RESUMO
Multiple Sclerosis (MS) is a heterogeneous inflammatory and neurodegenerative disease with an unpredictable course towards progressive disability. Treating progressive MS is challenging due to limited insights into the underlying mechanisms. We examined the molecular changes associated with primary progressive MS (PPMS) using a cross-tissue (blood and post-mortem brain) and multilayered data (genetic, epigenetic, transcriptomic) from independent cohorts. In PPMS, we found hypermethylation of the 1q21.1 locus, controlled by PPMS-specific genetic variations and influencing the expression of proximal genes (CHD1L, PRKAB2) in the brain. Evidence from reporter assay and CRISPR/dCas9 experiments supports a causal link between methylation and expression and correlation network analysis further implicates these genes in PPMS brain processes. Knock-down of CHD1L in human iPSC-derived neurons and knock-out of chd1l in zebrafish led to developmental and functional deficits of neurons. Thus, several lines of evidence suggest a distinct genetic-epigenetic-transcriptional interplay in the 1q21.1 locus potentially contributing to PPMS pathogenesis.
Assuntos
Encéfalo , Cromossomos Humanos Par 1 , Metilação de DNA , Proteínas de Ligação a DNA , Epigênese Genética , Peixe-Zebra , Humanos , Peixe-Zebra/genética , Animais , Metilação de DNA/genética , Cromossomos Humanos Par 1/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , DNA Helicases/genética , DNA Helicases/metabolismo , Neurônios/metabolismo , Esclerose Múltipla Crônica Progressiva/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Predisposição Genética para Doença , AdultoRESUMO
The biomedical research community addresses reproducibility challenges in animal studies through standardized nomenclature, improved experimental design, transparent reporting, data sharing, and centralized repositories. The ARRIVE guidelines outline documentation standards for laboratory animals in experiments, but genetic information is often incomplete. To remedy this, we propose the Laboratory Animal Genetic Reporting (LAG-R) framework. LAG-R aims to document animals' genetic makeup in scientific publications, providing essential details for replication and appropriate model use. While verifying complete genetic compositions may be impractical, better reporting and validation efforts enhance reliability of research. LAG-R standardization will bolster reproducibility, peer review, and overall scientific rigor.
Assuntos
Animais de Laboratório , Guias como Assunto , Animais , Animais de Laboratório/genética , Reprodutibilidade dos Testes , Projetos de Pesquisa , Experimentação Animal/normas , Pesquisa Biomédica/normasRESUMO
Individuals with mutations in CHD8 present with gastrointestinal complaints, yet the underlying mechanisms are understudied. Here, using a stable constitutive chd8 mutant zebrafish model, we found that the loss of chd8 leads to a reduced number of vagal neural crest cells (NCCs), enteric neural and glial progenitors, emigrating from the neural tube, and that their early migration capability was altered. At later stages, although the intestinal colonization by NCCs was complete, we found the decreased numbers of both serotonin-producing enterochromaffin cells and NCC-derived serotonergic neurons, suggesting an intestinal hyposerotonemia in the absence of chd8 Furthermore, transcriptomic analyses revealed an altered expression of key receptors and enzymes in serotonin and acetylcholine signaling pathways. The tissue examination of chd8 mutants revealed a thinner intestinal epithelium accompanied by an accumulation of neutrophils and the decreased numbers of goblet cells and eosinophils. Last, single-cell sequencing of whole intestines showed a global disruption of the immune balance with a perturbed expression of inflammatory interleukins and changes in immune cell clusters. Our findings propose a causal developmental link between chd8, NCC development, intestinal homeostasis, and autism-associated gastrointestinal complaints.
