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1.
Am J Hum Genet ; 104(4): 685-700, 2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30929737

RESUMO

Conventional genetic testing of individuals with neurodevelopmental presentations and congenital anomalies (ND/CAs), i.e., the analysis of sequence and copy number variants, leaves a substantial proportion of them unexplained. Some of these cases have been shown to result from DNA methylation defects at a single locus (epi-variants), while others can exhibit syndrome-specific DNA methylation changes across multiple loci (epi-signatures). Here, we investigate the clinical diagnostic utility of genome-wide DNA methylation analysis of peripheral blood in unresolved ND/CAs. We generate a computational model enabling concurrent detection of 14 syndromes using DNA methylation data with full accuracy. We demonstrate the ability of this model in resolving 67 individuals with uncertain clinical diagnoses, some of whom had variants of unknown clinical significance (VUS) in the related genes. We show that the provisional diagnoses can be ruled out in many of the case subjects, some of whom are shown by our model to have other diseases initially not considered. By applying this model to a cohort of 965 ND/CA-affected subjects without a previous diagnostic assumption and a separate assessment of rare epi-variants in this cohort, we identify 15 case subjects with syndromic Mendelian disorders, 12 case subjects with imprinting and trinucleotide repeat expansion disorders, as well as 106 case subjects with rare epi-variants, a portion of which involved genes clinically or functionally linked to the subjects' phenotypes. This study demonstrates that genomic DNA methylation analysis can facilitate the molecular diagnosis of unresolved clinical cases and highlights the potential value of epigenomic testing in the routine clinical assessment of ND/CAs.

2.
Nat Commun ; 9(1): 4885, 2018 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-30459321

RESUMO

Coffin-Siris and Nicolaides-Baraitser syndromes (CSS and NCBRS) are Mendelian disorders caused by mutations in subunits of the BAF chromatin remodeling complex. We report overlapping peripheral blood DNA methylation epi-signatures in individuals with various subtypes of CSS (ARID1B, SMARCB1, and SMARCA4) and NCBRS (SMARCA2). We demonstrate that the degree of similarity in the epi-signatures of some CSS subtypes and NCBRS can be greater than that within CSS, indicating a link in the functional basis of the two syndromes. We show that chromosome 6q25 microdeletion syndrome, harboring ARID1B deletions, exhibits a similar CSS/NCBRS methylation profile. Specificity of this epi-signature was confirmed across a wide range of neurodevelopmental conditions including other chromatin remodeling and epigenetic machinery disorders. We demonstrate that a machine-learning model trained on this DNA methylation profile can resolve ambiguous clinical cases, reclassify those with variants of unknown significance, and identify previously undiagnosed subjects through targeted population screening.

3.
Hum Mol Genet ; 26(21): 4278-4289, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28973161

RESUMO

Defects in neuronal migration cause brain malformations, which are associated with intellectual disability (ID) and epilepsy. Using exome sequencing, we identified compound heterozygous variants (p.Arg71His and p. Leu729ThrfsTer6) in TMTC3, encoding transmembrane and tetratricopeptide repeat containing 3, in four siblings with nocturnal seizures and ID. Three of the four siblings have periventricular nodular heterotopia (PVNH), a common brain malformation caused by failure of neurons to migrate from the ventricular zone to the cortex. Expression analysis using patient-derived cells confirmed reduced TMTC3 transcript levels and loss of the TMTC3 protein compared to parental and control cells. As TMTC3 function is currently unexplored in the brain, we gathered support for a neurobiological role for TMTC3 by generating flies with post-mitotic neuron-specific knockdown of the highly conserved Drosophila melanogaster TMTC3 ortholog, CG4050/tmtc3. Neuron-specific knockdown of tmtc3 in flies resulted in increased susceptibility to induced seizures. Importantly, this phenotype was rescued by neuron-specific expression of human TMTC3, suggesting a role for TMTC3 in seizure biology. In addition, we observed co-localization of TMTC3 in the rat brain with vesicular GABA transporter (VGAT), a presynaptic marker for inhibitory synapses. TMTC3 is localized at VGAT positive pre-synaptic terminals and boutons in the rat hypothalamus and piriform cortex, suggesting a role for TMTC3 in the regulation of GABAergic inhibitory synapses. TMTC3 did not co-localize with Vglut2, a presynaptic marker for excitatory neurons. Our data identified TMTC3 as a synaptic protein that is involved in PVNH with ID and epilepsy, in addition to its previously described association with cobblestone lissencephaly.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Heterotopia Nodular Periventricular/metabolismo , Adulto , Animais , Encéfalo/anormalidades , Córtex Cerebral/metabolismo , Drosophila melanogaster , Epilepsia/genética , Epilepsia/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Heterozigoto , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Masculino , Malformações do Sistema Nervoso/metabolismo , Neurônios/metabolismo , Linhagem , Heterotopia Nodular Periventricular/genética , Terminações Pré-Sinápticas , Ratos , Convulsões/metabolismo , Sinapses/metabolismo , Sequenciamento Completo do Exoma
4.
Invest Ophthalmol Vis Sci ; 53(13): 8333-43, 2012 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-23150625

