RESUMO
PURPOSE: This preliminary study aimed to analyze and identify differentially expressed miRNAs in Bulgarian patients with non-functioning pituitary neuroendocrine tumors (NFPitNET). The relationship between deregulated miRNAs and tumor invasiveness, recurrence, and size was determined. METHODS: Twenty patients with NFPitNET were selected and fresh pituitary tumor tissues were collected. RNA containing miRNAs were isolated using miRNAeasy mini kit and analyzed by quantitative real-time polymerase chain reaction (PCR) using LNA miRNA Cancer-Focus PCR Panel (Qiagen). RESULTS: Three miRNAs (miR-210-3p, miR-149-3p, and miR-29b-3p) were deregulated in invasive compared to non-invasive NFPitNETs. Differential expression of four-miRNA signatures - miRNA-17, miR-19, miR-106a, and miR-20, correlated with patient recurrence. CONCLUSION: This prospective pilot study selected a unique miRNA expression profile, that correlates with invasiveness and recurrence in non-functioning pituitary neuroendocrine tumors. Moreover, some of the selected miRNAs are reported for the first time in patients with this disease, shedding light on the molecular mechanisms involved in pituitary pathogenesis. The identified miRNAs demonstrate potential as biomarkers, deserving further investigation in a larger cohort to validate their clinical applicability.
Assuntos
MicroRNAs , Invasividade Neoplásica , Recidiva Local de Neoplasia , Tumores Neuroendócrinos , Neoplasias Hipofisárias , Humanos , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Neoplasias Hipofisárias/metabolismo , Masculino , Feminino , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/patologia , Tumores Neuroendócrinos/metabolismo , Recidiva Local de Neoplasia/genética , Adulto , Idoso , Projetos Piloto , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
We report on a unified rapid betaine-based-PCR protocol for amplification of the (CAG)n region in Huntington disease (HD) and the (CGG)n region in Fragile X syndrome (FXS), followed by an electrophoretic separation on automated sequencer for precise determination of the triplet numbers. The high betaine concentration (2.5 M betaine) permits precise amplification of the CAG and CGG repeats. Ten HD affected patients and 10 healthy individuals from HD families were re-evaluated. For FXS the CGG region in normal individuals and premutations of about 100 repeats were precisely amplified by this protocol. Ten unrelated FXS premutation carriers and 24 mentally retarded non-FXS affected boys were re-examined by this method. The results totally coincided with the previous ones. This protocol is a good choice as a fast screening test. Within 24 h we can have preliminary information on the patient's genetic status. Normal individuals, CGG premutation carriers up to 100 repeats, as well as HD patients carrying an expansion up to 50 CAG repeats can be easily clarified. This accounts for a relatively large proportion (about 90%) of the suspected HD and FXS patients, referred to our laboratory for genetic analysis. The calculation of the repeat's number is more accurate for the correct interpretation of the results, screening tests and genetic counselling.
Assuntos
Alelos , Síndrome do Cromossomo X Frágil/genética , Doença de Huntington/genética , Reação em Cadeia da Polimerase/métodos , Sequências Repetitivas de Ácido Nucleico , Repetições de Trinucleotídeos , Betaína/química , Síndrome do Cromossomo X Frágil/sangue , Humanos , Doença de Huntington/sangue , Masculino , Análise de Sequência de DNA/métodosRESUMO
Limb-girdle muscular dystrophy type 2A (LGMD2A) is caused by mutations in the calpain 3 (CAPN3) gene. The clinical diagnoses of these cases in Bulgaria are very complicated, no protein analysis on muscular biopsy is available in our country, and genetic tests are the only possibility to clarify the diagnoses in clinically ambiguous cases. We screened 48 unrelated Bulgarian cases with preliminary diagnoses of different types of muscular dystrophy for mutations in the CAPN3 gene. Altogether, 20 families (42%) were found to carry mutations in the CAPN3 gene. Several misdiagnosed cases were clarified. Three novel and six recurrent mutations were identified. In total, 40% of the patients are homozygous for c.550delA, and 70% carry it at least on one allele. The affected group of women in our sample shows later onset, milder clinical manifestation, slower progression, and later invalidization.
Assuntos
Calpaína/genética , Proteínas Musculares/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Mutação Puntual , Deleção de Sequência , Adolescente , Adulto , Substituição de Aminoácidos , Bulgária , Criança , Feminino , Humanos , MasculinoRESUMO
In eukaryotes, DNA damage elicits a multifaceted response that includes cell cycle arrest, transcriptional activation of DNA repair genes, and, in multicellular organisms, apoptosis. We demonstrate that in Saccharomyces cerevisiae, DNA damage leads to a 6- to 8-fold increase in dNTP levels. This increase is conferred by an unusual, relaxed dATP feedback inhibition of ribonucleotide reductase (RNR). Complete elimination of dATP feedback inhibition by mutation of the allosteric activity site in RNR results in 1.6-2 times higher dNTP pools under normal growth conditions, and the pools increase an additional 11- to 17-fold during DNA damage. The increase in dNTP pools dramatically improves survival following DNA damage, but at the same time leads to higher mutation rates. We propose that increased survival and mutation rates result from more efficient translesion DNA synthesis at elevated dNTP concentrations.