Association of a BMP 5 microsatellite with knee osteoarthritis : case-control study
Introduction: We aimed to explore the involvement of a multiallelic functional polymorphism in knee osteoarthritis (OA) susceptibility as a prototype of possible genetic factors escaping GWAS detection. Methods: OA patients and controls from three European populations (Greece, Spain and the UK) adding up to 1003 patients (716 women, 287 men) that had undergone total knee joint replacement (TKR) due to severe primary OA and 1543 controls (758 women, 785 men) lacking clinical signs or symptoms of OA were genotyped for the D6S1276 microsatellite in intron 1 of BMP5. Genotype and mutiallelic trend tests were used to compare cases and controls. Results: Significant association was found between the microsatellite and knee OA in women (P from 3.1 x10 to 4.1 x10 depending on the test), but not in men. Three of the alleles showed significant differences between patients and controls, one of them of increased risk and two of protection. The gender association and the allele direction of change were very concordant with those previously reported for hip OA. Conclusions: We have found association of knee OA in women with the D6S1276 functional microsatellite that modifies in cis the expression of BMP5 making this a sounder OA genetic factor and extending its involvement to other joints. This result also shows the interest of analysing other multiallelic polymorphisms. Introduction Investigation of the genetic component of osteoarthritis (OA) susceptibility has yielded the identification of several loci achieving genome-wide significance or consistent replication [1-3]. These loci are insufficient to account for the heritability of OA, which has been estimated as 49 to 78% for knee OA in twin and family studies [4-6]. Multiple possible causes of this discrepancy, which has become known as the missing heritability problem of complex diseases, have been discussed [7]. Some have a particular importance in OA, like the difficulties in discriminating between cases and controls, the variability in phenotype definitions and the relatively small size of genome wide association studies (GWAS) in which OA has been studied in comparison with other diseases [3,8]. Other causes are shared by most complex diseases. They include genetic variants that are not well ascertained with the current GWAS and interactions between genetic variants, and between genetic and environmental factors that are beyond the analysis and design of current studies. Among the variants escaping GWAS detection are rare-frequency variants, even if they have a large effect, and variants with multiple alleles. The latter group is made of large structural variants and small repetitive sequences. They could escape GWAS detection because their multiple alleles cannot have sufficient linkage disequilibrium (LD) with bi-allelic markers as the single nucleotide polymorphisms (SNPs) analyzed in GWAS. One of these multi-allelic polymorphisms, a variable number tandem repeat (VNTR) polymorphism in the gene for asporin, has shown consistent association with OA in Asians and less so in Europeans [9,10]. Other multi-allelic polymorphisms have also been found associated with OA, but none of these studies has been followed by an attempt of replication [11-14]. One of the OA-associated multi-allelic polymorphisms is an intronic microsatellite in BMP5 (bone morphogenetic * Correspondence: antonio.gonzalez.martinez-pedrayo@sergas.es Laboratorio Investigacion 10 & Rheumatology, Instituto de Investigacion Sanitaria Hospital Clinico Universitario de Santiago, Choupana s/n 15706, Santiago de Compostela, Spain Full list of author information is available at the end of the article Rodriguez-Fontenla et al. Arthritis Research & Therapy 2012, 14:R257 http://arthritis-research.com/content/14/6/R257 © 2012 Rodriguez-Fontenla et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. protein 5). It has been associated with hip OA in UK women and its allelic variants showed a regulatory effect on the BMP5 promoter in vitro [11]. BMP5 is a good candidate gene for OA because BMPs are members of the transforming growth factor (TGF)-beta superfamily that were identified by their involvement in cartilage and bone development. Now, they are known to give morphogenetic signals directing tissue organization throughout the body. In particular, BMP5 is implicated in bone morphogenesis and in the formation of the skeletal pattern, in addition to having roles in other organs [15]. It is expressed in proliferating zone chondrocytes of the growth plate and is very markedly increased by hypertrophic differentiation. This is in contrast with other BMPs [16]. In chondrocytes, upon binding to the BMP membrane receptors, BMP5 leads to phosphorylation of p38 MAP kinase, ERK and SMAD-1,-5, and -8. Nuclear accumulation of phosphor-SMADs leads to over-expression of genes involved in cartilage homeostasis as proteoglycan and of markers of hypertrophic differentiation [17]. At a cellular level, this translates into an increase of chondrocyte proliferation and in the synthesis of cartilage matrix [16]. In addition, BMP5 expression is decreased in OA synovia [18] and bone [19]. These changes in expression together with the BMP5 chondrogenic role and involvement in chondrocyte hypertrophy indicate BMP5 relevance for OA pathogenesis. We have genotyped the D6S1276 BMP5 microsatellite in a large set of knee OA cases and disease-free controls and found an association with knee OA in women. The associated alleles and the gender specificity are similar to those previously found in UK women with hip OA. This finding reinforces the need to explore the involvement of BMP5 genetic variation in OA and extends it to OA in other joints. It also shows that other multi-allelic variants are worthy of examination for their possible involvement in OA. Materials and methods Patients and controls OA patients and controls were recruited at Thessaly in Greece, Santiago de Compostela in Spain and Oxford in the UK. All were European Caucasians. There were 1,003 patients (716 women, 287 men) who had undergone total knee joint replacement (TKR) due to severe primary OA. The 1,543 controls (758 women, 785 men) lacked clinical signs of OA. Full details about the patients and the controls have been reported [20]. In the Greek collection, there were 369 TKR patients (298 women and 71 men) and 383 controls (241 women and 142 men). TKR patients had a Kellgren-Lawrence (K/L) grade > 2 prior to surgery and an age range of 40 to 90 years. Rheumatoid arthritis, other autoimmune diseases, chondrodysplasias, infection-induced OA, and post-traumatic OA were excluded. The controls had a K/L grade of 0 and had undergone treatment for injuries and fractures. They were from 46 to 88 years old. In the Spanish collection, there were 274 TKR patients (222 women and 52 men) and 462 controls (161 women and 301 men). Patients were selected as consecutive patients, of 55-80 years of age at the time of surgery, and undergoing TKR if a rheumatologist considered them to suffer from primary OA. Exclusion criteria were inflammatory, infectious, or traumatic joint pathology and lesions due to crystal deposition or osteonecrosis. Controls were older than 55 years of age and were selected during preoperative work-up for elective surgery other than joint surgery and without symptoms or signs of OA. The UK collection included 360 TKR patients (196 women and 164 men) of 56 to 85 years of age and 698 controls (356 women and 342 men) of 55 to 89 years of age. Patients showed symptoms and signs of OA of sufficient severity to require surgery, including a K/L grade ≥ 2 (> 90% of them had grade 3 or 4). Inflammatory arthritis (rheumatoid, polyarthritic or autoimmune disease) was excluded, as was post-traumatic or post-septic arthritis. Controls had no signs or symptoms of arthritis or joint disease (pain, swelling, tenderness or restriction of movement). All patients and controls gave their written informed consent and the use of their DNA for OA genetic studies was approved by the respective ethics committees.