WO1998044150A1 - Polymorphisms of an il-1 receptor antagonist gene - Google Patents

Abstract

A method of diagnosis comprises determining genotype of an IL-1 receptor antagonist gene, such as to determine whether an individual possesses a wild type IL-1 receptor antagonist gene or polymorphisms thereof, and the results may be used to diagnose disease such as osteoporosis or predisposition to osteoporosis and treat affected or predisposed individuals.

Description

POLYMORPHISMS OF AN IL-1 RECEPTOR ANTAGONIST GENE

This invention relates to diagnostic method and apparatus based upon polymorphisms of an IL-1 receptor antagonist gene (IL-1RN). More specifically, this invention relates to a method for diagnosis of pre-disposition to certain disease states, particularly osteoporosis, by screening for the presence of this polymorphism. The invention also relates to apparatus for screening for the polymorphism. The invention further relates to IL-1 receptor antagonist genes containing a polymorphism and to a probe therefor.

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 (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 (3) 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 (19,21). Twin studies in postmenopausal and elderly women also support a persistent and significant genetic influence on bone mass in later life (1,5). This may 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 (6,12,25), 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 (2,13).

Osteoporosis is a complex disease that is likely to have a polygenic aetiology (8), and candidate gene analysis has demonstrated that polymorphisms of the vitamin D receptor (VDR) locus (23) the oestrogen receptor (ER) locus (15) and the type I collagen alpha 1 (COL1A1) locus (7) are all potential genetic markers for bone mass and bone loss.

The search for further genetic markers for use in diagnosis of disease, 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 a IL-1 receptor antagonist gene.

The method of the invention typically comprises determining whether an individual is homozygous or heterozygous for a IL-1 receptor antagonist gene and a particular polymorphism thereof. The method is conveniently used to screen for an individual at risk of a condition or disease correlated with a polymorphism of this gene.

The DNA sequence of the IL-1 receptor antagonist gene region is known and has been published (Lennard A et al, Cytokine 1992; 4:83-89). This sequence is referred to hereafter as the wild type sequence or the published sequence. The method of the invention determines whether the individual being tested has a IL-1 receptor antagonist gene which is identical with the published sequence or whether that individual has a IL-1 receptor antagonist gene which differs from the published sequence, i.e. is a polymoφhism of the published sequence. In carrying out the invention, an individual's IL-1 receptor antagonist gene genotype is generally determined by analysis of a section of the IL-1 receptor antagonist 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 IL-1 receptor antagonist gene.

In use of a specific embodiment of the invention to be described below in further detail, an individual is screened to determine whether he or she possess a IL-1 receptor antagonist gene which is the published sequence or is a polymoφhism thereof in which there is a variable number of a 86bp repeat within intron 2 of the interleukin-1 receptor antagonist gene. In this specific embodiment, the presence of a polymoφhism in which there are 4 repeats correlates with a predisposition to osteoporosis.

Screening is carried out, for example, using PCR primers adapted to amplify a portion of the IL-1 receptor antagonist gene in the region of and including the repeat. It is preferred that the PCR primers are selected so as to amplify a region of the gene that surrounds the region of the repeat and includes at least six nucleotides on either side of this position. 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 IL-1 receptor antagonist gene. PCR techniques are described for example in EP-A-0200362 and EP-A- 0201184.

In a specific embodiment of the invention described in further detail below, the PCR primers have the nucleotide sequences :-

CTCAGCAACACTCCTAT (SEQ ID NO: 1) and TCCTGGTCTGCAGGTAA (SEQ ID NO: 2).

The screening is suitably carried out by amplifying a DNA fragment including the region of the repeat in the IL-1 receptor antagonist gene and determining the size of the PCR fragment or fragments obtained.

In a further embodiment of the invention, the diagnostic method comprises analysis of the IL- 1 receptor antagonist gene using single strand conformational polymoφhism (SSCP) mapping. It is preferred that the PCR primers are selected so as to be homologous with a region of the genome within 200bp of the repeat on the IL-1 receptor antagonist gene.

A second aspect of the invention provides diagnostic means comprising PCR primers adapted to amplify a region of a IL-1 receptor antagonist gene, preferably a DNA segment comprising intron 2.

In a specific embodiment of the invention described below, the PCR primers are SEQ ID NO: 1 and SEQ ID NO: 2.

Optionally, the diagnostic means further comprises means to determine how many repeats there are in the region of intron 2. The invention further provides a diagnostic kit comprising diagnostic means according to the second aspect of the invention, optionally within a container.

A third aspect of the invention provides DNA probes capable of distinguishing between a wild type IL-1 receptor antagomst gene, to which the probe does not bind, and a polymoφhism thereof, such as one containing 4 repeats of an 86bp sequence in intron 2 to which the probe does bind.

The present invention is based upon the discovery of an 86 bp repeated sequence polymoφhism in the IL-1 receptor antagonist gene. An aspect of the invention is that the polymoφhism is correlated with a predisposition to osteoporosis. The invention is of advantage in that by screening for the presence of the polymoφhism it is possible to identify individuals likely to have a genetic predisposition to this disease.

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.

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 IL-1 receptor antagonist gene, is heterozygous for the wild type and the 4-repeat polymoφhism, or is homozygous for the polymoφhism.

According to the invention, an individual who is homozygous for the risk polymoφhism 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-5441964, the contents of which are incoφorated 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 incoφorated 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, the use of salcatonin and administration of calcium supplements. In pharmaceutical treatment of osteoporosis, all routes of administration are suitable and include but are not limited to oral, injection intravenously, intraperitoneally, intramuscularly and subcutaneously, intranasal and topical administration. Typical dosages and durations of treatment are as described in clinician's textbooks such as British National Formulary, incoφorated 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, incoφorated herein by 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 IL-1 receptor antagonist gene polymoφhism 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.

A still further aspect of the invention is that a further polymoφhism may be so correlated with presence of the IL-1 receptor antagonist polymoφhism that the two polymoφhisms are in linkage disequilibrium. Thus, diagnosis of disease by determining genotype of the further polymoφhism may lead to a similarly reliable diagnosis of osteoporosis or predisposition thereto.

The invention thus also provides a method of identifying, and optionally treating, an individual predisposed or susceptible to osteoporosis, said method comprising determining genotype of a first gene in said individual, wherein genotype of said first gene is correlated with genotype of a IL-1 receptor antagonist gene in said individual.

The invention yet further provides a method of identifying a further polymoφhism correlated with predisposition to osteoporosis, comprising identifying in a cohort of individuals with polymoφhisms of the IL-1 receptor antagonist gene a further polymoφhism and determining whether that further polymoφhism is correlated with polymoφhism of the IL-1 receptor antagonist gene. The further polymoφhism may preferably be on the the same gene, but can also be on any gene.

