Objective:The phenotype and genotype of a pedigree with Glanzmann thrombasthenia caused by compound heterozygous mutation in the ITGA2B gene and its molecular pathogenesis were explored.Methods:The platelet aggregation rate of the proband and his family was detected by using a platelet aggregation test with adenosine diphosphate, collagen, epinephrine, arachidonic acid, and ristocetin. The expression levels of CD41 (αⅡb), CD61 (β3), and CD42b (GPⅠb) on the platelet surface was detected by flow cytometry. Gene sequencing technology was used for the genetic identification of the family. RT-PCR was used in the detection of mRNA splicing, and qRT-PCR was used in detecting the relative mRNA level of the ITGA2B gene. Bioinformatics analysis was used to evaluate the pathogenicity of mutation sites and their effects on protein structure and function. The expressions of total αⅡb and β3 in platelets were analyzed by Western blot.Results:Except ristocetin, the other four inducers could not induce platelet aggregation in the proband. Flow cytometry showed that the expression levels of αⅡb and β3 were only 0.25% and 9.76%, respectively, on the platelet surface of the proband, whereas GPⅠb expression was relatively normal. The expression levels of glycoproteins in the other family members were almost normal. c.480C>G and c.2929C>T mutations were detected in the proband through gene sequencing. The c.480C>G mutation was inherited from his mother, and the c.2929C>T mutation was inherited from his father. The RT-PCR and sequencing results showed that the c.480C>G mutation caused mRNA splicing in the proband and his mother, resulting in the deletion of 99 bases in c.476G-574A (p.S160-S192). qRT-PCR showed that the c.2929C>T variant reduced the mRNA level of the ITGA2B gene in the proband and his father. Bioinformatics analysis suggested that the c.480C>G mutation might form a binding sequence with hnRNP A1 protein and generate the 5′SS splice site. The three-dimensional structural model of the αⅡb subunit showed that the β-propeller domain of the p.S160-S192 deletion lost two β-strands and one α-helix in blade 2. The c.2929C>T nonsense mutation caused premature translation termination and produced a truncated protein with the deletion of p.R977-E1039, including the cytoplasmic domain, transmembrane domain, and a β chain of the extracellular Calf-2 domain. The total αⅡb expression of the proband was absent, and the relative expression of β3 was 11.36% of the normal level.Conclusion:The compound heterozygous mutation c.480C>G in exon 4 and c.2929C>T in exon 28 of the ITGA2B gene probably underlies Glanzmann thrombasthenia in this pedigree.

Objective:To analyze the clinical features and etiological results of neonatal central nervous system(CNS) infection and provide basis for optimization of pathogen detection strategy for CNS infection.Methods:We collected the clinical and laboratory data of hospitalized neonates with clinical diagnosis of CNS infection in the neonatal department at Hebei Provincial Children′s Hospital, from January 1, 2020 to August 31, 2021.The clinical manifestations of the enrolled neonates, as well as the cerebrospinal fluid(CSF)pathogens detected by conventional and molecular biological detection techniques were analyzed.Laboratory characteristics of different kinds of pathogen were compared.Results:A total of 101 eligible neonates were enrolled.The median gestational age was 38.8(36.2, 39.6)weeks, with a prematurity rate 26.7%.There were 68 boys.The median age of onset was 9(2, 14)days.Blood culture was positive in 19(18.8%) cases, including 17 cases of bacteria and two cases of fungus.Positive findings were found in CSF specimens of 33(32.7%)cases by various methods including 13 bacteria, 19 viruses and one fungi.Streptococcus group B and Escherichia coli were the first two bacteria in CSF.Enterovirus was the most common virus in CSF.In terms of detection methods of CSF pathogens, seven cases(7/101, 6.9%) were detected by CSF culture, two cases(2/21, 9.5%)by smear, 22 cases(22/45, 48.9%)by single-virus targeted/multiplex polymerase chain reaction and four cases(4/7, 57.1%)by metagenomic next-generation sequencing.The CSF white blood cell counts, protein levels and blood C-reactive protein levels were higher in the cases with bacteria/fungi detection from CNS infection than in those with virus detection( P<0.05). Almost all neonates(98/101, 97.0%)were clinically cured or significantly improved before discharge.Two neonates were discharged against medical advice and one neonate was transferred to the other hospital after clinical improvement. Conclusion:Combined use of conventional and molecular biological detection techniques can significantly improve the etiological positive rate of neonatal CNS infection.Viral infection is not rare in the neonatal population.Our study demonstrated the spectrum of organism causing neonatal CNS infection, which provided a basis for the optimization of pathogen detection strategy.

