CA2345938A1 - Polymorphism in a tgf-beta gene correlated to osteoporosis - Google Patents

Abstract

Methods and apparatus for diagnosis of disease and predisposition to disease are described. In particular, methods for diagnosis of osteoporosis and determination of bone mineral density in individuals are provided as well as treatments and therapies for delaying and preventing the onset of disease in at-risk individuals.

Description

POLYMORPHISM IN A TGF-SS GENE CORRELATED TO OSTEOPORPSIS
This invention relates to diagnostic method and apparatus based upon polymorphism of a TGF-f3 Gene. More specifically, this invention relates to a method for diagnosis of pre-disposition to disease by screening for the presence of a polymorphism. The invention also relates to apparatus for screening for the potymorphism. The invention further relates to TGF-t3 genes containing a polymorphism and to a probe therefor.
Osteoporosis is a common age-related condition characterised by reduced bone mineral density (BMD), deterioration in skeletal microarchitecture and an increased risk of fragility fracture. One in three Caucasian women will experience an osteoporotic fracture during their lifetime and it is estimated that the annual health cost of such fractures amounts to around USS14 billion in the USA alone.
Hormone replacement therapy is an established treatment for osteoporosis and has proved successful in halting further decline in bone density that is characteristic in women suffering from this disease. Hormone replacement therapy is generally not, however, able to bring about a reversal of osteoporosis, that is to say it is not capable of inducing an increase in the bone density of sufferers. The same criticism is made of other known treatments for osteoporosis.
It would, accordingly, be of particular advantage to be able to identify with increased accuracy those individuals having a predisposition or increased susceptibility to osteoporosis. Suitable therapy could then be put into place before the effects of osteoporosis set in.
Genetic factors play an important role in determining bone mineral density SUBSTIME SHEET (RULE 26) 22- ; 2-2060 ~ PCT/G 899/03446 (BMD) in later life, with the genetic influence mediated through effects on both peak mass and on age- and menopause- related bone loss. At the menopause there is an increase in the production and activity of various cytokines and growth factors within the bone microenvironment.
Bone mineral density (BMD) in later life is a strong predictor of subsequent osteoporotic fracture and is determined by both the peak value achieved during skeletal growth and by age- and menopause- related bone loss.
Family and twin studies suggest a strong genetic component to the determination of peak bone mass, with 50-85% of the population variance in BMD being attributable to genetic factors. Twin studies in postmenopausal and elderly women also support a persistent and significant genetic influence on bone mass in later life. This rnay represent either a strong residual effect from the genetic contribution to peak bone mass or an independent genetic effect on the regulation of bone loss.
Indirect assessment of bone turnover through biochemical markers suggests a genetic regulation of bone metabolism that may translate into differing effects on bone loss although to date only two twin studies have directly attempted to explore the genetic contribution to age- and menopause- related bone loss with conflicting and uncertain results.
Osteoporosis is a complex disease that is likely to have a polygenic aetiology, and candidate gene analysis has demonstrated that polymorphisms of the vitamin D receptor (uDR) locus the oestrogen receptor (ER) locus and the type I collagen alpha 1 (COL1 A1 ) locus are all potential genetic markers for bone mass and bone loss. WO-A-97/28280 describes two polymorphisms in the promoter region of the TGF-~B1 gene that have been shown to influence the amount of TGF-~1 protein in the blood and correlates these polymorphisms with predisposition to a number of disease states including hypertension, cancer and osteoporosis.
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AMENDED SHEET

22- ~ 2-20(i0 , PCT/GB99/03446 -2a-Langdahl et al. (Bone Vo1.20, No.3, 1997: 289-294) describes a sequence variation in an intron of a TGF-Beta 1 gene, the 713-SdeIC variation. The sequence variation is a single base deletion eight bases upstream of the start of intron 5. The 713-BdeIC variation is correlated with an increase in bone turnover but in both the non-osteoporotic normal control individuals and the total group of osteoporotic patients bone mass was unaffected by the presence of this variation.
Yamada et al. (J. Bone. Miner. Res. Vol. 13, No.10, 1998:1569-1676) relates to a T-~C transition at nucleotide 29 in the signal sequence region of the TGF-Beta 1 gene. Yamada et al. describe the correlation between this variation and susceptibility to osteoporosis in postmenopausal Japanese women.
The search for further genetic markers for use in diagnosis of disease, AMENDED SHEET

