遗传性耳聋分子诊断及梯级检测策略应用

遗传性耳聋是人类最常见的感觉障碍之一，具有高度遗传异质性。目前常用的遗传性耳聋分子诊断方法包括基因芯片、Sanger测序、靶向富集测序和全外显子组测序等，诊断率可达33.5%～56.67%，但还有相当一部分患者不能进行及时有效的分子病因诊断。考虑到患者家庭的经济负担及目前全外显子组/全基因组测序仍相对昂贵，根据患者情况提供包含多种检测手段的梯级诊断策略至关重要。因此，本文对遗传性耳聋分子诊断现状以及梯级检测在遗传性耳聋分子诊断中的应用进行综述，以期为诊断策略的选择提供参考。     

外显子组测序技术在人类疾病研究中的应用

外显子组测序是针对基因组中的蛋白质编码区,靶向富集外显子区域测序,以发现疾病相关遗传变异的技术。该技术近年越来越多地应用于发现人类基因组低频变异、鉴定单基因遗传病致病基因和肿瘤等复杂疾病易感基因研究,成为人类疾病相关变异研究的重要工具。综述了外显子组测序技术的基本原理及其在人类疾病相关基因研究中的应用。     

全基因组外显子测序及其应用

近年来,众多研究小组开展了大量的全基因组关联研究(Genome-wide association studies,GWAS),发现并鉴定了许多与复杂疾病/性状相关联的遗传变异,为复杂疾病发病机制的研究提供了重要线索。由于GWAS的结果存在假阳性、假阴性、检测到的单核苷酸多态性很少位于功能区以及对稀有变异和结构变异不敏感等问题,导致了其应用的局限性。而新一代测序技术的进步,促进了全基因组测序和全基因组外显子测序的快速发展,为解决上述问题提供了契机。全基因组外显子测序是利用序列捕获技术将全基因组外显子区域DNA捕捉并富集后进行高通量测序的基因组分析方法。由于其具有对常见和罕见变异高灵敏度,能发现外显子区绝大部分疾病相关变异以及仅需要对约1%的基因组进行测序等优点,促使全基因组外显子测序成为鉴定孟德尔疾病的致病基因最有效的策略,也被运用于复杂疾病易感基因的研究和临床诊断中。     

新一代测序技术在遗传性耳聋基因研究及诊断中的应用

超过50%的耳聋由遗传基因缺陷所致,伴随着基因组学技术的发展,耳聋分子遗传学研究逐渐成为耳科学研究的前沿领域。新一代高通量测序技术的出现,提供了以数据为导向的新的遗传性疾病研究模式,革新了人们对遗传性疾病的认识过程,使得对遗传性疾病的研究策略也发生了重大转变。近年来新一代测序技术(Next generation sequencing,NGS)在耳聋研究中的应用,大大加快了耳聋基因发现的速度,并逐渐向临床应用方向转化。文章总结了遗传性耳聋的特点和研究现状,以及新一代测序技术在耳聋研究中的应用和前景,以及基于NGS技术的耳聋基因研究和临床耳聋基因诊断的发展方向。     

基于全外显子组数据的ABO血型的基因判定

全外显子组测序研究已经应用在疾病、药物等方面,是临床研究中一种辅助分子诊断方法。该方法也逐步应用在临床ABO血型的判定中,由于目前临床血型判定的主要方法是血清学,疑难样本判定等问题无法解决,因此分子水平的基因测序方法提高了血型判定的准确性,比如PCR方法和基因芯片方法。为进一步提高ABO血型精细分型的准确性,通过分析全外显子组测序数据,开发了相关的VBA程序,能够快速自动化判定ABO精细分型,并且初步判定结果与临床判定结果一致,可以作为临床血型精确判定的辅助手段。     

