Objective:To explore the clinical features and molecular characteristics of myeloproliferative neoplasms (MPNs) patients with NFE2 gene mutations.Methods:Gene targeted sequencing was used to detect NFE2 gene mutation in 723 patients diagnosed with MPNs who were admitted to Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College between April 2021 and June 2023. The association between NFE2 gene mutations and clinical features and molecular characteristics of MPNs patients were retrospectively analyzed.Results:Among 723 patients with MPNs, NFE2 gene mutations were found in 41 cases (5.7%) . NFE2 gene mutations were predominantly frameshift mutations (44.4%) , followed by nonsense mutations (33.3%) . The median number of mutations in patients with NFE2 gene mutations (4 [2,5]) was higher compared to the group without NFE2 gene mutations (2, [1,3]) ( P<0.001) . NFE2 gene mutations frequently co-occurred with mutations in MPL, ATM, PPM1D, and TET1. NFE2 gene mutations were mostly sub-clonal events, with 80.5% occurring after MPNs driver mutations (JAK2, CALR, or MPL) . NFE2 mutations were correlated with older age [median age: 60 (54, 67) years vs 54 (41, 63) years, P=0.001]. Patients with NFE2 gene mutations had a higher incidence of pre-diagnosis thrombosis (39.0% vs 22.0%, P=0.012) and pre-diagnosis arterial thrombosis (36.6% vs 20.4%, P=0.014) . Using a logistic regression analysis model adjusting for age and comorbidities (including chronic infections, malignancies, and autoimmune diseases) , NFE2 gene mutation was identified as an independent determinant of elevated tumor necrosis factor-alpha (TNF-α) ( OR=2.747, 95% CI: 1.143-6.605, P=0.024) , interferon-gamma (IFN-γ) ( OR=2.689, 95% CI: 1.191-6.076, P=0.017) , IL-10 ( OR=3.219, 95% CI: 1.343-7.717, P=0.009) , IL-12P70 ( OR=3.397, 95% CI:1.003-11.508, P=0.049) , IL-17 ( OR=2.284, 95% CI: 1.017-5.127, P=0.045) . In polycythaemia vera (PV) patients with the NFE2 gene mutation, the proportion of those classified as high-risk is notably higher in both the IWG-PV and mutation-enhanced international prognostic systems for PV (MIPSS-PV) (66.7% vs 25.3% for IWG-PV, P=0.033; 22.2% vs 2.0% for MIPSS-PV, P=0.013) . Similarly, for essential thrombocythaemia (ET) patients, the proportion in the high-risk group of the mutation-enhanced international prognostic systems for ET (MIPSS-ET) is significantly higher (15.4% vs 6.1%, P=0.021) . No statistically significant differences were observed in overall survival or cumulative incidence of thrombosis between NFE2-mutated (38 cases) and non-mutated MPNs patients (671 cases, P>0.05) . Conclusion:NFE2 gene mutations in MPNs were predominantly frameshift mutations. NFE2 gene mutations were correlated with older age, elevated levels of several inflammatory factors (including TNF-α、IFN-γ、IL-10、IL-12P70、IL-17) , and they mostly occurred in late-stage of MPNs.

