MXPA98007040A - Determination of the collage genotype - Google Patents

Abstract

The present invention relates to a diagnostic means that determines the genotype of a collagen gene of an individual. A polymorphism in the collagen gene correlates with the predisposition to osteoporosis, thus the diagnosis of a predisposition is carried out using the diagnostic means. An isolated gene comprising the nucleotide substitution of the polymorphism is also described, although it is a diagnostic method based on the presence or absence of the polymorphism.

Description

DETERMINATION OF THE COLLAGEN GENOTYPE Field of the Invention This invention relates to a method and diagnostic apparatus based on a polymorphism in a collagen gene. More specifically, this invention relates to a method and apparatus for diagnosing the predisposition to certain disease states, when examining the presence of this polymorphism, and more specifically to diagnose the predisposition to osteoporosis. The invention also relates to a collagen gene that contains the polymorphism. BACKGROUND OF THE INVENTION Hormone replacement therapy is an established treatment for osteoporosis, and has demonstrated success in stopping additional bone density depletion, which is characteristic in women suffering from this disease. However, hormone replacement therapy is generally not able to cause an inversion of osteoporosis, that is, it is not able to induce an increase in the bone density of patients. In accordance with the foregoing, it would be of particular advantage to be able to identify with increased accuracy those individuals who have a predisposition, or increased susceptibility to osteoporosis. Then, the right therapy could be put in place, before the effects of osteoporosis were established. SUMMARY OF THE INVENTION It is an object of the present invention to provide a method and apparatus for detecting individuals who have a predisposition or susceptibility to certain disease states, in particular, osteoporosis. It is a further object of the invention to identify individuals who have such a predisposition or susceptibility, by identifying those individuals with an altered collagen gene. It is another object of the present invention to provide a therapy for those individuals who have a predisposition or susceptibility to certain disease states. Still a further object of the invention is to provide a therapy for those individuals who have a predisposition or susceptibility to osteoporosis. According to the above, a first aspect of the invention provides a diagnostic method comprising, determining whether an individual is homozygous or heterozygous for a collagen gene and a polymorphism thereof. In one embodiment of the invention, the diagnostic method is to examine an individual at risk of a condition or disease correlated with the presence of the polymorphism. DETAILED DESCRIPTION OF THE INVENTION In one embodiment of the invention, described in detail hereinafter, the diagnosis employs the polymerase chain reaction or the conformational analysis of the single-filament polymorphism, or both, and determines whether an individual it has a wild-type collagen gene or a polymorphism thereof. Each individual can be homozygous for the wild type, heterozygous for the wild type and a polymorphism, or homozygous for polymorphisms in the collagen gene. The presence of a polymorphism correlates with the predisposition to osteoporosis. In a particular embodiment of the invention, the polymorphism is located in a transcriptional control region of a collagen gene, and the method of the invention comprises analyzing the transcriptional control region to examine a polymorphism therein. The method further comprises the use of a reporter means to react to the presence of the polymorphism. The indicator means typically induces a perceptible signal in the presence of the polymorphism, and may induce a color change or coagulation, or induce a restriction site, detectable by additional analytical steps. Another indicator means comprises an antibody having agglutination affinity that distinguishes between a wild-type sequence and a polymorphism. A particular method of the invention comprises the examination of a polymorphism at a binding site for Spl, a nuclear binding protein, where the presence of polymorphism correlates with the predisposition to osteoporosis. In the use of a specific embodiment of the invention to be described below in more detail, an individual is examined to determine whether he or she possesses a binding site Spl in a collagen gene, which is a published sequence or is a polymorphism of the same, in which a guanosine nucleotide at position 1245 has been replaced by a thymidine nucleotide. In this specific modality, the presence of polymorphism, in which guanosine is replaced by thymidine at position 1245, correlates with a predisposition to osteoporosis. The examination is carried out, for example, using PCR primers adapted to amplify a portion of the binding site Spl, which includes the nucleotide at position 1245. It is preferred that the PCR primers are selected in order to amplify a region of the gene that surrounds position 1245, and includes at least six nucleotides on either side of this position.

