Improved genetic testing for monogenic diabetes using targeted next-generation sequencing.
Improved genetic testing for monogenic diabetes using targeted next-generation sequencing.
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DOI：
10.1007/s00125-013-2962-5
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发表时间：
2013-09
期刊：
影响因子：
8.2
通讯作者：
Caswell, R.
中科院分区：
文献类型：
--
作者：
Ellard, S.;Allen, H. Lango;De Franco, E.;Flanagan, S. E.;Hysenaj, G.;Colclough, K.;Houghton, J. A. L.;Shepherd, M.;Hattersley, A. T.;Weedon, M. N.;Caswell, R.
目前诊断单基因糖尿病的基因测试依赖于根据患者的表型选择适当的基因进行分析。下一代测序可以在一次测试中同时分析多个基因。我们的目标是开发一种有针对性的下一代测序分析，以检测所有已知的MODY和新生儿糖尿病基因的突变。我们选择了29个基因，这些基因的突变已被报道会导致新生儿糖尿病、MODY、母亲遗传性糖尿病和耳聋(MIDD)或家族性部分脂营养不良(FPLD)。外显子捕获分析被设计为包括编码区和剪接点。共检测了114例患者样本--32例有已知突变，82例以前检测过MODY(n = 33)或新生儿糖尿病(n = 49)，但没有发现突变。序列数据被分析是否存在碱基替换、小插入或缺失(Indels)和外显子缺失或复制。在32例阳性对照中，我们检测了所有已发现的变异(34个突变和36个多态)，包括55个碱基替换，10个小插入或缺失，5个部分/全部基因缺失/重复。在5名MODY患者(15%)和9名新生儿糖尿病患者(18%)中发现了以前未确定的突变。这些患者中的大多数(12/14)具有以前未经检测的基因突变。我们新的靶向下一代测序分析为同时分析所有单基因糖尿病基因提供了一种高度敏感的方法。这种单一的测试可以检测以前通过桑格测序或多重连接依赖的探针扩增剂量分析确定的突变。测试的基因数量增加导致了更高的突变检测率。这篇文章的在线版本(doi：10.1007/s00125-0132962-5)包含经同行审查但未经编辑的补充材料，授权用户可以使用。
Current genetic tests for diagnosing monogenic diabetes rely on selection of the appropriate gene for analysis according to the patient’s phenotype. Next-generation sequencing enables the simultaneous analysis of multiple genes in a single test. Our aim was to develop a targeted next-generation sequencing assay to detect mutations in all known MODY and neonatal diabetes genes. We selected 29 genes in which mutations have been reported to cause neonatal diabetes, MODY, maternally inherited diabetes and deafness (MIDD) or familial partial lipodystrophy (FPLD). An exon-capture assay was designed to include coding regions and splice sites. A total of 114 patient samples were tested—32 with known mutations and 82 previously tested for MODY (n = 33) or neonatal diabetes (n = 49) but in whom a mutation had not been found. Sequence data were analysed for the presence of base substitutions, small insertions or deletions (indels) and exonic deletions or duplications. In the 32 positive controls we detected all previously identified variants (34 mutations and 36 polymorphisms), including 55 base substitutions, ten small insertions or deletions and five partial/whole gene deletions/duplications. Previously unidentified mutations were found in five patients with MODY (15%) and nine with neonatal diabetes (18%). Most of these patients (12/14) had mutations in genes that had not previously been tested. Our novel targeted next-generation sequencing assay provides a highly sensitive method for simultaneous analysis of all monogenic diabetes genes. This single test can detect mutations previously identified by Sanger sequencing or multiplex ligation-dependent probe amplification dosage analysis. The increased number of genes tested led to a higher mutation detection rate. The online version of this article (doi:10.1007/s00125-013-2962-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
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