Búsqueda de variantes genéticas causales y estudio de sus implicaciones funcionales en pacientes con esclerosis múltiple

Abstract

Several genome-wide association studies (GWAS) have contributed to the characterization of the genetic component of multiple sclerosis (MS). These studies examine hundreds of thousands or millions of single nucleotide polymorphisms (SNPs) distributed throughout the whole genome. However, the causal genetic variants of the disease and the pathogenic mechanisms by which they are associated with the risk of developing MS remain unknown. In addition, GWAS are focused on common variants (minority allele frequency (MAF) ≥1%) and do not take into account the contribution of the rare genetic variants. In this context, the objective of this thesis was to identify genetic variants associated with MS susceptibility and to study their functional implications. DNA resequencing of the coding and regulatory regions of 14 genes linked to MS risk in a discovery cohort of 524 MS patients and 546 healthy controls comprised the first phase of the study. The analysis of the data was structured in three blocks: a) the identification of common variants associated with the risk of MS; b) the detection of genes that tend to accumulate rare variants in MS patients; and c) the finding of structural variants associated with the susceptibility to develop MS. Regarding the first block, 32 common SNPs differentially distributed between MS patients and healthy controls were identified, from which a selection was performed for their validation in an independent cohort of 3450 patients and 1688 controls by means of genotyping. Among the validated polymorphisms, two were chosen for additional studies: i) rs10892307 (CXCR5), for which a SNP was identified in high linkage disequilibrium (LD), rs11602393, whose MS risk allele was associated with a decreased CXCR5 promoter activity by a dual-luciferase reporter assay and with a reduced proportion of circulating regulatory T cells expressing CXCR5 on their surface by flow cytometry; and ii) rs2762943 (CYP24A1), for which it was found that MS patients carrying the risk allele showed diminished serum calcitriol levels and a trend for increased levels of CYP24A1 gene expression, which codes for an enzyme responsible for vitamin D degradation, after its in vitro stimulation with calcitriol in peripheral blood mononuclear cells (PBMCs). In reference to the second block, rare variant enrichments were detected more frequently in MS patients within specific regions of the FCRL1, RGS1, TRAF3 and CYP24A1 genes, of which the one reported in the whole sequence of RGS1 was associated with a decreased RGS1 gene expression in PBMCs, with a lower percentage of circulating B cells expressing RGS1 on their membrane, and with a lack of response to in vitro stimulation of PBMCs with interferon-β, all in MS patients. The third block uncovered a deletion of exons 4-10 in the FCRL1 gene in a patient with MS but, despite its experimental validation, no additional carrier individuals could be identified to tightly associate it with MS. Overall, the complete set of results led to conclude that i) the variant rs11602393 is associated with a decrease in circulating regulatory T cells with CXCR5-mediated migratory capacity towards CXCL13 chemokine enriched germinal centers; ii) the risk allele of the rs2762943 polymorphism could be related to a decrease in serum calcitriol levels influenced by a higher CYP24A1 enzyme activity; iii) that the presence of rare variants associated with the RGS1 gene can alter both the migratory capacity of B cells by reducing the proportion of them expressing RGS1 and the response to in vitro exposure of PBMCs to interferon-β treatment; and iv) that the FCRL1 deletion still cannot be associated with the risk of MS.

Date of Award	8 Nov 2018
Original language	Spanish
Supervisor	Manuel Comabella (Director), Javier Montalban Gairin (Director) & Sunny Malhotra (Co-director)