Frequency of allotype “b” in human platelet antigen 1 to 29 systems among blood donors in Japan estimated using high‐resolution melt analysis

Abstract

Background: Antibodies against human platelet antigens (HPAs) cause thrombocytopenias. It is thus important to know the frequency of "b" allotypes in each HPA system for the diagnosis and treatment of anti-HPA antibodymediated thrombocytopenia. Study Design and Methods: Genomic DNA was extracted from peripheral blood cells obtained from 2170 blood donors in Japan and was subjected to highresolution melt (HRM) analysis using polymerase chain reaction for each of the HPA genes, using 23 primer pairs. For genotyping, the resulting amplicons were classified based on their HRM curves. In some cases, direct sequence analysis was performed after HRM analysis to determine nucleotide substitutions. In cases where amino acid substitutions were predicted, protein expression levels were examined in a cell line using 293T cells. Results: The frequencies of each of the HPA-b genotypes were as follows: HPA-1b, 0.4%; HPA-2b, 11.8%; HPA-3b, 41.3%; HPA-4b, 0.8%; HPA-5b, 4.3%; HPA-6b, 1.9%; HPA-15b, 48.8%; HPA-21b, 0.6%; and "b" allotype in the other HPA systems, 0.0%. Twenty-eight variants were found; nine of them were predicted to cause amino acid substitution. However, expression analysis revealed that they did not affect protein expression levels on the cell surface. Conclusion: Nine HPA systems are of primary importance in Japan in potentially triggering thrombocytopenia via the HPA antibodies. Similar studies in other countries or races, together with ours, could provide basic information for clinicians in multiethnic societies.

Production and nonclinical evaluation of an autologous iPSC–derived platelet product for the iPLAT1 clinical trial

“…The relative frequency of the HPA-1b allele was 0.4% in Japan. Therefore, the HPA-1b/1b phenotype was calculated to be <0.002% in the Japanese population, 15 and there was no suitable blood donor in the Japanese Red Cross Society (JRC) registry. Written informed consent was obtained from the patient.…”

Current understanding and future perspectives for anti‐human platelet antigen‐15 antibodies in patients with alloimmune thrombocytopenia: History, laboratory testing, and clinical impact

“…45 One of the newly developed assays, PCR-HRM, is faster and more costeffective than PCR-SSP but requires expensive, specialized equipment. 44 In Japan, bead-based multiplex PCR-SSO is the most widely used method because it can genotype multiple HPAs in a single well and is commercially available.…”

“…A number of genotyping platforms have been developed for HPA‐15, including: sequence‐specific primer polymerase chain reaction (PCR‐SSP), 5 restriction fragment length pattern PCR (PCR‐RFLP), 5 real‐time PCR, 5 melting curve analysis, 35 sequence‐specific oligonucleotide (PCR‐SSO), 36 DNA sequencing‐based typing (PCR‐SBT), 37 matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS), 38 PCR‐single‐base extensions (PCR‐SBE), 39 DNA microarray, 40 next‐generation sequencing, 41,42 droplet digital PCR, 43 and high resolution melting PCR (PCR‐HRM) 44 (Table 1). While PCR‐SSP is the most popular method for HPA‐15 genotyping, current methods may be less time‐consuming, more cost‐effective, and may achieve high‐throughput 45 .…”

“…While PCR‐SSP is the most popular method for HPA‐15 genotyping, current methods may be less time‐consuming, more cost‐effective, and may achieve high‐throughput 45 . One of the newly developed assays, PCR‐HRM, is faster and more cost‐effective than PCR‐SSP but requires expensive, specialized equipment 44 . In Japan, bead‐based multiplex PCR‐SSO is the most widely used method because it can genotype multiple HPAs in a single well and is commercially available.…”

Genetic landscape of human platelet antigen variants in the Indian population analysed from 1029 whole genomes

“…In recent years, a large diversity of HPA genotypes have been reported from varied human ethnic groups from a variety of geographical locations (Azizi et al., 2021; Hayashi et al., 2020; Jovanovic Srzentic et al., 2021; Kazemi et al., 2020; Silva‐Malta et al., 2018; Yan et al., 2006). Many of these studies have suggested significant differences in the frequencies between populations.…”