Comprehensive prospective investigation of splicing variants in the coding region of RHD : Insights into phenotypic consequences including revision of RHD * weak D type 3

As splicing has been shown to be commonly altered by single nucleotide variants (SNVs) in the RHD gene, resulting in a mild to severe drop in the expression of the D antigen, we sought to identify prospectively those deleterious SNVs that may disrupt splicing. All potential substitutions affecting the coding deoxyribonucleic acid (DNA) sequence (CDS) of the RHD gene were scored by using two in silico tools: SpliceAI and SPiP. In the 25 top-score SNVs selected for functional analysis, 11 variants either partially (N = 8) or completely (3) disrupted splicing, suggesting that the quantitative expression of the D antigen may be negatively affected. Interestingly, the c.8C>G variant defining the RHD weak D type 3 allele was found to impair splicing dramatically both in vitro and in vivo, while the p.(Ser3Cys) amino acid substitution in the RhD protein is not responsible for the decrease in the membrane expression of the D antigen. Overall, our fundamental findings redefine the functional effect of the RHD weak D type 3 allele and emphasize the complex regulation of D antigen expression. This prospective work also illustrates how the combination of computational and experimental analyses is instrumental for rare variant interpretation and genotype-phenotype correlation studies.