Chromosome microarray analysis(CMA) is a cost-effective molecular cytogenetic technique that has been used as a first-line diagnostic test in neurodevelopmental disorders in the USA since 2011. The impact of CMA resul...Chromosome microarray analysis(CMA) is a cost-effective molecular cytogenetic technique that has been used as a first-line diagnostic test in neurodevelopmental disorders in the USA since 2011. The impact of CMA results on clinical practice in China is not yet well studied, so we aimed to better evaluate this phenomenon.We analyzed the CMA results from 434 patients in our clinic, and characterized their molecular diagnoses, clinical features, and follow-up clinical actions based on these results. The overall diagnostic yield for our patients was 13.6%(59 out of 434). This gave a detection rate of 14.7%for developmental delay/intellectual disability(DD/ID,38/259) and 12% for autism spectrum disorders(ASDs,21/175). Thirty-three recurrent(n≥2) variants were found, distributed at six chromosomal loci involving known chromosome syndromes(such as DiGeorge, Williams Beuren, and Angelman/Prader-Willi syndromes).The spectrum of positive copy number variants in our study was comparable to that reported in Caucasian populations, but with specific characteristics. Parental origin tests indicated an effect involving a significant maternal transmission bias to sons. The majority of patients with positive results(94.9%) had benefits, allowing earlier diagnosis(36/59), prioritized full clinical management(28/59), medication changes(7/59), a changed prognosis(30/59), and prenatal genetic counseling(15/59). Our results provide information on de novo mutations in Chinese children with DD/ID and/or ASDs. Our data showed that microarray testing provides immediate clinical utility for patients. It is expected that the personalized medical care of children with developmental disabilities will lead to improved outcomes in long-term developmental potential.We advocate using the diagnostic yield of clinically actionable results to evaluate CMA as it provides information of both clinical validity and clinical utility.展开更多
Pathological protein aggregation is a prevalent characteristic of major age-related neurodegenerative diseases,including Alzheimer's disease(AD),Huntington's disease(HD),amyotrophic lateral sclerosis(ALS),fron...Pathological protein aggregation is a prevalent characteristic of major age-related neurodegenerative diseases,including Alzheimer's disease(AD),Huntington's disease(HD),amyotrophic lateral sclerosis(ALS),frontotemporal dementia(FTD),and Parkinson's disease(PD),and serves as a crucial biomarker for the diagnosis and classification.Although the etiological role of protein aggregation in neurodegenerative diseases is under debate,it is believed to be associated with disease progression and clinical outcomes.1 Therefore,a comprehensive understanding of how protein aggregation forms and clears holds immense potential for discovering and developing therapeutic drugs against neurodegenerative diseases.展开更多
Direct in vivo conversion of astrocytes into functional new neurons induced by neural transcription factors has been recognized as a potential new therapeutic intervention for neural injury and degenerative disorders....Direct in vivo conversion of astrocytes into functional new neurons induced by neural transcription factors has been recognized as a potential new therapeutic intervention for neural injury and degenerative disorders. However, a few recent studies have claimed that neural transcription factors cannot convert astrocytes into neurons, attributing the converted neurons to pre-existing neurons mis-expressing transgenes. In this study, we overexpressed three distinct neural transcription factors––NeuroD1, Ascl1, and Dlx2––in reactive astrocytes in mouse cortices subjected to stab injury, resulting in a series of significant changes in astrocyte properties. Initially, the three neural transcription factors were exclusively expressed in the nuclei of astrocytes. Over time, however, these astrocytes gradually adopted neuronal morphology, and the neural transcription factors was gradually observed in the nuclei of neuron-like cells instead of astrocytes. Furthermore,we noted that transcription factor-infected astrocytes showed a progressive decrease in the expression of astrocytic markers AQP4(astrocyte endfeet signal), CX43(gap junction signal), and S100β. Importantly, none of these changes could be attributed to transgene leakage into preexisting neurons. Therefore, our findings suggest that neural transcription factors such as NeuroD1, Ascl1, and Dlx2 can effectively convert reactive astrocytes into neurons in the adult mammalian brain.展开更多
