二代测序技术在家族性非髓样甲状腺癌遗传易感基因筛查中的应用

发布时间：2018-08-14 13:18

【摘要】：目的:家族性非髓样甲状腺癌(familial non-medullary thyroid carcinoma,FNMTC)包括甲状腺乳头状癌、甲状腺滤泡癌、甲状腺未分化癌,其特异性致病基因尚不清楚。随着测序技术发展革新,二代测序技术以其高通量、平行测序及经济高效的特点得到广泛的应用推广。本研究通过筛选与甲状腺癌遗传易感性相关的基因,制备适用于非髓样甲状腺癌易感性筛查的二代测序试剂盒探针,应用于非髓样甲状腺癌遗传易感基因的筛查,探讨二代测序技术对非髓样甲状腺癌遗传易感性筛查的价值。方法:以甲状腺癌相关的31个基因的全外显子以及RET基因的部分内含子为目标区域制备二代测序试剂盒探针。收集47例FNMTC及16例散发性非髓样甲状腺癌(sporadic non-medullary thyroid carcinoma,SNMTC)患者外周血,提取DNA样本,利用探针,与全基因组DNA杂交,富集目标区域,再利用Illumina MiSeq第二代测序仪进行测序。测序数据分析采用BWA aligner 0.7.10,DNA易位分析采用Tophat2和Factera 1.4.3完成;用ExAC和ClinVar数据库分析、过滤,筛出高质量的突变位点,采用Sanger测序法验证突变位点。利用SPSS19.0软件对FNMTC和SNMTC两组患者的突变比例、突变组和未突变组患者的临床病理资料进行统计学分析,以P0.05作为差异有统计学意义。结果:1.在基于二代测序的FNMTC易感性检测中,每个样本的产生的总测序数据量约为1.3~2.9兆(M)测序读段(reads),平均为2.5M测序读段,平均98.9%的读段在基因组,54.3%的读段分布在二代测序的目标区域内;对于FNMTC检测的二代测序目标区域,平均测序深度为500×,最小和最大测序深度分别为157×和1505×;所有样本中,超过96%的目标测序区域的平均测序深度大于200×(测序深度是测序得到的碱基总量与基因组大小的比值,是评价测序量的指标之一)。通过不同样本间每个目标区域覆盖深度的关联性对探针捕获效率可重复性的评估显示,大多数成对的样本间关联系数大于0.8。2.在63例非髓样甲状腺癌患者中共发现45个高质量的杂合性胚系突变,其中包括38个单核苷酸变异,1个缺失突变,1个剪接供体变异,和5个框内插入或缺失突变;47例FNMTC患者共携带37个胚系突变,14例SNMTC患者共携带8个胚系突变。FNMTC患者中基因突变携带者的比率高于散发组(61.7%vs.37.5%,P=0.092);最常见的胚系突变基因是MSH6,其次为APC;在8个FNMTC家系中发现同一家系的患者携带相同的胚系突变位点,分别为APC L292F、A2778S,MSH6 G355S、A36V,MSH2 L719F,BRAF D22N,MEN1 G508D,BRCA1 SS955S,BRCA2 G2508S和GNAS P459PDAPADPDSGAAR。3.根据突变结果将非髓样甲状腺癌患者分为两组:突变组(n=29)和未突变组(n=18)。对患者的临床病理特征进行比较分析发现,突变组患者中央区淋巴结转移率较未突变组明显增高(68.6%vs.30.8%,P=0.003)。且在具有亲子关系的FNMTC患者中,子代患者的中央区淋巴结转移率高于亲代患者(100%vs.42.9%,P=0.019)。结论:本研究中的FNMTC易感性探针捕获效率稳定,得到的突变数据经全面质量控制(碱基变异分布、最小等位基因频数分布等)符合胚系突变单核苷酸多态性变异的典型特征,数据可靠性高。二代测序技术在FNMTC样本易感性基因筛查中的应用,帮助我们发现了与FNMTC相关的一些新突变位点;同时提示我们不同的FNMTC家系携带的胚系突变基因可能不同。携带有胚系突变的FNMTC患者更易于出现中央区淋巴结的转移,对其进行预防性中央区淋巴结清扫是必要的。因此,利用FNMTC易感性探针检测胚系基因突变,对FNMTC患者治疗方案的制定具有指导意义。
[Abstract]:Objective: The specific pathogenic genes of familial non-medullary thyroid carcinoma (FNMTC) including papillary thyroid carcinoma, follicular thyroid carcinoma and undifferentiated thyroid carcinoma are unknown. In this study, by screening genes related to genetic susceptibility to thyroid cancer, a second-generation sequencing kit probe for non-medullary thyroid cancer susceptibility screening was prepared, which was used to screen genes susceptible to non-medullary thyroid cancer, and to explore the second-generation sequencing technology for non-medullary thyroid cancer genetic susceptibility screening. Methods: DNA samples were extracted from peripheral blood of 47 FNMTC patients and 16 sporadic non-medullary thyroid carcinoma (SNMTC) patients. DNA samples were extracted from the peripheral blood of the patients with FNMTC and 16 sporadic non-medullary thyroid carcinoma (SNMTC). Genome-wide DNA hybridization was performed to enrich the target region, and then sequenced using Illumina MiSeq second-generation sequencer. BWA aligner 0.7.10 was used to analyze the sequence data. Tophat 2 and Factera 1.4.3 were used to analyze the DNA translocation. ExAC and CLINVAR databases were used to analyze, filter and screen out high-quality mutation sites. Sanger sequencing was used to verify the mutation sites. Results: 