TET2 and clonal hematopoiesis-related gene variants in patients with acquired pure red cell aplasia

TET2 and clonal hematopoiesis-related gene variants in patients with acquired pure red cell aplasia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article TET2 and clonal hematopoiesis-related gene variants in patients with acquired pure red cell aplasia Fumihiro Ishida, Toru Kawakami, Fumihiro Kawakami, Shuji Matsuzawa, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3834690/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Dysregulation of T cell-mediated immunity is considered a major pathophysiological mechanism of acquired pure red cell aplasia (PRCA), such as idiopathic PRCA, large granular lymphocytic leukemia-associated PRCA, and thymoma-associated PRCA. Although STAT3 mutations are frequently detected in PRCA patients, other mutational profiles and their involvement in the clinical characteristics are yet to be clarified. Whole-exome sequencing and targeted sequencing were performed using a custom-designed panel for PRCA (n = 53). The frequently mutated genes were NEB (40%), STAT3 (36%), PCLO (30%), TET2 (23%), and KMT2D (15%). Four of the 12 patients with mutations in TET2 had germline TET2 variants. Patients positive for TET2 variants had significantly more variants of lymphoid clonal hematopoiesis-related genes than those without TET2 variants (11/12 vs. 23/41, P = 0.038). Patients with TET2 variants relapsed after immunosuppressive therapy more frequently than those without TET2 variant (55% [6/11] vs. 11% [4/35], P = 0.0065). These data suggest that variants of clonal hematopoiesis-related genes, including TET2 , in addition to STAT3 , play important roles in the pathophysiology of PRCA. Biological sciences/Genetics/Clinical genetics/Disease genetics Biological sciences/Immunology/Immunological disorders/Lymphoproliferative disorders TET2 clonal hematopoiesis pure red cell aplasia bone marrow failure Figures Figure 1 Figure 2 INTRODUCTION Acquired pure red cell aplasia (PRCA) is an anemic disorder of bone marrow failure syndrome, defined by reticulocytopenia and marked reduction or absence of erythroid progenitors in the bone marrow [ 1 ]. PRCA develops via T-cell- or autoantibody-dependent immune mechanisms with a variety of underlying backgrounds. Regarding genetic background of PRCA, STAT3 mutations, a particularly frequent type of genetic alteration in large granular lymphocytic leukemia (LGLL), were detected in patients with various types of PRCA, including idiopathic LGLL- and thymoma-associated PRCA [ 2 , 3 ]. STAT3 mutations are restricted to CD8-positive (CD8 + ) T-cells. Other studies have identified variants of several genes, such as KMT2D , KDM6A and BCOR , with high variant allele frequencies (VAFs) [ 4 – 6 ]. However, these results have been inconsistent among reports, and many clinical questions remain, including regarding the relationships between mutational profiles and clinical characteristics of PRCA patients. To determine the genetic profile and its relationship with clinical information, we performed whole-exome sequencing (WES) and targeted sequencing analyses on a large number of PRCA patients using an originally designed gene panel. METHODS Patients PRCA patients were enrolled in this study, as were AA patients for a comparison; the diagnostic criteria for these diseases used in this study are summarized in Supplemental Table S1 [ 7 – 10 ]. Clinical data, including age, sex, underlying conditions, and laboratory data, were collected from medical records. Therapeutic medications for PRCA patients and their outcomes were obtained. A response criterion for PRCA [ 11 ] was also adopted. This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Shinshu University School of Medicine (approval number 723) and each participating center. Written informed consent was obtained from all patients and healthy controls. DNA extraction Mononuclear cells (MNCs) were isolated from peripheral blood or bone marrow using Ficoll gradient separation (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and stored at -80°C until DNA extraction. DNA was extracted using the QIAamp DNA Blood Mini Kit (QIAGEN, GmbH, Hilden, Germany) according to the manufacturer’s instructions. Sorting of cell subpopulations In select patients, target cell subpopulations were separated using a fluorescence-activated cell sorter (FACS). For example, CD3 + CD4 − CD8 + or CD3 + CD4 + CD8 − T cells were separated using antibodies against CD3 (APC, clone SP34-2; BD Biosciences, Franklin Lakes, NJ, USA), CD4 (PerCP, clone L200; BD Biosciences), and CD8 (PE, clone RPA-T8; BD Biosciences) with a FACSAria cell sorter (BD Biosciences) (Supplemental Figure S1 ). WES WES was performed using the Ion AmpliSeq technology. DNA was extracted from CD3 + CD8 − CD4-positive (CD4 + ) cells or CD3 + CD4 − CD8 + cells of PRCA patients. The libraries were prepared using the Ion AmpliSeq Exome RDY Kit according to the protocol for preparing Ion AmpliSeq libraries (Thermo Fisher Scientific, Waltham, MA, USA). DNA concentrations of the libraries were measured using an Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific). The libraries were subjected to WES on Ion S5 according to the manufacturer’s standard protocol using the Ion 540 Chip Kit (Thermo Fisher Scientific). Data were analyzed using the Torrent Suite software program (v5.12.1; Thermo Fisher Scientific) and Ion Reporter software program (v5.12; Thermo Fisher Scientific). The variants were called using the workflow “AmpliSeq Exome single sample (Somatic).” The main variant calling settings were as follows: variant frequency filter, 0.02; base quality Q-value, ≥ 6.5; minimum coverage depth, 20; and maximum strand bias, 0.9 (single nucleotide polymorphism [SNP]), 0.85 (INDEL). Variants considered SNPs or synonymous variants were eliminated. Variants with a VAF of 20%-60% from CD8 + cells were compared with CD4 + cells, and the variant characteristics of CD8 + cells were selected. Target sequencing Target sequencing was performed using the Ion AmpliSeq technology. Candidate genes were selected from the WES results, and genes related to clonal hematopoiesis of indeterminate potential (CHIP) or lymphoproliferative disorders were also included. Primers were designed to cover 97% of the coding sequences of the candidate genes using the AmpliSeq Designer system (Thermo Fisher Scientific). The analyzed genes are summarized in Supplemental Table S2 . The libraries were prepared using the Ion AmpliSeq Library Kit Plus according to the protocol for preparing Ion AmpliSeq libraries (Thermo Fisher Scientific). DNA concentrations of the libraries were measured using an Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific). The libraries were subjected to amplicon sequencing on the Ion GeneStudio S5 system according to the manufacturer’s standard protocol using an Ion 530 or 540 Chip (Thermo Fisher Scientific). Data were analyzed using the Torrent Suite software program (v5.8.0; Thermo Fisher Scientific). The main variant calling settings were as follows: variant frequency filter, 0.01; base quality Q-value, ≥ 20; minimum coverage depth, 1000; and maximum strand bias, 0.95 (SNP), 0.9 (INDEL). The called variants were annotated using wANNOVAR ( http://wannovar.wglab.org/index.php ), and variants considered SNPs or synonymous variants were eliminated. Validation of candidate somatic variants by Sanger sequencing PCR amplification was performed using primers for Sanger sequencing (Supplemental Table S3 ), and PCR products were purified by gel electrophoresis, followed by extraction with a QIAExII Gel Extraction kit (QIAGEN) or Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). The purified PCR products were then sequenced using a BigDye v1.1 Cycle Sequencing kit and an ABI Prism 3500 Genetic Analyzer (Thermo Fisher Scientific). Statistical analyses Comparisons between different groups were performed using Fisher’s exact test, a two-sided t -test, the Mann-Whitney U test, or a log-rank test, as appropriate. P -values of < 0.05. All statistical analyses were performed using the EZR software program (ver. 1.55) 23 . RESULTS Patient demographics A total of 53 PRCA patients were included in this study. Peripheral blood samples were collected from all patients. Ten AA patients and two healthy individuals were included as controls. The subtypes of PRCA were idiopathic disease (n = 11), T-LGLL-associated PRCA (n = 26), thymoma-associated PRCA (n = 10), autoimmune disease-associated PRCA (n = 3), and others (n = 3). The backgrounds of AA were idiopathic disease (n = 9) and paroxysmal nocturnal hemoglobinuria (n = 1). The clinical characteristics of the patients are summarized in Table 1. WES of CD4 + cells and CD8 + cells in PRCA The backgrounds