RESUMEN
PURPOSE: Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors of the gastrointestinal tract. Most (80 %) contain activating mutations in the KIT receptor tyrosine kinase, roughly 10 % in platelet-derived growth factor receptor-alpha (PDGFRA). In a small subset, BRAF mutations are an alternative molecular pathway. GISTs respond well to imatinib, but low response is seen in patients with wild-type KIT or PDGFRA. Resistance has also been reported as a result of mutations in downstream effectors such as BRAF. METHODS: We provide here a molecular characterization of a series of primary GISTs from Italian patients. Of 121 GIST cases diagnosed between 2000 and 2012, 83 were evaluated by PCR amplification and direct sequencing for mutations in KIT exons 8, 9, 11, 13, and 17, PDGFRA exons 12, 14, and 18, and BRAF exon 15. Eighty-one GISTs also underwent K-RAS testing. RESULTS: Sixty-four GISTs were positive: 55 had mutations in KIT and 9 in PDGFRA; 16 patients were mutation negative. Three samples came from NF1 patients and were KIT- and PDGFRA negative. Overall, we identified six novel mutations in KIT (p.K550_M552delinsL, p.Q556_W557delinsG p.Q556_G575del, p.W557_V559delinsQ p.P573_R588dup, p.G592_K593dup) and one novel mutation in PDGFRA (p.D842_N848delinsVDV), thus contributing to widening the spectrum of known mutations in GIST tumors and confirming the most frequently altered regions underlying GIST development. CONCLUSIONS: Among the 64 KIT- and PDGFRA-positive sporadic patients in our series, no BRAF or KRAS mutations were identified, suggesting that co-occurrence of these mutations is likely to be rare in the northwestern Italian population and not a frequent cause of primary resistance to imatinib in KIT-positive GIST patients.
Asunto(s)
Tumores del Estroma Gastrointestinal/genética , Mutación/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Proteínas ras/genética , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/uso terapéutico , Benzamidas/uso terapéutico , Biomarcadores de Tumor/genética , Femenino , Estudios de Seguimiento , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Humanos , Mesilato de Imatinib , Masculino , Persona de Mediana Edad , Piperazinas/uso terapéutico , Reacción en Cadena de la Polimerasa , Pronóstico , Proteínas Proto-Oncogénicas p21(ras) , Pirimidinas/uso terapéutico , Estudios RetrospectivosRESUMEN
Trichilemmal cysts (TCs) can occur as sporadic lesions or in hereditary-familial settings with autosomal dominant transmission. These entities have not been widely analyzed in their peculiar aspects yet. The aim of this study was to describe a cohort of patients with diagnosis of TCs through a clinical and biomolecular characterization, intended to highlight some effective diagnostic criteria for their identification. Among 149 cases of this study, 24 cases of TCs (16.1%) arose in patients with at least one first-degree relative with diagnosis of TCs. Peculiar findings concerning hereditary lesions included the multiple presentation with an early onset age. On the basis of clinical evaluation, we propose a panel of clinical and histologic criteria for the diagnosis of hereditary TCs, which includes: (i) the diagnosis of TCs in at least two first-degree relatives or in three first- or second-degree relatives in two consecutive generations; (ii) at least one of the patients with TCs diagnosed <45 years; and (iii) the diagnosis of multiple or giant (>5-cm lesions) or rare histopathologic features (proliferating and ossifying) TCs.
