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1.
Hum Mutat ; 40(9): 1557-1578, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31131967

RESUMO

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Biologia Computacional/métodos , Mutação de Sentido Incorreto , Neoplasias/diagnóstico , Processamento Alternativo , Detecção Precoce de Câncer , Feminino , Predisposição Genética para Doença , Humanos , Funções Verossimilhança , Masculino , Herança Multifatorial , Neoplasias/genética
2.
Am J Hum Genet ; 99(2): 423-9, 2016 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-27453577

RESUMO

Hypothalamic hamartoma (HH) with gelastic epilepsy is a well-recognized drug-resistant epilepsy syndrome of early life.(1) Surgical resection allows limited access to the small deep-seated lesions that cause the disease. Here, we report the results of a search for somatic mutations in paired hamartoma- and leukocyte-derived DNA samples from 38 individuals which we conducted by using whole-exome sequencing (WES), chromosomal microarray (CMA), and targeted resequencing (TRS) of candidate genes. Somatic mutations were identified in genes involving regulation of the sonic hedgehog (Shh) pathway in 14/38 individuals (37%). Three individuals had somatic mutations in PRKACA, which encodes a cAMP-dependent protein kinase that acts as a repressor protein in the Shh pathway, and four subjects had somatic mutations in GLI3, an Shh pathway gene associated with HH. In seven other individuals, we identified two recurrent and three single brain-tissue-specific, large copy-number or loss-of-heterozygosity (LOH) variants involving multiple Shh genes, as well as other genes without an obvious biological link to the Shh pathway. The Shh pathway genes in these large somatic lesions include the ligand itself (SHH and IHH), the receptor SMO, and several other Shh downstream pathway members, including CREBBP and GLI2. Taken together, our data implicate perturbation of the Shh pathway in at least 37% of individuals with the HH epilepsy syndrome, consistent with the concept of a developmental pathway brain disease.


Assuntos
Epilepsias Parciais/genética , Hamartoma/genética , Proteínas Hedgehog/metabolismo , Doenças Hipotalâmicas/genética , Mutação/genética , Transdução de Sinais/genética , Proteína de Ligação a CREB/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Exoma/genética , Feminino , Humanos , Fatores de Transcrição Kruppel-Like/genética , Perda de Heterozigosidade , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Proteína Gli2 com Dedos de Zinco , Proteína Gli3 com Dedos de Zinco
3.
Exp Neurol ; 249: 8-19, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23939410

RESUMO

Purkinje neurons are a sensitive and specialised cell type important for fine motor movement and coordination. Purkinje cell damage manifests as motor incoordination and ataxia - a prominent feature of many human disorders including spinocerebellar ataxia and Huntington's disease. A correlation between Purkinje degeneration and excess cerebellar levels of tissue-type plasminogen activator (tPA) has been observed in multiple genetically-distinct models of ataxia. Here we show that Purkinje loss in a mouse model of Huntington's disease also correlates with a 200% increase in cerebellar tPA activity. That elevated tPA levels arise in a variety of ataxia models suggests that tPA is a common mediator of Purkinje damage. To address the specific contribution of tPA to cerebellar dysfunction we studied the T4 mice line that overexpresses murine tPA in postnatal neurons through the Thy1.2 gene promoter, which directs preferential expression to Purkinje cells within the cerebellum. Here we show that T4 mice develop signs of cerebellar damage within 10 weeks of birth including atrophy of Purkinje cell soma and dendrites, astrogliosis, reduced molecular layer volume and altered gait. In contrast, T4 mice displayed no evidence of microgliosis, nor any changes in interneuron density, nor alteration in the cerebellar granular neuron layer. Thus, excess tPA levels may be sufficient to cause targeted Purkinje cell degeneration and ataxia. We propose that elevated cerebellar tPA levels exert a common pathway of Purkinje cell damage. Therapeutically lowering cerebellar tPA levels may represent a novel means of preserving Purkinje cell integrity and motor coordination across a wide range of neurodegenerative diseases.


