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1.
Am J Hum Genet ; 105(3): 493-508, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31447100

RESUMO

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

2.
Am J Hum Genet ; 104(6): 1223-1232, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31130282

RESUMO

Aberrant signaling through pathways controlling cell response to extracellular stimuli constitutes a central theme in disorders affecting development. Signaling through RAS and the MAPK cascade controls a variety of cell decisions in response to cytokines, hormones, and growth factors, and its upregulation causes Noonan syndrome (NS), a developmental disorder whose major features include a distinctive facies, a wide spectrum of cardiac defects, short stature, variable cognitive impairment, and predisposition to malignancies. NS is genetically heterogeneous, and mutations in more than ten genes have been reported to underlie this disorder. Despite the large number of genes implicated, about 10%-20% of affected individuals with a clinical diagnosis of NS do not have mutations in known RASopathy-associated genes, indicating that additional unidentified genes contribute to the disease, when mutated. By using a mixed strategy of functional candidacy and exome sequencing, we identify RRAS2 as a gene implicated in NS in six unrelated subjects/families. We show that the NS-causing RRAS2 variants affect highly conserved residues localized around the nucleotide binding pocket of the GTPase and are predicted to variably affect diverse aspects of RRAS2 biochemical behavior, including nucleotide binding, GTP hydrolysis, and interaction with effectors. Additionally, all pathogenic variants increase activation of the MAPK cascade and variably impact cell morphology and cytoskeletal rearrangement. Finally, we provide a characterization of the clinical phenotype associated with RRAS2 mutations.

3.
Cell Death Dis ; 10(3): 201, 2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30814510

RESUMO

The pressure towards innovation and creation of new model systems in regenerative medicine and cancer research has fostered the development of novel potential therapeutic applications. Kidney injuries provoke a high request of organ transplants making it the most demanding system in the field of regenerative medicine. Furthermore, renal cancer frequently threaten patients' life and aggressive forms still remain difficult to treat. Ethical issues related to the use of embryonic stem cells, has fueled research on adult, patient-specific pluripotent stem cells as a model for discovery and therapeutic development, but to date, normal and cancerous renal experimental models are lacking. Several research groups are focusing on the development of organoid cultures. Since organoids mimic the original tissue architecture in vitro, they represent an excellent model for tissue engineering studies and cancer therapy testing. We established normal and tumor renal cell carcinoma organoids previously maintained in a heterogeneous multi-clone stem cell-like enriching medium. Starting from adult normal kidney specimens, we were able to isolate and propagate organoid 3D-structures composed of both differentiated and undifferentiated cells while expressing nephron specific markers. Furthermore, we were capable to establish organoids derived from cancer tissues although with a success rate inferior to that of their normal counterpart. Cancer cultures displayed epithelial and mesenchymal phenotype while retaining tumor specific markers. Of note, tumor organoids recapitulated neoplastic masses when orthotopically injected into immunocompromised mice. Our data suggest an innovative approach of long-term establishment of normal- and cancer-derived renal organoids obtained from cultures of fleshly dissociated adult tissues. Our results pave the way to organ replacement pioneering strategies as well as to new models for studying drug-induced nephrotoxicity and renal diseases. Along similar lines, deriving organoids from renal cancer patients opens unprecedented opportunities for generation of preclinical models aimed at improving therapeutic treatments.