The axolotl is broadly used in regenerative, developmental, and evolutionary biology research. Targeted gene knock-in is crucial for precision transgenesis, enabling disease modeling, visualization, tracking, and func...The axolotl is broadly used in regenerative, developmental, and evolutionary biology research. Targeted gene knock-in is crucial for precision transgenesis, enabling disease modeling, visualization, tracking, and functional manipulation of specific cells or genes of interest(GOIs). Existing CRISPR/Cas9-mediated homology-independent method for gene knock-in often causes “scars/indels” at integration junctions.Here, we develop a CRISPR/Cas9-mediated semi-homology-directed recombination(HDR) knock-in method using a donor construct containing a single homology arm for the precise integration of GOIs.This semi-HDR approach achieves seamless single-end integration of the Cherry reporter gene and a large inducible Cre cassette into intronless genes like Sox2 and Neurod6 in axolotls, which are challenging to modify with the homology-independent method. Additionally, we integrate the inducible Cre cassette into intron-containing loci(e.g., Nkx2.2 and FoxA2) without introducing indels via semi-HDR. GOIs are properly expressed in F0 founders, with approximately 5%-10% showing precise integration confirmed by genotyping. Furthermore, using the Nkx2.2:CreER^(T2)line, we fate-map spinal cord p3 neural progenitor cells,revealing that Nkx2.2^(+) cells adopt different lineages in development and regeneration, preferentially generating motoneurons over oligodendrocytes during regeneration. Overall, this semi-HDR method balances efficiency and precision in the integration of GOIs, providing a valuable tool for generating knock-in axolotls and potentially extending to other species.展开更多
Over the past decade,a growing number of studies have reported transcription factor-based in situ reprogramming that can directly conve rt endogenous glial cells into functional neurons as an alternative approach for ...Over the past decade,a growing number of studies have reported transcription factor-based in situ reprogramming that can directly conve rt endogenous glial cells into functional neurons as an alternative approach for n euro regeneration in the adult mammalian central ne rvous system.Howeve r,many questions remain regarding how a terminally differentiated glial cell can transform into a delicate neuron that forms part of the intricate brain circuitry.In addition,concerns have recently been raised around the absence of astrocyte-to-neuron conversion in astrocytic lineage-tra cing mice.In this study,we employed repetitive two-photon imaging to continuously capture the in situ astrocyte-to-neuron conversion process following ecto pic expression of the neural transcription factor NeuroD1 in both prolife rating reactive astrocytes and lineage-tra ced astrocytes in the mouse cortex.Time-lapse imaging over several wee ks revealed the ste p-by-step transition from a typical astrocyte with numero us short,tapered branches to a typical neuro n with a few long neurites and dynamic growth cones that actively explored the local environment.In addition,these lineage-converting cells were able to migrate ra dially or to ngentially to relocate to suitable positions.Furthermore,two-photon Ca2+imaging and patch-clamp recordings confirmed that the newly generated neuro ns exhibited synchronous calcium signals,repetitive action potentials,and spontaneous synaptic responses,suggesting that they had made functional synaptic connections within local neural circuits.In conclusion,we directly visualized the step-by-step lineage conversion process from astrocytes to functional neurons in vivo and unambiguously demonstrated that adult mammalian brains are highly plastic with respect to their potential for neuro regeneration and neural circuit reconstruction.展开更多
1文献来源研究一:Rudin CM,Poirier JT,Byers LA,et al.Molecular subtypes of small cell lung cancer:A synthesis of human and mouse model data[J].Nat Rev Cancer,2019,19(5):289-297.研究二:Owonikoko TK,Dwivedi B,Chen ZJ,et a...1文献来源研究一:Rudin CM,Poirier JT,Byers LA,et al.Molecular subtypes of small cell lung cancer:A synthesis of human and mouse model data[J].Nat Rev Cancer,2019,19(5):289-297.研究二:Owonikoko TK,Dwivedi B,Chen ZJ,et al.YAP1 positive small-cell lung cancer subtype is associated with the T-cell inflamed gene expression profile and confers good prognosis and long term survival[J].J Clin Oncol,2020,38(15S):Abstr 9019.展开更多
基金supported by grants from the National Natural Science Foundation of China (81761128035 and 81781220701)the Shanghai Municipal Science and Technology Committee (17XD1403200 and 18dz2313505)+2 种基金the Research Physician Project of Shanghai Municipal Education Commission (20152234)the Shanghai Municipal Health and Family Planning Commission (GDEK201709, 2017ZZ02026, and 2017EKHWYX02)the Scientific Program of Shanghai Shenkang Hospital Development Center (16CR2025B) of China
