OA2-AM24-TU-15 - Four Novel ABO Enhancer Variants in Patients With Altered Phenotypes

The ABO blood group system was discovered more than a century ago and continues to have clinically relevant variants discovered. Many variants exist in the coding sequence and flanking intron regions which directly affect the function of the encoded glycosyltransferase. Other variants alter gene expression via regulatory elements in enhancer and promoter regions. This report describes four novel variants in the intron 1 enhancer element of the ABO gene found in five patients (S1-S5) with unusual serology.

Study

Design/Methods:

Serologic testing was performed by standard tube agglutination. DNA was isolated from WBCs (QIAGEN). Sanger sequencing included ABO promoter, intron 1 enhancer, exons 1 to 7 and flanking intron regions. Targeted next generation sequencing (NGS) was done to phase variants for samples S2-S4.

Results/Findings:

S1 was from a 36-year-old male who typed A_veryweakB. Sequencing revealed ABO*A1.01 and B.01 backgrounds and new variant c.28+5789T >C. S2 was from a 75-year-old male who displayed a B_veryweak phenotype. Sequencing showed ABO*B.01 and O.01.01 backgrounds and new variant c.28+5790A >G. NGS phased this change to ABO*B.01. S3 was from a 66-year-old male who tested strongly for the A antigen but displayed mixed-field agglutination. Sequencing revealed ABO alleles A2.01 and O.01.01 and intron 1 change: c.28+5882A >G. NGS analysis placed this change on the A2 allele. S4 was from a 50-year-old female who forward typed as O but reverse typed as A. Sanger sequencing detected changes consistent with ABO*A1.02 and O.01.28 and novel variant: c.28+5861T >G. The variant was phased to A1.02 with NGS. S5 was from a 61-year-old female. Her sample was grossly hemolyzed but displayed microscopic reactivity with Anti-A,B. Sequencing determined her ABO alleles as ABO*A1.02 and O.01.02(46A,189C,681G) and detected the same enhancer variant as S4: c.28+5861T >G.

Conclusions:

This report describes four novel ABO intron 1 variants resulting in very weak or altered ABO serology: c.28+5789T >C on A1, c.28+5790A >G on B, c.28+5882A >G on A2 and c.28+5861T >G on A1.02. All variants lie within the enhancer element located 5.7-6.0kb into intron 1, which recruits transcription factors to regulate ABO expression. Variants c.28+5789T >C and c.28+5790A >G alter an inverted GATA-1 transcription factor binding site possibly causing impaired transcriptional activity and downregulated antigen expression. Variant c.28+5861T >G may achieve similar effects by acting on a separate direct GATA-1 binding site. Variant c.28+5882A >G may alter A antigen expression by interfering with a RUNX1 transcription factor binding site. These novel alleles further showcase the importance of the enhancer region in regulating the expression of ABO antigens.