Asunto(s)
Quiste Folicular/diagnóstico , Quiste Folicular/genética , Enfermedades del Cabello/diagnóstico , Enfermedades del Cabello/genética , Receptores de Superficie Celular/genética , Adulto , Anciano , Secuencia de Bases , Quiste Epidérmico , Exones , Femenino , Quiste Folicular/patología , Quiste Folicular/cirugía , Enfermedades del Cabello/patología , Enfermedades del Cabello/cirugía , Humanos , Patrón de Herencia , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Receptores Patched , LinajeRESUMEN
A French and an Australian study have recently identified a rare germline functional variant in the microphthalmia-associated transcription factor (MITF) (E318K) that predisposes to familial and sporadic melanoma and to renal cell carcinoma (RCC), showing a new link between two tumour types with different risk factors and between deregulated sumoylation and cancer. The aim of this study was to test the prevalence of the MITF E318K mutation in 667 Italian melanoma patients. We observed significant associations between histological subtypes and family cancer history. Carriers exhibited a nearly threefold higher risk of developing melanoma compared with controls. Carriers were also more likely to have developed multiple primary melanomas (6.40-fold), compared with wt patients. Carriers with a personal and/or family history of pancreatic cancer and kidney cancer had a nearly 31- and eightfold higher risk of developing melanoma compared with wt patients. Our findings further support MITF as a medium-penetrance melanoma susceptibility gene, highlight a potential association with histological subtypes and suggest that MITF may predispose to pancreatic cancer.
Asunto(s)
Sustitución de Aminoácidos/genética , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal/genética , Melanoma/genética , Melanoma/patología , Factor de Transcripción Asociado a Microftalmía/genética , Neoplasias Cutáneas/genética , Estudios de Casos y Controles , Familia , Femenino , Humanos , Italia , Masculino , Linaje , Prevalencia , Neoplasias Cutáneas/patologíaRESUMEN
Pancreatic adenocarcinoma (PC) is the third most common cancer associated with BRCA mutations. Most notice has been given to BRCA2, while the association between BRCA1 and PC is less widely reported. Recently, PALB2 has been implicated in both PC and breast cancer (BC) susceptibility. We selected 29 Italian PC patients from a case-control study of PC according to their personal and family history of both PC and breast/ovarian cancer (BC/OC) and tested them for presence of germline mutations in BRCA1, BRCA2 and PALB2. We identified no germline mutations or deletions in PALB2, but detected 7 BRCA mutations (4 in BRCA1 and 3 in BRCA2). These findings suggest that PALB2 does not play a major role in PC susceptibility in our population. As we found an almost equal frequency of germline mutations in BRCA1 and BRCA2, germline alterations in either of these genes may explain a subset of Italian families presenting both PC and BC/OC. Moreover, as we began the observation of these families from probands who are affected by PC, we provide here a direct assessment of the role of PALB2 and BRCA mutations in PC susceptibility.
Asunto(s)
Adenocarcinoma/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Eliminación de Gen , Mutación de Línea Germinal/genética , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Proteínas Supresoras de Tumor/genética , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/genética , Estudios de Casos y Controles , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Femenino , Predisposición Genética a la Enfermedad , Humanos , Italia , Masculino , Persona de Mediana Edad , Neoplasias Ováricas/genética , LinajeRESUMEN
BACKGROUND: Carrying the cyclin-dependent kinase inhibitor 2A (CDKN2A) germline mutations is associated with a high risk for melanoma. Penetrance of CDKN2A mutations is modified by pigmentation characteristics, nevus phenotypes, and some variants of the melanocortin-1 receptor gene (MC1R), which is known to have a role in the pigmentation process. However, investigation of the associations of both MC1R variants and host phenotypes with melanoma risk has been limited. METHODS: We included 815 CDKN2A mutation carriers (473 affected, and 342 unaffected, with melanoma) from 186 families from 15 centers in Europe, North America, and Australia who participated in the Melanoma Genetics Consortium. In this family-based study, we assessed the associations of the four most frequent MC1R variants (V60L, V92M, R151C, and R160W) and the number of variants (1, ≥2 variants), alone or jointly with the host phenotypes (hair color, propensity to sunburn, and number of nevi), with melanoma risk in CDKN2A mutation carriers. These associations were estimated and tested using generalized estimating equations. All statistical tests were two-sided. RESULTS: Carrying any one of the four most frequent MC1R variants (V60L, V92M, R151C, R160W) in CDKN2A mutation carriers was associated with a statistically significantly increased risk for melanoma across all continents (1.24 × 10(-6) ≤ P ≤ .0007). A consistent pattern of increase in melanoma risk was also associated with increase in number of MC1R variants. The risk of melanoma associated with at least two MC1R variants was 2.6-fold higher than the risk associated with only one variant (odds ratio = 5.83 [95% confidence interval = 3.60 to 9.46] vs 2.25 [95% confidence interval = 1.44 to 3.52]; P(trend) = 1.86 × 10(-8)). The joint analysis of MC1R variants and host phenotypes showed statistically significant associations of melanoma risk, together with MC1R variants (.0001 ≤ P ≤ .04), hair color (.006 ≤ P ≤ .06), and number of nevi (6.9 × 10(-6) ≤ P ≤ .02). CONCLUSION: Results show that MC1R variants, hair color, and number of nevi were jointly associated with melanoma risk in CDKN2A mutation carriers. This joint association may have important consequences for risk assessments in familial settings.
