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1.
Int J Mol Sci ; 22(2)2021 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-33430070

RESUMEN

The nosocomial opportunistic Gram-negative bacterial pathogen Acinetobacter baumannii is resistant to multiple antimicrobial agents and an emerging global health problem. The polymyxin antibiotic colistin, targeting the negatively charged lipid A component of the lipopolysaccharide on the bacterial cell surface, is often considered as the last-resort treatment, but resistance to colistin is unfortunately increasing worldwide. Notably, colistin-susceptible A. baumannii can also develop a colistin dependence after exposure to this drug in vitro. Colistin dependence might represent a stepping stone to resistance also in vivo. However, the mechanisms are far from clear. To address this issue, we combined proteogenomics, high-resolution microscopy, and lipid profiling to characterize and compare A. baumannii colistin-susceptible clinical isolate (Ab-S) of to its colistin-dependent subpopulation (Ab-D) obtained after subsequent passages in moderate colistin concentrations. Incidentally, in the colistin-dependent subpopulation the lpxA gene was disrupted by insertion of ISAjo2, the lipid A biosynthesis terminated, and Ab-D cells displayed a lipooligosaccharide (LOS)-deficient phenotype. Moreover, both mlaD and pldA genes were perturbed by insertions of ISAjo2 and ISAba13, and LOS-deficient bacteria displayed a capsule with decreased thickness as well as other surface imperfections. The major changes in relative protein abundance levels were detected in type 6 secretion system (T6SS) components, the resistance-nodulation-division (RND)-type efflux pumps, and in proteins involved in maintenance of outer membrane asymmetry. These findings suggest that colistin dependence in A. baumannii involves an ensemble of mechanisms seen in resistance development and accompanied by complex cellular events related to insertional sequences (ISs)-triggered LOS-deficiency. To our knowledge, this is the first study demonstrating the involvement of ISAjo2 and ISAba13 IS elements in the modulation of the lipid A biosynthesis and associated development of dependence on colistin.

2.
Artículo en Inglés | MEDLINE | ID: mdl-31636899

RESUMEN

Background: Carbapenem-resistant Klebsiella pneumoniae are becoming increasingly common in hospital settings worldwide and are a source of increased morbidity, mortality and health care costs. The global epidemiology of carbapenem-resistant K. pneumoniae is characterized by different strains distributed geographically, with the strain ST258 being predominant in Europe and USA, and ST11 being most common in East Asia. ST15 is a less frequently occurring strain but has nevertheless been reported worldwide as a source of hospital outbreaks of carbapenem-resistant K. pneumoniae. Methods: In this study, whole-genome sequencing and antimicrobial susceptibility testing was used to characterize 57 clinical isolates of carbapenem-resistant K. pneumoniae belonging to a strain of ST15, which were collected at a Vietnamese pediatric hospital from February throughout September 2015. Results: Aside from the carbapenem resistance gene bla KPC-2, which was carried by all isolates, prevalence of resistance genes to other antibiotics including aminoglycosides, macrolides, quinolones, fosfomycin and trimethoprim, was also high. All isolates were multidrug-resistant. Susceptibility was highest to ceftazidime/avibactam (96%), gentamicin (91%) and tigecycline (82%). Notably, the colistin resistance rate was very high (42%). Single-nucleotide polymorphism analysis indicated that most isolates belonged to a single clone. Conclusions: The diverse variety of antibiotic resistance genes and the high antibiotic resistance rates to last-resort antibiotics such as carbapenems and colistin, is indicative of a highly adaptable strain. This emphasizes the importance of implementation of infection controls measures, continued monitoring of antibiotic resistance and prudent use of antibiotics to prevent further selection of resistant strains and the emergence of pan-resistant clones.


