RESUMO
Congenital diaphragmatic hernia (CDH), characterized by malformation of the diaphragm and hypoplasia of the lungs, is one of the most common and severe birth defects, and is associated with high morbidity and mortality rates. There is growing evidence demonstrating that genetic factors contribute to CDH, although the pathogenesis remains largely elusive. Single-nucleotide polymorphisms have been studied in recent whole-exome sequencing efforts, but larger copy number variants (CNVs) have not yet been studied on a large scale in a case control study. To capture CNVs within CDH candidate regions, we developed and tested a targeted array comparative genomic hybridization platform to identify CNVs within 140 regions in 196 patients and 987 healthy controls, and identified six significant CNVs that were either unique to patients or enriched in patients compared with controls. These CDH-associated CNVs reveal high-priority candidate genes including HLX, LHX1, and HNF1B We also discuss CNVs that are present in only one patient in the cohort but have additional evidence of pathogenicity, including extremely rare large and/or de novo CNVs. The candidate genes within these predicted disease-causing CNVs form functional networks with other known CDH genes and play putative roles in DNA binding/transcription regulation and embryonic development. These data substantiate the importance of CNVs in the etiology of CDH, identify CDH candidate genes and pathways, and highlight the importance of ongoing analysis of CNVs in the study of CDH and other structural birth defects.
Assuntos
Hibridização Genômica Comparativa/métodos , Variações do Número de Cópias de DNA , Marcadores Genéticos , Hérnias Diafragmáticas Congênitas/genética , Polimorfismo de Nucleotídeo Único , Estudos de Casos e Controles , Humanos , PrognósticoRESUMO
To improve ovarian cancer patient survival, effective treatments addressing chemoresistant recurrences are particularly needed. Mullerian inhibiting substance (MIS) has been shown to inhibit the growth of a stem-like population of ovarian cancer cells. We have recently engineered peptide modifications to human MIS [albumin leader Q425R MIS (LRMIS)] that increase production and potency in vitro and in vivo. To test this novel therapeutic peptide, serous malignant ascites from highly resistant recurrent ovarian cancer patients were isolated and amplified to create low-passage primary cell lines. Purified recombinant LRMIS protein successfully inhibited the growth of cancer spheroids in vitro in a panel of primary cell lines in four of six patients tested. Adeno-associated virus (AAV) -delivered gene therapy has undergone a clinical resurgence with a good safety profile and sustained gene expression. Therefore, AAV9 was used as a single i.p. injection to deliver LRMIS to test its efficacy in inhibiting growth of palpable tumors in patient-derived ovarian cancer xenografts from ascites (PDXa). AAV9-LRMIS monotherapy resulted in elevated and sustained blood concentrations of MIS, which significantly inhibited the growth of three of five lethal chemoresistant serous adenocarcinoma PDXa models without signs of measurable or overt toxicity. Finally, we tested the frequency of MIS type II receptor expression in a tissue microarray of serous ovarian tumors by immunohistochemistry and found that 88% of patients bear tumors that express the receptor. Taken together, these preclinical data suggest that AAV9-LRMIS provides a potentially well-tolerated and effective treatment strategy poised for testing in patients with chemoresistant serous ovarian cancer.
