RESUMO
At least 5% of cancer diagnoses are attributed to a causal pathogenic or likely pathogenic germline genetic variant (hereditary cancer syndrome-HCS). These individuals are burdened with lifelong surveillance monitoring organs for a wide spectrum of cancers. This is associated with substantial uncertainty and anxiety in the time between screening tests and while the individuals are awaiting results. Cell-free DNA (cfDNA) sequencing has recently shown potential as a non-invasive strategy for monitoring cancer. There is an opportunity for high-yield cancer early detection in HCS. To assess clinical validity of cfDNA in individuals with HCS, representatives from eight genetics centers from across Canada founded the CHARM (cfDNA in Hereditary and High-Risk Malignancies) Consortium in 2017. In this perspective, we discuss operationalization of this consortium and early data emerging from the most common and well-characterized HCSs: hereditary breast and ovarian cancer, Lynch syndrome, Li-Fraumeni syndrome, and Neurofibromatosis type 1. We identify opportunities for the incorporation of cfDNA sequencing into surveillance protocols; these opportunities are backed by examples of earlier cancer detection efficacy in HCSs from the CHARM Consortium. We seek to establish a paradigm shift in early cancer surveillance in individuals with HCSs, away from highly centralized, regimented medical screening visits and toward more accessible, frequent, and proactive care for these high-risk individuals.
Assuntos
Ácidos Nucleicos Livres , Síndromes Neoplásicas Hereditárias , Feminino , Humanos , Predisposição Genética para Doença , Síndromes Neoplásicas Hereditárias/diagnóstico , Síndromes Neoplásicas Hereditárias/genética , Síndromes Neoplásicas Hereditárias/epidemiologia , Testes Genéticos/métodos , Biópsia Líquida , Ácidos Nucleicos Livres/genéticaRESUMO
Patients with early-onset lysosomal storage diseases are ideal candidates for prenatal therapy because organ damage starts in utero. We report the safety and efficacy results of in utero enzyme-replacement therapy (ERT) in a fetus with CRIM (cross-reactive immunologic material)-negative infantile-onset Pompe's disease. The family history was positive for infantile-onset Pompe's disease with cardiomyopathy in two previously affected deceased siblings. After receiving in utero ERT and standard postnatal therapy, the current patient had normal cardiac and age-appropriate motor function postnatally, was meeting developmental milestones, had normal biomarker levels, and was feeding and growing well at 13 months of age.
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Doença de Depósito de Glicogênio Tipo II , Humanos , Lactente , Doença de Depósito de Glicogênio Tipo II/tratamento farmacológicoRESUMO
BACKGROUND: Physical literacy is a concept used to describe the combined physical, affective and cognitive capacities facilitating an active lifestyle. Physical activity participation is essential for children living with chronic medical conditions, but knowledge of physical literacy among this group is scarce. METHODS: An explanatory, sequential mixed methods design was used to comprehensively describe the physical literacies of children with chronic medical conditions (CMCs). Participants were recruited from paediatric cardiology, respirology/cystic fibrosis, neurology, haematology and endocrinology outpatient clinics. All participants completed the Canadian Assessment of Physical Literacy (2nd Edition), and those with higher and lower scores were invited to a semi-structured interview. A deductive-inductive thematic analysis was applied using Margaret Whitehead's conceptualization of physical literacy. RESULTS: Using normative strata, 80.0% of the 99 children assessed (mean age = 9.97 ± 1.3 years, 48% girls) were considered beginning or progressing in their overall physical literacy (mean score = 56.5 ± 13.8/100). Meanwhile, physical literacy informed participants' approach to new, active experiences and may have contributed to a strong sense of self. There was a significant difference between endocrinology and haematology patients on total physical literacy score (p = 0.03) but not domain scores. Participants scored high on motivation/confidence (mean = 22.9 ± 5.0/30) but obtained low physical competence (mean = 11.8 ± 5.6/30) and daily behaviour scores (n = 72, mean = 15.5 ± 7.1/30). Main themes represent salient experiences of children with CMCs within the domains of physical literacy, including their need to evaluate active contexts, self-regulate activity intensity and manage physical limitations. CONCLUSIONS: Children with CMCs can achieve recommended levels of physical literacy without meeting normative standards for physical competence. Participants would benefit from a physical literacy intervention that targets the development of bodily self-regulation skills and risk evaluation in active settings.
