HLA class I allele-lacking leukocytes predict rare clonal evolution to MDS/AML in patients with acquired aplastic anemia

Blood ◽  
2021 ◽  
Author(s):  
Kohei Hosokawa ◽  
Hiroki Mizumaki ◽  
Takeshi Yoroidaka ◽  
Hiroyuki Maruyama ◽  
Tatsuya Imi ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Clonal Evolution ◽  
Hla Class I ◽  
Class I

scholarly journals A brief, but comprehensive, guide to clonal evolution in aplastic anemia

Hematology ◽  
2018 ◽  
Vol 2018 (1) ◽  
pp. 457-466 ◽  
Author(s):  
Daria V. Babushok
Keyword(s):  
Aplastic Anemia ◽  
Somatic Mutations ◽  
Clonal Evolution ◽  
Clinical Care ◽  
Hla Class I ◽  
The Elderly ◽  
Hematologic Malignancies ◽  
Class I ◽  
Clonal Hematopoiesis ◽  
Immune Mediated

Abstract Acquired aplastic anemia (AA) is an immune-mediated bone marrow aplasia that is strongly associated with clonal hematopoiesis upon marrow recovery. More than 70% of AA patients develop somatic mutations in their hematopoietic cells. In contrast to other conditions linked to clonal hematopoiesis, such as myelodysplastic syndrome (MDS) or clonal hematopoiesis of indeterminate potential in the elderly, the top alterations in AA are closely related to its immune pathogenesis. Nearly 40% of AA patients carry somatic mutations in the PIGA gene manifested as clonal populations of cells with the paroxysmal nocturnal hemoglobinuria phenotype, and 17% of AA patients have loss of HLA class I alleles. It is estimated that between 20% and 35% of AA patients have somatic mutations associated with hematologic malignancies, most characteristically in the ASXL1, BCOR, and BCORL1 genes. Risk factors for evolution to MDS in AA include the duration of disease, acquisition of high-risk somatic mutations, and age at AA onset. Emerging data suggest that several HLA class I alleles not only predispose to the development of AA but may also predispose to clonal evolution in AA patients. Long-term prospective studies are needed to determine the true prognostic implications of clonal hematopoiesis in AA. This article provides a brief, but comprehensive, review of our current understanding of clonal evolution in AA and concludes with 3 cases that illustrate a practical approach for integrating results of next-generation molecular studies into the clinical care of AA patients in 2018.

scholarly journals Spectrum and Clinical Significance of HLA Class I Alleles and Their Somatic Mutations in Immune Aplastic Anemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3738-3738
Author(s):  
Yoshitaka Zaimoku ◽  
Sharon D. Adams ◽  
Bhavisha A Patel ◽  
Audrey Ai Chin Lee ◽  
Sachiko Kajigaya ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Surface ◽  
Aplastic Anemia ◽  
Clinical Significance ◽  
Somatic Mutations ◽  
Clonal Evolution ◽  
Hla Class I ◽  
Surface Expression ◽  
Class I ◽  
Cell Surface Expression

