用三个DNA探针对Wilson病进行基因诊断的研究

Wilson病(WD),又称肝豆状核变性,是一种常染色体隐性遗传病,该病主要由于铜代谢异常引起神经症状、肝硬化和角膜色素环等临床表现。发病年龄多在10岁左右,早期发现并治疗能有效地控制病情,否则可导致死亡。本实验室用三个位于WD基因两侧的探针对三个WD家系共14名成员进行了RFLP分析,预测出两个先证者的同胞为携带者、一例患者的子女为携带者、另一例患者的同胞属正常个体,这些结果与其它生化指标和临床症状基本相符。     

肝豆状核变性基因诊断的研究

肝豆状核变性又称Wilson氏病(WD),是一种较常见的以铜代谢障碍为特征常染色体隐性遗传病。其致病基因已定位于D1 3q 14.2-3。应用两个与WD基因紧密连锁的侧翼标记对9个WD家系进行单倍体检测,证实该单倍体可用于该病的症状前诊断和杂合子检出。     

糖原累积病Ⅳ型1例及其家系的临床和分子遗传分析

目的 研究1例糖原累积病Ⅳ型(GSD Ⅳ)患者及其家系的基因突变情况。方法 患儿,女,1岁5个月,因轻度黄疸,肝脾肿大2月,生长迟缓1年就诊。采集该家系先证者及其父母,两个哥哥的外周血,采用二代测序方法查找先证者致病基因及突变位点,Sanger测序进行突变验证。结果 该家系先证者为GBE1基因c.1571G>A纯合错义突变,双亲及一个哥哥为GBE1基因c.1571G>A杂合错义突变,另一个哥哥基因检测未见该突变,确诊为GSD Ⅳ型,建议行肝移植治疗,因家属拒绝,患儿于生后22个月死亡。结论 首次在国内报道了GSD Ⅳ型的家系及其分子遗传情况以及GBE1基因c.1571G>A纯合突变,丰富了GSD Ⅳ型在中国人群的突变谱。     

一例Gitelman综合征家系的临床特征及基因突变分析

目的 分析Gitelman综合征患者的临床特征及基因突变类型。方法 收集先证者及家系成员临床资料及实验室检查结果，采集其外周静脉血，经测序分析寻找相关突变位点，结合临床特征对其突变基因进行分析。结果 先证者的临床特征和实验室检查基本符合Gitelman综合征诊断。基因突变分析显示，先证者及其弟弟的SLC12A3基因第1号外显子存在c.248G>A(p.Arg83Gln)杂合错义的致病突变；先证者、先证者弟弟和先证者女儿的SLC12A3基因第7号内含子与第8号外显子之间存在c.965-1＿976delins-ACCGAAAATTTT缺失插入突变，该突变可导致NCCT蛋白在第7内含子和第8外显子之间的剪接位点异常，最终导致蛋白活性和功能受损。结论 Gitelman综合征患者起病隐匿，临床医师需详细问诊与完善实验室检验；SLC12A3基因c.248G>A(p.Arg83Gln)和c.965-1＿976delins-ACCG A A AATTTT突变是该Gitelman综合征家系的可能致病变异。     

X-连锁严重联合免疫缺陷综合征家系IL2RG基因突变研究

目的 对3个临床拟诊X-连锁严重联合免疫缺陷综合征(X-SCID)患儿及父母进行基因突变分析,为遗传咨询及产前诊断提供依据。方法 应用高通量测序和直接测序的方法对患儿及家系成员进行白细胞介素-2受体基因(IL2RG)突变检测,分析IL2RG基因外显子区及与外显子交界的部分内含子区域DNA序列改变情况,寻找可能的致病突变位点,并对其中一个家系进行羊水细胞产前诊断。结果 家系1、家系2患儿IL2RG基因检测到c.202G>A(p.Glu68Lys)突变,家系3患儿IL2RG基因检测到c.676C>T(p.Arg226Cys)突变,患儿母亲均为相应突变携带者。家系3先证者母亲进行产前诊断胎儿为女性,携带与先证者相同的致病基因,夫妇双方选择继续妊娠。结论 通过IL2RG基因检测明确诊断3例X-SCID患儿,并成功对1个X-SCID家系进行产前诊断,指导家系3夫妇选择妊娠。     

经典型苯丙酮尿症家系苯丙氨酸羟化酶基因突变分析及产前基因诊断

目的为经典型苯丙酮尿症(PKU)家系提供产前基因诊断。方法选取2008-2015年新生儿筛查中心确诊,并随访的经典型PKU家系中的4例,采用Sanger测序法对经典型PKU家系先证者的苯丙氨酸羟化酶(PAH)基因外显子及其侧翼序列进行突变分析,寻找致病突变位点,并在其父母中进行验证。联合PAH基因测序及PAH基因内部及附近的STR连锁分析,对其中3个家系的3例胎儿进行产前基因诊断。结果 4例经典型PKU家系经测序明确了8个突变等位基因,突变检出率达100%(8/8),鉴定了8个突变位点,分别是EX6-96 AG、E280K、D282G、R413P、R241C、R243Q、R252W、IVS6-1 GA。综合测序和连锁分析结果显示,3例胎儿均获得产前诊断,其中家系3和家系4胎儿为PKU患儿,家系2胎儿为杂合携带者。结论联合基因测序及多态性连锁分析降低了经典型PKU产前诊断风险,提高了诊断结果的可靠性。     

耳聋基因诊断技术作为新生儿听力筛查补充手段的应用价值

目的探索耳聋基因诊断技术作为新生儿听力筛查补充手段的应用价值,旨在为新生儿遗传性耳聋病例及其家系提供更科学的再发风险和再生育指导。方法收集2013-2016年温州市鹿城、瑞安、永嘉3个地区经新生儿听力筛查发现、确诊的耳聋病例,从中筛选疑似为遗传性耳聋的29例先证者及其家庭成员77名共同纳入研究,采集病史及家族史、结合听力及影像学检查,提取家系先证者及其家系成员的外周血基因组DNA,对GJB2、SLC26A4和12S rRNA基因的扩增产物进行DNA测序,并进一步对基因序列变异进行生物信息学分析和对比。结果共检出17个耳聋儿家庭携带常见耳聋致病基因突变,2例先证者及其父母均为GJB2纯合突变导致的遗传性耳聋,患儿父母再生育耳聋儿风险为100.00%。4例先证者为GJB2纯合突变导致的遗传性耳聋,其父母均为GJB2突变携带者;1例先证者为SLC26A4纯合突变导致的大前庭水管综合征,其父母均为SLC26A4突变携带者,2例先证者为GJB2 V371(c.109GA)纯合突变,该位点突变更倾向于致病突变;以上7个耳聋家族患儿父母再生育耳聋儿风险为25.00%。结论耳聋基因诊断技术作为听力筛查后续补充手段有利于明确新生儿疑似遗传性耳聋患儿的遗传病因,能科学客观地为患儿及其家系提供再发风险和再生育咨询与指导。     

全外显子组测序鉴定侏儒症家系基因突变及产前诊断

目的 揭示一侏儒症患者家系的遗传病因,为遗传咨询和产前诊断提供有效信息。方法 应用全外显子组测序(whole exome sequencing, WES)技术对侏儒症家系成员进行致病基因及突变位点筛查,结合临床表型,确定候选基因的致病位点,通过Sanger测序法对WES结果进行验证,排除二代测序假阳性位点。最后,利用鉴定得出的该家系致病基因的突变位点信息对患者胎儿进行产前诊断。结果 WES结果显示先证者、先证者父亲、先证者胎儿均存在FGFR3基因NM＿001163213.1:c.1626C>G(p.Asn542Lys)位点杂合变异,先证者妻子此未位点未发生突变。胎儿经超声检测股骨长度低于-2SD,有极大概率患有侏儒症。Sanger测序检测结果与上述WES结果一致,进一步证明了该检测结果的可靠性。结论 FGFR3基因NM＿001163213.1:c.1626C>G(p.Asn542Lys)位点杂合变异是侏儒症的重要发病原因,侏儒症患者3代的遗传咨询和产前诊断应重视该位点的检测。     

云南5例花偠傣地中海贫血的家系调查

目的 了解云南省新平县傣族地中海贫血先证者的家系调查的情况及遗传规律.方法 对先证者进行家系调查,用特康血细胞分析仪进行血细胞分析,pH 8.6缓冲液醋酸纤维薄膜做电泳分析,采用反向点杂交技术诊断β地贫基因突变,采用缺口聚合酶链反应技术检测α地贫缺失突变.结果 β-地贫先证者在家系调查中,在父或母均发现异常.结论 家系分析HbE为常染色体遗传,每代均有HbE,其儿女都会出现异常血红蛋白改变,说明遗传率较高,应重视预防,这对优生优育、提高人口素质具有重要意义.     

