WO2002092850A2 - Diagnostic method for osteoporosis and related disorders - Google Patents

Abstract

Provided are methods and materials for determining the susceptibility of an individual to a disorder which is associated with an abnormal (e.g. low) level of bone mineral density (BMD), the method comprising use of a Fos related antigen-1 gene (Fra-1) marker. The methods may be used e.g. for diagnosis of osteoporosis. Preferred Fra-1 markers include single nucleotide polymorphisms at positions 933, 1301, 4755, 1948, and 2394 of the Fra-1 gene.

Description

DIAGNOSTIC METHOD FOR OSTEOPOROSIS AND RELATED DISORDERS

The present invention relates to methods for diagnosis of disorders which are associated with abnormal levels of bone mineral density (BMD) , in particular osteoporosis, and to materials for use in such methods .

PRIOR ART

Osteoporosis is a common disease with a strong genetic component [25] characterised by reduced bone mass, microarchitectural deterioration of bone tissue and an increased risk of fracture [14] . Bone mineral density (BMD) is an important risk factor for osteoporotic fracture and osteoporosis is defined to exist when BMD values fall more than 2.5 standard deviations below the young adult mean. Studies in twins and families have indicated that that between 50%-85% of the variance in BMD is genetically determined depending on the site examined [9,23]. The genes responsible for these effects are incompletely defined. However, linkage studies in families and sibling pairs have identified several candidate loci for the regulation of BMD [5,13,15]. One of these candidate loci for regulation of BMD lies on chromosome llql2-13.

DISCLOSURE OF THE INVENTION

The present inventors have identified novel single nucleotide polymorphisms in the Fra-1 gene which is located in the chromosome region in llql2-13, and shown that these polymorphisms are associated with bone mineral density (BMD) .

Such single nucleotide polymorphisms that are highly associated with BMD are useful as genetic markers for identifying people with low BMD, so that these individuals could be targeted for treatment to prevent osteoporosis. Fra 1 is the Fos related antigen-1 gene (Fra-1) , and is a component of the transcription factor activator protein-1 (AP-1), which is composed of dimers of Fos proteins (c-Fos, FosB, Fra-1, and Fra-2) and Jun proteins (c-Jun, JunB, and JunD) .

Previous work by Jochum et al [12] showed that transgenic mice overexpressing Fra-1 in various organs develop a progressive increase in bone mass leading to osteosclerosis of the entire skeleton. Studies by Wang et al demonstrated that c-Fos knock out mice develop osteopetrosis due to an early differentiation block in the osteoclast lineage [27] and that Fra-1 could rescue the differentiation block caused by the lack of c-Fos in vitro and in vivo [19] . Furthermore, in vitro studies by Owens et al showed that Fra-1 plays a role in osteoclast differentiation which is distinct from that of c-Fos [22] . However, none of these studies has demonstrated that polymorphic variants of the Fra-1 gene are associated with bone mineral density (BMD) .

The present inventors determined the intron/exon structure of the Fra-1 gene and then screened the gene coding and regulatory regions for polymorphisms and related these to BMD in a population based association study. This was particularly difficult because of the presence of a novel Fra-1 pseudogene which made the selection and design of primers etc., complex (as discussed in more detail in the Examples below) .

At its most general, the present invention provides a method for determining the susceptibility of an individual to a disorder which is associated with an abnormal level of bone mineral density, the method comprising using a Fra-1 marker, particularly a polymorphic marker to assess the susceptibility of that individual for said disorder .

Disorders which are associated with abnormal levels of bone mineral density are hereinafter known as "BMD-associated disorders" . Abnormal levels of BMD may be low or high. Preferably, the present invention is concerned with disorders associated with low BMD, especially osteoporosis and related disorders .

For example, the methods of the present invention may be used to determine the risk of certain consequences of abnormal levels of BMD, such as to determine the risk of osteoporotic fracture (McGuigan et al (2001) Osteoporosis International, 12, 91-96).

Low levels of BMD are those which are greater than 1 standard deviation below the adult mean for BMD, particularly between 1 and 3 standard deviations below the adult mean. For example, in the case of osteoporosis, the level of BMD is less than -2.5 standard deviations below the young adult mean. For osteopenia, BMD values are between -1 and -2.5 standard deviations below the young adult mean .

The method may comprise first obtaining a sample of nucleic acid, preferably genomic DNA, from an individual, and then using a Fra-1 polymorphic marker to determine the susceptibility of that individual .

The DNA sample analysed may be all or part of the sample being obtained.

Where the polymorphism is not intronic the assessment may be performed using mRNA (or cDNA) , rather than genomic DNA.

Where the present invention relates to the analysis of nucleic acid of an individual, such an individual may be one who has a BMD- associated disorder, is considered at risk from BMD-associated disorder such as osteoporosis (e.g. by having a sibling with and/or family history of BMD-associated disorder), or may be symptomless.

The sample from the individual may be prepared from any convenient sample, for example from blood or skin tissue. A sample obtained from an individual may be analysed according to methods of the present invention. Methods of the present invention may therefore include providing a sample of nucleic acid obtained from an individual .

Alternatively, the assessment of the Fra-1 polymorphic marker may be performed or based on an historical DNA sample, by assessing the Fra-1 polymorphic marker in DNA sequences which are stored on a databank.

It is preferred that the polymorphic marker is a single nucleotide polymorphism (SNP) , which may be in an intron, exon or promoter sequence of the Fra-1 gene. Most preferred are SNPs which are directly responsible for the BMD phenotype. Preferred SNPs may be in an exon, or may be in an intron. Intronic SNPs may, for example, be situated in regions involved in gene transcripton. SNPs may be directly responsible for the BMD phenotype because of an effect on the amino acid coding, or by disruption of regulatory elements, e.g., which may regulate gene expression, or by disruption of sequences (which may be exonic or intronic) involved in regulation of splicing, such as exonic or splicing enhancers as discussed below.

Preferred SNPs are at the following positions in the genomic sequence of Fra-1 shown in Annex 1: 933 (SNP933); 1301 (SNP1301) ; 4755 (SNP4755) ; 1948 (SNP1948) ; or 2394 (SNP2394 ) , more preferred are SNPs at positions 933; 1301; or 4755, even more preferred are SNPs at positions 1301 or 4755, most preferred is the SNP at position 1301.

The polymorphic marker may be a polymorphic marker which is in linkage disequilibrium with any of the markers discussed above, e.g., in linkage disequilibrium with an exonic or intronic marker which may be directly responsible for the BMD phenotype. In particular, the polymorphic marker may be in linkage disequilibrium with SNP933; SNP1301; SNP4755; SNP1948; or SNP2394. Polymorphic markers which are in linkage disequilibrium with any of the polymorphic markers described herein may be identified by DNA sequencing, e.g., within the Fra-1 gene.

Accordingly, in one embodiment the method of the present invention comprises assessing in a genomic DNA sample obtained from an individual (which sample may be historical, as defined above) one or more Fra-1 SNPs selected from the SNPs at the following positions of the Fra-1 genomic sequence shown in Annex 1: 933; 1301; 4755; 1948; or 2394, or a polymorphism in linkage disequilibrium with one of said SNPs.

As is understood by the person skilled in the art, linkage disequilibrium is the non-random association of alleles. Further details may be found in Kruglyak (1999) Nature Genetics, Vol 22, page 139 and Boehnke (2001) Nature Genetics 25: 246-247). For example, results of recent studies indicate (summarised by Boehnke) that significant linkage disequilibrium may extend for between 0.1 to 0.2 centimorgans .

In a further embodiment the method may comprise assessing two or more Fra-1 SNPs. For example, the method may comprise assessing SNP1301 and one or more further SNPs selected from 933; 4755; 1948; or 2394, or a polymorphism in linkage disequilibrium with one of said SNPs. Any suitable combination of two or more markers may be used to determine the susceptibility of the individual for a BMD- associated disorder.

The method of the invention may comprise, in addition to assessing one or more Fra-1 SNPs, or one or more polymorphism in linkage disequilibrium with a Fra-1 SNP, the assessement of other polymorphisms which are linked or associated with a BMD-associated disorder.

Examples of such other polymorphisms include polymorphisms in the

VDR gene and the COLIA1 gene (Uitterlinden, et al . (2001) Journal of Bone and Mineral Research) . The assessment of an SNP may involve determining the identity of a nucleotide at the position of said single nucleotide polymorphism.

For the following SNPs : SNP933; SNP1301; SNP4755; SNP1948; or

SNP2394, the nucleotide at the position of the SNP may be as shown in Table 5. For example, for SNP 1301, an individual who is T/T homozygous for the polymorphism is classified as being at the highest risk; an individual who is C/T heterozygous is classified as having moderate risk; an individual who is C/C homozygous is in the lowest risk category.

Alternatively, or additionally the assessment of the SNP may be performed without determining the precise identity of the nucleotide at that positions. For example, where the assessment involves assessment based upon restriction enzyme sites as discussed elsewhere herein.