Objective:To explore the clinical features and molecular characteristics of myeloproliferative neoplasms (MPNs) patients with NFE2 gene mutations.Methods:Gene targeted sequencing was used to detect NFE2 gene mutation in 723 patients diagnosed with MPNs who were admitted to Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College between April 2021 and June 2023. The association between NFE2 gene mutations and clinical features and molecular characteristics of MPNs patients were retrospectively analyzed.Results:Among 723 patients with MPNs, NFE2 gene mutations were found in 41 cases (5.7%) . NFE2 gene mutations were predominantly frameshift mutations (44.4%) , followed by nonsense mutations (33.3%) . The median number of mutations in patients with NFE2 gene mutations (4 [2,5]) was higher compared to the group without NFE2 gene mutations (2, [1,3]) ( P<0.001) . NFE2 gene mutations frequently co-occurred with mutations in MPL, ATM, PPM1D, and TET1. NFE2 gene mutations were mostly sub-clonal events, with 80.5% occurring after MPNs driver mutations (JAK2, CALR, or MPL) . NFE2 mutations were correlated with older age [median age: 60 (54, 67) years vs 54 (41, 63) years, P=0.001]. Patients with NFE2 gene mutations had a higher incidence of pre-diagnosis thrombosis (39.0% vs 22.0%, P=0.012) and pre-diagnosis arterial thrombosis (36.6% vs 20.4%, P=0.014) . Using a logistic regression analysis model adjusting for age and comorbidities (including chronic infections, malignancies, and autoimmune diseases) , NFE2 gene mutation was identified as an independent determinant of elevated tumor necrosis factor-alpha (TNF-α) ( OR=2.747, 95% CI: 1.143-6.605, P=0.024) , interferon-gamma (IFN-γ) ( OR=2.689, 95% CI: 1.191-6.076, P=0.017) , IL-10 ( OR=3.219, 95% CI: 1.343-7.717, P=0.009) , IL-12P70 ( OR=3.397, 95% CI:1.003-11.508, P=0.049) , IL-17 ( OR=2.284, 95% CI: 1.017-5.127, P=0.045) . In polycythaemia vera (PV) patients with the NFE2 gene mutation, the proportion of those classified as high-risk is notably higher in both the IWG-PV and mutation-enhanced international prognostic systems for PV (MIPSS-PV) (66.7% vs 25.3% for IWG-PV, P=0.033; 22.2% vs 2.0% for MIPSS-PV, P=0.013) . Similarly, for essential thrombocythaemia (ET) patients, the proportion in the high-risk group of the mutation-enhanced international prognostic systems for ET (MIPSS-ET) is significantly higher (15.4% vs 6.1%, P=0.021) . No statistically significant differences were observed in overall survival or cumulative incidence of thrombosis between NFE2-mutated (38 cases) and non-mutated MPNs patients (671 cases, P>0.05) . Conclusion:NFE2 gene mutations in MPNs were predominantly frameshift mutations. NFE2 gene mutations were correlated with older age, elevated levels of several inflammatory factors (including TNF-α、IFN-γ、IL-10、IL-12P70、IL-17) , and they mostly occurred in late-stage of MPNs.

Objective:Molecular mechanisms underlying compound heterozygous mutations in a patient with inherited antithrombin (AT) deficiency.Methods:The proband was admitted to the First Affiliated Hospital of Wenzhou Medical University in November 2018 with a one-day history of sudden syncope and limb twitching. Peripheral venous blood was collected from the proband and members of his lineages, totaling nine persons across three generations, and a family lineage survey was conducted. AT activity (AT:A) was measured using a chromogenic substrate assay, while AT antigen (AT:Ag) was detected through an immunoturbidimetric assay. Mutation sites were identified by means of Sanger sequencing of the SERPINC1 gene, and silico tools were applied to predict the mutational conservation and hydrophobicity changes. Recombinant plasmid expression vectors were constructed and transfected into HEK293T cells for in vitro overexpression studies. The recombinant AT protein was characterized using Western Blotting, ELISA, and cellular immunofluorescence assays.Results:The proband was a 21-year-old man with type Ⅰ AT deficiency. His AT:A was 33%, along with a corresponding reduction in AT:Ag. The genetic analysis revealed there was a heterozygous insertion mutation at c.318_319insT (p.Asn107*) and a heterozygous missense mutation at c.922G>T (p.Gly308Cys) in exons 2 and 5, respectively. These mutation sites were entirely conserved among the homologous species. Additionally, hydrophobicity studies showed that the p.Gly308Cys mutation will decrease the hydrophilicity of amino acid residues 307-313. The in vitro expression studies indicated a reduction of approximately 46.98%±2.94% and 41.35%±1.48% in the amount of recombinant protein AT-G308C in transfected cell lysates and culture supernatants, respectively. Treatment with the proteasome inhibitor (MG132) restored the cytoplasmic levels of AT-G308C protein to a level similar to that of wild-type protein. However, neither cell lysate nor culture supernatant demonstrated the presence of the recombinant protein AT-N107*. Conclusions:The heterozygous insertion mutation of p.Asn107* and the heterozygous missense mutation of p.Gly308Cys have been associated with reduced AT levels in proband. The p.Asn107* heterozygous insertion mutation may initiate the degradation of mRNA via nonsense mutation-mediated mechanisms, which would remove the defective transcripts, as well as the p.The Gly308Cys heterozygous missense mutation may cause the AT protein to undergo proteasome-dependent degradation by modifying the hydrophobicity of nearby residues in the cytoplasm.

