title: A rare variant in the osteoarthritis-associated locus GDF5 is functional and reveals a site that can be manipulated to modulate GDF5 expression.
abstract: Osteoarthritis (OA) is a polygenic disease characterized by cartilage loss, with the single-nucleotide polymorphism (SNP) rs143383 (C/T) influencing OA susceptibility across a range of ethnic groups. The SNP resides within the 5'-UTR of the growth and differentiation factor 5 gene (GDF5), with the OA-associated T-allele mediating reduced GDF5 expression. As GDF5 codes for a cartilage anabolic protein, this reduced expression may explain why the T-allele of rs143383 is an OA risk factor. Our deep sequencing of GDF5 identified a C/A transversion located -41 bp relative to the gene's transcription start site. This promoter variant is predicted to affect transcription factor binding and it may therefore highlight a regulatory site that could be exploited to manipulate GDF5 expression and alleviate the detrimental effect mediated by the T-allele of rs143383. Here, we describe our functional assessment of the -41 bp variant. Using reporter constructs we demonstrated that the transversion leads to increased gene expression to such a degree that the A-allele is able to compensate for the reduced expression mediated by the T-allele of rs143383. Using electrophoretic mobility shift assays we identified YY1 as a trans-acting factor that differentially binds to the alleles of the -41 bp variant, with more avid binding to allele A. Knockdown of YY1 led to a significant reduction in GDF5 expression, supporting YY1 as a GDF5 activator. In conclusion, we demonstrated that the -41 bp variant is functional and we have identified a regulatory region of GDF5 that can be exploited to overcome the OA genetic deficit mediated by the T-allele of rs143383.
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Dodd
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Syddall
authors.forename: John
authors.initials: J
authors.lastname: Loughlin
mesh_terms: D045744:Cell Line, Tumor; D019902:Chondrocytes / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000235:genetics*; D055785:Gene Knockdown Techniques; D055428:Growth Differentiation Factor 5 / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008156:Luciferases; D010003:Osteoarthritis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D050986:YY1 Transcription Factor / Q000235:genetics / Q000378:metabolism
chemical_list: D017931:DNA Primers; C525893:GDF5 protein, human; D055428:Growth Differentiation Factor 5; D050986:YY1 Transcription Factor; C494698:YY1 protein, human; D008156:Luciferases