基因拷贝数异常的研究进展

基因拷贝数异常(copy number variations,CNVs)是广泛存在于人体基因组的一种结构变异现象,主要包括拷贝数的缺失、插入、重组以及多位点的复杂变异等。最初是在病人的基因组中发现,后来的研究表明在正常人体中也普遍存在。有关CNVs的研究将随机个体之间的基因组差异估计值大大提高,极大的改变了人们的认识。目前,关于CNVs的研究多处在初步探索阶段,CNVs如何导致疾病,以及如何引起基因等的改变而诱发疾病的机理也需更进一步的研究加以验证和证实。该文主要就近年来关于CNVs的研究进展作一综述。     

构建基于荧光多重cPCR的小鼠基因拷贝数检测方法

目的建立基于竞争性聚合酶链式反应(competitive polymerase chain reaction,cPCR)小鼠基因拷贝数变异(copy number variations,CNVs)的检测方法,用于检测野生小家鼠来源一号染色体替换系群体(population of specific chromosome 1 substitution strains,PCSSs)的CNVs。方法选取小鼠一号染色体上11个CNVs位点,及7、9和X染色体上各1个内对照位点,分别构建克隆质粒为竞争性粒模板,应用cPCR技术,建立荧光通用引物多重cPCR检测方法。结果多重cPCR方案适用于小鼠一号染色体上11个CNV位点的拷贝数检测,且能准确检测X染色体的拷贝数。结论实现小鼠快速、高通量的CNVs检测,可准确检测小鼠1号染色体中11个CNV位点的拷贝数变异。     

基因结构变异检测方法综述

在人类基因组中结构变异(SVs),拷贝数变化(CNVs),单核苷酸多态性(SNP)是非常普遍的,而且和人类健康与疾病密切相关,因此检测这些结构变异对于人类生命健康非常重要。基于第二代基因测序平台,目前已经有很多结构变异检测算法,这些算法主要分为五大类:微阵列方法、读对方法、读深方法、分裂读取方法、序列组装方法。本文系统地阐述了这五类方法的基本原理、优缺点以及使用范围,并简要介绍了每一种方法的经典检测算法及应用范围、检测性能等,并对未来检测算法的研究提出了展望。     

利用人类全基因组亚硫酸氢盐测序数据检测CNVs的研究

DNA甲基化异常可能导致拷贝数变异(copy number variants,CNVs)的发生,而CNVs的发生又可能改变DNA甲基化水平。全基因组亚硫酸氢盐测序(whole genome bisulfite sequencing,WGBS)技术能够获得DNA水平的测序数据,具有挖掘CNVs的潜力和优势,但利用WGBS数据挖掘CNVs的效果尚不清楚。本研究选取了5款检测CNVs不同策略的软件(BreakDancer、cn.mops、CNVnator、DELLY、Pindel),基于人类的真实(2.62 billion reads)和模拟(12.35 billion reads)测序数据,进行150次CNVs检测,评估CNVs检出数量、精确率、召回率、相对检出能力、内存占用和运行时间等指标,旨在讨论利用WGBS数据检测CNVs的最佳方案。基于真实WGBS数据,Pindel检出缺失型和重复型CNVs的数量最多,CNVnator对缺失型CNVs的检测精确率最高,cn.mops对重复型CNVs的检测精确率最高,Pindel对缺失型CNVs的召回率最高,cn.mops对重复型CNVs的召回率最高...     

拷贝数变异的全基因组关联分析

基因组拷贝数变异(copy number variations,CNVs)是指与基因组参考序列相比,基因组中≥1 kb的DNA片段插入、缺失和/或扩增,及其互相组合衍生出的复杂变异．由于其具有分布范围广、可遗传、相对稳定和高度异质性等特点,目前认为,CNVs是一种新的可以作为疾病易感标志的基因组DNA多态性,其变异引起的基因剂量改变可以导致表型改变．最近,一种基于CNVs的新的疾病易感基因鉴定策略——CNV全基因组关联分析开始出现,这一策略和传统的基于单核苷酸多态性的关联分析具有互补性,通过认识基因组结构变异可以认识复杂疾病的分子机制和遗传基础．     