Objective:To explore the correlation between early inflammation indicators and the severity of coronavirus disease 2019 (COVID-19).Methods:A retrospective study was conducted. Patients with COVID-19 admitted to Wenzhou Central Hospital from January 17 to February 14, 2020 were enrolled. The general information, chest CT before admission, the first laboratory parameters and chest CT within 24 hours after admission were collected. Patients were followed up for 30 days after the first onset of dyspnea or pulmonary imaging showed that the lesions progressed more than 50% within 24 to 48 hours (according to the criteria for severe cases) as the study endpoint. According to the endpoint, the patients were divided into two groups: mild type/common type group and severe/critical group, and the differences in general information and inflammation index of the two groups were compared. Logistic regression was used to analyze the inflammation index and the severity of COVID-19. Receiver operating characteristic (ROC) curve was draw to evaluate the predictive value of early inflammation indicators for severe/critical in patients with COVID-19.Results:A total of 140 patients with COVID-19 were included, 74 males and 66 females; the average age was (45±14) years old; 6 cases (4.3%) of mild type, 107 cases (76.4%) of common type, and 22 cases (15.7%) of severe type, 5 cases (3.6%) were critical. There were significantly differences in ages (years old: 43±13 vs. 57±13), the proportion of patients with one chronic disease (17.7% vs. 55.6%), C-reactive protein [CRP (mg/L): 7.3 (2.3, 21.0) vs. 40.1 (18.8, 62.6)], lymphocyte count [LYM (×10 9/L): 1.3 (1.0, 1.8) vs. 0.8 (0.7, 1.1)], the neutrophil/lymphocyte ratio [NLR: 2.1 (1.6, 3.0) vs. 3.1 (2.2, 8.8)] and multilobularinltration, hypo-lymphocytosis, bacterial coinfection, smoking history, hyper-tension and age [MuLBSTA score: 5.0 (3.0, 5.0) vs. 5.0 (5.0, 7.0)] between mild/common group and severe/critical group (all P < 0.05). Univariate Logistic regression analysis showed that CRP, NLR, MuLBSTA score, age, and whether chronic diseases were associated with the severity of COVID-19 [odds ratio ( OR) and 95% confidence interval (95% CI) were 1.037 (1.020-1.055), 1.374 (1.123-1.680), 1.574 (1.296-1.911), 1.082 (1.042-1.125), 6.393 (2.551-16.023), respectively, all P < 0.01]. Further multivariate Logistic regression analysis showed that CRP and MuLBSTA score were risk factors for the development of COVID-19 to severe/critical cases [OR and 95% CI were 1.024 (1.002-1.048) and 1.321 (1.027-1.699) respectively, both P < 0.05]. ROC curve analysis showed that the area under the curve for CRP and MuLBSTA score to predict severe/critical cases were both 0.818, and the best cut-off points were 27.4 mg/L and 6.0 points, respectively. Conclusion:CRP and MuLBSTA score are related to the severity of COVID-19, and may have good independent predictive ability for the development of severe/critical illness.

