肝豆状核变性患者ATP7B基因8-9、10-12号外显子突变分析研究

目的　对肝豆状核变性(Wilson disease, WD)ATP7B基因的外显子8-9、10-12进行DNA测序并分析。方法　50例WD患者和20例对照正常人提取基因组DNA, PCR扩增ATP7B第8-9（约1100bp）、10-12号外显子(约850bp),对所有患者及对照组DNA外显子-内含子扩增产物行直接双向测序。结果　50例WD患者, 11例为8号内含子nt53592A→G伴nt53671G→A的纯合性突变;4例为8号外显子Arg778Leu突变伴Leu770Leu同义突变; 10-12号外显子5例存在外显子11 Gly790Arg杂合错义突变; 11例存在外显子12 Arg952Lys杂合错义突变; 2例伴有外显子12IIe929Val多态性。结论　外显子8、12为中国人ATP7B基因突变热点,已验证其为中国人常见突变位点;8内含子突变与ATP7B表达减少及WD发病相同（正常人未见）;外显子11突变率占所有病人的10%,以前报道甚少,也值得做进一步做大病例研究。     

肝豆状核变性患者ATP7B基因8、12号外显子突变的DNA分析

目的 对肝豆状核变性(Wilson disease,WD)ATP7B基因的外显子8、12进行DNA测序分析并建立直接基因诊断方法.方法 102例WD患者和82例对照正常人提取基因组DNA,PCR扩增ATP7B第8、12号外显子,8号外显子及12号外显子扩增产物分别行Msp Ⅰ及Tail内切酶酶切分析;后对所有患者及对照组DNA外显子扩增产物行直接测序.结果 102例WD患者,8号外显子35例存在Msp Ⅰ酶切反应异常,测序示Arg778Leu纯合或杂合突变(占34.31%),其中12例伴Leu770Leu多态性;12号外显子13例存在Tail酶切反应异常,测序示Thr935Met杂合错义突变(占12.75%),1例存在Argg19Gly杂合错义突变,Arg952Lys在患者及对照组中均检出.结论ATPTB基因外显子8、12为WD突变热点,其中8号外显子以Arg778Leu、12号外显子以Thr935Met为主要突变形式,临床上可用PCR-Msp Ⅰ酶切、PCR-Tail酶切反应作为WD患者初步筛选方法.     

变性高效液相色谱在检测肝豆状核变性基因突变中的作用

目的　建立变性高效液相色谱 (denaturinghighperformanceliquidchromatography,DHPLC)技术检测肝豆状核变性 (WD)基因第 8外显子突变。方法　利用聚合酶链式反应 (PCR)扩增WD基因第 8外显子片段 ,扩增产物直接进行DHPLC分析 ;对峰型有改变的样品经测序分析确认突变。结果　在 5 1例WD先证者中共发现两种错义突变 (Arg778Leu和Arg778Gln)、一种插入突变 (Ins2 30 2C)和一种多态性位点 (C2 310G)。其中 12例先证者带有Arg778Leu杂合错义突变 ,3例为Arg778Leu纯合错义突变 ,1例为Arg778Gln杂合错义突变 ,1例为杂合 2 30 2C插入突变。多态位点C2 310G与Arg778Leu突变完全连锁。结论　WD基因第 8外显子阳性检出率为 33 3% (17/ 5 1) ,说明DHPLC技术是一种可用于临床WD基因诊断的高效、灵敏和操作简便的方法     

华中和华东地区肝豆状核变性患者ATP7B基因第13号外显子突变的研究

目的 对华中和华东地区肝豆状核变性(HLD)患者ATP7B基因第13号外显子突变进行研究.方法 应用PCR-DNA测序技术对来自我国华中和华东地区的139例非同源家系的HLD患者的ATP7B基因第13号外显子进行检测,并与52名正常对照者进行比较.结果 正常对照组测序未见异常.HLD组共检出6种ATP7B基因的突变类型和1种多态,其中Gly988Val杂合突变为新的发现.HLD组突变及多态总的检出率为29.49%(41/139),染色体突变频率为15.83%(44/278);其中Pro992Leu纯合或杂合突变的检出率为23.02%(32/139),其染色体突变频率为12.59%(35/278).结论 ATP7B基因第13号外显子是华中和华东地区HLD患者的一个基因突变热区,是筛选HID可疑患者时优先检测的外显子之一.     

