[Blood Typing and Pedigree Analysis of a Novel RHD Allele with c.179T>C and c.1154-31T>C Mutations]

To perform genotyping on a specimen serologically identified as a D variant, and conduct a family survey and analysis.

Serological methods were used to confirm RhD and detect RhD antigen epitopes in the proband and his family members' specimens. PCR-SSP method was applied to analyze RHD gene zygosity and perform preliminary RHD genotyping. Sanger sequencing and single-molecule real-time sequencing were used to perform sequencing analysis of RHD gene exons 1-10 and whole-genome sequencing for the proband and his family members' specimens. The tertiary structure models of the protein before and after mutation were constructed using AlphaFold 3 software to analyze the changes in interactions between the mutated amino acid and surrounding residues. Additionally, online tools were employed to predict the impact of the amino acid substitution due to the mutations on RhD protein function.

The serological testing of the proband's mother revealed an RhD-negative blood type with the antigen profile CCee, RHD exon sequencing identified a heterozygous genotype: RHD*01N.03/RHD*01N.01. Both the proband and his father exhibited serological D variants with the antigen profile CcEe, and preliminary PCR-SSP analysis indicated RhD heterozygosity. Sanger sequencing of exons 1-10 of the RHD gene and whole-genome sequencing of the proband and his father revealed a c.179T>C mutation in exon 2 and a c.1154-31T>C mutation in exon 9. AlphaFold 3 modeling predicted that the loss of hydrophobic contact between the Ile-60 and Val-56 residues leads to reduced protein stability. Among the three software tools used for predicting protein function damage, two suggested that this mutation might be deleterious to the function of the RhD protein.

The proband has a heterozygous genotype of RHD*01N.01/RHD*(c.179T>C, c.1154-31T>C). The p.Ile60Thr mutation leads to changes in intramolecular forces between amino acid residues, thereby causing partial changes in the structure and function of the mutated protein.