Unique Combination of 22q11 and 14qter Microdeletion Syndromes Detected Using Oligonucleotide Array-CGH.

We report an infant with a unique combination of 22q11 deletion syndrome and 14q terminal deletion syndrome. The proband had clinical symptoms compatible with diagnosis of 22q11 deletion syndrome: microcephaly, micrognathia, high-arched palate, hypertelorism, short palpebral fissures, square nasal root, prominent tubular nose, hypoplastic nasal alae, bulbous nasal tip, dysplastic low-set ears, short philtrum, and heart defect, but no cell-mediated immunodeficiency typical for the syndrome. G-banding and fluorescence in situ hybridization analyses revealed a karyotype 45,XY,der(14)t(14;22)(q32.3;q11.2),-22.ish del(14)(q32.33)(D14S1420-),del(22)(q11.2q11.2)(N25-). Subsequent analyses disclosed a translocation between chromosomes 14 and 22 in the proband's mother with a deleted 14q telomere. Using comparative genome hybridization on oligonucleotide-based microarray (array-CGH), the deletion at 22q11.21 in the size of ∼4.25 Mb was revealed in the proband as well as the deletion of the telomeric area at 14q32.33qter (∼3.24 Mb) in the proband and his mother. However, both the proband and his mother showed mild symptoms (microcephaly, thin lips, carp-shaped mouth) typical for patients with the described terminal 14q deletion syndrome.

Analysis of chromosomal aberrations in patients with mental retardation using the array-CGH technique: a single Czech centre experience.

Submicroscopic structural chromosomal aberrations (microduplications and microdeletions) are believed to be common causes of mental retardation. These so-called copy number variations can now be routinely detected using various platforms for array-based comparative genomic hybridization (array-CGH), which allow genome-wide identification of pathogenic genomic imbalances. In this study, oligonucleotide-based array-CGH was used to investigate a panel of 23 patients with mental retardation and developmental delay, dysmorphic features or congenital anomalies. Array-CGH confirmed or revealed 16 chromosomal aberrations in a total of 12 patients. Analysis of parental samples showed that five aberrations had occurred de novo: del(1)(p36.33p36.23), del(4)(p16.3p16.2) joined with dup(8)(p23.3p23.1), del(6)(q14.1q15), del(11)(q13.1q13.4). Three aberrations appeared to be inherited from an unaffected parent: dup(3)(q29), del(6)(q12), dup(16)(p13.11). Six aberrations appeared to be inherited from a parental carrier: del(1)(p36.33) joined with dup(12)(q24.32), del(21)(q22.2q22.3) joined with dup(11)(q24.2q25), del(X)(q22.3) and del(1)(q21.1). In two cases, parents were not available for testing: del(17)(q11.2q12) and del(2)(q24.3q31.1). Our results show that the use of oligonucleotide-based array- CGH in a clinical diagnostic laboratory increases the detection rate of pathogenic submicroscopic chromosomal aberrations in patients with mental retardation and congenital abnormalities, but it also presents challenges for clinical interpretation of the results (i.e., distinguishing between pathogenic and benign variants). Difficulties with analysis notwithstanding, the array-CGH is shown to be a sensitive, fast and reliable method for genome-wide screening of chromosomal aberrations in patients with mental retardation and congenital abnormalities.

Oligonucleotide-based array CGH as a diagnostic tool in multiple myeloma patients.

