Genetic analysis and functional evaluation of the C/T(-318) and A/G(-1661) polymorphisms of the CTLA-4 gene in patients affected with Graves' disease.

In this study, we evaluated the A/G(-1661), C/T(-318), A/G49 and A/G6230 single nucleotide polymorphisms (SNPs) of the cytotoxic T lymphocyte antigen 4 (CTLA-4) gene for association with Graves' disease (GD) in 126 Russian simplex families. The conditional TDT analysis revealed significant overtransmission of the A(-1661)G(-318) haplotype (P = 0.033) and undertransmission of the GT haplotype (P = 0.0043) from parents homozygous for both +49 and +6230 polymorphisms. Parents homozygous for both (-1661) and (-318) markers significantly overtransmitted the G49G6230 haplotype (P = 0.0013) and undertransmitted the AG haplotype (P = 0.035) to affected offspring. This suggests in favor of the independent genetic effects of the 3' and 5'ends of CTLA-4 in conferring the susceptibility to GD. Both SNPs located at the 5' untranslated region of CTLA-4 were functionally analyzed using the luciferase reporter assay. We observed differential activation of the C/T(-318) promoter variant when Jurkat T cells and HeLa cells were cotransfected with a plasmid expressing lymphoid enhancing factor 1 (LEF1) and various CTLA-4 promoter constructs. The (-318) SNP modifies a putative binding site for LEF1 so that it alters the stimulating effect of LEF1 on the expression ability of the CTLA-4 promoter. The (-1661) dimorphism modifies a potential binding site for myocyte enhancer factor 2 (MEF2). No significant correlation between the (-1661) SNP and MEF2 activity in cotransfection experiments was found. Observed data help for further understanding a functional role of CTLA-4 promoter polymorphisms in the pathogenic mechanism of GD.