title: Abnormal splicing of hepatocyte nuclear factor 1 alpha in maturity-onset diabetes of the young.
abstract: OBJECTIVE
  Mutations in the HNF-1 alpha gene result in maturity-onset diabetes of the young (MODY); an early-onset, dominantly inherited form of diabetes caused by pancreatic beta-cell dysfunction. Splice site mutations represent approximately 10% of reported HNF-1 alpha mutations. No studies to date have investigated the effect of splice site mutations on mRNA processing because the tissues with abundant HNF-1alpha expression (liver, pancreas, kidney and gut) are not easily accessible for analysis. This study aimed to define the pathogenic mechanism in three novel splice site mutations by analysing illegitimate transcripts.
  
  
  METHODS
  To assess the consequence of potential HNF-1 alpha splice site mutations we developed a nested reverse transcriptase PCR (RT-PCR) assay for the amplification of illegitimate HNF-1 alpha transcripts in Epstein Barr virus transformed lymphoblastoid cell lines.
  
  
  RESULTS
  Sequencing the illegitimate HNF-1 alpha transcripts showed that the splice donor site mutation IVS8nt+1G>A leads to complete skipping of exon 8, the splice acceptor site mutation IVS4nt-2A>G causes skipping of exon 5 with the recruitment of a cryptic splice acceptor site within intron 5 and the cryptic splice acceptor site mutation (IVS7nt-6G>A) resulted in the skipping of exon 7. All three changes are predicted to result in premature termination of the HNF-1alpha protein, providing further evidence for their role as pathogenic mutations.
  
  
  CONCLUSIONS
  We conclude that the sequencing of illegitimate transcripts from lymphoblastoid cell lines is helpful in the assessment of intronic variation in HNF-1 alpha that could alter splicing. This analysis of the mRNA is required to define mutational mechanisms and confirm pathogenic status.
authors.affiliation: Centre for Molecular Genetics, School of Postgraduate Medicine and Health Sciences, University of Exeter, Exeter EX2 5AX, UK.
authors.forename: M P
authors.initials: MP
authors.lastname: Bulman
authors.forename: L W
authors.initials: LW
authors.lastname: Harries
authors.forename: T
authors.initials: T
authors.lastname: Hansen
authors.forename: M
authors.initials: M
authors.lastname: Shepherd
authors.forename: W F
authors.initials: WF
authors.lastname: Kelly
authors.forename: A T
authors.initials: AT
authors.lastname: Hattersley
authors.forename: S
authors.initials: S
authors.lastname: Ellard
mesh_terms: D000328:Adult; D017398:Alternative Splicing; D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics*; D005091:Exons; D005190:Family; D005260:Female; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D007668:Kidney / Q000503:physiopathology; D008099:Liver / Q000503:physiopathology; D008297:Male; D009687:Nuclear Proteins; D010375:Pedigree; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta