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Objective:To study the clinical characteristics and gene mutations in a family with combined inherited antithrombin (AT) and factor Ⅶ (FⅦ) deficiency, and explore the relationship between AT gene, F7 gene mutations and diseases. Methods:Pedigree investigation. Blood samplesand clinical dataswere collected fromthe proband and her family members (a total of 16 people in 3 generations) who admitted to the First Affiliated Hospital of Wenzhou Medical University in November 2018. The prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), antithrombin activity (AT: A), antithrombin antigen (AT: Ag), protein C activity (PC: A), protein S activity (PS: A), FⅦ activity (FⅦ: C), FⅦ antigen (FⅦ: Ag) and other indicators were detectedto confirm the diagnosis. DNA direct sequencing analysis of all exons, flanking sequences, 5′ and 3′ untranslated regions of AT genes and F7 genes, and the mutation sites were confirmed by clone sequencingor reverse sequencing. Results:The AT: A and AT: Ag of the proband were 46% and 135 mg/L, respectively (reference range: 250-360 mg/L), some of her family members′ s (father, aunt, two cousins, younger brother and nephew) AT: A and AT: Ag were reduced to 50% of normal range. Her father (Ⅰ 2), aunt (Ⅰ 4), elder brother (Ⅱ 7), younger brother (Ⅱ 8), and nephew (Ⅲ 3)′s FⅦ: C were 45%, 50%, 48%, 47% and 48%, respectively; and their FⅦ:Ag was within the normal range. Genetic analysis revealed that the proband(Ⅱ 6) and some of her family members (father, aunt, two cousins, younger brother and nephew) took rs3138521 polymorphism in the 5′ untranslated region of AT gene. Her father (Ⅰ 2), aunt (Ⅰ 4), elder brother (Ⅱ 7), younger brother (Ⅱ 8), nephew (Ⅲ 3) took c.1091G>A heterozygous missense mutationin exon 8 of F7 gene, resulting in p.Arg304Gln. Conclusion:The rs3138521 in AT gene and c.1091G>A in F7 gene, which may be the molecular mechanism leading to combined inherited AT and FⅦ deficiency in this family.
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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.
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Humanos , Exones , Factor XII , Genética , Deficiencia del Factor XII , Genética , Heterocigoto , LinajeRESUMEN
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.
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Femenino , Humanos , Deficiencia del Factor XI , Genética , Variación Genética , Heterocigoto , Mutación , LinajeRESUMEN
OBJECTIVE@#To analyze the phenotype and genetic variants of a pedigree affected with hereditary protein C (PC) deficiency.@*METHODS@#The protein C activity (PC:A) of the proband and her family members (a four-generation pedigree including 11 individuals) were tested by chromogenic substrate method, and the protein C antigen (PC:Ag) was detected with an enzyme-linked immunosorbent assay(ELISA). The 9 exons and flanking sequences of the protein C (PROC) gene were amplified by PCR and directly sequenced. Suspected mutation was validated by clone sequencing and in other members of the family. MutationTaster and ClustalX-2.1-win was used to analyze the pathogenicity and conservation of the mutation site,respectively. Three-dimentional protein model and amino acids interaction were analyzed with Swiss-PdbViewer software.@*RESULTS@#The PC: A and PC: Ag of the proband were decreased to 46% (reference range: 70%-130%) and 50% (referencerange:70%-140%), respectively. Her grandmother,aunt, cousin and younger brother also showed declined PC:A and PC:Ag by approximately 50%. Genetic analysis revealed that the above individuals have all carried a deletional mutation c.1212-1212delG (p.Met364TrpfsX15) in exon 9 of the PROC gene which can cause replacement of Methionine at position 364 by Tryptophan and alteration of 15 downstream amino acids, and produce a premature stop codon at position 378. The score of MutationTaster was 1.000, indicating that the variant is pathogenic. Conservative analysis showed that the 15 altered amino acids are located in a conserved region across nine homologous species. Protein model analysis showed that the mutation has disrupted a catalytic domain of protein C thereby affected its function.@*CONCLUSION@#The heterozygous c.1212-1212delG deletional mutation in exon 9 of the PROC gene, which was unreported previously,probably accounts for the decrease of PC:A and PC:Ag in this pedigree.