The invention further again provides a method of predicting response to osteoporosis therapy, comprising diagnosing genotype of an IL-1 receptor antagonist 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 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 polymoφhism in an IL-1 receptor antagonist gene indicates that hormone replacement therapy, or an equivalent, is likely to be effective. More specifically, possessing two copies of the risk polymoφhism indicates an increased level of therapy is likely to be appropriate.

A specific embodiment of the invention is now described with reference to drawings in which:-

fig. 1 shows visualization of PCR products of polymoφhisms of the interleukin-1 receptor antagonist receptor gene; and

to the sequence listing in which:-

SEQ ID NO:s 1 and 2 are PCR primers according to the invention; and

SEQ ID NO: 3 shows the entire interleukin-1 receptor antagonist receptor gene sequence, having Genbank accession number X64532- HSILIRECA.

We examined the relationship between annual rates of change in BMD over 5 years and a 86 bp variable number tandem repeat polymoφhism of the IL-1RN gene in 109 early postmenopausal women. All women were within 5 years of a natural menopause at the study's onset, healthy and not on hormone replacement therapy or other medication known to affect bone metabolism. BMD was measured annually over the 5 year study period at the lumbar spine and femoral neck using dual energy xray absoφtiometry. Three alleles were identified (Al, A2, A3), with most subjects falling into three common genotypes: A1A1 (42.2%), A1A2 (44.9%o), A2A2 (6.4%). There was no significant relationship between the IL-1RN genotypes and baseline bone mass at either the spine or hip. Annual rates of bone loss were increased in those with the A1A1 genotype (mean ± SEM = -1.4 ± 0.2 %,/yr) compared to the Al A2 genoytpe (-0.7 ± 0.2 %/yr) at the lumbar spine, (p = 0.03). A similar trend was seen at the hip although this failed to reach statistical significance (p = 0.09). The presence of the Al allele was associated with increased bone loss at the spine when compared to presence of the A2 allele, with a recessive pattern of risk.

Patients and Methods

Subjects were selected from a large general population cohort of 1003 white, Caucasian women, with a mean age (± SD) for the total cohort of 54.2 ± 6.0 years (9). Women in the age range 45-64 had been selected from a large single general practice in Chingford, North- East London (total of 11,000 registered patients) to participate in a longitudinal epidemiological study of rheumatic diseases. 1,353 women were found to be in the age range specified, and of these 78% (1,003) agreed to participate. The area is predominantly middle class, 98% are white and the population similar to UK normals in terms of height, weight, smoking status, hysterectomy rates and use of hormone replacement therapy (HRT). From this total cohort, 125 women were selected for the present study if they were within 5 years of a natural menopause and currently not on HRT or other medication known to affect bone metabolism. If hormonal treatment was initiated during the study period, results were included up to that time point. Throughout the study no women received concurrent treatment with medication known to affect bone metabolism such as steroids, bisphosphonates, calcium or vitamin D. Postmenopausal status was defined as an absence of menstruation for at least 6 months and was confirmed by measurement of serum follicular stimulating hormone and oestradiol levels. All women were healthy with no history of physical illnesses known to affect bone. All subjects had completed a nurse-administered questionnaire detailing medical and gynaecological/obstetric histories, current and past medications including HRT, smoking status, alcohol consumption, dietary calcium intake and exercise levels. Informed consent was obtained from all women and the study protocol was approved by the local ethical committee.

BMD was measured at the lumbar spine (L1-L4) and femoral neck using dual energy X-ray absoφtiometry (Hologic QDR-1000; Hologic Inc., Waltham, MA). Short-term reproducibility, assessed by duplicate measurements in healthy volunteers, was 0.8 % at the lumbar spine and 1.6% at the femoral neck. Annual measurements of BMD were made at 0, 12, 24, 36 and 48 months. In calculating the rate of change in BMD it was assumed that the expected change in BMD is linear with time for each subject with variation in slopes and intercepts from subject to subject, and that the deviations of the measured BMD for a subject from the expected BMD have a zero mean and constant variance and are uncorrelated. Under these assumptions a linear regression equation, where BMD is a dependent variable expressed as a linear function with time (in years), was fitted for individual subjects. Under this model, the annual percentage change in BMD for each subject was then derived by dividing the regression slope by the intercept at time zero.

High molecular weight DNA was obtained from peripheral blood leucocytes using a phenol and chloroform extraction system. PCR was performed using 2 flanking primers to amplify an 86bp tandem repeat region in intron 2 of the IL1-RN gene (5'-CTC AGC AAC ACT CCT AT-3' and 5'-TCC TGG TCT GCA GGT AA-3'). PCR reactions were performed in a total volume of 20 μl under standard conditions (24) using a Corbett Research FTS-1 Thermocycler programmed as follows: 1 min at 96°, 30 cycles of 1 min at 94°, 1 min at 60°, 2 min at 70°, and a final extension of 3 min at 72. Final PCR products were separated by electrophoresis using 1.2%) agarose gels at 100 V for 1 hour. To viualise DNA, gels were stained with ethidium bromide and viewed under ultraviolet light. Alleles were sized relative to a 1 kb DNA marker and subsequently coded using published nomenclature.

Differences in demographic variables between the IL1-RN genotypes were initially compared using analysis of variance (ANOVA) for normally distributed variables and chi-squared test for categorical variables. Adjustment for potential confounders was performed using analysis of covariance (ANCOVA). Direct comparisons between the variable means of the common genotype groups was performed using two-sided Student t-test. All analysis was performed using the PC software statistical programme STATA™.

Results

Characteristics of the 125 women entered into the study are shown in Table 1. Full clinical and genotype results were available on 109 of these women. No significant differences were observed between subjects with genotype results and those where DNA was either unavailable or attempts at PCR unsuccessful. Analysis of the genotypes in the 109 women showed evidence of three alleles which corresponded both in size and frequency to those previously identified (Table 2). From the three observed alleles we were able to demonstrate five out of the six possible genotypes, with the majority of individuals falling into two genotypes groups (A1A1 and A1A2).

The relationship between the IL-1RN genotype groups is shown in Table 3. There was no significant difference between the genotype groups in anthropomoφhic or environmental variables known to influence bone mass. No demonstratable effect of IL-1RN genotype was observed on baseline BMD at either the lumbar spine or femoral neck. BMD at the lumbar spine tended, however, to be lower in subjects with the A2A2 genotype although this only approached significance when compared to the heterozygous A1A2 genotype group (P = 0.08). When examining the relationship between IL-1RN genotype and bone loss, subjects with the genotype A1A1 exhibited increased rates of bone loss at both the spine and the hip when compared to those with the Al A2 genotype. No evidence of an allele dose effect was observed as the genotype group A2A2 also had high rates of loss, although due to the sample size this did not differ significantly from the rates observed in the other genotype groups. Presence of the Al allele (A1A1 and A1A3 genotype groups combined) was associated with increased bone loss at the lumbar spine when compared to the A2 allele (A1A2, A2A2 and A2A3 combined), mean annual %> difference (95% confidence interval) of -0.62 %/yr (-1.20, - 0.03), P = 0.04. Similar findings were observed at the femoral neck although this was nonsignificant, mean annual % difference -0.67 % yr (-1.62, +0.28), P = 0.16. These data suggested that the pattern of risk associated with the IL-1RN genotypes was such that the A2 allele was dominant over the Al allele. These findings were essentially unaltered after adjustment for age, weight, smoking status and menopause duration.

