RESUMO
Cyclosporin A (CsA) induces DNA double-strand breaks in LIG4 syndrome fibroblasts, specifically upon transit through S-phase. The basis underlying this has not been described. CsA-induced genomic instability may reflect a direct role of Cyclophilin A (CYPA) in DNA repair. CYPA is a peptidyl-prolyl cis-trans isomerase (PPI). CsA inhibits the PPI activity of CYPA. Using an integrated approach involving CRISPR/Cas9-engineering, siRNA, BioID, co-immunoprecipitation, pathway-specific DNA repair investigations as well as protein expression interaction analysis, we describe novel impacts of CYPA loss and inhibition on DNA repair. We characterise a direct CYPA interaction with the NBS1 component of the MRE11-RAD50-NBS1 complex, providing evidence that CYPA influences DNA repair at the level of DNA end resection. We define a set of genetic vulnerabilities associated with CYPA loss and inhibition, identifying DNA replication fork protection as an important determinant of viability. We explore examples of how CYPA inhibition may be exploited to selectively kill cancers sharing characteristic genomic instability profiles, including MYCN-driven Neuroblastoma, Multiple Myeloma and Chronic Myelogenous Leukaemia. These findings propose a repurposing strategy for Cyclophilin inhibitors.
Assuntos
Hidrolases Anidrido Ácido , Proteínas de Ciclo Celular , Ciclofilina A , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA , Proteína Homóloga a MRE11 , Proteínas Nucleares , Humanos , Proteína Homóloga a MRE11/metabolismo , Proteína Homóloga a MRE11/genética , Ciclofilina A/metabolismo , Ciclofilina A/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Hidrolases Anidrido Ácido/metabolismo , Hidrolases Anidrido Ácido/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP/metabolismo , DNA Ligase Dependente de ATP/genética , Instabilidade GenômicaRESUMO
BACKGROUND: Severe congenital neutropenia presents with recurrent infections early in life as a result of arrested granulopoiesis. Multiple genetic defects are known to block granulocyte differentiation; however, a genetic cause remains unknown in approximately 40% of cases. OBJECTIVE: We aimed to characterize a patient with severe congenital neutropenia and syndromic features without a genetic diagnosis. METHODS: Whole exome sequencing results were validated using flow cytometry, Western blotting, coimmunoprecipitation, quantitative PCR, cell cycle and proliferation analysis of lymphocytes and fibroblasts and granulocytic differentiation of primary CD34+ and HL-60 cells. RESULTS: We identified a homozygous missense mutation in DBF4 in a patient with mild extra-uterine growth retardation, facial dysmorphism and severe congenital neutropenia. DBF4 is the regulatory subunit of the CDC7 kinase, together known as DBF4-dependent kinase (DDK), the complex essential for DNA replication initiation. The DBF4 variant demonstrated impaired ability to bind CDC7, resulting in decreased DDK-mediated phosphorylation, defective S-phase entry and progression and impaired differentiation of granulocytes associated with activation of the p53-p21 pathway. The introduction of wild-type DBF4 into patient CD34+ cells rescued the promyelocyte differentiation arrest. CONCLUSION: Hypomorphic DBF4 mutation causes autosomal-recessive severe congenital neutropenia with syndromic features.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Saccharomyces cerevisiae , Humanos , Proteínas de Ciclo Celular/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Mutação , FosforilaçãoRESUMO
Replicating the human genome efficiently and accurately is a daunting challenge involving the duplication of upward of three billion base pairs. At the core of the complex machinery that achieves this task are three members of the B family of DNA polymerases: DNA polymerases α, δ, and ε. Collectively these multimeric polymerases ensure DNA replication proceeds at optimal rates approaching 2 × 103 nucleotides/min with an error rate of less than one per million nucleotides polymerized. The majority of DNA replication of undamaged DNA is conducted by DNA polymerases δ and ε. The DNA polymerase α-primase complex performs limited synthesis to initiate the replication process, along with Okazaki-fragment synthesis on the discontinuous lagging strand. An increasing number of human disorders caused by defects in different components of the DNA-replication apparatus have been described to date. These are clinically diverse and involve a wide range of features, including variable combinations of growth delay, immunodeficiency, endocrine insufficiencies, lipodystrophy, and cancer predisposition. Here, by using various complementary approaches, including classical linkage analysis, targeted next-generation sequencing, and whole-exome sequencing, we describe distinct missense and splice-impacting mutations in POLA1 in five unrelated families presenting with an X-linked syndrome involving intellectual disability, proportionate short stature, microcephaly, and hypogonadism. POLA1 encodes the p180 catalytic subunit of DNA polymerase α-primase. A range of replicative impairments could be demonstrated in lymphoblastoid cell lines derived from affected individuals. Our findings describe the presentation of pathogenic mutations in a catalytic component of a B family DNA polymerase member, DNA polymerase α.
