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1.
Hum Mutat ; 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31343788

RESUMO

Alagille syndrome is an autosomal dominant disease with a known molecular etiology of dysfunctional Notch signaling caused primarily by pathogenic variants in JAGGED1 (JAG1), but also by variants in NOTCH2. The majority of JAG1 variants result in loss of function, however disease has also been attributed to lesser understood missense variants. Conversely, the majority of NOTCH2 variants are missense, though fewer of these variants have been described. In addition, there is a small group of patients with a clear clinical phenotype in the absence of a pathogenic variant. Here, we catalog our single-center study, which includes 401 probands and 111 affected family members amassed over a 27-year period, to provide updated mutation frequencies in JAG1 and NOTCH2 as well as functional validation of nine missense variants. Combining our cohort of 86 novel JAG1 and three novel NOTCH2 variants with previously published data (totaling 713 variants), we present the most comprehensive pathogenic variant overview for Alagille syndrome. Using this data set, we developed new guidance to help with the classification of JAG1 missense variants. Finally, we report clinically consistent cases for which a molecular etiology has not been identified and discuss the potential for next generation sequencing methodologies in novel variant discovery.

2.
Eur J Hum Genet ; 27(4): 612-620, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30626929

RESUMO

Clinical exome sequencing (CES) has become the preferred diagnostic platform for complex pediatric disorders with suspected monogenic etiologies. Despite rapid advancements, the major challenge still resides in identifying the casual variants among the thousands of variants detected during CES testing, and thus establishing a molecular diagnosis. To improve the clinical exome diagnostic efficiency, we developed Phenoxome, a robust phenotype-driven model that adopts a network-based approach to facilitate automated variant prioritization. Phenoxome dissects the phenotypic manifestation of a patient in concert with their genomic profile to filter and then prioritize variants that are likely to affect the function of the gene (potentially pathogenic variants). To validate our method, we have compiled a clinical cohort of 105 positive patient samples that represent a wide range of genetic heterogeneity. Phenoxome identifies the causative variants within the top 5, 10, or 25 candidates in more than 50%, 71%, or 88% of these exomes, respectively. Furthermore, we show that our method is optimized for clinical testing by outperforming the current state-of-art method. We have demonstrated the performance of Phenoxome using a clinical cohort and showed that it enables rapid and accurate interpretation of clinical exomes. Phenoxome is available at https://phenoxome.chop.edu/ .

3.
J Mol Diagn ; 21(1): 38-48, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30577886

RESUMO

Clinical exome sequencing (CES) has a reported diagnostic yield of 20% to 30% for most clinical indications. The ongoing discovery of novel gene-disease and variant-disease associations are expected to increase the diagnostic yield of CES. Performing systematic reanalysis of previously nondiagnostic CES samples represents a significant challenge for clinical laboratories. Here, we present the results of a novel automated reanalysis methodology applied to 300 CES samples initially analyzed between June 2014 and September 2016. Application of our reanalysis methodology reduced reanalysis variant analysis burden by >93% and correctly captured 70 of 70 previously identified diagnostic variants among 60 samples with previously identified diagnoses. Notably, reanalysis of 240 initially nondiagnostic samples using information available on July 1, 2017, revealed 38 novel diagnoses, representing a 15.8% increase in diagnostic yield. Modeling monthly iterative reanalysis of 240 nondiagnostic samples revealed a diagnostic rate of 0.57% of samples per month. Modeling the workload required for monthly iterative reanalysis of nondiagnostic samples revealed a variant analysis burden of approximately 5 variants/month for proband-only and approximately 0.5 variants/month for trio samples. Approximately 45% of samples required evaluation during each monthly interval, and 61.3% of samples were reevaluated across three consecutive reanalyses. In sum, automated reanalysis methods can facilitate efficient reevaluation of nondiagnostic samples using up-to-date literature and can provide significant value to clinical laboratories.