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Exercício Físico , Letramento em Saúde , Humanos , Criança , Feminino , Masculino , Doença Crônica , Exercício Físico/psicologia , Canadá , Desenvolvimento Infantil/fisiologia , Pesquisa QualitativaRESUMO
PURPOSE: Nuclear receptor binding SET domain protein 1, NSD1, encodes a histone methyltransferase H3K36. NSD1 is responsible for the phenotype of the reciprocal 5q35.2q35.3 microdeletion-microduplication syndromes. We expand the phenotype and demonstrate the functional role of NSD1 in microduplication 5q35 syndrome. METHODS: Through an international collaboration, we report nine new patients, contributing to the emerging phenotype, highlighting psychiatric phenotypes in older affected individuals. Focusing specifically on the undergrowth phenotype, we have modeled the effects of Mes-4/NSD overexpression in Drosophila melanogaster. RESULTS: The individuals (including a family) from diverse backgrounds with duplications ranging in size from 0.6 to 4.5 Mb, have a consistent undergrowth phenotype. Mes-4 overexpression in the developing wing causes undergrowth, increased H3K36 methylation, and increased apoptosis. We demonstrate that altering the levels of insulin receptor (IR) rescues the apoptosis and the wing undergrowth phenotype, suggesting changes in mTOR pathway signaling. Leucine supplementation rescued Mes-4/NSD induced cell death, demonstrating decreased mTOR signaling caused by NSD1. CONCLUSION: Given that we show mTOR inhibition as a likely mechanism and amelioration of the phenotype by leucine supplementation in a fly model, we suggest further studies should evaluate the therapeutic potential of leucine or branched chain amino acids as an adjunct possible treatment to ameliorate human growth and psychiatric phenotypes and propose inclusion of 5q35-microduplication as part of the differential diagnosis for children and adults with delayed bone age, short stature, microcephaly, developmental delay, and psychiatric phenotypes.
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Transtornos Cromossômicos/genética , Cromossomos Humanos Par 5 , Duplicação Gênica , Histona-Lisina N-Metiltransferase/genética , Serina-Treonina Quinases TOR/metabolismo , Adolescente , Adulto , Animais , Caspases/metabolismo , Morte Celular , Criança , Pré-Escolar , Regulação para Baixo , Drosophila melanogaster , Feminino , Humanos , Leucina/metabolismo , Leucina/farmacologia , Masculino , Linhagem , Fenótipo , Transdução de Sinais , Adulto JovemRESUMO
Natural products and secondary metabolites comprise an indispensable resource from living organisms that have transformed areas of medicine, agriculture, and biotechnology. Recent advances in high-throughput DNA sequencing and computational analysis suggest that the vast majority of natural products remain undiscovered. To accelerate the natural product discovery pipeline, cell-free metabolic engineering approaches used to develop robust catalytic networks are being repurposed to access new chemical scaffolds, and new enzymes capable of performing diverse chemistries. Such enzymes could serve as flexible biocatalytic tools to further expand the unique chemical space of natural products and secondary metabolites, and provide a more sustainable route to manufacture these molecules. Herein, we highlight select examples of natural product biosynthesis using cell-free systems and propose how cell-free technologies could facilitate our ability to access and modify these structures to transform synthetic and chemical biology.