Asunto(s)
Genes p16 , Heterocigoto , Melanoma/genética , Mutación , Receptor de Melanocortina Tipo 1/genética , Neoplasias Cutáneas/genética , Adulto , Australia , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Europa (Continente) , Femenino , Color del Cabello , Humanos , Masculino , Nevo/complicaciones , Nevo/genética , América del Norte , Fenotipo , Medición de Riesgo , Factores de Riesgo , Pigmentación de la Piel , Quemadura Solar/complicaciones , Población Blanca/genéticaRESUMEN
Lynch syndrome is an inherited cancer syndrome caused by germline mutations in mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. LS predisposes to high risk of early-onset colorectal, endometrial and other tumors. Patients with Lynch syndrome have also been shown to have an elevated risk for pancreatic cancer (PC). In this study, we aimed to estimate the frequency of suspected Lynch syndrome among a series of 135 PC patients. Further, we wanted to determine the frequency of MMR gene mutations in the suspected Lynch syndrome cases. We also aimed to verify the pathogenicity of any novel non-truncating variants we might detect with a functional assay. Based on personal and/or familial cancer history, 19 patients were classified as suspected Lynch syndrome cases. DNA material for mutation analysis was available for eleven of them. Four patients were found to carry a total of five MLH1 or MSH2 variants. Of these, MSH2-Q402X, MSH2-G322D, and MLH1-K618A had been previously reported, while the MSH2-E205Q and MSH2-V367I variants were novel. MSH2-Q402X is a known stop mutation and reported here for the first time here in association with PC. MLH1-K618A was found in the unaffected branch of a kindred, suggesting that it may be a polymorphism or a low penetrance variant. MSH2-G322D likely does not cause a MMR defect, although this variant has also been associated with breast cancer as indeed seen in our patient. The novel variants MSH2-E205Q and MSH2-V367I were found in the same patient. Both novel variants were however functional in the applied MMR assay. Our findings suggest that only a small subset of pancreatic cancer patients carry pathogenic MMR mutations.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Predisposición Genética a la Enfermedad , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Adulto , Secuencia de Bases , Neoplasias Colorrectales Hereditarias sin Poliposis/complicaciones , Reparación de la Incompatibilidad de ADN/genética , Análisis Mutacional de ADN , Femenino , Mutación de Línea Germinal , Humanos , Inmunohistoquímica , Italia , Masculino , Inestabilidad de Microsatélites , Persona de Mediana Edad , Homólogo 1 de la Proteína MutL , Linaje , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We evaluated the contribution of germline CDKN2A mutations and MC1R variants to the development of melanoma in a hospital-based study of single (SPM, n = 398) and multiple primary melanoma (MPM, n = 95). The overall frequency of CDKN2A mutations was 15.2%, and four-fold higher in MPM than in SPM cases (OR = 4.27; 95% CI 2.43-7.53). The likelihood of identifying a CDKN2A mutation increased with family history of melanoma and younger age at diagnosis in MPM cases. Compared to SPM patients, the risk of harboring a CDKN2A mutation rose as the number of primary melanomas increased and was not influenced by family history. The G101W and E27X founder mutations were the most common. Several other mutations (W15X, Q50X, R58X, A68L, A127P and H142R) were detected for the first time in Italian patients. One novel mutation, T77A, was identified. Several non-coding variants with unknown functional significance were also found (5'UTR -25C > T, -21C > T, -67G > C, IVS1 +37G > C); the novel 5'UTR -21C > T variant was not detected in controls. The CDKN2A A148T polymorphism was more frequent in MPM patients than in the control population (15.7% versus 6.6%). Compared to the SPM patients, MPM cases had a 2-fold increased probability of being MC1R variant carriers and a higher probability of carrying two or more variants. No specific association was observed between the type of variant and the number of melanomas, suggesting that the number rather than the type of MC1R variant increases the risk of MPM. We observed no interaction between CDKN2A status and the presence of MC1R variants. The high frequency of CDKN2A mutations in our MPM cases, independent of their family history, is of relevance to genetic counseling and testing in our population.