5.
Am J Hum Genet ; 102(5): 995-1007, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29656858

RESUMO

Developmental and epileptic encephalopathies (DEEs) represent a large clinical and genetic heterogeneous group of neurodevelopmental diseases. The identification of pathogenic genetic variants in DEEs remains crucial for deciphering this complex group and for accurately caring for affected individuals (clinical diagnosis, genetic counseling, impacting medical, precision therapy, clinical trials, etc.). Whole-exome sequencing and intensive data sharing identified a recurrent de novo PACS2 heterozygous missense variant in 14 unrelated individuals. Their phenotype was characterized by epilepsy, global developmental delay with or without autism, common cerebellar dysgenesis, and facial dysmorphism. Mixed focal and generalized epilepsy occurred in the neonatal period, controlled with difficulty in the first year, but many improved in early childhood. PACS2 is an important PACS1 paralog and encodes a multifunctional sorting protein involved in nuclear gene expression and pathway traffic regulation. Both proteins harbor cargo(furin)-binding regions (FBRs) that bind cargo proteins, sorting adaptors, and cellular kinase. Compared to the defined PACS1 recurrent variant series, individuals with PACS2 variant have more consistently neonatal/early-infantile-onset epilepsy that can be challenging to control. Cerebellar abnormalities may be similar but PACS2 individuals exhibit a pattern of clear dysgenesis ranging from mild to severe. Functional studies demonstrated that the PACS2 recurrent variant reduces the ability of the predicted autoregulatory domain to modulate the interaction between the PACS2 FBR and client proteins, which may disturb cellular function. These findings support the causality of this recurrent de novo PACS2 heterozygous missense in DEEs with facial dysmorphim and cerebellar dysgenesis.

6.
Am J Med Genet A ; 170(3): 699-702, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26494257

RESUMO

We present a patient with neonatal onset of hypertonia and seizures identified through whole exome sequencing to have compound heterozygous variants, c.294dupA (p.Leu99fs) and c.1925C>A (p.Ala642Glu), in the BRCA1-associated protein required for ATM activation-1 (BRAT1) gene. Variants in BRAT1 have been identified to cause lethal neonatal rigidity and multifocal seizure syndrome (OMIM# 614498), which consistently manifests a severe neurological phenotype that includes neonatal presentation of rigidity and hypertonia, microcephaly and arrested head growth, intractable seizures, absence of developmental progress, apneic episodes, and death usually by 6 months of age. Our patient initially had a similarly severe neurological picture but remains alive at 6 years of age, expanding the phenotype to include longer term survival and providing further insights into genotype-phenotype correlations and the natural history of this disease.


Assuntos
Estudos de Associação Genética , Proteínas Nucleares/genética , Alelos , Exoma , Feminino , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Microcefalia/genética , Fenótipo , Convulsões/genética
7.
Nat Commun ; 6: 8329, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26387913

RESUMO

Piezo1 ion channels are mediators of mechanotransduction in several cell types including the vascular endothelium, renal tubular cells and erythrocytes. Gain-of-function mutations in PIEZO1 cause an autosomal dominant haemolytic anaemia in humans called dehydrated hereditary stomatocytosis. However, the phenotypic consequence of PIEZO1 loss of function in humans has not previously been documented. Here we discover a novel role of this channel in the lymphatic system. Through whole-exome sequencing, we identify biallelic mutations in PIEZO1 (a splicing variant leading to early truncation and a non-synonymous missense variant) in a pair of siblings affected with persistent lymphoedema caused by congenital lymphatic dysplasia. Analysis of patients' erythrocytes as well as studies in a heterologous system reveal greatly attenuated PIEZO1 function in affected alleles. Our results delineate a novel clinical category of PIEZO1-associated hereditary lymphoedema.


Assuntos
Anemia Hemolítica Congênita/metabolismo , Hidropisia Fetal/metabolismo , Canais Iônicos/metabolismo , Doenças Linfáticas/metabolismo , Sequência de Aminoácidos , Anemia Hemolítica Congênita/genética , Pré-Escolar , Eritrócitos/metabolismo , Feminino , Genes Recessivos , Humanos , Hidropisia Fetal/genética , Lactente , Canais Iônicos/química , Canais Iônicos/genética , Doenças Linfáticas/genética , Masculino , Dados de Sequência Molecular , Mutação , Mutação de Sentido Incorreto , Alinhamento de Sequência
8.
Blood ; 125(21): 3263-72, 2015 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-25855602