OBJECTIVE: To explore the molecular pathogenesis for a pedigree affected with hereditary coagulation factor XII (FXII) deficiency. METHODS: Potential variant of the F12 gene was analyzed by PCR and Sanger sequencing. Expression plasmids were constructed by site-directed mutagenesis based on the wild-type and transiently transfected into 293T cells. FXII:C and FXII:Ag of the expression products were determined in the supernatant and cell lysate. Western blotting was used to verify the identify of the protein. RESULTS: Gene sequencing revealed that the proband has carried 46TT genetype and heterozygous p.Glu502Lys variants in exon 13, and a heterozygous p.Gly542Ser variant in exon 14 of the F12 gene. Transfection experiment suggested that the FXII:C and FXII:Ag of p.Glu502Lys variant in the supernatant were 28% and 24%, compared with the wild-type (100%) and FXII:Ag of cell lysates was 39% compared to the wild-type (100%). The FXII:C and FXII:Ag of p. Gly542Ser variant in the supernatant were 32% and 17% and the FXII:Ag of cell lysates was 59%. CONCLUSION The 46TT genetype, p.Glu502Lys and p.Gly542Ser variants of the F12 gene probably underlie the low FXII level in the proband. As shown by in vitro experiment, the p.Glu502Lys and p.Gly542Ser variants can both inhibit the synthesis and secrection of the FXII protein.
Exons ; Factor XII ; genetics ; Factor XII Deficiency ; genetics ; Heterozygote ; Humans ; Pedigree

OBJECTIVE: To analyze the phenotype and genetic basis for a pedigree affected with hereditary coagulation factor XI deficiency. METHODS: Activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen (FIB), FXI activity (FXI:C) and the antigen of FXI (FXI:Ag) were determined for the proband and members from his pedigree. Sanger sequencing was used to analyze all exons, exon-intronic boundaries, as well as the 5'- and 3'- untranslated regions of the F11 gene. Suspected variants were verified in her family members and confirmed by reverse sequencing. The impact of the variants on the protein function was predicted by using PolyPhen-2 and SIFT software. The protein structure and amino acid interaction were analyzed by using Swiss-PdbViewer. RESULTS: The APTT, FXI:C and FXI:Ag of the proband and her sister were significantly reduced to 73.0 s, 10.0%, 15.0% and 87.1 s, 2.0% and 11.5%, respectively. APTT of some family members was slightly prolonged, and FXI:C and FXI:Ag also decreased to various extents. DNA sequencing revealed that the proband and her sister have carried compound heterozygous variants of c.738G>A (p.Trp228stop) and c.938G>T (p.Ser295Ile) respectively in exons 7 and 9 of the F11 gene. Her father, sister and daughter were heterozygous for the c.738G>A (p.Trp228stop) variant, while her mother and nephew were heterozygous for the c.938G>T (p.Ser295Ile). Both PolyPhen-2 and SIFT predicted that the p.Ser295Ile variant is likely to be deleterious and can affect the protein function. Modeling analysis indicated that the p.Ser295Ile variant may lead to disruption of a hydrogen bond, resulting in alteration of protein structure and instability. CONCLUSION The compound heterozygous c.738G>A (p.Trp228stop) and c.938G>T (p.Ser295Ile) variants of the F11 gene probably underlie the decreased FXI level in this pedigree.
