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

Area of Science:

• Genetics
• Molecular Biology
• Immunology

Background:

• Splice-site variants in the ABO gene can disrupt ABO glycosyltransferase biosynthesis.
• This disruption leads to reduced expression of A or B antigens on red blood cells, causing weak antigen phenotypes.

Purpose of the Study:

• To characterize the impact of four specific intron 6 splice-site variants (three in A1 allele, one in ABO*B.01 allele) on ABO pre-mRNA splicing.
• To establish a genotype-splicing-phenotype association model for these ABO variants.

Main Methods:

• Serologic and molecular genetic analyses
• Bioinformatic predictions of splice site recognition
• Minigene assays to assess splicing efficiency
• Quantitative real-time PCR to measure transcript levels

Main Results:

• All four variants impaired 5' splice site recognition, reducing functional ABO transcripts.
• A allele variants (c.374+5G>A, c.374+4A>G, c.374+4A>T) resulted in 2.8% to 10.2% exon 6 retention.
• B allele variant (c.374+2_374+3insT) retained 4.7% exon 6 transcripts, allowing trace B glycosyltransferase expression.
• Transcriptional levels in an ABel subtype individual were 51.7% of controls.

Conclusions:

• Splice-site variants in the ABO gene reduce functional transcript abundance through altered transcriptional regulation.
• This leads to decreased ABO glycosyltransferase expression and variable weak antigen phenotypes.
• The study provides a genotype-splicing-phenotype model for understanding ABO blood group variations.