Multiplex-PCR technique to predict polymorphic antigens - M, N, S and s - and associations between their alleles and Mia-associated hybrid glycophorins

Abstract

Serological typing of MNS polymorphic antigens - M, N, S and s - remains a fundamental technique in transfusion medicine and prenatal care, providing essential information for matching blood donors and recipients and managing haemolytic disease. Although this method is well proven and routinely used, it is not a comprehensive solution, as it has several weaknesses. Alternatively, multiplex polymerase chain reaction (PCR) is a commonly used genotyping tool due to its potency and ability to amplify several DNA targets simultaneously in a single reaction. In this work, we aimed to develop multiplex PCR and evaluate its performance for GYPA*M, GYPA*N, GYPB*S, and GYPB*s allele identification using serological and DNA sequencing methods. We also aimed to investigate the correlation between these alleles and Mi^a-associated hybrid glycophorins (GPs). Remarkably, multiplex PCR was well optimised, and the results aligned with serological phenotyping and DNA sequencing data with maximum accuracy and reliability; this confirmed our findings on its validity in predicting MNSs phenotypes. In addition, this work strongly demonstrates, for the first time, a moderate correlation between the GYPA*M/M and GYPB*s/s genotypes and Mi^a-associated hybrid GPs among Thai donors. Individuals with the GYPA*M/M and GYPB*s/s genotypes, predicted M + N - S- s + phenotypes, will thus most likely to express the Mi(a+) antigen. Nevertheless, further studies are required to validate these results and elucidate the underlying correlations.

Keywords: Genetic variability; MNS blood group system; Mia-associated hybrid glycophorins; Multiplex PCR; Red cell genotyping.

Fig. 1

Schematic depiction of the GYPA (A) and GYPB (B) gene sections illustrating the locations of the polymorphisms (*) and the oligonucleotide primers used in this study. The intron is indicated by a thin line, and exon sequences belonging to the gene-based genomic reference sequences of GYPA (NG_007470.3) and GYPB (NG_007483.2) are displayed as blue and green boxes, respectively. The numbering scheme is contingent on the reference genes. The GYPB-R primer was used for DNA sequencing but was not included in the multiplex PCR testing

Fig. 2

Agarose gel electrophoresis results for multiplex PCR with DNA control samples of four phenotypes. Lanes 001A and 001B, M+ N− S− s+; Lanes 002A and 002B, M+ N+ S− s+; Lanes 003A and 003B, M− N+ S+ s−; Lanes 004A and 004B, M+ N+ S+ s+; Lanes NCA and NCB, non-template controls; Lane M: DNA ladder marker (GeneRuler 100 bp Plus; Fermentas, Carlsbad, CA, USA). Gene and allele markers corresponding to the amplified fragments are indicated on the right. Molecular sizes are displayed (in bp) on the left and right.

Fig. 3

The allele frequencies of the variants were compared between 12 distinct ethnic groups and Thais, using data obtained from previous studies. The symbols ●, ○, ▲, △, ■, □, ◆, ◇, ⬣, , ▼, ▽ and ★ denote allele frequencies of African American, American Native, Alaska Native/Aleut, Caucasian, Chinese, Filipino, Hawaiian/Pacific Islander, Hispanic, Japanese, Korean, South Asian, Southeast Asian, and Thai, respectively. The allele frequencies that were significantly different (p < .05) between Thai and other populations are displayed in red, while the non-statistically significant differences are displayed in blue

Fig. 4

Phi correlation heat map: Blue denotes a positive correlation (darker blue indicates a stronger correlation); green denotes a negative correlation. The phi index ranged from −1 to +1. Bold values denote statistical significance at the p < .05 level. NA = not available.