Ultra-sensitive droplet digital PCR for detecting a low-prevalence somatic GNAQ mutation in Sturge-Weber syndrome.
Ultra-sensitive droplet digital PCR for detecting a low-prevalence somatic GNAQ mutation in Sturge-Weber syndrome.
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DOI：
10.1038/srep22985
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发表时间：
2016-03-09
影响因子：
4.6
通讯作者：
Matsumoto N
中科院分区：
文献类型：
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作者：
Uchiyama Y;Nakashima M;Watanabe S;Miyajima M;Taguri M;Miyatake S;Miyake N;Saitsu H;Mishima H;Kinoshita A;Arai H;Yoshiura K;Matsumoto N
液滴数字PCR（ddPCR）是一种用于测量靶核酸序列量的方法，可用于使用TaqMan探针确定体细胞突变率。在这项研究中，通过ddPCR的测试DNA的拷贝数的检测限确定基于泊松分布。肽核酸（PNA）与靶病变强杂交，可抑制PCR靶扩增。因此，将PCR与PNA和ddPCR联用（PNA-ddPCR），可降低检测限。我们使用ddPCR和PNA-ddPCR重新分析了Sturge-Weber综合征（SWS）患者的体细胞GNAQ突变（c.548G > A）。重要的是，在先前通过下一代测序发现突变阴性的三名患者中，两名患者具有GNAQ突变，突变等位基因频率小于1%。此外，我们能够在40名SWS患者中的4名患者的血液白细胞或唾液DNA中发现相同的突变。血管异常和血液白细胞分别来源于内皮细胞和成血管细胞，两者都是中胚层来源。因此，血液白细胞可能含有GNAQ突变，这取决于获得体细胞突变的时间。这些数据表明，在一些SWS患者中使用血液DNA进行诊断的可能性。
Droplet digital PCR (ddPCR), a method for measuring target nucleic acid sequence quantity, is useful for determining somatic mutation rates using TaqMan probes. In this study, the detection limit of copy numbers of test DNA by ddPCR was determined based on Poisson distribution. Peptide nucleic acid (PNA), which strongly hybridises to target lesions, can inhibit target amplification by PCR. Therefore, by combination of PCR with PNA and ddPCR (PNA–ddPCR), the detection limit could be lowered. We reanalysed a somatic GNAQ mutation (c.548G > A) in patients with Sturge–Weber syndrome (SWS) using ddPCR and PNA–ddPCR. Importantly, among three patients previously found to be mutation negative by next–generation sequencing, two patients had the GNAQ mutation with a mutant allele frequency of less than 1%. Furthermore, we were able to find the same mutation in blood leukocyte or saliva DNA derived from four out of 40 SWS patients. Vascular anomalies and blood leukocytes originate from endothelial cells and haemangioblasts, respectively, which are both of mesodermal origin. Therefore, blood leukocytes may harbour the GNAQ mutation, depending on the time when the somatic mutation is acquired. These data suggest the possibility of diagnosis using blood DNA in some patients with SWS.