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1.
Nat Commun ; 11(1): 4673, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938917

RESUMO

RAS-MAPK signaling mediates processes critical to normal development including cell proliferation, survival, and differentiation. Germline mutation of RAS-MAPK genes lead to the Noonan-spectrum of syndromes. Here, we present a patient affected by a 6p-interstitial microdeletion with unknown underlying molecular etiology. Examination of 6p-interstitial microdeletion cases reveals shared clinical features consistent with Noonan-spectrum disorders including short stature, facial dysmorphia and cardiovascular abnormalities. We find the RAS-responsive element binding protein-1 (RREB1) is the common deleted gene in multiple 6p-interstitial microdeletion cases. Rreb1 hemizygous mice display orbital hypertelorism and cardiac hypertrophy phenocopying the human syndrome. Rreb1 haploinsufficiency leads to sensitization of MAPK signaling. Rreb1 recruits Sin3a and Kdm1a to control H3K4 methylation at MAPK pathway gene promoters. Haploinsufficiency of SIN3A and mutations in KDM1A cause syndromes similar to RREB1 haploinsufficiency suggesting genetic perturbation of the RREB1-SIN3A-KDM1A complex represents a new category of RASopathy-like syndromes arising through epigenetic reprogramming of MAPK pathway genes.


Assuntos
Proteínas de Ligação a DNA/genética , Haploinsuficiência , Sistema de Sinalização das MAP Quinases/genética , Síndrome de Noonan/etiologia , Fatores de Transcrição/genética , Proteínas ras/metabolismo , Anormalidades Múltiplas/genética , Animais , Deleção Cromossômica , Cromossomos Humanos Par 6 , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histonas/metabolismo , Humanos , Masculino , Metilação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo Correpressor Histona Desacetilase e Sin3/genética , Complexo Correpressor Histona Desacetilase e Sin3/metabolismo , Fatores de Transcrição/metabolismo , Proteínas ras/genética
2.
Nature ; 585(7824): 251-255, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848248

RESUMO

Mutation of C9orf72 is the most prevalent defect associated with amyotrophic lateral sclerosis and frontotemporal degeneration1. Together with hexanucleotide-repeat expansion2,3, haploinsufficiency of C9orf72 contributes to neuronal dysfunction4-6. Here we determine the structure of the C9orf72-SMCR8-WDR41 complex by cryo-electron microscopy. C9orf72 and SMCR8 both contain longin and DENN (differentially expressed in normal and neoplastic cells) domains7, and WDR41 is a ß-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and the DENN domain of SMCR8 drive the lysosomal localization of the complex in conditions of amino acid starvation. The structure suggested that C9orf72-SMCR8 is a GTPase-activating protein (GAP), and we found that C9orf72-SMCR8-WDR41 acts as a GAP for the ARF family of small GTPases. These data shed light on the function of C9orf72 in normal physiology, and in amyotrophic lateral sclerosis and frontotemporal degeneration.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteínas Relacionadas à Autofagia/química , Proteína C9orf72/química , Proteína C9orf72/genética , Proteínas de Transporte/química , Microscopia Crioeletrônica , Demência Frontotemporal/genética , Haploinsuficiência , Complexos Multiproteicos/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/ultraestrutura , Proteína C9orf72/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , Demência Frontotemporal/metabolismo , Humanos , Lisossomos/metabolismo , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Domínios Proteicos
3.
PLoS One ; 15(6): e0234910, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32559200

RESUMO

Aging in mammals is the gradual decline of an organism's physical, mental, and physiological capacity. Aging leads to increased risk for disease and eventually to death. Here, we show that Brd2 haploinsufficiency (Brd2+/-) extends lifespan and increases healthspan in C57B6/J mice. In Brd2+/- mice, longevity is increased by 23% (p<0.0001), and, relative to wildtype animals (Brd2+/+), cancer incidence is reduced by 43% (p<0.001). In addition, relative to age-matched wildtype mice, Brd2 heterozygotes show healthier aging including: improved grooming, extended period of fertility, and lack of age-related decline in kidney function and morphology. Our data support a role for haploinsufficiency of Brd2 in promoting healthy aging. We hypothesize that Brd2 affects aging by protecting against the accumulation of molecular and cellular damage. Given the recent advances in the development of BET inhibitors, our research provides impetus to test drugs that target BRD2 as a way to understand and treat/prevent age-related diseases.