Assuntos
Ataxia/metabolismo , Ataxia/fisiopatologia , Líquido Extracelular/metabolismo , Marcha/fisiologia , Células de Purkinje/metabolismo , Ativador de Plasminogênio Tecidual/fisiologia , Animais , Ataxia/enzimologia , Líquido Extracelular/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células de Purkinje/enzimologia , Células de Purkinje/patologia
4.
Cell Rep ; 2(4): 889-901, 2012 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-23041318

RESUMO

Cellular injury causes a myriad of processes that affect proteostasis. We describe nucleocytoplasmic coagulation (NCC), an intracellular disulfide-dependent protein crosslinking event occurring upon late-stage cell death that orchestrates the proteolytic removal of misfolded proteins. In vitro and in vivo models of neuronal injury show that NCC involves conversion of soluble intracellular proteins, including tubulin, into insoluble oligomers. These oligomers, also seen in human brain tissue following neurotrauma, act as a cofactor and substrate for the plasminogen-activating system. In plasminogen(-/-) mice, levels of misfolded ß-tubulin were elevated and its clearance delayed following neurotrauma, demonstrating a requirement for plasminogen in the removal of NCC constituents. While additional in vivo studies will further dissect this phenomenon, our study clearly shows that NCC, a process analogous to the formation of thrombi, generates an aggregated protein scaffold that limits release of cellular components and recruits clearance mechanisms to the site of injury.


Assuntos
Fibrinolisina/metabolismo , Neurônios/metabolismo , Animais , Apoptose , Células Cultivadas , Dissulfetos/química , Humanos , Linfócitos/imunologia , Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Plasminogênio/metabolismo , Proteólise/efeitos dos fármacos , Ativador de Plasminogênio Tecidual/farmacologia , Tubulina (Proteína)/metabolismo
5.
Brain ; 135(Pt 11): 3251-64, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22822039

RESUMO

The neurovascular unit provides a dynamic interface between the circulation and central nervous system. Disruption of neurovascular integrity occurs in numerous brain pathologies including neurotrauma and ischaemic stroke. Tissue plasminogen activator is a serine protease that converts plasminogen to plasmin, a protease that dissolves blood clots. Besides its role in fibrinolysis, tissue plasminogen activator is abundantly expressed in the brain where it mediates extracellular proteolysis. However, proteolytically active tissue plasminogen activator also promotes neurovascular disruption after ischaemic stroke; the molecular mechanisms of this process are still unclear. Tissue plasminogen activator is naturally inhibited by serine protease inhibitors (serpins): plasminogen activator inhibitor-1, neuroserpin or protease nexin-1 that results in the formation of serpin:protease complexes. Proteases and serpin:protease complexes are cleared through high-affinity binding to low-density lipoprotein receptors, but their binding to these receptors can also transmit extracellular signals across the plasma membrane. The matrix metalloproteinases are the second major proteolytic system in the mammalian brain, and like tissue plasminogen activators are pivotal to neurological function but can also degrade structures of the neurovascular unit after injury. Herein, we show that tissue plasminogen activator potentiates neurovascular damage in a dose-dependent manner in a mouse model of neurotrauma. Surprisingly, inhibition of activity following administration of plasminogen activator inhibitor-1 significantly increased cerebrovascular permeability. This led to our finding that formation of complexes between tissue plasminogen activator and plasminogen activator inhibitor-1 in the brain parenchyma facilitates post-traumatic cerebrovascular damage. We demonstrate that following trauma, the complex binds to low-density lipoprotein receptors, triggering the induction of matrix metalloproteinase-3. Accordingly, pharmacological inhibition of matrix metalloproteinase-3 attenuates neurovascular permeability and improves neurological function in injured mice. Our results are clinically relevant, because concentrations of tissue plasminogen activator: plasminogen activator inhibitor-1 complex and matrix metalloproteinase-3 are significantly elevated in cerebrospinal fluid of trauma patients and correlate with neurological outcome. In a separate study, we found that matrix metalloproteinase-3 and albumin, a marker of cerebrovascular damage, were significantly increased in brain tissue of patients with neurotrauma. Perturbation of neurovascular homeostasis causing oedema, inflammation and cell death is an important cause of acute and long-term neurological dysfunction after trauma. A role for the tissue plasminogen activator-matrix metalloproteinase axis in promoting neurovascular disruption after neurotrauma has not been described thus far. Targeting tissue plasminogen activator: plasminogen activator inhibitor-1 complex signalling or downstream matrix metalloproteinase-3 induction may provide viable therapeutic strategies to reduce cerebrovascular permeability after neurotrauma.