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Variación Genética/genética , Mutación de Línea Germinal/genética , Melanoma/genética , Receptor de Melanocortina Tipo 1/genética , Neoplasias Cutáneas/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Quinasa 4 Dependiente de la Ciclina/genética , Femenino , Humanos , Masculino , Melanoma/clasificación , Melanoma/patología , Persona de Mediana Edad , Factores de Riesgo , Neoplasias Cutáneas/clasificación , Neoplasias Cutáneas/patología , Adulto JovenRESUMEN
Mutations in the PTCH gene, the human homolog of the Drosophila patched gene, have been found to lead to the autosomal dominant disorder termed Nevoid Basal Cell Carcinoma Syndrome (NBCCS, also called Gorlin Syndrome). Patients display an array of developmental anomalies and are prone to develop a variety of tumors, with multiple Basal Cell Carcinomas occurring frequently. We provide here the results of molecular testing of a set of Italian Nevoid Basal Cell Carcinoma Syndrome patients. Twelve familial patients belonging to 7 kindreds and 5 unaffected family members, 6 non-familial patients and an additional set of 7 patients with multiple Basal Cell Carcinoma but no other criteria for the disease were examined for mutations in the PTCH gene. All of the Nevoid Basal Cell Carcinoma Syndrome patients were found to carry variants of the PTCH gene. We detected nine novel mutations (1 of which occurring twice): 1 missense mutation (c.1436T>G [p.L479R]), 1 nonsense mutation (c.1138G>T [p.E380X]), 6 frameshift mutations (c.323_324ins2, c.2011_2012dup, c.2535_2536dup, c.2577_2583del, c.3000_3005del, c.3050_3051del), 1 novel splicing variant (c.6552A>T) and 3 mutations that have been previously reported (c.3168+5G>A, c.1526G>T [p.G509V], and c.3499G>A [p.G1167R]). None of the patients with multiple Basal Cell Carcinoma but no other criteria for the syndrome, carried germline coding region mutations.
Asunto(s)
Síndrome del Nevo Basocelular/genética , Codón sin Sentido , Mutación del Sistema de Lectura , Mutación Missense , Mutación Puntual , Sitios de Empalme de ARN/genética , Receptores de Superficie Celular/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Niño , Secuencia de Consenso , Análisis Mutacional de ADN , Femenino , Humanos , Italia , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Receptores Patched , Receptor Patched-1 , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Roughly 40% of germinal mutations in melanoma families (MF) affect p16(INK4a) and p14(ARF). We investigated the association between INK4/ARF alterations and the occurrence of pancreatic cancer in MF and in sporadic pancreatic cancer (SPC) patients. PATIENTS AND METHODS: Forty-nine MF, 66 SPC cases and 54 controls were enrolled. The INK4/ARF locus was screened. RESULTS: As compared with the general population, the risk of pancreatic cancer (PC) was increased 9.4-fold [95% confidence interval (CI) 2.7-33.4] and 2.2-fold (95% CI 0.8-5.7) in G101W-positive and -negative MF, respectively, while mean ages at onset were 61 and 77 years, respectively. A 1.7 (95% CI 1.06-2.79) increased risk of cancer at any site was observed among first-degree relatives of SPC cases as compared with controls. The G101W founder mutation was detected in 4% of SPC cases but the rate increased to 13% when tumor clustering in either branch of families was taken into account. One G101W-positive PC patient with a melanoma in a first-degree relative harbored a germline deletion of the second allele, including exon 1B. CONCLUSIONS: The presence of a deletion including exon 1B in two PC patients points to the involvement of p14(ARF) in the development of PC and may suggest that the increased risk of PC in MF is caused by impairment of both loci.
