RESUMO
Human exocyst complex is an evolutionary conserved multimeric complex composed of proteins encoded by eight genes EXOC1-EXOC8. It is known that the exocyst complex plays a role in ciliogenesis, cytokinesis, cell migration, autophagy, and fusion of secretory vesicles. Recently, loss of function variants in EXOC7 and EXOC8 has been associated with abnormalities of cerebral cortical development leading to a neurodevelopmental phenotype. Neurodevelopmental disorders are a huge group of clinically and genetically heterogeneous disorders. In the present study, we recruited a large consanguineous family segregating a neurodevelopmental disorder in an autosomal recessive form. We performed clinical phenotyping by imaging the patient's brain followed by whole exome sequencing examining DNA from two affected individuals. The clinical phenotypes of the disease were suggestive of brain atrophy. Clinical examination revealed intellectual impairment with hypertonia and brisk reflexes. WES followed by Sanger sequencing revealed a novel homozygous nonsense mutation [EXOC8; NM_175876.5; c.1714G > T; p.(Glu572Ter)] in the DNA of affected individuals. Both parents of the patients were heterozygous for the identified mutation. All the pathogenicity prediction softwares predicted the identified variant as disease causing. This study reports a second protein-truncating variant in EXOC8. The findings confirm that loss of function variants in EXOC8 underlies a neurodevelopmental disorder. The identification of a protein-truncating variant in EXOC8 in the current study can be helpful in establishing genotype-phenotype correlations. Our results also provide new insights into genetic counseling and clinical management for the affected individuals.
Assuntos
Exoma , Transtornos do Neurodesenvolvimento , DNA , Homozigoto , Humanos , Transtornos do Neurodesenvolvimento/genética , Linhagem , Fenótipo , Sequenciamento do ExomaRESUMO
PURPOSE: The exocyst complex is a conserved protein complex that mediates fusion of intracellular vesicles to the plasma membrane and is implicated in processes including cell polarity, cell migration, ciliogenesis, cytokinesis, autophagy, and fusion of secretory vesicles. The essential role of these genes in human genetic disorders, however, is unknown. METHODS: We performed homozygosity mapping and exome sequencing of consanguineous families with recessively inherited brain development disorders. We modeled an EXOC7 splice variant in vitro and examined EXOC7 messenger RNA (mRNA) expression in developing mouse and human cortex. We modeled exoc7 loss-of-function in a zebrafish knockout. RESULTS: We report variants in exocyst complex members, EXOC7 and EXOC8, in a novel disorder of cerebral cortex development. In EXOC7, we identified four independent partial loss-of-function (LOF) variants in a recessively inherited disorder characterized by brain atrophy, seizures, and developmental delay, and in severe cases, microcephaly and infantile death. In EXOC8, we found a homozygous truncating variant in a family with a similar clinical disorder. We modeled exoc7 deficiency in zebrafish and found the absence of exoc7 causes microcephaly. CONCLUSION: Our results highlight the essential role of the exocyst pathway in normal cortical development and how its perturbation causes complex brain disorders.
Assuntos
Encefalopatias , Microcefalia , Animais , Proliferação de Células/genética , Homozigoto , Humanos , Camundongos , Microcefalia/genética , Peixe-Zebra/genéticaRESUMO
Ras is the most mutated oncoprotein in cancer. Among the three oncogenic effectors of Ras - Raf, PI3 Kinase and RalGEF>Ral - signalling through RalGEF>Ral (Ras-like) is by far the least well understood. A variety of signals and binding partners have been defined for Ral, yet we know little of how Ral functions in vivo. This review focuses on previous research in Drosophila that defined a function for Ral in apoptosis and established indirect relationships among Ral, the CNH-domain MAP4 Kinase misshapen, and the JNK MAP kinase basket. Most of the described signalling components are not essential in C. elegans, facilitating subsequent analysis using developmental patterning of the C. elegans vulval precursor cells (VPCs). The functions of two paralogous CNH-domain MAP4 Kinases were defined relative to Ras>Raf, Notch and Ras>RalGEF>Ral signalling in VPCs. MIG-15, the nematode ortholog of misshapen, antagonizes both the Ral-dependent and Ras>Raf-dependent developmental outcomes. In contrast, paralogous GCK-2, the C. elegans ortholog of Drosophila happyhour, propagates the 2°-promoting signal of Ral. Manipulations via CRISPR of Ral signalling through GCK-2 coupled with genetic epistasis delineated a Ras>RalGEF>Ral>Exo84>GCK-2>MAP3KMLK-1> p38PMK-1 cascade. Thus, genetic analysis using invertebrate experimental organisms defined a cascade from Ras to p38 MAP kinase.
Assuntos
Caenorhabditis elegans , Transdução de Sinais , Animais , Caenorhabditis elegans/metabolismo , Transdução de Sinais/genética , Fosfatidilinositol 3-Quinases/metabolismo , Drosophila/metabolismoRESUMO
C. elegans vulval precursor cell (VPC) fates are patterned by an epidermal growth factor (EGF) gradient. High-dose EGF induces 1° VPC fate, and lower dose EGF contributes to 2° fate in support of LIN-12/Notch. We previously showed that the EGF 2°-promoting signal is mediated by LET-60/Ras switching effectors, from the canonical Raf-MEK-ERK mitogen-activated protein (MAP) kinase cascade that promotes 1° fate to the non-canonical RalGEF-Ral that promotes 2° fate. Of oncogenic Ras effectors, RalGEF-Ral is by far the least well understood. We use genetic analysis to identify an effector cascade downstream of C. elegans RAL-1/Ral, starting with an established Ral binding partner, Exo84 of the exocyst complex. Additionally, RAL-1 signals through GCK-2, a citron-N-terminal-homology-domain-containing MAP4 kinase, and PMK-1/p38 MAP kinase cascade to promote 2° fate. Our study delineates a Ral-dependent developmental signaling cascade in vivo, thus providing the mechanism by which lower EGF dose is transduced.