N. A. P. Wood

THE INTERLEUKIN-10-1082 G/A POLYMORPHISM: ALLELE FREQUENCY IN DIFFERENT POPULATIONS AND FUNCTIONAL SIGNIFICANCE

Abstract. Genotypic variation in the human interleukin-10 (IL-10) promoter may account for marked inter-individual variation in IL-10 production and may influence susceptibility to autoimmune diseases. The G/A polymorphism at position -1082 has been linked to high/low IL-10 producer status. We directly tested the functional significance of this polymorphism using DNA-binding assays and reporter gene assays, examined allele frequencies in two geographically distinct populations and assessed intra- and inter-individual variation in IL-10 production in vitro according to genotype. Functional analyses showed that the -1082 region contains a putative ETS-like transcription factor-binding site, and nuclear factors from a monocyte cell line bind to this region. Transient transfection studies in an Epstein-Barr virus-transformed B cell line indicated that the -1082 A allele confers a two fold increase in transcriptional activity of the IL-10 promoter compared to the G allele. There was marked inter-individual variation in IL-10 production by peripheral blood mononuclear cells in vitro, with no consistent effect of genotype.

HLA‐DR/Dw MATCHING BY PCR FINGERPRINTING: THE ORIGIN OF PCR FINGERPRINTS AND FURTHER APPLICATIONS

Polymerase chain reaction (PCR) fingerprinting, a new method for the rapid matching of HLA‐Dr/Dw allotypes, involves the visual comparison of polymorphic HLA‐DRB gene second exon PCR products, resolved in non‐denaturing polyacrylamide gels (Bidwell & Hui, 1990). We show here that the satellite DNA bands within PCR fingerprints originate by heteroduplex formation between heterologous DNAs co‐amplified by a common PCR primer set. We also present two further applications of the technique which permit discrimination between unrelated HLA‐DR/Dw allotypes with similar PCR fingerprints.

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.

A bi-allelic VNTR in the human TNFR2 (p75) gene promoter.

We describe a bi-allelic VNTR polymorphism within a 42 bp region in the promoter of the tumour necrosis factor receptor 2 gene (TNFR2). Within this region there are one (Allele 1) or two (Allele 2) repeats of a 15 bp sequence, 5′-GCCGGGC AGGTGGAG-3′. Allele frequencies observed in a Caucasian population were 0.3 (Allele 1) and 0.7 (Allele 2).

Identification of novel single nucleotide polymorphisms in intron 1B and exon 1C of the human interleukin-1 receptor type I (IL-1RI) gene.

We have identified two novel single nucleotide polymorphisms in the 5′ region of the human IL-1RI gene: (1) A → G at position 52 in intron 1B (GenBank accession number AF146426), which creates an MspI restriction endonuclease site. Allele frequencies in a Caucasian population were 0.1 (A allele) and 0.9 (G allele). (2) A → T at position 140 in exon 1C (GenBank accession number AF146427). Allele frequencies in a Caucasian population were 0.27 (A allele) and 0.73 (T allele).