Assuntos
Transtorno Autístico , Crista Neural , Animais , Crista Neural/metabolismo , Peixe-Zebra , Serotonina/metabolismo , Movimento Celular/genética , Intestinos , HomeostaseRESUMO
Cerebellar dysfunction has been demonstrated in autism spectrum disorders (ASDs); however, the circuits underlying cerebellar contributions to ASD-relevant behaviors remain unknown. In this study, we demonstrated functional connectivity between the cerebellum and the medial prefrontal cortex (mPFC) in mice; showed that the mPFC mediates cerebellum-regulated social and repetitive/inflexible behaviors; and showed disruptions in connectivity between these regions in multiple mouse models of ASD-linked genes and in individuals with ASD. We delineated a circuit from cerebellar cortical areas Right crus 1 (Rcrus1) and posterior vermis through the cerebellar nuclei and ventromedial thalamus and culminating in the mPFC. Modulation of this circuit induced social deficits and repetitive behaviors, whereas activation of Purkinje cells (PCs) in Rcrus1 and posterior vermis improved social preference impairments and repetitive/inflexible behaviors, respectively, in male PC-Tsc1 mutant mice. These data raise the possibility that these circuits might provide neuromodulatory targets for the treatment of ASD.
Assuntos
Transtorno do Espectro Autista/fisiopatologia , Cerebelo/fisiopatologia , Vias Neurais/fisiopatologia , Córtex Pré-Frontal/fisiopatologia , Animais , Masculino , Camundongos , Camundongos MutantesRESUMO
KIF21B is a kinesin protein that promotes intracellular transport and controls microtubule dynamics. We report three missense variants and one duplication in KIF21B in individuals with neurodevelopmental disorders associated with brain malformations, including corpus callosum agenesis (ACC) and microcephaly. We demonstrate, in vivo, that the expression of KIF21B missense variants specifically recapitulates patients' neurodevelopmental abnormalities, including microcephaly and reduced intra- and inter-hemispheric connectivity. We establish that missense KIF21B variants impede neuronal migration through attenuation of kinesin autoinhibition leading to aberrant KIF21B motility activity. We also show that the ACC-related KIF21B variant independently perturbs axonal growth and ipsilateral axon branching through two distinct mechanisms, both leading to deregulation of canonical kinesin motor activity. The duplication introduces a premature termination codon leading to nonsense-mediated mRNA decay. Although we demonstrate that Kif21b haploinsufficiency leads to an impaired neuronal positioning, the duplication variant might not be pathogenic. Altogether, our data indicate that impaired KIF21B autoregulation and function play a critical role in the pathogenicity of human neurodevelopmental disorder.
Assuntos
Cinesinas/genética , Atividade Motora , Mutação/genética , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/fisiopatologia , Animais , Axônios/metabolismo , Movimento Celular , Proliferação de Células , Córtex Cerebral/embriologia , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Masculino , Camundongos , Mutação de Sentido Incorreto/genética , Rede Nervosa/patologia , Rede Nervosa/fisiopatologia , Neurônios/metabolismo , Tamanho do Órgão , Organogênese/genética , Linhagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/genéticaRESUMO
The neuro-oncological ventral antigen 2 (NOVA2) protein is a major factor regulating neuron-specific alternative splicing (AS), previously associated with an acquired neurologic condition, the paraneoplastic opsoclonus-myoclonus ataxia (POMA). We report here six individuals with de novo frameshift variants in NOVA2 affected with a severe neurodevelopmental disorder characterized by intellectual disability (ID), motor and speech delay, autistic features, hypotonia, feeding difficulties, spasticity or ataxic gait, and abnormal brain MRI. The six variants lead to the same reading frame, adding a common proline rich C-terminal part instead of the last KH RNA binding domain. We detected 41 genes differentially spliced after NOVA2 downregulation in human neural cells. The NOVA2 variant protein shows decreased ability to bind target RNA sequences and to regulate target AS events. It also fails to complement the effect on neurite outgrowth induced by NOVA2 downregulation in vitro and to rescue alterations of retinotectal axonal pathfinding induced by loss of NOVA2 ortholog in zebrafish. Our results suggest a partial loss-of-function mechanism rather than a full heterozygous loss-of-function, although a specific contribution of the novel C-terminal extension cannot be excluded.
