Harries L W, Ellard S, Jones R W A, Hattersley A T, Bingham C
Molecular Genetics, Institute of Biomedical and Clinical Science, Peninsula Medical School, Exeter, UK.
Diabetologia. 2004 May;47(5):937-42. doi: 10.1007/s00125-004-1383-x. Epub 2004 Apr 15.
AIMS/HYPOTHESIS: Mutations in the hepatocyte nuclear factor-1 beta ( HNF-1 beta) 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-1 beta mutations at the mRNA level. This is the aim of the present study.
We have developed a nested RT-PCR assay that exploits the presence of ectopic HNF-1 beta transcripts in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines derived from subjects carrying HNF-1 beta splice site mutations.
We report a fourth mutation of the intron 2 splice donor site, IVS2nt+2insT. Sequence analysis of ectopic HNF-1 beta 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-1 beta 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-1 beta gene by mRNA analysis. This type of approach is a useful and important tool to define mutational mechanisms and determine pathogenicity.
目的/假设：肝细胞核因子1β（HNF-1β）基因突变会导致肾脏发育紊乱，通常涉及肾囊肿和早发性糖尿病（即肾囊肿、糖尿病、胰腺发育不全综合征/青少年发病的成年型糖尿病5型）。已报道了16种突变，包括内含子2剪接供体位点的3种剪接突变。由于在活体受试者中，难以获取表达丰富（肾脏、肝脏、胰腺、肠道、肺和性腺）的组织进行分析，因此之前很难在mRNA水平确定HNF-1β突变的影响。本研究旨在解决这一问题。
我们开发了一种巢式逆转录聚合酶链反应（RT-PCR）检测方法，利用携带HNF-1β剪接位点突变的受试者的爱泼斯坦-巴尔病毒（EBV）转化的淋巴母细胞系中异位HNF-1β转录本的存在。
我们报道了内含子2剪接供体位点的第4种突变，即IVS2nt+2insT。对异位HNF-1β转录本的序列分析表明，IVS2nt+2insT和IVS2nt+1G>T均导致外显子2缺失，并预计会导致HNF-1β蛋白过早终止。突变转录本的丰度低于正常转录本，但没有证据表明存在无义介导的衰变。
结论/解读：这是第一项通过mRNA分析确定HNF-1β基因突变致病后果的研究。这种方法是定义突变机制和确定致病性的有用且重要的工具。
