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摘要: 目的 对临床送检的一例产生抗-D的RhD变异型个体进行RHD基因分析，预测与分析突变对RhD蛋白的影响，评估该变异D类型的临床意义。方法 采用聚合酶链反应(PCR)直接测序法对RHD基因10个外显子及侧翼序列进行序列分析。采用PCR-序列特异性引物对患者RHD基因进行合子型分析。使用PyMOL软件分析突变引起的RhD蛋白三维结构的变化。利用蛋白质变异效应分析、分类非容忍变异和多态性表型分型算法三种在线软件预测突变导致的氨基酸替换对RhD蛋白功能的影响。结果 该变异D个体为RHD(+)/RHD(－)杂合子型。测序结果显示在第5外显子有c.787G＞A突变，该突变导致263位甘氨酸被精氨酸替代。PyMOL分析结果显示，与野生型相比，精氨酸与相邻氨基酸之间的氢键变短、氢键数增加。3种蛋白功能损伤预测软件中，有2种提示p.G263R对RhD蛋白是"有害突变"。结论 该D变异型为弱D100型。c.787G＞A突变导致的氨基酸替换可能影响RhD蛋白的正确组装折叠，导致RhD抗原特征的改变。这提示该D变异型被正常D抗原免疫后具有产生抗-D的潜力。Abstract: Objective To perform genetic analysis of a RHD variant, predict and analyze the effect of the mutation on RhD antigen and evaluate the clinical significance of the variant type.Methods Ten exons and adjacent flanking intron regions of RHD gene were amplified and analyzed by direct sequencing. RHD zygosity test was performed using Polymerase Chain Reaction-Sequence Specific Primers (PCR-SSP). PyMOL software were used to analyze 3D structures of RhD caused by the variant alleles. The effect of non-synonymous substitution was predicted using Protein Variation Effect Analyzer (PROVEAN), Sorting Intolerant From Tolerant (SIFT) and Polymorphism Phenotyping algorithm (PolyPhen-2) software.Results The variant was hemizygous RHD(－). One single-nucleotide missense variant c.787G>A was detceted in exon 5 encoding a p.G263R substitution. PyMOL 3D structural simulation analysis showed that hydrogen bonds between arginine and adjacent amino acids became shorter and increased compared with wild type. Two of the three bioinformatics programs suggested that p. G263R substitution was deleterious to RhD protein function.Conclusion The RHD variant was classified as weak D100 type. The amino acid substitution caused by c.787G>A mutation may affect the normal assembly of tertiary structure, leading to changes in RhD antigen characteristics. These results suggest that the D variant has the potential to produce anti-D when immunized with normal D antigen.
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Key words:
- Rh blood group /
- D-variant /
- weak D type 100 /
- PyMOL /
- bioinformatics analysis
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表 1 三种软件对c.787G>A突变是否损伤RhD蛋白功能的分析
突变 氨基酸改变 预测结果 软件 分值 预测结果 SIFT 0.142 可以容忍 c.787G>A p.G263R PROVEAN -6.4 有害 Polyphen-2 0.525 突变可能有害 注：根据预测原理，SIFT预测得分≤0.05提示突变对蛋白有害，得分>0.05提示突变可以容忍。PROVEAN预测得分≤-2.5提示突变对蛋白有害，得分>-2.5提示突变对蛋白功能无影响。Polyphen-2预测得分在0~0.452为突变无害，得分在0.453~0.956为突变可能有害，得分在0.957~1.000为突变很可能有害。 
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