RESUMO
Incomplete penetrance is the rule rather than the exception in Mendelian disease. In syndromic monogenic disorders, phenotypic variability can be viewed as the combination of incomplete penetrance for each of multiple independent clinical features. Within genetically identical individuals, such as isogenic model organisms, stochastic variation at molecular and cellular levels is the primary cause of incomplete penetrance according to a genetic threshold model. By defining specific probability distributions of causal biological readouts and genetic liability values, stochasticity and incomplete penetrance provide information about threshold values in biological systems. Ascertainment of threshold values has been achieved by simultaneous scoring of relatively simple phenotypes and quantitation of molecular readouts at the level of single cells. However, this is much more challenging for complex morphological phenotypes using experimental and reductionist approaches alone, where cause and effect are separated temporally and across multiple biological modes and scales. Here I consider how causal inference, which integrates observational data with high confidence causal models, might be used to quantify the relative contribution of different sources of stochastic variation to phenotypic diversity. Collectively, these approaches could inform disease mechanisms, improve predictions of clinical outcomes and prioritize gene therapy targets across modes and scales of gene function. This article is part of a discussion meeting issue 'Causes and consequences of stochastic processes in development and disease'.
Assuntos
Variação Biológica da População , Humanos , Penetrância , Processos Estocásticos , Causalidade , FenótipoRESUMO
Dynein-2 is a large multiprotein complex that powers retrograde intraflagellar transport (IFT) of cargoes within cilia/flagella, but the molecular mechanism underlying this function is still emerging. Distinctively, dynein-2 contains two identical force-generating heavy chains that interact with two different intermediate chains (WDR34 and WDR60). Here, we dissect regulation of dynein-2 function by WDR34 and WDR60 using an integrative approach including cryo-electron microscopy and CRISPR/Cas9-enabled cell biology. A 3.9 Å resolution structure shows how WDR34 and WDR60 use surprisingly different interactions to engage equivalent sites of the two heavy chains. We show that cilia can assemble in the absence of either WDR34 or WDR60 individually, but not both subunits. Dynein-2-dependent distribution of cargoes depends more strongly on WDR60, because the unique N-terminal extension of WDR60 facilitates dynein-2 targeting to cilia. Strikingly, this N-terminal extension can be transplanted onto WDR34 and retain function, suggesting it acts as a flexible tether to the IFT "trains" that assemble at the ciliary base. We discuss how use of unstructured tethers represents an emerging theme in IFT train interactions.
Assuntos
Cílios , Dineínas , Dineínas/metabolismo , Microscopia Crioeletrônica , Transporte Biológico , Cílios/metabolismo , Flagelos/metabolismoRESUMO
Bardet-Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate.
Assuntos
Síndrome de Bardet-Biedl , Humanos , Camundongos , Animais , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/patologia , Camundongos Knockout , Proteínas/metabolismo , Cílios/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Idiopathic scoliosis (IS) is the deformation and/or abnormal curvature of the spine that develops progressively after birth. It is a very common condition, affecting approximately 4% of the general population, yet the genetic and mechanistic causes of IS are poorly understood. Here, we focus on PPP2R3B, which encodes a protein phosphatase 2A regulatory subunit. We found that PPP2R3B is expressed at sites of chondrogenesis within human foetuses, including the vertebrae. We also demonstrated prominent expression in myotome and muscle fibres in human foetuses, and zebrafish embryos and adolescents. As there is no rodent orthologue of PPP2R3B, we used CRIPSR/Cas9-mediated gene-editing to generate a series of frameshift mutations in zebrafish ppp2r3b. Adolescent zebrafish that were homozygous for this mutation exhibited a fully penetrant kyphoscoliosis phenotype which became progressively worse over time, mirroring IS in humans. These defects were associated with reduced mineralisation of vertebrae, resembling osteoporosis. Electron microscopy demonstrated abnormal mitochondria adjacent to muscle fibres. In summary, we report a novel zebrafish model of IS and reduced bone mineral density. In future, it will be necessary to delineate the aetiology of these defects in relation to bone, muscle, neuronal and ependymal cilia function.