Asunto(s)
Adenocarcinoma/genética , Genes p16 , Mutación de Línea Germinal , Neoplasias Pancreáticas/genética , Proteína p14ARF Supresora de Tumor/genética , Adenocarcinoma/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Análisis Mutacional de ADN , Femenino , Predisposición Genética a la Enfermedad , Heterocigoto , Humanos , Incidencia , Masculino , Melanoma/epidemiología , Melanoma/genética , Melanoma/fisiopatología , Persona de Mediana Edad , Datos de Secuencia Molecular , Neoplasias Pancreáticas/epidemiología , Linaje , Reacción en Cadena de la PolimerasaRESUMEN
Multiple primary cancers are one of the hallmarks of inherited predisposition. Outside the familial context, multiple primary tumors could be related either to germline de novo mutations or to low-penetrance mutations, in predisposing genes. We selected 100 patients who displayed multiple primary melanoma (MPM) without any known melanoma cases recorded within their families and looked for germline mutations in the two melanoma-predisposing genes identified to date, CDKN2A and CDK4 exon 2. Nine patients (9%) had germline mutations in CDKN2A, whereas none carried germline mutations in exon 2 of CDK4. Seven cases displayed a recurrent missense mutation, G101W, already described in more than 20 melanoma-prone families; one case carried a missense mutation never reported to date (P114S), and the last case was a carrier of a 6 bp insertion at nucleotide 57 resulting in a duplication of codons 18 and 19. To ascertain whether the G101W was a mutational hot spot for de novo mutations or a common founder mutation, we genotyped eight microsatellite markers flanking the CDKN2A gene. After allowing for recombination over time, haplotype sharing provided evidence for an original G101W mutation common to 6 out of 7 sporadic MPM cases. Therefore, it can be concluded that de novo germline CDKN2A mutations associated with MPM are rare.
Asunto(s)
Quinasas Ciclina-Dependientes/genética , Efecto Fundador , Genes p16 , Mutación de Línea Germinal/genética , Melanoma/genética , Neoplasias Primarias Múltiples/genética , Proteínas Proto-Oncogénicas , Neoplasias Cutáneas/genética , Adulto , Quinasa 4 Dependiente de la Ciclina , Análisis Mutacional de ADN/métodos , Femenino , Tamización de Portadores Genéticos , Predisposición Genética a la Enfermedad/genética , Genotipo , Humanos , Masculino , Persona de Mediana Edad , LinajeRESUMEN
Physical interaction between CDKN2A/p16 and CDK4 proteins regulates the cell cycle progression through the G1 phase and dysfunction of these proteins by gene mutation is implicated in genetic predisposition to melanoma. We analysed 15 Italian melanoma families for germ line mutations in the coding region of the CDKN2A gene and exon 2 of the CDK4 gene. One novel disease-associated mutation (P48T), 3 known pathological mutations (R24P, G101W and N71S) and 2 common polymorphisms (A148T and Nt500 G>C) were identified in the CDKN2A gene. In a family harbouring the R24P mutation, an intronic variant (IVS1, +37 G>C) of uncertain significance was detected in a non-carrier melanoma case. The overall incidence of CDKN2A mutations was 33.3%, but this percentage was higher in families with 3 or more melanoma cases (50%) than in those with only 2 affected relatives (25%). Noteworthy, functional analysis established that the novel mutated protein, while being impaired in cell growth and inhibition assays, retains some in vitro binding to CDK4/6. No variant in the p16-binding region of CDK4 was identified in our families. Our results, obtained in a heterogeneous group of families, support the view that inactivating mutations of CDKN2A contribute to melanoma susceptibility more than activating mutations of CDK4 and that other genetic factors must be responsible for melanoma clustering in a high proportion of families. In addition, they indicate the need for a combination of functional assays to determine the pathogenetic nature of new CDKN2A mutations.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Quinasas Ciclina-Dependientes/genética , Mutación de Línea Germinal , Melanoma/genética , Proteínas Proto-Oncogénicas , Neoplasias Cutáneas/genética , Adulto , Anciano , Ciclo Celular , División Celular , Quinasa 4 Dependiente de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Análisis Mutacional de ADN , ADN de Neoplasias/análisis , Susceptibilidad a Enfermedades , Exones , Femenino , Fase G1 , Ligamiento Genético , Pruebas Genéticas , Humanos , Italia/epidemiología , Masculino , Persona de Mediana Edad , Linaje , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Transcripción GenéticaRESUMEN