including diagnosis of osteoporosis and predisposition thereto, nevertheless continues.
It is an object of this invention to provide method and apparatus for detecting individuals having a predisposition or susceptibility to osteoporosis. It is a further object of the invention to identify individuals having such a predisposition or susceptibility by identifying those individuals on the basis of genotype. It is another object of the invention to provide a therapy for those individuals. Still further objects of the invention are to provide an isolated gene comprising a polymorphism indicative of predisposition to osteoporosis and probe therefor.
Accordingly, a first aspect of the invention provides a method of diagnosis comprising determining genotype of an intron of a TGF-f3 gene.
The method of the invention typically comprises determining whether an individual is homozygous or heterozygous for the gene and a particular polymorphism thereof and thereby determining if the individual possesses a risk polymorphism, the risk polymorphism being correlated with osteoporosis or disposition thereto. The method is conveniently used to screen for an individual at risk of a condition or disease correlated with a polymorphism of this gene. In a preferred embodiment of the invention the TGF-~B gene is a TGF-,B1 gene and the polymorphism is a T-~C
polymorphism located in intron 5 of said TGF-,81 gene. However, it is envisaged that other polymorphisms that are located within the same or a different intron may be so correlated with the presence of the intron 5 polymorphism of the invention that the two polymorphisms are in linkage disequilibrium. Thus, diagnosis of disease by determining genotype of the further polymorphism may lead to a similarly reliable diagnosis of osteoporosis or predisposition thereto.
The method of the invention determines whether the individual being tested has a TGF-f3 gene which is identical with the published sequence or whether that individual has a gene which differs from the published sequence, i.e. is a polymorphism of the published sequence. In carrying out the invention, an individual's TGF-f3 gene genotype is generally determined by analysis of a section of the gene, rather than by analysis of the entire gene. If the sequence of that section is found to be the same as the corresponding section in the wild type sequence, then that individual is classified as having the wild type gene.
In use of an embodiment of the invention to be described below in further detail, an individual is screened to determine whether he or she possess a TGF-f3 gene which is the published sequence or is a polymorphism thereof in which there is a polymorphism in one or more of its introns. In this specific embodiment, the' presence of a homozygous polymorphism in an intron of the gene correlates with a predisposition to osteoporosis.
In an example of the invention, described in more detail below, the presence of a BstUl restriction site in intron 5 of a TGF-,81 gene is correlated with increased risk of osteoporosis or predisposition to osteoporosis as well as increased bone turnover and reduced BMD. This novel restriction site polymorphism is produced as a result of a T->C
substitution introducing the 5'-CG ,~ CG-3' recognition site for BstUl.
Screening is carried out, for example, using PCR primers adapted to amplify a portion of gene in the region of and including the site of the polymorphism It is preferred that the PCR primers are selected so as to amplify a region of the gene that surrounds the region and includes at least six nucleotides on either side. PCR techniques are well known in the art and it would be within the ambit of a person of ordinary skill in this art to identify primers for amplifying a suitable section of the gene. PCR
techniques are described for example in EP-A-0200362 and EP-A-0201 i 84.