一个Waardenburg综合征家系的致病基因突变分析

目的分析一个Waardenburg综合征(WS)家系成员的临床表型和基因突变。方法收集一个WS综合征患者家系的临床资料,采用Sanger测序法对家系成员进行WS综合征相关基因的外显子测序分析。结果家系中共有2例患者,先证者及其弟弟具有WSⅡ的先天性感音神经性耳聋和虹膜色素异常的临床特征,均携带SOX10基因新发c.52GT(p.E18X)杂合致病突变;先证者父亲、母亲和姐姐SOX10基因序列测序分析均未见异常。结论在一个Waardenburg综合征家系中发现未见报道的SOX10基因新发突变,对于该病遗传咨询和产前诊断具有重要意义。     

ATP7B gene mutations in Hungarian patients with Wilson disease--case reports to illustrate the diverse clinical presentations

ATP7B gene mutations were examined in 70 Wilson patients from Hungary. 11 different mutations were found. In Hungary, similarly to other Central-Eastern European countries, the H1069Q was the most the frequent mutation, detected in 51 patients (73%) by semi-nested polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP) assay. 10 further mutations have been found by sequencing as follows: P767P-fs, R778G, K844K-fs, I857T, R969Q, T977M, E1064K, M769L, Y715H and P1273S. These latter three mutations have not been described before. Among the 11 mutations there are five, which have been published only in patients of Turkish, Italian or Albanian origin. It might be the genetic consequence of the 150 years long occupation of Hungary in the 16th and 17th century by Turks. The genotype-phenotype analysis showed that the Kayser-Fleischer ring was more frequent (10/12 = 83%), and the age at the diagnosis was higher in H1069Q homozygous patients than in compound heterozygous or negative patients. Diverse clinical presentation of the disease was demonstrated by case reports giving messages for the practitioners. The gene mutation analysis is of particular importance in siblings of the index patient, since the detection of two mutant allels confirm the diagnosis of the disease even in absence of symptoms. The clinical manifestation of the disease can be preceded by the treatment.     

Clinical symptoms, diagnosis and treatment of multiple endocrine neoplasia type 1. Results of genetic screening in Hungarian patients

Multiple endocrine neoplasia type 1 syndrome is an autosomal dominant disorder characterized by endocrinopathies involving the parathyroid glands, anterior pituitary gland, and pancreas. Also, it may be associated with foregut carcinoid, adrenocortical tumors and non-endocrine tumors. After reviewing the prevalence, genetic background, clinical symptoms, diagnosis and treatment of the disorder, the authors present their genetic screening method used for the detection of mutations of the MEN1 gene (prescreening of polymerase chain reaction amplified exons using temporal temperature gradient gel electrophoresis followed by direct DNA sequencing). Using this method, the authors identified disease-causing MEN1 gene mutations in 9 probands (small deletions in 2 cases, insertion in 2 cases, nonsense mutations in 2 cases and missense mutations in 3 cases). Of the 9 mutations, 4 proved to be novel mutation not reported in the literature. Family screening indicated de novo mutations in 2 probands. In addition to mutations, several sequence polymorphisms were also detected. The authors conclude that one of the major advantages of genetic screening in families with MEN1 syndrome was the identification of family members carrying the mutation who should be regularly screened for disease manifestations and those not carrying the mutation in whom clinical screening is unnecessary. Also, genetic screening may be useful in cases when MEN1 syndrome is suspected, but the clinical manifestations do not fully establish the diagnosis of MEN1 syndrome.     

尿素循环障碍患者家系的基因突变分析及产前诊断

目的:分析7例尿素循环障碍(urea cycle disorder,UCD)患者家系的基因突变情况,并对2例再生育家庭进行产前诊断。方法:联合应用高通量测序(panel)结合Sanger测序、长距离-聚合酶链反应(LD-PCR)以及多重连接探针扩增(multiplex ligation-dependent probe amplification,MLPA)等基因检测技术,对7例疑似UCD患者家系进行相关致病基因突变分析,并对其中2例高风险家系的胎儿羊水标本进行产前基因诊断。结果:7例疑似UCD患者均得到明确基因诊断:4例患儿为SLC25A13基因突变引起的新生儿肝内胆汁淤积症(neonatal intrahepatic cholestasis caused by citrin deficiency,NICCD),且1例同时患脊髓性肌萎缩症(spinal muscular atrophy,SMA);1例患儿为ASS1基因突变引起的瓜氨酸血症Ⅰ型(citrullinaemia typeⅠ,CTLN1);2例患儿为OTC基因突变引起的鸟氨酸氨甲酰转移酶缺乏症(ornithine transcarbamylase deficiency,OTCD)。2家系再生育时产前诊断结果:1例胎儿为父源SLC25A13致病基因携带者;1例为OTC正常的UCD胎儿。结论:基因诊断有助于可疑UCD患者的明确诊断以及分型,部分疑似UCD胎儿可能OTC正常。对于生育过UCD患儿的家系,再生育时进行产前基因诊断可积极预防出生缺陷。     

