RHD*95A型D变异体血型鉴定及分子生物学研究

Identification and Molecular Biology of Variant D Blood Group of RHD*95A Genotype

目的:探讨RHD*95A基因型的D变异体血型的分子生物学特征及其产生的遗传机制。方法:分析一家系3代共6个标本,采用试管法及微柱凝胶卡法鉴定RHD血型,应用聚合酶链式反应-序列特异性引物法(PCR-SSP)对RHD基因的10个外显子进行扩增及直接测序分析。利用同源建模的方法比较突变后的RHD蛋白与野生型RHD蛋白结构上的区别。结果:血清学鉴定先证者为D变异型,RHD基因测序分析发现第1外显子存在c.95C>A碱基突变,导致多肽链第32位氨基酸由苏氨酸Thr(T)转变成天冬氨酸Asn(N),其余外显子序列与正常RHD*01基因一致。该家系中发现先证者的父亲、叔叔、奶奶均携带相同的RHD*95A等位基因。蛋白建模结果提示,RHD基因多肽链发生p.T32N突变后,与第32位氨基酸残基相连的氢链发生改变,影响RHD蛋白结构稳定性。结论:在中国人群中发现RHD*95A基因的遗传家系,该家系中发现4例携带c.95C>A碱基突变的RHD基因,证明该突变可稳定遗传。该突变可引起RHD抗原表达减弱。对D变异型人群进行基因鉴定及蛋白结构分析有利于探索其形成的分子机制,保障输血安全。

Objective:To explore the molecular biology of D variant blood group with RHD*95 A genotype and the genetic mechanism of its generation.Methods:A total of 6 samples from 3 generations of a family were analyzed.RHD blood group was identified by saline test tube and microcolumn gel card method.10 exons of RHD gene were amplified by Polymerase Chain Reaction-Sequence Specific Primer(PCR-SSP)and analyzed by direct sequencing.Homology modeling was used to compare the structural differences between mutant RHD protein and wild-type RHD protein.Results:The proband was identified as D variant by serological identification,RHD gene sequencing directly detected a c.95 c>A mutation in exon 1 that leads to encoding the 32-bit amino acids by threonine Thr(T)into aspartic acid Asn(N),the rest of the exon sequences were normal compared with the normal RHD*01 gene.In the family,the proband′s father,grandmather and uncle were all carried the same RHD*95 A allele.Protein modeling results suggested that the hydrogen chain connected to the 32 nd amino acid residue was changed after p.T32 N mutation,which affected the structural stability of RHD protein.Conclusion:The first genetic lineage of the RHD*95 A gene was identified in a Chinese population.The c.95 C>A mutation in RHD gene was found in the family,which resulted in reduced expression of RHD antigen and showed D variant,the mutation could be stably inheritable.Gene identification and protein structure analysis of D variant population is helpful to explore the molecular mechanism of its formation and ensure the safety of blood transfusion.