4.
Neurology ; 91(4): e319-e330, 2018 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-29959261

RESUMO

OBJECTIVE: To characterize clinically and molecularly an early-onset, variably progressive neurodegenerative disorder characterized by a cerebellar syndrome with severe ataxia, gaze palsy, dyskinesia, dystonia, and cognitive decline affecting 11 individuals from 3 consanguineous families. METHODS: We used whole-exome sequencing (WES) (families 1 and 2) and a combined approach based on homozygosity mapping and WES (family 3). We performed in vitro studies to explore the effect of the nontruncating SQSTM1 mutation on protein function and the effect of impaired SQSTM1 function on autophagy. We analyzed the consequences of sqstm1 down-modulation on the structural integrity of the cerebellum in vivo using zebrafish as a model. RESULTS: We identified 3 homozygous inactivating variants, including a splice site substitution (c.301+2T>A) causing aberrant transcript processing and accelerated degradation of a resulting protein lacking exon 2, as well as 2 truncating changes (c.875_876insT and c.934_936delinsTGA). We show that loss of SQSTM1 causes impaired production of ubiquitin-positive protein aggregates in response to misfolded protein stress and decelerated autophagic flux. The consequences of sqstm1 down-modulation on the structural integrity of the cerebellum in zebrafish documented a variable but reproducible phenotype characterized by cerebellum anomalies ranging from depletion of axonal connections to complete atrophy. We provide a detailed clinical characterization of the disorder; the natural history is reported for 2 siblings who have been followed up for >20 years. CONCLUSIONS: This study offers an accurate clinical characterization of this recently recognized neurodegenerative disorder caused by biallelic inactivating mutations in SQSTM1 and links this phenotype to defective selective autophagy.

5.
Eur J Hum Genet ; 25(7): 823-831, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28594414

RESUMO

RASopathies comprise a group of disorders clinically characterized by short stature, heart defects, facial dysmorphism, and varying degrees of intellectual disability and cancer predisposition. They are caused by germline variants in genes encoding key components or modulators of the highly conserved RAS-MAPK signalling pathway that lead to dysregulation of cell signal transmission. Germline changes in the genes encoding members of the RAS subfamily of GTPases are rare and associated with variable phenotypes of the RASopathy spectrum, ranging from Costello syndrome (HRAS variants) to Noonan and Cardiofaciocutaneous syndromes (KRAS variants). A small number of RASopathy cases with disease-causing germline NRAS alterations have been reported. Affected individuals exhibited features fitting Noonan syndrome, and the observed germline variants differed from the typical oncogenic NRAS changes occurring as somatic events in tumours. Here we describe 19 new cases with RASopathy due to disease-causing variants in NRAS. Importantly, four of them harbored missense changes affecting Gly12, which was previously described to occur exclusively in cancer. The phenotype in our cohort was variable but well within the RASopathy spectrum. Further, one of the patients (c.35G>A; p.(Gly12Asp)) had a myeloproliferative disorder, and one subject (c.34G>C; p.(Gly12Arg)) exhibited an uncharacterized brain tumour. With this report, we expand the genotype and phenotype spectrum of RASopathy-associated germline NRAS variants and provide evidence that NRAS variants do not spare the cancer-associated mutation hotspots.


Assuntos
Síndrome de Costello/genética , Displasia Ectodérmica/genética , Insuficiência de Crescimento/genética , GTP Fosfo-Hidrolases/genética , Mutação em Linhagem Germinativa , Cardiopatias Congênitas/genética , Proteínas de Membrana/genética , Síndrome de Noonan/genética , Adolescente , Adulto , Criança , Pré-Escolar , Síndrome de Costello/patologia , Displasia Ectodérmica/patologia , Facies , Insuficiência de Crescimento/patologia , Feminino , Genótipo , Cardiopatias Congênitas/patologia , Humanos , Lactente , Recém-Nascido , Masculino , Mutação de Sentido Incorreto , Síndrome de Noonan/patologia , Fenótipo
6.
Hum Mutat ; 38(7): 798-804, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28390077

RESUMO

RASopathies are a group of rare, clinically related conditions affecting development and growth, and are caused by germline mutations in genes encoding signal transducers and modulators with a role in the RAS signaling network. These disorders share facial dysmorphia, short stature, variable cognitive deficits, skeletal and cardiac defects, and a variable predisposition to malignancies. Here, we report on a de novo 10-nucleotide-long deletion in HRAS (c.481_490delGGGACCCTCT, NM_176795.4; p.Leu163ProfsTer52, NP_789765.1) affecting transcript processing as a novel event underlying a RASopathy characterized by developmental delay, intellectual disability and autistic features, distinctive coarse facies, reduced growth, and ectodermal anomalies. Molecular and biochemical studies demonstrated that the deletion promotes constitutive retention of exon IDX, which is generally skipped during HRAS transcript processing, and results in a stable and mildly hyperactive GDP/GTP-bound protein that is constitutively targeted to the plasma membrane. Our findings document a new mechanism leading to altered HRAS function that underlies a previously unappreciated phenotype within the RASopathy spectrum.


Assuntos
Deficiências do Desenvolvimento/genética , Regulação Neoplásica da Expressão Gênica , Genes ras , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Transtorno Autístico/genética , Células COS , Membrana Celular/metabolismo , Cercopithecus aethiops , Criança , Pré-Escolar , Éxons , Facies , Deleção de Genes , Mutação em Linhagem Germinativa , Humanos , Deficiência Intelectual/genética , Masculino , Fenótipo , RNA Mensageiro/metabolismo , Transdução de Sinais
7.
Hum Mutat ; 38(4): 451-459, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28074573

RESUMO

Germline mutations in PTPN11, the gene encoding the Src-homology 2 (SH2) domain-containing protein tyrosine phosphatase (SHP2), cause Noonan syndrome (NS), a relatively common, clinically variable, multisystem disorder. Here, we report on the identification of five different PTPN11 missense changes affecting residues Leu261 , Leu262 , and Arg265 in 16 unrelated individuals with clinical diagnosis of NS or with features suggestive for this disorder, specifying a novel disease-causing mutation cluster. Expression of the mutant proteins in HEK293T cells documented their activating role on MAPK signaling. Structural data predicted a gain-of-function role of substitutions at residues Leu262 and Arg265 exerted by disruption of the N-SH2/PTP autoinhibitory interaction. Molecular dynamics simulations suggested a more complex behavior for changes affecting Leu261 , with possible impact on SHP2's catalytic activity/selectivity and proper interaction of the PTP domain with the regulatory SH2 domains. Consistent with that, biochemical data indicated that substitutions at codons 262 and 265 increased the catalytic activity of the phosphatase, while those affecting codon 261 were only moderately activating but impacted substrate specificity. Remarkably, these mutations underlie a relatively mild form of NS characterized by low prevalence of cardiac defects, short stature, and cognitive and behavioral issues, as well as less evident typical facial features.


Assuntos
Predisposição Genética para Doença/genética , Mutação , Síndrome de Noonan/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/genética , Modelos Moleculares , Mutação de Sentido Incorreto , Síndrome de Noonan/patologia , Ligação Proteica , Domínios Proteicos , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Domínios de Homologia de src
8.
Am J Hum Genet ; 99(4): 974-983, 2016 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-27666369

RESUMO

Tubulinopathies constitute a family of neurodevelopmental/neurodegenerative disorders caused by mutations in several genes encoding tubulin isoforms. Loss-of-function mutations in TBCE, encoding one of the five tubulin-specific chaperones involved in tubulin folding and polymerization, cause two rare neurodevelopmental syndromes, hypoparathyroidism-retardation-dysmorphism and Kenny-Caffey syndrome. Although a missense mutation in Tbce has been associated with progressive distal motor neuronopathy in the pmn/pmn mice, no similar degenerative phenotype has been recognized in humans. We report on the identification of an early-onset and progressive neurodegenerative encephalopathy with distal spinal muscular atrophy resembling the phenotype of pmn/pmn mice and caused by biallelic TBCE mutations, with the c.464T>A (p.Ile155Asn) change occurring at the heterozygous/homozygous state in six affected subjects from four unrelated families originated from the same geographical area in Southern Italy. Western blot analysis of patient fibroblasts documented a reduced amount of TBCE, suggestive of rapid degradation of the mutant protein, similarly to what was observed in pmn/pmn fibroblasts. The impact of TBCE mutations on microtubule polymerization was determined using biochemical fractionation and analyzing the nucleation and growth of microtubules at the centrosome and extracentrosomal sites after treatment with nocodazole. Primary fibroblasts obtained from affected subjects displayed a reduced level of polymerized α-tubulin, similarly to tail fibroblasts of pmn/pmn mice. Moreover, markedly delayed microtubule re-polymerization and abnormal mitotic spindles with disorganized microtubule arrangement were also documented. Although loss of function of TBCE has been documented to impact multiple developmental processes, the present findings provide evidence that hypomorphic TBCE mutations primarily drive neurodegeneration.


