Although it is known that human leukocyte antigen (HLA)-DPB1 disparity has a strong impact on outcomes in unrelated hematopoietic transplantation with induction of acute graft-versus-host disease ...(GVHD) and a graft-versus-leukemia (GVL) effect, its role in unrelated umbilical cord blood transplantation (UR-CBT) has yet to be fully clarified. Our current study is being conducted to elucidate the impact of HLA-DPB1 mismatch, along with the effect of other HLA loci mismatches at the allele level. HLA six loci alleles were retrospectively typed in 1157 Japanese donors and patients with leukemia or myelodysplastic syndrome who underwent transplantation with a single unit of cord blood. HLA-DPB1 mismatch was associated with a significant reduction in leukemia relapse (hazard ratio 0.61, P<0.001), whereas the other HLA loci allele-level mismatches did not. No significant effect of HLA-DPB1 mismatch was observed in the risk of acute GVHD, engraftment or mortality. This HLA-DPB1 GVL effect without induction of severe acute GVHD or deterioration of survival rate has not been reported in unrelated bone marrow or peripheral blood stem cell transplantations, suggesting apparent advantages of UR-CBT. Accordingly, selection of an HLA-DPB1 mismatch cord blood might be the preferable choice for single-unit UR-CBT.
We have developed a new high-throughput, high-resolution genotyping method for the detection of alleles at the human leukocyte antigen (HLA)-A, -B, -C, and -DRB1 loci by combining polymerase chain ...reaction (PCR) and sequence-specific oligonucleotide probes (SSOPs) protocols with the Luminex 100 xMAP flow cytometry dual-laser system to quantitate fluorescently labeled oligonucleotides attached to color-coded microbeads. In order to detect the HLA alleles with a frequency of more than 0.1% in the Japanese population, we created 48 oligonucleotide probes for the HLA-A locus, 61 for HLA-B, 34 for HLA-C, and 51 for HLA-DRB1. The accuracy of the PCR-SSOP-Luminex method was determined by comparing it to the nucleotide sequencing method after subcloning into the plasmid vector using 150 multinational control samples obtained from the International HLA DNA Exchange University of California Los Angeles. In addition, we performed the PCR-SSOP-Luminex method for HLA allele typing on DNA samples collected from 1,018 Japanese volunteers. Overall, the genotyping method exhibited an accuracy of 85.91% for HLA-A, 85.03% for HLA-B, 97.32% for HLA-C, and 90.67% for HLA-DRB1 using 150 control samples, and 100% for HLA-A and -C, 99.90% for HLA-B, and 99.95% for HLA-DRB1 in 1,018 Japanese samples. The PCR-SSOP-Luminex method provides a simple, accurate, and rapid approach toward multiplex genotyping of HLA alleles to the four-digit or higher level of resolution in the Japanese population. It takes only approximately 5 h from DNA extraction to the definition of HLA four-digit alleles at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci for 96 samples when handled by a single typist.
Leukocytes that lack HLA allelic expression are frequently detected in patients with acquired aplastic anemia (AA) who respond to immunosuppressive therapy (IST), although the exact mechanisms ...underlying the HLA loss and HLA allele repertoire likely to acquire loss-of-function mutations are unknown. We identified a common nonsense mutation at position 19 (c.19C>T, p.R7X) in exon 1 (Exon1mut) of different HLA-A and -B alleles in HLA-lacking granulocytes from AA patients. A droplet digital PCR (ddPCR) assay capable of detecting as few as 0.07% Exon1mut HLA alleles in total DNA revealed the mutation was present in 29% (101/353) of AA patients, with a median allele frequency of 0.42% (range, 0.071% to 21.3%). Exon1mut occurred in only 12 different HLA-A (n=4) and HLA-B (n=8) alleles, including B*40:02 (n=31) and A*02:06 (n=15), which correspond to 4 HLA supertypes (A02, A03, B07, and B44). The percentages of patients who possessed at least one of these 12 HLA alleles were significantly higher in the 353 AA patients (92%, P.
