The multifactorial and multistage pathogenesis of hepatocellular carcinoma(HCC)has fascinated a wide spectrum of scientists for decades.While a number of major risk factors have been identified,their mechanistic roles...The multifactorial and multistage pathogenesis of hepatocellular carcinoma(HCC)has fascinated a wide spectrum of scientists for decades.While a number of major risk factors have been identified,their mechanistic roles in hepatocarcinogenesis still need to be elucidated.Many tumor suppressor genes(TSGs)have been identified as being involved in HCC.These TSGs can be classified into two groups depending on the situation with respect to allelic mutation/loss in the tumors:the recessive TSGs with two required mutated alleles and the haploinsufficient TSGs with one required mutated allele.Hepatitis B virus(HBV)is one of the most important risk factors associated with HCC.Although mice cannot be infected with HBV due to the narrow host range of HBV and the lack of a proper receptor,one advantage of mouse models for HBV/HCC research is the numerous and powerfulgenetic tools that help investigate the phenotypic effects of viral proteins and allow the dissection of the dose-dependent action of TSGs.Here,we mainly focus on the application of mouse models in relation to HBV-associated HCC and on TSGs that act either in a recessive or in a haploinsufficient manner.Discoveries obtained using mouse models will have a great impact on HCC translational medicine.展开更多
Microphthalmia-associated transcription factor (MITF) controls melanocyte survival and differentiation through directly regulating the expression of the tyrosinase (TYR) and tyrosinase-related proteins 1 and 2 (TYRP1 ...Microphthalmia-associated transcription factor (MITF) controls melanocyte survival and differentiation through directly regulating the expression of the tyrosinase (TYR) and tyrosinase-related proteins 1 and 2 (TYRP1 and TYRP2) genes. MITF mutations have been reported to result in an abnormal melanocyte devel-opment and lead to Waardenburg syndrome type 2 (WS2), characterized by variable degrees of sensorineu-ral hearing loss and patchy regional distribution of hypopigmentation. Recently, MITF was also indicated as a causative gene for a more severe syndrome, the Tietz Syndrome (TS), characterized by generalized hy-popigmentation and complete hearing loss. However, few functional studies have been performed to com-pare the diseases-causing mutations. Here, we analyzed the in vitro activity of two recent identified WS2-as-sociated mutation (p.R217I and p.T192fsX18) and one TS-associated mutation p.N210K. The R217I MITF retained partial activity, normal DNA-binding ability and nuclear distribution, whereas the T192fsX18 MITF failed to activate TYR promoter due to loss of DNA-binding activity, and aberrant subcellular localization. The aberrant subcellular localization of T192fsX18 MITF may be caused by deletion of a putative nuclear localization signal (NLS) at aa 213-218 (ERRRRF). Indeed, MITF with deletion of the NLS fragment failed to translocate into the nucleus and activated the TYR promoter. Tagging this NLS to GFP promoted the green fluorescence protein (GFP) translocated into the nucleus. The surprising finding of our study is that a TS-as-sociated MITF mutation, N210K, showed comparable in vitro activity as WT. Thus, the possible involve-ment of MITF in TS and its underlying mechanisms still need further investigation.展开更多
Objective To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity,if any,during prenatal consultation.Methods Zebrafish was used as a model for generating mutant.The patte...Objective To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity,if any,during prenatal consultation.Methods Zebrafish was used as a model for generating mutant.The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization(WISH).CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion.Activity and light/dark tests were performed in arhgef10^(−/−),arhgef10^(+/−),and wild-type zebrafish larvae.ARHGEF10 was knocked down using small interferon RNA(siRNA)in the SH-SY5Y cell line,and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining,respectively.Results WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization(hpf)and in the hemopoietic system at 36-48 hpf.The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae.Moreover,arhgef10^(−/−) zebrafish had more severe symptoms than arhgef10^(+/-) zebrafish.Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis.Conclusion Based on our findings,ARHGEF10 appeared to have a haploinsufficiency effect.展开更多
