RESUMO
Elucidating how life history traits vary geographically is important to understanding variation in population dynamics. Because many aspects of ectotherm life history are climate-dependent, geographic variation in climate is expected to have a large impact on population dynamics through effects on annual survival, body size, growth rate, age at first reproduction, size-fecundity relationship, and reproductive frequency. The Eastern Massasauga (Sistrurus catenatus) is a small, imperiled North American rattlesnake with a distribution centered on the Great Lakes region, where lake effects strongly influence local conditions. To address Eastern Massasauga life history data gaps, we compiled data from 47 study sites representing 38 counties across the range. We used multimodel inference and general linear models with geographic coordinates and annual climate normals as explanatory variables to clarify patterns of variation in life history traits. We found strong evidence for geographic variation in six of nine life history variables. Adult female snout-vent length and neonate mass increased with increasing mean annual precipitation. Litter size decreased with increasing mean temperature, and the size-fecundity relationship and growth prior to first hibernation both increased with increasing latitude. The proportion of gravid females also increased with increasing latitude, but this relationship may be the result of geographically varying detection bias. Our results provide insights into ectotherm life history variation and fill critical data gaps, which will inform Eastern Massasauga conservation efforts by improving biological realism for models of population viability and climate change.
Assuntos
Mudança Climática , Variação Genética , Modelos Biológicos , Viperidae/fisiologia , Animais , Feminino , Great Lakes Region , MasculinoRESUMO
CONTEXT: Mutations of the proto-oncogene B-raf (BRAF) have been detected in melanocytic lesions and papillary carcinomas of the thyroid, and identification of these mutations could be useful in resolving some diagnostic problems. OBJECTIVE: To develop a method to evaluate mutations of BRAF that could provide results much more rapidly than conventional polymerase chain reaction and DNA sequencing assays. DESIGN: An assay using a LightCycler was developed to evaluate DNA sequences encoding amino acids within the activation loop of BRAF. RESULTS: Using this real-time polymerase chain reaction method, we analyzed 55 paraffin-embedded melanoma or nevus samples. The V600E mutation was found in 0 (0%) of 13 samples diagnosed histologically as Spitz nevi, 9 (24.3%) of 37 invasive melanomas, and 5 (100%) of 5 other melanocytic nevi. Two additional mutations, V600K and VK600-1E, also were identified in cases of invasive melanoma. We analyzed 14 paraffin-embedded papillary thyroid cancer (PTC) samples, 6 of which showed the V600E mutation. We found that our test worked efficiently with fine-needle aspirate specimens, and it identified 6 V600E mutations in 10 fine-needle aspirate specimens diagnosed as PTC. We also identified 4 V600E mutations in 6 specimens of PTC metastatic to lymph node. Unlike the melanocytic lesions, the PTC specimens yielded only V600E mutations. Comparison of our real-time polymerase chain reaction results with conventional polymerase chain reaction and DNA sequencing demonstrated 100% concordance. Surprisingly, we did not identify the previously reported VK600-1E or K601E mutations in our PTC specimens. CONCLUSIONS: Our results show that the real-time polymerase chain reaction method is a rapid and accurate method for identifying BRAF mutations, such as V600E, in both paraffin-embedded tissue and fine-needle aspirate specimens.
Assuntos
Carcinoma Papilar/genética , Melanoma/genética , Nevo Pigmentado/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Neoplasias da Glândula Tireoide/genética , Sequência de Bases , Biópsia por Agulha Fina , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , DNA de Neoplasias/genética , Humanos , Melanoma/metabolismo , Melanoma/patologia , Dados de Sequência Molecular , Mutação , Nevo Pigmentado/metabolismo , Nevo Pigmentado/patologia , Reação em Cadeia da Polimerase/métodos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas B-raf/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologiaRESUMO
The source of acellular specimens is not infrequently challenged, especially for toxicology specimens, but such specimens may not be amenable to conventional genetic testing to confirm the source. Our evaluation of genomic and mitochondrial DNA (mtDNA) amplicons using polymerase chain reaction (PCR) from centrifuged, filtered, or whole urine specimens demonstrated higher sensitivity of detection of mtDNA than genomic DNA and a higher detection rate for the mtDNA markers than genomic markers in all sample sets. The mitochondrial amplicons were sequenced to identify specific single nucleotide polymorphisms (SNPs). Subsequently, a real-time PCR technique using fluorescence resonance energy transfer (FRET) probes designed to hybridize to the published mtDNA sequence over known SNP locations present within the mitochondrial control region was developed. Our results demonstrate the feasibility of using a FRET-based assay of mitochondrial genotypes with acellular laboratory specimens to screen for specimen mix-ups or to confirm sources of controversial toxicology specimens.