Assuntos
Lesões Encefálicas/fisiopatologia , Permeabilidade Capilar/fisiologia , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Ativador de Plasminogênio Tecidual/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Albuminas/metabolismo , Animais , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Lesões Encefálicas/líquido cefalorraquidiano , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Injeções Intraventriculares , Masculino , Inibidores de Metaloproteinases de Matriz/uso terapêutico , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Inibidor 1 de Ativador de Plasminogênio/administração & dosagem , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Recuperação de Função Fisiológica/fisiologia , Ativador de Plasminogênio Tecidual/administração & dosagem , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Ativador de Plasminogênio Tecidual/metabolismo
6.
J Steroid Biochem Mol Biol ; 110(3-5): 236-43, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18515094

RESUMO

Androgen signaling, mediated by the androgen receptor (AR), is a critical factor influencing growth of normal and malignant breast cells. Given the increasing use of exogenous androgens in women, a better understanding of androgen action in the breast is essential. This study compared the effects of 5alpha-dihydrotestosterone (DHT) and a synthetic androgen, mibolerone, on estradiol (E(2))-induced proliferation of breast cancer cells. DHT modestly inhibited E(2)-induced proliferation and mibolerone significantly inhibited proliferation in T-47D cells. The effects of both androgens could be reversed by an AR antagonist, suggesting that their actions were mediated, in part, by AR. Whereas high physiological doses (10-100nM) of DHT reduced E(2)-mediated induction of the estrogen-regulated gene progesterone receptor (PR) to basal levels, mibolerone at lower doses (1nM) eliminated PR expression, suggesting that mibolerone may also act via the PR. In the AR positive, PR-negative MCF-7 cells, mibolerone had modest effects on E(2)-induced proliferation, but was a potent inhibitor of proliferation in the AR positive, PR positive MCF-7M11 PRA cells. The effects of mibolerone in breast cancer cells were similar to those of the progestin, medroxyprogesterone acetate. Our results demonstrate that mibolerone can have both androgenic and progestagenic actions in breast cancer cells.


Assuntos
Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Nandrolona/análogos & derivados , Receptores Androgênicos/fisiologia , Receptores de Progesterona/fisiologia , Androgênios , Di-Hidrotestosterona/farmacologia , Avaliação Pré-Clínica de Medicamentos , Estradiol/farmacologia , Humanos , Acetato de Medroxiprogesterona/farmacologia , Nandrolona/farmacologia , Progestinas/farmacologia , Congêneres da Testosterona/farmacologia , Células Tumorais Cultivadas
7.
Cancer Res ; 65(18): 8487-96, 2005 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16166329

RESUMO

Previously, we reported that androgen receptor (AR), but not estrogen receptor (ER) or progesterone receptor (PR), is predictive of response to the synthetic progestin, medroxyprogesterone acetate (MPA), in a cohort of 83 patients with metastatic breast cancer. To further investigate the role of AR in determining response to MPA in this cohort, we analyzed AR levels by immunohistochemistry with two discrete antisera directed at either the NH2 or the COOH termini of the receptor. Compared with tumors that responded to MPA (n = 31), there was a significant decrease in the intensity and extent of AR immunoreactivity with both AR antisera in tumors from nonresponders (n = 52). Whereas only a single AR immunostaining pattern was detected in responders to MPA, reflecting concordance of immunoreactivity with the two AR antisera, tumors from nonresponders exhibited four distinct AR immunostaining patterns: (a) concordance with the two antibodies (31%), (b) staining only with the COOH-terminal antibody (33%), (c) staining only with the NH2-terminal antibody (22%), or (d) no immunoreactivity with either NH2- or COOH-terminal antibody (14%). DNA sequencing and functional analysis identified inactivating missense gene mutations in the ligand-binding domain of the AR in tumors from two of nine nonresponders positive with the NH2-terminal AR antisera but negative for COOH-terminal immunoreactivity and lacking specific, high-affinity dihydrotestosterone binding in tumor cytosol fractions. Tumors with more AR than the median level (37 fmol/mg protein) had significantly lower levels of PR (30 fmol/mg protein) than tumors with low AR (PR; 127 fmol/mg protein) despite comparable levels of ER. Ligand-dependent activation of the AR in human T47D and MCF-7 breast cancer cells resulted in inhibition of estradiol-stimulated cell proliferation and a reduction in the capacity of the ER to induce expression of the PR. These effects could be reversed using a specific AR antisense oligonucleotide. Increasing the ratio of AR to ER resulted in a greater androgen-dependent inhibition of ER function. Collectively, these data suggest that reduced levels of AR or impaired AR function contribute to the failure of MPA therapy potentially due to abrogation of the inhibitory effect of AR on ER signaling.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Acetato de Medroxiprogesterona/farmacologia , Receptores Androgênicos/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Imuno-Histoquímica , Mutação , Pós-Menopausa , Conformação Proteica , Isoformas de Proteínas , Ensaio Radioligante , Receptores Androgênicos/genética , Receptores Estrogênicos/antagonistas & inibidores , Transdução de Sinais
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