罕见RhD变异型的分子机制及D抗原生物信息学分析

Molecular mechanism and bioinformatics analysis of 9 samples with rare RhD variants

目的研究9例罕见RhD变异型的分子机制及其D抗原表位和蛋白结构。方法在深圳地区210644名无偿献血者中筛选得到RhD变异型标本9例。采用常规血清学方法对9例标本进行Rh分型,间接抗球蛋白试验(IAT)确认RhD抗原以及抗体筛查。D-screen试剂分析标本的RhD抗原表位。分别利用序列特异性引物聚合酶联反应(PCR-SSP)法和Sanger测序方法分析RHD基因杂合性和RHD基因编码区及其侧翼区域序列。利用PROVEAN、SIFT、PolyPhen-2和MutationTaster软件分析预测突变对RhD蛋白质功能的影响。使用Robetta服务器和Swiss-PdbViewer 4.1.0进行RhD蛋白模型构建。结果9例标本的RHD基因型分别为:RHD^(*)weak D type 25、RHD^(*)weak D type 50、RHD^(*)weak D type 95、RHD*weak D type 12、RHD^(*)weak D type 128和4个新的RHD等位基因。生物信息学软件和三维结构预测结果显示突变影响了蛋白质的结构和功能。结论鉴定了5个罕见的RhD变异型和4个新的RHD等位基因。这些RhD变异型的表型和基因型结果丰富了RHD等位基因库的研究数据。通过对RhD抗原的生物信息学分析,有助于进一步研究RHD基因突变对蛋白结构和功能的影响。

Objective To study the molecular mechanism of 9 samples with rare RhD variants and their RhD epitopes and protein structure.Methods The 9 blood samples with rare RhD variants were collected from 210644 blood donors of Shenzhen Blood Center.Regular serological assaying was used for determination of Rh type for the 9 samples.Indirect antihuman globulin test(IAT)was used to confirm the RhD antigen and to screen the antibodies.D-screen reagent was sued to analyze the RhD epitopes of the samples.RHD zygosity testing of the samples was detected by PCR-SSP.The nucleotide sequences of all 9 exons and adjacent flanking intron regions of RHD gene were sequenced.The prediction of the effects of mutations on RhD protein function were analyzed using PROVEAN,SIFT,PolyPhen-2 and MutationTaster software.Robetta and Swiss-PdbViewer 4.1.0 were used for modeling the tertiary structures of RhD.Results A total of 9 individuals with rare RhD variants were identified as follows:RHD^(*)weak D type 25,RHD^(*) weak D type 50,RHD^(*) weak D type 95,RHD^(*) weak D type 12,RHD*weak D type 128 and four novel RHD alleles.The prediction of the tertiary structures showed that the RhD protein conformation was disrupted in the 9 rare RhD variants samples.Conclusion Five rare and four novel RHD alleles have been identified.Their phenotypic and genotypic descriptions enrich the database of reported RHD alleles.Bioinformatics analysis provided evidences for further study of the structure and functions of RhD protein.