Clonal hematopoiesis associated with loss of HLA class I alleles due to somatic mutations and/or 6p loss of heterozygosity (LOH) is frequent in immune aplastic anemia (AA). HLA-B*40:02 is more likely to be involved in HLA loss in Japanese AA patients, suggesting a role for this allele in immune pathophysiology (Zaimoku Y et al, Blood 2017). Mutations in non-B*40:02 HLA class I alleles have been reported in a limited number of patients from the United States (Babushok D et al, Blood Adv 2017) and Japan (Mizumaki H et al, 60th ASH meeting), but their prevalence and clinical significance are not well characterized. We investigated somatic mutations of HLA class I alleles, HLA allele frequencies, and their correlations with outcomes of therapy in a total of 532 AA patients, aged 2 years or older, treated on various Hematology Branch protocols (clinicaltrials.gov NCTs 00001964, 00061360, 00195624, 00260689, 00944749, 01193283, and 01623167). HLA allele-lacking (HLA-) monocytes from cryopreserved peripheral blood mononuclear cells were screened by flow cytometry after staining with allele-specific monoclonal antibodies for HLA-A and/or HLA-B (HLA-flow) in 172 AA patients. HLA- monocytes accounting for 0.5% to 100% (median 9.5%) of total monocytes were detected in 49 (28%) of the 172 patients and in 59 (15%) of 382 alleles analyzed (Figure 1). Loss of cell surface expression was frequent for HLA-B14 (46%), B27 (33%), B49 (33%), A68 (26%), A2 (23%), B40 (21%), and B8 (21%). One percent to 60% (median, 8.9%) of glycosylphosphatidylinositol-linked protein-negative (GPI-) monocytes were also present in 43% (21 of 49) of the patients with HLA- monocytes, but GPI- clones had normal HLA cell surface expression. Deep sequencing of HLA-A, HLA-B and HLA-C on sorted HLA- and HLA+GPI+ monocytes was performed in 42 of the 48 patients from whom adequate cells were available. Somatic mutations and/or LOH corresponding to the lacking alleles were detected in all 42 cases (Figure 1): 9 had both somatic mutations and LOH, 20 had somatic mutations only, and 13 had LOH only. Among the 13 patients who showed only LOH in the absent allele, 6 had somatic mutations in other alleles of HLA+ monocytes that was not analyzable of HLA expression, and 2 had a breakpoint of LOH between HLA-A and HLA-C, leading to loss of a single HLA-A allele. Somatic mutations or LOH involving only one allele were present in 37 patients among 6 HLA-A alleles (in 02:01 [7 patients], 02:05 [1], 02:06 [3], 02:11 [1], 68:01 [2], 68:02 [2]) and 10 HLA-B alleles (07:02 [1], 08:01 [4], 14:01 [1], 14:02 [7], 27:05 [1], 35:02 [1], 35:05 [1], 40:01 [1], 40:02 [3], 45:01 [1]), but were not found in HLA-C alleles. HLA allele frequencies in AA patients, including 271 white Americans, 120 African-Americans, and 99 Hispanics and Latinos, were compared with ethnicity-matched individuals in bone marrow donor datasets of the National Marrow Donor Program, and underwent random-effects meta-analyses. HLA-B*07, B*14, and B*40 were overrepresented in AA, while A*02, A*68, and B*08 frequencies were similar to those of healthy donors (Figure 2). In 164 severe AA patients who were initially treated with horse antithymocyte globulin (hATG), cyclosporine, and eltrombopag between 2012 and 2018, 36 and 79 were positive and negative for HLA- monocytes, respectively, and 49 were not tested by HLA-flow. There was no significant difference in overall and complete response rates at six months among the three groups (Figure 3). Clonal evolution, defined as acquisition of abnormal bone marrow cytogenetics or morphology, especially high-risk evolution to chromosome 7 abnormalities, complex cytogenetics, or morphological MDS/AML, tended to be more frequent in patients with HLA- monocytes, compared to the other two groups, but the difference did not reach statistical significance. Clinical outcomes were also assessed according to the presence of specific HLA alleles in 400 severe AA patients who were treated with hATG-based initial immunosuppressive therapy from 2000 to 2018: there was no significant differences in probabilities of response and clonal evolution according to the alleles associated with somatic mutations. Our study revealed that somatic mutations in HLA genes in AA are broadly distributed, but some alleles are preferentially affected. Inconsistent with previous studies, we found that outcomes of therapy did not significantly correlate with HLA gene mutations or with distinct HLA alleles. Disclosures No relevant conflicts of interest to declare.

scholarly journals HLA associations, somatic loss of HLA expression, and clinical outcomes in immune aplastic anemia

Blood ◽  
2021 ◽  
Author(s):  
Yoshitaka Zaimoku ◽  
Bhavisha A Patel ◽  
Sharon D Adams ◽  
Ruba N Shalhoub ◽  
Emma M Groarke ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Bone Marrow Cells ◽  
Late Onset ◽  
Clonal Evolution ◽  
Gene Mutations ◽  
Hla Class I ◽  
Class I ◽  
Hematopoietic Stem ◽  
Monosomy 7 ◽  
Hla Loss

