Prediction of JENU-Negative Phenotype by Genotyping among Thai Patients with Multiple Transfusions Open Access

In the MNS blood group system, M and N antigens are located on glycophorin A (GPA), whereas S and s antigens are located on glycophorin B (GPB); GPA and GPB are encoded by the GYPA and GYPB genes, respectively. Owing to the similar sequence between certain regions of those two genes, crossing-over or gene conversion events can result in hybrid formations, namely, hybrid GP variants [1‒3]. GP hybrid alleles resulting from gene conversion events, include GYP(A-B-A) and GYP(B-A-B) hybrid genes. The “repair mechanism” in the GYP(B-A-B) hybrids modifies the region in GYPB, corresponding to GYPA at exon 3, substituting an inactive splice site in GYPB with an active GYPA sequence. Consequently, a hybrid protein is produced from a partial GYPB nucleotide sequence encoded by the so-called GYPB hybrid exon [4].

Currently, eight hybrid GP variants express the Mi^a (MNS7) antigen including GP(A-B-A) hybrids, GP.Vw and GP.Hut; GP(B-A-B) hybrids, GP.Mur, GP.Hop, GP.Bun, GP.HF, and GP.Kip and GP(B-A), GP.MOT, respectively [5]. The Mi^a-positive phenotype, particularly GP.Mur, is commonly found in Southeast Asian populations with a frequency ranging from 4.5 to 22.3% [6‒10]. Consequently, anti-Mi^a (anti-Mur) caused by alloimmunisation is implicated in mild to severe cases of hemolytic transfusion reactions (HTRs) and hemolytic disease of the fetus and newborn [11‒15].

According to the Clinical Practice Guidelines for Diagnosis and Management of Thalassemia in Thailand, the antigen typing of Rh (C, c, E, and e) and Mi^a is a minimal requirement before the first transfusion. Additionally, for in patients with multiple antibodies, antigen typing of other blood group systems, such as Kidd, Duffy, Kell, MNS, Lewis, and P1PK, is helpful in predicting antibody specificities [16]. Moreover, antigen typing to provide phenotype-matched donors is recommended to reduce the risk of alloimmunisation, or HTRs.

In a related study, anti-E, -c, -Jk^b, -S, and a panreactive alloantibody, subsequently identified as anti-JENU, were found in a transfusion-dependent Thai patient with thalassemia. This antibody can be produced by homozygous GP.Mur individuals (also known as JENU-negative). JENU is an antigen that has SYISSQTN and GETG epitopes, encoded by GYPB exon 2 and exon 4. Only two of the 3,600 red blood cell (RBC) units randomly selected in the mass screening were compatible with this patient [17], which highlighted the difficulty of finding donors with the extremely rare JENU-negative phenotype.

In a recent study, the GYP*(B-A-B) hybrid alleles among 127 Thai blood donors with Mi^a-positive phenotypes were characterized. The GYP*Mur allele was the most prevalent; however, the GYP*Mur/GYPB genotype frequency (87.40%) was higher than the frequency of homozygous GYP*Mur/GYP*Mur genotype (3.15%). The remaining genotypes (9.45%) were heterozygous GYP*Thai/GYPB and GYP*Thai II/GYPB [7].

Multiple RBC transfusions can cause the production of alloantibodies against one or more red cell antigens. Of those, they may produce specific antibodies to GPB, including anti-JENU, therefore rendering it challenging to detect them and execute subsequent transfusions. In general, patients possessing Mi^a antigens who need RBC transfusions can receive them from either Mi(a+) or Mi(a–) donors. The patient was, on some occasions, a GP.Mur homozygote (GP.Mur/GP.Mur) and does not express normal GPB, thus being JENU-negative. These patients can acquire RBC antibodies with unknown specificity, which may be anti-JENU, following RBC transfusions with normal GPB, regardless of whether they possess Mi^a-positive or Mi^a-negative phenotypes. Unfortunately, typing of JENU antigen is not specified, neither in national nor international commercial reagent RBCs used for antibody detection. Therefore, it is possible to employ genotyping for GYP*(B-A-B) hybrid alleles with GP.Mur homozygote (GP.Mur/GP.Mur) to predict the JENU-negative phenotype in Thai patients who need RBC transfusions to be able to reduce the alloimmunisation risks in the aforementioned scenarios. This study aimed to predict the JENU-negative phenotype in multitransfused Thai patients using in-house molecular typing techniques.