A second aspect of the invention provides for an isolated DNA fragment comprising all or a part of the sequence of SEQ ID N0:1. The sequence in SEQ ID N0:1 is the last fifty bases of intron 5 of the human TGF-~1 gene including the T-~C substitution at position 31 of that sequence. Such a DNA fragment may suitably be inserted into a vector for transformation into prokaryotic or eukaryotic cells. Alternatively, the fragment may serve as a template for production of a nucleic acid probe sequence for use in applications such as northern or southern blotting or RNase protection.
Thus the invention provides DNA probes capable of distinguishing between a wild type gene, to which the probe does not bind, and a polymorphism thereof, such as one containing a polymorphism in intron 5 to which the probe does bind.
The invention is of advantage in that by screening for the presence of the polymorphism it is possible to identify individuals likely to have a genetic predisposition to this disease.
A third aspect of the invention provides diagnostic means comprising PCR
primers adapted to amplify a region of a TGF-f3 gene, preferably a DNA
segment comprising intron 5.
Suitable primers are recited in the example below and correspond to SEQ
ID NOS: 2 and 3. The invention further provides a diagnostic kit comprising PCR primers for use in amplification of an intron in a TGF ~ gene and reference means to enable determination of the genotype, optionally within a container. Such reference means typically include control reactions, a written manual or diagrammatic representations indicating positive and negative results.
The kit of the invention may also comprise an amount of the restriction enzyme BstUl enabling PCR amplimers of the intron 5 region of the TGF-~B1 gene to be analysed for the presence of the novel T->C polymorphism of the invention.
Accordingly, a fourth aspect of the invention provides a method of therapy comprising screening an individual for a predisposition to osteoporosis and, if a genetic predisposition is identified, treating that individual to delay or reduce or prevent the osteoporosis, wherein a predisposition to osteoporosis is correlated with a polymorphism in an intron of a TGF ~B1 gene.
A suitable treatment to prevent or reduce or delay osteoporosis is hormone replacement therapy. The use of this therapy is well known in the art.
According to the invention, hormone replacement therapy can thus be commenced in individuals likely to have a predisposition to osteoporosis but in whom osteoporosis has not yet begun to any significant extent.
It is believed that the use of hormone replacement therapy carries with it a concomitant increased risk of breast cancer. The invention offers the advantage that the increased risk of breast cancer associated with hormone replacement therapy can be accepted only by those women who are known to have a likelihood of predisposition to osteoporosis. In an embodiment of this aspect of the invention, the predisposition of an individual to osteoporosis is assessed by determining whether that individual is homozygous for the wild type TGF-f3 gene, is heterozygous for the wild type and the, or is homozygous for the polymorphism - indicating risk of predisposition to osteoporosis.
According to the invention, an individual who is homozygous for the risk polymorphism is classified as being at highest risk.
Another suitable treatment is use of bisphosphonates. Two specific treatments involve using xanthine oxidase inhibitors or substituted benzodiazepines and are described in US-A-5436258 and US-A-54419fi4, the contents of which are incorporated herein by reference. Still further treatments will be known to a person of skill in the art. Potential treatments are described, for example, in JP-A-09030977, WO-A-97/06254, JP-A-09025293, WO-A-97/04799, WO-A-97/03060 and JP-A-09012592, the contents of which are incorporated herein by reference.
Currently authorised treatments for osteoporosis include the use of oestrogens, with and without progestogen, the use of selective oestrogen receptor modulators, the use of anabolic steroids such as nandrolone, the use of the bisphosphonates alendronate and disodium etidronate. Further treatments suitably include the use of salcatonin, administration of calcium supplements and use of isoflavones or other plant derived steroids.
In pharmaceutical treatment of osteoporosis, all routes of administration are suitable and include but'' are not limited to oral, injection intravenously, intraperitoneally,intramuscularlyandsubcutaneously,intranasalandtopical administration. Typical dosages and durations of treatment are as described in clinician's textbooks such as British National Formulary, incorporated herein by reference.
Currently, none of the osteoporosis medications that have been approved by the Food and Drug Administration (FDA) for postmenopausal women have been approved for men.
Testosterone replacement therapy may be prescribed for a man with a low testosterone level.
Calcitonin is a medication that slows or stops bone loss and may relieve the pain of fractures in some patients. Calcitonin is approved by the FDA
for the treatment of osteoporosis in postmenopausal women. While its effect in men has not been studied, evidence suggests that it may work the same in men as in women. Calcitonin is available as an injection and as a nasal spray. Its use is described in US-A-5440012, incorporated herein by 8 PCTlGB99/03446 _g_ reference.
Bisphosphonates are a class of drugs that have been shown to help preserve and increase bone density by slowing or stopping bone loss. The FDA has approved the bisphosphonate known as alendronate for the treatment of postmenopausal osteoporosis in women; it is currently being studied for treatment of osteoporosis in men. There are other bisphosphonates under development - and in fact etidronate has been approved, though only outside the USA.
Sodium fluoride has recently been recommended for approval by an FDA
committee. Parathyroid hormone, calcitriol, and others are investigational drugs. It will be some time before research: findings are available on these preparations.
Decrease in bone mineral density can also be slowed by taken calcium supplements, and some suggested levels are 1,000 mg of calcium a day for women on oestrogen replacement therapy and 1,500 mg of calcium daily for women not receiving oestrogen therapy.
Thus, a range of treatments, for those suffering or predisposed to osteoporosis are known and all are believed suitable for use in combination with its diagnosis according to the present invention.
Optionally, the assessment of an individual's risk factor is calculated by reference both to the presence of a TGF-f3 gene polymorphism and also to other known genetic or physiological or dietary or other indications. The invention in this way provides further information on which measurement of an individual's risk can be based.
The invention thus also provides a method of identifying, and optionally treating, an individual predisposed or susceptible to osteoporosis, said WO 00/23b18 ~ PCTlGB99/0344b method comprising determining genotype of a first gene in said individual, wherein genotype of said first gene is correlated with genotype of a TGF-(3 gene in said individual.
The invention further again provides a method of predicting response to osteoporosis therapy, comprising diagnosing genotype of a TGF-f3 gene, in accordance with the first aspect of the invention.
This latter aspect of the invention thus enables informed choice of therapy, including choice of type of therapy and choice of amount or strength of therapeutic agent, to be made for a given individual predisposed to osteoporosis. Moreover, for a given individual already suffering from osteoporosis, the invention enables an assessment of whether the currently f prescribed therapy is likely to be effective in treating the disease or if an alternative therapy regime will be more successful: In a specific embodiment of the invention, diagnosis of a risk polymorphism in a TGF-f3 gene indicates that hormone replacement therapy, or an equivalent, is likely to be effective. More specifically, possessing two copies of the risk polymorphism indicates an increased level of therapy is likely to be appropriate.
