A Common HLA Allelic Mutation of exon1 in Leukocytes Defines Class I Alleles Responsible for Autoantigen Presentation of Acquired Aplastic Anemia
Authors: Mizumaki Hiroki , Hosomichi Kazuyoshi , Imi Tatsuya , Tsuji Noriaki , Mikoto Tanabe , Tran Dung Cao , Hosokawa Kohei Thi , Zaimoku Yoshitaka , Hosokawa Kohei , Katagiri Takamasa , Azuma Fumihiro , Tajima Atsushi , Nakao Shinji 
Published: November 2019

[Background] Acquired aplastic anemia (AA) is thought to be caused by cytotoxic T lymphocyte (CTL) attack specific to antigens presented by class I HLA alleles on hematopoietic stem cells (HSCs) although the target antigens on HSCs are still unknown. HLA alleles that are responsible for the auto-antigen presentation, which can be inferred from the presence of leukocytes that lack particular HLA class I alleles (HLA-lacking leukocytes [HLA-LLs]), may provide useful information on the identification of autoantigens in AA. We previously reported that deep sequencing of HLA class I genes of HLA-LLs revealed various loss-of-function mutations in alleles, including HLA-A*02:06 and B*40:02, suggesting that limited HLA-class I alleles are involved in the autoantigen presentation of AA (ASH 2018). Intriguingly, 60% of patients possessing HLA-LLs due to 6pLOH or several inactivating mutations in different HLA-A or HLA-B genes shared a nonsense mutation in exon 1 (Exon1mut) of which allelic frequency was very low (range 1.0-37.3%, median 4.5%). We hypothesized that the nonsense mutation, which efficiently lacks the corresponding HLA-allelic expression, might have been overlooked due to its low VAFs, and if we could establish a highly sensitive assay for detecting Exon1mut and determine the HLA alleles that undergo the mutation for a large number of AA patients, we might be able to define all HLA alleles that are involved in autoantigen presentation of AA.

[Objectives/Methods] To test this hypothesis, we developed a highly sensitive droplet digital PCR (ddPCR) assay for precisely detecting Exon1mut in the peripheral blood (PB) of AA patient. In brief, the exon 1 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-A and HLA-B alleles. The amplicons were subjected to a ddPCR assay using TaqMan probes complementary to wild-type (WT) and mutant-specific (MT) sequences, which were labeled with different fluorochromes (6-FAM for MT and HEX for WT). Peripheral blood leukocytes from 363 patients with AA (mean 64 [range, 11-93]) years of age, 134 with severe AA and 229 with non-severe AA; 173 males and 190 females; 84 6pLOH[+] and 279 6pLOH[-]) were subjected to the ddPCR assay. All blood samples were analyzed for 6pLOH by SNP array-based methods or a ddPCR assay as previously described. The HLA allele that underwent Exon1mut was determined by targeted deep sequencing using a unique molecular identifier (UMI), which enabled us to detect variant calling at a VAF as low as 0.1%, or deduced from the alleles contained in the lost haplotype, which are known to be the frequently lost alleles due to 6pLOH.

[Results] Using 2 different ddPCR mixtures for HLA-A and HLA-B, the presence of Exon1mut was evaluable in all 363 AA patients. The sensitivity of the ddPCR assay for detecting Exon1mutwas 0.07%. 6pLOH was detected in 84 (23.1%) of the 363 AA patients. Ninety-nine (27.3%) of the 363 patients with (55 [65.5%] of 84) or without (44 [15.8%] of 279) 6pLOH were found to be positive for Exon1mut. The median allele frequency of Exon1mut in DNA from the Exon1mut(+) patients was 0.6% (range, 0.074% to 21.3%). In 17 patients whose blood samples were serially available, Exon1mutwas persistently detected in 13 and disappeared in 4 patients for 10-77 months (Figure 1). Among 43 different HLA-A and HLA-B alleles carried by the Exon1mut(+) patients, those with Exon1mutcould be identified by targeted deep sequencing in 54 patients. In 13 of the remaining 42 patients with Exon1mut, the Exon1mut-involved HLA alleleswere deduced from the alleles contained in the lost haplotype due to 6pLOH. These were 12 alleles and included A*02:06 (n=11), A*31:01 (n=3), B*13:01 (n=2), B*40:01 (n=3), B*40:02 (n=26), B*40:03 (n=1), B*54:01 (n=6), A*02:01 (n=2), A*02:07 (n=1), B*44:03 (n=1), B*55:02 (n=2) and B*56:01 (n=1) (Figure 2). The last five infrequent alleles were newly identified as "risk alleles" using the Exon1mutdetection. Two-hundred and twenty (92%) of 239 patients with PNH-type cells possessed at least 1 of the 12 alleles, while 103 (85%) of 121 patients without PNH-type cells did (P=0.045).

[Conclusions] The Exon1mutdetection assay identified 12 HLA-alleles that are closely and exclusively involved in the autoantigen presentation of AA in Japanese patients. Similarity analyses of their antigen-presentation motifs may help to identify autoantigen peptides in AA.

Disclosures

Nakao: Takeda Pharmaceutical Company Limited: Honoraria; SynBio Pharmaceuticals: Consultancy; Ono Pharmaceutical: Honoraria; Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Celgene: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria.