Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 23
Filtrar
Mais filtros










Base de dados
Tipo de estudo
Intervalo de ano de publicação
1.
Am J Hum Genet ; 105(4): 836-843, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31564437

RESUMO

Osteogenesis imperfecta (OI) comprises a genetically heterogeneous group of skeletal fragility diseases. Here, we report on five independent families with a progressively deforming type of OI, in whom we identified four homozygous truncation or frameshift mutations in MESD. Affected individuals had recurrent fractures and at least one had oligodontia. MESD encodes an endoplasmic reticulum (ER) chaperone protein for the canonical Wingless-related integration site (WNT) signaling receptors LRP5 and LRP6. Because complete absence of MESD causes embryonic lethality in mice, we hypothesized that the OI-associated mutations are hypomorphic alleles since these mutations occur downstream of the chaperone activity domain but upstream of ER-retention domain. This would be consistent with the clinical phenotypes of skeletal fragility and oligodontia in persons deficient for LRP5 and LRP6, respectively. When we expressed wild-type (WT) and mutant MESD in HEK293T cells, we detected WT MESD in cell lysate but not in conditioned medium, whereas the converse was true for mutant MESD. We observed that both WT and mutant MESD retained the ability to chaperone LRP5. Thus, OI-associated MESD mutations produce hypomorphic alleles whose failure to remain within the ER significantly reduces but does not completely eliminate LRP5 and LRP6 trafficking. Since these individuals have no eye abnormalities (which occur in individuals completely lacking LRP5) and have neither limb nor brain patterning defects (both of which occur in mice completely lacking LRP6), we infer that bone mass accrual and dental patterning are more sensitive to reduced canonical WNT signaling than are other developmental processes. Biologic agents that can increase LRP5 and LRP6-mediated WNT signaling could benefit individuals with MESD-associated OI.

2.
Cancer Res ; 79(19): 4855-4868, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31405847

RESUMO

The advent of molecularly targeted therapeutic agents has opened a new era in cancer therapy. However, many tumors rely on nondruggable cancer-driving lesions. In addition, long-lasting clinical benefits from single-agent therapies rarely occur, as most of the tumors acquire resistance over time. The identification of targeted combination regimens interfering with signaling through oncogenically rewired pathways provides a promising approach to enhance efficacy of single-agent-targeted treatments. Moreover, combination drug therapies might overcome the emergence of drug resistance. Here, we performed a focused flow cytometry-based drug synergy screen and identified a novel synergistic interaction between GLUT1-mediated glucose transport and the cell-cycle checkpoint kinases ATR and CHK1. Combined inhibition of CHK1/GLUT1 or ATR/GLUT1 robustly induced apoptosis, particularly in RAS-mutant cancer cells. Mechanistically, combined inhibition of ATR/CHK1 and GLUT1 arrested sensitive cells in S-phase and led to the accumulation of genotoxic damage, particularly in S-phase. In vivo, simultaneous inhibition of ATR and GLUT1 significantly reduced tumor volume gain in an autochthonous mouse model of KrasG12D -driven soft tissue sarcoma. Taken together, these findings pave the way for combined inhibition of GLUT1 and ATR/CHK1 as a therapeutic approach for KRAS-driven cancers. SIGNIFICANCE: Dual targeting of the DNA damage response and glucose transport synergistically induces apoptosis in KRAS-mutant cancer, suggesting this combination treatment for clinical validation in KRAS-stratified tumor patients.

3.
Cell ; 176(3): 505-519.e22, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30612738

RESUMO

Genomic instability can be a hallmark of both human genetic disease and cancer. We identify a deleterious UBQLN4 mutation in families with an autosomal recessive syndrome reminiscent of genome instability disorders. UBQLN4 deficiency leads to increased sensitivity to genotoxic stress and delayed DNA double-strand break (DSB) repair. The proteasomal shuttle factor UBQLN4 is phosphorylated by ATM and interacts with ubiquitylated MRE11 to mediate early steps of homologous recombination-mediated DSB repair (HRR). Loss of UBQLN4 leads to chromatin retention of MRE11, promoting non-physiological HRR activity in vitro and in vivo. Conversely, UBQLN4 overexpression represses HRR and favors non-homologous end joining. Moreover, we find UBQLN4 overexpressed in aggressive tumors. In line with an HRR defect in these tumors, UBQLN4 overexpression is associated with PARP1 inhibitor sensitivity. UBQLN4 therefore curtails HRR activity through removal of MRE11 from damaged chromatin and thus offers a therapeutic window for PARP1 inhibitor treatment in UBQLN4-overexpressing tumors.