拷贝数变异: 基因组多样性的新形式

基因拷贝数变异是指DNA片段大小范围从kb到Mb的亚微观突变, 是一可能具有致病性、良性或未知临床意义的基因组改变。Fosmid末端配对序列比较策略、比较基因组杂交芯片是当前较多使用的检测手段。染色体非等位的同源重排、非同源突变和非b DNA结构是造成基因组拷贝数变异的重要原因。拷贝数变异可导致不同程度的基因表达差异, 对正常表型的构成及疾病的发生发展具有一定作用。文章在总结基因拷贝数变异的认识过程和研究策略的基础上, 分析了拷贝数变异的形成和作用机制, 介绍了第一代人类基因组拷贝数变异图谱, 阐述了拷贝数变异研究的临床意义, 提示在探索疾病相关的遗传变异时不能错失拷贝数变异这一基因组多样性的新形式。     

高通量测序数据的基因组拷贝数变异检测方法综述

拷贝数变异是指基因组中发生大片段的DNA序列的拷贝数增加或者减少。根据现有的研究可知,拷贝数变异是多种人类疾病的成因,与其发生与发展机制密切相关。高通量测序技术的出现为拷贝数变异检测提供了技术支持,在人类疾病研究、临床诊疗等领域,高通量测序技术已经成为主流的拷贝数变异检测技术。虽然不断有新的基于高通量测序技术的算法和软件被人们开发出来,但是准确率仍然不理想。本文全面地综述基于高通量测序数据的拷贝数变异检测方法,包括基于reads深度的方法、基于双末端映射的方法、基于拆分read的方法、基于从头拼接的方法以及基于上述4种方法的组合方法,深入探讨了每类不同方法的原理,代表性的软件工具以及每类方法适用的数据以及优缺点等,并展望未来的发展方向。     

家养动物基因组拷贝数变异研究进展及其育种应用展望

家养动物参考基因组组装的不断完善和群体重测序数据的持续增加促进了基因组中大量变异的发现。基因组上的变异主要包括单核苷酸变异(SNP)和拷贝数变异(CNV)两种类型。相对于数量众多,已经被广泛研究和用作分子育种标记SNP,目前已经被发现和经过实验验证其功能的CNV数量较少,鲜有被直接用作分子标记进行育种的报道。CNV片段长度大、在基因组中普遍存在且比SNP变异覆盖的基因组范围更广,所以可能对农艺性状造成很大影响,其在畜禽基因组研究和育种应用中具有广阔前景。重点讨论了家养动物CNV的研究进展,并对其在家养动物育种中的应用进行了分析展望。     

ABCC4与人类肿瘤

ABCC4（ATP-binding cassette transporter family class C4,ABCC4）是ABC蛋白家族成员,主要参与转运机体物质代谢中产生的有机阴离子和一些异型生物质等生物学功能。近年研究发现某些人类肿瘤存在Abcc4基因的拷贝数变异,主要表现为Abcc4基因拷贝数增加和ABCC4蛋白过表达,这些改变与肿瘤发生发展、耐药,以及治疗疗效具有相关性。该文综述了Abcc4基因的拷贝数变异和异常表达与肿瘤生物学特性的关系,探讨ABCC4在肿瘤发生发展中的作用机制。     

人类基因组结构变异

基因组结构变异通常是指基因组内大于1 kb的DNA片段缺失、插入、重复、倒位、易位以及DNA拷贝数目变化(CNVs)。人类基因组结构变异涉及数千片段不连续的基因组区域, 含数百万DNA碱基对, 可含数个基因及调控序列, 多种基因功能因此缺失或改变, 导致机体表型变化、疾病易感性改变或发生疾病。对基因组结构变异的研究, 有助于用动态的观点全面分析基因组遗传变异得到整合的基因型, 理解结构变异的潜在医学作用及机体整体功能的复杂性。文章从人类基因组结构变异的类型、研究方法, 对个体表型、疾病及生物进化的影响等方面综合阐述人类基因组结构变异的最新研究进展。     

