WilSon病蛋白及其基因在肝豆状核变性患者中的改变

目的探讨肝豆状核变性(WD)发病的分子生物学机制.方法通过外科手术获得3例WD患者、2例对照者的肝活检标本,利用胶原酶温育消化法分离肝细胞,并进行体外原代培养;肝细胞裂解离心后,使用6%聚丙烯酰胺凝胶行SDS-PAGE电泳,分离WD蛋白;以抗人WD蛋白抗体为第一抗体、辣根过氧化物酶标记的羊抗兔抗体为第二抗体,对肝细胞WD蛋白进行Westem-blot印迹检测,观察特异性条带数目、密度深浅和分子量大小;同时采用荧光PCR技术,筛查所有研究对象WD基因8号外显子(exon 8)的突变情况,对异常者进行DNA直接测序结果 WD患者及对照者肝细胞WD蛋白Westem-blot检测均表现为特异的155kDa条带,3例WD患者中1例无明显变化,另2例出现155kDa条带密度降低,仅有对照组密度的1/3～1/2,提示这两例患者的WD蛋白在肝内表达异常;荧光PCR筛查发现1例患者存在exon 8 Arg778 Leu突变,DNA直接测序证实为2273G→TG杂合突变.结论 WD基因蛋白产物在WD患者肝细胞的表达存在异常,这可能与WD基因Arg778Leu位点突变有关,直接检测WD基因蛋白产物有助于研究WD的发病机制.     

肝豆状核变性基因启动子区的多态和突变研究

目的　检测肝豆状核变性 (wilsondisease ,WD)基因启动子区的DNA序列 ,发现存在的多态和突变。方法　 2 0 0 1- 0 2～ 2 0 0 4 - 0 2检测 36个WD家系 71名成员 (其中 4 8例WD患者 )及 2 0名正常人的基因组DNA序列 ,进行分析。结果　在正常对照、患者一级亲属和WD患者的启动子区 - 190、- 78和 2 6 0位 (转录起始点为 1)均发现存在单个碱基的不同 ;在 4 8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。结论　启动子区在调控WD基因转录活性中起重要的作用 ,提示启动子区的突变是WD的分子发病机制之一。     

湖南地区肝豆状核变性基因突变热区的序列检测与分析

目的检测湖南地区汉族人群肝豆状核变性患者（WD）ATP7B基因常见突变种类和形式。方法提取22例WD患者外周血基因组DNA，聚合酶链反应（PCR）扩增ATP7B基因第5、8、12及13号外显子并进行DNA直接测序检测，应用在线BLAST软件分析。结果22例患者中共发现15例患者存在基因突变。其中10例患者存在8号外显子2273G→T杂合突变（即Arg778Leu），且均伴有2250C→G多态（即Leu770Leu，均为杂合子），未发现纯合突变。12号外显子中共发现2855G→A多态（即Arg952Lys）7例（杂合突变4例，纯合突变3例），其中1例合并12号外显子2828G→A杂合突变（即Gly943Asp），另3例合并Arg778Leu杂合突变。13号外显子2975C→T杂合突变（即Pro992Leu）1例。5号外显子未发现突变。结论Arg778Leu是湖南地区汉族WD患者的突变热点，5号外显子为非突变热点。     

肝豆状核变性发病机理和致病基因的研究近况

肝豆状核变性(HLD)的铜代谢异常主要表现为胆汁排铜减少和铜蓝蛋白合成障碍。前者与大分子量铜结合蛋白缺乏或金属巯基蛋白异常有关,后者由铜蓝蛋白基因转录水平降低等原因所致。HLD 致病基因造成上述两种改变,从而引起体内铜的大量沉积。该基因被定位于13q14～q21,应用该基因附近 DNA 标记的 RFLP 连锁分析,是进行症状前诊断和杂合子检测的可靠方法。     

肝豆状核变性患者肝组织WD基因mRNA的表达特征

采用原位杂交结合图象分析的半定量方法研究肝豆状核变性(WD)基因mRNA的表达特征,并对WD患者WD基因全部21个外显子进行DNA序列测定,探索WD的发病机制。 一、材料与方法 1.实验对象:WD患者4例,病例符合WD的临床诊断标准。(1)具有肝病史、肝病体征或锥体外系征;(2)血清铜蓝蛋白降低和肝铜升高;(3)角膜K-F环。行脾切除术时获取肝组织标本;对照组1例,男,32岁,来自肝脏外伤修补术的肝组织标本,经各项检查排除各种肝炎、肝硬化、肝肿瘤疾病。     

