A new form of familial platelet disorder caused by germline mutations in RUNX1 in a pedigree / 中华内科杂志

Objective: To investigate the clinical and biological characteristics of familial platelet disorder (FPD) with germline Runt-related transcription factor (RUNX) 1 mutations. Methods: Patients diagnosed with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) with RUNX1 mutations from February 2016 to December 2021 in Wuhan No.1 Hospital underwent pedigree analysis and were screened for gene mutations (somatic and germline). Patients diagnosed with FPD with germline RUNX1 mutations were enrolled and evaluated in terms of clinical characteristics and biological evolution. Bioinformatics analysis was used to assess the pathogenicity of mutations and to analyze the effect of mutated genes on the function of the corresponding protein. Results: Germline RUNX1 mutations were detected in three out of 34 patients suffering from MDS/AML who had RUNX1 mutations. A pedigree of FPD with RUNX1 (RUNX1-FPD) c.562A>C and RUNX1 c.1415T>C mutations was diagnosed, and the mutations were of patrilineal origin. Bioinformatics analysis indicated that the locus at positions 188 and 472 in the AML-1G type of RUNX1 was highly conserved across different species, and that variations might influence functions of the proteins. The mutations were evaluated to be highly pathogenic. Of the nine cases with germline RUNX1 mutations: two patients died due AML progression; one case with AML survived without leukemia after transplantation of hemopoietic stem cells; four patients showed mild-to-moderate thrombocytopenia; two cases had no thrombocytopenia. During the disease course of the proband and her son, mutations in RUNX1, NRAS and/or CEBPA and KIT appeared in succession, and expression of cluster of differentiation-7 on tumor cells was enhanced gradually. None of the gene mutations correlated with the tumor were detected in the four cases not suffering from MDS/AML, and they survived until the end of follow-up. Conclusions: RUNX1-FPD was rare. The mutations c.562A>C and c.1415T>C of RUNX1 could be the disease-causing genes for the family with RUNX1-FPD, and these mutations could promote malignant transformation. Biological monitoring should be carried out regularly to aid early intervention for family members with RUNX1-FPD.

Establishment of leukemia cell model with inducible AML1-ETO expression and its effect on fatty acid metabolism in leukemia cells / 中华血液学杂志

Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.

Analysis of Coexisting Gene with NRAS in Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the coexisting mutations and clinical significance of Homo sapiens neuroblastoma RAS viral oncogene homolog (NRAS) gene in acute myeloid leukemia (AML) patients.@*METHODS@#High-throughput DNA sequencing and Sanger sequencing were used to detect 51 gene mutations. The occurrence, clinical characteristics and treatment efficacy of coexisting genes with NRAS were investigated.@*RESULTS@#A total of 57 NRAS mutations (17.5%) were detected in 326 patients with AML. Compared with the patients in NRAS non-mutation group, patients in the mutant group were younger (P=0.018) and showed lower platelet count (P=0.033), but there was no significant difference in peripheral leukocyte count, hemoglobin, and sex. For FAB classification, NRAS mutation and M2 subtype showed mutually exclusive (P=0.038). Among 57 patients carried with NRAS mutation, 51 (89.5%) patients carried with other gene mutations, 25 (43.9%) carried with double gene mutations, 10 (17.5%) carried with 3 gene mutations, and 16 (28.1%) corried with ≥ 4 gene mutations. The most common coexisting gene mutation was KRAS (24.6%, 14/57), followed by FLT3-ITD (14.0%, 8/57), RUNX1 (12.3%, 7/57), NPM1 (10.5%, 6/57), PTPN11 (10.5%, 6/57), DNMT3A (10.5%, 6/57) and so on. The age (P=0.013, P=0.005) and peripheral platelet count (P=0.007, P=0.021) of patients with NPM1 or DNMT3A mutations were higher than those of the patients with wild type, but there was no significant difference in peripheral leukocyte count and hemoglobin. Also, there was no significant difference in age, peripheral leukocyte count, hemoglobin, and peripheral platelet count between the patients in KRAS, FLT3-ITD, RUNX1 or PTPN11 mutant group and the wild group. Patients with FLT3-ITD mutations showed a lower complete remission (CR) rate (P=0.044). However, there was no significant difference in CR rate between the patients with KRAS, NPM1, RUNX1, PTPN11 or DNMT3A mutations and the wild group. The CR rate of the patents with single gene mutation, double gene mutations, 3 gene mutations, and≥ 4 gene mutations were decreased gradually, and there was no significant difference in CR rate between pairwise comparisons.@*CONCLUSION@#The mutation rate of NRAS mutation is 17.5%, 89.5% of AML patients with NRAS mutation coexist with additional gene mutations. The type of coexisting mutations has a certain impact on clinical characteristics and CR rate of patients with AML.