Assuntos
Encefalopatias/complicações , Encefalopatias/genética , Chaperonas Moleculares/genética , Atrofia Muscular Espinal/complicações , Atrofia Muscular Espinal/genética , Mutação/genética , Adolescente , Idade de Início , Animais , Criança , Feminino , Fibroblastos , Heterozigoto , Homozigoto , Humanos , Lactente , Recém-Nascido , Itália , Masculino , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/patologia , Chaperonas Moleculares/metabolismo , Nocodazol/farmacologia , Fuso Acromático/metabolismo , Fuso Acromático/patologia , Tubulina (Proteína)/metabolismo , Adulto Jovem
9.
Am J Hum Genet ; 99(4): 962-973, 2016 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-27666370

RESUMO

Microtubules are dynamic cytoskeletal elements coordinating and supporting a variety of neuronal processes, including cell division, migration, polarity, intracellular trafficking, and signal transduction. Mutations in genes encoding tubulins and microtubule-associated proteins are known to cause neurodevelopmental and neurodegenerative disorders. Growing evidence suggests that altered microtubule dynamics may also underlie or contribute to neurodevelopmental disorders and neurodegeneration. We report that biallelic mutations in TBCD, encoding one of the five co-chaperones required for assembly and disassembly of the αß-tubulin heterodimer, the structural unit of microtubules, cause a disease with neurodevelopmental and neurodegenerative features characterized by early-onset cortical atrophy, secondary hypomyelination, microcephaly, thin corpus callosum, developmental delay, intellectual disability, seizures, optic atrophy, and spastic quadriplegia. Molecular dynamics simulations predicted long-range and/or local structural perturbations associated with the disease-causing mutations. Biochemical analyses documented variably reduced levels of TBCD, indicating relative instability of mutant proteins, and defective ß-tubulin binding in a subset of the tested mutants. Reduced or defective TBCD function resulted in decreased soluble α/ß-tubulin levels and accelerated microtubule polymerization in fibroblasts from affected subjects, demonstrating an overall shift toward a more rapidly growing and stable microtubule population. These cells displayed an aberrant mitotic spindle with disorganized, tangle-shaped microtubules and reduced aster formation, which however did not alter appreciably the rate of cell proliferation. Our findings establish that defective TBCD function underlies a recognizable encephalopathy and drives accelerated microtubule polymerization and enhanced microtubule stability, underscoring an additional cause of altered microtubule dynamics with impact on neuronal function and survival in the developing brain.


Assuntos
Alelos , Encefalopatias/genética , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Mutação , Dobramento de Proteína , Tubulina (Proteína)/metabolismo , Adolescente , Idade de Início , Encéfalo/metabolismo , Encéfalo/patologia , Encefalopatias/patologia , Proliferação de Células , Pré-Escolar , Feminino , Fibroblastos , Humanos , Lactente , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/patologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica , Fuso Acromático/metabolismo , Fuso Acromático/patologia , Tubulina (Proteína)/química
10.
Cell Rep ; 13(3): 504-515, 2015 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-26456833

RESUMO

Somatic PTPN11 mutations cause juvenile myelomonocytic leukemia (JMML). Germline PTPN11 defects cause Noonan syndrome (NS), and specific inherited mutations cause NS/JMML. Here, we report that hematopoietic cells differentiated from human induced pluripotent stem cells (hiPSCs) harboring NS/JMML-causing PTPN11 mutations recapitulated JMML features. hiPSC-derived NS/JMML myeloid cells exhibited increased signaling through STAT5 and upregulation of miR-223 and miR-15a. Similarly, miR-223 and miR-15a were upregulated in 11/19 JMML bone marrow mononuclear cells harboring PTPN11 mutations, but not those without PTPN11 defects. Reducing miR-223's function in NS/JMML hiPSCs normalized myelogenesis. MicroRNA target gene expression levels were reduced in hiPSC-derived myeloid cells as well as in JMML cells with PTPN11 mutations. Thus, studying an inherited human cancer syndrome with hiPSCs illuminated early oncogenesis prior to the accumulation of secondary genomic alterations, enabling us to discover microRNA dysregulation, establishing a genotype-phenotype association for JMML and providing therapeutic targets.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Leucemia Mielomonocítica Juvenil/metabolismo , Células Mieloides/citologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucemia Mielomonocítica Juvenil/genética , Leucemia Mielomonocítica Juvenil/patologia , MicroRNAs/genética , Mutação , Células Mieloides/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Regulação para Cima
11.
J Biol Chem ; 290(48): 29022-34, 2015 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-26446793