74例肝豆状核变性患者中ATP7B基因七种新突变的发现

目的 分析中国人肝豆状核变性（Wilson disease,WD）患者ATP7B基因突变的分布特征,建立利用变性高效液相色谱（DHPLC）技术对Wilson病进行基因诊断的方法,并评价其在临床的应用价值.方法 对临床确诊为Wilson病的74例患者及50名健康人抽取外周静脉血提取基因组DNA.以患者和健康人的DNA为模板,分别对ATP7B基因的21个外显子进行PCR扩增.取PCR产物应用DHPLC技术在部分变性条件下检测突变并DNA测序证实突变位点.结果 利用DHPLC技术筛查并经测序证实,共发现22种ATP7B基因突变类型,其中7种是新发现的,同时发现11种多态,其中3种是新发现的.第8外显子Arg778Leu突变率最高,其频率为25.0%.其次,2356-2A＞G突变频率为3.4%,Arg919Gly突变频率为2.7%,其他外显子突变频率均在1.0%～2.0%之间.结论 中国人的WD基因突变具有多样性特点,热点突变是第8外显子Arg778Leu.DHPLC具有高通量、敏感、准确且经济的特点,适合于大样本的筛查且能够发现未知的突变,是一种有临床应用价值的基因诊断技术.     

肝豆状核变性患者MURR1基因全长编码序列的突变检测和分析

目的 研究我国肝豆状核变性（WD）患者MURR1基因的突变特征及其与WD患者临床表型的关系.方法 根据MURR1基因全长3个外显子及其毗邻内含子序列,自行设计合成引物,聚合酶链反应（PCR）扩增61例经ATP7B基因诊断确诊的WD患者的MURR1基因序列,PCR产物纯化后直接测序检测MURR1基因突变和多态,并以60名健康人作为对照.结果 1例患者在5＇非翻译区检出较为罕见的-43T→C杂合新多态,因例数太少,无法分析基因型和临床表型的关系.27例患者检出2号内含子的IVS2＋63C→G杂合多态,1例为纯合多态,频率为23.8%（29/122）;60名健康对照中,26名检出此杂合多态,3名为纯合多态,频率为26.7%（32/120）.通过χ^2检验,IVS2＋63C→G在健康对照和WD患者中的频率基本相等（χ^2=0.269,P＞0.1）,表明这是一个群体多态.在MURR1基因编码区和剪接位置均未发现突变或多态.结论 MURR1基因突变在我国WD患者中是罕见的,与我国WD患者的临床表型无相关性.     

肝豆状核变性ATP7B基因Arg778Leu突变型与临床表现的相关性研究

目的:探讨肝豆状核变性(Hepatolenticular Degeneration,HLD)患者ATP7B基因Arg778Leu突变型与临床表现之间的相关性。方法:采用PCR和DNA测序技术检测91例HLD患者ATPTB基因8号外显子Arg778Leu突变,将91例患者分为纯合突变组、杂合突变组和无突变组,并与临床表现(性别、起病年龄、临床表型)进行相关分析。结果:在91例HLD患者中检出26例Arg778Leu纯合子和40例杂合子,其余25例无此突变。患者性别、起病年龄、临床表型与该突变型均无相关性。结论:Arg778Leu突变与患者性别、起病年龄及临床表型无关。     

Wilson病ATP7B基因启动子区的研究

目的 检测肝豆状核变性（Wilson disease，WD）ATP7B基因启动子区的DNA序列，分析其结构，发现存在的突变，通过报告基因瞬时表达研究突变对启动子功能的影响。方法 检测36个WD家系71名成员（其中48例WD患者）及20名正常人的基因组DNA序列并进行分析。对发现的突变，进行荧光素酶报告基因瞬时表达研究。结果 （1）在正常对照、患者一级亲属和WD患者的启动子区-190、-78和＋260位（转录起始点为＋1）均发现存在单个碱基的不同；（2）在48例病人中发现3例存在-183位C→T突变，其中2例为纯合突变，另1例为杂合突变，在正常对照、患者一级亲属中未发现此改变；（3）通过荧光素酶报告基因瞬时表达研究，发现-183位C→T突变不影响ATP7B基因启动子的功能。结论 本研究在ATP7B基因启动子区未发现存在致病突变，提示ATP7B基因启动子区存在致病突变在中国人群中是不常见的。     

