Allelic expression analysis of the osteoarthritis susceptibility locus that maps to chromosome 3p21 reveals cis-acting eQTLs at GNL3 and SPCS1.

BACKGROUND: An osteoarthritis (OA) susceptibility locus has been mapped to chromosome 3p21, to a region of high linkage disequilibrium encompassing twelve genes. Six of these genes are expressed in joint tissues and we therefore assessed whether any of the six were subject to cis-acting regulatory polymorphisms active in these tissues and which could therefore account for the association signal. METHODS: We measured allelic expression using pyrosequencing assays that can distinguish mRNA output from each allele of a transcript single nucleotide polymorphism. We assessed RNA extracted from the cartilage and other joint tissues of OA patients who had undergone elective joint replacement surgery. A two-tailed Mann-Whitney exact test was used to test the significance of any allelic differences. RESULTS: GNL3 and SPCS1 demonstrated significant allelic expression imbalance (AEI) in OA cartilage (GNL3, mean AEI = 1.04, p = 0.0002; SPCS1, mean AEI = 1.07, p < 0.0001). Similar results were observed in other tissues. Expression of the OA-associated allele was lower than that of the non-associated allele for both genes. CONCLUSIONS: cis-acting regulatory polymorphisms acting on GNL3 and SPCS1 contribute to the OA association signal at chromosome 3p21, and these genes therefore merit further investigation.

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

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.

Allelic expression analysis of the osteoarthritis susceptibility gene COL11A1 in human joint tissues.

BACKGROUND: The single nucleotide polymorphism (SNP) rs2615977 is associated with osteoarthritis (OA) and is located in intron 31 of COL11A1, a strong candidate gene for this degenerative musculoskeletal disease. Furthermore, the common non-synonymous COL11A1 SNP rs1676486 is associated with another degenerative musculoskeletal disease, lumbar disc herniation (LDH). rs1676486 is a C-T transition mediating its affect on LDH susceptibility by modulating COL11A1 expression. The risk T-allele of rs1676486 leads to reduced expression of the COL11A1 transcript, a phenomenon known as allelic expression imbalance (AEI). We were keen therefore to assess whether the effect that rs1676486 has on COL11A1 expression in LDH is also observed in OA and whether the rs2615977 association to OA also marked AEI. METHODS: Using RNA from OA cartilage, we assessed whether either SNP correlated with COL11A1 AEI by 1) measuring COL11A1 expression and stratifying the data by genotype at each SNP; and 2) quantifying the mRNA transcribed from each allele of the two SNPs. We also assessed whether rs1676486 was associated with OA susceptibility using a case-control cohort of over 18,000 individuals. RESULTS: We observed significant AEI at rs1676486 (p < 0.0001) with the T-allele correlating with reduced COL11A1 expression. This corresponded with observations in LDH but the SNP was not associated with OA. We did not observe AEI at rs2615977. CONCLUSIONS: COL11A1 is subject to AEI in OA cartilage. AEI at rs1676486 is a risk factor for LDH, but not for OA. These two diseases therefore share a common functional phenotype, namely AEI of COL11A1, but this appears to be a disease risk only in LDH. Other functional effects on COL11A1 presumably account for the OA susceptibility that maps to this gene.

Increased type II deiodinase protein in OA-affected cartilage and allelic imbalance of OA risk polymorphism rs225014 at DIO2 in human OA joint tissues.

OBJECTIVE: Genetic variation at the type II deiodinase (D2) gene (DIO2) was previously identified as osteoarthritis (OA) risk factor. To investigate mechanisms possibly underlying this association, we assessed D2 protein in healthy and OA-affected cartilage and investigated allelic balance of the OA risk polymorphism rs225014 at DIO2 in human OA joints. METHODS: Immunohistochemical staining of healthy and OA-affected cartilage was performed for D2. We then assessed allelic balance of DIO2 mRNA within OA-affected cartilage both at and away from the lesion, ligaments and subchondral bone. Allelic balance was measured by the amount of alleles 'C' and 'T' of the intragenic OA risk polymorphism rs225014 in heterozygous carriers. RESULTS: A markedly higher amount of D2 positive cells and staining intensity was observed in OA cartilage. A significant, 1.3-fold higher presence was observed for the OA-associated rs225014 'C' allele relative to the 'T' allele of DIO2, which was significant in 28 of 31 donors. CONCLUSION: In OA cartilage, D2 protein presence is increased. The allelic imbalance of the DIO2 mRNA transcript, with the OA risk allele 'C' of rs225014 more abundant than the wild-type 'T' allele in heterozygote carriers provides a possible mechanism by which genetic variation at DIO2 confers OA risk.

Allelic expression analysis of the osteoarthritis susceptibility locus that maps to MICAL3.

BACKGROUND: A genome-wide association scan with subsequent replication study that involved over 67,000 individuals of European ancestry has produced evidence of association of single nucleotide polymorphism rs2277831 to primary osteoarthritis (OA) with a P-value of 2.9 × 10(-5). rs2277831, an A/G transition, is located in an intron of MICAL3. This gene is located on chromosome 22q11.21 and the association signal encompasses two additional genes, BCL2L13 and BID. It is becoming increasingly apparent that many common complex traits are mediated by cis-acting regulatory polymorphisms that influence, in a tissue-specific manner, gene expression or transcript stability. METHODS: We used total and allelic expression analysis to assess whether the OA association to rs2277831 is mediated by an influence on MICAL3, BCL2L13 or BID expression. Using RNA extracted from joint tissues of 60 patients who had undergone elective joint replacement surgery, we assessed whether rs2277831 correlated with allelic expression of either of the three genes by: 1) measuring the expression of each gene by quantitative PCR and then stratifying the data by genotype at rs2277831 and 2) accurately discriminating and quantifying the mRNA synthesised from the alleles of OA patients using allelic-quantitative PCR. RESULTS: We found no evidence for a correlation between gene expression and genotype at rs2277831, with P-values of 0.09 for BCL2L13, 0.07 for BID and 0.33 for MICAL3. In the allelic expression analysis we observed several examples of significant (p < 0.05) allelic imbalances, with an allelic expression ratio of 2.82 observed in BCL2L13 (P = 0.004), 2.09 at BID (P = 0.001) and the most extreme case being at MICAL3, with an allelic expression ratio of 5.47 (P = 0.001). However, there was no correlation observed between the pattern of allelic expression and the genotype at rs2277831. CONCLUSIONS: In the tissues that we have studied, our data do not support our hypothesis that the association between rs2277831 and OA is due to the effect this SNP has on MICAL3, BCL2L13 or BID gene expression. Instead, our data point towards other functional effects accounting for the OA associated signal.