RESUMO

JAK1 and JAK3 are recurrently mutated in acute lymphoblastic leukemia. These tyrosine kinases associate with heterodimeric cytokine receptors such as IL-7 receptor or IL-9 receptor, in which JAK1 is appended to the specific chain, and JAK3 is appended to the common gamma chain. Here, we studied the role of these receptor complexes in mediating the oncogenic activity of JAK3 mutants. Although JAK3(V674A) and the majority of other JAK3 mutants needed to bind to a functional cytokine receptor complex to constitutively activate STAT5, JAK3(L857P) was unexpectedly found to not depend on such receptor complexes for its activity, which was induced without receptor or JAK1 co-expression. Introducing a mutation in the FERM domain that abolished JAK-receptor interaction did not affect JAK3(L857P) activity, whereas it inhibited the other receptor-dependent mutants. The same cytokine receptor independence as for JAK3(L857P) was observed for homologous Leu(857) mutations of JAK1 and JAK2 and for JAK3(L875H). This different cytokine receptor requirement correlated with different functional properties in vivo and with distinct sensitivity to JAK inhibitors. Transduction of murine hematopoietic cells with JAK3(V674A) led homogenously to lymphoblastic leukemias in BALB/c mice. In contrast, transduction with JAK3(L857P) induced various types of lymphoid and myeloid leukemias. Moreover, ruxolitinib, which preferentially blocks JAK1 and JAK2, abolished the proliferation of cells transformed by the receptor-dependent JAK3(V674A), yet proved much less potent on cells expressing JAK3(L857P). These particular cells were, in contrast, more sensitive to JAK3-specific inhibitors. Altogether, our results showed that different JAK3 mutations induce constitutive activation through distinct mechanisms, pointing to specific therapeutic perspectives.


Assuntos
Janus Quinase 3 , Mutação de Sentido Incorreto , Inibidores de Proteínas Quinases/farmacologia , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Humanos , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo
12.
Nat Genet ; 47(6): 661-7, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25915598

RESUMO

Zimmermann-Laband syndrome (ZLS) is a developmental disorder characterized by facial dysmorphism with gingival enlargement, intellectual disability, hypoplasia or aplasia of nails and terminal phalanges, and hypertrichosis. We report that heterozygous missense mutations in KCNH1 account for a considerable proportion of ZLS. KCNH1 encodes the voltage-gated K(+) channel Eag1 (Kv10.1). Patch-clamp recordings showed strong negative shifts in voltage-dependent activation for all but one KCNH1 channel mutant (Gly469Arg). Coexpression of Gly469Arg with wild-type KCNH1 resulted in heterotetrameric channels with reduced conductance at positive potentials but pronounced conductance at negative potentials. These data support a gain-of-function effect for all ZLS-associated KCNH1 mutants. We also identified a recurrent de novo missense change in ATP6V1B2, encoding the B2 subunit of the multimeric vacuolar H(+) ATPase, in two individuals with ZLS. Structural analysis predicts a perturbing effect of the mutation on complex assembly. Our findings demonstrate that KCNH1 mutations cause ZLS and document genetic heterogeneity for this disorder.


