A novel RHD*01N allele harboring a c.634+1G>A splice site variant results in the RhD-negative phenotype in a Chinese blood donor.

Background: The D-negative phenotype demonstrates significant ethnic diversity in its molecular background. This study reports the identification of a novel RHD*01 N allele resulting from a splicing site variation observed in a Chinese blood donor.

Study design and methods: The D blood group phenotype was determined using serological techniques, including the saline method, and the indirect antiglobulin test (IAT) performed by both tube and microcolumn gel methods. Screening for the RHD gene was performed by directly amplifying exon 10 of the RHD gene from whole blood samples. Upon confirming the presence of the RHD gene in the sample, further detailed analysis was performed using Sanger sequencing and third-generation single-molecule real-time (SMRT) sequencing technologies. Additionally, the deep learning-based tool SpliceAI (Illumina, USA) was used to evaluate the potential impact of an unreported variant on splicing.

Results: The D antigen in the proband's blood sample was detected as negative by both automated and manual methods. Exon 10 of the RHD gene was detected as positive. Sanger sequencing and third-generation single-molecule real-time (SMRT) sequencing were utilized for the detection of the RHD gene, and the results demonstrated that the sample carried a single RHD haplotype harboring a c.634+1G>A splice site variant. The SpliceAI prediction indicates that the c.634+1G>A splice site variant has a significant impact on splicing.

Conclusion: A novel RHD allele harboring the c.634+1G>A splice site variant, which results in the RhD-negative phenotype, was identified and characterized in a Chinese blood donor.