RESUMO
Colorectal cancer (CRC) is a heterogeneous disease presenting with a wide spectrum of morphological and molecular characteristics sometimes even within the same patient. To understand the mechanisms of oscillations in the KRAS status we evaluated the collective of CRC patients tested using allele-specific PCR and Sanger-sequencing. Mutant KRAS allele was observed in 43.3% of cases. Repeated analysis of KRAS status in recurrent tumors or metastases was performed in 18/665 cases and a total of 6 cases with different KRAS status was found. In three cases the histological pattern of the tumor was identical. In one patient different histology and molecular status was seen between the primary and the recurrent tumor samples. In two further cases localization, histological type and KRAS mutational status all supported the occurrence of synchron/metachron colorectal tumors. In conclusion, both the progression of the original disease but also multiple tumor formation may contribute to mutation status differences during the course of colorectal carcinoma.
Assuntos
Adenocarcinoma/genética , Códon/genética , Neoplasias Colorretais/genética , Éxons/genética , Mutação/genética , Recidiva Local de Neoplasia/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , DNA de Neoplasias/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Proteínas Proto-Oncogênicas p21(ras) , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Langerhans cell histiocytosis (LCH) is a rare neoplastic disease originating from cells characterized by antigen-presenting Langerhans cell phenotype. The clinical spectrum of LCH is highly variable including localized and disseminated forms mostly occurring in children. Recently, about 60% of LCHs were reported to carry the activating BRAF mutation V600E. In our retrospective study, we evaluated the occurrence and prognostic impact of the V600E mutation in formaldehyde-fixed, paraffin-embedded samples from 15 pediatric LCH cases treated at our institution. Allele-specific polymerase chain reaction (PCR) and direct sequencing were used to demonstrate the presence of V600E mutation, and immunohistochemistry (IHC) using the mutant protein-specific VE1 antibody clone was performed to confirm mutant BRAF protein expression. Eight of 15 (53.3%) cases proved to be BRAF mutants by any of the methods applied, with a single case showing a discrepancy (PCR negative/IHC positive). Four of the BRAF-mutant cases (50.0%) showed refractory disease and progressed to death within 43 months, whereas the remaining mutant cases were stable and responded well to therapy. Wild-type BRAF cases (7/15, 46.6%) with generally comparable initial presentation were all treated successfully. In conclusion, activating V600E BRAF mutation can be frequently demonstrated in pediatric LCH by both allele-specific PCR and IHC. Unfavorable risk cases potentially also responding to BRAF-inhibitory therapy can be identified by mutation testing using archival formaldehyde-fixed, paraffin-embedded tumor samples.
Assuntos
Histiocitose de Células de Langerhans/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Adolescente , Alelos , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Estudos RetrospectivosRESUMO
BACKGROUND: Von Hippel-Lindau disease is an autosomal dominantly inherited highly penetrant tumor syndrome predisposing to retinal and central nervous system hemangioblastomas, renal cell carcinoma and phaeochromocytoma among other less frequent complications. METHODS: Molecular genetic testing of the VHL gene was performed in five unrelated families affetced with type I VHL disease, including seven patients and their available family members. RESULTS: Molecular genetic investigations detected three novel (c.163 G > T, c.232A > T and c.555C > A causing p.Glu55X, p.Asn78Tyr and p.Tyr185X protein changes, respectively) and two previously described (c.340 + 1 G > A and c.583C > T, resulting in p.Gly114AspfsX6 and p.195GlnX protein changes, respectively) germline point mutations in the VHL gene. Molecular modeling of the VHL-ElonginC-HIF-1alpha complex predicted that the p.Asn78Tyr amino acid exchange remarkably alters the 77-83 loop structure of VHL protein and destabilizes the VHL-HIF-1alpha complex suggesting that the mutation causes type I phenotype and has high risk to associate to renal cell carcinoma. The novel p.55X nonsense mutation associated to bilateral RCC and retinal angioma in a 15-year-old male patient. CONCLUSION: We describe the earliest onset renal cell carcinoma in VHL disease reported so far in a 15-year-old boy with a nonsense VHL mutation. Individual tailoring of screening schedule based on molecular genetic status should be considered in order to diagnose serious complications as early as possible. Our observations add to the understanding of genotype-phenotype correlation in VHL disease and can be useful for genetic counseling and follow-up of VHL patients.
Assuntos
Carcinoma de Células Renais/genética , Códon sem Sentido , Mutação em Linhagem Germinativa , Neoplasias Renais/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Doença de von Hippel-Lindau/genética , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Criança , Primers do DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Proteína Supressora de Tumor Von Hippel-Lindau/químicaRESUMO
Natural killer (NK) cells host in the human endometrium with dedicated role in reproductive physiology. Interestingly, malignant transformation of these specialized cells has not been presented thus far. Here we report a primary endometrial NK-cell lymphoma of a 48 year-old patient presenting with irregular bleeding. The endometrial curetting showed a dense lymphomatous infiltrate demonstrating highly infiltrative aggressive features with characteristic angiocentric, partially angiodestructive growth pattern and accompanying focal necroses. The lymphoma cells displayed a CD3ε/CD56/TIA-1/granzyme-B-positive and CD5/CD4/CD8/TCRγδ-negative immunophenotype, proved to be positive for Epstein-Barr virus by EBER in situ hybridization, and revealed no clonal T-cell receptor gene rearrangement. The diagnosis of uterine extranodal NK-cell lymphoma, nasal-type was made. Clinically, the disease was limited to the uterus at diagnosis, but progressed rapidly, and the patient died within 5 months due disseminated lymphoma, irrespective of intensive chemotherapy. Genuine NK-cell lymphomas occurring in the uterus as primary site seem to be rare making the therapeutic decisions extremely complicated.
Assuntos
Neoplasias do Endométrio/diagnóstico , Células Matadoras Naturais/patologia , Linfoma de Células T/diagnóstico , Neoplasias Nasais/diagnóstico , Neoplasias do Colo do Útero/diagnóstico , Diagnóstico Diferencial , Neoplasias do Endométrio/tratamento farmacológico , Evolução Fatal , Feminino , Humanos , Imunofenotipagem , Linfoma de Células T/tratamento farmacológico , Pessoa de Meia-Idade , Neoplasias Nasais/tratamento farmacológico , Neoplasias do Colo do Útero/tratamento farmacológicoRESUMO
Mono-ADP-ribosylation is the enzymatic transfer of ADP-ribose from NAD(+) to acceptor proteins catalyzed by ADP-ribosyltransferases. Using m-aminophenylboronate affinity chromatography, 2D-gel electrophoresis, in-gel digestion and MALDI-TOF analysis we have identified eight in vitro ADP-ribosylated proteins in Streptomyces coelicolor, which can be classified into three categories: (i) secreted proteins; (ii) metabolic enzymes using NAD(+)/NADH or NADP(+)/NADPH as coenzymes; and (iii) other proteins. The secreted proteins could be classified into two functional categories: SCO2008 and SC05477 encode members of the family of periplasmic extracellular solute-binding proteins, and SCO6108 and SC01968 are secreted hydrolases. Dehydrogenases are encoded by SC04824 and SC04771. The other targets are GlnA (glutamine synthetase I., SC02198) and SpaA (starvation-sensing protein encoded by SC07629). SCO2008 protein and GlnA had been identified as ADP-ribosylated proteins in previous studies. With these results we provided experimental support for a previous suggestion that ADP-ribosylation may regulate membrane transport and localization of periplasmic proteins. Since ADP-ribosylation results in inactivation of the target protein, ADP-ribosylation of dehydrogenases might modulate crucial primary metabolic pathways in Streptomyces. Several of the proteins identified here could provide a strong connection between protein ADP-ribosylation and the regulation of morphological differentiation in S. coelicolor.
Assuntos
Adenosina Difosfato Ribose/análise , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Streptomyces coelicolor/química , Sequência de Aminoácidos , Cromatografia de Afinidade , Eletroforese em Gel Bidimensional , Dados de Sequência Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
OBJECTIVE: Von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome caused by mutations or deletions of the VHL tumor-suppressor gene. Germline VHL gene alterations may be also present in patients with apparently sporadic pheochromocytoma (ASP), although a wide variation in mutation frequencies has been reported in different patient cohorts. DESIGN: Herein, we report the analysis of the VHL gene in Hungarian families with VHL disease and in those with ASP. METHODS: Seven families (35 members) with VHL disease and 37 unrelated patients with unilateral ASP were analyzed. Patients were clinically evaluated and the VHL gene was analyzed using direct sequencing, multiplex ligation-dependent probe amplification, and real-time PCR with SYBR Green chemistry. RESULTS: Disease-causing genetic abnormalities were identified in each of the seven VHL families and in 3 out of the 37 patients with ASP (one nonsense and six missense mutations, two large gene deletions and one novel 2 bp deletion). Large gene deletions and other genetic alterations resulting in truncated VHL protein were found only in families with VHL type 1, whereas missense mutations were associated mainly, although not exclusively, with VHL type 2B and type 2C. CONCLUSIONS: The spectrum of VHL gene abnormalities in the Hungarian population is similar to that observed in Western, Japanese, or Chinese VHL kindreds. The presence of VHL gene mutations in 3 out of the 37 patients with ASP suggests that genetic testing is useful not only in patients with VHL disease but also in those with ASP.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Mutação em Linhagem Germinativa , Feocromocitoma/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Neoplasias das Glândulas Suprarrenais/complicações , Adulto , Criança , Análise Mutacional de DNA , Família , Feminino , Testes Genéticos , Humanos , Hungria , Masculino , Pessoa de Meia-Idade , Feocromocitoma/complicações , Adulto Jovem , Doença de von Hippel-Lindau/complicações , Doença de von Hippel-Lindau/genéticaRESUMO