Any suitable method may be used for assessment of a SNP. Examples of such methods will now be discussed in more detail.

Tests may be carried out on preparations containing genomic DNA, cDNA and/or mRNA. Testing of cDNA or mRNA may be used where the SNP is exonic.

The method of assessment of the polymorphism may comprise determining the binding of an oligonucleotide probe to the nucleic acid sample. The probe may comprise a nucleic acid sequence which binds specifically to a particular allele of a polymorphism and does not bind specifically to other alleles of the polymorphism.

Suitable probes may comprise all or part of the sequence shown in Annex I (or complement thereof) , or all or part of a polymorphic form of the sequence shown in Annex I (or complement thereof (e.g., containing one or more of the polymorphisms shown in Table 5) . Under suitably stringent conditions, specific hybridisation of such a probe to the test nucleic acid is indicative of the presence or absence of a sequence alteration (polymorphism) in the test nucleic acid. More than one probe may be used if desired.

The oligonucleotide probe may comprise a label and hybridisation of the probe may be determined by detecting the presence of that label.

A suitable method may include hybridisation of one or more (e.g. two) oligonucleotide probes or primers to target nucleic acid.

Where the nucleic acid is double- stranded DNA, hybridisation will generally be preceded by denaturation to produce single-stranded DNA. A screening procedure, chosen from the many available to those skilled in the art, is used to identify successful hybridisation events and isolated hybridised nucleic acid.

Binding of a probe to target nucleic acid (e.g. DNA) may be measured using any of a variety of techniques at the disposal of those skilled in the art. For instance, probes may be radioactively, fluorescently or enzymatically labelled.

Other methods not employing labelling of probe include examination of restriction fragment length polymorphisms, amplification using PCR, RN'ase cleavage and allele specific oligonucleotide probing. Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined.

Those skilled in the art are well able to employ suitable conditions of the desired stringency for selective hybridisation, taking into account factors such as oligonucleotide length and base composition, temperature and so on. Suitable selective hybridisation conditions for oligonucleotides of 17 to 30 bases include hybridization overnight at 42°C in 6X SSC and washing in 6X SSC at a series of increasing temperatures from 42°C to 65°C.

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is (Sambrook et al . , 1989): T_m = 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex. As an illustration of the above formula, using [Na+] =

[0.368] and 50-% formamide, with GC content of 42% and an average probe size of 200 bases, the T_m is 57°C. The T_m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C. Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention.

Other suitable conditions and protocols are described in Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al . , 1989, Cold Spring Harbor Laboratory Press and Current Protocols in Molecular Biology, Ausubel et al . eds . , John Wiley & Sons, 1992.

The hybridisation of such a probe may be part of a PCR or other amplification procedure. Accordingly, in one embodiment the method of assessing the polymorphism includes the step of amplifying a portion of the Fra-1 locus, which portion comprises at least one polymorphism.

The assessment of the polymorphism in the amplification product may then be carried out by any suitable method, e.g., as described herein. An example of such a method is a combination of PCR and low stringency hybridisation with a suitable probe. Unless stated otherwise, the methods of assessing the polymorphism described herein may be performed on a genomic DNA sample, or on an amplification product thereof. Where the method involves PCR, or other amplification procedure, any suitable PCR primers may be used. The person skilled in the art is able to design such primers, examples of which are shown in Table 3.

An oligonucleotide for use in nucleic acid amplification may be about 30 or fewer nucleotides in length (e.g. 18, 21 or 24). Generally specific primers are upwards of 14 nucleotides in length, but need not be than 18-20. Those skilled in the art are well versed in the design of primers for use processes such as PCR. Various techniques for synthesizing oligonucleotide primers are well known in the art, including phosphotriester and phosphodiester synthesis methods.

Suitable polymerase chain reaction (PCR) methods are reviewed, for instance, in "PCR protocols; A Guide to Methods and Applications", Eds. Innis et al, 1990, Academic Press, New York, Mullis et al, Cold Spring Harbor Symp. Quant. Biol . , 51:263, (1987), Ehrlich (ed) , PCR technology, Stockton Press, NY, 1989, and Ehrlich et al, Science, 252:1643-1650, (1991)). PCR comprises steps of denaturation of template nucleic acid (if double-stranded) , annealing of primer to target, and polymerisation.

As mentioned earlier, an amplification method may be a method other than PCR. Such methods include strand displacement activation, the QB replicase system, the repair chain reaction, the ligase chain reaction, rolling circle amplification and ligation activated transcription. For convenience, and because it is generally preferred, the term PCR is used herein in contexts where other nucleic acid amplification techniques may be applied by those skilled in the art. Unless the context requires otherwise, reference to PCR should be taken to cover use of any suitable nucleic amplification reaction available in the art.

The polymorphism may be assessed or confirmed by nucleotide sequencing of a nucleic acid sample to determine the identity of a polymorphic allele. The identity may be determined by comparison of the nucleotide sequence obtained with a sequence shown in the Annex, Figures and Tables herein. In this way, the allele of the polymorphism in the test sample may be compared with the alleles which are shown to be associated with susceptibility for osteoporosis .

Nucleotide sequence analysis may be performed on a genomic DNA sample, or amplified part thereof, or RNA sample as appropriate, using methods which are standard in the art.

Where an amplified part of the genomic DNA sample is used, the genomic DNA sample may be subjected to a PCR amplification reaction using a pair of suitable primers. In this way the region containing a particular polymorphism or polymorphisms may be selectively amplified (PCR methods and primers are discussed in more detail above) . The nucleotide sequence of the amplification product may then be determined by standard techniques.

Other techniques which may be used are single base extension techniques and pyrosequencing.

The assessment of the polymorphism may be performed by single strand conformation polymorphism analysis (SSCP) . In this technique, PCR products from the region to be tested are heat denatured and rapidly cooled to avoid the reassociation of complementary strands. The single strands then form sequence dependent conformations that influence gel mobility. The different mobilities can then be analysed by gel electrophoresis .

Assessment may be by heteroduplex analysis. In this analysis, the DNA sequence to be tested is amplified, denatured and renatured to itself or to known wild-type DNA. Heteroduplexes between different alleles contain DNA "bubbles" at mismatched basepairs that can affect mobility through a gel. Therefore, the mobility on a gel indicates the presence of sequence alterations.

Where an SNP creates or abolishes a restriction site, the assessment may be made using RFLP analysis. In this analysis, the DNA is mixed with the relevant restriction enzyme (i.e., the enzyme whose restriction site is created or abolished) . The resultant DNA is resolved by gel electrophoresis to distinguish between DNA samples having the restriction site, which will be cut at that site, and DNA without that restriction site, which will not be cut.

Where the SNP does not create or abolish a restriction site the SNP may be assessed in the following way. A mutant PCR primer may be designed which introduces a mutation into the amplification product, such that a restriction site is created when one of the polymorphic variants is present but not when another polymorphic variant is present. After PCR amplification using this primer (and another suitable primer) , the amplification product is admixed with the relevant restriction enzyme and the resultant DNA analysed by gel electrophoresis to test for digestion. Further details of this method are given in the examples .

Further methods for assessment of the polymorphism are reviewed by Schafer and Hawkins, (Nature Biotechnology (1998)16, 33-39, and references referred to therein) and include: denaturing gradient gel electrophoresis, RNase cleavage, chemical cleavage of mismatch, T4 endonuclease VII cleavage, multiphoton detection, cleavase fragment length polymorphism, E. coli mismatch repair enzymes, denaturing high performance liquid chromatography, (MALDI-TOF) mass spectrometry, analysing the melting characteristics for double stranded DNA fragments as described by Akey et al (2001) Biotechniques 30; 358- 367.

The assessment of the polymorphism may be carried out on a DNA microchip, if appropriate. One example of such a microchip system may involve the synthesis of microarrays of oligonucleotides on a glass support. Fluorescently - labelled PCR products may then be hybridised to the oligonucleotide array and sequence specific hybridisation may be detected by scanning confocal microscopy and analysed automatically. Clearly, the invention embraces methods for determining the presence or absence in a test sample of a polymorphic marker in the Fra-1 sequence which is an SNP as described above e.g. by use of the probes or primers described herein.

In another aspect, the present invention provides a method for mapping further polymorphisms which are associated, or are in linkage disequilibrium with a Fral polymorphism, as described herein. Such a method may preferably be used to identify a polymorphism having a causative role in BMD-associated disorders. Such a method may comprise screening genomic DNA samples, e.g., from patients with low and high BMD using a genetic sequence derived from one or more of the Fra-1 markers described herein and identifying further polymorphic associated with said Fra-1 marker.

Such a method may involve sequencing of the Fra-1 gene, or may involve sequencing regions upstream and downstream of the Fra-1 gene for associated polymorphisms.

In a further aspect, the present invention provides a method of identifying open reading frames which are involved in BMD-associated disorders. Such a method may comprise screening a genomic sample with an oligonucleotide sequence derived from a Fra-1 polymorphic marker as described herein and identifying open reading frames proximal to that genetic sequence.

