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1.
Am J Hum Genet ; 111(5): 863-876, 2024 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-38565148

RESUMO

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and, with new innovative methods, can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the Genomics Research to Elucidate the Genetics of Rare Diseases consortium and analyzed using the seqr platform. The addition of CNV detection to exome analysis identified causal CNVs for 171 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb. The causal CNVs consisted of 140 deletions, 15 duplications, 3 suspected complex structural variants (SVs), 3 insertions, and 10 complex SVs, the latter two groups being identified by orthogonal confirmation methods. To classify CNV variant pathogenicity, we used the 2020 American College of Medical Genetics and Genomics/ClinGen CNV interpretation standards and developed additional criteria to evaluate allelic and functional data as well as variants on the X chromosome to further advance the framework. We interpreted 151 CNVs as likely pathogenic/pathogenic and 20 CNVs as high-interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher-resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.


Assuntos
Variações do Número de Cópias de DNA , Sequenciamento do Exoma , Exoma , Doenças Raras , Humanos , Variações do Número de Cópias de DNA/genética , Doenças Raras/genética , Doenças Raras/diagnóstico , Exoma/genética , Masculino , Feminino , Estudos de Coortes , Testes Genéticos/métodos
2.
N Engl J Med ; 390(21): 1985-1997, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38838312

RESUMO

BACKGROUND: Genetic variants that cause rare disorders may remain elusive even after expansive testing, such as exome sequencing. The diagnostic yield of genome sequencing, particularly after a negative evaluation, remains poorly defined. METHODS: We sequenced and analyzed the genomes of families with diverse phenotypes who were suspected to have a rare monogenic disease and for whom genetic testing had not revealed a diagnosis, as well as the genomes of a replication cohort at an independent clinical center. RESULTS: We sequenced the genomes of 822 families (744 in the initial cohort and 78 in the replication cohort) and made a molecular diagnosis in 218 of 744 families (29.3%). Of the 218 families, 61 (28.0%) - 8.2% of families in the initial cohort - had variants that required genome sequencing for identification, including coding variants, intronic variants, small structural variants, copy-neutral inversions, complex rearrangements, and tandem repeat expansions. Most families in which a molecular diagnosis was made after previous nondiagnostic exome sequencing (63.5%) had variants that could be detected by reanalysis of the exome-sequence data (53.4%) or by additional analytic methods, such as copy-number variant calling, to exome-sequence data (10.8%). We obtained similar results in the replication cohort: in 33% of the families in which a molecular diagnosis was made, or 8% of the cohort, genome sequencing was required, which showed the applicability of these findings to both research and clinical environments. CONCLUSIONS: The diagnostic yield of genome sequencing in a large, diverse research cohort and in a small clinical cohort of persons who had previously undergone genetic testing was approximately 8% and included several types of pathogenic variation that had not previously been detected by means of exome sequencing or other techniques. (Funded by the National Human Genome Research Institute and others.).


Assuntos
Variação Genética , Doenças Raras , Sequenciamento Completo do Genoma , Feminino , Humanos , Masculino , Estudos de Coortes , Exoma , Sequenciamento do Exoma , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/etnologia , Doenças Genéticas Inatas/genética , Testes Genéticos , Genoma Humano , Fenótipo , Doenças Raras/diagnóstico , Doenças Raras/etnologia , Doenças Raras/genética , Análise de Sequência de DNA , Criança , Adolescente , Adulto Jovem , Adulto
3.
Hum Mol Genet ; 32(20): 2981-2995, 2023 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-37531237

RESUMO

Protein phosphatase 1 regulatory subunit 3F (PPP1R3F) is a member of the glycogen targeting subunits (GTSs), which belong to the large group of regulatory subunits of protein phosphatase 1 (PP1), a major eukaryotic serine/threonine protein phosphatase that regulates diverse cellular processes. Here, we describe the identification of hemizygous variants in PPP1R3F associated with a novel X-linked recessive neurodevelopmental disorder in 13 unrelated individuals. This disorder is characterized by developmental delay, mild intellectual disability, neurobehavioral issues such as autism spectrum disorder, seizures and other neurological findings including tone, gait and cerebellar abnormalities. PPP1R3F variants segregated with disease in affected hemizygous males that inherited the variants from their heterozygous carrier mothers. We show that PPP1R3F is predominantly expressed in brain astrocytes and localizes to the endoplasmic reticulum in cells. Glycogen content in PPP1R3F knockout astrocytoma cells appears to be more sensitive to fluxes in extracellular glucose levels than in wild-type cells, suggesting that PPP1R3F functions in maintaining steady brain glycogen levels under changing glucose conditions. We performed functional studies on nine of the identified variants and observed defects in PP1 binding, protein stability, subcellular localization and regulation of glycogen metabolism in most of them. Collectively, the genetic and molecular data indicate that deleterious variants in PPP1R3F are associated with a new X-linked disorder of glycogen metabolism, highlighting the critical role of GTSs in neurological development. This research expands our understanding of neurodevelopmental disorders and the role of PP1 in brain development and proper function.


Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Deficiência Intelectual , Transtornos do Neurodesenvolvimento , Masculino , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/complicações , Proteína Fosfatase 1/genética , Transtorno do Espectro Autista/genética , Transtorno Autístico/genética , Glucose , Glicogênio , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/complicações
4.
Ann Neurol ; 2024 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-39301775

RESUMO

OBJECTIVE: De novo variants in cullin-3 ubiquitin ligase (CUL3) have been strongly associated with neurodevelopmental disorders (NDDs), but no large case series have been reported so far. Here, we aimed to collect sporadic cases carrying rare variants in CUL3, describe the genotype-phenotype correlation, and investigate the underlying pathogenic mechanism. METHODS: Genetic data and detailed clinical records were collected via multicenter collaboration. Dysmorphic facial features were analyzed using GestaltMatcher. Variant effects on CUL3 protein stability were assessed using patient-derived T-cells. RESULTS: We assembled a cohort of 37 individuals with heterozygous CUL3 variants presenting a syndromic NDD characterized by intellectual disability with or without autistic features. Of these, 35 have loss-of-function (LoF) and 2 have missense variants. CUL3 LoF variants in patients may affect protein stability leading to perturbations in protein homeostasis, as evidenced by decreased ubiquitin-protein conjugates in vitro. Notably, we show that 4E-BP1 (EIF4EBP1), a prominent substrate of CUL3, fails to be targeted for proteasomal degradation in patient-derived cells. INTERPRETATION: Our study further refines the clinical and mutational spectrum of CUL3-associated NDDs, expands the spectrum of cullin RING E3 ligase-associated neuropsychiatric disorders, and suggests haploinsufficiency via LoF variants is the predominant pathogenic mechanism. ANN NEUROL 2024.

5.
Am J Hum Genet ; 108(8): 1450-1465, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34186028

RESUMO

The genetic causes of global developmental delay (GDD) and intellectual disability (ID) are diverse and include variants in numerous ion channels and transporters. Loss-of-function variants in all five endosomal/lysosomal members of the CLC family of Cl- channels and Cl-/H+ exchangers lead to pathology in mice, humans, or both. We have identified nine variants in CLCN3, the gene encoding CIC-3, in 11 individuals with GDD/ID and neurodevelopmental disorders of varying severity. In addition to a homozygous frameshift variant in two siblings, we identified eight different heterozygous de novo missense variants. All have GDD/ID, mood or behavioral disorders, and dysmorphic features; 9/11 have structural brain abnormalities; and 6/11 have seizures. The homozygous variants are predicted to cause loss of ClC-3 function, resulting in severe neurological disease similar to the phenotype observed in Clcn3-/- mice. Their MRIs show possible neurodegeneration with thin corpora callosa and decreased white matter volumes. Individuals with heterozygous variants had a range of neurodevelopmental anomalies including agenesis of the corpus callosum, pons hypoplasia, and increased gyral folding. To characterize the altered function of the exchanger, electrophysiological analyses were performed in Xenopus oocytes and mammalian cells. Two variants, p.Ile607Thr and p.Thr570Ile, had increased currents at negative cytoplasmic voltages and loss of inhibition by luminal acidic pH. In contrast, two other variants showed no significant difference in the current properties. Overall, our work establishes a role for CLCN3 in human neurodevelopment and shows that both homozygous loss of ClC-3 and heterozygous variants can lead to GDD/ID and neuroanatomical abnormalities.


