Abnormal splicing of hepatocyte nuclear factor 1 alpha in maturity-onset diabetes of the young
Abnormal splicing of hepatocyte nuclear factor 1 alpha in maturity-onset diabetes of the young
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DOI：
10.1007/s00125-002-0919-1
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发表时间：
2002-10-01
期刊：
影响因子：
8.2
通讯作者：
Ellard, S
中科院分区：
文献类型：
--
作者：
Bulman, MP;Harries, LW;Ellard, S
目的/假设。HNF-1 α基因的突变导致年轻人的成熟型糖尿病（MODY）;一种由胰腺β细胞功能障碍引起的早发性、显性遗传性糖尿病。剪接位点突变约占报告的HNF-1 α突变的10%。迄今为止，还没有研究调查剪接位点突变对mRNA加工的影响，因为具有丰富HNF-1 α表达的组织（肝脏、胰腺、肾脏和肠道）不容易进行分析。本研究的目的是通过分析非法转录物来确定三个新的剪接位点突变的致病机制。为了评估潜在的HNF-1 α剪接位点突变的后果，我们开发了一种巢式逆转录酶PCR（RT-PCR）检测法，用于扩增Epstein巴尔病毒转化的淋巴母细胞系中的非法HNF-1 α转录本。对非法HNF-1 α转录物进行测序显示，剪接供体位点突变IVS 8 nt +1G>A导致外显子8的完全跳跃，剪接受体位点突变IVS 4 nt-2A>G导致外显子5的跳跃，并在内含子5内募集隐蔽剪接受体位点，隐蔽剪接受体位点突变（IVS 7 nt-6 G>A）导致外显子7的跳跃。所有这三个变化预计会导致HNF-1 α蛋白的提前终止，为它们作为致病突变的作用提供了进一步的证据。我们的结论是，从淋巴母细胞系的非法成绩单的测序是有帮助的，在HNF-1 α的内含子变异，可以改变剪接的评估。mRNA的这种分析是定义突变机制和确认致病状态所必需的。
Aims/hypothesis. Mutations in the HNF-1alpha 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-1alpha 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-1alpha splice site mutations we developed a nested reverse transcriptase PCR (RT-PCR) assay for the amplification of illegitimate HNF-1alpha transcripts in Epstein Barr virus transformed lymphoblastoid cell lines.Results. Sequencing the illegitimate HNF-1alpha 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.Conclusion/interpretation. We conclude that the sequencing of illegitimate transcripts from lymphoblastoid cell lines is helpful in the assessment of intronic variation in HNF-1alpha that could alter splicing. This analysis of the mRNA is required to define mutational mechanisms and confirm pathogenic status.