Assuntos
Longevidade/genética , Fatores de Transcrição/genética , Animais , Feminino , Fertilidade , Asseio Animal , Haploinsuficiência , Rim/crescimento & desenvolvimento , Rim/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
4.
Nat Commun ; 11(1): 3018, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32541668

RESUMO

Chronic pancreatitis represents a risk factor for the development of pancreatic cancer. We find that heterozygous loss of histone H2A lysine 119 deubiquitinase BAP1 (BRCA1 Associated Protein-1) associates with a history of chronic pancreatitis and occurs in 25% of pancreatic ductal adenocarcinomas and 40% of acinar cell carcinomas. Deletion or heterozygous loss of Bap1 in murine pancreata causes genomic instability, tissue damage, and pancreatitis with full penetrance. Concomitant expression of KrasG12D leads to predominantly intraductal papillary mucinous neoplasms and mucinous cystic neoplasms, while pancreatic intraepithelial neoplasias are rarely detected. These lesions progress to metastatic pancreatic cancer with high frequency. Lesions with histological features mimicking Acinar Cell Carcinomas are also observed in some tumors. Heterozygous mice also develop pancreatic cancer suggesting a haploinsufficient tumor suppressor role for BAP1. Mechanistically, BAP1 regulates genomic stability, in a catalytic independent manner, and its loss confers sensitivity to irradiation and platinum-based chemotherapy in pancreatic cancer.


Assuntos
Neoplasias Pancreáticas/metabolismo , Pancreatite Crônica/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Estudos de Coortes , Regulação Neoplásica da Expressão Gênica , Haploinsuficiência , Humanos , Camundongos , Neoplasias Pancreáticas/genética , Pancreatite Crônica/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética
5.
Am J Hum Genet ; 107(1): 164-172, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32553196

RESUMO

CNOT1 is a member of the CCR4-NOT complex, which is a master regulator, orchestrating gene expression, RNA deadenylation, and protein ubiquitination. We report on 39 individuals with heterozygous de novo CNOT1 variants, including missense, splice site, and nonsense variants, who present with a clinical spectrum of intellectual disability, motor delay, speech delay, seizures, hypotonia, and behavioral problems. To link CNOT1 dysfunction to the neurodevelopmental phenotype observed, we generated variant-specific Drosophila models, which showed learning and memory defects upon CNOT1 knockdown. Introduction of human wild-type CNOT1 was able to rescue this phenotype, whereas mutants could not or only partially, supporting our hypothesis that CNOT1 impairment results in neurodevelopmental delay. Furthermore, the genetic interaction with autism-spectrum genes, such as ASH1L, DYRK1A, MED13, and SHANK3, was impaired in our Drosophila models. Molecular characterization of CNOT1 variants revealed normal CNOT1 expression levels, with both mutant and wild-type alleles expressed at similar levels. Analysis of protein-protein interactions with other members indicated that the CCR4-NOT complex remained intact. An integrated omics approach of patient-derived genomics and transcriptomics data suggested only minimal effects on endonucleolytic nonsense-mediated mRNA decay components, suggesting that de novo CNOT1 variants are likely haploinsufficient hypomorph or neomorph, rather than dominant negative. In summary, we provide strong evidence that de novo CNOT1 variants cause neurodevelopmental delay with a wide range of additional co-morbidities. Whereas the underlying pathophysiological mechanism warrants further analysis, our data demonstrate an essential and central role of the CCR4-NOT complex in human brain development.