Bone mineral density (BMD) in later life is a major determinant of osteoporotic fracture risk and has been shown to be under strong genetic influence. Segregation analysis within families and data from twin studies has suggested that this genetic effect on BMD is probably mediated by a number of genes each having small individual effects. Transforming growth factor 13 (TGF-f3) is an important regulatory cytokine and is found in high concentrations in the bone matrix. TGF-f3 is therefore a plausible candidate for the genetic regulation of BMD. In total 91 1 DZ pairs and 386 MZ pairs (age range 18-76 years) were studied, with measurements of BMD using DXA and calcaneal ultrasound. In accordance with the present invention, a novel T-~C polymorphism was identified in intron 5 with an allele frequency of 0.25 within the DZ subjects. Comparison of the variance in femoral neck BMD between the MZ and DZ twins showed a heritability of 62% at this site. BMD at the femoral neck was 5% lower in subjects homozygous for the presence of the TGF-f3 polymorphism when compared to the other two genotype groups. No effect was seen at the lumbar spine, ultradistal radius, or with ultrasound measurements. Results were unaffected after adjustment for potential confounders. Linkage analysis within the DZ twin pairs confirmed the significance of this polymorphism on hip BMD.
BMD is one of the strongest predictors of fracture risk (Cummings et al.
(1993)) and a large number of twin and family studies have suggested a strong genetic influence on this trait, with up to 85% of the population variance in BMD being attributable to genetic factors (Seeman et al. ( 1989);
Arden et al. (1996); Jouanny et al. (1995).
Normal skeletal morphogenesis is dependent on a complex interaction between osteoblasts, osteoclasts, and local growth factors. During growth and development the processes of osteoblastic bone formation and osteoclastic bone resorption appear coupled, thereby maintaining skeletal integrity and preserving bone mass and shape. Non-invasive measures of bone turnover have shown that after the menopause there is an increase in bone resorption, leading to a loss of bone and subsequent development of osteoporosis. Several studies have also shown that in women with osteoporotic fracture the remodelling balance is more negative when compared to age-matched women with no fracture history. It is believed that this postmenopausal increase in bone resorption in the oestrogen deficient state is mediated in part by cytokines (interleukins 1 and fi, tumour necrosis factor and various growth factors (transforming growth factor, insulin-like growth factors). Twin studies have also suggested that there is a genetic influence on the general process of bone turnover in pre-and post-menopausal women, with higher correlations for biochemical markers of both bone formation and resorption seen in identical compared to non-identical twins (Kelly et al. ( 1991 ); Garnero et al. ( 1996)1.
Transforming growth factor f3 (TGF-f3) is synthesised by osteoblasts and osteoclasts in vivo and has three isoforms. High concentrations of all these isoforms can be extracted from the mineralised bone matrix, and although TGF-(3 is found in a variety of tissues, the concentration of TGF-f3 appears to be highest in the bone matrix. Osteoblasts produce TGF-f3 largely as a matrix-bound latent complex composed of 390 amino acids. It is only released during bone resorption, with subsequent activation in the acidic environment below the ruffled border of the resorbing osteoclast. This suggests that TGF-f3 may play a central regulatory role in the coupling that exists between bone formation and resorption. Active TGF-f3 is formed of two identical disulphide-linked polypeptide chains consisting of the 112 amino acids from the C-terminal part of the precursor protein. The TGF-(3 gene maps to chromosome 19q 13 and contains 7 exons. The active component of TGF-f3 is encoded by part of exon 5, exon 6, and part of axon 7. The TGF-f3 gene may therefore be an important candidate gene for the development of osteoporosis, with variation at this locus being associated with differences in BMD and therefore risk of fracture.
A further aspect of the invention provides for a method of diagnosing osteoarthritis and predisposition to osteoarthritis in an individual comprising determining the genotype of intron 5 in a TGF-~1 gene, wherein absence of a BstUl restriction site is correlated with risk of osteoarthritis.
It is known that predisposition to osteoarthritis and osteoporosis may be linked in that the process of bone deposition seen in osteoarthritis is under the control of similar signalling factors to those that control the process of bone resorption, as seen in osteoporosis. The activities of osteoblasts (bone forming cells) and osteoclasts (bone resorbing cells) are to an extent controlled by the same group cytokines including TGF-~3. Accordingly, a WO 00/2361$ PGT/GB99/03446 further aspect of the present invention provides for a method of diagnosing osteoarthritis and predisposition to osteoarthritis in an individual comprising determining the genotype of intron 5 in a TGF-~1 gene. The risk of disease or predisposition thereto is inversely related to risk of or predisposition to osteoporosis, thus for example absence of a BstUl restriction site is correlated with risk of osteoarthritis, and mutatis mutandis for all other embodiments of the invention.
The present invention is now illustrated by way of the following example.
Example Methods Subjects The subjects studied were Caucasian, female monozygous (MZ) and dizygous (DZ) twins (age range 18-76 years) recruited after national media campaigns. All subjects were healthy and did not suffer from diseases specifically affecting bone, and were broadly representative of the normal United Kingdom population as previously described. Twins completed a nurse-administered questionnaire detailing medical, obstetric and gynaecological histories, full drug histories, dietary calcium assessment, exercise levels, smoking status, and alcohol intake. Twin zygosity was determined by questionnaire and in doubtful cases this was confirmed with multiplex DNA fingerprinting.
Measurements BMD was measured at the lumbar spine (L1-4), non-dominant hip (femoral neck, total hip), and non-dominant ultradistal radius using DXA on a Hologic QDR-2000. Reproducibility as assessed by the coefficient of variation (CV%) from duplicate measures in healthy volunteers was between 0.8% and 1.6% at the skeletal sites measured. Subjects were classified as having osteoporosis according to the World Health Organisation diagnostic criteria if their BMD measurement was 2.5 standard deviations (SD) below the mean peak young adult value (i.e. T -score < -2.5).
Calcaneal ultrasound was measured using a McHue Cuba Clinical scanner.
This produced two output variables: broadband ultrasound attenuation (BUA) and velocity of sound (VOS). Reproducibility as assessed by the CV% in duplicate measures on 30 subjects was 2.5% (BUA) and 0.44%
(VOS).
Polvmorahism identification DNA was prepared for each subject from peripheral blood leucocytes using a standard phenol extraction method. Common single nucleotide polymorphisms (SNPs) in the TGF-f3 gene were detected by sequence analysis of 24 unrelated dizygotic (DZ) individuals and comparison made with the published sequence (Accession no. Y00112). This strategy was chosen to identify polymorphisms with an allele frequency of at least 0.1 within the study group. Oligonucleotide primer pairs were designed to cover the TGF-f31 coding regions, and the promoter and 5'-untranslated region up to position 1363. Following amplification by the polymerase chain reaction (PCR) the products were purified with the Advanced Genetic Technologies Corp 96 well PCR purification system and sequenced using PE Applied Biosystems dRhodamine Terminator cycle sequencing kit. The sequencing reactions were analyzed on an ABI 377 DNA sequencer.
Restriction enzyme digests Polymorphism screening within the DZ group was performed using PCR-restriction fragment length polymorphism (RFLP) based methods with restriction enzyme digest.