RESUMO

Hypoxia-inducible factors (HIFs) are master regulators of the transcriptional response to low oxygen and play essential roles in embryonic development, tissue homeostasis, and disease. Recent studies have demonstrated that hematopoietic stem cells (HSCs) within the bone marrow localize to a hypoxic niche and that HIF-1α promotes HSC adaptation to stress. Because the related factor HIF-2α is also expressed in HSCs, the combined role of HIF-1α and HIF-2α in HSC maintenance is unclear. To this end, we have conditionally deleted the HIF-α dimerization partner, the aryl hydrocarbon receptor nuclear translocator (ARNT) in the hematopoietic system to ablate activity of both HIF-1α and HIF-2α and assessed the functional consequence of ARNT deficiency on fetal liver and adult hematopoiesis. We determined that ARNT is essential for adult and fetal HSC viability and homeostasis. Importantly, conditional knockout of both Hif-1α and Hif-2α phenocopied key aspects of these HSC phenotypes, demonstrating that the impact of Arnt deletion is primarily HIF dependent. ARNT-deficient long-term HSCs underwent apoptosis, potentially because of reduced B-cell lymphoma 2 (BCL-2) and vascular endothelial growth factor A (VEGF-A) expression. Our results suggest that HIF activity may regulate HSC homeostasis through these prosurvival factors.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/fisiologia , Sobrevivência Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real
9.
Am J Med Genet A ; 164A(11): 2701-6, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25123577

RESUMO

The 22q11.2 deletion syndrome (22q11.2DS) is a common microdeletion disorder. Most of the patients show the common 3 Mb deletion but proximal 1.5 Mb deletion and unusual deletions located outside the common deleted region, have been detected particularly with the advance of comparative cytogenomic microarray technologies. The individuals reported in the literature with unusual deletions involving the 22q11 region, showed milder facial phenotypes, decreased incidence of cardiac anomalies, and intellectual disability. We describe two sibs with an atypical 0.8 Mb microdeletion of chromosome 22q11 who both showed myelomeningocele and mild facial dysmorphisms. The association between neural tube defect and the clinical diagnosis of Di George anomaly/velocardiofacial syndrome is well documented in the literature, but not all cases had molecular studies to determine breakpoint regions. This report helps to narrow a potential critical region for neural tube defects associated with 22q11 deletions.


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 22 , Defeitos do Tubo Neural/genética , Exoma , Facies , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Cariotipagem , Imagem por Ressonância Magnética , Masculino , Meningomielocele/diagnóstico , Meningomielocele/genética , Defeitos do Tubo Neural/diagnóstico , Fenótipo , Irmãos
10.
PLoS One ; 9(8): e104661, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25144710

RESUMO

Specification and development of the apical membrane in epithelial cells requires the function of polarity proteins, including Pard3 and an atypical protein kinase C (PrkC). Many epithelial cells possess microtubule-based organelles, known as cilia, that project from their apical surface and the membrane surrounding the cilium is contiguous with the apical cell membrane. Although cilia formation in cultured cells required Pard3, the in vivo requirement for Pard3 in cilia development remains unknown. The vertebrate photoreceptor outer segment represents a highly specialized cilia structure in which to identify factors necessary for apical and ciliary membrane formation. Pard3 and PrkC localized to distinct domains within vertebrate photoreceptors. Using partial morpholino knockdown, photo-morpholinos, and pharmacological approaches, the function of Pard3 and PrkC were found to be required for the formation of both the apical and ciliary membrane of vertebrate photoreceptors. Inhibition of Pard3 or PrkC activity significantly reduced the size of photoreceptor outer segments and resulted in mislocalization of rhodopsin. Suppression of Pard3 or PrkC also led to a reduction in cilia size and cilia number in Kupffer's Vesicle, which resulted in left-right asymmetry defects. Thus, the Par-PrkC complex functions in cilia formation in vivo and this likely reflects a general role in specifying non-ciliary and ciliary compartments of the apical domain.


Assuntos
Proteínas de Transporte/metabolismo , Cílios/metabolismo , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Western Blotting , Proteínas de Transporte/genética , Eletroforese em Gel de Poliacrilamida , Imuno-Histoquímica , Hibridização In Situ , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
11.
J Immunol ; 192(4): 1661-70, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24403534

RESUMO

The cytokine IL-10 has an important role in limiting inflammation in many settings, including toxoplasmosis. In the present studies, an IL-10 reporter mouse was used to identify the sources of this cytokine following challenge with Toxoplasma gondii. During infection, multiple cell types expressed the IL-10 reporter but NK cells were a major early source of this cytokine. These IL-10 reporter(+) NK cells expressed high levels of the IL-12 target genes T-bet, KLRG1, and IFN-γ, and IL-12 depletion abrogated reporter expression. However, IL-12 signaling alone was not sufficient to promote NK cell IL-10, and activation of the aryl hydrocarbon receptor (AHR) was also required for maximal IL-10 production. NK cells basally expressed the AHR, relevant chaperone proteins, and the AHR nuclear translocator, which heterodimerizes with the AHR to form a competent transcription factor. In vitro studies revealed that IL-12 stimulation increased NK cell AHR levels, and the AHR and AHR nuclear translocator were required for optimal production of IL-10. Additionally, NK cells isolated from T. gondii-infected Ahr(-/-) mice had impaired expression of IL-10, which was associated with increased resistance to this infection. Taken together, these data identify the AHR as a critical cofactor involved in NK cell production of IL-10.