Assuntos
Mutação da Fase de Leitura/genética , Proteínas do Tecido Nervoso/genética , Transtornos do Neurodesenvolvimento/genética , Neurônios/fisiologia , Splicing de RNA/genética , Proteínas de Ligação a RNA/genética , Processamento Alternativo/genética , Animais , Orientação de Axônios/genética , Sequência de Bases/genética , Células Cultivadas , Pré-Escolar , Regulação para Baixo/genética , Feminino , Heterozigoto , Humanos , Deficiência Intelectual/genética , Transtornos do Desenvolvimento da Linguagem/genética , Masculino , Camundongos , Hipotonia Muscular/genética , Antígeno Neuro-Oncológico Ventral , Peixe-Zebra/genéticaRESUMO
Progeroid syndromes are a group of rare genetic disorders, which mimic natural aging. Unraveling the molecular defects in such conditions could impact our understanding of age-related syndromes such as Alzheimer's or cardiovascular diseases. Here we report a de novo heterozygous missense variant in the intermediate filament vimentin (c.1160 T > C; p.(Leu387Pro)) causing a multisystem disorder associated with frontonasal dysostosis and premature aging in a 39-year-old individual. Human vimentin p.(Leu387Pro) expression in zebrafish perturbed body fat distribution, and craniofacial and peripheral nervous system development. In addition, studies in patient-derived and transfected cells revealed that the variant affects vimentin turnover and its ability to form filaments in the absence of wild-type vimentin. Vimentin p.(Leu387Pro) expression diminished the amount of peripilin and reduced lipid accumulation in differentiating adipocytes, recapitulating key patient's features in vivo and in vitro. Our data highlight the function of vimentin during development and suggest its contribution to natural aging.
Assuntos
Progéria/genética , Vimentina/genética , Células 3T3-L1 , Adipócitos/metabolismo , Adiposidade , Adulto , Animais , Células Cultivadas , Genes Dominantes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células MCF-7 , Masculino , Camundongos , Mutação , Neurogênese , Perilipina-1/metabolismo , Progéria/patologia , Vimentina/metabolismo , Peixe-ZebraRESUMO
"An International Meeting on Wolf-Hirschhorn Syndrome (WHS)" was held at The University Hospital La Paz in Madrid, Spain (October 13-14, 2017). One hundred and twenty-five people, including physicians, scientists and affected families, attended the meeting. Parent and patient advocates from the Spanish Association of WHS opened the meeting with a panel discussion to set the stage regarding their hopes and expectations for therapeutic advances. In keeping with the theme on therapeutic development, the sessions followed a progression from description of the phenotype and definition of therapeutic endpoints, to definition of genomic changes. These proceedings will review the major points of discussion.
Assuntos
Cromossomos Humanos Par 4/imunologia , Deficiências do Desenvolvimento/genética , Convulsões/genética , Síndrome de Wolf-Hirschhorn/genética , Deleção Cromossômica , Cromossomos Humanos Par 4/genética , Deficiências do Desenvolvimento/epidemiologia , Deficiências do Desenvolvimento/patologia , Feminino , Humanos , Fenótipo , Convulsões/epidemiologia , Convulsões/terapia , Espanha/epidemiologia , Síndrome de Wolf-Hirschhorn/epidemiologia , Síndrome de Wolf-Hirschhorn/terapiaRESUMO
The 16p11.2 BP4-BP5 deletion and duplication syndromes are associated with a complex spectrum of neurodevelopmental phenotypes that includes developmental delay and autism spectrum disorder, with a reciprocal effect on head circumference, brain structure and body mass index. Mouse models of the 16p11.2 copy number variant have recapitulated some of the patient phenotypes, while studies in flies and zebrafish have uncovered several candidate contributory genes within the region, as well as complex genetic interactions. We evaluated one of these loci, KCTD13, by modeling haploinsufficiency and complete knockout in mice. In contrast to the zebrafish model, and in agreement with recent data, we found normal brain structure in heterozygous and homozygous mutants. However, recapitulating previously observed genetic interactions, we discovered sex-specific brain volumetric alterations in double heterozygous Kctd13xMvp and Kctd13xLat mice. Behavioral testing revealed a significant deficit in novel object recognition, novel location recognition and social transmission of food preference in Kctd13 mutants. These phenotypes were concomitant with a reduction in density of mature spines in the hippocampus, but potentially independent of RhoA abundance, which was unperturbed postnatally in our mutants. Furthermore, transcriptome analyses from cortex and hippocampus highlighted the dysregulation of pathways important in neurodevelopment, the most significant of which was synaptic formation. Together, these data suggest that KCTD13 contributes to the neurocognitive aspects of patients with the BP4-BP5 deletion, likely through genetic interactions with other loci.
Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Transtornos da Memória/genética , Transtornos da Memória/psicologia , Memória de Curto Prazo , Complexos Ubiquitina-Proteína Ligase/deficiência , Animais , Comportamento Animal , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Modelos Animais de Doenças , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Marcação de Genes , Loci Gênicos , Genótipo , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Deleção de Sequência , Fatores SexuaisRESUMO
RLIM, also known as RNF12, is an X-linked E3 ubiquitin ligase acting as a negative regulator of LIM-domain containing transcription factors and participates in X-chromosome inactivation (XCI) in mice. We report the genetic and clinical findings of 84 individuals from nine unrelated families, eight of whom who have pathogenic variants in RLIM (RING finger LIM domain-interacting protein). A total of 40 affected males have X-linked intellectual disability (XLID) and variable behavioral anomalies with or without congenital malformations. In contrast, 44 heterozygous female carriers have normal cognition and behavior, but eight showed mild physical features. All RLIM variants identified are missense changes co-segregating with the phenotype and predicted to affect protein function. Eight of the nine altered amino acids are conserved and lie either within a domain essential for binding interacting proteins or in the C-terminal RING finger catalytic domain. In vitro experiments revealed that these amino acid changes in the RLIM RING finger impaired RLIM ubiquitin ligase activity. In vivo experiments in rlim mutant zebrafish showed that wild type RLIM rescued the zebrafish rlim phenotype, whereas the patient-specific missense RLIM variants failed to rescue the phenotype and thus represent likely severe loss-of-function mutations. In summary, we identified a spectrum of RLIM missense variants causing syndromic XLID and affecting the ubiquitin ligase activity of RLIM, suggesting that enzymatic activity of RLIM is required for normal development, cognition and behavior.
Assuntos
Deficiência Intelectual Ligada ao Cromossomo X/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Transtorno da Conduta/genética , Feminino , Genes Ligados ao Cromossomo X , Células HEK293 , Humanos , Recém-Nascido , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Camundongos , Pessoa de Meia-Idade , Mutação , Linhagem , Fatores de Transcrição/genética , Ubiquitinação , Inativação do Cromossomo X , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Kabuki Syndrome (KS) is a rare disorder characterized by distinctive facial features, short stature, skeletal abnormalities, and neurodevelopmental deficits. Previously, we showed that loss of function of RAP1A, a RAF1 regulator, can activate the RAS/MAPK pathway and cause KS, an observation recapitulated in other genetic models of the disorder. These data suggested that suppression of this signaling cascade might be of therapeutic benefit for some features of KS. To pursue this possibility, we performed a focused small molecule screen of a series of RAS/MAPK pathway inhibitors, where we tested their ability to rescue disease-relevant phenotypes in a zebrafish model of the most common KS locus, kmt2d. Consistent with a pathway-driven screening paradigm, two of 27 compounds showed reproducible rescue of early developmental pathologies. Further analyses showed that one compound, desmethyl-Dabrafenib (dmDf), induced no overt pathologies in zebrafish embryos but could rescue MEK hyperactivation in vivo and, concomitantly, structural KS-relevant phenotypes in all KS zebrafish models (kmt2d, kmd6a and rap1). Mass spectrometry quantitation suggested that a 100 nM dose resulted in sub-nanomolar exposure of this inhibitor and was sufficient to rescue both mandibular and neurodevelopmental defects. Crucially, germline kmt2d mutants recapitulated the gastrulation movement defects, micrognathia and neurogenesis phenotypes of transient models; treatment with dmDf ameliorated all of them significantly. Taken together, our data reinforce a causal link between MEK hyperactivation and KS and suggest that chemical suppression of BRAF might be of potential clinical utility for some features of this disorder.
