nucleotides in length, up to 40 nucleotides in length, or more conveniently up to 30 nucleotides in length, such as for example, from about 8 to about 25 nucleotides in length or from about 8 to about 15 nucleotides in length.

In general, suitable PCR primers will comprise sequences entirely complementary to the corresponding sequence to be amplified. However, if required, one or more, for example up to 3, up to 5 or up to 8 mismatches may be introduced, for example to introduce a convenient restriction enzyme site, provided that such mismatches do not unduly affect the ability of the primer to hybridize to its target sequence. Suitable primers may carry one or more labels to facilitate detection.

The presence of a T residue at position 89 of the U3 region of the PTHRl gene shown in SEQ ID NO: 3, the presence of a T residue at position 311 of the M6 to M7 region shown in SEQ ID NO: 5, an A residue at position 317 of SEQ ID NO: 6, an A residue at position 86 of SEQ ID NO: 8, an A residue at position 88 of SEQ ID NO: 9, a G residue at position 108 of SEQ ID NO: 10, a G residue at position 86 of SEQ ID NO: 11, a T residue at position 99 of SEQ ID NO: 12, a T residue at position 73 of SEQ ID NO: 13, a C residue at positionl26 of SEQ ID NO: 13 and/or an A residueiat position 68 of SEQ ID NO: 16 of the PTHRl gene of the subject is indicative of predisposition or susceptibility to osteoporosis.

The invention also provides a polynucleotide comprising the sequence set out in SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 15 or 16 or a fragment thereof. Preferably the polynucleotide encompasses position 89 of SEQ ID NO: 3 and more preferably the nucleotide at position 89 of SEQ ID NO: 3 is a T residue, as shown in SEQ ID NO: 4. Other preferred polynucleotides encompass position 86 of SEQ ID NO: 8, preferably an A, position 88 of SEQ ID NO: 9, preferably an A, position 108 of SEQ ID NO: 10 preferably a G, position 86 of SEQ ID NO: 11, preferably a G, position 99 of SEQ ID NO: 12, preferably a T, position 73 and/or 126 of SEQ ID NO: 13, preferably a T and/or C and/or position 68 of SEQ ID NO: 16, preferably an A. A polynucleotide comprising the sequence set out in SEQ ID NO: 7 or a fragment thereof will preferably comprise the tetranucleotide repeat (AAAG)_n as located from position 440 of SEQ ID NO:7. A polynucleotide which does not encompass any one of the specified positions is preferably a primer which is part of a primer pair wherein the two primers are on complementary strands and which are on opposite sides of the polymoφhic position. The invention also provides polynucleotides complementary to a polynucleotide of the invention. The polynucleotide may be R A or DNA including genomic DNA, synthetic

DNA or cDNA. Preferably the nucleotide sequence is a DNA sequence. Nucleotide sequence information is provided in SEQ ID NOs: 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 15 ^■ and 16. Such nucleotides can be isolated from human cells or synthesised according to methods well known in the art, as described by way of example in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

A variant of a polynucleotide comprising the sequence set out in SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 15 or 16 or a fragment thereof is also provided. A variant polynucleotide of the invention can hydridize to the sequence or complement of the sequence set out in SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 15 or 16 at a level significantly above background. Background hybridization may occur, for example, because of other DNAs present in a DNA library. The signal level generated by the interaction between a polynucleotide of the invention and the sequence set out in SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 15 or 16 is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the sequence set out in SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 15 or 16. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with ³²P. Selective hybridisation may typically be achieved using conditions of medium stringency and preferably high stringency (for example, 2 x SSC [0.15M sodium chloride and 0.015M sodium citrate] at about 50°C to about 60 °C). However, such hybridisation may be carried out under any suitable conditions known in the art (see Sambrook et al, 1989, supra). For example, if high stringency is required suitable conditions include from 0.1 to 0.2 x SSC at about 60 °C to about 65 °C. If lower stringency is required suitable conditions include 2 x SSC at 60°C.

The sequence of SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 15 or 16 may be modified by nucleotide substitutions, for example from 1, 2 or 3 to 10, 25, 50 or 100 substitutions provided that the modified sequence can still be used to detect a polymoφhism under highly stringent conditions. The polynucleotide of SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 15 or 16 may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends.

A nucleotide sequence which is capable of selectively hybridizing to the complement of the sequence set out in SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 15 or 16 will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to that sequence over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, more preferably at least 100 contiguous nucleotides or most preferably over the full length the sequence. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analyses is publicly available through the National Centre for Biotechnology Information (hτtp://www.ncbi.nlm.nih.gov . This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the . cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

Any combination of the above mentioned degrees of sequence identity and minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i.e. higher sequence identity over longer lengths) being preferred. Thus, for example a polynucleotide which has at least 90% sequence identity over 25, preferably over 30 nucleotides forms one aspect of the invention, as does a polynucleotide which has at least 95% sequence identity over 40 nucleotides. Polynucleotides of the invention may be fragments of the sequence set out in