Assuntos
Interleucina-10/biossíntese , Interleucina-12/metabolismo , Células Matadoras Ativadas por Linfocina/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Toxoplasma/imunologia , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/biossíntese , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Dimerização , Genes Reporter , Inflamação/imunologia , Interferon gama/biossíntese , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Receptores de Hidrocarboneto Arílico/deficiência , Receptores de Hidrocarboneto Arílico/genética , Receptores Imunológicos/biossíntese , Transdução de Sinais/imunologia , Proteínas com Domínio T/biossíntese , Toxoplasmose Animal/imunologia
12.
Carcinogenesis ; 35(5): 1067-77, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24408928

RESUMO

Hypoxia-inducible factors (HIFs) accumulate in both neoplastic and inflammatory cells within the tumor microenvironment and impact the progression of a variety of diseases, including colorectal cancer. Pharmacological HIF inhibition represents a novel therapeutic strategy for cancer treatment. We show here that acriflavine (ACF), a naturally occurring compound known to repress HIF transcriptional activity, halts the progression of an autochthonous model of established colitis-associated colon cancer (CAC) in immunocompetent mice. ACF treatment resulted in decreased tumor number, size and advancement (based on histopathological scoring) of CAC. Moreover, ACF treatment corresponded with decreased macrophage infiltration and vascularity in colorectal tumors. Importantly, ACF treatment inhibited the hypoxic induction of M-CSFR, as well as the expression of the angiogenic factor (vascular endothelial growth factor), a canonical HIF target, with little to no impact on the Nuclear factor-kappa B pathway in bone marrow-derived macrophages. These effects probably explain the observed in vivo phenotypes. Finally, an allograft tumor model further confirmed that ACF treatment inhibits tumor growth through HIF-dependent mechanisms. These results suggest pharmacological HIF inhibition in multiple cell types, including epithelial and innate immune cells, significantly limits tumor growth and progression.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Acriflavina/administração & dosagem , Acriflavina/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Transdução de Sinais/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Nat Immunol ; 14(6): 611-8, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23644504

RESUMO

Natural T helper 17 (nTH17) cells are a population of interleukin 17 (IL-17)-producing cells that acquire effector function in the thymus during development. Here we demonstrate that the serine/threonine kinase Akt has a critical role in regulating nTH17 cell development. Although Akt and the downstream mTORC1-ARNT-HIFα axis were required for generation of inducible TH17 (iTH17) cells, nTH17 cells developed independently of mTORC1. In contrast, mTORC2 and inhibition of Foxo proteins were critical for development of nTH17 cells. Moreover, distinct isoforms of Akt controlled the generation of TH17 cell subsets, as deletion of Akt2, but not of Akt1, led to defective generation of iTH17 cells. These findings define mechanisms regulating nTH17 cell development and reveal previously unknown roles of Akt and mTOR in shaping subsets of T cells.


Assuntos
Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/imunologia , Células Th17/imunologia , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/imunologia , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Citometria de Fluxo , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Immunoblotting , Interleucina-17/imunologia , Interleucina-17/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Complexos Multiproteicos/imunologia , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo , Células Th17/metabolismo
14.
J Clin Invest ; 122(4): 1427-43, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22426208

