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

Abstract

Background: Although P1 and Xg^a 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 Xg^a 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 Xg^a (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 Xg^a 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 Xg^a , 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 Xg^a . Although this enabled molecular typing of these antigens, the Y chromosome PAR1 region interfered with Xg^a 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.

Figure 1.

WGS Based approach for determination of molecular basis for P1 and for Xg^a.

Figure 2.

Identification of the P1 Genetic Basis. Two approaches used to identify A4GALT changes associated with P1 expression. (A) The frequency approach used whole genome sequences of the A4GALT region to correlate nt changes with expected P1 antigen frequency. The percentages represent the antigen phenotype frequencies for each nt change when used to simulate typing for the antigen from whole genomes. (B) The serology approach used whole genome sequences of the A4GALT region to correlate nt changes with serologic typing. Changes also found in the frequency approach shown in A are colored green. (C) Potential nt changes responsible for P1 expression designated by chromosomal coordinates and by rs database numbering. Note: A4GALT is transcribed in reverse to its direction in the reference genome, as such larger genomic positions correspond to smaller gene positions. The nt bases have been reverse complimented from the human reference genome.

Figure 3.

Identification of the Xg^a Genetic Basis. Two approaches used to identify XG changes associated with Xg^a expression. (A) The frequency approach used whole genome sequences of the XG region to correlate nt changes with expected Xg^a antigen frequency. The percentages represent the antigen phenotype frequencies for each nt change when used to simulate typing for the antigen from whole genomes. (B) The serology approach used whole genome sequences of the XG region to correlate nt changes with serologic typing. Changes also found in the frequency approach shown in A are colored green. (C) Potential nt change responsible for Xg^a expression. (D) Xg^a nt changes in males and the corresponding misplaced chromosome Y PAR1 nt changes.