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Produtos Biológicos/metabolismo , Produtos Biológicos/química , Sequenciamento de Nucleotídeos em Larga Escala , Engenharia Metabólica , Estrutura MolecularRESUMO
We demonstrate the novel spiroannulation of exo-imines with 1,3-dipoles, for the first time, leading to 3D spirocycles with a secondary amine (NH) in the spiro-ring. The synthetic method described herein allows access to these previously unexplored heterospirocyclic cores that have application in the discovery of functional molecules for medicinal and materials science. This was demonstrated by discovering an unprecedented class of heterospirocycles with antimalarial activity against the human protozoan P. falciparum.
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Antimaláricos , Iminas , Antimaláricos/farmacologia , Reação de Cicloadição , HumanosRESUMO
Capicua, encoded by the gene CIC, is an evolutionarily conserved high-mobility group-box transcription factor downstream of the receptor tyrosine kinase and mitogen-activated protein kinase pathways. It was initially discovered and studied in Drosophila. Recurrent mutations in CIC were first identified in oligodendroglioma, a subtype of low-grade glioma. Subsequent studies have identified CIC aberrations in multiple types of cancer and have established CIC as a potent tumour suppressor involved in regulating pathways related to cell growth and proliferation, invasion and treatment resistance. The most well-known and studied targets of mammalian CIC are the oncogenic E-Twenty Six transcription factors ETV1/4/5, which have been found to be elevated in cancers with CIC aberrations. Here, we review the role of CIC in normal mammalian development, oncogenesis and tumour progression, and the functional interactors that mediate them. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Supressores de Tumor/fisiologia , Receptores Proteína Tirosina Quinases/genética , Proteínas Repressoras/genética , Animais , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVES: To describe current practices and safety concerns regarding cardiac emergency medications in cardiac anesthesia. DESIGN: An anonymous survey with multiple-choice questions. SETTINGS: Online survey using Opinio platform. PARTICIPANTS: Cardiac anesthesiologists from United States and Canada. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Response rate was 12% (nâ¯=â¯320), with 78% of respondents from the United States and 22% from Canada. The majority of the respondents were experienced (66%), academic (60%), and worked in large cardiac institutions (81%). Most cardiac emergency medications were prepared in the operating room (53.4%), followed by the pharmacy (34%) and industry (8.2%). American respondents had more medications prepared by a pharmacy (53%) versus Canadian (10%, p < 0.001). The majority (85%) considered expiration time of cardiac medications prepared in the operating room to be more than 12 hours. Familiarity with the American Society of Anesthesiologists guidelines on labeling was 58%, other medication safety guidelines 25%, and 34% were not familiar with any guidelines. The majority used color-coded labeling (95%), and a minority (11%) used bar-code systems. Most respondents (69%) agreed that lack of availability of preprepared medications could compromise patient safety. Having to prepare medications by themselves was a concern for respondents based on distractions (66%), lack of availability for emergencies (53%), labeling errors (41%), incorrect concentration (36%), sterility (33%), and stability (30%). CONCLUSION: This survey found that cardiac emergency medications commonly are prepared in the operating room. The authors identified gaps in familiarity with parenteral medications safety guidelines. Most safety concerns could be addressed with the application of current medication safety guidelines.
