Nakayama Manabu, Oda Hirotsugu, Nakagawa Kenji, Yasumi Takahiro, Kawai Tomoki, Izawa Kazushi, Nishikomori Ryuta, Heike Toshio, Ohara Osamu
Department of Technology Development, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan.
Laboratory of Pharmacogenomics, Graduate School of Pharmaceutical Sciences, Chiba University, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan.
Biochem Biophys Rep. 2016 Dec 23;9:146-152. doi: 10.1016/j.bbrep.2016.12.002. eCollection 2017 Mar.
Autoinflammatory diseases occupy one of a group of primary immunodeficiency diseases that are generally thought to be caused by mutation of genes responsible for innate immunity, rather than by acquired immunity. Mutations related to autoinflammatory diseases occur in 12 genes. For example, low-level somatic mosaic NLRP3 mutations underlie chronic infantile neurologic, cutaneous, articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID). In current clinical practice, clinical genetic testing plays an important role in providing patients with quick, definite diagnoses. To increase the availability of such testing, low-cost high-throughput gene-analysis systems are required, ones that not only have the sensitivity to detect even low-level somatic mosaic mutations, but also can operate simply in a clinical setting. To this end, we developed a simple method that employs two-step tailed PCR and an NGS system, MiSeq platform, to detect mutations in all coding exons of the 12 genes responsible for autoinflammatory diseases. Using this amplicon sequencing system, we amplified a total of 234 amplicons derived from the 12 genes with multiplex PCR. This was done simultaneously and in one test tube. Each sample was distinguished by an index sequence of second PCR primers following PCR amplification. With our procedure and tips for reducing PCR amplification bias, we were able to analyze 12 genes from 25 clinical samples in one MiSeq run. Moreover, with the certified primers designed by our short program-which detects and avoids common SNPs in gene-specific PCR primers-we used this system for routine genetic testing. Our optimized procedure uses a simple protocol, which can easily be followed by virtually any office medical staff. Because of the small PCR amplification bias, we can analyze simultaneously several clinical DNA samples with low cost and can obtain sufficient read numbers to detect a low level of somatic mosaic mutations.
自身炎症性疾病属于原发性免疫缺陷病的范畴，这类疾病通常被认为是由负责先天免疫的基因突变引起的，而非获得性免疫。与自身炎症性疾病相关的突变发生在12个基因中。例如，慢性婴儿神经、皮肤、关节综合征（CINCA），也称为新生儿多系统炎症性疾病（NOMID），其发病基础是低水平的体细胞镶嵌NLRP3突变。在当前临床实践中，临床基因检测在为患者提供快速、明确的诊断方面发挥着重要作用。为了提高此类检测的可及性，需要低成本的高通量基因分析系统，这些系统不仅要具备检测甚至低水平体细胞镶嵌突变的灵敏度，还要能在临床环境中简便操作。为此，我们开发了一种简单的方法，该方法采用两步加尾PCR和NGS系统（MiSeq平台）来检测与自身炎症性疾病相关的12个基因的所有编码外显子中的突变。使用这种扩增子测序系统，我们通过多重PCR共扩增了来自12个基因的234个扩增子。这是在一个试管中同时完成的。每个样本在PCR扩增后通过第二条PCR引物的索引序列进行区分。通过我们的程序和减少PCR扩增偏差的技巧，我们能够在一次MiSeq运行中分析25个临床样本中的12个基因。此外，通过我们简短程序设计的经过验证的引物——该程序可检测并避免基因特异性PCR引物中常见的单核苷酸多态性（SNP）——我们将此系统用于常规基因检测。我们优化后的程序采用了简单的方案，几乎任何门诊医务人员都能轻松遵循。由于PCR扩增偏差小，我们可以低成本同时分析多个临床DNA样本，并能获得足够的读数以检测低水平的体细胞镶嵌突变。