Immune aplastic anemia (AA) features somatic loss of HLA class I allele expression on bone marrow cells, consistent with a mechanism of escape from T cell-mediated destruction of hematopoietic stem and progenitor cells. The clinical significance of HLA abnormalities has not been well characterized. We examined somatic loss of HLA class I alleles, and correlated HLA loss and mutation-associated HLA genotypes with clinical presentation and outcomes after immunosuppressive therapy in 544 AA patients. HLA class I allele loss was detected in 92 (22%) of the 412 patients tested, in whom there were 393 somatic HLA gene mutations and 40 instances of loss of heterozygosity. Most frequently affected was HLA-B*14:02, followed by HLA-A*02:01, HLA-B*40:02, HLA-B*08:01, and HLA-B*07:02. HLA-B*14:02, HLA-B*40:02, and HLA-B*07:02 were also overrepresented in AA. High-risk clonal evolution was correlated with HLA loss, HLA-B*14:02 genotype, and older age, which yielded a valid prediction model. In two patients, we traced monosomy 7 clonal evolution from preexisting clones harboring somatic mutations in HLA-A*02:01 and HLA-B*40:02. Loss of HLA-B*40:02 correlated with higher blood counts. HLA-B*07:02 and HLA-B*40:01 genotypes and their loss correlated with late onset of AA. Our results suggest the presence of specific immune mechanisms of molecular pathogenesis with clinical implications. HLA genotyping and screening for HLA loss may be of value in the management of immune AA. This study was registered at clinicaltrials.gov as NCT00001964, NCT00061360, NCT00195624, NCT00260689, NCT00944749, NCT01193283, and NCT01623167.

scholarly journals Somatic HLA mutations expose the role of class I–mediated autoimmunity in aplastic anemia and its clonal complications

2017 ◽  
Vol 1 (22) ◽  
pp. 1900-1910 ◽  
Author(s):  
Daria V. Babushok ◽  
Jamie L. Duke ◽  
Hongbo M. Xie ◽  
Natasha Stanley ◽  
Jamie Atienza ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Clonal Evolution ◽  
Gene Mutations ◽  
Therapy Response ◽  
Hla Class I ◽  
Class I ◽  
Key Points ◽  
Inactivating Mutations ◽  
I Gene

Key Points Somatic HLA class I gene mutations are frequent in aAA and define HLA class I restricted autoimmunity in aAA. HLA alleles targeted by inactivating mutations are overrepresented in aAA and correlate with poor therapy response and clonal evolution.

scholarly journals Identification of an HLA Class I Allele Closely Involved in the Pathogenesis of Acquired Aplastic Anemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 729-729
Author(s):  
Yoshitaka Zaimoku ◽  
Hiroyuki Takamatsu ◽  
Kazuyoshi Hosomichi ◽  
Tatsuhiko Ozawa ◽  
Noriharu Nakagawa ◽  
...  
Keyword(s):  
T Cell ◽  
Aplastic Anemia ◽  
Missense Mutation ◽  
Hla Class I ◽  
The Other ◽  
Class I ◽  
Variant Allele ◽  
Cytotoxic T Cell ◽  
Hematopoietic Stem

