A functional analysis of the association of Type 1 Diabetes with the cytotoxic T-lymphocyte antigen-4 (CTLA4) gene /

Type 1 Diabetes (T1D) is characterized by the autoimmune destruction of the insulin producing beta cells of the pancreas. Linkage with Type 1 Diabetes has been reported on chromosome 2q31-33. Within this 23 cM interval, T1D association with CTLA4 polymorphisms has been localized to a linkage disequilibrium (LD) block covering the D2S72- CTLA4-D2S116 interval. Within this interval, T1D is associated with a haplotype in the CTLA4 gene, containing several potentially functional polymorphisms. Due to the extent of LD in the region, genetics studies alone are not sufficient to identify the causative polymorphism(s). To this end, we undertook a systematic analysis of each of these polymorphisms and their functional relevance in the pathogenesis of Diabetes.We investigated the role of the only coding polymorphism in the CTLA4 gene, a Threonine to Alanine substitution in the N-terminal signal sequence. The Ala17 (+49G) allele is associated with increased risk to T1D. Since the major function of the signal sequence is to direct trafficking of the pre-protein to the Endoplasmic Reticulum (ER), we hypothesized that a substitution could alter these events. The Ala17 is inefficiently N-glycosylated in the ER and its expression is lower than the Thr17 at the cell surface in COS1 cells.Using the family-based transmission disequilibrium test, (TDT) we report an association of the -318C-allele at the proximal promoter polymorphism and T1D. Conditional TDT analysis dissected the contributions of the linked -318C>T and +6230G>A polymorphisms, as independent contributors to the genetic effect. We report transcriptional effects of the promoter alleles in vitro , and in vivo with the -318T allele inducing stronger transcriptional activity. This effect may be mediated by alternative transcription factor binding at the Single Nucleotide Polymorphism (SNP).We confirmed association of the 3'+6230G>A SNP with T1D. In evaluating its contribution to the expression of soluble and full-length CTLA4 isoforms in an allele-dependent manner, we found no correlation between alleles at +6230 and levels of either isoform. The 3' SNP does not alter in cis the expression of the downstream ICOS gene.We propose that susceptibility at the CTLA4 locus is mediated by a haplotype exerting both transcriptional and post-translational effects