RESUMO
OBJECTIVES: The congenital diaphragmatic hernia (CDH), characterized by malformation of the diaphragm and lung hypoplasia, is a common and severe birth defect that affects around 1 in 4000 live births. However, the etiology of most cases of CDH remains unclear. The aim of this study was to perform a retrospective analysis of copy number variations (CNVs) using a high-resolution array comparative genomic hybridization (array-CGH) in a cohort of fetuses and newborns with CDH. METHODS: Forty seven fetuses and newborns with either isolated or syndromic CDH were analyzed by oligonucleotide-based array-CGH Agilent 180K technique. RESULTS: A mean of 10.2 CNVs was detected by proband with a total number of 480 CNVs identified based on five categories: benign, likely benign, of uncertain signification, likely pathogenic, and pathogenic. Diagnostic performance was estimated at 19.15% (i.e., likely pathogenic and pathogenic CNVs) for both CDH types. We identified 11 potential candidate genes: COL25A1, DSEL, EYA1, FLNA, MECOM, NRXN1, RARB, SPATA13, TJP2, XIRP2, and ZFPM2. CONCLUSION: We suggest that COL25A1, DSEL, EYA1, FLNA, MECOM, NRXN1, RARB, SPATA13, TJP2, XIRP2, and ZFPM2 genes may be related to CDH occurrence. Thus, this study provides a possibility for new methods of a positive diagnosis.
Assuntos
Hérnias Diafragmáticas Congênitas , Recém-Nascido , Humanos , Hérnias Diafragmáticas Congênitas/genética , Variações do Número de Cópias de DNA , Hibridização Genômica Comparativa/métodos , Estudos Retrospectivos , Feto , Fatores de Transcrição/genéticaRESUMO
Goldberg-Shprintzen syndrome (GOSHS, MIM #609460) is an autosomal recessive disorder of intellectual disability, specific facial gestalt and Hirschsprung's disease (HSCR). In 2005, homozygosity mapping in a large consanguineous family identified KIAA1279 as the disease-causing gene. KIAA1279 encodes KIF-binding protein (KBP), whose function is incompletely understood. Studies have identified either the mitochondria or the cytoskeleton as the site of KBP localization and interactions. To better delineate the KIAA1279-related clinical spectrum and the molecular mechanisms involved in GOSHS, we studied five new patients from three different families. The homozygous KIAA1279 mutations in these patients (p.Arg90X, p.Ser200X or p.Arg202IlefsX2) led to nonsense-mediated mRNA decay and loss of KBP function. Despite the absence of functional KBP, respiratory chain complex activity in patient fibroblasts was normal. KBP did not co-localize with mitochondria in control human fibroblasts, but interacted with the actin and tubulin cytoskeleton. KBP expression directly affected neurite growth in a neuron-like cell line (human neuroblastoma SH-SY5Y), in keeping with the central (polymicrogyria) and enteric (HSCR) neuronal developmental defects seen in GOSHS patients. The KBP interactions with actin filaments and microtubules (MTs) demonstrated in our study constitute the first evidence that an actin MT cross-link protein is involved in neuronal development in humans.
Assuntos
Anormalidades Craniofaciais/metabolismo , Doença de Hirschsprung/metabolismo , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Actinas/genética , Actinas/metabolismo , Adolescente , Adulto , Criança , Anormalidades Craniofaciais/genética , Feminino , França , Doença de Hirschsprung/genética , Humanos , Lactente , Iraque , Masculino , Microtúbulos/genética , Mutação , Proteínas do Tecido Nervoso/genética , Linhagem , Ligação Proteica , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , População Branca/genéticaRESUMO
Mowat-Wilson syndrome (MWS) is a severe intellectual disability (ID)-distinctive facial gestalt-multiple congenital anomaly syndrome, commonly associating microcephaly, epilepsy, corpus callosum agenesis, conotruncal heart defects, urogenital malformations and Hirschsprung disease (HSCR). MWS is caused by de novo heterozygous mutations in the ZEB2 gene. The majority of mutations lead to haplo-insufficiency through premature stop codons or large gene deletions. Only three missense mutations have been reported so far; none of which resides in a known functional domain of ZEB2. In this study, we report and analyze the functional consequences of three novel missense mutations, p.Tyr1055Cys, p.Ser1071Pro and p.His1045Arg, identified in the highly conserved C-zinc-finger (C-ZF) domain of ZEB2. Patients' phenotype included the facial gestalt of MWS and moderate ID, but no microcephaly, heart defects or HSCR. In vitro studies showed that all the three mutations prevented binding and repression of the E-cadherin promoter, a characterized ZEB2 target gene. Taking advantage of the zebrafish morphant technology, we performed rescue experiments using wild-type (WT) and mutant human ZEB2 mRNAs. Variable, mutation-dependent, embryo rescue, correlating with the severity of patients' phenotype, was observed. Our data provide evidence that these missense mutations cause a partial loss of function of ZEB2, suggesting that its role is not restricted to repression of E-cadherin. Functional domains other than C-ZF may play a role in early embryonic development. Finally, these findings broaden the clinical spectrum of ZEB2 mutations, indicating that MWS ought to be considered in patients with lesser degrees of ID and a suggestive facial gestalt, even in the absence of congenital malformation.
