Wilson病ATP7B基因突变检测

目的:研究Wilson病(WD)患者基因突变的类型和发生情况,为本病的早期诊断提供理论依据。方法:提取13例WD患儿的基因组DNA,用聚合酶链反应法,对其ATP7B基因的21个外显子进行扩增,并用DNA测序方法对扩增产物进行测序分析。并选择100名健康儿童的样本作为对照,以排除发现的基因突变为多态性的可能。结果:13例WD患儿中,共发现7种错义突变,2种无义突变,其中最常见的突变方式为c.2333 G>T(p.Arg778Leu),占46.15%;其次为c.2975 C>T(p.Pro992Leu),占15.38%。21个外显子中,8号外显子发生突变的频率最高,为53.85%;其次为13号外显子,其突变频率为26.92%。结论:ATP7B基因的8、13号外显子是WD的突变热点,其中8号外显子以Arg778Leu、13号外显子以Pro992Leu为主要突变形式,基因检测可对WD患儿作出快速、准确的诊断。     

中国汉人iWlson病基因Arg919lyG点突变与中医证候相关性研究

目的:探讨中国汉人Wilson病基因Arg919Gly点突变与中医证候的相关性.方法:收集先证者临床资料,应用聚合酶链式反应(PCR)方法、高效液相色谱技术等检测Wilson病ATP7B基因第12外显子突变.结果:在全部203例患者中,32例存在第12外显子Arg919Gly杂合错义突变,6例存在Arg919Gly纯合突变,165例未检出突变.WD患者Arg919Gly点突变检出率为17.7%(38/203),基因突变频率为10.8%(38/406).38例Arg919Gly点突变患者中,涉及脑损害者28例(77.8%),其发病年龄延迟并与神经症状关系密切,与阴虚风动、气血两虚证有一定相关性,经统计学处理有显著差异.结论:WD基因外显子12是ATP7B基因的第二突变热区.中国汉人Wilson病基因Arg919Gly点突变与中医阴虚风动、气血两虚证密切相关.     

中国汉人Wilson病基因Arg919Gly点突变与中医证候相关性研究

目的:探讨中国汉人Wilson病基因Arg919Gly点突变与中医证候的相关性.方法:收集先证者临床资料,应用聚合酶链式反应(PCR)方法、高效液相色谱技术等检测Wilson病ATP7B基因第12外显子突变.结果:在全部203例患者中,32例存在第12外显子Arg919Gly杂合错义突变,6例存在Arg919Gly纯合突变,165例未检出突变.WD患者Arg919Gly点突变检出率为17.7%(38/203),基因突变频率为10.8%(38/406).38例Arg919Gly点突变患者中,涉及脑损害者28例(77.8%),其发病年龄延迟并与神经症状关系密切,与阴虚风动、气血两虚证有一定相关性,经统计学处理有显著差异.结论:WD基因外显子12是ATP7B基因的第二突变热区.中国汉人Wilson病基因Arg919Gly点突变与中医阴虚风动、气血两虚证密切相关.     

中国人肝豆状核变性基因突变热点区的快速检测

目的 探讨中国人肝豆状核变性(Wilson's disease,WD)患者和携带者基因突变热点区的简便、准确的检测方法。方法 采用聚合酶链反应．限制性酶切多态性技术(PCR-RFLP)对35例WD患者和50例健康体检者检测中国WD基因——P型铜转运三磷酸腺苷酶(ATP7B)外显子8的密码子778位基因突变。结果 35例WD患者共检出4例778位突变纯合子和ll例杂合子,纯合子检出率11．4％,杂合子检出率31．4％。50例正常对照中无一例突变。结论 ATP7B基因8号外显子778位是中国人WD的高频突变点,PCR-RFLP法具有简便、准确的优点。     