Abstract [Background] The frequent loss of heterozygosity of the HLA haplotype in the short arm of chromosome 6 (6pLOH) in leukocytes is thought to offer compelling evidence of cytotoxic T cell (CTL) involvement in the development of acquired aplastic anemia (AA) because it represents the escape of hematopoietic stem/progenitor cells (HSPCs) with 6pLOH from the attack of CTLs that are specific to autoantigens presented by the lacked HLA class I allele. Although our previous study suggested that HLA-B*40:02 is the major allele involved in this phenomenon, the exact role of B*40:02 remained unclear because 6pLOH involving this allele is always associated with a lack of HLA-A and C alleles in the haplotype, and the presence of B*40:02-missing leukocytes were unable to be shown due to the lack of monoclonal antibodies (mAbs) specific to B61, the HLA-B antigen that corresponds to B*40:02. We recently succeeded in generating a mAb specific for HLA-B61 that enabled us to explore the role of B*40:02 in the development of AA. [Methods] Using the new mAb, we examined peripheral blood samples of 28 AA (12 with 6pLOH and 16 without 6pLOH) patients carrying this allele for the presence of B61(-) leukocytes using flow cytometry. HLA genes were enriched by sequence capture, a hybridization-based gene enrichment method, from genomic DNA of sorted B61(-) granulocytes, and were subjected to deep sequencing using an NGS (MiSeq). B61(+) granulocytes or T cells were used as controls. Potential mutations responsible for the B61-missing were identified when 10 or more variant reads were found only in B61(-) granulocytes. Thereafter, HLA-B alleles carrying those mutations were determined taking advantage of the nearest allele-specific SNPs. [Results] Among the 12 6pLOH(+) patients, 10 (83%) possessed 0.5%-60% B61-missing granulocytes that were not lacking HLA-A, in addition to 12% to 99% 6pLOH(+) granulocytes that lacked both B61 and an HLA-A allele on the same haplotype (Figure 1). B61(-) granulocytes that accounted for 0.5%-99% of the total granulocytes were detected in 9 (56%) of the 16 6pLOH(-) patients. The prevalence of missing B61 in the 28 AA patients was 21/28 (75%), much more frequent than those of the 3 other major alleles (A*02:01, 32%; A*02:06, 30%; A*24:02, 6%). B61(-) granulocytes were available for mutation analyses of HLA-B alleles in 15 of the 19 patients who possessed B61(-) granulocytes. The mean coverage of HLA-B gene was 426x. In total, 43 somatic mutations of HLA-B were identified in B61(-) granulocytes, all of which were present in B*40:02 but not in any of the other HLA-B alleles. Median variant allele frequency was 4.8% (range, 1.0% - 43%) and the number of mutations in each patient was 1 to 6 (Figure 2). Thirty-nine mutations were exonic while 4 were intronic. Exonic mutations included frameshift insertions (n=12), frameshift deletions (n=16), non-frameshift deletions (n=2), nonsense mutations (n=7), a missense mutation (n=1) and a start codon mutation (n=1). All four intronic mutations were considered to be a splice site mutation; two mutations deactivated 5' and 3' splice sites, whereas the other two were single base substitutions within intron 3, making alternative 5' splicing site with strong consensus sequence: GGC [A>G] TGAGT and TTC [C>G] TGAGT. Surprisingly, missense mutations in the alpha-2 and alpha-3 chain-coding region of HLA-B*40:02 were detected exclusively in the B61(+) granulocytes of two patients possessing B61(-) granulocytes, suggesting the inability of the mutant HSPCs to interact with CTLs. Variant allele frequencies of the two missense mutation were 40% and 45%, respectively. As a result of the mutation, virtually all granulocytes of the two patients were affected by B*40:02 mutations that allowed the HSPCs to escape the T cell attack. [Conclusions] The markedly high prevalence of leukocytes lacking HLA-B*40:02 as a result of either or both 6pLOH or structural gene mutations clearly indicates that antigen presentation by HSPCs to CTLs via the HLA-B allele plays a critical role in the pathogenesis of AA. Disclosures Takamatsu: Celgene: Honoraria; Janssen Pharmaceuticals: Honoraria. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.

scholarly journals Identification of an HLA class I allele closely involved in the autoantigen presentation in acquired aplastic anemia

Blood ◽  
2017 ◽  
Vol 129 (21) ◽  
pp. 2908-2916 ◽  
Author(s):  
Yoshitaka Zaimoku ◽  
Hiroyuki Takamatsu ◽  
Kazuyoshi Hosomichi ◽  
Tatsuhiko Ozawa ◽  
Noriharu Nakagawa ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Antigen Presentation ◽  
Somatic Mutations ◽  
Critical Role ◽  
Hla Class I ◽  
Class I ◽  
Key Points

Key Points Somatic mutations of HLA-B*40:02 are very frequently detected in granulocyte of patients with acquired aplastic anemia. Antigen presentation via HLA-B4002 may play a critical role in the pathophysiology of acquired aplastic anemia.

scholarly journals Bystander Proliferation of Piga-Mutated Hematopoietic Progenitor Cells in Acquired Aplastic Anemia Patients Possessing HLA Class I Allele-Lacking Leukocytes

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1308-1308 ◽  
Author(s):  
Takeshi Yoroidaka ◽  
Kohei Hosokawa ◽  
Tatsuya Imi ◽  
Takamasa Katagiri ◽  
Fumihiro Azuma ◽  
...  
Keyword(s):  
T Cells ◽  
Aplastic Anemia ◽  
Progenitor Cells ◽  
Hla Class I ◽  
Survival Advantage ◽  
Class I ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Lineage Diversity

