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Functional characterisation of the osteoarthritis genetic risk locus that resides at the gene ALDH1A2, coding for the retinoic acid synthesis enzyme RALDH2, identifies rs4646636 and rs12915901 as key target SNPs

Lookup NU author(s): Dr Colin Shepherd, Dr Dongxing Zhu, Andrew Skelton, Dr Louise Reynard, Professor John Loughlin

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Abstract

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, 16 OA risk loci have been identified at genome wide significance. A hand OA risk locus was discovered in an Icelandic cohort marked by the single nucleotide polymorphism (SNP) rs12907038, and replicated in European cohorts using two further SNPs: rs3204689 and rs4238326. The region encompasses the ALDH1A2 gene, which encodes an enzyme responsible for retinoic acid production, RALDH2. This enzyme is essential for mammalian skeletal development processes and is key to cartilage homeostasis. No protein-coding SNPs correlate with the association signal. Evidence suggests instead that an expression quantitative trait locus (eQTL) operates at ALDH1A2 and is responsible for the association signal, with the risk allele at rs3204689 correlating with reduced ALDH1A2 expression in knee and hip cartilage. We set out to further characterise this eQTL and to identify the causal SNP/s.Methods: Nucleic acid was extracted from cartilage, infrapatellar fat pad, synovial membrane lining and trabecular bone from a total of 196 patients who had undergone knee or hip joint replacement surgery due to primary OA. We extracted nucleic acid from the thumb bone of hand OA patients who had undergone a trapeziectomy. ALDH1A2 expression was quantified by qPCR and the eQTL at rs3204689 by pyrosequencing. We investigated correlations between mRNA expression of ALDH1A2 and key chondrocyte genes by siRNA treatment of ex vivo cultured chondrocytes and by RNA-seq. We quantified allelic imbalance in ex vivo chondrocytes during a time-course in the presence of actinomycin D to assess the effect of rs3204689 on transcript stability. To investigate the transcriptional regulatory properties of SNPs within the linkage disequilibrium block we used publically available bioinformatics databases, luciferase reporter assays and electrophoretic mobility shift assays (EMSAs).Results:ALDH1A2 mRNA was detected at highest levels in cartilage and infrapatellar fat pad, followed by synovial membrane tissue, with lower levels detected in bone. We found a mean allelic expression imbalance (AEI) ratio at rs3204689 in cartilage of 0.72 (n = 31, P < 0.0001), 0.85 in infrapatellar fat pad (n = 24, P < 0.0001) and 0.85 in bone (n = 16, P = 0.001). Allelic imbalance was witnessed in ex vivo cultured chondrocytes (P = 0.0096) and in trapezium samples. In all cases the risk allele correlated with decreased expression. There was no AEI in synovial tissue thus the eQTL is not ubiquitous. Significant correlations between ALDH1A2 and key chondrocyte genes (SOX9, P = 0.002; ADAMTS5, P = 0.007; VEGFA, P = 0.004) were established by qPCR following siRNA depletion of ALDH1A2 in chondrocytes and by RNA-seq in 16 cartilage tissue samples. ALDH1A2 mRNA stability in chondrocytes was unaffected by genotype at rs3204689, indicating that the eQTL operates at the transcriptional level. We identified 39 functional SNPs within the rs3204689 and rs4238326 LD blocks using RegulomeDB and subjected these to luciferase reporter assays, revealing that rs4646636 and rs12915901 mimic the allelic imbalance observed in patient samples. Differential allelic transcription factor binding to these SNPs was witnessed by EMSA.Conclusions: Our data demonstrate that the underlying mechanism for the OA risk locus at ALDH1A2 is altered transcription factor binding across genetic variants at this site, with SNPs rs4646636 (intron 12 of the gene) and rs12915901 (intron 7) key targets. The risk correlates with reduced expression of ALDH1A2 in multiple joint tissues, which impacts upon expression of key genes. Furthermore, we have for the first time characterised the eQTL as acting in hand tissue from OA patients, the skeletal site pivotal to the association signal. We provide mechanistic insight into the effects of genotype at this OA risk locus.


Publication metadata

Author(s): Shepherd C, Zhu D, Skelton AJ, Combe J, Reynard LN, Loughlin J

Publication type: Conference Proceedings (inc. Abstract)

Publication status: Published

Conference Name: Osteoarthritis Research Society International 2017

Year of Conference: 2017

Pages: S9-S9

Online publication date: 18/04/2017

Acceptance date: 18/04/2017

Publisher: Elsevier

URL: https://doi.org/10.1016/j.joca.2017.02.031

DOI: 10.1016/j.joca.2017.02.031

Series Title: Osteoarthritis and Cartilage


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