The role of RUNX1 in the apoptosis of epithelial cells in nasal polyps / 中华耳鼻咽喉头颈外科杂志

Objective: To explore the expression of Runt-related transcription factor 1 (RUNX1) in nasal polyps (NPs) tissues and the potential role on apoptosis of primary human nasal epithelial cells (pHNECs) in NPs. Methods: The expression level of RUNX1 in NPs tissues was determined by Western blot (WB) and immunohistochemical staining (IHC). In vitro, TNF-α (20 ng/ml) was used to stimulate pHNECs to establish the apoptosis injury model. Hoechst staining was performed to observe pHNECs apoptosis by kit. Subsequently, quantitative real-time PCR (qRT-PCR) and WB were utilized to detect the expression of apoptosis-related proteins B-cell lymphoma-2 (BCL-2), BCL2-associated X (BAX) and cysteinyl aspartate specific proteinase-3 (Caspase-3) to assess the level of apoptosis. The plasmid of sh-RUNX1-6 was transfected into the pHNECs apoptosis model, then the effect of RUNX1 silence on apoptosis was evaluated by WB and flow cytometry. Statistical analysis was performed by the SPSS 19.0 and GraphPad Prism5 software. Results: The expression of RUNX1 in NPs tissue was significantly higher than that in inferior turbinates, and the difference was statistically significant (0.274±0.042 vs 0.110±0.027, t=9.675, P<0.05). Compared with the inferior turbinates, BAX and Caspase-3 expressions were increased whereas BCL-2 was decreased in NPs, and the differences were statistically significant (BAX 0.346±0.032 vs 0.302±0.037, Caspase-3 0.228±0.061 vs 0.158±0.065, BCL-2 0.090±0.047 vs 0.276±0.057, t value was 2.680, 2.361 and 7.575, respectively, all P<0.05). The expression levels of RUNX1 and apoptosis in pHNECs increased in a time-dependent manner after TNF-α exposure (P<0.05). Plasmid of sh-RUNX1-6 transfected silenced the expression of RUNX1 in pHNECs treated by TNF-α. After silencing RUNX1 in pHNECs apoptosis model, the protein levels of BAX and Caspase-3 were decreased, while the expression of BCL-2 was increased, the rate of apoptosis was decreased (P<0.05). Conclusions: RUNX1 is increased in NPs. Silencing RUNX1 can inhibit the apoptosis and reduce cell inflammatory damage of pHNECs induced by TNF-α.

Clinical Characteristics of Acute Myeloid Leukemia Patients with RUNX1 Gene Mutation / 中国实验血液学杂志

OBJECTIVE@#To investigate the incidence of Runt-related transcription factor 1 (RUNX1) gene and its associated gene mutations in patients with acute myeloid leukemia (AML), and analyze its clinical characteristics and prognosis.@*METHODS@#The genomic DNA-PCR method was used to detect the exon of RUNX1 gene, and the gene mutations were analyzed by genetic sequencing. NPM1, DNMT3A, FLT3-ITD, IDH1/2, K/N-RAS, CEPBA, TET2, and WT1 co-mutations were also detected. Patients were followed up to determine efficacy and prognosis.@*RESULTS@#Among 171 patients, the RUNX1 gene mutation was detected in 17 cases, and the mutation rate was 9.9%. The type of RUNX1 gene mutation was 9 missense mutations, 4 frameshift mutations, and 4 nonsense mutations. The peripheral blood leukocyte count of the patients in mutation group was 3 (1-101) ×10@*CONCLUSION@#AML patients with RUNX1 gene mutation shows unique clinical and biological characteristics, RUNX1 mutation can be regarded as a molecular marker of poor prognosis in AML patients.