Abstract [Background] Hematopoietic stem progenitor cells (HSPCs) with PIGA mutations are thought to acquire a survival advantage over normal HSPCs under immune attack against HSPCs and produce glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells in patients with acquired aplastic anemia (AA). Various underlying mechanisms of the survival advantage of PIGA-mutated HSPCs have been proposed; however, it remains still unclear how PIGA-mutated HSPCs are immunologically selected in AA. Approximately 15% of AA patients with increased GPI(-) cells possess another aberrant leukocyte subset that lacks the expression of the HLA-class I allele due to a copy number-neutral loss of heterozygosity of the HLA haplotype, which occurs in the short arm of chromosome 6 (6pLOH) as a result of uniparental disomy, or HLA allelic mutations. The presence of HLA-class I allele-lacking leukocytes (HLA-LLs) is considered to be the most compelling evidence to support the involvement of cytotoxic T lymphocytes (CTLs) in the development of bone marrow failure. Charactering GPI(-) leukocytes and platelets in AA patients with HLA-LLs may provide an insight into the mechanism underlying the immune selection of PIGA-mutated HSPCs. [Patients and Methods] We investigated the presence of GPI(-) leukocytes, erythrocytes, and platelets in 63 patients with AA using high-sensitivity flow cytometry (FCM). For the platelet analysis, platelet rich plasma (PRP) was obtained by centrifuging anticoagulated blood at 1000 rpm for 7 minutes with the brake turned off. Thirty microliters of PRP was incubated with monoclonal antibodies specific to CD55-PE, CD59-PE, CD41a-APC and HLA-A2 or A24-FITC for 20 minutes at room temperature in the dark. To prevent doublets, samples were diluted 1 to 100 in PBS and filtered with mesh immediately before the FCM analysis. Thirty of the 63 patients were heterozygous for the HLA-A allele with A24 and A2, and thus the presence of both HLA-LLs and HLA-A allele-lacking platelets could be evaluated by FCM. The lack of the HLA-A allele due to 6pLOH or allelic mutations in all HLA-LL(+) patients was confirmed by a droplet digital PCR or deep sequencing. [Results] Increased GPI(-) granulocytes, which accounted for 0.01-99.8% of the total granulocytes, were detected in 37 (58.7%) patients while HLA-A24 or A2-lacking granulocytes accounted for 0.39-98.3% of the total granulocytes in 20 (66.7%) of the 30 patients. Eight patients possessed both GPI(-) cells and HLA-LLs. In all 8 of these patients, the two aberrant cell populations were mutually exclusive. The analyses of different cell lineages revealed HLA-A allele-lacking cells in all lineages of cells, including granulocytes (Gs), monocytes (Ms), T cells (Ts), B cells (Bs), NK cells (NKs), and platelets (Ps) in 7 of the 8 patients; the remaining one patient had the GMTP pattern. In contrast, the lineage diversity of GPI(-) in the 8 patients was more restricted; GMTBNKP was only detected in 2 patients; the combinations in the other 6 patients were GT (n=1), GMBNKP (n=2), GMTNKP (n= 1) and GMTBP (n= 2). In Case 1, GPI(-) cells were not detected in T cells while HLA-A24(-) cells were detected in all lineages of cells including T cells (Figure 1). The limited lineage diversity of GPI(-) cells was also evident in 6 patients who did not possess HLA-LLs (GMP, GMBP, GMBNKP, GMTNKP, GMTBP) with GPI(-) granulocytes>10% while the GMTBNKP pattern was common in 10 HLA-LL(+) patients who did not possess GPI(-) cells, regardless of their percentage of HLA-A allele-lacking granulocytes. Longitudinal follow-up of 5 patients over a period of 8-27 years showed a decline in the percentage of GPI(-) granulocytes (39.2 to 0.00%, 11.4 to 0.04%, 3.50 to 0.30%, 1.77 to 0.00% and 0.79 to 0.11%) and a reciprocal increase in the percentage of HLA-A allele-lacking granulocytes (80.0 to 95.2%, 92.0 to 99.1%, 24.0 to 24.4%) in 3 patients who had been placed under observation; in two patients (Cases 2 and 3) whose GPI(-) granulocyte percentages had been >10%, the PNH clones were completely replaced by HLA-LL clones during 6 and 8 years, respectively (Figure 2). [Conclusions] The limited diversity of the blood cell lineage and spontaneous decline of GPI(-) cells that coexisted with HLA-LLs suggest that GPI(-) cells are derived from the PIGA-mutated hematopoietic progenitor cells that were allowed to proliferate as a bystander in the environment where the CTL attack against HSPCs is taking place. Disclosures Nakao: Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria.

scholarly journals Loss-of-Function Mutations in HLA-Class I Alleles in Acquire Aplastic Anemia: Evidence for the Involvement of Limited Class I Alleles in the Auto-Antigen Presentation of Aplastic Anemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2584-2584
Author(s):  
Hiroki Mizumaki ◽  
Kazuyoshi Hosomichi ◽  
Tanabe Mikoto ◽  
Takeshi Yoroidaka ◽  
Tatsuya Imi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Monoclonal Antibodies ◽  
Aplastic Anemia ◽  
Antigen Presentation ◽  
Deep Sequencing ◽  
Research Funding ◽  
Hla Class I ◽  
Class I ◽  
Loss Of Function ◽  
Targeted Deep Sequencing