Assuntos
DNA Polimerase I/genética , DNA Primase/genética , Doenças Genéticas Ligadas ao Cromossomo X/etiologia , Transtornos do Crescimento/etiologia , Hipogonadismo/etiologia , Deficiência Intelectual/etiologia , Microcefalia/etiologia , Mutação , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Genótipo , Transtornos do Crescimento/patologia , Humanos , Hipogonadismo/patologia , Lactente , Deficiência Intelectual/patologia , Masculino , Microcefalia/patologia , Pessoa de Meia-Idade , Linhagem , Sequenciamento do ExomaRESUMO
The role of somatic genetic variants in the pathogenesis of intracranial-aneurysm formation is unknown. We identified a 23-year-old man with progressive, right-sided intracranial aneurysms, ipsilateral to an impressive cutaneous phenotype. The index individual underwent a series of genetic evaluations for known connective-tissue disorders, but the evaluations were unrevealing. Paired-sample exome sequencing between blood and fibroblasts derived from the diseased areas detected a single novel variant predicted to cause a p.Tyr562Cys (g.149505130T>C [GRCh37/hg19]; c.1685A>G) change within the platelet-derived growth factor receptor ß gene (PDGFRB), a juxtamembrane-coding region. Variant-allele fractions ranged from 18.75% to 53.33% within histologically abnormal tissue, suggesting post-zygotic or somatic mosaicism. In an independent cohort of aneurysm specimens, we detected somatic-activating PDGFRB variants in the juxtamembrane domain or the kinase activation loop in 4/6 fusiform aneurysms (and 0/38 saccular aneurysms; Fisher's exact test, p < 0.001). PDGFRB-variant, but not wild-type, patient cells were found to have overactive auto-phosphorylation with downstream activation of ERK, SRC, and AKT. The expression of discovered variants demonstrated non-ligand-dependent auto-phosphorylation, responsive to the kinase inhibitor sunitinib. Somatic gain-of-function variants in PDGFRB are a novel mechanism in the pathophysiology of fusiform cerebral aneurysms and suggest a potential role for targeted therapy with kinase inhibitors.
Assuntos
Aneurisma/genética , Aneurisma Intracraniano/genética , Mutação , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Adolescente , Adulto , Sequência de Aminoácidos , Aneurisma/patologia , Criança , Estudos de Coortes , Feminino , Humanos , Aneurisma Intracraniano/patologia , Masculino , Homologia de Sequência , Adulto JovemRESUMO
Nucleotide metabolism is a complex pathway regulating crucial cellular processes such as nucleic acid synthesis, DNA repair and proliferation. This study shows that impairment of the biosynthesis of one of the building blocks of DNA, dTTP, causes a severe, early-onset neurodegenerative disease. Here, we describe two unrelated children with bi-allelic variants in DTYMK, encoding dTMPK, which catalyzes the penultimate step in dTTP biosynthesis. The affected children show severe microcephaly and growth retardation with minimal neurodevelopment. Brain imaging revealed severe cerebral atrophy and disappearance of the basal ganglia. In cells of affected individuals, dTMPK enzyme activity was minimal, along with impaired DNA replication. In addition, we generated dtymk mutant zebrafish that replicate this phenotype of microcephaly, neuronal cell death and early lethality. An increase of ribonucleotide incorporation in the genome as well as impaired responses to DNA damage were observed in dtymk mutant zebrafish, providing novel pathophysiological insights. It is highly remarkable that this deficiency is viable as an essential component for DNA cannot be generated, since the metabolic pathway for dTTP synthesis is completely blocked. In summary, by combining genetic and biochemical approaches in multiple models we identified loss-of-function of DTYMK as the cause of a severe postnatal neurodegenerative disease and highlight the essential nature of dTTP synthesis in the maintenance of genome stability and neuronal survival.
