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ИННОВАЦИОННЫЕ ТЕХНОЛОГИИ В ПЕДИАТРИИ И ДЕТСКОЙ ХИРУРГИИ
otide and SNP arrays. In the prenatal case we found mosaicism for an sSMC(4), sSMC(6), sSMC(9), sSMC(14) and sSMC(22),while the postnatal case had an sSMC(4), sSMC(8) and sSMC(11). SNP markers indicated that sSMC(4) resulted from a maternal meiosis II error in the prenatal case and SNP-marker-segregation was consistent with maternal meiosis I error of sSMC(8) in the postnatal case. In the latter, a boy with developmental/psychomo-tor delay, autism, hyperactivity, speech delay and hypotonia, the sSMC(8) was present at the highest frequency in blood and the only mitotically stable sSMC in cultured lymphoblastoid B-cell lines. By comparison with other patients with corresponding duplication a minimal region of overlap was identified, with CHRNB3 and CHRNA6 as dosage-sensitive candidate genes for the phenotype. These genes encode subunits of nicotinic acetylcholine receptors (nAChRs). We propose that overproduction of these sub-units leads to perturbed component stoichiometries with dominant negative effects on the function of nAChRs, as it was shown by others in vitro. With the limitation that in each case only one sSMC could be studied, our findings demonstrate that different meiotic errors lead to multiple sSMCs. Age-related aneuploidy in female meiosis can be a reason here and predivision-sister-chromatid-separation during meiosis I or II, or both seem to generates multiple sSMCs. Part of this research was supported by the Else Kroner-Fresenius-Stiftung (2011_A42).
GENE EXPRESSION PROFILING OF MULTIDRUG RESISTANT CEM/ADR-5000 AND SENSITIVE CCRF-CEM LEUKEMIA CELLS TO SHED LIGHT ON DRUG RESISTANCE MECHANISMS OF TUMORS
Kadioglu O.1, Cao J.1, Kosyakova N.2, Mrasek K.2, Liehr T.2, Efferth T.1
'Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Mainz, Germany
Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Jena, Germany
In order to evaluate differential gene expression profiles, chromosomal alterations and aberrations, multidrug resistant CEM/ADR5000 and sensitive CCRF-CEM leukemia cells were analyzed by RNA sequencing, multicolor fluorescence in situ hybridization (mFISH) and array-comparative genomic hybridization (array-CGH). The gene expression profiles of those leukemia cell lines have not been described well so far and in this study we identified differential gene expression profile of multidrug-resistant (CEM/ADR5000) and sensitive (CCRF-CEM) leukemia cell lines via RNA-sequencing (Illumina HiSeq 2000). Deregulated genes were identified by using the reads per kilobase of exon model per million mapped reads (RPKM) values and they were subjected to downstream pathway and network analysis using Ingenuity Software. Many ATP-binding cassette transporters (ABC) transporters were observed to be deregulated in CEM/ADR5000 cells and deeper analysis showed that pathways and networks linked with multidrug resistance and carcinogenesis were influenced in CEM/ADR5000 cells.
Chromosomal aberrations, translocations, amplifications and deletions were detected by mFISH and array-CGH analyses. The later results were used to validate the RNA-sequencing results. In addition to deletion at chromosome 8, translocation between chromosome 8 and 9 and trisomy at chromosome 20 observed in CCRF-CEM cell line, translocations between chromosomes 3 and 10, 10 and 16, 7 and 18, 9 and 22 and 5,18 and 21, and loss of one X-chromosome were observed in the CEM/ADR5000 cell line. Aberrations were detected within 3q27.1 in CEM/ADR5000 causing slight down-regulation in expression of ABCC5, ABCF3, and within Xq28 causing slight down-regulation in expression of ABCD1. ABCB1, being the critical protein playing role in multidrug resistance, was observed in RNA-seq analyses to be 400-fold up-regulated as a result of an amplification in 7q21.12 observed in array-CGH analyses. This study showed that genomic rearrangements changed gene expression in CEM/ADR5000. Several genes involved in rearrangements were linked to multidrug resistance and carcinogenesis characterizing CEM/ADR5000 cells.