Assuntos
Escoliose , Peixe-Zebra , Animais , Adolescente , Humanos , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Escoliose/genética , Sistemas CRISPR-Cas , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , MutaçãoRESUMO
BACKGROUND: Biliary atresia (BA) is the most common obstructive cholangiopathy in neonates, often progressing to end-stage cirrhosis. BA pathogenesis is believed to be multifactorial, but the genetic contribution, especially for nonsyndromic BA (common form: > 85%) remains poorly defined. METHODS: We conducted whole exome sequencing on 89 nonsyndromic BA trios to identify rare variants contributing to BA etiology. Functional evaluation using patients' liver biopsies, human cell and zebrafish models were performed. Clinical impact on respiratory system was assessed with clinical evaluation, nasal nitric oxide (nNO), high speed video analysis and transmission electron microscopy. FINDINGS: We detected rare, deleterious de novo or biallelic variants in liver-expressed ciliary genes in 31.5% (28/89) of the BA patients. Burden test revealed 2.6-fold (odds ratio (OR) [95% confidence intervals (CI)]= 2.58 [1.15-6.07], adjusted p = 0.034) over-representation of rare, deleterious mutations in liver-expressed ciliary gene set in patients compared to controls. Functional analyses further demonstrated absence of cilia in the BA livers with KIF3B and TTC17 mutations, and knockdown of PCNT, KIF3B and TTC17 in human control fibroblasts and cholangiocytes resulted in reduced number of cilia. Additionally, CRISPR/Cas9-engineered zebrafish knockouts of KIF3B, PCNT and TTC17 displayed reduced biliary flow. Abnormally low level of nNO was detected in 80% (8/10) of BA patients carrying deleterious ciliary mutations, implicating the intrinsic ciliary defects. INTERPRETATION: Our findings support strong genetic susceptibility for nonsyndromic BA. Ciliary gene mutations leading to cholangiocyte cilia malformation and dysfunction could be a key biological mechanism in BA pathogenesis. FUNDING: The study is supported by General Research Fund, HMRF Commissioned Paediatric Research at HKCH and Li Ka Shing Faculty of Medicine Enhanced New Staff Start-up Fund.
Assuntos
Atresia Biliar/etiologia , Cílios/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Mutação , Fenótipo , Animais , Atresia Biliar/diagnóstico , Sistemas CRISPR-Cas , Linhagem Celular , Biologia Computacional/métodos , Edição de Genes , Técnicas de Silenciamento de Genes , Ontologia Genética , Estudos de Associação Genética/métodos , Heterogeneidade Genética , Loci Gênicos , Humanos , Fígado/metabolismo , Fígado/patologia , Análise de Sequência de DNA , Sequenciamento do Exoma , Peixe-ZebraRESUMO
Aminoacyl-tRNA synthetases (ARSs) are ubiquitous, ancient enzymes that charge amino acids to cognate tRNA molecules, the essential first step of protein translation. Here, we describe 32 individuals from 21 families, presenting with microcephaly, neurodevelopmental delay, seizures, peripheral neuropathy, and ataxia, with de novo heterozygous and bi-allelic mutations in asparaginyl-tRNA synthetase (NARS1). We demonstrate a reduction in NARS1 mRNA expression as well as in NARS1 enzyme levels and activity in both individual fibroblasts and induced neural progenitor cells (iNPCs). Molecular modeling of the recessive c.1633C>T (p.Arg545Cys) variant shows weaker spatial positioning and tRNA selectivity. We conclude that de novo and bi-allelic mutations in NARS1 are a significant cause of neurodevelopmental disease, where the mechanism for de novo variants could be toxic gain-of-function and for recessive variants, partial loss-of-function.
