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1.
Clin Genet ; 97(2): 264-275, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31573083

RESUMO

Children with neurofibromatosis type 1 (NF1) may exhibit an incomplete clinical presentation, making difficult to reach a clinical diagnosis. A phenotypic overlap may exist in children with other RASopathies or with other genetic conditions if only multiple café-au-lait macules (CALMs) are present. The syndromes that can converge in these inconclusive phenotypes have different clinical courses. In this context, an early genetic testing has been proposed to be clinically useful to manage these patients. We present the validation and implementation into diagnostics of a custom NGS panel (I2HCP, ICO-IMPPC Hereditary Cancer Panel) for testing patients with a clinical suspicion of a RASopathy (n = 48) and children presenting multiple CALMs (n = 102). We describe the mutational spectrum and the detection rates identified in these two groups of individuals. We identified pathogenic variants in 21 out of 48 patients with clinical suspicion of RASopathy, with mutations in NF1 accounting for 10% of cases. Furthermore, we identified pathogenic mutations mainly in the NF1 gene, but also in SPRED1, in more than 50% of children with multiple CALMs, exhibiting an NF1 mutational spectrum different from a group of clinically diagnosed NF1 patients (n = 80). An NGS panel strategy for the genetic testing of these two phenotype-defined groups outperforms previous strategies.

3.
Stem Cell Reports ; 12(2): 411-426, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30713041

RESUMO

Neurofibromatosis type 1 (NF1) is a tumor predisposition genetic disease caused by mutations in the NF1 tumor suppressor gene. Plexiform neurofibromas (PNFs) are benign Schwann cell (SC) tumors of the peripheral nerve sheath that develop through NF1 inactivation and can progress toward a malignant soft tissue sarcoma. There is a lack of non-perishable model systems to investigate PNF development. We reprogrammed PNF-derived NF1(-/-) cells, descendants from the tumor originating cell. These NF1(-/-)-induced pluripotent stem cells (iPSCs) captured the genomic status of PNFs and were able to differentiate toward neural crest stem cells and further to SCs. iPSC-derived NF1(-/-) SCs exhibited a continuous high proliferation rate, poor myelination ability, and a tendency to form 3D spheres that expressed the same markers as their PNF-derived primary SC counterparts. They represent a valuable model to study and treat PNFs. PNF-derived iPSC lines were banked for making them available.

4.
Hum Mutat ; 39(8): 1112-1125, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29774626

RESUMO

Plexiform neurofibromas (PNFs) are benign peripheral nerve sheath tumors involving large nerves present in 30%-50% Neurofibromatosis type 1 (NF1) patients. Atypical neurofibromas (ANF) are distinct nodular lesions with atypical features on histology that arise from PNFs. The risk and timeline of malignant transformation in ANF is difficult to assess. A recent NIH workshop has stratified ANFs and separated a subgroup with multiple atypical features and higher risk of malignant transformation termed atypical neurofibromatous neoplasms with uncertain biological potential (ANNUBP). We performed an analysis of intratumor heterogeneity on eight PNFs to link histological and genomic findings. Tumors were homogeneous although histological and molecular heterogeneity was identified. All tumors were 2n, almost mutation-free and had a clonal NF1(-/-) origin. Two ANFs from the same patient showed atypical features on histology and deletions of CDKN2A/B. One of the ANFs exhibited different areas in which the degree of histological atypia correlated with the heterozygous or homozygous loss of the CDKN2A/B loci. CDKN2A/B deletions in different areas originated independently. Results may indicate that loss of a single CDKN2A/B copy in NF1(-/-) cells is sufficient to start ANF development and that total inactivation of both copies of CDKN2A/B is necessary to form an ANNUBP.