Assuntos
Doenças Neurodegenerativas/genética , Núcleosídeo-Fosfato Quinase/genética , Animais , Feminino , Humanos , Masculino , Microcefalia/genética , Mutação , Peixe-ZebraRESUMO
Defects in OFD1 underlie the clinically complex ciliopathy, Oral-Facial-Digital syndrome Type I (OFD Type I). Our understanding of the molecular, cellular and clinical consequences of impaired OFD1 originates from its characterised roles at the centrosome/basal body/cilia network. Nonetheless, the first described OFD1 interactors were components of the TIP60 histone acetyltransferase complex. We find that OFD1 can also localise to chromatin and its reduced expression is associated with mis-localization of TIP60 in patient-derived cell lines. TIP60 plays important roles in controlling DNA repair. OFD Type I cells exhibit reduced histone acetylation and altered chromatin dynamics in response to DNA double strand breaks (DSBs). Furthermore, reduced OFD1 impaired DSB repair via homologous recombination repair (HRR). OFD1 loss also adversely impacted upon the DSB-induced G2-M checkpoint, inducing a hypersensitive and prolonged arrest. Our findings show that OFD Type I patient cells have pronounced defects in the DSB-induced histone modification, chromatin remodelling and DSB-repair via HRR; effectively phenocopying loss of TIP60. These data extend our knowledge of the molecular and cellular consequences of impaired OFD1, demonstrating that loss of OFD1 can negatively impact upon important nuclear events; chromatin plasticity and DNA repair.
Assuntos
Cromatina/metabolismo , Cílios/patologia , Reparo do DNA/genética , Síndromes Orofaciodigitais/genética , Síndromes Orofaciodigitais/patologia , Proteínas/metabolismo , Recombinação Genética/genética , Acetilação , Pontos de Checagem do Ciclo Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina/genética , Cílios/enzimologia , Quebras de DNA de Cadeia Dupla , Fibroblastos , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Síndromes Orofaciodigitais/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/genética , RNA Interferente Pequeno/genéticaRESUMO
SHORT syndrome is a rare, recognizable syndrome resulting from heterozygous mutations in PIK3R1 encoding a regulatory subunit of phosphoinositide-3-kinase (PI3K). The condition is characterized by short stature, intrauterine growth restriction, lipoatrophy and a facial gestalt involving a triangular face, deep set eyes, low hanging columella and small chin. PIK3R1 mutations in SHORT syndrome result in reduced signaling through the PI3K-AKT-mTOR pathway. We performed whole exome sequencing for an individual with clinical features of SHORT syndrome but negative for PIK3R1 mutation and her parents. A rare de novo variant in PRKCE was identified. The gene encodes PKCε and, as such, the AKT-mTOR pathway function was assessed using phospho-specific antibodies with patient lymphoblasts and following ectopic expression of the mutant in HEK293 cells. Kinase analysis showed that the variant resulted in a partial loss-of-function. Whilst interaction with PDK1 and the mTORC2 complex component SIN1 was preserved in the mutant PKCε, it bound to SIN1 with a higher affinity than wild-type PKCε and the dynamics of mTORC2-dependent priming of mutant PKCε was altered. Further, mutant PKCε caused impaired mTORC2-dependent pAKT-S473 following rapamycin treatment. Reduced pFOXO1-S256 and pS6-S240/244 levels were also observed in the patient LCLs. To date, mutations in PIK3R1 causing impaired PI3K-dependent AKT activation are the only known cause of SHORT syndrome. We identify a SHORT syndrome child with a novel partial loss-of-function defect in PKCε. This variant causes impaired AKT activation via compromised mTORC2 complex function.
Assuntos
Transtornos do Crescimento/genética , Hipercalcemia/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Doenças Metabólicas/genética , Nefrocalcinose/genética , Proteína Quinase C-épsilon/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Nanismo/genética , Feminino , Transtornos do Crescimento/metabolismo , Células HEK293 , Humanos , Hipercalcemia/metabolismo , Doenças Metabólicas/metabolismo , Mutação , Nefrocalcinose/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Proteína Quinase C-épsilon/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Encephalocraniocutaneous lipomatosis (ECCL) is a sporadic condition characterized by ocular, cutaneous, and central nervous system anomalies. Key clinical features include a well-demarcated hairless fatty nevus on the scalp, benign ocular tumors, and central nervous system lipomas. Seizures, spasticity, and intellectual disability can be present, although affected individuals without seizures and with normal intellect have also been reported. Given the patchy and asymmetric nature of the malformations, ECCL has been hypothesized to be due to a post-zygotic, mosaic mutation. Despite phenotypic overlap with several other disorders associated with mutations in the RAS-MAPK and PI3K-AKT pathways, the molecular etiology of ECCL remains unknown. Using exome sequencing of DNA from multiple affected tissues from five unrelated individuals with ECCL, we identified two mosaic mutations, c.1638C>A (p.Asn546Lys) and c.1966A>G (p.Lys656Glu) within the tyrosine kinase domain of FGFR1, in two affected individuals each. These two residues are the most commonly mutated residues in FGFR1 in human cancers and are associated primarily with CNS tumors. Targeted resequencing of FGFR1 in multiple tissues from an independent cohort of individuals with ECCL identified one additional individual with a c.1638C>A (p.Asn546Lys) mutation in FGFR1. Functional studies of ECCL fibroblast cell lines show increased levels of phosphorylated FGFRs and phosphorylated FRS2, a direct substrate of FGFR1, as well as constitutive activation of RAS-MAPK signaling. In addition to identifying the molecular etiology of ECCL, our results support the emerging overlap between mosaic developmental disorders and tumorigenesis.
