title: Next-generation sequencing reveals deep intronic cryptic ABCC8 and HADH splicing founder mutations causing hyperinsulinism by pseudoexon activation.
abstract: 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.
authors.affiliation: Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter EX2 5DW, UK.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Flanagan
authors.forename: Weijia
authors.initials: W
authors.lastname: Xie
authors.forename: Richard
authors.initials: R
authors.lastname: Caswell
authors.forename: Annet
authors.initials: A
authors.lastname: Damhuis
authors.forename: Christine
authors.initials: C
authors.lastname: Vianey-Saban
authors.forename: Teoman
authors.initials: T
authors.lastname: Akcay
authors.forename: Feyza
authors.initials: F
authors.lastname: Darendeliler
authors.forename: Firdevs
authors.initials: F
authors.lastname: Bas
authors.forename: Ayla
authors.initials: A
authors.lastname: Guven
authors.forename: Zeynep
authors.initials: Z
authors.lastname: Siklar
authors.forename: Gonul
authors.initials: G
authors.lastname: Ocal
authors.forename: Merih
authors.initials: M
authors.lastname: Berberoglu
authors.forename: Nuala
authors.initials: N
authors.lastname: Murphy
authors.forename: Maureen
authors.initials: M
authors.lastname: O'Sullivan
authors.forename: Andrew
authors.initials: A
authors.lastname: Green
authors.forename: Peter E
authors.initials: PE
authors.lastname: Clayton
authors.forename: Indraneel
authors.initials: I
authors.lastname: Banerjee
authors.forename: Peter T
authors.initials: PT
authors.lastname: Clayton
authors.forename: Khalid
authors.initials: K
authors.lastname: Hussain
authors.forename: Michael N
authors.initials: MN
authors.lastname: Weedon
authors.forename: Sian
authors.initials: S
authors.lastname: Ellard
mesh_terms: D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000235:genetics*; D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D002460:Cell Line; D005091:Exons; D006801:Humans; D006946:Hyperinsulinism / Q000235:genetics*; D007438:Introns; D008297:Male; D009154:Mutation; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics*; D022821:RNA Splice Sites; D011955:Receptors, Drug / Q000235:genetics*; D017422:Sequence Analysis, DNA; D064233:Sulfonylurea Receptors
chemical_list: D018528:ATP-Binding Cassette Transporters; D024661:Potassium Channels, Inwardly Rectifying; D022821:RNA Splice Sites; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D015094:3-Hydroxyacyl CoA Dehydrogenases