Bias of selection on human copy-number variants

Although large-scale copy-number variation is an important contributor to conspecific genomic diversity, whether these variants frequently contribute to human phenotype differences remains unknown. If they have few functional consequences, then copy-number variants (CNVs) might be expected both to be distributed uniformly throughout the human genome and to encode genes that are characteristic of the genome as a whole. We find that human CNVs are significantly overrepresented close to telomeres and centromeres and in simple tandem repeat sequences. Additionally, human CNVs were observed to be unusually enriched in those protein-coding genes that have experienced significantly elevated synonymous and nonsynonymous nucleotide substitution rates, estimated between single human and mouse orthologues. CNV genes encode disproportionately large numbers of secreted, olfactory, and immunity proteins, although they contain fewer than expected genes associated with Mendelian disease. Despite mouse CNVs also exhibiting a significant elevation in synonymous substitution rates, in most other respects they do not differ significantly from the genomic background. Nevertheless, they encode proteins that are depleted in olfactory function, and they exhibit significantly decreased amino acid sequence divergence. Natural selection appears to have acted discriminately among human CNV genes. The significant overabundance, within human CNVs, of genes associated with olfaction, immunity, protein secretion, and elevated coding sequence divergence, indicates that a subset may have been retained in the human population due to the adaptive benefit of increased gene dosage. By contrast, the functional characteristics of mouse CNVs either suggest that advantageous gene copies have been depleted during recent selective breeding of laboratory mouse strains or suggest that they were preferentially fixed as a consequence of the larger effective population size of wild mice. It thus appears that CNV differences among mouse strains do not provide an appropriate model for large-scale sequence variations in the human population.     

Whole Genome Distribution and Ethnic Differentiation of Copy Number Variation in Caucasian and Asian Populations

Although copy number variation (CNV) has recently received much attention as a form of structure variation within the human genome, knowledge is still inadequate on fundamental CNV characteristics such as occurrence rate, genomic distribution and ethnic differentiation. In the present study, we used the Affymetrix GeneChip® Mapping 500K Array to discover and characterize CNVs in the human genome and to study ethnic differences of CNVs between Caucasians and Asians. Three thousand and nineteen CNVs, including 2381 CNVs in autosomes and 638 CNVs in X chromosome, from 985 Caucasian and 692 Asian individuals were identified, with a mean length of 296 kb. Among these CNVs, 190 had frequencies greater than 1% in at least one ethnic group, and 109 showed significant ethnic differences in frequencies (p<0.01). After merging overlapping CNVs, 1135 copy number variation regions (CNVRs), covering approximately 439 Mb (14.3%) of the human genome, were obtained. Our findings of ethnic differentiation of CNVs, along with the newly constructed CNV genomic map, extend our knowledge on the structural variation in the human genome and may furnish a basis for understanding the genomic differentiation of complex traits across ethnic groups.     

人工诱变技术在植物抗病育种中的应用(综述)

介绍人工诱变技术在植物抗病育种中的主要成就,并探讨其发展方向及前景。人工诱变技术与杂交育种、基因转移及离体筛选等手段相结合,提高了育种效率,拓宽了抗病育种的范围。该技术在植物抗病育种中的成功应用,将有利于培育植物抗病新品种,促进农业的增产增收及可持续发展。     

Replication stress and mechanisms of CNV formation

Copy number variants (CNVs) are widely distributed throughout the human genome, where they contribute to genetic variation and phenotypic diversity. De novo CNVs are also a major cause of numerous genetic and developmental disorders. However, unlike many other types of mutations, little is known about the genetic and environmental risk factors for new and deleterious CNVs. DNA replication errors have been implicated in the generation of a major class of CNVs, the nonrecurrent CNVs. We have found that agents that perturb normal replication and create conditions of replication stress, including hydroxyurea and aphidicolin, are potent inducers of nonrecurrent CNVs in cultured human cells. These findings have broad implications for identifying CNV risk factors and for hydroxyurea-related therapies in humans.     

Origins and breakpoint analyses of copy number variations: up close and personal

Array-based methods have enabled the detection of many genomic gains and losses. These are stated as copy number variants (CNVs) and comprise up to 13% of the human genome. Based on their breakpoints and modes of formation CNVs are termed recurrent or nonrecurrent. Recurrent CNVs are flanked by low copy repeats and are of a fixed size. They arise as a result of misalignment during meiosis by a mechanism named nonallelic homologous recombination. Several of such recurrent CNVs have been linked to human diseases. Nonrecurrent CNVs, which are not flanked by low copy repeats, are of variable size and may arise via mechanisms like nonhomologous end joining and replication-based mechanisms described by the fork stalling and template switching and microhomology-mediated break-induced replication models. It is becoming clear that most disease-causing CNVs are nonrecurrent and generally arise via replication-based mechanisms. Furthermore, it is now appreciated that genomic features other than low copy repeats play a role in the formation of nonrecurrent CNVs. This review will discuss the different mechanisms of CNV formation and how high resolution analyses of CNV breakpoints have added to our knowledge of their precise structure.     