Assuntos
Anormalidades Múltiplas/genética , Anormalidades Craniofaciais/genética , Canais de Potássio Éter-A-Go-Go/genética , Fibromatose Gengival/genética , Deformidades Congênitas da Mão/genética , ATPases Vacuolares Próton-Translocadoras/genética , Animais , Células CHO , Códon sem Sentido , Cricetinae , Cricetulus , Feminino , Estudos de Associação Genética , Humanos , Masculino , Potenciais da Membrana , Modelos Moleculares , Mutação de Sentido Incorreto , Linhagem , Conformação Proteica , Xenopus laevis
13.
Blood ; 124(26): 3924-31, 2014 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-25352124

RESUMO

The acquisition of growth signal self-sufficiency is 1 of the hallmarks of cancer. We previously reported that the murine interleukin-9-dependent TS1 cell line gives rise to growth factor-independent clones with constitutive activation of the Janus kinase (JAK)- signal transducer and activator of transcription (STAT) pathway. Here, we show that this transforming event results from activating mutations either in JAK1, JAK3, or in both kinases. Transient and stable expression of JAK1 and/or JAK3 mutants showed that each mutant induces STAT activation and that their coexpression further increases this activation. The proliferation of growth factor-independent TS1 clones can be efficiently blocked by JAK inhibitors such as ruxolitinib or CMP6 in short-term assays. However, resistant clones occur upon long-term culture in the presence of inhibitors. Surprisingly, resistance to CMP6 was not caused by the acquisition of secondary mutations in the adenosine triphosphate-binding pocket of the JAK mutant. Indeed, cells that originally showed a JAK1-activating mutation became resistant to inhibitors by acquiring another activating mutation in JAK3, whereas cells that originally showed a JAK3-activating mutation became resistant to inhibitors by acquiring another activating mutation in JAK1. These observations underline the cooperation between JAK1 and JAK3 mutants in T-cell transformation and represent a new mechanism of acquisition of resistance against JAK inhibitors.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Janus Quinase 1/genética , Janus Quinase 3/genética , Inibidores de Proteínas Quinases/química , Trifosfato de Adenosina/química , Animais , Linhagem Celular , Proliferação de Células , Transformação Celular Neoplásica , Células HEK293 , Humanos , Janus Quinases/antagonistas & inibidores , Camundongos , Mutação de Sentido Incorreto , Mutação Puntual , Estrutura Terciária de Proteína , Pirazóis/química , Transdução de Sinais
14.
Nat Genet ; 46(6): 635-639, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24777450

RESUMO

Dilated cardiomyopathy (DCM) is a highly heterogeneous trait with sarcomeric gene mutations predominating. The cause of a substantial percentage of DCMs remains unknown, and no gene-specific therapy is available. On the basis of resequencing of 513 DCM cases and 1,150 matched controls from various cohorts of distinct ancestry, we discovered rare, functional RAF1 mutations in 3 of the cohorts (South Indian, North Indian and Japanese). The prevalence of RAF1 mutations was ~9% in childhood-onset DCM cases in these three cohorts. Biochemical studies showed that DCM-associated RAF1 mutants had altered kinase activity, resulting in largely unaltered ERK activation but in AKT that was hyperactivated in a BRAF-dependent manner. Constitutive expression of these mutants in zebrafish embryos resulted in a heart failure phenotype with AKT hyperactivation that was rescued by treatment with rapamycin. These findings provide new mechanistic insights and potential therapeutic targets for RAF1-associated DCM and further expand the clinical spectrum of RAF1-related human disorders.


Assuntos
Cardiomiopatia Dilatada/genética , Mutação , Proteínas Proto-Oncogênicas c-raf/genética , Adulto , Idade de Início , Idoso , Sequência de Aminoácidos , Animais , Cardiomiopatia Dilatada/etnologia , Estudos de Casos e Controles , Estudos de Coortes , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Células HEK293 , Humanos , Índia , Japão , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fenótipo , Prevalência , Homologia de Sequência de Aminoácidos , Sirolimo/química , Peixe-Zebra
15.
Hum Mol Genet ; 23(16): 4315-27, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24705357

