人恶性胸膜间皮瘤DNA从头测序及基因突变分析

楚雄师范学院学报 ›› 2024, Vol. 39 ›› Issue (3): 38-49.

人恶性胸膜间皮瘤DNA从头测序及基因突变分析

邱璐^1,*, 王播勇^1,2, 田梦丽¹, 高顺玉¹, 熊海波¹, 熊伟²

1.楚雄师范学院资源环境与化学学院,云南楚雄 675000;
2.大理大学基础医学院,云南大理 671000

出版日期:2024-05-20 发布日期:2024-06-05

DNA De Novo Sequencing and Gene Mutation Analysis of Human Malignant Pleural Mesothelioma

Lu Qiu^1,*, Boyong Wang^1,2,#, Mengli Tian^1,#, Shunyu Gao¹, Haibo Xiong¹, Wei Xiong²

1. School of Resources, Environment and Chemistry, Chuxiong Normal University, Yunnan 675000;
2. Basic Medical College of Dali University, Dali Yunnan 671000

Online:2024-05-20 Published:2024-06-05
Contact: Qiu Lu, female, second grade Professor, mainly engaged in the study of molecular mechanism of tumor. E-mail： qiulu @cxtc.edu.cn, Tel. 18908787795
About author:^#Co-first author： Boyong Wang, Tian Mengli.
Supported by:
National Natural Science Foundation of China （No.11864002）

摘要/Abstract

摘要： 通过DNA从头测序分析人胸膜间皮瘤发生的高关联度突变基因。提取恶性胸膜间皮瘤（MPM）组织和正常胸膜组织DNA,构建基因文库,用Illumina HiSeqX Ten PE 150平台测序,将测序结果与人类基因组数据库的参考序列进行比对、注释,并对测序结果进行过滤、错误率分布检查、GC含量分布检查分析。MPM组织DNA平均过滤37829946 bp,错误率小于0.12%,GC含量占41.17%,而正常胸膜组织DNA平均过滤39089681 bp,错误率小于0.1%,GC含量占41.7%,两者测序质量均在Q 30（≥80%）以上,MPM为87.43%,正常胸膜为88.36%。以上高质量测序数据通过 BWA比对到参考基因组（GRCh 37/hg 19）,得到最初比对序列,利用重复标记后的比对序列进行覆盖度、深度等统计,覆盖深度达到10 X以上该突变位点可信。结果显示,实验病例XL14覆盖深度达到10 X的占 98.59 %,覆盖率达到99.83 %;对照病例Z5占98.50 %,覆盖率达到99.79 %。对该序列进行基因注释分析,发现一系列单核苷酸多态性、基因插入缺失、基因结构变异、基因拷贝数变异,筛选出总变异位点数29277个,可能致病的变异位点数22个,致病性的变异位点数5个,不确定变异有害性的位点数为3353个,其余变异位点均为良性。进一步对突变基因进行富集、关联性分析,预测出突变基因TXNDC2与人胸膜间皮瘤的发生高度相关,相关系数达到0.8以上;突变基因PIEN、ABCC1、UGT1A7、UGT1A3、UGT1A4、UGT1A9、ALDH3B1、UGT1A5等与人胸膜间皮瘤有一定关联性,关联度在0~0.2之间。基因TXNDC2、PIEN、ABCC1、UGT1A7、UGT1A3、UGT1A4、UGT1A9、ALDH3B1、UGT1A5的变异可能与人胸膜间皮瘤的发生发展有关。本实验为人胸膜间皮瘤分子诊断提供了参考。

关键词: 人胸膜间皮瘤, 从头测序, 单核苷酸多态性, 基因插入缺失, 基因结构变异, 基因拷贝数变异

Abstract: To analyze the high correlation mutation genes of human pleural mesothelioma by DNA de novo sequencing, DNA was extracted from malignant pleural mesothelioma （MPM） tissues and normal pleural tissues, and a gene library was constructed. The Illumina HiSeqX Ten PE 150 platform was used for sequencing. The sequencing results were compared and annotated with the reference sequence of the human genome database. The sequencing results were filtered, the error rate distribution was checked, and the GC content distribution was checked and analyzed. The average filtering of MPM tissue DNA was 37829946 bp, the error rate was less than 0.12 %, and the GC content accounted for 41.17 %. The average filtering of normal pleural tissue DNA was 39089681 bp, the error rate was less than 0.1 %, and the GC content accounted for 41.7 %. The sequencing quality of both was above Q30 （≥ 80 %）, MPM was 87.43 %, and normal pleura was 88.36 %. The above high-quality sequencing data were compared to the reference genome （GRCh 37 / hg19） by BWA, and the initial alignment sequence was obtained. The coverage and depth of the alignment sequence after repeated labeling were used for statistics. The mutation site was credible when the coverage depth reached 10 X or more. The results showed that 98.59 % of the experimental cases XL14 had a coverage depth of 10 X, and the coverage rate reached 99.83 %. The control case Z5 accounted for 98.50 %, and the coverage rate reached 99.79 %. Gene annotation analysis of the sequence revealed a series of single nucleotide polymorphisms, gene insertion and deletion, gene structure variation, and gene copy number variation. The total number of variation sites was 29277, and the number of possible pathogenic variation sites was 22. The number of pathogenic variation sites was 5, the number of sites with uncertain variation was 3353, and the remaining variation sites were benign. Further enrichment and correlation analysis of mutant genes were carried out to obtain the correlation map and predict the mutation.