In an attempt to identify genes encoding triple-helical DNA-binding proteins, we performed South-Western screening of a human keratinocyte cDNA expression library using a purine (Pu)-rich triplex DNA probe. We isolated two independent clones containing part of the loricrin gene. Both were translated with a different reading frame than that of the loricrin protein, the major component of the cell envelope during normal keratinocyte cornification. The affinity of the encoded polypeptide for Pu-triplex DNA was confirmed by electrophoretic mobility shift assays using a bacterially expressed N-terminal loricrin deletion fused with the maltose-binding protein (MBP-LOR3ARF). Interactions between Pu-triplex DNA and MBP-LOR3ARF are characterized by a distribution of four increasingly slower mobility complexes, suggesting that multiple MBP-LOR3ARF molecules can recognize a single triplex. Binding was also observed between MBP-LOR3ARF and a pyrimidine-motif triplex DNA, although at lower affinity than Pu-triplex DNA. No apparent binding was observed between MBP-LOR3ARF and double-stranded DNA, suggesting that MBP-LOR3ARF is a bona fide Pu-triplex binding protein. Finally, purified specific rabbit antibodies against LORARF detected four human proteins with apparent molecular masses of 210, 110, 68, and 66 kDa on Western blot analysis. The 210-, 110-, and 68-kDa proteins also showed specific Pu-triplex DNA binding in a South-Western experiment, suggesting that LORARF shares common domains with other human Pu-triplex DNA-binding proteins.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de la Membrana/genética , Sistemas de Lectura/genética , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Secuencia de Bases , Clonación Molecular , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/inmunología , Células HeLa , Humanos , Queratinocitos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Sondas de Oligonucleótidos , Unión Proteica , Proteínas Recombinantes de Fusión , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunologíaRESUMEN
The formation of triple-helical DNA has been implicated in several cellular processes, including transcription, replication and recombination. While there is no direct evidence for triplexes in vivo, cellular proteins that specifically recognize triplex DNA have been described. Using a purine-motif triplex probe and southwestern library screening, we isolated five independent clones expressing the same C-terminal 210 amino acids of the Saccharomyces cerevisiae protein Cdp1p fused with beta-galactosidase. In electrophoretic mobility shift assays, recombinant Cdp1pDelta1-867 bound Pu-motif triplex DNAs with high affinity (K:(d) approximately 5 nM) and bound Py-motif triplex, duplex and single-stranded DNAs with far lower affinity (0.5-5.0 microM). Genetic analyses revealed that the CDP1 gene product was required for proper chromosome segregation. The possible involvement of triplex DNA in this process is discussed.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Unión Competitiva , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , División Celular , Segregación Cromosómica , Clonación Molecular , ADN/química , ADN/genética , Sondas de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Biblioteca Genómica , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Eliminación de Secuencia/genética , Especificidad por Sustrato , Factores de Elongación TranscripcionalRESUMEN
Germline mutations within the coding region of CDKN2A have been observed in affected members of melanoma-prone families. G101W is the most common CDKN2A missense mutation identified to date. It has been reported in several families from around the world, with a particularly high occurrence in France and Italy. Given the frequency of this mutation, we were interested in determining whether the mutation resulted from a single origin or represented a mutational hotspot in the CDKN2A gene. In addition, given the geographical distribution of the mutation, we examined the date of origination of the mutation and its migratory spread. We examined 10 families from Italy, 4 families from the United States, and 6 families from France with the G101W mutation. The following eight markers were employed for the haplotype analysis: IFNA, D9S736, D9S1749, D9S942, D9S1748, D9S1604, D9S171, and D9S126. Our findings showed no significant evidence for mutational heterogeneity, suggesting that all studied families derived from a single ancestral haplotype on which the mutation arose. Using maximum-likelihood methods, we estimated the mutation to have arisen 97 generations ago (1-LOD-unit support interval 70-133 generations) providing some explanation for the wide geographical spread of this common mutation, particularly in southwestern Europe. The presence of a founder mutation in a defined geographic area can facilitate carrier detection and genetic counseling and can provide an opportunity to study disease penetrance and the effect of environmental factors on the background of a common genetic susceptibility.