Assuntos
Neurofibroma Plexiforme/genética , Neurofibroma/genética , Neurofibromatose 1/genética , Adulto , Idoso , Criança , Pré-Escolar , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Genômica/métodos , Humanos , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética
5.
Am J Med Genet A ; 176(5): 1258-1269, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29681099

RESUMO

Organized and hosted by the Children's Tumor Foundation (CTF), the Neurofibromatosis (NF) conference is the premier annual gathering for clinicians and researchers interested in neurofibromatosis type 1 (NF1), neurofibromatosis type 2 (NF2), and schwannomatosis (SWN). The 2016 edition constituted a blend of clinical and basic aspects of NF research that helped in clarifying different advances in the field. The incorporation of next generation sequencing is changing the way genetic diagnostics is performed for NF and related disorders, providing solutions to problems like genetic heterogeneity, overlapping clinical manifestations, or the presence of mosaicism. The transformation from plexiform neurofibroma (PNF) to malignant peripheral nerve sheath tumor (MPNST) is being clarified, along with new management and treatments for benign and premalignant tumors. Promising new cellular and in vivo models for understanding the musculoskeletal abnormalities in NF1, the development of NF2 or SWN associated schwannomas, and clarifying the cells that give rise to NF1-associated optic pathway glioma were presented. The interaction of neurofibromin and SPRED1 was described comprehensively, providing functional insight that will help in the interpretation of pathogenicity of certain missense variants identified in NF1 and Legius syndrome patients. Novel promising imaging techniques are being developed, as well as new integrative and holistic management models for patients that take into account psychological, social, and biological factors. Importantly, new therapeutic approaches for schwannomas, meningiomas, ependymomas, PNF, and MPNST are being pursued. This report highlights the major advances that were presented at the 2016 CTF NF conference.


Assuntos
Neurilemoma/diagnóstico , Neurilemoma/etiologia , Neurofibromatoses/diagnóstico , Neurofibromatoses/etiologia , Neurofibromatose 1/diagnóstico , Neurofibromatose 1/etiologia , Neurofibromatose 2/diagnóstico , Neurofibromatose 2/etiologia , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/etiologia , Animais , Gerenciamento Clínico , Modelos Animais de Doenças , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Técnicas de Diagnóstico Molecular , Neurilemoma/terapia , Neurofibromatoses/terapia , Neurofibromatose 1/terapia , Neurofibromatose 2/terapia , Neoplasias Cutâneas/terapia , Pesquisa Médica Translacional
6.
JAMA Dermatol ; 154(3): 341-346, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29322178

RESUMO

Importance: Neurofibromatosis type 2 (NF2) is a devastating genetic condition characterized by the development of multiple tumors of the nervous system. An early diagnosis of individuals with NF2 would facilitate treatment and reduction of disease impact because most severe effects of the disease do not usually develop before adolescence. Little attention has traditionally been paid to dermatological signs in NF2. However, skin plaques are commonly seen in patients with NF2, normally appearing either at birth or early childhood, providing an opportunity for early NF2 detection and testing. Objective: To determine the clinical utility of skin plaque identification and characterization in children for reaching an early diagnosis of patients with NF2 and to evaluate their molecular pathogenesis and their use in the genetic diagnostics of NF2. Design, Setting, and Participants: Diagnostic test study by the histological and genetic characterization of skin plaques from patients with NF2. Patients were 7 individuals with NF2 or clinical suspicion of NF2 treated at the Spanish Reference Center on Phakomatoses. Main Outcomes and Measures: Histological evaluation of all skin plaques was performed. Fresh skin plaques were cultured to obtain Schwann cells and the NF2 gene was genetically analyzed. For all 7 patients, NF2 clinical history was reviewed. Results: In all 7 patients (4 male and 3 female), all skin plaques analyzed were histologically characterized as plexiform schwannomas. Genetic analysis of primary Schwann cell cultures derived from them allowed the identification of a constitutional and a somatic NF2 mutation. Genetic testing allowed the early diagnosis of NF2 in a child only exhibiting the presence of skin plaques. Most of the patients with NF2 analyzed had an early presentation of skin plaques and a severe NF2 phenotype. Conclusions and Relevance: This work emphasizes the clinical utility of a careful dermatological inspection and the correct identification of skin plaques in children for an early diagnosis of NF2. We show for the first time that Schwann cells derived from skin plaque plexiform schwannomas bear the double inactivation of the NF2 gene and thus constitute an excellent source of tissue for genetic testing, especially in the context of mosaicism.


