肝豆状核变性疾病的基因分析及基因诊断的研究

华中科技大学

硕士学位论文

肝豆状核变性疾病的基因分析及基因诊断的研究

姓名：王平

申请学位级别：硕士

专业：临床检验学

指导教师：吴健民

20070501

华　中　科　技　大　学　硕　士　学　位　论　文

第一部分　

ＡＴＰ７Ｂ和ＣＯＭＭＤ１基因与肝豆状核变性疾病　

临床表型相关性的研究　

摘　要　

目的检测 37例中国肝豆状核变性患者的ATP7B基因和COMMD1基因，探讨2个基因在WD致病和患者临床表现多样性中所起的作用。方法PCR法选择性扩增ATP7B基因的突变热区；扩增COMMD1基因的3个外显子包括和内含子的交界区，并对扩增产物测序。再将基因型结果与临床表型进行相关分析。结果37例标本中检测到12例有ATP7BArg778Leu突变，其中有9例表现为肝脏症状首发（75%）；2例有Thr935Met 突变；COMMD1基因上没有发现任何能引起密码子和剪切位点改变的突变，检测到了1个多态性IVS2+63C>G。结论 ATP7B仍是目前唯一确定的WD致病基因，其Arg778Leu突变与肝脏症状表现为首发症状有关。没有证据支持COMMD1基因与中国人群的肝豆状核变性有关。

关键词 肝豆状核变性 ＡＴＰ７Ｂ基因 ＣＯＭＭＤ１基因 临床表现　

Abstract

Aim To study a cohort of 37 Chinese Han people with Wilson disease to illuminate the role of ATP7B and COMMD1 in the pathogenesis of WD as well as the influence to clinical manifestation diversity in WD. Methods The three exons of the COMMD1 gene including the intron–exon boundaries and hot area of ATP7B gene were amplified by polymerase chain reaction (PCR) and analyzed by direct sequencing. Results Our study detected ATP7B Arg778Leu mutation in 12 patients, 9 of them show liver symptom at the onset of disease. Thr935Met were detected in 2 patients. We did not reveal any mutations leading to an amino acid or splicing site change in the COMMD1 sequence. A polymorphism at IVS2+63C>G was confirmed. Conclusions There is no evidence to

华　中　科　技　大　学　硕　士　学　位　论　文

第二部分

四引物扩增受阻突变体系快速检测Ｗｉｌｓｏｎ病　

基因Ａｒｇ７７８Ｌｅｕ突变　

摘要

目的建立一种不需要限制性片段长度多态性或直接测序的新方法检测肝豆状核变性（WD）病人突变热点ATP7B 基因的Arg778Leu突变基因型。方法用四引物扩增受阻突变体系聚合酶链反应（Tetra-primer amplification refractory mutation system-PCR, tetra -primer ARMS-PCR）检测中国37例WD患者和30例正常对照的ATP7B基因Arg778Leu突变，并用测序进行验证。结果 37例WD患者中检出Arg778Leu纯合突变3例，杂合突变9例，总检出率32.43%(12/37)；30例正常对照未发现突变；DNA测序结果与四引物ARMS-PCR结果完全一致。结论 ATP7B基因Arg778Leu突变是中国人WD突变热点；四引物ARMS-PCR法适用于Arg778Leu 突变检测，有快速、简便、准确的优点，可以区分等位基因是纯合子或杂合子，易于大量本的人群筛查。

关键词肝豆状核变性；四引物扩增受阻突变体系；基因诊断

Abstract

Aim The aim of this study was to establish a new method for genotyping ATP7B Arg778Leu gene mutation that does not require RFLP PCR or sequencing. Design and Method 4-primer ARMS- PCR was performed to screen the Arg778Leu mutation in 37 unrelated WD patients and 30 unrelated healthy controls. And direct sequencing was used to confirm the results specific amplification products. Results PCR products were visualized on agarose gel electrophoresis. Among the 37 WD patients, 3 were homozygous and 9 were heterozygous for this mutation. The mutation rate was totally 32.43%(12/37).The results of direct sequencing completely consisted with the results of