Asunto(s)
Enterobacteriaceae Resistentes a los Carbapenémicos/aislamiento & purificación , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/aislamiento & purificación , Secuenciación Completa del Genoma/métodos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Enterobacteriaceae Resistentes a los Carbapenémicos/clasificación , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Niño , Farmacorresistencia Bacteriana Múltiple , Femenino , Hospitales Pediátricos , Humanos , Klebsiella pneumoniae/clasificación , Klebsiella pneumoniae/genética , Masculino , Fenotipo , Polimorfismo de Nucleótido Simple , Vietnam
3.
Future Microbiol ; 13: 1647-1655, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30489158

RESUMEN

AIM: To characterize extended-spectrum ß-lactamase-producing Escherichia coli harboring the colistin resistance gene mcr-1 from human fecal samples collected in 2012 in a rural area of Shandong province, PR China. MATERIALS & METHODS: Whole-genome sequencing and antimicrobial susceptibility testing was performed on 25 mcr-1-positive isolates to determine carriage of antibiotic resistance and virulence genes, diversity and antibiotic resistance profiles. RESULTS: The isolates were highly genetically diverse and carried a large variety of different antibiotic resistance genes. The multidrug-resistance rate was high (96%). Virulence genes associated with intestinal pathogenic E. coli were carried by 32% of the isolates. CONCLUSION: Further monitoring of the epidemiological situation is necessary to ensure a preparedness for potential emergence of novel, difficult-to-treat strains and awareness of available treatment options.


Asunto(s)
Toxinas Bacterianas/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Heces/microbiología , Epidemiología Molecular , beta-Lactamasas/metabolismo , Antibacterianos/farmacología , Toxinas Bacterianas/clasificación , China , Colistina/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Escherichia coli/enzimología , Escherichia coli/metabolismo , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/clasificación , Humanos , Pruebas de Sensibilidad Microbiana , Filogenia , Plásmidos , Población Rural , Virulencia/genética , Secuenciación Completa del Genoma
5.
Int J Antimicrob Agents ; 51(5): 789-793, 2018 May.
Artículo en Inglés | MEDLINE | ID: mdl-29180281

RESUMEN

Resistance among Klebsiella pneumoniae to the last-resort antibiotics carbapenems and colistin is increasing worldwide. In this study, whole-genome sequencing was used to determine the colistin resistance mechanisms in clinical isolates of carbapenem- and colistin-resistant K. pneumoniae from Vietnam. Alterations in the regulatory gene mgrB, via mutations and insertion sequence transpositions, were found in 30 of 31 isolates, emphasising the importance of this resistance mechanism in colistin-resistant K. pneumoniae.


Asunto(s)
Proteínas Bacterianas/genética , Carbapenémicos/farmacología , Colistina/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Klebsiella pneumoniae/efectos de los fármacos , Elementos Transponibles de ADN , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Hospitales Pediátricos , Humanos , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Mutación Puntual , Vietnam
6.
World J Surg ; 42(2): 482-489, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29159601

RESUMEN

INTRODUCTION: Pheochromocytomas are neuroendocrine tumors of the adrenal glands. Up to 40% of the cases are caused by germline mutations in one of at least 15 susceptibility genes, making them the human neoplasms with the highest degree of heritability. Recurrent somatic alterations are found in about 50% of the more common sporadic tumors with NF1 being the most common mutated gene (20-25%). In many sporadic tumors, however, a genetic explanation is still lacking. MATERIALS AND METHODS: We investigated the genomic landscape of sporadic pheochromocytomas with whole-exome sequencing of 16 paired tumor and normal DNA samples and extended confirmation analysis in 2 additional cohorts comprising a total of 80 sporadic pheochromocytomas. RESULTS: We discovered on average 33 non-silent somatic variants per tumor. One of the recurrently mutated genes was FGFR1, encoding the fibroblast growth factor receptor 1, which was recently revealed as an oncogene in pediatric brain tumors. Including a subsequent analysis of a larger cohort, activating FGFR1 mutations were detected in three of 80 sporadic pheochromocytomas (3.8%). Gene expression microarray profiling showed that these tumors clustered with NF1-, RET,- and HRAS-mutated pheochromocytomas, indicating activation of the MAPK and PI3K-AKT signal transduction pathways. CONCLUSION: Besides RET and HRAS, FGFR1 is only the third protooncogene found to be recurrently mutated in pheochromocytomas. The results advance our biological understanding of pheochromocytoma and suggest that somatic FGFR1 activation is an important event in a subset of sporadic pheochromocytomas.


Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Feocromocitoma/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Activación Enzimática , Femenino , Perfilación de la Expresión Génica , Mutación de Línea Germinal , Humanos , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas p21(ras)/genética
7.
JAMA Oncol ; 3(9): 1204-1212, 2017 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-28384794

RESUMEN

Importance: Effective cancer prevention is based on accurate molecular diagnosis and results of genetic family screening, genotype-informed risk assessment, and tailored strategies for early diagnosis. The expanding etiology for hereditary pheochromocytomas and paragangliomas has recently included SDHA, TMEM127, MAX, and SDHAF2 as susceptibility genes. Clinical management guidelines for patients with germline mutations in these 4 newly included genes are lacking. Objective: To study the clinical spectra and age-related penetrance of individuals with mutations in the SDHA, TMEM127, MAX, and SDHAF2 genes. Design, Setting, and Patients: This study analyzed the prospective, longitudinally followed up European-American-Asian Pheochromocytoma-Paraganglioma Registry for prevalence of SDHA, TMEM127, MAX, and SDHAF2 germline mutation carriers from 1993 to 2016. Genetic predictive testing and clinical investigation by imaging from neck to pelvis was offered to mutation-positive registrants and their relatives to clinically characterize the pheochromocytoma/paraganglioma diseases associated with mutations of the 4 new genes. Main Outcomes and Measures: Prevalence and spectra of germline mutations in the SDHA, TMEM127, MAX, and SDHAF2 genes were assessed. The clinical features of SDHA, TMEM127, MAX, and SDHAF2 disease were characterized. Results: Of 972 unrelated registrants without mutations in the classic pheochromocytoma- and paraganglioma-associated genes (632 female [65.0%] and 340 male [35.0%]; age range, 8-80; mean [SD] age, 41.0 [13.3] years), 58 (6.0%) carried germline mutations of interest, including 29 SDHA, 20 TMEM127, 8 MAX, and 1 SDHAF2. Fifty-three of 58 patients (91%) had familial, multiple, extra-adrenal, and/or malignant tumors and/or were younger than 40 years. Newly uncovered are 7 of 63 (11%) malignant pheochromocytomas and paragangliomas in SDHA and TMEM127 disease. SDHA disease occurred as early as 8 years of age. Extra-adrenal tumors occurred in 28 mutation carriers (48%) and in 23 of 29 SDHA mutation carriers (79%), particularly with head and neck paraganglioma. MAX disease occurred almost exclusively in the adrenal glands with frequently bilateral tumors. Penetrance in the largest subset, SDHA carriers, was 39% at 40 years of age and is statistically different in index patients (45%) vs mutation-carrying relatives (13%; P < .001). Conclusions and Relevance: The SDHA, TMEM127, MAX, and SDHAF2 genes may contribute to hereditary pheochromocytoma and paraganglioma. Genetic testing is recommended in patients at clinically high risk if the classic genes are mutation negative. Gene-specific prevention and/or early detection requires regular, systematic whole-body investigation.


Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Predisposición Genética a la Enfermedad , Neoplasias de Cabeza y Cuello/genética , Neoplasias Primarias Secundarias/genética , Paraganglioma Extraadrenal/genética , Feocromocitoma/genética , Adolescente , Neoplasias de las Glándulas Suprarrenales/diagnóstico por imagen , Adulto , Edad de Inicio , Anciano , Anciano de 80 o más Años , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Niño , Análisis Mutacional de ADN , Detección Precoz del Cáncer/métodos , Complejo II de Transporte de Electrones/genética , Femenino , Pruebas Genéticas , Genotipo , Mutación de Línea Germinal , Neoplasias de Cabeza y Cuello/diagnóstico por imagen , Humanos , Estudios Longitudinales , Imagen por Resonancia Magnética , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Proteínas Mitocondriales/genética , Paraganglioma Extraadrenal/diagnóstico por imagen , Penetrancia , Feocromocitoma/diagnóstico por imagen , Estudios Prospectivos , Sistema de Registros , Adulto Joven
8.
Nat Rev Endocrinol ; 13(4): 233-247, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-27857127

