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

Variants in the Human Insulin Gene That Affect Pre-mRNA Splicing: Is -23HphI a Functional Single Nucleotide Polymorphism at IDDM2?
Variants in the Human Insulin Gene That Affect Pre-mRNA Splicing: Is -23HphI a Functional Single Nucleotide Polymorphism at IDDM2?
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.
0012-1797
260-264
Kralovicova, Jana
b3e0c1e7-05ed-445d-b3d9-ace11e3b4878
Gaunt, Tom R.
ff4bc39d-405c-4ba1-896b-7e7d2f747387
Rodriguez, Santiago
f235ea2b-b6f3-45e4-9fc3-5a0383689ed6
Wood, Peter J.
30039979-9541-4a0a-8aef-0dfe53114e02
Day, Ian N.M.
b749b30a-1f4c-40eb-af0e-a50427388b39
Vorechovsky, Igor
7245de2f-8c9b-4034-8935-9a451d9b682e
Kralovicova, Jana
b3e0c1e7-05ed-445d-b3d9-ace11e3b4878
Gaunt, Tom R.
ff4bc39d-405c-4ba1-896b-7e7d2f747387
Rodriguez, Santiago
f235ea2b-b6f3-45e4-9fc3-5a0383689ed6
Wood, Peter J.
30039979-9541-4a0a-8aef-0dfe53114e02
Day, Ian N.M.
b749b30a-1f4c-40eb-af0e-a50427388b39
Vorechovsky, Igor
7245de2f-8c9b-4034-8935-9a451d9b682e

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).

Record type: Article

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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Published date: January 2006
Additional Information: Brief genetics report

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Local EPrints ID: 24815
URI: http://eprints.soton.ac.uk/id/eprint/24815
ISSN: 0012-1797
PURE UUID: b1666ca7-88b4-4d3c-a1a3-e095c31cc356
ORCID for Igor Vorechovsky: ORCID iD orcid.org/0000-0002-6740-6502

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Date deposited: 04 Apr 2006
Last modified: 16 Mar 2024 03:32

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Contributors

Author: Jana Kralovicova
Author: Tom R. Gaunt
Author: Santiago Rodriguez
Author: Peter J. Wood
Author: Ian N.M. Day

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