Determination of cytokine regulatory haplotypes by induced heteroduplex analysis of DNA

Multiple single nucleotide polymorphisms (SNP) in the promoter region of th e human interleukin-10 (n-10) gene and in the signal/leader sequence of the human transforming growth factor beta 1 (TGF-beta1) gene, have been associ ated with susceptibility, severity and clinical outcome for a number of dis eases. One common explanation for this, is that different haplotypes of the se SNPs regulate the expression of the respective cytokines. Therefore, acc urate determination of haplotypes by physical linkage analysis represents a n important tool in investigating the pathogenesis of such diseases. Here, we demonstrate that the use of induced heteroduplex generators (IHGs) may b e used to identify haplotypes within target sequences in the IL-10 and TGF- beta1 genes. Four haplotypes were observed within the IL-10 promoter region , consisting of -1082, -851, -819 and -592 SNPs. For the TGF-beta1 signal/l eader sequence, we observed three haplotypes of the T869C (Leu10Pro) and G9 15C (Arg25Pro) SNPs. In both cases, all combinations of these haplotypes co uld be resolved unequivocally with a single IHG reagent. (C) 2001 Elsevier Science B.V. All rights reserved.