Genotyping for Glycophorin GYP(B-A-B) Hybrid Genes Using a Single Nucleotide Polymorphism-Based Algorithm by Matrix-Assisted Laser Desorption/Ionisation, Time-of-Flight Mass Spectrometry

Abstract

The genetic basis for five GP(B-A-B) MNS system hybrid glycophorin blood group antigens results from rearrangement between the homologous GYPA and GYPB genes. Each hybrid glycophorin displays a characteristic profile of antigens. Currently, no commercial serological reagents are currently available to serologically type for these antigens. The aim of this study was to develop a single nucleotide polymorphism (SNP) mapping genotyping technique to allow characterisation of various GYP(B-A-B) hybrid alleles. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS) assays were designed to genotype five GYP(B-A-B) hybrid alleles. Eight nucleotide positions were targeted and incorporated into the SNP mapping protocol. The allelic frequencies were calculated using peak areas. Sanger sequencing was performed to resolve a GYP*Hop 3' breakpoint. Observed allelic peak area ratios either coincided with the expected ratio or were skewed (above or below) from the expected ratio with switching occurring at and after the expected break point to generate characteristic mass spectral plots for each hybrid. Sequencing showed that the GYP*Hop crossover in the intron 3 region, for this example, was identical to that for GYP*Bun reference sequence. An analytical algorithm using MALDI-TOF MS genotyping platform defined GYPA inserts for five GYP(B-A-B) hybrids. The SNP mapping technique described here demonstrates proof of concept that this technology is viable for genotyping hybrid glycophorins, GYP(A-B-A), GYP(A-B) and GYP(B-A), and addresses the gap in current typing technologies.

Genotyping analysis of MNS blood group GP(B‐A‐B) hybrid glycophorins in the Chinese Southern Han population using a high‐resolution melting assay

“…All control samples were fully phenotyped and genotyped by DNA sequencing. DNA samples for GYP*Hop/GYPB heterozygote and GYP*Kip/GYPB heterozygote, described in our previous studies, were not used in this study due to its limited supply …”

“…DNA samples for GYP*Hop/GYPB heterozygote and GYP*Kip/GYPB heterozygote, described in our previous studies, were not used in this study due to its limited supply. 5,7 PCR and HRM genotyping assay A HRM assay detecting GYP(B-A-B) hybrid alleles was used in this study as previously described. 22 One pair of primers was designed to amplify the Pseudoexon 3 of GYPB gene and the Exon 3 of GYP(B-A-B) hybrid genes.…”

“…GP(B‐A‐B) hybrid glycophorins include GP.Mur, GP.Bun, GP.Hop, GP.HF, and GP.Kip, which arise from the insertion of a homologous segment from the Exon 3 region of the GYPA gene (including the 5′ end of Intron 3) into the GYPB gene. For all the five GP(B‐A‐B) hybrid glycophorins, the breakpoints for the insertion of the GYPA segment are all located in the GYPB Pseudoexon 3 and Intron 3 region, which is considered to be a hotspot for gene conversion events .…”

“…Some of these methods either are laborious and time‐consuming, such as the need for gel electrophoresis, or can only be used for screening but are unable to identify the exact type of hybrid glycophorin. In a previous study, we developed a matrix‐assisted laser desorption/ionization, time‐of‐flight mass spectrometry (MALDI‐TOF MS) assay that targets eight nucleotide positions in GYPA and GYPB to genotype for GYP(B‐A‐B) hybrid glycophorins . MALDI‐TOF MS technology can accurately distinguish five kinds of GYP(B‐A‐B) hybrid alleles and reliably detect gene copy number, but needs an expensive mass spectrometer.…”

“…In a previous study, we developed a matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) assay that targets eight nucleotide positions in GYPA and GYPB to genotype for GYP(B-A-B) hybrid glycophorins. 5 MALDI-TOF MS technology can accurately distinguish five kinds of GYP(B-A-B) hybrid alleles and reliably detect gene copy number, 5,20 but needs an expensive mass spectrometer.…”