Assuntos
Oftalmopatias/genética , Lipomatose/genética , Síndromes Neurocutâneas/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Adolescente , Linhagem Celular Tumoral , Neoplasias do Sistema Nervoso Central/diagnóstico , Neoplasias do Sistema Nervoso Central/genética , Pré-Escolar , Exoma , Olho/fisiopatologia , Oftalmopatias/diagnóstico , Feminino , Humanos , Lactente , Lipomatose/diagnóstico , Masculino , Mutação , Mutação de Sentido Incorreto , Síndromes Neurocutâneas/diagnóstico , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Convulsões/genética , Análise de Sequência de DNARESUMO
Mutations in ATR(ataxia telangiectasia and RAD3-related) cause Seckel syndrome (ATR-SS), a microcephalic primordial dwarfism disorder. Hitherto, the clinical manifestation of ATR deficiency has been attributed to its canonical role in DNA damage response signalling following replication fork stalling/collapse. Here, we show that ATR regulates cilia-dependent signalling in a manner that can be uncoupled from its function during replication. ATR-depleted or patient-derived ATR-SS cells form cilia of slightly reduced length but are dramatically impaired in cilia-dependent signalling functions, including growth factor and Sonic hedgehog signalling. To better understand the developmental impact of ATR loss of function, we also used zebrafish as a model. Zebrafish embryos depleted of Atr resembled ATR-SS morphology, showed a modest but statistically significant reduction in cilia length and other morphological features indicative of cilia dysfunction. Additionally, they displayed defects in left-right asymmetry including ambiguous expression of southpaw, incorrectly looped hearts and randomized localization of internal organs including the pancreas, features typically conferred by cilia dysfunction. Our findings reveal a novel role for ATR in cilia signalling distinct from its canonical function during replication and strengthen emerging links between cilia function and development.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Cílios/patologia , Nanismo/patologia , Microcefalia/patologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular , Cílios/metabolismo , Replicação do DNA , Modelos Animais de Doenças , Nanismo/genética , Fácies , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Microcefalia/genética , Transdução de Sinais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Accurate and efficient replication of the human genome occurs in the context of an array of constitutional barriers, including regional topological constraints imposed by chromatin architecture and processes such as transcription, catenation of the helical polymer and spontaneously generated DNA lesions, including base modifications and strand breaks. DNA replication is fundamentally important for tissue development and homeostasis; differentiation programmes are intimately linked with stem cell division. Unsurprisingly, impairments of the DNA replication machinery can have catastrophic consequences for genome stability and cell division. Functional impacts on DNA replication and genome stability have long been known to play roles in malignant transformation through a variety of complex mechanisms, and significant further insights have been gained from studying model organisms in this context. Congenital hypomorphic defects in components of the DNA replication machinery have been and continue to be identified in humans. These disorders present with a wide range of clinical features. Indeed, in some instances, different mutations in the same gene underlie different clinical presentations. Understanding the origin and molecular basis of these features opens a window onto the range of developmental impacts of suboptimal DNA replication and genome instability in humans. Here, I will briefly overview the basic steps involved in DNA replication and the key concepts that have emerged from this area of research, before switching emphasis to the pathological consequences of defects within the DNA replication network; the human disorders. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Transformação Celular Neoplásica , Reparo do DNA , Replicação do DNA , Instabilidade Genômica/genética , Mutação/genética , Animais , Cromatina/genética , HumanosRESUMO
Mutations of genes within the phosphatidylinositol-3-kinase (PI3K)-AKT-MTOR pathway are well known causes of brain overgrowth (megalencephaly) as well as segmental cortical dysplasia (such as hemimegalencephaly, focal cortical dysplasia and polymicrogyria). Mutations of the AKT3 gene have been reported in a few individuals with brain malformations, to date. Therefore, our understanding regarding the clinical and molecular spectrum associated with mutations of this critical gene is limited, with no clear genotype-phenotype correlations. We sought to further delineate this spectrum, study levels of mosaicism and identify genotype-phenotype correlations of AKT3-related disorders. We performed targeted sequencing of AKT3 on individuals with these phenotypes by molecular inversion probes and/or Sanger sequencing to determine the type and level of mosaicism of mutations. We analysed all clinical and brain imaging data of mutation-positive individuals including neuropathological