N6-methyladenosine (m 6 A) is the most abundant internal modification in mammalian mRNA and recent studies have highlighted the importance of m 6 A levels in tumor development. In this study, we investigated the expression of methyltransferase-like 3 (METTL3) and 14 (METTL14), components of the RNA m 6 A methyltransferase complex, in samples from 89 patients with acute myeloid leukemia (AML), and followed the survival of 75 of these patients. Our results show that METTL3 and METTL14 are highly expressed in most of the patients with AML (except those with APL), and high levels of METTL3 and/or METTL14 correlated to shorter survival in the patients. In leukemia cell lines K562 and kasumi-1, both METTL3 and METTL14 promote cell proliferation and cell cycle, and the knockdown of METTL3 and METTL14 inhibits proliferation, and induces apoptosis and differentiation. Notably, the knockdown of METTL3 and METTL14 in K562 cell line leads to several changes in the expression of p53 signal pathway, including the upregulation of p53, cyclin dependent kinase inhibitor 1A (CDKN1A/p21), and downregulation of mdm2. Importantly, the m 6 A level of mdm2 mRNA was significant lower after knock-down of METTL3 and METTL14 examined by m 6 A-RIP and mdm2 qPCR assay, and the half-life of mdm2 under actinomycin-D treatment became shorter. Taken together, our study demonstrates that the lower m 6 A levels of mdm2 mRNA mediated by the knockdown of METTL3 and METTL14 could lead to the low stability of mdm2 mRNA transcripts and low expression of MDM2, in the end, activate p53 signal pathway. Both METTL3 and METTL14 play an oncogenic role in AML by targeting mdm2/p53 signal pathway.

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Background Increasing germline gene mutations have been discovered in haematological malignancies with the development of next-generation sequencing (NGS), which is critical for proper clinical management and long-term follow-up of affected individuals. Tet methylcytosine dioxygenase 2 (TET2) is one of the most common mutations in haematological neoplasms. We aimed to compare the clinical characteristics of patients with germline and somatic TET2 mutations in haematological diseases and to analyse whether germline TET2 mutations have a family aggregation and tumour predisposition. Methods Out of 612 patients who underwent NGS of 34 recurrently mutated genes in haematological diseases, 100 haematological patients with TET2 mutations were selected for further study. Somatic mutations were detected by NGS in bone marrow/peripheral blood genomic DNA (gDNA). Germline TET2 mutations were validated in nail/hair gDNA by Sanger sequencing. Digital data were extracted from the haematology department of the West China Hospital of Sichuan University. TET2 mutation results were analysed by referencing online public databases (COSMIC and ClinVar). Results One hundred patients were studied, including 33 patients with germline and 67 patients with somatic TET2 mutations. For germline TET2 mutations, the variant allele frequency (VAF) was more stable (50.58% [40.5–55], P < 0.0001), and mutation sites recurrently occurred in three sites, unlike somatic TET2 mutations. Patients with germline TET2 mutations were younger (median age 48, 16–82 years) (P = 0.0058) and mainly suffered from myelodysplastic syndromes (MDS) (n = 13, 39.4%), while patients with somatic TET2 mutations were mainly affected by acute myeloid leukemia (AML) (n = 26, 38.8%) (P = 0.0004). Germline TET2 mutation affected the distribution of cell counts in the peripheral blood and bone marrow (P < 0.05); it was a poor prognostic factor for MDS patients via univariate analysis (HR = 5.3, 95% CI: 0.89–32.2, P = 0.0209) but not in multivariate analysis using the Cox regression model (P = 0.062). Conclusions Germline TET2 mutation might have a family aggregation, and TET2 may be a predisposition gene for haematological malignancy under the other gene mutations as the second hit. Germline TET2 mutation may play a role in the proportion of blood and bone marrow cells and, most importantly, may be an adverse factor for MDS patients.

