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1.
Genet Med ; 23(9): 1636-1647, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34145395

RESUMO

PURPOSE: Much of the heredity of melanoma remains unexplained. We sought predisposing germline copy-number variants using a rare disease approach. METHODS: Whole-genome copy-number findings in patients with melanoma predisposition syndrome congenital melanocytic nevus were extrapolated to a sporadic melanoma cohort. Functional effects of duplications in PPP2R3B were investigated using immunohistochemistry, transcriptomics, and stable inducible cellular models, themselves characterized using RNAseq, quantitative real-time polymerase chain reaction (qRT-PCR), reverse phase protein arrays, immunoblotting, RNA interference, immunocytochemistry, proliferation, and migration assays. RESULTS: We identify here a previously unreported genetic susceptibility to melanoma and melanocytic nevi, familial duplications of gene PPP2R3B. This encodes PR70, a regulatory unit of critical phosphatase PP2A. Duplications increase expression of PR70 in human nevus, and increased expression in melanoma tissue correlates with survival via a nonimmunological mechanism. PPP2R3B overexpression induces pigment cell switching toward proliferation and away from migration. Importantly, this is independent of the known microphthalmia-associated transcription factor (MITF)-controlled switch, instead driven by C21orf91. Finally, C21orf91 is demonstrated to be downstream of MITF as well as PR70. CONCLUSION: This work confirms the power of a rare disease approach, identifying a previously unreported copy-number change predisposing to melanocytic neoplasia, and discovers C21orf91 as a potentially targetable hub in the control of phenotype switching.


Assuntos
Melanoma , Nevo , Neoplasias Cutâneas , Humanos , Imuno-Histoquímica , Melanoma/genética , Fenótipo , Neoplasias Cutâneas/genética
2.
Hum Mol Genet ; 27(11): 1927-1940, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29635513

RESUMO

Mutations in SNX14 cause the autosomal recessive cerebellar ataxia 20 (SCAR20). Mutations generally result in loss of protein although several coding region deletions have also been reported. Patient-derived fibroblasts show disrupted autophagy, but the precise function of SNX14 is unknown. The yeast homolog, Mdm1, functions in endoplasmic reticulum (ER)-lysosome/vacuole inter-organelle tethering, but functional conservation in mammals is still required. Here, we show that loss of SNX14 alters but does not block autophagic flux. In addition, we find that SNX14 is an ER-associated protein that functions in neutral lipid homeostasis and inter-organelle crosstalk. SNX14 requires its N-terminal transmembrane helices for ER localization, while the Phox homology (PX) domain is dispensable for subcellular localization. Both SNX14-mutant fibroblasts and SNX14KO HEK293 cells accumulate aberrant cytoplasmic vacuoles, suggesting defects in endolysosomal homeostasis. However, ER-late endosome/lysosome contact sites are maintained in SNX14KO cells, indicating that it is not a prerequisite for ER-endolysosomal tethering. Further investigation of SNX14- deficiency indicates general defects in neutral lipid metabolism. SNX14KO cells display distinct perinuclear accumulation of filipin in LAMP1-positive lysosomal structures indicating cholesterol accumulation. Consistent with this, SNX14KO cells display a slight but detectable decrease in cholesterol ester levels, which is exacerbated with U18666A. Finally, SNX14 associates with ER-derived lipid droplets (LD) following oleate treatment, indicating a role in ER-LD crosstalk. We therefore identify an important role for SNX14 in neutral lipid homeostasis between the ER, lysosomes and LDs that may provide an early intervention target to alleviate the clinical symptoms of SCAR20.