WO 00/23618 ~ PCT/GB99/03446 BstUl PCR-RFLP analysis of the intron 5 SNP
PCR amplification was performed using the ~PCR primers SEQ ID NOS: 2 and 3. Reactions were performed in 25,u1 with the following composition:
0.3NM primers, 0.2mM dNTPs, 1 mM MgCL2, 1 X Taqgold buffer, 1.25 units Taqgold (P.E. Applied Biosystems) and 50ng genomic DNA.
Thermocycling was performed on a MJ Research DNA Engine Tetrad PTC-225 thermal cycler using the following conditions: 95°C for 14 minutes, 35 cycles of 94°C for 15 seconds, 60°C for 15 seconds, 72°C for 30 seconds, followed by final extension of 72°C for 10 minutes. The 230 by PCR product was digested with 3 units of BstUl (New England Biolabs) at 60°C for 2 hours producing polymorphic fragments of 202 and 28 bp.
Products were analysed by agarose gel electrophoresis with size determination after transillumination under ultra-violet light. Alleles were coded as A 1 = presence of restriction site, and AO = absence of site.
Statistical analysis Differences in the mean value in each variable for the three TGF-f3 intron 5 genotypes (AOAO, AOA1 and A1A1) were tested using generalised estimating equations (GEE)(Zeger et al. (1986)). This method takes into account the dependence of measurements within twin pairs in estimating the significance of the differences. The GEE models were extended to include potential confounding variables (age, menopausal status).
Significant GEE results were followed up using a quantitative genetic modelling approach in which additional phenotypic data on MZ twins were used. A model was specified which provided estimates for the genetic and environmental variance components and estimates for the means of each possible genotype (AOAO, AOA1 and A1A1). This enabled us to estimate the percentage of the genetic variation in the phenotype that could be attributed to the TGF-f3 polymorphism. Model fitting was performed using Mx. Parameters were estimated by normal-theory maximum-likelihood, where the model were fitted to the raw data. Linkage analysis within the DZ twins was also analysed using the MAPMAKER/SIBS programme lversion 1.0). This calculates the maximum likelihood sharing probabilities for each DZ pair and as parental information was not available this utilised estimated allele frequencies from within the total DZ group. Evidence for linkage of the polymorphism to the trait was taken as a nominal lod score >_ 1.00 or a nominal P value S 0.05.