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OBJECTIVE@#To analyze the phenotype and F5 gene variant in a pedigree affected with hereditary coagulation factor V (FV) deficiency.@*METHODS@#All of the exons, flanking sequences, and 5' and 3' untranslated regions of the F5 gene were subjected to PCR and direct sequencing. Suspected variant sites were confirmed by clone sequencing. Influence of the variants was predicted by using software including ClustalX and Mutation Taster.@*RESULTS@#The prothrombin time (PT) and activated partial thromboplastin time (APTT) of the proband were prolonged to 20.3 s and 59.2 s, respectively, while FV activity (FV:C) and FV antigen (FV:Ag) were reduced by 13% and 17%, respectively. The FV:C and FV:Ag of his father, sister and daughter were decreased to 35%, 37%, 29% and 42%, 46%, 35%, respectively. The proband was found to carry a heterozygous c.2851delT variant in exon 13 of the F5 gene, which caused a frameshift and resulted in a truncated protein (p.Ser923LeufsX8). In addition, a heterozygous c.1538G to A (p.Arg485Lys) variant was found in exon 10. The father, sister and daughter of the proband all carried the p.Ser923LeufsX8 variant, while his mother and son carried the heterozygous p.Arg485Lys polymorphism. His younger brother and wife were of the wild type. Bioinformatic analysis showed that p.Ser923 was highly conserved across various species. Mutation Taster scored 1.00 for the p.Ser923LeufsX8 variant, and the result has predicted a corresponding disease.@*CONCLUSION@#A heterozygous deletional mutation c.2851delT in exon 13 of the F5 gene and a heterozygous c.1538G to A polymorphism harbored by the proband may be associated with the decreased FV level in this pedigree.
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Femenino , Humanos , Masculino , Factor V , Genética , Deficiencia del Factor V , Genética , Eliminación de Gen , Pruebas Genéticas , Heterocigoto , Mutación , Linaje , FenotipoRESUMEN
Objective To investigate the inhibitory effect of small interference RNA (siRNA) targeted against transforming growth factor β1 (TGFβ1) in mice with hepatic fibrosis infected with Schistosoma japonicum.Methods Three short hairpin RNAs (shRNA) targeting different positions of TGFβ1 and one unrelated control sequence (HK) were designed and cloned to a plasmid pGenesil-1 respectively to obtain four recombinant expression vectors.Thirty male BALB/c mice were randomly divided into six groups,including normal group,model group,control group (pGenesil-HK) and three treatment groups (pGenesil-TGFβ1-m1,pGenesil-TGFβ1-m2 and pGenesil-TGFβ1-m3) and each group had five mice.The hepatic fibrosis animal models infected with Schistosoma japonicum were constructed.The levels of hydroxyproline (HYP) in liver tissue were examined by biochemistry.Liver histopathology was examined by hematoxylin-eosin and Masson staining.The mRNA expression and protein expression levels of TGFβ1,mothers against decapentaplegic homolog (Smad) 3,Smad 7 and α-smooth muscle actin (α-SMA) in the livers were detected by quantitative real time polymerase chain reaction (RT-qPCR) and Western blot.Two independent samples t test was used to compare the measurement data between groups.Results The liver fibrogenesis was obviously improved in all treatment groups compared with model group.The levels of HYP of liver tissue in all treatment groups were significantly lower than that in model group (t =14.870,7.097 and 10.741,respectively,all P < 0.01).The mRNA expression levels of TGFβ1,Smad 3 and α-SMA(model group vs pGenesil-TGFβ1-m1 group,t =3.235,5.141 and 10.026,respectively;model group vs pGenesil-TGFβ1-m2 group,t =3.396,5.145 and 4.951,respectively;model group vs pGenesil-TGFβ1-m3 group,t =3.511,5.429 and 6.485,respectively) and protein (model group vs pGenesil-TGFβ1-m1 group,t =8.847,8.044 and 10.746,respectively;model group vs pGenesil-TGFβ1-m2 group,t =9.709,7.484 and 10.847,respectively;model group vs pGenesil-TGFβ1-m3 group,t =9.672,8.766 and 11.508,respectively) were significantly decreased in all treatment groups compared with model group (all P < 0.01),while the levels of Smad 7 mRNA and protein were significantly increased in all treatment groups compared with model group(t=11.742 and 11.211,respectively in pGenesil-TGFβ1-m1 group;t =14.446 and 13.736,respectively in pGenesil-TGFβ1-m2 group;t =10.892 and 10.908,respectively in pGenesil-TGFβ1-m3 group,all P < 0.01).Conclusions Specific siRNA targeting TGFβ1 could significantly inhibit the liver fibrogenesis in mice infected with Schistosoma japonicum.The anti-fibrosis mechanisms of the siRNA maybe associated with the down-regulation of TGFβ1,Smad 3 and α-SMA expressions and up-regulation of Smad 7 expression in liver tissue,which results in suppressing the activation of hepatic stellate cells.