IL-1RN is a 17-kD protein that binds to IL-1 receptors and competes with both IL-l and IL- lβ without detectable IL-1 agonist effects. Monocytes of patients with "high turnover" osteoporosis, the histological hallmark of postmenopausal osteoporosis, secrete increased amounts of IL-1 (18). Subsequent observations have shown that this postmenopausal increase in IL-1 activity results from an effect of oestrogen on the production of both IL-1 and its receptor antagonist IL-1RN. Animal studies have also revealed that treatment with IL-1RN blocks the ex vivo osteoclast formation induced by ovariectomy (14). The human IL-1RN gene maps to chromosome 2ql3-14 and consists of 4 exons (16). The VNTR is due to a 86 bp repeat within intron 2 of the gene and this may be of functional significance as the sequence is reported to contain three potential protein binding sites; an α-interferon silencer A, a β-interferon silencer B and an acute phase response element (24). Polymoφhic VNTR repeats have been shown to influence gene expression at the insulin locus (26), although it is unknown if the IL-1RN VNTR has any direct effect on rates of transciption or message stability. A preliminary study in inflammatory bowel disease has also suggested an association between the A2 allele of the IL-1RN locus and ulcerative colitis (17). In the absence of any functional significance associated with the VNTR locus, it is possible that the polymoφhism may be in linkage disequilibrium with another disease locus on chromosome 2. Multipoint linkage analysis around the IL-1RN locus would then be required to test this hypothesis and to map these novel candidate loci.

The invention thus provides method and apparatus for identification and treatment of individuals having a IL-1 receptor antagonist polymoφhism, correlated with a predisposition to osteoporosis.

References

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3. Cummings, S.R., Black, D.M., Nevitt, M.C., Browner, W., Cauley, J., Ensrud, K., Genant, H.K., Palermo, L., Scott, J. and Vogt, T.M. Bone density at various sites for prediction of hip fractures. Lancet 341:72-75; 1993.

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7. Grant, S.F., Reid, D.M., Blake, G., Herd, R, Fogelman, I. and Ralston, S.H. Reduced bone density and osteoporosis associated with a polymoφhic Spl binding site in the collagen type I alpha 1 gene. Nature Genet 14:203-205; 1996.

8. Gueguen, R., Jouanny, P., Guillemin, F., Kuntz, C, Pourel, J. and Siest, G. Segregation analysis and variance components analysis of bone mineral density in healthy families. J Bone Miner Res 10:2017-2022; 1995.

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11. Keen, R.W., Nguyen, T., Sobnack, R, Perry, L.A., Thompson, P.W. and Spector, T.D. Can biochemical markers predict bone loss at the hip and spine?: a 4-year prospective study of 141 early postmenopausal women. Osteoporosis Int 6:399-406; 1996.

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Table 1

Mean (± SD) characteristics of 125 women

Variable Value

Age (yrs) 53.1 (3.5)

Age at menopause (yrs) 50.4 (3.1)

Menopause duration (yrs) 2.7 (1.7)

BMI (kg/m²) 26.0 (4.8)

No. ever smoking (%) 54 (43)

Lumbar spine BMD (g/cm²) 0.969 (0.144)

Femoral neck BMD (g/cm²) 0.763 (0.130)

Change in lumbar spine BMD (%/yr) -1.1 (1.5)

Change in femoral neck BMD (%/yr) -0.50 (2.2)

Table 2

IL-IRN alleles identified through DNA ampiication of 109 unrelated individuals with comparison with published data

"Current study. N = 109 subjects

^bTarlow et al. N = 70 subjects Table 3

Characteristics of subjects according to IL-IRN genotype

A1A1 A1A2 A2A2 A1A3 A2A3

(n=46) (n=49) (n=7) (n=3) (n=4)

Age (yrs) 52.2 53.2 52.6 53.0 55.8

(3.4) (4.0) (2.8) (4.0) (1.5)

Age at menopause (yrs) 49.8 50.2 50.6 50.0 51.5

(3.1) (3.9) (3.4) (4.0) (1.0)

Menopause duration (yrs) 2.4 3.0 2.0 3.0 4.3

(1.8) (1.6) (1.8) (0.0) (0.5)

BMI (kg/m²) 26.4 26.1 25.4 24.7 23.8

(4.7) (5.3) (3.9) (3.1) (2.1)

No. ever smoking (%) 20 23 0 1 3

(43) (47) (0) (33) (75)

Lumbar spine BMD (g/cm²) 0.960 0.985 0.888 0.982 0.912

(0.145) (0.139) (0.118) (0.082) (0.123)

Femoral neck BMD (g cm²) 0.748 0.775 0.699 0.741 0.743

(0.129) (0.131) (0.100) (0.170) (0.110)

Change in lumbar spine BMD (%/yr) -1.4 -0.7^a -1.6 -1.8 -0.9

(1.5) (1-5) (0.8) (0-5) (1.9)

Change in femoral neck BMD (%/yr) -0.9 0.0 -1.2 -0.4 -2.0^b

(2.3) (2.2) (0.9) (1.4) (-)

P =0.03 vs A1A1 only 1 subject in this group with annual rate of hip loss data SEQUENCE LISTING

(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: KEEN; Richard William

(B) STREET: St Thomas' Hospital, Lambeth Palace Road

(C) CITY: London

(E) COUNTRY: GB

(F) POSTAL CODE (ZIP) : SE1 7EH

(A) NAME: SPECTOR: Timothy David

(B) STREET: St Thomas' Hospital, Lambeth Palace Road

(C) CITY: London

(E) COUNTRY: GB

(F) POSTAL CODE (ZIP) : SE1 7EH

(ii) TITLE OF INVENTION: POLYMORPHISMS OF AN IL-1 RECEPTOR ANTAGONIST GENE

(iii) NUMBER OF SEQUENCES: 3

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(v) CURRENT APPLICATION DATA:

APPLICATION NUMBER: WO [NOT KNOWN]

(2) INFORMATION FOR SEQ ID NO : 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1: CTCAGCAACA CTCCTAT 17

(2) INFORMATION FOR SEQ ID NO : 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2: TCCTGGTCTG CAGGTAA 17

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12565 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3:

