Abnormal splicing of hepatocyte nuclear factor-1 beta in the renal cysts and diabetes syndrome

Abstract

Aims/hypothesis. Mutations in the hepatocyte nuclear factor-1 beta (HNF-1β) gene result in disorders of renal development, typically involving renal cysts and early-onset diabetes (the RCAD syndrome/ MODY5). Sixteen mutations have been reported, including three splicing mutations of the intron 2 splice donor site. Because tissues showing abundant expression (kidney, liver, pancreas, gut, lung and gonads) are not easily accessible for analysis in living subjects, it has previously proven difficult to determine the effect of HNF-1β mutations at the mRNA level. This is the aim of the present study. Methods. We have developed a nested RT-PCR assay that exploits the presence of ectopic HNF-1β transcripts in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines derived from subjects carrying HNF-1β splice site mutations.Results. We report a fourth mutation of the intron 2 splice donor site, IVS2nt+2insT. Sequence analysis of ectopic HNF-1β transcripts showed that both IVS2nt+2insT and IVS2nt+1G>T result in the deletion of exon 2 and are predicted to result in premature termination of the HNF-1β protein. Mutant transcripts were less abundant than the normal transcripts but there was no evidence of nonsense-mediated decay. Conclusions/interpretation. This is the first study to define the pathogenic consequences of mutations within the HNF-1β gene by mRNA analysis. This type of approach is a useful and important tool to define mutational mechanisms and determine pathogenicity.Keywords HNF-1β · Illegitimate transcription · MODY5 · RCAD · RT-PCR · Splicing. Abbreviations: Ct, crossing point · EBV, Epstein-Barr virus · HNF-1β, hepatocyte nuclear factor beta · MODY5, maturity-onset diabetes of the young subtype 5 · NMD, nonsense-mediated decay · RCAD, renal cysts and diabetes syndrome Diabetologia (2004) 47:937-942

Hnf1b haploinsufficiency differentially affects developmental target genes in a new renal cysts and diabetes mouse model

“…However, despite the conservation of the exon-2 cryptic splice donor site in humans ( ), variants lacking the last 32 bp of exon-2 have not been described ( Harries et al, 2004 ).…”

“…The genetic discrepancies between mouse models and human disease further suggest that mouse mutant models generated so far do not correctly represent the human mutations or, alternatively, that mice are less sensitive to haploinsufficiency. To explore these possibilities and obtain a more comprehensive view of HNF1B function in organ development and disease in the context of the whole animal, we generated a novel RCAD mouse model by introducing a previously identified human hotspot mutation at the intron-2 splice donor site (<IVS2nt+1G>T) ( Bingham et al, 2003 ; Harries et al, 2004 ). Moreover, because patients with these mutations exhibited the typical features of HNF1B mutations ( Bingham et al, 2003 ), this model could be representative of a large number of HNF1B intragenic disease-causing mutations.…”

Association of Agenesis of the Dorsal Pancreas With HNF1B Heterozygote Mutation: A Case Report

“…Early reports demonstrated an association between HNF1B mutations and maturity-onset diabetes of the young (MODY). These patients often presented with renal cysts and were initially considered as renal cysts and diabetes (RCAD) syndrome (15). It is now evident that mutation of this gene results in a panel of HNF1B-related manifestations.…”

From glucose sensing to exocytosis: takes from maturity onset diabetes of the young

“…MODY5 or renal cyst-diabetes syndrome occurs due to loss-of-function mutations and is characterized by phenotypic heterogeneity regarding the organ involvement and higher penetrance of renal anomalies than diabetes even among same family members ( 118 ). Intron 2 is the hot spot for splice-site mutations, which result in complete loss of exon 2 through yet-unidentified mechanisms and consequent truncated inactive protein ( 119 ). Most single amino acid substitutions are located in the DBD and disrupt the bonds with DNA directly or indirectly via perturbing local environment such as creating superfluous interactions with neighboring residues.…”