Assuntos
Canais de Cloreto/genética , Modelos Animais de Doenças , Canais Iônicos/fisiologia , Mutação , Transtornos do Neurodesenvolvimento/patologia , Fenótipo , Adolescente , Animais , Criança , Pré-Escolar , Feminino , Homozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos , Camundongos Knockout , Transtornos do Neurodesenvolvimento/etiologia , Transtornos do Neurodesenvolvimento/metabolismo
6.
N Engl J Med ; 385(22): 2059-2065, 2021 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-34818480

RESUMO

The integration of genomic testing into clinical care enables the use of individualized approaches to the management of rare diseases. We describe the use of belzutifan, a potent and selective small-molecule inhibitor of the protein hypoxia-inducible factor 2α (HIF2α), in a patient with polycythemia and multiple paragangliomas (the Pacak-Zhuang syndrome). The syndrome was caused in this patient by somatic mosaicism for an activating mutation in EPAS1. Treatment with belzutifan led to a rapid and sustained tumor response along with resolution of hypertension, headaches, and long-standing polycythemia. This case shows the application of a targeted therapy for the treatment of a patient with a rare tumor-predisposition syndrome. (Funded by the Morin Family Fund for Pediatric Cancer and Alex's Lemonade Stand Foundation.).


Assuntos
Neoplasias das Glândulas Suprarrenais/tratamento farmacológico , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Indenos/uso terapêutico , Paraganglioma/tratamento farmacológico , Policitemia/tratamento farmacológico , Adolescente , Neoplasias das Glândulas Suprarrenais/genética , Glândulas Suprarrenais/diagnóstico por imagem , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Biomarcadores/sangue , Cromograninas/sangue , Feminino , Mutação com Ganho de Função , Humanos , Indenos/efeitos adversos , Imageamento por Ressonância Magnética , Normetanefrina/sangue , Paraganglioma/genética , Policitemia/genética , Transdução de Sinais , Síndrome , Sequenciamento Completo do Genoma
7.
Am J Med Genet A ; 194(5): e63509, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38158391

RESUMO

Advances in bioinformatic tools paired with the ongoing accumulation of genetic knowledge and periodic reanalysis of genomic sequencing data have led to an improvement in genetic diagnostic rates. Candidate gene variants (CGVs) identified during sequencing or on reanalysis but not yet implicated in human disease or associated with a phenotypically distinct condition are often not revisited, leading to missed diagnostic opportunities. Here, we revisited 33 such CGVs from our previously published study and determined that 16 of them are indeed disease-causing (novel or phenotype expansion) since their identification. These results emphasize the need to focus on previously identified CGVs during sequencing or reanalysis and the importance of sharing that information with researchers around the world, including relevant functional analysis to establish disease causality.


Assuntos
Biologia Computacional , Genômica , Humanos , Sequenciamento do Exoma , Fenótipo , Genômica/métodos , Biologia Computacional/métodos , Alelos
8.
PLoS Genet ; 17(7): e1009639, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34232960

RESUMO

ARHGAP42 encodes Rho GTPase activating protein 42 that belongs to a member of the GTPase Regulator Associated with Focal Adhesion Kinase (GRAF) family. ARHGAP42 is involved in blood pressure control by regulating vascular tone. Despite these findings, disorders of human variants in the coding part of ARHGAP42 have not been reported. Here, we describe an 8-year-old girl with childhood interstitial lung disease (chILD), systemic hypertension, and immunological findings who carries a homozygous stop-gain variant (c.469G>T, p.(Glu157Ter)) in the ARHGAP42 gene. The family history is notable for both parents with hypertension. Histopathological examination of the proband lung biopsy showed increased mural smooth muscle in small airways and alveolar septa, and concentric medial hypertrophy in pulmonary arteries. ARHGAP42 stop-gain variant in the proband leads to exon 5 skipping, and reduced ARHGAP42 levels, which was associated with enhanced RhoA and Cdc42 expression. This is the first report linking a homozygous stop-gain variant in ARHGAP42 with a chILD disorder, systemic hypertension, and immunological findings in human patient. Evidence of smooth muscle hypertrophy on lung biopsy and an increase in RhoA/ROCK signaling in patient cells suggests the potential mechanistic link between ARHGAP42 deficiency and the development of chILD disorder.