A region which is described as 'proximal' to a polymorphic marker may be within about lOOOkb of the marker, preferably within about 500kb away, and more preferably within about lOOkb, more preferably within 50 kb, more preferably within 10 kb of the marker.

In a further aspect, the present invention provides a Fra-1 nucleic acid sequence, or polypeptide, for use in the treatment or prevention of a BMD- associated disorder.

The Fra-1 nucleic acid sequence may be that shown in Annex 1, or may have some or all of the polymorphisms shown in Table 5. Further aspects include the use of such a nucleic acid sequence in the manufacture of a medicament for the treatment or prevention of a BMD-associated disorder, and methods of treatment for a BMD- associated disorder comprising the administration of a Fra-1 nucleic acid molecule to an individual .

For example, the such a Fra-1 nucleic acid sequence may be used in a method of gene therapy to treat or prevent a BMD-associated disorder, such as osteoporosis.

Specifically, one or more copies of a Fra-1 nucleic acid sequence (e.g., the sequence shown in Annexl, polymorphic variant thereof, or fragment of either) may be inserted into the appropriate cells within a patient, using vectors that include, but are not limited to adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes. The person skilled in the art is readily able to produce such a gene therapy vectors. For an example see, Anderson, U.S. Pat. No. 5,399,349.

The Fra-1 polypeptide is preferably the polypeptide encoded by the nucleotide sequence shown in Annex 1.

The Fra-1 nucleic acid, polypeptide or antibodies thereto may be used in the diagnosis or prognosis of osteoporosis. For example, the presence or absence of alleles of the nucleic acid may be determined in an individual, the levels of expression of nucleic acid, or an amount of protein in the cells of an individual may be determined, which may lead to the diagnosis or prognosis of osteoporosis. Levels of expression may be determined by any known technique in the art, which may involve analysis of RNA or protein levels.

In a further aspect the present invention provides oligonucleotide sequences, probes and primers which may be suitable for use in the methods of the invention. For example, suitable probes may comprise all or part of the sequence shown in Annex I, or all or part of a polymorphic form of the sequence shown in Annexl (e.g., containing one or more of the polymorphisms shown in Table 5) . Suitable PCR primer pairs may comprise a first and second primers which hybridise to DNA in regions flanking (or in certain embodiments described herein, including) the polymorphism of interest, as is well understood by the person skilled in the art. Examples of preferred primer sequences are shown in Table 3.

The nucleic acid for use in the methods of the invention may be a variant, fragment or derivative of the sequences disclosed herein, e.g., a variant, fragment or derivative of the sequence shown in Annex 1 or polymorphic variant thereof, or variant, fragment or derivative of a probe or primer sequence disclosed herein.

A variant nucleic acid molecule shares homology with, or is identical to, all or part of the coding sequence discussed above.

Thus a variant may be a distinctive part or fragment (however produced) corresponding to a portion of the sequences described here, e.g., may relate to a particular functional part of the nucleic acid sequence or encoded protein.

Also included are nucleic acids which have been extended at the 3' or 5' terminus. This may be particularly useful in producing a labelled probe.

Homology (i.e. similarity or identity) may be as defined using sequence comparisons are made using FASTA and FASTP (see Pearson & Lipman, 1988. Methods in Enzymology 183: 63-98). Parameters are preferably set, using the default matrix, as follows: Gapopen (penalty for the first residue in a gap) : -12 for proteins / -16 for DNA; Gapext (penalty for additional residues in a gap) : -2 for proteins / -4 for DNA; KTUP word length: 2 for proteins / 6 for DNA. Homology may be at the nucleotide sequence and/or encoded amino acid sequence level. Preferably, the nucleic acid and/or amino acid sequence shares at least about 60%, or 70%, or 80% homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% homology with Fra-1. The homology of a particular sequence may be determined using hybridisation assays, at an appropriate stringency as would be understood by the person skilled in the art.

Changes to a sequence, to produce a derivative, may be by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, which may lead to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide.

Changes may be desirable for a number of reasons, including introducing or removing the following features: restriction endonuclease sequences; 3' or 5' extensions, e.g., may be added for use in probes; leader or other targeting sequences (e.g. hydrophobic anchoring regions) may be added or removed from the expressed protein to determine its location following expression.

Oligonucleotides for use in probing or amplification reactions may be fragments of the sequence shown in Annex I, or polymorphic variant thereof, such oligonucleotides are discussed elsewhere herein.

Nucleic acid for use in the methods of the present invention, such as an oligonucleotide probe and/or pair of amplification primers, may be provided as part of a kit, e.g. in a suitable container such as a vial in which the contents are protected from the external environment. The kit may include instructions for use of the nucleic acid, e.g. in PCR and/or a method for determining the presence of nucleic acid of interest in a test sample. A kit wherein the nucleic acid is intended for use in PCR may include one or more other reagents required for the reaction, such as poly erase, nucleosides, buffer solution etc. The nucleic acid may be labelled. A kit for use in determining the presence or absence of nucleic acid of interest may include one or more articles and/or reagents for performance of the method, such as means for providing the test sample itself, e.g. a swab for removing cells from the buccal cavity or a syringe for removing a blood sample (such components generally being sterile) .

The various embodiments of the invention described above may also apply to the following: a diagnostic means for determing the risk of a BMD-associated disorder (e .g. osteoporosis); a diagnostic kit comprising such a diagnostic means; a method of osteoporosis therapy, which may include the step of screening an individual for a genetic predisposition to osteoporosis, wherein the predisposition is correlated with a Fra-1 polymorphic marker, and if a predisposition is identified, treating that individual to prevent or reduce the onset of osteoporosis (such a method may comprise the treatment of the individual by hormone replacement therapy) ; and the use, in the manufacture of means for assessing whether an individual has a predisposition to osteoporosis, of sequences (e.g., PCR primers) to amplify a region of the Fra-1 gene.

The invention will now be further described with reference to the following non-limiting Examples and Annexes. Other embodiments of the invention will occur to those skilled in the art in the light of these.

Figures, Tables and Sequence Annex

Figure 1 shows C933T polymorphism in Promoter of Fra-1.

Figure 2 shows the exonic Polymorphisms of Fra 1

Figure 3 shows the summary of Fra-1 polymorphisms

Figure 4 shows the design of a mutagenic PCR based assay to detect the C1301T polymorphism

Annex 1 shows the complete genomic sequence of the human Fra-1 gene,

Table 1 shows the intron/exon structure of the Fra-1 gene. Table 2 shows the positions of the primers used in searching for polymorphisms in the Fra-1 gene

Table 3 shows the oligonucleotide sequences of the primers used in searching for polymorphisms in the Fra-1 gene.

Table 4 shows clinical details of the individuals from which the DNA samples were taken for screening for polymorphisms.

Table 5 shows a summary of the polymorphisms identified in the Fra-1 gene .

Table 6 shows Hardy Weinberg Equilibrium Analysis for Fra-1 Pstl Genotypes for the C933T polymorphism.

Table 7 shows characteristics of the study subjects for the investigation of the clinical association for the C1301T polymorphism

Table 8 shows the Hardy Weinberg Equilibrium Analysis for Fra-1 EcoRI Genotypes for C1301T polymorphism

Table 9 shows characteristics of the EcoRI Genotypes in 1991/1992

Table 10 shows characteristics of the EcoRI Genotypes in 2000

Table 11 shows multiple Linear regression analysis of BMD and EcoRI Genotypes in (1991/1992) .

Table 12 shows multiple Linear regression analysis of BMD and EcoRI Genotypes in 2000

EXAMPLES

Example 1- Genomic Organisation of the Human Fra-1 Gene The intron/exon structure of Fra-1 was determined using combination of vectorette PCR and long PCR. During these experiments, a Fra-1 pseudogene was also identified, characterised and mapped to chromosome 4. Details of the experimental procedures are published elsewhere (Albagha PhD thesis, (2001) University of Aberdeen). The complete genomic sequence of the human fra-1 gene is shown in Annex 1. The intron/exon structure in relation to the sequence shown in Annex 1 is shown in Table 1.

Example 2 -Mutation Screening of Fra-1

The coding region, the promoter region, part of the first intron, and part of 3'UTR of the human Fra-1 gene were screened for novel polymorphisms using a combination of SSCP and direct DNA sequencing.

The identification of a Fra-1 pseudogene with high homology to the mRNA sequence of the functional gene made primer design more complicated. To ensure amplification of the functional gene only, oligonucleotide primers were designed using intronic sequences flanking each exon.

Table 2 summarises the primers used in mutation screening, their location in reference to the sequence shown in Annex I and the PCR product size. The primer sequences are shown in Table 3.

DNA from 10 patients with high BMD and 10 patients with low BMD were used to screen for novel polymorphisms. The clinical details and other relevant information for these is shown in Table 4.

The nine fragments of DNA spanning the coding region, part of the promoter region, part of the 3"UTR, and part of the first intron were amplified by PCR for further scanning for mutation by SSCP or direct DNA sequencing. The promoter segment 1, promoter segment 2, and Exon 3 fragments were analysed by SSCP whereas promoter segment 3 - exon 1, intron 1 segment 1, intron 1 segment 2, Exon 2, Exon 4, and part of the 3^"UTR were analysed by direct DNA sequencing. SSCP analysis identified one putative polymorphism in the promoter segment and DNA sequencing showed that this was a C — > T transition at position 933 in reference to the sequence humfra-1. seq. (Figure 1) . Note that this polymorphism creates a recognition site for the enzyme Pstl (CTGCAG) . However, it was only observed in 2 samples out of the 20 samples which were used for screening.

Direct DNA sequencing identified four other polymorphisms: C1301T, G1948C, T2394C, and A4755G located in exonl, intron 1 segment 1, intron 1 segment 2, and exon 2, respectively. Information on these polymorphisms summarised in Figures 2-3 and Table 5.

The Exon 1 polymorphism (C1301T) did not create or abolish a recognition site for any of commercially available restriction enzymes. However, a mutant primer was designed so that a recognition site for the restriction enzyme EcoRI (G AATTC) would be created when the C variant is present (Figure 4) . This was achieved by changing the 19^th nucleotide of the forward primer from C to A. Screening for this polymorphism was then performed by PCR amplification followed by digestion with EcoRI . Genotypes were represented as EE, Ee, and ee with the uppercase letter signifying the absence of, and lowercase letter the presence of the EcoRI restriction Site (i.e. "E" is the T variant and "e" is the C variant) .

Other polymorphisms were identified by DNA sequencing of the fra-1 gene as summarised in Figure 3. However so far, we have studied clinical associations of the promoter polymorphism (C933T) and the exon 1 polymorphism (C1301T) as discussed below.

Example 3 -Clinical Associations of Promoter (C933T) polymorphism.

DNA from 191 women from a population based sample of postmenopausal women derived from the Grampian region were screened for the promoter polymorphism by PCR-RFLP assay and the results showed that only 6 subjects had the polymorphism in the heterozygous state (C/T) . The homozygote T variant (T/T) was not observed in this population of samples. Genotype frequencies were in hardy Weinberg equilibrium as shown in Table 6. This polymorphism had a heterozygosity value of 0.031 and therefore it was not investigated further in relation to BMD. However, this polymorphism could still be used as a diagnostic.

Example 4- Clinical Associations of exon 1 (C1301T) Polymorphism

The relationship between Fra-1 exon 1 (C1301T) polymorphism was investigated in relation to BMD in 501 perimenopausal women randomly selected from the Grampian region. Relevant anthropometric and clinical details of the study subjects are shown in table 7.

Bone mineral density measurements were carried out by dual energy x- ray absorptiometry using Norland xR26 or XR36 densitometers .

Analysis was performed on data collected in 1991/1992 (Baseline data - Table 9) , and repeated on data collected in 1998-2000 (Table 10) to assess the relationship between genotypes and bone loss. The mean bone loss over the 8 year follow-up period were 3.38% and 4.95% for LS and FN BMD, respectively.

Genotype frequencies showed a slight excess of heterozygotes, but overall, the results did not deviate significantly from Hardy Weinberg equilibrium (p value = 0.051, Table 8). This polymorphism was more polymorphic than the Pstl polymorphism (heterozygosity = 0.49) .

Next the relationship between the three genotypes of the EcoRI polymorphism and BMD was investigated. Data on the frequency of the EcoRI polymorphism in relation to BMD and other relevant clinical and anthropometric variables are shown in Tables 9 and 10.

Women with the genotype EE had significantly lower lumbar spine BMD than those with Ee or ee genotype. A similar trend was observed at the femoral neck, however, the relationship at the lumbar spine was more highly significant (p < 0.001) than the femoral neck (p = 0.12). The association with EcoRI genotypes remained significant after 8 years at both sites in 285 woman where follow up data were available, however, the significance at FN increased (p = 0.009). There was no significant relationship between the EcoRI genotypes and bone loss over 8 years and no other variable differed between the three genotype groups .

The relation between the EcoRI genotypes and BMD was next investigated in a multiple linear regression analysis model including other predictors of BMD such as weight, age, height, and YSM. The effect of the "B" allele on BMD appeared to be recessive to that of the "e" allele. Therefore, genotypes were coded as 1 and 2; corresponding to the genotype EE and the combined Ee & ee genotypes, respectively. Results are shown in tables 11 and 12.

The EcoRI genotype predicted BMD at both lumbar spine (p = 0.001) but not femoral neck (p = 0.199) in this regression model. Weight accounted for the majority of the variance in LS (8.4%) and FN (14.8%) BMD. The EcoRI genotypes accounted for 1.5% of the total variance in BMD at LS and FN, respectively.

Later in life of the individual, the contribution of weight to the total variance of BMD appears to increase at both LS (6.3% -→ 9.7%) and FN (13.9 → 14.2%) . The effect of EcoRI genotype declined with age at the LS (4.1% → 3.5%) but not FN (1.4% → 1.5%) BMD.

Discussion

We have identified novel polymorphisms in the Fral gene which is located in chromosome region llql2-13 and shown their association with osteoporosis. In order to screen the Fra-1 gene for polymorphisms, we have first determined the intron/exon structure so that a further 20-50 intronic nucleotides are included in the mutation screening to ensure that splice mutations are not missed. The human Fra-1 gene demonstrated high homology to the mouse gene in terms of genomic organisation as well as DNA sequence indicating conservation of this gene during mammalian evolution. During the studies we also identified a novel Fra-1 pseudogene { Ψ Fra-1) by PCR amplification from genomic DNA. PCR products obtained using different trans-intronic primer pairs showed sequences of high homology to the Fra-1 gene, however, PCR product sizes for these sequences were similar to what was expected from Fra-1 cDNA indicating that these sequences have no introns . Although These sequences were highly homologous to Fra-1 cDNA sequence (no introns) , multiple mutations were observed including one mutation that introduces a premature stop codon which would result in premature truncation of the translated message and would likely generate a non-functional protein. These data suggest that these sequences represent a processed Fra-1 pseudogene that might have arisen by reverse transcription of Fra-1 mRNA and re-insertion into the chromosomal DNA [29] . PCR primers used in mutation screening were designed based on intronic sequences to avoid co-amplification of the pseudogene .

Mutation screening identified 5 novel polymorphisms in the Fra-1 gene. The polymorphism located in the promoter was very rare (heterozygosity = 0.031) and since osteoporosis is a common disease, further investigation were focused on the other two common polymorphisms (exonl and exon 2 polymorphisms) .

We have used the exon 1 polymorphism for further study. We have screened 285 individuals followed -up for 8 years for the EcoRI polymorphism. EcoRI Genotype frequencies were at borderline deviation from Hardy-Weinberg equilibrium (HWE) . Geneticists usually consider deviation from HWE as an evidence for a technical problem or genotyping error. However, when no such problems exist, deviations from HWE may be indicative of genetic association of the given marker with a disease gene, or linkage disequilibrium (LD) [21] . In this study, genotyping error and technical problems can be excluded because three control samples (representing the EE, Ee, and ee genotypes) verified by DNA sequencing were run with every experiment. Furthermore, genotyping was repeated for 25 samples randomly selected from the original 285 samples. All genotypes were identical to those obtained from the first round genotyping experiment. Therefore, the deviation from HWE observed for the EcoRI genotypes might be an indication for possible association with BMD either directly or through linkage disequilibrium. Analysis of the relationship between this polymorphism and BMD revealed strong association with LS BMD. Individuals bearing the EE genotype had significantly lower LS and FN BMD than those without this genotype. Although LS and FN BMD are highly correlated, the relationship with LS BMD was highly significant compared to the FN which advocates the notion that genes contribute differentially to the determination of BMD at various skeletal sites.

In order to investigate the possibility of gene dose effect on BMD, EcoRI genotypes were entered in a multiple linear regression analysis which included other possible predictors of BMD such as weight, height, age, YSM, and HRT use. Genotypes were coded as 1 and 2; corresponding to the EE and the combined Be & ee genotype groups, respectively. The Ee and ee genotype groups were combined in the analysis because the effect of the "E" allele on BMD appeared to be recessive to that of the "e" allele.

The EcoRI genotypes significantly predicted BMD and accounted for 4.1 % and 1.4% of the total variance in LS and FN BMD, respectively. The effect of EcoRI polymorphism appeared to decline with age suggesting that other factors appear to modify the effect of this polymorphism on BMD later in life. This is consistent with the absence of significant association between the Ecoi?T polymorphism and bone loss over a period of 8 years .

One possible explanation for the association of EcoRI polymorphism with BMD is that the EE genotype might be in LD with another mutation nearby that causes decrease in Fra-1 transcription levels leading to reduced bone mass .