Assuntos
Alelos , Doença de Hirschsprung/genética , Proteínas de Homeodomínio/genética , Deficiência Intelectual/genética , Microcefalia/genética , Mutação de Sentido Incorreto , Proteínas Repressoras/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , DNA/metabolismo , Modelos Animais de Doenças , Fácies , Feminino , Ordem dos Genes , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Fenótipo , Ligação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Transcrição Gênica , Peixe-Zebra , Homeobox 2 de Ligação a E-box com Dedos de Zinco , Dedos de Zinco/genéticaRESUMO
Pitt-Hopkins syndrome (PTHS), characterized by severe intellectual disability and typical facial gestalt, is part of the clinical spectrum of Rett-like syndromes. TCF4, encoding a basic helix-loop-helix (bHLH) transcription factor, was identified as the disease-causing gene with de novo molecular defects. While PTHS appears to be a recognizable clinical entity, it seems to remain underdiagnosed, especially when facial gestalt is less typical. With the aim to facilitate the diagnosis of PTHS and to increase its rate and specificity, we have investigated 33 novel patients and defined a Clinical Diagnosis Score. Analysis of 112 individuals (79 previously reported and 33 novel patients) allowed us to delineate the TCF4 mutational spectrum, with 40% point mutations, 30% small deletions/insertions, and 30% deletions. Most of these were private mutations and generated premature stop codons. Missense mutations were localized in the bHLH domain, which is a mutational hotspot. No obvious difference was observed between patients harboring truncating, missense mutations, or deletions, further supporting TCF4 haploinsufficiency as the molecular mechanism underlying PTHS. In this study, we have summarized the current knowledge of TCF4 molecular pathology, reported all the mutations in the TCF4 database (http://www.LOVD.nl/TCF4), and present a novel and comprehensive diagnostic strategy for PTHS.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Cromossomos Humanos Par 18/genética , Hiperventilação/diagnóstico , Deficiência Intelectual/diagnóstico , Fatores de Transcrição/genética , Adolescente , Criança , Pré-Escolar , Cromossomos Humanos Par 18/química , Bases de Dados Genéticas , Fácies , Feminino , Estudos de Associação Genética , Variação Genética , Genótipo , Haploinsuficiência , Haplótipos , Humanos , Hiperventilação/genética , Lactente , Deficiência Intelectual/genética , Masculino , Mutação de Sentido Incorreto , Fenótipo , Estrutura Terciária de Proteína , Deleção de Sequência , Inversão de Sequência , Índice de Gravidade de Doença , Fator de Transcrição 4RESUMO
Despite increasing insight into the genetics of infertility, the developmental disease processes remain unclear due to the lack of adequate experimental models. The advent of induced pluripotent stem cell (iPSC) technology has provided a unique tool for in vitro disease modeling enabling major advances in our understanding of developmental disease processes. We report the full characterization of complex genetic abnormalities in two infertile patients with either azoospermia or XX male syndrome and we identify genes of potential interest implicated in their infertility. Using the erythroblasts of both patients, we generated primed iPSCs and converted them into a naive-like pluripotent state. Naive-iPSCs were then differentiated into primordial germ-like cells (PGC-LCs). The expression of early PGC marker genes SOX17, CD-38, NANOS3, c-KIT, TFAP2C, and D2-40, confirmed progression towards the early germline stage. Our results demonstrate that iPSCs from two infertile patients with significant genetic abnormalities are capable of efficient production of PGCs. Such in vitro model of infertility will certainly help identifying causative factors leading to early germ cells development failure and provide a valuable tool to explore novel therapeutic strategies.
