Frequencies and subtypes of glycophorin GYP(B-A-B) hybrids among northern Thais, Burmese, and Karen with a previous history of malaria infection: a study in the Thailand-Myanmar border area

Abstract

Background: Evidence indicates that genetic variations in the GYP(B-A-B) hybrid genes are associated with protection against malaria. Therefore, this study aims to characterize the GYP(B-A-B) hybrid alleles among northern Thais, Burmese, and Karen with and without a previous history of malaria infection.

Methods: A total of 709 DNA samples were genotyped to identify GYP(B-A-B) hybrids using PCR-sequence specific primers (PCR-SSP) combined with Sanger sequencing. Additionally, some DNA samples (n = 243) were also tested with high-resolution melting (HRM) analysis.

Results: In our sampled populations, 14/87 (16.0%), 3/34 (8.8%), 0/16 (0%), and 1/18 (5.6%) of northern Thais, Burmese, Karen, and other minorities in Myanmar with a previous history of malaria infection, respectively, were identified with GYP(B-A-B) hybrid genes, whereas individuals without a history of malaria infection were 24/155 (15.5%), 5/183 (2.6%), and 4/216 (1.9%) in northern Thais, Burmese, and Karen, respectively. In the latter groups, DNA sequences showed that 17/155 (11.0%) northern Thais were GYP*Mur/GYPB heterozygotes and the other 6/155 (3.9%) were GYP*Thai/GYPB heterozygotes. The remaining one (0.6%) sample was a GYP*Mur/GYP*Mur homozygote. Among Burmese, 3/183 (1.6%) were GYP*Mur/GYPB heterozygotes and 1/183 (0.5%) was GYP*Thai/GYPB heterozygote. The remaining one (0.5%) sample being a GYP*Mur/GYP*Mur homozygote. Among Karen samples, all four were GYP*Mur/GYPB heterozygotes.

Conclusion: Across all studied populations, GYP*Mur was the predominant allele, followed by GYP*Thai. In addition, genotyping results obtained by HRM were consistent with PCR-SSP combined with Sanger sequencing. A statistically non-significant association was noted for the glycophorin GYP(B-A-B) hybrids and malaria infection. Our findings provide insight into genetic variations of GYP(B-A-B) hybrid alleles among populations in the Thailand-Myanmar border area. This information could be used as a guideline to identify compatible blood products for transfusion and to prevent alloimmunization.

Keywords: Blood group genotyping; GYP(B-A-B) hybrid alleles; Malaria; Mia antigen.

Figure 1. Map of the study location.

Mae Sot district, Tak Province of Thailand shares the international border with the Kayin state of Myanmar. Map data ©2025 Esri, USGS — NOSTRA, Esri, TomTom, Garmin, FAO, NOAA, USGS. Created using ArcGIS Online Map Viewer (https://www.arcgis.com/apps/mapviewer/index.html).

Figure 2. Screening results of glycophorin GYP(B-A-B) hybrids using PCR-SSP.

Lane M represents a 100-bp DNA molecular weight ladder (VC 100 bp Plus DNA Ladder, NL1407, Vivantis). Lanes #1, #2, and #3 show positive GYP(B-A-B) hybrid samples amplified using two primer sets: specific primers (GYP*HF, GYP*Mur, GYP*Bun, GYP*Hop, GYP*Kip, and GYP*Hut) and internal control primers (human growth hormone, HGH, gene), resulting in PCR bands of 158 bp and 434 bp, respectively. Lanes #4, #5, and #6 represent negative GYP(B-A-B) hybrid samples, showing only the internal control band with 434 bp.

Figure 3. Nucleotide sequence alignment of GYPA, GYPB, and GYP(B-A-B).

The nucleotide sequences of exon 3, part of intron 3, and exon 4 of GYPA, GYPB, and GYP(B-A-B) were retrieved from GenBank and were aligned using Unipro UGENE: a unified bioinformatics toolkit. The figure displays the consensus sequences and the polymorphic positions at c.140, c.160, c.165, c.166, c.167, c.170, c.173, c.174, c.178, c.203, c.212, c.223, c.226, c.230, IVS3+1, IVS3+25, IVS3+55, IVS3+89, IVS3+110, IVS3+246, IVS3+252, and c.239. Each GYP(B-A-B) subtype has distinct GYPA nucleotide insertions (indicated by highlighted parts). GYP*HF shows a 98 bp insertion from c.160 to IVS3+25. GYP*Mur has a 55 bp insertion from c.203 to IVS3+25. GYP*Bun and GYP*Hop share an identical 131 bp insertion from c.212 to IVS3+110. To distinguish GYP*Bun from GYP*Hop, a single nucleotide polymorphism (SNP) located in exon 4 needed to be observed (c.239C = GYP*Bun, c.239T = GYP*Hop). GYP*Thai has a 22 bp insertion from c.212 to IVS3+1. Whereas GYP*Kip shows a 35 bp insertion from c.223 to IVS3+25.

Figure 4. DNA sequence chromatogram of exon 3 and part of intron 3 regions of GYP(B-A-B) hybrids.

(A) The GYP*Mur/ GYP*Mur homozygote sample displays single unambiguous peaks of the chromatogram of the GYPA sequence at c.203, c.212, c.223, c.226, c.230, IVS3+1, and IVS3+25 (red arrow). (B) The GYP*Mur/GYPB heterozygote sample consists of 2 alleles, GYP*Mur and GYPB, which display double peaks at c.203, c.212, c.223, c.226, c.230, IVS3+1, and IVS3+25 (red arrow).

Figure 5. Melting curve profiles of wild-type GYPB/GYPB and GYP(B-A-B) hybrids by HRM analysis.

Fluorescence intensity for all samples was plotted in relative fluorescence units (RFU). (A) The normalized melt curve was adjusted to relative values of 1.0 and 0 to remove the background fluorescence and enhance the visibility of subtle melt profile differences. (B) The difference curve was plotted to visually amplify differences between melt profiles of different subtypes. Four different subtypes ((GYPB/GYPB (red), GYP*Mur/GYP*Mur (yellow), GYP*Mur/GYPB (blue), and GYP*Thai/GYPB (green)) were distinguished regarding both melting temperature shifts and curve shapes. Homozygous variants are characterized primarily by the temperature shift, while heterozygotes are commonly identified by a change in melt curve shape due to base-pairing mismatches.