Objective: To explore radiosensitivity-associated genes in esophageal squamous cell carcinoma by targeted sequencing panel. Methods: The peripheral blood from 22 esophageal squamous cell carcinoma (ESCC) patients received radiotherapy alone were collected, respectively. The genomic DNA (gDNA) of peripheral blood was extracted and used to create a library of gDNA restriction fragments. The gDNA restriction fragments were hybridized to the HaloPlex probe capture library, which comprises 356 cancer genes selected from the Catalogue of Somatic Mutations in Cancer (Cosmic) database of 2011 updated edition. The sequencing data were aligned by the Genome Analysis Toolkit GATK (version 3.0) and Picar. The single nucleotide polymorphism and inserted-deletion (SNP/InDel) variations were annotated by online database. The pathway enrichment was analyzed by Ingenuity Pathway analysis (IPA). Moreover, according to the short-period curative effect, 22 patients were divided into two groups: the radiation- sensitivity group (CR+ PR) and the radiation-resistant group (PD+ SD). The nonsynonymous mutation sites were statistically analyzed and the genes associated with radiosensitivity of ESCC were screened. Results: More than 97% sequencing reads were aligned to human genome reference sequence and more than 90% sequencing reads were the target sequences. SNP/InDel database annotation results showed that the mutations of 22 cases mainly distributed in exons, and the mutant types were mainly missense and synonymous single nucleotide variant (SNV). There were 23 genes of high-frequency mutation associated with esophageal cancer. Pathway enrichment by IPA showed that 3 pathways were associated with the development of esophageal cancer, which were roles of BRCA1 in DNA damage response pathway, DNA double-strand break repair by non-homologous end joining pathway and ATM signaling pathway. According to the curative effect, five genes including mismatch repair system component (PMS1), fibronectin 1(FN1), mutL homolog 1 (MLH1), B-Raf proto-oncogene, serine/threonine kinase (BRAF), patched 1 (PTCH1) and cytochrome P450 family 2 subfamily C member 19 (CYP2C19) were associated with radiosensitivity of ESCC patients.Moreover, the PTCH1 was mutated in all of 22 ESCC patients, while the variations of rs199476092 and rs202111971 sites of PTCH1 were only identified in the radiation-resistant group. Conclusions: We find that the variations of rs199476092 and rs202111971 in the encoding region of PTCH1 gene are significantly associated with radiosensitivity of ESCC patients.
目的： 通过高通量靶向基因检测(TPS)技术探查与食管癌放射敏感性相关的基因。 方法： 收集22例单纯放疗的食管癌患者外周血，提取DNA，利用Haloplex方法对356种已知恶性肿瘤相关基因进行文库捕获，基于Illumina MiSeq技术平台进行TPS，测序数据进行单核苷酸多态性/插入缺失标记(SNP/InDel)位点数据库注释和通路富集分析。将22例患者根据放疗近期疗效分为放射敏感组(CR＋PR)和放射抗拒组(PD＋SD)，对其中的非同义突变位点进行统计分析，筛选与食管癌放射敏感性相关基因。 结果： 22例患者的测序数据中，97%以上的reads与人类基因组序列相匹配，数据相当可靠。SNP/InDel数据库注释结果显示，22例患者的突变位点主要分布在外显子区域，其对应的功能分布主要为错义与同义单核苷酸变异(SNV)。进一步筛选出与食管癌相关的高频突变基因有23个。IPA通路富集结果显示，与食管癌发生发展相关的通路有3条，分别为BRCA1基因相关的DNA修复通路、DNA双链断裂修复的非同源末端连接修复通路和ATM信号通路。根据放疗疗效筛选出与食管癌放射敏感性相关的基因分别为错配修复基因蛋白1(PMS1)、纤维连接蛋白1(FN1)、MLH1、鼠类肉瘤滤过性毒菌致癌同源体B1(BRAF)、人类同源的果蝇片段基因1(PTCH1)和CYP2C19。进一步统计分析显示，PTCH1在22例患者中均有突变，其中rs199476092和rs202111971突变位点仅见于放射抗拒组。 结论： PTCH1基因编码区内rs199476092和rs202111971突变位点与食管癌患者的放射敏感性密切相关。.
Keywords: Esophageal neoplasms; Radiosensitivity-associated gene; Target sequencing panel.