Factor XI Deficiency ; genetics ; Female ; Genetic Variation ; Heterozygote ; Humans ; Mutation ; Pedigree

Objective:To explore the genetic characteristics, clinical features, and prognostic values of RAS mutations in patients with myelofibrosis (MF) .Methods:We analyzed 112-gene targeted sequencing data from 226 patients who had a diagnosis of either primary myelofibrosis (PMF) or post-polycythemia vera/post-essential thrombocythemia (post-PV MF and post-ET MF) from December 2011 to December 2019. A retrospective analysis of the genetic characteristics, clinical features, and prognosis of RAS mutations was performed.Results:Among 266 patients diagnosed PMF or post-PV/ET MF, RAS mutations were found in 14 (6.2%) cases, including 9 (4.0%) cases of NRAS mutations, 8 (3.5%) cases of KRAS mutations, and 3 (1.3%) cases of both NRAS and KRAS mutations. All of the NRAS mutations were located in codons 12 and 13. The median VAFs of RAS mutations were significantly lower than those of the driver mutations, confirming that they represent sub-clonal events that are acquired during the disease course. SETBP1, SRSF2, and MPL tended to be clustered with RAS mutations. Patients with RAS mutations had a higher number of additional oncogenic mutations (median, 3.36 vs 1.17, P<0.001) . RAS mutations had a statistically significant association with elevated monocyte cell counts ( P=0.003) , lower platelet counts ( P=0.026) , higher bone marrow blasts ( P=0.022) , splenomegaly ( P=0.005) , and very high-risk (VHR) karyotype abnormality percentage ( P=0.031) . In univariate analysis, the OS of patients with NRAS mutations were significantly inferior in the entire MF and PMF cohorts ( P=0.001, P=0.008) . In a multivariate model, NRAS retained an independent negative prognostic factor in PMF. Conclusion:RAS gene mutations were constantly related to elevated monocyte cell counts, lower platelet counts, higher bone marrow blasts, and VHR karyotype abnormality percentage that usually defined high-risk disease and often occurred as sub-clonal events. NRAS mutation is an independent poor prognostic factor in PMF.

Objective To observe the effects of insulin caliper for blood glucose control on glycemic central tendency, fluctuation and incidence of hypoglycemia in patients with sepsis, and evaluate its application value. Methods One hundred sepsis patients with significant hyperglycemia from December 2015 to December 2017 were selected. All patients needed continuous intravenous insulin infusion to maintain blood glucose. The patients were divided into caliper group and conventional group by random digits table method with 50 cases each, patients of 2 groups adopted an insulin dose modification scheme based on insulin caliper for blood glucose control and paper-based insulin dose modification scheme respectively to control blood glucose. Finally, 92 cases completed the study, including 47 cases in caliper group and 45 cases in conventional group. Blood glucose was measured every 2 hours 0 to 12 hours after intravenous insulin and every 4 hours 16 to 72 hours after intravenous insulin. The incidence of hypoglycemia, insulin dose, ICU time, total hospital stay and hospitalization cost were observed. The proportion of hypoglycemia to total blood glucose measurement, proportion of achieving the glucose control target at each time point, glycemic coefficient of variance, glycemic lability index (GLI) and mean amplitude of glycemic excursion (MAGE) were calculated. Results A total of 1 379 blood glucose values were obtained in caliper group, and a total of 1 332 blood glucose values were obtained in conventional group. There were no statistical difference in blood glucose values 0 to 12 hours after intravenous insulin between 2 groups (P＞0.05). The blood glucose values 16 to 72 hours after intravenous insulin in caliper group were significantly lower than those in conventional group, and there were statistical differences (P＜0.01 or ＜0.05). There were no statistical differences in glycemic coefficient of variance, insulin dose, incidence of hypoglycemia and proportion of hypoglycemia to total blood glucose measurement between 2 groups (P＞0.05). The GLI and MAGE in caliper group were significantly lower than those in conventional group: 12.96 (8.73, 19.58) vs. 23.27 (13.07, 44.61) and (0.66 ± 0.22) mmol/L vs. (0.87 ± 0.28) mmol/L, the proportion of achieving the glucose control target at each time point was significantly higher than that in conventional group: 41.99% (579/1 379) vs. 27.18% (362/1 332), and there were statistical differences (P＜0.01). There were no statistical differences in ICU time, total hospital stay, hospitalization cost, nosocomial infection rate and prognosis between 2 groups (P＞0.05). Conclusions For emergent and critical patients with sepsis, insulin caliper for blood glucose control presents favorable application value for achieving glucose control target, reducing glycemic fluctuation, lowering the incidence of hypoglycemia, low cost and good operability.