RUNX1 gene mutations are associated with adverse prognosis of patients with acute myeloidleukemia / 南方医科大学学报

OBJECTIVE@#To explore the rate and distribution of Runt- related transcription factor 1 (RUNX1) gene mutations in patients with acute myeloid leukemia (AML) and the correlation of these mutations with the clinical characteristics and survival outcomes of the patients.@*METHODS@#The genomic DNA extracted from the bone marrow of 158 patients with newly diagnosed AML for PCR amplification of RUNX1 gene and sequence analysis to identify the mutations. The mutations of ASXL1, DNMT3A, TET2, FLT3, CEBPA, NPM1, IDH2, NRAS and c-KIT genes were also examined to analyze their association with RUNX1 gene mutations.@*RESULTS@#Among the 158 AML patients, 19 (12.0%) were found to have RUNX1 mutations in A166G (9 cases), A142T (6 cases) and A162L (4 cases). RUNX1 mutations were more frequent in elderly patients (@*CONCLUSIONS@#RUNX1 gene mutations are associated with an adverse prognosis of patients with AML.

A Case of Chronic Myeloid Leukemia With Rare Variant ETV6/ABL1 Rearrangement

No abstract available.

A Novel KIT INDEL Mutation in Acute Myeloid Leukemia With t(8;21)(q22;q22); RUNX1-RUNX1T1

No abstract available.

Frequency and Clinical Characteristics of Intrachromosomal Amplification of Chromosome 21 in Korean Childhood B-lineage Acute Lymphoblastic Leukemia

BACKGROUND: Intrachromosomal amplification of chromosome 21 (iAMP21) is known to be associated with poor prognosis in B-cell ALL (B-ALL). To determine the frequency and clinical characteristics of iAMP21 in Korean B-ALL patients, we performed FISH and multiplex ligation-dependent probe amplification (MLPA) analyses. METHODS: A total of 102 childhood B-ALL patients were screened with ETV6-RUNX1 FISH probes (Abbott Molecular, USA). The presence of an iAMP21 was confirmed by using MLPA P327 iAMP21-ERG probemix (MRC Holland, The Netherlands). RESULTS: iAMP21 was detected in one of the screened B-ALL patients (1/102 patients, 1.0%) who presented the ALL immunophenotype and complex karyotype at initial diagnosis. The patient relapsed twice after bone marrow transplantation. MLPA showed 12.5-Mb and 4.28-Mb regions of amplification and deletion, respectively. CONCLUSIONS: The frequency of iAMP21 is considerable in Korean pediatric patients. Our report suggests that iAMP21 in childhood B-ALL has very unfavorable impact on patient's prognosis. Additional methods such as MLPA analysis is essential to rule out patients with equivocal interphase FISH results.

Diagnostic Utility of Multiprobe Fluorescence in situ Hybridization Assay for Detecting Cytogenetic Aberrations in Acute Leukemia

BACKGROUND: Specific cytogenetic aberrations detected by conventional karyotyping or FISH play a major role in the diagnosis, prognosis, and treatment of patients with acute leukemia. The FISH technique enhances the capacity of conventional karyotyping to detect subtle chromosomal aberrations. Multiprobe FISH assay (Cytocell, UK) can hybridize multiple probes to a single slide, thereby increasing the detection rate of cytogenetic aberrations. This study aimed to evaluate multiprobe FISH in detecting cytogenetic abnormalities in acute leukemia. METHODS: Thirty newly diagnosed acute leukemia patients who attended the hematology clinic at Dong-A University Hospital from October 2008 to October 2012 were enrolled in the study. The multiprobe FISH results were compared with those of G-banding. RESULTS: Multiprobe FISH detected the chromosomal aberrations identified by G-banding, as well as additional aberrations in 6 of 30 (20.0%) cases, which included ETV6/RUNX1 translocation, p16 deletion, TP53 deletion, and IGH break-apart. CONCLUSIONS: The multiprobe FISH assay was a more sensitive and reliable technique compared with G-banding. It was also more cost-effective and yielded faster results.