PCR techniques are well known in the art, and would be within the scope of a person of ordinary skill in this art, to identify primers to amplify a suitable section of the binding site Spl, including position 1245. PCR techniques are described for example in EP-A-0200362 and EP-A-0201184. In a further embodiment of the invention, the diagnostic method comprises the analysis of the binding site Spl in the collagen gene, using the correlation of the single filament conformational polymorphism (SSCP). It is preferred that PCR primers for this purpose be selected in order to be homologous with a region of the genome within 200bp of position 1245 in the collagen gene. It is further preferred that the PCR primers are selected such that position 1245 is substantially toward the middle of the amplified DNA segment. A second aspect of the invention provides the diagnostic means adapted to determine the genotype of a collagen gene; in this way, the diagnostic means diagnoses the presence of a polymorphisms in a collagen gene. The diagnostic means preferably comprises PCR primers adapted to amplify a region of a transcriptional control region in a collagen gene, preferably a DNA segment comprising a nucleotide at position 1245 in a binding site Spl in a collagen gene . It is preferred that the PCR primers are adapted to amplify a DNA segment that is up to 1 kb in length, more preferably up to 500 bp in length. In a particular embodiment of the invention, the segment is approximately 260 bp in length. Optionally, the diagnostic means further comprises the means for determining which nucleotide is at position 1245 at a binding site Spl in the collagen gene. An example is a restriction endonuclease capable of cleaving the gene, only if the polymorphism is present, alternatively, capable of splitting only the gene, or a normal variant thereof, in which the nucleotide at position 1245 is guanosine. The invention further provides a diagnostic equipment comprising the diagnostic means according to the second aspect of the invention, optionally within a container. A third aspect of the invention provides a collagen gene, or a fragment thereof, in which a guanosine nucleotide at position 1245 is replaced with a thymidine nucleotide. The fragment comprises position 1245, and is at least 30 nucleotides in length. The present invention is based on the discovery of a single base polymorphism at a binding site Spl of a human collagen gene; there are four Spl agglutination sites in the first intron. An additional aspect of the discovery is that the polymorphism correlates with a predisposition to osteoporosis. The invention is in advantage, in that when examining the presence of the polymorphism it is possible to identify the individuals that probably have this genetic predisposition. According to the above, a fourth aspect of the invention provides a method of therapy comprising, examining an individual for predisposition to osteoporosis and, if a genetic predisposition is identified, treating that individual to delay or reduce or prevent the osteoporosis. An adequate treatment to avoid 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 be started in this way in individuals who are likely to have a predisposition to osteoporosis, but in whom osteoporosis has not yet started to any significant degree. Another suitable treatment is the use of bisphosphonates, and additional treatments for a person skilled in the art will still be known. It is known that the use of hormone replacement therapy, it can be carried out with an increased concomitant 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 know that they have a predisposition to osteoporosis. In an embodiment of the fourth aspect of the invention, the predisposition of an individual to osteoporosis is assessed by determining whether that individual is homozygous for a collagen gene, in which nucleotide 1245 is guanosine, is heterozygous for this gene, and polymorphism in which the guanosine at position 1245 is replaced by thymidine, or is homozygous for the polymorphism. According to the invention, an individual who is homozygous T / T for the polymorphism is classified as being at higher risk. An individual being heterozygous G / T is classified as having moderate risk. An individual being homozygous G / G is classified as being in the lowest risk category. Optionally, the assessment of an individual risk factor is calculated by reference both to the presence of a polymorphism of the collagen gene, as well as to other dietary or physiological or genetic or other indications. In this way, this invention provides additional information on which the measurement of an individual risk can be based. A specific embodiment of the invention is the nucleotide sequence of SEQ ID NO: 1, listed in