Assuntos
Anormalidades Múltiplas/prevenção & controle , Face/anormalidades , Doenças Hematológicas/prevenção & controle , Imidazóis/farmacologia , Oximas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Doenças Vestibulares/prevenção & controle , Peixe-Zebra/crescimento & desenvolvimento , Anormalidades Múltiplas/patologia , Animais , Anormalidades Craniofaciais/prevenção & controle , Face/patologia , Doenças Hematológicas/patologia , Imidazóis/efeitos adversos , Imidazóis/química , Anormalidades Maxilomandibulares/prevenção & controle , Sistema de Sinalização das MAP Quinases , Oximas/efeitos adversos , Oximas/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Testes de Toxicidade , Doenças Vestibulares/patologia , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Endoglin, a TGF-ß coreceptor predominantly expressed in endothelial cells, plays an important role in vascular development and tumor-associated angiogenesis. However, the mechanism by which endoglin regulates angiogenesis, especially during tip cell formation, remains largely unknown. In this study, we report that endoglin promoted VEGF-induced tip cell formation. Mechanistically, endoglin interacted with VEGF receptor (VEGFR)-2 in a VEGF-dependent manner, which sustained VEGFR2 on the cell surface and prevented its degradation. Endoglin mutants deficient in the ability to interact with VEGFR2 failed to sustain VEGFR2 on the cell surface and to promote VEGF-induced tip cell formation. Further, an endoglin-targeting monoclonal antibody (mAb), TRC105, cooperated with a VEGF-A targeting mAb, bevacizumab, to inhibit VEGF signaling and tip cell formation in vitro and to inhibit tumor growth, metastasis, and tumor-associated angiogenesis in a murine tumor model. This study demonstrate a novel mechanism by which endoglin initiates and regulates VEGF-driven angiogenesis while providing a rationale for combining anti-VEGF and anti-endoglin therapy in patients with cancer.-Tian, H., Huang, J. J., Golzio, C., Gao, X., Hector-Greene, M., Katsanis, N., Blobe, G. C. Endoglin interacts with VEGFR2 to promote angiogenesis.
Assuntos
Endoglina/metabolismo , Neoplasias Experimentais/metabolismo , Neovascularização Patológica/metabolismo , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Bevacizumab/farmacologia , Células COS , Linhagem Celular , Chlorocebus aethiops , Endoglina/antagonistas & inibidores , Endoglina/genética , Humanos , Camundongos , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Proteólise/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Copy-number changes in 16p11.2 contribute significantly to neuropsychiatric traits. Besides the 600 kb BP4-BP5 CNV found in 0.5%-1% of individuals with autism spectrum disorders and schizophrenia and whose rearrangement causes reciprocal defects in head size and body weight, a second distal 220 kb BP2-BP3 CNV is likewise a potent driver of neuropsychiatric, anatomical, and metabolic pathologies. These two CNVs are engaged in complex reciprocal chromatin looping, intimating a functional relationship between genes in these regions that might be relevant to pathomechanism. We assessed the drivers of the distal 16p11.2 duplication by overexpressing each of the nine encompassed genes in zebrafish. Only overexpression of LAT induced a reduction of brain proliferating cells and concomitant microcephaly. Consistently, suppression of the zebrafish ortholog induced an increase of proliferation and macrocephaly. These phenotypes were not unique to zebrafish; Lat knockout mice show brain volumetric changes. Consistent with the hypothesis that LAT dosage is relevant to the CNV pathology, we observed similar effects upon overexpression of CD247 and ZAP70, encoding members of the LAT signalosome. We also evaluated whether LAT was interacting with KCTD13, MVP, and MAPK3, major driver and