GENTOPHEN · Linking genotype to phenotype for the rheumatoid arthritis susceptibility locus 6q23: beyond genome wide association studies

Linking genotype to phenotype for the rheumatoid arthritis susceptibility locus 6q23: beyond genome wide association studies

Genome wide association studies have been extremely successful in identifying regions of the genome that predispose to rheumatoid arthritis (RA). However, for the majority of these disease loci, the genes responsible for the predisposition have not been identified yet. The majority of associated variants map outside coding regions, so their impact on phenotype might be the result of an ability to differentially regulate gene expression. An intergenic region upstream TNFAIP3 is one of the most important loci in RA. My work aims to investigate the mechanism by which variation in this region predisposes to RA. rs6920220 is the most strongly associated variant in the locus and is in perfect linkage disequilibrium (LD) with 7 other SNPs. Among them, Bioinformatics searches indicated that rs6927172 shows more evidence of regulatory activity. Therefore, I prioritized this variant for functional studies. I tested whether rs6927172 was associated with impaired expression of TNFAIP3, but found no differences in mRNA levels between individuals carrying the different alleles of the variant in blood. However, I am now testing whether TNFAIP3 expression is modulated by the SNP in isolated primary cell types. I detected differential protein binding to the SNP using electrophoretic mobility shift assays (EMSA). Bioinformatics searches predicted binding of NFkB, an important mediator of RA pathogenesis, so antibodies against this transcription factor were used in chromatin immunoprecipitation studies to test whether the predicted binding occurred and whether there are allele specific differences in the binding. Preliminary data shows an increase in NFkB binding in cells carrying the risk allele. Hence, the intergenic associated region shows evidence of transcriptional regulatory activity, but as it lies at a large distance from any gene, I am using a modification of the chromosome conformation capture technique to test whether the associated variant physically interacts with TNFAIP3 when the DNA assumes its natural 3D conformation. Preliminary data shows that the promoter of TNFAIP3 interacts with a 5’ intergenic region in close proximity to the RA associated SNPs and also with a 3’ intergenic region where additional SNPs associated with RA map. These results suggest that rs6927172 is a functional causal variant in the TNFAIP3 loci, by impaired regulation of TNFAIP3 expression through physical interaction with the gene promoter and altered binding of NFkB. One of the major goals of the field of complex diseases genetics is to identify the specific genetic variants from a risk-associated locus that accounts for phenotypic differences based on the functional biology it modulates. The work carried out during my MC-ERG is aimed at developing a strategy to identify causal variants and genes in intergenic disease associated regions, and therefore represents advancement beyond the state-of-the-art in the field and has the potential to have a wider impact in the unravelling of the causes of complex diseases. My MC-ERG has helped me obtain a prestigious Wellcome Trust Research Career Development Fellowship which will have a great impact in my future career and boost my opportunities to secure further funding.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз