广州地区无偿献血者的CD36缺失频率及其分子基础研究

The Study on the frequency and molecular analysis of the CD36 deficiency in Guangzhou blood donor

目的了解正常无偿献血人群中CD36缺失频率及其分子基础。方法选取2015年5月—2016年12月选取广州地区1000名18—55周岁,符合国家献血者健康标准的无偿捐献血小板的健康献血者,采集其外周血3—5mL/人(份),从中分离出血小板和单核细胞,用以做CD36表达水平的流式检测,筛选出CD36缺失者并计算缺失率。提取单核细胞基因组DNA,应用聚合酶链反应-测序分型方法(PCR-SBT)扩增CD36基因的12个编码区序列片段并测序,所得序列与GenBank数据库获取的CD36核酸序列(ACCESSION:NG_008192)做比对分析,确认有无新的基因突变。结果本组广州地区无偿献血者的CD36缺失率为2.1%(21/1000),其中CD缺失Ⅰ型占23.81%(5/21)、Ⅱ型占76.19%(16/21)。将全部缺失型做测序分析后发现1个新的基因变异型:194-201delTTTGGATC;克隆测序确认该个体(标本)CD36cDNA从第194位发生TTTGGATC碱基串的缺失;生物信息学分析:该突变型可能导致CD36阅读框发生框移,产生终止密码子,导致编码蛋白提前结束。结论广州无偿献血人群中存在一定的CD36缺失频率。发现的1例CD36新突变(GenBank:KY972554),其可以促使成熟蛋白氨基酸序列提前结束,可能是引起血小板CD36缺失的原因。

Objective To screen CD36 deficient individuals from the blood donor pool,and investigate the molecular mechanisms of CD36 deficiency.Methods 1,000 Whole blood samples were collected during May 2015 to December 2016.Healthy blood donors aged 18—55 years in the Guangzhou area fulfilling the National health standards for blood donors were sorted out. samples of CD36 deficiency were detected by CD36 antigen using flow cytometry on the PLTs and monocytes. The deficiency rate was calculated. The extracted DNAs were sent for sequencing the CD36 gene coding fragment by polymerase chain reaction-sequencing typing( PCR-SBT). The results were compared with standard CD36 nucleic acid sequences( ACCESSION: NG008192) from the Gen Bank database to confirm the gene mutation.Results The CD36 deficiency rate of free blood donors in the Guangzhou area was 2. 1 %( 21/1,000),of which the type I accounted for 23. 81 %( 5/21) and type II accounted for 76. 19 %( 16/21). Analyzed all the CD36 deficiency individuals by sequencing with a novel mutation type:194—201 del TTTGGATC. Cloning sequencing confirmed the absence of TTTGGATC base strings from the 194 loci of the individual( specimen) CD36 c DNA; After analysis of bioinformatics,we found the deletion of TTTGGATC lead to a frameshift and the appearance of a termination codon,which finally induce the premature termination of the coding protein. Conclusion There is a certain frequency of CD36 deficiency in the group of blood donors in Guangzhou. One case of a novel CD36 mutation( Gen Bank: KY972554) was identified,which is potentially capable of premature termination of the coding protein. This maybe account for CD36 deficiency in platelets.