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

Aims/hypothesis. Mutations in the hepatocyte nuclear factor-1 beta (HNF-1beta) 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-1beta 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-1beta transcripts in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines derived from subjects carrying HNF-1beta splice site mutations. Results. We report a fourth mutation of the intron 2 splice donor site, IVS2nt+2insT. Sequence analysis of ectopic HNF-1beta 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-1beta 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-1beta gene by mRNA analysis. This type of approach is a useful and important tool to define mutational mechanisms and determine pathogenicity.