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Genotype at the MGP OA risk locus correlates with an expression quantitative trait locus operating on MGP in cartilage and with DNA methylation at a cluster of regulatory CpGs within ARHGDIB

Lookup NU author(s): Dr Colin Shepherd, Dr Louise Reynard, Professor John LoughlinORCiD


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Purpose: Osteoarthritis (OA) is a complex, polygenic disease, where multiple genetic risk alleles each contribute a small but cumulative increase in disease susceptibility. To date, over 20 OA risk loci have been identified at genome wide significance including two hand OA signals: the rs3204689 ALDH1A2 locus and the recently reported rs4764133MGP locus on chromosome 12p12.3. Despite a highly correlated single nucleotide polymorphism (SNP) at the 12p12.3 locus causing a non-synonymous amino acid change within the MGP protein, evidence suggests that this region operates as an expression quantitative trait locus (eQTL) where the OA risk allele correlates with decreased MGP expression. This eQTL was reported in cartilage and bone tissue samples taken from OA patients. MGP encodes matrix Gla protein, an extracellular matrix component that inhibits calcification. It is suggested, therefore, that OA risk at this locus is a consequence of an impaired ability to inhibit cartilage calcification. Our aim was to further characterise the MGP eQTL in joint tissues and to investigate genetic and epigenetic factors which may contribute to OA risk at this locus.Methods: The Infinium HumanMethylation450 BeadChip (Illumina) was used to profile genome-wide DNA methylation in the bisulphite-converted cartilage DNA of 87 patients who had undergone joint replacement surgery. The HumanOmniExpress BeadChip (Illumina) was used for genotyping. Methylation levels were correlated with genotype at the proxy SNPrs12316046 (r2=0.99 with rs4764133) for CpGs within 1 Mb up- or downstream of this SNP by logistic regression. Nucleic acid was extracted from the trapezium of patients who had undergone a trapeziectomy due to hand OA and was used to quantify MGP expression by quantitative PCR (qPCR) or allelic expression imbalance (AEI) by pyrosequencing. MGPexpression was also quantified in RNA extracted from the intact articular cartilage, infrapatellar fat pad, synovium, trabecular bone and blood samples from knee and hip OA patients. RNA-sequencing and in-silico analyses were performed to characterise the expression of additional genes and to investigate regulatory elements, respectively, pertaining to this locus.Results: Genotype at rs12316046 correlated with DNA methylation at 11 CpG sites across the 12p12.3 locus including a cluster of five CpGs within a 500 bp region encompassing the first exon of ARHGDIB (nominal P<0.05). This region of ARHGDIB is predicted to have regulatory activity. RNA-sequencing data revealed expression of both MGPand ARHGDIB in cartilage. ARHGDIB expression was 2.5-fold higher in OA compared to control hip cartilage (P<0.01), while there was no differential expression of MGP (P>0.05). Of the nine ARHGDIBtranscript isoforms predicted by Ensembl, only one was expressed in cartilage: the protein coding isoform ARHGDIB-201. All four predicted MGP transcript isoforms were detected (MGP-201, -202, -203 and -204), three of which are protein coding. MGP mRNA was detected by qPCR in abundance in OA knee and hip cartilage, and at lower levels in OA fat pad, synovium, blood, bone and trapezium samples. AEI analysis confirmed the presence of an eQTL operating on MGP in cartilage (mean AEI = 0.67, P<0.05) correlating with reduced expression of the risk allele. No evidence of an MGP eQTL was found in trabecular bone or trapezium samples (mean AEI=1.01, P>0.05 for both tissues).Conclusions: Our data confirm that OA risk at this locus correlates with reduced expression of MGP mRNA in cartilage. This eQTL is specific to cartilage in our cohort of patient samples. The discovery of a novel methylation quantitative trait locus (mQTL) correlating with the OA signal and residing within a functional region of nearby ARHGDIB implies an interplay between genetic susceptibility and epigenetic changes and highlights a second gene whose function may also be modulated by the OA risk-conferring allele at the rs4764133 locus.

Publication metadata

Author(s): Shepherd C, Reynard LN, Loughlin J

Publication type: Conference Proceedings (inc. Abstract)

Publication status: Published

Conference Name: Osteoarthritis Research Society International 2018

Year of Conference: 2018

Pages: S154-S155

Online publication date: 16/04/2018

Acceptance date: 16/04/2018

ISSN: 1063-4584

Publisher: Osteoarthritis & Cartilage


DOI: 10.1016/j.joca.2018.02.334