Assuntos
Genes da Neurofibromatose 2 , Neurilemoma/genética , Neurofibromatose 2/diagnóstico , Neurofibromatose 2/genética , Neoplasias Cutâneas/genética , Adolescente , Adulto , Pré-Escolar , Feminino , Testes Genéticos , Humanos , Masculino , Mutação , Neurilemoma/patologia , Células de Schwann , Neoplasias Cutâneas/patologia
7.
Bioinformatics ; 33(19): 3088-3090, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28575171

RESUMO

Motivation: Data visualization is a crucial tool for data exploration, analysis and interpretation. For the visualization of genomic data there lacks a tool to create customizable non-circular plots of whole genomes from any species. Results: We have developed karyoploteR, an R/Bioconductor package to create linear chromosomal representations of any genome with genomic annotations and experimental data plotted along them. Plot creation process is inspired in R base graphics, with a main function creating karyoplots with no data and multiple additional functions, including custom functions written by the end-user, adding data and other graphical elements. This approach allows the creation of highly customizable plots from arbitrary data with complete freedom on data positioning and representation. Availability and implementation: karyoploteR is released under Artistic-2.0 License. Source code and documentation are freely available through Bioconductor (http://www.bioconductor.org/packages/karyoploteR) and at the examples and tutorial page at https://bernatgel.github.io/karyoploter_tutorial. Contact: bgel@igtp.cat.


Assuntos
Genômica/métodos , Software , Gráficos por Computador , Genoma
8.
Sci Rep ; 7: 37984, 2017 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-28050010

RESUMO

Next generation sequencing panels have been developed for hereditary cancer, although there is some debate about their cost-effectiveness compared to exome sequencing. The performance of two panels is compared to exome sequencing. Twenty-four patients were selected: ten with identified mutations (control set) and fourteen suspicious of hereditary cancer but with no mutation (discovery set). TruSight Cancer (94 genes) and a custom panel (122 genes) were assessed alongside exome sequencing. Eighty-three genes were targeted by the two panels and exome sequencing. More than 99% of bases had a read depth of over 30x in the panels, whereas exome sequencing covered 94%. Variant calling with standard settings identified the 10 mutations in the control set, with the exception of MSH6 c.255dupC using TruSight Cancer. In the discovery set, 240 unique non-silent coding and canonic splice-site variants were identified in the panel genes, 7 of them putatively pathogenic (in ATM, BARD1, CHEK2, ERCC3, FANCL, FANCM, MSH2). The three approaches identified a similar number of variants in the shared genes. Exomes were more expensive than panels but provided additional data. In terms of cost and depth, panels are a suitable option for genetic diagnostics, although exomes also identify variants in non-targeted genes.


Assuntos
Predisposição Genética para Doença , Testes Genéticos/métodos , Síndromes Neoplásicas Hereditárias/diagnóstico , Benchmarking , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Síndromes Neoplásicas Hereditárias/genética , Sequenciamento Completo do Exoma/métodos
9.
Sci Rep ; 7: 39348, 2017 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-28051113

RESUMO

We wanted to implement an NGS strategy to globally analyze hereditary cancer with diagnostic quality while retaining the same degree of understanding and control we had in pre-NGS strategies. To do this, we developed the I2HCP panel, a custom bait library covering 122 hereditary cancer genes. We improved bait design, tested different NGS platforms and created a clinically driven custom data analysis pipeline. The I2HCP panel was developed using a training set of hereditary colorectal cancer, hereditary breast and ovarian cancer and neurofibromatosis patients and reached an accuracy, analytical sensitivity and specificity greater than 99%, which was maintained in a validation set. I2HCP changed our diagnostic approach, involving clinicians and a genetic diagnostics team from panel design to reporting. The new strategy improved diagnostic sensitivity, solved uncertain clinical diagnoses and identified mutations in new genes. We assessed the genetic variation in the complete set of hereditary cancer genes, revealing a complex variation landscape that coexists with the disease-causing mutation. We developed, validated and implemented a custom NGS-based strategy for hereditary cancer diagnostics that improved our previous workflows. Additionally, the existence of a rich genetic variation in hereditary cancer genes favors the use of this panel to investigate their role in cancer risk.