Objective To examine the influencing factors related to clinical efficacy and outcomes of adult primary immune thrombocytopenia (ITP).Methods The clinical data of 161 cases of ITP admitted in the Second Hospital of Shanxi Medical University from June 2013 to March 2017 were collected.The influencing factors related to clinical efficacy and prognosis of adult ITP patients were analyzed.Results There were 60 males and 101 females with a M/F ratio of 0.59∶1 and a median age of 45 years (18-84 years).There were 109 newly diagnosed ITP cases,14 persistent ITP cases and 38 chronic ITP cases in this series.Seventy nine patients received intravenous immunoglobulin g (IVIg) treatment and 82 patients received high dose-dexamethasone treatment.There were no significant differences in clinical efficacy [91.13％(72/79) vs.87.80％(72/82),x2=0.181,P=0.914] and relapse rate [36.11％(26/72) vs.30.55％(22/72),x2=0.189,P=0.910] between IVIg and high dose-dexamethosone groups.Multivariate regression analysis showed that bleeding score ≥2 was the independent risk factor for the lower clinical efficacy (RR=1.415,95％CI:1.008-1.986,P＜0.05).Patients were followed up for a median of 9.0 months (0.5-55.0 months),48 patients relapsed with a relapse rate of 33.33％ and a median relapse time of 1.8 months (0.5-24.0 months).Conclusions IVIg and high dose-dexamethasone have the similar clinical efficacy and relapse rate for treatment of adult ITP.The patients with the bleeding score ≥2 are more likely to get lower remission rate.

Objective To explore the clinical characteristics of renal transplantation in patients with diabetes mellitus (DM)-induced end-stage nephropathy.Methods The clinical data of 408 cases who underwent renal transplantation in our center from 2009 to 2013 were retrospectively analyzed.The patients were divided into DM group (n =82) and non-DM group (n =326).The postoperative infection,delayed graft function (DGF),adverse events,and the survival rate of patients/kidneys were comparatively analyzed.Results The incidence of postoperative infection,DGF and adverse events was significantly higher in DM group than in non-DM group (23.2％ vs.15.6％,P =0.04;17.1％ vs.8.6 ％,P =0.04;13.4％ vs.8.3 ％,P =0.03).No significant difference was found in the 1-,2-,and 3-year survival rate of patients and kidneys between the two groups after operation (P＞ 0.05).Conclusion The incidence of postoperative infection,DGF and adverse events is higher in DM patients.The DM does not affect the survival rate of patients/kidneys through appropriate treatment.It is important to prevent complications in DM patients after renal transplantation.

Objective To investigate the mutations of epigenetic regulation factor ASXL1 gene in myelodysplastic syndrome(MDS).Methods Mutation analysis of ASXL1 gene in 53 de novo MDS patients and 20 healthy persons was performed by using polymerase chain reaction(PCR)followed by sequence analysis at DNA level.The clinical and laboratory characteristics were compared in MDS patients with ASXL1 gene mutation and ASXL1 wild type.ASXL1 mutation in mRNA level was detected by using reverse transcription PCR(RT-PCR)followed by sequence analysis.Results ASXL1 gene mutations were observed in 9 cases(16.9%)of 53 MDS patients.6 mutation types were detected,including 4 frameshift mutations types(2 cases with p.Glu635ArgfsX15,3 cases with p.Gly646TrpfsX12,1 case with p.Ala640GlyfsX14 and 1 case with p.Gly790TrpfsX10)and 2 nonsense mutation types(1 case with p.Gln1063X and 1 case with p.Gln695X).All the mutations were heterozygous,and p.Gly790TrpfsX10 and p.Gln695X were new mutation types.In addition,a single nucletide polymorphism(SNP)p.Gly652Ser was also detected in 4 cases with MDS.5 cases of p.G652S SNP and 1 case of p.Leu1173Leu SNP were detected in 20 healthy people.Frameshift mutation(p.Gly646TrpfsX12)could be detected at mRNA level by using RT-PCR.Differences were not observed in red blood cell counts,white blood cell counts,platelet counts,hemoglobin levels,reticulocyte,neutrophil granulocyte,the peripheral blood lymphocytes percentage,T-cell subsets in the peripheral blood,the proportion of primitive cell in the marrow and MDS types between the patients with ASXL1 gene mutation and ASXL1 wild type patients(P >0.05).Conclusion There is a high frequency of ASXL1 gene mutation in MDS patients,which can be detected at mRNA level.

Objective To analyze the clinical features and risk factors for an emerging infection during the first induction chemotherapy in elderly patients with acute leukemia.Methods A retrospective analysis of clinical data of 79 elderly patients with newly diagnosed acute leukemia was performed in Second Hospital of Shanxi Medical University from January 2014 to May 2016.Results The 70 cases among 79 elderly patients with acute leukemia were suffered from infection with infection incidence rate of 88.6％ (70/79)during first induction chemotherapy.The infection-related fatality rate was 8.6 ％ (6/70).Being clear about sites of infection accounted for 90.0 ％ (63/70),and the top three infection sites were the lungs,gastrointestinal tract and the bloodstream.113 pathogenic strains were detected,including gram-negative bacilli accounting for 42.5 ％ (48/113),Gram-positive cocci for 30.1％ (34/113),fungi for 24.8％ (28/113),the virus for 2.7％ (3/113).Based on clinically and confirmatively diagnosis,the invasive fungal diseases mostly as Candida accounted for 30.4 ％ (24/79),mixed infections accounted for 34.3％ (24/70).Univariate analysis showed agranulocytosis and AML were risk factors for infection.Logistic multivariate regression analysis showed that agranulocytosis was a risk factor for infection (OR=12.010,95％CI:2.346-107.973,P=0.000).The infection does not affect a complete remission rate of acute leukemia (x2 =0.001,P=0.983).Conclusions For newly diagnosed elderly acute leukemia patients,an emerging infection during the first induction chemotherapy is characterized by a high incidence,high fungal infection rate,most common site in lung,Gram-negative bacteria as most common pathogen,and an increased infection rate by agranulocytosis.The infection does not affect the remission rate of acute leukemia.

Objective: To investigate the effect of biology and mTOR pathway activity of down-regulated TSC2 gene expression on U937 leukemia cells. Methods: Gene expression was down-regulated by lentivirus induced RNA interference on TSC2 high expressed U937 cell line; the proliferation, apoptosis and differentiation were detected by CCK-8 assay, colony formation assay and flow cytometry; the gene expression level and protein kinase activity were detected by qRT-PCR and Western blot. Results: Down-regulated expression of TSC2 gene promoted U937 cell proliferation and colony formation ability (P<0.05) . The proportion in G₀/G₁ phase of TSC2 down-regulated U937 cell was much lower than that of the control cells [ (52.53±3.75) % vs (75.10±4.33) %, t=6.829, P=0.002], the S phase [ (22.43±1.00) % vs (15.47±1.20) %, t=-5.581, P=0.019] and G₂/M phase [ (25.03±4.34) % vs (14.33±0.91) %, t=-5.413, P=0.013] was remarkably higher than that of the control cells (P<0.05) . There were no statistically significant differences in cell apoptosis and differentiation (P>0.05) . Down-regulation of TSC2 led to the increased activity of mTOR, 4EBP1 and S6K1, but did not influence the activity of AKT. The expressions of proliferation related cyclinD1, c-myc and PTEN were also up-regulated after TSC2 silenced, but the expressions of P27KIP and BCL-XL were not changed. Conclusion Downregulation of TSC2 could promote the proliferation of U937 cells through up-regulation of mTOR activity.

Objective To construct pIRES2-ZsGreen1/FⅨ expression vector,using the pcDNA/ FⅨ plasmid containing FⅨ cDNA as template,and express in HEK-293 cells.Methods The total ORF of F Ⅸ gene was amplified from pcDNA/F Ⅸ plasmid,then the amplified fragment was clonded into the pIRES2-ZsGreen1 vector using the Infusion enzyme.The positive clones of eukaryotic expression vector of pIRES2-ZsGreen1/F Ⅸ were screened and expanded after transfection,then were constructed and confirmed by PCR and sequencing.Transient expression experiments were performed using HEK-293 cells transfected with the expression vectors and observed the expression of ZsGreenl protein by confocal laser microscope.The relative expression levels of F Ⅸ mRNA,protein and F Ⅸ activity (F Ⅸ ∶ C) were detected by real time PCR (RT-PCR),immunofluorescence microscopy,One-Stage method,respectively.Results The expression vector,pIRES2-ZsGreen1/F Ⅸ,was successfully constructed and expressed in HEK-293 cells.RT-PCR detected the expression of F Ⅸ mRNA in HEK-293 cells and the immunofluorescence microscopy showed F Ⅸ protein distributed in the surrounding of nucleus.F Ⅸ ∶ C of cell lysates and cell culture fluid transfected with the expression vectors were (92.03 ± 0.29)％ and (86.89 ± 8.78)％,respectively;while both F Ⅸ ∶ C of cell lysates and cell culture fluid transfected with or without the expression vectors were 0.Conclusion The experimental results showed the expression vector,pIRES2-ZsGreen1/F Ⅸ,was successfully constructed,which provided experiment basement for the follow study on the location,function and molecular pathology of hemophilia B.