Assuntos
Hormônio Antimülleriano/genética , Hormônio Antimülleriano/uso terapêutico , Dependovirus/metabolismo , Terapia Genética , Neoplasias Ovarianas/terapia , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Ascite/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Músculos/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Receptores de Peptídeos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/genética , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Análise Serial de Tecidos , Transgenes , Tropismo , Carga TumoralRESUMO
Congenital Diaphragmatic Hernia (CDH) is a common and often lethal birth defect characterized by diaphragmatic structural defects and pulmonary hypoplasia. CDH is isolated in 60% of newborns, but may also be part of a complex phenotype with additional anomalies. We performed whole exome sequencing (WES) on 87 individuals with isolated or complex CDH and on their unaffected parents, to assess the contribution of de novo mutations in the etiology of diaphragmatic and pulmonary defects and to identify new candidate genes. A combined analysis with 39 additional trios with complex CDH, previously published, revealed a significant genome-wide burden of de novo variants compared to background mutation rate and 900 control trios. We identified an increased burden of likely gene-disrupting (LGD, i.e. nonsense, frameshift, and canonical splice site) and predicted deleterious missense (D-mis) variants in complex and isolated CDH patients. Overall, an excess of predicted damaging de novo LGD and D-mis variants relative to the expected frequency contributed to 21% of complex cases and 12% of isolated CDH cases. The burden of de novo variants was higher in genes expressed in the developing mouse diaphragm and heart. Some overlap with genes responsible for congenital heart defects and neurodevelopmental disorders was observed in CDH patients within our cohorts. We propose that de novo variants contribute significantly to the development of CDH.
Assuntos
Estudo de Associação Genômica Ampla , Hérnias Diafragmáticas Congênitas/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Ligação ProteicaRESUMO
Congenital diaphragmatic hernia (CDH) is a common and severe birth defect. Despite its clinical significance, the genetic and developmental pathways underlying this disorder are incompletely understood. In this study, we report a catalog of variants detected by a whole exome sequencing study on 275 individuals with CDH. Predicted pathogenic variants in genes previously identified in either humans or mice with diaphragm defects are enriched in our CDH cohort compared with 120 size-matched random gene sets. This enrichment was absent in control populations. Variants in these critical genes can be found in up to 30.9% of individuals with CDH. In addition, we filtered variants by using genes derived from regions of recurrent copy number variations in CDH, expression profiles of the developing diaphragm, protein interaction networks expanded from the known CDH-causing genes, and prioritized genes with ultrarare and highly disruptive variants, in 11.3% of CDH patients. These strategies have identified several high priority genes and developmental pathways that likely contribute to the CDH phenotype. These data are valuable for comparison of candidate genes generated from whole exome sequencing of other CDH cohorts or multiplex kindreds and provide ideal candidates for further functional studies. Furthermore, we propose that these genes and pathways will enhance our understanding of the heterogeneous molecular etiology of CDH.
Assuntos
Hérnias Diafragmáticas Congênitas/etiologia , Hérnias Diafragmáticas Congênitas/genética , Animais , Estudos de Coortes , Biologia Computacional , Variações do Número de Cópias de DNA , Diafragma/embriologia , Exoma , Variação Genética , Hérnias Diafragmáticas Congênitas/embriologia , Humanos , Camundongos , Mapas de Interação de ProteínasRESUMO
Chromosome 8p23.1 is a common hotspot associated with major congenital malformations, including congenital diaphragmatic hernia (CDH) and cardiac defects. We present findings from high-resolution arrays in patients who carry a loss (n = 18) or a gain (n = 1) of sub-band 8p23.1. We confirm a region involved in both diaphragmatic and heart malformations. Results from a novel CNVConnect algorithm, prioritizing protein-protein interactions between products of genes in the 8p23.1 hotspot and products of previously known CDH causing genes, implicated GATA4, NEIL2, and SOX7 in diaphragmatic defects. Sequence analysis of these genes in 226 chromosomally normal CDH patients, as well as in a small number of deletion 8p23.1 patients, showed rare unreported variants in the coding region; these may be contributing to the diaphragmatic phenotype. We also demonstrated that two of these three genes were expressed in the E11.5-12.5 primordial mouse diaphragm, the developmental stage at which CDH is thought to occur. This combination of bioinformatics and expression studies can be applied to other chromosomal hotspots, as well as private microdeletions or microduplications, to identify causative genes and their interaction networks.
