Application of targeted nanopore sequencing for the screening and determination of structural variants in patients with Lynch syndrome
Application of targeted nanopore sequencing for the screening and determination of structural variants in patients with Lynch syndrome
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DOI：
10.1038/s10038-021-00927-9
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发表时间：
2021-05-06
影响因子：
3.5
通讯作者：
Ikenoue, Tsuneo
中科院分区：
文献类型：
--
作者：
Yamaguchi, Kiyoshi;Kasajima, Rika;Ikenoue, Tsuneo
林奇综合征是一种遗传性疾病，其特点是患结直肠癌和其他癌症的风险增加。错配修复（MMR）基因的种系变异是导致这种疾病的原因。此前，我们在符合改良的Amsterdam II标准的结直肠癌患者中筛选了MMR基因，多重结扎依赖探针扩增（MPLA）在17例患者中鉴定出了MLH1和MSH2的11个结构变体（SVs）。在这项研究中，我们测试了长读测序与目标富集相结合的方法对sv及其断点的测定效果。通过阵列探针捕获DNA，与包括四个MMR基因在内的靶区杂交，然后使用纳米孔测序平台MinION对DNA进行测序。与其他地区相比，目标地区的覆盖率约为1000倍。应用该系统对17例患者中的4例测试用例进行了正确的断点映射。此外，在没有致病单核苷酸变异的病例中，我们新发现了MSH2的84 kb区域的缺失。这些数据表明，长读测序结合基于杂交的富集是鉴定sv及其断点的有效方法。这一策略可能取代MLPA在遗传性疾病中筛选SVs。
Lynch syndrome is a hereditary disease characterized by an increased risk of colorectal and other cancers. Germline variants in the mismatch repair (MMR) genes are responsible for this disease. Previously, we screened the MMR genes in colorectal cancer patients who fulfilled modified Amsterdam II criteria, and multiplex ligation-dependent probe amplification (MPLA) identified 11 structural variants (SVs) of MLH1 and MSH2 in 17 patients. In this study, we have tested the efficacy of long read-sequencing coupled with target enrichment for the determination of SVs and their breakpoints. DNA was captured by array probes designed to hybridize with target regions including four MMR genes and then sequenced using MinION, a nanopore sequencing platform. Approximately, 1000-fold coverage was obtained in the target regions compared with other regions. Application of this system to four test cases among the 17 patients correctly mapped the breakpoints. In addition, we newly found a deletion across an 84 kb region of MSH2 in a case without the pathogenic single nucleotide variants. These data suggest that long read-sequencing combined with hybridization-based enrichment is an efficient method to identify both SVs and their breakpoints. This strategy might replace MLPA for the screening of SVs in hereditary diseases.