RESUMO

RASopathies, a family of disorders characterized by cardiac defects, defective growth, facial dysmorphism, variable cognitive deficits and predisposition to certain malignancies, are caused by constitutional dysregulation of RAS signalling predominantly through the RAF/MEK/ERK (MAPK) cascade. We report on two germline mutations (p.Gly39dup and p.Val55Met) in RRAS, a gene encoding a small monomeric GTPase controlling cell adhesion, spreading and migration, underlying a rare (2 subjects among 504 individuals analysed) and variable phenotype with features partially overlapping Noonan syndrome, the most common RASopathy. We also identified somatic RRAS mutations (p.Gly39dup and p.Gln87Leu) in 2 of 110 cases of non-syndromic juvenile myelomonocytic leukaemia, a childhood myeloproliferative/myelodysplastic disease caused by upregulated RAS signalling, defining an atypical form of this haematological disorder rapidly progressing to acute myeloid leukaemia. Two of the three identified mutations affected known oncogenic hotspots of RAS genes and conferred variably enhanced RRAS function and stimulus-dependent MAPK activation. Expression of an RRAS mutant homolog in Caenorhabditis elegans enhanced RAS signalling and engendered protruding vulva, a phenotype previously linked to the RASopathy-causing SHOC2(S2G) mutant. Overall, these findings provide evidence of a functional link between RRAS and MAPK signalling and reveal an unpredicted role of enhanced RRAS function in human disease.


Assuntos
Carcinogênese/genética , Mutação/fisiologia , Fenótipo , Proteínas ras/genética , Animais , Caenorhabditis elegans , Estudos de Coortes , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mielomonocítica Juvenil/genética , MAP Quinase Quinase Quinases/metabolismo , Síndrome de Noonan/genética , Proteína Oncogênica v-akt/metabolismo , Transdução de Sinais/genética , Proteínas ras/química , Proteínas ras/metabolismo
16.
J Med Genet ; 50(8): 493-9, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23687348

RESUMO

BACKGROUND: Kaufman oculocerebrofacial syndrome (KOS) is a developmental disorder characterised by reduced growth, microcephaly, ocular anomalies (microcornea, strabismus, myopia, and pale optic disk), distinctive facial features (narrow palpebral fissures, telecanthus, sparse and laterally broad eyebrows, preauricular tags, and micrognathia), mental retardation, and generalised hypotonia. KOS is a rare, possibly underestimated condition, with fewer than 10 cases reported to date. Here we investigate the molecular cause underlying KOS. METHODS: An exome sequencing approach was used on a single affected individual of an Italian consanguineous family coupled with mutation scanning using Sanger sequencing on a second unrelated subject with clinical features fitting the disorder. RESULTS: Exome sequencing was able to identify homozygosity for a novel truncating mutation (c.556C>T, p.Arg186stop) in UBE3B, which encodes a widely expressed HECT (homologous to the E6-AP carboxyl terminus) domain E3 ubiquitin-protein ligase. Homozygosity for a different nonsense lesion affecting the gene (c.1166G>A, p.Trp389stop) was documented in the second affected subject, supporting the recessive mode of inheritance of the disorder. Mutation scanning of the entire UBE3B coding sequence on a selected cohort of subjects with features overlapping, in part, those recurring in KOS did not reveal disease-causing mutations, suggesting phenotypic homogeneity of UBE3B lesions. DISCUSSION: Our data provide evidence that KOS is caused by UBE3B loss of function, and further demonstrate the impact of misregulation of protein ubiquitination on development and growth. The available clinical records, including those referring to four UBE3B mutation-positive subjects recently described as belonging to a previously unreported entity, which fits KOS, document the clinical homogeneity of this disorder.


Assuntos
Anormalidades do Olho/enzimologia , Anormalidades do Olho/genética , Deficiência Intelectual/enzimologia , Deficiência Intelectual/genética , Deformidades Congênitas dos Membros/enzimologia , Deformidades Congênitas dos Membros/genética , Microcefalia/enzimologia , Microcefalia/genética , Ubiquitina-Proteína Ligases/genética , Sequência de Bases , Criança , Exoma , Facies , Feminino , Homozigoto , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Linhagem
17.
Am J Med Genet A ; 161A(7): 1706-13, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23686761