modifiers of the proximal 16p11.2 600 kb BP4-BP5 syndromes, respectively. Co-injected embryos exhibited an increased microcephaly, suggesting the presence of genetic interaction. Correspondingly, carriers of 1.7 Mb BP1-BP5 rearrangements that encompass both the BP2-BP3 and BP4-BP5 loci showed more severe phenotypes. Taken together, our results suggest that LAT, besides its well-recognized function in T cell development, is a major contributor of the 16p11.2 220 kb BP2-BP3 CNV-associated neurodevelopmental phenotypes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Transtorno Autístico/genética , Encéfalo/patologia , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 16 , Variações do Número de Cópias de DNA , Deficiência Intelectual/genética , Proteínas de Membrana/genética , Microcefalia/genética , Microcefalia/patologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Transtorno Autístico/imunologia , Transtorno Autístico/patologia , Encéfalo/metabolismo , Criança , Pré-Escolar , Deleção Cromossômica , Transtornos Cromossômicos/imunologia , Transtornos Cromossômicos/patologia , Cromossomos Humanos Par 16/genética , Cromossomos Humanos Par 16/imunologia , Estudos de Coortes , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Lactente , Deficiência Intelectual/imunologia , Deficiência Intelectual/patologia , Masculino , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Fenótipo , Fosfoproteínas/fisiologia , Transdução de Sinais , Adulto Jovem , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND: The ciliopathies represent an umbrella group of >50 clinical entities that share both clinical features and molecular etiology underscored by structural and functional defects of the primary cilium. Despite the advances in gene discovery, this group of entities continues to pose a diagnostic challenge, in part due to significant genetic and phenotypic heterogeneity and variability. We consulted a pediatric case from asymptomatic, non-consanguineous parents who presented as a suspected ciliopathy due to a constellation of retinal, renal, and skeletal findings. RESULTS: Although clinical panel sequencing of genes implicated in nephrotic syndromes yielded no likely causal mutation, an oligo-SNP microarray identified a ~20-Mb region of homozygosity, with no altered gene dosage, on chromosome 16p13. Intersection of the proband's phenotypes with known disease genes within the homozygous region yielded a single candidate, IFT140, encoding a retrograde intraflagellar transport protein implicated previously in several ciliopathies, including the phenotypically overlapping Mainzer-Saldino syndrome (MZSDS). Sanger sequencing yielded a maternally inherited homozygous c.634G>A; p.Gly212Arg mutation altering the exon 6 splice donor site. Functional studies in cells from the proband showed that the locus produced two transcripts: a majority message containing a mis-splicing event that caused a premature termination codon and a minority message homozygous for the p.Gly212Arg allele. Zebrafish in vivo complementation studies of the latter transcript demonstrated a loss of function effect. Finally, we conducted post-hoc trio-based whole exome sequencing studies to (a) test the possibility of other causal loci in the proband and (b) explain the Mendelian error of segregation for the IFT140 mutation. We show that the proband harbors a chromosome 16 maternal heterodisomy, with segmental isodisomy at 16p13, likely due to a meiosis I error in the maternal gamete. CONCLUSIONS: Using clinical phenotyping combined with research-based genetic and functional studies, we have characterized a recurrent IFT140 mutation in the proband; together, these data are consistent with MZSDS. Additionally, we report a rare instance of a uniparental isodisomy unmasking a deleterious mutation to cause a ciliary disorder.
