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H. Mizumaki et al.
lacking leukocytes: HLA-LL) are often detected in the peripheral blood of AA patients.8,9 The presence of HLA- LL represents compelling evidence to support the involve- ment of cytotoxic T lymphocytes specific to HSPC in the development of bone marrow failure, and the detection of these leukocytes would be useful for diagnosing immune pathophysiology in patients with AA and other types of bone marrow failure, including low-risk myelodysplastic syndrome. However, assays for detecting HLA-LL, such as flow cytometry using monoclonal antibodies specific to HLA-A or HLA-B alleles and single nucleotide polymor- phism (SNP) arrays, have not been popularized because of the need for HLA typing and low frequencies of copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH).10,11
6pLOH was considered the most common way for HSPC to lose HLA alleles.10-15 We recently reported that, using targeted deep sequencing with a next-generation sequencer, somatic loss-of-function mutations of HLA- B*40:02 were frequently detected in granulocytes of AA patients possessing HLA-B*40:02. These results strongly suggested that antigen presentation by HSPC via HLA- B4002 plays a critical role in the pathogenesis of AA.9 Loss-of-function mutations in HLA class I alleles other than HLA-B*40:02 were also detected in patients with AA. Babushok et al. identified mutations in several HLA class I alleles in leukocytes of AA patients, including HLA- A*33:03, A*68:01 and HLA-B*14:02.14 We recently ana- lyzed leukocytes of AA patients with 6pLOH and detect- ed somatic loss-of-function mutations in HLA-A*02:06 and B*54:01.16,17 However, HLA class I alleles responsible for autoantigen presentation in AA patients without HLA- B*40:02, who account for approximately 80% of all AA patients, are largely unknown due to the limited number of AA patients who have been studied for loss-of-function mutations in HLA class I alleles.
To identify HLA class I alleles other than HLA-B*40:02 that are involved in the autoantigen presentation of AA, we performed targeted next-generation sequencing in AA patients with HLA-LL who had HLA class I alleles other than HLA-B*40:02. During the course of the mutation analysis, we identified a nonsense mutation at codon 19 (c.19C>T, p.R7X) in exon 1 (Exon1mut) of some HLA-A or HLA-B alleles. Surprisingly, Exon1mut was shared by different HLA-A or HLA-B alleles and was prevalent in AA patients although their variant allelic frequencies (VAF) were very low. A sensitive assay that can detect Exon1mut could help to identify HLA-A or HLA-B alleles that are responsible for autoantigen presentation and provide insight into the immune pathophysiology of bone marrow failure.
Against this backdrop, we developed a highly sensitive droplet digital PCR (ddPCR) assay for detecting Exon1mut, and determined the prevalence of Exon1mut and HLA alleles likely to acquire this mutation in AA patients.
Methods
Detailed information on the materials and methods are provid- ed in the Online Supplementary Data.
Patients
Twenty Japanese AA patients with HLA-LL who did not have an HLA-B*40:02 allele were analyzed for the presence of loss-of- function mutations in HLA alleles. We studied a total of 353
Table 1. Baseline characteristics of the patients with aplastic anemia.
Characteristics
Total
Age in years, median (range)
Sex, male/female
Severity
Non-severe AA Severe/very severe AA
Patients with increased GPI– granulocytes, n (%) None (<0.003%)
0.003-1.0%
>1.0%
IST prior to sampling, n (%) CsA±TPO-RA
rATG+CsA±TPO-RA
N. of patients
353
63 (11-93) 167/186
202
151
245 (69.4) 108 177
68
61 (17.2) 31
30
GPI–: glycosylphosphatidylinositol-anchored proteins deficient; AA: aplastic anemia; IST: immunosuppressive therapy; CsA: cyclosporine; TPO-RA: thrombopoietin receptor agonist; ATG: antithymocyte globulin.
Japanese AA patients, including the 20 patients who were further analyzed for the prevalence and clinical significance of Exon1mut in AA between 2010 and 2018 (Table 1). A schematic of the experi- ments is provided in Online Supplementary Figure S1. All patients were genotyped for HLA-A, HLA-B, HLA-C, and HLA-DRB1 alle- les using the PCR sequence-specific oligonucleotide method. All patients provided consent to participation in this study, which was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of the Kanazawa University Institute of Medical, Pharmaceutical, and Health Sciences.
Detection of glycosylphosphatidylinositol-deficient cells and cells with 6p loss of heterozygosity
Glycosylphosphatidylinositol-anchored protein-deficient (GPI–) cells were detected using high sensitivity flow cytometry, as pre- viously described.18 6pLOH was assessed using a SNP array-based method with GeneChip 500K arrays (Affymetrix, Japan) or a ddPCR assay with a QX200 AutoDG Droplet Digital PCR System (Bio-Rad, CA, USA), as previously described.9,10
Deep sequencing of HLA class I genes
From peripheral blood samples of the 20 patients with HLA-LL, which were stained with anti-HLA-allele-specific and lineage-spe- cific monoclonal antibodies, paired fractions of granulocytes and CD3+ T cells were sorted and were subjected to DNA extraction (Online Supplementary Figure S2). The monoclonal antibodies used in this study are summarized in Online Supplementary Table S1. Nucleotide sequences of HLA-A and HLA-B genes in sorted gran- ulocytes of patients with HLA-LL were determined using a next- generation sequencer (Miseq; Illumina, CA, USA).
Digital droplet polymerase chain reaction assay for detecting Exon1mut
We developed a sensitive ddPCR assay for precise detection of Exon1mut in the peripheral blood of AA patients using the QX200 ddPCR system. Briefly, we designed two different sets of primer pairs complementary to the consensus sequences of HLA-A and HLA-B alleles, and locked nucleic acid-based probes complemen- tary to wild-type and mutant-specific sequences (Online Supplementary Table S2). Detailed protocols for ddPCR are provid- ed in the Online Supplementary Methods.
Determination of HLA alleles that acquired Exon1mut HLA alleles that acquired Exon1mut were determined by deep sequencing with the next-generation sequencer, or deduced from
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