Objective: To establish a visualized nomogram with early predictive value for the severity of first-onset acute pancreatitis (AP). Methods: 706 cases of first-onset AP patients admitted to the First Affiliated Hospital of Wenzhou Medical University within 72 hours from January 2013 to January 2016 were collected. According to the revised Atlanta classification of AP in 2012, AP patients was divided into non-severe pancreatitis (NSAP, also called mild acute pancreatitis and moderately severe acute pancreatitis) group and severe acute pancreatitis (SAP) group. The demographic data (age, body mass index and admission time, etc) and laboratory tests (serum amylase, blood sugar, albumin, white blood cells, creatinine, urea nitrogen) were collected and statistically analyzed. Logistic univariate and multivariant regression analysis were performed based on the relevant clinical indicators. The statistically significant indicators were used to obtain regression equations. The R-language software was used to obtain the visualized nomogram via LR model, which was further validated by ROC curve analysis. Results: In univariate logistic regression analysis, the OR (95% CI) values of blood glucose, creatinine at admission and 24 h after admission, urea nitrogen at admission and 24 h after admission, white blood cell, albumin in NSAP group and SAP group were 1.132(1.080-1.186), 1.019(1.013-1.025), 1.026(1.020-1.033), 1.066(1.035-1.099), 1.333(1.241-1.432), 1.083(1.032-1.136), and 0.853(0.811-0.889), and all the differences were statistically significant (all P values <0.05). Multivariate logistic regression analysis showed that the regression equation of the LR model was Y=-2.657-0.116×albumin(g/L)+ 0.082×white blood cell(×10⁹/L)+ 0.118× glycemia(mmol/L)+ 0.022× 24 h after admission creatinine (μmol/L). A total score of more than 60 points on the nomogram predicted the possibility of SAP. If the total score exceeded 130, the possibility of SAP may be up to 14% or more. Furthermore, the ROC curve analysis confirmed that the sensitivity and specificity of LR model established in this study for predicting of SAP were superior to those of urea nitrogen, creatinine and BISAP score alone by AUC, respectively. Conclusions This nomogram may be a useful clinical tool for predicting the severity of the first-onset acute pancreatitis.

Objective To explore the effects of nursing based on psychological nursing procedure in patients with chronic obstructive pulmonary disease (COPD). Methods A total of 280 COPD patients in the Respiratory Department of the First Affiliated Hospital of Xi'an Jiaotong University from January 2016 to January 2017 were selected by simple random sampling method and divided into two groups, with 140 cases in each group. The control group was treated with routine nursing. The observation group was treated according to the psychological nursing procedure on the basis of the control group. The intervention effects were compared using Hamilton Anxiety Scale (HAMA), Hamilton Depression Scale (HAMD), General Self-Efficiency Scale (GSES) and Drug Attitude Inventory (DAI). The recovery of patients' lung function were evaluated and compared with 1 s forced expiratory volume (FEV1), forced vital capacity (FVC) and ratio (FEV1/FVC). Results After 3 months' intervention, the scores of HAMA and HAMD of the observation group were (8.74±3.21) and (7.94±2.17) respectively, which were lower than those of the control group, with statistical significance (t=5.980, 9.920; P<0.01). The total score of GSES and DAI of the observation group were (102.43±26.42) and (17.72±4.53), which were higher than those of the control group, with statistical significance (t=4.162, 5.969; P< 0.01). The FEV1/FVC of the observation group was (69.73±6.78)%, which was higher than that of the control group (57.74±5.16)%, and the difference was statistically significant (t=2.764, P< 0.01). Conclusions The nursing intervention based on psychological care procedure can alleviate COPD patients with anxiety and depression, improve self-efficacy and treatment compliance, which is worthy of clinical promotion.