the sequence listing below. In SEQ ID NO: 1, the polymorphism lies in T instead of G, at position 1245 in the gene sequence. For reference, this polymorphism at base 1245 in the collagen gene sequence corresponds to base 1240 in the published Bornstein PNAS 1987 sequence; 84: 8869-8873. The binding site Spl is found at 1210-1219 and 1245-1254 in the sequence of the collagen gene. In addition, SEQ ID NO: 1 comprises 5 other differences consistent with those reported by Bornstein. Two additional polymorphisms have also been found in our studies (a T-base insertion +498 in Bornstein's paper, and a C-G substitution in base 811 in Bornstein's paper). These polymorphisms form additional aspects of the invention. In additional specific embodiments of the invention, the diagnostic means comprises primers of SEQ ID N0: s 2 and 3, the sequences of which are shown in the sequence listing below. The 5 'end of SEQ ID NO: 2 is agglutinated at position 1006. The 3' end of SEQ ID NO: 3 is agglutinated at position 1246. The preparation of additional suitable primers to determine the genotype of a Collagen will be within the scope of a person of ordinary experience in the field. Osteoporosis is a common condition characterized by reduced bone mass, icroarchitectural deterioration of bone tissue, and increased risk of fracture. The risk of osteoporotic fracture is related to bone mineral density, (BMD) which, in turn, is under strong genetic control. Although the genes that regulate bone mass are incompletely defined, the type I collagen genes (C0LIA1 and C0LIA2), are important candidates, since mutations that affect their coding regions give rise to osteogenesis imperfecta (01), a rare disease characterized by severe osteoporosis and multiple fractures. However, the extensive DNA sequence in the osteoporotic individuals has not revealed abnormality of the COLI coding regions. In . a specific embodiment of the invention described in detail below, PCR-SSCP is used to examine a polymorphisms in the transcriptional control region of COLIA 1, to determine the presence or absence of a G / T polymorphism in the first intron at a site of recognition for nuclear binding protein Spl- an important regulator of gene transcription. BRIEF DESCRIPTION OF THE DRAWINGS Now follows the description of the specific embodiments of the invention, illustrated in the figures in which: Figure 1 shows a polymorphism in intron 1 of the C0LIA1 gene in a binding site Spl. Panel A: EMSA analysis was performed with double-filament labeled oligonucleotides that include the putative polymorphic Spl site in the first intron of COLIA1 as a test. The change of band (signaled) was canceled by the excess oligo Spl cold, while the oligos API and NFkB had no effect consistent with the presence of a Spl site. The position of a non-specific band is indicated. Panel B shows the effect of a specific monoclonal antibody Spl (0.5 mg) on the band change shown in Panel A. The antibody Spl attenuated the change, and this effect was canceled by coincubation with an excess of the peptide Spl (0.5 mg), confirming the specificity for the agglutination Spl; Figure 2 shows a polymorphic Spl site in the COLIA1 gene, which is related to bone mineral density. The adjusted BMD values in the spine were significantly related to the COLIA1 genotype, with evidence of a gene-dose effect. A similar (but not significant) trend was observed for BMD in the femoral neck (hip). The difference between the values of the BMD in the spine in the groups "SS" and "Ss" was significant (p <0.02), as was the difference between the combined groups "SS" and "Ss" / "ss "(p < 0.02); Figure 3 shows a Spl polymorphism of COLIA1, and bone mineral density: menopause effect. Panel A: adjusted BMD values in the spine, and Panel B: adjusted BMD values in the hip, are related to the menopausal age and the COLIA1 genotype. The BMD data for the "Ss" and "ss" groups were combined in view of the small number of "ss" individuals (4 perimenopausal and 5 late postmopausic), and because the BMD values were similar in these subgroups . Although the difference in BMD between genotypes was small in early premenopausal and postmenopausal women (0-5 years), there was a significant difference in spinal BMD in late postmenopausal women (> 5 years). A similar trend was observed for the BMD of the hip, but this failed to reach importance (p = 0.06); and Figure 4 shows an overrepresentation of the "s" alleles of COLIA1 in patients with osteoporotic vertebral fracture. The analysis of the C0LIA1 genotypes in patients with osteoporotic vertebral fracture (n = 55), and control subjects of the same age (n = 55) showed the significant overrepresentation of the "s" alleles in the group with osteoporotic vertebral fracture, equivalent to the relative