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 casecontrol 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 1). 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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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
References
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