等位基因特异-荧光定量PCR检测胱硫醚-β-合成酶基因T833C/G919A点突变及其与糖尿病肾病的关联性初步观察

目的建立胱硫醚-β-合成酶（CBS）基因T833C、G919A点突变的等位基因特异-荧光定量PCR（TaqMan—ARMS）测定方法，探讨2型糖尿病肾病患者CBS基因T833C、Gg19A点突变率及与糖尿病肾病（DN）的相互关系。方法采用PCR时引物3’端错配扩增阻滞（ARMS）原理，结合荧光定量PCR技术（TaqMan探针），制备野生和突变质粒标准品，建立野生引物和突变引物2个反应体系，根据荧光定量PCR时野生引物循环阈值（Wct）与突变引物循环阈值（Mct）的比值△ct（△ct=Wct／Mct）及扩增有无指数增长期出现，建立点突变等位基因型的判定标准，并对94例DN患者（组1）和140例尿微量白蛋白正常的非糖尿病对照者（组2）的CBS基因T833C、Gg19A点突变进行检测。结果建立的TaqMan-ARMS方法检测T833C野生等位基因型的判定标准为Act〈0．72或Met〉40，杂合突变型为0．9〈Act〈1．10，纯合突变型为1．40〈Act或Wet〉40；G919A野生等位基因型判定标准为Act〈0．74或Mct〉40，杂合突变型为0．92〈Act〈1．03，纯合突变型为1．26〈Act或Wet〉40。T833C点突变TY、TC、CC3种基因型在DN组分布频率分别为98．94％、1．06％和0，组2中分别是99．29％、0．71％和0；两组人群833C等位基因频率分别为0．53％和0．36％，基因型和等位基因频率差异均无统计学意义。各组中未发现G919A等位基因杂合突变和纯合突变型。结论成功建立了测定CBS基因T833C、G919A点突变的TaqMan—ARMS方法，该方法准确、特异，适合高通量点突变的检测。本研究未观察到CBS基因T833C、G919A点突变为DN的遗传危险因素。     

中国东北地区非综合征性耳聋患者GJB2基因的致聋突变分析

目的分析58例中国东北地区非综合征性耳聋（nonsyndromic hearing impairment，NSm）患儿GJB2基因突变类型和频率。方法收集吉林省吉林市聋哑学校的58例非综合征性耳聋患儿（分别来自57个家庭）及37例听力正常家属的血样，经聚合酶链反应（Dolymerase chain reaction，PCR）扩增GJB2基因编码区。用酶切方法初步分析已知的233—235位点，进一步行DNA测序证实酶切结果并发现新的突变类型；同时对部分家属进行DNA测序，区分并证实致病突变位点或多态性改变。结果58例患儿中发现11例（18．97％）为233—235delC突变，其中7例（12．07％）为233—235delC纯合突变，4例（6．90％）为233—235delC杂合突变；发现1例35delG杂合突变；4例235delC杂合突变及1例35delG杂合突变者均伴有299—300delAT杂合突变。在患儿及听力正常的家属中均发现有G79A杂合及A341G杂合复合变异、G79A纯合及A341G纯合复合变异。结论东北地区NSHI患儿的GJB2突变热点为233—235delC，突变率为18．97％。233—235delC杂合及299—300delAT杂合复合突变、35delG杂合及299—300delAT杂合复合突变为致病突变。G79A杂合及A341G杂合复合变异、G79A纯合及A341G纯合复合变异为多态性改变。     

建立一种EGFR突变检测的凸环引物荧光PCR新方法

目的建立一种能快速、有效检测EGFR突变的凸环引物荧光PCR新方法。方法同时用凸环引物荧光PCR技术法和Sanger测序法对混有不同比例的EGFR突变型质粒的样本进行对照检测,以对比两者灵敏度;采用两种方法对93例肺癌组织的EGFR基因进行热突变位点E746一A750del和L858R的检测,并统计其符合率。结果在93例肺癌组织的DNA中,凸环引物荧光PCR技术法和Sanger测序法检测到EGFR突变分别为29例（E746．A750del15例,L858R14例）和28例（E746-A750del15例,L858R13例）,符合率达96．6％,且凸环引物荧光PCR技术法能检测出混有5％突变质粒型的样本,其灵敏度达5％,比Sanger测序法高。结论凸环引物荧光PCR技术法比测序法更为简便,且灵敏度更高,易于实现,2小时内即可得出准确结果。     

7例肝豆状核变性患儿ATP7B基因突变的检测与分析

目的 探讨肝豆状核变性患儿ATP7B基因全长外显子基因突变类型及其与临床表型的相关性.方法 对7例肝豆状核变性患儿的ATP7B基因进行全外显子检测.结果 7例肝豆状核变性患儿中1例ATP7B基因未检测出突变,6例检测出6种突变,分别为R778L、G711W、N1270S、A887LfsX14、de1930I、V1121A,R778L和V1121A检出率为28.6％,其他突变检出率均为14.2％.结论 肝豆状核变性患儿ATP7B基因突变呈多样性,R778L突变与临床表型不存在明确相关性.     