analysis in one instance. We performed ex vivo kinase assays on AKT3 engineered with the patient mutations and examined the phospholipid binding profile of pleckstrin homology domain localizing mutations. We identified 14 new individuals with AKT3 mutations with several phenotypes dependent on the type of mutation and level of mosaicism. Our comprehensive clinical characterization, and review of all previously published patients, broadly segregates individuals with AKT3 mutations into two groups: patients with highly asymmetric cortical dysplasia caused by the common p.E17K mutation, and patients with constitutional AKT3 mutations exhibiting more variable phenotypes including bilateral cortical malformations, polymicrogyria, periventricular nodular heterotopia and diffuse megalencephaly without cortical dysplasia. All mutations increased kinase activity, and pleckstrin homology domain mutants exhibited enhanced phospholipid binding. Overall, our study shows that activating mutations of the critical AKT3 gene are associated with a wide spectrum of brain involvement ranging from focal or segmental brain malformations (such as hemimegalencephaly and polymicrogyria) predominantly due to mosaic AKT3 mutations, to diffuse bilateral cortical malformations, megalencephaly and heterotopia due to constitutional AKT3 mutations. We also provide the first detailed neuropathological examination of a child with extreme megalencephaly due to a constitutional AKT3 mutation. This child has one of the largest documented paediatric brain sizes, to our knowledge. Finally, our data show that constitutional AKT3 mutations are associated with megalencephaly, with or without autism, similar to PTEN-related disorders. Recognition of this broad clinical and molecular spectrum of AKT3 mutations is important for providing early diagnosis and appropriate management of affected individuals, and will facilitate targeted design of future human clinical trials using PI3K-AKT pathway inhibitors.
Assuntos
Deficiências do Desenvolvimento/genética , Megalencefalia/genética , Mutação/genética , Proteínas Proto-Oncogênicas c-akt/genética , Encéfalo/diagnóstico por imagem , Criança , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/patologia , Feminino , Estudos de Associação Genética , Células HEK293 , Humanos , Imunoprecipitação , Imageamento por Ressonância Magnética , Masculino , Megalencefalia/diagnóstico por imagem , Megalencefalia/patologia , Mutagênese Sítio-Dirigida/métodos , Fosfatidilinositóis/metabolismo , TransfecçãoRESUMO
Activating somatic PIK3CA mutations underlie a growing heterogeneous spectrum of segmental overgrowth disorders. We report the identification and evaluation of a novel de novo constitutional PIK3CA mutation (NM_006218.2:c.335T>A, p.Ile112Asn) in a child with congenital megalencephaly and macrosomia. Functional characterization of patient cells using a variety of endpoints demonstrates increased phosphatidylinositol-3-kinase (PI3K) activity. The mutation lies in a linker region adjacent to the p85 (PIK3R2) binding domain of the p110α (PIK3CA) catalytic subunit of PI3K. We show that altered stoichiometry within the p85-p110 complex likely underlies the hyperactive PI3K-AKT-mTOR signaling in this instance. Our findings expand upon the recently proposed "PIK3CA-related overgrowth spectrum" associated with PIKC3A mutations and PI3K hyperactivation, adding constitutional PIK3CA mutations as an underlying cause of megalencephaly and macrosomia in newborns.
Assuntos
Fosfatidilinositol 3-Quinases/genética , Criança , Classe I de Fosfatidilinositol 3-Quinases , Humanos , Masculino , Megalencefalia/genética , MutaçãoRESUMO
SHORT syndrome is a rare, multisystem disease characterized by short stature, anterior-chamber eye anomalies, characteristic facial features, lipodystrophy, hernias, hyperextensibility, and delayed dentition. As part of the FORGE (Finding of Rare Disease Genes) Canada Consortium, we studied individuals with clinical features of SHORT syndrome to identify the genetic etiology of this rare disease. Whole-exome sequencing in a family trio of an affected child and unaffected parents identified a de novo frameshift insertion, c.1906_1907insC (p.Asn636Thrfs*18), in exon 14 of PIK3R1. Heterozygous mutations in exon 14 of PIK3R1 were subsequently identified by Sanger sequencing in three additional affected individuals and two affected family members. One of these mutations, c.1945C>T (p.Arg649Trp), was confirmed to be a de novo mutation in one affected individual and was also identified and shown to segregate with the phenotype in an unrelated family. The other mutation, a de novo truncating mutation (c.1971T>G [p.Tyr657*]), was identified in another affected individual. PIK3R1 is involved in the phosphatidylinositol 3 kinase (PI3K) signaling cascade and, as such, plays an important role in cell growth, proliferation, and survival. Functional studies on lymphoblastoid cells with the PIK3R1 c.1906_1907insC mutation showed decreased phosphorylation of the downstream S6 target of the PI3K-AKT-mTOR pathway. Our findings show that PIK3R1 mutations are the major cause of SHORT syndrome and suggest that the molecular mechanism of disease might involve downregulation of the PI3K-AKT-mTOR pathway.
Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/genética , Mutação da Fase de Leitura , Transtornos do Crescimento/genética , Hipercalcemia/genética , Doenças Metabólicas/genética , Nefrocalcinose/genética , Adolescente , Criança , Pré-Escolar , Análise Mutacional de DNA/métodos , Exoma , Éxons , Feminino , Triagem de Portadores Genéticos , Heterozigoto , Humanos , Recém-Nascido , Masculino , Linhagem , Fenótipo , Fosforilação , Transdução de SinaisRESUMO
Mutations in ORC1, ORC4, ORC6, CDT1, and CDC6, which encode proteins required for DNA replication origin licensing, cause Meier-Gorlin syndrome (MGS), a disorder conferring microcephaly, primordial dwarfism, underdeveloped ears, and skeletal abnormalities. Mutations in ATR, which also functions during replication, can cause Seckel syndrome, a clinically related disorder. These findings suggest that impaired DNA replication could underlie the developmental defects characteristic of these disorders. Here, we show that although origin licensing capacity is impaired in all patient cells with mutations in origin licensing component proteins, this does not correlate with the rate of progression through S phase. Thus, the replicative capacity in MGS patient cells does not correlate with clinical manifestation. However, ORC1-deficient cells from MGS patients and siRNA-mediated depletion of origin licensing proteins also have impaired centrosome and centriole copy number. As a novel and unexpected finding, we show that they also display a striking defect in the rate of formation of primary cilia. We demonstrate that this impacts sonic hedgehog signalling in ORC1-deficient primary fibroblasts. Additionally, reduced growth factor-dependent signaling via primary cilia affects the kinetics of cell cycle progression following cell cycle exit and re-entry, highlighting an unexpected mechanism whereby origin licensing components can influence cell cycle progression. Finally, using a cell-based model, we show that defects in cilia function impair chondroinduction. Our findings raise the possibility that a reduced efficiency in forming cilia could contribute to the clinical features of MGS, particularly the bone development abnormalities, and could provide a new dimension for considering developmental impacts of licensing deficiency.
Assuntos
Replicação do DNA/genética , Nanismo/genética , Transtornos do Crescimento/genética , Microcefalia/genética , Micrognatismo/genética , Complexo de Reconhecimento de Origem/genética , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Centríolos/genética , Centríolos/metabolismo , Cílios/genética , Cílios/fisiologia , Microtia Congênita , Orelha/anormalidades , Fácies , Transtornos do Crescimento/etiologia , Humanos , Micrognatismo/etiologia , Patela/anormalidades , Proteínas Serina-Treonina Quinases/genética , Fase S/genéticaRESUMO
ATR (ataxia telangiectasia and Rad3 related) is an essential regulator of genome integrity. It controls and coordinates DNA-replication origin firing, replication-fork stability, cell-cycle checkpoints, and DNA repair. Previously, autosomal-recessive loss-of-function mutations in ATR have been demonstrated in Seckel syndrome, a developmental disorder. Here, however, we report on a different kind of genetic disorder that is due to functionally compromised ATR activity, which translates into an autosomal-dominant inherited disease. The condition affects 24 individuals in a five-generation pedigree and comprises oropharyngeal cancer, skin telangiectases, and mild developmental anomalies of the hair, teeth, and nails. We mapped the disorder to a â¼16.8 cM interval in chromosomal region 3q22-24, and by sequencing candidate genes, we found that ATR contained a heterozygous missense mutation (c.6431A>G [p.Gln2144Arg]) that segregated with the disease. The mutation occurs within the FAT (FRAP, ATM, and TRRAP) domain-which can activate p53-of ATR. The mutation did not lead to a reduction in ATR expression, but cultured fibroblasts showed lower p53 levels after activation of ATR with hydroxyurea than did normal control fibroblasts. Moreover, loss of heterozygosity for the ATR locus was noted in oropharyngeal-tumor tissue. Collectively, the clinicopathological and molecular findings point to a cancer syndrome and provide evidence implicating a germline mutation in ATR and susceptibility to malignancy in humans.