Objective: To analyse germ line and acquired somatic mutations of 30 high frequency genes and explore their clinical significance in hematologic malignancies. Methods: A total of 254 patients with hematologic diseases (145 males and 109 females) from Jan 2017 to Apr 2018 in the Southwest China were included and the samples from bone marrow and oral mucosa were collected, and the gene mutations of germ line and acquired somatic cells were examined by the next-generation sequencing technology or PCR. The frequency and locus of germ line and acquired somatic mutations of 30 high frequency genes, as well as their clinical significance to the diagnosis and classification in hematologic diseases were analysed. Results: 1.The frequency and locus of germ line and acquired somatic mutations, and their clinical significance: no mutation was detected in 24 cases (9.4%), and a total of 659 genetic locus mutations were detected in 230 cases (90.6%), including 520 somatic cell mutations (78.9%) and 139 germ line mutations (21.1%). The results of gene mutations are listed in order: the number of acquired somatic mutation: the number of germ line mutation: common mutation locus: the type of hematological disease for the patients diagnosed in the West China hospital, as described in Table 1. 2.Characteristics of acquired somatic and germ line mutations: the genes of WT1, U2AF1, TP53, TET2, STAG2, RUNX1, PIGA, NRAS, KIT, IDH2, EZH2, DNMT3A, CBL and BCOR were dominated by acquired somatic mutations, while the genes of ZRSR2, JAK2 and BCORL1 were dominated by germ line mutations, the genes of SRSF2, SF3B1, SETBP1, PTPN11, NPM1, PHF6, KRAS, IDH1, FLT3-ITD, ETV6 and CEBPA were detected only in acquired somatic mutations, BCOR and ASXL1 have a similar proportion of acquired somatic and germ line mutation; only 42-4C>A of ZRSR2 mutation was existed in both somatic and germ line, however, the mutation sites of the acquired somatic and germ line of other genes have not been found to be existed at the same time. 3.The clinical significance of the acquired somatic and germ line mutation: It was found that the genes of TP53, SF3B1, EZH2 and DNMT3A tend to appear in MDS, the genes of ZRSR2, WT1, U2AF1, TET2, RUNX1, NRAS, NPM1, KRAS, KIT, IDH1/2, FLT3-ITD, CEBPA and BCOR tend to appear in AML, and the similar probability of the STAG2, PTPN11, PHF6, ETV6, CBL, BCORL1, ASXL1 and NF1 genes appear in AML and MDS, while the genes of PIGA tend to appear in PNH, and BCOR in AA. Conclusion: The acquired somatic and germ line mutations are significantly different in the frequency, mutation site and the type of hematological disease, and the further research will clarify their potential roles in the pathogenesis of hematological tumors, and have important clinical significance for the classification and prognosis of the hematological diseases. Key words: gene mutation, somatic, germ line, hematologic malignancies Table 1. Table 1. Disclosures No relevant conflicts of interest to declare.

Background: More and more germline gene mutations have been discovered in hematological malignances with the development of next gene sequencing (NGS). Tet methylcytosine dioxygenase 2 (TET2) is one of the most common mutation genes in hematological neoplasms. We aimed to analyzed whether germline TET2 mutation has a family aggregation or is a tumor predisposition gene. Further we compared its impact with somatic TET2 mutation in hematological diseases.Methods: A total cohort of 103 hematological patients with TET2 mutation were included from December 2016 to December 2019. Data were extracted from hematology department of West China Hospital of Sichuan University. Bone marrow (BM) or peripheral blood (PB) as somatic DNA origin to be detected by next-generation sequencing (NGS), and nails and hairs as germline DNA origin to be detected by Sanger sequencing, respectively. Further, we compared the clinical characteristics between the patients with germline and somatic TET2 mutation.Results: 103 patients were included, including 33 (32.03%) patients with germline TET2 mutation and 70 (67.97%) patients with somatic TET2 mutation. Variant allele frequency (VAF) of germline TET2 mutation was more stable in our study, ranging from 40% to 55% and mutation sites were more concentrated. Patients with germline TET2 mutation were younger with median age 48 (range, 16-82) (P=0.0078). Further, patients with germline TET2 mutation were mainly myelodysplastic syndromes (MDS) (n=13, 39.4%), while patients with somatic TET2 mutation were acute myeloid leukemia (AML) (n=28, 40.0%) (P=0.0003). Germline TET2 mutation affected the distribution of peripheral blood cell count and the proportion in bone marrow (P<0.05). Germline TET2 mutation was a poor prognosis factor in MDS patients via univariate analysis (HR=5.3, 95%CI: 0.89-32.2, P=0.0209), but not in multivariate analysis by Cox regression model (P=0.062).Conclusions: Some family members were asymptomatic carriers, which indicated germline TET2 mutation might have a family aggregation. More importantly, TET2 gene may be a predisposition gene of hematological malignant when the other gene mutations as the second hit. The VAF of germline TET2 mutation is more stable. At the same time, the germline TET2 mutation may be an adverse factor for the MDS patients.