4.
J Med Genet ; 55(12): 837-846, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30323018

RESUMO

BACKGROUND: Wiedemann-Rautenstrauch syndrome (WRS) is a form of segmental progeria presenting neonatally, characterised by growth retardation, sparse scalp hair, generalised lipodystrophy with characteristic local fatty tissue accumulations and unusual face. We aimed to understand its molecular cause. METHODS: We performed exome sequencing in two families, targeted sequencing in 10 other families and performed in silico modelling studies and transcript processing analyses to explore the structural and functional consequences of the identified variants. RESULTS: Biallelic POLR3A variants were identified in eight affected individuals and monoallelic variants of the same gene in four other individuals. In the latter, lack of genetic material precluded further analyses. Multiple variants were found to affect POLR3A transcript processing and were mostly located in deep intronic regions, making clinical suspicion fundamental to detection. While biallelic POLR3A variants have been previously reported in 4H syndrome and adolescent-onset progressive spastic ataxia, recurrent haplotypes specifically occurring in individuals with WRS were detected. All WRS-associated POLR3A amino acid changes were predicted to perturb substantially POLR3A structure/function. CONCLUSION: Biallelic mutations in POLR3A, which encodes for the largest subunit of the DNA-dependent RNA polymerase III, underlie WRS. No isolated functional sites in POLR3A explain the phenotype variability in POLR3A-related disorders. We suggest that specific combinations of compound heterozygous variants must be present to cause the WRS phenotype. Our findings expand the molecular mechanisms contributing to progeroid disorders.

5.
Cell Rep ; 25(4): 1027-1039.e6, 2018 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-30355482

RESUMO

Cdkn1a, which encodes p21, functions as a major route for p53-mediated cell-cycle arrest. However, the consequence of Cdkn1a gene dosage on tumor suppression has not been systematically investigated. Here, we employed BAC transgenesis to generate a Cdkn1aSUPER mouse, which harbors an additional Cdkn1a allele within its natural genomic context. We show that these mice display enhanced cell-cycle arrest and reduced apoptosis in response to genotoxic stress. Furthermore, using a chemically induced skin cancer model and an autochthonous Kras-driven lung adenocarcinoma model, we show that Cdkn1aSUPER mice display a cancer protection phenotype that is indistinguishable from that observed in Tp53SUPER animals. Moreover, we demonstrate that Tp53 and Cdkn1a cooperate in mediating cancer resistance, using a chemically induced fibrosarcoma model. Overall, our Cdkn1aSUPER allele enabled us to assess the contribution of Cdkn1a to Tp53-mediated tumor suppression.

6.
Sci Rep ; 7(1): 15511, 2017 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-29138515

RESUMO

Small cell lung cancer (SCLC) is a difficult to treat subtype of lung cancer. One of the hallmarks of SCLC is its almost uniform chemotherapy sensitivity. However, chemotherapy response is typically transient and patients frequently succumb to SCLC within a year following diagnosis. We performed a transcriptome analysis of the major human lung cancer entities. We show a significant overexpression of genes involved in the DNA damage response, specifically in SCLC. Particularly CHEK1, which encodes for the cell cycle checkpoint kinase CHK1, is significantly overexpressed in SCLC, compared to lung adenocarcinoma. In line with uncontrolled cell cycle progression in SCLC, we find that CDC25A, B and C mRNAs are expressed at significantly higher levels in SCLC, compared to lung adenocarcinoma. We next profiled the efficacy of compounds targeting CHK1 and ATR. Both, ATR- and CHK1 inhibitors induce genotoxic damage and apoptosis in human and murine SCLC cell lines, but not in lung adenocarcinoma cells. We further demonstrate that murine SCLC tumors were highly sensitive to ATR- and CHK1 inhibitors, while Kras G12D -driven murine lung adenocarcinomas were resistant against these compounds and displayed continued growth under therapy. Altogether, our data indicate that SCLC displays an actionable dependence on ATR/CHK1-mediated cell cycle checkpoints.

