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Expression analysis of the osteoarthritis genetic susceptibility locus mapping to an intron of the MCF2L gene and marked by the polymorphism rs11842874.

ABSTRACT: BACKGROUND:Osteoarthritis (OA) is a painful, debilitating disease characterised by loss of articular cartilage with concurrent changes in other tissues of the synovial joint. Genetic association studies have shown that a number of common variants increase the risk of developing OA. Investigating their activity can uncover novel causal pathways and potentially highlight new treatment targets. One of the reported OA association signals is marked by the single nucleotide polymorphism (SNP) rs11842874 at chromosome 13q34. rs11842874 is positioned within a small linkage disequilibrium (LD) block within intron 4 of MCF2L, a gene encoding guanine-nucleotide exchange factor DBS. There are no non-synonymous SNPs that correlate with this association signal and we therefore set out to assess whether its effect on OA susceptibility is mediated by alteration of MCF2L expression. METHODS:Nucleic acid was extracted from cartilage, synovial membrane or infrapatellar fat pad tissues from OA patients. Expression of MCF2L was measured by quantitative PCR and RNA-sequencing whilst the presence of DBS was studied using immunohistochemistry. The functional effect of SNPs within the 13q34 locus was assessed using public databases and in vitro using luciferase reporter analysis. RESULTS:MCF2L gene and protein expression are detectable in joint tissues, with quantitative differences in the expression of the gene and in the transcript isoforms expressed between the tissues tested. There is an expression quantitative trait locus (eQTL) operating within synovial membrane tissue, with possession of the risk-conferring A allele of rs11842874 correlating with increased MCF2L expression. SNPs within the rs11842874 LD block reside within transcriptional regulatory elements and their direct analysis reveals that several show quantitative differences in regulatory activity at the allelic level. CONCLUSIONS:MCF2L is subject to a cis-acting eQTL in synovial membrane that correlates with the OA association signal. This signal contains several functional SNPs that could account for the susceptibility and which therefore merit further investigation. As far as we are aware, this is the first example of an OA susceptibility locus operating as an eQTL in synovial membrane tissue but not in cartilage.

SUBMITTER: Shepherd C

PROVIDER: S-EPMC4653905 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Expression analysis of the osteoarthritis genetic susceptibility locus mapping to an intron of the MCF2L gene and marked by the polymorphism rs11842874.

Shepherd Colin C Skelton Andrew J AJ Rushton Michael D MD Reynard Louise N LN Loughlin John J

BMC medical genetics 20151119

<h4>Background</h4>Osteoarthritis (OA) is a painful, debilitating disease characterised by loss of articular cartilage with concurrent changes in other tissues of the synovial joint. Genetic association studies have shown that a number of common variants increase the risk of developing OA. Investigating their activity can uncover novel causal pathways and potentially highlight new treatment targets. One of the reported OA association signals is marked by the single nucleotide polymorphism (SNP) ...[more]

PMID: 26584642

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Project description:BackgroundThe arcOGEN genome-wide association study reported the rs9350591 C/T single nucleotide polymorphism (SNP) as marking a region on chromosome 6q14.1 that is associated with hip osteoarthritis (OA) in Europeans, with an odds ratio (OR) of 1.18 and a p-value of 2.42 × 10(-9). rs9350591 is an intergenic SNP surrounded by seven genes within 1 Mb. Six of the genes are expressed in cartilage. We sought to characterise this signal to assess whether the association of rs9350591 with OA is mediated by modulating gene expression.MethodsTotal RNA was extracted from hip or knee cartilage of 161 OA patients and from hip cartilage of 29 non-OA patients who had undergone hip replacements as a result of neck-of-femur (NOF) fractures. We used quantitative PCR (qPCR) to measure overall gene expression, and pyrosequencing to assess allelic expression of the genes. A mesenchymal stem cell (MSC) differentiation model was used to assess gene expression during chondrogenesis.ResultsWe identified a significant decrease in the expression of SENP6 (p = 0.005) and MYO6 (p = 0.026) in OA hip cartilage relative to the non-OA hip control cartilage. However, we found no evidence for a correlation between gene expression and rs9350591 genotype for any of the six genes. In addition, we identified expression quantitative trait loci (eQTLs) operating on COL12A1, TMEM30A, SENP6 and MYO6, although these were not relevant to the OA associated signal. Finally, all genes were dynamically expressed during chondrogenesis.ConclusionsThe regulation of gene expression at this locus is complex, highlighted by the down-regulation of SENP6 and MYO6 in OA hip cartilage and by eQTLs operating on four of the genes at the locus. However, modulation of gene expression in the end-stage OA cartilage that we have investigated is not the mechanism by which this association signal is operating. As implied by the dynamic patterns of gene expression throughout chondrogenesis, the association signal marked by rs9350591 could instead be exerting its effects during joint development.

Project description:BackgroundOsteoarthritis (OA) is a common disease of older individuals that impacts detrimentally on the quality and the length of life. It is characterised by the painful loss of articular cartilage and is polygenic and multifactorial. Genome-wide association scans have highlighted over 90 osteoarthritis genetic signals, some of which reside within or close to highly plausible candidate genes. An example is an association to polymorphisms within and adjacent to the matrix Gla protein gene MGP. We set out to undertake a functional study of this gene.MethodsNucleic acid was extracted from cartilage, infrapatellar fat pad, synovium, trabecular bone, trapezium and peripheral whole blood from OA patients and also from mesenchymal stem cells (MSCs) subjected to chondrogenesis. Expression of MGP was measured by quantitative PCR (qPCR), RNA-sequencing and allelic expression imbalance (AEI) analysis. Matrix Gla protein was depleted in chondrocytes by knocking down MGP expression using RNA interference (RNAi) and the effect on a range of genes assessed by qPCR.ResultsMGP is expressed in joint tissues, blood and chondrocytes cultured from MSCs. There is a higher expression in diseased versus non-diseased cartilage. Polymorphisms that are associated with OA also correlate with the expression of MGP, with the OA risk-conferring allele showing significantly reduced expression in cartilage, fat pad and synovium but increased expression in blood. Depletion of Matrix Gla protein had a significant effect on the majority of genes tested, with an increased expression of catabolic genes that encode enzymes that degrade cartilage.ConclusionsMGP expression is subject to cis-acting regulators that correlate with the OA association signal. These are active in a range of joint tissues but have effects which are particularly strong in cartilage. An opposite effect is observed in blood, highlighting the context-specific nature of the regulation of this gene's expression. Recapitulation of the genetic deficit in cartilage chondrocytes is pro-catabolic.

Project description: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.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.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.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.

Dataset Information

Expression analysis of the osteoarthritis genetic susceptibility locus mapping to an intron of the MCF2L gene and marked by the polymorphism rs11842874.

Publications

Expression analysis of the osteoarthritis genetic susceptibility locus mapping to an intron of the MCF2L gene and marked by the polymorphism rs11842874.

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