Assuntos
Deficiências do Desenvolvimento/genética , Expressão Gênica/genética , Transtornos do Neurodesenvolvimento/genética , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , RNA/genética , Receptores CCR4/genética , Fatores de Transcrição/genética , Alelos , Feminino , Variação Genética/genética , Haploinsuficiência/genética , Heterozigoto , Humanos , Masculino , Malformações do Sistema Nervoso/genética , Fenótipo , Estabilidade Proteica
6.
Nat Commun ; 11(1): 2289, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385263

RESUMO

The osteoblast differentiation capacity of skeletal stem cells (SSCs) must be tightly regulated, as inadequate bone formation results in low bone mass and skeletal fragility, and over-exuberant osteogenesis results in heterotopic ossification (HO) of soft tissues. RUNX2 is essential for tuning this balance, but the mechanisms of posttranslational control of RUNX2 remain to be fully elucidated. Here, we identify that a CK2/HAUSP pathway is a key regulator of RUNX2 stability, as Casein kinase 2 (CK2) phosphorylates RUNX2, recruiting the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP), which stabilizes RUNX2 by diverting it away from ubiquitin-dependent proteasomal degradation. This pathway is important for both the commitment of SSCs to osteoprogenitors and their subsequent maturation. This CK2/HAUSP/RUNX2 pathway is also necessary for HO, as its inhibition blocked HO in multiple models. Collectively, active deubiquitination of RUNX2 is required for bone formation and this CK2/HAUSP deubiquitination pathway offers therapeutic opportunities for disorders of inappropriate mineralization.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Ossificação Heterotópica/metabolismo , Osteogênese , Adulto , Idoso , Animais , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Diferenciação Celular , Displasia Cleidocraniana/genética , Displasia Cleidocraniana/patologia , Feminino , Deleção de Genes , Haploinsuficiência/genética , Membro Posterior/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Ossificação Heterotópica/genética , Ossificação Heterotópica/patologia , Osteoblastos/metabolismo , Fosforilação , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo
7.
PLoS Biol ; 18(4): e3000717, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32353004

RESUMO

Extensive evidence links Glutamate receptor, ionotropic, NMDA2B (GRIN2B), encoding the GluN2B/NR2B subunit of N-methyl-D-aspartate receptors (NMDARs), with various neurodevelopmental disorders, including autism spectrum disorders (ASDs), but the underlying mechanisms remain unclear. In addition, it remains unknown whether mutations in GluN2B, which starts to be expressed early in development, induces early pathophysiology that can be corrected by early treatments for long-lasting effects. We generated and characterized Grin2b-mutant mice that carry a heterozygous, ASD-risk C456Y mutation (Grin2b+/C456Y). In Grin2b+/C456Y mice, GluN2B protein levels were strongly reduced in association with decreased hippocampal NMDAR currents and NMDAR-dependent long-term depression (LTD) but unaltered long-term potentiation, indicative of mutation-induced protein degradation and LTD sensitivity. Behaviorally, Grin2b+/C456Y mice showed normal social interaction but exhibited abnormal anxiolytic-like behavior. Importantly, early, but not late, treatment of young Grin2b+/C456Y mice with the NMDAR agonist D-cycloserine rescued NMDAR currents and LTD in juvenile mice and improved anxiolytic-like behavior in adult mice. Therefore, GluN2B-C456Y haploinsufficiency decreases GluN2B protein levels, NMDAR-dependent LTD, and anxiety-like behavior, and early activation of NMDAR function has long-lasting effects on adult mouse behavior.


Assuntos
Ansiedade/genética , Hipocampo/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Receptores de N-Metil-D-Aspartato/genética , Animais , Ansiedade/fisiopatologia , Comportamento Animal/efeitos dos fármacos , Ciclosserina/farmacologia , Potenciais Pós-Sinápticos Excitadores/genética , Técnicas de Introdução de Genes , Haploinsuficiência/genética , Heterozigoto , Hipocampo/metabolismo , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Camundongos Mutantes , Mutação , Proteínas do Tecido Nervoso/metabolismo , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/metabolismo
8.
PLoS Genet ; 16(5): e1008832, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463833