Assuntos
Retículo Endoplasmático/genética , Metabolismo dos Lipídeos/genética , Nexinas de Classificação/genética , Ataxias Espinocerebelares/genética , Autofagia/genética , Retículo Endoplasmático/metabolismo , Endossomos , Técnicas de Inativação de Genes , Células HEK293 , Homeostase/efeitos dos fármacos , Humanos , Proteínas de Filamentos Intermediários/genética , Gotículas Lipídicas/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Mutação , Ácido Oleico/farmacologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Nexinas de Classificação/deficiência , Nexinas de Classificação/metabolismo , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/fisiopatologia
3.
J Invest Dermatol ; 144(4): 811-819.e4, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-37802293

RESUMO

Mosaic variants in genes GNAQ or GNA11 lead to a spectrum of vascular and pigmentary diseases including Sturge-Weber syndrome, in which progressive postnatal neurological deterioration led us to seek biologically targeted therapeutics. Using two cellular models, we find that disease-causing GNAQ/11 variants hyperactivate constitutive and G-protein coupled receptor ligand-induced intracellular calcium signaling in endothelial cells. We go on to show that the aberrant ligand-activated intracellular calcium signal is fueled by extracellular calcium influx through calcium-release-activated channels. Treatment with targeted small interfering RNAs designed to silence the variant allele preferentially corrects both the constitutive and ligand-activated calcium signaling, whereas treatment with a calcium-release-activated channel inhibitor rescues the ligand-activated signal. This work identifies hyperactivated calcium signaling as the primary biological abnormality in GNAQ/11 mosaicism and paves the way for clinical trials with genetic or small molecule therapies.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Subunidades alfa de Proteínas de Ligação ao GTP , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Mutação , Cálcio , Células Endoteliais/metabolismo , Mosaicismo , Sinalização do Cálcio/genética , Ligantes
4.
J Invest Dermatol ; 144(3): 593-600.e7, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37716647

RESUMO

Among children with multiple congenital melanocytic nevi, 25% have no established genetic cause, of whom many develop a hyperproliferative and severely pruritic phenotype resistant to treatment. Gene fusions have been reported in individual cases of congenital melanocytic nevi. We studied 169 patients with congenital melanocytic nevi in this study, 38 of whom were double wild type for pathogenic NRAS/BRAF variants. Nineteen of these 38 patients had sufficient tissue to undergo RNA sequencing, which revealed mosaic BRAF fusions in 11 of 19 patients and mosaic RAF1 fusions in 1 of 19. Recurrently, fusions involved the loss of the 5´ regulatory domain of BRAF or RAF1 but preserved the kinase domain. We validated all cases and detected the fusions in two separate nevi in 5 of 12 patients, confirming clonality. The absence of the fusion in blood in 8 of 12 patients indicated mosaicism. Primary culture of BRAF-fusion nevus cells from 3 of 12 patients demonstrated highly increased MAPK activation, despite only mildly increased BRAF expression, suggesting additional mechanisms of kinase activation. Trametinib quenched MAPK hyperactivation in vitro, and treatment of two patients caused rapid improvement in bulk tissue, improving bodily movement and reducing inflammation and severe pruritus. These findings offer a genetic diagnosis to an additional group of patients and trametinib as a treatment option for the severe associated phenotypes.


Assuntos
Nevo de Células Epitelioides e Fusiformes , Nevo Pigmentado , Neoplasias Cutâneas , Criança , Humanos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Mutação , Nevo Pigmentado/tratamento farmacológico , Nevo Pigmentado/genética , Nevo Pigmentado/congênito
5.
J Invest Dermatol ; 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38897541

RESUMO

RAS proteins regulate cell division, differentiation, and apoptosis through multiple downstream effector pathways. Oncogenic RAS variants are the commonest drivers in cancers; however, they also drive many benign lesions predisposing to malignancy, such as melanocytic nevi, thyroid nodules, and colonic polyps. Reversal of these benign lesions could reduce cancer incidence; however, the effects of oncogenic RAS have been notoriously difficult to target with downstream pathway inhibitors. In this study, we show effective suppression of oncogenic and currently undruggable NRASQ61K in primary cells from melanocytic nevi using small interfering RNA targeted to the recurrent causal variant. This results in striking reduction in expression of ARL6IP1, a known inhibitor of endoplasmic reticulum stress-induced apoptosis not previously linked to NRAS. We go on to show that a single dose of small interfering RNA in primary cells triggers an apoptotic cascade, in contrast to treatment with a MAPK/extracellular signal-regulated kinase kinase inhibitor. Protective packaging of the targeted small interfering RNA into lipid nanoparticles permits successful delivery into a humanized mouse model of melanocytic nevi and results in variant NRAS knockdown in vivo. These data show that RAS-induced protection from apoptosis is involved in persistence of NRAS-driven melanocytic nevi and anticipate that targeted small interfering RNA could form the basis of clinical trials for RAS-driven benign tumors.