Assuntos
Detecção Precoce de Câncer/métodos , Testes Genéticos/métodos , Técnicas de Diagnóstico Molecular/métodos , Síndromes Neoplásicas Hereditárias/diagnóstico , Síndromes Neoplásicas Hereditárias/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Sensibilidade e Especificidade
10.
Bioinformatics ; 32(2): 289-91, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26424858

RESUMO

MOTIVATION: Statistically assessing the relation between a set of genomic regions and other genomic features is a common challenging task in genomic and epigenomic analyses. Randomization based approaches implicitly take into account the complexity of the genome without the need of assuming an underlying statistical model. SUMMARY: regioneR is an R package that implements a permutation test framework specifically designed to work with genomic regions. In addition to the predefined randomization and evaluation strategies, regioneR is fully customizable allowing the use of custom strategies to adapt it to specific questions. Finally, it also implements a novel function to evaluate the local specificity of the detected association. AVAILABILITY AND IMPLEMENTATION: regioneR is an R package released under Artistic-2.0 License. The source code and documents are freely available through Bioconductor (http://www.bioconductor.org/packages/regioneR). CONTACT: rmalinverni@carrerasresearch.org.


Assuntos
Variação Genética , Genoma Humano , Genômica/métodos , Software , Humanos , Anotação de Sequência Molecular , Linguagens de Programação , Análise de Sequência de DNA
11.
EMBO Mol Med ; 7(5): 608-27, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25810463

RESUMO

Malignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas that can arise either sporadically or in association with neurofibromatosis type 1 (NF1). These aggressive malignancies confer poor survival, with no effective therapy available. We present the generation and characterization of five distinct MPNST orthoxenograft models for preclinical testing and personalized medicine. Four of the models are patient-derived tumor xenografts (PDTX), two independent MPNSTs from the same NF1 patient and two from different sporadic patients. The fifth model is an orthoxenograft derived from an NF1-related MPNST cell line. All MPNST orthoxenografts were generated by tumor implantation, or cell line injection, next to the sciatic nerve of nude mice, and were perpetuated by 7-10 mouse-to-mouse passages. The models reliably recapitulate the histopathological properties of their parental primary tumors. They also mimic distal dissemination properties in mice. Human stroma was rapidly lost after MPNST engraftment and replaced by murine stroma, which facilitated genomic tumor characterization. Compatible with an origin in a catastrophic event and subsequent genome stabilization, MPNST contained highly altered genomes that remained remarkably stable in orthoxenograft establishment and along passages. Mutational frequency and type of somatic point mutations were highly variable among the different MPNSTs modeled, but very consistent when comparing primary tumors with matched orthoxenografts generated. Unsupervised cluster analysis and principal component analysis (PCA) using an MPNST expression signature of ~1,000 genes grouped together all primary tumor-orthoxenograft pairs. Our work points to differences in the engraftment process of primary tumors compared with the engraftment of established cell lines. Following standardization and extensive characterization and validation, the orthoxenograft models were used for initial preclinical drug testing. Sorafenib (a BRAF inhibitor), in combination with doxorubicin or rapamycin, was found to be the most effective treatment for reducing MPNST growth. The development of genomically well-characterized preclinical models for MPNST allowed the evaluation of novel therapeutic strategies for personalized medicine.