Asunto(s)
Sustitución de Aminoácidos/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Efecto Fundador , Mutación de Línea Germinal/genética , Melanoma/genética , Femenino , Francia , Frecuencia de los Genes/genética , Heterogeneidad Genética , Marcadores Genéticos/genética , Predisposición Genética a la Enfermedad/genética , Genotipo , Haplotipos/genética , Humanos , Italia , Funciones de Verosimilitud , Masculino , Linaje , Factores de Tiempo , Estados UnidosRESUMEN
Germline mutations of CDKN2A, at 9p21, are responsible for predisposition to melanoma in some families. However, evidence of linkage to 9p21 has been demonstrated in a significant proportion of kindreds with no detectable mutations in CDKN2A. It is possible that mutations in noncoding regions may be responsible for predisposition to melanoma in these families. We have analyzed approximately 1 kb of the CDKN2A promoter upstream of the start codon in an attempt to identify causal mutations in 107 melanoma families. Four sequence variants were detected. Two of these (A-191G and A-493T) did not segregate with disease and were present in a control population at a comparable frequency, indicating that they are unlikely to predispose to melanoma. The A-493T variant appeared to be in linkage disequilibrium with the previously described CDKN2A polymorphism Ala148Thr. The variant G-735A was detected in the control population, but segregation of this variant with melanoma within families could not be discounted. The fourth variant (G-34T), located in the 5' UTR, creates an aberrant initiation codon. This variant appeared to segregate with melanoma and was not detected in a control population. G-34T has recently been identified in a subset of Canadian melanoma families and was concluded to be associated with predisposition to melanoma. The creation of an aberrant initiation site in the 5' UTR may have an important role in carcinogenesis in a small percentage of families; however, mutations in the CDKN2A promoter appear to have a limited role in predisposition to melanoma.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Pruebas Genéticas/métodos , Melanoma/genética , Regiones Promotoras Genéticas/genética , Adolescente , Adulto , Anciano , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal/genética , Guanina/análisis , Humanos , Desequilibrio de Ligamiento/genética , Linaje , Mutación Puntual , Timina/análisisRESUMEN
Germline mutations impairing the p16(INK4)-function have previously been demonstrated to be responsible for genetic predisposition in at least one half of melanoma-prone kindreds of North European origin. Familial melanoma kindreds have also been found to present an increased risk of pancreatic cancer and other cancers, but results relative to more common neoplasias incidence, in particular, are heterogeneous. We report here a clinical-epidemiological study, including the presence of additional neoplasias, in 14 apparently unrelated kindreds coming from a small geographic region of Northern Italy (Liguria), having therefore lived for generations in similar environmental conditions. We identified the common p16 missense mutation (Gly101Trp) reported in several previously studied kindreds, in 7 of 14 families, whereas the remaining 7 families had no detectable mutations in the coding region of p16 gene. Median age at diagnosis and other melanoma features were studied. When compared with the expected figures, based on regional incidence rates, a significant excess of pancreatic cancer, with 4 cases diagnosed, and of breast cancer, with 7 cases, was observed. The 7 families without apparent CDKN2A involvement were also negative for hot-spot exon 2 mutation of CDK4. Environmental factors do not appear to play a role in the excess of non-melanoma neoplasia in our families, as somewhat substantiated by the control group, composed of spouses and members of non-affected branches; they do not reveal any increased cancer incidence compared with the general population. Furthermore, given the proven significance of interaction between the melanoma susceptibility gene and the propensity to sunburns and other environmental risk factors, our results, obtained from a small but homogeneous sample, may have important implications for further risk assessment studies.
