Document Detail

Next-generation sequencing reveals deep intronic cryptic ABCC8 and HADH splicing founder mutations causing hyperinsulinism by pseudoexon activation.
MedLine Citation:
PMID:  23273570     Owner:  NLM     Status:  MEDLINE    
Next-generation sequencing (NGS) enables analysis of the human genome on a scale previously unachievable by Sanger sequencing. Exome sequencing of the coding regions and conserved splice sites has been very successful in the identification of disease-causing mutations, and targeting of these regions has extended clinical diagnostic testing from analysis of fewer than ten genes per phenotype to more than 100. Noncoding mutations have been less extensively studied despite evidence from mRNA analysis for the existence of deep intronic mutations in >20 genes. We investigated individuals with hyperinsulinaemic hypoglycaemia and biochemical or genetic evidence to suggest noncoding mutations by using NGS to analyze the entire genomic regions of ABCC8 (117 kb) and HADH (94 kb) from overlapping ~10 kb PCR amplicons. Two deep intronic mutations, c.1333-1013A>G in ABCC8 and c.636+471G>T HADH, were identified. Both are predicted to create a cryptic splice donor site and an out-of-frame pseudoexon. Sequence analysis of mRNA from affected individuals' fibroblasts or lymphoblastoid cells confirmed mutant transcripts with pseudoexon inclusion and premature termination codons. Testing of additional individuals showed that these are founder mutations in the Irish and Turkish populations, accounting for 14% of focal hyperinsulinism cases and 32% of subjects with HADH mutations in our cohort. The identification of deep intronic mutations has previously focused on the detection of aberrant mRNA transcripts in a subset of disorders for which RNA is readily obtained from the target tissue or ectopically expressed at sufficient levels. Our approach of using NGS to analyze the entire genomic DNA sequence is applicable to any disease.
Sarah E Flanagan; Weijia Xie; Richard Caswell; Annet Damhuis; Christine Vianey-Saban; Teoman Akcay; Feyza Darendeliler; Firdevs Bas; Ayla Guven; Zeynep Siklar; Gonul Ocal; Merih Berberoglu; Nuala Murphy; Maureen O'Sullivan; Andrew Green; Peter E Clayton; Indraneel Banerjee; Peter T Clayton; Khalid Hussain; Michael N Weedon; Sian Ellard
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2012-12-27
Journal Detail:
Title:  American journal of human genetics     Volume:  92     ISSN:  1537-6605     ISO Abbreviation:  Am. J. Hum. Genet.     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-01-14     Completed Date:  2013-03-12     Revised Date:  2014-03-19    
Medline Journal Info:
Nlm Unique ID:  0370475     Medline TA:  Am J Hum Genet     Country:  United States    
Other Details:
Languages:  eng     Pagination:  131-6     Citation Subset:  IM    
Copyright Information:
Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.
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MeSH Terms
3-Hydroxyacyl CoA Dehydrogenases / genetics*
ATP-Binding Cassette Transporters / genetics*
Cell Line
Hyperinsulinism / genetics*
Potassium Channels, Inwardly Rectifying / genetics*
RNA Splice Sites
Receptors, Drug / genetics*
Sequence Analysis, DNA
Sulfonylurea Receptors
Grant Support
098395//Wellcome Trust; //Medical Research Council
Reg. No./Substance:
0/Potassium Channels, Inwardly Rectifying; 0/RNA Splice Sites; 0/Receptors, Drug; 0/Sulfonylurea Receptors; EC 1.1.1.-/3-Hydroxyacyl CoA Dehydrogenases

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