A rapid NGS strategy for comprehensive molecular diagnosis of Birt-Hogg-Dubé syndrome in patients with primary spontaneous pneumothorax.
A rapid NGS strategy for comprehensive molecular diagnosis of Birt-Hogg-Dubé syndrome in patients with primary spontaneous pneumothorax.
复制标题
原发性自发性气胸患者 Birt-Hogg-Dube 综合征综合分子诊断的快速 NGS 策略
DOI：
10.1186/s12931-016-0377-9
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发表时间：
2016-05-27
影响因子：
5.8
通讯作者：
Yi L
中科院分区：
文献类型：
--
作者：
Zhang X;Ma D;Zou W;Ding Y;Zhu C;Min H;Zhang B;Wang W;Chen B;Ye M;Cai M;Pan Y;Cao L;Wan Y;Jin Y;Gao Q;Yi L
原发性自发性气胸（PSP）或肺囊肿是由FLCN基因杂合突变引起的Birt-Hogg-Dube综合征（BHDS）的表现之一。大多数突变是SNV和小插入缺失，并且还存在大约10%的大基因内缺失和突变的重复。这些分子结果通常通过不同的方法获得，包括临床实验室中的桑格测序和多重连接依赖性探针扩增。此外，作为一种遗传异质性疾病，PSP可能是由多个基因突变引起的，包括FBN 1、COL 3A 1、CBS、SERPINA 1和TSC 1/TSC 2基因。对于鉴别诊断，这些基因也应该进行筛选，这使得诊断程序更加耗时和劳动密集型。40例PSP患者分为2组。试验组为19例经常规桑格测序和MLPA鉴定为FLCN不同致病性突变的患者，21例未经任何基因筛查的随机PSP患者为盲法样本组。设计FLCN、FBN 1、COL 3A 1、CBS、SERPINA 1和TSC 1/TSC 2 7个PSP基因，采用Haloplex系统富集，在Miseq平台上测序，并对40例患者进行分析，评价靶向NGS方法的性能。我们证明了与气胸相关的全谱基因，包括FLCN基因突变，可以在多重序列数据中同时识别。值得注意的是，通过我们对序列数据的内部拷贝数分析，我们不仅可以检测基因内缺失，还可以同时确定近似的缺失连接。基于NGS的Haloplex靶点富集技术可同时检测外显子和内含子SNV、小插入缺失、大基因内缺失和PSP相关基因缺失连接，可替代桑格测序和MLPA，是PSP患者BHDS综合分子诊断的一种快速、经济的筛查策略。
Primary spontaneous pneumothorax (PSP) or pulmonary cysts is one of the manifestations of Birt-Hogg-Dube syndrome (BHDS) that is caused by heterozygous mutations in FLCN gene. Most of the mutations are SNVs and small indels, and there are also approximately 10 % large intragenic deletions and duplications of the mutations. These molecular findings are generally obtained by disparate methods including Sanger sequencing and Multiple Ligation-dependent Probe Amplification in the clinical laboratory. In addition, as a genetically heterogeneous disorder, PSP may be caused by mutations in multiple genes include FBN1, COL3A1, CBS, SERPINA1 and TSC1/TSC2 genes. For differential diagnosis, these genes should also be screened which makes the diagnostic procedure more time-consuming and labor-intensive. Forty PSP patients were divided into 2 groups. Nineteen patients with different pathogenic mutations of FLCN previously identified by conventional Sanger sequencing and MLPA were included in test group, 21 random PSP patients without any genetic screening were included in blinded sample group. 7 PSP genes including FLCN, FBN1, COL3A1, CBS, SERPINA1 and TSC1/TSC2 were designed and enriched by Haloplex system, sequenced on a Miseq platform and analyzed in the 40 patients to evaluate the performance of the targeted-NGS method. We demonstrated that the full spectrum of genes associated with pneumothorax including FLCN gene mutations can be identified simultaneously in multiplexed sequence data. Noteworthy, by our in-house copy number analysis of the sequence data, we could not only detect intragenic deletions, but also determine approximate deletion junctions simultaneously. NGS based Haloplex target enrichment technology is proved to be a rapid and cost-effective screening strategy for the comprehensive molecular diagnosis of BHDS in PSP patients, as it can replace Sanger sequencing and MLPA by simultaneously detecting exonic and intronic SNVs, small indels, large intragenic deletions and determining deletion junctions in PSP-related genes.