7.
Nat Commun ; 8(1): 153, 2017 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-28751718

RESUMO

Chronic lymphocytic leukemia (CLL) remains an incurable disease. Two recurrent cytogenetic aberrations, namely del(17p), affecting TP53, and del(11q), affecting ATM, are associated with resistance against genotoxic chemotherapy (del17p) and poor outcome (del11q and del17p). Both del(17p) and del(11q) are also associated with inferior outcome to the novel targeted agents, such as the BTK inhibitor ibrutinib. Thus, even in the era of targeted therapies, CLL with alterations in the ATM/p53 pathway remains a clinical challenge. Here we generated two mouse models of Atm- and Trp53-deficient CLL. These animals display a significantly earlier disease onset and reduced overall survival, compared to controls. We employed these models in conjunction with transcriptome analyses following cyclophosphamide treatment to reveal that Atm deficiency is associated with an exquisite and genotype-specific sensitivity against PARP inhibition. Thus, we generate two aggressive CLL models and provide a preclinical rational for the use of PARP inhibitors in ATM-affected human CLL.ATM and TP53 mutations are associated with poor prognosis in chronic lymphocytic leukaemia (CLL). Here the authors generate mouse models of Tp53- and Atm-defective CLL mimicking the high-risk form of human disease and show that Atm-deficient CLL is sensitive to PARP1 inhibition.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Modelos Animais de Doenças , Leucemia Linfocítica Crônica de Células B/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Deleção Cromossômica , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 17/genética , Ciclofosfamida/farmacologia , Perfilação da Expressão Gênica/métodos , Humanos , Immunoblotting , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Análise de Sobrevida , Proteína Supressora de Tumor p53/genética
8.
Hum Mol Genet ; 26(15): 2923-2932, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28472301

RESUMO

CACNA1D encodes the pore-forming α1-subunit of Cav1.3, an L-type voltage-gated Ca2+-channel. Despite the recent discovery of two de novo missense gain-of-function mutations in Cav1.3 in two individuals with autism spectrum disorder (ASD) and intellectual disability CACNA1D has not been considered a prominent ASD-risk gene in large scale genetic analyses, since such studies primarily focus on likely-disruptive genetic variants. Here we report the discovery and characterization of a third de novo missense mutation in CACNA1D (V401L) in a patient with ASD and epilepsy. For the functional characterization we introduced mutation V401L into two major C-terminal long and short Cav1.3 splice variants, expressed wild-type or mutant channel complexes in tsA-201 cells and performed whole-cell patch-clamp recordings. Mutation V401L, localized within the channel's activation gate, significantly enhanced current densities, shifted voltage dependence of activation and inactivation to more negative voltages and reduced channel inactivation in both Cav1.3 splice variants. Altogether, these gating changes are expected to result in enhanced Ca2+-influx through the channel, thus representing a strong gain-of-function phenotype. Additionally, we also found that mutant channels retained full sensitivity towards the clinically available Ca2+ -channel blocker isradipine. Our findings strengthen the evidence for CACNA1D as a novel candidate autism risk gene and encourage experimental therapy with available channel-blockers for this mutation. The additional presence of seizures and neurological abnormalities in our patient define a novel phenotype partially overlapping with symptoms in two individuals with PASNA (congenital primary aldosteronism, seizures and neurological abnormalities) caused by similar Cav1.3 gain-of-function mutations.


Assuntos
Transtorno do Espectro Autista/genética , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Criança , Epilepsia/genética , Epilepsia/metabolismo , Mutação com Ganho de Função/genética , Humanos , Ativação do Canal Iônico/fisiologia , Masculino , Mutação , Mutação de Sentido Incorreto , Técnicas de Patch-Clamp , Convulsões/genética
9.
Hum Genet ; 136(7): 821-834, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28393272

