Neural crest E-cadherin loss drives cleft lip/palate by epigenetic modulation via pro-inflammatory gene-environment interaction

Gene-environment interactions are believed to play a role in multifactorial phenotypes, although poorly described mechanistically. Cleft lip/palate (CLP), the most common craniofacial malformation, has been associated with both genetic and environmental factors, with little gene-environment interaction experimentally demonstrated. Here, we study CLP families harbouring CDH1/E-Cadherin variants with incomplete penetrance and we explore the association of pro-inflammatory conditions to CLP. By studying neural crest (NC) from mouse, Xenopus and humans, we show that CLP can be explained by a 2-hit model, where NC migration is impaired by a combination of genetic (CDH1 loss-of-function) and environmental (pro-inflammatory activation) factors, leading to CLP. Finally, using in vivo targeted methylation assays, we demonstrate that CDH1 hypermethylation is the major target of the pro-inflammatory response, and a direct regulator of E-cadherin levels and NC migration. These results unveil a gene-environment interaction during craniofacial development and provide a 2-hit mechanism to explain cleft lip/palate aetiology

Effects of antipsychotics with different weight gain liabilities on human in vitro models of adipose tissue differentiation and metabolism

AbstractWeight gain and metabolic abnormalities are serious side effects associated with the use of several second generation antipsychotics (SGA). The adipose tissue has been considered a direct SGA target involved in the development of these adverse effects. Recent studies, mainly using murine cells, have suggested that SGA increase both adipogenesis of preadipocytes and lipid accumulation in mature adipocytes. However, to date there has been little research comparing the effects of antipsychotics with different propensities to induce weight gain on human in vitro models of white adipose tissue neoformation and metabolism. The present study aimed to investigate the effects of antipsychotics either strongly associated with weight gain, such as the SGA clozapine and olanzapine, or not, such as the SGA ziprasidone and the classical antipsychotic haloperidol, on proliferation and adipocyte differentiation of human adipose-derived stem cells (ADSCs) and lipogenesis in human mature adipocytes. Whereas ziprasidone induced elevated levels of cell death during adipogenesis and could not be investigated further, we observed that clozapine, olanzapine and haloperidol had slight stimulatory effects on the transcriptional program of ADSCs adipogenesis. However, the observed changes in adipocyte-specific genes were not accompanied by a significant increase in triglyceride accumulation within differentiated adipocytes. Our data also showed that these three antipsychotics displayed inhibitory effects on the proliferation rates of undifferentiated ADSCs. Regarding mature adipocyte metabolism, we observed that olanzapine slightly inhibited insulin-stimulated lipogenesis at the highest concentration used, and haloperidol exerted the strongest inhibitory effects on both basal and insulin-stimulated lipogenesis. Taken together, our results suggest that a direct and potent effect of clozapine and olanzapine on adipose tissue biology is not an important mechanism by which these SGA induce metabolic disturbances in humans. On the other hand, the haloperidol-mediated downregulation of the lipogenic capacity of human adipose tissue may be a possible mechanism contributing to its lower propensity to induce serious metabolic side effects

Array comparative genomic hybridization in confirmation of the deleted genes in a patient with subterminal deletion of the long arm of chromosome 10 associated with sagittal craniosynostosis and dysmorphic features

5th Congress of the Brazilian Biotechnology Society (SBBIOTEC), Florianópolis, Brazil. 10-14 November 2013Craniosynostosis results from premature ossification of one or more cranial sutures and leads to alterations in the shape of the skull and/or premature closure of cranial fontanels, causing impairment of brain perfusion, vision and hearing, airway obstruction, learning difficulties, severe cosmetic deformities and high intracranial pressure [1]. To date, the genetic mechanisms leading to sagittal craniosynostosis are poorly known. The identification of candidate genes underlying this condition may contribute to elucidation of the etiology of this common malformation. The aim of this study was to associate genotype-phenotype of a patient with a deletion in the long arm of chromosome 10 and craniosynostosis.EEA La ConsultaFil: Faria, Ágatha Cristhina. Universidade Federal do Espírito Santo; BrasilFil: Atique, Ferraz de Rodrigo Toledo. Universidade de São Paulo. Instituto de Biociências; BrasilFil: Rebouças, Maria Regina Galvêas Oliveira. Hospital Infantil Nossa Senhora da Glória (HINSG); BrasilFil: Cavagnaro, Pablo Federico. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria La Consulta; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza; ArgentinaFil: Rabbi-Bortolini, Eliete. Associação Educacional de Vitória (AEV/FAESA). BrasilFil: Passos-Bueno, Maria Rita. Universidade de São Paulo. Instituto de Biociências; BrasilFil: Errera, Flávia Imbroisi Valle. Escola Superior de Ciências da Santa Casa de Misericórdia de Vitória. Centro de Pesquisa (EMESCAM); Brasi

Effects of different liposuction techniques on the isolation of mesenchymal stem cells