纤维蛋白原Bβ-链复合杂合突变导致的遗传性无纤维蛋白原血症

遗传性无纤维蛋白原血症是一种由于纤维蛋白原基因缺陷所致常染色体隐性遗传病.为了对1例遗传性无纤维蛋白原血症家系进行表型和基因型分析,采集了该家系三代10人外周血,吸取上层血浆用血凝仪检测活化部分凝血活酶时间（APTT）、凝血酶原时间（PT）和凝血酶时间（TI）;纤维蛋白原（Fg）含量分别用Clauss法和免疫比浊法进行检测;以常规酚-氯仿法抽提家系所有成员外周血基因组DNA,PCR扩增Fg基因FGA、FGB和FGG所有外显子及其侧翼序列和启动子区,PCR产物纯化后直接测序以检测基因突变.102例健康献血者作为正常对照.结果表明：先证者表型诊断为无纤维蛋白原血症;基因型呈Fg B β-链Arg17stop和Gly347Arg复合杂合突变,前者来源于母系,后者来源于父系.结论：Fg B β-链Arg17 stop和Gly347Arg复合杂合突变是引起该家系先证者产生无纤维蛋白原血症的原因.     

小脑性共济失调症患者谷氨酸受体δ2基因12号外显子突变研究

目的探讨小脑性共济失调症患者谷氨酸受体82（GluRδ2）基因12号外显子突变的情况。方法采用PCR、琼脂糖凝胶电泳及DNA测序方法检测24例小脑性共济失调症患者（有家族史者17例、散发者7例）及其16名无症状家系成员和10名正常人的GIuRδ2基因12号外显子突变情况。结果经PCR扩增和琼脂糖凝胶电泳后,24例患者、16名无症状家系成员及10名正常人12号外显子均可见一长度为222bp片段,未见该外显子纯合缺失突变。DNA测序结果显示,24例患者未发现类似h05J小鼠的突变碱基缺失及Lurcher小鼠的突变碱基置换。结论小脑性共济失调症患者中不存在GluRδ2基因12号外显子纯合缺失突变,也不存在碱基突变或缺失,提示GIuRδ2基因12号外显子与本病发病机制可能无关。     

肝豆状核变性的临床特征及其分子诊断

目的:观察肝豆状核变性患者的多样性临床表现,探讨建立分子诊断的方法。方法:采集62例肝豆状核变性患者的临床资料,包括临床表现、实验室检查和其他特征等,进行统计分析。应用序列分析方法检测ATP7B基因突变,并对p.Arg778Leu基因突变的基因型与表型的相关性进行分析。结果:27例(43.5%)患者诊断时年龄<8岁,且均缺乏典型的临床表现,仅有2例出现K-F环,而59.3%的患者仅表现为肝功能异常。与表现为肝脏疾病的患者相比,以神经系统为表现的患者诊断时年龄较大(P     

Genetic studies discover novel coding and non‐coding mutations in patients with Wilson's disease in China