GTCGACCTGC AGGTCAACGG ATCTGAGAGG AGAGTAGCTT CTTGTAGATA ACAGTTGGAT 60

TATATACCAT GTCCTGATCC CCTTCATCAT CCAGGAGAGC AGAGGTGGTC ACCCTGATAG 120

CAGCAAGCCT GGGGGCTGCA GCTTGGTGGG TAGAGGTACT CAGGGGTACA GATGTCTCCA 180

AACCTGTCCT GCTGCCTTAG GGAGCTTCTA ATAAGTTGAT GGATTTGGTT AAAATTAACT 240

TGGCTACTTG GCAGGACTGG GTCAGTGAGG ACCAACAAAA AGAAGACATC AGATTATACC 300

CTGGGGGTTT GTATTTCTTG TGTTTCTTTC TCTTCTTTGT ACTAAAATAT TTACCCATGA 360

CTGGGAAAGA GCAACTGGAG TCTTTGTAGC ATTATCTTAG CAAAAATTTA CAAAGTTTGG 420

AAAACAATAT TGCCCATATT GTGTGGTGTG TCCTGTGACA CTCAGGATTC AAGTGTTGGC 480

CGAAGCCACT AAATGTGAGA TGAAGCCATT ACAAGGCAGT GTGCACATCT GTCCACCCAA 540

GCTGGATGCC AACATTTCAC AAATAGTGCT TGCGTGACAC AAATGCAGTT CCAGGAGGCC 600

CAAATGAAAA TGTTTGTACT GAAATTTGTT AAAGCTTCCC GACAAACTAG ATTTΛTCAGT 660

AAGGATTGTT TTCTGCAAGG GGGATGAAAC TTGTGGGGTG AGCCATTTGG GCTGAGGAGG 720

AGGGAGGTTG GAGCTGAGAA ATGTGGAGAC AATTTCCCTT TAGAAGGACT GAATCTCCCT 780

GCCTCTCTGG GGTGCGGCAG CCAGCAGGAT CCAATGGTGT ATATGTCTCC CCAGCTCCCC 840

ATTCAGTGAT ATCATGTCAG TAGCTTGAAA TTATCCGTGG TGGGAGTATT ATGTCATGGA 900

AATTGGCAAA TGGAAACTTT TATTGGAGAT TCAATTGTTA AACTTTTACC AGCACAACAC 960

TGCCCTGCCT TCAGAGTCAA TGACCCTATC CAAGTTTAAT CCATCTGTCC ACTGTCTCCA 1020

ACACGATCTT TATAAAACAC ACCTGACAAC ATTACCCTTT TATTCAGTTT TTTAAAAGAT 1080

AAGTTTCCAG CTCATCGGGG TGGCTTTAAA GGCCATTTCT CCTCTGGACC TCACCCAACT 1140

TTTCAAATCA CTTTTCCTAC CCCTACCTCT AAATGCTACT CAAACTCCAG CCATCCTGAA 1200

TAATAAGACT TTTGAAAAGT AGATTATGGG CTGGGCACAG TGGCTCACAC CTGTAATCCC 1260

AGCACTTTGG GAGGCCAAGA TGGGTGGATC ACCTGAGGTC GGGAGTTCGA GACCAGCCTG 1320

ACTAACATAG TGAAACCCTG TCTCTACTAA AAATACAAAA TTAGTTGGGG GTGGTGGCAC 1380

AAGCCTGTAA TCCCAGCTAC TCAGGAGGTT GAGGCAGGGG AATTGCTTGA ACCTGGGAGG 1440 CGGAGGTTGC GGTGAGCCTA GATTGCTCCA CTGCACTCCA GCCTGGGCAA CAAGAGCGAA 1500