Assuntos
Anestesia em Procedimentos Cardíacos , Adulto , Anestesiologistas , Canadá , Emergências , Humanos , Inquéritos e Questionários , Estados UnidosRESUMO
BACKGROUND: Aberrations in Capicua (CIC) have recently been implicated as a negative prognostic factor in a multitude of cancer types through the derepression of targets downstream of the mitogen-activated protein kinase (MAPK) signaling cascade, such as oncogenic E26 transformation-specific (ETS) transcription factors. The Ataxin-family protein ATXN1L has previously been reported to interact with CIC in both developmental and disease contexts to facilitate the repression of CIC target genes and promote the post-translational stability of CIC. However, little is known about the mechanisms at the base of ATXN1L-mediated CIC post-translational stability. RESULTS: Functional in vitro studies utilizing ATXN1LKO human cell lines revealed that loss of ATXN1L leads to the accumulation of polyubiquitinated CIC protein, promoting its degradation through the proteasome. Although transcriptomic signatures of ATXN1LKO cell lines indicated upregulation of the mitogen-activated protein kinase pathway, ERK activity was found to contribute to CIC function but not stability. Degradation of CIC protein following loss of ATXN1L was instead observed to be mediated by the E3 ubiquitin ligase TRIM25 which was further validated using glioma-derived cell lines and the TCGA breast carcinoma and liver hepatocellular carcinoma cohorts. CONCLUSIONS: The post-translational regulation of CIC through ATXN1L and TRIM25 independent of ERK activity suggests that the regulation of CIC stability and function is more intricate than previously appreciated and involves several independent pathways. As CIC status has become a prognostic factor in several cancer types, further knowledge into the mechanisms which govern CIC stability and function may prove useful for future therapeutic approaches.
Assuntos
Sistema de Sinalização das MAP Quinases , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Linhagem Celular , Humanos , Proteólise , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Healthy active lifestyles are critically important for children with complex heart problems (CHP) that affect heart structure, rhythm or function. They are at increased risk for morbidities such as atherosclerosis, obesity, anxiety and depression. Educating children with CHP and their families about the relevance of healthy lifestyles is an important part of clinical care. DESIGN: This study used a collaborative approach among six patients/family members and 22 health professionals to develop a series of knowledge-to-action tools suitable for counselling children with CHP and their families about their healthy lifestyle needs. METHODS: After development of the knowledge-to-action tools had been completed, one cardiologist and one research assistant implemented one or more of these new resources during each clinic visit as appropriate for each patient. Thirty-nine parents and eight children completed post-clinic interviews to explore their perceptions of the new resources. The nine resources developed included brochures and websites addressing physical activity with a heart condition, body contact restrictions, exercise test results, emotional health, finding community resources, encouragement for asking healthy lifestyle questions and a brief, in-clinic healthy lifestyle assessment. RESULTS: Families found the resources useful and helpful for clarifying their specific concerns. They also provided suggestions to improve the content and delivery of the resources so that they would be suitable for a variety of settings-schools, community and sports. CONCLUSION: Future research is required to evaluate the effectiveness of these resources for raising awareness and knowledge about healthy active lifestyles among children with CHP and the impact of these resources for changing healthy lifestyle behaviours.
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Estilo de Vida Saudável , Pais , Criança , Exercício Físico , Família , Humanos , Instituições AcadêmicasRESUMO
We report a rare delayed complication of de novo pseudoaneurysm formation and rupture after stereotactic radiotherapy for cerebral arteriovenous malformation. The patient presented with intracerebral haemorrhage due to rupture of a pseudoaneurysm in the previously irradiated field, which was excised for histological examination. The literature was reviewed for similar cases.
Assuntos
Falso Aneurisma , Malformações Arteriovenosas Intracranianas , Radiocirurgia , Falso Aneurisma/diagnóstico por imagem , Falso Aneurisma/etiologia , Falso Aneurisma/cirurgia , Hemorragia Cerebral/diagnóstico por imagem , Hemorragia Cerebral/etiologia , Humanos , Malformações Arteriovenosas Intracranianas/diagnóstico por imagem , Malformações Arteriovenosas Intracranianas/cirurgia , Radiocirurgia/efeitos adversosRESUMO
PURPOSE: We investigated the value of transcriptome sequencing (RNAseq) in ascertaining the consequence of DNA variants on RNA transcripts to improve the diagnostic rate from exome or genome sequencing for undiagnosed Mendelian diseases spanning a wide spectrum of clinical indications. METHODS: From 234 subjects referred to the Undiagnosed Diseases Network, University of California-Los Angeles clinical site between July 2014 and August 2018, 113 were enrolled for high likelihood of having rare undiagnosed, suspected genetic conditions despite thorough prior clinical evaluation. Exome or genome sequencing and RNAseq were performed, and RNAseq data was integrated with genome sequencing data for DNA variant interpretation genome-wide. RESULTS: The molecular diagnostic rate by exome or genome sequencing was 31%. Integration of RNAseq with genome sequencing resulted in an additional seven cases with clear diagnosis of a known genetic disease. Thus, the overall molecular diagnostic rate was 38%, and 18% of all genetic diagnoses returned required RNAseq to determine variant causality. CONCLUSION: In this rare disease cohort with a wide spectrum of undiagnosed, suspected genetic conditions, RNAseq analysis increased the molecular diagnostic rate above that possible with genome sequencing analysis alone even without availability of the most appropriate tissue type to assess.
