RESUMO
Chromosome 15q11-q13.1 duplication is a common copy number variant associated with autism spectrum disorder (ASD). Most cases are de novo, maternal in origin and fully penetrant for ASD. Here, we describe a unique family with an interstitial 15q11.2-q13.1 maternal duplication and the presence of somatic mosaicism in the mother. She is typically functioning, but formal autism testing showed mild ASD. She had several congenital anomalies, and she is the first 15q Duplication case reported in the literature to develop unilateral renal carcinoma. Her two affected children share some of these clinical characteristics, and have severe ASD. Several tissues in the mother, including blood, skin, a kidney tumor, and normal kidney margin tissues were studied for the presence of the 15q11-q13.1 duplication. We show the mother has somatic mosaicism for the duplication in several tissues to varying degrees. A growth competition assay in two types of stem cells from duplication 15q individuals was also performed. Our results suggest that the presence of this interstitial duplication 15q chromosome may confer a previously unknown growth advantage in this particular individual, but not in the general interstitial duplication 15q population.
RESUMO
Dental pulp stem cells (DPSCs) provide an exciting new avenue to study neurogenetic disorders. DPSCs are neural crest-derived cells with the ability to differentiate into numerous tissues including neurons. The therapeutic potential of stem cell-derived lines exposed to culturing ex vivo before reintroduction into patients could be limited if the cultured cells acquired tumorigenic potential. We tested whether DPSCs that spontaneously immortalized in culture acquired features of transformed cells. We analyzed immortalized DPSCs for anchorage-independent growth, genomic instability, and ability to differentiate into neurons. Finally, we tested both spontaneously immortalized and human telomerase reverse transcriptase (hTERT)-immortalized DPSC lines for the ability to form tumors in immunocompromised animals. Although we observed increased colony-forming potential in soft agar for the spontaneously immortalized and hTERT-immortalized DPSC lines relative to low-passage DPSC, no tumors were detected from any of the DPSC lines tested. We noticed some genomic instability in hTERT-immortalized DPSCs but not in the spontaneously immortalized lines tested. We determined that immortalized DPSC lines generated in our laboratory, whether spontaneously or induced, have not acquired the potential to form tumors in mice. These data suggest cultured DPSC lines that can be differentiated into neurons may be safe for future in vivo therapy for neurobiological diseases.
Assuntos
Polpa Dentária/transplante , Crista Neural/transplante , Neurônios/citologia , Transplante de Células-Tronco/efeitos adversos , Animais , Diferenciação Celular/genética , Transformação Celular Neoplásica , Polpa Dentária/citologia , Humanos , Camundongos , Telomerase/farmacologiaRESUMO
BACKGROUND/AIM: Activating mutations in the KRAS gene are found in more than 30% of colorectal tumors, where they are associated with a poor response to anti-epidermal growth factor receptor therapies. Mutation testing techniques have therefore become an urgent concern. Several methods for KRAS mutation detection have been described in the literature. Most of these are laboratory developed tests and only a few commercial assays are currently available. MATERIALS AND METHODS: We studied the performance characteristics of a KRAS mutation detection assay on the ABI-3130XL genetic analyzer using a new commercial mutation detection kit based on shifted termination assay technology. Samples were analyzed in parallel by different reference laboratories using alternative methodologies. Various sample types were used including formalin-fixed paraffin-embedded tissue, fine-needle aspirates, and cyst fluid specimens. RESULTS: A high level of agreement (100% correlation for formalin-fixed paraffin-embedded tissue and fine-needle aspirate samples and 93% correlation for cyst fluid specimens) was obtained despite the use of different methodologies. CONCLUSION: Shift termination assay is a simple, robust, and sensitive method for the identification of KRAS mutations in a wide variety of specimen types.