SEQ ID NO: 2, 3 or 4, wherein the fragments comprise a T nucleotide at position 89 as defined by reference to SEQ ID NO: 3. Fragments may be fragments of SEQ ID NO: 15 which fragments span positions 101 and 102. Polynucleotides of the invention may be fragments of the sequence set out in SEQ ID NO: 7, wherein the fragments comprise at least part of a tetranucleotide repeat from position 440 as defined by reference to SEQ ID NO: 7. Polynucleotides of the invention may be fragments of the sequence set out in SEQ ID NO: 8 comprising an A residue at position 86 as desired by reference to SEQ ID NO: 8. Polynucleotides of the invention may be fragments of the sequence set out in SEQ ID NO: 9 comprising an A residue at position 88 as desired by reference to SEQ ID NO: 9. Polynucleotides of the invention may be fragments of the sequence set out in SEQ ID NO: 10 comprising a G residue at position 108 as defined by reference to SEQ ID NO: 10. Polynucleotides of the invention may be fragments of the sequence set out in SEQ ID NO: 11 comprising a G residue at position 86 as defined by reference to SEQ ID NO: 11. Polynucleotides of the invention may be fragments of the sequence set out in SEQ ID NO: 12 comprising a G residue at position 99 as defined by reference to SEQ ID NO: 12. Polynucleotides of the invention may be fragments of the sequence set out in SEQ ID NO: 13 comprising a T residue at position 73 as defined by reference to SEQ ID NO: 13. Polynucleotides of the invention may be fragments of the sequence set out in SEQ ID NO: 13 comprising a C residue at position 126 as defined by reference to SEQ ID NO: 13. Polynucleotides of the invention may be fragments of the sequence set out in SEQ ID NO: 16 comprising an A residue at position 68 as defined by reference to SEQ ID NO: 16. Such fragments will preferably be at least 10, preferably at least 25, or at least 50, for example at least 100, at least 200, or at least 500, or at least 1000 nucleotides in length. They will typically be up to 40, 50, 60, 70, 100 or 150 nucleotides in length.

A polynucleotide of the invention is preferably a probe or primer suitable for use in a method of diagnosis according to the invention. Such a probe or primer is thus capable of selectively detecting a polymoφhism in the PTHRl gene. A probe or primer may be from 10 to 50 base pairs, preferably from 15 to 30 and more preferably from 20 to 25 base pairs long. A primer is preferably in a region surrounding the polymoφhic position or encompassing the polymoφhism, for example, surrounding or including the polymoφhism at position 89 of SEQ ID NO: 3, position 317 of SEQ ID NO: 6, position 86 of SEQ ID NO: 8, position 88 of SEQ ID NO: 9, position 108 of SEQ ID NO: 10, position 86 of SEQ ID NO: 11, position 99 of SEQ ID NO: 12, position 73 of SEQ ID NO: 13, position 126 of SEQ ID NO: 13, positions 100 to 101 of SEQ ID NO: 15, position 68 of SEQ ID NO: 16 or all or part of the tetranucleotide repeat from position 440 of SEQ ID NO:7. Where the primer encompasses the polymoφhic position the nucleotide at one end of the primer is preferably complementary to the polymoφhic residue.

Preferably the primer is capable of binding to the PTHRl gene of a subject when a T residue is present at position 89 of SEQ ID NO: 3, a T residue is present at position 311 of SEQ ID NO: 5, an A residue is present at position 317 of SEQ ID NO: 6, an A residue is present at position 86 of SEQ ID NO: 8, an A residue is present at position 88 of SEQ ID NO: 9, a G residue is present at position 108 of SEQ ID NO: 10, a G residue is present at position 86 of SEQ ID NO: 11, a T residue is present at position 99 of SEQ ID NO: 12, a T residue is present at position 73 of SEQ ID NO: 13, a C residue is present at positionl26 of SEQ ID NO: 13 and/or an A residue is present at position 68 of SEQ ID NO: 16. Other preferred primers are capable of selectively binding to SEQ ID NO: 15 but not to SEQ ID NO: 14. A probe is preferably capable of hybridising to a region including the polymoφhic position, for example position 89 of SEQ ID NO: 3, position 317 of SEQ ID NO: 6, position 86 of SEQ ID NO: 8, position 88 of SEQ ID NO: 9, position 108 of SEQ ID NO: 10, position 86 of SEQ ID NO: 11, position 99 of SEQ ID NO: 12, position 73 of SEQ ID NO: 13, position 126 of SEQ ID NO: 13, positions 100 to 101 of SEQ ID NO: 15, position 68 of SEQ ID NO: 16 or all or part of the tetranucleotide repeat (AAAG)_n from position 440 of SEQ ID NO:7.

Preferably the probe is capable of binding to the PTHRl gene of a subject when a T residue is present at position 89 of SEQ ID NO: 3, a T residue is present at position 311 of SEQ ID NO: 5, an A residue is present at position 317 of SEQ ID NO: 6, an A residue is present at position 86 of SEQ ID NO: 8, an A residue is present at position 88 of SEQ ID NO: 9, a G residue is present at position 108 of SEQ ID NO: 10, a G residue is present at position 86 of SEQ ID NO: 11, a T residue is present at position 99 of SEQ ID NO: 12, a T residue is present at position 73 of SEQ ID NO: 13, a C residue is present at positionl26 of SEQ ID NO: 13 and/or an A residue is present at position 68 of SEQ ID NO: 16. Other preferred probes are capable of selectively binding to SEQ ID NO: 15 but not to SEQ ID NO: 14.

The invention also provides a test kit for use in a method for determining the susceptibility of a subject to a bone disorder such as osteoporosis. A test kit of the invention comprises means for determining the identity of the nucleotide at a polymoφhic position of the PTHRl gene of the subject which means is typically a probe or primer of the invention. Preferred test kits of the invention comprise means for determining the identity of the nucleotide at the polymoφhic position, for example, the nucleotide at position 89 of the U3 promoter region of the PTHRl gene, which position is defined by reference to SEQ ID NO: 3, position 317 of SEQ ID NO: 6, position 86 of SEQ ID NO: 8, position 88 of SEQ ID NO: 9, position 108 of SEQ ID NO: 10, position 86 of SEQ ID NO: 11, position 99 of SEQ ID NO: 12, position 73 of SEQ ID NO: 13, position 126 of SEQ ID NO: 13, positions 100 to 101 of SEQ ID NO: 15 or position 68 of SEQ ID NO: 16. Alternative test kits comprise means for identifying the tetranucleotide repeat (AAAG)_n from position 440 of the U4 region defined with reference to SEQ ID NO: 7. A kit of the invention may comprise a probe or primer capable of selectively hybridising under stringent conditions to the M6-M7 exon region, or a part thereof, only if a T residue is present at nucleotide position 311. In another preferred embodiment the probe comprises at least the nucleotide at position 311 and preferably the nucleotides at positions 299 to 303 of SEQ ID NO: 5 or is the complement thereof.