A total of 861 Thai patients with multiple transfusions aged 13 years and older, receiving treatment and follow-up at the Sirinthorn Hospital, Bangkok and Thammasat University Hospital, Pathum Thani, Thailand, were included in this study. All these patients had recently received at least three units of donor RBCs within 3–12 weeks. Leftover EDTA-anticoagulated blood samples from multitransfused patients were collected from January 2024 to August 2024. Patients’ records were evaluated retrospectively during the study for evidence of transfusion reaction occurring during the transfusion of the final unit.

This study was approved by the Committee on Human Rights Related to Research Involving Human Subjects, Thammasat University, Pathum Thani, Thailand (COE No. 073/2567), according to the Declaration of Helsinki. All patients signed a written informed consent.

The Mi^a antigen typing in all samples was performed by a conventional tube technique using human monoclonal IgM anti-Mi^a (National Blood Centre Thai Red Cross Society, Bangkok, Thailand).

ABO grouping, Rh(D) typing, antibody screening, and crossmatching were performed by column agglutination technology using an automated ORTHO VISION Max Analyzer (Ortho-Clinical Diagnostics, Raritan, NJ, USA). In case of patients’ plasma showing positive antibody screening, antibody identification was performed using 11-panel cells (National Blood Centre, Thai Red Cross Society, Bangkok, Thailand) along with an autocontrol. The direct antiglobulin test was performed in all cases with positive autocontrol. All tests were performed according to standard operating procedures and manufacturer instructions.

Genomic DNA was isolated from the samples using the DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions and stored at −20°C until further analysis.

The PCR-sequence-specific primer (PCR-SSP) was used to predict Mi^a, S, and s antigens at the molecular level. Specific PCR amplification of the GYPB*S and GYPB*s alleles in the GYPB gene was performed. A co-amplification of the human growth hormone gene was run as the internal control. The PCR amplification conditions and primers used were previously described [6, 18]. In addition, two primer sets targeting the GYP(A-B-A) and GYP(B-A-B) hybrid genes were used along with human growth hormone-specific control primers. The PCR products of GYP*Hut, GYP*Mur, GYP*Hop, GYP*Bun, and GYP*HF, and the other set of GYP*Vw were amplified to determine possibly predicted GP variants in conditions previously reported [6, 7].

A genomic region in GYPB exons 2 and 3 was amplified by PCR to characterize GYP*(B-A-B) hybrid alleles. Identifying the GYP*Mur, GYP*Bun, GYP*Bun-like (GYP*Thai and GYP*Thai II), GYP*Hop, GYP*HF, and GYP*Kip variants using fast next-generation sequencing was performed as described [7], with modifications. Briefly, the PCR amplification reaction mixture was as follows: template DNA 150 ng/μL, forward primer (GYPB-2564-F: 5′-CCC​TAG​CAG​ATG​GAG​ACA​CTG-3′) 3 µmol/L, reverse primer (GYPB-2564-R: 5′-CTT​TGT​CTT​TAC​AAT​TTC​GTG​TGA​A-3′) 3 µmol/L, 2X PCR reaction mixture (Ultra HiFidelity PCR Kit, TIANGEN BIOTECH, Beijing, China) 5 U/μL, for a total volume of 30 μL. The PCR condition was optimized at 98°C as initial denaturation for 30 s, 98°C for 10 s, and 68°C for 1 min, for 10 cycles, 98°C for 10 s, 64°C for 50 s, and 72°C for 1 min 30 s, for 25 cycles, and finally 72°C for 5 min. Thereafter, amplified PCR products (2,564 bp) were separated from 1.5% agarose gel containing SYBR Safe DNA Gel Stain (Invitrogen, Paisley, UK), electrophoresed in a 1× TBE buffer at 100 volts, and visualized under a blue light transilluminator. The PCR bands were cut and purified, following the instructions provided with a gel extraction kit (QIAquick Gel Extraction Kit, QIAGEN GmbH, QIAGEN, Hilden, Germany) and the eluted fragments were then sequenced using sequencing-based technology on the Illumina Platform (FastNGS Service, U2Bio, Bangkok, Thailand).