Ⅱ型糖尿病的家庭聚集性研究

目的　通过对Ⅱ型糖尿病家庭聚集性的分析,探讨遗传因素在糖尿病患者一级亲属成员发病中所起的作用及其相对危险度。方法　采用以人群为基础的遗传流行病学病例对照研究方法,对３６３ 例Ⅱ型糖尿病先证者家系及２９１ 例人群对照家系进行了调查。结果　先证者家系一级亲属糖尿病的患病率为３ ．９４ ％ ,对照组一级亲属为１ ．０９ ％ ,相对危险度为３ ．６２ ,各组血缘亲属的相对危险度均在３ ．０ 以上;糖尿病先证者诊断时年龄越轻,其一级亲属的糖尿病患病率和相对危险度越高,其家系内发生多例糖尿病病例的可能性越大。结论　Ⅱ型糖尿病具有明显的家庭聚集倾向,其一级亲属对糖尿病的遗传易感性较高,是糖尿病预防和控制的重点人群。     

Comparison of ascertainment-bias correction schemes for pedigrees ascertained through multiple probands

Assessing the genetic causation of a disease, which is of prime importance in medical genetics, is usually done by analysing pedigree data. When gathering such data, it is often more practical to adopt a non-random sampling strategy. However, unless suitable corrections for non-random sampling are made at the time of data analysis, inferences may be grossly affected. For pedigree data ascertained through multiple probands, various correction schemes have been suggested, although the efficiencies of these schemes are unknown. This paper compares such schemes, using Monte Carlo simulation techniques, under a simple genetic model, for pedigrees of fixed sizes and structures and for probands of two types of relationship--parent-offspring, and a pair of siblings. It is found that gene frequencies are grossly overestimated and the penetrance value of heterozygotes slightly underestimated whether or not any correction for non-random sampling of pedigrees is made. Knowledge of the population value of the gene frequency improves the estimate of the penetrance parameter.     

非胰岛素依赖型糖尿病的遗传方式分析

为了解人群中非胰岛素依赖型糖尿病(NIDDM)的遗传方式,以遗传流行病学方法,对在人群糖尿病抽样调查中发现的204例NIDDM先证者进行家系调查,获含先证者在内的核心家系304个,对其进行分离分析.常染色体显性遗传(AD)检验结果拒绝AD假设.根据核心家系的确认概率采用不同的矫正公式计算分离比,经与理论分离比比较,结果亦拒绝该病常染色体隐性遗传的假设.表明该人群的NIDDM不符合单基因遗传规律.     

2个范德伍德综合征家系的IRF6基因突变检测

目的对2个中国范德伍德综合征(Van der Woude syndrome,VWS)家系进行临床和遗传特点分析,并进行IRF6基因的突变检测,明确中国人VWS致病基因IRF6的突变情况,并发现可疑的突变热点区域。方法通过先证者及现场家系调查、临床检查和系谱分析收集2个VWS家系成员24人的外周血样本,提取DNA用于IRF6基因的聚合酶链扩增反应(polymerase chain reaction,PCR)。采用Sanger测序法对所有VWS家系成员的IRF6基因的编码区7个外显子进行测序,使用mutation surveyor软件对测序结果与参考序列进行比对分析。结果 2个家系24人中受累患者6名,5名(83.3%)患者有唇腭裂,5名(83.3%)患者有唇瘘,6名患者均无牙齿发育不全表现,唇瘘伴唇腭裂表型常见,无其他组织器官畸形。24名成员均进行了IRF6基因第3至第9外显子的测序,6名患者均存在基因突变,家系1的3名患者均携带位于第9外显子的c.1234CT杂合突变;家系2的3名患者均携带位于第9外显子的c.1210GA杂合突变;未发现其他非患者的家系成员携带IRF6基因突变。结论 VWS综合征存在遗传异质性和临床表型复杂性,患者的表型与致病突变有关。通过基因检测可以对VWS患者进行遗传分析和产前诊断,弥补产前超声容易漏诊的不足。     

