A whole genome approach for discovering the genetic basis of blood group antigens: independent confirmation for P1 and Xg^a

Abstract

BACKGROUND Although P1 and Xga are known to be associated with the A4GALT and XG genes, respectively, the genetic basis of antigen expression has been elusive. Recent reports link both P1 and Xga expression with nucleotide changes in the promotor regions and with antigen‐negative phenotypes due to disruption of transcription factor binding. STUDY DESIGN AND METHODS Whole genome sequencing was performed on 113 individuals as part of the MedSeq Project with serologic RBC antigen typing for P1 (n = 77) and Xga (n = 15). Genomic data were analyzed by two approaches, nucleotide frequency correlation and serologic correlation, to find A4GALT and XG changes associated with P1 and Xga expression. RESULTS For P1, the frequency approach identified 29 possible associated nucleotide changes, and the serologic approach revealed four among them correlating with the P1+/P1– phenotype: chr22:43,115,523_43,115,520AAAG/delAAAG (rs66781836); chr 22:43,114,551C/T (rs8138197); chr22:43,114,020 T/G (rs2143918); and chr22:43,113,793G/T (rs5751348). For Xga, the frequency approach identified 82 possible associated nucleotide changes, and among these the serologic approach revealed one correlating with the Xg(a+)/Xg(a–) phenotype: chrX:2,666,384G/C (rs311103). CONCLUSION A bioinformatics analysis pipeline was created to identify genetic changes responsible for RBC antigen expression. This study, in progress before the recently published reports, independently confirms the basis for P1 and Xga. Although this enabled molecular typing of these antigens, the Y chromosome PAR1 region interfered with Xga typing in males. This approach could be used to identify and confirm the genetic basis of antigens, potentially replacing the historical approach using family pedigrees as genomic sequencing becomes commonplace.

Blood group P1 prediction using multiplex PCR genotyping of A4GALT among Thai blood donors

“…Moreover, two other SNVs (rs2143918 and rs5751348), which are tightly linked to rs8138197, showed 100% correlation with P1 expression in different populations 20‐22 . In agreement with the aforementioned studies, it was recently reported that four SNVs (rs66781836, rs8138197, rs2143918 and rs5751348) correlated with P 1 and P 2 phenotypes 21 . Studies examining the gene regulation of those SNVs showed that rs5751348 contains potential transcription factor‐binding sites for runt‐related transcription factor 1 (RUNX1) and early growth response 1 (EGR1), suggesting that these proteins play a role in creating P 1 and P 2 phenotypes 22,23 …”

“…Previous studies have found that a single‐nucleotide variant (SNV) in A4GALT (rs8138197) correlated with P1 expression in 99.5% and 100% of Swedish 18 and Taiwanese 19 blood donors, respectively. Moreover, two other SNVs (rs2143918 and rs5751348), which are tightly linked to rs8138197, showed 100% correlation with P1 expression in different populations 20‐22 . In agreement with the aforementioned studies, it was recently reported that four SNVs (rs66781836, rs8138197, rs2143918 and rs5751348) correlated with P 1 and P 2 phenotypes 21 .…”

“…In 2011, the rs8138197 showed 99.5% concordance with P 1 Swedish donor cohort 18 and, 3 years later, reported the other two SNVs (rs2143918 and rs5751348), tightly linked to rs8138197 to show 100% correlation in the four populations 20 . In 2019, four SNVs in A4GALT —rs66781836, rs8138197, rs2143918 and rs5751348—were reported to correlate with P 1 and P 2 phenotypes 21 . In this study, the SNV rs66781836 located at chr22:43115523_43,115520delAAAG, a 4‐nucleotide deletion, was not found in all Thai donors, and it was thus excluded from further PCR development.…”

“…Genomic DNA samples were PCR‐amplified for SNVs present in the non‐coding regions of A4GALT , particularly rs66781836, rs8138197, rs2143918 and rs5751348 20‐23 . Primer pair sequences for each amplicon are shown in Table 1.…”

Automated typing of red blood cell and platelet antigens from whole exome sequences

“…We estimated the effect of this limitation on RBC and PLT antigen typing by reviewing the antigen allele tables for nonexonic nucleotide changes that impact antigen phenotype predictions. Other than the recently published transcription factor binding regions deep within A4GALT1 Intron 1 and upstream of XG that control the expression the P1 and Xg a antigens , all other nonexonic changes associated with antigenic variation are located within 13 bp of an exon boundary. In fact, analysis of WES data from all 75 participants showed a similar depth of coverage pattern to previous WGS efforts, with overall adequate sequencing coverage of all exons, but with some low‐sequence coverage regions in nonantigen allele nucleotide positions within CR1 , C4A , C4B , and several other genes (Fig.…”