外显子测序技术在疾病研究中的应用

新一代测序技术的发展为疾病研究带来了新的机遇.在现阶段,外显子测序与全基因组测序相比,不仅费用更低,数据的阐释也更为简单.通过对疾病样本进行外显子组捕获测序,不仅能快速发现罕见遗传疾病的致病基因,也能用于研究多基因引起的常见疾病,如癌症、糖尿病、高血压等,来揭示这些疾病的遗传致病机理.外显子测序技术是当前的热点技术,极...     

全外显子测序产前诊断Walker-Warburg综合征

目的利用全外显子测序技术对患有脑积水和先天性心脏病的胎儿进行产前诊断,并指导再次妊娠。方法提取胎儿羊水细胞DNA以及父母外周血DNA,采用外显子测序技术进行检测,经过建库、杂交捕获、测序等过程,将得到的数据经过比对、软件分析、查找数据库及文献等,根据美国医学遗传学与基因组学学会(ACMG,2015)原则寻找胎儿的致病基因,并利用Sanger测序对致病位点进行验证。结果全外显子测序技术检测结果发现患儿POMT1基因发生复合杂合突变,分别遗传自母亲的经典剪切位点突变c.605+1GA(IVS7)和遗传自父亲的移码突变c.1367_c.1368(exon 15)ins GA,p.L456Lfs*80;Sanger测序验证结果与外显子捕获测序结果一致。胎儿为Walker-Warburg综合征患者。胎儿父母最终决定终止妊娠。结论利用全外显子测序技术可快速可靠地诊断Walker-Warburg综合征患者,在临床决策及产前咨询中发挥重要作用。     

全外显子及靶向文库捕获测序在多囊肾病基因诊断中的应用比较

目的 比较全外显子和靶向文库捕获高通量测序对多囊肾病相关基因的检测效率.方法 对6份多囊肾病标本(包括一份低比例嵌合变异标本)分别进行全外显子捕获或靶目标捕获2种方法建立文库,Illumina HiSeq 2000仪器连续双向测序.结果 以PKD1、PKD2和PKHD1 3个基因作为目标序列,靶向捕获法平均测序深度为190倍,5倍以上测序深度占全部有效序列的85.59％,靶区域覆盖度95％以上,但PKD1第一外显子仍有200～300 bp区域不能覆盖;全外显子捕获法平均测序深度为28.34倍,5倍以上测序深度占全部有效序列的55.35％,目标区域覆盖度较低,PKD1外显子覆盖度小于40％. 结论 靶向文库捕获测序法具有较高的敏感性、准确性,更适合PKD基因变异的检测,但高通量测序技术对PKD1基因检测仍有不足之处.     

全基因组与外显子组测序的应用评估

随着新一代测序技术的不断成熟完善,人类基因组和外显子组测序得到了迅猛发展。测序技术的高通量性致使测序价格越来越低廉,使全基因组和外显子组测序真正用于临床诊断成为现实。作为医学预测和个体化医疗的重要工具之一,全基因组和外显子组测序应用范围广泛,该文针对其在遗传性疾病基因变异中的诊断进行讨论,同时为临床医生如何申请测序,测序报告解释,以及如何与患者沟通检测结果提供了新视角。     

第二代测序技术及其在急性髓系白血病和骨髓增生异常综合征中的运用

第2代测序技术（next-generation sequencing,NGS）悄然取代经典的Sanger测序技术,成为科研人员发掘人类肿瘤疾病遗传学秘密的最实用、最可靠的方法。随着技术的不断更新及完善,进行全基因组测序不再遥不可及。近年来,部分科学家将此项技术运用于血液系统恶性肿瘤的研究,积极推动急性髓系白血病、骨髓增生异常综合征的全基因组测序的进程,期待从源头上找出部分血液系统恶性肿瘤的致病机理。本文就第2代测序技术及全基因组测序、外显子基因组测序和转录基因组测序在急性髓系白血病、骨髓增生异常综合征两种疾病中的运用进行综述。     

第二代测序技术及其在急性髓系白血病和骨髓增生异常综合征中的运用

第2代测序技术(next-generation sequencing,NGS)悄然取代经典的Sanger测序技术,成为科研人员发掘人类肿瘤疾病遗传学秘密的最实用、最可靠的方法.随着技术的不断更新及完善,进行全基因组测序不再遥不可及.近年来,部分科学家将此项技术运用于血液系统恶性肿瘤的研究,积极推动急性髓系白血病、骨髓增生异常综合征的全基因组测序的进程,期待从源头上找出部分血液系统恶性肿瘤的致病机理.本文就第2代测序技术及全基因组测序、外显子基因组测序和转录基因组测序在急性髓系白血病、骨髓增生异常综合征两种疾病中的运用进行综述.     