Assuntos
Aspartato-tRNA Ligase/genética , Mutação com Ganho de Função/genética , Mutação com Perda de Função/genética , Transtornos do Neurodesenvolvimento/genética , Aminoacil-RNA de Transferência/genética , Alelos , Aminoacil-tRNA Sintetases/genética , Linhagem Celular , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Linhagem , RNA de Transferência/genética , Células-Tronco/fisiologiaRESUMO
Mutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. Studies implicating the phenotypic consequences of SNX14 mutations to be consequences of subcellular disruption to autophagy and lipid metabolism have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) have also been conducted, demonstrated an important biochemical role during lipid biogenesis. In this study we report the effect of loss of SNX14 in mice, which resulted in embryonic lethality around mid-gestation due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. In contrast to other vertebrates, zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation were both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the consequences of loss of function varies between species. Mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo.
Assuntos
Viabilidade Fetal/genética , Metabolismo dos Lipídeos/genética , Placenta/anormalidades , Nexinas de Classificação/genética , Ataxias Espinocerebelares/genética , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Desenvolvimento Embrionário/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Fenótipo , Fosfolipídeos/sangue , Gravidez , Trofoblastos/citologia , Peixe-ZebraRESUMO
Gene editing using the CRISPR/Cas9 system is an extremely efficient approach for generating mutations within the genomic DNA of immortalized cell lines. This procedure begins with a straightforward cloning step to generate a single plasmid encoding the Cas9 enzyme as well as a synthetic guide RNA (sgRNA) which is selected to target specific sites within the genome. This plasmid is transfected into cells either alone, in order to generate random insertion-deletion alleles ("indels") at the desired locus via the nonhomologous end-joining pathway, or in conjunction with a homology-directed repair template oligonucleotide to generate a specific point mutation. Here we describe a procedure to perform gene editing in IMCD3 and HEK293 cells and to subsequently isolate clonal cell lines carrying mutations of interest.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Alelos , Separação Celular/métodos , Clonagem Molecular/métodos , Citometria de Fluxo/métodos , Células HEK293 , Humanos , Mutação INDEL , RNA Guia de CinetoplastídeosRESUMO
Background: Lenz-Majewski syndrome (LMS) is characterized by osteosclerosis and hyperostosis of skull, vertebrae and tubular bones as well as craniofacial, dental, cutaneous, and digit abnormalities. We previously found that LMS is caused by de novo dominant missense mutations in the PTDSS1 gene, which encodes phosphatidylserine synthase 1 (PSS1), an enzyme that catalyses the conversion of phosphatidylcholine to phosphatidylserine. The mutations causing LMS result in a gain-of-function, leading to increased enzyme activity and blocking end-product inhibition of PSS1. Methods: Here, we have used transpose-mediated transgenesis to attempt to stably express wild-type and mutant forms of human PTDSS1 ubiquitously or specifically in chondrocytes, osteoblasts or osteoclasts in zebrafish. Results: We report multiple genomic integration sites for each of 8 different transgenes. While we confirmed that the ubiquitously driven transgene constructs were functional in terms of driving gene expression following transient transfection in HeLa cells, and that all lines exhibited expression of a heart-specific cistron within the transgene, we failed to detect PTDSS1 gene expression at either the RNA or protein levels in zebrafish. All wild-type and mutant transgenic lines of zebrafish exhibited mild scoliosis with variable incomplete penetrance which was never observed in non-transgenic animals. Conclusions: Collectively the data suggest that the transgenes are silenced, that animals with integrations that escape silencing are not viable, or that other technical factors prevent transgene expression. In conclusion, the incomplete penetrance of the phenotype and the lack of a matched transgenic control model precludes further meaningful investigations of these transgenic lines.