凋亡相关基因程序化细胞死亡基因5在新生儿缺氧缺血性脑病中的表达

目的探讨程序化细胞死亡基因5(programmed cell death 5,PDCD5)在新生儿缺氧缺血性脑病(hypoxicischcmic encephalopathy,HIE)血清中的表达及与细胞凋亡的关系,分析其与血清高敏C反应蛋白(high-sensitivity C-reactive protein hsCRP)水平及临床分度之间的相关性,为临床早期诊断及病情判断提供理论依据。方法选择70例HIE患儿,按临床分度(轻度、中度、重度)分为三个亚组,轻度组30例,中度组20例,重度组20例,同期出生的足月健康新生儿30例为对照组,采用酶联免疫吸附双抗体夹心ELISA法检测生后1、3、7d血清PDCD5水平,并检测其生后3、7d血清hsCRP水平。结果 HIE各组生后1d血清PDCD5开始升高,3d达高峰,7d下降。血清PDCD5水平在HIE各组中呈现据病情分度依次升高的变化规律,组间差异具有统计学意义(P0.01)。HIE病情越重,PDCD5升高越明显,HIE各组生后1d(r=0.851)、3d(r=0.932)、7d(r=0.773)血清PDCD5水平与病情分度呈正相关(P均0.001)。HIE各组生后3d和7d血清hsCRP水平均较对照组明显升高,差异有统计学意义(P0.01)。HIE患儿生后3d(r=0.604)、7d(r=0.379)血清PDCD5水平与血清hsCRP水平呈正相关(P均0.001)。结论 PDCD5可能参与了新生儿HIE中细胞凋亡的发生与发展。动态检测HIE患儿清中PDCD5和hsCRP水平对判断新生儿HIE的病情严重程度、评估预后有一定的临床价值。     

Genetic mutations in patients with possible familial hypercholesterolaemia in South East Scotland

Familial hypercholesterolaemia (FH) is one of the most common genetic disorders in the general population. Genetic testing of this condition is increasingly available in the UK to confirm its diagnosis, but the strategies of genetic testing vary. In this pilot study, we sought to investigate whether a strategy that focuses on the low-density lipoprotein receptor (LDLR) and apolipoprotein B (APOB) genes can identify the majority of genetic variants in patients with possible FH in South East Scotland. Forty patients with a clinical diagnosis of possible FH according to the Simon Broome criteria were recruited in a lipid clinic serving South East Scotland. All 18 exons of the LDLR gene were sequenced and multiplex ligation probe amplification was performed to identify major deletions and duplications. Variants of the APOB gene at codon 3527 were investigated by direct sequencing. Genetic mutations were detected in 45% of the patients. Sixteen patients (40%) were found to have mutations in their LDLR gene, whereas two other patients (5%) were identified as heterozygous for the APOB variant commonly associated with FH (c.10580G>A; p.R3527Q). None of these genetic variants were detected in more than two patients. Multiple genetic mutations are associated with a clinical phenotype of FH in South East Scotland. A genetic testing strategy which focuses on a limited number of mutations is unlikely to confirm the diagnosis of FH in the majority of patients in this part of Scotland.     

Heritability of plasma amyloid beta in typical late-onset Alzheimer's disease pedigrees.

Plasma amyloid beta42 peptide (Abeta42) levels are significantly elevated in all genetic forms of early-onset Alzheimer's disease caused by familial Alzheimer's disease mutations or Down's syndrome. Moreover, recent studies have determined that both plasma Abeta42 and Abeta40 levels are significantly elevated in late-onset Alzheimer's disease (LOAD) patients, their cognitively normal first-degree relatives, and members of typical LOAD families when compared to appropriate controls. To determine the magnitude of the genetic component affecting plasma Abeta levels, we estimated the heritability of plasma Abeta42 and Abeta40 in 15 extended, multigenerational LOAD pedigrees, using a variance components method. Heritability estimates as high as 73 and 54% were found for plasma Abeta42 and Abeta40 levels, respectively. Inclusion of the ApoE epsilon4 dosage as a covariate was not found to have a significant effect on the heritability of these traits. These results suggest that genetic determinants other than ApoE account for a very substantial percentage of the phenotypic variance in plasma Abeta levels. The high heritability and the significant elevation of these traits in LOAD pedigrees suggest that at least some of the genetic determinants of plasma Abeta levels may lead to elevated Abeta and LOAD in these families. Thus, we suggest that plasma Abeta levels are quantitative traits that may be excellent surrogate markers for use in linkage analysis to identify loci that are important in typical LOAD.