RESUMO

Dysregulation of CDK8 (Cyclin-Dependent Kinase 8) and its regulatory partner CycC (Cyclin C), two subunits of the conserved Mediator (MED) complex, have been linked to diverse human diseases such as cancer. Thus, it is essential to understand the regulatory network modulating the CDK8-CycC complex in both normal development and tumorigenesis. To identify upstream regulators or downstream effectors of CDK8, we performed a dominant modifier genetic screen in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in developing wing imaginal discs. We identified 26 genomic loci whose haploinsufficiency can modify these CDK8- or CycC-specific phenotypes. Further analysis of two overlapping deficiency lines and mutant alleles led us to identify genetic interactions between the CDK8-CycC pair and the components of the Decapentaplegic (Dpp, the Drosophila homolog of TGFß, or Transforming Growth Factor-ß) signaling pathway. We observed that CDK8-CycC positively regulates transcription activated by Mad (Mothers against dpp), the primary transcription factor downstream of the Dpp/TGFß signaling pathway. CDK8 can directly interact with Mad in vitro through the linker region between the DNA-binding MH1 (Mad homology 1) domain and the carboxy terminal MH2 (Mad homology 2) transactivation domain. Besides CDK8 and CycC, further analyses of other subunits of the MED complex have revealed six additional subunits that are required for Mad-dependent transcription in the wing discs: Med12, Med13, Med15, Med23, Med24, and Med31. Furthermore, our analyses confirmed the positive roles of CDK9 and Yorkie in regulating Mad-dependent gene expression in vivo. These results suggest that CDK8 and CycC, together with a few other subunits of the MED complex, may coordinate with other transcription cofactors in regulating Mad-dependent transcription during wing development in Drosophila.


Assuntos
Ciclina C/genética , Quinase 8 Dependente de Ciclina/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Transcrição/metabolismo , Animais , Ciclina C/metabolismo , Quinase 8 Dependente de Ciclina/metabolismo , Drosophila , Regulação da Expressão Gênica no Desenvolvimento , Haploinsuficiência , Discos Imaginais/crescimento & desenvolvimento , Discos Imaginais/metabolismo , Transdução de Sinais , Transcrição Genética
9.
Nature ; 581(7809): 452-458, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32461655

RESUMO

The acceleration of DNA sequencing in samples from patients and population studies has resulted in extensive catalogues of human genetic variation, but the interpretation of rare genetic variants remains problematic. A notable example of this challenge is the existence of disruptive variants in dosage-sensitive disease genes, even in apparently healthy individuals. Here, by manual curation of putative loss-of-function (pLoF) variants in haploinsufficient disease genes in the Genome Aggregation Database (gnomAD)1, we show that one explanation for this paradox involves alternative splicing of mRNA, which allows exons of a gene to be expressed at varying levels across different cell types. Currently, no existing annotation tool systematically incorporates information about exon expression into the interpretation of variants. We develop a transcript-level annotation metric known as the 'proportion expressed across transcripts', which quantifies isoform expression for variants. We calculate this metric using 11,706 tissue samples from the Genotype Tissue Expression (GTEx) project2 and show that it can differentiate between weakly and highly evolutionarily conserved exons, a proxy for functional importance. We demonstrate that expression-based annotation selectively filters 22.8% of falsely annotated pLoF variants found in haploinsufficient disease genes in gnomAD, while removing less than 4% of high-confidence pathogenic variants in the same genes. Finally, we apply our expression filter to the analysis of de novo variants in patients with autism spectrum disorder and intellectual disability or developmental disorders to show that pLoF variants in weakly expressed regions have similar effect sizes to those of synonymous variants, whereas pLoF variants in highly expressed exons are most strongly enriched among cases. Our annotation is fast, flexible and generalizable, making it possible for any variant file to be annotated with any isoform expression dataset, and will be valuable for the genetic diagnosis of rare diseases, the analysis of rare variant burden in complex disorders, and the curation and prioritization of variants in recall-by-genotype studies.


Assuntos
Doença/genética , Haploinsuficiência/genética , Mutação com Perda de Função/genética , Anotação de Sequência Molecular , Transcrição Genética , Transcriptoma/genética , Transtorno do Espectro Autista/genética , Conjuntos de Dados como Assunto , Deficiências do Desenvolvimento/genética , Éxons/genética , Feminino , Genótipo , Humanos , Deficiência Intelectual/genética , Masculino , Anotação de Sequência Molecular/normas , Distribuição de Poisson , RNA Mensageiro/análise , RNA Mensageiro/genética , Doenças Raras/diagnóstico , Doenças Raras/genética , Reprodutibilidade dos Testes , Sequenciamento Completo do Exoma
10.
PLoS One ; 15(5): e0232789, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32407325