Assuntos
DNA Mitocondrial/análise , Sequência de Bases , DNA Mitocondrial/urina , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Sensibilidade e EspecificidadeRESUMO
A 62-year-old woman presented with recurring right upper quadrant pain and underwent a routine laparoscopic cholecystectomy. A large gallstone was found impacted in the fundus of the gall bladder. Interestingly, aside from noting mild inflammation of the gall bladder wall, microscopic examination of the specimen identified 2 fragments of benign thyroid tissue. Given the routine nature of the surgical procedure and lack of abnormality detected during the operation, the attending pathologist suspected extraneous tissue contamination ("floater") of the pathology specimen and submitted the block and slides to Molecular Pathology. The thyroid tissue-containing fragments and gallbladder wall were independently isolated and subjected to genetic fingerprinting using a standard forensic DNA identification panel. All fragments showed the identical fingerprint, strongly suggesting that they belonged to the same patient. The results indicated that the thyroid tissue was from an ectopic rest adjacent to the gall bladder, which has been reported only very rarely in the previous literature and illustrates the unusual use of molecular genetic testing to confirm the presence of ectopic tissue versus contamination.
Assuntos
Coristoma/diagnóstico , Impressões Digitais de DNA/métodos , Vesícula Biliar/patologia , Cálculos Biliares/diagnóstico , Glândula Tireoide , Coristoma/genética , Coristoma/cirurgia , Feminino , Vesícula Biliar/metabolismo , Vesícula Biliar/cirurgia , Cálculos Biliares/genética , Cálculos Biliares/cirurgia , Humanos , Repetições de Microssatélites , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
CONTEXT: It is uncertain whether extensive prostate-specific antigen (PSA) testing and extended biopsies currently performed will increase the incidence of no residual cancer on subsequent prostatectomy. OBJECTIVE: To identify the incidence of cases with no residual cancer on prostatectomy after a positive 10-core biopsy and to review the clinical, biopsy, and prostatectomy findings and the results of specimen identity analysis. DESIGN: We identified 9 patients with no residual cancer in 1351 consecutive prostatectomies and we reviewed the clinical, biopsy, and prostatectomy data from our institutional database. In 6 cases encountered after 2003, we also performed a polymerase chain reaction-based microsatellite analysis on formalin-fixed tissue to confirm the identity of the biopsies and prostatectomies. RESULTS: All patients had positive biopsies in 1 or 2 cores: 1 in 7 and 2 in 2 patients (1 each, unilateral and bilateral). Mean total cancer length on biopsy measured 2.5 mm, which represented 1.7% of the total biopsy tissue. Gleason score 6 was found in 8 of 9 patients and 1 patient had Gleason score 9. Patients' age was 60.3 years, preoperative PSA was 6.0 ng/mL, and PSA density was 0.1 (all means). In 6 cases tested for microsatellite identity, the patient identity was confirmed. CONCLUSIONS: Incidence of no residual cancer on prostatectomy of 0.67% after 10-core positive biopsy is higher than previously reported. In most cases, finding no residual cancer on prostatectomy after exhaustive work-up may indicate minimal patient disease. Microsatellite analysis provides a useful and cost-effective test in establishing specimen identity.
Assuntos
Adenocarcinoma/cirurgia , Sistemas de Identificação de Pacientes , Complicações Pós-Operatórias , Prostatectomia , Neoplasias da Próstata/cirurgia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Idoso , Biópsia , DNA de Neoplasias/análise , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Neoplasia Residual/patologia , Neoplasia Residual/cirurgia , Reação em Cadeia da Polimerase , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/patologia , Complicações Pós-Operatórias/cirurgia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Manejo de EspécimesRESUMO
PURPOSE: To determine the role of methylation of HIC1, a candidate tumor suppressor gene on 17p13.3, in various types of pediatric tumors. EXPERIMENTAL DESIGN: We examined the methylation status of the HIC1 promoter by methylation specific PCR in 157 pediatric tumors and 27 nonmalignant tissues. We correlated methylation with mRNA expression by reverse transcription-PCR in eight tumor-derived cell lines. RESULTS: HIC1 methylation was frequent in medulloblastomas (80%, 12 of 15), retinoblastomas (67%, 6 of 9), rhabdomyosarcomas (59%, 13 of 22), germ cell tumors (55%, 6 of 11), and neurouroblastic tumors (36%, 14 of 39); neuroblastomas (43%, 12 of 28); ganglioneuromas (17%, 1 of 6); and ganglioneuroblastomas (20%, 1 of 5). In contrast, a low incidence of methylation was observed in osteosarcomas (17%, 2 of 12), Ewing's tumors (9%, 1 of 11), Wilms' tumors (3%, 1 of 31), and hepatoblastomas (0%, 0 of 7). HIC1 methylation was more frequent in the aggressive alveolar subtype of rhabdomyosarcomas (100%, 8 of 8) than the embryonal subtype (33%, 4 of 12; P < 0.005) and was rare in the nonmalignant tissues examined. Methylation was also demonstrated by sequencing in nine randomly selected tumor samples. Seven of eight pediatric tumor cell lines examined were methylated and showed loss or reduced HIC1 mRNA. Expression was strongly induced in all cell lines by treatment with the demethylating agent 5-aza 2'deoxycytidine. CONCLUSIONS: Our data suggest that aberrant methylation of HIC1 may play a role in the pathogenesis of specific pediatric tumors.
