RHD*845A/1227A变异型个体的分子机制

Molecular Mechanism of a Rhesus D Variant Individual with RHD*845A/1227A

目的:研究1例罕见的RhD变异型个体的基因突变机制。方法:采用常规血清学方法对样本进行Rh分型,间接抗人球蛋白试验确认RhD抗原以及抗体筛查。D-screen试剂分析样本的RhD抗原表位。利用序列特异性引物聚合酶链反应法检测RHD基因以及RhD杂合型分析,Sanger测序方法分析RHD基因编码区序列。应用逆转录聚合酶链反应检测样本RHD mRNA,对RHD cDNA进行TA克隆和序列分析。结果:血清学检测该样本RhD抗原为弱阳性,Rh分型为CcDEe,抗体筛查结果为阴性。经D-screen试剂检测样本表现出部分D的抗原表位分布。经RHD基因编码序列分析发现,RHD基因第6外显子第845位和第9外显子第1227位碱基均显示G/A杂合。TA克隆得到3种剪接异构体。结论:该研究对象为RHD基因845G>A和1227G>A突变个体,该杂合突变导致样本红细胞RhD抗原减弱。

Objective:To explore the genetic mutation mechanism of a rare Rhesus D variant individual.Methods:Regular serological assay was used for determination of Rh type for the sample.Indirect anti-human globulin test(IAT)was used to confirm the RhD antigen and screen the antibodies.D-screen reagent was used to analyze the RhD epitopes of the sample.RHD genotype and RHD zygosity testing of the sample were detected by palymerase chain reaction with sequence-specific primers(PCR-SSP).The full length coding region of RHD gene was sequenced.RHD mRNA was detected using reverse transcription polymerase chain reaction(RT-PCR).The PCR products were cloned and sequenced.Results:The RhD blood group of the sample was determined as weak D,and the Rh phenotype was CcDEe.The antibody screening was negative.The sample tested with all monoclonal anti-Ds in D-screen showed the D epitope profiles as partial D types.The analysis of RHD gene sequence indicated that the individual with RHD c.845G/A and RHD c.1227G/A base heterozygosis.Three kinds of alternative splicing isoforms were obtained by TA cloning and sequencing.Conclusion:The object has RHD c.845G/A and RHD c.1227G/A mutation.This heterozygous mutation is responsible for the low expression of RhD antigen on the red blood cells of the sample.