risk of presence of the fracture of 2.95 95% Cl = (1.63-9.56). Example 1 The study was based on a regional reference center for bone disease, which caters to a white Caucasian population of approximately 0.5 million from a geographically isolated region in the United Kingdom. The study group comprised 200 women, of which 108 (54%) were consecutive references for clinical evaluation; the rest was drawn at random from the local population. Individuals with diseases known to affect bone metabolism (corticosteroid use, pituitary disease, immobilization, primary hyperparathyroidism, neoplasia, thyrotoxicosis) were excluded. The age range was 45-88 years, with an intermediate age (+ sem) of 60.1 + 0.73 years. One hundred and seventy-four women (87%) were postmenopausal with an average of 13.3 + 0.78 years after menopause (defined as the absence of menstruation for 6 months). 18. According to the criteria of the World Health Organization ( WHO), 22% of the study group was normal, 32% were osteopaenic, and 46% were osteoporotic. These proportions are expected in a group of predominantly postmenopausal women, since the prevalence of osteoporosis increases with age. A subgroup of 55 individuals (27.5%) had vertebral compression fractures, diagnosed clinically and by the presence of one or more biconcave or wedge vertebral deformities on x-rays of the spine. The examination of the transcriptional control regions of COLIA1 (promoter and first intron 20,21) by PCR-SSCP, showed evidence of 3 polymorphisms, of which all were in the first intron; two of these were rare (4% and 3% of allele frequency) and one common (22% of allele frequency). Because osteoporosis is a common disease, additional studies focused on the common polymorphism, which was characterized by a DNA sequence as a G / T substitution at the first base of a recognition site for the transcription factor Spl . The electrophoretic mobility shift (EMSA) analysis confirmed that the polymorphism lies within a Spl agglutination site since the band change corresponding to the Spl DNA complex was canceled by the excess of Oligonucleotide Spl, but without being affected by the oligonucleotides API or NFkB (Figure 1; Panel A). Additional confirmation was provided by superchange analysis, which showed attenuation of the gel change with a Spl specific monoclonal antibody, and its restoration by the addition of the excess peptide Spl (Figure 1, Panel B). A rapid PCR-based test for polymorphism was devised (see methods), and the distribution of the allele was studied in a group of 200 women. The frequencies of the allele were consistent with those predicted by the Hardy-Weinberg equation; 9"ss" homozygotes (4.5%); 71"Ss" heterozygotes (35.5%) and 120 SS homozygotes (60%). The analysis of these genotypes in relation to bone mass showed that adjusted BMD values in the lumbar spine were significantly reduced in both the "Ss" and "ss" individuals, when compared with the "SS" individuals with the evidence of a gene-dose effect (Figure 2). The findings were similar when the BMD was expressed as the result Z (Spine with the result Z = -0.3 + 0.11 of "SS", Vs -0.74 + 0.15 of "Ss" Vs -1.12 + 0.34 of the "ss"; < 0.03 ANOVA). BMD values of the femoral neck followed a similar pattern, but the difference between genotypes was not significant, possibly reflecting the greater importance of environmental factors in the determination of BMD at that site. In contrast, there was no significant difference between the genotypes in the following anthropomorphic and environmental variables: height (mean + sem; SS Vs Ss Vs ss, respectively): 1.58 + 0.06 m Vs 1.57 + 0.07 m Vs 1.61 + 0.10 m; weight: 64.4 + 1.2 kg Vs 61.5 + 1.4 Vs 64.2 + 2.8; users of hormone replacement therapy: 11.5% Vs 7.2% Vs 12.5%; Current smokers 15.9% Vs 15.9% Vs 12%; taking calcium: 446 + 38 mg Vs 531 + 49 mg Vs 563 + 157 mg. Because the BMD in this age group reflects a balance between maximum bone mass and the proportion of bone loss 1, we will next analyze the internal relationship between the alleles of C0LIA1 and the enopausal state, since this is an important determinant of BMD in women (Figure 3). In the present, adjusted BMD values are recalculated, excluding menopausal age as a co-variable. In the figure, the data were grouped from the "Ss" and "ss" groups because the values of the BMD were similar in these groups, and in view of the small number of "ss" homozygotes. This analysis showed that the difference in BMD between genotypes was accentuated in women with postmenopause from 5 to more years in both the spine and the hip. • Although this was a cross-sectional study, the above observation raises the possibility that the "s" allele may act as a marker for increased postmenopausal bone loss, rather than maximum bone mass. In this