Assuntos
Doenças Genéticas Inatas/diagnóstico , Patologia Molecular , Doenças Raras/diagnóstico , Transcriptoma/genética , Exoma/genética , Doenças Genéticas Inatas/genética , Testes Genéticos/normas , Humanos , Mutação/genética , RNA-Seq/normas , Doenças Raras/genética , Análise de Sequência de DNA/normas , Sequenciamento do Exoma/normas , Sequenciamento Completo do Genoma/normasRESUMO
OBJECTIVE: Individuals with urea cycle disorders (UCDs) often present with intellectual and developmental disabilities. The major aim of this study was to evaluate the impact of diagnostic and therapeutic interventions on cognitive outcomes in UCDs. METHODS: This prospective, observational, multicenter study includes data from 503 individuals with UCDs who had comprehensive neurocognitive testing with a cumulative follow-up of 702 patient-years. RESULTS: The mean cognitive standard deviation score (cSDS) was lower in symptomatic than in asymptomatic (p < 0.001, t test) individuals with UCDs. Intellectual disability (intellectual quotient < 70, cSDS < -2.0) was associated with the respective subtype of UCD and early disease onset, whereas height of the initial peak plasma ammonium concentration was inversely associated with neurocognitive outcomes in mitochondrial (proximal) rather than cytosolic (distal) UCDs. In ornithine transcarbamylase and argininosuccinate synthetase 1 deficiencies, we did not find evidence that monoscavenger therapy with sodium or glycerol phenylbutyrate was superior to sodium benzoate in providing cognitive protection. Early liver transplantation appears to be beneficial for UCDs. It is noteworthy that individuals with argininosuccinate synthetase 1 and argininosuccinate lyase deficiencies identified by newborn screening had better neurocognitive outcomes than those diagnosed after the manifestation of first symptoms. INTERPRETATION: Cognitive function is related to interventional and non-interventional variables. Early detection by newborn screening and early liver transplantation appear to offer greater cognitive protection, but none of the currently used nitrogen scavengers was superior with regard to long-term neurocognitive outcome. Further confirmation could determine these variables as important clinical indicators of neuroprotection for individuals with UCDs. ANN NEUROL 2019.