一个常染色体显性遗传非综合征型耳聋家系致病基因及可能性分子遗传学机制研究

对一个中国常染色体显性遗传非综合征型耳聋家系(HBXG-712家系)进行了考察,主要采用了微阵列芯片法、全外显子组测序(whole exome sequencing,WES)技术以及Sanger测序法等方法检测该家系致病基因,另通过对蛋白产物进行三维重建进行生物信息学分析。经WES测序,本次研究共获得51个候选基因,通过将Sanger测序结果与标准序列进行比对及验证基因型与表型的一致性,确定EYA4为该家系致病基因,同时位于该基因上的错义突变c.T1301A(p.I434K)为其分子病因基础,且生物信息学分析认为该突变具有致病性。本研究结果提示:EYA4(c.T1301A;p.I434K)为HBXG-712家系耳聋的致病基因,提示发生于eya同源区域的错义突变可导致非综合征型耳聋的发生。     

一种结合单张芯片序列捕获和高通量测序技术测序外显子组的方法

随着高通量测序技术的发展,全外显子测序已经成为一种研究人类疾病的重要方法.本文展示了一种通过Nimblegen2.1M芯片进行外显子DNA序列捕获和高通量测序的方法,包括两步法文库制备.测序的平均覆盖深度达33倍时,95.6%的34M目标区域得到均衡覆盖,特异性达到80%.对比全基因组鸟枪法测序的结果,此方法在检测SNP时的假阳性率为0.97%,假阴性率为6.27%.本方法对于全基因组扩增的DNA也适用.结果显示,全外显子测序技术可以在大规模的群体研究和医学研究中起到重要作用.     

外显子组测序在人类疾病中的应用

据估计,约85%的人类遗传变异集中在蛋白编码区,因此对全部的蛋白编码区（外显子组）进行重测序,可以快速、有效地鉴定人类疾病遗传变异。以往鉴定孟德尔遗传病的致病基因多采用连锁分析结合候选定位克隆的方法,不仅耗时长,而且成功率低。2009年,科学家第一次应用外显子组测序在4名弗里曼谢尔登综合征（常染色体显性遗传病）中发现了位于MYH3中的点突变,显示出外显子组测序在孟德尔遗传病致病基因鉴定中的强大功效。就复杂疾病而言,传统的关联研究,包括全基因组关联研究（GWAS）,虽然鉴定了大量的常见变异,但对低频变异和罕见变异的检测能力十分有限;深度测序的发展为解决上述问题提供了良好的契机。本文就外显子组测序在人类疾病中的应用作一简要综述。     

应用全外显子测序和显微切割技术识别1 例肺癌患者高频突变基因的异质性探索

目的:通过对一例肺鳞癌患者全外显子测序来识别这例肺癌的可能致病基因,并通过显微切割初步探索这例肺癌肿瘤细胞的起源与演化。方法:利用全外显子测序技术对肺癌肿瘤组织和相应癌旁组织测序;用COSMIC肿瘤数据库比较分析统计出肺癌可能致病基因;用激光显微切割技术提取五个不同部位肿瘤细胞;巢式PCR扩增,一代测序验证基因分型。结果:发现了这例肺癌病人的7个高频突变基因:LPHN2、TP53、MYH2、TGM2、C10orf137、MS4A3和EP300;这些基因在10×镜下和20×镜下经显微切割的肺癌组织的5个不同部位上的基因分型不同。结论:我们通过全外显子测序发现了这例肺癌的7个可能致病基因,并初步探索了这例肺癌肿瘤细胞是多克隆起源的。     

定位于5q31-5q32的DFNA52的20个候选基因的突变筛查

为了克隆定位于5号染色体微卫星标记D5S2056和D5S638之间约8.8 cM的区间内的非综合征性常染色体显性遗传性耳聋 DFNA52 (OMIM: 607683)的致病基因, 文章根据基因在耳蜗组织的表达情况, 筛选出20个候选基因, 设计合成了扩增20个基因外显子及外显子与内含子交界的引物, 用DNA直接测序法进行序列变异分析。结果显示, 在基因外显子及侧翼区共发现了45个单核苷酸多态, 其中42个变异在多态数据库已报道, 其余3个为新发现的单核苷酸多态, 序列变异与疾病表型无共分离现象, 排除了这些基因外显子突变导致遗传性耳聋的可能性。     

