Functional Analysis of Four Splice-Site Variants, including a Novel Variant, on Antigen Expression and ABO Subgroup...

Splice-site variants within the ABO gene have the potential to impair ABO glycosyltransferase biosynthesis, leading to decreased expression of A or B antigens on the surface of red blood cells. This study characterized how four intron 6 splice-site variants-three in the A1 allele (c.374+4A>T, c.374+4A>G, and c.374+5G>A) and one in the ABO∗B.01 allele (c.374+2_374+3insT)-affect ABO pre-mRNA splicing. A combination of serologic, molecular genetic, bioinformatic, and minigene assays established a genotype-splicing-phenotype association model. Bioinformatics predictions showed that all variants impaired the recognition of the 5' splice site. Specifically, the A allele variant c.374+5G>A induced approximately 2.8% exon 6 retention, whereas the c.374+4A>G (A_finn/A_weak) and c.374+4A>T (A_weak) variants preserved approximately 6.9% and 10.2% functional transcripts, respectively. The novel B allele insertion variant c.374+2_374+3insT (B_el subtype) retained approximately 4.7% exon 6-containing transcripts, sufficient for trace B glycosyltransferase expression. Quantitative real-time PCR analysis confirmed that the transcriptional levels of the ABO gene in the AB_el subtype individual carrying the c.374+2_374+3insT variant were approximately 51.7% of those in the ABO∗A1.02/ABO∗B.01 control. This study demonstrated that splice-site variants in the ABO gene reduce the abundance of functional transcripts via altered transcriptional regulation, which, in turn, leads to decreased ABO glycosyltransferase expression, ultimately resulting in weak antigen phenotypes of varying intensity.