Assuntos
Modelos Animais de Doenças , Neurilemoma/patologia , Neurilemoma/terapia , Medicina de Precisão/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Humanos , Camundongos Nus , Pacientes
12.
BMC Med Genomics ; 8: 2, 2015 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-25739810

RESUMO

BACKGROUND: A clinical overlap exists between mosaic Neurofibromatosis Type 2 and sporadic Schwannomatosis conditions. In these cases a molecular analysis of tumors is recommended for a proper genetic diagnostics. This analysis is challenged by the fact that schwannomas in both conditions bear a somatic double inactivation of the NF2 gene. However, SMARCB1-associated schwannomas follow a four-hit, three-step model, in which both alleles of SMARCB1 and NF2 genes are inactivated in the tumor, with one of the steps being always the loss of a big part of chromosome 22 involving both loci. CASE PRESENTATION: Here we report a 36-year-old woman who only presented multiple subcutaneous schwannomas on her right leg. To help discriminate between both possible diagnoses, an exhaustive molecular genetic and genomic analysis was performed on two schwannomas of the patient, consisting in cDNA and DNA sequencing, MLPA, microsatellite multiplex PCR and SNP-array analyses. The loss of a big part of chromosome 22 (22q12.1q13.33) was identified in both tumors. However, this loss involved the NF2 but not the SMARCB1 locus. SNP-array analysis revealed the presence of the same deletion breakpoint in both schwannomas, indicating that this alteration was actually the first NF2 inactivating hit. In addition, a distinct NF2 point mutation in each tumor was identified, representing independent second hits. In accordance with these results, no deletions or point mutations in the SMARCB1 gene were identified. None of the mutations were present in the blood. Two of the patient's children inherited chromosome 22 deleted in schwannomas of the mother, but in its wild type form. CONCLUSIONS: These results conclusively confirm the segmental mosaic NF2 nature of the clinical phenotype presented.


Assuntos
Perna (Membro) , Técnicas de Diagnóstico Molecular , Neurilemoma/genética , Neurofibromatose 2/diagnóstico , Neurofibromatose 2/genética , Adulto , Sequência de Bases , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Diagnóstico Diferencial , Feminino , Genes da Neurofibromatose 2 , Humanos , Repetições de Microssatélites/genética , Neurilemoma/diagnóstico , Polimorfismo de Nucleotídeo Único , Proteína SMARCB1 , Fatores de Transcrição/genética
13.
Brain ; 136(Pt 7): 2262-78, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23748155

RESUMO

The number of Schwann cells is fitted to axonal length in peripheral nerves. This relationship is lost when tumorigenic stimuli induce uncontrolled Schwann cell proliferation, generating tumours such us neurofibromas and schwannomas. Schwann cells also re-enter the cell cycle following nerve injury during the process of Wallerian degeneration. In both cases proliferation is finally arrested. We show that in neurofibroma, the induction of Jmjd3 (jumonji domain containing 3, histone lysine demethylase) removes trimethyl groups on lysine-27 of histone-H3 and epigenetically activates the Ink4a/Arf-locus, forcing Schwann cells towards replicative senescence. Remarkably, blocking this mechanism allows unrestricted proliferation, inducing malignant transformation of neurofibromas. Interestingly, our data suggest that in injured nerves, Schwann cells epigenetically activate the same locus to switch off proliferation and enter the senescence programme. Indeed, when this pathway is genetically blocked, Schwann cells fail to drop out of the cell cycle and continue to proliferate. We postulate that the Ink4a/Arf-locus is expressed as part of a physiological response that prevents uncontrolled proliferation of the de-differentiated Schwann cell generated during nerve regeneration, a response that is also activated to avoid overproliferation after tumorigenic stimuli in the peripheral nervous system.