意大利蜜蜂染色体DNA非编码区抗白垩病相关的SNP的筛选与验证

【目的】筛选验证意大利蜜蜂Apis mellifera ligustica染色体DNA非编码区与抗白垩病相关的SNP。【方法】本研究将蜜蜂球囊菌Ascosphaera apis孢子接种于人工饲养的意大利蜜蜂3日龄幼虫,根据是否存在白垩病症状进而筛选出抗病个体和易感个体。基于前期重测序结果中意大利蜜蜂第2和11号染色体DNA非编区与抗白垩病相关的SNP信息,利用PCR测序的方法筛选并验证意大利蜜蜂幼虫第2和11号染色体DNA非编码区与幼虫抗白垩病相关的55个SNP。【结果】发现位于意大利蜜蜂第11号染色体LOC100578413基因5′端的非编码区的SNP(T14570310C)在抗病个体中T等位基因频率高于C等位基因频率,且抗病个体中的T等位基因频率显著高于易感幼虫中的T等位基因频率,表明该SNP位点与抗白垩病相关。该分子标记对抗性个体和易感个体的判断结果与前期筛选的编码区SNP(C2587245T)分子标记的结果一致。【结论】筛选并验证意大利蜜蜂第11号染色体DNA非编码区的SNP(T14570310C)与抗白垩病相关。该位点为抗白垩病分子辅助选育提供新的分子标记,在意大利蜜蜂白垩病早期检测和培育白垩病抗性的蜂种方面具有重要意义。     

High-resolution copy-number variation map reflects human olfactory receptor diversity and evolution

Olfactory receptors (ORs), which are involved in odorant recognition, form the largest mammalian protein superfamily. The genomic content of OR genes is considerably reduced in humans, as reflected by the relatively small repertoire size and the high fraction ( approximately 55%) of human pseudogenes. Since several recent low-resolution surveys suggested that OR genomic loci are frequently affected by copy-number variants (CNVs), we hypothesized that CNVs may play an important role in the evolution of the human olfactory repertoire. We used high-resolution oligonucleotide tiling microarrays to detect CNVs across 851 OR gene and pseudogene loci. Examining genomic DNA from 25 individuals with ancestry from three populations, we identified 93 OR gene loci and 151 pseudogene loci affected by CNVs, generating a mosaic of OR dosages across persons. Our data suggest that approximately 50% of the CNVs involve more than one OR, with the largest CNV spanning 11 loci. In contrast to earlier reports, we observe that CNVs are more frequent among OR pseudogenes than among intact genes, presumably due to both selective constraints and CNV formation biases. Furthermore, our results show an enrichment of CNVs among ORs with a close human paralog or lacking a one-to-one ortholog in chimpanzee. Interestingly, among the latter we observed an enrichment in CNV losses over gains, a finding potentially related to the known diminution of the human OR repertoire. Quantitative PCR experiments performed for 122 sampled ORs agreed well with the microarray results and uncovered 23 additional CNVs. Importantly, these experiments allowed us to uncover nine common deletion alleles that affect 15 OR genes and five pseudogenes. Comparison to the chimpanzee reference genome revealed that all of the deletion alleles are human derived, therefore indicating a profound effect of human-specific deletions on the individual OR gene content. Furthermore, these deletion alleles may be used in future genetic association studies of olfactory inter-individual differences.     

A study of CNVs as trait-associated polymorphisms and as expression quantitative trait loci

We conducted a comprehensive study of copy number variants (CNVs) well-tagged by SNPs (r(2)≥ 0.8) by analyzing their effect on gene expression and their association with disease susceptibility and other complex human traits. We tested whether these CNVs were more likely to be functional than frequency-matched SNPs as trait-associated loci or as expression quantitative trait loci (eQTLs) influencing phenotype by altering gene regulation. Our study found that CNV-tagging SNPs are significantly enriched for cis eQTLs; furthermore, we observed that trait associations from the NHGRI catalog show an overrepresentation of SNPs tagging CNVs relative to frequency-matched SNPs. We found that these SNPs tagging CNVs are more likely to affect multiple expression traits than frequency-matched variants. Given these findings on the functional relevance of CNVs, we created an online resource of expression-associated CNVs (eCNVs) using the most comprehensive population-based map of CNVs to inform future studies of complex traits. Although previous studies of common CNVs that can be typed on existing platforms and/or interrogated by SNPs in genome-wide association studies concluded that such CNVs appear unlikely to have a major role in the genetic basis of several complex diseases examined, our findings indicate that it would be premature to dismiss the possibility that even common CNVs may contribute to complex phenotypes and at least some common diseases.