Assuntos
Neoplasias Colorretais/genética , DNA de Neoplasias/análise , Testes Genéticos/métodos , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas/análise , Proteínas ras/análise , Biópsia por Agulha Fina , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , DNA de Neoplasias/genética , Humanos , Mutação , Neoplasias Pancreáticas/patologia , Inclusão em Parafina , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo , Fixação de Tecidos , Proteínas ras/genéticaRESUMO
AIMS: To determine whether KRAS mutations occur in primary bladder adenocarcinoma. METHODS AND RESULTS: Twenty-six cases of primary urinary bladder adenocarcinoma were analysed. DNA was extracted from formalin-fixed, paraffin-embedded tissue and amplified with shifted termination assay technology, which recognizes wild-type or mutant target sequences and selectively extends detection primers with labelled nucleotides. A mutation in KRAS was found in three (11.5%) of 26 primary bladder adenocarcinomas. Two of these three cases exhibited a G13D mutation, whereas the remaining case contained a mutation in G12V. None of the ten cases of urothelial carcinoma with glandular differentiation displayed KRAS mutation. Colonic adenocarcinoma contained a KRAS mutation in 18 (33%) of 55 cases. There was no distinct difference with regard to grade, stage or outcome according to the limited clinicopathological data available. However, the two youngest patients, aged 32 and 39 years, in our study group, with a mean population age of 61 years, were found to have mutations in KRAS. CONCLUSIONS: KRAS mutations are present in a small subset of primary urinary bladder adenocarcinomas. Future clinical trials for treatment of bladder adenocarcinoma, employing targeted therapies similar to those used for treatment of colon cancer, may also benefit from the predictive implications of KRAS mutational testing.
Assuntos
Adenocarcinoma/classificação , Adenocarcinoma/genética , Mutação/genética , Proteínas Proto-Oncogênicas/genética , Neoplasias da Bexiga Urinária/classificação , Neoplasias da Bexiga Urinária/genética , Proteínas ras/genética , Adenocarcinoma/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA de Neoplasias/genética , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inclusão em Parafina , Valor Preditivo dos Testes , Prognóstico , Proteínas Proto-Oncogênicas p21(ras) , Neoplasias da Bexiga Urinária/diagnósticoRESUMO
Genetically engineered mice have been generated to model cerebral ß-amyloidosis, one of the hallmarks of Alzheimer disease (AD) pathology, based on the overexpression of a mutated cDNA of the amyloid-ß precursor protein (AßPP) or by knock-in of the murine Aßpp gene alone or with presenilin1 mutations. Here we describe the generation and initial characterization of a new mouse line based on the presence of 2 copies of the human genomic region encoding the wild-type AßPP and the L166P presenilin 1 mutation. At â¼6 mo of age, double-mutant mice develop amyloid pathology, with signs of neuritic dystrophy, intracellular Aß accumulation, and glial inflammation, an increase in AßPP C-terminal fragments, and an 8 times increase in Aß42 levels with a 40% decrease in Aß40 levels, leading to a significant increase (14 times) of Aß42/Aß40 ratios, with minimal effects on presenilin or the Notch1 pathway in the brain. We conclude that in mice, neither mutations in AßPP nor overexpression of an AßPP isoform are a prerequisite for Aß pathology. This model will allow the study of AD pathogenesis and testing of therapeutic strategies in a more relevant environment without experimental artifacts due to the overexpression of a single-mutant AßPP isoform using exogenous promoters.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Substituição de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Encéfalo/patologia , Cromossomos Artificiais de Levedura/genética , DNA Complementar/genética , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Receptores Notch/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Fanconi anemia (FA) is an inherited chromosomal instability syndrome characterized by bone marrow failure, myelodysplasia (MDS), and acute myeloid leukemia (AML). Eight FA proteins associate in a nuclear core complex to monoubiquitinate FANCD2/FANCI in response to DNA damage. Additional functions have been described for some of the core complex proteins; however, in vivo genetic proof has been lacking. Here we show that double-mutant Fancc(-/-);Fancg(-/-) mice develop spontaneous hematologic sequelae including bone marrow failure, AML, MDS and complex random chromosomal abnormalities that the single-mutant mice do not. This genetic model provides evidence for unique core complex protein function independent of their ability to monoubiquitinate FANCD2/FANCI. Importantly, this model closely recapitulates the phenotypes found in FA patients and may be useful as a preclinical platform to evaluate the molecular pathogenesis of spontaneous bone marrow failure, MDS and AML in FA.
Assuntos
Medula Óssea/fisiopatologia , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Leucemia Mieloide Aguda/genética , Mutação , Síndromes Mielodisplásicas/genética , Animais , Aberrações Cromossômicas , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The vertical transmission of a prion disease from infected mothers to their offspring is believed to be one of the routes for the natural spread of animal prion diseases. Supporting this notion is the observation that prion infectivity occurs in the placenta of infected ewes. Furthermore, the prion protein (PrP), both in its cellular form (PrP(C)) and its pathological isoform (PrP(Sc)), has been observed at the fetal-maternal interface of scrapie-infected sheep. However, whether these features of prion infectivity also hold true for human prion diseases is currently unknown. To begin to address such an important question, we examined PrP in the uterus as well as gestational tissues, including the placenta and amniotic fluid, in a pregnant woman with sporadic Creutzfeldt-Jakob disease (CJD). Although the proteinase K (PK)-resistant prion protein, PrP27-30, was present in the brain tissues of the mother, the PrP detected in the uterus, placenta, and amniotic fluid was sensitive to PK digestion. Unlike PrP(C) in the brain and adjacent cerebrospinal fluid, the predominant PrP species in the reproductive and gestational tissues were N-terminally truncated, similar to urine PrP. Our study did not detect abnormal PrP in the reproductive and gestational tissues in this case of CJD. Nevertheless, examination by a highly sensitive bioassay is ongoing to ascertain possible prion infectivity from CJD in the amniotic fluid.
