Kay Chapman

Identification of new susceptibility loci for osteoarthritis (arcOGEN): A genome-wide association study

Summary Background Osteoarthritis is the most common form of arthritis worldwide and is a major cause of pain and disability in elderly people. The health economic burden of osteoarthritis is increasing commensurate with obesity prevalence and longevity. Osteoarthritis has a strong genetic component but the success of previous genetic studies has been restricted due to insufficient sample sizes and phenotype heterogeneity. Methods We undertook a large genome-wide association study (GWAS) in 7410 unrelated and retrospectively and prospectively selected patients with severe osteoarthritis in the arcOGEN study, 80% of whom had undergone total joint replacement, and 11 009 unrelated controls from the UK. We replicated the most promising signals in an independent set of up to 7473 cases and 42 938 controls, from studies in Iceland, Estonia, the Netherlands, and the UK. All patients and controls were of European descent. Findings We identified five genome-wide significant loci (binomial test p≤5·0×10−8) for association with osteoarthritis and three loci just below this threshold. The strongest association was on chromosome 3 with rs6976 (odds ratio 1·12 [95% CI 1·08–1·16]; p=7·24×10−11), which is in perfect linkage disequilibrium with rs11177. This SNP encodes a missense polymorphism within the nucleostemin-encoding gene GNL3. Levels of nucleostemin were raised in chondrocytes from patients with osteoarthritis in functional studies. Other significant loci were on chromosome 9 close to ASTN2, chromosome 6 between FILIP1 and SENP6, chromosome 12 close to KLHDC5 and PTHLH, and in another region of chromosome 12 close to CHST11. One of the signals close to genome-wide significance was within the FTO gene, which is involved in regulation of bodyweight—a strong risk factor for osteoarthritis. All risk variants were common in frequency and exerted small effects. Interpretation Our findings provide insight into the genetics of arthritis and identify new pathways that might be amenable to future therapeutic intervention. Funding arcOGEN was funded by a special purpose grant from Arthritis Research UK.

Mutations in the region encoding the von Willebrand factor A domain of matrilin-3 are associated with multiple epiphyseal dysplasia

Multiple epiphyseal dysplasia (MED) is a relatively mild and clinically variable osteochondrodysplasia, primarily characterized by delayed and irregular ossification of the epiphyses and early-onset osteoarthritis. Mutations in the genes encoding cartilage oligomeric matrix protein (COMP) and type IX collagen (COL9A2 and COL9A3) have previously been shown to cause different forms of MED (refs. 4–13). These dominant forms of MED (EDM1–3) are caused by mutations in the genes encoding structural proteins of the cartilage extracellular matrix (ECM); these proteins interact with high affinity in vitro. A recessive form of MED (EDM4) has also been reported; it is caused by a mutation in the diastrophic dysplasia sulfate transporter gene (SLC26A). A genomewide screen of family with autosomal-dominant MED not linked to the EDM1–3 genes provides significant genetic evidence for a MED locus on the short arm of chromosome 2 (2p24–p23), and a search for candidate genes identified MATN3 (ref. 18), encoding matrilin-3, within the critical region. Matrilin-3 is an oligomeric protein that is present in the cartilage ECM. We have identified two different missense mutations in the exon encoding the von Willebrand factor A (vWFA) domain of matrilin-3 in two unrelated families with MED (EDM5). These are the first mutations to be identified in any of the genes encoding the matrilin family of proteins and confirm a role for matrilin-3 in the development and homeostasis of cartilage and bone.

Large‐scale analysis of association between GDF5 and FRZB variants and osteoarthritis of the hip, knee, and hand