RESUMO

BAHD1 is a heterochomatinization factor recently described as a component of a multiprotein complex associated with histone deacetylases HDAC1/2. The physiological and patho-physiological functions of BAHD1 are not yet well characterized. Here, we examined the consequences of BAHD1 deficiency in the brains of male mice. While Bahd1 knockout mice had no detectable defects in brain anatomy, RNA sequencing profiling revealed about 2500 deregulated genes in Bahd1-/- brains compared to Bahd1+/+ brains. A majority of these genes were involved in nervous system development and function, behavior, metabolism and immunity. Exploration of the Allen Brain Atlas and Dropviz databases, assessing gene expression in the brain, revealed that expression of the Bahd1 gene was limited to a few territories and cell subtypes, particularly in the hippocampal formation, the isocortex and the olfactory regions. The effect of partial BAHD1 deficiency on behavior was then evaluated on Bahd1 heterozygous male mice, which have no lethal or metabolic phenotypes. Bahd1+/- mice showed anxiety-like behavior and reduced prepulse inhibition (PPI) of the startle response. Altogether, these results suggest that BAHD1 plays a role in chromatin-dependent gene regulation in a subset of brain cells and support recent evidence linking genetic alteration of BAHD1 to psychiatric disorders in a human patient.


Assuntos
Ansiedade/genética , Encéfalo/metabolismo , Proteínas Cromossômicas não Histona/genética , Reflexo de Sobressalto/genética , Animais , Ansiedade/fisiopatologia , Encéfalo/patologia , Cromatina/genética , Regulação da Expressão Gênica/genética , Haploinsuficiência/genética , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Humanos , Camundongos , Camundongos Knockout , Fenótipo , Análise de Sequência de RNA
11.
Zhonghua Er Ke Za Zhi ; 58(3): 218-222, 2020 Mar 02.
Artigo em Chinês | MEDLINE | ID: mdl-32135594

RESUMO

Objective: To explore the clinical phenotype, immunological features, pathogenesis and gene variation of a case with A20 haploinsufficiency (HA20). Methods: A patient diagnosed with tumor necrosis factor α-induced protein 3 (TNFAIP3) mutated HA20 was admitted into Shenzhen Children's Hospital in May,2019.The clinical data was analyzed. Flow cytometry was used to detect the patient's peripheral blood lymphocyte subsets, and also, the percentage of follicular helper T cell (TFH) cells in the patient and thirteen healthy controls. After the construction of empty vector, wild-type and mutant plasmid vectors, a wild-type or mutant overexpression system of the TNFAIP3 gene was established in 293T cells and Hela cells. Then, the expression level of A20 in 293T cells and the expression of inhibitor K binding α (IKBα) in green fluorescent protein (GFP)+Hela cells before and after tumor necrosis factor α (TNF-α) stimulation were measured, to verify the pathogenicity of this variation. Results: A 5 years and 11 months old boy, presented with recurrent oral ulcer, abdominal pain, joint swelling and arthralgia. Oral ulcer, chronic skin rashes, knee joint swelling were observed. The levels of inflammatory markers were increased. Colonoscopy showed congestion of mucosa and multiple ulcers in terminal ileum and ileocecus. The absolute number of naive B cells was 124×10(6) cells/L (reference range 147×10(6)-431×10(6) cells/L), accounting for 0.430 of the total B cells (reference range 0.484-0.758). Compared to healthy controls (0.016-0.071), the percentage of TFH cells in CD4(+)T cells was much lower (0.008).A heterozygous mutation of TNFAIP3 gene (c.909_913 del, p.L303fs) was identified by genetic analysis. In vitro study showed that truncated A20 protein was expressed in TNFAIP3 mutant overexpressed 293T cells, which verified the pathogenicity of this variation. Besides, after TNF-α stimulation, the degradation rate of IkBα protein in mutant overexpressed Hela cells (35%) was between the other two groups (15% in the wild-type group and 57% in the non-loaded group). Conclusions: This case with HA20 due to a de novo TNFAIP3 gene mutation presents with early onset Behcet-like autoinflammatory syndrome. This variation leads to expression of truncated A20 protein, enhanced degradation of IkBα, and further activation of nuclear factor κB signaling pathway.


