A Mitochondrial DNA A8701G Mutation Associated with Maternally Inherited Hypertension and Dilated Cardiomyopathy in a Chinese Pedigree of a Consanguineous Marriage

Background:

Cardiovascular diseases, including dilated cardiomyopathy (DCM) and hypertension, are the leading cause of death worldwide. The role of mitochondrial DNA (mtDNA) in the pathogenesis of these diseases has not been completely clarified. In this study, we evaluate whether A8701G mutation is associated with maternally inherited hypertension and DCM in a Chinese pedigree of a consanguineous marriage.

Methods:

Fourteen subjects in a three-generation Han Chinese family with hypertension and DCM, in which consanguineous marriage was present in the parental generation, were interviewed. We divided all the family members into case (7 maternal members) and control group (7 nonmaternal members) for comparison. Clinical evaluations and sequence analysis of mtDNA were obtained from all participants. Frequency differences between maternal and nonmaternal members were tested to locate the disease-associated mutations.

Results:

The majority of the family members presented with a maternal inheritance of hypertension and DCM. Sequence analysis of mtDNA in this pedigree identified eight mtDNA mutations. Among the mutations identified, there was only one significant mutation: A8701G (P = 0.005), which is a homoplasmic mitochondrial missense mutation in all the matrilineal relatives. There was no clear evidence for any synergistic effects between A8701G and other mutations.

Conclusions:

A8701G mutation may act as an inherited risk factor for the matrilineal transmission of hypertension and DCM in conjunction with genetic disorders caused by consanguineous marriage.     

扩张型心肌病与δ-肌聚糖基因突变

目的 :探讨中国人扩张型心肌病患者中δ-肌聚糖 (δ- sarcoglycan,SGCD)基因的突变情况。方法 :通过聚合酶链反应 -单链构象多态性分析 ,结合 DNA测序 ,对 60例扩张型心肌病患者和 60例对照 SGCD基因的所有 9个外显子进行分析。结果 :该基因外显子 9m发现 3种类型的单链构象多态性图谱 ;DNA测序证实其中一种为正常野生型 ,另外两种为832 G/A和 848A/G突变杂合子。 832 G/A突变 ( 2 78Ala→ Thr)仅在对照组检出 1例 ,对照组 A等位基因频率 0 .0 0 8;848A/G突变 ( 2 83Gln→ Arg)共检出 5例 ,G等位基因频率在扩张型心肌病组和对照组中差异无显著性 ( P>0 .2 5 ) ,对照组 G等位基因频率 0 .0 33。结论 :发现 SGCD基因 2个新的突变位点 ;未能证实这 2个位点的突变与扩张型心肌病相关     

全外显子组测序发现一家族性扩张型心肌病致病基因

 目的 对一扩张型心肌病(dilated cardiomyopathy, DCM)家系行全基因组外显子测序以寻找该家系的致病基因。方法 收集在复旦大学附属中山医院就诊的1位DCM患者及其家系成员的临床资料,采集相关家系成员外周血并抽提DNA,对该家系5名成员行全基因组外显子测序,寻找致病基因,用Sanger测序对家系其他成员进行验证。结果 通过对家系患者与正常人测序结果比对分析,同时经过多个生物数据库数据过滤,发现LMNA基因6号外显子上存在的杂合突变 c.961 C>T (p.Arg321Ter)为该家系的可能致病基因突变。LMNA c.961 C>T无义突变导致LMNA编码蛋白质过程提前终止,相应蛋白质功能异常,进而导致该家系中此突变基因的携带者出现心功能异常。结论 本研究应用全基因组外显子测序从一DCM家系中发现其致病基因及突变位点:LMNA c.961 C>T (p.Arg321Ter),突变导致该家系相关成员心功能异常。此位点在汉族人群中尚属首次报道。     

LHON患者携带新的线粒体多态位点

目的:寻找此Leber遗传性视神经病变(leber′s hereditary optic neuropathy,LHON)家系的致病突变位点。方法:采集静脉血,提取全基因组DNA,聚合酶链反应(PCR)扩增目的片段,酶切和直接测序反应寻找碱基改变位点。结果:患者mtDNA序列存在G11778A突变,发现4个多态性位点,其中G14476A为未报道的多态位点,该位点未引起编码蛋白质的改变,属无义突变。该多态位点在100例正常人中所占比例为3%。结论:该家系以G11778A为致病突变,G14476A为新的线粒体多态位点。     