肝豆状核变性的分子生物学研究进展

肝豆状核变性，又称Ｗｉｌｓｏｎ病（ＷＤ），是一种以铜代谢障碍为特征的常染色体隐性遗传病，其致病基因定位于１３ｑ１４．３，已克隆分离出ＷＤ基因。序列分析及家系突变研究表明，该基因编码与铜离子活化有关的Ｐ型ＡＴＰ酶。基因的突变形式多种多样，在３８种已发现的突变中至少有２３种突变影响酶的功能，但突变热点仍在进一步筛选之中。     

肝豆状核变性3例

目的提高对不典型肝豆状核变性病的诊断能力.方法本文将我们在临床中收治的3例肝豆状核变性病做了简要的回顾分析.男2例,女1例,年龄分别为13岁、10岁、15岁.误诊时间分别为12mo、1mo、2mo.主要症状分别为反复乏力、腹胀、尿黄,阵发性腹痛;双下肢痉挛.误诊病种为神经病变,肝硬变,肾结石.其1例父母为近亲结婚.结果肝豆状核变性(WILson病)是一种可累及多系统病变的疾病.多以青少年为主.有一定的遗传性,易误诊.结论结合文献提出凡出现原因未明的儿童肝硬变,儿童期学习成绩下降,长期乏力、腹胀、腹痛、振颤及精神症状等表现,均应考虑本病的可能,作出相应的检查,尽早作出诊断.     

肝豆状核变性39例临床分析

目的 探讨肝豆状核变性的临床特点。方法 回顾性分析39例Wilson病患者的临床表现。结果 39例患者中，精神、神经异常者22例，乏力、腹胀、纳差13例，其他4例。K-F环阳性31例。神经型19例、肝型13例、肝神经型7例。血清铜蓝蛋白(CER)、血清铜(Cu^2 )均降低；血清尿酸低于正常下限37例。尿隐血、蛋白阳性30例。头颅CT显示双尾状核、豆状核低密度灶26例，CT阴性患者MRI阳性8例。结论 肝豆状核变性多见于青少年，神经系统及肝损害为主要临床表现，常伴有肾脏损害，角膜K-F环是重要阳性体征，头颅CT或MRI可作为辅助诊断手段。     

金属硫蛋白与肝豆状核变性

肝豆状核变性发病的异常蛋白学说目前正受到重视。本文概述了近年来金属硫蛋白的研究进展,并介绍了肝豆状核变性中金属硫蛋白的异常变化以及金属硫蛋白在肝豆状核变性发病机理中的作用。     

Copper transportion of WD protein in hepatocytes from Wilson disease patients in vitro

AIM:To study the effect of copper transporting P-typeATPase in copper metabolism of hepstocyte andpathogenesis of Wilson disease(WD).METHODS:WD copper transporting properties in someorganelles of the cultured hepstocytes were studied fromWD patients and normal controls.These culturedhepstocytes were incubated in the media of copper 15mg·L~(-1) only,copper 15 mg·L~(-1) with vincristine(agonist of P-type ATPase)0.5mg·L~(-1),or copper 15 mg·L~(-1) withvanadate(antagonist of P-type ATPase)18 39mg.L~(-1)separately.Mlcrosome(endoplasmic reticulum and Golgiapparatus),lysosome,mitochondria,and cytosol wereisolated by differential centrifugstion.Copper contents inthese organelles were measured with atomic absorptionspectrophotometer,and the influence in copper transportionof these organelles by vanadate and vincristine werecomparatively analyzed between WD patients and controls.WD copper transporting P-type ATPase was detected bySDS-PAGE in conjunction with Western blot in liver samplesof WD patients and controls.RESULTS:The specific WD proteins(Mr155 000 lanes)wereexpressed in human hepatocytes,including the control andWD patients.After incubation with medium containingcopper for 2 h or 24 h,the microsome copper concentrationIn WD patients was obviously lower than that of controls,and the addtion of vanadste or vincristine would change thecopper transporting of microsomes obviously.Whenincubated with vincristine,levels of copper in microsomewere significantly increased,while incubated with vanadate,the copper corcentrations in microsome were obviouslydecreased.The results indicated that there were WDproteins,the copper tmnsportion P-type ATPase in themicrosome of hepstocytes.WD patients possessedabnormal copper transporting function of WD protein in themicrosome,and the agonist might correct the defect of copper transportion by promoting the activity of coppertransportion P-type ATPase.CONCLUSION:Copper tmnsportion P-type ATPase plays animportant role in hepatocytic copper metabolism.Dysfunction of hepatocytic WD protein copper transportionmight be one of the most important factors for WD.     

Gastropathy in patients with Wilson disease

Abstract

Background: Gastrointestinal symptoms are common in patients with Wilson disease (WD) and may be related to the disease itself or to adverse drug reactions (ADRs).