RESUMO

Pathogenic variants in genes encoding subunits of the spliceosome are the cause of several human diseases, such as neurodegenerative diseases. The RNA splicing process is facilitated by the spliceosome, a large RNA-protein complex consisting of small nuclear ribonucleoproteins (snRNPs), and many other proteins, such as heterogeneous nuclear ribonucleoproteins (hnRNPs). The HNRNPU gene (OMIM *602869) encodes the heterogeneous nuclear ribonucleoprotein U, which plays a crucial role in mammalian development. HNRNPU is expressed in the fetal brain and adult heart, kidney, liver, brain, and cerebellum. Microdeletions in the 1q44 region encompassing HNRNPU have been described in patients with intellectual disability (ID) and other clinical features, such as seizures, corpus callosum abnormalities (CCA), and microcephaly. Recently, pathogenic HNRNPU variants were identified in large ID and epileptic encephalopathy cohorts. In this study, we provide detailed clinical information of five novels and review two of the previously published individuals with (likely) pathogenic de novo variants in the HNRNPU gene including three non-sense and two missense variants, one small intragenic deletion, and one duplication. The phenotype in individuals with variants in HNRNPU is characterized by early onset seizures (6/7), severe ID (6/6), severe speech impairment (6/6), hypotonia (6/7), and central nervous system (CNS) (5/6), cardiac (4/6), and renal abnormalities (3/4). In this study, we broaden the clinical and mutational HNRNPU-associated spectrum, and demonstrate that heterozygous HNRNPU variants cause epilepsy, severe ID with striking speech impairment and variable CNS, cardiac, and renal anomalies.


Assuntos
Epilepsia/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/genética , Heterozigoto , Deficiência Intelectual/genética , Idade de Início , Agenesia do Corpo Caloso/genética , Sistema Nervoso Central/anormalidades , Sistema Nervoso Central/patologia , Deleção Cromossômica , Cromossomos Humanos Par 1 , Epilepsia/diagnóstico , Feminino , Variação Genética , Humanos , Lactente , Deficiência Intelectual/diagnóstico , Rim/anormalidades , Masculino , Microcefalia/diagnóstico , Microcefalia/genética , Hipotonia Muscular/diagnóstico , Hipotonia Muscular/genética , Fenótipo , Processamento de RNA , Ribonucleoproteínas Nucleares Pequenas/genética , Convulsões/diagnóstico , Convulsões/genética
10.
Am J Med Genet A ; 173(1): 264-267, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27753196

RESUMO

Heterozygous germline mutations in MTOR have been shown to underlie Smith-Kingsmore syndrome, a rare autosomal dominant syndrome characterized by macrocephaly, developmental delay, and dysmorphic facial features. Recently, two unrelated families with the MTOR mutation, c.5395G>A p.(Glu1799Lys), were reported. Here, we describe siblings from a non-consanguineous German family in whom we identified the same heterozygous missense mutation in MTOR. Remarkably, in all reported families with Smith-Kingsmore syndrome and the MTOR c.5395G>A mutation, including the family described herein, healthy parents of recurrently affected children do not have detectable levels of the mutation in tested tissues, lending credence to gonadal mosaicism as the underlying mechanism. Furthermore, the glutamic acid at position 1799 was shown to present a recurrent somatic mutation site in several cancers, including colon cancer, pointing to a somatic mutational hotspot in MTOR. Importantly, we highlight the occurrence of multiple intestinal polyps in the older sibling. Further patients are required to establish definitively whether polyp formation forms part of the SKS clinical spectrum. © 2016 Wiley Periodicals, Inc.


Assuntos
Alelos , Estudos de Associação Genética , Mosaicismo , Mutação , Fenótipo , Serina-Treonina Quinases TOR/genética , Substituição de Aminoácidos , Pré-Escolar , Códon , Facies , Feminino , Genótipo , Mutação em Linhagem Germinativa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Linhagem , Análise de Sequência de DNA , Irmãos , Síndrome
11.
Am J Med Genet A ; 170(12): 3282-3288, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27530281

RESUMO

Kabuki syndrome (KS) is a rare developmental disorder characterized by multiple congenital malformations, postnatal growth retardation, intellectual disability, and recognizable facial features. It is mainly caused by mutations in either KMT2D or KDM6A. We describe a 14-year-old boy with KS presenting with an unusual combination of bilateral microphthalmia with orbital cystic venous lymphatic malformation and neonatal cholestasis with bile duct paucity, in addition to the typical clinical features of KS. We identified the novel KMT2D mutation c.10588delC, p.(Glu3530Serfs*128) by Mendeliome (Illumina TruSight One®) sequencing, a next generation sequencing panel targeting 4,813 genes linked to human genetic disease. We analyzed the Mendeliome data for additional mutations which might explain the exceptional clinical presentation of our patient but did not find any, leading us to suspect that the above named symptoms might be part of the KMT2D-associated spectrum of anomalies. We thus extend the range of KS-associated malformations and propose a hypothetical connection between KMT2D and Notch signaling. © 2016 Wiley Periodicals, Inc.