Von Hippel-Lindau disease (VHL) is a rare autosomal dominant disease characterized by development of cystic and tumorous lesions at multiple sites, including the brain, spinal cord, kidneys, adrenals, pancreas, epididymis and eyes. The clinical phenotype results from molecular abnormalities of the VHL tumor suppressor gene, mapped to human chromosome 3p25-26. The VHL gene encodes two functionally active VHL proteins due to the presence of two translational initiation sites separated by 53 codons. The majority of disease-causing mutations have been detected downstream of the second translational initiation site, but there are conflicting data as to whether few mutations located in the first 53 codons, such as the Pro25Leu could have a pathogenic role. In this paper we report a large Hungarian VHL type 2 family consisting of 32 members in whom a disease-causing AGT80AAT (Ser80Ile) c.239G>A, p.Ser80Ile mutation, but not the concurrent CCT25CTT (Pro25Leu) c.74C>T, p.Pro25Leu variant co-segregated with the disease. To our knowledge, the Ser80Ile mutation has not been previously described in VHL type 2 patients with high risk of pheochromocytoma and renal cell cancer. Therefore, this finding represents a novel genotype-phenotype association and VHL kindreds with Ser80Ile mutation will require careful surveillance for pheochromocytoma. We concluded that the Pro25Leu variant is a rare, neutral variant, but the presence such a rare gene variant may make genetic counseling difficult.
Assuntos
Mutação , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Doença de von Hippel-Lindau/genética , Sequência de Aminoácidos , Feminino , Humanos , Masculino , Linhagem , Prolina/genética , Estrutura Secundária de Proteína , Alinhamento de Sequência , Serina/genética , Proteína Supressora de Tumor Von Hippel-Lindau/químicaRESUMO
We explore the possibilities offered by flow cytometric microbead analysis to develop high throughput methods for the detection of deletions/insertions and single-strand DNA lesions. The products of PCR reactions derived from reference and test samples are denatured and reannealed, then exposed to enzymatic or chemical treatments distinguishing homoduplices from heteroduplices. The biotin- and dye labeled reaction products are immobilized on microbeads and the homo- and heteroduplices are assessed in separate fluorescence channels, by flow cytometry. Using a model system based on the mixed lineage leukemia gene breakpoint cluster region, we demonstrate that deletions and insertions in genomic DNA can be detected, using S1 nuclease and chemical cleavage to distinguish hetero- from homoduplices, or a restriction enzyme cleaving only the homoduplices. Single-strand discontinuities can also be detected, by combining nick-translation, using labeled nucleotide, and flow cytometric microbead analysis. The methodical approaches demonstrated are applicable in a versatile manner in basic cell and molecular biological research and also promise direct application for high throughput screening of genetic diseases and lesions, including insertions or deletions of short sequence elements and single-strand lesions formed at hypersensitive sites in response to apoptotic stimuli.
Assuntos
DNA de Cadeia Simples/análise , Citometria de Fluxo/métodos , Deleção de Genes , Análise Heteroduplex/métodos , Microesferas , Mutagênese Insercional/métodos , DNA de Cadeia Simples/genética , HumanosRESUMO
PURPOSE: To identify mutations in the Transforming Growth Factor Beta Induced (TGFBI) gene in Hungarian patients with corneal dystrophy and to characterize histological features of their corneal buttons excised during penetrating keratoplasty. METHODS: Exons of TGFBI were sequenced in 38 members of 15 unrelated families with corneal dystrophy and exon 12 was also sequenced in 100 healthy controls from the same population. Immunohistological analysis of available corneal buttons excised during penetrating keratoplasty was also performed. RESULTS: Molecular genetic analysis revealed a heterozygous R124C mutation in 18 patients with lattice type I dystrophy. A R555W heterozygous mutation was detected in five patients with granular Groenouw type I corneal dystrophy and a R555Q heterozygous mutation was found in four patients clinically diagnosed with Reis-Bücklers (one patient) and Thiel-Behnke (three patients) dystrophy. Three patients with "atypical granular" dystrophy later diagnosed as Avellino dystrophy were heterozygous for the R124H mutation. A novel heterozygous mutation (T1640C) causing a F547S amino acid exchange was detected in a patient with polymorphic corneal amyloidosis. Immunohistochemistry showed the presence of BIGH3 protein deposits in all examined corneal buttons. Electron microscopy confirmed the presence of amyloid fibrils in the case of the novel mutation. CONCLUSIONS: Our results indicate that molecular genetic analysis is required to confirm the diagnosis of corneal dystrophies. We report the first cases of Avellino dystrophy from Central-Eastern Europe. We conclude that the novel F547S mutation causes polymorphic corneal amyloidosis since no other mutations were detected in the TGFBI gene of this patient and the novel mutation could not be found in healthy controls.