of PRCA analyzed with WES were as follows: idiopathic (n = 2), thymoma or thymic cancer (n = 5), and autoimmune diseases (n = 2). The median numbers of CD4 + cells and CD8 + cells in the peripheral blood of the patients were 0.54×10 9 /L (0.44–0.68×10 9 /L) and 1.08×10 9 /L (0.29–2.25×10 9 /L), respectively. In this sequencing analysis, the median depth of coverage was 84x (range: 31–123) for CD4 + cells and 136x (range: 114–205) for CD8 + cells. The detected variants with VAFs of 20%-60% are shown in Supplemental Table S4 and Supplemental Figure S2 . The median number of mutated genes detected was 37 (range: 19–42) for CD4 + cells and 31 (range: 4–52) for CD8 + cells. None of the mutated genes was shared across samples. We included ORAI1 , HIPK4 , MUC1 , and SPAG5 as candidate mutated genes in CD8 + cells and subjected them to a target sequencing panel. We also added several mutated genes in both samples derived from CD4 + and CD8 + cells, including TET2 , HCFC1 , and NHS , for the panel. Landscape of mutations in PRCA To obtain further insight into the genetic profiles of PRCA patients, we examined MNC-derived DNA from patients with PRCA or AA and healthy controls using amplicon sequencing with a custom panel. In this sequencing analysis, the median depth of coverage was 3,368x (range: 1,665-5,454). MUC17 and IGFN1 were omitted from further analyses because of their high false-positive rates. The landscape of gene mutations is shown in Fig. 1 and Supplemental Figure S3 . The detected mutations are summarized in Supplemental Table S5. Fifty-two patients (98%) with PRCA had at least 1 variant out of the 50 genes in the panel. Variants in 31 genes were detected in multiple cases. The top 5 recurrently mutated genes were NEB (40%), STAT3 (36%), PCLO (30%), TET2 (23%), and KMT2D (15%) (Supplementary Figure S4). Frequent mutated genes in each subtype were as follows: PCLO (45%), NEB (36%), and STAT3 (27%) in idiopathic PRCA; PCLO (50%), and NEB (30%) in thymoma-associated PRCA; and STAT3 (54%), NEB (42%), TET2 (27%), PCLO (19%), and BRCA2 (19%) in LGLL-associated PRCA. STAT3 and TET2 variants were not detected in AA patients. We classified TET2 , DNMT3A , and CUX1 as myeloid CHIP (M-CHIP)-related genes and NEB , PCLO , and KMT2D as lymphoid CHIP (L-CHIP)-related genes according to Niroula et al. [ 12 ]. In PRCA patients, variants of L-CHIP- and M-CHIP-related genes were detected in 62% and 45% of cases, respectively. Conversely, variants of those genes were less frequently detected in AA patients, where the mutation-positive rates of L-CHIP-related genes and M-CHIP-related genes were 30% and 10%, respectively. These results strongly suggest the unique mutational profiles of PRCA. TET2 variants in PRCA TET2 variants were found in 12 PRCA patients, and the median VAF was 19.6% (range: 1%-51.9%). TET2 variants detected in this cohort are summarized in Fig. 2 and Supplementary Table S5. Five patients had TET2 variants, with high VAFs of > 40%. The median VAF of TET2 variants when excluding those with a VAF ≥ 40% was 8.1% (n = 8, range: 1.1–20.9). The breakdown of the patients with TET2 variants was as follows: idiopathic PRCA (n = 1), thymoma-associated PRCA (n = 1), LGLL-associated PRCA (n = 7), and autoimmune disease-associated PRCA (n = 3). Among the patients with VAF > 40% for TET2 variants, we performed Sanger sequencing of DNA derived from the buccal mucosa in UPN 9, 11, and 35, and all were positive for the corresponding variants, which strongly suggests germline TET2 variants in these three cases. Of the patients with germline TET2 variants, UPN 11 had a parent-child relationship with UPN 20. UPN 9 and 35 did not have any family history of cytopenia or hematological malignancies. TET2 germline variants (F387Y, N813S, and R881W) have not been previously cited with reference to COSMIC (v98, released 2023-MAY-23) or other previously reported germline variants [ 13 – 15 ]. Some variants with lower VAFs (C1135Y, R1516X and I1873T) were reported in COSMIC as myeloid malignancies-related mutations; however, none of the TET2 -mutated PRCA patients had dysplastic features in bone marrow cells or abnormal karyotypes. Three patients with TET2 variants (25%) had STAT3 variant comutations (Fig. 1 ). TET2- mutated patients had significantly more variants of L-CHIP-related genes than patients without TET2 variants (11/12 vs. 23/41 patients, P = 0.038). None of the 12 patients with TET2 variants developed myeloid malignancy. Variants of L-CHIP-related genes in PRCA Variants of L-CHIP-related genes were also frequently detected in PRCA patients from various backgrounds, and the mutation positivity rates were as follows: NEB , 40%; PCLO , 30%; and KMT2D , 15%. VAFs of each gene often exceeded 40%: 15 of 21 (71%) NEB variants, 4 of 16 (25%) PCLO variants, and 4 of 9 (44%) KMT2D variants, which suggested germline variants or somatic mutations of high VAF. Interestingly, NEB A4716N variants were found in 4 patients. NEB A4716N is registered in dbSNP (rs796065338), but its frequencies have not been described. Thirty-three patients (62%) were positive for variants in L-CHIP-related genes. Thirty-six percent of patients with variants of L-CHIP-related genes had STAT3 variants (12/33), and 30% of them (10/33) had TET2 variants. Variants of L-CHIP-related genes did not differ among PRCA subtypes. Variants of M-CHIP-related genes in PRCA DNMT3A and CUX1, M-CHIP-related genes besides TET2 , were detected in 9% (5/53) and 13% (7/53) of PRCA, respectively. Two patients with DNMT3A variants and three with CUX1 variants had high-VAF (> 40%) variants. None of the patients with DNMT3A or CUX1 variants developed myeloid malignancies. Differences in the clinical characteristics of PRCA patients according to genetic profiles We examined the relationship between clinical characteristics and mutated genes, especially those related to L-CHIP or M-CHIP, including STAT3 , TET2 , DNMT3A , CUX1 , NEB , PCLO , and KMT2D . Patients with NEB variants were significantly younger than those without variants (median: 42 vs. 63 years old, P = 0.00074), and patients with DNMT3A variants were significantly older than those without variants (median: 72 vs. 49 years old, P = 0.034). In addition, TET2 variant (+) patients relapsed after first-line immunosuppressive therapies significantly more frequently than TET2 variant (-) patients (55% [6/11] vs. 11% [4/35], P = 0.0065) (Table 2). The chronological analysis of recurrently mutated genes Serial blood samples from three PRCA patients were available for targeted sequencing (Table 3). The first patient with thymoma-associated PRCA (UPN 1) underwent thymectomy and achieved PRCA remission. Variants of TET2 and NEB were not detected in the second sample after five years, and new variants of PTPN23 , PCLO , and CARD10 developed. The second patient with autoimmune disorder-associated PRCA (UPN 9) received immunosuppressive therapy and achieved remission, after which he experienced relapse. The TET2 R1516X variant disappeared after therapy; however, TET2 F387Y variant was persistently detected. The third patient with LGLL-associated PRCA (UPN 30) showed refractoriness to CsA and responded to CY. After CY therapy, the TET2 C1193Y variant became undetectable, and the STAT3 R618R variant was newly detected. DISCUSSION In this study, we revealed a detailed mutation profile of PRCA. Notably, TET2 variants were detected in 23% of PRCA patients, regardless of the subtype. Five of these variants had a VAF ≥ 40%, 3 cases were confirmed as germline variants, and 1 patient with the N813S variant was also a member of a family with N813S germline variants. Although somatic TET2 variants are frequently associated with myeloid malignancies [ 16 – 20 ], T-cell lymphoma [ 21 , 22 ], and CHIP [ 23 ], germline TET2 variants have also been reported in various diseases, although rare, including myeloid malignancies [ 13 – 15 ], lymphoid malignancies [ 24 ], immune dysregulation syndromes [ 25 , 26 ], and pulmonary arterial hypertension [ 27 , 28 ]. Loss of function variants of TET2 lead to DNA hypermethylation, which can cause enhanced inflammation [ 27 ] or immune dysregulation [ 24 – 26 ]. López et al. reported that heterozygous TET2 loss-of-function variants could induce increased methylation in CD8 + cells. Loss of Tet2 causes overexpression of IL-1, leading to increased inflammation or leukemogenesis [ 29 – 33 ]. Furthermore, increased IL-1 levels induce clonal expansion of Tet2 +/- hematopoietic cells [ 34 ]. Gene expression and blood concentrations of IL-1β are increased in T-LGLL [ 35 ]. In addition, Tet2 -knockout hematopoietic stem and progenitor cells can activate the IL-6/Stat3 pathway in response to inflammatory stress [ 36 ]. In T-LGLL, IL-6 mRNA is highly expressed and contributes