Assuntos
Anormalidades Múltiplas/diagnóstico , Ductos Biliares/anormalidades , Colestase/diagnóstico , Face/anormalidades , Doenças Hematológicas/diagnóstico , Microftalmia/diagnóstico , Fenótipo , Doenças Vestibulares/diagnóstico , Adolescente , Consanguinidade , Exoma , Facies , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imagem por Ressonância Magnética/métodos , Masculino , Exame Físico , Locos de Características Quantitativas
12.
Elife ; 52016 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-27240166

RESUMO

The molecular pathways underlying tumor suppression are incompletely understood. Here, we identify cooperative non-cell-autonomous functions of a single gene that together provide a novel mechanism of tumor suppression in basal keratinocytes of zebrafish embryos. A loss-of-function mutation in atp1b1a, encoding the beta subunit of a Na,K-ATPase pump, causes edema and epidermal malignancy. Strikingly, basal cell carcinogenesis only occurs when Atp1b1a function is compromised in both the overlying periderm (resulting in compromised epithelial polarity and adhesiveness) and in kidney and heart (resulting in hypotonic stress). Blockade of the ensuing PI3K-AKT-mTORC1-NFκB-MMP9 pathway activation in basal cells, as well as systemic isotonicity, prevents malignant transformation. Our results identify hypotonic stress as a (previously unrecognized) contributor to tumor development and establish a novel paradigm of tumor suppression.


Assuntos
Carcinoma Basocelular/fisiopatologia , Queratinócitos/enzimologia , Queratinócitos/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Pressão Osmótica , ATPase Trocadora de Sódio-Potássio/deficiência , Peixe-Zebra
13.
Int J Dermatol ; 55(8): 898-902, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27062382

RESUMO

Papillon-Lefèvre syndrome is an autosomal recessive genodermatosis typically manifesting with the constellation of palmoplantar keratoderma and progressive early-onset periodontitis. The cutaneous phenotype can be strikingly psoriasiform, possibly posing a diagnostic challenge. This rare disorder is caused by loss-of-function mutations in the CTSC gene, which encodes cathepsin C. We report six patients with Papillon-Lefèvre syndrome from five consanguineous Turkish families, in whom genetic analysis of the CTSC gene revealed four recurrent mutations (c.415G>A; c.1015C>T; c.1019A>G; and c.103-105delCTG) and a novel missense mutation (c.117G>T) in the homozygous state.


Assuntos
Catepsina C/genética , Triagem de Portadores Genéticos , Mutação de Sentido Incorreto , Doença de Papillon-Lefevre/genética , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Doença de Papillon-Lefevre/diagnóstico , Linhagem , Doenças Raras , Amostragem , Índice de Gravidade de Doença , Turquia , Adulto Jovem
14.
Am J Med Genet A ; 170(3): 728-33, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26640080

RESUMO

Using whole-exome sequencing, we identified a homozygous acceptor splice-site mutation in intron 6 of the KATNB1 gene in a patient from a consanguineous Turkish family who presented with congenital microcephaly, lissencephaly, short stature, polysyndactyly, and dental abnormalities. cDNA analysis revealed complete loss of the natural acceptor splice-site resulting either in the usage of an alternative, exonic acceptor splice-site inducing a frame-shift and premature protein truncation or, to a minor extent, in complete skipping of exon 7. Both effects most likely lead to complete loss of KATNB1 function. Homozygous and compound heterozygous mutations in KATNB1 have very recently been described as a cause of microcephaly with brain malformations and seizures. We extend the KATNB1 associated phenotype by describing a syndrome characterized by primordial dwarfism, lissencephaly, polysyndactyly, and dental anomalies, which is caused by a homozygous truncating KATNB1 mutation.


