人体基因组嵌合突变鉴定与定量的生物信息新方法开发及突变规律挖掘

Genomic mosaicism results from postzygotic mutations that occur during development or aging that give rise to two or more cell populations with distinct genome sequences within one individual. Postzygotic mosaicisms have long been known to play critical roles in cancer, and have been found to be involved in an increasing number of non-cancer diseases in the past decade. Recent research by our and other groups have discovered postzygotic mosaicisms in whole genome sequences of healthy individuals, affecting the somatic cells, germ cells, or both. However the mutation rate, mutation profile, tissue distribution, mutation origination, and selection of postzygotic mosaicism in healthy human individuals had remained unknown. Although case reports from our and other groups have identified genomic mosaicism in the parents of children with severe genetic disorders such as Dravet Syndrome, it remained unknown how many more of the “de novo” mutations causing these disorders might in fact have been inherited from parental mosaicism undetectable by current technology.. Here we propose to study genomic mosaicism with the following three specific aims:.(1).Develop new bioinformatics methods for the detection and quantification of genomic mosaicism without matched control tissues from whole-genome sequencing data, whole-exome sequencing data, and amplicon re-sequencing data, respectively, by designing Bayesian models..(2).Sequence the whole genomes of 33 postmortem tissues from 6 healthy donors, and compare the genomic mosaicism patterns in different tissues, ages, and chromosomal regions. Reconstruct tissue lineages tree and reverse-engineer the origination and propagation of the mutations, and elucidate the selection pressure of postzygotic mutations in different tissues..(3).Detect and quantify mosaicism in parents of 200 children with Dravet Syndrome using amplicon resequencing and our new bioinformatics methods. Identify what percentage of the “de novo” mutations might in fact be inherited from parental mosaicism undetectable by current Sanger-based technology. Elucidate the patterns of mosaicism in the sperm samples of the fathers.. We have established a strong computational hardware and software platform as well as a genomic and molecular biology wet lab. We has extensive research experience with next-generation sequencing analysis. We have already acquired the tissue samples from healthy donors and our co-PI, Dr. Yuehua Zhang, have collected blood samples from ~400 children with Dravet Syndrome and multiple samples from ~100 families, all with IRB approval. These can ensure the successful execution of the proposed research.. Postzygotic mosaicism is a fundamental yet under-investigated phenomenon in human genetics. Our proposed research and technology may not only elucidate the postzygotic mutation process in physiological states, but also provide important data for genetic counseling.

基因组嵌合是一种人体广泛存在的重要现象。然而由于技术局限，之前主要集中在对癌症病人的研究，对正常人多为个别突变的个案报道，只在近三、四年才在全基因组水平有所研究。申请人于2014年在国际上首次报道了在正常人外周血的全基因组序列中鉴定出单碱基嵌合突变，并发现两例Dravet综合征(DS)患儿的正常父母携带致病位点的嵌合突变。但是对体内不同组织的单碱基嵌合突变规律基本未知，DS新发突变有多少其实来源于父母嵌合突变也未知。本项目拟（1）开发生物信息学新方法，基于新一代全基因组测序、全外显子测序、扩增子测序、不依赖对照组织样本对嵌合突变进行鉴定与定量；（2）通过对6个正常捐献者尸体共33个组织的全基因组测序及分析，研究嵌合突变在不同组织、基因组区域、及年龄的突变率、突变规律、及选择压力；（3）在合作申请人临床收集的200位DS患儿的父母血液及父亲精液中鉴定嵌合突变的存在与频率，为遗传咨询提供指导。

合子后突变是指在受精卵形成后的发育或衰老过程中发生的DNA变异，之前的研究发现其在癌症发生中有至关重要的作用。但是，目前对于非癌症状况下合子后突变的分布和功能研究尚不清楚，主要原因是在基因组尺度下在非癌症样本中鉴定合子后突变存在很大技术挑战。本申请人的研究主要聚焦在开发新的基于新一代高通量测序鉴定和验证合子后突变的生物信息学方法，以及研究关于合子后突变在正常人类发育过程和多种神经发育疾病中的生物学问题。本申请人的研究组开发了1）第一个可以在没有对照样本的情况下从非癌症样本中精确鉴定单碱基合子后突变的生物信息学算法MosaicHunter；2）基于重测序的高通量验证单碱基合子后突变的方法PASM；3）关于非癌症疾病和健康人群中单碱基合子后突变的知识库MosaicBase；4）富集和鉴定合子后转座子插入突变的建库和生物信息学分析流程HAT-seq。以上面这些生物信息学方法和数据库，本研究组1）分析了单碱基合子后突变在健康人群不同组织和器官的基因组和空间分布，并发现了“早期胚胎发育”和“克隆扩张”是单碱基合子后突变的两种不同来源；2）使用数学建模的方法研究了单碱基合子后突变在胚胎发育中的发生率以及突变等位基因从父母到孩子的传递概率；3）揭示了单碱基合子后突变在多种神经发育疾病中的致病贡献，包括Dravet综合征、早发癫痫脑病、儿童交替性偏瘫、雷特综合征等；4）通过分析2361个来自Simons Foundation的孤独症单发家系，发现了单碱基合子后突变作为一种新的遗传致病因素可以解释大约6%的孤独症病例；5）系统研究了合子后转座子插入突变在雷特综合征和健康对照的大脑和其他器官中的基因组分布和特征，并发现了合子后转座子插入突变与雷特综合征的发生和症状的关联。上述的工作以12篇研究文章的形式均发表于遗传学、基因组学或生物信息学领域权威杂志，这些工作揭示了合子后突变这一过去被忽视的重要遗传因素在人类正常发育和疾病发生中所发挥的生物学作用。