Background:CSF1R-related leukoencephalopathy,also known as hereditary diffuse leukoencephalopathy with spheroids(HDLS),is a rare white-matter encephalopathy characterized by motor and neuropsychiatric symptoms due to ...Background:CSF1R-related leukoencephalopathy,also known as hereditary diffuse leukoencephalopathy with spheroids(HDLS),is a rare white-matter encephalopathy characterized by motor and neuropsychiatric symptoms due to colony-stimulating factor 1 receptor(CSF1R)gene mutation.Few of CSF1R mutations have been functionally testified and the pathogenesis remains unknown.Methods:In order to investigate clinical and pathological characteristics of patients with CSF1R-related leukoencephalopathy and explore the potential impact of CSF1R mutations,we analyzed clinical manifestations of 15 patients from 10 unrelated families and performed brain biopsy in 2 cases.Next generation sequencing was conducted for 10 probands to confirm the diagnosis.Sanger sequencing,segregation analysis and phenotypic reevaluation were utilized to substantiate findings.Functional examination of identified mutations was further explored.Results:Clinical and neuroimaging characteristics were summarized.The average age at onset was 35.9±6.4 years(range 24–46 years old).Younger age of onset was observed in female than male(34.2 vs.39.2 years).The most common initial symptoms were speech dysfunction,cognitive decline and parkinsonian symptoms.One patient also had marked peripheral neuropathy.Brain biopsy of two cases showed typical pathological changes,including myelin loss,axonal spheroids,phosphorylated neurofilament and activated macrophages.Electron microscopy disclosed increased mitochondrial vacuolation and disorganized neurofilaments in ballooned axons.A total of 7 pathogenic variants(4 novel,3 documented)were identified with autophosphorylation deficiency,among which c.2342C>T remained partial function of autophosphorylation.Western blotting disclosed the significantly lower level of c.2026C>T(p.R676*)than wild type.The level of microtubule associated protein 1 light chain 3-II(LC3-II),a classical marker of autophagy,was significantly lower in mutants expressed cells than wild type group by western blotting and immunofluorescence staining.Conclusions:Our findings support the loss-of-function and haploinsufficiency hypothesis in pathogenesis.Autophagy abnormality may play a role in the disease.Repairing or promoting the phosphorylation level of mutant CSF1R may shed light on therapeutic targets in the future.However,whether peripheral polyneuropathy potentially belongs to CSF1R-related spectrum deserves further study with longer follow-up and more patients enrolled.展开更多
STXBP1 encephalopathy(STXBP1-E)is a rare neurodevelopmental disorder that includes epilepsy;it is caused by de novo STXBP1 mutations.In clinical settings,pharmaceutical interventions to treat STXBP1-E predominantly co...STXBP1 encephalopathy(STXBP1-E)is a rare neurodevelopmental disorder that includes epilepsy;it is caused by de novo STXBP1 mutations.In clinical settings,pharmaceutical interventions to treat STXBP1-E predominantly concentrate on seizure control.However,effective treatments for seizure recurrence,treatment resistance,and common comorbidities remain scarce.Patients with STXBP1-E display a wide range of pathogenic variations that manifest as loss-of-function,gain-of-function,or dominant-negative effects.However,recent studies have primarily investigated the pathogenic mechanisms resulting from loss-of-function mutations using STXBP1 haploinsufficiency models.This approach fails to accurately assess the impact of disease-causing mutations.Moreover,to evaluate new syntaxin-binding protein 1(STXBP1)-targeting drugs,novel models that incorporate disease-causing mutations or even the genetic backgrounds of patients are needed.Here,we discuss the clinical symptoms of STXBP1-E and the relationship between this disorder and STXBP1 mutations.We also review recent progress toward understanding the biological function of STXBP1 and its deficiency-induced cellular defects.We then discuss recent discoveries concerning the pathogenesis of STXBP1-E and the limitations and challenges associated with the current research model.Additionally,we underscore the value of leveraging stem cell technology to study the pathogenic mechanisms of STXBP1-E,and review stem cell transplantation as a potential approach for treating this disorder.We also discuss potential future research directions that need to be resolved.展开更多
基金Supported by Research grants from the Ministry of Science and Technology(MOST)in Taiwan,No.NSC99-2628-B-010-001-MY3,MOST 103-2321-B-010-003,MOST 103-2633-H-010-001,MOST 103-2633-B-400-002 and MOST104-3011-B-010-001a grant from the Ministry of Education,Aim for the Top University Plan
文摘The multifactorial and multistage pathogenesis of hepatocellular carcinoma(HCC)has fascinated a wide spectrum of scientists for decades.While a number of major risk factors have been identified,their mechanistic roles in hepatocarcinogenesis still need to be elucidated.Many tumor suppressor genes(TSGs)have been identified as being involved in HCC.These TSGs can be classified into two groups depending on the situation with respect to allelic mutation/loss in the tumors:the recessive TSGs with two required mutated alleles and the haploinsufficient TSGs with one required mutated allele.Hepatitis B virus(HBV)is one of the most important risk factors associated with HCC.Although mice cannot be infected with HBV due to the narrow host range of HBV and the lack of a proper receptor,one advantage of mouse models for HBV/HCC research is the numerous and powerfulgenetic tools that help investigate the phenotypic effects of viral proteins and allow the dissection of the dose-dependent action of TSGs.Here,we mainly focus on the application of mouse models in relation to HBV-associated HCC and on TSGs that act either in a recessive or in a haploinsufficient manner.Discoveries obtained using mouse models will have a great impact on HCC translational medicine.