Assuntos
CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferase , Transferases de Grupos Nitrogenados/genética , Síndrome de Costela Curta e Polidactilia , Peixe-Zebra , Animais , Animais Geneticamente Modificados , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferase/genética , Linhagem da Célula , Células HeLa , Humanos , TransgenesRESUMO
Neural crest stem/progenitor cells (NCSCs) populate a variety of tissues, and their dysregulation is implicated in several human diseases including craniosynostosis and neuroblastoma. We hypothesised that small molecules that inhibit NCSC induction or differentiation may represent potential therapeutically relevant drugs in these disorders. We screened 640 FDA-approved compounds currently in clinical use for other conditions to identify those which disrupt development of NCSC-derived skeletal elements that form the zebrafish jaw. In the primary screen, we used heterozygous transgenic sox10:gfp zebrafish to directly visualise NCSC-derived jaw cartilage. We noted partial toxicity of this transgene in relation to jaw patterning, suggesting that our primary screen was sensitised for NCSC defects, and we confirmed 10 novel, 4 previously reported, and 2 functional analogue drug hits in wild-type embryos. Of these drugs, 9/14 and 7/14, respectively, are known to target pathways implicated in osteoarthritis pathogenesis or to cause reduced bone mineral density/increased fracture risk as side effects in patients treated for other conditions, suggesting that our screen enriched for pathways targeting skeletal tissue homeostasis. We selected one drug that inhibited NCSC induction and one drug that inhibits bone mineralisation for further detailed analyses which reflect our initial hypotheses. These drugs were leflunomide and cyclosporin A, respectively, and their functional analogues, teriflunomide and FK506 (tacrolimus). We identified their critical developmental windows of activity, showing that the severity of defects observed related to the timing, duration, and dose of treatment. While leflunomide has previously been shown to inhibit NCSC induction, we demonstrate additional later roles in cartilage remodelling. Both drugs altered expression of extracellular matrix metalloproteinases. As proof-of-concept, we also tested drug treatment of disease-relevant mammalian cells. While leflunomide treatment inhibited the viability of several human NCSC-derived neuroblastoma cell lines coincident with altered expression of genes involved in ribosome biogenesis and transcription, FK506 enhanced murine calvarial osteoblast differentiation and prevented fusion of the coronal suture in calvarial explants taken from Crouzon syndrome mice.
RESUMO
Oligomeric assemblies of intraflagellar transport (IFT) particles build cilia through sequential recruitment and transport of ciliary cargo proteins within cilia. Here we present the 1.8 Å resolution crystal structure of the Chlamydomonas IFT-B protein IFT80, which reveals the architecture of two N-terminal ß-propellers followed by an α-helical extension. The N-terminal ß-propeller tethers IFT80 to the IFT-B complex via IFT38 whereas the second ß-propeller and the C-terminal α-helical extension result in IFT80 homo-dimerization. Using CRISPR/Cas to create biallelic Ift80 frameshift mutations in IMCD3 mouse cells, we demonstrate that IFT80 is absolutely required for ciliogenesis. Structural mapping and rescue experiments reveal that human disease-causing missense mutations do not cluster within IFT80 and form functional IFT particles. Unlike missense mutant forms of IFT80, deletion of the C-terminal dimerization domain prevented rescue of ciliogenesis. Taken together our results may provide a first insight into higher order IFT complex formation likely required for IFT train formation.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cílios/química , Cílios/metabolismo , Biogênese de Organelas , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Chlamydomonas/fisiologia , Cristalografia por Raios X , Mutação da Fase de Leitura , Edição de Genes , Multimerização ProteicaRESUMO
Mutations in SNX14 cause the autosomal recessive cerebellar ataxia 20 (SCAR20). Mutations generally result in loss of protein although several coding region deletions have also been reported. Patient-derived fibroblasts show disrupted autophagy, but the precise function of SNX14 is unknown. The yeast homolog, Mdm1, functions in endoplasmic reticulum (ER)-lysosome/vacuole inter-organelle tethering, but functional conservation in mammals is still required. Here, we show that loss of SNX14 alters but does not block autophagic flux. In addition, we find that SNX14 is an ER-associated protein that functions in neutral lipid homeostasis and inter-organelle crosstalk. SNX14 requires its N-terminal transmembrane helices for ER localization, while the Phox homology (PX) domain is dispensable for subcellular localization. Both SNX14-mutant fibroblasts and SNX14KO HEK293 cells accumulate aberrant cytoplasmic vacuoles, suggesting defects in endolysosomal homeostasis. However, ER-late endosome/lysosome contact sites are maintained in SNX14KO cells, indicating that it is not a prerequisite for ER-endolysosomal tethering. Further investigation of SNX14- deficiency indicates general defects in neutral lipid metabolism. SNX14KO cells display distinct perinuclear accumulation of filipin in LAMP1-positive lysosomal structures indicating cholesterol accumulation. Consistent with this, SNX14KO cells display a slight but detectable decrease in cholesterol ester levels, which is exacerbated with U18666A. Finally, SNX14 associates with ER-derived lipid droplets (LD) following oleate treatment, indicating a role in ER-LD crosstalk. We therefore identify an important role for SNX14 in neutral lipid homeostasis between the ER, lysosomes and LDs that may provide an early intervention target to alleviate the clinical symptoms of SCAR20.