regard, it is of interest that osteogenesis imperfecta is also associated with an increased rate of fracture after menopause, presumably because the defect in collagen production accentuates the bone loss that normally occurs postmenopausally. Next, we sought to determine if there was over representation of the "s" allele in patients with severe osteoporosis, who had vertebral compression fractures. In this analysis, the 55 patients with osteoporotic vertebral fractures were compared with the 55 controls of equal age and sex. In accordance with the BMD data, the significant over-representation of the "Ss" and "ss" genotypes was found in patients with vertebral fracture (54% Vs 27%; chi-squared = 7.42; df = l; p < 0.01), equivalent to the relative risk of presence of vertebral fracture of 2.97 (95% confidential interval 1.63-9.56) Methods The bone mineral density (BMD) was assessed by double-energy X-ray absorptiometry as previously described (by Garton , M., Reid, I., Loveridge, N., Robins, S., Murchison, L., Beckett, G., and Reid, DM, Bone mineral density, and metabolism in premenopausal women taking thyroxine, Endocrinology - l Clinical 41: 747-755, 1995), and was expressed as the T-result and the Z-result, which refer to the actual bone density (g / cm squared) for the average of the normal population in healthy individuals , young locally derived, and in the controls of the same age; respectively. Adjusted BMD values (g / cm2) were also calculated by multiple linear regression analysis, as previously described, to correct confusing factors that may influence BMD, including age, menopausal age, height, weight, use of hormone replacement therapy, calcium diet intake (by questionnaire) and smokers. RESULTS Preliminary analyzes showed that according to the previous 18 studies, the only variables that significantly influenced BMD were age, menopausal age, and body weight. The adjusted BMD values shown are correct for these variables, with the exception of the data in Figure 3, where the menopausal age was excluded as a co-variable in the calculation. Polymorphisms in the promoter and intron 1 of COLIA1 were searched by PCR-SSCP in the DNA extracted from whole blood using a computer (Nucleon, Scotlab, United Kingdom). Oligonucleotide primer sets (Oswell DNA services, University of Edinburgh, United Kingdom); sequences available from the authors in question), were designed to produce PCR fragments of coating 10 of 262-336bp that extends over the promoter (bases -751 to +39), and the first itron (bases +174 to +1805) of the human gene COLIA1 (Figure 1). The PCR (incorporating 2 (Ci 32P Dctp in each reaction), was carried out under standard conditions using 0.1 ng of DNA as model The products were reduced in native 5% polyacrylamide gels run at 20 degrees C with 5% of glycerol, and detected by autoradiography for 2-3 days In all cases, the putative polymorphisms identified by SSCP were confirmed by direct sequencing (DNA sequencer 373A Applied Biosystems, Perkin Elmer, USA) Polymorphisms were detected Subsequently, mutagenesis was directed to a PCR-mediated site with an unequal primer that introduced a restriction site for the polymorphic alleles in amplified fragments, Ectophoretic mobility shift analysis (EMSA) was carried out using nuclear extracts prepared from MG63 cells. (a human osteoblast as a cell line), using standard methods as previously described. A double-stranded labeled oligonucleotide (32 P dATP) incorporating the polymorphic Spl binding site was used as a test for EMSA with unlabeled double-stranded oligonucleotides for Spl, API and NFkB in 100-fold molar excess as competitors. A specific monoclonal antibody from Spl and a peptide Spl (Biotechnology Santa Cruz) were used in a final concentration of 0.5 (g / tube in the superchange analysis). CONCLUSIONS In this way, in a clinical study of 200 women, it was found that individuals homozygous or heterozygous for the substitution of T (designated as "Ss" and "ss", respectively), have significantly lower BMD of the spine. to homozygotes for the substitution of G (designated as "SS"), with evidence of a gene-dose effect of the "s" allele on the bone mass. Additional analyzes showed that the difference in BMD between genotypes was accentuated in postmenopausal women for more than 5 years, raising the possibility that the "s" genotype could be associated with postmenopausal bone loss. Consistent with this hypothesis, unfavorable "Ss" and "ss" genotypes were significantly overrepresented in postmenopausal patients with severe osteoporosis and vertebral fractures, as compared to controls of equal age and sex (54.7% Vs 27.2%; (2 = 7.4; df = l; p < 0.01), equivalent to a relative risk of 2.97 (95% confidence interval 1.63-9.56) for the presence of vertebral fracture.