Assuntos
Proteínas Ativadoras de GTPase/genética , Hipertensão/genética , Doenças Pulmonares Intersticiais/genética , Animais , Criança , Feminino , Homozigoto , Humanos , Leucocitose/genética , Leucocitose/imunologia , Doenças Pulmonares Intersticiais/patologia , Linfocitose/genética , Linfocitose/imunologia , Masculino , Camundongos , Linhagem , Sequenciamento do Exoma , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
9.
Genet Med ; 24(6): 1297-1305, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35341654

RESUMO

PURPOSE: As genomic sequencing becomes more common, medically actionable secondary findings will increasingly be returned to health care providers (HCPs), who will be faced with managing the resulting patient care. These findings are generally unsolicited, ie, unrelated to the sequencing indication and/or ordered by another clinician. METHODS: To understand the impact of receiving unsolicited results, we interviewed HCPs who received genomic results for patients enrolled in the Electronic Medical Records and Genomics (eMERGE) Phase III Network, which returned results on >100 actionable genes to eMERGE participants and HCPs. RESULTS: In total, 16 HCPs across 3 eMERGE sites were interviewed about their experience of receiving a positive (likely pathogenic or pathogenic), negative, or variant of uncertain significance result for a patient enrolled in eMERGE Phase III and about managing their patient on the basis of the result. Although unsolicited, HCPs felt responsible for managing the patient's resulting medical care. HCPs indicated that clinical utility depended on the actionability of results, and whereas comfort levels varied, confidence was improved by the availability of subspecialist consults. HCPs were concerned about patient anxiety, insurability, and missing an actionable result in the electronic health record. CONCLUSION: Our findings help inform best practices for return of unsolicited genomic screening findings in the future.


Assuntos
Registros Eletrônicos de Saúde , Genoma , Genômica , Pessoal de Saúde , Humanos , Grupos Populacionais
10.
Genet Med ; 23(10): 1901-1911, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34113008

RESUMO

PURPOSE: ADP ribosylation factor guanine nucleotide exchange factors (ARFGEFs) are a family of proteins implicated in cellular trafficking between the Golgi apparatus and the plasma membrane through vesicle formation. Among them is ARFGEF1/BIG1, a protein involved in axon elongation, neurite development, and polarization processes. ARFGEF1 has been previously suggested as a candidate gene for different types of epilepsies, although its implication in human disease has not been well characterized. METHODS: International data sharing, in silico predictions, and in vitro assays with minigene study, western blot analyses, and RNA sequencing. RESULTS: We identified 13 individuals with heterozygous likely pathogenic variants in ARFGEF1. These individuals displayed congruent clinical features of developmental delay, behavioral problems, abnormal findings on brain magnetic resonance image (MRI), and epilepsy for almost half of them. While nearly half of the cohort carried de novo variants, at least 40% of variants were inherited from mildly affected parents who were clinically re-evaluated by reverse phenotyping. Our in silico predictions and in vitro assays support the contention that ARFGEF1-related conditions are caused by haploinsufficiency, and are transmitted in an autosomal dominant fashion with variable expressivity. CONCLUSION: We provide evidence that loss-of-function variants in ARFGEF1 are implicated in sporadic and familial cases of developmental delay with or without epilepsy.


Assuntos
Epilepsia , Fatores de Troca do Nucleotídeo Guanina , Haploinsuficiência , Deficiência Intelectual , Epilepsia/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Heterozigoto , Humanos , Deficiência Intelectual/genética
11.
Clin Genet ; 99(2): 313-317, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33156547

RESUMO

Phosphatidylinositol Glycan Anchor Biosynthesis class H (PIGH) is an essential player in the glycosylphosphatidylinositol (GPI) synthesis, an anchor for numerous cell membrane-bound proteins. PIGH deficiency is a newly described and rare disorder associated with developmental delay, seizures and behavioral difficulties. Herein, we report three new unrelated families with two different bi-allelic PIGH variants, including one new variant p.(Arg163Trp) which seems associated with a more severe phenotype. The common clinical features in all affected individuals are developmental delay/intellectual disability and hypotonia. Variable clinical features include seizures, autism spectrum disorder, apraxia, severe language delay, dysarthria, feeding difficulties, facial dysmorphisms, microcephaly, strabismus, and musculoskeletal anomalies. The two siblings homozygous for the p.(Arg163Trp) variant have severe symptoms including profound psychomotor retardation, intractable seizures, multiple bone fractures, scoliosis, loss of independent ambulation, and delayed myelination on brain MRI. Serum iron levels were significantly elevated in one individual. All tested individuals with PIGH deficiency had normal alkaline phosphatase and CD16, a GPI-anchored protein (GPI-AP), was found to be decreased by 60% on granulocytes from one individual. This study expands the PIGH deficiency phenotype range toward the severe end of the spectrum with the identification of a novel pathogenic variant.