文摘Chromosome microarray analysis(CMA) is a cost-effective molecular cytogenetic technique that has been used as a first-line diagnostic test in neurodevelopmental disorders in the USA since 2011. The impact of CMA results on clinical practice in China is not yet well studied, so we aimed to better evaluate this phenomenon.We analyzed the CMA results from 434 patients in our clinic, and characterized their molecular diagnoses, clinical features, and follow-up clinical actions based on these results. The overall diagnostic yield for our patients was 13.6%(59 out of 434). This gave a detection rate of 14.7%for developmental delay/intellectual disability(DD/ID,38/259) and 12% for autism spectrum disorders(ASDs,21/175). Thirty-three recurrent(n≥2) variants were found, distributed at six chromosomal loci involving known chromosome syndromes(such as DiGeorge, Williams Beuren, and Angelman/Prader-Willi syndromes).The spectrum of positive copy number variants in our study was comparable to that reported in Caucasian populations, but with specific characteristics. Parental origin tests indicated an effect involving a significant maternal transmission bias to sons. The majority of patients with positive results(94.9%) had benefits, allowing earlier diagnosis(36/59), prioritized full clinical management(28/59), medication changes(7/59), a changed prognosis(30/59), and prenatal genetic counseling(15/59). Our results provide information on de novo mutations in Chinese children with DD/ID and/or ASDs. Our data showed that microarray testing provides immediate clinical utility for patients. It is expected that the personalized medical care of children with developmental disabilities will lead to improved outcomes in long-term developmental potential.We advocate using the diagnostic yield of clinically actionable results to evaluate CMA as it provides information of both clinical validity and clinical utility.
基金supported by the National Natural Science Foundation of China(32270892 and 32070708).
文摘Pathological protein aggregation is a prevalent characteristic of major age-related neurodegenerative diseases,including Alzheimer's disease(AD),Huntington's disease(HD),amyotrophic lateral sclerosis(ALS),frontotemporal dementia(FTD),and Parkinson's disease(PD),and serves as a crucial biomarker for the diagnosis and classification.Although the etiological role of protein aggregation in neurodegenerative diseases is under debate,it is believed to be associated with disease progression and clinical outcomes.1 Therefore,a comprehensive understanding of how protein aggregation forms and clears holds immense potential for discovering and developing therapeutic drugs against neurodegenerative diseases.
基金supported by the Key Project of Guangzhou City,No.202206060002Science and Technology Project of Guangdong Province,No.2018B030332001Guangdong Provincial Pearl River Project,No.2021ZT09Y552 (all to GC)。
文摘Direct in vivo conversion of astrocytes into functional new neurons induced by neural transcription factors has been recognized as a potential new therapeutic intervention for neural injury and degenerative disorders. However, a few recent studies have claimed that neural transcription factors cannot convert astrocytes into neurons, attributing the converted neurons to pre-existing neurons mis-expressing transgenes. In this study, we overexpressed three distinct neural transcription factors––NeuroD1, Ascl1, and Dlx2––in reactive astrocytes in mouse cortices subjected to stab injury, resulting in a series of significant changes in astrocyte properties. Initially, the three neural transcription factors were exclusively expressed in the nuclei of astrocytes. Over time, however, these astrocytes gradually adopted neuronal morphology, and the neural transcription factors was gradually observed in the nuclei of neuron-like cells instead of astrocytes. Furthermore,we noted that transcription factor-infected astrocytes showed a progressive decrease in the expression of astrocytic markers AQP4(astrocyte endfeet signal), CX43(gap junction signal), and S100β. Importantly, none of these changes could be attributed to transgene leakage into preexisting neurons. Therefore, our findings suggest that neural transcription factors such as NeuroD1, Ascl1, and Dlx2 can effectively convert reactive astrocytes into neurons in the adult mammalian brain.
基金supported by the National Key R&D Program of China(2021YFA0805000,2023YFA1800600,2019YFE0106700)the National Natural Science Foundation of China(92268114,31970782,32070819)+1 种基金the High-level Hospital Construction Project of GuangdongProvincial People'sHospital(DFJHBF202103 and KJ012021012)BGI grant(BGIRSZ20210002).