Assuntos
Líquido Amniótico/metabolismo , Síndrome de Creutzfeldt-Jakob/metabolismo , Placenta/metabolismo , Proteínas PrPSc/metabolismo , Útero/metabolismo , Adulto , Autopsia , Biópsia , Western Blotting , Encéfalo/metabolismo , Síndrome de Creutzfeldt-Jakob/patologia , Endopeptidase K/farmacologia , Feminino , Humanos , GravidezRESUMO
Increased iron levels and iron-mediated oxidative stress play an important role in the pathogenesis of many neurodegenerative diseases. The finding that mutations in the ferritin light polypeptide (FTL) gene cause a neurodegenerative disease known as neuroferritinopathy or hereditary ferritinopathy (HF) provided a direct connection between abnormal brain iron storage and neurodegeneration. HF is characterized by a severe movement disorder and by the presence of nuclear and cytoplasmic ferritin inclusion bodies in glia and neurons throughout the CNS and in tissues of multiple organ systems. Here we report that the expression in transgenic mice of a human FTL cDNA carrying a thymidine and cytidine insertion at position 498 (FTL498-499InsTC) leads to the formation of nuclear and cytoplasmic ferritin inclusion bodies. As in HF, ferritin inclusions are seen in glia and neurons throughout the CNS as well as in cells of other organ systems. Our studies show histological, immunohistochemical, and biochemical similarities between ferritin inclusion bodies found in transgenic mice and in individuals with HF. Expression of the transgene in mice leads to a significant decrease in motor performance and a shorter life span, formation of ferritin inclusion bodies, misregulation of iron metabolism, accumulation of ubiquitinated proteins, and incorporation of elements of the proteasome into inclusions. This new transgenic mouse represents a relevant model of HF in which to study the pathways that lead to neurodegeneration in HF, to evaluate the role of iron mismanagement in neurodegenerative disorders, and to evaluate potential therapies for HF and related neurodegenerative diseases.
Assuntos
Ferritinas/genética , Expressão Gênica/genética , Sobrecarga de Ferro/genética , Mutação/genética , Doenças Neurodegenerativas/genética , Animais , Apoferritinas , Comportamento Animal , Encéfalo/patologia , Encéfalo/ultraestrutura , Modelos Animais de Doenças , Humanos , Ferro/metabolismo , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Sobrecarga de Ferro/fisiopatologia , Imageamento por Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão/métodos , Atividade Motora/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologiaRESUMO
Recent in situ hybridization studies showed that mRNA levels of OLIG1 and OLIG2 transcription factors are elevated in oligodendrogliomas. We raised polyclonal antibodies against a synthetic peptide homologous to the human transcription factor Olig1 and studied by immunohistochemistry the expression of Olig1 in 84 brain tumors and in non-neoplastic brain tissues. All oligodendrogliomas, oligoastrocytomas, and dysembryoplastic neuroepithelial tumors showed moderate to strong intranuclear immunoreactivity in cells morphologically identified as oligodendrocytes. In addition, some astrocytomas showed a slight to moderate intranuclear immunoreactivity. None of the other neuroepithelial and non-neuroepithelial tumors showed nuclear immunoreactivity. Double immunostaining of oligodendrogliomas, oligoastrocytomas, and glioblastoma multiforme (GBM) using antibodies against Olig1 and GFAP showed the presence of 3 different cell populations: 1) immunopositive for Olig1 and immunonegative for GFAP, histologically identified as oligodendrocytes; 2) immunopositive only for GFAP, histologically identified as astrocytes; and 3) immunonegative for both antibodies ("null cells"), histologically observed as a population of cells usually with round nuclei and a small amount of cytoplasm. The use of double immunostaining facilitated the distinction among these 3 different tumors. In summary, the use of immunohistochemistry using Olig1 antibodies alone or in combination with anti-GFAP antibody, which can be performed in the routine diagnostic setting, may help in the diagnosis of neuroepithelial tumors.