Assuntos
Hérnias Diafragmáticas Congênitas , Animais , Deleção Cromossômica , Cromossomos Humanos Par 8/genética , Cromossomos Humanos Par 8/metabolismo , DNA/sangue , DNA/genética , DNA Glicosilases/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Feminino , Fator de Transcrição GATA4/genética , Cardiopatias Congênitas/sangue , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Hérnia Diafragmática/sangue , Hérnia Diafragmática/genética , Hérnia Diafragmática/metabolismo , Humanos , Cariotipagem , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Gravidez , Mapas de Interação de Proteínas , Fatores de Transcrição SOXF/genéticaRESUMO
BACKGROUND: Thiazolidinediones (TZD) have been reported to improve early stages of diabetic nephropathy independent of glycaemic control. Since blockade of the renin-angiotensin system (RAS) is known to reduce the risk of nephropathy, we hypothesised that the renal effect of TZDs might be related to a favourable effect on the intrarenal RAS. We aimed to determine if the TZD rosiglitazone could reduce RAS activation. METHODS: We studied adult type 2 diabetic patients and placed them on rosiglitazone for three months. We have previously used the renal haemodynamic response to angiotensin-converting enzyme (ACE) inhibition to demonstrate the state of RAS activation, and thus measured renal plasma flow (RPF) and glomerular filtration rate (GFR) before and after administration of captopril at 0 month and at three months. Plasma renin activity (PRA), active renin, aldosterone and natriuretic peptides were analysed. RESULTS: The RPF response to ACE inhibition was not altered. There was no change in GFR, PRA, active renin and aldosterone levels. Two patients developed oedema one had an elevated baseline active renin and another had an elevated baseline aldosterone level. CONCLUSION: The favourable effects of TZDs on diabetic nephropathy is likely not related to an influence on the RAS.
Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Hipoglicemiantes/administração & dosagem , PPAR gama/agonistas , Circulação Renal/efeitos dos fármacos , Sistema Renina-Angiotensina/efeitos dos fármacos , Tiazolidinedionas/administração & dosagem , Adulto , Idoso , Feminino , Taxa de Filtração Glomerular/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , RosiglitazonaRESUMO
CD44 is a transmembrane hyaluronic acid receptor gene that encodes over 100 different tissue-specific protein isoforms. The most ubiquitous, CD44 standard, has been used as a cancer stem cell marker in ovarian and other cancers. Expression of the epithelial CD44 variant containing exons v8-10 (CD44v8-10) has been associated with more chemoresistant and metastatic tumors in gastrointestinal and breast cancers, but its role in ovarian cancer is unknown; we therefore investigated its use as a prognostic marker in this disease. The gene expression profiles of 254 tumor samples from The Cancer Genome Atlas RNAseqV2 were analyzed for the presence of CD44 isoforms. A trend for longer survival was observed in patients with high expression of CD44 isoforms that include exons v8-10. Immunohistochemical (IHC) analysis of tumors for presence of CD44v8-10 was performed on an independent cohort of 210 patients with high-grade serous ovarian cancer using a tumor tissue microarray. Patient stratification based on software analysis of staining revealed a statistically significant increase in survival in patients with the highest levels of transmembrane protein expression (top 10 or 20%) compared to those with the lowest expression (bottom 10 and 20%) (p = 0.0181, p = 0.0262 respectively). Expression of CD44v8-10 in primary ovarian cancer cell lines was correlated with a predominantly epithelial phenotype characterized by high expression of epithelial markers and low expression of mesenchymal markers by qPCR, Western blot, and IHC. Conversely, detection of proteolytically cleaved and soluble extracellular domain of CD44v8-10 in patient ascites samples was correlated with significantly worse prognosis (p<0.05). Therefore, presence of transmembrane CD44v8-10 on the surface of primary tumor cells may be a marker of a highly epithelial tumor with better prognosis while enzymatic cleavage of CD44v8-10, as detected by presence of the soluble extracellular domain in ascites fluid, may be indicative of a more metastatic disease and worse prognosis.