Assuntos
Retículo Endoplasmático/genética , Metabolismo dos Lipídeos/genética , Nexinas de Classificação/genética , Ataxias Espinocerebelares/genética , Autofagia/genética , Retículo Endoplasmático/metabolismo , Endossomos , Técnicas de Inativação de Genes , Células HEK293 , Homeostase/efeitos dos fármacos , Humanos , Proteínas de Filamentos Intermediários/genética , Gotículas Lipídicas/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Mutação , Ácido Oleico/farmacologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Nexinas de Classificação/deficiência , Nexinas de Classificação/metabolismo , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/fisiopatologiaRESUMO
3MC syndrome is an autosomal recessive heterogeneous disorder with features linked to developmental abnormalities. The main features include facial dysmorphism, craniosynostosis and cleft lip/palate; skeletal structures derived from cranial neural crest cells (cNCC). We previously reported that lectin complement pathway genes COLEC11 and MASP1/3 are mutated in 3MC syndrome patients. Here we define a new gene, COLEC10, also mutated in 3MC families and present novel mutations in COLEC11 and MASP1/3 genes in a further five families. The protein products of COLEC11 and COLEC10, CL-K1 and CL-L1 respectively, form heteromeric complexes. We show COLEC10 is expressed in the base membrane of the palate during murine embryo development. We demonstrate how mutations in COLEC10 (c.25C>T; p.Arg9Ter, c.226delA; p.Gly77Glufs*66 and c.528C>G p.Cys176Trp) impair the expression and/or secretion of CL-L1 highlighting their pathogenicity. Together, these findings provide further evidence linking the lectin complement pathway and complement factors COLEC11 and COLEC10 to morphogenesis of craniofacial structures and 3MC etiology.
Assuntos
Anormalidades Múltiplas/genética , Fissura Palatina/genética , Colectinas/genética , Anormalidades Craniofaciais/genética , Craniossinostoses/genética , Mutação , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/patologia , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Fissura Palatina/metabolismo , Colectinas/metabolismo , Anormalidades Craniofaciais/metabolismo , Craniossinostoses/metabolismo , Exoma/genética , Saúde da Família , Feminino , Predisposição Genética para Doença/genética , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Análise de Sequência de DNA/métodos , SíndromeRESUMO
Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine.