Results Sequence analysis of 24 unrelated individuals identified a novel polymorphism in the TGF-f3 gene. The novel polymorphism was a T-~C
substitution in intron 5, 20bp upstream of exon 6. This substitution in intron 5 introduces a BstUl site (recognition sequence 5'-CG 1 CG-3'), and subsequent screening for this within the whole DZ cohort using the BstUl PCR-RFLP showed the allele frequencies to be 0.75 and 0.25, with the genotype distributions being in Hardy Weinberg equilibrium.
In total, data was available on 911 DZ and 386 MZ twin pairs. Intron 5 genotype results were available on 1664 of the DZ subjects. Reasons for absence of genetic results included inadequate DNA extraction or failure of the PCR-RFLP assay. No 'significant differences were observed, however, between subjects with and without TGF-f3 genotype results.
Characteristics of the study population are shown in Table 1. In comparison with the DZ twins, the MZ twins were on average slightly older by 2 years and had a higher percentage of women who were postmenopausal. Within the DZ group there were no differences in age, height, weight, smoking status or hormone replacement (HRT) use between the three TGF-f3 intron 5 genotype groups. There were however, small differences in the proportion of postmenopausal women (Table 1 ).
The TGF-f3 intron 5 genotype A1A1 was associated with hip BMD when compared to the other 2 genotypes, with a 5% reduction in femoral neck BMD and a 3.8% reduction in total hip BMD (Table 2). These findings were unaltered after adjustment for the small difference observed in menopausal status. These results at the hip, suggest a recessive pattern of risk associated with carriage of the rarer A1 TGF-f3 intron 5 allele. No genotypic association was seen at the lumbar spine, ultradistal radius or in the calcaneal ultrasound parameters of BUA and VOS. The prevalence of clinically defined osteoporosis (T-score <-2.5) at the femoral neck in the genotype group A1A1 was 18% compared to 10% in the AOAO and AOA1 groups. This demonstrates a 70% increased risk for a subject with the A1A1 genotype having femoral neck osteoporosis when compared to the other 2 genotypes, with an odds ratio (95% confidence interval) of 1.71 (0.95, 3.07), P = 0.07.
Having demonstrated an association between TGF-f3 intron 5 genotype and hip BMD, twin modelling and variance component analysis was utilised to estimate the proportion of the genetic variance explained by this polymorphism. This analysis confirmed previous studies showing high heritabilities for BMD and ultrasound parameters at the various skeletal sites. At the femoral neck the estimated heritability was 0.62, with the proportion of the population genetic variance explained by the intron 5 TGF-f3 polymorphism at this site being 0.60%.
The results of the single point linkage analysis are shown in Table 3. These demonstrate linkage between the intron 5 polymorphism and femoral neck BMD. For the measurement of BUA the nominal P value approaches 0.05 although the lod score does not exceed 1.00.
Thus, in accordance with the present invention, a novel T-~C polymorphism in a TGF-f3 intron, specifically intron 5 in an embodiment of the invention, of the human TGF-f3 gene. Our data demonstrate both association and linkage between this polymorphism and hip BMD in a large group of unselected, normal female twins. The polymorphic C allele was present at a relatively high allelic frequency in the female population (0.25), and there appeared to be a recessive pattern of risk associated with this allelic variant. Hip BMD was 3-5% lower in women who were homozygous for the carriage of the polymorphic allele, when compared to women who were either heterozygous or homozygous for the commoner allele. Women who were homozygous for the polymorphism also had a 70% increased risk of having osteoporosis at the femoral neck in comparison to the other two genotypes.
These cross-sectional data indicate that subjects who are homozygous for an intronic polymorphism of the TGF-f3 gene have reduced BMD at the femoral neck and total hip. The observation that these findings were confined to the hip (and predominantly at the femoral neck), rather than being seen at the spine and distal radius suggest a site-specific association.
The absence of any genotype association with bone ultrasound measures also suggests that the TGF-f3 intron 5 polymorphism has a predominant effect on BMD rather than on bone quality or structure. Although the proportion of population variance in BMD attributable to this polymorphism appears low, this reflects the recessive risk associated with this locus as only approximately 6% of the population would be expected to be of the genotype A1A1. Our data indicate, however that these subjects have a 70% increased risk of having osteoporosis and therefore face significantly increased risk of fracture. TGF-f3 genotype therefore identifies an at risk sub-group of women who would benefit from targeted intervention. This is also illustrated by recent findings from a large population study examining the relationship between a polymorphic variant in the promoter region of the type I collagen 1 a gene and osteoporosis. In this study the polymorphic allele was associated with a 2-fold increased risk of fracture despite only explaining 0.3 to 0.4% of the population variance in BMD.
There is currently a large amount of data suggesting that TGF-f3 may play a central role in the regulation of BMD and bone turnover. In vivo studies have shown that local injection of TGF-f3 under the periosteum stimulates cartilage and bone formation (Noda et al. (1989)) and that systemic injection of TGF-f32 also leads to a generalised increase in osteoblastic activity. In vitro, TGF-f3 induces extracellular matrix secretion by osteoblasts, inhibits matrix mineralisation, and modulates osteoprogenitor cell proliferation. The rate of bone formation is altered in the TGF-(3 knockout mouse (Geiser et al. ( 1996)) and administration of TGF-13 corrects the bone density deficiency in elderly mice with osteoporosis. Oestrogen action on bone also appears to be mediated via effects on TGF-f3 and induction of osteoclast apoptosis. Allelic variation at the TGF-f3 locus may therefore be important in determining the therapeutic response of the oestrogen and selective oestrogen receptor modulators on bone and other tissues.
The functional significance of our findings on TGF-f3 activity is uncertain, although as the polymorphism is 20 by upstream of exon 6 it could have some influence on the active component of the TGF-f3 protein. The lack of coding sequence variation identified in this study and others suggests that the amino acid sequence of the active form of TGF-f3 is highly conserved with strong selective pressures against variant proteins. It has been reported that up to 15% of human diseases are caused by point-sequence variation in splice regions resulting in either exon skipping or cryptic splicing and although the intron 5 polymorphism is not located in a splice donor or acceptor site it could affect a branch point. The intronic polymorphic sequence change may also affect messenger RNA stability and further studies will be required to examine these possibilities. If this polymorphism is not functional, then these findings may suggest that the TGF-f3 polymorphism is actually in linkage disequilibrium with a novel disease locus mapping to this chromosomal region. The finding of a positive linkage result at this locus with hip BMD would also implicate this chromosomal region and multipoint linkage analysis will be required to refine the location of the putative disease locus.
The invention thus provides method and apparatus for diagnosis of osteoporosis or predisposition thereto.