ACTCCATCTC AAAAAAATAA ATAAATAAAT AAAGTAGATT ACATCAGATA CCTCTGGCCT 1560

AGGTTGTTTA TGACCAACTC TCCTGCTGAG AATAACTAGA AAAGCTAGAC AAAACATATT 1620

TCCAAAAGAT CTCTTTGGAG GCATCAGAGA ATGGCCAAGG CTGTAAGGAA CTGCCTGAGC 1680

CCAGAGAGGT GGAGCCCAGC ACTGGTGCCC TTTACTCCTG GGGACATGTG CTGGTTTCAA 1740

AAACTTCAGC TGAGCTTTTG AGCATTCATG GAACTTGGTG GGGGAGATGA AATTTGTACC 1800

TTAAATCCTG CCTACAGGGA GGGTCCCTGA TAATCCCCAC CCAATTTGGA AATCTGGGTC 1860

AGCCTTCACA GGTACTGAAG CCCTCCTCTG AATGATCTCA AGTCCTGCTA GGGTAGAGGT 1920

TACCTGCTTT TGAAAGGCTC CTGGCCTACC TGTGCAGCAG GAGCAAAAGT GAACCATCTC 1980

AGGGTACAGA TAACAATCAT CCAGAGCCTT GAATGACCTC TACTGTGCTT AATATATAGT 2040

ATTCAGCAGT CAGTAAAAAG GATTTAGGCA CATGCAAGAT GACCTGTGTA TCAGGGAGAA 2100

ATAGGCAATA AATTGAGATC CAGCAGGGAT TTGAATCATG GATTTGAATC AGGGGCAGCC 2160

TTCGAAAGAA CTATGGAGAA TATACTCAGA TTTAAAACAT AAGATTGGAA TTTTTGGCAG 2220

AGAACTAACA ACTGTACAAA AAAGGAACCA AATGGAAATC CTAGAACTGA AAGATGCAAT 2280

TAACCGATGT TGAGAAATAG CCAACATCTA TTGAACACTT CCCATGTGGA CAGCTGTGCT 2340

AAACACTTTA CAGGCATCAA CATAAGATGT GTCCCCTTAC AGCAGTGCAG TGTCCCTCCT 2400

AAGACATGGA CAGCCTGGTT TCCCTATCTC TCTGCTTCAT CAAAACCCCT TTACGTGGGG 2460

CTTAGACACT CCTGTTGTCT CTAGTGTCTA GTAGCACAGG GCTCAGCACA TGGAAGCCAC 2520

TAGATACAAT TTGATGACCA GGACCTCCGA TGAAAGCCAT GGGTGCTGAT TGGGAAGGCA 2580

TTGTCTTTTA TGTGCTATGG TCTTAAAGCT TCATCCAGGA AGCAGAACTC GGGGGGTGCT 2640

GAGGACCCAG AACCGAGAAT AAGATTAGTC AGAGATTTCC TGTGGGCAGA AATCATAAGG 2700

ACGCCAACTG TTTGGGTGAG ATAAGACGAA ACCAAGAGTG GACTTGTGGC CAGAAGCGTG 2760

AGGAAGAGGG AGAGAGCTTC CCTTGTCCCC TTTCTTCCTC TCCCTAAGCC ACAGTGATTG 2820

ACAGCCCCCC CGCTTTGGAG TCAGAGCAGG CTTGAGACTG GACTGGGAAA GGAGGGTGGG 2880

TCAGGATACA GAGCAGGAAG GCTGGGAGTG CAGGGCAGGA GCAAGGGGCT GGGGCATTCA 2940

TTGTGCCTGA TCTCTCCCAC TTTACCTGGG GTAAAGAAGC ATATGCAAAA GCCACGGTGT 3000

GAGTATTTCC CAAGTGCCAG GGTCAGGGCA TGATTCATCA CGTGCAGCAT TTCATTCAAT 3060

CCTTATAGTA ACCGATGATG TGGCTTCTAT TATTAGCTCT ATCAGATAAT GAAACTGAGA 3120

CCAAGACAGG CTCTGCACAT TGTGTGGGGT AATGACACAG GGGGATTCAG ACCTAGACTC 3180

CATAACTCCT GCCCCAGGGA CCACCCCCAC CCTCACCCTG TGCATGTCGA CAAAGGACAG 3240

ACTGGGCCAC TTCTCAGGAC ACAGCGGGGA AATGACACAG AGCAGGGAGG TTCCAGGAGC 3300

CCCGAGCGTC TTTTCTCCAG GAGAATACTC TCTGAATTCA GACTGGGGTC AGAGAAACAT 3360

TTACCCAGGA GCCGCAGTGT GGGTGGGGCT TTTTACTTGA AACGCTGTCT GAAGGCAGTG 3420

GCAGGATGAA CTCTCCACCC TACCTTGGCA AGCCACTTCT CTTCTGCAAT CTGTAAGGAC 3480 ATTGTTGAGA GAATTATGGT CTTCCAATTC CGGAGGGTTG AAGAAAGACA AATAGGAGAG 3540

AACCTATCAT AGTCAGGTGC TAGCTGCCTT CTCTTTCAGA GAGTGTGAGA ATAAAGTGAT 3600

ACACTTGATT ATTAGCAAAT ACTTTGGAAA TTTTAAACGC TAATATTCAA CACACTCTGG 3660

AAGAGGCAAA TAAGTAGACA GGTTCATATA CATCATCTCC TTCAGCTAGT CCTCACAAAA 3720

ACAAACAAAT GAATAAACAA AATTCTTCTT TGGCCCTCAT AGGAAGACAC TGTTTCTTGA 3780

ACGTGTTTCA AAAAGGATGG GTGACTCACT CAAGGTCACA CTGTTTATGA GGACAGTACA 3840

GGAATACAGA CATGCCATTT TGCCTGAAAA AATCCATCAC CCAGGGAGGT GACACAATTT 3900

TGCAGAAATG TTCTATTTCC TCTGAAGGAT ACATTCTTTA AACCTTTGGG AAATTCATTC 3960

ATAGTCTTCC TCCTTTGAAG GATTACTCTC TGGACACAAA GTGTTTGATT CTGATTTGTT 4020

GGTTGGAAGA TGTGTTGGTT GAGAGAAAGA TTCTGATTTG TTGGTTGAAA ATAGACTCAT 4080

CAAGATCAAC TGCTGTAGTA GTAAATATTT TGACATTTTG TCTGTATTCC TGTGCTGCCC 4140

TCACAAGCTG CATCACCTTG AGTGAGTCAT TCATACTTTT TTGTTTGTTT TTGTTTTGGA 4200

GATGGAGTCT TACTCTGTTG CCTAGGCTGG AGTGCGGTGG CGTGATCTTG GCTCACTGCG 4260

ACCTCCATCT CCTGGGTTCA AGTGATCCTC CTGCCTCAGC CTCCCGAGTA GCTGGGATTA 4320

CAGGCACATG CCACCATCCC TGCTAATTTT TGCATTTTCA GTAGAGACGG AGTTTCACCA 4380

TGTTGGTCAG GTTGGTCTTG AACTCCTGAC CTCAGGTGAT CCGCCCACCT CAGCCTCCCC 4440

AAGTGCTGGG ATTACAGGTG TGAGCCACCG TGCCCAGCCC AGCCATCATT TTTGAAACAC 4500

GTTTGAGAAA TAGTGTCTTC CTTTGAGGGC CAAGGAGACA TTTTTTTTGT TTATTTGTTT 4560

GTTTTTGTGA GGACTAGCTG AAGGGGGTGA TGTATATTAA CCTGCCTACT TATTTGCCTC 4620

TTCCCAGAGT GTGATGAATA TTAGGGTTTA AAGTTTCTGA AGCATTTGTT AATAAAGCCC 4680

GGGGCTGGAG GTCAGAAGAC CTGGATTTCT CTGCATACTT TTGCCATCAG CAAGCTGTGT 4740

GACCTTGGAC AGATCCCTTT TTTGTCTAAA TCTTTCTGAG TCTTCTTGAA AACAATGCCA 4800

GGTTGGGACA GGATGATTGC CAAGCTCCCG TCCAGCTCTA AAACACTGCA ACGTATGCTT 4860

CTGCACCAGC ACTGTCCATC CTGTAGATCA TGCAGAAATT CTCTTCAACT TTTTCCTACC 4920

CATAAAATAG GAGCATGCTT ACCTTTTTCC TAATGTTCCA GGCCCCGGGT CTAGATATTG 4980

TAAGTAAGGA AGTTAATGTG TATCAGAGCC CATTATGGGC CAGAAGTTCT CCTCTTCCTT 5040

CCTACACCTG CTTCCTCCCT CCCTCCCTCC CTCTTTCCCT TCCTTCCTTC CATCCATTTG 5100

TGAAGAAGAC ATGATCACCC TCATTCTGAG AGTGAAGAGA CAGAGGCTCA ACTAATGAAA 5160

TGATTTGTTC AAGGTCACAC GGGTGGCACA AGGCAAGTGG CAGAGGTTGA ATTTAGACCC 5220

ATTCCTGTCC AAATGCTGAG TTTATGTCAT CGTCCCGAGA CCATAACTTT AAAGATGTAA 5280

GATAGTGGGA AAAGAGTTGA TTTCAAAGCA CCTCTCAGAA GGACTCACTT TACATCAGGG 5340

GTCAGCAGAC TCAGGCCAAA TCCGGTCCAT TCCCCGCTTT TGCAAAGAAA GTTGTAGTGG 5400

AACACAGCTA GGCTTATTGA TTTATGGATT GCCAACGTCC TTTTGTGAAA CAGACAGCTG 5460

AGCTGAGTAA TCGTGGCGCA CAAAACCTAA AATATTTACT ATCTCGTCCT TTACAGAATG 5520 TTTGCCAATC TATGGTCCGG AGTCCAAGGC TGTCCATTTT TCAAAGAACA CAAAGTGACA 5580