Assuntos
Linfócitos B/patologia , Proteínas de Transporte/genética , Ataxia Cerebelar/genética , Mutação de Sentido Incorreto , Retinose Pigmentar/genética , Animais , Linfócitos B/metabolismo , Células Cultivadas , Ataxia Cerebelar/patologia , Pré-Escolar , Cromossomos Humanos Par 16 , Éxons , Feminino , Homozigoto , Humanos , Masculino , Linhagem , Fenótipo , Retinose Pigmentar/patologia , Dissomia Uniparental , Peixe-Zebra/metabolismoRESUMO
Autosomal-dominant tubulo-interstitial kidney disease (ADTKD) encompasses a group of disorders characterized by renal tubular and interstitial abnormalities, leading to slow progressive loss of kidney function requiring dialysis and kidney transplantation. Mutations in UMOD, MUC1, and REN are responsible for many, but not all, cases of ADTKD. We report on two families with ADTKD and congenital anemia accompanied by either intrauterine growth retardation or neutropenia. Ultrasound and kidney biopsy revealed small dysplastic kidneys with cysts and tubular atrophy with secondary glomerular sclerosis, respectively. Exclusion of known ADTKD genes coupled with linkage analysis, whole-exome sequencing, and targeted re-sequencing identified heterozygous missense variants in SEC61A1-c.553A>G (p.Thr185Ala) and c.200T>G (p.Val67Gly)-both affecting functionally important and conserved residues in SEC61. Both transiently expressed SEC6A1A variants are delocalized to the Golgi, a finding confirmed in a renal biopsy from an affected individual. Suppression or CRISPR-mediated deletions of sec61al2 in zebrafish embryos induced convolution defects of the pronephric tubules but not the pronephric ducts, consistent with the tubular atrophy observed in the affected individuals. Human mRNA encoding either of the two pathogenic alleles failed to rescue this phenotype as opposed to a complete rescue by human wild-type mRNA. Taken together, these findings provide a mechanism by which mutations in SEC61A1 lead to an autosomal-dominant syndromic form of progressive chronic kidney disease. We highlight protein translocation defects across the endoplasmic reticulum membrane, the principal role of the SEC61 complex, as a contributory pathogenic mechanism for ADTKD.
Assuntos
Anemia/genética , Heterozigoto , Nefropatias/genética , Mutação , Canais de Translocação SEC/genética , Adulto , Idoso , Alelos , Sequência de Aminoácidos , Animais , Biópsia , Criança , Doença Crônica , Progressão da Doença , Retículo Endoplasmático/metabolismo , Exoma/genética , Feminino , Retardo do Crescimento Fetal/genética , Genes Dominantes , Complexo de Golgi/metabolismo , Humanos , Recém-Nascido , Nefropatias/patologia , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Mutação de Sentido Incorreto/genética , Neutropenia/genética , Linhagem , Fenótipo , RNA Mensageiro/análise , RNA Mensageiro/genética , Canais de Translocação SEC/química , Síndrome , Adulto Jovem , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
The endothelial receptor tyrosine kinase (RTK) Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1's role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A) in zebrafish (Danio rerio) significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its RTK properties. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1) and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1 mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis.
Assuntos
Neovascularização Fisiológica/fisiologia , Fosfotreonina/metabolismo , Processamento de Proteína Pós-Traducional , Receptor de TIE-1/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Angiopoietina-1/fisiologia , Animais , Vasos Sanguíneos/embriologia , Colágeno , Combinação de Medicamentos , Endotélio Vascular/metabolismo , Ativação Enzimática , Células Endoteliais da Veia Umbilical Humana , Humanos , Laminina , Morfogênese , Mutagênese Sítio-Dirigida , Neovascularização Fisiológica/genética , Fosforilação , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteoglicanas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
Patterns of amino acid conservation have served as a tool for understanding protein evolution. The same principles have also found broad application in human genomics, driven by the need to interpret the pathogenic potential of variants in patients. Here we performed a systematic comparative genomics analysis of human disease-causing missense variants. We found that an appreciable fraction of disease-causing alleles are fixed in the genomes of other species, suggesting a role for genomic context. We developed a model of genetic interactions that predicts most of these to be simple pairwise compensations. Functional testing of this model on two known human disease genes revealed discrete cis amino acid residues that, although benign on their own, could rescue the human mutations in vivo. This approach was also applied to ab initio gene discovery to support the identification of a de novo disease driver in BTG2 that is subject to protective cis-modification in more than 50 species. Finally, on the basis of our data and models, we developed a computational tool to predict candidate residues subject to compensation. Taken together, our data highlight the importance of cis-genomic context as a contributor to protein evolution; they provide an insight into the complexity of allele effect on phenotype; and they are likely to assist methods for predicting allele pathogenicity.