Example 2 Methods 185 Caucasian women (age range 44-64 years) of an unselected sample from a large general practice in Northeast London were studied as part of the Chingford Population Study. Bone mineral density was measured in the lumbar spine (L1-L4) and the femoral neck with double-energy X-ray absorptiometry (Hologic QDR-1000). The self-reported personal history of fracture was taken for the period of 10 years preceding the start of the study (1978-88), and was subsequently validated with hospital records and radiographs. Lateral X-rays on the spine were obtained in all women, and lumbar and thoracic fractures were defined morphometrically, using validated methods. The genotype analysis was carried out using previously described methods, and the alleles were coded as "S" (absence of the Bal I restriction site) and "s" (presence of the site). The degradants of urinary collagen (pyridinoline and deoxypyridinoline) were measured in urine samples taken early in a subgroup of women, using liquid chromatography of high pressure. RESULTS The genotype frequencies observed in the total group were in Hardy-Weinberg equilibrium, and were similar to those previously reported in the United Kingdom: SS 61.1% (n = 113); Ss 36.2% (n = 67); ss 2.7% (n = 5). Given the small number of "ss" homozygotes, the results of this group were grouped with the heterozygous genotype for the subsequent analysis. Bone mineral density was significantly reduced in the lumbar spine in subjects with the "s" allele, the mean difference (95% Cl) 0.047 g / cm2 (0.001, 0.093). A similar trend was also observed in the femoral neck, although this difference was not significant, mean difference (95% Cl) 0.026 g / cm2 (-0.013, 0.065). There were 55 validated fractures in the total group, 28 vertebral and 27 appendiceal. The frequency of the "Ss / ss" genotypes increased significantly in the cases of fractures (49%) compared to the controls (33%), with a disparity ratio (95% of Cl) of 1.95. (1.01, 3.78). The examination of prevalent fractures in the two most common sites of the vertebra and wrist (Colles), showed a similar tendency in the genotype distributions in each skeletal site, although the sizes of the samples were too small for the importance to be demonstrated. The degrading results of urinary collagen were available in a subset of 82 subjects. Levels of pyridinoline, but not deoxypyridinoline, were elevated in subjects with the "s" allele (p 0.05).

Conclusions Our data show an association between the C0LIA1 polymorphism, the low bone density and the fracture. Example 3 Methods The study group comprised a sample stratified by random age of 895 postmenopausal women aged 55-80 years from the Rotterdam study, a population, based on a study group of chronic diseases in old age. The polymorphism was determined by a polymerase chain reaction based on the analysis, and the genotype data were related to bone density, the rate of bone loss and the presence of vertebral fractures. RESULTS Bone mineral density (BMD) was reduced in heterozygotes G / T (designated as "Ss"), in comparison with homozygotes G / G ("SS") significant in the femoral neck (p = 0.0009), triangle Ward (p = 0.02), and just above the level of importance in the spine (p = 0.06). The rare T / T homozygotes ("ss") had a lower BMD still consistent with a gene-dose effect on bone mass. A significant difference in the rate of decrease in BMD with age was reflected, by means of large genotype-dependent differences in the BMD of women in the larger groups, suggesting that the alleles of COLIA1 may act as a marker for increased bone loss related with the age. Longitudinal studies in a subgroup of 684 women supported this view by showing significantly faster rates of bone loss in the Ward triangle in the Ss and ss genotypes over a 2-year period., when compared with SS homozygotes (p = 0.04). Finally, the Ss and ss genotypes associated with low BMD were significantly overrepresented in 43 patients, who had vertebral fractures compared to controls (Chi-square = 4.1, p = 0.04), which corresponds to a relative risk of 1.9 (95 percent) of confidence interval 1.0-3.5) for the occurrence of fracture in carriers of allele "a". Conclusions These findings suggest that the Spl polymorphism of COLIA1 is an important marker of reduced bone mass and osteoporotic fracture. They also indicate the allele associated with low BMD to determine bone loss related to age. An osteoporotic fracture risk function that includes the Spl polymorphism of COLIA1 can help in clinical practice to identify individuals at high risk of osteoporotic fracture for prophylactic therapy, before substantial bone loss has occurred. Studies on the genetic basis of osteoporosis are important in the identification of regulatory genes that can act as therapeutic targets, and in helping to develop methods to identify individuals at risk of disease. Our studies have revealed a strong association between bone mass and a common polymorphism that affects a regulatory motif in the control-transcriptional region of the C0LIA1 gene. We have also demonstrated the significant overrepresentation of the unfavorable "s" alleles in a group of patients with the clinically important fracture-vertebral condition. Although the molecular mechanism by which this polymorphism affects bone density will require further study, our data identify COLIA1 as an important candidate gene for the genetic regulation of bone mass, and raise the possibility that the genotype at the Spl site may be of value in target individuals, who are at risk of osteoporosis for early prophylactic therapy.