Aim: To investigate gastroscopy findings in patients with WD and to analyze the risk of gastropathy in the context of different manifestations and treatments of WD as well as Helicobacter pylori infection status.

Methods: This cross-sectional study included patients diagnosed or monitored for WD between 2007 and 2017. All enrolled patients were examined with gastroscopy and checked for infection with a urease test. Based on predominant manifestations, WD was classified as pre-symptomatic, hepatic (only liver symptoms) or neurological. Patients were divided into three treatment groups: untreated, treated with d-penicillamine (DPA) or zinc sulfate therapy.

Results: Of 115 patients, 58 were male and the median age was 30 years. Gastropathy was observed in 65.2% of all patients. Factors that increased the risk of gastropathy were zinc sulfate (odds ratio [OR]?=?3.01; 95% confidence interval [CI]: 1.12–8.09, p?=?.03), H. pylori infection (OR?=?2.96; 95%CI: 1.34–6.56, p?=?.01) and neurological manifestations (OR?=?2.55; 95%CI: 1.16–5.60, p?=?.02). In total, 9.6% of patients had gastric or duodenal ulcers and 29.6% had esophageal varices but no difference was seen by treatment status. In multivariate analysis, zinc sulfate remained associated with higher risk of gastropathy compared with no treatment (OR?=?4.57; 95%CI: 1.21–17.19; p?=?.03) and DPA (OR?=?6.28; 95%CI: 1.43–27.56; p?=?.01).

Conclusions: Our results show that gastropathy in WD may be influenced by the treatment used.

• Keypoints

• In a retrospective study of 115 patients with Wilson’s disease, gastric injury was frequent.

• Patients receiving zinc sulfate had increased gastropathy risk compared with those receiving no treatment or d-penicillamine.

     

肝豆状核变性基因表达产物的初步研究

目的 对肝豆状核变性（WD）基因编码产物WD蛋白进行检测,探讨WD的发病机制,方法 应用Western-blot蛋白印迹技术对诊断为WD的患才进行WD蛋白的研究。结果 发现患者WD在肝内表达缺失或者含量改变。结论 WD患者可能同时存在有WD基因exon8和exon5的突变,直接检测WD基因产物为进一步研究WD的病理机制奠定了基础。     

Clinical and molecular characterization of Wilson disease in Spanish patients

Wilson disease (WD) results when specific mutations occur at the ATP7B gene. The presence of mutations in the ATP7B gene was studied in the coding region and the intron-exon boundaries in 15 WD Spanish patients, and their first-degree relatives when possible. A total of 20 nucleotide sequence changes were detected, 18 missense and two splicing mutations. Six of these variants were classified as disease-causing mutations, five missense, and one splicing; four of them have been previously described (M645R, A1065P, H1069Q, and 3060 + 5G > T), whereas two were novel (P768L and A990P). No mutation was clearly prevalent, although the H1069Q mutation predominated, nor did a good phenotype-genotype correlation exist. The two new mutations described were manifested as an asymptomatic increase in serum transaminases. The remaining 14 changes were classified as polymorphisms and their potential effects on protein function are discussed. The identification of mutations in the ATP7B gene has allowed a conclusive diagnosis to be made of WD in patients presenting neurological phenotype or neurological of hepatic phenotype, who would otherwise not have been diagnosed using classical criteria. WD patients could start chelating treatment earlier on and possibly modify the natural progression of the disease.     

血清C反应蛋白水平在克罗恩病中的改变

目的 比较血清C反应蛋白(CRP)水平在不同肠道疾病、不同病变部位和不同活动度中的变化,评估CRP作为疾病活动度指标的价值.方法 收集CD患者42例,分为结肠组25例和小肠组17例,另收集溃疡性结肠炎(UC)患者23例和肠易激综合征(IBS)患者26例作为对照组.胶乳增强免疫透射比浊法检测血清高敏(hs)-CRP水平.结果 ①CD、UC、IBS组hs-CRP均值分别为(9.9±7.8)mg/L、(3.6±4.5)mg/L、(1.1±1.8)mg/L,CD组显著高于UC组和IBS组,差异有统计学意义(P＜0.01),三组hs-CRP超过正常值患者的比率分别为76.2%、30.4%和7.7%,CD组亦显著高于UC组和IBS组,差异有统计学意义(P＜0.01).②在CD患者中,结肠组的hs-CRP显著高于小肠组[(11.9±7.6)mg/L比(6.8±7.2)mg/L,P=0.04);结肠组hs-CRP超过正常值患者的比率较小肠组高,但差异无统计学意义(P＞0.05).③CD患者中,hs-CRP≥10 mg/L者18例,其中疾病缓解(疾病活动指标＜150)4例(4/17),轻度(150～220)3例(3/11),中度(221～450)10例(10/13),重度(＞450)1例(1/1);hs-CRP与CD疾病活动指标、红细胞沉降率有显著相关性(r分别为0.52和0.70,P值均＜0.01).结论 CRP作为疾病活动指标主要用于CD,病变仅累及小肠者的CRP升高程度显著低于结肠病变者,CD病情愈重CRP升高愈明显.     