Assuntos
Anormalidades Múltiplas/genética , Doenças do Desenvolvimento Ósseo/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Transtornos do Neurodesenvolvimento/genética , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Linhagem , Fenótipo , Adulto Jovem
12.
Am J Med Genet A ; 185(1): 203-207, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33037779

RESUMO

Inherited optic neuropathies (IONs) are neurodegenerative disorders characterized by optic atrophy with or without extraocular manifestations. Optic atrophy-10 (OPA10) is an autosomal recessive ION recently reported to be caused by mutations in RTN4IP1, which encodes reticulon 4 interacting protein 1 (RTN4IP1), a mitochondrial ubiquinol oxydo-reductase. Here we report novel compound heterozygous mutations in RTN4IP1 in a male proband with developmental delay, epilepsy, optic atrophy, ataxia, and choreoathetosis. Workup was notable for transiently elevated lactate and lactate-to-pyruvate ratio, brain magnetic resonance imaging with optic atrophy and T2 signal abnormalities, and a nondiagnostic initial genetic workup, including chromosomal microarray and mitochondrial panel testing. Exome sequencing identified a paternally inherited missense variant (c.263T>G, p.Val88Gly) predicted to be deleterious and a maternally inherited deletion encompassing RTN4IP1. To our knowledge, this is the first report of a non-single nucleotide pathogenic variant associated with OPA10. This case highlights the expanding phenotypic spectrum of OPA10, the association between "syndromic" cases and severe RTN4IP1 mutations, and the importance of nonbiased genetic testing, such as ES, to analyze multiple genes and variants types, in patients suspected of having genetic disease.


Assuntos
Proteínas de Transporte/genética , Deficiências do Desenvolvimento/genética , Epilepsia/genética , Proteínas Mitocondriais/genética , Atrofia Óptica/genética , Ataxia/diagnóstico por imagem , Ataxia/genética , Ataxia/patologia , Proteínas de Transporte/ultraestrutura , Pré-Escolar , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/patologia , Epilepsia/diagnóstico por imagem , Epilepsia/patologia , Exoma/genética , Feminino , Predisposição Genética para Doença , Testes Genéticos/métodos , Humanos , Lactente , Recém-Nascido , Imageamento por Ressonância Magnética , Masculino , Proteínas Mitocondriais/ultraestrutura , Mutação/genética , Atrofia Óptica/diagnóstico por imagem , Atrofia Óptica/patologia , Linhagem , Conformação Proteica , Relação Estrutura-Atividade , Sequenciamento do Exoma
13.
Genet Med ; 22(4): 736-744, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31780822

RESUMO

PURPOSE: To investigate the impact of rapid-turnaround exome sequencing in critically ill neonates using phenotype-based subject selection criteria. METHODS: Intensive care unit babies aged <6 months with hypotonia, seizures, a complex metabolic phenotype, and/or multiple congenital malformations were prospectively enrolled for rapid (<7 day) trio-based exome sequencing. Genomic variants relevant to the presenting phenotype were returned to the medical team. RESULTS: A genetic diagnosis was attained in 29 of 50 (58%) sequenced cases. Twenty-seven (54%) patients received a molecular diagnosis involving known disease genes; two additional cases (4%) were solved with pathogenic variants found in novel disease genes. In 24 of the solved cases, diagnosis had impact on patient management and/or family members. Management changes included shift to palliative care, medication changes, involvement of additional specialties, and the consideration of new experimental therapies. CONCLUSION: Phenotype-based patient selection is effective at identifying critically ill neonates with a high likelihood of receiving a molecular diagnosis via rapid-turnaround exome sequencing, leading to faster and more accurate diagnoses, reducing unnecessary testing and procedures, and informing medical care.


Assuntos
Estado Terminal , Exoma , Idoso , Exoma/genética , Testes Genéticos , Humanos , Lactente , Recém-Nascido , Fenótipo , Estudos Prospectivos , Sequenciamento do Exoma
14.
Int J Mol Sci ; 21(21)2020 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-33126568

