Determination of cytokine regulatory haplotypes by induced heteroduplex analysis of DNA.
Multiple single nucleotide polymorphisms (SNP) in the promoter region of the human interleukin-10 (IL-10) gene and in the signal/leader sequence of the human transforming growth factor beta 1 (TGF-beta1) gene, have been associated with susceptibility, severity and clinical outcome for a number of diseases. One common explanation for this, is that different haplotypes of these SNPs regulate the expression of the respective cytokines. Therefore, accurate determination of haplotypes by physical linkage analysis represents an important tool in investigating the pathogenesis of such diseases. Here, we demonstrate that the use of induced heteroduplex generators (IHGs) may be 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/leader sequence, we observed three haplotypes of the T869C (Leu10Pro) and G915C (Arg25Pro) SNPs. In both cases, all combinations of these haplotypes could be resolved unequivocally with a single IHG reagent.
Department of Pathology and Microbiology, University of Bristol, Homoeopathic Hospital Site, Cotham, BS6 6JU, Bristol, UK. nigel.wood@bristol.ac.uk
N A
NA
Wood
L J
LJ
Keen
L A
LA
Tilley
J L
JL
Bidwell
D001483:Base Sequence; D016207:Cytokines / Q000235:genetics* / Q000276:immunology; D004247:DNA / Q000032:analysis* / Q000235:genetics; D004586:Electrophoresis; D006239:Haplotypes; D006801:Humans; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity
D016207:Cytokines; D004247:DNA