A test kit of the invention may optionally comprise, appropriate buffer(s), enzymes, for example a thermostable polymerase such as Taq polymerase and/or control polynucleotides. A kit of the invention may also comprise appropriate packaging and instructions for use in a method for determining the susceptibility of a subject to osteoporosis. A test kit of the invention may also comprise an agent which reduces the risk of osteoporosis.

The invention allows diagnosis of subjects at risk from osteoporosis before the onset of disease symptoms or before the onset of severe symptoms. The risk of osteoporosis can thus be reduced, prevented or delayed by administration of treatment of therapy in advance of osteoporosis appearance, for example before reduced BMD is apparent. Suitable treatments to reduce the risk of osteoporosis include a change of lifestyle. Such changes of lifestyle include for example, reduction of smoking, an increase in exercise and an increase in calcium in the diet. Alternatively, the treatment can be pharmaceutical, in which case any suitable agent can be used which is known to reduce the risk of osteoporosis.

Agents which reduce the risk of osteoporosis may also be used in the manufacture of a medicament for use in a method of treatment of a subject identified as susceptible to osteoporosis. Thus, the condition of a subject identified as susceptible to osteoporosis can be improved by administration of an agent which reduces the risk of osteoporosis. A therapeutically effective amount of an agent which reduces^' the risk of osteoporosis may be given to a human patient identified as susceptible to osteoporosis. Accordingly, the present invention provides the use of a substance or composition in the manufacture of a medicament for use in the treatment or prevention of osteoporosis in a subject, which subject has been identified as being susceptible to osteoporosis using a method of the invention.

The invention also provides a method of treating or preventing osteoporosis in a subject, which method comprises: (i) determining whether the subject has a polymoφhism in the PTHRl gene associated with reduced BMD, thereby determining whether the subject is susceptible to osteoporosis; and

(ii) administering to a subject identified in (i) as being susceptible to osteoporosis a therapeutically effective amount of an anti-osteoporosis agent. Examples of agents which can be used to reduce the risk of osteoporosis include bisphosphonates, calcium supplements, parathyroid hormone (PTH), parathyroid hormone related peptide (PTHrP), osteoprotegrin (OPG), hormone replacement therapy, selective estrogen receptor modulation (e.g. raloxifene) and calcitonin. The invention further provides a method of treating a bone disorder, such as osteoporosis, comprising providing an agent that complements a polymoφhism in the PTHRl gene that is associated with low bone mineral density, such as any of the polymoφhisms described above, or any candidate polymoφhisms identifiable by the polymoφhism identification methods of the invention. An agent which complements such a polymoφhism may be in any one of a number of forms. For example, an agent may be, or comprise, a DNA such as a DNA which can bind in the region of the polymoφhism.

An agent suitable for use in the treatment of a bone disorder, such as an agent which reduces the risk of osteoporosis, may be administered in a variety of dosage forms. Thus, they can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. The agent which reduces the risk of bone disorders such as osteoporosis may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. The may also be administeredi as suppositories. A physician will be able to determine the required route of administration for each particular patient.

An agent identified by a method of the invention may be used in the manufacture of a medicament for the treatment of a bone disorder, such as osteoporosis. A therapeutically effective amount of such an agent may be administered to a subject having polymoφhism in the PTHRl gene associated with low BMD.

The formulation of an agent which reduces the risk of osteoporosis for use in preventing or treating osteoporosis will. depend upon factors such as the nature of the exact agent, whether a pharmaceutical or veterinary use is intended, etc. An agent which reduces the risk of osteoporosis may be formulated for simultaneous, separate or sequential use.

Products containing means for determining the identity of the nucleotide at a polymoφhic position of the PTHRl gene of a subject, and an agent which reduces the risk of osteoporosis as a combined preparation for simultaneous, separate or sequential use in a method of treatment of the human or animal body by therapy.

Thus, such a product may comprise both means for diagnosis and means for therapy. An agent which reduces the risk of osteoporosis is typically formulated for administration in the present invention with a pharmaceutically acceptable carrier or diluent. The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film-coating processes.

Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions. A therapeutically effective amount of an agent which reduces the risk of osteoporosis is administered to a patient. A therapeutically effective amount of an agent is typically an amount which prevents or lessens a reduction in BMD or which reverses a reduction in BMD. Thus, a therapeutically effective amount is an amount which rectifies abnormal BMD levels. The therapeutically effective amount of the agent may be an amount which ameliorates the symptoms of a subject having low bone mineral density, i.e. having a bone disorder such as osteoporosis.

The dose of an agent which reduces the risk of osteoporosis may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.01 to 50 mg per kg of body weight, according to the activity of the specific inliibitor, the age, weight and condition of the subject to be treated, the type and severity of the degeneration and the frequency and route of administratiqn. Preferably, daily dosage levels are from 20 to 40 micrograms per day.

The effectiveness of particular anti-osteoporosis agents may be affected by or dependent on whether the individual has particular polymoφhisms in the PTHRl gene. Thus the invention can allow the determination of whether an individual will respond to a particular anti-osteoporosis agent by determining whether the individual has a polymoφhism which affects the effectiveness of that agent. The invention includes a method of treating a patient who has been identified as being able to respond to the agent, which method comprises administering the agent to the patient. Similarly certain anti-osteoporosis agents may produce side effects in individuals with particular polymoφhisms in the PTHRl gene. Thus the invention can also allow the identification of a patient who is at increased risk of suffering side effects due to such an anti-osteoporosis agent by identifying whether an individual has such a polymoφhism. The polymoφhism may therefore have the greatest effect on the efficacy of drugs designed to modulate the activity of the PTHRl or other components in its signaling pathway. However, the polymoφhisms may also affect the response to agents acting on other biochemical pathways regulated by the PTHR. The invention may therefore be useful both to predict the clinical response to such agents and to determine therapeutic dose.

