title: Variants in the human insulin gene that affect pre-mRNA splicing: is -23HphI a functional single nucleotide polymorphism at IDDM2?
abstract: 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.
authors.affiliation: University of Southampton School of Medicine, Human Genetics Division, Duthie Building, MP808, Tremona Road, Southampton SO16 6YD, U.K.
authors.forename: Jana
authors.initials: J
authors.lastname: Královicová
authors.forename: Tom R
authors.initials: TR
authors.lastname: Gaunt
authors.forename: Santiago
authors.initials: S
authors.lastname: Rodriguez
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Wood
authors.forename: Ian N M
authors.initials: IN
authors.lastname: Day
authors.forename: Igor
authors.initials: I
authors.lastname: Vorechovsky
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D002460:Cell Line; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics*; D006801:Humans; D007328:Insulin / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011384:Proinsulin / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D007328:Insulin; D012333:RNA, Messenger; D011384:Proinsulin