INTRODUÇÃO: O enxerto de gordura nos últimos anos voltou a ter destaque como aliado dos cirurgiões plásticos no preenchimento de partes moles, no rejuvenescimento facial volumétrico, nos refinamentos de reconstruções mamárias e por ser rica fonte de células-tronco de comportamento mesenquimal (células-tronco adipoderivadas). Considerando que essas células têm importante papel na angiogênese e na diferenciação adipogênica, com impacto direto na sobrevivência dos enxertos de gordura, determinar parâmetros que otimizem a sua obtenção é imperativo. Nesse contexto, o objetivo deste trabalho é avaliar e comparar dois métodos de obtenção do tecido adiposo da região abdominal quanto ao número de células viáveis presentes na fração vásculo-estromal e analisar a expressão de marcadores de superfície. MÉTODO: Foram selecionadas 9 pacientes do sexo feminino submetidas a lipoaspiração. O tecido adiposo foi obtido da região abdominal infraumbilical. Da metade direita foram coletados 20 ml de gordura, empregando-se cânula acoplada a uma seringa, cujo êmbolo foi tracionado de 2 cc em 2 cc, gerando baixas pressões de aspiração (grupo manual). O mesmo processo foi repetido na metade esquerda, entretanto a cânula estava acoplada a um coletor intermediário estéril e esse a uma máquina de vácuo sob pressão negativa constante de 350 mmHg (grupo a vácuo). As amostras foram centrifugadas e a gordura da camada intermediária dos dois grupos foi submetida a contagem celular, estabelecimento de culturas e posterior imunofenotipagem. RESULTADOS: Este estudo demonstrou que, apesar de não haver diferença estatisticamente significativa, a obtenção da gordura da região abdominal empregando-se lipoaspirador com pressão negativa de 350 mmHg proporcionou maior número de células presentes na fração vásculo-estromal quando comparado à obtenção por meio de seringas de 10 ml, com baixas pressões de aspiração. CONCLUSÕES: O emprego de pressão negativa de 350 mmHg é seguro para a obtenção das células-tronco adipoderivadas e o rendimento celular entre os dois grupos não apresentou diferença estatisticamente significativa

Array comparative genomic hybridization in confirmation of the deleted genes in a patient with subterminal deletion of the long arm of chromosome 10 associated with sagittal craniosynostosis and dysmorphic features

We thank the Institute of Biosciences of USP for technical support and PPSUS - SESA/FAPES/CNPq for financial suppor

Using a combination of MLPA kits to detect chromosomal imbalances in patients with multiple congenital anomalies and mental retardation is a valuable choice for developing countries

Conventional karyotyping detects anomalies in 3-15% of patients with multiple congenital anomalies and mental retardation (MCA/MR). Whole-genome array screening (WGAS) has been consistently suggested as the first choice diagnostic test for this group of patients, but it is very costly for large-scale use in developing countries. We evaluated the use of a combination of Multiplex Ligation-dependent Probe Amplification (MLPA) kits to increase the detection rate of chromosomal abnormalities in MCA/MR patients. We screened 261 MCA/MR patients with two subtelomeric and one microdeletion kits. This would theoretically detect up to 70% of all submicroscopic abnormalities. Additionally we scored the de Vries score for 209 patients in an effort to find a suitable cut-off for MLPA screening. Our results reveal that chromosomal abnormalities were present in 87 (33.3%) patients, but only 57 (21.8%) were considered causative. Karyotyping detected 15 abnormalities (6.9%), while MLPA identified 54 (20.7%). Our combined MLPA screening raised the total detection number of pathogenic imbalances more than three times when compared to conventional karyotyping. We also show that using the de Vries score as a cutoff for this screening would only be suitable under financial restrictions. A decision analytic model was constructed with three possible strategies: karyotype, karyotype + MLPA and karyotype + WGAS. Karyotype + MLPA strategy detected anomalies in 19.8% of cases which account for 76.45% of the expected yield for karyotype + WGAS. Incremental Cost Effectiveness Ratio (ICER) of MLPA is three times lower than that of WGAS, which means that, for the same costs, we have three additional diagnoses with MLPA but only one with WGAS. We list all causative alterations found, including rare findings, such as reciprocal duplications of regions deleted in Sotos and Williams-Beuren syndromes. We also describe imbalances that were considered polymorphisms or rare variants, such as the new SNP that confounded the analysis of the 22q13.3 deletion syndrome. (C) 2011 Elsevier Masson SAS. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CEPID (Centro de Pesquisa, Inovacao e Difusao)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ São Paulo, Inst Biociencias, Dept Genet & Biol Evolut, Ctr Estudos Genoma Humano, BR-05508900 São Paulo, BrazilUniv São Paulo, Fac Med, Dept Oncol, BR-05508 São Paulo, BrazilUniv Fed Campina Grande, Campina Grande, PB, BrazilUniversidade Federal de São Paulo, Dept Ginecol, Lab Ginecol Mol, São Paulo, BrazilAssoc Beneficente Coleta Sangue, São Paulo, BrazilUniv São Paulo, Fac Med, Inst Crianca, BR-05508 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ginecol, Lab Ginecol Mol, São Paulo, BrazilWeb of Scienc

Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome

Purpose: To facilitate future diagnosis of Knobloch syndrome (KS) and better understand its etiology, we sought to identify not yet described COL18A1 mutations in KS patients. In addition, we tested whether mutations in this gene lead to absence of the COL18A1 gene product and attempted to better characterize the functional effect of a previously reported missense mutation. Methods: Direct sequencing of COL18A1 exons was performed in KS patients from four unrelated pedigrees. We used immunofluorescent histochemistry in skin biopsies to evaluate the presence of type XVIII collagen in four KS patients carrying two already described mutations: c. 3277C>T, a nonsense mutation, and c. 3601G>A, a missense mutation. Furthermore, we determined the binding properties of the mutated endostatin domain p.A1381T (c.3601G>A) to extracellular matrix proteins using ELISA and surface plasmon resonance assays. Results: We identified four novel mutations in COL18A1, including a large deletion involving exon 41. Skin biopsies from KS patients revealed lack of type XVIII collagen in epithelial basement membranes and blood vessels. We also found a reduced affinity of p.A1381T endostatin to some extracellular matrix components. Conclusions: COL18A1 mutations involved in Knobloch syndrome have a distribution bias toward the coding exons of the C-terminal end. Large deletions must also be considered when point mutations are not identified in patients with characteristic KS phenotype. We report, for the first time, lack of type XVIII collagen in KS patients by immunofluorescent histochemistry in skin biopsy samples. As a final point, we suggest the employment of this technique as a preliminary and complementary test for diagnosis of KS in cases when mutation screening either does not detect mutations or reveals mutations of uncertain effect, such as the p.A1381T change.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) - CEPIDConselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

Contribution of polymorphisms in genes associated with craniofacial development to the risk of nonsyndromic cleft lip and/or palate in the Brazilian population

Background and Objective: Nonsyndromic cleft lip and/or palate (NSCL/P) is a complex disease associated with both genetic and environmental factors. One strategy for identifying of possible NSCL/P genetic causes is to evaluate polymorphic variants in genes involved in the craniofacial development. Design: We carried out a case-control analysis of 13 single nucleotide polymorphisms in 9 genes related to craniofacial development, including TBX1, PVRL1, MID1, RUNX2, TP63, TGFB3, MSX1, MYH9 and JAG2 , in 367 patients with NSCL/P and 413 unaffected controls from Brazil to determine their association with NSCL/P. Results: Four out of 13 polymorphisms (rs28649236 and rs4819522 of TBX1, rs7940667 of PVRL1 and rs1057744 of JAG2 ) were presented in our population. Comparisons of allele and genotype frequencies revealed that the G variant allele and the AG/GG genotypes of TBX1 rs28649236 occurred in a frequency significantly higher in controls than in the NSCL/P group (OR: 0.41; 95% CI: 0.25-0.67; p=0.0002). The frequencies of rs4819522, rs7940667 and rs1057744 minor alleles and genotypes were similar between control and NSCL/P group, without significant differences. No significant associations among cleft types and polymorphisms were observed. Conclusion: The study suggests for the first time evidences to an association of the G allele of TBX1 rs28649236 polymorphism and NSCL/P

Deletion of the Basement Membrane Heparan Sulfate Proteoglycan Type XVIII Collagen Causes Hypertriglyceridemia in Mice and Humans

Background: Lipoprotein lipase (Lpl) acts on triglyceride-rich lipoproteins in the peripheral circulation, liberating free fatty acids for energy metabolism or storage. This essential enzyme is synthesized in parenchymal cells of adipose tissue, heart, and skeletal muscle and migrates to the luminal side of the vascular endothelium where it acts upon circulating lipoproteins. Prior studies suggested that Lpl is immobilized by way of heparan sulfate proteoglycans on the endothelium, but genetically altering endothelial cell heparan sulfate had no effect on Lpl localization or lipolysis. The objective of this study was to determine if extracellular matrix proteoglycans affect Lpl distribution and triglyceride metabolism. Methods and Findings: We examined mutant mice defective in collagen XVIII (Col18), a heparan sulfate proteoglycan present in vascular basement membranes. Loss of Col18 reduces plasma levels of Lpl enzyme and activity, which results in mild fasting hypertriglyceridemia and diet-induced hyperchylomicronemia. Humans with Knobloch Syndrome caused by a null mutation in the vascular form of Col18 also present lower than normal plasma Lpl mass and activity and exhibit fasting hypertriglyceridemia. Conclusions: This is the first report demonstrating that Lpl presentation on the lumenal side of the endothelium depends on a basement membrane proteoglycan and demonstrates a previously unrecognized phenotype in patients lacking Col18.National Institute of Health (NIH)[HL087228]National Institute of Health (NIH)[GM33063]National Institute of Health (NIH)[HL67255]CEPID/FAPESPCNPqUniversity of Colorado Denver Department of MedicineLeducq FoundationAmerican Heart Association[0735038N

Genetics and genomics in Brazil: a promising future

The authors would like to express their gratitude to Celia P. Koiffmann for a critical review of the manuscript. M. R. P. B., D. B., and L. A. B. are funded by FAPESP and CNPq