PRRT2 c.649C>T突变导致智力障碍以及蛋白质表达异常

摘要:目的?利用全外显子测序技术寻找1例智力障碍合并癫痫及运动障碍患者的遗传学病因，并探究其致病机制。方法?提取患儿及父母双方的外周血DNA，使用外显子测序技术寻找患儿的致病基因，并使用Sanger测序验证致病位点。利用小鼠cDNA诱导克隆该致病基因并构建质粒，采用western blot检测致病基因蛋白与野生型蛋白的表达情况。结果?全外显子测序检测发现该患者PRRT2基因存在无义突变(c.649C>T;p.R217X)，遗传自母亲；Sanger测序验证结果与外显子捕获测序结果相一致。western blot证实小鼠PRRT2?R223X突变蛋白(对应人PRRT2 R217X)不能正常表达。结论?PRRT2基因c.649C>T突变可能是该例患者智力障碍的致病原因。突变的PRRT2?R217X蛋白不能正常表达。     

全基因组外显子测序捕获黏多糖贮积症Ⅶ型的致病基因GUSB

目的鉴定黏多糖贮积症Ⅶ型患者的致病基因,为产前诊断提供新的方法。方法应用全基因外显子组技术捕获该患者致病基因,结合现代生物信息学方法,采用Sanger测序验证基因突变位点是否存在该患者家系成员和健康人基因组内。结果发现该患者基因β-葡萄糖醛酸酶(GUSB)(g.65444706GA)突变,健康人未见该突变,该碱基的突变导致苏氨酸变成丝氨酸,从而导致GUSB活性缺乏或GUSB量的不足。结论基因GUSB(g.65444706GA)突变是该患者的致病基因,全基因组外显子测序能解决传统定位克隆技术常常面临该病家系患者太少、病例散发、基因位点的异质性、外显不全及候选基因太多等难题,为该病产前诊断提供了新的途径。     

运用全外显子组测序技术诊断非综合征性耳聋基因变异

目的 评价全外显子组测序(whole-exome sequencing,WES)技术在非综合征性耳聋患者基因变异诊断临床应用的可行性. 方法 利用Illumina Hiseq2000平台对15例耳聋患者标本进行全外显子捕获测序,重点关注80个耳聋相关基因的序列捕获效率及变异检测情况并运用常规测序验证. 结果 目标区域(region of interested,ROI)20X以上平均覆盖度为98％;在15份耳聋患者标本中均发现致病性变异,涉及GJB2、SLC26A4、DSPP、TECTA和CHD7基因,其中,无义突变2个,移码突变4个,剪切位点变异1个,其余为错义突变.经常规Sanger测序验证,结果一致. 结论 全外显子组测序技术可快速可靠诊断非综合征性耳聋患者基因变异.     

二代测序技术在儿童T淋巴母细胞淋巴瘤全外显子测序中的应用

目的建立从石蜡包埋的儿童T淋巴母细胞淋巴瘤（T-LBL）病理组织中提取基因组DNA,采用二代测序技术行全外显子测序检测的方法并分析其可行性。方法从10例儿童T-LBL石蜡包埋组织中提取基因组DNA,PCR反应扩增后采用二代测序技术行全外显子测序检测并确定致病位点,所获得的致病突变采用Sanger测序法测序确认,比较两种方法的一致性。结果10例石蜡包埋T-LBL组织标本中均成功提取到基因组DNA,采用二代测序技术开展全外显子测序测序均检测到致病突变;二代测序检测结果经Sanger测序法验证,证实致病突变确实存在,且与Sanger测序结果一致。结论基于二代测序技术的全外显子测序测序可用于检测石蜡包埋的儿童T-LBL病理标本的致病突变。     

胃转流术的动物模型选择及现状

2型糖尿病是严重危害人类健康的慢性疾病,经过多年的临床及基础研究,证实了胃转流术(GBP)对2型糖尿病具有很好的治疗作用,但其机制尚未完全阐明。为了明确GBP治疗2型糖尿病的具体机制,运用动物模型研究其机制是必不可少的,而选择适宜的动物模型显得尤为重要。与广泛运用于动物研究中的2型糖尿病大鼠模型相比较,非啮齿类2型糖尿病动物模型具有以下优点:更加贴近人类生理,能够更好地模拟2型糖尿病的发病过程,更适宜外科手术操作。非啮齿类2型糖尿病动物模型主要通过动物本身的基因缺陷(自发性)、基因敲除技术、高脂饮食诱导、化学药物诱导四种方法建立。该篇综述着眼于GBP术的基础研究需求,总结了较为常用的2型糖尿病动物模型资料。     