Multiple myeloma (MM) is a hematological disease caused by malignant proliferation of clonal plasma cells (PCs) known for its clinical and biological heterogeneity. Identification of chromosomal changes in genome of PCs plays a key role in MM pathogenesis and is supposed to have important prognostic significance for MM patients. There are two major genetic entities in MM. Hyperdiploid tumors (H-MM), which include about 50% of MM tumors, often have multiple trisomies involving chromosomes 3, 5, 7, 9, 11, 15, 19, and 21 and a substantially lower prevalence of IgH translocations. Nearly half of tumors are non-hyperdiploid (NH-MM), and mostly have one of five recurrent IgH translocations: 11ql13 (CCND1), 6p21 (CCND3), 16q23 (MAF), 20q12 (MAFB), and 4p16 (FGFR3 and MMSET). The development and expanded use of new technologies, such as genome-wide array-based comparative genomic hybridization (aCGH) has accelerated genomic research in MM. This technique is a powerful tool to globally analyze recurrent copy number changes in tumor genome in a single reaction and to study cancer biology and clinical behaviors. It widely overcame routinely used cytogenetic techniques (G-banding, FISH) both in minimal resolution of chromosomal changes and amount of obtained genomic data important for further analyses and clinical applications. Array CGH technique is now used to better understanding of molecular phenotypes, sensitivity to particular chemotherapeutic agents, and prognosis of these diseases. This paper brings brief literature and methodic overview of oligonucleotide-based array-CGH technique in MM diagnosis.

Efficacy of high-resolution comparative genomic hybridization (HR-CGH) in detection of chromosomal abnormalities in children with acute leukaemia.

The efficient detection of chromosomal aberrations in childhood acute leukaemias presents a significant component in the diagnostics of this frequent malignant disease. We used comparative genomic hybridization (CGH) and high-resolution comparative genomic hybridization (HR-CGH) to determine the frequency of chromosomal changes in 33 children with acute leukaemia (AL). The yields of chromosomal abnormalities were compared with the results obtained using conventional cytogenetics (G-banding) and fluorescence in situ hybridization (FISH). Conventional cytogenetics revealed chromosomal changes in 17 (52 %) of studied patients. The employment of FISH together with G-banding analysis identified chromosomal changes in 27 (82 %) of the AL patients investigated. CGH detected changes in DNA copy numbers in 24 (73 %) patients, 40 losses and 67 gains were found in total. HR-CGH disclosed 98 losses and 97 gains in 26 (79 %) patients. In comparison with CGH, HR-CGH analyses unveiled 88 new chromosomal aberrations: 58 losses and 30 gains. The most commonly gained chromosomes were 21 (22.5 %), X (15 %), 18 (12,5 %) and 17 (10 %). The most common losses involved sub-regions or arms of chromosomes 7 (15 %), 9 (12.5 %), 16, 19 and 1 (10 % each). Cytogenetic and molecular cytogenetic analyses of 33 childhood acute leukaemias revealed chromosomal changes in total 31 (94 %) patients. The evaluation of HR-CGH sensitivity proved that the minimal cell population of malignant cells in which a certain chromosomal change could be found was close to the 20 - 30 % level. Our results confirm the benefits of HR-CGH in detecting chromosomal changes in childhood AL. Supplementing G-banding and FISH with the HR-CGH diagnostic method increases the detection of unbalanced structural chromosomal rearrangements and can reveal small cell clones with gains and losses of whole chromosomes in hyperdiploid AL.

Bio-available amino acids and mineral nitrogen forms in soil of moderately mown and abandoned mountain meadows.

The abandonment of traditional mowing methods of mountain meadows in the Czech Republic at the end of the last century has resulted in secondary re-colonization of these areas. Altered accumulation of plant biomass resulted in a deceleration of N turnover. A mountain meadow may be regarded as a N-limited ecosystem in which plant nutrition is dependent on direct uptake of soil amino acids. The composition and distribution of ammonium ions, nitrate ions and the 16 bio-available proteinaceous amino acids were investigated in the top 7 cm of the Ah horizon of a Gleyic Luvisol in a long-term moderately mown meadow and an eleven year old, abandoned or uncut meadow. Ammonium N has a dominant role in both ecosystems. The moderately mown meadow showed accelerated N-turnover and higher net ammonization. The plant community showed a dependence on this form. Plant utilization of nitrates and amino acids appeared to be negligible. The uncut or abandoned meadow showed net ammonization from May (start of the experiment) through August, after which plant N-uptake consisted only of amino acids due to microbial immobilization. The release of bio-available nitrogen from spring until the beginning of summer in the Ah horizon was too low to explain total plant N-uptake. Glutamic acid, arginine and aspartic acids had the highest concentrations of any of the amino acids analyzed.