OBJECTIVE GDF5 and FRZB have been proposed as genetic loci conferring susceptibility to osteoarthritis (OA); however, the results of several studies investigating the association of OA with the rs143383 polymorphism of the GDF5 gene or the rs7775 and rs288326 polymorphisms of the FRZB gene have been conflicting or inconclusive. To examine these associations, we performed a large-scale meta-analysis of individual-level data. METHODS Fourteen teams contributed data on polymorphisms and knee, hip, and hand OA. For rs143383, the total number of cases and controls, respectively, was 5,789 and 7,850 for hip OA, 5,085 and 8,135 for knee OA, and 4,040 and 4,792 for hand OA. For rs7775, the respective sample sizes were 4,352 and 10,843 for hip OA, 3,545 and 6,085 for knee OA, and 4,010 and 5,151 for hand OA, and for rs288326, they were 4,346 and 8,034 for hip OA, 3,595 and 6,106 for knee OA, and 3,982 and 5,152 for hand OA. For each individual study, sex-specific odds ratios (ORs) were calculated for each OA phenotype that had been investigated. The ORs for each phenotype were synthesized using both fixed-effects and random-effects models for allele-based effects, and also for haplotype effects for FRZB. RESULTS A significant random-effects summary OR for knee OA was demonstrated for rs143383 (1.15 [95% confidence interval 1.09-1.22]) (P=9.4x10(-7)), with no significant between-study heterogeneity. Estimates of effect sizes for hip and hand OA were similar, but a large between-study heterogeneity was observed, and statistical significance was borderline (for OA of the hip [P=0.016]) or absent (for OA of the hand [P=0.19]). Analyses for FRZB polymorphisms and haplotypes did not reveal any statistically significant signals, except for a borderline association of rs288326 with hip OA (P=0.019). CONCLUSION Evidence of an association between the GDF5 rs143383 polymorphism and OA is substantially strong, but the genetic effects are consistent across different populations only for knee OA. Findings of this collaborative analysis do not support the notion that FRZB rs7775 or rs288326 has any sizable genetic effect on OA phenotypes.

Osteoarthritis-Susceptibility Locus on Chromosome 11q, Detected by Linkage

We present a two-stage genomewide scan for osteoarthritis-susceptibility loci, using 481 families that each contain at least one affected sibling pair. The first stage, with 272 microsatellite markers and 297 families, involved a sparse map covering 23 chromosomes at intervals of approximately 15 cM. Sixteen markers that showed evidence of linkage at nominal P</=.05 were then taken through to the second stage, with an additional 184 families. This second stage confirmed evidence of linkage for markers on chromosome 11q. Additional markers from this region were then typed to create a denser map. We obtained a maximum single-point LOD score, at D11S901, of 2.40 (P=.0004) and a maximum multipoint-LOD score of 3.15, between markers D11S1358 and D11S35. A subset of 196 of the 481 families, comprising affected female sibling pairs, generated a corrected LOD score of 2.54 (P=.0003) for marker D11S901, with evidence for linkage extending 12 cM proximal to this marker. When we stratified for affected male sibling pairs there was no evidence of linkage to chromosome 11. Our data suggest that a female-specific susceptibility gene for idiopathic osteoarthritis is located on chromosome 11q.

Meta-analysis of genome-wide association studies confirms a susceptibility locus for knee osteoarthritis on chromosome 7q22

Objectives Osteoarthritis (OA) is the most prevalent form of arthritis and accounts for substantial morbidity and disability, particularly in older people. It is characterised by changes in joint structure, including degeneration of the articular cartilage, and its aetiology is multifactorial with a strong postulated genetic component. Methods A meta-analysis was performed of four genome-wide association (GWA) studies of 2371 cases of knee OA and 35 909 controls in Caucasian populations. Replication of the top hits was attempted with data from 10 additional replication datasets. Results With a cumulative sample size of 6709 cases and 44 439 controls, one genome-wide significant locus was identified on chromosome 7q22 for knee OA (rs4730250, p=9.2×10−9), thereby confirming its role as a susceptibility locus for OA. Conclusion The associated signal is located within a large (500 kb) linkage disequilibrium block that contains six genes: PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, β), HPB1 (HMG-box transcription factor 1), COG5 (component of oligomeric golgi complex 5), GPR22 (G protein-coupled receptor 22), DUS4L (dihydrouridine synthase 4-like) and BCAP29 (B cell receptor-associated protein 29). Gene expression analyses of the (six) genes in primary cells derived from different joint tissues confirmed expression of all the genes in the joint environment.

Insights into the genetic architecture of osteoarthritis from stage 1 of the arcOGEN study

Objectives The genetic aetiology of osteoarthritis has not yet been elucidated. To enable a well-powered genome-wide association study (GWAS) for osteoarthritis, the authors have formed the arcOGEN Consortium, a UK-wide collaborative effort aiming to scan genome-wide over 7500 osteoarthritis cases in a two-stage genome-wide association scan. Here the authors report the findings of the stage 1 interim analysis. Methods The authors have performed a genome-wide association scan for knee and hip osteoarthritis in 3177 cases and 4894 population-based controls from the UK. Replication of promising signals was carried out in silico in five further scans (44 449 individuals), and de novo in 14 534 independent samples, all of European descent. Results None of the association signals the authors identified reach genome-wide levels of statistical significance, therefore stressing the need for corroboration in sample sets of a larger size. Application of analytical approaches to examine the allelic architecture of disease to the stage 1 genome-wide association scan data suggests that osteoarthritis is a highly polygenic disease with multiple risk variants conferring small effects. Conclusions Identifying loci conferring susceptibility to osteoarthritis will require large-scale sample sizes and well-defined phenotypes to minimise heterogeneity.