Analysis of Downs syndrome with molecular techniques for future diagnoses

Down syndrome (DS) is a genetic disorder appeared due to the presence of trisomy in chromosome 21 in the G-group of the acrocentric region. DS is also known as non-Mendelian inheritance, due to the lack of Mendel’s laws. The disorder in children is identified through clinical symptoms and chromosomal analysis and till now there are no biochemical and molecular analyses. Presently, whole exome sequencing (WES) has largely contributed in identifying the new disease-causing genes and represented a significant breakthrough in the field of human genetics and this technique uses high throughput sequencing technologies to determine the arrangement of DNA base pairs specifying the protein coding regions of an individual’s genome. Apart from this next generation sequencing and whole genome sequencing also contribute for identifying the disease marker. From this review, the suggestion was to perform the WES is DS children to identify the marker region.     

Whole Exome Sequencing Reveals Homozygous Mutations in RAI1, OTOF,and SLC26A4 Genes Associated with Nonsyndromic Hearing Loss in Altaian Families (South Siberia)

Hearing loss (HL) is one of the most common sensorineural disorders and several dozen genes contribute to its pathogenesis. Establishing a genetic diagnosis of HL is of great importance for clinical evaluation of deaf patients and for estimating recurrence risks for their families. Efforts to identify genes responsible for HL have been challenged by high genetic heterogeneity and different ethnic-specific prevalence of inherited deafness. Here we present the utility of whole exome sequencing (WES) for identifying candidate causal variants for previously unexplained nonsyndromic HL of seven patients from four unrelated Altaian families (the Altai Republic, South Siberia). The WES analysis revealed homozygous missense mutations in three genes associated with HL. Mutation c.2168A>G (SLC26A4) was found in one family, a novel mutation c.1111G>C (OTOF) was revealed in another family, and mutation c.5254G>A (RAI1) was found in two families. Sanger sequencing was applied for screening of identified variants in an ethnically diverse cohort of other patients with HL (n = 116) and in Altaian controls (n = 120). Identified variants were found only in patients of Altaian ethnicity (n = 93). Several lines of evidences support the association of homozygosity for discovered variants c.5254G>A (RAI1), c.1111C>G (OTOF), and c.2168A>G (SLC26A4) with HL in Altaian patients. Local prevalence of identified variants implies possible founder effect in significant number of HL cases in indigenous population of the Altai region. Notably, this is the first reported instance of patients with RAI1 missense mutation whose HL is not accompanied by specific traits typical for Smith-Magenis syndrome. Presumed association of RAI1 gene variant c.5254G>A with isolated HL needs to be proved by further experimental studies.     

Functional consequences of Genetics variant in TMC1 and TMC2 within a United Arab Emirates family with Pre-lingual hearing loss

Hearing loss (HL) is the most prevalent sensory disorder whose etiology comes from environmental and/or genetic factors. Approximately 60 % of HL cases are due to mutations in genes responsible for maintaining a normal hearing function. Despite the monogenic inheritance of hereditary hearing loss (HHL), its diagnosis is challenging as both clinical and genetic heterogeneity characterizes it. Through the development of next-generation sequencing (NGS) techniques, the number of identified mutations responsible for HHL has increased exponentially during the last decade. Mutations in the TMC1 have been reported in several patients with nonsyndromic hereditary hearing loss (NSHHL), more precisely in cases with an autosomal recessive inheritance pattern. In this study, we conducted whole-exome sequencing (WES) analysis of a United Arabs Emirates (UAE) family with autosomal recessive nonsyndromic hearing loss (ARNSHL). This analysis revealed segregation of the TMC1 missense mutation c.596A > T (p.Asn199Ile) with the disease. Bioinformatics analysis supported the pathogenic effect of this mutation and predicted its impact at the proteomics level. Molecular docking analysis of TMC2WT, TMC2R123K, TMC2Q205R, and TMC2R123K + Q205R. Finally, protein docking results suggest a role for TMC2 variants in the phenotypic variability observed within the investigated family.     