to continuous STAT3 activation [ 37 ]. Abnormalities in IL-1 or IL-6 signaling may play a role in the pathogenesis of PRCA, including the LGLL-associated type. TET2 variants are thought to be pre-leukemic events, and second-hit variants, including DNMT3A , STAT3 KMT2D [ 38 ] and RHOA cause lymphomagenesis [ 21 , 22 , 39 ]. In our study, 25% of patients with TET2 variants (3/12) had STAT3 variants, and 92% (11/12) had variants of L-CHIP-related genes, suggesting that interweaving of these genes might be involved in the pathophysiology of PRCA, although our study is limited by its lack of functional analyses, including DNA methylation and gene expression studies. Although variants of STAT3 or CHIP-related genes have been previously reported in PRCA [ 2 – 6 , 40 ], this is the first study to clarify the high frequency of co-mutation of TET2 , STAT3 and L-CHIP-related genes. In previous studies with TET2 mutational analyses in PRCA [ 4 – 6 , 41 ]. TET2 variants were detected in 4 of 101 cases (4%). The variants reported by Fujishima et al. were P288fs, G641fs, T759fs, H1904R, and R1465X, and two patients had two variants. Single-nucleotide variants (SNVs) registered in public databases were excluded from previous reports, as were SNVs with frequencies greater than 0.1%. We included SNVs registered in public databases with a frequency < 1% in public database in this analysis because the frequencies of TET2 SNVs detected in our study were extremely low (Supplemental Table S6), which ruled out the possibility of SNPs. The detection sensitivity of sequencing methods and patient backgrounds, including causes of PRCA and ethnic composition, might also have influenced the results. According to Niroula et al., the detection rates of L-CHIP-related genes were much lower than those of M-CHIP-related genes in the general population [ 12 ]. In our study, patients with TET2 variants had L-CHIP-related gene variants significantly more frequently than M-CHIP-related gene variants, suggesting a strong relationship between TET2 and L-CHIP-related genes. Originally, L-CHIP-related genes were detected mainly in chronic lymphocytic leukemia, a mature B-cell neoplasm [ 12 ]. In fact, NEB or PCLO are not frequently mutated genes in T-cell lymphomas [ 42 ]. In a study on T-LGLL by Cheon et al., mutations in NEB and PCLO genes were found in 1/93 (1%) and 4/93 (4%) patients, respectively [ 43 ]. These results suggest the existence of variants of L-CHIP-related genes in the T cell population. Compared to PRCA, frequently detected variants (> 5%) tend to be classified as M-CHIP-related genes in AA [ 44 ] or myelodysplastic neoplasms (MDS) [ 16 ] (Supplemental Table S7). In contrast, KMT2D is classified as an L-CHIP-related gene, whereas KDM6A and TET2 are M-CHIP in T-LGLL [ 43 ]. Among M-CHIP-related genes, TET2 variants are most frequently detected in PRCA, whereas DNMT3A was the most frequently mutated gene in AA [ 44 ] and age-related CHIP [ 45 ]. These findings suggest a unique pattern of CHIP in PRCA. Based on the VAFs of the mutated genes, there might be two evolutionary patterns of mutations in TET2 -mutated PRCA (Supplemental Figure S5). In type 1, high-VAF variants of NEB , PCLO , KMT2D or TET2 occur first, followed by the addition of somatic variants of TET2 are secondly added. In type 2, germline variants of TET2 exist, and clonal development and expansion of somatic variants of other genes occur as late events. Type 1 mutations are recognized in autoimmune-, thymoma-associated, LGLL-associated, and idiopathic PRCA, whereas type 2 mutations are mostly found in LGLL-associated PRCA. TET2 variants have been reported in T-LGLL or NK-LGLL [ 43 , 46 , 47 ]. The frequency of TET2 variants was 5% in T-LGLL and 28%-34% in NK-LGLL. No hotspot loci in TET2 were detected in either relevant study. Pastoret et al. showed that TET2 variants were found in myeloid and NK cells of three of four patients with NK-LGLL, suggesting TET2 mutations might be an early event in the pathogenesis of NK-LGLL [ 47 ]. This finding suggests a possible mechanism for the type 2 TET2 mutation in PRCA. STAT3 variants are restricted to CD8 + cells [ 2 ]; however, details of the involved cell lineages and other somatic variants remain unclear. Some TET2 variant-positive clones decreased considerably after immunosuppressive therapy or thymectomy (Table 3), suggesting the possibility of the involvement of somatic TET2 variants in T cells. We assumed that the pathophysiology of PRCA includes dysregulation of cellular immunity and impairment of erythropoiesis, which might be partially affected by germline or somatic variants of TET2 . PRCA and AA share several clinical features, including T-cell abnormalities, efficacy of immunosuppressive treatments, and decreased hematopoietic progenitor cells among bone marrow failure syndromes; however, several differences also exist, such as impaired cell lineage, backgrounds of disease, and frequency of progression to myeloid malignancies. None of the AA patients in our cohort had STAT3 or TET2 variants. Our sequencing panel did not contain PIGA , BCOR or ASXL1 , which are frequently mutated in AA; however, they were rarely found in other studies of PRCA [ 4 – 6 , 41 ]. A better understanding of the genetic alterations characteristic of PRCA would lead to a better understanding of the mechanisms underlying the development of refractoriness to immunosuppressive therapy in PRCA. In conclusion, germline or somatic variants of TET2 and variants of CHIP-related genes were recurrently found in PRCA patients, and mutations in these genes may play important roles in the pathophysiology of PRCA. Declarations COMPETING INTERESTS The authors declare no competing financial interests. AUTHOR CONTRIBUTIONS Contribution: T.K. designed the study, performed experiments, and analyzed the data. F.K., S.M., T.Y., Y.M., A.A., D.H., S.M., and Y.H. performed experiments. S.N., H.S., Y.K., and H.N. collected samples and clinical data. F.I. conceived and designed the study, analyzed the data, and supervised the research. T.K., H.N. and F.I. wrote the manuscript. ACKNOWLEDGEMENTS The authors thank Dr. Yujiro Ito of Hamamatsu University School of Medicine, Dr. Eiko Oya of Matsusaka Chuo General Hospital, Dr. Atsushi Isoda of Hoshi Clinic, Dr. Toshimitsu Ueki of Nagano Red Cross Hospital, Dr. Toshiro Ito of Matsumoto Medical Center, Dr. Taizo Shimomura of Kumamoto Shinto General Hospital, Dr. Masao Hagihara of Eiju General Hospital, Dr. Yoshiki Akatsuka of Fujita Health University, Dr. Tatsuya Imi and Hiroyuki Takamatsu of Kanazawa University, Dr. Takayuki Takahashi of Shinko Hospital, and Dr. Go Aoki of Komatsu Municipal Hospital for providing the patient data. We also thank Ms. Natsumi Ida and Ms. Masae Maruyama for technical assistance. 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Zhang X, Shi Y, Song L, Shen C, Cai Q, Zhang Z, et al. Identification of mutations in patients with acquired pure red cell aplasia. Acta Biochim Biophys Sin (Shanghai) 2018; 50: 685–692. Moffitt AB, Dave SS. Clinical Applications of the Genomic Landscape of Aggressive Non-Hodgkin Lymphoma. J Clin Oncol 2017; 35: 955–962. Cheon H, Xing JC, Moosic KB, Ung J, Chan V, Chung DS, et al. Genomic Landscape of TCR Alpha-Beta and TCR Gamma-Delta T-Large Granular Lymphocyte Leukemia. Blood 2022. Yoshizato T, Dumitriu B, Hosokawa K, Makishima H, Yoshida K, Townsley D, et al. Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia. N Engl J Med 2015; 373: 35–47. Jaiswal S, Fontanillas P, Flannick J, Manning A, Grauman PV, Mar BG, et al. Age-related clonal hematopoiesis associated with adverse outcomes. N Engl J Med 2014; 371: 2488–2498. Olson TL, Cheon H, Xing JC, Olson KC, Paila U, Hamele CE, et al. Frequent somatic TET2 mutations in chronic NK-LGL leukemia with distinct patterns of cytopenias. Blood 2021; 138: 662–673. Pastoret C, Desmots F, Drillet G, Le Gallou S, Boulland ML, Thannberger A, et al. Linking the KIR phenotype with STAT3 and TET2 mutations to identify chronic lymphoproliferative disorders of NK cells. Blood 2021; 137: 3237–3250. Tables Tables 1-3 is available in the Supplementary Files section. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files table1clinicalinformationv2.xlsx Table 1 table2TET2comparison.xlsx Table 2 table3serialanalyses.xlsx Table 3 TableS4.xls Supplemental Table S4 TableS5.xls Supplemental Table S5 SupInfov2.docx Supplemental Materials Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3834690","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":265409596,"identity":"07279327-0fe3-4351-9cdd-3e71846cc527","order_by":0,"name":"Fumihiro Ishida","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIie2QsQrCMBCGrwh2SZxPFPsKSkEEB1/FTB3EyV0Lwrn4AIKDr1BxcUzJ4OIDOLi4uAm6iNlM1dG2dBPJR3JkyHd/LgAWyw/ihgDSCbEB6IRg1geWrjD5VvxiSnJThPg+5MNwIGK97QSr5ZROequgKUunK9SPGcowUnxv6jGe+XyfKGUfgZ1TlV6iOGQqCqo5pCaRhLZppTJTYk0YeAtBVU1JinvPVSQn7MNBEPKXwnJS2MXMQtiKjFLjFEBVsRH2M2Zh7nBz0zT2vEVwNg/rQmU3W19v8/Qf+0LJbDGXRZQXj+KKxWKx/C1PPnZXoxAnv1gAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0525-7636","institution":"Shinshu University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Fumihiro","middleName":"","lastName":"Ishida","suffix":""},{"id":265409597,"identity":"bede2e21-8a7d-482a-91bd-c1a46ce47244","order_by":1,"name":"Toru Kawakami","email":"","orcid":"https://orcid.org/0000-0003-0178-5332","institution":"Shinshu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Toru","middleName":"","lastName":"Kawakami","suffix":""},{"id":265409598,"identity":"59b9a200-23ae-4e92-af67-76e1e89e4862","order_by":2,"name":"Fumihiro Kawakami","email":"","orcid":"","institution":"Shinshu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Fumihiro","middleName":"","lastName":"Kawakami","suffix":""},{"id":265409599,"identity":"c0cf87cf-a4fa-4925-8c1d-0fb11f9431a8","order_by":3,"name":"Shuji Matsuzawa","email":"","orcid":"","institution":"Shinshu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuji","middleName":"","lastName":"Matsuzawa","suffix":""},{"id":265409600,"identity":"d0fe5bfd-4576-48e4-979f-5aa6617ddc8b","order_by":4,"name":"Taku Yamane","email":"","orcid":"","institution":"Shinshu University 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Medicine","correspondingAuthor":false,"prefix":"","firstName":"Daigo","middleName":"","lastName":"Higano","suffix":""},{"id":265409604,"identity":"283953db-9d29-4580-a195-7e1690fcd05a","order_by":8,"name":"Shotaro Miyairi","email":"","orcid":"","institution":"Shinshu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shotaro","middleName":"","lastName":"Miyairi","suffix":""},{"id":265409605,"identity":"bc1f0ef1-c9cf-4238-afb5-8bdf29d882fa","order_by":9,"name":"Kaoko Sakai","email":"","orcid":"","institution":"Shinshu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kaoko","middleName":"","lastName":"Sakai","suffix":""},{"id":265409606,"identity":"d6764ee7-1336-4767-8c64-3924460a32c9","order_by":10,"name":"Sayaka NIshina","email":"","orcid":"","institution":"Shinshu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sayaka","middleName":"","lastName":"NIshina","suffix":""},{"id":265409607,"identity":"d5a4d07b-d1cc-4949-b83e-bc202eca92b6","order_by":11,"name":"Hitoshi Sakai","email":"","orcid":"","institution":"Shinshu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hitoshi","middleName":"","lastName":"Sakai","suffix":""},{"id":265409608,"identity":"67202fea-1010-4495-9271-e1f62135af5b","order_by":12,"name":"Yasushi Kubota","email":"","orcid":"https://orcid.org/0000-0001-7785-1362","institution":"Saga University","correspondingAuthor":false,"prefix":"","firstName":"Yasushi","middleName":"","lastName":"Kubota","suffix":""},{"id":265409609,"identity":"866e28ee-22a6-4f8b-89de-4b33cf918ac8","order_by":13,"name":"Yumiko Higuchi","email":"","orcid":"","institution":"Shinshu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yumiko","middleName":"","lastName":"Higuchi","suffix":""},{"id":265409610,"identity":"64243d2f-796d-4689-8e35-ee884f4c3d91","order_by":14,"name":"Hideyuki Nakazawa","email":"","orcid":"","institution":"Shinshu University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hideyuki","middleName":"","lastName":"Nakazawa","suffix":""}],"badges":[],"createdAt":"2024-01-04 13:42:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3834690/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3834690/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49332949,"identity":"060dea1a-8633-400f-96b4-5dcb414f497a","added_by":"auto","created_at":"2024-01-08 19:35:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":62920,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOncoplot of PRCA. \u003c/strong\u003eEach row represents a gene mutated in more than one case, and the columns represent individual patients. The backgrounds of PRCA are color-coded at the top. The variant allele frequency is indicated by the colored boxes; black indicates ≥40%, and gray indicates \u0026lt;40%. The clinical phenotypes are shown above the heat map. The bar graph on the right shows the total number of mutations detected in each gene. PRCA, pure red cell aplasia; UPN, unique patient number; LGLL, large granular lymphocytic leukemia-associated PRCA; idiopathic, idiopathic PRCA; thymoma, thymoma-associated PRCA; autoimmune, autoimmune disease-associated PRCA; M-CHIP, myeloid clonal hematopoiesis of indeterminate potential-related genes; L-CHIP, lymphoid clonal hematopoiesis of indeterminate potential-related genes.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3834690/v1/45e762202029951625a9ca08.png"},{"id":49333272,"identity":"490ba9ac-43bb-4327-9821-9d6e974bb4b1","added_by":"auto","created_at":"2024-01-08 19:43:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":32528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe distribution of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTET2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e variants.\u003c/strong\u003e Each circle represents variants with variant allele frequencies (VAFs) ≥40%, and squares represent variants with VAFs \u0026lt;40%. DSBH, double-stranded b helix\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3834690/v1/cffbc833b13368a4752b79c2.png"},{"id":50310465,"identity":"d6b058c0-926c-4c43-824c-840b4009b90d","added_by":"auto","created_at":"2024-01-29 14:30:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":594114,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3834690/v1/274e286f-cc97-48cb-bff0-6e69ca4d37a1.pdf"},{"id":49333273,"identity":"b4dfe37d-558f-421d-88c2-a95243b35a01","added_by":"auto","created_at":"2024-01-08 19:43:48","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10786,"visible":true,"origin":"","legend":"Table 1","description":"","filename":"table1clinicalinformationv2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3834690/v1/13998f00b468eb0d13467549.xlsx"},{"id":49333271,"identity":"f4ea1b56-0e20-490c-a29a-f4b11a6d2b76","added_by":"auto","created_at":"2024-01-08 19:43:48","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10766,"visible":true,"origin":"","legend":"Table 2","description":"","filename":"table2TET2comparison.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3834690/v1/f9ff14b6abd4d7ac4699f5ac.xlsx"},{"id":49333416,"identity":"c37d36d8-5080-49ee-9b53-57d429bcb8fd","added_by":"auto","created_at":"2024-01-08 19:51:48","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10457,"visible":true,"origin":"","legend":"Table 3","description":"","filename":"table3serialanalyses.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3834690/v1/93ad1e0dad1271df25bb0728.xlsx"},{"id":49333274,"identity":"6c81bf4e-2f42-4506-ab90-a874d1f226e7","added_by":"auto","created_at":"2024-01-08 19:43:48","extension":"xls","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":141824,"visible":true,"origin":"","legend":"Supplemental Table S4","description":"","filename":"TableS4.xls","url":"https://assets-eu.researchsquare.com/files/rs-3834690/v1/9516f06acca69019f8fc5297.xls"},{"id":49332954,"identity":"1653000b-7b21-4142-9030-0c3d2b71bd9d","added_by":"auto","created_at":"2024-01-08 19:35:48","extension":"xls","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":139264,"visible":true,"origin":"","legend":"Supplemental Table S5","description":"","filename":"TableS5.xls","url":"https://assets-eu.researchsquare.com/files/rs-3834690/v1/6d5f1799b1fd8b5792a2d199.xls"},{"id":49332951,"identity":"c2018abf-aeda-400b-a40f-dd8df44c620c","added_by":"auto","created_at":"2024-01-08 19:35:48","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":665716,"visible":true,"origin":"","legend":"Supplemental Materials","description":"","filename":"SupInfov2.docx","url":"https://assets-eu.researchsquare.com/files/rs-3834690/v1/1d41227ccde65a6ffcee3418.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"\u003ci\u003eTET2\u003c/i\u003e and clonal hematopoiesis-related gene variants in patients with acquired pure red cell aplasia","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAcquired pure red cell aplasia (PRCA) is an anemic disorder of bone marrow failure syndrome, defined by reticulocytopenia and marked reduction or absence of erythroid progenitors in the bone marrow [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. PRCA develops via T-cell- or autoantibody-dependent immune mechanisms with a variety of underlying backgrounds.