Assessment of Osteoarthritis Candidate Genes in a Meta-Analysis of Nine Genome-Wide Association Studies

To assess candidate genes for association with osteoarthritis (OA) and identify promising genetic factors and, secondarily, to assess the candidate gene approach in OA.

A meta-analysis of genome-wide association studies identifies novel variants associated with osteoarthritis of the hip

Objectives Osteoarthritis (OA) is the most common form of arthritis with a clear genetic component. To identify novel loci associated with hip OA we performed a meta-analysis of genome-wide association studies (GWAS) on European subjects. Methods We performed a two-stage meta-analysis on more than 78 000 participants. In stage 1, we synthesised data from eight GWAS whereas data from 10 centres were used for ‘in silico’ or ‘de novo’ replication. Besides the main analysis, a stratified by sex analysis was performed to detect possible sex-specific signals. Meta-analysis was performed using inverse-variance fixed effects models. A random effects approach was also used. Results We accumulated 11 277 cases of radiographic and symptomatic hip OA. We prioritised eight single nucleotide polymorphism (SNPs) for follow-up in the discovery stage (4349 OA cases); five from the combined analysis, two male specific and one female specific. One locus, at 20q13, represented by rs6094710 (minor allele frequency (MAF) 4%) near the NCOA3 (nuclear receptor coactivator 3) gene, reached genome-wide significance level with p=7.9×10−9 and OR=1.28 (95% CI 1.18 to 1.39) in the combined analysis of discovery (p=5.6×10−8) and follow-up studies (p=7.3×10−4). We showed that this gene is expressed in articular cartilage and its expression was significantly reduced in OA-affected cartilage. Moreover, two loci remained suggestive associated; rs5009270 at 7q31 (MAF 30%, p=9.9×10−7, OR=1.10) and rs3757837 at 7p13 (MAF 6%, p=2.2×10−6, OR=1.27 in male specific analysis). Conclusions Novel genetic loci for hip OA were found in this meta-analysis of GWAS.

Stratification Analysis of an Osteoarthritis Genome Screen—Suggestive Linkage to Chromosomes 4, 6, and 16