RESUMO

High-grade serous carcinoma (HGSC), the most lethal subtype of epithelial ovarian cancer (EOC), is characterized by widespread TP53 mutations (>90%), most of which are missense mutations (>70%). The objective of this study was to investigate differential transcriptional targets affected by a common germline P72R SNP (rs1042522) in two p53 hotspot mutants, R248Q and R248W, and identify the mechanism through which the P72R SNP affects the neomorphic properties of these mutants. Using isogenic cell line models, transcriptomic analysis, xenografts, and patient data, we found that the P72R SNP modifies the effect of p53 hotspot mutants on cellular morphology and invasion properties. Most importantly, RNA sequencing studies identified CXCL1 a critical factor that is differentially affected by P72R SNP in R248Q and R248W mutants and is responsible for differences in cellular morphology and functional properties observed in these p53 mutants. We show that the mutants with the P72 SNP promote a reversion of the EMT phenotype to epithelial characteristics, whereas its R72 counterpart promotes a mesenchymal transition via the chemokine CXCL1. These studies reveal a new role of the P72R SNP in modulating the neomorphic properties of p53 mutants via CXCL1, which has significant implications for tumor invasion and metastasis.


Assuntos
Biomarcadores Tumorais/metabolismo , Quimiocina CXCL1/metabolismo , Transição Epitelial-Mesenquimal , Mutação , Neoplasias Ovarianas/patologia , Polimorfismo Genético , Proteína Supressora de Tumor p53/genética , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Quimiocina CXCL1/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Invasividade Neoplásica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Fenótipo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Biol Reprod ; 96(2): 491-501, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28203708

RESUMO

Phosphoramide mustard (PM) destroys rapidly dividing cells and activates the DNA double strand break marker, γH2AX, and DNA repair in rat granulosa cells and neonatal ovaries. The effects of PM exposure on DNA damage and activation of DNA damage repair in lean and obese female mice were investigated. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice received sesame oil or PM (95%; 25 mg/kg; intraperitoneal injection). Obesity increased (P < 0.05) hepatic and spleen but decreased (P < 0.05) uterine weight. PM exposure reduced (P < 0.05) spleen weight regardless of body composition, however, decreased (P < 0.05) ovarian and hepatic weight were observed in the obese PM-exposed females. PM decreased (P < 0.05) primordial and primary follicle number in lean females. Obesity and PM increased (P < 0.05) γH2AX protein. DNA damage repair genes Prkdc, Parp1, and Rad51 mRNA were unaltered by obesity, however, Atm and Xrcc6 mRNA were increased (P < 0.05) while Brca1 was reduced (P < 0.05). Obesity reduced (P < 0.05) PRKDC, XRCC6 and but increased (P < 0.05) ATM protein. ATM, BRCA1 and RAD51 protein levels were increased (P < 0.05) by PM exposure in both lean and obese mice, while PM-induced increased (P < 0.05) XRCC6 and PARP1 were observed only in lean mice. Thus, PM induces ovarian DNA damage in vivo; obesity alters DNA repair response gene mRNA and protein level; the ovary activates DNA repair proteins in response to PM; but obesity compromises the ovarian PM response.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Obesidade/patologia , Ovário/patologia , Mostardas de Fosforamida/toxicidade , Animais , Biomarcadores , Feminino , Camundongos , Camundongos Endogâmicos , Ovário/efeitos dos fármacos , RNA Mensageiro
16.
Biol Reprod ; 96(2): 478-490, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28203716

RESUMO

Mechanisms underlying obesity-associated reproductive impairment are ill defined. Hyperinsulinemia is a metabolic perturbation often observed in obese subjects. Insulin activates phosphatidylinositol 3-kinase (PI3K) signaling, which regulates ovarian folliculogenesis, steroidogenesis, and xenobiotic metabolism. The impact of progressive obesity on ovarian genes encoding mRNA involved in insulin-mediated PI3K signaling and xenobiotic biotransformation [insulin receptor (Insr), insulin receptor substrate 1 (Irs1), 2 (Irs2), and 3 (Irs3); kit ligand (Kitlg), stem cell growth factor receptor (Kit), protein kinase B (AKT) alpha (Akt1), beta (Akt2), forkhead transcription factor (FOXO) subfamily 1 (Foxo1), and subfamily 3 (Foxo3a), microsomal epoxide hydrolase (Ephx1), cytochrome P450 family 2, subfamily E, polypeptide 1 (Cyp2e1), glutathione S-transferase (GST) class Pi (Gstp1) and class mu 1 (Gstm1)] was determined in normal wild-type nonagouti (a/a; lean) and lethal yellow mice (KK.CG-Ay/J; obese) at 6, 12, 18, or 24 weeks of age. At 6 weeks, ovaries from obese mice had increased (P < 0.05) Insr and Irs3 but decreased (P < 0.05) Kitlg, Foxo1, and Cyp2e1 mRNA levels. Interestingly, at 12 weeks, an increase (P < 0.05) in Kitlg and Kit mRNA, pIRS1Ser302, pAKTThr308, EPHX1, and GSTP1 protein level was observed due to obesity, while Cyp2e1 mRNA and protein were reduced. A phosphoramide mustard (PM) challenge increased (P < 0.05) ovarian EPHX1 protein abundance in lean but not obese females. In addition, lung tissue from PM-exposed animals had increased (P < 0.05) EPHX1 protein with no impact of obesity thereon. Taken together, progressive obesity affected ovarian signaling pathways potentially involved in obesity-associated reproductive disorders.