Incidence of the main genetic markers in glioblastoma multiforme is independent of tumor topology.

Glioblastoma multiforme (GBM) is the most common as well as the most aggressive type of primary brain tumor of astrocytic origin in adults. GBM is characterized by a high degree of intratumoral heterogeneity both in histomorphology and genetic changes. Trisomy/polysomy of chromosome 7, monosomy of chromosome 10, EGFR gene amplification and p53 deletion have been described as the typical genetic markers for tumor classification and prediction of possible response to therapy. Our work was based on detection of these four main genetic changes both in central and peripheral parts of the tumors to evaluate possible differences in the topological incidence of these genetic markers. Chromosomal abnormalities in tumor samples from a group of 21 patients surgically treated for GBM were characterized by means of the interphase-fluorescence in situ hybridization (I-FISH) technique using sets of centromere and locus-specific DNA probes. In addition, we performed a detailed analysis of one selected tumor sample using a genomic microarray system (GenoSensor Array 300) to characterize copy number changes of specific sequences and refine results obtained by I-FISH. However, the data show no significant differences in occurrence of the described genetic markers in either part of the tumor.

Malignant fibrous histiocytoma of the parotid gland.

We described a rare malignant fibrous histiocytoma of the parotid gland (MFH) in a 63-year-old woman. During six months the tumour size became 10 cm in diameter with skin ulceration. The tumour was examined morphologically, by immunohistochemistry and molecular biology methods - FASAY and CGH. The histology revealed a storiform-pleomorphic type of MFH with high mitotic rate. The FASAY method identified a non-mutated p53 gene. The chromosomal changes were identified by the CGH method and 6 cytogenetic changes were found in the tumour cells (deletions at 8p12-p22, 13q32-qter, 14q24-qter, and gains of chromosomal material at 5p, 8q12-q23, and Xq25-qter). The patient died shortly after the beginning of chemotherapy. Autopsy revealed brain and cerebellar haemorrhage. No other tumour foci were proved. In view of short course of disease we lack the data about the influence of the non-mutated p53 gene on the prognosis and therapy.

The effect of extractants on degradation of L-glutamate and L-arginine in the course of shaking and filtration at low temperature.

The effects of demineralized water (DEMI H(2)O) and 0.5 M ammonium acetate (0.5 M AAc) on losses of L-glutamic acid and L-arginine in the course of shaking and filtration at low temperature (6 degrees C) were tested. The concentration of L-glutamic acid decreased by 6.3% in DEMI H(2)O and by 4.9% in 0.5 M AAc, whereas the L-arginine concentration decreased by 6.0% (DEMI H(2)O) and 10.7% (0.5 M AAc). We found a significantly (P < 0.05) higher degradation of L-arginine in 0.5 M AAc compared with that of DEMI H(2)O.

[Comparison of standard prognostic factors with the deletion of 13q14 detected by interphase fluorescence in situ hybridization on separated and unseparated bone marrow cells in multiple myeloma]. / Porovnání standardních prognostických faktoru u pacientu s mnohocetným myelomem s delecí 13q14 stanovenou metodou interfázní fluorescencní in situ hybridizace na separovaných a neseparovaných bunkách kostní direne.