Assuntos
Azoospermia , Células-Tronco Pluripotentes Induzidas , Azoospermia/genética , Azoospermia/metabolismo , Diferenciação Celular/genética , Eritroblastos , Células Germinativas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , MasculinoRESUMO
BACKGROUND: Terminal deletions of the long arm of chromosome 7 are well known and frequently associated with syndromic holoprosencephaly due to the involvement of the SHH (aliases HHG1, SMMCI, TPT, TPTPS, and MCOPCB5) gene region. However, interstitial deletions including CNTNAP2 (aliases Caspr2, KIAA0868, and NRXN4) and excluding the SHH region are less common. METHODS: We report the clinical and molecular characterization associated with pure 7q35 and 7q35q36.1 deletion in two unrelated patients as detected by oligonucleotide-based array-CGH analysis. RESULTS: The common clinical features were abnormal maternal serum screening during first-trimester pregnancy, low occipitofrontal circumference at birth, hypotonia, abnormal feet, developmental delay, impaired language development, generalized seizures, hyperactive behavior, friendly personality, and cranio-facial dysmorphism. Both deletions occurred de novo and sequencing of CNTNAP2, a candidate gene for epilepsy and autism showed absence of mutation on the contralateral allele. CONCLUSION: Combined haploinsufficiency of GALNTL5 (alias GalNAc-T5L), CUL1, SSPO (aliases SCO-spondin, KIAA0543, and FLJ36112), AOC1 (alias DAO), RHEB, and especially KMT2C (alias KIAA1506 and HALR) with monoallelic disruption of CNTNAP2 may explain neurologic abnormalities, hypotonia, and exostoses. Haploinsufficiency of PRKAG2 (aliases AAKG, AAKG2, H91620p, WPWS, and CMH6) and KCNH2 (aliases Kv11.1, HERG, and erg1) genes may be responsible of long QT syndrome observed for one patient.
Assuntos
Deleção Cromossômica , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 7/genética , Anormalidades Craniofaciais/genética , Proteínas de Ligação a DNA/genética , Deficiências do Desenvolvimento/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Pré-Escolar , Transtornos Cromossômicos/patologia , Anormalidades Craniofaciais/patologia , Deficiências do Desenvolvimento/patologia , Haploinsuficiência , Humanos , Masculino , Teste Pré-Natal não Invasivo , FenótipoRESUMO
Cystic fibrosis is mainly caused by mutations that interfere with the biosynthetic folding of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The aim of this study was to find cellular proteins interacting with CFTR and regulating its processing. We have used a genetic screen in yeast to identify such proteins and identified CSN5 that interacted with the third cytoplasmic loop of CFTR. CSN5 is the 5th component of the COP9 signalosome, a complex of eight subunits that shares significant homologies to the lid subcomplex of the 26S proteasome and controls the stability of many proteins. The present study shows that CSN5 associates with the core-glycosylated form of CFTR and suggests that this association targets misfolded CFTR to the degradative pathway. Identifying CSN5 as a new component of the degradative pathway is an important step towards the goal of unraveling the sorting between misfolded and correctly folded CFTR proteins.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeo Hidrolases/metabolismo , Dobramento de Proteína , Western Blotting , Complexo do Signalossomo COP9 , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Glicosilação , Células HeLa , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Rim/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Peptídeo Hidrolases/genética , Ligação Proteica , Proteínas Tirosina Quinases/antagonistas & inibidores , Saccharomyces cerevisiae , Estilbenos/farmacologia , Frações Subcelulares , Técnicas do Sistema de Duplo-HíbridoRESUMO
Proximal 19p13.12 microdeletion has been rarely reported. Only five postnatal cases with intellectual disability, facial dysmorphism, branchial arch defects and overlapping deletions involving proximal 19p13.12 have been documented. Two critical intervals were previously defined: a 700 kb for branchial arch defects and a 350 kb for hypertrichosis-synophrys-protruding front teeth. We describe the first prenatal case, a fetal death in utero at 39 weeks of gestation. Agilent 180K array-CGH analysis identified a heterozygous interstitial 745 kb deletion at 19p13.12 chromosome region, encompassing both previously reported critical intervals, including at least 6 functionally relevant genes: NOTCH3, SYDE1, AKAP8, AKAP8L, WIZ and BRD4. Quantitative PCR showed that the deletion occurred de novo with a median size of 753 kb. NOTCH3 and SYDE1 were candidate genes for placental pathology whilst AKAP8, AKAP8L, WIZ and BRD4 were highly expressed in the branchial arches. Molecular characterization and sequencing of candidate genes for placental pathology and branchial arch defects were carried out in order to correlate the genotype-phenotype relationship and unravel the underlying mechanism of proximal 19p13.12 microdeletion syndrome. This case also contributes to define the novel critical interval and expand the clinical phenotype spectrum of proximal 19p13.12 microdeletion syndrome.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 19 , Doenças Fetais/diagnóstico , Doenças Fetais/genética , Anormalidades Múltiplas/genética , Adulto , Região Branquial/anormalidades , Hibridização Genômica Comparativa , Feminino , Morte Fetal , Genótipo , Heterozigoto , Humanos , Deficiência Intelectual/genética , Fenótipo , Reação em Cadeia da Polimerase , Gravidez , Terceiro Trimestre da Gravidez , Diagnóstico Pré-Natal , SíndromeRESUMO
Interstitial 17q24.1 or 17q24.2 deletions were reported after conventional cytogenetic analysis or chromosomal microarray analysis in patients presenting intellectual disability, facial dysmorphism, and/or malformations. We report on a fetus with craniofacial dysmorphism, talipes equinovarus, and syndactyly associated with a de novo 2.5 Mb 17q24.1q24.2 deletion. Among the deleted genes, KPNA2 and PSMD12 are discussed for the correlation with the fetal phenotype. This is the first case of prenatal diagnosis of 17q24.1q24.2 deletion.