\u003c/p\u003e \u003cp\u003eRegarding genetic background of PRCA, \u003cem\u003eSTAT3\u003c/em\u003e mutations, a particularly frequent type of genetic alteration in large granular lymphocytic leukemia (LGLL), were detected in patients with various types of PRCA, including idiopathic LGLL- and thymoma-associated PRCA [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. \u003cem\u003eSTAT3\u003c/em\u003e mutations are restricted to CD8-positive (CD8\u003csup\u003e+\u003c/sup\u003e) T-cells. Other studies have identified variants of several genes, such as \u003cem\u003eKMT2D\u003c/em\u003e, \u003cem\u003eKDM6A\u003c/em\u003e and \u003cem\u003eBCOR\u003c/em\u003e, with high variant allele frequencies (VAFs) [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, these results have been inconsistent among reports, and many clinical questions remain, including regarding the relationships between mutational profiles and clinical characteristics of PRCA patients.\u003c/p\u003e \u003cp\u003eTo determine the genetic profile and its relationship with clinical information, we performed whole-exome sequencing (WES) and targeted sequencing analyses on a large number of PRCA patients using an originally designed gene panel.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003ePRCA patients were enrolled in this study, as were AA patients for a comparison; the diagnostic criteria for these diseases used in this study are summarized in Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Clinical data, including age, sex, underlying conditions, and laboratory data, were collected from medical records. Therapeutic medications for PRCA patients and their outcomes were obtained. A response criterion for PRCA [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] was also adopted.\u003c/p\u003e \u003cp\u003e This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Shinshu University School of Medicine (approval number 723) and each participating center. Written informed consent was obtained from all patients and healthy controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction\u003c/h2\u003e \u003cp\u003eMononuclear cells (MNCs) were isolated from peripheral blood or bone marrow using Ficoll gradient separation (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and stored at -80\u0026deg;C until DNA extraction. DNA was extracted using the QIAamp DNA Blood Mini Kit (QIAGEN, GmbH, Hilden, Germany) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSorting of cell subpopulations\u003c/h2\u003e \u003cp\u003eIn select patients, target cell subpopulations were separated using a fluorescence-activated cell sorter (FACS). For example, CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e\u0026minus;\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e or CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e\u0026minus;\u003c/sup\u003e T cells were separated using antibodies against CD3 (APC, clone SP34-2; BD Biosciences, Franklin Lakes, NJ, USA), CD4 (PerCP, clone L200; BD Biosciences), and CD8 (PE, clone RPA-T8; BD Biosciences) with a FACSAria cell sorter (BD Biosciences) (Supplemental Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWES\u003c/h2\u003e \u003cp\u003eWES was performed using the Ion AmpliSeq technology. DNA was extracted from CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e\u0026minus;\u003c/sup\u003eCD4-positive (CD4\u003csup\u003e+\u003c/sup\u003e) cells or CD3\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e\u0026minus;\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e cells of PRCA patients. The libraries were prepared using the Ion AmpliSeq Exome RDY Kit according to the protocol for preparing Ion AmpliSeq libraries (Thermo Fisher Scientific, Waltham, MA, USA). DNA concentrations of the libraries were measured using an Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific). The libraries were subjected to WES on Ion S5 according to the manufacturer\u0026rsquo;s standard protocol using the Ion 540 Chip Kit (Thermo Fisher Scientific). Data were analyzed using the Torrent Suite software program (v5.12.1; Thermo Fisher Scientific) and Ion Reporter software program (v5.12; Thermo Fisher Scientific). The variants were called using the workflow \u0026ldquo;AmpliSeq Exome single sample (Somatic).\u0026rdquo; The main variant calling settings were as follows: variant frequency filter, 0.02; base quality Q-value, \u0026ge;\u0026thinsp;6.5; minimum coverage depth, 20; and maximum strand bias, 0.9 (single nucleotide polymorphism [SNP]), 0.85 (INDEL). Variants considered SNPs or synonymous variants were eliminated. Variants with a VAF of 20%-60% from CD8\u003csup\u003e+\u003c/sup\u003e cells were compared with CD4\u003csup\u003e+\u003c/sup\u003e cells, and the variant characteristics of CD8\u003csup\u003e+\u003c/sup\u003e cells were selected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTarget sequencing\u003c/h2\u003e \u003cp\u003eTarget sequencing was performed using the Ion AmpliSeq technology. Candidate genes were selected from the WES results, and genes related to clonal hematopoiesis of indeterminate potential (CHIP) or lymphoproliferative disorders were also included. Primers were designed to cover 97% of the coding sequences of the candidate genes using the AmpliSeq Designer system (Thermo Fisher Scientific). The analyzed genes are summarized in Supplemental Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. The libraries were prepared using the Ion AmpliSeq Library Kit Plus according to the protocol for preparing Ion AmpliSeq libraries (Thermo Fisher Scientific). DNA concentrations of the libraries were measured using an Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific). The libraries were subjected to amplicon sequencing on the Ion GeneStudio S5 system according to the manufacturer\u0026rsquo;s standard protocol using an Ion 530 or 540 Chip (Thermo Fisher Scientific). Data were analyzed using the Torrent Suite software program (v5.8.0; Thermo Fisher Scientific). The main variant calling settings were as follows: variant frequency filter, 0.01; base quality Q-value, \u0026ge;\u0026thinsp;20; minimum coverage depth, 1000; and maximum strand bias, 0.95 (SNP), 0.9 (INDEL). The called variants were annotated using wANNOVAR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://wannovar.wglab.org/index.php\u003c/span\u003e\u003cspan address=\"http://wannovar.wglab.org/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and variants considered SNPs or synonymous variants were eliminated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eValidation of candidate somatic variants by Sanger sequencing\u003c/h2\u003e \u003cp\u003ePCR amplification was performed using primers for Sanger sequencing (Supplemental Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), and PCR products were purified by gel electrophoresis, followed by extraction with a QIAExII Gel Extraction kit (QIAGEN) or Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). The purified PCR products were then sequenced using a BigDye v1.1 Cycle Sequencing kit and an ABI Prism 3500 Genetic Analyzer (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eComparisons between different groups were performed using Fisher\u0026rsquo;s exact test, a two-sided \u003cem\u003et\u003c/em\u003e-test, the Mann-Whitney U test, or a log-rank test, as appropriate. \u003cem\u003eP\u003c/em\u003e-values of \u0026lt;\u0026thinsp;0.05. All statistical analyses were performed using the EZR software program (ver. 1.55)\u003csup\u003e23\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePatient demographics\u003c/h2\u003e \u003cp\u003eA total of 53 PRCA patients were included in this study. Peripheral blood samples were collected from all patients. Ten AA patients and two healthy individuals were included as controls. The subtypes of PRCA were idiopathic disease (n\u0026thinsp;=\u0026thinsp;11), T-LGLL-associated PRCA (n\u0026thinsp;=\u0026thinsp;26), thymoma-associated PRCA (n\u0026thinsp;=\u0026thinsp;10), autoimmune disease-associated PRCA (n\u0026thinsp;=\u0026thinsp;3), and others (n\u0026thinsp;=\u0026thinsp;3). The backgrounds of AA were idiopathic disease (n\u0026thinsp;=\u0026thinsp;9) and paroxysmal nocturnal hemoglobinuria (n\u0026thinsp;=\u0026thinsp;1). The clinical characteristics of the patients are summarized in Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWES of CD4\u003csup\u003e+\u003c/sup\u003e cells and CD8\u003csup\u003e+\u003c/sup\u003e cells in PRCA\u003c/h2\u003e \u003cp\u003eThe backgrounds of PRCA analyzed with WES were as follows: idiopathic (n\u0026thinsp;=\u0026thinsp;2), thymoma or thymic cancer (n\u0026thinsp;=\u0026thinsp;5), and autoimmune diseases (n\u0026thinsp;=\u0026thinsp;2). The median numbers of CD4\u003csup\u003e+\u003c/sup\u003e cells and CD8\u003csup\u003e+\u003c/sup\u003e cells in the peripheral blood of the patients were 0.54\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L (0.44\u0026ndash;0.68\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L) and 