Genome-wide screen in obese pedigrees with type 2 diabetes mellitus from a defined Dutch population

A genome scan was performed in obese type 2 diabetes mellitus pedigrees to identify susceptibility loci involved in obesity-driven type 2 diabetes mellitus. We studied the 20% most obese diabetes pedigrees from a confined Dutch population from around the town of Breda. Previously we, and others, have already shown that a susceptibility locus influencing obesity in diabetes may reside on chromosome 18p11. We now report evidence to also suggest linkage for type 2 diabetes in these obese pedigrees on chromosome regions 11p (genome-wide P-value 相似文献   

Monogenic diabetes: a gateway to precision medicine in diabetes

Monogenic diabetes refers to diabetes mellitus (DM) caused by a mutation in a single gene and accounts for approximately 1%–5% of diabetes. Correct diagnosis is clinically critical for certain types of monogenic diabetes, since the appropriate treatment is determined by the etiology of the disease (e.g., oral sulfonylurea treatment of HNF1A/HNF4A-diabetes vs. insulin injections in type 1 diabetes). However, achieving a correct diagnosis requires genetic testing, and the overlapping of the clinical features of monogenic diabetes with those of type 1 and type 2 diabetes has frequently led to misdiagnosis. Improvements in sequencing technology are increasing opportunities to diagnose monogenic diabetes, but challenges remain. In this Review, we describe the types of monogenic diabetes, including common and uncommon types of maturity-onset diabetes of the young, multiple causes of neonatal DM, and syndromic diabetes such as Wolfram syndrome and lipodystrophy. We also review methods of prioritizing patients undergoing genetic testing, and highlight existing challenges facing sequence data interpretation that can be addressed by forming collaborations of expertise and by pooling cases.     

脂联素单核苷酸多态性+45位点的表达在2型糖尿病中的研究

目的研究脂联素单核苷酸多态性SNP＋45位点基因在广州汉族2型糖尿病患者中的分布,评价该基因是否为2型糖尿病的易感基因。方法采用聚合酶链式反应-限制性内切酶长度多态性技术,对2型糖尿病（T2DM）组和健康对照（CON）组人群进行脂联素基因SNP＋45多态位点基因的检测。结果T2DM组人群脂联素SNP＋45位点的基因型分布：TT型56．4％、GT型34．9％、GG型8．7％。CON组脂联素SNP＋45位点的基因型分布：TT型42．0％、GT型48．2％、GG型9．8％。结论广州地区T2DM组与CON组脂联素SNP＋45位点的基因型差异有统计学意义。T2DM组T／T型基因明显增多;CON组GT＋GG型基因增多。提示脂联素基因第2外显子45位点多态性与2型糖尿病相关,TT基因型者具有较高的2型糖尿病易感性。     

中国人群脂联素基因+45位点基因多态性与2型糖尿病相关性的Meta分析

目的通过Meta分析评估中国人群脂联素基因外显子二+45位点单核苷酸多态性与2型糖尿病的相关性。方法计算机检索PubMed、Ovid、CBM、VIP、CNKI和WanFang Data中关于中国人群脂联素基因SNP45与2型糖尿病相关性的病例-对照研究,并辅以文献追溯。检索时限均从建库至2012年6月。由2名评价者按纳入排除标准独立选择文献、提取资料、评价质量后采用Stata 11.0软件进行Meta分析,采用分层和敏感性分析评估结果稳定性,并用Begg’s漏斗图和Egger’s法评估文献的发表偏倚。结果共纳入21篇文献(含22个研究),其中2型糖尿病患者3 273例,对照人群2 597例。Meta分析结果显示,无论在显性、隐性或加性遗传模式下,两组差异均有统计学意义[OR(95%CI)分别为1.36(1.04,1.78)、2.07(1.55,2.75)、2.44(1.59,3.75)]。结论脂联素基因外显子二+45位点基因多态性与中国人群的2型糖尿病具有相关性。不论是在显性、隐性还是在加性遗传模式下,G等位基因均为2型糖尿病的危险因素。     

The presence of family history and the development of type 2 diabetes mellitus risk factors in rural children.