Assuntos
Nanismo/genética , Homozigoto , Lisencefalia/genética , Microcefalia/genética , Mutação , Sindactilia/genética , Pré-Escolar , Análise Mutacional de DNA , Nanismo/diagnóstico , Exoma , Feminino , Ordem dos Genes , Loci Gênicos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lisencefalia/diagnóstico , Imagem por Ressonância Magnética , Microcefalia/diagnóstico , Linhagem , Fenótipo , Sindactilia/diagnóstico , Síndrome
15.
J Clin Invest ; 125(9): 3585-99, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26280580

RESUMO

The genetic disorder Kabuki syndrome (KS) is characterized by developmental delay and congenital anomalies. Dominant mutations in the chromatin regulators lysine (K)-specific methyltransferase 2D (KMT2D) (also known as MLL2) and lysine (K)-specific demethylase 6A (KDM6A) underlie the majority of cases. Although the functions of these chromatin-modifying proteins have been studied extensively, the physiological systems regulated by them are largely unknown. Using whole-exome sequencing, we identified a mutation in RAP1A that was converted to homozygosity as the result of uniparental isodisomy (UPD) in a patient with KS and a de novo, dominant mutation in RAP1B in a second individual with a KS-like phenotype. We elucidated a genetic and functional interaction between the respective KS-associated genes and their products in zebrafish models and patient cell lines. Specifically, we determined that dysfunction of known KS genes and the genes identified in this study results in aberrant MEK/ERK signaling as well as disruption of F-actin polymerization and cell intercalation. Moreover, these phenotypes could be rescued in zebrafish models by rebalancing MEK/ERK signaling via administration of small molecule inhibitors of MEK. Taken together, our studies suggest that the KS pathophysiology overlaps with the RASopathies and provide a potential direction for treatment design.


Assuntos
Anormalidades Múltiplas/genética , Exoma , Face/anormalidades , Doenças Hematológicas/genética , Sistema de Sinalização das MAP Quinases/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Proteínas de Ligação a Telômeros/genética , Doenças Vestibulares/genética , Proteínas de Peixe-Zebra/genética , Anormalidades Múltiplas/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Bovinos , Criança , Pré-Escolar , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Doenças Hematológicas/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Masculino , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ratos , Proteínas de Ligação a Telômeros/metabolismo , Doenças Vestibulares/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
16.
Hum Mol Genet ; 24(13): 3708-17, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25839420

RESUMO

DNA double-strand breaks (DSBs) are highly toxic lesions, which, if not properly repaired, can give rise to genomic instability. Non-homologous end-joining (NHEJ), a well-orchestrated, multistep process involving numerous proteins essential for cell viability, represents one major pathway to repair DSBs in mammalian cells, and mutations in different NHEJ components have been described in microcephalic syndromes associated, e.g. with short stature, facial dysmorphism and immune dysfunction. By using whole-exome sequencing, we now identified in three affected brothers of a consanguineous Turkish family a homozygous mutation, c.482G>A, in the XRCC4 gene encoding a crucial component of the NHEJ pathway. Moreover, we found one additional patient of Swiss origin carrying the compound heterozygous mutations c.25delG (p.His9Thrfs*8) and c.823C>T (p.Arg275*) in XRCC4. The clinical phenotype presented in these patients was characterized by severe microcephaly, facial dysmorphism and short stature, but they did not show a recognizable immunological phenotype. We showed that the XRCC4 c.482G>A mutation, which affects the last nucleotide of exon 4, induces defective splicing of XRCC4 pre-mRNA mainly resulting in premature protein truncation and most likely loss of XRCC4 function. Moreover, we observed on cellular level that XRCC4 deficiency leads to hypersensitivity to DSB-inducing agents and defective DSB repair, which results in increased cell death after exposure to genotoxic agents. Taken together, our data provide evidence that autosomal recessive mutations in XRCC4 induce increased genomic instability and cause a NHEJ-related syndrome defined by facial dysmorphism, primary microcephaly and short stature.