RESUMO

The formation of ectopic calcifications in soft tissues can occur either sporadically or as a genetically determined condition, and is seen only infrequently. We report on a girl in whom widespread, rapidly progressive ectopic calcifications were detected shortly after birth. Calcifications became present around all joints, tendons and ligaments, but did not involve internal organs and skin, and eventually caused almost complete immobility of the child at 2 years. There were no other health problems and cognitive development was normal. We compare the manifestations in the child to the characteristics of known entities causing ectopic calcifications and conclude the child differs to each. Laboratory evaluation failed to identify autoimmune phenomena as well as calcium metabolism or other biochemical abnormalities; molecular studies did not identify occurrence of mutations in disease genes known to be involved in ectopic calcifications. We conclude the manifestations in the child represent an unreported entity of hitherto unknown etiology.


Assuntos
Calcinose/etiologia , Receptores de Ativinas Tipo I/genética , Calcinose/genética , Pré-Escolar , Cromograninas , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Lactente , Ligamentos/patologia , Imagem por Ressonância Magnética , Masculino , Mutação , Tendões/patologia
19.
J Biol Chem ; 287(32): 27066-77, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22711529

RESUMO

Activating mutations in PTPN11 cause Noonan syndrome, the most common nonchromosomal disorder affecting development and growth. PTPN11 encodes SHP2, an Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase that positively modulates RAS function. Here, we characterized functionally all possible amino acid substitutions arising from single-base changes affecting codons 62 and 63 to explore the molecular mechanisms lying behind the largely invariant occurrence of the Y62D and Y63C substitutions recurring in Noonan syndrome. We provide structural and biochemical data indicating that the autoinhibitory interaction between the N-SH2 and protein-tyrosine phosphatase (PTP) domains is perturbed in both mutants as a result of an extensive structural rearrangement of the N-SH2 domain. Most mutations affecting Tyr(63) exerted an unpredicted disrupting effect on the structure of the N-SH2 phosphopeptide-binding cleft mediating the interaction of SHP2 with signaling partners. Among all the amino acid changes affecting that codon, the disease-causing mutation was the only substitution that perturbed the stability of the inactive conformation of SHP2 without severely impairing proper phosphopeptide binding of N-SH2. On the other hand, the disruptive effect of the Y62D change on the autoinhibited conformation of the protein was balanced, in part, by less efficient binding properties of the mutant. Overall, our data demonstrate that the selection-by-function mechanism acting as driving force for PTPN11 mutations affecting codons 62 and 63 implies balancing of counteracting effects operating on the allosteric control of the function of SHP2.


Assuntos
Síndrome de Noonan/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética
20.
Hum Mutat ; 33(4): 703-9, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22253195

RESUMO

Noonan syndrome (NS) is among the most common nonchromosomal disorders affecting development and growth. NS is genetically heterogeneous, being caused by germline mutations affecting various genes implicated in the RAS signaling network. This network transduces extracellular signals into intracellular biochemical and transcriptional responses controlling cell proliferation, differentiation, metabolism, and senescence. To explore the transcriptional consequences of NS-causing mutations, we performed global mRNA expression profiling on peripheral blood mononuclear cells obtained from 23 NS patients carrying heterozygous mutations in PTPN11 or SOS1. Gene expression profiling was also resolved in five subjects with Noonan-like syndrome with loose anagen hair (NS/LAH), a condition clinically related to NS and caused by an invariant mutation in SHOC2. Robust transcriptional signatures were found to specifically discriminate each of the three mutation groups from 21 age- and sex-matched controls. Despite the only partial overlap in terms of gene composition, the three signatures showed a notable concordance in terms of biological processes and regulatory circuits affected. These data establish expression profiling of peripheral blood mononuclear cells as a powerful tool to appreciate differential perturbations driven by germline mutations of transducers involved in RAS signaling and to dissect molecular mechanisms underlying NS and other RASopathies.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Síndrome de Noonan/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína SOS1/genética , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica , Granuloma de Células Gigantes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucócitos Mononucleares/fisiologia , Masculino , Mutação , Síndrome de Noonan/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína SOS1/metabolismo , Transdução de Sinais , Transcrição Genética , Proteínas ras/metabolismo
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