Assuntos
Proteínas de Ciclo Celular/genética , Transtornos Cromossômicos/genética , Mutação em Linhagem Germinativa , Neoplasias Orofaríngeas/genética , Proteínas Serina-Treonina Quinases/genética , Adulto , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia , Criança , Pré-Escolar , Cromossomos , Feminino , Fibroblastos/metabolismo , Genes p53/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla/métodos , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação de Sentido Incorreto , LinhagemRESUMO
A homozygous mutational change in the Ataxia-Telangiectasia and RAD3 related (ATR) gene was previously reported in two related families displaying Seckel Syndrome (SS). Here, we provide the first identification of a Seckel Syndrome patient with mutations in ATRIP, the gene encoding ATR-Interacting Protein (ATRIP), the partner protein of ATR required for ATR stability and recruitment to the site of DNA damage. The patient has compound heterozygous mutations in ATRIP resulting in reduced ATRIP and ATR expression. A nonsense mutational change in one ATRIP allele results in a C-terminal truncated protein, which impairs ATR-ATRIP interaction; the other allele is abnormally spliced. We additionally describe two further unrelated patients native to the UK with the same novel, heterozygous mutations in ATR, which cause dramatically reduced ATR expression. All patient-derived cells showed defective DNA damage responses that can be attributed to impaired ATR-ATRIP function. Seckel Syndrome is characterised by microcephaly and growth delay, features also displayed by several related disorders including Majewski (microcephalic) osteodysplastic primordial dwarfism (MOPD) type II and Meier-Gorlin Syndrome (MGS). The identification of an ATRIP-deficient patient provides a novel genetic defect for Seckel Syndrome. Coupled with the identification of further ATR-deficient patients, our findings allow a spectrum of clinical features that can be ascribed to the ATR-ATRIP deficient sub-class of Seckel Syndrome. ATR-ATRIP patients are characterised by extremely severe microcephaly and growth delay, microtia (small ears), micrognathia (small and receding chin), and dental crowding. While aberrant bone development was mild in the original ATR-SS patient, some of the patients described here display skeletal abnormalities including, in one patient, small patellae, a feature characteristically observed in Meier-Gorlin Syndrome. Collectively, our analysis exposes an overlapping clinical manifestation between the disorders but allows an expanded spectrum of clinical features for ATR-ATRIP Seckel Syndrome to be defined.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Nanismo/genética , Transtornos do Crescimento , Micrognatismo , Proteínas Serina-Treonina Quinases , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Códon sem Sentido , Microtia Congênita , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Nanismo/patologia , Orelha/anormalidades , Orelha/patologia , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/patologia , Regulação da Expressão Gênica , Transtornos do Crescimento/genética , Transtornos do Crescimento/patologia , Heterozigoto , Humanos , Masculino , Microcefalia/genética , Microcefalia/patologia , Micrognatismo/genética , Micrognatismo/patologia , Osteocondrodisplasias/genética , Osteocondrodisplasias/patologia , Patela/anormalidades , Patela/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Splicing de RNA , Transdução de Sinais/genéticaRESUMO
A 0.8 kb intronic duplication in MAGT1 and a single base pair deletion in the last exon of ATRX were identified using a chromosome X-specific microarray and exome sequencing in a family with five males demonstrating intellectual disability (ID) and unusual skin findings (e.g., generalized pruritus). MAGT1 is an Mg²âº transporter previously associated with primary immunodeficiency and ID, whereas mutations in ATRX cause ATRX-ID syndrome. In patient cells, the function of ATRX was demonstrated to be abnormal based on altered RNA/protein expression, hypomethylation of rDNA, and abnormal cytokinesis. Dysfunction of MAGT1 was reflected in reduced RNA/protein expression and Mg²âº influx. The mutation in ATRX most likely explains the ID, whereas MAGT1 disruption could be linked to abnormal skin findings, as normal magnesium homeostasis is necessary for skin health. This work supports observations that multiple mutations collectively contribute to the phenotypic variability of syndromic ID, and emphasizes the importance of correlating clinical phenotype with genomic and cell function analyses.
Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Deficiência Intelectual Ligada ao Cromossomo X/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Prurido/genética , Cromossomos Humanos X , Citocinese , Metilação de DNA , DNA Ribossômico/metabolismo , Exoma , Feminino , Genes Duplicados , Humanos , Íntrons , Magnésio/metabolismo , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Deficiência Intelectual Ligada ao Cromossomo X/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Linhagem , Fenótipo , Mutação Puntual , Prurido/patologia , Análise de Sequência de DNA , Síndrome , Proteína Nuclear Ligada ao XRESUMO
The underlying etiologies of genetic congenital microcephaly are complex and multifactorial. Recently, with the exponential growth in the identification and characterization of novel genetic causes of congenital microcephaly, there has been a consolidation and emergence of certain themes concerning underlying pathomechanisms. These include abnormal mitotic microtubule spindle structure, numerical and structural abnormalities of the centrosome, altered cilia function, impaired DNA repair, DNA Damage Response signaling and DNA replication, along with attenuated cell cycle checkpoint proficiency. Many of these processes are highly interconnected. Interestingly, a defect in a gene whose encoded protein has a canonical function in one of these processes can often have multiple impacts at the cellular level involving several of these pathways. Here, we overview the key pathomechanistic themes underlying profound congenital microcephaly, and emphasize their interconnected nature.
Assuntos
Microcefalia/genética , Animais , Cílios/fisiologia , Dano ao DNA , Reparo do DNA , Replicação do DNA , Humanos , Microcefalia/patologia , Mitose , MutaçãoRESUMO
Wolf-Hirschhorn syndrome (WHS) is a contiguous gene deletion disorder associated with the distal part of the short arm of chromosome 4 (4p16.3). Employing a unique panel of patient-derived cell lines with differing-sized 4p deletions, we provide evidence that haploinsufficiency of SLBP and/or WHSC2 (NELF-A) contributes to several novel cellular phenotypes of WHS, including delayed progression from S-phase into M-phase, reduced DNA replication in asynchronous culture and altered higher order chromatin assembly. The latter is evidenced by reduced histone-chromatin association, elevated levels of soluble chaperone-bound histone H3 and increased sensitivity to micrococcal nuclease digestion in WHS patient-derived cells. We also observed increased camptothecin-induced inhibition of DNA replication and hypersensitivity to killing. Our work provides a novel pathogenomic insight into the aetiology of WHS by describing it, for the first time, as a disorder of impaired chromatin reorganization. Delayed cell-cycle progression and impaired DNA replication likely underlie or contribute to microcephaly, pre- and postnatal growth retardation, which constitute the core clinical features of WHS.
Assuntos
Proteínas Nucleares/genética , Fatores de Elongação da Transcrição/genética , Síndrome de Wolf-Hirschhorn/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Deleção Cromossômica , Cromossomos Humanos Par 4/genética , Dano ao DNA , Haploinsuficiência , Humanos , Fenótipo , Síndrome de Wolf-Hirschhorn/patologiaRESUMO
Defects in centrosome, centrosomal-associated and spindle-associated proteins are the most frequent cause of primary microcephaly (PM) and microcephalic primordial dwarfism (MPD) syndromes in humans. Mitotic progression and segregation defects, microtubule spindle abnormalities and impaired DNA damage-induced G2-M cell cycle checkpoint proficiency have been documented in cell lines from these patients. This suggests that impaired mitotic entry, progression and exit strongly contribute to PM and MPD. Considering the vast protein networks involved in coordinating this cell cycle stage, the list of potential target genes that could underlie novel developmental disorders is large. One such complex network, with a direct microtubule-mediated physical connection to the centrosome, is the kinetochore. This centromeric-associated structure nucleates microtubule attachments onto mitotic chromosomes. Here, we described novel compound heterozygous variants in CENPE in two siblings who exhibit a profound MPD associated with developmental delay, simplified gyri and other isolated abnormalities. CENPE encodes centromere-associated protein E (CENP-E), a core kinetochore component functioning to mediate chromosome congression initially of misaligned chromosomes and in subsequent spindle microtubule capture during mitosis. Firstly, we present a comprehensive clinical description of these patients. Then, using patient cells we document abnormalities in spindle microtubule organization, mitotic progression and segregation, before modeling the cellular pathogenicity of these variants in an independent cell system. Our cellular analysis shows that a pathogenic defect in CENP-E, a kinetochore-core protein, largely phenocopies PCNT-mutated microcephalic osteodysplastic primordial dwarfism-type II patient cells. PCNT encodes a centrosome-associated protein. These results highlight a common underlying pathomechanism. Our findings provide the first evidence for a kinetochore-based route to MPD in humans.