Type 2 diabetes mellitus is reaching epidemic proportions among children and adolescents. School health fairs offer an opportunity to identify children with risk factors for the development of type 2 diabetes mellitus. This study identified selected risk factors (i.e., high-risk racial/ethnic group, obesity, elevated blood pressure, elevated casual blood glucose, elevated total cholesterol, and the presence of acanthosis nigricans) for development of type 2 diabetes mellitus in rural children with or without a family history of diabetes during annual school health fairs. Of the children screened, 40% (673) presented with two or more of the identified risk factors for type 2 diabetes mellitus. The presence of multiple risk factors in participants reporting a positive family history of diabetes mellitus versus those with no family history was not statistically significant. Based on the study results, factors other than family history may be more predictive for the development of type 2 diabetes mellitus in rural school children.     

HLA antigens in insulin-dependent diabetes mellitus.

Diabetes mellitus is largely determined by genetic factors but environmental factors are necessary to convert genetic susceptibility into overt disease. Studies of twins show that the genetic impact in non-insulin-dependent diabetes mellitus is stronger than in insulin-dependent diabetes mellitus. The genetic factors involved in non-insulin-dependent diabetes mellitus are not known and the outcome of molecular genetic research has so far been disappointing. The major genetic susceptibility to insulin-dependent diabetes mellitus is conferred by genes in the HLA region on chromosome 6. Despite many advances in molecular genetics in insulin-dependent diabetes mellitus the serologically detectable HLA antigens and haplotypes are still the best available markers. This review describes the important developments in immunogenetics in insulin-dependent diabetes mellitus and summarises the main findings from earlier studies. Genetically the potential for primary prevention of insulin-dependent diabetes mellitus already exists and will become a reality as soon as the environmental determinants are identified. A wide application of immunogenetic methods will be needed in the prevention of insulin-dependent diabetes mellitus.     

HLA Antigens in Insulin-Dependent Diabetes Mellitus

Diabetes mellitus is largely determined by genetic factors but environmental factors are necessary to convert genetic susceptibility into overt disease. Studies of twins show that the genetic impact in non-insulin-dependent diabetes mellitus is stronger than in insulin-dependent diabetes mellitus. The genetic factors involved in non-insulindependent diabetes mellitus are not known and the outcome of molecular genetic research has so far been disappointing. The major genetic susceptibility to insulindependent diabetes mellitus is conferred by genes in the HLA region on chromosome 6. Despite many advances in molecular genetics in insulindependent diabetes mellitus the serologically detectable HLA antigens and haplotypes are still the best available markers. This review describes the important developments in immunogenetics in insulindependent diabetes mellitus and summarises the main findings from earlier studies. Genetically the potential for primary prevention of insulin-dependent diabetes mellitus already exists and will become a reality as soon as the environmental determinants are identified. A wide application of immunogenetic methods will be needed in the prevention of insulin-dependent diabetes mellitus.     

大连地区2型糖尿病危险因素的非条件Logistic回归分析

背景糖尿病是环境因素、遗传因素等多种因素所导致的慢性全身性代谢疾病,其病因仍不十分清楚.探讨上述危险因素在糖尿病发病中的作用,对制定糖尿病的一、二级预防措施有重要意义.目的探讨2型糖尿病危险因素,为制定适宜的干预措施提供依据.设计以诊断为依据的横断面研究.单位一所大学的流行病学教研室、一所大学医院的内分泌科.对象采取分层、整群的随机抽样方法,在大连城乡居住5年以上、年龄在40岁以上的常住居民中,按城市和乡村分两层,每层抽取1 250人,两层共抽取2 500人.被抽中的原糖尿病患者亦予以统计.方法问卷调查,同时进行身高、体质量、腰围、臀围、血压及血糖等项目的体检.以2型糖尿病患者做为病例组,正常人做为对照组,采用单因素及多因素非条件Logistic回归分析方法进行分析.主要观察指标与2型糖尿病发病相关因素的单因素及多因素非条件Logistic回归分析.结果糖尿病家族史(OR=2.339)、肥胖(体质量指数,OR=1.462)、收缩压升高(OR=1.016)、高血脂(OR=1.615)、年龄(OR=1.043)是2型糖尿病的主要危险因素.结论糖尿病家族史、收缩压升高、肥胖、高血脂及年龄是2型糖尿病的危险因素,对高危人群及全人群采取措施适当控制体质量、血压等是预防该病发生的重要手段.