RESUMO

PURPOSE: Diabetic retinopathy entails proliferation of vascular endothelial cells (ECs) and unregulated angiogenesis. We have previously shown that ECs increase the expression of an embryonic variant of fibronectin (FN), called extra domain-B FN (ED-B FN) in response to high glucose. We also showed that ED-B FN regulates EC tube morphogenesis, possibly through vascular endothelial growth factor (VEGF). In the present study, we have attempted to decipher the mechanisms by which ED-B FN may modulate EC phenotype. METHODS: We hypothesized that ED-B FN regulates VEGF expression in ECs through interaction with selected integrin receptors. To test this hypothesis, we first cultured ECs in high levels of glucose to investigate for any alteration. We then used integrin-specific matrix mimetic peptides, neutralizing antibodies, and RNAi to identify the integrin(s) involved in VEGF expression. Finally, we used an animal model of diabetes to study whether these in vitro mechanisms also take place in the retina. RESULTS: Our results show that exposure of ECs to high levels of glucose increased VEGF expression. ED-B FN mediated this increase since knockdown of ED-B FN completely prevented glucose-induced VEGF expression. We then identified ß1 integrin as the essential receptor involved in high glucose-induced VEGF expression. We also showed that diabetes increased ß1 integrin and VEGF expression in the retina, which normalized upon ED-B knockdown. CONCLUSIONS: These findings showed that high levels of glucose in diabetes increased VEGF expression in ECs through ED-B FN and ß1 integrin interaction. These results provide novel mechanistic basis of increased VEGF expression in diabetes.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Células Endoteliais/efeitos dos fármacos , Fibronectinas/metabolismo , Regulação da Expressão Gênica/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Anticorpos Neutralizantes , Western Blotting , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Células Endoteliais/metabolismo , Fibronectinas/genética , Técnica Indireta de Fluorescência para Anticorpo , Glucose/toxicidade , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrina alfa2beta1/antagonistas & inibidores , Integrina alfa2beta1/metabolismo , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfa5beta1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Vasos Retinianos/citologia , Transfecção
5.
J Diabetes Investig ; 2(2): 123-31, 2011 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-24843471

RESUMO

UNLABELLED: Aims/Introduction: In diabetes, increased oxidative stress as a result of damage to the electron transport chain can lead to tissue injury through upregulation of multiple vasoactive factors and extracellular matrix proteins. Benfotiamine, a lipid soluble thiamine derivative, through reducing mitochondrial superoxide production, blocks multiple pathways leading to tissue damage in hyperglycemia. We investigated if treatment with benfotiamine can prevent diabetes-induced production of vasoactive factors and extracellular matrix proteins, and whether such effects are tissue-specific. We also examined whether effects of benfotiamine are mediated through a nuclear mechanism. MATERIALS AND METHODS: Retinal, renal and cardiac tissues from the streptozotocin-induced diabetic rats were examined after 4 months of follow up. mRNA levels were quantified using real-time RT-PCR. Protein levels were quantified using western blot and ELISA. Cellular expressions of 8-Hydroxy-2'-deoxyguanosine, a marker of nuclear DNA damage and Phospho-H2AX were also examined. RESULTS: Diabetic animals showed hyperglycemia, glucosuria, increased urinary albumin/creatine ratio and loss of bodyweight. In the kidneys, heart and retina, diabetes caused increased production of endothelin-1, transforming growth factor-ß1, vascular endothelial growth factor and augmented extracellular matrix proteins (collagen, fibronectin [FN] and its splice variant extradomain B containing FN), along with evidence of structural alterations, characteristic of diabetes-induced tissue damage. Such changes were prevented by benfotiamine. Furthermore, benfotiamine prevented diabetes-induced oxidative DNA damage and upregulation of p300, a histone acetylator and a transcription coactivator. CONCLUSIONS: Data from the present study suggest that benfotiamine is effective in preventing tissue damage in diabetes and at the transcriptional level such effects are mediated through prevention of p300 upregulation. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00077.x, 2010).