线粒体tRNATyrA5834G突变与Leber遗传性视神经病变的相关性

目的：探讨线粒体tRNATyrA5834G突变与Leber遗传性视神经病变（LHON）的相关性。方法：对2个携带tRNATyrA5834G突变的LHON家系进行临床和分子遗传学特征等分析评估,对家系先证者和其他成员进行详细的眼科相关检查、线粒体全基因组分析、种系发生学分析及单体型分析。结果：先证者的临床症状及眼科相关检查均符合典型LHON表现,家系其他成员无异常。2个家系均未携带ND1 G3460A和ND4 G11778A及ND6 T14484C这3个原发突变位点,多态性变异位点均属于东亚单体型M7b。2个先证者均携带具有高度保守性的A5834G和T12811C突变位点,其中A5834G在17个物种中的保守系数为87.5%。结论：线粒体tRNATyrA5834G突变可能是与LHON相关的mtDNA突变位点,同时低外显率提示其他因素,如核修饰基因、环境等可能影响这2个家系的表型表达。     

R25G mutation in exon 1 of LMNA gene is associated with dilated cardiomyopathy and limb-girdle muscular dystrophy 1B

Background Mutations of the LMNA gene encoding lamin A and C are associated with dilated cardiomyopathy （DCM）, conduction system defects and skeletal muscle dystrophy. Here we report a family with a mutation of the LMNA gene to identify the relationship between genotype and phenotype. Methods All 30 members of the family underwent clinical and genetic evaluation. A mutation analysis of the LMNA gene was performed. All of the 12 exons of LMNA gene were extended with polymerase chain reaction （PCR） and the PCR products were screened for gene mutation by direct sequencing. Results Ten members of the family had limb-girdle muscular dystrophy （LGMD） and 6 are still alive. Two patients suffered from DCM. Cardiac arrhythmias included atrioventricular block and atrial fibrillation; sudden death occurred in 2 patients. The pattern of inheritance was autosomal dominant. Mutation c.73C〉G （R25G） in exon 1 encoding the globular domains was confirmed in all of the affected members, resulting in the conversion of arginine （Arg） to glycine （Gly）. Conclusions The mutation R25G in exon 1 of LMNA gene we reported here in a Chinese family had a phenotype of malignant arrhythmia and mild LGMD, suggesting that patients with familial DCM, conduction system defects and skeletal muscle dystrophy should be screened by genetic testing for the LMNA gene.     

中国汉族家族性肥厚型心肌病肌球蛋白重链基因G823E突变分析

目的 研究中国人家族性肥厚型心肌病(HCM)的致病基因突变位点,分析基因型与临床表型的相互关系.方法 在1个中国汉族HCM家系中进行心脏肌钙蛋白T基因(TNNT2)、心脏肌球蛋白结合蛋白C基因(MYBPC3)和心脏β-肌球蛋白重链基因(MYH7)的突变筛查,聚合酶链反应(PCR)扩增基因功能区外显子片段,并对PCR产物进行测序分析.以120名正常志愿者为对照组.结果 在该家系接受调查的12名成员中有4名成员携带MYH7基因G823E杂合突变,该突变位点位于MYH7基因的22号外显子并使823位的甘氨酸(G)转换为谷氨酸(E),该突变在西方人中未见报道,其导致的临床表型在家系内部呈现较明显的异质性,携带该突变的家族成员发病年龄和临床症状差异较大.该家系成员TNNT2及MYBPC3基因未发现突变且对照组相同位置未发现异常.结论 MYH7基因为我国家族性HCM的致病基因之一,G823E突变所致肥厚型心肌病呈现明显的个体异质性表型.     