Assuntos
Doença/genética , Genômica , Mutação de Sentido Incorreto/genética , Supressão Genética/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Alelos , Animais , Evolução Molecular , Genoma Humano/genética , Humanos , Proteínas Imediatamente Precoces/genética , Microcefalia/genética , Proteínas Associadas aos Microtúbulos , Fenótipo , Proteínas/genética , Alinhamento de Sequência , Proteínas Supressoras de Tumor/genéticaRESUMO
The cerebral cortex is built during embryonic neurogenesis, a period when excitatory neurons are generated from progenitors. Defects in neurogenesis can cause acute neurodevelopmental disorders, such as microcephaly (reduced brain size). Altered dosage of the 1q21.1 locus has been implicated in the etiology of neurodevelopmental phenotypes; however, the role of 1q21.1 genes in neurogenesis has remained elusive. Here, we show that haploinsufficiency for Rbm8a, an exon junction complex (EJC) component within 1q21.1, causes severe microcephaly and defective neurogenesis in the mouse. At the onset of neurogenesis, Rbm8a regulates radial glia proliferation and prevents premature neuronal differentiation. Reduced Rbm8a levels result in subsequent apoptosis of neurons, and to a lesser extent, radial glia. Hence, compared to control, Rbm8a-haploinsufficient brains have fewer progenitors and neurons, resulting in defective cortical lamination. To determine whether reciprocal dosage change of Rbm8a alters embryonic neurogenesis, we overexpressed human RBM8A in two animal models. Using in utero electroporation of mouse neocortices as well as zebrafish models, we find RBM8A overexpression does not significantly perturb progenitor number or head size. Our findings demonstrate that Rbm8a is an essential neurogenesis regulator, and add to a growing literature highlighting roles for EJC components in cortical development and neurodevelopmental pathology. Our results indicate that disruption of RBM8A may contribute to neurodevelopmental phenotypes associated with proximal 1q21.1 microdeletions.
Assuntos
Córtex Cerebral/embriologia , Desenvolvimento Embrionário/fisiologia , Haploinsuficiência/fisiologia , Microcefalia/genética , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Animais , Células Cultivadas , Córtex Cerebral/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microcefalia/metabolismo , Organogênese/fisiologiaRESUMO
The 16p11.2 600 kb copy-number variants (CNVs) are associated with mirror phenotypes on BMI, head circumference, and brain volume and represent frequent genetic lesions in autism spectrum disorders (ASDs) and schizophrenia. Here we interrogated the transcriptome of individuals carrying reciprocal 16p11.2 CNVs. Transcript perturbations correlated with clinical endophenotypes and were enriched for genes associated with ASDs, abnormalities of head size, and ciliopathies. Ciliary gene expression was also perturbed in orthologous mouse models, raising the possibility that ciliary dysfunction contributes to 16p11.2 pathologies. In support of this hypothesis, we found structural ciliary defects in the CA1 hippocampal region of 16p11.2 duplication mice. Moreover, by using an established zebrafish model, we show genetic interaction between KCTD13, a key driver of the mirrored neuroanatomical phenotypes of the 16p11.2 CNV, and ciliopathy-associated genes. Overexpression of BBS7 rescues head size and neuroanatomical defects of kctd13 morphants, whereas suppression or overexpression of CEP290 rescues phenotypes induced by KCTD13 under- or overexpression, respectively. Our data suggest that dysregulation of ciliopathy genes contributes to the clinical phenotypes of these CNVs.