1. In FNMTC susceptibility testing based on second-generation sequencing, the total amount of sequencing data produced by each sample was about 1.3-2.9 trillion (M) measurements. The average reads were 2.5M, 98.9% in genome and 54.3% in the target region of the second-generation sequencing. For the second-generation sequencing target region detected by FNMTC, the average sequencing depth was 500 x, and the minimum and maximum sequencing depth were 157 X and 1505 x, respectively. The average sequencing depth is more than 200 * (the ratio of the total number of bases to the genome size is one of the criteria for evaluating the sequencing quantity). The correlation between the coverage depth of each target area among different samples shows that the correlation coefficient between most pairs of samples is greater than 0.8. 2. A total of 45 high-quality heterozygous embryoline mutations were found in 63 patients with non-medullary thyroid carcinoma, including 38 single nucleotide mutations, 1 deletion mutation, 1 splice donor mutation, and 5 intra-frame insertion or deletion mutations; 47 patients with FNMTC carried 37 embryoline mutations, and 14 patients with SNMTC carried 8 embryoline mutations. The frequency of gene mutation carriers was higher than that of sporadic group (61.7% vs. 37.5%, P = 0.092); the most common embryonic line mutation gene was MSH6, followed by APC; the same embryonic line mutation sites were found in eight FNMTC families, namely APC L292F, A2778S, MSH6 G355S, A36V, MSH2 L719F, BRAF D22N, MEN1 G508D, BRCA1 SS955S, BRCA2 2050, BRCA2 G355S 8S and GNAS P459 PDAPAD PDSGAAR.3. Patients with non-medullary thyroid carcinoma were divided into two groups according to the mutation results: mutation group (n=29) and non-mutation group (n=18). A comparative analysis of the clinical and pathological characteristics showed that the central lymph node metastasis rate of the mutation group was significantly higher than that of the non-mutation group (68.6% vs. 30.8%, P = 0.003). The central lymph node metastasis rate in the offspring of FNMTC patients was higher than that in the parental group (100% vs. 42.9%, P = 0.019). Conclusion: The FNMTC susceptibility probe capture efficiency in this study was stable, and the mutation data obtained by total quality control (base mutation distribution, minimum allele frequency distribution, etc.) conformed to the single nucleotide polymorphism of embryonic line mutation. The application of second-generation sequencing technology in FNMTC sample susceptibility gene screening has helped us find some new mutation sites related to FNMTC, and suggested that different FNMTC families may carry different mutation genes of embryonic line. Preventive central lymph node dissection is necessary for central lymph node metastasis. Therefore, the detection of embryonic gene mutation by FNMTC susceptibility probe is of guiding significance for the treatment of FNMTC patients.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R736.1

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