RESUMO
Despite progress in human reproductive biology, the cause of male infertility often remains unknown, due to the lack of appropriate and convenient in vitro models of meiosis. Induced pluripotent stem cells (iPSCs) derived from the cells of infertile patients could provide a gold standard model for generating primordial germ cells and studying their development and the process of spermatogenesis. We report the characterization of a complex chromosomal rearrangement (CCR) in an azoospermic patient, and the successful generation of specific-iPSCs from PBMC-derived erythroblasts. The CCR was characterized by karyotype, fluorescence in situ hybridization and oligonucleotide-based array-comparative genomic hybridization. The CCR included five breakpoints and was caused by the inverted insertion of a chromosome 12 segment into the short arm of one chromosome 7 and a pericentric inversion of the structurally rearranged chromosome 12. Gene mapping of the breakpoints led to the identification of a candidate gene, SYCP3. Erythroblasts from the patient were reprogrammed with Sendai virus vectors to generate iPSCs. We assessed iPSC pluripotency by RT-PCR, immunofluorescence staining and teratoma induction. The generation of specific-iPSCs from patients with a CCR provides a valuable in vitro genetic model for studying the mechanisms by which chromosomal abnormalities alter meiosis and germ cell development.
Assuntos
Eritroblastos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Infertilidade Masculina/patologia , Proteínas Nucleares/genética , Vírus Sendai/genética , Espermatócitos/fisiologia , Testículo/patologia , Adulto , Atrofia , Proteínas de Ciclo Celular , Diferenciação Celular , Células Cultivadas , Técnicas de Reprogramação Celular , Inversão Cromossômica/genética , Cromossomos Humanos Par 12/genética , Hibridização Genômica Comparativa , Proteínas de Ligação a DNA , Feminino , Estudos de Associação Genética , Humanos , Hibridização in Situ Fluorescente , Infertilidade Masculina/genética , Cariotipagem , Masculino , Meiose/genéticaRESUMO
Generation of gametes derived in vitro from pluripotent stem cells holds promising prospects for future reproductive applications. Indeed, it provides information on molecular and cellular mechanisms underlying germ cell (GC) development and could offer a new potential treatment for infertility. Great progress has been made in derivation of gametes from embryonic stem cells, despite ethical issues. Induced pluripotent stem cells (iPSCs) technology allows the reprogramming of a differentiated somatic cell, possibly emanating from the patient, into a pluripotent state. With the emergence of iPSCs, several studies created primordial GC stage to mature gamete-like cells in vitro in mice and humans. Recent findings in GC derivation suggest that in mice, functional gametes can be generated in vitro. This strengthens the idea that it might be possible in the future to generate functional human sperm and oocytes from pluripotent stem cells in culture.
Assuntos
Gametogênese , Infertilidade/terapia , Células-Tronco Pluripotentes/citologia , Transplante de Células-Tronco , Animais , Feminino , Humanos , Masculino , Células-Tronco Pluripotentes/metabolismoRESUMO
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial anion channel. Morbidity is mainly due to lung disease, which is characterized by chronic neutrophilic inflammation. Deregulation of inflammatory pathways is observed in the airways of CF patients, as evidenced by exaggerated NF-κB activity, causing an increase in the local release of pro-inflammatory cytokines such as IL-8. COMMD1, a pleiotropic protein, was recently shown to interact with CFTR and to promote CFTR cell surface expression. The effect of COMMD1 on the NF-κB pathway was assessed in CF and non-CF bronchial epithelial cells by knockdown and overexpression experiments. Results showed that (i) COMMD1 knockdown induced NF-κB-dependent transcription, (ii) COMMD1 overexpression inhibited NF-κB activity and was associated with a decrease in IL-8 transcript level and protein secretion. These data demonstrate the anti-inflammatory properties of COMMD1 in bronchial epithelial cells and open new therapeutic avenues in CF.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fibrose Cística/complicações , Fibrose Cística/metabolismo , Inflamação/complicações , Inflamação/metabolismo , Brônquios/patologia , Linhagem Celular , Fibrose Cística/patologia , Regulação para Baixo , Células Epiteliais/metabolismo , Humanos , Inflamação/patologia , Interleucina-8/genética , Interleucina-8/metabolismo , Modelos Biológicos , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Fator de Transcrição AP-1/metabolismo , Transcrição GênicaRESUMO
The CFTR (cystic fibrosis transmembrane conductance regulator) protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel) and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis.