A systematic study of single-nucleotide polymorphisms in the A4GALT gene suggests a molecular genetic basis for the P1/P2 blood groups.

BACKGROUND: The molecular mechanism for the formation of the P1/P2 blood groups remains unsolved. It has been shown that the P1/P2 polymorphism is connected to the different A4GALT gene expression levels in P1 and P2 red blood cells. STUDY DESIGN AND METHODS: The present investigation conducted a pilot investigation that involved the detailed and stepwise screening of single-nucleotide polymorphisms (SNPs) in the A4GALT gene, followed by a larger-scale association study. The transcription-inducing activity by the different genotypes of SNPs was analyzed using reporter assays. RESULTS: A total of 416 different SNP sites in the A4GALT genes from four P1 and four P2 individuals were analyzed in the pilot investigation, and 11 SNP sites, distributed in the A4GALT Intron 1 region, exhibited an association with the P1/P2 phenotypes. In the follow-up association study, the genotypes at the 11 SNPs of a total of 338 individuals across four different ethnic populations were determined, and the results show that two SNPs, rs2143918 and rs5751348, are consistently associated with the P1/P2 phenotypes. Reporter assays demonstrated significantly higher transcription-inducing activity by the SNPs bearing the P(1)-allele genotype than by the SNPs bearing the P(2)-allele genotype and that the difference in transcriptional activity was determined by the different genotypes at SNP rs5751348. CONCLUSION: The results of this investigation demonstrate a consistent association of A4GALT SNPs rs2143918 and rs5751348 with the P1/P2 phenotypes and suggest that SNP rs5751348 may lead to allelic variations in A4GALT gene expression and consequently leads to the formation of the P1/P2 phenotypes.

A direct blood polymerase chain reaction approach for the determination of GP.Mur (Mi.III) and other Hil+ Miltenberger glycophorin variants.

BACKGROUND: GP.Mur (Mi.III) is a glycophorin B-A-B hybrid sialoglycoprotein expressing several potent immunogens, including Mi(a), Mur, and Hil. GP.Mur is considered one of the most important red blood cell (RBC) phenotypes in blood banking in Southeast Asia. However, there are no antibodies commercially available for the screening of GP.Mur RBCs. STUDY DESIGN AND METHODS: To develop a direct blood polymerase chain reaction (PCR) approach for the screening of GP.Mur cells, we first confirmed the genomic sequence differences among four GP.Mur and three Mi(a-) samples by sequencing their GYP.Mur and GYPB genes. With these data, we designed PCR primers that best discriminate GYPB and GYP.Mur. Our primer design also allows the detection of other Hil+ glycophorin variants. We also constructed two plasmids--pGBi2i3 and pMiIIIi2i3--which serve as the negative and positive control DNA, respectively, for the PCR procedure. Additionally, we designed a control PCR to be run side by side with the typing PCR. RESULTS: Because of the high specificity of our primers, we found it unnecessary to extract DNA from blood samples for PCR. We have tested this PCR method on 379 fresh and frozen blood samples. The results were further validated by serology and DNA sequencing and were shown to be completely accurate in our hand. We also found that the rapid genotyping method--high-resolution melting--can be a timesaving alternative for DNA sequencing. CONCLUSION: This direct blood PCR approach for determination of GP.Mur and related Hil+ phenotypes is reliable and economical and is expected to be useful for blood banking in Southeast Asia.

The molecular genetic background leading to the formation of the human erythroid-specific Xga/CD99 blood groups.

The Xga and CD99 antigens of the human Xg blood group system show a unique and sex-specific phenotypic relationship. The phenotypic relationship is believed to result from transcriptional coregulation of the XG and CD99 genes, which span the pseudoautosomal boundary of the X and Y chromosomes. However, the molecular genetic background responsible for these blood groups has remained undetermined. During the present investigation, we initially conducted a pilot study aimed at individuals with different Xga/CD99 phenotypes; this used targeted next-generation sequencing of the genomic areas relevant to XG and CD99 This was followed by a large-scale association study that demonstrated a definite association between a single nucleotide polymorphism (SNP) rs311103 and the Xga/CD99 blood groups. The G and C genotypes of SNP rs311103 were associated with the Xg(a+)/CD99H and Xg(a-)/CD99L phenotypes, respectively. The rs311103 genomic region with the G genotype was found to have stronger transcription-enhancing activity by reporter assay, and this occurred specifically with erythroid-lineage cells. Such activity was absent when the same region with the C genotype was investigated. In silico analysis of the polymorphic rs311103 genomic regions revealed that a binding motif for members of the GATA transcription factor family was present in the rs311103[G] region. Follow-up investigations showed that the erythroid GATA1 factor is able to bind specifically to the rs311103[G] region and markedly stimulates the transcriptional activity of the rs311103[G] segment. The present findings identify the genetic basis of the erythroid-specific Xga/CD99 blood group phenotypes and reveal the molecular background of their formation.