Paired analysis of tumor mutation burden calculated by targeted deep sequencing panel and whole exome sequencing in non-small cell lung cancer

Owing to rapid advancements in NGS (next generation sequen-cing), genomic alteration is now considered an essential pre-dictive biomarkers that impact the treatment decision in many cases of cancer. Among the various predictive biomarkers, tumor mutation burden (TMB) was identified by NGS and was con-sidered to be useful in predicting a clinical response in cancer cases treated by immunotherapy. In this study, we directly com-pared the lab-developed-test (LDT) results by target sequencing panel, K-MASTER panel v3.0 and whole-exome sequencing (WES) to evaluate the concordance of TMB. As an initial step, the reference materials (n = 3) with known TMB status were used as an exploratory test. To validate and evaluate TMB, we used one hundred samples that were acquired from surgically resected tissues of non-small cell lung cancer (NSCLC) patients. The TMB of each sample was tested by using both LDT and WES methods, which extracted the DNA from samples at the same time. In addition, we evaluated the impact of capture re-gion, which might lead to different values of TMB; the evalu-ation of capture region was based on the size of NGS and target sequencing panels. In this pilot study, TMB was evalu-ated by LDT and WES by using duplicated reference samples; the results of TMB showed high concordance rate (R² = 0.887). This was also reflected in clinical samples (n = 100), which showed R² of 0.71. The difference between the coding sequence ratio (3.49%) and the ratio of mutations (4.8%) indicated that the LDT panel identified a relatively higher number of mutations. It was feasible to calculate TMB with LDT panel, which can be useful in clinical practice. Furthermore, a customized approach must be developed for calculating TMB, which differs according to cancer types and specific clinical settings.     

Whole exome sequencing,in silico and functional studies confirm the association of the GJB2 mutation p.Cys169Tyr with deafness and suggest a role for the TMEM59 gene in the hearing process

The development of next generation sequencing techniques has facilitated the detection of mutations at an unprecedented rate. These efficient tools have been particularly beneficial for extremely heterogeneous disorders such as autosomal recessive non-syndromic hearing loss, the most common form of genetic deafness. GJB2 mutations are the most common cause of hereditary hearing loss. Amongst them the NM_004004.5: c.506G > A (p.Cys169Tyr) mutation has been associated with varying severity of hearing loss with unclear segregation patterns. In this study, we report a large consanguineous Emirati family with severe to profound hearing loss fully segregating the GJB2 missense mutation p.Cys169Tyr. Whole exome sequencing (WES), in silico, splicing and expression analyses ruled out the implication of any other variants and confirmed the implication of the p.Cys169Tyr mutation in this deafness family. We also show preliminary murine expression analysis that suggests a link between the TMEM59 gene and the hearing process. The present study improves our understanding of the molecular pathogenesis of hearing loss. It also emphasizes the significance of combining next generation sequencing approaches and segregation analyses especially in the diagnosis of disorders characterized by complex genetic heterogeneity.     

Whole genome sequencing and its applications in medical genetics

Fundamental improvement was made for genome sequencing since the next-generation sequencing (NGS) came out in the 2000s. The newer technologies make use of the power of massively-parallel short-read DNA sequencing, genome alignment and assembly methods to digitally and rapidly search the genomes on a revolutionary scale, which enable large-scale whole genome sequencing (WGS) accessible and practical for researchers. Nowadays, whole genome sequencing is more and more prevalent in detecting the genetics of diseases, studying causative relations with cancers, making genome-level comparative analysis, reconstruction of human population history, and giving clinical implications and instructions. In this review, we first give a typical pipeline of whole genome sequencing, including the lab template preparation, sequencing, genome assembling and quality control, variants calling and annotations. We compare the difference between whole genome and whole exome sequencing (WES), and explore a wide range of applications of whole genome sequencing for both mendelian diseases and complex diseases in medical genetics. We highlight the impact of whole genome sequencing in cancer studies, regulatory variant analysis, predictive medicine and precision medicine, as well as discuss the challenges of the whole genome sequencing.      