MT-ND1 3571C>T 突变与Leber遗传性视神经病变相关研究

目的 探讨3个携带m.3571C＞T突变的中国Leber遗传性视神经病变家系（Leber＇s hereditary optic neuropathy,LHON）的临床和分子遗传学特征.方法 对3个LHON家系中66名成员和116例正常对照者线粒体DNA（mitochondrial DNA,mtDNA）相关区域进行PCR扩增突变、纯化及测序,并对测序结果进行生物信息学分析.结果 线粒体DNA序列分析结果显示,3个家系所有受试者和正常对照者均未发现m.3460G＞A、m.11778G＞A和m.14484T＞C这3个常见的原发突变位点,而3个先证者及其母系成员均携带m.3497C＞T和m.3571C ＞T突变位点,非母系成员和116例正常对照均不携带m.3497C＞T和m.3571C＞T突变位点.m.3497C＞T是已知与LHON相关的突变位点.结论 m.3497C＞T和m.3571C＞T突变可能协同增加LHON的发生,考虑为LHON的易感位点.     

Molecular basis of von Hippel-Lindau syndrome in Chinese patients

Background  Von Hippel-Lindau (VHL) syndrome is an autosomal dominant familial cancer syndrome predisposing the affected individuals to multiple tumours in various organs. The genetic basis of VHL in Southern Chinese is largely unknown. In this study, we characterized the mutation spectrum of VHL in nine unrelated Southern Chinese families.

Methods  Nine probands with clinical features of VHL, two symptomatic and eight asymptomatic family members were included in this study. Prenatal diagnosis was performed twice for one proband. Two probands had only isolated bilateral phaeochromocytoma. The VHL gene was screened for mutations by polymerase chain reaction, direct sequencing and multiplex ligation-dependent probe amplification (MLPA).

Results  The nine probands and the two symptomatic family members carried heterozygous germline mutations. Eight different VHL mutations were identified in the nine probands. One splicing mutation, NM_000551.2: c.463+1G>T, was novel. The other seven VHL mutations, c.233A>G [p.Asn78Ser], c.239G>T [p.Ser80Ile], c.319C>G [p.Arg107Gly], c.481C>T [p.Arg161X], c.482G>A [p.Arg161Gln], c.499C>T [p.Arg167Trp] and an exon 2 deletion, had been previously reported. Three asymptomatic family members were positive for the mutation and the other five tested negative. In prenatal diagnosis, the fetuses were positive for the mutation.

Conclusions  Genetic analysis could accurately confirm VHL syndrome in patients with isolated tumours such as sporadic phaeochromocytoma or epididymal papillary cystadenoma. Mutation detection in asymptomatic family members allows regular tumour surveillance and early intervention to improve their prognosis. DNA-based diagnosis can have an important impact on clinical management for VHL families. 

     

11个携带线粒体tRNASer(UCN) G7444A突变的中国汉族非综合征型耳聋家系分析评估

目的：通过对11个携带线粒体tRNASer(UCN) G7444A突变的中国汉族非综合征型耳聋家系进行临床和分子遗传学特征等分析评估,探讨线粒体tRNASer(UCN) G7444A突变在母系遗传非综合征型耳聋发生发展中的作用。方法：PCR扩增2 650例中国汉族非综合征型耳聋样本的线粒体12S rRNA、线粒体tRNASer(UCN)基因以及GJB2基因。对11个携带线粒体tRNASer(UCN) G7444A突变的中国汉族非综合征型耳聋家系进行听力学检测、耳聋相关热点基因突变检测以及家系资料等综合分析。结果：在2 650例中国汉族非综合征型耳聋患者中,14例携带线粒体tRNASer(UCN) G7444A突变,突变率为0.53%。在这11个携带线粒体tRNASer(UCN) G7444A突变的家系中,同时携带线粒体tRNASer(UCN) G7444A突变和线粒体12S rRNA A1555G、12S rRNA C1494T或GJB2 c.235delC的家系分别为3、1和2个。临床资料分析表明,这11个中国汉族非综合征型耳聋家系母系成员在听力损失严重程度、发病年龄以及耳聋外显率方面存在较大差异。同时携带线粒体tRNASer(UCN) G7444A突变和线粒体12S rRNA A1555G、C1494T或GJB2 c.235delC突变家系的耳聋平均外显率分别为29.4%、42.9%和19.0%。前两者的外显率明显高于只携带线粒体tRNASer(UCN) G7444A突变家系的耳聋平均外显率（为14.0%）。结论：线粒体tRNASer(UCN) G7444A突变可能与线粒体12S rRNA A1555G和C1494T原发突变存在协同作用,共同影响非综合征型耳聋的表型。     