Assuntos
Insulina/metabolismo , Obesidade , Ovário/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Mostardas de Fosforamida/toxicidade , Transdução de Sinais/fisiologia , Animais , Feminino , Camundongos , Ovário/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/genética
17.
Toxicol Appl Pharmacol ; 276(3): 179-87, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24576726

RESUMO

Chronic exposure to the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA), generated during combustion of organic matter including cigarette smoke, depletes all ovarian follicle types in the mouse and rat, and in vitro models mimic this effect. To investigate the mechanisms involved in follicular depletion during acute DMBA exposure, two concentrations of DMBA at which follicle depletion has (75 nM) and has not (12.5 nM) been observed were investigated. Postnatal day four F344 rat ovaries were maintained in culture for four days before a single exposure to vehicle control (1% DMSO; CT) or DMBA (12 nM; low-concentration or 75 nM; high-concentration). After four or eight additional days of culture, DMBA-induced follicle depletion was evaluated via follicle enumeration. Relative to control, DMBA did not affect follicle numbers after 4 days of exposure, but induced large primary follicle loss at both concentrations after 8 days; while, the low-concentration DMBA also caused secondary follicle depletion. Neither concentration affected primordial or small primary follicle number. RNA was isolated and quantitative RT-PCR performed prior to follicle loss to measure mRNA levels of genes involved in xenobiotic metabolism (Cyp2e1, Gstmu, Gstpi, Ephx1), autophagy (Atg7, Becn1), oxidative stress response (Sod1, Sod2) and the phosphatidylinositol 3-kinase (PI3K) pathway (Kitlg, cKit, Akt1) 1, 2 and 4 days after exposure. With the exception of Atg7 and cKit, DMBA increased (P < 0.05) expression of all genes investigated. Also, BECN1 and pAKT(Thr308) protein levels were increased while cKIT was decreased by DMBA exposure. Taken together, these results suggest an increase in DMBA bioactivation, add to the mechanistic understanding of DMBA-induced ovotoxicity and raise concern regarding female low concentration DMBA exposures.


Assuntos
9,10-Dimetil-1,2-benzantraceno/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Ovário/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Autofagia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Epóxido Hidrolases/genética , Feminino , Glutationa Transferase/genética , Folículo Ovariano/efeitos dos fármacos , Ovário/metabolismo , Ovário/patologia , Estresse Oxidativo , Fosfatidilinositol 3-Quinases/genética , Ratos , Ratos Endogâmicos F344
18.
Toxicol Appl Pharmacol ; 277(1): 1-7, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24642057

RESUMO

The finite ovarian follicle reserve can be negatively impacted by exposure to chemicals including the anti-neoplastic agent, cyclophosphamide (CPA). CPA requires bioactivation to phosphoramide mustard (PM) to elicit its therapeutic effects however; in addition to being the tumor-targeting metabolite, PM is also ovotoxic. In addition, PM can break down to a cytotoxic, volatile metabolite, chloroethylaziridine (CEZ). The aim of this study was initially to characterize PM-induced ovotoxicity in growing follicles. Using PND4 Fisher 344 rats, ovaries were cultured for 4 days before being exposed once to PM (10 or 30 µM). Following eight additional days in culture, relative to control (1% DMSO), PM had no impact on primordial, small primary or large primary follicle number, but both PM concentrations induced secondary follicle depletion (P<0.05). Interestingly, a reduction in follicle number in the control-treated ovaries was observed. Thus, the involvement of a volatile, cytotoxic PM metabolite (VC) in PM-induced ovotoxicity was explored in cultured rat ovaries, with control ovaries physically separated from PM-treated ovaries during culture. Direct PM (60 µM) exposure destroyed all stage follicles after 4 days (P<0.05). VC from nearby wells depleted primordial follicles after 4 days (P<0.05), temporarily reduced secondary follicle number after 2 days, and did not impact other stage follicles at any other time point. VC was determined to spontaneously liberate from PM, which could contribute to degradation of PM during storage. Taken together, this study demonstrates that PM and VC are ovotoxicants, with different follicular targets, and that the VC may be a major player during PM-induced ovotoxicity observed in cancer survivors.