Whole exome sequencing of a Saudi family and systems biology analysis identifies CPED1 as a putative causative gene to Celiac Disease

Celiac disease (CD) is a gastrointestinal disorder whose genetic basis is not fully understood. Therefore, we studied a Saudi family with two CD affected siblings to discover the causal genetic defect. Through whole exome sequencing (WES), we identified that both siblings have inherited an extremely rare and deleterious CPED1 genetic variant (c.241 A > G; p.Thr81Ala) segregating as autosomal recessive mutation, suggesting its putative causal role in the CD. Saudi population specific minor allele frequency (MAF) analysis has confirmed its extremely rare prevalence in homozygous condition (MAF is 0.0004). The Sanger sequencing analysis confirmed the absence of this homozygous variant in 100 sporadic Saudi CD cases. Genotype-Tissue Expression (GTEx) data has revealed that CPED1 is abundantly expressed in gastrointestinal mucosa. By using a combination of systems biology approaches like protein 3D modeling, stability analysis and nucleotide sequence conservation analysis, we have further established that this variant is deleterious to the structural and functional aspects of CPED1 protein. To the best of our knowledge, this variant has not been previously reported in CD or any other gastrointestinal disease. The cell culture and animal model studies could provide further insight into the exact role of CPED1 p.Thr81Ala variant in the pathophysiology of CD. In conclusion, by using WES and systems biology analysis, present study for the first-time reports CPED1 as a potential causative gene for CD in a Saudi family with potential implications to both disease diagnosis and genetic counseling.     

下一代半导体测序技术在遗传性心肌病分子诊断中的应用

遗传性心肌病(Inherited cardiomyopathy, ICM)是一种常见的遗传性心脏疾病,主要由基因突变所致,是青少年和年轻运动员猝死的最主要原因之一。到目前为止,已经发现约100个基因和其致病有关,这些基因相关的变异位点具有不同的致病机制。随着临床遗传检测在遗传疾病诊断中的应用,对遗传性心肌病的分子遗传学特性及其致病机制进行深入了解,是对该病遗传诊断的关键。下一代半导体测序仪在2010年底由美国Life Technologies公司发布,其以布满微孔的高密度半导体芯片为测序基础,具有快速、经济、灵敏性好、准确率高等特点,已经应用于遗传疾病的突变筛查。文章主要对遗传性心肌病的分子遗传学特性和下一代半导体测序技术在遗传性心肌病遗传检测中的应用以及面临的挑战进行了概括总结,有助于遗传性心肌病的诊断、预防和治疗。     

Mutations in glucokinase and other genes detected in neonatal and type 1B diabetes patient using whole exome sequencing may lead to disease-causing changes in protein activity

Monogenic diabetes is caused by mutations that reduce β-cell function. While Sanger sequencing is the standard method used to detect mutated genes. Next-generation sequencing techniques, such as whole exome sequencing (WES), can be used to find multiple gene mutations in one assay. We used WES to detect genetic mutations in both permanent neonatal (PND) and type 1B diabetes (T1BD).A total of five PND and nine T1BD patients were enrolled in this study. WES variants were assessed using VarioWatch, excluding those identified previously. Sanger sequencing was used to confirm the mutations, and their pathogenicity was established via the literature or bioinformatic/functional analysis. The PND and T1BD patients were diagnosed at 0.1–0.5 and 0.8–2.7?years of age, respectively. Diabetic ketoacidosis was present at diagnosis in 60% of PND patients and 44.4% of T1BD patients. We found five novel mutations in five different genes. Notably, patient 602 had a novel homozygous missense mutation c.1295C?>?A (T432?K) in the glucokinase (GCK) gene. Compared to the wild-type recombinant protein, the mutant protein had significantly lower enzymatic activity (2.5%, p?=?0.0002) and V_max (1.23?±?0.019 vs. 0.33?±?0.016, respectively; p?=?0.005). WES is a robust technique that can be used to unravel the etiologies of genetically heterogeneous forms of diabetes. Homozygous inactivating mutations of the GCK gene may have a significant role in PND pathogenesis.