Assuntos
Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica/genética , Regeneração Nervosa/fisiologia , Neurofibroma/patologia , Células de Schwann/fisiologia , Degeneração Walleriana/patologia , Fatores Etários , Animais , Animais Recém-Nascidos , Axônios/patologia , Axônios/ultraestrutura , Células Cultivadas , Senescência Celular/genética , Imunoprecipitação da Cromatina , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/genética , Modelos Animais de Doenças , Progressão da Doença , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Epigenômica , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Regeneração Nervosa/genética , Neuregulina-1/genética , Neurofibroma/genética , Neurofibroma/fisiopatologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Células de Schwann/patologia , Células de Schwann/ultraestrutura , Nervo Isquiático/citologia , Transdução de Sinais/genética , Transfecção , Proteína Supressora de Tumor p53/deficiência , Degeneração Walleriana/etiologia , Degeneração Walleriana/fisiopatologia
14.
Nat Genet ; 45(7): 756-66, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23685747

RESUMO

Malignant peripheral nerve sheath tumors (MPNSTs) are sarcomas of Schwann cell lineage origin that occur sporadically or in association with the inherited syndrome neurofibromatosis type 1. To identify genetic drivers of MPNST development, we used the Sleeping Beauty (SB) transposon-based somatic mutagenesis system in mice with somatic loss of transformation-related protein p53 (Trp53) function and/or overexpression of human epidermal growth factor receptor (EGFR). Common insertion site (CIS) analysis of 269 neurofibromas and 106 MPNSTs identified 695 and 87 sites with a statistically significant number of recurrent transposon insertions, respectively. Comparison to human data sets identified new and known driver genes for MPNST formation at these sites. Pairwise co-occurrence analysis of CIS-associated genes identified many cooperating mutations that are enriched in Wnt/ß-catenin, PI3K-AKT-mTOR and growth factor receptor signaling pathways. Lastly, we identified several new proto-oncogenes, including Foxr2 (encoding forkhead box R2), which we functionally validated as a proto-oncogene involved in MPNST maintenance.


Assuntos
Transformação Celular Neoplásica/genética , Genes Neoplásicos , Testes Genéticos/métodos , Neoplasias da Bainha Neural/genética , Animais , Linhagem Celular Tumoral , Análise Mutacional de DNA , Elementos de DNA Transponíveis/genética , Genes Neoplásicos/fisiologia , Estudos de Associação Genética , Humanos , Camundongos , Camundongos Transgênicos , Mutação/fisiologia , Neurofibroma/genética , Transdução de Sinais/genética
15.
Clin Chem ; 59(6): 928-37, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23386700

RESUMO

BACKGROUND: About 5% of patients with neurofibromatosis type 1 (NF1) bear constitutional microdeletions that encompass NF1 (neurofibromin 1) and neighboring genes. These patients are characterized by the development of a high number of dermal neurofibromas (dNFs), mental retardation, and an increased risk of developing a malignant peripheral nerve sheath tumor (MPNST). Additionally, 10% of somatic second hits identified in dNFs are caused by deletions involving the NF1 gene. To detect constitutional and somatic deletions, we developed a probe-based quantitative PCR (qPCR) assay for interrogating the copy number status of 11 loci distributed along a 2.8-Mb region around the NF1 gene. METHODS: We developed the qPCR assay with Universal ProbeLibrary technology (Roche) and designed a Microsoft Excel spreadsheet to analyze qPCR data for copy number calculations. The assay fulfilled the essential aspects of the MIQE (minimum information for publication of quantitative real-time PCR experiments) guidelines and used the qBase relative quantification framework for calculations. RESULTS: The assay was validated with a set of DNA samples with known constitutional or somatic NF1 deletions. The assay showed high diagnostic sensitivity and specificity and distinguished between Type-1, Type-2, and atypical constitutional microdeletions in 14 different samples. It also identified 16 different somatic deletions in dNFs. These results were confirmed by multiplex ligation-dependent probe amplification. CONCLUSIONS: The qPCR assay provides a methodology for detecting constitutional NF1 microdeletions that could be incorporated as an additional technique in a genetic-testing setting. It also permits the identification of somatic NF1 deletions in tissues with a high percentage of cells bearing 2 copies of the NF1 gene.