肝豆状核变性基因型与临床表型相关性的研究进展

肝豆状核变性(WD)主要是由于编码P型ATP酶的基因突变,导致铜代谢障碍而发病。国内外应用分子生物学等技术对ATP7B基因突变进行了广泛研究。近年来对基因型-表型的相关性的研究日益受到重视,并取得一定进展,本文就基因突变的类型与WD患者的发病年龄、症状、铜代谢生化异常程度及治疗疗效的相关性作一综述。     

一个肌张力障碍-帕金森综合征的家系调查及致病基因研究

目的初步分析一个肌张力障碍-帕金森综合征家系的可能致病基因。方法对一个肌张力障碍-帕金森综合征的家系进行家系调查。在收集先证者相关血液生化指标、超声波、MRI影像学和PET核医学检查等临床资料基础上,对先证者及其家系三代共8人先后进行了目标基因以及全外显子基因测序加可疑位点Sanger测序验证。结果先证者临床表现为眼睑、口面、下肢肌张力异常,伴动作慢、肌张力高和震颤等典型肌张力障碍-帕金森综合征症状,多巴胺能药物不敏感及服药后罕见的阴道出血不良反应。血生化、铜蓝蛋白、肝脏超声及角膜裂隙灯照相、头颈及腰段脊髓MRI扫描均正常,头颅¹⁸F-dopa PET/MRI显像提示双侧豆状核多巴胺神经元受损。该家系三代共9人中出现症状2例(1例已死亡),基因检测显示先证者及另外3人携带父源的RYR1基因36号外显子c.5972C>T (p.T1911I)杂合突变;另有SETX基因外显子10的c.2439A>C (p.E813D)杂合突变;GCDH基因外显子10的c.1060G>A (p.G354S)杂合突变;ATP7B基因外显子18的c.3792G>C ...     

Using Fluorescence PCR Analysis For Gene Diagnosis and Carrier Detection of Chinese Wilson's Disease

Objective To develop a noval gene diagnostic method for detecting the high frequency spot of gene mutation in Chinese Wilson's disease by using the most advanced fluorescence PCR in order to make an early diagnosis and carrier detection. Methods 66 Chinese WD patients from 58 families had typical nanifestations of WD, and significant low levels of serum ceruioplasmin (CP), low levels of serum copper., high levels of urine copper. 55 family members (parents 33 and siblings 22) from 42 families of 58 WD families were normal phenotype with normal levels of CP. 30 in patients suffering from acute cerebrovascular disease, vertigo and headache had no blood relationship to be the control group. We got 5ml blood from each object to collect DNA, and designed two fluorcscent gene probes to hybridize with thc normal and mutant sequence of Arg778Leu respectively. The content of probe hybridization was concordant with the fluoresccin which was released during PCR process. The homozygote, heterozygote of WD and normal were identified by thc results of fluorescence PCR and through analysis we obtained the mutation rate of Arg778Leu. After that we selected 3 random samples (2 from WD patients, I from control group) for direct DNA sequencing in exon 8 of WD gencto testify the accuracy of fluorescence PCR. Results Among 66 Chinese WD patients, homozygous for mutation of Arg778Leu had been found in 5 cases and compound heterozygous found in 21 cases. and the mutation rate of Arg778Leu in our study was totally 39.4%. Of 55 normal phenotype family members. 12 individuals incluing parents 7 and siblings 5 were detected as heterozyous in which 11 (7 parents and 4 siblings) had been confirmed as WD gene carriers but not pre-symptomatic patients according to the throughtout examination and the normal CP. There were no mutation of Arg778Leu in all 30 control cases. Thc results of direct DNA sequencing of 3 at random samples were consilient to those results detected by fluorescence PCR. Conclusion The viewpant which the Arg778Leu mutation is the high frequency spot of Chincse WD gene should be supported by our study. Fluorescence PCR analysis is a rapid. accurate gene diagnostic method and demonstrates a high detecting rate. therefore. it is now the most advanced gene diagnostic method for Wilson's disease.     