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Objective@#To investigate the inhibitory effect of small interference RNA (siRNA) targeted against transforming growth factor β1 (TGFβ1) in mice with hepatic fibrosis infected with Schistosoma japonicum.@*Methods@#Three short hairpin RNAs (shRNA) targeting different positions of TGFβ1 and one unrelated control sequence (HK) were designed and cloned to a plasmid pGenesil-1 respectively to obtain four recombinant expression vectors. Thirty male BALB/c mice were randomly divided into six groups, including normal group, model group, control group (pGenesil-HK) and three treatment groups (pGenesil-TGFβ1-m1, pGenesil-TGFβ1-m2 and pGenesil-TGFβ1-m3) and each group had five mice. The hepatic fibrosis animal models infected with Schistosoma japonicum were constructed. The levels of hydroxyproline (HYP) in liver tissue were examined by biochemistry. Liver histopathology was examined by hematoxylin-eosin and Masson staining. The mRNA expression and protein expression levels of TGFβ1, mothers against decapentaplegic homolog (Smad) 3, Smad 7 and α-smooth muscle actin (α-SMA) in the livers were detected by quantitative real time polymerase chain reaction (RT-qPCR) and Western blot. Two independent samples t test was used to compare the measurement data between groups.@*Results@#The liver fibrogenesis was obviously improved in all treatment groups compared with model group.The levels of HYP of liver tissue in all treatment groups were significantly lower than that in model group (t=14.870, 7.097 and 10.741, respectively, all P<0.01). The mRNA expression levels of TGFβ1, Smad 3 and α-SMA(model group vs pGenesil-TGFβ1-m1 group, t=3.235, 5.141 and 10.026, respectively; model group vs pGenesil-TGFβ1-m2 group, t=3.396, 5.145 and 4.951, respectively; model group vs pGenesil-TGFβ1-m3 group, t=3.511, 5.429 and 6.485, respectively)and protein (model group vs pGenesil-TGFβ1-m1 group, t=8.847, 8.044 and 10.746, respectively; model group vs pGenesil-TGFβ1-m2 group, t=9.709, 7.484 and 10.847, respectively; model group vs pGenesil-TGFβ1-m3 group, t=9.672, 8.766 and 11.508, respectively) were significantly decreased in all treatment groups compared with model group (all P< 0.01), while the levels of Smad 7 mRNA and protein were significantly increased in all treatment groups compared with model group(t=11.742 and 11.211, respectively in pGenesil-TGFβ1-m1 group; t=14.446 and 13.736, respectively in pGenesil-TGFβ1-m2 group; t=10.892 and 10.908, respectively in pGenesil-TGFβ1-m3 group, all P< 0.01).@*Conclusions@#Specific siRNA targeting TGFβ1 could significantly inhibit the liver fibrogenesis in mice infected with Schistosoma japonicum. The anti-fibrosis mechanisms of the siRNA maybe associated with the down-regulation of TGFβ1, Smad 3 and α-SMA expressions and up-regulation of Smad 7 expression in liver tissue, which results in suppressing the activation of hepatic stellate cells.
