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

Objectives: This study aimed to investigate single-nucleotide variants (SNVs) associated with P1 expression among Thai blood donors and develop a genotyping method using multiplex polymerase chain reaction (PCR) to predict P1 blood group status.

Background: The α1,4-galactosyltransferase (A4GALT), also called Gb3/CD77 synthase or P1/P^k synthase enzyme, is encoded by the A4GALT gene and catalyses the transfer of galactose from uridine diphosphate-galactose to lactosylceramide, creating the P^k antigen (Gb3). The same enzyme synthesises the P1 antigen by adding terminal galactose to paragloboside. The A4GALT transcripts are elevated in P₁ , and different SNVs in transcription factor-binding regions of A4GALT correlate with P₁ and P₂ phenotypes.

Material and methods: A total of 218 blood samples from Thai blood donors at the Thammasat University Hospital were tested for the P1 antigen using the conventional tube technique. Genomic DNA was extracted, and non-coding regions of A4GALT were sequenced and analysed. A multiplex PCR assay was developed and validated to identify P1-associated SNVs and was subsequently tested on 1022 Thai DNA samples of unknown P1 antigen status.

Results: In the tested cohort (n = 218), P₁ and P₂ phenotypes were found in 24.77% and 75.23% of donors, respectively. Moreover, three SNVs-rs8138197 (C/T), rs2143918 (T/G) and rs5751348 (G/T)-correlated 100% with both phenotypes. Finally, findings agreed with serological phenotyping and DNA sequencing results, confirming their validity for predicting P1 antigen positivity.

Conclusions: This study confirmed that three SNVs also correlated with P₁ /P₂ phenotypes among Thais, as expected. A multiplex PCR found that SNVs rs2143918 (T) and rs5751348 (G) predicted blood group P1 and is an accurate, reproducible, cost-effective and less time-consuming alternative to traditional methods.