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: GEMINI INTERNATIONAL HOLDINGS LIMITED (B) STREET: Le Regina, 13-15 Boulevard des Moulins (C) CITY: Monaco (E) COUNTRY: Monaco (F) POSTAL CODE (ZIP): MC 98000 (A) NAME: RALSTON; Stuart Hamilton (B) STREET: University of Aberdeen, AURIS, Business Center (C) CITY: 23 St Machar Drive, Aberdeen (E) COUNTRY: UK (F) POSTAL CODE (ZIP): AB2 1RY (A) NAME: GRANT, Struan Frederick Airth (B) STREET: University of Aberdeen, AURIS, Business Center (C) CITY: 23 St Machar Drive, Aberdeen (E) COUNTRY: UK (F) POSTAL CODE (ZIP): AB2 1RY (ii) TITLE OF THE INVENTION: METHOD AND DIAGNOSTIC APPARATUS (iii) NUMBER OF SEQUENCES: 3 (iv) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: FLEXIBLE MAGNETIC DISC (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH : 318 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TAACTTCTGG ACTATTTGCG GACTTTTTGG TTCTTTGGCT AAAAGTGACC TGGAGGCATT 60 GGCTGGCTTT GGGGGACTGG GGATGGCCCC GAGAGCGGGC TTTTAAGATG TCTAGGTGCT 120 GGAGGTTAGG GTGTCTCCTA ATTTTGAGGT ACATTTCAAG TCTTGGGGGG GCCTCCCTTC 180 CAATCAGCCG CTCCCATTCT CCTAGCCCCG CCCCCGCCAC CCCACCTGCC CAGGGAATGT 240 GGGCGGGATG AGGGCTGGAC CTCCCTTCTC TCCTCCCTCG CCCTCCTCCT GTCTCTACCA 300 CGCAGCCACT CCCCACGA 318 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: - 2í TAACTTCTGG ACTATTTGCG 20 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) FILAMENTO: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: CCGGTCCTAC TCCCGACCTG 20

Claims (18)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and therefore the property described in the following claims is claimed as property. 1. A diagnostic means for determining the genotype of a transcriptional control sequence of a collagen gene, for the diagnosis of osteoporosis or predisposition to osteoporosis.
2. 2. Diagnostic means according to claim 1, characterized in that the collagen gene is Col 1 alpha
3. 3. A diagnostic means according to claim 1 or 2, characterized in that the transcriptional control sequence comprises a binding site Spl.
4. 4. Diagnostic means according to any of claims 1-3, characterized in that it comprises PCR primers adapted to amplify a region of a transcriptional control sequence in a collagen gene.
5. 5. Diagnostic means according to claim 4, characterized in that it comprises PCR primers adapted to amplify a DNA segment comprising a nucleotide at position 1245 in a Col 1 alpha gene.
6. 6. Diagnostic means according to claim 4 or 5, characterized in that the PCR primers are SEQ ID NO 2 and SEQ ID NO 3.
7. 7. A method for diagnosing osteoporosis or predisposition to osteoporosis, characterized in that it comprises determining whether an individual It has a polymorphism in the transcriptional control sequence of a collagen gene.
8. 8. A diagnostic method according to claim 7, characterized in that it comprises determining whether an individual possesses a wild type transcriptional control sequence in a collagen gene, or a variant that differs from the wild type by a substitution of the single nucleotide.
9. A method according to claim 8, characterized in that the variant comprises a thymidine residue at position 1245.
10. 10. A method according to any of claims 7-9, characterized in that it comprises amplifying a portion of that collagen gene of the individual, using PCR techniques.
11. 11. DNA comprising a 1 alpha collagen gene, characterized in that the guanosine at position 1245 is replaced by thymidine.
12. 12. An isolated DNA characterized in that it comprises SEQ ID NO: 13.
13. A method of therapy for osteoporosis characterized in that it comprises: screening an individual for a genetic predisposition to osteoporosis; and if such a predisposition is identified, treat that individual to avoid or reduce osteoporosis or delay the onset of osteoporosis, wherein the predisposition to osteoporosis correlates with a polymorphism in a transcriptional control sequence of a collagen gene.
14. A method according to claim 13, characterized in that a predisposition to osteoporosis correlates with a collagen gene, in which at position 1245 a guanosine nucleotide is replaced by thymidine.
15. 15. A method according to claim 13 or 14, characterized in that it comprises the treatment of an individual by means of hormone replacement therapy.
16. 16. A diagnostic equipment characterized in that it comprises the means according to any of claims 1-6.
17. 17. The use, in the development of means for assessing whether an individual has a predisposition to osteoporosis, of PCR primers adapted to amplify a region of a transcriptional control sequence in a collagen gene. The use according to claim 17, characterized in that the PCR primers are adapted to amplify a DNA segment comprising position 1245 in a Col 1 alpha gene.