To the Editor: We have previously carried out a two-stage genomewide linkage screen for osteoarthritis (MIM 165720) susceptibility loci, using an affected-sibling-pair approach (Chapman et al. 1999). In stage 1 of this screen, we tested 272 microsatellite markers in 297 families, each of which contained at least one pair of siblings who had undergone hip-, knee-, or hip and knee–replacement surgery for primary osteoarthritis. Loci that demonstrated evidence for linkage at nominal P=.05 were then taken through to stage 2, in which they were tested against a further 184 families. Sixteen markers within nine genomic regions from stage 1 had evidence of linkage, at P=.05. When the data for stages 1 and 2 were combined, the P value decreased for 3 of the 16 loci (D2S202, D11S907, and D11S903) and was constant for a 4th (D11S901). We subsequently concentrated our analysis on the chromosome regions to which these markers map. To test these linkages further, we genotyped additional markers and obtained maximum multipoint LOD scores (MLSs) of 1.2 for chromosome 2 and 3.1 for chromosome 11. Because there is evidence, from epidemiological, twin, and segregation studies, that the genetic contribution to osteoarthritis differs between the sexes and between different joint groups (Lindberg 1986; Cooper et al. 1994; Kaprio et al. 1996; Chitnavis et al. 1997; Felson et al. 1998), we stratified our chromosomes 2 and 11 linkage data according to sex and site of osteoarthritis (hip or knee). This stratification indicated that the suggestion of linkage to chromosome 2 was principally accounted for by affected sibling pairs with hip osteoarthritis (MLS 2.2), whereas the suggestion of linkage to chromosome 11 was restricted to affected female pairs (MLS 2.8). Because this analysis highlighted substantial differences between the strata tested, we have now reanalyzed stage 1 of our genome screen, for the remaining 20 autosomes, to determine whether any regions harbor susceptibility loci that are obscured in the unstratified data set. We stratified our stage 1 data into the same six strata tested in our analysis of chromosomes 2 and 11: affected females only (132 families), affected males only (60 families), hips only (194 families), knees only (34 families), female hip (85 families), and male hip (44 families). (A more detailed breakdown of these families can be found in the study by Chapman et al. [1999].) We did not stratify for female knee or male knee, because the number of families was too small (16 and 4, respectively) to allow reliable inference of linkage. Multipoint linkage analysis was performed on the stratified data by means of the ASPEX program. Ten of the 20 autosomes have one or more multipoint peaks with uncorrected MLS⩾1.0 for one or more of the six strata tested (table 1). The highest MLS is 3.9, for chromosome 4q in the female-hip strata, followed by 2.9, for chromosome 6 in the hip-only strata, and 2.1, for chromosome 16 in the female-hip strata. When we adjust MLS values to correct for the seven models tested (one unstratified analysis and six stratified analyses), by deducting from the original values (Kidd and Ott 1984), chromosome 4 has an MLS value of 3.1, chromosome 6 has an MLS value of 2.1, and chromosome 16 has an MLS value of 1.3. The uncorrected multipoint plots of these three chromosomes are shown in figure 1. Figure 1 Multipoint analysis. A, Chromosome 4, female hip (n=85 families) and female only (n=132 families). B, Chromosome 6, hip only (n=194 families). C, Chromosome 16, female hip (n=85 families) and female only (n=132 families). Table 1 Stratified MLSs The suggestion of linkage on chromosome 4 is centered on 4q12–4q21.2 and is restricted to female pairs with hip disease. Roby et al. (1999) have recently reported linkage of chromosome 4q to severe early-onset hip osteoarthritis in a large pedigree of Dutch origin. This locus maps to the telomeric end of 4q (4q35), placing it >50 cM distal to the linkage that we have observed. It is therefore unlikely that the two linkages have detected the same locus. More than 50 cM of chromosome 6 has an uncorrected MLS⩾2.0 in the hip-only stratum, between markers D6S257 and D6S262. This region of chromosome 6 contains a strong candidate gene for osteoarthritis, COL9A1 (6q12–6q13). This gene maps within the 11-cM interval between D6S257 and D6S286 and encodes the α1 chain of type IX collagen. This collagen is a quantitatively minor cartilage collagen that decorates the type II collagen fibril and that interacts with extrafibrillar macromolecules (Ayad et al. 1994). Two transgenic mouse models have demonstrated that mutations in the equivalent mouse gene can result in an osteoarthritis phenotype. In the first model, a truncated form of the gene resulted in mice with a mild osteochondrodysplasia phenotype and secondary osteoarthritis (Nakata et al. 1993). In the second model, a knockout mouse had no congenital abnormality but developed a severe osteoarthritis that was comparable, in timing and pathology, to human primary osteoarthritis (Fassler et al. 1994). A more detailed analysis of this second model revealed that the synthesis of the α1 polypeptide chain was necessary for type IX collagen assembly (Hagg et al. 1997). Chromosome 16 does not contain any known genes that can be considered as strong candidates for osteoarthritis susceptibility. As more genes are mapped, candidate loci on this chromosome may become apparent. Overall, the stratification of our genome screen has revealed additional chromosomal regions that may harbor susceptibility loci for osteoarthritis. Stratification increases the level of genetic homogeneity and can therefore assist in the mapping of loci for complex traits. Our analysis highlights the potential utility of this approach for osteoarthritis.

Finer linkage mapping of a primary hip osteoarthritis susceptibility locus on chromosome 6

Primary osteoarthritis (OA) is a common late-onset disease that exhibits complex genetic transmittance. A previous genome-wide linkage scan of OA affected sibling pair families (ascertained by total joint replacement surgery) identified a region of suggestive linkage on chromosome 6, with a maximum multipoint-LOD score (MLS) of 2.9 in 194 families containing sibling pairs concordant for total hip replacement (THR-families). However, up to 50 cM of the chromosome had a multipoint-LOD score >2.0, vvindicating that the susceptibility locus was poorly mapped. We have now genotyped chromosome 6 to a higher density in an expanded cohort of 378 THR-families. We obtained an MLS of 2.8 to an 11.4 cM interval defined by markers D6S452 and 509-8B2, which map between 70.5 to 81.9 cM from the 6p-telomere. Stratification by gender revealed that this linkage was completely accounted for by female THR-families (n=146), with an MLS of 4.0 and with the highest two-point LOD score being 4.6 for marker D6S1573 (75.9 cM). The 11.4 cM interval just encompasses the candidate gene COL9A1 (81.9 cM). We identified and then genotyped twenty common single nucleotide polymorphisms (SNPs) from within COL9A1 in the 146 probands from our female THR-families and in 215 age-matched female controls. No SNP allele, genotype or haplotype demonstrated association to disease. Overall, we have narrowed the chromosome 6 OA susceptibility locus to a point at which linkage disequilibrium/association analysis is feasible, we have demonstrated that this locus is female specific, and found no evidence that COL9A1 encodes for the susceptibility.