Variants within the CBLB gene are associated with multiple sclerosis

Multiple sclerosis (MS) is a multi-factorial neuroinflammatory and autoimmune disorder. A primary cause of disability in young adults, it results from interactions between unknown environmental factors and alleles of many susceptibility loci across the genome. Recent investigations of the genetics of MS have resulted in important advances, driven largely by completion of the first genome-wide association scans (GWAS). To detect additional loci, we performed a GWAS in 882 Sardinian Multiple Sclerosis (MS) cases and 872 controls genotyped with the Affymetrix 6.0 chip, using 575,678 SNPs that passed quality checks. We then successfully imputed 6,031,588 SNPs using haplotypes available from HapMap II, HapMap III and 1000 Genomes projects, and tested for association ~6.6 million variants. The strongest signal (OR=2.05, p=1.45x10-20) was observed at a SNP tag for the HLADRB1* 0301-DQB1*0201 allele. We then selected 9 SNPs outside of the HLA locus for validation and follow-up, based on their level of significance, proximity to functional candidate genes, and quality of imputation. Of those, SNP rs9657904 on chr3q13 was successfully confirmed in the GWAS samples and replicated in an independent set of 1,775 MS cases and 2,005 controls (p=9.4x10-6). Notably, this variant is absent from the HapMap II reference panel, and we were able to fully assess it only after imputation with HapMap III and 1000 Genomes haplotypes. Combining all available genotypes, the observed pvalue was 1.6x10-10 (OR=1.40). The most associated variants at this locus fall in the promoter of a gene, CBLB, which encodes a negative regulator of adaptive immune responses. In support of its involvement in MS, mice lacking the ortholog are prone to experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. The strongest associated variant was also replicated in 1,441 cases and 1,465 controls from central-northern Italy with a similar effect size, hence confirming the role of this marker in increased risk for multiple sclerosis in another southern European population. Finally, we sequenced by the Sanger method the coding regions and promoter of the gene in 96 patients, observing novel variants that are potentially causative, and will now be assessed with focused biological studies.