For example, the causative polymorphism may be intronic. Intragenic regulatory elements have been reported to play a significant role in gene regulation. Work by Bergers et al showed that basal and AP-1 regulated expression of the Fra-1 gene depends on regulatory sequences present in the first intron of the rat Fra-1 gene [1] . These elements were identified as a consensus AP-1 site and two API-like elements.

The EcoRI polymorphism is a silent mutation in the Fra-1 gene. Although linkage disequilibrium might be one explanation for the association of EcoRI with BMD, the possibility of this polymorphism being functional cannot be excluded. Particularly in the light of the recent reports which showed that exonic point mutations can cause skipping of one or more exons, presumably during pre-mRNA splicing in the nucleus [10,18,20,28]. Exonic point mutations in some genes such as BRCA1 [17] and dystrophin gene [24] were found to disrupt sequence motifs called Exonic Splicing Enhancers (ESEs) leading to exon skipping. ESEs are discrete but degenerate sequence motifs of approximately 6-8 nucleotides [2] recognised by a group of proteins called SR proteins (which are modular splicing factors that are involved in mRNA splicing) . Disruption of these motifs by point mutation causes inefficient splicing of exons leading to exon skipping and hence a truncated protein. Whether the association between the EcoRI polymorphism and BMD was due to a direct effect or through linkage disequilibrium, this polymorphism might be useful as a genetic marker for identifying people with low LS BMD.

In conclusion, The present study is the first to report 5 novel polymorphisms in the human Fra-1 gene and also the first to find an association between one of these polymorphisms (EcoRI) and BMD. The EcoRI polymorphism described here might be acting as a marker for a nearby mutation causing low bone mass which makes it a useful polymorphism for identifying individuals with low spine bone mass, or the marker may itself be functional, as discussed above. Reference List

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Table 1

Intron Exon structure of human Fra-1 gene in relation to the sequence of Annex 1 :

Table 2 - Oligonucleotide primers used in searching the human Fra-1 gene for novel polymorphisms

Numbers are in reference to the sequence "humfra-1.seq" shown in Annex 1. Table 3 Oligonucleotide sequences of the primers used in mutation screening

Table 4 - DNA samples used in SSCP analysis

VF: vertebral fracture; OF: osteoporotic fracture. SEM = standard error of the mean; LS = Lumbar Spine; FN = Femoral neck; Z = BMD Z score. NA; not available. Table 5 Summary of the polymorphisms identified in the Fra-1 gene,

^*Nucleotide numbers are in reference to the sequence "humfra-1. seq" . "Calculation is based on the 20 samples used in mutation screening.

Table 6 Hardy Weinberg Equilibrium Analysis for Fra-1 Pstl Genotypes .

Total 100.00 191.00 191.00 0.048

Heterozygo 0.031

^*p value = 0.825,-. n = numbers

Table 7 Characteristics of the study subjects (baseline data)

Characteristic Mean ± sem (1991/1992)

Number 501

Age (yrs) 47.91 ± 0.07

Height (cm) 161.3 ± 0.3

Weight (kg) 67.3 ± 0.6

Years Since Menopause

(YSM) 1.6 ± 0.2

Postmenopausal Women 107 (37.5%)

Lumbar Spine BMD

(g/cm²) 1.069 ± 0.007

Femoral Neck BMD

(g/cm²) 0.892 ± 0.006^"

Lumbar Spine Z-Score 0.328 ± 0.048 Femoral Neck Z-Score 0.052 ± 0.047 Hysterectomy (%) 90 (17.9%) Ever Used HRT (%) 72 (14.4%)

Table 8. Hardy Weinberg Equilibrium Analysis for Fra-1 EcoRI Genotypes .

Genotype Frequency Observed Expected _χ ² Allele Frequen

EE 16.37 82.00 92.70 1.23 E 43

Ee 53.29 267.00 245.61 1.86 e 57 ee 30.34 152.00 162.70 0.70

Total 100.00 501 501.00 3.80^*

Heterozygo 0.490

^*p value = 0.051;. n = numbers Table 9 - Characteristics of the EcoRI Genotypes (baseline data)

EcoRI Genotype Characteristic ^{EE Ee ee} p-value'

Number (%) 82 (16.4) 267 (53.3) 152 (30.3)

Age (y) 48.00 ± 0.18 47.88 ± 0.10 47.91 ± 0.13 0.845 Height (m) 1.60 ± 0.06 1.61 ± 0.04 1.62 ± 0.06 0.427 Weight (kg) 67.2 ± 1.5 67.6 ± 0.7 67.0 + 1.1 0.904 YSM^** (y) 1.29 ± 0.34 1.93 ± 0.27 1.21 ± 0.24 0.136

LS BMD (Z- 0.038 ± 0.108 0.479 ± 0.065 0.220 ± 0.086 <0.001 score)

FN BMD (Z- -0.078 ± 0.102 0.142 ± 0.068 -0.035 ± 0.081 0.120 score)

LS BMD (g/cm²) 1.028 ± 0.015 1.090 + 0.009 1.054 ± 0.012 <0.001

FN BMD (g/cm²) 0.876 ± 0.012 0.903 ± 0.008 0.882 ± 0.009 0.112

Ever used HRT _{8 9 1 ) 47 (17}._{6) 17} (n.₂) 0.085 (%)

HRT duration 0.13 ± 0.06 0.46 ± 0.09 0.25 + 0.09 0.078

(y⁾

Hysterectomy ₁₃ (₁₅.₈) 55 (20.6) 22 (14.5) 0.251

(%)

^*p-value from ANOVA/chi-square analysis.

^**YSM = years since menopause. Premenopausal women were entered as YSM = 0 in the analysis

Table 10 - Characteristics of the EcoRI Genotypes in. 2000 EcoRI Genotype p-

Characteristic EE Ee ee value^*

Number (%) 46 (16.1) 157 (55.1) 82 (28.8)

Age (y) 54.46 ± 0.28 54.62 ± 0.14 54.70 ± 0.19 0.749

Height (m) 1.59 ± 0.08 1.61 ± 0.05 1.61 ± 0.07 0.199

Weight (kg) 69.6 ± 2.3 68.8 ± 0.9 69.5 ± 1.5 0.875

5.57 ± 0.82 6.47 ± 0.56 5.50 ± 0.62 YSM (y) 0.466

(33) (112) (58)

LS BMD (Z- -0.442 ± -0.057 ±

0.280 ± 0.098 <0.001 score) 0.181 0.122

FN BMD (Z- -0.596 ± -0.129 ± -0.494 ±

0.009 score) 0.142 0.095 0.109

LS BMD (g/cm²) 0.960 ± 0.0261.061 ± 0.0141.013 ± 0.017<0.001

FN BMD (g/cm²) 0.814 ± 0.0180.870 ± 0.0110.825 ± 0.0130.009

LS-Bone Loss

-3.71 ± 1.17 -3.07 ± 0.61 -3.81 ± 0.87 0.749

(%)

FN-Bone Loss

-5.42 ± 0.96 -4.42 ± 0.55 -5.73 ± 0.78 0.336

(%)

Ever used HRT

18 (39.1) 86 (54.8) 41 (50.0) 0.172

(%) HRT duration

1.77 ± 0.39 2.76 ± 0.28 2.68 ± 0.38 0.216

(y⁾

Hysterectomy

10 (21.7) 49 (31.2) 19 (23.2) 0.269 (%)

^*p-vaϊue from ANOVA/chi- square analysis.

^**YSM = years since menopause, numbers in brackets represents number of postmenopausal women. Premenopausal women were entered as YSM = 0 in the analysis Table 11- Multiple Linear regression analysis of BMD and EcoRI Genotypes in (baseline data).

Lumbar Spine BMD Femoral Neck BMD

Predictor Coefficient R- value Predictor Coefficient R^~ p value

Weight (kg) 0.0031 0.084 <0.001 Weight (kg) 0.0036 0.148 <0.001 EcoRI genotype^* 0.0468 0.099 0.007 Age (y) - 0.0061 0.156 0.057 YSM - 0.0034 0.108 0.044 Height (cm) 0.0012 0.160 0.170 EcoRI 0.199

Height (cm) 0.0023 0 . 117 0.034 genotype^* 0.0181 0.162 Age (y) - 0.0054 0 . 120 0.164 HRT use - 0.0036 0.164 0.377 HRT use 0.0016 0 . 120 0.743 YSM - 0.0011 0.165 0.425 Constant 0.6600 0.011 Constant 0.7097 0.001

Final Adjusted R² = 0.11 zsted R² = 0.155

^*Study subjects were classified into 2 genotype groups: 1 and 2 corresponding to individuals with EE and the combined Be & ee genotypes, respectively

Table 12 - Multiple Linear regression analysis of BMD and EcoRI Genotypeis in 2000.

