Identification and characterisation of a novel GHR defect disrupting the polypyrimidine tract and resulting in GH insensitivity
Identification and characterisation of a novel GHR defect disrupting the polypyrimidine tract and resulting in GH insensitivity
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DOI：
10.1530/eje-09-0583
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发表时间：
2010-01-01
影响因子：
5.8
通讯作者：
Metherell, L. A.
中科院分区：
文献类型：
--
作者：
David, A.;Miraki-Moud, F.;Metherell, L. A.
目的：GH不敏感（GHI）在大多数情况下是由GH受体（GHR）功能受损引起的。除了一个已知的GHR突变外，其他所有突变都位于编码序列或外显子/内含子边界。我们发现并描述了一个严重GHI患者GHR多聚嘧啶管中出现的第一个内含子缺陷。设计：我们使用体外剪接实验和细胞转染系统研究了这种新缺陷对mRNA剪接的影响。方法：直接测序法测定GHR。为了评估新缺陷的影响，通过将GHR外显子8及其侧翼的野生型或突变型内含子序列插入到特征良好的剪接报告基因（Adml-par L1-L2）中，产生了两个异源小基因（野生型和突变型L1-GHR8-L2）。p -32标记的pre-mRNA由两种构建体生成，并在HeLa核提取物或HEK293细胞中孵育。结果：对GHR的测序显示，在内含子7 （IVS7-6T > a）的多嘧啶通道中存在一种新的纯合缺陷。这种碱基变化不涉及高度保守的剪接位点序列，也不影响GHR mRNA剪接。然而，在体外剪接实验和转染的HEK293细胞中，突变体L1-GHR8-L2 mRNA的外显子8被清楚地证明了跳跃。结论：GHR多嘧啶通道的破坏导致mRNA剪接异常，导致GHR蛋白突变。据预测，这是缺乏其跨膜和胞内结构域，因此，不能转导生长激素信号。
Objective: GH insensitivity (GHI) is caused in the majority of cases by impaired function of the GH receptor (GHR). All but one known GHR mutation are in the coding sequence or the exon/intron boundaries. We identified and characterised the first intronic defect occurring in the polypyrimidine tract of the GHR in a patient with severe GHI.Design: We investigated the effect of the novel defect on mRNA splicing using an in vitro splicing assay and a cell transfection system.Methods: GHR was analysed by direct sequencing. To assess the effect of the novel defect, two heterologous minigenes (wild-type and mutant L1-GHR8-L2) were generated by inserting GHR exon 8 and its flanking wild-type or mutant intronic sequences into a well-characterised splicing reporter (Adml-par L1-L2). P-32-labelled pre-mRNA was generated from the two constructs and incubated in HeLa nuclear extracts or HEK293 cells.Results: Sequencing of the GHR revealed a novel homozygous defect in the polypyrimidine tract of intron 7 (IVS7-6T > A). This base change does not involve the highly conserved splice site sequences, and is not predicted in silico to affect GHR mRNA splicing. Nevertheless, skipping of exon 8 from the mutant L1-GHR8-L2 mRNA was clearly demonstrated in the in vitro splicing assay and in transfected HEK293 cells.Conclusion: Disruption of the GHR polypyrimidine tract causes aberrant mRNA splicing leading to a mutant GHR protein. This is predicted to lack its transmembrane and intracellular domains and, thus, be incapable of transducing a GH signal.