1.08\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L (0.29\u0026ndash;2.25\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L), respectively. In this sequencing analysis, the median depth of coverage was 84x (range: 31\u0026ndash;123) for CD4\u003csup\u003e+\u003c/sup\u003e cells and 136x (range: 114\u0026ndash;205) for CD8\u003csup\u003e+\u003c/sup\u003e cells. The detected variants with VAFs of 20%-60% are shown in Supplemental Table S4 and Supplemental Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. The median number of mutated genes detected was 37 (range: 19\u0026ndash;42) for CD4\u003csup\u003e+\u003c/sup\u003e cells and 31 (range: 4\u0026ndash;52) for CD8\u003csup\u003e+\u003c/sup\u003e cells. None of the mutated genes was shared across samples. We included \u003cem\u003eORAI1\u003c/em\u003e, \u003cem\u003eHIPK4\u003c/em\u003e, \u003cem\u003eMUC1\u003c/em\u003e, and \u003cem\u003eSPAG5\u003c/em\u003e as candidate mutated genes in CD8\u003csup\u003e+\u003c/sup\u003e cells and subjected them to a target sequencing panel. We also added several mutated genes in both samples derived from CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e cells, including \u003cem\u003eTET2\u003c/em\u003e, \u003cem\u003eHCFC1\u003c/em\u003e, and \u003cem\u003eNHS\u003c/em\u003e, for the panel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLandscape of mutations in PRCA\u003c/h2\u003e \u003cp\u003eTo obtain further insight into the genetic profiles of PRCA patients, we examined MNC-derived DNA from patients with PRCA or AA and healthy controls using amplicon sequencing with a custom panel. In this sequencing analysis, the median depth of coverage was 3,368x (range: 1,665-5,454). \u003cem\u003eMUC17\u003c/em\u003e and \u003cem\u003eIGFN1\u003c/em\u003e were omitted from further analyses because of their high false-positive rates. The landscape of gene mutations is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplemental Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e. The detected mutations are summarized in Supplemental Table S5. Fifty-two patients (98%) with PRCA had at least 1 variant out of the 50 genes in the panel. Variants in 31 genes were detected in multiple cases. The top 5 recurrently mutated genes were \u003cem\u003eNEB\u003c/em\u003e (40%), \u003cem\u003eSTAT3\u003c/em\u003e (36%), \u003cem\u003ePCLO\u003c/em\u003e (30%), \u003cem\u003eTET2\u003c/em\u003e (23%), and \u003cem\u003eKMT2D\u003c/em\u003e (15%) (Supplementary Figure S4). Frequent mutated genes in each subtype were as follows: \u003cem\u003ePCLO\u003c/em\u003e (45%), \u003cem\u003eNEB\u003c/em\u003e (36%), and \u003cem\u003eSTAT3\u003c/em\u003e (27%) in idiopathic PRCA; \u003cem\u003ePCLO\u003c/em\u003e (50%), and \u003cem\u003eNEB\u003c/em\u003e (30%) in thymoma-associated PRCA; and \u003cem\u003eSTAT3\u003c/em\u003e (54%), \u003cem\u003eNEB\u003c/em\u003e (42%), \u003cem\u003eTET2\u003c/em\u003e (27%), \u003cem\u003ePCLO\u003c/em\u003e (19%), and \u003cem\u003eBRCA2\u003c/em\u003e (19%) in LGLL-associated PRCA. \u003cem\u003eSTAT3\u003c/em\u003e and \u003cem\u003eTET2\u003c/em\u003e variants were not detected in AA patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe classified \u003cem\u003eTET2\u003c/em\u003e, \u003cem\u003eDNMT3A\u003c/em\u003e, and \u003cem\u003eCUX1\u003c/em\u003e as myeloid CHIP (M-CHIP)-related genes and \u003cem\u003eNEB\u003c/em\u003e, \u003cem\u003ePCLO\u003c/em\u003e, and \u003cem\u003eKMT2D\u003c/em\u003e as lymphoid CHIP (L-CHIP)-related genes according to Niroula et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In PRCA patients, variants of L-CHIP- and M-CHIP-related genes were detected in 62% and 45% of cases, respectively. Conversely, variants of those genes were less frequently detected in AA patients, where the mutation-positive rates of L-CHIP-related genes and M-CHIP-related genes were 30% and 10%, respectively. These results strongly suggest the unique mutational profiles of PRCA.\u003c/p\u003e \u003cp\u003eTET2 \u003cem\u003evariants in PRCA\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eTET2\u003c/em\u003e variants were found in 12 PRCA patients, and the median VAF was 19.6% (range: 1%-51.9%). \u003cem\u003eTET2\u003c/em\u003e variants detected in this cohort are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Supplementary Table S5. Five patients had \u003cem\u003eTET2\u003c/em\u003e variants, with high VAFs of \u0026gt;\u0026thinsp;40%. The median VAF of \u003cem\u003eTET2\u003c/em\u003e variants when excluding those with a VAF\u0026thinsp;\u0026ge;\u0026thinsp;40% was 8.1% (n\u0026thinsp;=\u0026thinsp;8, range: 1.1\u0026ndash;20.9). The breakdown of the patients with \u003cem\u003eTET2\u003c/em\u003e variants was as follows: idiopathic PRCA (n\u0026thinsp;=\u0026thinsp;1), thymoma-associated PRCA (n\u0026thinsp;=\u0026thinsp;1), LGLL-associated PRCA (n\u0026thinsp;=\u0026thinsp;7), and autoimmune disease-associated PRCA (n\u0026thinsp;=\u0026thinsp;3). Among the patients with VAF\u0026thinsp;\u0026gt;\u0026thinsp;40% for \u003cem\u003eTET2\u003c/em\u003e variants, we performed Sanger sequencing of DNA derived from the buccal mucosa in UPN 9, 11, and 35, and all were positive for the corresponding variants, which strongly suggests germline \u003cem\u003eTET2\u003c/em\u003e variants in these three cases. Of the patients with germline \u003cem\u003eTET2\u003c/em\u003e variants, UPN 11 had a parent-child relationship with UPN 20. UPN 9 and 35 did not have any family history of cytopenia or hematological malignancies. \u003cem\u003eTET2\u003c/em\u003e germline variants (F387Y, N813S, and R881W) have not been previously cited with reference to COSMIC (v98, released 2023-MAY-23) or other previously reported germline variants [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Some variants with lower VAFs (C1135Y, R1516X and I1873T) were reported in COSMIC as myeloid malignancies-related mutations; however, none of the \u003cem\u003eTET2\u003c/em\u003e-mutated PRCA patients had dysplastic features in bone marrow cells or abnormal karyotypes. Three patients with \u003cem\u003eTET2\u003c/em\u003e variants (25%) had \u003cem\u003eSTAT3\u003c/em\u003e variant comutations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eTET2-\u003c/em\u003emutated patients had significantly more variants of L-CHIP-related genes than patients without \u003cem\u003eTET2\u003c/em\u003e variants (11/12 vs. 23/41 patients, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.038). None of the 12 patients with \u003cem\u003eTET2\u003c/em\u003e variants developed myeloid malignancy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eVariants of L-CHIP-related genes in PRCA\u003c/h2\u003e \u003cp\u003eVariants of L-CHIP-related genes were also frequently detected in PRCA patients from various backgrounds, and the mutation positivity rates were as follows: \u003cem\u003eNEB\u003c/em\u003e, 40%; \u003cem\u003ePCLO\u003c/em\u003e, 30%; and \u003cem\u003eKMT2D\u003c/em\u003e, 15%. VAFs of each gene often exceeded 40%: 15 of 21 (71%) \u003cem\u003eNEB\u003c/em\u003e variants, 4 of 16 (25%) \u003cem\u003ePCLO\u003c/em\u003e variants, and 4 of 9 (44%) \u003cem\u003eKMT2D\u003c/em\u003e variants, which suggested germline variants or somatic mutations of high VAF. Interestingly, \u003cem\u003eNEB\u003c/em\u003e A4716N variants were found in 4 patients. \u003cem\u003eNEB\u003c/em\u003e A4716N is registered in dbSNP (rs796065338), but its frequencies have not been described. Thirty-three patients (62%) were positive for variants in L-CHIP-related genes. Thirty-six percent of patients with variants of L-CHIP-related genes had \u003cem\u003eSTAT3\u003c/em\u003e variants (12/33), and 30% of them (10/33) had \u003cem\u003eTET2\u003c/em\u003e variants. Variants of L-CHIP-related genes did not differ among PRCA subtypes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eVariants of M-CHIP-related genes in PRCA\u003c/h2\u003e \u003cp\u003e \u003cem\u003eDNMT3A\u003c/em\u003e and CUX1, M-CHIP-related genes besides \u003cem\u003eTET2\u003c/em\u003e, were detected in 9% (5/53) and 13% (7/53) of PRCA, respectively. Two patients with \u003cem\u003eDNMT3A\u003c/em\u003e variants and three with \u003cem\u003eCUX1\u003c/em\u003e variants had high-VAF (\u0026gt;\u0026thinsp;40%) variants. None of the patients with \u003cem\u003eDNMT3A\u003c/em\u003e or \u003cem\u003eCUX1\u003c/em\u003e variants developed myeloid malignancies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDifferences in the clinical characteristics of PRCA patients according to genetic profiles\u003c/h2\u003e \u003cp\u003eWe examined the relationship between clinical characteristics and mutated genes, especially those related to L-CHIP or M-CHIP, including \u003cem\u003eSTAT3\u003c/em\u003e, \u003cem\u003eTET2\u003c/em\u003e, \u003cem\u003eDNMT3A\u003c/em\u003e, \u003cem\u003eCUX1\u003c/em\u003e, \u003cem\u003eNEB\u003c/em\u003e, \u003cem\u003ePCLO\u003c/em\u003e, and \u003cem\u003eKMT2D\u003c/em\u003e. Patients with \u003cem\u003eNEB\u003c/em\u003e variants were significantly younger than those without variants (median: 42 vs. 63 years old, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00074), and patients with \u003cem\u003eDNMT3A\u003c/em\u003e variants were significantly older than those without variants (median: 72 vs. 49 years old, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.034). In addition, \u003cem\u003eTET2\u003c/em\u003e variant (+) patients relapsed after first-line immunosuppressive therapies significantly more frequently than \u003cem\u003eTET2\u003c/em\u003e variant (-) patients (55% [6/11] vs. 11% [4/35], \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0065) (Table\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eThe chronological analysis of recurrently mutated genes\u003c/h2\u003e \u003cp\u003eSerial blood samples from three PRCA patients were available for targeted sequencing (Table\u0026nbsp;3). The first patient with thymoma-associated PRCA (UPN 1) underwent thymectomy and achieved PRCA remission. Variants of \u003cem\u003eTET2\u003c/em\u003e and \u003cem\u003eNEB\u003c/em\u003e were not detected in the second sample after five years, and new variants of \u003cem\u003ePTPN23\u003c/em\u003e, \u003cem\u003ePCLO\u003c/em\u003e, and \u003cem\u003eCARD10\u003c/em\u003e developed. The second patient with autoimmune disorder-associated PRCA (UPN 9) received immunosuppressive therapy and achieved remission, after which he experienced relapse. The \u003cem\u003eTET2\u003c/em\u003e R1516X variant disappeared after therapy; however, \u003cem\u003eTET2\u003c/em\u003e F387Y variant was persistently detected. The third patient with LGLL-associated PRCA (UPN 30) showed refractoriness to CsA and responded to CY. After CY therapy, the \u003cem\u003eTET2\u003c/em\u003e C1193Y variant became undetectable, and the \u003cem\u003eSTAT3\u003c/em\u003e R618R variant was newly detected.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we revealed a detailed mutation profile of PRCA. Notably, \u003cem\u003eTET2\u003c/em\u003e variants were detected in 23% of PRCA patients, regardless of the subtype. Five of these variants had a VAF\u0026thinsp;\u0026ge;\u0026thinsp;40%, 3 cases were confirmed as germline variants, and 1 patient with the N813S variant was also a member of a family with N813S germline variants. Although somatic \u003cem\u003eTET2\u003c/em\u003e variants are frequently associated with myeloid malignancies [\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], T-cell lymphoma [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and CHIP [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], germline \u003cem\u003eTET2\u003c/em\u003e variants have also been reported in various diseases, although rare, including myeloid malignancies [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], lymphoid malignancies [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], immune dysregulation syndromes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and pulmonary arterial hypertension [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Loss of function variants of \u003cem\u003eTET2\u003c/em\u003e lead to DNA hypermethylation, which can cause enhanced inflammation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] or immune dysregulation [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. L\u0026oacute;pez et al. reported that heterozygous \u003cem\u003eTET2\u003c/em\u003e loss-of-function variants could induce increased methylation in CD8\u003csup\u003e+\u003c/sup\u003e cells. Loss of \u003cem\u003eTet2\u003c/em\u003e causes overexpression of IL-1, leading to increased inflammation or leukemogenesis [\u003cspan additionalcitationids=\"CR30 CR31 CR32\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Furthermore, increased IL-1 levels induce clonal expansion of Tet2 +/- hematopoietic cells [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Gene expression and blood concentrations of IL-1β are increased in T-LGLL [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In addition, \u003cem\u003eTet2\u003c/em\u003e-knockout hematopoietic stem and progenitor cells can activate the IL-6/Stat3 pathway in response to inflammatory stress [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In T-LGLL, IL-6 mRNA is highly expressed and contributes to continuous STAT3 activation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Abnormalities in IL-1 or IL-6 signaling may play a role in the pathogenesis of PRCA, including the LGLL-associated type. \u003cem\u003eTET2\u003c/em\u003e variants are thought to be pre-leukemic events, and second-hit variants, including \u003cem\u003eDNMT3A\u003c/em\u003e, \u003cem\u003eSTAT3 KMT2D\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and \u003cem\u003eRHOA\u003c/em\u003e cause lymphomagenesis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In our study, 25% of patients with \u003cem\u003eTET2\u003c/em\u003e variants (3/12) had \u003cem\u003eSTAT3\u003c/em\u003e variants, and 92% (11/12) had variants of L-CHIP-related genes, suggesting that interweaving of these genes might be involved in the pathophysiology of PRCA, although our study is limited by its lack of functional analyses, including DNA methylation and gene expression studies. Although variants of \u003cem\u003eSTAT3\u003c/em\u003e or CHIP-related genes have been previously reported in PRCA [\u003cspan additionalcitationids=\"CR3 CR4 CR5\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], this is the first study to clarify the high frequency of co-mutation of \u003cem\u003eTET2\u003c/em\u003e, \u003cem\u003eSTAT3\u003c/em\u003e and L-CHIP-related genes.\u003c/p\u003e \u003cp\u003eIn previous studies with \u003cem\u003eTET2\u003c/em\u003e mutational analyses in PRCA [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. \u003cem\u003eTET2\u003c/em\u003e variants were detected in 4 of 101 cases (4%). The variants reported by Fujishima et al. were P288fs, G641fs, T759fs, H1904R, and R1465X, and two patients had two variants. Single-nucleotide variants (SNVs) registered in public databases were excluded from previous reports, as were SNVs with frequencies greater than 0.1%. We included SNVs registered in public databases with a frequency\u0026thinsp;\u0026lt;\u0026thinsp;1% in public database in this analysis because the frequencies of \u003cem\u003eTET2\u003c/em\u003e SNVs detected in our study were extremely low (Supplemental Table S6), which ruled out the possibility of SNPs. The detection sensitivity of sequencing methods and patient backgrounds, including causes of PRCA and ethnic composition, might also have influenced the results.\u003c/p\u003e \u003cp\u003eAccording to Niroula et al., the detection rates of L-CHIP-related genes were much lower than those of M-CHIP-related genes in the general population [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In our study, patients with \u003cem\u003eTET2\u003c/em\u003e variants had L-CHIP-related gene variants significantly more frequently than M-CHIP-related gene variants, suggesting a strong relationship between \u003cem\u003eTET2\u003c/em\u003e and L-CHIP-related genes. Originally, L-CHIP-related genes were detected mainly in chronic lymphocytic leukemia, a mature B-cell neoplasm [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In fact, \u003cem\u003eNEB\u003c/em\u003e or \u003cem\u003ePCLO\u003c/em\u003e are not frequently mutated genes in T-cell lymphomas [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In a study on T-LGLL by Cheon et al., mutations in \u003cem\u003eNEB\u003c/em\u003e and \u003cem\u003ePCLO\u003c/em\u003e genes were found in 1/93 (1%) and 4/93 (4%) patients, respectively [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. These results suggest the existence of variants of L-CHIP-related genes in the T cell population. Compared to PRCA, frequently detected variants (\u0026gt;\u0026thinsp;5%) tend to be classified as M-CHIP-related genes in AA [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] or myelodysplastic neoplasms (MDS) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] (Supplemental Table S7). In contrast, \u003cem\u003eKMT2D\u003c/em\u003e is classified as an L-CHIP-related gene, whereas \u003cem\u003eKDM6A\u003c/em\u003e and \u003cem\u003eTET2\u003c/em\u003e are M-CHIP in T-LGLL [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Among M-CHIP-related genes, \u003cem\u003eTET2\u003c/em\u003e variants are most frequently detected in PRCA, whereas \u003cem\u003eDNMT3A\u003c/em\u003e was the most frequently mutated gene in AA [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and age-related CHIP [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. These findings suggest a unique pattern of CHIP in PRCA.