RESUMEN

Phaeochromocytomas and paragangliomas (PPGLs) are neural-crest-derived tumours of the sympathetic or parasympathetic nervous system that are often inherited and are genetically heterogeneous. Genetic testing is recommended for patients with these tumours and for family members of patients with hereditary forms of PPGLs. Due to the large number of susceptibility genes implicated in the diagnosis of inherited PPGLs, next-generation sequencing (NGS) technology is ideally suited for carrying out genetic screening of these individuals. This Consensus Statement, formulated by a study group comprised of experts in the field, proposes specific recommendations for the use of diagnostic NGS in hereditary PPGLs. In brief, the study group recommends target gene panels for screening of germ line DNA, technical adaptations to address different modes of disease transmission, orthogonal validation of NGS findings, standardized classification of variant pathogenicity and uniform reporting of the findings. The use of supplementary assays, to aid in the interpretation of the results, and sequencing of tumour DNA, for identification of somatic mutations, is encouraged. In addition, the study group launches an initiative to develop a gene-centric curated database of PPGL variants, with annual re-evaluation of variants of unknown significance by an expert group for purposes of reclassification and clinical guidance.


Asunto(s)
Neoplasias de las Glándulas Suprarrenales/diagnóstico , Conferencias de Consenso como Asunto , Pruebas Genéticas/tendencias , Paraganglioma/diagnóstico , Feocromocitoma/diagnóstico , Neoplasias de las Glándulas Suprarrenales/genética , Predisposición Genética a la Enfermedad/genética , Pruebas Genéticas/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/tendencias , Humanos , Paraganglioma/genética , Feocromocitoma/genética
9.
Genes Chromosomes Cancer ; 55(5): 452-9, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26773571

RESUMEN

Pheochromocytomas (PCC) and abdominal paragangliomas (PGL) display a highly diverse genetic background and recent gene expression profiling studies have shown that PCC and PGL (together PPGL) alter either kinase signaling pathways or the pseudo-hypoxia response pathway dependent of the genetic composition. Recurrent mutations in the Harvey rat sarcoma viral oncogene homolog (HRAS) have recently been verified in sporadic PPGLs. In order to further establish the HRAS mutation frequency and to characterize the associated expression profiles of HRAS mutated tumors, 156 PPGLs for exon 2 and 3 hotspot mutations in the HRAS gene was screened, and compared with microarray-based gene expression profiles for 93 of the cases. The activating HRAS mutations G13R, Q61R, and Q61K were found in 10/142 PCC (7.0%) and a Q61L mutation was revealed in 1/14 PGL (7.1%). All HRAS mutated cases included in the mRNA expression profiling grouped in Cluster 2, and 21 transcripts were identified as altered when comparing the mutated tumors with 91 HRAS wild-type PPGL. Somatic HRAS mutations were not revealed in cases with known PPGL susceptibility gene mutations and all HRAS mutated cases were benign. The HRAS mutation prevalence of all PPGL published up to date is 5.2% (49/950), and 8.8% (48/548) among cases without a known PPGL susceptibility gene mutation. The findings support a role of HRAS mutations as a somatic driver event in benign PPGL without other known susceptibility gene mutations. HRAS mutated PPGL cluster together with NF1- and RET-mutated tumors associated with activation of kinase-signaling pathways.


Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Regulación Neoplásica de la Expresión Génica , Genes ras , Mutación , Feocromocitoma/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven
10.
Genes Chromosomes Cancer ; 54(9): 542-54, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26032282

