Page 82 - 2021_06-Haematologica-web

H. Mizumaki et al.
Results
Identification of Exon1mut in different HLA-A and HLA-B alleles in HLA-lacking leukocytes from patients with aplastic anemia
To identify HLA class I alleles other than HLA-B*40:02 that are critically involved in autoantigen presentation in AA, we sequenced HLA-A and HLA-B alleles of sorted granulocytes from 20 patients with HLA-LL not possess- ing HLA-B*40:02. The clinical characteristics of these 20 patients are shown in Online Supplementary Table S3. HLA- A– granulocytes or HLA-A+B– granulocytes, accounting for 2.4-99.8% of all granulocytes, were detected in these patients (Online Supplementary Figure S5, Online Supplementary Table S3). Median read depths of the HLA- A and HLA-B alleles were 925 and 1,012 for targeted deep sequencing and 43,013 and 35,267 for amplicon sequenc- ing, respectively. Of the 20 AA patients assessed, six had 6pLOH alone, ten had various loss-of-function mutations in addition to 6pLOH, and four had only somatic loss-of- function mutations in HLA-A. Three (UPN 210, 335, and 348) of the 14 patients with loss-of-function mutations had the mutations in HLA-B of sorted HLA-A+ granulo-
A
cytes. Of note, 12 of 14 patients with loss-of-function mutations had Exon1mut in HLA-A (A*02:06, n=7; A*31:01, n=1) and HLA-B (B*13:01, n=1; B*40:01, n=2; and B*54:01, n=1). The other two patients (UPN 335 and UPN 210) had different loss-of-function mutations from Exon1mut in HLA- B*40:03 and HLA-B*54:01, respectively. Interestingly, a frameshift mutation of HLA-B*54:01 also occurred at codon 19 (c.19delC, p.R7Efs) in exon 1 (Figure 1A and B, Online Supplementary Table S4).
Exon1mut detection using a sensitive droplet digital polymerase chain reaction assay
To detect Exon1mut with high sensitivity and specificity, we established a ddPCR assay that allows for precise measurement of mutant allele frequency without the need for HLA typing. Tested samples containing a fixed amount of wild-type DNA and serial dilutions of Exon1mut template DNA revealed a detection limit of 0.07% for both HLA-A and HLA-B (Online Supplementary Figure S6). The ddPCR assay yielded 0% to 0.042% (median, 0.009%) positive dots in peripheral blood of 24 healthy individuals, validat- ing the cut-off value of 0.07%. The ddPCR assay was able to detect Exon1mut, which had an allelic frequency of <1.0%
B
Figure 2. Detection of Exon1mut using the droplet digital polymerase chain
(A) Representative droplet digi- tal polymerase chain reac- tion (ddPCR) plots of Exon1mut in two patients with aplastic anemia. The ddPCR assay detected 0.92% Exon1mut DNA in HLA- A*02:06 of UPN 299 and 0.98% Exon1mut DNA in HLA- B*40:01 of UPN 211. (B) A minor population of HLA- allele-lacking leukocytes in UPN 280 detected by flow cytometry and the ddPCR assay. The ddPCR assay detected 0.27% Exon1mut in whole blood where granulo- cytes and monocyte with Exon1mut were diluted with lymphocytes without Exon1mut. The percentage of Exon1mut -positive cells was consistent with the percent- age of HLA-A2– monocytes (0.6%) detected by flow cytometry. UPN: unique patient number; VAF: vari- ant allele frequency; FCM:
reaction assay.
flow cytometry.
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haematologica | 2021; 106(6)