WO 00/23618 PCTlGB99/03446 Table 1 Mean ( t SD) characteristics of female twin subjects variable MZ total DZ totalDZ subjects 724) ~" ~ according ~ ~58~ to TGF-~1 intron 5 genotype HVHU 1 "j--(n = 848) (n = 609) (n =
Age (yrs) 50.1 47.5 47.6 47.5 g0) 49.1 ( 13.4) ( 11.3) ( 11.0) ( 1 1.9) (9.6) Height (cm) 162 (6) 163 (6) 162 (6) 163 (6) 162 (6) Weight (kg) 63.9 65.9 66.0 65.4 65.4 (10.7j (12.3) (12.1) (12.1) (12.7) S a b j a c t s 67 54 51 56 62 postmenopausal (%) Subjects ever smoking44 48 45 49 51 (%) Subjects ever use 30 30 28 31 31 of HRT (%) SUBSTITUTE SHEET (RULE 26~

Table 2 Mean ( t SD) BMD at lumbar spine, hip and forearm, and calcaneal ultrasound measurements in Dt subjects according to their TGF ~f39 intron 5 genotype Variable T F X81 Intron 5 Genotype 0 n = A n = n =

848) L a m b a r s 0.998 (0.147)1.011 (0.149) 1.005 (0.146) p i n a BMD (g/cm2) Femoral neck 0.812 (0.131)0.810 (0.130) 0.770 (0.112)*' BMD (g/cm2) Total hip BMD 0.924 (0.132)0.925 (0.131 0.889 (0.117) ) ~

(g/cm2) ' Ultradistal radius0.455 (0.068)0.458 (0.069) 0.451 (0.068) BMD (g/cmz) VOS (m/sec) 1659 (52) 1660 (52) 1661 (51 ) BUA (dB/MHz/cm) 78 ( 19) 78 ( 19) 76 ( 17) * AlAl vs AOAO, P = 0.005; AlAl vs AOA1, P = 0.04 $ AlAl vs AOA1, P = 0.05 Table 3 Single point lod scores for TGF-~1 intron 5 genotype and BMD/IJltrasound measures in DZ twin pairs.
Variable TGF-,81 Intron 5 Lod score Nominal P-va ue Lumbar spine 0.365 0.195 BMD (g/cm2) Femoral neck 1.091 0.025 BMD (g/cm2) Total hip BMD 0.479 0.138 (9/cmz Ultradistal radius 0.570 0.105 BMD (g/cm2) VOS (m/sec) 0.159 0.392 BUA (dB/MHz/cm) 0.785 0.057 References:
Arden NK, Baker J, Hogg C, Baan K, Spector TD 1996. The heritability of bone mineral density, ultrasound of the calcaneus and hip axis length: a study of postmenopausal twins. J Bone Miner Res 11: 530-534.
Cummings SR, Black DM, Nevitt MC, Browner W, Cauley J, ~nsrud K, Genant HK, Palermo L, Scott J, Vogt TM 1993. Bone density at various sites for prediction of hip fractures. The study of osteoporotic fractures research group. Lancet 341:
72-75.
Garnero P, Arden NK, Griffiths GO, Delmas PD, Spector TD 1996 Genetic influences on bone turnover in postmenopausal tv~ins. J Clin Endocrinol Metab 81: 140-146.
Geiser AG, Helvering LM, Zeng QQ, Sato M 1996 Bone growth in the TGF ~1 knockout mouse. J Bone Miner Res 11: S378. (Abstr) Jouanny P, Guillemin F, Kuntz C, Jeandel C, Pourel J 1995. Environmental and genetic factors affecting bone mass: similarity of bone density among members of healthy families. Arthritis Rheum 38: 61-67.
Kelly PJ, Hopper JL, Macaskill GT, Pocock NA, Sambrook PN, Eisman JA 1991 Genetic factors in bone turnover. J Clin Endocrinol Metab 72: 808-813.
Noda M, Camilliere JJ 1989 In vivo stimulation of bone formation by transforming growth factor,8. Endocrinology 124: 2991-2994.
Seeman E, Hopper JL, Bach L, Cooper ME, Parkinson E, McKay J, Jerums G
1989. Reduced bone mass in daughters of women with osteoporosis. N Engl J
Med 320: 554-558.

Zeger SL, Liang KY 1986 Longitudinal data analysis for discrete and continuous outcomes. Biometrics 42: 121-30 SEQUENCE LISTING
- <110> GEMINI RESEARCH LIMITED
SPECTOR, TIMOTHY D
KEEN, RICHARD W
GIBSON, FERNANDO
MOLLOY, HELEN R
<120> DIAGNOSTIC METHOD AND APPARATUS BASED ON POLYMORPHISM IN
A TGF-BETA GENE
<130> GWS/DJC/20648 <140>
<141>
<160> 3 <170> PatentIn Ver. 2.1 <210> 1 <211> 50 <212> DNA
<213> human <400> 1 tctatggtgg tagcccctcc ctgcccctga cgcgtctctc ctgcctgcag 50 <210> 2 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: PCR PRIMER
<400> 2 atggtggtag cccctccct 19 <210> 3 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: PCR PRIMER
<400> 3 gcatctcgta gcccggtgg SUBSTITUTE SHEET (RULE 26~

Claims (23)

Claims:

1. A method of diagnosing osteoporosis or predisposition to osteoporosis in an individual comprising determining the genotype of intron 5 in a TGF-.beta.
gene.

2, A method according to claim 1 wherein presence of a BstUl restriction site in said intron 5 is correlated with increased risk of osteoporosis or predisposition to osteoporosis.

3. A method according to claims 1 and 2, wherein said TGF-.beta. gene is the TGF-.beta.1 gene.

4. A method according any previous claim, comprising amplifying a portion of said individual's TGF-.beta. gene, said portion including at least a part of said intron 5, by using PCR techniques.

5. An isolated DNA molecule comprising the sequence of SEQ ID NO: 1.

6. A kit for diagnosis of osteoporosis or predisposition to osteoporosis comprising PCR primers far use in amplification of intron 5 in a TGF-.beta.
gene, and reference means to enable determination of the genotype of said intron 5.

7. A kit according to claim 6 wherein said TGF-.beta. gene is a TGF-.beta.1 gene.

8. A kit according to claims 6 and 7 wherein said PCR primers comprise SEQ
ID NO: 2.

9. A kit according to any of claims 6-8 wherein said PCR primers comprise SEQ
ID NO:3.

10. A kit according to any of claims 6-9 further comprising the restriction enzyme BstUl.

11. A method of osteoporosis therapy comprising:-screening an individual for a genetic predisposition to osteoporosis; and if such a predisposition is identified, treating that individual to prevent osteoporosis or to delay onset of osteoporosis, wherein a predisposition to osteoporosis is correlated with a polymorphism in intron 5 of a TGF-.beta.1 gene.

12. A method according to claim 11 in which a predisposition to osteoporosis is correlated with the presence of a BstUl restriction site in said intron 5 of a TGF-.beta.1 gene.

13. A method according to claims 11 and 12 in which a predisposition to osteoporosis is correlated with a substitution of a thymidine with a cytosine at position 31 in SEQ 1D NO:1.

14. A method of treating an individual predisposed or susceptible to osteoporosis said method comprising:-(a) determining the genotype of intron 5 in a TGF-.beta.1 gene in said individual in order to identify a risk genotype in said TGF-.beta.1 gene; and (b) administering to said individual an effective dose of a therapeutic composition suitable to delay, reduce, or prevent osteoporosis in said animal.

15. The method of claim 14 comprising administering to said individual an effective dose of said therapeutic composition selected from a group consisting of steroid hormones, isoflavones, calcium supplements, bisphosphonates, calcitonin, sodium fluoride, parathyroid hormone and calcitriol,

16. A method for predicting response to osteoporosis therapy, comprising diagnosing the genotype of intron 5 in a TGF-.beta. gene according to the method of claim 1.

17. Use, in the manufacture of means for assessing whether an individual has a predisposition to osteoporosis, of PCR primers adapted to amplify a region of a TGF-.beta. gene, said region including intron 5.

18. Use according to Claim 17 wherein said TGF-.beta. gene is a TGF-,.beta.1 gene,

19. A method of determining bone mineral density in an individual comprising determining the genotype of intron 5 in a TGF-.beta. gene.

20. A method of determining risk of fracture in an individual comprising determining the genotype of intron 5 in a TGF-.beta. gene.

21. A method of determining bone turnover in an individual comprising determining the genotype of intron 5 in a TGF-.beta. gene.

22. A method according to any of claims 19-21, wherein identification of a BstUl restriction site in said intron 5 is diagnostic of increased risk and absence of said BstUl restriction site is diagnostic of reduced risk.

23. A method of diagnosing osteoarthritis and predisposition to osteoarthritis in an individual comprising determining the genotype of intron 5 in a TGF-.beta.1 gene.