Assuntos
Doenças Autoimunes , Haploinsuficiência , NF-kappa B , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/genética , Pré-Escolar , Haploinsuficiência/genética , Células HeLa , Heterozigoto , Humanos , Masculino , Fenótipo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética
12.
Am J Pathol ; 190(3): 602-613, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32113662

RESUMO

Casitas B-lineage lymphoma (c-Cbl) is a recently identified ubiquitin ligase of nuclear ß-catenin and a suppressor of colorectal cancer (CRC) growth in cell culture and mouse tumor xenografts. We hypothesized that reduction in c-Cbl in colonic epithelium is likely to increase the levels of nuclear ß-catenin in the intestinal crypt, augmenting CRC tumorigenesis in an adenomatous polyposis coli (APCΔ14/+) mouse model. Haploinsufficient c-Cbl mice (APCΔ14/+ c-Cbl+/-) displayed a significant (threefold) increase in atypical hyperplasia and adenocarcinomas in the small and large intestines; however, no differences were noted in the adenoma frequency. In contrast to the APCΔ14/+ c-Cbl+/+ mice, APCΔ14/+ c-Cbl+/- crypts showed nuclear ß-catenin throughout the length of the crypts and up-regulation of Axin2, a canonical Wnt target gene, and SRY-box transcription factor 9, a marker of intestinal stem cells. In contrast, haploinsufficiency of c-Cbl+/- alone was insufficient to induce tumorigenesis regardless of an increase in the number of intestinal epithelial cells with nuclear ß-catenin and SRY-box transcription factor 9 in APC+/+ c-Cbl+/- mice. This study demonstrates that haploinsufficiency of c-Cbl results in Wnt hyperactivation in intestinal crypts and accelerates CRC progression to adenocarcinoma in the milieu of APCΔ14/+, a phenomenon not found with wild-type APC. While emphasizing the role of APC as a gatekeeper in CRC, this study also demonstrates that combined partial loss of c-Cbl and inactivation of APC significantly contribute to CRC tumorigenesis.


Assuntos
Adenocarcinoma/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Neoplasias do Colo/genética , Neoplasias Colorretais/genética , Haploinsuficiência , Linfoma/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Adenocarcinoma/patologia , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Carcinogênese , Neoplasias do Colo/patologia , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Linfoma/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
13.
Metabolism ; 107: 154215, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32209360

RESUMO

OBJECTIVE: PTEN haploinsufficiency plays an important role in prostate cancer development in men. However, monoallelic deletion of Pten gene failed to induce high prostate intraepithelial neoplasia (PIN) until Pten+/- mice aged or fed a high-calorie diet. Because CEACAM1, a cell adhesion molecule with a potential tumor suppression activity, is induced in Pten+/- prostates, the study aimed at examining whether the rise of CEACAM1 limited neoplastic progression in Pten+/- prostates. METHODS: Pten+/- were crossbred with Cc1-/- mice harboring a null deletion of Ceacam1 gene to produce Pten+/-/Cc1-/- double mutants. Prostates from 7-month old male mice were analyzed histologically and biochemically for PIN progression. RESULTS: Deleting Ceacam1 in Pten+/- mice caused an early development of high-grade PIN in parallel to hyperactivation of PI3 kinase/Akt and Ras/MAP kinase pathways, with an increase in cell proliferation, epithelial-to-mesenchymal transition, angiogenesis and inflammation relative to Pten+/- and Cc1-/- individual mutants. It also caused a remarkable increase in lipogenesis in prostate despite maintaining insulin sensitivity. Concomitant Ceacam1 deletion with Pten+/- activated the IL-6/STAT3 signaling pathways to suppress Irf-8 transcription that in turn, led to a decrease in the expression level of promyelocytic leukemia gene, a well characterized tumor suppressor in prostate. CONCLUSIONS: Ceacam1 deletion accelerated high-grade prostate intraepithelial neoplasia in Pten haploinsufficient mice while preserving insulin sensitivity. This demonstrated that the combined loss of Ceacam1 and Pten advanced prostate cancer by increasing lipogenesis and modifying the STAT3-dependent inflammatory microenvironment of prostate.