Lumbar Spine BMD Femoral Neck BMD

Predictor Coefficient R- p value Predictor Coefficient R² p value

Weight (kg) 0.0040 0.097 <0.001 Weight (kg) 0.0038 0.142 <0.001

EcoRI genotype^* 0.0838 0.132 0.001 Age (y) - 0.0099 0.158 0.024 EcoRI

Age (y) - 0.0103 0.147 0.067 genotype^* 0.0430 0.173 0.034

YSM - 0.0027 0.154 0.079 HRT use 0.0017 0.175 0.458

HRT use 0.0046 0.162 0.110 Height (cm) 0.0007 0.176 0.575

Height (cm) 0.0014 0.164 0.406 YSM - 0.0005 0.176 0.643

Constant 0.9381 0.019 Constant 0.9284 0.003

Final Adj usted R² - 0. 146 Final Adjusted R² = 0. .159

^*Study subjects were classified into 2 genotype groups: 1 and 2 corresponding to individuals with EE and the combined Ee & ee genotypes, respectively.

Annex 1

Human Fra-1 gene sequence

001 catctatgat ggaagtaatc agagagaagc tttgggatgg tgcccagcac

051 taacattctg gatgtgcgac aaggtgtctg tgaaggtgta agggctttga

101 aaatcttggc tgagcctggg cttggagtgc ccacggctgt gacttggcaa

151 cctgtgccag ctactcactt agcgacttca agcaattcat ttcagcatct

201 cccagcctca gtttccccat ctgtaaaaca gggatgagga tgatacctgc

251 ctcacattca ctcacctttg acatactctc caaacaagtc tggtggccac

301 aagcctccga aaggcactga ccgtaatgaa gatggcgttt atcagacctc

351 aggacacctg tagtggccct gaattaaaac tcgttatagc tcctgaaatt

401 atcctgagta atactattat attgcatttt atgtggggtt agttcaaagc

451 attaccttat cgcaaacatt taaaatatac cacccagcct gggcaatacg

501 gcgagaccca atccctacaa aaaacacaaa aattacctgg gtgtggtgac

551 gcgcgcctgt agtcccactt acttggaggg ggcgctgagg caggagaatc

601 cctttagctc aggaagttga gcctgcagtg cgccgagatc gagccactgc

651 actccagcct gggtgacaga gacccggtct caaaaatata aagtaaaata

701 aaatatgcca ccatttttgt aaccctggtt tgtcatttat tcacttttca

751 tatactcgaa gccttaaaac aaggccagtg gaaagacctc actccacgaa

801 gctttgggtg gcggttggcg tggctcctag agatgtgatt ctttcgtgct

851 attttgtggg agcagaaacg gaggttagcc caggcctcga gagggctggg

901 gcggggcgcg ggctctggca ggtgcgtcag tccgcagggg aacccggggc

951 tccacctggg cgcggcgagg aagttacacc atgtatgggc agctacgtca

1001 ggggggcggg cccgcagccg ccggggaacg ccgagccggg cccatcccgg

1051 tgaaaaggct gcagccggac ttggggaggc gtcgccaagt tcgggaccga

1101 cgggccaagg cggcgcgtct cgggggtgga gcctggaggt gaccgcgccg

1151 ctgcaacgcc cccacccccc gcggtcgcag tggttcagcc cgagaacttt

1201 tcattcataa aaagaaaaga ctccgcacgg cgcgggtgag tcagaaccca 1251 gcagccgtgt accccgcaga gccgccagcc ccgggcatgt tccgagactt

1301 cggggaaccc ggcccgagct ccgggaacgg cggcgggtac ggcggccccg 1351 cgcagccccc ggccgcagcg caggcagccc agcaggtgag tggggcccga 1401 cgccggtggt cccacgggag gaccgcgcgt gggaggccgg ggtcaactcc

1451 cgcacgctcc gggactggag cggggaaccg gagtcggggc cgggctgggg

1501 gctggggcga ggatcctgcc cctcgccgtc cggggccagt tgcccccacc

1551 gcgcagcggg cagcagagac ccgcgaactc cccttctccc agccgccccc

1601 ttctccctcg ccgtccgcgt ccgtccctcc gtctccgtgc ccgctttctc

1651 tcctcctgcc cacctctgac ttctgcgtcc accctgcttc agaaggggcg

1701 tgggcgccta gggtggtcgc ctcgaaggct aggagggtct ctccgcttcg

1751 ggctgaattc ctgggatctg gagtctgggc tggggtgagg gtccccacac

1801 gggagggagg cgggatccgt cgggagagca gctgggccag ggtgggagtc

1851 ccggcccggt ctgcgaccct ccgcgtgtcc gtccgtctgt ctgtccttcg

1901 cccctccgtc gccaagtccc tatcgtttct cggcctgggg agtccagggg

1951 ggtccgggca gtggaacctg ccaggtcccc ggagaccggc cctcgatccc

2001 tttgccgaat gcggcgaata gagacctcga ggttccggga cgtgcgcggg

2051 gtcaggctgc gggcgactct cgcgacccca ctctcagccc ctaacggcgc

2101 ccgctgcccg ccggggggca cccgggaggc tgcaggtgcc catttcctgt

2151 cgaggggctg cgagcacgtg tcgacagggg agggaaggag gggcgtccgt

2201 tccgccgagt cacggcggcc gagtcacggc ggctgagtca cccggggcgt

2251 ccctcccttc ctggcctgga gcggcccggg cccgggagtg ggagatgccg

2301 ccggcggtgc ctcggacccg gaactggggt cactgggacg ccgggcccgc

2351 agcgcttggg ctcgggctac cgctgtcggg acttgcctgg gcttgtgggc

2401 gcttcccgga cgattgggcg cccacaccca gggcccagcg gcagagctag

2451 aacccacttc tcccagaaca ctgagcccca tcccaaccca ctcctcagat

2501 agctggctga tagaatggga acagggacgt tgtgctgggc gcttgctaca

2551 tcatttacca tatttcacgg aatcgaagac ctcatcgatt gaaaaaaaaa

2601 cccactataa ctttatgcca ctaattttct ttccttctct ttcttttttt

2651 ttttttttct gagacagggt ctccctctgt tgcccagact atagagcagt

2701 ggtgcaatca cagctcactg cagcctcagc ctcctgggct caagcaatcc

2751 tactgagatc ctagctggga agcgacctgg ccacaggcac gcaccaccac

2801 acccaactaa ttttttaatt tttttgtaga aatgaagtct cactgtgttg

2851 cccggtgtga cctcaaactc ctgagctcaa gtgatccttc tacctcagcc 2901 tcccaaagtg ctgggtatga gccaccatgc ctagtccact aaaatttttt

2951 aaattgccaa ttaaactatg atttcagagc tattaaaggg tacttcttaa

3001 catcagtgaa atcctgtaat cctcacctgt aacaatatta aaaatgagga

3051 aactgaggca catggaggtt aaataacttg cccatggtca cacagccagt

3101 aagcagctga gagctgagat tagaattggg acagcctgcc atgccaggtg

3151 gctaaagcct gtcatctcaa cactttggga ggctgaggtg ggtggactgc

3201 ttcagctcag gaatttgaga ccaccctggg caacctagtg agaccctgtc

3251 tctacaaaaa aatttaaaaa aaattagcca ggccgtgcgc ggtgcctcat

3301 gcctataatc ccagcactgt gggaggccaa ggcgggtgga tcaccagagg

3351 tcaggagttc aagaccagcc tggccaacat agtgaaaccc tgtctctact

3401 aaaaatacaa aaattagctg ggcacagttg tgggcgcctg tagtcccagc

3451 tactcgggag gctgaggcag gagaattgct tgaatccagg aggtggaggt

3501 tgcagtgagg cgagatcggg ctactgcact ccaacctggg tgacaaagca

3551 agactgtgtc tcaaaaaaaa aaaaaaaaaa aaaacggaaa agaaaaaaaa

3601 aattagccaa atgtggtggt gcacgtcggt agtcccagct acttgggagg

3651 ctgaggtggg aggattgctt gagcccgcag gtcactgcac tccaacctgg

3701 gtgaaagagt gagaccctgt ctcaagaaaa aaaaaaaaaa aagcaatgga

3751 atggcctgaa cccagagcag tgactctttc ctgcctcagc ccctctgcac

3801 tgggcacctg ccacttacca gactgtcgca cacattctct cacctgattc

3851 tcacagcctc cccttgagct gggagctgta attatcctct tcgtgcaaac

3901 aggtgcagga gtgggtaaga gcccagctgc tgggctcatt ggctctgtgt

3951 ctgcggacaa ctgttctccc gtctgaaaat gaagatacta ggctgggcgc

4001 tgtggttcac gcctgtaatc ccagcacttt gggaggccga ggcgggtgga

4051 tcacctgagg ttgggagctg gagaccagcc tggcaaacat ggtgaaaccc

4101 tgtctctacc acaaatacaa aaattagccg ggcatggtgg tgcacgccta

4151 taatcccagc tactcaggag gctggggcag gaggatcact cgaacctggg

4201 aggcgcaggt tgcagtgagc tgagatcgtg ccactacact ccagcctggg

4251 caacagagtg agactccatc tgaaaaaaaa aaaagaaaaa aaagaaagaa

4301 aagaaaatga agatattact gtgccaatga tatgagtcag tatttgtaaa

4351 gtggttagaa cagttcctgg cagtttgtgc tttataaatg ttagttctaa 4401 tggggaaatg ggggcacaga gaggttatgt gacctgcccc aaagccacac