TGAGACTGTC CCATGTGCAG GGAGCCCTAT CATTTTATTA TGAAAAAACG GCCTTTCTGC 5640

TCAAATCTGT TTTTTAAAAA GTCAACAAAC AGACTCTGGG TACCTGTCAG GAACAGTAGG 5700

GAGTTTGGTT TCCATTGTGC TCTTCTTCCC AGGAACTCAA TGAAGGGGAA ATAGAAATCT 5760

TAATTTTGGG GAAATTGCAC AGGGGAAAAA GGGGAGGGAA TCAGTTACAA CACTCCATTG 5820

CGACACTTAG TGGGGTTGAA AGTGACAACA GCAAGGGTTT CTCTTTTTGG AAATGCGAGG 5880

AGGGTATTTC CGCTTCTCGC AGTGGGGCAG GGTGGCAGAC GCCTAGCTTG GGTGAGTGAC 5940

TATTTCTTTA TAAACCACAA CTCTGGGCCC GCAATGGCAG TCCACTGCTT GCTGCAGTCA 6000

CAGAATGGAA ATCTGCAGAG GCCTCCGCAG TCACCTAATC ACTCTCCTCC TCTTCCTGTT 6060

CCATTCAGAG ACGATCTGCC GACCCTCTGG GAGAAAATCC AGCAAGATGC AAGCCTTCAG 6120

GTAAGGCTAC CCCAAGGAGG AGAAGGTGAG GGTGGATCAG CTGGAGACTG GAAACATATC 6180

ACAGCTGCCA GGGCTGCCAG GCCAGAGGGC CTGAGAACTG GGTTTGGGCT GGAGAGGATG 6240

TCCATTATTC AAGAAAGAGG CTGTTACATG CATGGGCTTC AGGACTTGTG TTTCAAAATA 6300

TCCCAGATGT GGATAGTGCG ACCGGAGGGC TGTCTTACTT TCCCAGAGAC TCAGGAACCC 6360

AGTGAGTAAT AGATGCATGC CAAGGAGTGG GACTGCGATT CAGGCCTAGT TGAATGTGCT 6420

GACAGAGAAG CAGAGAGGGG CACCAGGGGC ACAGCCCGAA GGCCCAGACT GATATGGGCA 6480

AGGCCTGTCT GTGCTGACAT GTCGGAGGGT CCCACTCTCC AGGGACCTTG GTTTCCCCGT 6540

CTGTGACATC TGTGACATGA GAGTCACGAT AACTCCTTGT GTGCCTTACA GGGTTGTTGT 6600

GAAAATTAAA TGCACAGATA ATAGCGTAAC AGTATTCCGT GCATTGTAAA GAGCCTGAAA 6660

ACCATTATGA TTTGAAAATG GAATCGGCTT TGTGAGACCA TCACTATTGT AAAGATGTGA 67 0

TGCTGATAGA AATGACAGGA CTGCTTGTGC ATGCCCTCTG CAGTGTGACA TTCCAGCAGT 6780

GAAATCATGT TGGGGTGACT TCTCCCCCAC TCTGACCTTT ATGTTTGTCT GGGCCGAGGC 6840

TGCAAGTCGG GCTCTGTGGG TGTATGAGTG ACAAGTCTCT CCCTTCCAGA TATGGGGACT 6900

GTCTGCTTCC CTAGGTTGCC TCTCCCTGCT CTGATCAGCT AGAAGCTCCA GGAGATCCTC 6960

CTGGAGGCCC CAGCAGGTGA TGTTTATCCC TCCAGACTGA GGCTAAATCT AGAAACTAGG 7020

ATAATCACAA ACAGGCCAAT GCTGCCATAT GCAAAGCACT TTGGTTTGCC TGGCCACCCC 7080

TCGTCGAGCA TGTGGGCTCT TCAGAGCACC TGATGAGGTG GGTACAGTTA GCCACACTTC 7140

ACAGGTGAAG AGGTGAGGCA CAGGTCCCAG GTCAGGCTGG CCGGAGCTCT GTTTATTACG 7200

TCTCACAGCT TTGAGTCCTG CTCTCAACCA GAGAGGCCCT TTACCAAGAA GAAAGGATTG 7260

GGACCCAGAA TCAGGTCACT GGCTGAGGTA GAGAGGAAGC CGGGTTGTTC CCAAGGGTAG 7320

CTGCTCCTGC AGGACTCTGA GCAGGTCACC AGCTAATGGA GGAAAGGCTC TAGGGAAAGA 7380

CCCTTCTGGT CTCAGACTCA GAGCGAGTTA GCTGCAAGGT GTTCCGTCTC TTGAAACTTC 7440

TACCTAGGTG CTATGGTAGC CACTAGTCTC AGGTGGCTAT TTAAATTTAT ACTTAAATGA 7500

ATGAAAATAG AAGAAAATTT AAAATCCAGA CCCTTGGTCA CACTATCCAC ATTTAAAGAG 7560 GTCAATAGCC ACATGTGGTT AGTGGCCACC CTATTGGGCA GTGCAGCTAC AGAACATTTT 7620