6.
JCI Insight ; 7(3)2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35132964

RESUMO

Norrie disease is caused by mutation of the NDP gene, presenting as congenital blindness followed by later onset of hearing loss. Protecting patients from hearing loss is critical for maintaining their quality of life. This study aimed to understand the onset of pathology in cochlear structure and function. By investigating patients and juvenile Ndp-mutant mice, we elucidated the sequence of onset of physiological changes (in auditory brainstem responses, distortion product otoacoustic emissions, endocochlear potential, blood-labyrinth barrier integrity) and determined the cellular, histological, and ultrastructural events leading to hearing loss. We found that cochlear vascular pathology occurs earlier than previously reported and precedes sensorineural hearing loss. The work defines a disease mechanism whereby early malformation of the cochlear microvasculature precedes loss of vessel integrity and decline of endocochlear potential, leading to hearing loss and hair cell death while sparing spiral ganglion cells. This provides essential information on events defining the optimal therapeutic window and indicates that early intervention is needed. In an era of advancing gene therapy and small-molecule technologies, this study establishes Ndp-mutant mice as a platform to test such interventions and has important implications for understanding the progression of hearing loss in Norrie disease.


Assuntos
Cegueira/congênito , Gerenciamento Clínico , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Previsões , Doenças Genéticas Ligadas ao Cromossomo X/fisiopatologia , Perda Auditiva Neurossensorial/fisiopatologia , Audição/fisiologia , Doenças do Sistema Nervoso/fisiopatologia , Degeneração Retiniana/fisiopatologia , Espasmos Infantis/fisiopatologia , Adolescente , Adulto , Animais , Cegueira/complicações , Cegueira/fisiopatologia , Cegueira/terapia , Criança , Pré-Escolar , Modelos Animais de Doenças , Feminino , Seguimentos , Doenças Genéticas Ligadas ao Cromossomo X/complicações , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/etiologia , Humanos , Masculino , Camundongos , Camundongos Mutantes , Doenças do Sistema Nervoso/complicações , Doenças do Sistema Nervoso/terapia , Degeneração Retiniana/complicações , Degeneração Retiniana/terapia , Espasmos Infantis/complicações , Espasmos Infantis/terapia , Adulto Jovem
7.
Sci Rep ; 10(1): 13763, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792680

RESUMO

Mutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. Studies implicating the phenotypic consequences of SNX14 mutations to be consequences of subcellular disruption to autophagy and lipid metabolism have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) have also been conducted, demonstrated an important biochemical role during lipid biogenesis. In this study we report the effect of loss of SNX14 in mice, which resulted in embryonic lethality around mid-gestation due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. In contrast to other vertebrates, zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation were both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the consequences of loss of function varies between species. Mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo.


Assuntos
Viabilidade Fetal/genética , Metabolismo dos Lipídeos/genética , Placenta/anormalidades , Nexinas de Classificação/genética , Ataxias Espinocerebelares/genética , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Desenvolvimento Embrionário/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Fenótipo , Fosfolipídeos/sangue , Gravidez , Trofoblastos/citologia , Peixe-Zebra
8.
F1000Res ; 8: 273, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31231513