RESUMEN

As subsets of pheochromocytomas (PCCs) lack a defined molecular etiology, we sought to characterize the mutational landscape of PCCs to identify novel gene candidates involved in disease development. A discovery cohort of 15 PCCs wild type for mutations in PCC susceptibility genes underwent whole-exome sequencing, and an additional 83 PCCs served as a verification cohort for targeted sequencing of candidate mutations. A low rate of nonsilent single nucleotide variants (SNVs) was detected (6.1/sample). Somatic HRAS and EPAS1 mutations were observed in one case each, whereas the remaining 13 cases did not exhibit variants in established PCC genes. SNVs aggregated in apoptosis-related pathways, and mutations in COSMIC genes not previously reported in PCCs included ZAN, MITF, WDTC1, and CAMTA1. Two somatic mutations and one constitutional variant in the well-established cancer gene lysine (K)-specific methyltransferase 2D (KMT2D, MLL2) were discovered in one sample each, prompting KMT2D screening using focused exome-sequencing in the verification cohort. An additional 11 PCCs displayed KMT2D variants, of which two were recurrent. In total, missense KMT2D variants were found in 14 (11 somatic, two constitutional, one undetermined) of 99 PCCs (14%). Five cases displayed somatic mutations in the functional FYR/SET domains of KMT2D, constituting 36% of all KMT2D-mutated PCCs. KMT2D expression was upregulated in PCCs compared to normal adrenals, and KMT2D overexpression positively affected cell migration in a PCC cell line. We conclude that KMT2D represents a recurrently mutated gene with potential implication for PCC development.


Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Proteínas de Unión al ADN/genética , Exoma , Mutación , Proteínas de Neoplasias/genética , Feocromocitoma/genética , Neoplasias de las Glándulas Suprarrenales/etiología , Línea Celular Tumoral , Estudios de Cohortes , Proteínas de Unión al ADN/metabolismo , Femenino , Dosificación de Gen , Humanos , Masculino , Proteínas de Neoplasias/metabolismo , Feocromocitoma/etiología , Feocromocitoma/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Análisis de Secuencia de Proteína , Transcriptoma
11.
Endocr Pathol ; 26(1): 9-14, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25403449

RESUMEN

Pheochromocytomas (PCCs) are tumors originating from the adrenal medulla displaying a diverse genetic background. While most PCCs are sporadic, about 40 % of the tumors have been associated with constitutional mutations in one of at least 14 known susceptibility genes. As 25 % of sporadic PCCs harbor somatic neurofibromin 1 gene (NF1) mutations, NF1 has been established as the most recurrently mutated gene in PCCs. To be able to pinpoint NF1-related pheochromocytoma (PCC) disease in clinical practice could facilitate the detection of familial cases, but the large size of the NF1 gene makes standard DNA sequencing methods cumbersome. The aim of this study was to examine whether mutations in the NF1 gene could be predicted by immunohistochemistry as a method to identify cases for further genetic characterization. Sixty-seven PCCs obtained from 67 unselected patients for which the somatic and constitutional mutational status of NF1 was known (49 NF1 wild type, 18 NF1 mutated) were investigated for NF1 protein immunoreactivity, and the results were correlated to clinical and genetic data. NF1 immunoreactivity was absent in the majority of the PCCs (44/67; 66 %), including 13 out of 18 cases (72 %) with a somatic or constitutional NF1 mutation. However, only a minority of the NF1 wild-type PCCs (18/49; 37 %) displayed retained NF1 immunoreactivity, thereby diminishing the specificity of the method. We conclude that NF1 immunohistochemistry alone is not a sufficient method to distinguish between NF1-mutated and non-mutated PCCs. In the clinical context, genetic screening therefore remains the most reliable tool to detect NF1-mutated PCCs.


Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Neurofibromina 1/análisis , Neurofibromina 1/genética , Feocromocitoma/genética , Neoplasias de las Glándulas Suprarrenales/patología , Análisis Mutacional de ADN , Marcadores Genéticos , Humanos , Inmunohistoquímica , Mutación/genética , Neurofibromina 1/metabolismo , Feocromocitoma/patología
12.
Oral Oncol ; 50(9): 832-9, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24993889