The present invention thus provides a method for determining the efficacy of, or side effects of, an agent useful in the treatment of a bone disorder in a subject having a polymoφhism associated with reduced BMD in the PTHRl gene, which method comprises: (i) providing a PTHRl gene having said polymoφhism or a protein encoded thereby;

(ii) contacting said gene or protein with said agent; and (iii) determining whether said agent interacts with said protein or gene. Preferably the gene or protein is the endogenous gene or protein in a subject having tl e polymoφhism. Preferably step (iii) comprises monitoring the effect of administering the agent on bone mineral density of the subject.

In a further aspect, the invention can be used to assess the predisposition and /or susceptibility of an individual to bone diseases mediated by the PTHRl. Polymoφhisms may be particularly relevant to the development of such diseases. The present invention may be used to recognise individuals who are particularly at risk from developing these conditions.

In a further aspect, the invention may further be used in the development of new drug therapies which selectively target one or more allelic variants of the PTHRl gene (i.e. which have different polymoφhisms). Identification of a link between a particular allelic variant and predisposition to disease development or response to drug therapy may have a significant impact on the design of new drugs. Drugs may be designed to regulate the biological activity of the variants implicated in the disease process while minimising effects on other variants. The invention provides a method for identifying an agent which modulates the expression of the PTHRl gene in a subject being a polymoφhism in a promoter region of the PTHRl gene, such as a SNP at position 89 of SEQ ID NO: 3, position 86 of SEQ ID NO: 8, position 88 of SEQ ID NO: 9, position 108 of SEQ ID NO: 10, position 86 of SEQ ID NO: 11 or a polymoφhic repeat as in SEQ ID NO: 7. The method typically comprises:

(i) providing a cell comprising the promoter region of the PTHRl gene having said polymoφhism, wherein said promoter region is operably linked to the coding sequence of a gene; (ii) contacting said cell with a test agent; and (iii) monitoring the expression of said gene, thereby determining whether the test agent modulates the expression of the PTHRl gene in the subject. The cell may be any suitable cell, for example a cell line in culture, for example HEK293 cells, COS cells, HeLa cells or BHK cells. Preferably the cell is a bone cell such as an osteoblast or an osteoclast. Thei coding sequence may encode a reporter gene such as gene fluorescent protein (GFP), β-galactosidase or luciferase. Alternatively, the coding sequence may be that of the PTHRl gene.

The test agent may modultate expression of the gene coding sequence operably linked to the PTHRl polymoφhic promoter sequence by up regulating or down regulating expression.

An agent which modulates the expression of the PTHRl gene identified by a method of the invention may be used in the manufacture of a medicament for the treatment of a bone disorder, such as osteoporosis. A therapeutically effective amount of such an agent may be administered to a subject having polymoφhism in the PTHRl gene associated with low BMD.

The invention also provides a method of formulating a pharmaceutical composition, which method comprises:

(i) identifying an agent that modulates the expression of the PTHRl gene in a subject having a polymoφhism in the promoter region of the

PTHRl gene associated with reduced BMD; and (ii) formulating the agent identified in (i) with a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition typically comprises a therapeutically effective amount of the agent.

A method of identifying an agent which interacts with a PTHRl protein encoded by a PTHRl gene having a polymoφhism associated with reduced BMD is also provided. The polymoφhism may be a polymoφhism which alters the amino acid sequence of the PTHRl protein or may be a polymoφhism which has no effect on the amino acid sequence of the PTHRl protein but which is co-inherited with a second polymoφhism, which may be unknown, that does result in a codon change(s). The method typically comprises:

(i) providing a protein as defined above; (ii) contacting the protein with a test agent; and (iii) monitoring any interaction between the protein and the test agent, thereby determining whether the test agent interacts with a PTHRl protein encoded by a PTHRl gene having a polymoφhism associated with reduced BMD.

An agent identified by this method may be administered to a subject having the polymoφhism in a method of treating osteoporosis in the subject.

The following Examples illustrate the invention.

Example 1: Fine Mapping of the PTHRl-Containing Region

This Example describes linkage mapping of the PTHRl gene to show that the PTHRl gene lies within the boundaries of the previously observed area of linkage with BMD.

To ensure that the demonstrated linkage of BMD with markers d3sl289 and d3s3559, located at 62.7 cM and 69.1 cM respectively from the p telomere on chromosome 3, was not due to chance characteristics of these markers alone and to establish boundaries for the area of linkage, a further set of markers from chromosome 3p was amplified, genotyped and analysed for linkage with BMD.

Fourteen additional microsatellite markers in the area of observed linkage were selected, using the Whitehead Institute STS database (http ://www. enome.wi.mit.edu) to establish chromosomal position. The markers selected are presented in Table 1, with genetic distance in Kosambi cM.

Microsatellite markers were amplified using MJ Thermal cyclers (MJ Research, Watertown, MA, U.S.A.) and pooled in sets according to their length and fluorescent colour tag. Products were then separated by electrophoresis in 6% polyacrilamide gels using ABI 373 semiautomated genotypers (Applied Biosytems, Warrington, UK). Gels were analysed using Genescan Analysis (Applied Biosystems,

Warrington, UK), and allele sizes determined using Genotyper version 1.1 (Applied Biosystems, Warrington, UK). Size data were converted to discrete allele numbers, and Mendelian inheritance verified using programs 'GAS' version 2.0 (Dr A. Young, unpublished). Table 1 : Markers used for further linkage mapping of PTHRl region

Statistical Analysis

Allele numbers and frequencies were calculated from the observed data. BMD was expressed as z scores (adjusted for age and gender). BMD was also adjusted for effects of body size by regressing BMD against BMI.