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Objective@#To analyze the phenotype and F5 gene variant in a pedigree affected with hereditary coagulation factor Ⅴ (FⅤ) deficiency.@*Methods@#All of the exons, flanking sequences, and 5′ and 3′ untranslated regions of the F5 gene were subjected to PCR and direct sequencing. Suspected variant sites were confirmed by clone sequencing. Influence of the variants was predicted by using software including ClustalX and Mutation Taster.@*Results@#The prothrombin time (PT) and activated partial thromboplastin time (APTT) of the proband were prolonged to 20.3 s and 59.2 s, respectively, while FⅤ activity (FⅤ∶C) and FⅤ antigen (FⅤ∶Ag) were reduced by 13% and 17%, respectively. The FⅤ∶C and FⅤ∶Ag of his father, sister and daughter were decreased to 35%, 37%, 29% and 42%, 46%, 35%, respectively. The proband was found to carry a heterozygous c. 2851delT variant in exon 13 of the F5 gene, which caused a frameshift and resulted in a truncated protein (p.Ser923LeufsX8). In addition, a heterozygous c. 1538G>A (p.Arg485Lys) variant was found in exon 10. The father, sister and daughter of the proband all carried the p. Ser923LeufsX8 variant, while his mother and son carried the heterozygous p. Arg485Lys polymorphism. His younger brother and wife were of the wild type. Bioinformatic analysis showed that p. Ser923 was highly conserved across various species. Mutation Taster scored 1.00 for the p. Ser923LeufsX8 variant, and the result has predicted a corresponding disease.@*Conclusion@#A heterozygous deletional mutation c. 2851delT in exon 13 of the F5 gene and a heterozygous c. 1538G>A polymorphism harbored by the proband may be associated with the decreased FⅤ level in this pedigree.
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Objective To analyze the consistency of individual examination diagnosis with terminologies expressed in the conclusion report of physical examinations by the chief inspection physician. Methods Based on the clinical classifications used in the Guidelines on Prevention and Treatment of Blood Lipid Abnormality for Chinese Adults, and related terminology descriptions of Dyslipidemias that were actually used in four-item blood lipid examinations diagnosis, a lexicon database of Hyperlipidemias was constructed with 39 terms used in the four-item blood lipid examination diagnosis and chief physician conclusions. Totally 11953 electronic chief physician inspection reports from 8 health check-up institutions were included. We investigated the terms of lexicon database using word frequency analysis method, calculated the positive rate in the diagnosis of four single examinations of serum lipid and the positive rate in the chief physician's conclusion. Consistency of chief physician's conclusion with single examination diagnosis was analyzed by Kappa test. Results (1) Among the 39 terms of lexicon database, there are 18 nonstandard terms used in single examination diagnosis, accounted for 46% of all terms; (2) In word frequency analysis, there are only 1% of terms that corresponded to clinical classifications of Hyperlipidemias accurately;(3) The positive rate of Hyperlipidemias in serum lipid four single examinations diagnosis was 47%, the positive rate of physician diagnosis was 35%. The consistency analysis of chief physician conclusions with single examination diagnosis showed Kappa=0.71(P0.75 may be suggested as a favorable value.
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Clustered regularly interspaced short palindromic repeats (CRISPR) is an acquired immune system existing in archaea and bacteria with the long-term process of evolutionary.CRISPR/Cas9 gene editing system is a new type of gene editing technology developed based on the system.CRISPR/Cas9 is a more efficient method for gene targeting than the previous methods.It has been successfully applied for gene-modified of eukaryotes since 2012,but the reports about pathogenic microorgaisms are rarely.Here,the research progress in the structure,mechanism of CRISPR/Cas9 system and its applications on pathogenic microorgaisms is reviewed.
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Objective@#To investigate the protective effect of ACY1215 (Rocilinostat), a histone deacetylase inhibitor, against brain edema in mice with acute liver failure.@*Methods@#Lipopolysaccharide combined with D-galactosamine was used to establish a mouse model of acute liver failure, and ACY1215 was used for intervention. The effect of ACY1215 on histopathological changes of the liver was observed after 24 hours, as well as the changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood ammonia, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), brain water content, blood-brain barrier structure, NF-κB-p65, histone, acetylated histone, and TNF-α mRNA in brain tissue.@*Results@#The mice with acute liver failure had marked pathological damage in liver tissue, as well as significant increases in the levels of ALT, AST, blood ammonia, TNF-α, and IFN-γ (t≥5.367, all P < 0.05). ACY1215 significantly improved the pathological damage in liver tissue and reduced the serum levels of ALT, AST, blood ammonia, TNF-α, and IFN-γ (t≤-3.515, all P < 0.05). ACY1215 also significantly reduced the expression of NF-κB-p65 (t = -5.871, P = 0.004) and the mRNA expression of TNF-α (t = -11.913, P < 0.01) in brain tissue and brain water content (t = -2.355, P < 0.01). According to the results of electron microscopy, the model group had an abnormal blood-brain barrier structure, and the ACY1215 group had slighter damage than the model group. Compared with the normal group, the model group had significant increases in the acetylation level of histone H3 and H4 in brain tissue (t≥3.009, both P < 0.05), while ACY1215 further upregulated the acetylation levels of histone H3 and H4 (t≥6.682, both P < 0.05).@*Conclusion@#ACY1215 exerts a protective effect against brain edema in mice with acute liver failure, possibly by regulating histone acetylation and inhibiting inflammation.