Assuntos
Cílios/metabolismo , Ciliopatias/genética , Nanismo/genética , Hipotonia Muscular/genética , Mapas de Interação de Proteínas , Proteínas/metabolismo , Coluna Vertebral/anormalidades , Transporte Biológico/fisiologia , Cromatografia de Afinidade/métodos , Ciliopatias/patologia , Ciliopatias/terapia , Análise Mutacional de DNA , Conjuntos de Dados como Assunto , Nanismo/patologia , Nanismo/terapia , Fibroblastos , Células HEK293 , Humanos , Espectrometria de Massas , Terapia de Alvo Molecular/métodos , Hipotonia Muscular/patologia , Hipotonia Muscular/terapia , Mapeamento de Interação de Proteínas/métodos , Proteínas/genética , Proteínas/isolamento & purificação , Proteômica/métodos , Coluna Vertebral/patologia , Análise de SistemasRESUMO
Moebius syndrome (MBS) is a congenital, non-progressive facial and abducens nerve palsy in the presence of full vertical gaze and may be associated with limb abnormalities and craniofacial dysmorphisms. MBS is now defined as a disorder of rhombencephalic maldevelopment and recent gene discoveries have shown this to be a dominant disorder in a subset of patients. Accurate diagnosis and management by a multidisciplinary team with expertise in congenital facial palsy is paramount.
Assuntos
Síndrome de Möbius/diagnóstico , Síndrome de Möbius/terapia , Criança , Deficiências do Desenvolvimento/etiologia , Diagnóstico Diferencial , Predisposição Genética para Doença , Humanos , Síndrome de Möbius/complicações , Síndrome de Möbius/genética , Transtornos da Motilidade Ocular/etiologiaRESUMO
Intellectual disability and cerebellar atrophy occur together in a large number of genetic conditions and are frequently associated with microcephaly and/or epilepsy. Here we report the identification of causal mutations in Sorting Nexin 14 (SNX14) found in seven affected individuals from three unrelated consanguineous families who presented with recessively inherited moderate-severe intellectual disability, cerebellar ataxia, early-onset cerebellar atrophy, sensorineural hearing loss, and the distinctive association of progressively coarsening facial features, relative macrocephaly, and the absence of seizures. We used homozygosity mapping and whole-exome sequencing to identify a homozygous nonsense mutation and an in-frame multiexon deletion in two families. A homozygous splice site mutation was identified by Sanger sequencing of SNX14 in a third family, selected purely by phenotypic similarity. This discovery confirms that these characteristic features represent a distinct and recognizable syndrome. SNX14 encodes a cellular protein containing Phox (PX) and regulator of G protein signaling (RGS) domains. Weighted gene coexpression network analysis predicts that SNX14 is highly coexpressed with genes involved in cellular protein metabolism and vesicle-mediated transport. All three mutations either directly affected the PX domain or diminished SNX14 levels, implicating a loss of normal cellular function. This manifested as increased cytoplasmic vacuolation as observed in cultured fibroblasts. Our findings indicate an essential role for SNX14 in neural development and function, particularly in development and maturation of the cerebellum.
Assuntos
Ataxia Cerebelar/genética , Deficiência Intelectual/genética , Nexinas de Classificação/genética , Sequência de Bases , Ataxia Cerebelar/patologia , Mapeamento Cromossômico , Códon sem Sentido/genética , Feminino , Fibroblastos/ultraestrutura , Redes Reguladoras de Genes/genética , Genes Recessivos/genética , Humanos , Deficiência Intelectual/patologia , Masculino , Microscopia Eletrônica , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Lenz-Majewski syndrome (LMS) is a syndrome of intellectual disability and multiple congenital anomalies that features generalized craniotubular hyperostosis. By using whole-exome sequencing and selecting variants consistent with the predicted dominant de novo etiology of LMS, we identified causative heterozygous missense mutations in PTDSS1, which encodes phosphatidylserine synthase 1 (PSS1). PSS1 is one of two enzymes involved in the production of phosphatidylserine. Phosphatidylserine synthesis was increased in intact fibroblasts from affected individuals, and end-product inhibition of PSS1 by phosphatidylserine was markedly reduced. Therefore, these mutations cause a gain-of-function effect associated with regulatory dysfunction of PSS1. We have identified LMS as the first human disease, to our knowledge, caused by disrupted phosphatidylserine metabolism. Our results point to an unexplored link between phosphatidylserine synthesis and bone metabolism.