受磷蛋白基因突变与中国成都地区扩张型心肌病的相关性研究

目的研究扩张型心肌病(DCM)患者的肌浆网受磷蛋白(phospholamban,PLN)基因突变情况,探讨该基因与中国DCM患者发病的关系。方法采用聚合酶链反应-单链构象多态性(PCR-SSCP)方法并结合核苷酸序列测定对89例DCM患者及110例对照组的PLN基因进行分析。结果病例组及对照组中均未发现存在异常构象,通过DNA测序未发现存在PLN基因第25核苷酸位点的C→T错义突变及116核苷酸位点的T→G突变。结论本研究未发现中国成都地区DCM患者PLN基因第25和116核苷酸位点存在突变。     

应用遗传连锁分析法对伴传导功能障碍的扩张型心肌病家系致病基因的定位

目的: 对一个常染色体显性遗传扩张型心肌病(familial dilated cardiomyopathy,FDCM)家系进行基因定位。方法: 收集FDCM 家系, 对该家系成员进行详细心血管内科检查确诊为扩张型心肌病,且伴发有传导功能障碍;采集外周血3～5 mL,并抽提基因组DNA;选取与该表型相关的已定位区间CMD1A(1q21.2-q21.3),CMD1H(2q14-q22), CMD1E(3p22-p25)和CMD1F(6q22-23)内的共计18个微卫星DNA标记,在该家系中进行排除性定位分析;最后,进行全基因组扫描及连锁分析。结果：①已定位区间的18个微卫星DNA标记位点的LOD值均<-2,证实该家系与已知DCM位点不连锁;②全基因组扫描及两点连锁分析结果显示,该家系致病基因位点与遗传标记D3S1614(3q26)连锁, 在θ= 0时得到最大LOD值2.68。结论: 该家系与已知的4个DCM位点均不连锁,其致病基因位于D3S1614（3q26）附近的一个新位点。     

Fv Leiden突变与我国汉族人布-加综合征的相关性及其临床意义

目的 研究Ｆｖ Ｌｅｉｄｅｎ突变（ＦｖＬ突变）与我国汉族人散发性布－加综合征（ＢＣＳ）、家族性ＢＣＳ的相关性,以及ＦｖＬ突变在家族性ＢＣＳ发病中的意义。方法 对２５例散发ＢＣＳ、６例家庭性ＢＣＳ（来自Ａ、Ｂ两个家系）及其家系成员（３９人）、３１名健康对照者进行ＦｖＬ突变分析。并对两个ＢＣＳ家系进行病因调查。结果 ２５例我国汉族人散发ＢＣＳ中无１例ＦｖＬ突变阳性。６例家族性ＢＣＳ中,４例ＦｖＬ突变阳     

X染色体连锁显性遗传先天性眼球震颤家系的致病基因突变研究

目的对4个X染色体连锁显性遗传先天性眼球震颤(XL-CIN)家系进行候选致病基因FRMD7突变筛查。方法采集家系成员外周血5 ml,提取基因组DNA;以4个家系的先证者基因组DNA为模板,聚合酶链反应(PCR)扩增FRMD7基因的全部外显子及其外显子-内含子拼接部的序列,DNA直接测序筛查突变位点;一旦发现突变致病性位点,则采用DNA双向测序方法在其他家系成员进行疾病与致病突变共分离分析,以及进一步确认突变,将患者的FRMD7基因外显子8和10的扩增产物克隆至TA克隆载体测序。结果 4个XL-CIN家系皆为X染色体连锁显性遗传伴外显不全,其中2个家系携带FRMD7基因已知致病性突变:XL-CIN 02家系存在c.G886C/GGT>CGT(p.G296R)错义突变,位于FRMD7基因外显子8;XL-CIN 03家系存在c.C910T/CGA>TGA(p.R304X)无义突变,位于外显子10。结论 FRMD7 G296R和R304X是导致XL-CIN 02和XL-CIN 03家系致病的主要原因。     