This data was analysed in nuclear families using MapMaker/Sibs (Kxuglyak & Lander, Am J Hum Genet 1995; 57: 439-454). The original 115 families were divided into 165 nuclear units containing at least one sibling pair for analysis, using the programme "GAS" (A. Young, unpublished). Nonparametric two-point and multipoint linkage analyses were performed using both Haseman-Elston (traditional and expectation maximisation methods) and variance components analysis. Analyses were performed both with and without correction for the number of sibling pairs drawn from each family. Single results presented below are for the "all pairs" setting both with and without correction. Where a range of LOD scores is presented, the lower value is for the "all pairs" setting with correction, and the higher value for no correction.

Results

Two point linkage results of additional microsatellite markers on Chromosome 3 and BMD at both LS and FN are presented in Table 2. Multipoint linkage results are shown graphically in Figure 3. All results refer to variance components analysis as in all cases this was more significant than the corresponding Haseman-Elston analyses (both traditional and expectation maximisation methods).

Table 2: Two Point LOP Scores for Additional Markers in PTHRl region

Microsatellite marker position is measured from the first marker used. Correction is for number of sibling pairs drawn from each family and is over- conservative.

Discussion

Additional linkage mapping with further markers on Chromosome 3 established that the PTHRl locus was within the boundaries of the observed area of linkage with BMD. However this does not establish the gene/s responsible for the observed linkage. Linkage mapping is inefficient in identifying genes determining complex traits and may be only able to isolate a disease-causing gene to .an area as broad as 1 cM (depending upon the relative risk of disease conferred by that gene and the number of sibling pairs sampled) (Kruglyak & Lander, Am J Hum Genet 1995; 57: 439-454). To localise a gene causing a five-fold increase in risk of a qualitative disease to an offspring even to a 1 cM chromosomal area requires on average 200 sibling pairs, whilst for a locus increasing risk two-fold 700 sibling pairs are needed (Kruglyak & Lander, Am J Hum Genet 1995; 56: 1212-1223). Further, Terwilliger and colleagues have shown with simulation studies that true peaks tend to be longer than false peaks (Terwilliger et al, Am J Hum Genet 1997; 61 : 430-438). It is thus improbable that linkage mapping will "pinpoint" a disease-causing gene. Therefore to further isolate such loci, other approaches are needed. Linkage varied markedly between the markers examined, even for adj acent markers, and the multipoint curve did not contain a single shaφ peak. This is not usual in linkage studies, as mentioned above. Observed linkage depends on many factors including the PIC of the markers chosen, chance variation in allele sharing of the markers chosen within the families in the sample and ancestral haplotypic effects. In summary, the further linkage mapping confirmed the boundaries for the area of linkage observed on chromosome 3p. The area included the PTHRl locus. It was therefore reasonable to study this gene further in association studies to assess whether polymoφhisms of PTHRl were responsible for the population variance of BMD.

Example 2: Mutation Screening of the PTHR Locus

This Example describes the screening for mutations in the PTHRl gene and demonstrates the linkage of a polymoφhism in the M6-M7 exon region and the identification of a novel polymoφhism in the U3 promoter region. Polymoφhisms .of the PTHRl gene and the promoter regions were sought using denaturing high performance liquid chromatography (DHPLC) as an efficient means of screening for mutations. Individual samples containing polymoφhisms were then sequenced to establish exact base changes and restriction fragment length polymoφhism (RFLP) assays designed for efficient genotyping in larger scale family-based and population-based association studies. Methods , Family Members

Samples from 36 unrelated patients with low BMD at femoral neck (FN) were selected from the family cohort. Mean FM z score was -2.45 (range -3.31 to - 1.96). 23 samples were from women, 13 from men.

Mutation Screening of the PTHRl Locus using DHPLC

Heteroduplex detection with DHPLC was used to screen for the presence of polymoφhisms in the 14 coding exons, introns, intron-exon boundaries and the promoter regions of PTHRl. Primer sequences for this work were obtained from previous publications (Shipani et al, J Clin Endocrinol Metab 1995; 80: 1611-1621, Bettoun et al, J Clin Endocrinol Metab 1997; 82: 1031-1040) or were redesigned using the available published sequence (http://www.ncbi.nih.gov/Genbank).

Sequencing

Regions containing heteroduplexes were sequenced to identify the polymoφhisms responsible. Sequencing data was compared with previously published sequence (Shipani et al, J Clin Endocrinol Metab 1995; 80: 1611-1621, Bettoun et al, J Clin Endocrinol Metab 1997; 82: 1031-1040, Bettoun et al, J Clin Invest 1998; 102: 958-967) and with genomic sequence released by the Human Genome Project (http://genome.uscs.edu), using the programmes BLAST 2 (Tatusova & Madden, FEMS Microbiol Lett 1999; 174: 247-250) and Sequence Navigator™ (Perkin Elmer, Applied Biosystems, Warrington, UK).

Results

Heteroduplexes were initially detected in three exons. Heteroduplexes of the M6-7 fragment were detected in 22/36 samples and heteroduplexes of the Tl fragment in 2/36 samples. Heterozygosity was also demonstrated in the U3 fragment (second promoter region), detected in 4/36 samples. Sequencing of these and additional fragments in which heterozygosity was demonstrated was carried out to determine the polymoφhic bases responsible for the heteroduplexes.

M6-7

A C/T change at position 311 (as defined by reference to SEQ ID NO: 5) was responsible for the observed heteroduplexes. This corresponds to position 61407 of the publically available PTHRl clone (AC094020). The frequency of the allele containing a T at this position is 68/184 chromosomes. Although in a coding sequence the mutationis silent (Asn463Asn).

Tl

An A/G base pair (G62133A) change at position 317 (as defined by reference to SEQ ID NO: 6) was found, resulting in a coding change in amino acid 546 (Glu to Lys). This corresponds to position 62133 of the publically available PTHRl clone (AC094020). The frequency of the allele containing an A at this position is 1/30 chromosomes.

U3 fin P3 A novel polymoφhism (C40850T) was found in U3 in 5/62 chromosomes and is shown in position 89 of SEQ ID NO:3, being a C/T base pair change. Further, sequencing of this promoter region (shown in SEQ ID NO: 3) from both heterozygote and homozygote samples showed several consistent differences with the previously published sequence (Bettoun et al, J Clin Endocrinol Metab 1997; 82: 1031-1040). When compared with genomic sequence data published by the Human Genome Project (http://genome.ucsc.edu^') (Figure 2) the sequencing obtained in these experiments was a closer match than was the previously published sequence. Between Uliand U2 fin P2

Two novel polymoφhisms were found in the P2 promoter region (between Ul and U2). The first (G36506A) is a G/A base pair change at position 36506 of the publically available PTHRl clone (AC094020) and is shown at position 86 of SEQ ID NO: 8. The frequency of the allele containing an A at this position is 1/48 chromosomes. The second (G36808A) is a G/A base pair change at position 36808 of the publically available PTHRl clone (AC094020) and is shown at position 88 of SEQ ID NO: 9. The frequency of the allele containing an A at this position is 6/48 chromosomes.

P3

Three further novel polymoφhisms were found in the P3 promoter region (between U2 and U3). The first (C40428G) is a C/G base pair change at position 40428 of the publically available PTHRl clone (AC094020) and is shown at position 108 of SEQ ID NO: 10. The frequency of the allele containing a G at this position is 1/32 chromosomes. The second (C41426G) is a C/G base pair change at position 41426 of the publically available PTHRl clone (AC094020) and is shown at position 86 of SEQ ID NO: 11. The frequency of the allele containing a G at this position is 2/30 chromosomes.

Intronic

Three novel SNPs were found in intronic sequences. The first (G52659T) is a G/T base pair change at position 52659 of the publically available PTHRl clone (AC094020) and is shown at position 99 of SEQ ID NO: 12. The frequency of the allele containing a T at this position is 7/32 chromosomes. The second (C52813T) is a C/T base pair change at position 52816 of the publically available PTHRl clone (AC094020) and is shown at position 73 of SEQ ID NO: 13. The frequency of the allele containing a T at this position is 8/20 chromosomes. The third (A52866C) is an A C base pair change at position 52866 of the publically available PTHRl clone (AC094020)ιand is shown at position 126 of SEQ ID NO: 13. The frequency of the allele containing a C at this position is 8/20 chromosomes.

A novel four base pair insertion/deletion polymoφhism was also found in the intronic sequences. This polymoφhism (54821-54824-GACA-DEL) is shown in SEQ ID NO: 14 (wild-type sequence) and in SEQ ID NO: 15 (mutant sequence). The deleted base pairs are shown at positions 101 to 104 of SEQ ID NO: 14. The frequency of the mutant allele (deletion) is 19/32 chromosomes.

Coding A novel polymoφhism (C56708A) was also found in the coding sequence at position 56708 of the publically available PTHRl clone (AC094020). This C/A base pair change is shown at position 68 of SEQ ID NO: 16. The frequency of the allele containing an A at this position is 2/32 chromosomes. Although present in the coding sequence this polymoφhism is synonymous (i.e. silent), Argl46Arg.

Genotyping

The polymoφhisms listed were genotyped in families with osteoporosis recruited through probands with low bone density for their age (T score <2.5, Z-score <2.0) and association tested using the program QTDT (Abecassis et al. Am. J. Hum. Gen. 66(1); 279-292, 2000).

Discussion

Polymoφhisms had previously been demonstrated in both M6-7 and T-l exons (Schipiani et al, J Clin Endocrinol Metab 1995; 80: 1611-1621, Hustmeyer et al, Hum Mol Genet 1993; 2: 1330). However the other polymoφhisms are novel.

The M6-7 exon polymoφhism detected in the osteoporotic population used in this study has also been observed in Caucasian, Black and Asian populations but has not previously been associated with osteoporosis (Hustmeyer et al, Hum Mol Genet 1993; 2: 1330). The frequency of heterozygotes in the osteoporotic group (22/36) appeared excessive. The presence of this polymoφhism was associated with a decreased bone density of approximately 0.5 standard deviations compared to those not carrying the variant. When assessed by Fisher's exact test there was no significant deviation from Hardy- Weinberg equilibrium (p=0.17). Although this polymoφhism does not result in an amino acid coding change, it may be in linkage disequilibrium with a polymoφhism or mutation with functional effect. This mechanism is thought to underlie the association of intronic Vitamin D receptor polymoφhisms and BMD.

In summary, several polymoφhisms of PTHRl were detected in a small population of osteoporotic patients, including several novel polymoφhisms and we have evidence to support the linkage of the polymoφhisms in the PTHRl gene to BMD and hence to bone disease and osteoporosis.

Claims

, CLAIMS

1. A method of diagnosing the existence of or susceptibility to a bone disorder, which method comprises determining whether the subject has a polymoφhism associated with low bone mineral density (BMD) in the parathyroid hormone receptor- 1 (PTHRl) gene, thereby determining the susceptibility of the subject to a bone disorder.

2. A method according to claim 1, wherein the polymoφhism is in a promoter region of the PTHRl gene.

3. A method according to claim 2, wherein the polymoφhism is at position 89 of U3 promoter region of the PTHRl gene, which position is defined by reference to SEQ ID NO: 3, or the polymoφhism is in linkage disequilibrium with said polymoφhism.

4. A method according to claim 3, wherein absence or presence of T at position 89 is determined, the presence of T indicating susceptibility of the subject to a bone disorder.

5. A method according to claim 2, wherein the polymoφhism is at position 86 of the P2 promoter region shown in SEQ ID NO: 8, or the polymoφhism is in linkage disequilibrium with said polymoφhism.

6. A method according to claim 5, wherein absence or presence of A at position 89 is determined, the presence of A indicating that the subject is susceptible to a bone disorder.

7. i A method according to claim 2, wherein the polymoφhism is at position 88 of the P2 promoter region shown in SEQ ID NO: 9, or the polymoφhism is in linkage disequilibrium with said, polymoφhism.

8. A method according to claim 7, wherein absence or presence of A at position 88 is determined, the presence of A indicating that the subject is susceptible to a bone disorder.

9. A method according to claim 2, wherein the polymoφhism is at position 108 of the P3 promoter region shown in SEQ ID NO : 10, or the polymoφhism is in linkage disequilibrium with said polymoφhism.

10. A method according to claim 9, wherein absence or presence of G at position 108 is determined, the presence of G indicating that the subject is susceptible to a bone disorder.

11. A method according to claim 2, wherein the polymoφhism is at position 86 of the P3 promoter region shown in SEQ ID NO: 11, or the polymoφhism is in linkage disequilibrium with said polymoφhism.

12. A method according to claim 11 , wherein absence or presence of G at position 86 is determined, the presence of G indicating that the subject is susceptible to a bone disorder.

13. A method according to claim 2 wherein the polymoφhism is the tetranucleotide repeat (AAAG)_n from position 440 of the U4 region of the PTHRl gene, which position is defined by reference to SEQ ID NO: 7, or the polymoφhism is in linkage disequilibrium with said polymoφhism.

14. _t A method according to claim 1, wherein the polymoφhism is in a coding region of the PTHRl gene.

15. A method according to claim 14, wherein the polymoφhism is at position 311 of exon M6-7 of the PTHRl gene, which position is defined by reference to SEQ ID NO: 5, or the polymoφhism is in linkage disequilibrium with said polymoφhism.

16. A method according to claim 15, wherein absence or presence of T at position 311 is determined, the presence of T indicating susceptibility of the subj ect to a bone disorder.

17. A method according to claim 14, wherein the polymoφhism is at position 317 of the sequence shown in SEQ ID NO: 6, or the polymoφhism is in linkage disequilibrium with said polymoφhism.

18. A method according to claim 17, wherein absence or presence of A at position 317 is determined, the presence of A indicating susceptibility of the subject to a bone disorder.

19. A method according to claim 14, wherein the polymoφhism is at position 68 of the sequence shown in SEQ ID NO: 16, or the polymoφhism is in linkage disequilibrium with said polymoφhism.

20. A method according to claim 19, wherein absence or presence of A at position 68 is determined, the presence of A indicating susceptibility of the subject to a bone disorder.

21. A method according to claim 1 , wherein the polymoφhism is in a intron of the PTHRl gene.

22. i A method according to claim 21, wherein the polymoφhism is at position 99 of the sequence shown in SEQ ID NO: 12, or the polymoφhism is in linkage disequilibrium with said polymoφhism.

23. A method according to claim 21 , wherein the polymoφhism is at position 73 or 126 of the sequence shown in SEQ ID NO: 13, or the polymoφhism is in linkage disequilibrium with said polymoφhism.

24. A method according to claim 22, wherein the absence or presence of T at position 73 and/or the absence or presence of C at position 126 is determined, wherein the presence of a T at position 73 and/or the presence of a C at position 126 indicating susceptibility of the subject to a bone disorder.

25. A method according to claim 21 , wherein the polymoφhism is a deletion of the GACA sequence at positions 101 to 104 of SEQ ID NO : 14, or the polymoφhism is in linkage disequilibrium with said polymoφhism.

26. A method according to any one of the preceding claims wherein the polymoφhism of the PTHRl gene associated with low BMD is determined for both alleles of the subject.

27. A method according to any one of the preceding claims wherein the bone disorder is osteoporosis.

28. Use of a substance or composition in the manufacture of a medicament for use in the treatment or prevention of a bone disorder in a subject, which subject has been identified as being susceptible to a bone disorder using a method according to any one of claims 1 to 27.

29. i Use according to claim 28 wherein the substance is parathyroid hormone (PTH), parathyroid hormone related peptide (PTHrP) or osteoprotegrin (OPG), or wherein the composition comprises PTH, PTHrP and/or OPG.

30. Use according to claim 28 or 29, wherein the bone disorder is osteoporosis.

31. A method of treating or preventing a bone disorder in a subject, which method comprises: (i) determining in accordance with a method of any one of claims 1 to 27 whether the subject has a polymoφhism associated with low BMD in the PTHRl gene, thereby determining whether the subject is susceptible to a bone disorder; and (ii) administering to a subject identified in (i) as susceptible to a bone disorder a therapeutically effective amount of an agent which reduces the risk of a bone disorder.

32. A method according to claim 31 , wherein said treating is prophylactic treatment of said bone disorder.

33. A method according to claim 31 or 32, wherein the agent is PTH, PTHrP or OPG.

34. A method according to anyone one of claims 31 to 33, wherein the bone disorder is osteoporosis.

35. A probe or primer for use in a method of diagnosis according to any one of claims 1 to 27, which probe or primer is capable of selectively detecting a polymoφhism in the PTHRl gene associated with low BMD.

36. _i A probe or primer according to claim 35 comprising the sequence set out in SEQ ID NO: 4 or a fragment or derivative thereof.

37. A probe according to claim 36, which is capable of selectively hybridising to a fragment of SEQ ID NO: 4 comprising a T residue at position 89 or the complement thereof.

38. A probe or primer according to claim 35, which is capable of selectively hybridising to a fragment of SEQ ID NO: 7 comprising at least part of the tetranucleotide repeat (AAAG)_n from position 440 or the complement thereof.

39. A probe or primer according to claim 35, which is capable of selectively hybridising to a fragment of SEQ ID NO: 6 comprising an A residue at position 317 or the complement thereof.

40. A probe or primer according to claim 35, which is capable of selectively hybridising to a fragment of SEQ ID NO: 8 comprising a G residue at position 86 or the complement thereof.

41. A probe or primer according to claim 35, which is capable of selectively hybridising to a fragment of SEQ ID NO: 9 comprising an A residue at position 88 or the complement thereof.

42. A probe or primer according to claim 35, which is capable of selectively hybridising to a fragment of SEQ ID NO: 10 comprising a G residue at position 108 or the complement thereof.

43. A probe or primer according to claim 35, which is capable of selectively hybridising to a fragment of SEQ ID NO: 11 comprising a G residue at position 86 or the complement thereof.

44. i A probe or primer according to claim 35, which is capable of selectively hybridising to a fragment of SEQ ID NO: 12 comprising a T residue at position 99 or the complement thereof.

45. A probe or primer according to claim 35, which is capable of selectively hybridising to a fragment of SEQ ID NO: 13 comprising a T residue at position 73 and/or a C residue at position 126 or the complement thereof.

46. A probe or primer according to claim 35, which is capable of selectively hybridising to a fragment of SEQ ID NO: 15 but not to SEQ ID NO: 14.

47. A probe or primer according to claim 35, which is capable of selectively hybridising to a fragment of SEQ ID NO: 16 comprising an A residue at position 68 or the complement thereof.

48. A kit suitable for use in a method for determining the susceptibility of a subject to a bone disorder, which kit comprises a probe or primer as defined in any one of claims 35 to 47.

49. A kit suitable for use in a method for determining the susceptibility of a subject to a bone disorder which kit comprises a primer or probe which selectively binds to the PTHRl gene, or a fragment thereof, only if a T residue is present at position 311 of exon M6-7 of the PTHRl gene, which position is defined by reference to SEQ ID NO: 5.

50. A method of identifying a polymoφhism which can be used to determine whether an individual has or is susceptible to a bone disorder, the method comprising screening the PTHRl gene of one or more individuals.

51. i A method of determining whether a candidate polymoφhism in the PTHRl gene can be typed to determine whether an individual has or is susceptible to a bone disorder, comprising determining whether the candidate polymoφhism is (i) associated with low bone mineral density or (ii) is in linkage disequilibrium with a polymorphism which is associated with low bone mineral density, and thereby determining whether the polymoφhism can be typed to determine whether an individual has or is susceptible to a bone disorder.

52. A method for identifying an agent which modulates the expression of the PTHRl gene in a subject having a polymoφhism as defined in claim 2, which method comprises:

(i) providing a cell comprising the promoter region of the PTHRl gene having said polymoφhism, wherein said promoter region is operably linked to the coding sequence of a gene; (ii) contacting said cell with a test agent;

(iii) monitoring the expression of said gene, thereby determining whether the test agent modulates the expression of PTHRl gene in said subject.

53. A method according to claim 52, wherein said cell is an osteoblast or an osteoclast.

54. A method according to claim 52 or 53, wherein said gene is the PTHRl gene.

55. A method according to claim 52 or 53, wherein said gene is a reporter gene.

56. A method according to any one of claims 52 to 55, wherein said agent up regulates PTHRl expression.

57. , A method according to any one of claims 52 to 55, wherein said agent down regulates PTHRl expression.

58. A method according to any one of claims 52 to 57 which further comprises synthesising an agent thus identified which modulates the expression of the PTHRl gene and/or incoφorating said agent into a pharmaceutical composition.

59. An agent identified by a method according to any one of claims 52 to 57 which modulates expression of the PTHRl gene in a subject having a polymoφhism as defined in claim 2.

60. Use of an agent according to claim 59 in the manufacture of a medicament for use in a method of treating a bone disorder.

61. A method of treating a bone disorder, which method comprises:

(i) identifying an agent that modulates the expression of the

PTHRl gene in a subject having a polymoφhism associated with low BMD in a promoter region of the PTHRl gene; and (ii) administering a therapeutically effective amount of an agent identified in (i) to a subject as defined in (i).

62. A method of treating a bone disorder, which method comprises providing an agent that complements a polymoφhism in the PTHRl gene that is associated with low bone mineral density

63. A method according to claim 62 wherein said agent is a DNA

64. A method of screening for an agent that will complement a polymoφhism in the PTHRl gene that is associated with low bone mineral density, wliich method comprises providing a PTHRl gene having said polymoφhism and determining whether a candidate agent will complement said polymoφhism.

65. An agent identified by a method according to claim 64.

66. A method according to claim 64 which further comprises synthesising an agent thus identified which modulates the expression of the PTHRl gene and/or incorporating said agent into a pharmaceutical composition.

- 67. A method for determining the efficacy of an agent useful in the treatment of a bone disorder in a subject having a polymoφhism associated with low BMD in the PTHRl gene, which method comprises:

(i) providing a PTHRl gene having said polymoφhism, or a protem encoded thereby; (ii) contacting said protein or gene with said agent; and

(iii) determining whether said protein or gene interacts with said agent.

68. A method according to claim 67, wherein said gene or protein is in a subject having said polymoφhism and step (iii) comprises monitoring the effect of administering the agent on BMD of said subject.