RESUMEN
BACKGROUND: Wilson disease (WD) is a serious autosomal recessive disorder caused by mutations in ATP7B-gene which encodes a copper-specific ATPase. WD patients suffer from impaired biliary excretion of copper from organism and its' accumulation in body organs. Molecular diagnostics of WD is an important part of correct diagnosis statement. The aim of the study was to design and validate a genotyping DNA microarray which enables to analyze 87 mutations and 17 polymorphisms in ATP7B gene, simultaneously. METHODS AND RESULTS: 97 WD patients with known genotypes and 46 samples prepared by mutagenesis were tested in the first phase of chip validation. All analyzed sequence variants were detected with 100% accuracy. Samples from WD suspected patients were tested in the second phase of validation. We have analyzed 58 unrelated patients, yet. The diagnosis of WD was confirmed in 10 patients, 13 patients were heterozygous for some mutation and 35 had no mutation in ATP7B gene. Samples with one or no mutation found by microarray analysis were sequenced directly and no further causal mutation was revealed. CONCLUSIONS: Wilson chip seems to be a fast and reliable method for screening of mutations in ATP7B gene.
Asunto(s)
Degeneración Hepatolenticular/diagnóstico , Análisis de Secuencia por Matrices de Oligonucleótidos , Degeneración Hepatolenticular/genética , HumanosRESUMEN
Wilson disease (WD) is an autosomal recessive inherited disorder of copper metabolism that is caused by mutations in the ATP7B gene. To date, more than 300 mutations have been described in this gene. Molecular diagnostics of WD utilizes restriction enzyme digestion, multiplex ligation-dependent probe amplification or a direct sequencing of the whole gene. To simplify and speed up the screening of ATP7B mutations, we have developed a genotyping microarray for the simultaneous detection of 87 mutations and 17 polymorphisms in the ATP7B gene based on the arrayed primer extension reaction. The patient's DNA is amplified in four multiplex polymerase chain reactions, fragmented products are annealed to arrayed primers spotted on a chip, which enables DNA polymerase extension reactions with fluorescently labeled dideoxynucleotides. The Wilson microarray was validated by screening 97 previously genetically confirmed WD patients. In total, we detected 43 mutations and 15 polymorphisms that represent a majority of the common mutations occurring in the Czech and Slovak populations. All screened sequence variants were detected with 100% accuracy. The Wilson chip appears to be a rapid, sensitive and cost-effective tool, representing the prototype of a disease chip that facilitates and speeds up the screening of potential WD patients.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Transporte de Catión/genética , Degeneración Hepatolenticular/genética , Análisis por Micromatrices/métodos , Mutación Puntual , ATPasas Transportadoras de Cobre , Análisis Mutacional de ADN , Tamización de Portadores Genéticos/métodos , Genotipo , Degeneración Hepatolenticular/diagnóstico , Heterocigoto , Humanos , Análisis por Micromatrices/instrumentación , MutaciónRESUMEN
The progression of colorectal cancer involves accumulation of various genetic and epigenetic events that dramatically change gene expression. The aim of this study was to investigate a possible new approach to the diagnosis of colorectal carcinoma patients, based on their gene expression profiles. Human 19K cDNA microarrays were used to analyze the gene expression profiles of 18 colorectal carcinoma patients. Transcriptome maps (TMs) were analyzed to detect chromosomal regions that could serve as potential diagnostic markers for colon cancer. A comparison of TMs showed chromosome regions with conserved changes of gene expression typical of colorectal cancer in general, and also patient-specific variable regions. We identified 195 genes with significantly altered expression in colon cancer. Functional analysis of the regulated genes distinguished three main categories: biological processes, cellular components, and molecular functions. We found that different patients had chromosome regions characterized by very similar changes of gene expression, probably linked to the most fundamental events in carcinogenesis. On the other hand, variable chromosome regions can be patient-specific. The variable regions may provide further information on the individual pathogenesis and prognosis of the patient. Comparison of TMs is proposed as a tool to facilitate diagnosis and treatment planning for individual patients.
Asunto(s)
Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Biomarcadores de Tumor/genética , Mapeo Cromosómico , ADN de Neoplasias/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
Bacillus subtilis 115 grew in a medium with amino acids and glucose with the maximum specific growth rates mu of 1.20-1.10/h in the temperature range of 45-48 degrees C. Activity of the extracellular neutral proteinase excreted by 1.3 mg/mL dry mass during 8 h of the postexponential and stationary growth phases decreased from its maximum value of 0.23 TU/mL at 40 degrees C to 0.13 and 0.06 TU/mL at 45 and 48 degrees C, respectively. Formation of the extracellular serine proteinase decreased even more - from 0.18 TU/mL at 40 degrees C to 0.06 and 0.03 TU/mL at 45 and 48 degrees C, respectively. Sporulation, expressed as the portion of sporangia with refractile spores at the 6th h of the stationary phase decreased from 46% at 40 degrees C to 17 and 3% at 45 and 48 degrees C, respectively.