Assuntos
Aziridinas/toxicidade , Ovário/efeitos dos fármacos , Mostardas de Fosforamida/toxicidade , Animais , Antineoplásicos/farmacocinética , Aziridinas/farmacologia , Ciclofosfamida/farmacocinética , Relação Dose-Resposta a Droga , Feminino , Técnicas In Vitro , Folículo Ovariano/efeitos dos fármacos , Mostardas de Fosforamida/farmacocinética , Ratos
19.
Toxicol Appl Pharmacol ; 267(1): 49-56, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23274565

RESUMO

4-Vinylcyclohexene diepoxide (VCD) destroys ovarian primordial and small primary follicles via apoptosis. In mice, VCD exposure induces ovarian mRNA expression of glutathione S-transferase (GST) family members, including isoform mu (Gstm). Extra-ovarian GSTM negatively regulates pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1) through protein complex formation, which dissociates during stress, thereby initiating ASK1-induced apoptosis. The present study investigated the ovarian response of Gstm mRNA and protein to VCD. Induction of Ask1 mRNA at VCD-induced follicle loss onset was determined. Ovarian GSTM:ASK1 protein complex formation was investigated and VCD exposure effects thereon evaluated. Phosphatidylinositol-3 kinase (PI3K) regulation of GSTM protein was also studied. Postnatal day (PND) 4 rat ovaries were cultured in control media ± 1) VCD (30 µM) for 2-8 days; 2) VCD (30 µM) for 2 days, followed by incubation in control media for 4 days (acute VCD exposure); or 3) LY294002 (20 µM) for 6 days. VCD exposure did not alter Gstm mRNA expression, however, GSTM protein increased (P<0.05) after 6 days of both the acute and chronic treatments. Ask1 mRNA increased (0.33-fold; P<0.05) relative to control after 6 days of VCD exposure. Ovarian GSTM:ASK1 protein complex formation was confirmed and, relative to control, the amount of GSTM bound to ASK1 increased 33% (P<0.05) by chronic but with no effect of acute VCD exposure. PI3K inhibition increased (P<0.05) GSTM protein by 40% and 71% on d4 and d6, respectively. These findings support involvement of GSTM in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1.


Assuntos
Cicloexenos/toxicidade , Glutationa Transferase/fisiologia , MAP Quinase Quinase Quinase 5/metabolismo , Ovário/efeitos dos fármacos , Ovário/metabolismo , Compostos de Vinila/toxicidade , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Ovário/enzimologia , Ratos , Ratos Endogâmicos F344
20.
Artigo em Inglês | MEDLINE | ID: mdl-37230770

RESUMO

Pathogenic variants in MECOM, a gene critical to the self-renewal and proliferation of hematopoietic stem cells, are known to cause a rare bone marrow failure syndrome associated with amegakaryocytic thrombocytopenia and bilateral radioulnar synostosis known as RUSAT2. However, the spectrum of disease seen with causal variants in MECOM is broad, ranging from mildly affected adults to fetal loss. We report two cases of infants born preterm who presented at birth with symptoms of bone marrow failure including severe anemia, hydrops, and petechial hemorrhages; radioulnar synostosis was not observed in either patient, and, unfortunately, neither infant survived. In both cases, genomic sequencing revealed de novo variants in MECOM considered to be responsible for their severe presentations. These cases add to the growing body of literature that describe MECOM-associated disease, particularly MECOM as a cause of fetal hydrops due to bone marrow failure in utero. Furthermore, they support the use of a broad sequencing approach for perinatal diagnosis, as MECOM is absent from available targeted gene panels for hydrops, and highlight the importance of postmortem genomic investigation.


Assuntos
Anemia , Hidropisia Fetal , Recém-Nascido , Gravidez , Lactente , Feminino , Adulto , Humanos , Hidropisia Fetal/genética , Hidropisia Fetal/diagnóstico , Fatores de Transcrição , Transtornos da Insuficiência da Medula Óssea/complicações , Proteína do Locus do Complexo MDS1 e EVI1
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