关键词: RHD基因, 弱D, 蛋白结构, 254C＜A位点突变

Abstract: Objective The serological identification of a sample with weak D phenotype were carried out, and the mutation sites were identified. Further the impact of all mutations at this site on protein structure and function as well as molecular mechanism were studied. Methods The Rh phenotype was identified by serological methods. The RHD zygosity analysis was analyzed by PCR-SSP, and the RHD exon sequence was analyzed by direct PCR sequencing. SOPMA was used for prediction of RhD structure. Constructing 3D simulated structures of proteins using AlphaFold. The protein stability was analyzed by Mupro. PROVEAN and PolyPhen-2 analysis were used to investigate the impact on protein function. Results The antibodies from different clones exhibit weak reactivity with sample red blood cells, and the Rh phenotype is ccEe; RHD gene sequencing revealed a novel mutation c.254C>A (p.Ala85Gly) in exon 2; Retrieval revealed the presence of two other base T and G mutations at position 254. Analysis and prediction suggested that the protein secondary structure did not undergo significant changes with different mutations at position 254. However, c.254C>G and c.254C>A mutations affect the stability of the protein and lead to functional impairment. Conclusion This specimen is a rare case of weak D phenotype caused by c.254C>A. Although the 254 site is located in the transmembrane region, it is a key site of the RHD gene and has four common base forms. Mutations can lead to decreased protein stability and impaired function.

Key words: RHD gene, Weak D phenotype, Protein structure, 254C＜A mutation