帕金森病患者线粒体DNA突变位点G1719A、G4580A、C7028T分析

目的研究有关线粒体DNA（mtDNA）点突变与我国人群散发性帕金森病（Parkinson disease,PD）的关系。方法采用经典的酚／氯仿抽提法对88例PD患者（研究组）和60例健康体检者（对照组）的全血基因组DNA进行抽提,针对与PD发病有关的mtDNA突变位点G1719A、G4580A、C7028T设计特异的引物,经过PCR鉴定和基因测序,在NCBI基因库上进行BLAST比对分析,发现突变位点。结果PD患者有10例在C7028T位点发现突变,8例在G1719A位点发现突变,3例在G4580A位点发现突变,对照组未发现突变位点。结论散发性PD患者存在线粒体基因的点突变,线粒体基因的点突变可能参与PD的发病过程。     

两个线粒体12S rRNA C1494T突变及药物性耳聋家系的分子遗传学研究

目的:探讨2个氨基糖甙类药物性耳聋及非综合征型耳聋家系的分子遗传学特征?方法:收集家系成员外周血样,常规方法提取基因组DNA?首先,利用基因芯片对中国人4个常见耳聋基因的9个突变热点进行分子筛查,9个位点分别为:GJB2基因的35 delG?176 del16?235 delC和299 delAT;GJB3基因的538 C>T;PDS基因的IVS7-2 A>G和2168 A>G以及mtDNA 12S rRNA基因的1494 C>T和1555 A>G?然后,对两家系的先证者分别进行线粒体DNA全序列及核基因TRMU和MTO1编码区的PCR扩增和测序分析?结果:芯片检测发现两家系的7名母系成员均存在同质性mtDNA 12S rRNA C1494T突变?与修正的剑桥参考序列相比,2名先证者的mtDNA全序列分析共检测到53个碱基变异,但除已知的12S rRNA C1494T突变外,其余52个碱基变异均为已报道的多态性位点;两家系先证者线粒体单体型分别是D4和D5a;TRMU和MTO1基因序列分析无异常发现?结论:线粒体DNA 12S rRNA C1494T突变是两个家系耳聋发生的主要分子基础,而氨基糖甙类抗生素的应用增强了该突变的表型表达;未能证实线粒体单体型以及核基因TRMU和MTO1对家系成员C1494T突变的表型具有修饰作用?     

Clinical and laboratory survey of 65 Chinese patients with Leigh syndrome

Background Leigh syndrome is an inherited neurodegenerative disease that emerges in infancy and childhood and presents with a clinically heterogeneous variety of neuromuscular and non-neuromuscular disorders. It can result from the inheritance of mutations in either nuclear or mitochondrial DNA. In the current study, we performed a retrospective study in 65 patients in order to investigate the clinical and genetic characteristics of Leigh syndrome in Chinese patients. Methods Sixty-five unrelated cases （35 men and 30 women） who were hospitalized in the past 12 years were reviewed. Diagnosis was based on both the clinical presentation and the characteristic neuropathologic findings of bilateral symmetric necrotizing lesions in the basal ganglia and brain stem as detected using cranial computed tomography （CT） scan or magnetic resonance imaging （MRI）. The differential diagnosis of organic acidurias and fatty acid IS-oxidation defects were performed. Specific point mutations and deletions in mitochondrial DNA （T8993G, T8993C, T9176C, A8344G, A3243G） were screened by PCR-restriction analysis and Southern blot. The SURF1 gene was sequenced. Skeletal muscle biopsies were performed in 17 （26.2%） of the patients. The diagnosis was confirmed by autopsy in 6 （9.2%） patients. Results The patients had various forms of metabolic encephalomyopathy. Filly-nine （90.8%） of the patients had the typical neuroradiological features of Leigh syndrome, including symmetrical necrotizing lesions scattered within the basal ganglia, thalamus and brain stem. Twenty （30.8%） patients were confirmed by genetic, biochemical analysis and autopsy. Specific point mutations in mitochondrial DNA were found in 5 cases （7.7%）. Of these, the A8344G mutation was detected in 2 patients. The T8993G T8993C, and A3243G point mutations were identified in 3 other patients, respectively. SURF1 mutations associated with cytochrome c oxidase deficiency were identified in 8 （12.3%） families by DNA sequencing. A G604C mutation was identified in 6 （9.2%） patients. The genotypes of 52 patients remained unknown. Conclusions Leigh syndrome presents as a diverse array of clinical features and can result from specific mutations in nuclear or mitochondrial DNA. In this study, SURF1 mutations associated with cytochrome c oxidase deficiency were identified in 8 （12.3%） out of 65 patients with Leigh syndrome. It indicates that SURF1 mutations might be a common cause of Leigh syndrome in China. The etiology of Leigh syndrome in Chinese patients represents a persistent challenge to clinicians.     