Assuntos
Estatura , Proteínas de Ligação a DNA/genética , Instabilidade Genômica , Microcefalia/genética , Mutação Puntual , Adolescente , Criança , Feminino , Humanos , Lactente , Masculino , Microcefalia/fisiopatologia , Fenótipo , Turquia
17.
Am J Hum Genet ; 96(3): 432-9, 2015 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-25683121

RESUMO

As a result of a whole-exome sequencing study, we report three mutant alleles in SEC24D, a gene encoding a component of the COPII complex involved in protein export from the ER: the truncating mutation c.613C>T (p.Gln205(∗)) and the missense mutations c.3044C>T (p.Ser1015Phe, located in a cargo-binding pocket) and c.2933A>C (p.Gln978Pro, located in the gelsolin-like domain). Three individuals from two families affected by a similar skeletal phenotype were each compound heterozygous for two of these mutant alleles, with c.3044C>T being embedded in a 14 Mb founder haplotype shared by all three. The affected individuals were a 7-year-old boy with a phenotype most closely resembling Cole-Carpenter syndrome and two fetuses initially suspected to have a severe type of osteogenesis imperfecta. All three displayed a severely disturbed ossification of the skull and multiple fractures with prenatal onset. The 7-year-old boy had short stature and craniofacial malformations including macrocephaly, midface hypoplasia, micrognathia, frontal bossing, and down-slanting palpebral fissures. Electron and immunofluorescence microscopy of skin fibroblasts of this individual revealed that ER export of procollagen was inefficient and that ER tubules were dilated, faithfully reproducing the cellular phenotype of individuals with cranio-lentico-sutural dysplasia (CLSD). CLSD is caused by SEC23A mutations and displays a largely overlapping craniofacial phenotype, but it is not characterized by generalized bone fragility and presented with cataracts in the original family described. The cellular and morphological phenotypes we report are in concordance with the phenotypes described for the Sec24d-deficient fish mutants vbi (medaka) and bulldog (zebrafish).


Assuntos
Craniossinostoses/genética , Anormalidades do Olho/genética , Hidrocefalia/genética , Osteogênese Imperfeita/genética , Proteínas de Transporte Vesicular/genética , Alelos , Animais , Osso e Ossos/patologia , Criança , Retículo Endoplasmático/metabolismo , Feminino , Heterozigoto , Humanos , Masculino , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Conformação Proteica , Análise de Sequência de DNA , Proteínas de Transporte Vesicular/metabolismo , Peixe-Zebra/genética
18.
Hum Mol Genet ; 24(8): 2267-73, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25561690

RESUMO

Colobomatous macrophthalmia with microcornea syndrome (MACOM, Online Mendelian Inheritance in Man (OMIM) 602499) is an autosomal dominantly inherited malformation of the eye, which is characterized by microcornea with increased axial length, coloboma of the iris and of the optic disc, and severe myopia. We performed whole-exome sequencing (WES) in two affected individuals from the 2p23-p16-linked MACOM family, which includes 13 affected individuals in 3 generations. As no shared novel variation was found on the linked haplotype, we performed copy number variation (CNV) analysis by comparing the coverage of all exons in the WES data sets of the 2 patients with the coverage of 26 control exomes. We identified a heterozygous deletion predicted to span 22 kb including exons 14-17 of CRIM1 (cysteine-rich transmembrane bone morphogenetic protein (BMP) regulator 1). Quantitative PCR (qPCR) analysis confirmed the deletion, which was present in 11 affected individuals. Split-read analysis of WES data followed by breakpoint PCR and Sanger sequencing determined both breakpoints flanked by a 4-bp microhomology (CTTG). In the mouse, Crim1 is a growth-factor-binding protein with pleiotropic roles in the development of multiple organs, including the eye. To investigate the role of Crim1 during eye development in mice, we crossed a Crim1(flox) mouse line with the Ap2α-cre mouse line, which expresses Cre in the head surface ectoderm. Strikingly, we observed alterations of eye development in homozygous mice leading to severe anatomical and morphological changes overlapping with the anomalies observed in MACOM patients. Taken together, these findings identify CRIM1 as the causative gene for MACOM syndrome and emphasize the importance of CRIM1 in eye development.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Doenças da Córnea/genética , Anormalidades do Olho/genética , Olho/crescimento & desenvolvimento , Haploinsuficiência , Proteínas de Membrana/metabolismo , Adulto , Animais , Sequência de Bases , Receptores de Proteínas Morfogenéticas Ósseas/genética , Doenças da Córnea/metabolismo , Doenças da Córnea/fisiopatologia , Variações do Número de Cópias de DNA , Éxons , Olho/anatomia & histologia , Olho/metabolismo , Anormalidades do Olho/metabolismo , Anormalidades do Olho/fisiopatologia , Feminino , Homozigoto , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Linhagem , Adulto Jovem
19.
Am J Hum Genet ; 95(5): 622-32, 2014 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-25439729

