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Variants in the Human Insulin Gene That Affect Pre-mRNA Splicing

Is –23HphI a Functional Single Nucleotide Polymorphism at IDDM2?

Jana Královiová, Tom R. Gaunt, Santiago Rodriguez, Peter J. Wood, Ian N.M. Day, and Igor Voechovsk

From the Human Genetics Division, University of Southampton School of Medicine, Southampton, U.K

Address correspondence and reprint requests to Igor Voechovsk, University of Southampton School of Medicine, Human Genetics Division, Duthie Building, MP808, Tremona Road, Southampton SO16 6YD, U.K. E-mail: i.vorechovsky{at}soton.ac.uk

Abbreviations: EST, expressed sequence tag; PPT, polypyrimidine tract; SNP, single nucleotide polymorphism; SR, serine/arginine rich; UTR, untranslated region

Predisposition to type 1 diabetes and juvenile obesity is influenced by the susceptibility locus IDDM2 that includes the insulin gene (INS). Although the risk conferred by IDDM2 has been attributed to a minisatellite upstream of INS, intragenic variants have not been ruled out. We examined whether INS polymorphisms affect pre-mRNA splicing and proinsulin secretion using minigene reporter assays. We show that IVS1-6A/T (–23HphI+/–) is a key INS variant that influences alternative splicing of intron 1 through differential recognition of its 3' splice site. The A allele resulted in an increased production of mature transcripts with a long 5' leader in several cell lines, and the extended mRNAs generated more proinsulin in culture supernatants than natural transcripts. The longer mRNAs were significantly overrepresented among ß-cell-expressed sequenced tags containing the A allele as compared with those with T alleles. In addition, we show that a rare insertion/deletion polymorphism IVS1+5insTTGC (IVS-69), which is exclusively present in Africans, activated a downstream cryptic 5' splice site, extending the 5' leader by 30 bp. These results indicate that –23HphI and IVS-69 are the most important INS variants affecting pre-mRNA splicing and suggest that –23HphI+/– is a common functional single nucleotide polymorphism at IDDM2.

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