BACKGROUND: Cytogenetic abnormalities of chromosome 13 are emerging as important prognostic factors in multiple myeloma and have been associated with poor prognosis. METHODS AND RESULTS: The occurrence of 13q14 deletion and other standard laboratory parameters were determined in 40 patients with multiple myeloma. We found that interphase fluorescence in situ hybridization using a locus specific probe for RB1 gene on immunomagnetically selected myeloma cells was more sensitive than non selected cells. The 13q14 deletion was found in 10 of 40 (25.0%) of bone marrow samples without cell selection and in 25 of 40 (62.5%) of samples with CD138+ enriched myeloma cells. Negative correlation was found between albumin and the 13q14 deletion in separated (p = 0.003) as well as in cells without selection (p = 0.010). No significant correlation was found in overall survival of separated and unseparated cells (p = 0.830; p = 0.260) and a similar result was obtained for treatment response after transplantation of separated cells (p = 0.520) or non-separated cells (0.190). CONCLUSIONS: Our results confirm that immunomagnetic selection of CD138+ cells increases the probability of detection of the 13q14 deletion in bone marrow samples. The correlation was found between albumin and the 13q14 deletion in both of type of cells.

A significance of additional chromosomal aberrations and other variables on post transplantation outcome of patients with CML.

Chronic myeloic leukemia (CML) is a malignant disease of hematopoietic stem cell characterized by the bcr/abl gene rearrangement. Allogeneic transplantation of stem cells (SCT) is a routinely used treatment method of patients with this diagnosis and remains the only curative mode of treatment. From January 1990 to December 2002, 78 patients with CML underwent allogeneic transplantation and were examined at the Department of Genetics in the National Cancer Institute in Bratislava. Using conventional cytogenetic and FISH 6 patients (7.7%) showed additional chromosomal changes before SCT. These patients had statistically worse post transplantation prognosis compared to the patients without additional changes before SCT (mean survival in month+/-standard error (58.08 (+/-6.70) vs. 5.17 (+/-0.98), p-value=0.001), patient mortality (67% vs. 31%)). In addition five other variables were evaluated for transplant outcome, namely, patient's age at the time of transplantation, sibling or non-sibling donor, higher than 1st chronic phase CML, time from diagnosis to transplantation and sex of donor and recipient. Only the comparison of HLA-identical sibling transplantation to unrelated donor transplantation was statistically significant (mean survival in month- 56.6 (+/-7.2) vs. 13 (+/-0.0), patient mortality 31% vs. 67%).

Bio-available amino acids extraction from soil by demineralized water and 0.5 M ammonium acetate.

The extraction and comparison of soil bio-available amino acids using either demineralised water (DEMI H(2)O) or 0.5 M ammonium acetate (0.5 M AAc) solution is reported. Results show that the extraction by 0.5 M AAc is a better method to assess the concentration of bio-available amino acids in soil than DEMI H(2)O due to higher extraction efficiency and better amino acid protection against microbial degradation during processing.

[Is the information we give women prior to invasive prenatal procedures adequate?]. / Jsou informace, které poskytujeme tehotným zenám pred invazivním prenatálním vysetrením, dostatecné?

BACKGROUND: The aim was to evaluate the psychological impact on women undergoing invasive procedures of prenatal diagnosis. SETTING: Department of Gynecology and Obstetrics, 1st Medical Faculty, Charles University, Prague. MATERIAL AND METHODS: A questionnaire was given to 200 pregnant women and to 160 midwives and students. The acquired data were statistically evaluated using the non-parametric chi 2 test for a 5% confidence interval and the Kruskal-Wallis test (analysis of non-normal distribution of random variables). RESULTS: We found that 85% of pregnant patients were satisfied with the information given by their obstetrician prior to the procedure, 53% of the patients were distressed about the procedure. The largest percentage of patients feared complications of the procedure, while fear of the results of the procedure took second place. CONCLUSION: We found that only some of the patients and midwives had complete information about the actual method of performing these procedures, about the risk, and about the time it takes to obtain results. Most patients receive their information from a doctor-geneticist, which is in agreement with our system. The patient's distress regarding the procedure is not dependent on the level of education. From the acquired data, it follows that greater significance should be placed on the informing patients as well as midwives about all aspects of performing invasive procedure of prenatal diagnosis. According to our study, neither the patient nor the midwife have an adequate perception of the benefits and risk of prenatal diagnostic examinations.