RESUMO

Filippi syndrome is a rare, presumably autosomal-recessive disorder characterized by microcephaly, pre- and postnatal growth failure, syndactyly, and distinctive facial features, including a broad nasal bridge and underdeveloped alae nasi. Some affected individuals have intellectual disability, seizures, undescended testicles in males, and teeth and hair abnormalities. We performed homozygosity mapping and whole-exome sequencing in a Sardinian family with two affected children and identified a homozygous frameshift mutation, c.571dupA (p.Ile191Asnfs(∗)6), in CKAP2L, encoding the protein cytoskeleton-associated protein 2-like (CKAP2L). The function of this protein was unknown until it was rediscovered in mice as Radmis (radial fiber and mitotic spindle) and shown to play a pivotal role in cell division of neural progenitors. Sanger sequencing of CKAP2L in a further eight unrelated individuals with clinical features consistent with Filippi syndrome revealed biallelic mutations in four subjects. In contrast to wild-type lymphoblastoid cell lines (LCLs), dividing LCLs established from the individuals homozygous for the c.571dupA mutation did not show CKAP2L at the spindle poles. Furthermore, in cells from the affected individuals, we observed an increase in the number of disorganized spindle microtubules owing to multipolar configurations and defects in chromosome segregation. The observed cellular phenotypes are in keeping with data from in vitro and in vivo knockdown studies performed in human cells and mice, respectively. Our findings show that loss-of-function mutations in CKAP2L are a major cause of Filippi syndrome.


Assuntos
Proteínas do Citoesqueleto/genética , Transtornos do Crescimento/genética , Deficiência Intelectual/genética , Microcefalia/genética , Sindactilia/genética , Animais , Sequência de Bases , Análise Citogenética , Facies , Mutação da Fase de Leitura/genética , Componentes do Gene , Genes Recessivos/genética , Transtornos do Crescimento/patologia , Humanos , Deficiência Intelectual/patologia , Itália , Masculino , Camundongos , Microcefalia/patologia , Microscopia Confocal , Dados de Sequência Molecular , Análise de Sequência de DNA , Sindactilia/patologia
20.
Hum Genet ; 133(1): 29-39, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23982343

RESUMO

Proteoglycan (PG) synthesis begins with the sequential addition of a "linker chain", made up of four sugar residues, to a specific region of a core protein. Defects in the enzymes catalyzing steps two to four of the linker chain synthesis have been shown to cause autosomal recessive human phenotypes while no mutation has yet been reported in humans for the xylosyltransferases 1 and 2 (XT1 and XT2), the initiating enzymes in the linker chain formation. Here, we present a consanguineous Turkish family with two affected individuals presenting with short stature, distinct facial features, alterations of fat distribution, and moderate intellectual disability. X-rays showed only mild skeletal changes in the form of a short femoral neck, stocky and plump long bones and thickened ribs. Using a combination of whole-exome sequencing (WES), determination of homozygous stretches by WES variants, and classical linkage analysis, we identified the homozygous missense mutation c.C1441T in XYLT1, encoding XT1, within a large homozygous stretch on chromosome 16p13.12-p12.1. The mutation co-segregated with the phenotype in the family, is not found in over 13,000 alleles in the exome variant server and is predicted to change a highly conserved arginine at position 481 (p.R481W) located in the putative catalytical domain. Immunostaining of primary patient fibroblasts showed a loss of predominance of Golgi localization in mutant cells. Moreover, western blot analysis of decorin in cell culture supernatant demonstrated glycosylation differences between patient and control cells. Our data provide evidence that functional alterations of XT1 cause an autosomal recessive short stature syndrome associated with intellectual disability.


Assuntos
Nanismo/genética , Deficiência Intelectual/genética , Pentosiltransferases/genética , Consanguinidade , Exoma , Feminino , Genes Recessivos , Ligação Genética , Homozigoto , Humanos , Masculino , Mutação de Sentido Incorreto , Linhagem , Pentosiltransferases/metabolismo , Fenótipo , Proteoglicanas/metabolismo , Análise de Sequência de DNA , Síndrome , Turquia , Raios X
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA