[Introduction of DNA microarray in molecular diagnostics of Wilson disease]. / Zavedení DNA cipu do molekulární diagnostiky Wilsonovy choroby.

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.

Genotyping microarray as a novel approach for the detection of ATP7B gene mutations in patients with Wilson disease.

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.

[Tumor biopsy--a trivial matter or a basic procedure?]. / Biopsie nádoru--banalita, nebo zásadní výkon?

The surgeon is frequently faced with the task of bioptic sampling of tissues from patients with tumourous diseases. The importance of such frequently minor procedures is incorrectly underrated. The importance of biopsy is in the foreground in particular at present at a time of individualization of treatment of malignant tumours, based on the development of new diagnostic-therapeutic methods. The bioptic specimen is the fundamental link in the basic decision--benignity or malignity--and also a unique source for assessment of prognostic and predictive factors on the basis of which we select the optimal therapeutic procedure. Contrary to current histopathological examination where the principles of correct collection of tissues specimens are generally known, in recent years the importance of molecular examinations is increasing where the surgeon must respect certain different principles to make the sampling successful. The authors working in a department of surgical oncology along with authors from specialized laboratories formulate rules of correct implementation of biopsies and transport of biological material in conjunction with recent laboratory methods, and based on examples of their own practice, they demonstrate how the initial approach of the surgeon can influence in a decisive way the correct diagnosis and therapeutic procedure in oncological patients.

Differential antileukemic activity of prednisolone and dexamethasone in freshly isolated leukemic cells.

This study was designed to compare the antileukemic activity of prednisolone and dexamethasone in childhood acute lymphoblastic leukemia (ALL) under in vitro conditions. The chemoresistance of leukemic cells was ascertained by means of a MTT assay in 69 ALL children at diagnosis and the concentration killing 50% of leukemic cells (LCS50) was determined. The children were treated using the protocol ALL-BFM 90/95. Statistical correlations were made among prednisolone (PRED) and/or dexamethasone (DEX) LCS50 and absolute number of blast cells (ANB) on day 0/8 and a new parameter named blast cells clearance (BCC, BCC8 [%] = ANB8: ANB0 x 100) on day 8. Despite the previously published results of Ito et al. (J. Clin. Oncol. 14: 2370-2376, 1996) and Kaspers et al. (MPO 27: 114-121, 1996) on a positive correlation of DEX versus PRED LCS50 (p < 0.002), in our study, we identified 30% of children (21/69) with differential in vitro responsiveness to PRED and DEX. 16% of patients (11/69) were highly sensitive to DEX and resistant to PRED, while 14% of them (10/69) were resistant to DEX and highly sensitive to PRED. The major difference found in our and the other studies was in the processing of leukemic cells. These results were confirmed in a model experiment using the CCRF-CEM line, where we showed that sensitivity to PRED and DEX, but not to other anti-cancer drugs critically depends on manipulation with tumor cells (cryopreservation). Correlation of PRED/DEX in vitro sensitivity values with parameters of in vivo patient's response to PRED monotherapy identified significant association of PRED LCS50 with BCC8 (p < 0.02). It indicates strong linkage of in vitro sensitivity to PRED with percentage of blast cells eliminated from patient blood within the first 8 days of PRED monotherapy.