Functional analysis of the osteoarthritis susceptibility locus residing at the carbohydrate sulfotransferase 11 gene CHST11

Purpose: CHST11 encodes an enzyme that catalyses the transfer of sulphate groups to chondroitin to form chondroitin-4-sulphate, a major component of glycosaminoglycan (GAG) chains in the cartilage extracellular matrix. The single nucleotide polymorphism (SNP) rs835487 that is located within intron 2 of CHST11 is associated with hip OA, at the genome-wide significance level, with a p-value of 1.64x10-8. The aim of this study was to 1) assess the role of CHST11 in cartilage development and homeostasis, and 2) identify the causal allele or alleles that account for the OA association signal and determine how this acts to increase susceptibility to the disease. Methods: The CHST11 open reading frame (ORF) was sequenced in 200 hip OA patients. CHST11 expression in normal and osteoarthritic cartilage was assessed using quantitative PCR. CHST11 protein was depleted or overexpressed in human iliac crest derived mesenchymal stem cells (MSCs) followed by chondrogenic differentiation using siRNAs and lentivirus transduction, respectively. SNPs in linkage disequilibrium (LD) with the association SNP rs835487 were identified and their function tested using luciferase enhancer assays in the human chondrosarcoma cell line SW1353. Allelic differences in enhancer activity were analysed further using electrophoretic mobility shift assays (EMSAs) and transcription factor prediction tools to identify trans-acting factors that bind to these functional SNPs. Results: There are no common amino acid changes within CHST11, indicating that the OA susceptibility has to be mediated by a cis-acting regulatory polymorphism that influences CHST11 expression. CHST11 is upregulated 2.6 fold in OA hip cartilage relative to non-OA hip cartilage (p = 0.007). CHST11 depletion caused a 27% reduction (p = 0.005) and CHST11 overexpression a 22% increase (p = 0.015) in the wet weight of cartilage discs formed by chondrogenic differentiation of MSCs. Expression of SOX9, COL2A1 and ACAN were down-regulated and RUNX2, MMP13 and ADAMTS5 up-regulated in cartilage discs formed from CHST11 knock-down MSCs relative to the control non-targeting siRNA. Micromass cultures of MSCs after CHST11 protein knock-down also had reduced GAG sulfation as determined by quantification of Alcian Blue staining. We identified six candidate SNPs within the OA association interval which is located within a region predicted to have transcriptional enhancer activity. There was differential enhancer activity of the two alleles of the rs835487 and rs835488 SNPs; these two SNPs are in perfect LD and our data suggests that they have an additive or synergistic effect on enhancer activity. EMSA supershift studies demonstrated that the transcription factors SP1, SP3 and SUB1 bind to both rs835487 and rs835488, with YY1 binding specifically over the rs835488 SNP. These proteins bind more avidly to the OA-associated alleles of rs835487 and rs835488 than the non-OA associated alleles. Conclusions: CHST11 plays an important role in chondrogenesis, with loss of the protein leading to decreased matrix synthesis and increased chondrocyte hypertrophy. rs835487 and rs835488 are the top candidate SNPs for the OA susceptibility, with the disease associated GT haplotype of the two SNPs having significantly different enhancer activity relative to the AC haplotype in-vitro. Furthermore, several transcription factors bind more strongly to the disease-associated alleles. We conclude that the OA susceptibility residing within CHST11 is mediated by differential protein binding to the alleles of the rs835487 and rs835488 SNPs, which are located within a region that functions as a cis-acting enhancer of CHST11 transcription.