Assuntos
Deleção de Genes , Genes da Neurofibromatose 1 , Testes Genéticos/métodos , Neoplasias/diagnóstico , Neoplasias/genética , Reação em Cadeia da Polimerase em Tempo Real , Humanos , Sensibilidade e Especificidade
16.
Eur J Hum Genet ; 21(7): 769-73, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23188051

RESUMO

Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder affecting about 1:33 000 newborns, mainly characterized by the development of tumors of the nervous system and ocular abnormalities. Around 85% of germline NF2 mutations are point mutations. Among them, ∼25% affect splicing and are associated with a variable disease severity. In the context of our NF2 Multidisciplinary Clinics, we have identified a patient fulfilling clinical criteria for the disease and exhibiting a severe phenotype. The patient carries a deep intronic mutation (g. 74409T>A, NG_009057.1) that produces the insertion of a cryptic exon of 167pb in the mature mRNA between exons 13 and 14, resulting in a truncated merlin protein (p.Pro482Profs*39). A mutation-specific antisense phosphorodiamidate morpholino oligomer was designed and used in vitro to effectively restore normal NF2 splicing in patient-derived primary fibroblasts. In addition, merlin protein levels were greatly recovered after morpholino treatment, decreasing patient's fibroblasts in vitro proliferation capacity and restoring cytoeskeleton organization. To our knowledge, this is the first NF2 case caused by a deep intronic mutation in which an in vitro antisense therapeutic approximation has been tested. These results open the possibility of using this approach in vivo for this type of mutation causing NF2.


Assuntos
Morfolinos/administração & dosagem , Neurofibromatose 2/genética , Neurofibromatose 2/terapia , Neurofibromina 2/genética , Adolescente , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Mutação em Linhagem Germinativa/genética , Humanos , Íntrons/genética , Morfolinos/genética , Morfolinos/uso terapêutico , Neurofibromatose 2/patologia , RNA Antissenso/genética
17.
Eur J Hum Genet ; 21(8): 864-70, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23249957

RESUMO

Next-generation sequencing (NGS) is changing genetic diagnosis due to its huge sequencing capacity and cost-effectiveness. The aim of this study was to develop an NGS-based workflow for routine diagnostics for hereditary breast and ovarian cancer syndrome (HBOCS), to improve genetic testing for BRCA1 and BRCA2. A NGS-based workflow was designed using BRCA MASTR kit amplicon libraries followed by GS Junior pyrosequencing. Data analysis combined Variant Identification Pipeline freely available software and ad hoc R scripts, including a cascade of filters to generate coverage and variant calling reports. A BRCA homopolymer assay was performed in parallel. A research scheme was designed in two parts. A Training Set of 28 DNA samples containing 23 unique pathogenic mutations and 213 other variants (33 unique) was used. The workflow was validated in a set of 14 samples from HBOCS families in parallel with the current diagnostic workflow (Validation Set). The NGS-based workflow developed permitted the identification of all pathogenic mutations and genetic variants, including those located in or close to homopolymers. The use of NGS for detecting copy-number alterations was also investigated. The workflow meets the sensitivity and specificity requirements for the genetic diagnosis of HBOCS and improves on the cost-effectiveness of current approaches.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Síndrome Hereditária de Câncer de Mama e Ovário/diagnóstico , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Algoritmos , Análise Custo-Benefício , DNA de Neoplasias/química , DNA de Neoplasias/genética , Feminino , Testes Genéticos/economia , Testes Genéticos/métodos , Humanos , Mutação , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
18.
PLoS One ; 7(8): e42682, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22916147

RESUMO

The study of somatic genetic alterations in tumors contributes to the understanding and management of cancer. Genetic alterations, such us copy number or copy neutral changes, generate allelic imbalances (AIs) that can be determined using polymorphic markers. Here we report the development of a simple set of calculations for analyzing microsatellite multiplex PCR data from control-tumor pairs that allows us to obtain accurate information not only regarding the AI status of tumors, but also the percentage of tumor-infiltrating normal cells, the locus copy-number status and the mechanism involved in AI. We validated this new approach by re-analyzing a set of Neurofibromatosis type 1-associated dermal neurofibromas and comparing newly generated data with results obtained for the same tumors in a previous study using MLPA, Paralog Ratio Analysis and SNP-array techniques.Microsatellite multiplex PCR analysis (MMPA) should be particularly useful for analyzing specific regions of the genome containing tumor suppressor genes and also for determining the percentage of infiltrating normal cells within tumors allowing them to be sorted before they are analyzed by more expensive techniques.


Assuntos
Alelos , Dosagem de Genes , Repetições de Microssatélites/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Neoplasias/genética , Humanos , Neoplasias/patologia
19.
Clin Cancer Res ; 18(18): 5020-30, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22811580

RESUMO

PURPOSE: Patients with neurofibromatosis type 1 (NF1) develop malignant peripheral nerve sheath tumors (MPNST), which are often inoperable and do not respond well to current chemotherapies or radiation. The goal of this study was to use comprehensive gene expression analysis to identify novel therapeutic targets. EXPERIMENTAL DESIGN: Nerve Schwann cells and/or their precursors are the tumorigenic cell types in MPNST because of the loss of the NF1 gene, which encodes the RasGAP protein neurofibromin. Therefore, we created a transgenic mouse model, CNP-HRas12V, expressing constitutively active HRas in Schwann cells and defined a Ras-induced gene expression signature to drive a Bayesian factor regression model analysis of differentially expressed genes in mouse and human neurofibromas and MPNSTs. We tested functional significance of Aurora kinase overexpression in MPNST in vitro and in vivo using Aurora kinase short hairpin RNAs (shRNA) and compounds that inhibit Aurora kinase. RESULTS: We identified 2,000 genes with probability of linkage to nerve Ras signaling of which 339 were significantly differentially expressed in mouse and human NF1-related tumor samples relative to normal nerves, including Aurora kinase A (AURKA). AURKA was dramatically overexpressed and genomically amplified in MPNSTs but not neurofibromas. Aurora kinase shRNAs and Aurora kinase inhibitors blocked MPNST cell growth in vitro. Furthermore, an AURKA selective inhibitor, MLN8237, stabilized tumor volume and significantly increased survival of mice with MPNST xenografts. CONCLUSION: Integrative cross-species transcriptome analyses combined with preclinical testing has provided an effective method for identifying candidates for molecular-targeted therapeutics. Blocking Aurora kinases may be a viable treatment platform for MPNST.


Assuntos
Neoplasias da Bainha Neural/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Transcriptoma , Animais , Aurora Quinase A , Aurora Quinases , Azepinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Neoplasias da Bainha Neural/metabolismo , Neoplasias da Bainha Neural/terapia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Pirimidinas/farmacologia , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Hum Mutat ; 32(7): 705-9, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21394830

RESUMO

Here we analyze the genetic and molecular basis responsible for a very benign phenotype observed in an NF1 patient. Quantification of cells carrying the NF1 mutation in different samples derived from the three embryonic layers revealed mosaicism. Furthermore, the construction of a minigene with patient's mutation (c.3198 - 314G>A) confirmed its benign nature due to the leakiness of the splicing mechanism that generated a proportion of correctly spliced transcripts. Hence, we concluded that the mild phenotype observed in this patient is the result of the presence of mosaicism together with the benign nature of a leaky NF1-splice mutation. Finally, with the aim of developing a personalized therapeutic approach for this patient, we demonstrated correction of the splicing defect by using specific antisense morpholino oligomers. Our results provide an example of the molecular complexity behind disease phenotypes and highlight the importance of using comprehensive genetic approaches to better assess phenotype-genotype correlations.


Assuntos
Mosaicismo , Neurofibromatose 1/genética , Neurofibromatose 1/patologia , Neurofibromina 1/genética , Adulto , Feminino , Estudos de Associação Genética , Genótipo , Humanos , Mutação/genética , Oligorribonucleotídeos Antissenso , Fenótipo , Medicina de Precisão , Isoformas de Proteínas/genética , Processamento de RNA/genética
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