4451 agcttatgag cagggatttg ccaccaggtc agtctctgac tgccaagctg

4501 tgctctttct gttcccagag ctagcctgta cattgaaggt agacagcagt

4551 gtagcctctg cccagtggaa gcccctcaat ccacacggac ctcatctgtg

4601 gccttccatt tcttattcct tagaagttcc acctggtgcc aagcatcaac

4651 accatgagtg gcagtcagga gctgcagtgg atggtacagc ctcatttcct

4701 ggggcccagc agttacccca ggcctctgac ctaccctcag tacagccccc

4751 cacaaccccg gccaggagtc atccgggccc tggggccgcc tccaggggta

4801 cgtcgaaggc cttgtgaaca ggtaaggcac agaggcattg cagtggttcc

4851 gaccccgccc aggagtgcta gggtgcagag aagctcctgc cagaaaggag

4901 agcacaggct taggaggggg taaggtaggc tacaggtgac tactgcccaa

4951 ggctgaaagt gataggagcc ccacgggagg cccagagggt acaacagtca

5001 cttccagatc agggaaagct tcctggaaaa ggtggcatat tagcctagaa

5051 ggatgggtag gactggcaga ggcagagagg aaggaaagga cattccaggt

5101 agcgggggtg gcaggagcaa ggatggaggc ggaggcaaga acatgtaagg

5151 tgcgcacacg gaatggtgag ggggtctcca cctgctgaca tgcatagagg

5201 gggcgcttat gttgtctggg agagaacagg aacaagaggg ttcccttcag

5251 ctactccctc agtctcagtt tccctgtctc atttaatcct cacaaaatct

5301 ctctggggaa tgtactctgg ttttccccat cttccaccaa agaaatgaag

5351 gcacagaagg gtagtcactt gcccaagatc acacaagtag tgagtaatga

5401 cacaggactg gccttggccc caggtctata caactctaaa gtcagatcct

5451 aaccaccaaa ccagggccag gtgcagtagc tcatgcctgt aatcccagca

5501 ctttgagaga cctaggcagg aggatcactt gaggccagga gttcaagact

5551 aggctgggca acatagagag accctgtctc taccgaaaaa acaaaataaa

5601 ataaaacccc caaaactaat caccaagcca cgctgtctca ctggaagcta

5651 acagaaggct caaagtagag ggaactgaca attcttggct ccccaaagcc

5701 cttaccaaaa taagtgagta agagatggcg agtctttaaa ggagtggctc

5751 atctttcctc tccctggggc attttggtgt gggagactac aggggatgag

5801 gttaaaaagc ttggtcggca ggtagaggat ggggagagag gttagggccc

5851 tgggaaaggt gagagatcag ccagagacag gtttcccaga acagaatgtc 5901 tggcctttgt ggtgaggagg gactgtggta tgagccgcag aagcgggcca

5951 ggggtaaacc ctcctgtgcg tccttccttc agcctggtcc tgagggtgac

6001 cctttgatcc tgggttctcc aggtagggct gtgagctgtg agttggatcc

6051 ttttggtgaa atggtctctc tcatctggcc tgtcactcaa tgtggaatag

6101 agtgagtgag ttctatgggt tctaagtcct gctctggaac cataagtaag

6151 ttatcctctc tgggcttcag tttttcatgg aaagttgcgt taagaatcta

6201 gtttaaggcc aggcatggtg gctcacgcct gtaatcccag cactttggga

6251 ggccaaggaa ggtggatcat gaggtcagga gatcgagacc atcctggcta

6301 acatgatgaa acggtgtctc tactaaaaaa tacaaaaaat tagctgggtg

6351 tggtggaagg cacctgtagt cgcagctact cgggaggctg aggcaggaga

6401 atggcgtgaa ccccggaggc ggagcttgca gtgagccgag atggcaccac

6451 tgcactccag cctgggcaac agagtgagac tccgtctcaa aaaaaaaaaa

6501 aaaaaaagaa tctatgtttt tttaaaaaag aggccagctg tggtgactcc

6551 ccctggtaat ccaaacattc taggaggcca gtgcaggagg atcccttgag

6601 cccatgagtt ccagaccagc ctgggcaaca cagtgagacc ccgtcactac

6651 acaaaaaaaa aaaaggaaaa aaaaggccga gcacagtggc tcatgcctgt

6701 aatcccagca ctttgggagg ctgaggaggg tggatcacct gaggttaaga

6751 gttcaagacc agcctggcca acatggtgaa acctcgtctc tactaaaaat

6801 acaaaaatta gccaggtgtg gtggtacatg cctgtaatcc cagctacttg

6851 ggaggctgag gcacgagaat cacttgaacc cagtaggtgg aggttgcagt

6901 gagccaagat tgggccaatg cactccagcc tgggaaagag tgagactctg

6951 tctaaaaaaa ataaaataaa ataaaataaa atctttgcat gttctcagtg

7001 aaaaacaaga atctacgtaa agtacttggc tggcacatag tagttttcca

7051 ataaattgca acttaatcag cagcactcat tagcccctcc tagtgcttac

7101 agctcatgga agaggagact tggttgtcca cagatccaga aggatgaggg

7151 tcctgcaaag agggtgctga gaaggaaagg tcacttctgg ctggggaata

7201 agacggcttc ctggaggcag gagcatctta gttgagtcct gggagatgag

7251 gagggttagg gtttgcggag ggggagggca ttatgggaga ggaatgtgca

7301 ctaaagctag gtgcgaggag gaaattttac caatcaaagc agacagcgcc

7351 tcagtgcagg gaactggttg ggtgcagtga tccctaaatc accggctgag 7401 ggggccctgc catgacctct gccagggtcc taccactggg ccaccgtgag

7451 gggcgagttc caggcagcca gtagcccagg gtcctcctca ctgcgggtct

7501 cacccccaga tcagcccgga ggaagaggag cgccgccgag taaggcgcga

7551 gcggaacaag ctggctgcgg ccaagtgcag gaaccggagg aaggaactga

7601 ccgacttcct gcaggcggtg agcaccagcc ctggtccacc gagctcagag

7651 ggaccctgtc tcccagagcc cccagagccc cagtatatgt agccccctcc

7701 tcaccatcct agctccttgt ctgaggggca gtccttctgg gaaaatgatg

7751 aggaaaaatt gattagaaac tggctgcagc atgtctcact cacagcagag

7801 ctgaagctca gccagcaatc tccttttttt tttgagacag agtctcgctc

7851 tgtcacccag gctggagtgc agtgatgaga tctcggctca tggctcactg

7901 tgacatctgt ctcccaggct ccagcaattc ttatgcctca gcctgctgag

7951 tacctggggc tacaggcgcc tgcctcccta cccgactaat ttttgtattt

8001 ttagtagaga cggggtttcg ccaagttagc caggctggtc tcaaactcct

8051 ggtctcaagt gatccgcccg cctcagcctc ccaaagtgct gggattacag

8101 gccggagtca ctgcctggtg ccagcaatct cctaaaacgt ctgggcctga

8151 tgggagttgt gcccattttc tcccaggggc ttatgggagt tgtagtccag

8201 acttgctggg gactctccac tgccccccgc tctccccccg cccaacccca

8251 gctcctcaga accctgagtc caaacgtctg cggctctggg gtattcagcc

8301 ctcacctttc tgactcctct cctgatcttc tacaggagac tgacaaactg

8351 gaagatgaga aatctgggct gcagcgagag attgaggagc tgcagaagca

8401 gaaggagcgc ctagagctgg tgctggaagc ccaccgaccc atctgcaaaa

8451 tcccggaagg agccaaggag ggggacacag gcagtaccag tggcaccagc

8501 agcccaccag ccccctgccg ccctgtacct tgtatctccc tttccccagg

8551 gcctgtgctt gaacctgagg cactgcacac ccccacactc atgaccacac

8601 cctccctaac tcctttcacc cccagcctgg tcttcaccta ccccagcact

8651 cctgagcctt gtgcctcagc tcatcgcaag agtagcagca gcagcggaga

8701 cccatcctct gacccccttg gctctccaac cctcctcgct ttgtgaggcg

8751 cctgagccct actccctgca gatgccaccc tagccaatgt ctcctcccct

8801 tcccccaccg gtccagctgg cctggacagt atcccacatc caactccagc

8851 aacttcttct ccatccctct aatgagactg accatattgt gcttcacagt 8901 agagccagct tggggccacc aaagctgccc actgtttctc ttgagctggc

8951 ctctctagca caatttgcac taaatcagag acaaaatatt tcccatttgt

9001 gccagaggaa tcctggcagc ccagagactt tgtagatcct tagaggtcct

9051 ctggagccct aaccccttcc agatcactgc cacactctcc atcaccctct

9101 tcctgtgatc cacccaaccc tatctcctga cagaaggtgc cactttaccc

9151 acctagaaca ctaactcacc agccccactg ccagcagcag caggtgattg

9201 gaccaggcca ttctgccgcc ccctcctgaa ccgcacagct caggaggcgc

9251 ccttggcttc tgtgatgagc tgatctgcgg atctcagctc tgagaagcct

9301 tcagctccag ggaatccaag cctccacagc gagggcagct gctatttatt

9351 ttcctaaaga gagtattttt atacaaacct accaaaatgg aataaaaggc

9401 ttgaagctgt ggcctgagtg cctcactgga cccagaggcc aatgggagag

9451 tatttggagc cctaggtccc agccttagct ctacagactc actgcatgac

Claims

Claims

1 A method for determining the susceptibility of an individual to a disorder which is associated with an abnormal level of bone mineral density (BMD) , the method comprising use of a Fos related antigen-1 gene (Fra-1) marker.