The patients’ sequencing results were analyzed and compared with those in the GenBank database (GenBank reference: GYPA, NG_007470.3; GYPB, NG_007483.2) with BioEdit software (UNT Computing for Arts and Sciences, Denton, TX, USA).

Altogether, 861 multitransfused Thai patients were included in this study. Among them, 68 patients (7.89%) possessed Mi^a-positive phenotypes.

Regarding 68 patients with Mi^a-positive phenotypes, their ages ranged from 13 years to 93 years, with a mean age of 53 years. They totaled 27 males and 41 females, and the male-to-female ratio was 0.7:1. The most common ABO blood group among 68 patients was group O (41.18%), followed by group B (35.29%), group A (19.12%), and group AB (4.41%), respectively. For the Rh blood group, all patients were Rh(D) positive. From 5 to over 50 donor, RBC units were transfused to patients at the time of sample collection, and alloantibodies were observed among 5 female patients. One patient was transfused with 5 RBC units and had anti-E. The other 4 thalassemia patients, receiving more than forty RBC units, had multiple antibodies, as shown in Table 1.

All 68 patient samples with Mi^a-positive phenotypes using serological testing were positive with only the set of primers specific for GYP*Hut, GYP*Mur, GYP*Hop, GYP*Bun, and GYP*HF. No GYP*Vw allele was identified using PCR-SSP. Among 68 Mi^a-positive patients, the most frequent genotype (predicted phenotype) was GYPB*s/GYPB*s (S−s+), 89.71%, followed by GYPB*S/GYPB*s (S+s+), 10.29%. The genotype of GYPB*S/GYPB*S (S+s−) was not found.

Identifying GYP*(B-A-B) hybrid alleles were analysed using Sanger DNA sequencing to predict JENU-negative phenotype. The differences in nucleotide positions of GYPA, GYPB, and GYP(B-A-B) hybrids had been reported [7, 19]. The GYP(B-A-B) hybrid genes include GYP*Mur, GYP*Bun, GYP*Thai, GYP*Thai II, GYP*Hop, GYP*HF, and GYP*Kip.

A single nucleotide polymorphism (SNP) of GYP*Mur at GYPB c.200G>C compared with GYP*Bun, GYP*Thai, GYP*Thai II, GYP*Hop and GYP*Kip, respectively. Moreover, GYP*Bun and GYP*Mur differed at SNP GYPB c.229 + 89G>A, and GYP*HF and GYP*Mur differed at SNP GYPB c.160G>C. The SNPs, GYPB c.200, and c.209 were compared with normal GYPB/GYPB (JENU-positive) to distinguish between homozygous GYP*Mur/GYP*Mur (JENU-negative) and heterozygous GYP*Mur/GYPB (JENU-positive); according to Figure 1, normal GYPB/GYPB at SNPs, GYPB c.200 and c.209 were CC and CC. Interestingly, homozygous GYP*Mur/GYP*Mur at SNPs, GYPB c.200 and c.209 were GG and AA, while heterozygous GYP*Mur/GYPB were GC and AC, as shown in Figure 1.

The frequency of GYP*(B-A-B) hybrid alleles analyzed using Sanger DNA sequencing is shown in Table 2. Among 68 Mi(a+) GYP(B-A-B) hybrid samples, 60/68 (88.24%) patients carried the GYP*Mur, of which 59/68 (86.76%) were GYP*Mur/GYPB heterozygotes, 1/68 (1.47%) was GYP*Mur/GYP*Mur homozygote 1/68 (1.47%). The remaining 8 patients included different GYP*Bun-like alleles; 7/8 (10.29%) were GYP*Thai/GYPB heterozygotes, and 1/8 (1.47%) was GYP*Thai II/GYPB heterozygotes. The GYP*Mur allele was the predominant hybrid allele and was found in 61/136 (44.85%) alleles. The GYP*Bun, GYP*HF, GYP*Hop, and GYP*Kip alleles were not observed in this study.

A 75-year female patient with homozygous GYP*Mur/GYP*Mur was anticipated to have a JENU-negative phenotype, and the predicted phenotype for S/s was S−s+. She received multiple transfusions within 1 year including 24 red cell units, 2 units of single donor platelets, and 5 units of leukocyte-poor platelet concentrates. The antibody screening test remained negative during the therapy for malignant neoplasm of the thyroid gland.