Assuntos
Metilação de DNA , Neoplasias/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Adolescente , Adulto , Linhagem Celular Tumoral , Criança , Pré-Escolar , Humanos , Lactente , Fatores de Transcrição Kruppel-Like , RNA/química , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Methylation of the promoter regions of CpG-rich sites in genes is the major mechanism for the silencing of many genes in tumors. Methylation of the key apoptosis-related gene caspase 8 (CASP8) has been reported in some childhood tumors and in neuroendocrine lung tumors. We examined the methylation status of 181 pediatric tumors and found frequent methylation in rhabdomyosarcomas (83%), medulloblastomas (81%), retinoblastomas (59%), and neuroblastomas (52%). Methylation frequencies were low in Wilms' tumors (19%) and absent in hepatoblastomas, acute leukemias, osteosarcomas, Ewing's sarcomas, and ganglioneuromas and in normal tissues. Methylation of CASP8 and the tumor suppressor gene RASSF1A were highly significantly correlated in all tumor types by both the chi(2) and the Fisher's exact tests (P < 0.0001 for both tests). Because the region of the gene examined by us and others is not located in the promoter region and lacks features of a CpG island, we explored the relationship between methylation and gene silencing in detail using 23 pediatric tumor cell lines. Studies included relating the methylation of the region to gene expression at mRNA and protein levels, enzymatic assays of gene function, clonal analysis of PCR amplicons of the region, and exposure to a demethylating agent. These studies indicated that methylation correlated with the loss of gene function in most cases; however, other mechanisms of gene inactivation were present in some cases. Posttranscriptional inactivation of the closely related gene caspase 10 was present in many cell lines. Our results suggest that deregulation of the death receptor pathway to apoptosis is frequent in many types of pediatric tumors and their cell lines.
Assuntos
Azacitidina/análogos & derivados , Caspases/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias/enzimologia , Proteínas Supressoras de Tumor , Azacitidina/farmacologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Caspase 10 , Caspase 8 , Caspase 9 , Caspases/genética , Criança , Metilação de DNA , Decitabina , Inibidores Enzimáticos/farmacologia , Amplificação de Genes , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Genes myc , Humanos , Ácidos Hidroxâmicos/farmacologia , Proteínas de Neoplasias/genética , Neoplasias/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Células Tumorais CultivadasRESUMO
Aberrant promoter methylation of tumor suppressor genes has not been fully investigated in pediatric tumors. Therefore, we examined the methylation status of nine genes (p16(INK4A), MGMT, GSTP1, RASSF1A, APC, DAPK, RARbeta, CDH1 and CDH13) in 175 primary pediatric tumors and 23 tumor cell lines using methylation-specific PCR. We studied the major forms of pediatric tumors--Wilms' tumor, neuroblastoma, hepatoblastoma, medulloblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, retinoblastoma and acute leukemia. The most frequently methylated gene in both primary tumors and cell lines was RASSF1A (40, 86%, respectively). However, the rates of RASSF1A methylation in individual tumor types varied from 0 to 88%. RASSF1A methylation was tumor specific and was absent in adjacent non-malignant tissues. Methylation of the other genes was relatively rare in tumors and non-malignant tissues (less than 5%). Neuroblastoma patients with methylation of RASSF1A were significantly older than patients without methylation (P=0.008). There was no relationship between methylation status and other clinico-pathologic parameters. We treated six cell lines lacking RASSF1A mRNA with 5-aza-2'deoxycytidine to examine the relationship between methylation and transcriptional silencing. In five of six cell lines, restoration of RASSF1A mRNA was confirmed by RT-PCR. Our findings indicate that aberrant promoter methylation of RASSF1A may contribute to the pathogenesis of many different forms of pediatric tumors.