Assuntos
Antígeno Carcinoembrionário/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/genética , Animais , Progressão da Doença , Haploinsuficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Proteína Oncogênica v-akt/genética , Fosfatidilinositol 3-Quinases/genética , Neoplasias da Próstata/patologia , Fator de Transcrição STAT3/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
15.
BMC Biol ; 18(1): 27, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32164660

RESUMO

BACKGROUND: CRISPR gene drive systems allow the rapid spread of a genetic construct throughout a population. Such systems promise novel strategies for the management of vector-borne diseases and invasive species by suppressing a target population or modifying it with a desired trait. However, current homing-type drives have two potential shortcomings. First, they can be thwarted by the rapid evolution of resistance. Second, they lack any mechanism for confinement to a specific target population. In this study, we conduct a comprehensive performance assessment of several new types of CRISPR-based gene drive systems employing toxin-antidote (TA) principles, which should be less prone to resistance and allow for the confinement of drives to a target population due to invasion frequency thresholds. RESULTS: The underlying principle of the proposed CRISPR toxin-antidote gene drives is to disrupt an essential target gene while also providing rescue by a recoded version of the target as part of the drive allele. Thus, drive alleles tend to remain viable, while wild-type targets are disrupted and often rendered nonviable, thereby increasing the relative frequency of the drive allele. Using individual-based simulations, we show that Toxin-Antidote Recessive Embryo (TARE) drives targeting an haplosufficient but essential gene (lethal when both copies are disrupted) can enable the design of robust, regionally confined population modification strategies with high flexibility in choosing promoters and targets. Toxin-Antidote Dominant Embryo (TADE) drives require a haplolethal target gene and a germline-restricted promoter, but they could permit faster regional population modification and even regionally confined population suppression. Toxin-Antidote Dominant Sperm (TADS) drives can be used for population modification or suppression. These drives are expected to spread rapidly and could employ a variety of promoters, but unlike TARE and TADE, they would not be regionally confined and also require highly specific target genes. CONCLUSIONS: Overall, our results suggest that CRISPR-based TA gene drives provide promising candidates for flexible ecological engineering strategies in a variety of organisms.


Assuntos
Antídotos/farmacologia , Antitoxinas/farmacologia , Sistemas CRISPR-Cas , Tecnologia de Impulso Genético/métodos , Genes Essenciais , Haploinsuficiência , Modelos Genéticos
16.
PLoS Genet ; 16(1): e1008558, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31923184

RESUMO

Autophagy, particularly with BECN1, has paradoxically been highlighted as tumor promoting in Ras-driven cancers, but potentially tumor suppressing in breast and ovarian cancers. However, studying the specific role of BECN1 at the genetic level is complicated due to its genomic proximity to BRCA1 on both human (chromosome 17) and murine (chromosome 11) genomes. In human breast and ovarian cancers, the monoallelic deletion of these genes is often co-occurring. To investigate the potential tumor suppressor roles of two of the most commonly deleted autophagy genes in ovarian cancer, BECN1 and MAP1LC3B were knocked-down in atypical (BECN1+/+ and MAP1LC3B+/+) ovarian cancer cells. Ultra-performance liquid chromatography mass-spectrometry metabolomics revealed reduced levels of acetyl-CoA which corresponded with elevated levels of glycerophospholipids and sphingolipids. Migration rates of ovarian cancer cells were increased upon autophagy gene knockdown. Genomic instability was increased, resulting in copy-number alteration patterns which mimicked high grade serous ovarian cancer. We further investigated the causal role of Becn1 haploinsufficiency for oncogenesis in a MISIIR SV40 large T antigen driven spontaneous ovarian cancer mouse model. Tumors were evident earlier among the Becn1+/- mice, and this correlated with an increase in copy-number alterations per chromosome in the Becn1+/- tumors. The results support monoallelic loss of BECN1 as permissive for tumor initiation and potentiating for genomic instability in ovarian cancer.


