Abstract
Osteoarthritis (OA) is an age-related disease characterized by articular cartilage degeneration. It is largely heritable, and genetic screening has identified single-nucleotide polymorphisms (SNPs) marking genomic risk loci. One such locus is marked by the G>A SNP rs75621460, downstream of TGFB1. This gene encodes transforming growth factor β1, the correct expression of which is essential for cartilage maintenance. This study investigated the regulatory activity of rs75621460 to characterize its impact on TGFB1 expression in disease-relevant patient samples (n = 319) and in Tc28a2 immortalized chondrocytes. Articular cartilage samples from human patients were genotyped, and DNA methylation levels were quantified using pyrosequencing. Gene reporter and electrophoretic mobility shift assays were used to determine differential nuclear protein binding to the region. The functional impact of DNA methylation on TGFB1 expression was tested using targeted epigenome editing. The analyses showed that SNP rs75621460 was located within a TGFB1 enhancer region, and the OA risk allele A altered transcription factor binding, with decreased enhancer activity. Protein complexes binding to A (but not G) induced DNA methylation at flanking CG dinucleotides. Strong correlations between patient DNA methylation levels and TGFB1 expression were observed, with directly opposing effects in the cartilage and the synovium at this locus. This demonstrated biologic pleiotropy in the impact of the SNP within different tissues of the articulating joint. The OA risk SNP rs75621460 impacts TGFB1 expression by modulating the function of a gene enhancer. We propose a mechanism by which the SNP impacts enhancer function, providing novel biologic insight into one mechanism of OA genetic risk, which may facilitate the development of future pharmacologic therapies.
Highlights
Transforming growth factor-β (TGF-β) signalling plays vital developmental and homeostatic roles in mammalian cell differentiation, proliferation, and extracellular matrix (ECM) production[1]
A role for aberrant TGF-β signalling in common musculoskeletal pathologies is supported by the genetic association of single nucleotide polymorphisms (SNPs) residing at chr19p13.2, the genomic location of TGFB1, with a spectrum of phenotypes including osteophytosis[11], osteoporosis[12], cleft palate[13], and, most recently, osteoarthritis (OA)[14]
The region encompassing rs75621460 is a gene enhancer OA risk SNP rs756215460 is an intergenic variant at chromosome 19q12, positioned between CCDC97 and TGFB1 (Fig.1a)
Summary
Transforming growth factor-β (TGF-β) signalling plays vital developmental and homeostatic roles in mammalian cell differentiation, proliferation, and extracellular matrix (ECM) production[1]. TGF-β signalling is widespread in mammalian tissues and the effects can be cell-type specific, displaying distinct, and sometimes paradoxical, effects[2,3]. The disease hallmark is the thinning and loss of articular cartilage, often accompanied by a low-grade synovial inflammation within the affected joint[16]. This leads to chronic impairment of joint function, with a resultant increased risk of premature death due to secondary co-morbidities[17,18]. There are no disease-modifying OA drugs and novel treatments are urgently required
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