Variants in the Human Insulin Gene That Affect Pre-mRNA Splicing: Is -23HphI a Functional Single Nucleotide Polymorphism at IDDM2?

Kralovicova, Jana, Gaunt, Tom R., Rodriguez, Santiago, Wood, Peter J., Day, Ian N.M. and Vorechovsky, Igor (2006) Variants in the Human Insulin Gene That Affect Pre-mRNA Splicing: Is -23HphI a Functional Single Nucleotide Polymorphism at IDDM2? Diabetes, 55 (1), 260-264. (doi:10.2337/diabetes.55.01.06.db05-0773).

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 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.
Type 1 diabetes results from the autoimmune destruction of the insulin-producing pancreatic ß-cells. In addition to a major susceptibility locus in the HLA region, termed IDDM1, genetic predisposition to this disease is conferred by a locus on chromosome 11, designated IDDM2 (1). The genetic risk at IDDM2 has been attributed to the INS minisatellite (2–6), which is composed of a variable number of tandem repeat sequences. However, reanalysis of allelic association data in type 1 diabetes did not rule out intragenic variants, including a single nucleotide polymorphism (SNP) –23HphI (7), which is located in position –6 relative to the 3' splice site of intron 1 (IVS1-6A/T). This SNP has been used as a surrogate marker for INS genotyping in a large number of studies to infer minisatellite haplotypes (class I/III) in disease susceptibility (2,3,6 and refs. therein). IVS1-6A/T is located in the polypyrimidine tract (PPT), a splicing signal of central importance for vertebrate 3' splice site recognition, and in a position exhibiting a great depletion of purine residues in the PPT (8). Since uridine is the preferred PPT nucleotide in pre-mRNA (9), the A allele, which reduces the Shapiro-Senapathy matrix score and other splicing prediction scores for the acceptor splice site of intron 1 (online appendix Table 1, available at http://diabetes.diabetesjournals.org), is likely to weaken efficient splicing of this intron. As intron 1 separates noncoding and coding exons, differential utilization of its 3' splice site by the uridine- and adenosine-containing pre-mRNAs would be predicted to result in distinct representation of mature transcripts with short and long 5' untranslated regions (UTRs) with potential effects on translation.

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Contributors

Author: Igor Vorechovsky

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