【作者】 王平；

【导师】 吴健民；

【作者基本信息】 华中科技大学 ， 临床检验学， 2007， 硕士

【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。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。更多还原

【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 support that there is a correlation between the COMMD1 gene and WD in patients of Chinese Han population. ATP7B is the only disease causing gene of WD. ATP7B Arg778Leu mutation is associated with the onset of liver symptom 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 4-primer ARMS- PCR. Conclusions The ATP7B Arg778Leu gene mutation was a hot spot in Chinese WD patients. 4-primer ARMS- PCR is a faster, convenient and accurate method for typing mutation in high throughput population screening. This approach can be used to detect other point mutations.更多还原