Association of the Cytotoxic T Lymphocyte-Associated Antigen 4 Gene with Type 1 Diabetes: Evidence for Independent Effects of Two Polymorphisms on the Same Haplotype Block

Abstract

A recent study mapped the known association of type 1 diabetes with the cytotoxic T lymphocyte-associated antigen 4 gene to a polymorphism at the 3'end (+6230G>A), but could not rule out additional contribution from the 5' end of the gene. To examine this possibility, we analyzed four polymorphisms at the 5'-flanking region for effects independent of +6230G>A. We confirm, by the transmission disequilibrium test, in 496 family trios overtransmission of the susceptibility allele (G) at +6230 (217/168; P = 0.013). Of the four promoter polymorphisms, one (-319C>T) showed overtransmission of the C allele (97/58; P = 0.0017). Because the undertransmitted T at the promoter is in linkage disequilibrium with the overtransmitted G at +6230G>A, the effect observed at the promoter cannot be accounted for by linkage disequilibrium with the +6230G>A. We confirm this by showing that parents heterozygous at the promoter but homozygous at +6230 overtransmit the C promoter allele even more significantly (53/24; P = 9 x 10(-4)). In vitro, the T promoter allele directs higher luciferase expression in Jurkat cells by 42% (P = 0.006), a difference also found in lymphocyte mRNA from eight individuals heterozygous at the promoter, but homozygous at +6230 (P = 1.3 x 10(-4)). Thus, the +6230G>A cannot be the sole functional variant. Either the two polymorphisms define a haplotype carrying the (yet unexamined) functional variant or the -319C>T contributes to the genetic association independently, a possibility suggested by the functional evidence we present.

Allelic effects on gene regulation at the autoimmunity-predisposing CTLA4 locus: a re-evaluation of the 3′ +6230G>A polymorphism

“…We had previously reported B40% higher expression of the À318T allele vs the À318C allele in mRNA without distinguishing between full-length and soluble CTLA4 transcripts. 9 In this study, we confirm a significantly higher expression from the À318T; þ 49A; þ 6230G haplotype than À318C; þ 49G; þ 6230G in flCTLA4 transcripts, where the mean allelic ratio of A/G is 1.3670.06 (7s.e.m.) (95% CI, 1.24-1.47) vs that in genomic DNA, 1.0370.01 (7s.e.m.)…”

“…This effect is consistent with the independent T1D association with À318C4T but does not explain the genetic effect of þ 6230G4A. 9 Characterization of the þ 6230G4A-containing transcript In the study by Ueda et al, 5 there are two transcripts detected by Northern blot analysis in resting PBMCs: a 4.1 and 2 kb transcript. Although it is possible that the larger transcript contains the 3 0 SNP, this is not clear from this experiment.…”

“…7 We found that this genetic effect is independent of the þ 6230 SNP and we confirmed a previous report 8 of increased in vitro transcription of the À318T allele. 9 This transcriptional effect is relevant in vivo as we showed that, in heterozygous subjects, there was increased CTLA4 mRNA transcribed from the T allele, 9 an effect we hypothesized may be mediated by the disruption of the predicted TCF1/LEF1 site.…”

CTLA4 −1661A/G and 3′UTR long repeat polymorphisms are associated with ulcerative colitis and influence CTLA4 mRNA and protein expression

“…It has been reported that five single-nucleotide polymorphisms of the CTLA4 gene (namely À1661A/G, À658C/T, À318C/T, þ 49A/G and þ 6230A/G) are in strong linkage disequilibrium. 39 The CTLA4 À318C/T polymorphism could affect transcriptional efficiency by modifying a transcription factor binding site for LEF1, and thereby regulate CTLA4 expression. 39 Ueda et al 40 found lower CTLA4 expression levels from the G allele at the þ 6230A/G polymorphism.…”

“…39 The CTLA4 À318C/T polymorphism could affect transcriptional efficiency by modifying a transcription factor binding site for LEF1, and thereby regulate CTLA4 expression. 39 Ueda et al 40 found lower CTLA4 expression levels from the G allele at the þ 6230A/G polymorphism. Hence, the CTLA4 À1661A/G polymorphism, could be associated with UC and effect gene expression as part of a haplotype with combined effects of neighboring polymorphic markers, such as À318C/T or þ 6230A/G.…”