Platelet function and coagulation in patients with Wilson disease.

Sixteen patients with Wilson disease (hepatolenticular degeneration) were studied from the hemostatic point of view, particularly with regard to platelet function. Five of the patients had a mild bleeding tendency that was characterized by easy bruising. Moderate thrombocytopenia was observed in three of the five bleeders and in two of the others. One bleeder was thrombocytotic and hyperfibrinogenemic. Bleeding times, platelet retention and prothrombin consumption were abnormal rarely. However, 15 of the 16 patients had some abnormality of platelet aggregation: one when adenosine diphosphate was added to platelet rich plasma, three when epinephrine was added, and the remainder when collagen was added. The collagen abnormalities were delayed or absent aggregation (five patients, four of whom were bleeders) and absence of a change of shape (12 of the 16 patients). Platelet aggregation was completely normal in only one patient.     

血清超敏C-反应蛋白、视黄醇结合蛋白4及血浆结合珠蛋白水平在冠心病患者中的变化及其意义

目的探讨冠心病(CHD)患者血清超敏C-反应蛋白(hs-CRP)、视黄醇结合蛋白4(RBP4)及血浆结合珠蛋白(Hp)水平的变化及其临床意义。方法选取2014年2月至2017年2月广安市人民医院心血管内科经冠状动脉造影检查确诊的CHD患者140例作为CHD组,另选取同期在我院心血管内科住院的非CHD患者60例作为对照组。将CHD组患者根据临床表现分为3个亚组:急性心肌梗死(AMI)组(n=38)、不稳定型心绞痛(UAP)组(n=62)和稳定型心绞痛(SAP)组(n=40)。依据病变支数分为3个亚组:单支病变组(n=27)、双支病变组(n=54)和三支病变组(n=59)。检测各组患者的血清hs-CRP、RBP4、Gensini评分及血浆Hp水平,并分析各指标与病情严重程度的关系。采用SPSS 16.0软件进行数据处理。依据数据类型,组间比较分别采用t检验和x~2检验。相关性分析采用Pearson线性相关分析法。结果 CHD组患者的血清hs-CRP[(7.29±3.65)vs(2.84±1.16)mg/L]、RBP4[(49.64±8.32)vs(42.07±5.51)ng/ml]及血浆Hp[(95.26±19.14)vs(70.13±16.33)mg/L]水平均显著高于对照组(P0.05)。AMI亚组患者的血清hs-CRP、RBP4、Gensini评分及血浆Hp水平均显著高于SAP亚组和UAP亚组患者(P0.05);UAP亚组患者的血清hs-CRP、RBP4、Gensini评分及血浆Hp水平均显著高于SAP亚组患者(P0.05)。三支病变亚组患者的血清hs-CRP、RBP4、Gensini评分及血浆Hp水平均显著高于双支病变和单支比病变亚组患者(P0.05);双支病变亚组患者的血清hs-CRP、RBP4、Gensini评分及血浆Hp水平均显著高于单支病变亚组患者(P0.05)。CHD患者的Gensini评分与血清hs-CRP(r=0.619)、RBP4(r=0.554)及血浆Hp(r=0.592)水平均呈显著正相关(P0.05)。结论 CHD患者血清hs-CRP、RBP4及血浆Hp水平显著升高,且与冠状动脉病变严重程度密切相关。     

Copper toxicosis gene MURRl is not changed in Wilson disease patients with normal blood ceruloplasmin levels

AIM: To analyze our Wilson disease patient cohort (n = 106) for alterations in the gene coding for MURR1. METHODS: Patients with an established diagnosis of Wilson disease but normal ceruloplasmin blood levels were chosen for our study (n = 14). Patients with two known disease-causing mutations in the ATP7B gene were not included. The three exons of the human MURR1 gene were sequenced after amplification of the genomic DNA by polymerase chain reaction. RESULTS: Our study did not reveal any mutations leading to an amino acid change in the MURR1 sequence of Wilson disease patients. A polymorphism at 472 bp of the coding sequence could be confirmed. CONCLUSION: The MURR1 gene plays no role in the pathogenesis of Wilson disease patients with normal serum ceruloplasmin levels.