42个携带线粒体基因组A3243G突变核心家系临床表型分析

目的 研究携带A3243G突变的家系成员的临床症状特点以及与A3243G突变负荷的关系.方法 收集42个携带A3243G突变的核心家系,对他们的临床表现、实验室检查和线粒体DNA 3243位点点突变检测结果进行分析.结果 (1)肌无力、癫痫发作、多毛、头痛、认知障碍、消瘦和身材矮小是A3243G突变家族中先证者最常见的临床症状,而且这些临床症状多同时存在.在实验室检查中,血乳酸、丙酮酸及MRI检查多有异常;(2)A3243G突变家族中的携带者大多表现正常,肌无力、消瘦和身材矮小是他们最常见的临床症状;(3)在先证者组中,尿液A3243G突变负荷高于血液(t=-15.06,P＜0.001),在先证者母亲组中,尿液A3243G突变负荷也高于血液(z=-6.241,P＜0.001);(4)先证者组血液和尿液中的A3243G突变负荷约是母亲组的2倍.结论 携带A3243G突变患者表型差异很大,先证者组的临床表现和实验室检查结果均较母亲组严重,可能与A3243G突变负荷有一定关系.     

3个遗传性对称性色素异常症家系中DSRAD基因的突变

目的:检测国内3个遗传性对称性色素异常症家系中DSRAD基因的突变.方法:PCR扩增3个家系中成员DSRAD基因的全部外显子,并行DNA测序.以100例无关正常人作对照.结果:PCR结合DNA测序发现3个家系中患者均存在DSRAD基因的异常:家系A中第3220位碱基发生了C→T的杂合突变,对应1074位的精氨酸被半胱氨酸替代;家系B与家系C中发现的突变相同,为第3325位碱基发生了G→T的杂合突变,对应1109位的天冬氨酸被酪氨酸替代.家系中未患病者及无关正常人未发现相应突变.结论:此3个遗传性对称性色素异常症家系中存在DSRAD基因的特异性突变,突变可能使蛋白功能缺陷,导致临床上出现皮肤色素异常.     

扩张型心肌病与Desmin及血小板活化因子乙酰水解酶基因相关性研究

目的探讨中国扩张型心肌病（dilated cardiomyopathy,DCM）人群中是否存在Desmin基因外显子8上的一个错意突变（Ile451Met）及检测血小板活化因子乙酰水解酶（platelet—activating factor acetylhydrolase,PAF-AH）基因G^99s→T错义突变发生频率,以及它们与DCM的关系。方法通过聚合酶链反应、限制性内切酶酶切、聚丙烯酰胺凝胶电泳及银染技术,结合DNA测序分析89例DCM患者和110例对照组Desmin基因及PAFAH基因可能突变位点（11e451Met,Va1279Phe）。结果通过上述方法检测后未能发现两组中存在Desmin基因（Ile451Met）突变,PAF—AH基因突变（Val279Phe）发生频率在病例组及对照组问的差异无统计学意义（P〉0．05）。结论在小样本中国成都地区扩张型心肌病患者中,未发现Desmin基因第8外显子存在Ile451Met突变,并且与PAF-AH基因突变（Val279Phe）无明显相关。