Dataset Information

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.

SUBMITTER: Dodd AW

PROVIDER: S-EPMC3641375 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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A rare variant in the osteoarthritis-associated locus GDF5 is functional and reveals a site that can be manipulated to modulate GDF5 expression.

Dodd Andrew W AW Syddall Catherine M CM Loughlin John J

European journal of human genetics : EJHG 20120829 5

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 ...[more]

PMID: 22929025

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Project description:ObjectiveSingle-nucleotide polymorphism (SNP) rs143383 (T to C) in the 5'-untranslated region (5'-UTR) of GDF5 has recently been reported to be associated with osteoarthritis (OA) susceptibility, with lower expression of the risk-associated T allele observed in vitro and in vivo. The in vivo studies were performed on cartilage tissue from OA patients. The present study was undertaken to expand the analysis of the effect of this SNP on GDF5 allelic expression to more joint tissue types, to investigate for cis and trans factors that interact with the SNP, and to examine novel cis-acting GDF5 regulatory polymorphisms.MethodsTissue samples were collected from OA patients undergoing joint replacement of the hip or knee. Nucleic acid was extracted, and, using rs143383 and an assay that discriminates and quantifies allelic expression, the relative amount of GDF5 expression from the T and C alleles was measured. Additional common variants in the GDF5 transcript sequence were interrogated as potential regulatory elements using allelic expression and luciferase reporter assays, and electrophoretic mobility shift assays were used to search for trans factors binding to rs143383.ResultsWe observed a consistent allelic expression imbalance of GDF5 in all tissues tested, implying that the functional effect mediated by rs143383 on GDF5 expression is joint-wide. We identified a second polymorphism, located in the 3'-UTR of GDF5, that influenced allelic expression of the gene independent of rs143383. Finally, we observed differential binding of deformed epidermal autoregulatory factor 1 (DEAF-1) to the 2 alleles of rs143383.ConclusionThese findings show that the OA susceptibility mediated by polymorphism in GDF5 is not restricted to cartilage, emphasizing the need to consider the disease as involving the whole joint. The existence of an additional cis-acting regulatory polymorphism highlights the complexity of the regulation of expression of this important OA susceptibility locus. DEAF-1 is a trans-acting factor that merits further investigation as a potential tool for modulating GDF5 expression.

Project description:A genetic deficit mediated by SNP rs143383 that leads to reduced expression of GDF5 is strongly associated with large-joint osteoarthritis. We speculated that this deficit could be attenuated by the application of exogenous GDF5 protein and as a first step we have assessed what effect such application has on primary osteoarthritis chondrocyte gene expression. Chondrocytes harvested from cartilage of osteoarthritic patients who had undergone joint replacement were cultured with wildtype recombinant mouse and human GDF5 protein. We also studied variants of GDF5, one that has a higher affinity for the receptor BMPR-IA and one that is insensitive to the GDF5 antagonist noggin. As a positive control, chondrocytes were treated with TGF-β1. Chondrocytes were cultured in monolayer and micromass and the expression of genes coding for catabolic and anabolic proteins of cartilage were measured by quantitative PCR. The expression of the GDF5 receptor genes and the presence of their protein products was confirmed and the ability of GDF5 signal to translocate to the nucleus was demonstrated by the activation of a luciferase reporter construct. The capacity of GDF5 to elicit an intracellular signal in chondrocytes was demonstrated by the phosphorylation of intracellular Smads. Chondrocytes cultured with TGF-β1 demonstrated a consistent down regulation of MMP1, MMP13 and a consistent upregulation of TIMP1 and COL2A1 with both culture techniques. In contrast, chondrocytes cultured with wildtype GDF5, or its variants, did not show any consistent response, irrespective of the culture technique used. Our results show that osteoarthritis chondrocytes do not respond in a predictable manner to culture with exogenous GDF5. This may be a cause or a consequence of the osteoarthritis disease process and will need to be surmounted if treatment with exogenous GDF5 is to be advanced as a potential means to overcome the genetic deficit conferring osteoarthritis susceptibility at this gene.

Dataset Information

A rare variant in the osteoarthritis-associated locus GDF5 is functional and reveals a site that can be manipulated to modulate GDF5 expression.

Publications

A rare variant in the osteoarthritis-associated locus GDF5 is functional and reveals a site that can be manipulated to modulate GDF5 expression.

Similar Datasets

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