基金funded by grants from National Nature Science Foundationof China(81260160 and 81170923)
文摘Microphthalmia-associated transcription factor (MITF) controls melanocyte survival and differentiation through directly regulating the expression of the tyrosinase (TYR) and tyrosinase-related proteins 1 and 2 (TYRP1 and TYRP2) genes. MITF mutations have been reported to result in an abnormal melanocyte devel-opment and lead to Waardenburg syndrome type 2 (WS2), characterized by variable degrees of sensorineu-ral hearing loss and patchy regional distribution of hypopigmentation. Recently, MITF was also indicated as a causative gene for a more severe syndrome, the Tietz Syndrome (TS), characterized by generalized hy-popigmentation and complete hearing loss. However, few functional studies have been performed to com-pare the diseases-causing mutations. Here, we analyzed the in vitro activity of two recent identified WS2-as-sociated mutation (p.R217I and p.T192fsX18) and one TS-associated mutation p.N210K. The R217I MITF retained partial activity, normal DNA-binding ability and nuclear distribution, whereas the T192fsX18 MITF failed to activate TYR promoter due to loss of DNA-binding activity, and aberrant subcellular localization. The aberrant subcellular localization of T192fsX18 MITF may be caused by deletion of a putative nuclear localization signal (NLS) at aa 213-218 (ERRRRF). Indeed, MITF with deletion of the NLS fragment failed to translocate into the nucleus and activated the TYR promoter. Tagging this NLS to GFP promoted the green fluorescence protein (GFP) translocated into the nucleus. The surprising finding of our study is that a TS-as-sociated MITF mutation, N210K, showed comparable in vitro activity as WT. Thus, the possible involve-ment of MITF in TS and its underlying mechanisms still need further investigation.
基金supported by the National Natural Science Foundation of China[NSFCno.81741021]the National Key Research and Development Program of China[no.2016YFC1000501,2016YFC1000500]。
文摘Objective To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity,if any,during prenatal consultation.Methods Zebrafish was used as a model for generating mutant.The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization(WISH).CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion.Activity and light/dark tests were performed in arhgef10^(−/−),arhgef10^(+/−),and wild-type zebrafish larvae.ARHGEF10 was knocked down using small interferon RNA(siRNA)in the SH-SY5Y cell line,and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining,respectively.Results WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization(hpf)and in the hemopoietic system at 36-48 hpf.The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae.Moreover,arhgef10^(−/−) zebrafish had more severe symptoms than arhgef10^(+/-) zebrafish.Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis.Conclusion Based on our findings,ARHGEF10 appeared to have a haploinsufficiency effect.
基金This work was partially supported by the Cohort Study of Cerebral White Matter Change(SWATCH)Multicenter Network(ChiCTR1800015295)This study was supported by the grants from the National Natural Science Foundation of China(No.81571086,81870889,81600978,81200965 and 81430022)+6 种基金National Key R&D Program of China(No.2016YFC1305804 and 2017YFC1310200)Doctoral Innovation Fund of Shanghai Jiao Tong University School of Medicine(No.BXJ201913)Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support(No.20161401)Interdisciplinary Project of Shanghai Jiao Tong University(No.YG2016MS64)the Research Fund for the Doctoral Program of Higher Education(No.20110073120088)Natural Science Foundation of Science and Technology of Shanghai(No.15ZR1426700)Guang Ci Qing Nian Grant(No.GCQN-2017-A03).
文摘Background:CSF1R-related leukoencephalopathy,also known as hereditary diffuse leukoencephalopathy with spheroids(HDLS),is a rare white-matter encephalopathy characterized by motor and neuropsychiatric symptoms due to colony-stimulating factor 1 receptor(CSF1R)gene mutation.Few of CSF1R mutations have been functionally testified and the pathogenesis remains unknown.Methods:In order to investigate clinical and pathological characteristics of patients with CSF1R-related leukoencephalopathy and explore the potential impact of CSF1R mutations,we analyzed clinical manifestations of 15 patients from 10 unrelated families and performed brain biopsy in 2 cases.Next generation sequencing was conducted for 10 probands to confirm the diagnosis.Sanger sequencing,segregation analysis and phenotypic reevaluation were utilized to substantiate findings.Functional examination of identified mutations was further explored.Results:Clinical and neuroimaging characteristics were summarized.The average age at onset was 35.9±6.4 years(range 24–46 years old).Younger age of onset was observed in female than male(34.2 vs.39.2 years).The most common initial symptoms were speech dysfunction,cognitive decline and parkinsonian symptoms.One patient also had marked peripheral neuropathy.Brain biopsy of two cases showed typical pathological changes,including myelin loss,axonal spheroids,phosphorylated neurofilament and activated macrophages.Electron microscopy disclosed increased mitochondrial vacuolation and disorganized neurofilaments in ballooned axons.A total of 7 pathogenic variants(4 novel,3 documented)were identified with autophosphorylation deficiency,among which c.2342C>T remained partial function of autophosphorylation.Western blotting disclosed the significantly lower level of c.2026C>T(p.R676*)than wild type.The level of microtubule associated protein 1 light chain 3-II(LC3-II),a classical marker of autophagy,was significantly lower in mutants expressed cells than wild type group by western blotting and immunofluorescence staining.Conclusions:Our findings support the loss-of-function and haploinsufficiency hypothesis in pathogenesis.Autophagy abnormality may play a role in the disease.Repairing or promoting the phosphorylation level of mutant CSF1R may shed light on therapeutic targets in the future.However,whether peripheral polyneuropathy potentially belongs to CSF1R-related spectrum deserves further study with longer follow-up and more patients enrolled.
基金funded by the Natural Science Foundation of Sichuan Province,grant number 2022NSFSC0607.
文摘STXBP1 encephalopathy(STXBP1-E)is a rare neurodevelopmental disorder that includes epilepsy;it is caused by de novo STXBP1 mutations.In clinical settings,pharmaceutical interventions to treat STXBP1-E predominantly concentrate on seizure control.However,effective treatments for seizure recurrence,treatment resistance,and common comorbidities remain scarce.Patients with STXBP1-E display a wide range of pathogenic variations that manifest as loss-of-function,gain-of-function,or dominant-negative effects.However,recent studies have primarily investigated the pathogenic mechanisms resulting from loss-of-function mutations using STXBP1 haploinsufficiency models.This approach fails to accurately assess the impact of disease-causing mutations.Moreover,to evaluate new syntaxin-binding protein 1(STXBP1)-targeting drugs,novel models that incorporate disease-causing mutations or even the genetic backgrounds of patients are needed.Here,we discuss the clinical symptoms of STXBP1-E and the relationship between this disorder and STXBP1 mutations.We also review recent progress toward understanding the biological function of STXBP1 and its deficiency-induced cellular defects.We then discuss recent discoveries concerning the pathogenesis of STXBP1-E and the limitations and challenges associated with the current research model.Additionally,we underscore the value of leveraging stem cell technology to study the pathogenic mechanisms of STXBP1-E,and review stem cell transplantation as a potential approach for treating this disorder.We also discuss potential future research directions that need to be resolved.