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Cognição/fisiologia , Testes de Estado Mental e Demência , Distúrbios Congênitos do Ciclo da Ureia/diagnóstico , Distúrbios Congênitos do Ciclo da Ureia/terapia , Adolescente , Adulto , Criança , Pré-Escolar , Estudos Transversais , Feminino , Seguimentos , Glicerol/análogos & derivados , Glicerol/farmacologia , Glicerol/uso terapêutico , Humanos , Lactente , Recém-Nascido , Transplante de Fígado/métodos , Masculino , Triagem Neonatal/métodos , Fenilbutiratos/farmacologia , Fenilbutiratos/uso terapêutico , Estudos Prospectivos , Distúrbios Congênitos do Ciclo da Ureia/psicologia , Adulto JovemRESUMO
AIMS: Dedifferentiated chondrosarcoma (DDCHS) is an aggressive type of chondrosarcoma that results from high-grade transformation of a low-grade chondrosarcoma. Mutations in the isocitrate dehydrogenase (IDH) 1 gene and the IDH2 gene that lead to increased d-2-hydroxyglutarate (2HG) oncometabolite production, promoting tumorigenesis, have been recently described in low-grade cartilaginous neoplasms. The aims of this study were to examine the prevalence of IDH mutations in a single-institution cohort of DDCHS cases and correlate 2HG levels with mutation status. METHODS AND RESULTS: We examined a series of 21 primary DDCHS cases by using Sanger sequencing and quantitative polymerase chain reaction genotyping to look for IDH1/IDH2 mutations, and evaluated the 2HG levels in formalin-fixed paraffin-embedded tumour and matched normal tissue samples by using a fluorometric assay. Seventy-six per cent of DDCHS cases (16/21) harboured a heterozygous IDH1 or IDH2 mutation. Six of 14 IDH-mutated DDCHS cases showed elevated 2HG levels in tumour tissue relative to matched normal tissue. There were no consistent histological or disease-specific survival differences between IDH-mutated tumours and wild-type tumours. CONCLUSIONS: Our study confirms the frequent presence of a variety of IDH1 and IDH2 mutation variants, indicating that a sequencing-based approach is required for DDCHS if IDH is to be used as a diagnostic marker. Similarly to other IDH-mutated tumour types, IDH-mutated DDCHS cases show elevated 2HG levels, indicating that the oncometabolite activity of 2HG may contribute to DDCHS oncogenesis and progression.
Assuntos
Neoplasias Ósseas , Condrossarcoma , Glutaratos/metabolismo , Isocitrato Desidrogenase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Transformação Celular Neoplásica/genética , Condrossarcoma/genética , Condrossarcoma/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
INTRODUCTION: Glycerol phenylbutyrate (GPB) is currently approved for use in the US and Europe for patients of all ages with urea cycle disorders (UCD) who cannot be managed with protein restriction and/or amino acid supplementation alone. Currently available data on GPB is limited to 12â¯months exposure. Here, we present long-term experience with GPB. METHODS: This was an open-label, long-term safety study of GPB conducted in the US (17 sites) and Canada (1 site) monitoring the use of GPB in UCD patients who had previously completed 12 months of treatment in the previous safety extension studies. Ninety patients completed the previous studies with 88 of these continuing into the long-term evaluation. The duration of therapy was open ended until GPB was commercially available. The primary endpoint was the rate of adverse events (AEs). Secondary endpoints were venous ammonia levels, number and causes of hyperammonemic crises (HACs) and neuropsychological testing. RESULTS: A total of 45 pediatric patients between the ages of 1 to 17â¯years (median 7â¯years) and 43 adult patients between the ages of 19 and 61 years (median 30â¯years) were enrolled. The treatment emergent adverse events (TEAE) reported in ≥10% of adult or pediatric patients were consistent with the TEAEs reported in the previous safety extension studies with no increase in the overall incidence of TEAEs and no new TEAEs that indicated a new safety signal. Mean ammonia levels remained stable and below the adult upper limit of normal (<35⯵mol/L) through 24â¯months of treatment in both the pediatric and adult population. Over time, glutamine levels decreased in the overall population. The mean annualized rate of HACs (0.29) established in the previously reported 12-month follow-up study was maintained with continued GPB exposure. CONCLUSION: Following the completion of 12-month follow-up studies with GPB treatment, UCD patients were followed for an additional median of 1.85 (range 0 to 5.86) years in the present study with continued maintenance of ammonia control, similar rates of adverse events, and no new adverse events identified.