2 A method as claimed in claim 1 wherein the disorder is associated with a low lower lumbar spine BMD.

3 A method as claimed in claim 2 wherein the disorder is osteoporosis .

4 A method as claimed in any one of the preceding claims wherein the method comprises

(i) obtaining a sample of nucleic acid from an individual, and (ii) assessing a polymorphic marker in the Fra-1 sequence of the nucleic acid to determine the susceptibility of that individual.

5 A method as claimed in claim 4 wherein the polymorphic marker is a single nucleotide polymorphism (SNP) and the identity of the nucleotide at the SNP is assessed.

6 A method as claimed in claim 4 or claim 5 wherein the nucleic acid is genomic DNA.

7 A method as claimed in claim 5 or claim 6 wherein the SNP is selected from the group consisting of the following positions numbered in accordance with the Fra-1 genomic sequence of Annex I: (i) 933 (SNP933),

(ii) 1301 (SNP1301),

(iii) 4755 (SNP4755) ,

(iv) 1948 (SNP1948),

(v) 2394 (SNP2394) , or a polymorphic marker which is in linkage disequilibrium with any of these. 8 A method as claimed in claim 7 wherein the identity of the nucleotide at the SNP is shown in Table 5.

9 A method as claimed in claim 8 wherein the SNP is SNP1301.

10 A method as claimed in claim 9 wherein an individual who is T/T homozygous for SNP 1301 is classified as being at the highest risk; an individual who is C/T heterozygous is classified as having moderate risk; an individual who is C/C homozygous is classified as having lowest risk, of susceptibility to a disorder which is associated with an abnormally low BMD.

11 A method as claimed in any one of claims 5 to 10 wherein two or more of said Fra-1 SNPs are assessed.

12 A method as claimed in any of claims 4 to 11 wherein the Fra-1 sequence in assessed by determining the binding of an oligonucleotide probe to the nucleic acid sample, wherein the probe comprises all or part of (i) the Fra-1 genomic sequence of Annex I, or (ii) a polymorphic form of the Fra-1 genomic sequence shown in Annex I, or (iii) the complement of either.

13 A method as claimed in claim 12 wherein the probe comprise a nucleic acid sequence which binds under stringent conditions specifically to one particular allele of the Fra-1 polymorphic marker and does not bind specifically to another alleles of the Fra- 1 polymorphic marker.

14 A method as claimed in claim 13 wherein the probe is labelled and binding of the probe is determined by presence of the label.

15 A method as claimed in any of claims 4 to 14 wherein the method comprises amplifying a region of the Fra-1 sequence comprising at least one polymorphic marker.

16 A method as claimed in claim 15 wherein a region of the Fra-1 sequence is amplified by use of two oligonucleotide primers. 17 A method as claimed in claim 16 wherein at least one of said primers binds under stringent conditions specifically to one particular allele of the Fra-1 polymorphic marker and does not bind specifically to another alleles of the Fra-1 polymorphic marker.

18 A method as claimed in claim 16 or claim 17 wherein at least one of said primers is a mutagenic primer which introduces a restriction site into said amplified region of the Fra-1 sequence.

19 A method as claimed in claim 18 wherein the mutagenic primer hybridises to SNP1301 numbered in accordance with the Fra-1 genomic sequence of Annex I and introduces a recognition site for the restriction enzyme EcoRI (G^VAATTC) where a C allele is present.

20 A method as claimed in any one of claim 16 to 18 wherein at least one of said primers is a primer shown in Table 3.

21 A method as claimed in any one of claims 4 to 11 wherein the Fra-1 sequence in assessed by a method selected from the group consisting of: strand conformation polymorphic marker analysis; heteroduplex analysis; RFLP analysis.

22 A method as claimed in any one of claims 4 to 21 wherein the polymorphic marker is assessed or confirmed by nucleotide sequencing,

23 A method as claimed in any one of the preceding claims wherein the individual is considered at risk from BMD-associated disorder.

24 A method as claimed in any one of the preceding claims for assessing the risk of osteoporotic fracture in an individual.

25 A method as claimed in claim 24 wherein non Fra-1 polymorphic markers which are linked or associated with a BMD-associated disorder are also assessed. 26 A method as claimed in claim 25 wherein the non Fra-1 polymorphic markers are polymorphic markers in the VDR gene and the C0LIA1 gene .

27 A method of determining the presence or absence in a test sample of a polymorphic marker in the Fra-1 sequence which is a single nucleotide polymorphism (SNP) selected from the group consisting of the following positions numbered in accordance with the Fra-1 genomic sequence of Annex I: (i) 933 (SNP933),

(ii) 1301 (SNP1301), (iii) 4755 (SNP4755) , (iv) 1948 (SNP1948), (v) 2394 (SNP2394) , which method comprises determining the binding of an oligonucleotide probe to the nucleic acid sample, wherein the probe comprises all or part of (i) the Fra-1 genomic sequence of Annex I, or; (ii) a polymorphic form of the Fra-1 genomic sequence shown in Annex I, or; (iii) the complement of either.

28 A method of determining the presence or absence in a test sample of a polymorphic marker in the Fra-1 sequence which is a single nucleotide polymorphism (SNP) selected from the group consisting of the following positions numbered in accordance with the Fra-1 genomic sequence of Annex I: (i) 933 (SNP933), (ii) 1301 (SNP1301), (iii) 4755 (SNP4755) , (iv) 1948 (SNP1948) , (v) 239 (SNP2394) , which method comprises use of two oligonucleotide primers capable of amplifying a portion of the Fra-1 sequence which portion comprises at least one of said SNPs.

29 A method for mapping polymorphic markers which are associated with a disorder which is associated with an abnormal level of bone mineral density (BMD) , the method comprising identifying polymorphic markers which are in linkage disequilibrium with an SNP which is selected from the group consisting of the following positions numbered in accordance with the Fra-1 genomic sequence of Annex I:

(i) 933 (SNP933), (ii) 1301 (SNP1301) ,

(iii) 4755 (SNP4755) ,

(iv) 1948 (SNP1948),

(v) 2394 (SNP2394) .

30 An oligonucleotide probe for use in a method of any one of claims 12 to 14 or claim 27.

31 An oligonucleotide probe as claimed in claim 30 which comprises a Fra-1 polymorphic marker which is a single nucleotide polymorphism (SNP) selected from the group consisting of the following positions numbered in accordance with the Fra-1 genomic sequence of Annex I:

(i) 933 (SNP933),

(ii) 1301 (SNP1301) ,

(iii) 4755 (SNP4755) , (iv) 1948 (SNP1948) ,

(v) 2394 (SNP2394) .

32 An oligonucleotide probe as claimed in claim 30 or claim 31 which comprises a label.

33 A PCR primer pair for use in a method of any one of claims 15 to 20 or claim 28 which primer pair comprises first and second primers which hybridise to DNA in regions or including flanking the Fra-1 polymorphic marker.

34 A PCR primer pair as claimed in claim 33 wherein the Fra-1 polymorphic marker is a single nucleotide polymorphism (SNP) selected from the group consisting of the following positions numbered in accordance with the Fra-1 genomic sequence of Annex I: (i) 933 (SNP933),

(ii) 1301 (SNP1301), (iii) 4755 (SNP4755) , ( iv) 1948 ( SNP1948 ) , (v) 2394 ( SNP2394 ) .

35 A PCR primer pair as claimed in claim 34 wherein at least one primer is selected from Table 3.

36 A kit comprising a probe and\or primer of any one of claims 30 to 35.

37 A method of osteoporosis therapy, which method includes the step of screening an individual for a genetic predisposition to osteoporosis in accordance with the method of any one of claims 24 to 26, whereby the predisposition is correlated with a Fra-1 polymorphic marker, and if a predisposition is identified, treating that individual to prevent or reduce the onset of osteoporosis.

38 A method as claimed in claim 37 wherein said treatment comprises hormone replacement therapy.