Asunto(s)
Neoplasias de la Mama/genética , Genes p16 , Melanoma/genética , Neoplasias Pancreáticas/genética , Neoplasias Cutáneas/genética , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/epidemiología , Niño , Análisis Mutacional de ADN , Exposición a Riesgos Ambientales , Femenino , Humanos , Incidencia , Italia/epidemiología , Masculino , Melanoma/epidemiología , Persona de Mediana Edad , Neoplasias Primarias Secundarias/epidemiología , Neoplasias Primarias Secundarias/genética , Neoplasias Pancreáticas/epidemiología , Linaje , Medición de Riesgo , Factores Sexuales , Neoplasias Cutáneas/epidemiologíaRESUMEN
OBJECTIVES: The purpose of this study was to analyse the presence of the granulocyte-macrophage colony-stimulating factor (GM-CSF) in human cerebrospinal fluid (SF) of patients affected by multiple sclerosis (MS) in comparison with non-inflammatory neurological diseases. MATERIAL AND METHODS: All SFs were collected from 59 patients for diagnostic purpose. The presence of GM-CSF was revealed by measuring its activity and by immunoassay. The data obtained were statistically evaluated. RESULTS: We found that GM-CSF is constitutively present in human SF; this presence was confirmed by its stimulating activity of colony-forming-unit granulocyte-macrophage (CFU-GM) production. No significant changes of the GM-CSF concentration in the SFs were observed among different neurological disorders (degenerative or vascular) and MS. CONCLUSION: Our data suggest that GM-CSF is a constitutive component of human SF, relatively uninfluenced by the different morbid conditions of the nervous system.
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Factor Estimulante de Colonias de Granulocitos y Macrófagos/líquido cefalorraquídeo , Esclerosis Múltiple/líquido cefalorraquídeo , Western Blotting , HumanosRESUMEN
The expression of intercellular adhesion molecule-1 (ICAM-1) and granulocyte-macrophage colony stimulating factor (GM-CSF) was investigated in 25 melanoma patients by evaluating 34 fresh biopsy specimens. ICAM-1 in situ hybridization and immunochemistry for ICAM-1 and GM-CSF were performed. Most of the metastatic melanoma samples (12 out of 18) and a few of the primary melanoma lesions (three out of 16) showed ICAM-1 expression. The expression of ICAM-1 was significantly (P < 0.01) higher in metastatic lesions than in primary tumours. GM-CSF mRNA and protein were detected in 10 of the 18 metastatic samples and in two of the 15 primary lesions. A significantly high degree (P < 0.0002) of concordance between ICAM-1 and GM-CSF expression was observed: the samples that were negative or positive for ICAM-1 expression were correspondingly negative or positive for GM-CSF. Correlation with clinical and histological parameters was examined. The expression of both molecules in metastatic samples was found to be significantly (P < 0.001) associated with a shorter recurrence-free period. These findings, if confirmed by a wider number of patients, could suggest the prognostic value of the simultaneous, and probably co-ordinated, expression of ICAM-1 and GM-CSF. They also highlight the importance of preventive molecular and biochemical characterization of neoplastic cell cytokine receptors, specifically focusing on the particular cytokine to be used as anticancer therapy and/or as adjunct to chemotherapy.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Melanoma/metabolismo , Neoplasias Cutáneas/metabolismo , Adulto , Edad de Inicio , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Ganglios Linfáticos/metabolismo , Masculino , Melanocitos/citología , Persona de Mediana Edad , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine involved in haematopoiesis and host defence. Production of GM-CSF has been detected in tumour cells including the U87MG astrocytoma cell line. Previous studies have been focused on the regulatory role of the proximal region of the GM-CSF promoter. Our studies on the distal region of the promoter in U87MG cells identify a negative cis element (-1377/-1298) which contains a AP1-like site able to bind c-jun and c-fos transcription factors, according to the results of DNA/protein binding assays. Mutagenesis of the AP1-like site eliminates AP1 binding and the negative effect on promoter activity.