RESUMEN

OBJECTIVES: Targeted therapy against the epidermal growth factor receptor (EGFR) only variably represents a therapeutic advance in head and neck squamous cell carcinoma (HNSCC). This study addresses the need of biomarkers of treatment response to the EGFR-targeting antibody cetuximab (Erbitux®). MATERIALS AND METHODS: The intrinsic cetuximab sensitivity of HNSCC cell lines was assessed by a crystal violet assay. Gene copy number analysis of five resistant and five sensitive cell lines was performed using the Affymetrix SNP 6.0 platform. Quantitative real-time PCR was used for verification of selected copy number alterations and assessment of mRNA expression. The functional importance of the findings on the gene and mRNA level was investigated employing siRNA technology. The data was statistically evaluated using Mann-Whitney U-test and Spearman's correlation test. RESULTS: Analysis of the intrinsic cetuximab sensitivity of 32 HNSCC cell lines characterized five and nine lines as cetuximab sensitive or resistant, respectively. Gene copy number analysis of five resistant versus five sensitive cell lines identified 39 amplified protein-coding genes, including YAP1, in the genomic regions 11q22.1 or 5p13-15. Assessment using qPCR verified that YAP1 amplification associated with cetuximab resistance. Amplification of YAP1 correlated to higher mRNA levels, and RNA knockdown resulted in increased cetuximab sensitivity. Assessment of several independent clinical data sets in the public domain confirmed YAP1 amplifications in multiple tumor types including HNSCC, along with highly differential expression in a subset of HNSCC patients. CONCLUSION: Taken together, we provide evidence that YAP1 could represent a novel biomarker gene of cetuximab resistance in HNSCC cell lines.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anticuerpos Monoclonales Humanizados/uso terapéutico , Biomarcadores/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Bases , Carcinoma de Células Escamosas/tratamiento farmacológico , Línea Celular Tumoral , Cetuximab , Cartilla de ADN , Resistencia a Antineoplásicos , Silenciador del Gen , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Humanos , Fosfoproteínas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción
13.
J Clin Endocrinol Metab ; 99(7): E1352-60, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24694336

RESUMEN

CONTEXT: Pheochromocytomas and paragangliomas have a highly diverse genetic background, with a third of the cases carrying a germline mutation in 1 of 14 identified genes. OBJECTIVE: This study aimed to evaluate next-generation sequencing for more efficient genetic testing of pheochromocytoma and paraganglioma and to establish germline and somatic mutation frequencies for all known susceptibility genes. DESIGN: A targeted next-generation sequencing approach on an Illumina MiSeq instrument was used for a mutation analysis in 86 unselected pheochromocytoma and paraganglioma tumor samples. The study included the genes EGLN1, EPAS1, KIF1Bß, MAX, MEN1, NF1, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL. RESULTS were verified in tumor and constitutional DNA with Sanger sequencing. RESULTS: In all cases with clinical syndromes or known germline mutations, a mutation was detected in the expected gene. Among 68 nonfamilial tumors, 32 mutations were identified in 28 of the samples (41%), including germline mutations in EGLN1, KIF1Bß, SDHA, SDHB, and TMEM127 and somatic mutations in EPAS1, KIF1Bß, MAX, NF1, RET, and VHL, including one double monoallelic EPAS1 mutation. CONCLUSIONS: Targeted next-generation sequencing proved to be fast and cost effective for the genetic analysis of pheochromocytoma and paraganglioma. More than half of the tumors harbored mutations in the investigated genes. Notably, 7% of the apparently sporadic cases carried germline mutations, highlighting the importance of comprehensive genetic testing. KIF1Bß, which previously has not been investigated in a large cohort, appears to be an equally important tumor suppressor as MAX and TMEM127 and could be considered for genetic testing of these patients.


Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Mutación de Línea Germinal , Secuenciación de Nucleótidos de Alto Rendimiento , Paraganglioma/genética , Feocromocitoma/genética , Neoplasias de las Glándulas Suprarrenales/epidemiología , Adulto , Análisis Mutacional de ADN/métodos , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad/genética , Pruebas Genéticas , Humanos , Masculino , Persona de Mediana Edad , Paraganglioma/epidemiología , Feocromocitoma/epidemiología , Suecia/epidemiología , Adulto Joven
14.
Endocr Relat Cancer ; 21(3): 495-504, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24741025