Spectra of Chromosomal Aberrations in 325 Leukemia Patients and Implications for the Development of New Molecular Detection Systems

This study investigated the spectrum of chromosomal abnormalities in 325 leukemia patients and developed optimal profiles of leukemic fusion genes for multiplex RT-PCR. We prospectively analyzed blood and bone marrow specimens of patients with acute leukemia. Twenty types of chromosomal abnormalities were detected in 42% from all patients by commercially available multiplex RT-PCR for detecting 28 fusion genes and in 35% by cytogenetic analysis including FISH analysis. The most common cytogenetic aberrations in acute myeloid leukemia patients was PML/PARA, followed by AML1/MGT8 and MLL1, and in acute lymphoid leukemia patients was BCR/ABL, followed by TEL/AML1 and MLL1 gene rearrangement. Among the negative results for multiplex RT-PCR, clinically significant t(3;3)(q21;q26.2), t(8;14)(q24;q32) and i(17)(q10) were detected by conventional cytogenetics. The spectrum and frequency of chromosomal abnormalities in our leukemia patients are differed from previous studies, and may offer optimal profiles of leukemic fusion genes for the development of new molecular detection systems.

Prognostic Significance of TEL/AML1 Rearrangement and Its Additional Genetic Changes in Korean Childhood Precursor B-Acute Lymphoblastic Leukemia / 대한진단검사의학회지

BACKGROUND: TEL (ETV6)/AML1 (RUNX1) rearrangement is observed in approximately 20-25% of childhood precursor B-ALL and is associated with a favorable outcome. Additional genetic changes, associated with TEL/AML1, are frequently found. We evaluated the prevalence and prognostic significance of TEL/AML1 rearrangement and additional genetic changes in the TEL and AML1 genes in Korean childhood precursor B-ALL. METHODS: We performed FISH using LSITEL/AML1 ES probe (Vysis, USA) in 123 children diagnosed as having precursor B-ALL and assessed clinical relevance of the TEL/AML1 rearrangement and additional genetic abnormalities. RESULTS: The frequency of TEL/AML1 was 17.1% (21/123) in patients with precursor B-ALL. TEL/ AML1-positive group showed male predominance (P=0.012) and younger age of onset than TEL/ AML1-negative group by 1.6 yr (P=0.013). The outcome of TEL/AML1-positive group tended to show lower incidences of relapse (1/21 vs 20/102), death (1/21 vs 17/102) and longer event free survival. Among TEL/AML1-positive patients, unrearranged TEL deletion, AML1 gain, and unrearranged TEL deletion combined with AML1 gain were detected in 61.9%, 23.8%, and 9.5%, respectively. There were no significant differences in the clinical features and outcome according to the presence or absence of additional genetic changes. CONCLUSIONS: The frequency of TEL/AML1 and additional genetic changes in TEL and AML1 is higher than previous studies in Korean children, and in close agreement with usually reported one, 20-25%. TEL/AML1-positive group showed a tendency toward better prognosis. Further study is needed to clarify the prognostic significance of additional changes in TEL and AML1 based on a large sample size.

Clinical Utility of Fluorescence in-situ Hybridization Profile Test in Detecting Genetic Aberrations in Acute Leukemia / 대한진단검사의학회지