Assuntos
Anormalidades Múltiplas/genética , Mutação , Transferases de Grupos Nitrogenados/genética , Adolescente , Animais , Células Cultivadas , Criança , Nanismo , Embrião não Mamífero , Feminino , Fibroblastos/metabolismo , Humanos , Hiperostose , Masculino , Dados de Sequência Molecular , Transferases de Grupos Nitrogenados/metabolismo , Fosfatidilserinas/biossíntese , Fosfatidilserinas/genética , Síndrome , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Primary cilia are cellular appendages important for signal transduction and sensing the environment. Bardet-Biedl syndrome proteins form a complex that is important for several cytoskeleton-related processes such as ciliogenesis, cell migration and division. However, the mechanisms by which BBS proteins may regulate the cytoskeleton remain unclear. We discovered that Bbs4- and Bbs6-deficient renal medullary cells display a characteristic behaviour comprising poor migration, adhesion and division with an inability to form lamellipodial and filopodial extensions. Moreover, fewer mutant cells were ciliated [48% ± 6 for wild-type (WT) cells versus 23% ± 7 for Bbs4 null cells; P < 0.0001] and their cilia were shorter (2.55 µm ± 0.41 for WT cells versus 2.16 µm ± 0.23 for Bbs4 null cells; P < 0.0001). While the microtubular cytoskeleton and cortical actin were intact, actin stress fibre formation was severely disrupted, forming abnormal apical stress fibre aggregates. Furthermore, we observed over-abundant focal adhesions (FAs) in Bbs4-, Bbs6- and Bbs8-deficient cells. In view of these findings and the role of RhoA in regulation of actin filament polymerization, we showed that RhoA-GTP levels were highly upregulated in the absence of Bbs proteins. Upon treatment of Bbs4-deficient cells with chemical inhibitors of RhoA, we were able to restore the cilia length and number as well as the integrity of the actin cytoskeleton. Together these findings indicate that Bbs proteins play a central role in the regulation of the actin cytoskeleton and control the cilia length through alteration of RhoA levels.
Assuntos
Actinas/metabolismo , Síndrome de Bardet-Biedl/metabolismo , Cílios/metabolismo , Cílios/ultraestrutura , Chaperoninas do Grupo II/genética , Proteínas/genética , Proteínas de Peixe-Zebra/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Animais , Síndrome de Bardet-Biedl/genética , Células Cultivadas , Proteínas do Citoesqueleto , Células Epiteliais/metabolismo , Adesões Focais/metabolismo , Chaperoninas do Grupo II/metabolismo , Humanos , Medula Renal/citologia , Medula Renal/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos , Células NIH 3T3 , Fenótipo , Polimerização , Multimerização Proteica , Proteínas/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Carpenter syndrome is an autosomal-recessive multiple-congenital-malformation disorder characterized by multisuture craniosynostosis and polysyndactyly of the hands and feet; many other clinical features occur, and the most frequent include obesity, umbilical hernia, cryptorchidism, and congenital heart disease. Mutations of RAB23, encoding a small GTPase that regulates vesicular transport, are present in the majority of cases. Here, we describe a disorder caused by mutations in multiple epidermal-growth-factor-like-domains 8 (MEGF8), which exhibits substantial clinical overlap with Carpenter syndrome but is frequently associated with abnormal left-right patterning. We describe five affected individuals with similar dysmorphic facies, and three of them had either complete situs inversus, dextrocardia, or transposition of the great arteries; similar cardiac abnormalities were previously identified in a mouse mutant for the orthologous Megf8. The mutant alleles comprise one nonsense, three missense, and two splice-site mutations; we demonstrate in zebrafish that, in contrast to the wild-type protein, the proteins containing all three missense alterations provide only weak rescue of an early gastrulation phenotype induced by Megf8 knockdown. We conclude that mutations in MEGF8 cause a Carpenter syndrome subtype frequently associated with defective left-right patterning, probably through perturbation of signaling by hedgehog and nodal family members. We did not observe any subject with biallelic loss-of function mutations, suggesting that some residual MEGF8 function might be necessary for survival and might influence the phenotypes observed.