Apolipoprotein E genotype analysis in Chinese Han ethnic children with Wilson's disease, with a concentration on those homozygous for R778L

Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism caused by a large number of different mutations in the ATP7B gene. R778L mutation is mostly observed in Chinese, Japanese and Korean patients, whereas the H1069Q point mutation in the ATP7B gene is the most frequent mutation in European patients with WD. In our previous study we did not find a significant correlation between genotype and phenotype (age of onset and clinical presentation) in patients homozygous (37 patients) or heterozygous (52 patients) for R778L. It was reported that European patients homozygous for H1069Q who were also homozygous for the ApoE genotype ε3/3 developed clinical symptoms 5–11 years later than did patients with genotypes other than ApoE ε3/3. In the present study (i) we firstly observed that ApoE ε3/3 did not delay the onset of WD; (ii) no association between ApoE genotype and WD clinical presentation in Chinese Han children, including those patients homozygous for R778L. Thus we conclude that the onset of WD in Chinese children is not related to ApoE ε3/3, although the high frequency of ApoE ε3/3 in Chinese Han children with WD was not significantly different from that in controls.     

Novel ATP7B gene mutations in Chinese Han patients with hepatolenticular degeneration

BACKGROUND: ATP7B gene exon 8 Arg778Leu and exon 12 Arg952Lys are gene mutation hot spots in Chinese Han patients with hepatolenticular degeneration, or Wilson's disease (WD). However, the gene fragments are too short for detection and the mutation detection rate remains low.OBJECTIVE: To analyze DNA sequences of ATP7B gene exon 8-exon 9 and exon 10-exon 12 sections. DESIGN, TIME AND SETTING: A concurrent, non-randomized, controlled, genetic polymorphism study was performed at the Anhui Medical Genetics Center, Anhui, China from March to July in 2009.PARTICIPANTS: Fifty patients, who were admitted to the Department of Neurology at the First Affiliated Hospital of Anhui Traditional Chinese Medical College between March and July in 2009, were diagnosed with WD. The WD group comprised 32 males and 18 females, with an average age of (18.8 ± 8.3) years. WD was confirmed by clinical observation, as well as physical, imaging, and biochemical examinations, including testing for serum copper, ceruloplasmin, and copper oxidase. The control group comprised 20 normal subjects, who underwent physical examination at the First Affiliated Hospital of Anhui Traditional Chinese Medical College, and included 13 males and 7 females, with an average age of (27.9 ± 2.4) years. All subjects were Chinese Han population.METHODS: Genomic DNA was extracted from 50 WD patients and 20 normal controls. Polymerase chain reaction amplification of ATP7B gene exon 8-exon 9 (about 1 100 bp) and exon 10-exon 12 (about 850 bp) segments was performed. DNA exon-intron amplification products from all subjects were processed through direct bidirectional sequencing, and sequencing results were analyzed. MAIN OUTCOME MEASURES: Sequence changes of ATP7B gene exon 8-exon 9 and exon 10-exon 12 segments.RESULTS: In the 50 included WD patients, ATP7B gene intron 8 nt53592A → G with nt53671G → A homozygous mutation was detected between exon 8-exon 9 in seven cases; exon 8 Arg778Leu mutations with Leu770Leu synonymous mutation was detected in four cases; exon 11 Gly790Arg heterozygous missense mutation between exon 10-exon 12 was found in four cases; exon 12 Arg952Lys heterozygous missense mutation was seen in 11 cases; and two additional cases were associated with exon 12IIe929Val polymorphism.CONCLUSION: ATP7B gene intron 8 mutation is a possible pathogenic mutation that is associated with WD pathogenesis. The exon 11 mutation rate accounts for 8% of all WD patients, and the very few previously reported cases deserve further study.     

