CD36基因新突变T538C（Trp180Arg）导致的CD36缺失和序列特异性引物PCR的基因分型技术的建立  

A novel CD.36 mutation T538C （Trp180Arg） results in CD36 deficiency and establishment of a genotyping method for the novel mutation based on sequence-specific primer PCR

作　　者：李丽兰 何保仁 周燕 钟周琳 黎海燕 卢芳 刘金莲 申卫东 李恒聪 蒋丽红 吴国光 

机构地区：[1]南宁输血医学研究所,530007 [2]南宁中心血站,530007

出　　处：《中华医学遗传学杂志》2016年第5期619-624,共6页Chinese Journal of Medical Genetics

基　　金：南宁市科学研究与技术开发计划项目[（科技惠民重大科技专项）（20133141）,（20133194）];广西科学研究与技术开发计划项目[（主席基金）（08160-05）,（桂科攻1355005-2-4）]

摘　　要：目的对广西一位女性个体CD36缺失的分子基础和特征及其基因分布进行研究。方法采用血小板抗原单克隆抗体特异性免疫固定试验（monoelonal antibody immobilization of platelet antigens assay, MAIPA） 和流式细胞技术（flowcytometry，FCM）检测确定广西地区一位汉族女性个体的CD36表型，应用CD36外显子测序、cDNA克隆测序对其cD36缺失发生的分子基础展开研究，构建表达CD36突变基因的细胞株[CD36（MT）细胞株]和表达CD36野生型基因的细胞株[CD36（wT）细胞株]，应用Western印迹检测细胞株CD36蛋白表达情况。建立序列特异性引物聚合酶链反应（polymerase chain reaction-sequence specific primer, PCR-SSP）检测技术对所发现的突变基因进行进一步检测确认，在1010名随机无血缘关系的中国广西健康人中进行该CD36突变基因的多态性调查。结果本研究对象经MAIPA和FCM表型检测结果为Ⅱ型CD36缺失，CD36外显子测序发现其具有T538C（Trp180Arg）突变杂合子，该突变点位于CD36第6外显子，克隆测序发现其CD36cDNA538位点T被c所替代，该突变可导致CD36成熟蛋白氨基酸序列发生Trp180Arg改变。应用所建立的CD36T538CPCR—SSP检测结果显示DNA分型检测结果与测序结果完全一致；Western印迹结果显示CD36在CD36（MT）细胞株上不表达，而在CD36（wT）细胞株上有表达。对1010名中国广西人群进行cD36T538C的基因多态性调查发现该CD36538T和538C等位基因频率分别为1．000和0．000，在本次调查人群中未发现具有538C等位基因的个体。结论发现了一个CD36基因新突变T538C（Trp180Arg）（GenBank：HM217022．1），并建立了鉴定该基因突变的PCR—SSP的技术。该基因新突变是中国广西人群中一个罕见型，可导致Ⅱ型CD36缺失的发生。本研究结果为了解中国地区人群CD36缺失发生的分子基础和特征以及CD36基因新突变T538C的人群调查提供了实验基础�Objective To explore the molecular basis for a CD36 deficiency individual and distribution of CD36 gene mutation in Guangxi population. Methods A female individual was studied. CD36 phenotype was detected by monoclonal antibody immobilization of platelet antigens assay （MAIPA） and flow cytometry （FCM）. The coding regions of the CD36 gene were sequenced. A DNA-based polymerase chain reaction- sequence specific primer （PCR-SSP） assay was used to verify the identified mutation. Cell lines expressing the mutant and wild-type CD36 [CD36 （MT） and CD36 （WT）] were established, with the expression of CD36 determined by Western blotting. The distribution of CD36 gene mutation was investigated among 1010 unrelated individuals with the PCR-SSP assay. Results Both MAIPA and FCM assays showed that the patient had type Ⅱ CD36 deficiency. DNA sequencing showed that she has carried a heterozygous mutation T538C （TrplSOArg） in the exon 6 of CD36. Sequencing of cDNA clone confirmed that there was a nucleotide substitution at position 538 （538T〉C）. Western blotting also confirmed that the CD36 did not express on the CD36 （MT） cell line that expressed the 538C mutant, but did express on the CD36 （WT） cell line. The novel CD36 mutation T538C was further verified with 100% concordance of genotyping results by DNA- based PCR-SSP assay and 1010 unrelated individuals. No CD36 538C allele was detected among the 1010 individuals. Conclusion This study has identified a novel CD36 mutation T538C（Trp180Arg） （GenBanc： HM217022.1）, and established a genotyping method for the novel sequence-specific primer PCR. The novel mutation is rare in Guangxi and can cause type II CD36 deficiency.

关 键 词：CD36缺失 基因突变 广西人群 血小板 

分 类 号：R394[医药卫生—医学遗传学]