TGCATCCCAG AAAGTTCTTT TGGATGTTGC TGCTCTACAG CATGCTTTGC TGAAACAGAA 7680

GTGCCTTCCC TGGGAATCTC AGATGGGAAG CAAGTAAGGA GGGGAGTCAA ATGTGGGCTC 7740

ACTGCTCACC AGCTGTGAGG GTTGGGCCTG CCTCTTAACC ATTGTCAGCC TCAGTCTTCT 7800

CATCCATGCA TGCCGTGGGT ATACTAAAAT ACTATACCCC TGGAAGAGCT GGATGCAAAT 7860

TTGACAAGTT CTGGGGGACA CAGGAAGGTG CCAAGCACAA GGCTGGGCAC ATGGTGGCTG 7920

TGCACTACAG CTGAGTCCTT TTCCTTTTCA GAATCTGGGA TGTTAACCAG AAGACCTTCT 7980

ATCTGAGGAA CAACCAACTA GTTGCTGGAT ACTTGCAAGG ACCAAATGTC AATTTAGAAG 8040

GTGAGTGGTT GCCAGGAAAG CCAATGTATC TGGGCATCAC GTCACTTTGC CCGTCTGTCT 8100

GCAGCAGCAT GGCCTGCCTG CACAAACCCT AGGTGCAATG TCCTAATCCT TGTTGGGTCT 8160

TTGTATTCAA GTTTGAAGCT GGGAGGGCCT GGCTACTGAA GGGCACATAT GAGGGTAGCC 8220

TGAAGAGGGT GTGGAGAGGT AGAGTCTAGG TCAGAGGTCA GTGCCTATAG GCAAGTGGTC 8280

CCAGGGCCAC AGCTGGGAAG GGCAAATACC AGAAGGCAAG GTTGACCATT CCCTTCCTCA 8340

AGTGCCTATT AAGGCTCCAT GTTCCTATGT TGTTCAAACC CTAACTCAAT CCCAAATTAA 8400

TCCACCATGT ATAAGGTTGA GCTATGTCTC TTATTCCTGG ACACCATACT CAGCCATATC 8460

TGGTCCACAC ATTAACAGCT GGATGACCTT GAAGAAGCTT CACCCACTCT GTTCCTCAGC 8520

TTTCCCTTCA GTGGGATGAT ATCAACTGGA CAACAGGATG TGCGATTCTT TTAGTTCCAG 8580

CCTTCCAGGA TGTTTTCACT CCCCTGTTTG TTGTTGTAGG ATGGTATTAC CTCCACCTTC 8640

CCACCTTCCC TATGCCCTGG TTCTGTCTCC TGTGCCTCGC TCTGAAAGTG GATGAGACCT 8700

ACAATTCCTG TCCTGGTAGT TCTCCTAATG AACACACTGA AGCACGAGGA AGCTGAGATT 8760

TTTGTTGCTA CATGAGAGCA TGGAGGCCTC TTAGGGAGAG AGGAGGTTCA GAGACTCCTA 8820

GGCTCCTGGT GGAGCCCCAC TCATGGCCTT GTTCATTTTC CCTGCCCCTC AGCAACACTC 8880

CTATTGACCT GGAGCACAGG TATCCTGGGG AAAGTGAGGG AAATATGGAC ATCACATGGA 8940

ACAACATCCA GGAGACTCAG GCCTCTAGGA GTAACTGGGT AGTGTGCATC CTGGGGAAAG 9000

TGAGGGAAAT ATGGACATCA CATGGAACAA CATCCAGGAG ACTCAGGCCT CTAGGAGTAA 9060

CTGGGTAGTG TGCATCCTGG GGAAAGTGAG GGAAATATGG ACATCACATG GAACAACATC 9120

CAGGAGACTC AGGCCTCTAG GAGTAACTGG GTAGTGTGCA TCCTGGGGAA AGTGAGGGAA 9180

ATATGGACAT CACATGGAAC AACATCCAGG AGACTCAGGC CTCTAGGAGT AACTGGGTAG 9240

TGTGCTTGGT TTAATCTTCT ATTTACCTGC AGACCAGGAA GATGAGACCT CTCTGCCCTT 9300

CTGACCTCGG GATTTTAGTT TTGTGGGGAC CAGGGGAGAT AGAAAAATAC CCGGGGTCTC 9360

TTCATTATTG CTGCTTCCTC TTCTATTAAC CTGACCCTCC CCTCTGTTCT TCCCCAGAAA 9420

AGATAGATGT GGTACCCATT GAGCCTCATG CTCTGTTCTT GGGAATCCAT GGAGGGAAGA 9480

TGTGCCTGTC CTGTGTCAAG TCTGGTGATG AGACCAGACT CCAGCTGGAG GTAAAAACAT 9540

GCTTTGGATC TCAAATCACC CCAAAACCCA GTGGCTTGAA ACAACCAAAA TTTTTTCTTA 9600 TGATTCTGTG GGTTGACCAG GATTAGCTGG GTAGTTCTGT TCCATGTGGT GGAACATGCT 9660

