[Two Novel Mutations at the CD36 Gene Splicing Sites and Their Molecular Basis for the CD36 Deficiency].

To study two novel CD36 gene mutations at the CD36 splicing sites found in Guangxi population, as well as the molecular basis and population incidence of them.DNA sequencing and cDNA clonal sequencing were used to detect CD36 exon sequence and the protein coding region sequence of CD36 mRNA for 2 CD36 deficient individuals (HHC and WGM) found in Guangxi population. Eukaryotic expression cell lines were established for the discovery of CD36 mRNA abnormal transcripts and Western blot assay was used to verify the effect of abnormal CD36 mRNA transcripts on CD36 expression. A DNA PCR-SSP genotyping method was established for the two CD36 novel mutations, and the population distribution was investigated among 110 CD36 deficient individuals in Guangxi region and 296 random individuals in Guangxi population.Novel mutation of c.430 -1G>C was found at the CD36 splicing site in HHC and WGM individuals, and novel mutation of c.1006 +2T>G at the CD36 splicing site was also found in the WGM individual. CD36 cDNA clonal sequencing showed that CD36 c.430 -1G>C could lead to the production of the two CD36 mRNA transcript variants: c.429_430ins［430-17_430-2;C］(p.Ala144fsTer1) and c.430_609del(p.Ala144_Pro203del)(GenBank:HM 217023.1); and CD36 c.1006 +2T>G could lead to the production of CD36 mRNA transcript variant of c.819_1006 del (p.Ser274GlufsTer16) (GenBank: HM217025.1). It was verified that all the three transcript variants could lead to CD36 deficiency by establishment of eukaryotic expression cell lines and Western blot assay. A study of the population incidence of two novel CD36 splicing site mutations found showed that in 110 CD36 deficient individuals and in 296 random individuals in Guangxi region, the mutation rate of CD36 c.430 -1G>C was 10.91% (12/110) and 1.35% (4/296), respectively, while CD36 c.1006 +2T>G was 2.73% (3/110) and 0 (0/296), respectively.This study identifies two novel CD36 mutations at CD36 splicing site, and preliminary clarified their molecular basis for the CD36 deficiency and the distribution characteristics in Guangxi population as well. It provides an experimental and theoretical basis for studying the molecular mechanism and characteristics of CD36 deficiency in Chinese population.位于CD36基因剪切位点的2个新突变及其导致CD36缺失的分子基础.对在广西人群中发现的2个位于CD36剪切位点的新基因突变，以及它们导致CD36缺失的分子基础和在人群中的发生率进行研究.对在广西人群中发现的2例CD36缺失个体（HHC和WGM）使用DNA测序和cDNA克隆测序，检测他们的CD36外显子序列及mRNA蛋白编码区序列，对所发现的CD36 mRNA异常转录本建立真核表达细胞株并用Western blot实验验证异常转录本对CD36表达的影响。对所发现的新突变基因建立DNA PCR-SSP基因分型方法，在广西地区的110名CD36缺失个体群和广西地区随机的296名人群间展开人群分布调查.在HHC和WGM个体中，发现了CD36剪切位点新突变CD36 c.430 -1G>C，且在WGM个体中还发现了CD36 c.1006 +2T>G的剪切位点新突变。cDNA克隆测序显示，CD36 c.430 -1G>C可导致c.429_430ins［430-17_430-2;C］（p.Ala144fsTer1）和c.430_609del(p.Ala144_Pro203del，GenBank注册号:HM217023.1）2种CD36 mRNA异常转录本的产生，而CD36 c.1006 +2T>G可导致c.819_1006del（p.Ser274GlufsTer16）(GenBank注册号:HM217025.1）CD36 mRNA异常转录本的产生。通过建立的真核表达细胞株及Western blot实验验证了以上3种异常转录本均可导致CD36表达缺失。对所发现的2个剪切位点突变基因展开的人群发生率的研究显示，在110名CD36缺失个体群和随机的296名人群中，CD36 c.430 -1G>C突变发生率分别为10.91%（12/110）和1.35%（4/296），而CD36 c.1006 +2T>G突变的发生率分别为2.73%（3/110）和0（0/296）.本研究发现了2个位于CD36基因剪切位点的新突变，初步阐明了它们导致CD36缺失的分子基础及在广西人群中的分布特征，为中国人群CD36缺失的分子机制及特征研究提供了实验和理论依据.