RESUMO

Localized tissue hypoxia is a consequence of vascular compromise or rapid cellular proliferation and is a potent inducer of compensatory angiogenesis. The oxygen-responsive transcriptional regulator hypoxia-inducible factor 2α (HIF-2α) is highly expressed in vascular ECs and, along with HIF-1α, activates expression of target genes whose products modulate vascular functions and angiogenesis. However, the mechanisms by which HIF-2α regulates EC function and tissue perfusion under physiological and pathological conditions are poorly understood. Using mice in which Hif2a was specifically deleted in ECs, we demonstrate here that HIF-2α expression is required for angiogenic responses during hindlimb ischemia and for the growth of autochthonous skin tumors. EC-specific Hif2a deletion resulted in increased vessel formation in both models; however, these vessels failed to undergo proper arteriogenesis, resulting in poor perfusion. Analysis of cultured HIF-2α-deficient ECs revealed cell-autonomous increases in migration, invasion, and morphogenetic activity, which correlated with HIF-2α-dependent expression of specific angiogenic factors, including delta-like ligand 4 (Dll4), a Notch ligand, and angiopoietin 2. By stimulating Dll4 signaling in cultured ECs or restoring Dll4 expression in ischemic muscle tissue, we rescued most of the HIF-2α-dependent EC phenotypes in vitro and in vivo, emphasizing the critical role of Dll4/Notch signaling as a downstream target of HIF-2α in ECs. These results indicate that HIF-1α and HIF-2α fulfill complementary, but largely nonoverlapping, essential functions in pathophysiological angiogenesis.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Circulação Colateral/fisiologia , Células Endoteliais/metabolismo , Membro Posterior/irrigação sanguínea , Isquemia/fisiopatologia , Neovascularização Patológica/fisiopatologia , Neoplasias Cutâneas/irrigação sanguínea , Angiopoietina-2/genética , Angiopoietina-2/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Hipóxia Celular , Movimento Celular , Células Cultivadas/citologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/deficiência , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neovascularização Fisiológica/fisiologia , Receptores Notch/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Recuperação de Função Fisiológica , Neoplasias Cutâneas/induzido quimicamente , Cicatrização/fisiologia
15.
Genes Cancer ; 2(12): 1117-33, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22866203

RESUMO

The vascular network delivers oxygen (O(2)) and nutrients to all cells within the body. It is therefore not surprising that O(2) availability serves as a primary regulator of this complex organ. Most transcriptional responses to low O(2) are mediated by hypoxia-inducible factors (HIFs), highly conserved transcription factors that control the expression of numerous angiogenic, metabolic, and cell cycle genes. Accordingly, the HIF pathway is currently viewed as a master regulator of angiogenesis. HIF modulation could provide therapeutic benefit for a wide array of pathologies, including cancer, ischemic heart disease, peripheral artery disease, wound healing, and neovascular eye diseases. Hypoxia promotes vessel growth by upregulating multiple pro-angiogenic pathways that mediate key aspects of endothelial, stromal, and vascular support cell biology. Interestingly, recent studies show that hypoxia influences additional aspects of angiogenesis, including vessel patterning, maturation, and function. Through extensive research, the integral role of hypoxia and HIF signaling in human disease is becoming increasingly clear. Consequently, a thorough understanding of how hypoxia regulates angiogenesis through an ever-expanding number of pathways in multiple cell types will be essential for the identification of new therapeutic targets and modalities.

16.
Invest Ophthalmol Vis Sci ; 50(11): 5463-71, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19474410

RESUMO

PURPOSE: Anterograde intraflagellar transport (IFT) is essential for photoreceptor outer segment formation and maintenance, as well as for opsin trafficking. However, the role of retrograde IFT in vertebrate photoreceptors remains unclear. The purpose of this study was to evaluate zebrafish photoreceptors lacking the retrograde IFT motor, cytoplasmic dynein-2. METHODS: Morpholino oligonucleotides against the heavy chain (dync2-h1), light intermediate chain (dync2-li1), and intermediate chain (dync2-i1) subunits of cytoplasmic dynein-2 were injected into zebrafish embryos. Retinas and ciliated cells of these zebrafish morphants were analyzed by immunohistochemistry and transmission electron microscopy. Whole-field electroretinograms (ERGs) were performed on dynein morphants at 5 to 6 days after fertilization (dpf). RESULTS: Zebrafish lacking cytoplasmic dynein-2 function exhibited small eyes, kidney cysts, and short photoreceptor outer segments, some of which were disorganized with accumulated vesicles. Morphant photoreceptor connecting cilia were swollen, but neither opsin nor arrestin was mislocalized, although IFT88 accumulated in the distal region of the connecting cilium. Nasal cilia were shortened and displayed cytoplasmic swelling along the axoneme. Loss of cytoplasmic dynein-2 function resulted in a significant reduction in the amplitude of ERG a-, b-, and d-waves but no change in threshold response. CONCLUSIONS: Retrograde IFT is essential for outer segment extension and IFT protein recycling in vertebrate photoreceptors. The results show, for the first time, that the dync2-i1 subunit of cytoplasmic dynein-2 is necessary for retrograde IFT. In addition, arrestin translocation does not require retrograde IFT. Finally, the ERG results indicate that loss of cytoplasmic dynein-2 reduces the photoreceptor light response.


