CD36 c.C275T基因突变导致CD36缺失的研究

Study of CD36 deficiency caused by CD36 c.C275T gene mutation

目的研究在广西CD36缺失个体中检测到的CD36基因突变c.C275T,是否可导致CD36缺失和在人群中的发生率。方法采用血小板抗原单克隆抗体特异性免疫固定试验(MAIPA)和流式细胞技术检测确定广西地区1位汉族男性个体(XXX)的CD36表型,应用CD36外显子测序、cDNA克隆测序对其CD36缺失的分子基础展开研究,构建真核表达细胞株及应用蛋白免疫印迹试验(Western blot)验证所检测到的CD36突变基因对CD36表达的影响。应用所建立的针对该突变基因的DNA PCR-SSP基因分型方法,在随机的无血缘关系的1026名广西地区人群中进行人群分布调查。结果经MAIPA和流式细胞技术检测鉴定XXX的CD36表型为Ⅱ型CD36缺失。CD36外显子测序发现其具有CD36 c.C275T(p.Thr92Met)突变,为突变杂合子,CD36 cDNA克隆测序发现该个体具有c.275C>T(p.Thr92Met)突变型和CD36野生型2种CD36 mRNA转录本。通过建立的真核表达细胞株及Western blot验证结果显示CD36 c.275C>T异常转录本可导致CD36表达缺失。该基因突变在人群的发生率研究显示,在随机的1026名广西人群中,CD36 c.275C>T突变的人群发生率为0。结论CD36基因突变c.275C>T(p.Thr92Met)可导致CD36缺失,并初步阐明了其导致CD36缺失的分子基础及在广西地区人群中的分布特征,为了解中国人群CD36缺失的分子基础和特征提供了实验和理论依据。

Objective To study whether CD36 gene mutation c.C275T which was detected in individuals with CD36 deficiency in Guangxi could lead to CD36 deficiency and the population incidence of the mutation.Methods The CD36 phenotype of a Han Chinese male individual(XXX)in Guangxi was detected by monoclonal antibody immobilization of platelet antigens assay(MAIPA)and flow cytometry(FCM).CD36 exons sequencing and cDNA cloning sequencing were used to study the molecular basis for CD36 deficiency.Eukaryotic expression cell lines were established for the CD36 mutation that was detected and Western blot was used to verify the effect of CD36 expression.The population incidence of the CD36 mutation was investigated among 1026 random and unrelated individuals in Guangxi population by DNA-based polymerase chain reaction with sequence specific primer(PCR-SSP).Results MAIPA and FCM assay showed that the individual was typeⅡCD36 deficiency.CD36 exons sequencing showed that the individual had carried a heterozygous mutation CD36 c.C275T(p.Thr92Met),and CD36 cDNA cloning sequencing confirmed that there were two kinds of CD36 mRNA transcripts:CD36 mRNA transcript variant c.275C>T(p.Thr92Met)and wild-type CD36 mRNA transcript.It was verified that CD36 mRNA transcript variant c.275C>T could lead to CD36 deficiency by the establishment of eukaryotic expression cell lines and Western blot assay.The population incidence of CD36 mutation c.275C>T was 0(0/1026)in Guangxi randomized population.Conclusion This study identifies that CD36 mutation c.275C>T(p.Thr92Met)can result in CD36 deficiency,and preliminary clarifies the molecular basis of CD36 deficiency and distribution characteristics of the mutation in Guangxi population.It help to provide experimental and theoretical basis for exploring the molecular basis and characteristics of CD36 deficiency in Chinese population.