Genetic association analysis of BMP5 as a potential osteoarthritis susceptibility gene

L. Southam, K. Chapman and J. Loughlin

University of Oxford, Institute of Musculoskeletal Sciences, Botnar Research Centre, Oxford, UK

SIR, A number of twin-pair and sibling risk studies have revealed a major genetic component to primary osteoarthritis (OA), which best fits into the oligogenic multifactorial class of human diseases [1]. We have linkage mapped an OA susceptibility locus to an 11.4 cM interval at chromosome 6p12.3-q13 in a cohort of 146 affected female sibling pair families ascertained by total hip replacement (female-THR families) for primary OA, with a maximum multipoint LOD score of 4.0 [2]. This interval encompasses the candidate gene BMP5 (6p12.1), which encodes for bone morphogenetic protein 5. BMPs are bone-derived factors that can induce new bone formation. BMP1 is a protease involved in the maturation of fibrillar collagens, whilst the remaining BMPs, including BMP5, are secreted molecules belonging to the transforming growth factor-ß family of growth and differentiation factors [3]. The normal development and repair of the synovial joint is influenced by the activity of BMPs, so it is reasonable to speculate that variation in the activity or action of these molecules could influence the development of arthritic phenotypes [4, 5]. Using genetic association analysis, we have tested BMP5 as the chromosome 6 OA susceptibility gene.

The association analysis was performed on a case–control cohort. The cases were the female probands from the 146 female-THR families used in the linkage analysis. Detailed information regarding these families has been reported elsewhere [2]. Their primary OA status was supported by clinical, radiological, operative and histological findings. The control cohort comprised 215 age-matched, unrelated females.

The controls had not undergone joint-replacement surgery or required any clinical treatment for OA. The average age of the controls when recruited into the study was 73 yr. All patients and all controls were of UK Caucasian origin. Ethical approval for the study was obtained from the Central Oxford Research Ethics Committee, and informed consent was obtained from all subjects.

The BMP5 gene sequence was accessed through the Ensembl genome browser (http://www.ensembl.org/). In 48 of the female patients, 1100 bp upstream of the 5' UTR, the 5' UTR, all seven exons (including acceptor and donor splice sites), the 3' UTR and 2 kb downstream of the translation termination codon were scanned by direct DNA sequencing. This scan identified seven common single nucleotide polymorphisms (SNPs) with minor allele frequencies >5%, a common indel dimorphism comprising the insertion/deletion of the tetranucleotide TTTG, and two [GT]n microsatellite polymorphisms (Table 1Go). Two of the seven SNPs were exonic and synonymous. Of the two [GT]n microsatellites, one was located in intron 5 whilst the second was located downstream of BMP5 and was dimorphic, with alleles composed of either 10 or 11 copies of the [GT] dinucleotide. The SNPs and the indel dimorphism were genotyped by polymerase chain reaction (PCR)-restriction enzyme analysis whilst the [GT]2 microsatellite dimorphism was amplified by PCR using fluorescently labelled primers, as described previously [2]. We did not complete the genotyping of SNP exon 1 (+111) as it was found to be in complete linkage disequilibrium with SNP upstream (-1407), and we were unable to genotype the intron 5 microsatellite owing to a run of 39 [T] residues preceding the [GT] repeat. Further information regarding the genotyping conditions can be obtained from the authors.


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TABLE 1. BMP5 sequence variants detected by direct DNA sequencing

 
All variants were in Hardy–Weinberg equilibrium in the control cohort. Association with OA was evaluated for each variant by {chi}2 using standard contingency table analysis. None of the variant alleles were associated at P ≤ 0.05 (Table 1Go), nor were any of the variant genotypes (data not shown). We subsequently stratified our families using their individual non-parametric linkage (NPL) scores taken from the Genehunter two-point linkage analysis (http://waldo.wi.mit.edu/ftp/distribution/software/genehunter/gh2/). This analysis was performed against marker D6S1573, which is located <2 Mb from BMP5 and which gave the highest two-point LOD score of 4.6 in the linkage analysis [2]. Seventy six of the 146 female-THR families showed a positive contribution to the linkage at D6S1573 (positive NPL scores). When the variant allele and genotype frequencies were compared between the probands from these 76 families and the controls, none showed a significant frequency difference (data not shown). Finally, haplotype frequencies amongst SNPs were determined using the estimate haplotype program EH-PLUS (http://www.iop.kcl.ac.uk/IoP/Departments/PsychMed/GEpiBST/software.stm) and the haplotype frequency differences between the probands and the controls were then compared using {chi}2 and contingency table analysis. Again, no frequency differences were observed (data not shown).

The identification of the genes encoding for susceptibility to a complex trait is an arduous exercise. However, there have recently been a number of encouraging breakthroughs [6]. From an association viewpoint, two broad strategies can be adopted: (i) the targeted study of candidate genes and (ii) a systematic analysis. Choosing candidates is inevitably prone to bias. However, for most diseases it is possible to identify plausible candidates. We took that approach with BMP5, but have found no evidence to support this gene as encoding for the primary OA susceptibility that we have linkage mapped to chromosome 6. It is possible that the susceptibility is mediated through BMP5 but is encoded within upstream or downstream cis controlling elements that regulate its expression. Such regulatory sequences are positioned several hundred kilobases from the mouse Bmp5 gene [7]. Our specific analysis of variants within or very close to BMP5 would have failed to account for any variation at equivalent regulatory sequences in the human gene.

We had previously found no evidence of association of the type IX collagen gene COL9A1 (6q12-q13) to primary OA [2]. We can therefore provisionally exclude two strong candidate genes (COL9A1 and BMP5) from within our chromosome 6 linkage interval. This suggests to us that the nature of OA genetic susceptibility is not predictable and that a more fruitful strategy for identifying OA susceptibility genes from within defined intervals may be the systematic analysis approach.

Notes

Correspondence to: J. Loughlin, University of Oxford, Institute of Musculoskeletal Sciences, Botnar Research Centre, Nuffield Orthopaedic Centre, Windmill Road, Oxford OX3 7LD, UK. E-mail: john.loughlin{at}ndcls.ox.ac.uk Back

References

  1. Loughlin J. Genetic epidemiology of primary osteoarthritis. Curr Opin Rheumatol 2001;13:111–6.[CrossRef][ISI][Medline]
  2. Loughlin J, Mustafa Z, Dowling B et al. Finer linkage mapping of a primary hip osteoarthritis susceptibility locus on chromosome 6. Eur J Hum Genet 2002;10:562–8.[CrossRef][ISI][Medline]
  3. Ducy P, Karsenty G. The family of bone morphogenetic proteins. Kidney Int 2000;57:2207–14.[CrossRef][ISI][Medline]
  4. Edwards CJ, Francis-West PH. Bone morphogenetic proteins in the development and healing of synovial joints. Semin Arthritis Rheum 2001;31:33–42.[CrossRef][ISI][Medline]
  5. Reddi AH. Interplay between bone morphogenetic proteins and cognate binding proteins in bone and cartilage development: noggin, chordin and DAN. Arthritis Res 2001;3:1–5.[CrossRef][ISI][Medline]
  6. Hugot J-P, Chamaillard M, Zouali H et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature 2001;411:599–603.[CrossRef][ISI][Medline]
  7. DiLeone RJ, Russell LB, Kingsley DM. An extensive 3' regulatory region controls expression of Bmp5 in specific anatomical structures of the mouse embryo. Genetics 1998;148:401–8.[Abstract/Free Full Text]
Accepted 28 November 2002





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