Abstract [Background] Acquired aplastic anemia (AA) is a rare syndrome characterized by pancytopenia and bone marrow hypoplasia. The cytotoxic T lymphocyte (CTL) attack against autologous hematopoietic stem progenitor cells (HSPCs) is thought to be responsible for bone marrow failure in the majority of AA cases; however, little is known about the target antigens of the CTLs. HLA class I-allele lacking leukocytes (HLA-LL) due to copy-number neutral loss of heterozygosity in the short arm of chromosome 6 (6pLOH) or somatic loss-of-function mutations in HLA class I genes are detected in approximately 20% of patients with newly diagnosed AA, and the presence of HLA-LL represents compelling evidence to support that CTLs specific to HSPCs are involved in the development of AA. Our recent studies using single nucleotide polymorphism array (SNP-A) genotyping and droplet digital polymerase chain reaction (ddPCR) revealed that HLA-B*40:02 is the most frequently lost among all class I alleles that are lost as a result of 6pLOH (Zaimoku, et al. Blood 2017). Various somatic loss-of-function mutations in B*40:02 revealed by deep sequencing in the study substantiated the important role of HLA-B4002 in the autoantigen presentation of AA. However, in the other 6pLOH(+) AA patients who did not possess HLA-B4002, which accounted for 20% of the total AA cases involving patients possessing HLA-LL, the allele in the missing haplotype that was responsible for the autoantigen presentation was largely unknown because the lost fragment of chromosome 6p usually contained 2 or more HLA class I alleles. [Objectives/Methods] To identify class I alleles other than HLA-B*40:02 that are critically involved in the auto-antigen presentation of AA, we screened a total of 624 patients for the presence of HLA-LL using monoclonal antibodies specific to class I HLA alleles, SNP-A, and ddPCR, and performed targeted deep sequencing of HLA class I genes by using SeqCap EZ Choice pobes (Roche) and MiSeq sequencer (Illumina). The paired fractions, including granulocytes that lacked an HLA-A allele and granulocytes that retained the HLA-A allele, as well as CD3+ T cells, were sorted using monoclonal antibodies specific to HLA-A alleles with a BD FACSAria Fusion system (BD Biosciences), and were subjected to DNA extraction. All DNA samples of granulocytes and control cells (CD3+ T cells or buccal mucosa cells) were prepared for targeted deep sequencing. [Results] One hundred and fourteen patients were found to be positive for HLA-LL and 62 (54.4%) of the 114 HLA-LL(+) patients did not carry B*40:02 (severe, n=30; non-severe, n=32; male, n=38; female, n=24; median age, 62 [range, 6-93] years). Apart from B*40:02 (45.6%), A*02:06 (24.6%) was the second-most frequent HLA class I allele in the lost haplotype. The targeted deep sequencing of 20 patients with HLA-LL revealed 6pLOH alone in 11 patients, and somatic loss-of-function mutations plus 6pLOH in 9 patients; none of the patients were positive for somatic loss-of-function mutations alone. Of note, somatic loss-of-function mutations were found in only 5 alleles (A*02:06 in four, B*40:01 in two, B*40:03, A*31:01, and B*54:01 in one each) out of 27 different alleles contained in the lost haplotype. Among the 9 patients with somatic loss-of-function mutations, the median number of mutations per patient was 1 (range, 1-2); these included a missense mutation (n=1), frameshift deletions (n=3) and nonsense mutations (n=7) (Figure). Four patients had a breakpoint of 6pLOH in between the HLA-A and C loci; their lost alleles were A*02:06 (n=2) and A*31:01 (n=2), and the occurrence of 6pLOH in the four patients was therefore attributed to the two HLA-A alleles. Sixty-six percent of the HLA-LL(+) B*40:02(-) patients had at least one of the five alleles in the lost haplotypes. The frequencies of each "high risk" allele found in patients possessing HLA-LL are summarized in Table. [Conclusions] In addition to B*40:02, five class I alleles including HLA-A*02:06, A*31:01, B*54:01, B*40:03 and B*40:01 are thought to play an essential role in the auto-antigen presentation by the HSPCs of Japanese AA patients. The frequencies of the six class I alleles in general Japanese population are much higher than those in the general Caucasian populations but similar to the frequencies in East Asian populations. The higher frequencies of the six alleles in comparison to Caucasian countries may account for the higher incidence of AA in East Asia. Disclosures Takamatsu: Ono: Research Funding; Bristol-Myers Squibb: Research Funding; Janssen: Honoraria; Celgene: Honoraria, Research Funding. Nakao:Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria.

