Advances in Alport syndrome diagnosis using next-generation sequencing
Advances in Alport syndrome diagnosis using next-generation sequencing
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DOI：
10.1038/ejhg.2011.164
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发表时间：
2012-01-01
影响因子：
5.2
通讯作者：
Bruttini, Mirella
中科院分区：
文献类型：
--
作者：
Artuso, Rosangela;Fallerini, Chiara;Bruttini, Mirella
Alport综合征（ATS）是一种遗传性肾病，常伴有感觉神经性听力减退和眼部异常。COL 4A 5基因突变导致X连锁ATS。在常染色体隐性和常染色体显性ATS中均报告了COL 4A 4和COL 4A 3基因突变。通过DHPLC和/或桑格测序进行的常规突变筛选是耗时的并且具有相对高的成本，这是因为缺乏热点并且每个基因的外显子数目高：51（C 0 L 4A 5）、48（C 0 L 4A 4）和52（C 0 L 4A 3）。通常需要几个月才能完成诊断，特别是在信息量较少的家系中。为了克服这些限制，我们设计了下一代测序（NGS）方案，能够同时检测三个基因中所有可能的变异。我们使用了一种将选择性扩增与454 Roche DNA测序平台（Genome Sequencer junior）偶联的方法。该技术的应用使我们能够在两名ATS患者中鉴定第二个突变（COL 4A 3中的p.Ser1147Phe和COL 4A 4中的p.Arg1682Trp），并重新考虑第三名患者中ATS的诊断。因此，本研究说明了成功的应用NGS基因座异质性的孟德尔疾病的突变筛查。European Journal of Human Genetics（2012）20，50-57; doi：10.1038/ejhg.2011.164; 2011年9月7日在线发表
Alport syndrome (ATS) is a hereditary nephropathy often associated with sensorineural hypoacusis and ocular abnormalities. Mutations in the COL4A5 gene cause X-linked ATS. Mutations in COL4A4 and COL4A3 genes have been reported in both autosomal recessive and autosomal dominant ATS. The conventional mutation screening, performed by DHPLC and/or Sanger sequencing, is time-consuming and has relatively high costs because of the absence of hot spots and to the high number of exons per gene: 51 (COL4A5), 48 (COL4A4) and 52 (COL4A3). Several months are usually necessary to complete the diagnosis, especially in cases with less informative pedigrees. To overcome these limitations, we designed a next-generation sequencing (NGS) protocol enabling simultaneous detection of all possible variants in the three genes. We used a method coupling selective amplification to the 454 Roche DNA sequencing platform (Genome Sequencer junior). The application of this technology allowed us to identify the second mutation in two ATS patients (p.Ser1147Phe in COL4A3 and p.Arg1682Trp in COL4A4) and to reconsider the diagnosis of ATS in a third patient. This study, therefore, illustrates the successful application of NGS to mutation screening of Mendelian disorders with locus heterogeneity. European Journal of Human Genetics (2012) 20, 50-57; doi:10.1038/ejhg.2011.164; published online 7 September 2011