Key words: human pleural mesothelioma, de novo sequencing, single nucleotide polymorphism, gene insertion deletion, gene structure variation, gene copy number variation.

中图分类号:

R734.3

邱璐, 王播勇, 田梦丽, 高顺玉, 熊海波, 熊伟. 人恶性胸膜间皮瘤DNA从头测序及基因突变分析[J]. 楚雄师范学院学报, 2024, 39(3): 38-49.

Lu Qiu, Boyong Wang, Mengli Tian, Shunyu Gao, Haibo Xiong, Wei Xiong. DNA De Novo Sequencing and Gene Mutation Analysis of Human Malignant Pleural Mesothelioma[J]. Journal of Chuxiong Normal University, 2024, 39(3): 38-49.

参考文献

[1] Emami H, Ilbeigi A, Khodadad K.An overview of asbestos and malignant pleural mesothelioma: an iranian perspective[J]. Asian Pacific journal of Cancer Prevention, 2017,18(10): 2619-2623.
[2] Brims F.Epidemiology and clinical aspects of malignant pleural mesothelioma[J]. Cancers, 2021,13:4149.
[3] Carbone M, Adusumilli P S, Alexander H R, et al. Mesothelioma: scientific clues for prevention, diagnosis, and therapy[J]. CA: A Cancer Journal For Clinicians, 2019,69(5): 402-429.
[4] Bibby A C, Tsim S, Kanellakis N, et al. Malignant pleural mesothelioma: an update on investigation, diagnosis and treatment[J]. European Respiratory Review, 2016,25(142): 472-486.
[5] Sinn K, Mosleh B, Hoda M A.Malignant pleural mesothelioma: recent developments[J]. Current Opinion In Oncology, 2020,33(1): 80-86.
[6] Husain A N, Colby T, Ordonez N, et al. Guidelines for pathologic diagnosis of malignant mesothelioma: 2012 update of the consensus statement from the International Mesothelioma Interest Group[J]. Archives of Pathology & Laboratory Medicine, 2013,137(5): 647-667.
[7] Bueno R, Stawiski E W, Goldstein L D, et al. Comprehensive genomic analysis of malignant pleural mesothelioma identifies recurrent mutations, gene fusions and splicing alterations[J]. Nature Genetics, 2016,48(4): 407-416.
[8] Woolhouse I, Bishop L, Darlison L, et al. British thoracic society guideline for the investigation and management of malignant pleural mesothelioma[J]. Thorax, 2018,73(Suppl 1): i1-i30.
[9] David R, Valerie R, Harvey P, et al. Recommendations for uniform definitions of surgical techniques for malignant pleural mesothelioma: a Consensus Report of the International Association for the Study of Lung Cancer International Staging Committee and the International Mesothelioma Interest Group[J]. Journal of Thoracic Oncology, 2011,6(8): 1304-1312.
[10] Yap T A, Aerts J G, Popat S, et al. Novel insights into mesothelioma biology and implications for therapy[J]. Nature Reviews. Cancer, 2017,17(8): 475-488.
[11] Sanger F, Nicklen S, Coulson A R.DNA sequencing with chain-terminating inhibitors[J]. Proceedings of the National Academy of Sciences of the United States of America, 1977,74(12): 5463-5467.
[12] Fedurco M, Romieu A, Williams S, et al. BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies[J]. Nucleic Acids Research, 2006,34(3): e22.
[13] Gerardo T, Anthony R, Milan F, et al. A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis[J]. Nucleic Acids Research, 2008,36(4): e25.
[14] Clarke J, Wu H, Jayasinghe L, et al. Continuous base identification for single-molecule nanopore DNA sequencing[J]. Nature Nanotechnology, 2009,4(4): 265-270.
[15] Nicole R.Cheap third-generation sequencing[J]. Nature Methods: Techniques for Life Scientists and Chemists, 2009,6(4): 244-245.
[16] Kimura K, Koike A.Analysis of genomic rearrangements by using the Burrows-Wheeler transform of short-read data[J]. BMC Bioinformatics, 2015,16(Suppl 18): S5.
[17] Kong Z Q, Ni L, Zhu M, et al. Analysis report of whole genome sequencing of a group of familial hereditary diseases[J]. Journal of Nanjing Medical University (Natural Science Edition), 2015,35(12): 1757-1760.