The prevalence of thalassemia, particularly β-thalassemia/HbE, in Southeast Asia, Southern China, and South Asia is high. The severity of thalassemia diseases ranges from a mild, asymptomatic anemia to transfusion-dependent development from early life [20]. The distribution of blood group antigen frequencies varies in different populations. For example, the prevalence of Rh-negative individuals is higher in Caucasians than in Asian population. On the contrary, Asian populations have a higher prevalence of the Mi^a antigen than other populations. Hence, patients requiring multiple and repeated transfusions should be typed for ABO, Rh (D, C, c, E, and e), and Mi^a antigens before the first transfusion and selected antigen-negative RBC units for compatibility testing [16, 21]. The possibility of alloimmunisation depends on the number and frequency of transfusions, antigen immunogenicity and the immune status of the recipient. Moreover, the effects of ethnic and antigenic pattern variations between donors and recipients have been reported [22, 23].

This study comprised 68 multitransfused patients with Mi^a-positive phenotypes. Approximately 60% were female, and 4 female patients receiving at least forty RBC units, had produced alloantibodies, including mixtures of two and three specificities. Among 5 patients with alloantibodies, anti-E was the most frequent, followed by anti-c, anti-Fy^b, anti-Di^a, and anti-Jk^b. One patient had unidentified antibodies. The prevalence of antibody specificity was consistent with a related study among multitransfused Thai patients [24]. Similar to a related study, our results confirm the association between alloimmunisation and female patients [25]. The relationship between the quantity of RBC units transfused and alloimmunisation in thalassemia remains unknown [26]. Our findings indicate that alloimmunisation is more likely to occur among patients who receiving more blood units, consistent with related studies [27, 28].

In this study, we characterized GYP*(B-A-B) hybrid alleles among multitransfused Thai patients with Mi^a-positive phenotypes using PCR-based coupled DNA sequencing. The most common alleles were GYP*Mur (44.85%) followed by GYP*Thai (5.89%) including GYP*Thai and GYP*Thai II. The GYP*Bun, GYP*HF, GYP*Hop, and GYP*Kip alleles were not found, similar to related studies among Thai blood donors [7, 19]. Regarding the results of the GYPB*S and GYPB*s allele detection by PCR-SSP, the most common predicted phenotype was S−s+ (91.17%), followed by S+s+ (8.82%) of the GYP*Mur/GYPB, and 1 patient having heterozygous GYP*Thai/GYPB(1.47%). Related studies among Thai blood donors revealed that all of the GYP*Mur/GYPB individuals possessed S−s+ phenotypes [7, 19].

Notably, a female Thai patient with JENU-negative phenotype (homozygous GYP*Mur) was transfused with 24 RBC units without alloantibody production. This might have been due to the number of RBC units or her disease status. She had malignant neoplasm of the thyroid gland with azacitidine therapy. This demethylating agent is a chemical analog of the cytosine nucleoside, which can inhibit DNA and RNA synthesis. In addition, its adverse effects include anemia, neutropenia, and thrombocytopenia [29]. This action may lead to the interference of antibody-producing cells and result in the absence of alloantibody detection, even when patients receive multiple RBC transfusions.

In conclusion, multiple transfusions can induce alloantibody production. Therefore, providing phenotype-match RBC transfusion is beneficial for patients with long-term transfusion therapy. In Asian populations, the Mi^a-positive phenotype should be further investigated for the JENU-negative phenotype to avoid anti-JENU production.

This study protocol was approved by the Committee on Human Rights Related to Research Involving Human Subjects, Thammasat University, Pathum Thani, Thailand (COE No. 073/2567), and written consent was provided by each participant.

All authors declare no conflict of interests.

The research presented in this article was financed exclusively by the National Research Council of Thailand, Bangkok, Thailand. No external funding or donations were provided for this research.

O.N. and P.R.: design and acquisition of data. P.R., W.S., and P.K.: selection and blood collection from patients for serological and molecular testing. K.I. and O.N.: data analysis, interpretation of data, and assistance with compiling of the results. O.N. and O.K.: drafting of this manuscript and critical revision of important intellectual content. All of the authors approved the final version of this manuscript for publication.

All data generated all analysed during this study are included in this article. Further inquiries can be directed to the corresponding author.