RESUMEN

Pheochromocytomas are neuroendocrine tumors arising from the adrenal medulla. While heritable mutations are frequently described, less is known about the genetics of sporadic pheochromocytoma. Mutations in genes involved in the cellular hypoxia response have been identified in tumors, and recently EPAS1, encoding HIF2α, has been revealed to be a new gene involved in the pathogenesis of pheochromocytoma and abdominal paraganglioma. The aim of this study was to further characterize EPAS1 alterations in non-familial pheochromocytomas. Tumor DNA from 42 adrenal pheochromocytoma cases with apparently sporadic presentation, without known hereditary mutations in predisposing genes, were analyzed for mutations in EPAS1 by sequencing of exons 9 and 12, which contain the two hydroxylation sites involved in HIF2α degradation, and also exon 2. In addition, the copy number at the EPAS1 locus as well as transcriptome-wide gene expression were studied by DNA and RNA microarray analyses, respectively. We identified six missense EPAS1 mutations, three in exon 9 and three in exon 12, in five of 42 pheochromocytomas (12%). The mutations were both somatic and constitutional, and had no overlap in 11 cases (26%) with somatic mutations in NF1 or RET. One sample had two different EPAS1 mutations, shown by cloning to occur in cis, possibly indicating a novel mechanism of HIF2α stabilization through inactivation of both hydroxylation sites. One of the tumors with an EPAS1 mutation also had a gain in DNA copy number at the EPAS1 locus. All EPAS1-mutated tumors displayed a pseudo-hypoxic gene expression pattern, indicating an oncogenic role of the identified mutations.


Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Exones/genética , Mutación/genética , Paraganglioma/genética , Feocromocitoma/genética , Neoplasias de las Glándulas Suprarrenales/patología , Adulto , Secuencia de Aminoácidos , Biomarcadores de Tumor/genética , Estudios de Cohortes , Variaciones en el Número de Copia de ADN , Femenino , Estudios de Seguimiento , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Estadificación de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Paraganglioma/patología , Feocromocitoma/patología , Pronóstico , Homología de Secuencia de Aminoácido
16.
World J Surg ; 38(3): 724-32, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24322175

RESUMEN

BACKGROUND: Pheochromocytomas (PCCs) develop from the adrenal medulla and are often part of a hereditary syndrome such as von Hippel-Lindau (VHL) syndrome. In VHL, only about 30 % of patients with a VHL missense mutation develop PCCs. Thus, additional genetic events leading to formation of such tumors in patients with VHL syndrome are sought. SDHAF2 (previously termed SDH5) and SDHD are both located on chromosome 11q and are required for the function of mitochondrial complex II. While SDHAF2 has been shown to be mutated in patients with paragangliomas (PGLs), SDHD mutations have been found both in patients with PCCs and in patients with PGLs. MATERIALS AND METHODS: Because loss of 11q is a common event in VHL-associated PCCs, we aimed to investigate whether SDHAF2 and SDHD are targets. In the present study, 41 VHL-associated PCCs were screened for mutations and loss of heterozygosity (LOH) in SDHAF2 or SDHD. Promoter methylation, as well as mRNA expression of SDHAF2 and SDHD, was studied. In addition, immunohistochemistry (IHC) of SDHB, known to be a universal marker for loss of any part the SDH complex, was conducted. RESULTS AND CONCLUSIONS: LOH was found in more than 50 % of the VHL-associated PCCs, and was correlated with a significant decrease (p < 0.05) in both SDHAF2 and SDHD mRNA expression, which may be suggestive of a pathogenic role. However, while SDHB protein expression as determined by IHC in a small cohort of tumors was lower in PCCs than in the surrounding adrenal cortex, there was no obvious correlation with LOH or the level of SDHAF2/SDHD mRNA expression. In addition, the lack of mutations and promoter methylation in the investigated samples indicates that other events on chromosome 11 might be involved in the development of PCCs in association with VHL syndrome.


Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Biomarcadores de Tumor/genética , Pérdida de Heterocigocidad , Proteínas Mitocondriales/genética , Feocromocitoma/genética , Succinato Deshidrogenasa/genética , Enfermedad de von Hippel-Lindau/complicaciones , Neoplasias de las Glándulas Suprarrenales/etiología , Estudios de Cohortes , Metilación de ADN , Marcadores Genéticos , Técnicas de Genotipaje , Humanos , Mutación Missense , Feocromocitoma/etiología , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Enfermedad de von Hippel-Lindau/genética
17.
PLoS One ; 8(12): e83761, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24358309

RESUMEN

Species identification can be interesting in a wide range of areas, for example, in forensic applications, food monitoring and in archeology. The vast majority of existing DNA typing methods developed for species determination, mainly focuses on a single species source. There are, however, many instances where all species from mixed sources need to be determined, even when the species in minority constitutes less than 1 % of the sample. The introduction of next generation sequencing opens new possibilities for such challenging samples. In this study we present a universal deep sequencing method using 454 GS Junior sequencing of a target on the mitochondrial gene 16S rRNA. The method was designed through phylogenetic analyses of DNA reference sequences from more than 300 mammal species. Experiments were performed on artificial species-species mixture samples in order to verify the method's robustness and its ability to detect all species within a mixture. The method was also tested on samples from authentic forensic casework. The results showed to be promising, discriminating over 99.9 % of mammal species and the ability to detect multiple donors within a mixture and also to detect minor components as low as 1 % of a mixed sample.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mamíferos/clasificación , Mamíferos/genética , Animales , Dermatoglifia del ADN/métodos , Genética Forense , Humanos , Filogenia , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/métodos , Especificidad de la Especie
18.
Endocr Relat Cancer ; 20(4): C13-7, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23756430

RESUMEN

Patients suffering from the neurofibromatosis type 1 syndrome, which is caused by germline mutations in the NF1 gene, have a tiny but not negligible risk of developing pheochromocytomas. It is, therefore, of interest that the NF1 gene has recently been revealed to carry somatic, inactivating mutations in a total of 35 (21.7%) of 161 sporadic pheochromocytomas in two independent tumor series. A majority of the tumors in both studies displayed loss of heterozygosity at the NF1 locus and a low NF1 mRNA expression. In view of previous findings that many sporadic pheochromocytomas cluster with neurofibromatosis type 1 syndrome-associated pheochromocytomas instead of forming clusters of their own, NF1 inactivation appears to be an important step in the pathogenesis of a large number of sporadic pheochromocytomas. A literature and public mutation database review has revealed that pheochromocytomas are among those human neoplasms in which somatic NF1 alterations are most frequent.


Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Neoplasias/genética , Neurofibromina 1/genética , Feocromocitoma/genética , Mutación de Línea Germinal , Humanos
20.
Hum Mol Genet ; 21(26): 5406-16, 2012 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-23010473

RESUMEN

Pheochromocytomas are neuroendocrine tumors of the adrenal medulla which can occur either sporadically or in the context of hereditary tumor syndromes. Whereas the genetic background of hereditary pheochromocytomas is becoming rather well-defined, very little is known about the more common sporadic form of the disease which constitutes ∼70% of all cases. In this study, we elucidate some of the molecular mechanisms behind sporadic pheochromocytoma by performing a comprehensive analysis of copy number alterations, gene expression, promoter methylation and somatic mutations in the genes RET, VHL, NF1, SDHA, SDHB, SDHC, SDHD, SDHAF2, KIF1Bß, TMEM127 and MAX, which have been associated with hereditary pheochromocytoma or paraganglioma. Our genomic and genetic analyses of 42 sporadic pheochromocytomas reveal that a large proportion (83%) has an altered copy number in at least one of the known susceptibility genes, often in association with an altered messenger RNA (mRNA) expression. Specifically, 11 sporadic tumors (26%) displayed a loss of one allele of the NF1 gene, which significantly correlated with a reduced NF1 mRNA expression. Subsequent sequencing of NF1 mRNA, followed by confirmation in the corresponding genomic DNA (gDNA), revealed somatic truncating mutations in 10 of the 11 tumors with NF1 loss. Our results thus suggest that the NF1 gene constitutes the most frequent (24%) target of somatic mutations so far known in sporadic pheochromocytomas.


Asunto(s)
Genes de Neurofibromatosis 1 , Mutación de Línea Germinal , Feocromocitoma/genética , Alelos , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Heterocigoto , Humanos , Inmunohistoquímica , Análisis por Micromatrices/métodos , Feocromocitoma/patología , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo
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