The Clinical Significance of Some Specific HLA Class I Alleles in Japanese Patients with Bone Marrow Failure (BMF).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1040-1040
Author(s):  
Hideyoshi Noji ◽  
Tsutomu Shichishima ◽  
Kazuhiko Ikeda ◽  
Akiko Nakamura ◽  
Kazuko Akutsu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Lactate Dehydrogenase ◽  
T Lymphocytes ◽  
Aplastic Anemia ◽  
Clinical Significance ◽  
Bone Marrow Failure ◽  
Hla Class I ◽  
Japanese Patients ◽  
Class I ◽  
Immune Mechanisms

Abstract Autoreactive T lymphocytes are implicated in the immune mechanisms involved in the bone marrow failure (BMF) syndrome, including aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH), and myelodysplastic syndrome (MDS). However, the significance of the HLA class I alleles remains unknown in the BMF syndrome. Nevertheless, from many clinical and basic studies, it is certain that CD8+ T lymphocytes are implicated in some of the immune mechanisms involved in the occurrence of AA. To clarify some clinical significance of the HLA class I alleles in the BMF syndrome, we investigated the alleles using a high-resolution method of genotyping in 78 Japanese patients with BMF, including 32 AA, 24 PNH, and 22 MDS patients. Subsequently, we compared various clinical findings, including age, sex, white blood cell counts, absolute neutrophil counts, hemoglobin concentrations, reticulocyte counts, platelet counts, values of lactate dehydrogenase, durations of illness, chromosomal findings, and proportions of CD55− and CD59− erythrocytes, between the groups with and without some alleles. The diagnosis and grading of the severity of AA were based on the criteria of the International Agranulocytosis and Aplastic Anemia Study Group (Blood1987; 70: 1718–21) and that of Frickhofen et al (N Engl J Med1991; 324: 1297–304), respectively. A patient with a CD55− and CD59− population of more than 1% was judged to have PNH erythrocytes (Blood1996; 87: 5332–40). The diagnosis of MDS was determined according to the FAB criteria (Br J Haematol1982; 51: 189–99). The frequencies of the HLA-B* 4002 allele in AA patients (21.9%) and of the HLA-A* 0206 allele in PNH patients (22.9%) were significantly different from those in controls (n=371; 8.6%, p<0.002 and 7.7%, p<0.001, respectively), while we found no specific HLA class I alelles in MDS patients. The frequency of the HLA-DRB1*1501 allele in PNH patients (31.3%) was significantly higher than that in controls (6.1%, p<0.0001), while we could not find the high frequencies of the HLA-DRB1*1501 (10.9%) and *1502 (10.9%) alleles in AA patients. The proportion of severe or very severe AA patients with the HLA-B* 4002 allele (10/17, 58.8%) was significantly higher than that of non-severe AA patients with the allele (3/15, 20%; p<0.05). In contrast, the proportion of severe or very severe AA patients (4/17; 23.5%) with the HLA-DRB1* 1501 allele was not different from that of non-severe AA patients (3/15; 20%) with the allele. Subsequently, the reticulocyte counts (138 ± 73 x 10 9/L) and values of lactate dehydrogenase (2399 ± 235 IU/L) at the time of examination in PNH patients (n=10) with the HLA-A* 0206 allele were significantly higher than those in PNH patients (n=14) without the allele (78 ± 34 x 109/L, p<0.02 and 972 ± 770 IU/L, p<0.05, respectively). In addition, the frequency of PNH patients with over 30% of complement-sensitive erythrocytes, consisting of intermediate and negative populations of CD55 and CD59 expressions on erythrocytes by flow cytometry, was significantly higher in PNH patients with the HLA-A* 0206 than in those without the allele (80% versus 28.6%, p<0.05). In conclusions, our results suggest that the HLA-B* 4002 allele in AA or the HLA-A* 0206 allele in PNH is related to grading of the severity of AA or grading of hemolysis of PNH, respectively.

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