文摘The axolotl is broadly used in regenerative, developmental, and evolutionary biology research. Targeted gene knock-in is crucial for precision transgenesis, enabling disease modeling, visualization, tracking, and functional manipulation of specific cells or genes of interest(GOIs). Existing CRISPR/Cas9-mediated homology-independent method for gene knock-in often causes “scars/indels” at integration junctions.Here, we develop a CRISPR/Cas9-mediated semi-homology-directed recombination(HDR) knock-in method using a donor construct containing a single homology arm for the precise integration of GOIs.This semi-HDR approach achieves seamless single-end integration of the Cherry reporter gene and a large inducible Cre cassette into intronless genes like Sox2 and Neurod6 in axolotls, which are challenging to modify with the homology-independent method. Additionally, we integrate the inducible Cre cassette into intron-containing loci(e.g., Nkx2.2 and FoxA2) without introducing indels via semi-HDR. GOIs are properly expressed in F0 founders, with approximately 5%-10% showing precise integration confirmed by genotyping. Furthermore, using the Nkx2.2:CreER^(T2)line, we fate-map spinal cord p3 neural progenitor cells,revealing that Nkx2.2^(+) cells adopt different lineages in development and regeneration, preferentially generating motoneurons over oligodendrocytes during regeneration. Overall, this semi-HDR method balances efficiency and precision in the integration of GOIs, providing a valuable tool for generating knock-in axolotls and potentially extending to other species.
基金supported by the National Natural Science Foundation of China,No.31970906(to WLei)the Natural Science Foundation of Guangdong Province,No.2020A1515011079(to WLei)+4 种基金Key Technologies R&D Program of Guangdong Province,No.2018B030332001(to GC)Science and Technology Projects of Guangzhou,No.202206060002(to GC)the Youth Science Program of the National Natural Science Foundation of China,No.32100793(to ZX)the Pearl River Innovation and Entrepreneurship Team,No.2021ZT09 Y552Yi-Liang Liu Endowment Fund from Jinan University Education Development Foundation。
文摘Over the past decade,a growing number of studies have reported transcription factor-based in situ reprogramming that can directly conve rt endogenous glial cells into functional neurons as an alternative approach for n euro regeneration in the adult mammalian central ne rvous system.Howeve r,many questions remain regarding how a terminally differentiated glial cell can transform into a delicate neuron that forms part of the intricate brain circuitry.In addition,concerns have recently been raised around the absence of astrocyte-to-neuron conversion in astrocytic lineage-tra cing mice.In this study,we employed repetitive two-photon imaging to continuously capture the in situ astrocyte-to-neuron conversion process following ecto pic expression of the neural transcription factor NeuroD1 in both prolife rating reactive astrocytes and lineage-tra ced astrocytes in the mouse cortex.Time-lapse imaging over several wee ks revealed the ste p-by-step transition from a typical astrocyte with numero us short,tapered branches to a typical neuro n with a few long neurites and dynamic growth cones that actively explored the local environment.In addition,these lineage-converting cells were able to migrate ra dially or to ngentially to relocate to suitable positions.Furthermore,two-photon Ca2+imaging and patch-clamp recordings confirmed that the newly generated neuro ns exhibited synchronous calcium signals,repetitive action potentials,and spontaneous synaptic responses,suggesting that they had made functional synaptic connections within local neural circuits.In conclusion,we directly visualized the step-by-step lineage conversion process from astrocytes to functional neurons in vivo and unambiguously demonstrated that adult mammalian brains are highly plastic with respect to their potential for neuro regeneration and neural circuit reconstruction.
文摘1文献来源研究一:Rudin CM,Poirier JT,Byers LA,et al.Molecular subtypes of small cell lung cancer:A synthesis of human and mouse model data[J].Nat Rev Cancer,2019,19(5):289-297.研究二:Owonikoko TK,Dwivedi B,Chen ZJ,et al.YAP1 positive small-cell lung cancer subtype is associated with the T-cell inflamed gene expression profile and confers good prognosis and long term survival[J].J Clin Oncol,2020,38(15S):Abstr 9019.