Assuntos
Arrestina/metabolismo , Dineínas do Citoplasma/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Western Blotting , Encéfalo/embriologia , Eletroforese em Gel de Poliacrilamida , Eletrorretinografia , Embrião não Mamífero/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Hibridização In Situ , Rim/embriologia , Células Fotorreceptoras de Vertebrados , Transporte Proteico , Segmento Externo das Células Fotorreceptoras da Retina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Peixe-Zebra
17.
J Cell Sci ; 121(11): 1907-15, 2008 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-18492793

RESUMO

Defects in protein transport within vertebrate photoreceptors can result in photoreceptor degeneration. In developing and mature photoreceptors, proteins targeted to the outer segment are transported through the connecting cilium via the process of intraflagellar transport (IFT). In studies of vertebrate IFT, mutations in any component of the IFT particle typically abolish ciliogenesis, suggesting that IFT proteins are equally required for IFT. To determine whether photoreceptor outer segment formation depends equally on individual IFT proteins, we compared the retinal phenotypes of IFT57 and IFT88 mutant zebrafish. IFT88 mutants failed to form outer segments, whereas IFT57 mutants formed short outer segments with reduced amounts of opsin. Our phenotypic analysis revealed that IFT57 is not essential for IFT, but is required for efficient IFT. In co-immunoprecipitation experiments from whole-animal extracts, we determined that kinesin II remained associated with the IFT particle in the absence of IFT57, but IFT20 did not. Additionally, kinesin II did not exhibit ATP-dependent dissociation from the IFT particle in IFT57 mutants. We conclude that IFT20 requires IFT57 to associate with the IFT particle and that IFT57 and/or IFT20 mediate kinesin II dissociation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cílios/metabolismo , Cinesina/metabolismo , Mutação/genética , Células Fotorreceptoras/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cílios/ultraestrutura , Microscopia Eletrônica de Transmissão , Fenótipo , Células Fotorreceptoras/anormalidades , Células Fotorreceptoras/ultraestrutura , Transporte Proteico/genética , Opsinas de Bastonetes/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
18.
Proc Natl Acad Sci U S A ; 104(11): 4600-5, 2007 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-17360570

RESUMO

Choroideremia is an X-linked hereditary retinal degeneration resulting from mutations in the Rab escort protein-1 (REP1). The Rep1 protein facilitates posttranslational modification of Rab proteins, which regulate intracellular trafficking in the retinal pigment epithelium (RPE) and photoreceptors and are likely involved in the removal of outer segment disk membranes by the RPE. A critical question for potential treatment of choroideremia is whether photoreceptor degeneration results from autonomous defects in opsin transport within the photoreceptor or as a nonautonomous and secondary consequence of RPE degeneration. To address this question, we have characterized the retinal pathology in zebrafish rep1 mutants, which carry a recessive nonsense mutation in the REP1 gene. Zebrafish rep1 mutants exhibit degeneration of the RPE and photoreceptors and complete loss of visual function as measured by electroretinograms. In the mutant RPE, photoreceptor outer segment material was not effectively eliminated, and large vacuoles were observed. However, opsin trafficking in photoreceptors occurred normally. Mosaic analysis revealed that photoreceptor degeneration was nonautonomous and required contact with the mutant RPE as mutant photoreceptors were rescued in wild-type hosts and wild-type photoreceptors degenerated in mutant hosts. We conclude that mutations in REP1 disrupt cellular processes in the RPE, which causes photoreceptor death as a secondary consequence. These results suggest that therapies that correct the RPE may successfully rescue photoreceptor loss in choroideremia.


Assuntos
Coroideremia/genética , Mutação , Células Fotorreceptoras de Vertebrados/patologia , Alelos , Animais , Coroideremia/patologia , Modelos Animais de Doenças , Eletrorretinografia , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Mosaicismo , Epitélio Pigmentado Ocular/metabolismo , Degeneração Retiniana/genética , Opsinas de Bastonetes/metabolismo , Fatores de Tempo , Peixe-Zebra
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