\u003c/p\u003e \u003cp\u003eBased on the VAFs of the mutated genes, there might be two evolutionary patterns of mutations in \u003cem\u003eTET2\u003c/em\u003e-mutated PRCA (Supplemental Figure S5). In type 1, high-VAF variants of \u003cem\u003eNEB\u003c/em\u003e, \u003cem\u003ePCLO\u003c/em\u003e, \u003cem\u003eKMT2D\u003c/em\u003e or \u003cem\u003eTET2\u003c/em\u003e occur first, followed by the addition of somatic variants of \u003cem\u003eTET2\u003c/em\u003e are secondly added. In type 2, germline variants of \u003cem\u003eTET2\u003c/em\u003e exist, and clonal development and expansion of somatic variants of other genes occur as late events. Type 1 mutations are recognized in autoimmune-, thymoma-associated, LGLL-associated, and idiopathic PRCA, whereas type 2 mutations are mostly found in LGLL-associated PRCA. \u003cem\u003eTET2\u003c/em\u003e variants have been reported in T-LGLL or NK-LGLL [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The frequency of \u003cem\u003eTET2\u003c/em\u003e variants was 5% in T-LGLL and 28%-34% in NK-LGLL. No hotspot loci in \u003cem\u003eTET2\u003c/em\u003e were detected in either relevant study. Pastoret et al. showed that \u003cem\u003eTET2\u003c/em\u003e variants were found in myeloid and NK cells of three of four patients with NK-LGLL, suggesting \u003cem\u003eTET2\u003c/em\u003e mutations might be an early event in the pathogenesis of NK-LGLL [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This finding suggests a possible mechanism for the type 2 \u003cem\u003eTET2\u003c/em\u003e mutation in PRCA. \u003cem\u003eSTAT3\u003c/em\u003e variants are restricted to CD8\u003csup\u003e+\u003c/sup\u003e cells [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]; however, details of the involved cell lineages and other somatic variants remain unclear. Some \u003cem\u003eTET2\u003c/em\u003e variant-positive clones decreased considerably after immunosuppressive therapy or thymectomy (Table\u0026nbsp;3), suggesting the possibility of the involvement of somatic \u003cem\u003eTET2\u003c/em\u003e variants in T cells. We assumed that the pathophysiology of PRCA includes dysregulation of cellular immunity and impairment of erythropoiesis, which might be partially affected by germline or somatic variants of \u003cem\u003eTET2\u003c/em\u003e.\u003c/p\u003e \u003cp\u003ePRCA and AA share several clinical features, including T-cell abnormalities, efficacy of immunosuppressive treatments, and decreased hematopoietic progenitor cells among bone marrow failure syndromes; however, several differences also exist, such as impaired cell lineage, backgrounds of disease, and frequency of progression to myeloid malignancies. None of the AA patients in our cohort had \u003cem\u003eSTAT3\u003c/em\u003e or \u003cem\u003eTET2\u003c/em\u003e variants. Our sequencing panel did not contain \u003cem\u003ePIGA\u003c/em\u003e, \u003cem\u003eBCOR\u003c/em\u003e or \u003cem\u003eASXL1\u003c/em\u003e, which are frequently mutated in AA; however, they were rarely found in other studies of PRCA [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. A better understanding of the genetic alterations characteristic of PRCA would lead to a better understanding of the mechanisms underlying the development of refractoriness to immunosuppressive therapy in PRCA.\u003c/p\u003e \u003cp\u003eIn conclusion, germline or somatic variants of \u003cem\u003eTET2\u003c/em\u003e and variants of CHIP-related genes were recurrently found in PRCA patients, and mutations in these genes may play important roles in the pathophysiology of PRCA.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e \u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAUTHOR CONTRIBUTIONS\u003c/h2\u003e \u003cp\u003eContribution: T.K. designed the study, performed experiments, and analyzed the data. F.K., S.M., T.Y., Y.M., A.A., D.H., S.M., and Y.H. performed experiments. S.N., H.S., Y.K., and H.N. collected samples and clinical data. F.I. conceived and designed the study, analyzed the data, and supervised the research. T.K., H.N. and F.I. wrote the manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eThe authors thank Dr. Yujiro Ito of Hamamatsu University School of Medicine, Dr. Eiko Oya of Matsusaka Chuo General Hospital, Dr. Atsushi Isoda of Hoshi Clinic, Dr. Toshimitsu Ueki of Nagano Red Cross Hospital, Dr. Toshiro Ito of Matsumoto Medical Center, Dr. Taizo Shimomura of Kumamoto Shinto General Hospital, Dr. Masao Hagihara of Eiju General Hospital, Dr. Yoshiki Akatsuka of Fujita Health University, Dr. Tatsuya Imi and Hiroyuki Takamatsu of Kanazawa University, Dr. Takayuki Takahashi of Shinko Hospital, and Dr. Go Aoki of Komatsu Municipal Hospital for providing the patient data. We also thank Ms. Natsumi Ida and Ms. Masae Maruyama for technical assistance. This research was supported in part by Kaken20K080709 and 21K16302 from a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.\u003c/p\u003e\u003ch2\u003eDATA AVAILABILITY\u003c/h2\u003e \u003cp\u003eSequencing data were deposited in the Japanese Genotype-phenotype Archive (JGA) under Accession Code JGAS000658.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMeans RT, Jr. Pure red cell aplasia. Blood 2016; 128: 2504\u0026ndash;2509.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKawakami T, Sekiguchi N, Kobayashi J, Imi T, Matsuda K, Yamane T, \u003cem\u003eet al.\u003c/em\u003e Frequent STAT3 mutations in CD8(+) T cells from patients with pure red cell aplasia. 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Blood 2021; 137: 3237\u0026ndash;3250.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1-3 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"TET2, clonal hematopoiesis, pure red cell aplasia, bone marrow failure","lastPublishedDoi":"10.21203/rs.3.rs-3834690/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3834690/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDysregulation of T cell-mediated immunity is considered a major pathophysiological mechanism of acquired pure red cell aplasia (PRCA), such as idiopathic PRCA, large granular lymphocytic leukemia-associated PRCA, and thymoma-associated PRCA. Although \u003cem\u003eSTAT3\u003c/em\u003e mutations are frequently detected in PRCA patients, other mutational profiles and their involvement in the clinical characteristics are yet to be clarified. Whole-exome sequencing and targeted sequencing were performed using a custom-designed panel for PRCA (n\u0026thinsp;=\u0026thinsp;53). The frequently mutated genes were \u003cem\u003eNEB\u003c/em\u003e (40%), \u003cem\u003eSTAT3\u003c/em\u003e (36%), \u003cem\u003ePCLO\u003c/em\u003e (30%), \u003cem\u003eTET2\u003c/em\u003e (23%), and \u003cem\u003eKMT2D\u003c/em\u003e (15%). Four of the 12 patients with mutations in \u003cem\u003eTET2\u003c/em\u003e had germline \u003cem\u003eTET2\u003c/em\u003e variants. Patients positive for \u003cem\u003eTET2\u003c/em\u003e variants had significantly more variants of lymphoid clonal hematopoiesis-related genes than those without \u003cem\u003eTET2\u003c/em\u003e variants (11/12 vs. 23/41, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.038). Patients with \u003cem\u003eTET2\u003c/em\u003e variants relapsed after immunosuppressive therapy more frequently than those without \u003cem\u003eTET2\u003c/em\u003e variant (55% [6/11] vs. 11% [4/35], \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0065). These data suggest that variants of clonal hematopoiesis-related genes, including \u003cem\u003eTET2\u003c/em\u003e, in addition to \u003cem\u003eSTAT3\u003c/em\u003e, play important roles in the pathophysiology of PRCA.\u003c/p\u003e","manuscriptTitle":"TET2 and clonal hematopoiesis-related gene variants in patients with acquired pure red cell aplasia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-08 19:35:43","doi":"10.21203/rs.3.rs-3834690/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"464632dd-0808-4382-88c3-6c38cc8825f3","owner":[],"postedDate":"January 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":27975281,"name":"Biological sciences/Genetics/Clinical genetics/Disease genetics"},{"id":27975282,"name":"Biological sciences/Immunology/Immunological disorders/Lymphoproliferative disorders"}],"tags":[],"updatedAt":"2024-01-29T14:22:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-08 19:35:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3834690","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3834690","identity":"rs-3834690","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}