Background:
The presence of HLA class I allele‐lacking leukocytes due to the copy‐number neutral loss of heterozygosity in the short arm of chromosome 6 (6pLOH) or HLA allelic mutations is compelling ...evidence supporting the notion that cytotoxic‐T lymphocyte attack on hematopoietic stem and progenitor cells (HSPCs) underlies the development of acquired aplastic anemia (AA). We previously reported that deep sequencing of HLA class I genes of HLA‐class I allele‐lacking granulocytes revealed various loss‐of‐function mutations in alleles such as HLA‐B
∗
40:02, A
∗
02:06, and A
∗
31:01, suggesting that limited HLA‐class I alleles are involved in the autoantigen presentation of AA (Zaimoku, et al. Blood. 2017;129:2908–16; Mizumaki, et al. Blood. 2018;132:2584). Of interest, our previous studies have revealed a common nonsense mutation in exon1 of the HLA‐A and B genes (Exon1
mut
) leading to the loss of specific HLA alleles across different class I alleles in AA patients harboring 6pLOH. If the Exon1
mut
is frequently detected with a sensitive assay in AA patients regardless of the presence of 6pLOH, it would serve as a useful indicator of the presence of an immune pathophysiology of bone marrow failure.
Aims:
To determine the prevalence and clinical significance of Exon1
mut
in AA, we established a novel droplet digital polymerase chain reaction (ddPCR) assay for precisely detecting Exon1
mut
in the peripheral blood of AA patients and investigated the correlation of the mutated sequence with the response to immunosuppressive therapy (IST).
Methods:
Exon1 regions of HLA‐A and HLA‐B alleles were amplified using two different sets of primer pairs that are complementary to the consensus sequences of the HLA class I alleles. The amplicons were subjected to a ddPCR assay using TaqMan probes with substituted locked nucleic acid bases complementary to wild‐type (WT) and Exon1
mut
sequences, which were labeled with different fluorochromes (FAM and HEX). The fractional abundance of the mutant allele was obtained by dividing the number of copies per microliter of the mutant allele (FAM) by the total copies per microliter of the WT allele (HEX) plus the mutant allele (FAM). Peripheral blood leukocytes from 248 patients with AA who were 14–93 years of age (97 with severe AA and 151 with non‐severe AA; 115 males and 133 females; 80 6pLOH+ and 168 6pLOH‐) were subjected to the ddPCR assay, and the correlation between the presence of Exon1
mut
and response to immunosuppressive therapy in newly diagnosed AA patients was examined.
Results:
Mixing experiments showed the ddPCR assay was able to detect as few as 0.07% leukocytes with Exon1
mut
in the background WT leukocytes. Exon1
mut
was detected in 51 (64%) of the 80 6pLOH(+) patients; their lost alleles were A
∗
02:06 (n = 11), A
∗
31:01 (n = 3), B
∗
40:02 (n = 23), B
∗
54:01 (n = 6), and other alleles (n = 8). Exon1
mut
was also detected in 35 (21%) of the 168 6pLOH(‐) patients. The median variant allele frequencies of Exon1
mut
was 0.59% (range, 0.074% to 8.70%). Overall, 86 (35%) of the 248 patients with or without 6pLOH were positive for Exon1
mut
. Fifty Exon1
mut
(+) patients were treated with IST (antithymocyte globulin+cyclosporine CsA, n = 30 or CsA alone, n = 20), and 43 (86%) showed a response.
Summary/Conclusion:
HLA allelic loss due to Exon1
mut
is common in AA patients. The presence or absence of this mutated allele can be determined using the ddPCR assay within 8 h without any information about patients’ HLA genotypes. This assay is therefore thought to be a powerful tool for diagnosing an immune pathophysiology of bone marrow failure.
A simple and novel genotyping method was developed to detect alleles at the swine leukocyte antigen (SLA)‐DRB1 and ‐DQB1 class II loci by using polymerase chain reaction (PCR)–fluorescently labeled ...sequence‐specific oligonucleotide probes (SSOPs) and Luminex 100 xMAP detection. The PCR–SSOP–Luminex method exhibited accuracy of 95% for both SLA‐DRB1 and ‐DQB1 in 6 homozygous and 16 heterozygous pig samples as confirmed by sequencing the PCR products of the same samples. In addition, 12 low‐resolution SLA class II haplotypes consisting of 7 and 9 DRB1 and DQB1 alleles were identified, respectively, in one population of 283 Landrace pigs. This genotyping method facilitates the rapid and accurate identification of two‐ or four‐digit alleles at the SLA‐DRB1 and ‐DQB1 loci.
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