Assuntos
Glicerol/análogos & derivados , Fenilbutiratos/uso terapêutico , Distúrbios Congênitos do Ciclo da Ureia/tratamento farmacológico , Adolescente , Adulto , Canadá , Criança , Pré-Escolar , Gerenciamento Clínico , Feminino , Seguimentos , Glicerol/efeitos adversos , Glicerol/uso terapêutico , Humanos , Hiperamonemia/induzido quimicamente , Lactente , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Fenilbutiratos/efeitos adversos , Estados Unidos , Adulto JovemRESUMO
The hedgehog (Hh) pathway is highly conserved and required for embryonic patterning and determination. Mutations in the Hh pathway are observed in sporadic tumors as well as under syndromic conditions. Common to these syndromes are the findings of polydactyly/syndactyly and brain overgrowth. The latter is also a finding most commonly observed in the cases of mutations in the PI3K/AKT/mTOR pathway. We have identified novel Hh pathway mutations and structural copy number variations in individuals with somatic overgrowth, macrocephaly, dysmorphic facial features, and developmental delay, which phenotypically closely resemble patients with phosphatase and tensin homolog (PTEN) mutations. We hypothesized that brain overgrowth and phenotypic overlap with syndromic overgrowth syndromes in these cases may be due to crosstalk between the Hh and PI3K/AKT/mTOR pathways. To test this, we modeled disease-associated variants by generating PTCH1 and Suppressor of Fused (SUFU) heterozygote cell lines using the CRISPR/Cas9 system. These cells demonstrate activation of PI3K signaling and increased phosphorylation of its downstream target p4EBP1 as well as a distinct cellular phenotype. To further investigate the mechanism underlying this crosstalk, we treated human neural stem cells with sonic hedgehog (SHH) ligand and performed transcriptional analysis of components of the mTOR pathway. These studies identified decreased expression of a set of mTOR negative regulators, leading to its activation. We conclude that there is a significant crosstalk between the SHH and PI3K/AKT/mTOR. We propose that this crosstalk is responsible for why mutations in PTCH1 and SUFU lead to macrocephaly phenotypes similar to those observed in PTEN hamartoma and other overgrowth syndromes associated with mutations in PI3K/AKT/mTOR pathway genes.
Assuntos
Proteínas Hedgehog/metabolismo , Megalencefalia/genética , Megalencefalia/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Pré-Escolar , Feminino , Deleção de Genes , Haploinsuficiência , Humanos , Lactente , Masculino , Megalencefalia/diagnóstico , Modelos Biológicos , Células-Tronco NeuraisRESUMO
Protein O-fucosyltransferase-1 (POFUT1) adds O-fucose monosaccharides to epidermal growth factor-like (EGF) repeats found on approximately 100 mammalian proteins, including Notch receptors. Haploinsufficiency of POFUT1 has been linked to adult-onset Dowling Degos Disease (DDD) with hyperpigmentation defects. Homozygous deletion of mouse Pofut1 results in embryonic lethality with severe Notch-like phenotypes including defects in somitogenesis, cardiogenesis, vasculogenesis and neurogenesis, but the extent to which POFUT1 is required for normal human development is not yet understood. Here we report a patient with a congenital syndrome consisting of severe global developmental delay, microcephaly, heart defects, failure to thrive and liver disease with a previously unreported homozygous NM_015352.1: c.485C>T variant (p.Ser162Leu) in POFUT1 detected by exome sequencing. Both parents are heterozygotes and neither manifests any signs of DDD. No other detected variant explained the phenotype. This variant eliminated a conserved N-glycosylation sequon at Asn160 in POFUT1 and profoundly decreased POFUT1 activity in patient fibroblasts compared to control fibroblasts. Purified p.Ser162Leu mutant protein also showed much lower POFUT1 activity with a lower affinity for EGF acceptor substrate than wild type POFUT1. Eliminating the N-glycan sequon by replacing Asn160 with Gln had little effect on POFUT1 activity, suggesting that loss of the glycan is not responsible for the defect. Furthermore, the p.Ser162Leu mutant showed weaker ability to rescue Notch activity in cell-based assays. These results suggest that this N-glycan of POFUT1 is not required for its proper enzymatic function, and that the p.Ser162Leu mutation of POFUT1 likely causes global developmental delay, microcephaly with vascular and cardiac defects.