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Objective To investigate the effect of MS-275, an histone deacetylase ( HDAC ) inhibitor, on acute liver failure ( ALF ) induced by D-galactosamine and lipopolysaccharide in mice. Methods Thirty specific pathogen free (SPF) C57BL/6 male mice were randomly and equally divided into control, ALF model and MS-275 groups. ALF model was induced by D-galactosamine ( D-Gal ) and lipopolysaccharide (LPS), and the mice in MS-275 group received MS-275 (1 mg/kg) at 2 h before the induction of ALF.Serum and liver samples of mice were obtained at 24 h after ALF induction.The serum levels of ALT, AST, TBil and tumor necrosis factor-alpha ( TNF-α) , interferon γ( IFNγ) , interleukin ( IL )-1β, high mobility group box 1 ( HMGB1 ) were tested by biochemical methods or ELISA kit, respectively.The expression of HDAC1, HDAC3, acetylation of histone H3, H4, P65, acetylation and phosphorylation of P65 in liver were detected by Western blotting.The changes of histology in liver was detected by HE staining, and the translocation of P65 in liver was detected by immunohistochemistry. Comparison of variables among the groups was performed using t test.Results MS-275 inhibited the infiltration of inflammatory cells and improved the pathological changes of liver tissue.Compared with ALF group, serum ALT, AST, TBil levels were decreased in MS-275 group ( t =-22.215, -11.914 and-12.160, all P<0.05), but still higher than those in the control group (t=14.852, 11.692 and 8.333, all P<0.05); serum TNF-α, IFNγ, IL-1β, HMGB1 levels were also significantly decreased in MS-275 group (t=-7.926, -3.427, -2.475 and -5.920, all P<0.05), but TNF-αand IFNγwere still higher than those in the control group (t=5.541 and 5.514, all P<0.05).Compared with control group, the expression of class I HDAC in liver tissue was significantly decreased in MS-275 group ( t=-3.676 and-10.576, P<0.05), while the expressions of acetylation of histone H3, H4 and P65 were significantly increased (t=3.976, 5.559 and 4.588, all P<0.05).MS-275 inhibited the translocation of P65 from cytoplasm to the nucleus.Conclusion MS-275 can protect liver from acute failure in mice through enhancing the acetylation levels of non-histones.
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Antiviral therapy is the most important thing in the treatment of chronic hepatitis B (CHB). Pegylated interferon (PEG-IFN) has both antiviral and immunomodulatory effects, and after drug withdrawal, 30%-40% of patients can achieve HBeAg seroconversion and sustained virologic response. Many studies have shown that HBsAg quantification can be used as an index to predict the anti-HBV effect of PEG-IFN and sustained immune control after drug withdrawal. This article reviews the relationship between HBsAg levels before, during, and after PEG-IFN therapy and antiviral effect to clarify the significance of HBsAg quantification in the PEG-IFN treatment of CHB and to guide the regimen of antiviral therapy.
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Objective To find out the general status about the traditional Chinese medicine therapies that were included into medicare among the selected hospitals in the country's public hospital reform project, and to investigate the reasons for those not included and make recommendations.Methods Collecting relevant information through questionnaires, experts interviews and discussions.Results There are still some TCM therapies not included into the range of reimbursement.Most Of them are external treatment of traditional Chinese medicine, acupuncture, massage, special treatments of traditional Chinese medicine and comprehensive therapies of TCM.Conclusions The establishment of negotiations department about medicare in the field of traditional Chinese medicine was needed.Medicare should be in favor of TCM therapies, and traditional Chinese medicine hospitals should strengthen training of personnel and hiring.At the same time, a reasonable increase of the proportion of TCM experts involved in the making health insurance directory was needed.
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The construction of biologic functional surfaces of materials, from the visual angle of material science, is aimed to make the biomaterials adapted by tissues, and to endow them with dynamic conformity; moreover, from the view-point of clinical applications, it is the functional surface to join the environmental tissues with the implanted material, playing the role of artificial extracellular matrix (ECM). The architecture of biologic functional surface is very important in tissue engineering science. Here the primary concepts of biological surface science and the construction and application of biofunctional surfaces in tissue engineering are reviewed.