Assuntos
Proteína Beclina-1/genética , Instabilidade Cromossômica , Haploinsuficiência , Proteínas Associadas aos Microtúbulos/genética , Neoplasias Ovarianas/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular , Feminino , Metaboloma , Camundongos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia
18.
Clin Dysmorphol ; 29(2): 97-100, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31929334

RESUMO

Deletion of the 1q43q44 chromosomal region has been related to a clinical syndrome characterized by neurodevelopmental delay, intellectual disability, microcephaly, congenital abnormality of the corpus callosum, and epilepsy and dysmorphic features. A wide variability of the clinical features have been linked to the contiguous deleted genes and incomplete penetrance has been observed too. Here, we report a 4-years-old boy with microcephaly, neurodevelopmental delay, and cardiac atrial septal defect, who had a de-novo 117 Kb 1q43-q44 microdeletion. The deleted chromosomal region encompassed the two genes SDCCAG8 and AKT3. The characteristics of the deletion and the clinical condition of the patient suggest a pathogenic role of the 1q43-q44 deletion, supporting a pivotal role of AKT3 gene in the expression of the clinical phenotype.


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 1 , Haploinsuficiência/genética , Microcefalia/diagnóstico , Microcefalia/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transtornos Psicomotores/diagnóstico , Transtornos Psicomotores/genética , Pré-Escolar , Hibridização Genômica Comparativa , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino
19.
Int J Mol Sci ; 21(2)2020 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-31963842

RESUMO

Mammalian telomere lengths are primarily regulated by telomerase, a ribonucleoprotein consisting of a reverse transcriptase (TERT) and an RNA subunit (TERC). TERC is constitutively expressed in all cells, whereas TERT expression is temporally and spatially regulated, such that in most adult somatic cells, TERT is inactivated and telomerase activity is undetectable. Most tumor cells activate TERT as a mechanism for preventing progressive telomere attrition to achieve proliferative immortality. Therefore, inactivating TERT has been considered to be a promising means of cancer therapy. Here we applied the CRISPR/Cas9 gene editing system to target the TERT gene in cancer cells. We report that disruption of TERT severely compromises cancer cell survival in vitro and in vivo. Haploinsufficiency of TERT in tumor cells is sufficient to result in telomere attrition and growth retardation in vitro. In vivo, TERT haploinsufficient tumor cells failed to form xenograft after transplantation to nude mice. Our work demonstrates that gene editing-mediated TERT knockout is a potential therapeutic option for treating cancer.


Assuntos
Técnicas de Inativação de Genes/métodos , Telomerase/genética , Telomerase/metabolismo , Neoplasias do Colo do Útero/patologia , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Feminino , Haploinsuficiência , Células HeLa , Humanos , Mutação INDEL , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo
20.
Pediatr Hematol Oncol ; 37(2): 176-183, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31971458

RESUMO

Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) haploinsufficiency (HI) is an immune dysregulation disorder characterized by T lymphocyte hyperactivation, and generalized lymphoproliferative and autoimmune disorders. Patients with CTLA4 haploinsufficiency are also prone to developing various malignancies. Due to the rare nature of this condition, the longitudinal evolution of CTLA4 haploinsufficiency clinical manifestations remains to be described. We report on over a decade-long course of a young adult men patient with known CTLA4 haploinsufficiency, who sequentially developed multiple autoimmune conditions along with two distinct episodes of classical Hodgkin Lymphoma (cHL) of unique histologies. Notably, the patient had serological and molecular evidence of Epstein Barr virus (EBV) infection at the time of diagnosis of both cHL. The allogeneic stem cell transplantation consolidated durable remission of cHL and CTLA-4 haploinsufficiency-related clinical manifestations, but failed to enable the patient to permanently suppress EBV viremia, which raises concerns for the development of other EBV infection-related conditions in the future. This case report adds on the current understanding of the natural clinical history of patients with CTLA4 haploinsufficiency, and their unique susceptibility to developing cHL.


Assuntos
Antígeno CTLA-4/genética , Doença de Hodgkin/genética , Adulto , Criança , Predisposição Genética para Doença , Haploinsuficiência , Doença de Hodgkin/patologia , Humanos , Masculino
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