GGGGTCACTT TGGAAGCTGC ATTCAGCAGA GTGCCAGGCT TGCGCTGGGC ATCCAAGGTG 9720

GTCCCTCATC CTCCAGGCTC TCTTTCCATG TGATCTCTCA GTGTTTAAGA GTTAGTTGGA 9780

GCTTCCTTAC AGCATGGCGG CTGACTTCCA AAAGGGATTA TTCCAAAAAG AGCCTCAACA 9840

TGCAGGCGCT TATTATGACT TCTGCTTGCA TCATCCTATT GGCCAAAGCC AGTCACGTGG 9900

CTAAGTCTAG CCCCCTGTGA GAGGAGACTG CATAAGAGTG TGAACACCAG GAGACACGGT 9960

CACTGGGGGC CACCACTGTA ACCATCTACC ACAGGACCTG AATCTCTGTG TGCTACTCCC 10020

TTGCTCAAGG GCCCCCCTAC CCACGCAGAC CTGCTGTCTT CTAGCAAAGC CCATCCTCAG 10080

GACCTTTCTC TTCCAATCCT TATTGACTCA AATTGATTAG TTGGTGCTCC ACCCAGAGCC 10140

CTGTGCTCCT TTATCTCATG TAATGTTAAT GGGTTTCCCA GCCCTGGGAA AACATGGCTT 10200

TGTCTCAGGG GCTTGCTGGA TGCAACCTTA ACCTCAATGT GAGTGGCCAT ACTGTGGCAC 10260

TGTCCCATCC CTCACCAGGG ACACTGTTCT GGAGGGTGAC TGCCTGTTCT GTGAGGAGTG 10320

GGGATGGCTA GGACATTGCA TGGAACACAC CACCACCCCA TCTTCTCAGA GCTCAAACCC 10380

TGACAGAACA CCAGCTCCAC AGGCCTTGGC TTCTGCTGAT GGTGCCGTGT ATTTACCAGA 10440

CTTAGTGGTC CAAGGCCAGA GTGGCAGATT TCCCAAAGTC AAGGTGTGAC AGTGGGACAG 10500

CCTCTTTGTG TCTTTGCTGT CCTAAGAAAC CTGGGCCAGG CCAGGCGCAG TGGCTCACGC 10560

CTTGTAATCC CAGCACTTTG AGAGGCCAAG GTGGGCAGAT CACGAGGTCA GGAGTTTGAG 10620

ACCAGCCTGG CCAACATTGG TGAAACCCTG TCTCTATTAA AAATAGAAAA CATTAGACAG 10680

GTGTGGTGGT GCATGCCTGT AATCCCAGCT ACTCAGGAGG CTGAGGCAGG AGAATCGCTT 10740

GAACCCAGGA GGTGGAGGTT GCAGTGAGCC GAGATTGTGC CACTGCACTC CAGCCTAGGC 10800

GACAGAGCAA GACTCCGTCT CGGGAAAATT AATTAATAAA TAAATAAACC TAGGTCCCAG 10860

AGTCCCACAG AATGGCAGAC AGGAGCACCT GGGGGCTTTT AGGGTATGGC ATTTCCCCTG 10920

TACTAACTCT GGGCTGTCCA GAGGCGATTT CATGGCGTGG AGTGGAGAGG GAGGCAGCAC 10980

AGGACTTCCT AGGCCTCAGC TCTCACCTGC CCATCTTTTG ATTTCCAGGC AGTTAACATC 11040

ACTGACCTGA GCGAGAACAG AAAGCAGGAC AAGCGCTTCG CCTTCATCCG CTCAGACAGT 11100

GGCCCCACCA CCAGTTTTGA GTCTGCCGCC TGCCCCGGTT GGTTCCTCTG CACAGCGATG 11160

GAAGCTGACC AGCCCGTCAG CCTCACCAAT ATGCCTGACG AAGGCGTCAT GGTCACCAAA 11220

TTCTACTTCC AGGAGGACGA GTAGTACTGC CCAGGCCTGC CTGTTCCCAT TCTTGCATGG 11280

CAAGGACTGC AGGGACTGCC AGTCCCCCTG CCCCAGGGCT CCCGGCTATG GGGGCACTGA 11340

GGACCAGCCA TTGAGGGGTG GACCCTCAGA AGGCGTCACA ACAACCTGGT CACAGGACTC 11400

TGCCTCCTCT TCAACTGACC AGCCTCCATG CTGCCTCCAG AATGGTCTTT CTAATGTGTG 11460

AATCAGAGCA CAGCAGCCCC TGCACAAAGC CCTTCCATGT CGCCTCTGCA TTCAGGATCA 11520

AACCCCGACC ACCTGCCCAA CCTGCTCTCC TCTTGCCACT GCCTCTTCCT CCCTCATTCC 11580

ACCTTCCCAT GCCCTGGATC CATCAGGCCA CTTGATGACC CCCAACCAAG TGGCTCCCAC 11640 ACCCTGTTTT ACAAAAAAGA AAAGACCAGT CCATGAGGGA GGTTTTTAAG GGTTTGTGGA 11700

AAATGAAAAT TAGGATTTCA TGATTTTTTT TTTTCAGTCC CCGTGAAGGA GAGCCCTTCA 11760

TTTGGAGATT ATGTTCTTTC GGGGAGAGGC TGAGGACTTA AAATATTCCT GCATTTGTGA 11820

AATGATGGTG AAAGTAAGTG GTAGCTTTTC CCTTCTTTTT CTTCTTTTTT TGTGATGTCC 11880

CAACTTGTAA AAATTAAAAG TTATGGTACT ATGTTAGCCC CATAATTTTT TTTTTCCTTT 11940

TAAAACACTT CCATAATCTG GACTCCTCTG TCCAGGCACT GCTGCCCAGC CTCCAAGCTC 12000

CATCTCCACT CCAGATTTTT TACAGCTGCC TGCAGTACTT TACCTCCTAT CAGAAGTTTC 12060

TCAGCTCCCA AGGCTCTGAG CAAATGTGGC TCCTGGGGGT TCTTTCTTCC TCTGCTGAAG 12120

GAATAAATTG CTCCTTGACA TTGTAGAGCT TCTGGCACTT GGAGACTTGT ATGAAAGATG 12180

GCTGTGCCTC TGCCTGTCTC CCCACCAGGC TGGGAGCTCT GCAGAGCAGG AAACATGACT 12240

CGTATATGTC TCAGGTCCCT GCAGGGCCAA GCACCTAGCC TCGCTCTTGG CAGGTACTCA 12300

GCGAATGAAT GCTGTATATG TTGGGTGCAA AGTTCCCTAC TTCCTGTGAC TTCAGCTCTG 12360

TTTTACAATA AAATCTTGAA AATGCCTATA TTGTTGACTA TGTCCTTGGC CTTGACAGGC 12420

TTTGGGTATA GAGTGCTGAG GAAACTGAAA GACCAATGTG TYTTYCTTAC CCCAGAGGCT 12480

GGCGCCTGGC CTCTTCTCTG AGAGTTCTTT TCTTCCTTCA GCCTCACTCT CCCTGGATAA 12540

CATGAGAGCA AATCTCTCTG CGGGG 12565

Claims

CLAEMS

1. A method of diagnosis comprising determining genotype of a IL- 1 receptor antagomst gene.

2. A method of diagnosis according to Claim 1 comprising determining whether an individual possesses a wild type IL-1 receptor antagonist gene.

3. A method of diagnosis according to Claim 1 or 2 comprising determining whether an individual possesses a wild type IL-1 receptor antagonist gene or a variant gene that contains 4 repeats of an 86bp sequence in intron 2.

4. A method according to any of Claims 1-3 comprising amplifying a portion of that individual's IL-1 receptor antagonist gene using PCR techniques.

5. A method according to any preceding claims for diagnosis of osteoporosis.

6. Diagnostic means, comprising means for determining genotype of a IL-1 receptor antagonist gene.

7. Diagnostic means according to Claim 6, comprising PCR primers adapted to amplify a region of an IL-1 receptor antagonist gene.

8. Diagnostic means according to Claim 7 comprising PCR primers adapted to amplify a portion of intron 2 of an IL-1 receptor antagonist gene.

9. Diagnostic means according to Claim 8 wherein the PCR primers are selected from SEQ ID NO: 1 and SEQ ID NO: 2.

10. 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 or reduce osteoporosis or to delay the onset of osteoporosis.

11. A method according to Claim 10 wherein a predisposition to osteoporosis is correlated with a IL-1 receptor antagonist gene which varies from the wild type gene sequence.

12. A method according to Claim 10 or 11 in which a predisposition to osteoporosis is correlated with a IL-1 receptor antagonist gene in which there is a twice repeated 86bp sequence in intron 2.

13. A method according to any of Claims 10-12 comprising treating the individual by hormone replacement therapy.

14. A diagnostic kit comprising means according to any of claims 6-9.

15. 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 IL-1 receptor antagonist gene.

16. A method of identifying an individual predisposed or susceptible to osteoporosis, said method comprising determining genotype of a first gene in said individual, wherein genotype of said first gene is correlated with genotype of a IL-1 receptor antagonist gene in said individual.

17. A method of identifying a further polymoφhism correlated with predisposition to osteoporosis, comprising identifying in a cohort of individuals with polymoφhisms of the IL-1 receptor antagonist gene a further polymoφhism and determining whether that further polymoφhism is correlated with polymoφhism of the IL-1 receptor antagonist gene.

18. A method of predicting response to osteoporosis therapy, comprising diagnosing genotype of an IL-1 receptor antagonist gene.

19. A method according to Claim 18 further comprising prescribing therapy according to its predicted effectiveness.

20. A method according to Claim 18 or 19 comprising predicting response to therapy according to the number of copies of a polymoφhism of the gene.

21. A method according to any of Claims 18 to 20 comprising prescribing hormone replacement, or an equivalent, therapy.