Variants in the human insulin gene that affect pre-mRNA splicing : Is -23HphI a functional single nucleotide polymorphism at IDDM2?

• Published in: Diabetes (New York, N.Y.) Vol. 55; no. 1; pp. 260 - 264
• Main Authors: KRALOVICOVA, Jana, GAUNT, Tom R, RODRIGUEZ, Santiago, WOOD, Peter J, DAY, Ian N. M, VORECHOVSKY, Igor
• Format: Journal Article
• Language: English
• Published: Alexandria, VA American Diabetes Association 2006
• Subjects: Alternative Splicing - genetics; Biological and medical sciences; Care and treatment; Cell Line; Diabetes; Diabetes Mellitus, Type 1 - genetics; Diabetes research; Diabetes. Impaired glucose tolerance; Diagnosis; Endocrine pancreas. Apud cells (diseases); Endocrinopathies; Etiopathogenesis. Screening. Investigations. Target tissue resistance; Health aspects; Humans; Insulin; Insulin - genetics; Medical sciences; Mutation; Polymorphism; Polymorphism, Single Nucleotide - genetics; Proinsulin - genetics; Risk factors; RNA, Messenger - genetics; RNA, Messenger - metabolism; Type 1 diabetes; United States; Endocrinopathy; Human; Variant; Pancreatic hormone; Splicing; Gene; Diabetes mellitus; Insulin; Polymorphism
• ISSN: 0012-1797; 1939-327X
• DOI: 10.2337/diabetes.55.01.06.db05-0773

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 beta-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.