6.
J Ophthalmol ; 2010: 465824, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20671964

RESUMO

Diabetic retinopathy is one of the most common causes of blindness in North America. Several signaling mechanisms are activated secondary to hyperglycemia in diabetes, leading to activation of vasoactive factors. We investigated a novel pathway, namely extracellular signal regulated kinase 5 (ERK5) mediated signaling, in modulating glucose-induced vascular endothelial growth factor (VEGF) expression. Human microvascular endothelial cells (HMVEC) were exposed to glucose. In parallel, retinal tissues from streptozotocin-induced diabetic rats were examined after 4 months of follow-up. In HMVECs, glucose caused initial activation followed by deactivation of ERK5 and its downstream mediators myocyte enhancing factor 2C (MEF2C) and Kruppel-like factor 2 (KLF2) mRNA expression. ERK5 inactivation further led to augmented VEGF mRNA expression. Furthermore, siRNA mediated ERK5 gene knockdown suppressed MEF2C and KLF2 expression and increased VEGF expression and angiogenesis. On the other hand, constitutively active MEK5, an activator of ERK5, increased ERK5 activation and ERK5 and KLF2 mRNA expression and attenuated basal- and glucose-induced VEGF mRNA expression. In the retina of diabetic rats, depletion of ERK5, KLF2 and upregulation of VEGF mRNA were demonstrated. These results indicated that ERK5 depletion contributes to glucose induced increased VEGF production and angiogenesis. Hence, ERK5 may be a putative therapeutic target to modulate VEGF expression in diabetic retinopathy.

7.
Am J Physiol Endocrinol Metab ; 298(1): E127-37, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19903865

RESUMO

Sustained hyperglycemia in diabetes causes alteration of a large number of transcription factors and mRNA transcripts, leading to tissue damage. We investigated whether p300, a transcriptional coactivator with histone acetyl transferase activity, regulates glucose-induced activation of transcription factors and subsequent upregulation of vasoactive factors and extracellular matrix (ECM) proteins in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated in varied glucose concentrations and were studied after p300 small interfering RNA (siRNA) transfection, p300 overexpression, or incubation with the p300 inhibitor curcumin. Histone H2AX phosphorylation and lysine acetylation were examined for oxidative DNA damage and p300 activation. Screening for transcription factors was performed with the Luminex system. Alterations of selected transcription factors were validated. mRNA expression of p300, endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), and fibronectin (FN) and its splice variant EDB(+)FN and FN protein production were analyzed. HUVECs in 25 mmol/l glucose showed increased p300 production accompanied by increased binding of p300 to ET-1 and FN promoters, augmented histone acetylation, H2AX phosphorylation, activation of multiple transcription factors, and increased mRNA expression of vasoactive factors and ECM proteins. p300 overexpression showed a glucose-like effect on the mRNA expression of ET-1, VEGF, and FN. Furthermore, siRNA-mediated p300 blockade or chemical inhibitor of p300 prevented such glucose-induced changes. Similar mRNA upregulation was also seen in the organ culture of vascular tissues, which was prevented by p300 siRNA transfection. Data from these studies suggest that glucose-induced p300 upregulation is an important upstream epigenetic mechanism regulating gene expression of vasoactive factors and ECM proteins in endothelial cells and is a potential therapeutic target for diabetic complications.


Assuntos
Complicações do Diabetes/genética , Células Endoteliais/fisiologia , Glucose/farmacologia , Hiperglicemia/genética , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação/efeitos dos fármacos , Animais , Aorta/fisiologia , Divisão Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Dano ao DNA/fisiologia , Complicações do Diabetes/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Endotelina-1/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Histonas/metabolismo , Humanos , Hiperglicemia/metabolismo , Masculino , Técnicas de Cultura de Órgãos , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Veias Umbilicais/citologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fatores de Transcrição de p300-CBP/genética
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