BACKGROUND: Cytogenetic abnormalities are one of the most reliable prognostic factors in acute leukemia. Combination of conventional chromosome analysis (CCA) and FISH provides higher sensitivity in detecting these genetic abnormalities, and it is effective to apply several FISH probes as a profile test. The objective of this study was to investigate the utility of FISH profile analyses in the initial diagnosis of acute leukemia. METHODS: Two hundred and forty one de novo acute leukemia patients diagnosed from January, 2002 to November, 2007 were included. For acute lymphoblastic leukemia profile test, FISH probes for BCR/ABL, TEL/AML1, MLL gene rearrangement and CDKN2A deletion were used. For acute myeloid leukemia profile test, probes for AML1/ETO, MLL and CBFbeta gene rearrangement were used. The results of CCA and FISH profile tests were collected, and the positive rates were compared. RESULTS: ALL FISH profile tests revealed additional genetic aberrations not detected by chromosome analysis in 48.6% (67/138) of cases, including those with normal karyotypes or no mitotic cells (37%, 51/138). Among these 51 cases, TEL/AML1 abnormalities were detected in 44.3%, followed by the abnormal CDKN2A signal (24.6%) and hyperdiploidy (18.0%). AML FISH profile tests revealed additional genetic abnormalities in 7.8% (8/103) of cases. CONCLUSIONS: FISH analysis as a profile test detected additional genetic aberrations in a significant proportion of acute leukemia, and was effective especially in detecting cryptic translocations, submicroscopic deletions and complex karyotypes. Our study supports the need to incorporate FISH profile test at initial work up in acute leukemia.

Near-tetraploidy Acute Myeloid Leukemia with RUNX1-RUNX1T1 Rearrangement Due to Cryptic t(8;21) / 대한진단검사의학회지

Tetraploidy or near-tetraploidy is a rare cytogenetic abnormality found in AML, and is divided into primary and secondary forms. The secondary tetraploidy or near-tetraploidy found in AML is known to be specifically associated with t(8;21). In this case report, FISH analysis detected RUNX1-RUNX1T1 gene rearrangement in the absence of cytogenetic abnormality of t(8;21), which suggests the presence of unvailed t(8;21). This is the first case report of tetraploidy or near-tetraploidy AML with cryptic RUNX1/RUNX1T1 in Korea. Although the prognosis of tetraploidy or near- tetraploidy with t(8;21) is known to be poor, this patient shows a relatively good clinical course compared to other reported cases.

AML1-ETO positive AML: first report from India.

Translocation (8;21) is associated with few typical morphological features and favorable prognosis. All patients of AML and MDS with increased blasts (N = 35) according to FAB criteria, presenting (between Jan 2004 to June 2005) to the Department of Hematology, AIIMS were studied. RT-PCR was done for the AML1-ETO fusion transcript in all cases. Overall incidence of AML1-ETO was 28.57% and no correlation was found between AML1-ETO positivity and clinical or hematological parameters except for a direct correlation with absolute blast count (ABC) (a lower ABC in the AML1-ETO positive cases). Interestingly, 1/3 MDS cases were positive for the same fusion transcript and thus, it appears worthwhile to look for AML1-ETO in all cases of MDS with increased blasts. Objective morphological evaluation using a scoring system based on morphological features was not helpful in predicting positivity for AML1-ETO. The effect of this translocation on long-term survival could not be determined by the present study.

Putative association of RUNX1 polymorphisms with IgE levels in a Korean population

RUNX1, a member of the runt domain gene family of transcription factors, encodes a heterodimeric transcription factor and regulates the expression of various genes related to hematopoiesis and myeloid differentiation. RUNX1 has been one of the target genes for research into various autoimmune diseases due to its properties as a transcription factor and functional distribution for chromosomal translocation. In an effort to identify additional gene polymorphisms in which variants have been implicated in asthma, we investigated the genetic polymorphisms in RUNX1 to evaluate it as a potential candidate gene for a host genetic study of asthma and IgE production. We identified 19 sequence variants by direct DNA sequencing in 24 individuals of which four common variants were selected for genotyping in our asthma cohort (1,055 asthmatic patients, 384 normal controls). Using logistic regression analysis for association with the risk of asthma, while controlling for age, gender, and smoking status as covariates, no significant associations with the risk of asthma were detected. However, two polymorphisms in the promoter region (-2084G>C and -1282G>A) showed a marginal association with total IgE levels (0.03 and 0.03 in recessive models, respectively). Our findings suggest that polymorphisms in RUNX1 might be one of the genetic factors for the regulation of IgE production.