Assuntos
Biomarcadores Tumorais/genética , Receptores de Hialuronatos/genética , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Isoformas de Proteínas/genética , Adulto , Idoso , Processamento Alternativo/genética , Carcinoma Epitelial do Ovário , Éxons/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/patologia , Prognóstico , Análise Serial de TecidosRESUMO
Hyperglycaemia-induced activation of the renin-angiotensin system (RAS) has been observed in normal and diabetic humans. Our main objective was to determine whether the mechanism involved a physical or metabolic effect of glucose. First, Sprague-Dawley rats of the CD strain were given sequential intravenous (i.v.) doses of 0.01, 0.1, 1.0, and 3.0 mg/kg candesartan 30 minutes apart, in the presence of a continuous i.v. infusion of dextrose 20% in water (D20W). The 0.1 mg/kg dose produced a maximal renal blood flow (RBF) response and was used thereafter. Another set of animals then received an infusion of either normal saline (NS), dextrose 5% in water (D5W) or dextrose 20% in water (D20W) for 2 hours, followed by candesartan 0.1 mg/kg i.v. Finally, the response to candesartan 0.1 mg/kg i.v. during D20W infusion was compared with that during infusion of 2-deoxyglucose (2DG), a glucose analogue that competitively inhibits the glycolytic enzyme, hexokinase. RBF (electromagnetic flowmeter), blood pressure (BP), blood glucose, and urine glucose were monitored. There was no significant RBF response to candesartan on either NS (6.01 to 0.48 to 6.20 to 0.49 ml/minute/g kidney; p=0.216) or D5W (7.63 to 1.20 to 7.58 to 1.39 ml/minute/g kidney; p=0.965), whereas there was a significant response to D20W (6.64 to 0.59 to 7.46 to 0.67 ml/minute/g kidney; p=0.002). The RBF response was significantly enhanced by D20W compared with 2DG (change in RBF: 0.82 to 0.22 vs. -0.04 to 0.26; p=0.05), despite similar BP, blood glucose, and urine glucose. Glucose acts, at least in part, through intracellular utilisation to induce RAS activation, as manifested by an enhanced renal vascular response to an angiotensin II antagonist.
Assuntos
Hiperglicemia/metabolismo , Hiperglicemia/fisiopatologia , Rim/metabolismo , Rim/fisiopatologia , Sistema Renina-Angiotensina/fisiologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Antimetabólitos/farmacologia , Benzimidazóis/farmacologia , Compostos de Bifenilo , Pressão Sanguínea/efeitos dos fármacos , Desoxiglucose/farmacologia , Relação Dose-Resposta a Droga , Glucose/farmacologia , Hematócrito , Hiperglicemia/sangue , Rim/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Circulação Renal/efeitos dos fármacos , Sistema Renina-Angiotensina/efeitos dos fármacos , Tetrazóis/farmacologiaRESUMO
A low sodium diet has often been implicated in the protection of low blood pressure populations from hypertension, but several other dietary factors, including those as yet unidentified, may also be involved. The Kuna Indians of Panama are free of hypertension and cardiovascular disease, but this is changing with migration to urban areas. We compared the indigenous diet of Kuna Indians living on remote islands in Panama (Ailigandi), whose lifestyle is largely hunter-gatherer, with those who have moved to a suburb of Panama City (Vera Cruz). Between April and October 1999, members of a Kuna research team administered a 118-item food frequency questionnaire to133 adult Kuna from Ailigandi and 183 from Vera Cruz. Single 24-hour urine collections and nonfasting blood samples were obtained. The Kuna in Ailigandi reported consuming a 10-fold higher amount of cocoa-containing beverages, 4 times the amount of fish, and twice the amount of fruit as urban Kuna (P<0.05 by t test). Salt added was ample among those living in Ailigandi and Vera Cruz according to both self-report (7.1+/-1.1 and 4.6+/-0.3 tsp weekly) and urinary sodium levels (177+/-9 and 160+/-7 mEq Na/g creatinine), respectively. The low blood pressure of island-dwelling Kuna does not seem to be related to a low salt diet. Among dietary factors that varied among migrating Kuna, the notably higher intake of flavanol-rich cocoa is a potential candidate for further study.