RESUMO

Background: Lenz-Majewski syndrome (LMS) is characterized by osteosclerosis and hyperostosis of skull, vertebrae and tubular bones as well as craniofacial, dental, cutaneous, and digit abnormalities. We previously found that LMS is caused by de novo dominant missense mutations in the  PTDSS1 gene, which encodes phosphatidylserine synthase 1 (PSS1), an enzyme that catalyses the conversion of phosphatidylcholine to phosphatidylserine. The mutations causing LMS result in a gain-of-function, leading to increased enzyme activity and blocking end-product inhibition of PSS1. Methods: Here, we have used transpose-mediated transgenesis to attempt to stably express wild-type and mutant forms of human PTDSS1 ubiquitously or specifically in chondrocytes, osteoblasts or osteoclasts in zebrafish. Results: We report multiple genomic integration sites for each of 8 different transgenes. While we confirmed that the ubiquitously driven transgene constructs were functional in terms of driving gene expression following transient transfection in HeLa cells, and that all lines exhibited expression of a heart-specific cistron within the transgene, we failed to detect PTDSS1 gene expression at either the RNA or protein levels in zebrafish. All wild-type and mutant transgenic lines of zebrafish exhibited mild scoliosis with variable incomplete penetrance which was never observed in non-transgenic animals. Conclusions: Collectively the data suggest that the transgenes are silenced, that animals with integrations that escape silencing are not viable, or that other technical factors prevent transgene expression. In conclusion, the incomplete penetrance of the phenotype and the lack of a matched transgenic control model precludes further meaningful investigations of these transgenic lines.


Assuntos
CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferase , Transferases de Grupos Nitrogenados/genética , Síndrome de Costela Curta e Polidactilia , Peixe-Zebra , Animais , Animais Geneticamente Modificados , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferase/genética , Linhagem da Célula , Células HeLa , Humanos , Transgenes
9.
Sci Rep ; 7(1): 11900, 2017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28928414

RESUMO

Histone Deacetylase 11 (HDAC11) is highly expressed in the central nervous system where it has been reported to have roles in neural differentiation. In contrast with previous studies showing nuclear and cytoplasmic localisation, we observed synaptic enrichment of HDAC11. Knockout mouse models for HDACs 1-9 have been important for guiding the development of isoform specific HDAC inhibitors as effective therapeutics. Given the close relationship between HDAC11 and neural cells in vitro, we examined neural tissue in a previously uncharacterised Hdac11 knockout mouse (Hdac11 KO/KO). Loss of HDAC11 had no obvious impact on brain morphology and neural stem/precursor cells isolated from Hdac11 KO/KO mice had comparable proliferation and differentiation characteristics. However, in differentiating neural cells we observed decreased expression of schizophrenia-associated gene Fez1 (fasciculation and elongation protein zeta 1), a gene previously reported to be regulated by HDAC11 activity. FEZ1 has been associated with the dendritic growth of neurons and risk of schizophrenia via its interaction with DISC1 (disrupted in schizophrenia 1). Examination of cortical, cerebellar and hippocampal tissue reveal decreased Fez1 expression specifically in the hippocampus of adult mice. The results of this study demonstrate that loss of HDAC11 has age dependent and brain-region specific consequences.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Regulação da Expressão Gênica , Hipocampo/metabolismo , Histona Desacetilases/genética , Proteínas do Tecido Nervoso/genética , Esquizofrenia/genética , Envelhecimento , Animais , Linhagem Celular , Hipocampo/ultraestrutura , Camundongos , Camundongos Knockout , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese
10.
Neuroreport ; 22(4): 161-5, 2011 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-21304326

RESUMO

Attentional awakening (AA) is an impairment in the identification of target stimuli that are presented early in a rapidly presented sequence of visual stimuli. Here we investigate whether AA is related to resource allocation, measured as amplitude of the P3 event-related potential and/or variance in the focus of temporal attention, measured as P3 phase distribution. We observed a relationship between P3 amplitude and AA that depended strongly on targets' a posteriori probabilities. Evidence was found for a link between performance and P3 phase distributions, but a relationship between AA and P3 phase distributions was not evident. These findings suggest that resource allocation is a relevant factor for AA whereas the variance in the focus of temporal attention contributes only little to AA.


Assuntos
Nível de Alerta/fisiologia , Atenção/fisiologia , Reconhecimento Visual de Modelos/fisiologia , Mascaramento Perceptivo/fisiologia , Percepção do Tempo/fisiologia , Adulto , Feminino , Humanos , Masculino , Adulto Jovem
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