ObjectivesWilson disease (WD) is a rare autosomal recessive genetic disorder associated with various mutations in the ATP7B gene and leads to significant disability or death if untreated. Early diagnosis and proper therapy usually predict a good prognosis, especially in pre‐symptomatic WD. Genetic testing provides an accurate and effective diagnostic method for the early diagnosis of WD.MethodsWe recruited 18 clinically diagnosed WD patients from 16 unrelated families and two independent individuals. The next‐generation sequencing of the ATP7B gene was performed. The 293T cell lines were divided into wild‐type (WT) ATP7B and mutated ATP7B groups. Cell proliferation was determined by Cell Counting Kit‐8 (CCK‐8) assay and apoptosis was detected by Annexin V/propidium iodide (PI) assays.ResultsPedigree analysis showed that compound heterozygous variants (17/18, 94.44%) were present in the majority of WD patients. A total of 33 ATP7B gene variants were identified, including three variants with uncertain significance (VUS) [two splice mutations (c.51+2T>G, c.1543+40G>A) and one frameshift mutation (c.3532_3535del)]. The CCK‐8 and apoptosis assays demonstrated that the VUS of ATP7B could significantly affect the transportation of copper.ConclusionsThe study revealed genetic defects of 16 Chinese families and two independent individuals with WD, which enriched the mutation spectrum of the ATP7B gene worldwide and provided valuable information for studying the mutation types of ATP7B in the Chinese populations. Genetic testing in WD patients is necessary to shorten the time to initiate therapy, reduce damage to the liver and improve the prognosis.     

Allele dropout in PCR-based diagnosis of Wilson disease: mechanisms and solutions

BACKGROUND: We investigated the mechanisms leading to allele dropout-the nonamplification of 1 of the alleles-in PCR-based diagnosis of Wilson disease (WD). METHODS: We extracted genomic DNA from blood samples from 6 WD patients (P1-P6) with allele dropouts detected in a previous study of WD in a Hong Kong Chinese population. We amplified the ATP7B gene by PCR and performed direct DNA sequencing of all exons of the ATP7B gene. To support the proposed mechanism of allele dropout, we used proofreading DNA polymerase, primer design avoiding single-nucleotide polymorphism sites, and duplex PCR. RESULTS: Patients P1-P4 were all apparently homozygous for a known disease-causing mutation, c.2975C > T (p.P992L) in exon 13. Patient P5 was apparently homozygous for a novel mutation, c.2524G > A, and patient P6 was apparently homozygous for another known mutation, c.522_523insA (p.K175K-fs). In all cases, we determined that the patients were actually heterozygous for these mutations. CONCLUSION: Our results confirm that allele dropout is the mechanism causing apparent homozygosity of heterozygous mutations in these WD patients.     

Molecular characterization of antithrombin III (ATIII) variants using polymerase chain reaction. Identification of the ATIII Charleville as an Ala 384 Pro mutation.

The genes of seven structural mutants of antithrombin III (ATIII), presenting either defective serine protease reactivity or abnormal heparin binding, were analyzed. The polymerase chain reaction (PCR) was used to amplify the corresponding gene exon and the mutation was identified by either dot blot analysis using a battery of allele-specific oligonucleotide probes or sequencing. Variants Paris and Paris 2 were identified as Arg 47 Cys mutations, and Clichy, Clichy 2, and Franconville were found to be Pro 41 Leu mutations. All five are heparin binding-site variants. ATIII Avranches is an Arg 393 His mutation and ATIII Charleville is an Ala 384 Pro mutation. These two mutations impair the reactive site of the molecule. ATIII Charleville is a new mutation of the reactive center, as predicted by previous biochemical data. The position of this new mutation, together with the other previously described mutations of the reactive center, sheds light on the molecular function of this site in inhibiting thrombin. Finally, genomic amplification by PCR is a powerful technique for the fast identification of antithrombin III mutations and their homozygous/heterozygous status, and should be useful for predicting thrombotic risk.     

Evaluation of a new efficient procedure for single-nucleotide polymorphism genotyping: tetra-primer amplification refractory mutation system-polymerase chain reaction.

Tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) is a new efficient method for single-nucleotide polymorphism (SNP) genotyping. To determine the optimal conditions for ARMS-PCR we attempted to genotype ten SNPs. DNA was extracted from the peripheral blood of 168 unrelated healthy Japanese volunteers. Two problems inhibited uniform efficiency of the amplification of three bands. The first problem was the lower amplification efficiency of the shorter and allele-specific products compared with the largest product. This phenomenon was overcome by increasing the relative concentration of the inner primers. The second problem was non-specific amplification of the shorter products. To reduce the amplification of these non-specific bands, adjusting any one of the following PCR conditions was effective: i) reducing the ratio of the inner primer concentration relative to that of the outer primers; ii) increasing the annealing temperature for the initial 5-10 cycles; iii) hot start PCR. With these procedures all ten of the SNPs were successfully genotyped. Our present data may be useful in the further application of tetra-primer ARMS-PCR to SNP genotyping.     

Mutation analysis of the ATP7B gene in a new group of Wilson's disease patients: contribution to diagnosis

Wilson's disease (WD), an autosomal recessive disorder of copper transport with a broad range of genotypic and phenotypic characteristics, results from mutations in the ATP7B gene. Herein we report the results of mutation analysis of the ATP7B gene in a group of 118 Wilson disease families (236 chromosomes) prevalently of Italian origin. Using DNA sequencing we identified 83 disease-causing mutations. Eleven were novel, while twenty one already described mutations were identified in new populations in this study. In particular, mutation analysis of 13 families of Romanian origin showed a high prevalence of the p.H1069Q mutation (50%). Detection of new mutations in the ATP7B gene in new populations increases our capability of molecular analysis that is essential for early diagnosis and treatment of WD.     

Molecular diagnosis of Wilson disease using prevalent mutations and informative single-nucleotide polymorphism markers

BACKGROUND: Wilson disease (WD) is an autosomal recessive disorder caused by defects in the ATPase, Cu(2+) transporting, beta-polypeptide gene (ATP7B) resulting in accumulation of copper in liver and brain. WD can be thwarted if detected at a presymptomatic stage, but occasional recombination during carrier detection with dinucleotide repeat markers flanking the WD locus may lead to faulty diagnosis. We examined the use of intragenic single-nucleotide polymorphism (SNP) markers to avoid this limitation. METHODS: We prepared genomic DNA from the peripheral blood of Indian WD patients. By use of PCR, we amplified the exons and flanking regions of the WD gene and then performed sequencing to identify the nucleotide variants. We genotyped the SNPs in 1871 individuals by use of the Sequenom mass array system. We made linkage disequilibrium plots using Haploview software. RESULTS: We identified 1 mutation accounting for 11% (19 of 174) of WD chromosomes among patients in addition to 4 prevalent mutations characterized previously. Among 24 innocuous allelic variants identified, we selected 3 SNPs found to have high heterozygosity (>0.40) for the detection of mutant WD chromosomes. On analyzing these SNPs in 28 test individuals, who were sibs to 17 unrelated WD patients, we obtained unequivocal genotyping in 25 cases (approximately 89%). The remaining 3 cases were genotyped by dinucleotide repeat marker (D13S133). CONCLUSION: Sets of SNP markers are highly heterozygous across most world populations and could be used in combination with analysis of prevalent mutations as a comprehensive strategy for determining presymptomatic and carrier sibs of WD patients.     

7个家系Wilson病同胞ATP7B基因突变分析及临床表型的研究

目的对Wilson病(WD)患病同胞进行ATP7B基因外显子测序,分析其突变的特点并探讨基因型与表型的关系。方法收集WD患病同胞的临床资料,并留取患者的外周血,提取基因组DNA,并对外显子扩增产物进行直接测序。结果 7个家系中7对同胞,共14例WD患者均检出致病突变,发现8种致病突变,包括5种错义突变,1种剪接位点突变,1种移码突变以及1种无义突变,其中,c.3851_3876del为新突变。7个家系同胞之间均为相同的基因突变形式,但同胞间临床分型不全相同,先证者临床症状多较其同胞明显,3个家系中表现为弟妹先发病。先证者与其同胞间血清铜及铜蓝蛋白水平无统计学差异。结论 WD患病同胞间基因突变形式一样,但临床分型、症状的轻重程度及发病时间可不同,WD的临床表型除了与基因突变形式有关外,还可能与其他因素相关。     

1,3-D-半乳糖基转移酶p.Arg187Cys突变导致形成ABx亚型

目的 既往在中国人群中发现的1,3-D-半乳糖基转移酶p.Arg187Cys突变个体表型为正常B型,本文探究该突变导致AB亚型的可能分子机制.方法 利用免疫血清学方法鉴定一名中国籍个体的血型血清型,PCR方法扩增ABO基因增强子、启动子和所有7个外显子及其侧翼序列,并对扩增产物进行测序分析;采用Chimira软件构建3...