经DNA测序证实的肝豆状核变性基因突变热区的研究

目的研究我国肝豆状核变性（ＷＤ）基因突变的特征。方法应用聚合酶链反应－单链构像多态（ＰＣＲ－ＳＳＣＰ）技术，结合ＤＮＡ测序技术，筛查４０个ＷＤ家系的５６例患者及无亲缘关系的６０名正常人的ＷＤ基因第５、８、１４号外显子（ｅｘｏｎ５，ｅｘｏｎ８，ｅｘｏｎ１４）的突变及多态。结果２１例患者（来自１５个ＷＤ家系）在ｅｘｏｎ８检出２种错义突变，占３７．５％（１５／４０），其中２例（来自同一家系）发生Ａｒｇ７７８Ｇｌｎ纯合子突变（２．５％），８例（分别来自８个ＷＤ家系）发生Ａｒｇ７７８Ｌｅｕ纯合子突变（２０．０％），余１１例（来自６个ＷＤ家系）发生Ａｒｇ７７８Ｌｅｕ杂合子突变（１５．０％，６／４０）。此外，在第８号外显子区域发现了两种多态；在ｅｘｏｎ５和ｅｘｏｎ１４侧翼的内含子区域各发现了１种新多态。这是我国首次经ＤＮＡ测序证实的ＷＤ基因突变热区。结论ｅｘｏｎ８为我国ＷＤ病人基因突变的热区之一，这对于建立准确快速的ＷＤ直接基因诊断方法具有重要意义，该测序方法对明确基因变异的具体部位和内容的具有一定的重要性。     

Pathogenic compound heterozygous ATP7B mutations with hypoceruloplasminaemia without clinical features of Wilson’s disease

The authors report a 44-year-old man with a history of attention deficit and hyperactivity disorder, obsessive compulsive behaviour, vocal tics, depression, and anxiety, in whom a compound heterozygous ATP7B mutation was found, associated with hypoceruloplasminemia, but without clinical or pathological manifestation of Wilson’s disease (WD). Genetic testing revealed a compound heterozygous ATP7B mutation already described in WD, p.Met645Arg (C1934TG/c.51 + 4A → T). Hypoceruloplasminaemia was detected but no clinical manifestations (hepatic or central nervous system) of WD were present. The authors conclude that patients can carry a heterozygous mutation of the ATP7B gene that is associated with hypoceruloplasminaemia and display no overt clinical hepatic and/or central nervous system manifestations of WD.     

New novel mutation of the ATP7B gene in a family with Wilson disease

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. The WD gene codes for a copper transporting P-type ATPase (ATP7B) are located on chromosome 13q14.3. Mutation of this gene disrupts copper homeostasis, resulting in the accumulation of copper in the liver, brain, kidneys and corneas and copper toxication at these sites. Since the detection of the WD gene in 1993, approximately 300 disease-specific muations have been identified. We recently evaluated a Korean family with WD. The proband, a 17-year-old boy, visited our hospital due to abnormal behaviors including generalized slow movement, dysphagia, drooling and ataxia. Laboratory results revealed decreases in serum copper and ceruloplasmin and an increase in urinary excretion of copper. He had liver cirrhosis, brain lesions and Kayser-Fleischer corenal rings. Molecular genetic analysis of the ATP7B gene demonstrated that he was heterozygous for deletion mutation c.2697_2723del27 in exon 11. Further study of family members revealed that his father and younger brother had the same mutation. The c.2697_2723del27 deletion mutation in exon 11 has not yet been reported as a causative muation of WD and is an in-frame deletion not expected to lead to a frame shift. Therefore, we report a novel mutation of the ATP7B gene in a family with WD.     

ARSA gene mutations in five Chinese metachromatic leukodystrophy patients

The objective was to identify arylsulfatase A mutations, if any, in five Chinese patients with metachromatic leukodystrophy. This would be the first such study in China. All eight exons and exon-intron boundaries of the arylsulfatase A gene (ARSA) were amplified with polymerase chain reaction, which was followed by direct DNA sequencing. Patient 1 exhibited a homozygous mutation at c.954G>A (p.W318X) in exon 5. Patient 2 exhibited compound heterozygous mutations, identified as one allele with the c.862C>T (p.R288C) missense mutation in exon 5 and the other allele with the c.1338dupC frameshift mutation in exon 8. Patient 3 exhibited only a c.179_180dupCA frameshift mutation in exon 1 in one allele. Patients 4 and 5 exhibited identical compound heterozygous mutations, identified as one allele with the c.296G>T (p.G99V) missense mutation and the other allele with the c.251G>A (p.R84Q) missense mutation in exon 2. Six DNA variants of the arylsulfatase A gene were identified: two novel frameshift mutations (c.179_180dupCA and c.1338dupC), one known nonsense mutation (p.W318X), and three known missense mutations (p.R84Q, p.G99V, and p.R288C).