Ian N.M. Day, Jana Kralovicova, Tom R. Gaunt, Santiago Rodriguez, Peter J. Wood, Igor Vorechovsky
Human Genetics Division, University of Southampton, School of Medicine, Duthie Building (MP808), Southampton General Hospita, Tremona Road, Southampton SO16 6YD, UK
Predisposition to type 1 diabetes and juvenile obesity is infuenced by the susceptibility locus IDDM2 that includes the insulin gene (INS). The risk conferred by IDDM2 has been attributed to, although never formally proven to be determined by, a minisatellite (VNTR) upstream of INS in the proximal promoter region. INS RFLP '-23HphI' has long been used as a convenient genotyping tag in perfect linkage disequilibrium with the short (class I, high diabetes risk) and long (class III) alleles of the VNTR. In silico predictions suggested to us that '-23HphI' might affect splicing efficiency for the 5' noncoding intron I as it actually represents an A/T substitution (A tagging VNTR class I) in the polypyrimidine tract at position IVS1-6, i.e. near the 3' splice site. We examined whether INS polymorphisms affect pre-mRNA splicing and proinsulin secretion using minigene reporter assays. The A allele resulted in an increased production of mature transcripts with a long 5' leader representing intron 1 retention, in several cell lines. Furthermore, the extended mRNAs generated sixfold more proinsulin in culture supernatants than natural transcripts. The longer mRNAs were also significantly over-represented among databased beta-cell expressed sequence tags containing the A allele as compared with those with T alleles. These results indicate that '-23HphI' is an important INS variant affecting pre-mRNA splicing, and secondarily exerting major effect on translation efficiency. It is possible that splicing and translation, instead of or as well as promoter function, represent the important functional aspect of IDDM2 disease associated haplotypes.
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