X-LINKED INTELLECTUAL DISABILITY-RELATED GENES DISRUPTED BY BALANCED X-AUTOSOME TRANSLOCATIONS
Moyses-Oliveira M.1, Guilherme R.S.14, Meloni V.A.1, Di B.A.1, de Melo C.B.2, Bragagnolo S.1, Moretti-Ferreira D.3, Kosyakova N.4, Liehr T.4, Carvalheira G.M.1, Melaragno M. I.1
!Genetics Division, Department of Morphology and Genetics, Universidade Federal de Sao Paulo, Sao Paulo, Brazil
2Psychobiology Department,
Universidade Federal de Sao Paulo, Sao Paulo, Brazil 3Departament of Genetics, Instituto de Biociencias de Botucatu, Universidade Estadual de Sao Paulo, Sao Paulo, Brazil
Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Jena, Germany
Detailed molecular characterization of chromosomal rearrangements involving X-chromosome has been a key strategy in identifying X-linked intellectual disability-causing genes. We fine-mapped the breakpoints in four women with balanced X-autosome translocations and variable pheno-types in order to investigate the corresponding genetic contribution to intellectual disability. Three patients presented with cognitive impairment, confirming the association between the disrupted genes (TSPAN7-MRX58, KIAA2022-MRX98 and IL1RAPL1 -MRX21/34) and intellectual disability. For TSPAN7 and KIAA202 gene expression analysis showed absence of functional copies in the patient. In the third patient gene expression experiments suggested a fusion transcript ZNF611-IL1RAPL1 under the control of the ZNF611 promoterwith a ZNF611 gene disrupted at the autosomal breakpoint. The X-chromosomal breakpoint in the fourth patient, a woman with normal intellectual abilities, revealed disruption of the ZDHHC15 gene (MRX91). The expression assays did not detect ZDHHC15 gene expression in the patient, thus questioning the involvement of ZDH-
РОССИЙСКИЙ ВЕСТНИКПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 4, 2015
Раздел 2 Педиатрия
HC15 gene in intellectual impairment. Overall, revealing the disruption of an X-linked intellectual disability-related gene in patients with balanced X-autosome translocation is a useful tool for the better characterization of critical genes for neurodevelopment. This approach might be a valuable resource when applied not only in patients with cognitive impairment, but also in subjects with normal cognition.
A NEW IGH@ GENE REARRANGEMENT ASSOCIATED WITH CDKN2A/B DELETION IN A YOUNG ADULT B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (BALL)
Othman M.A.K.1, Grygalewicz B.2, Pienkowska-Grela B.3, Ejduk A.3, Rincic M.1-4, Melo J.5-6, Carreira I. M.5-6, Meyer B.7, Marzena W.8, Liehr T.1 'Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Jena, Germany 2Cytogenetic Laboratory, Maria Sklodowska-Curie Memorial Cancer Centre and Institute, Warsaw, Poland 3Department of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland "Croatian Institute of Brain Research, Zagreb, Croatia 5Laboratory of Cytogenetics and Genomics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal 6CIMAGO, Centro de Investigagao em Meio Ambiente, Geneticae Oncobiologia 7ZytoVision GmbH, Bremerhaven, Germany 8Department of Haematology and Bone Marrow Transplantation, Holycross Cancer Center, Kielce, Poland
Acquired copy number changes are common in acute leukemia. They are reported as recurrent amplification or deletion and may be indicative for involvement of oncogenes or tumor suppressor genes in the acquired disease and can serve as potential biomarkers for prognosis or even as a target for molecular therapy. Here, we report a gain of copy numbers of 14ql3 to 14q32 leading to an IGH@ locus splitting in a young adult female, present as a yet unreported rearrangement in B-cell acute lymphoblastic leukemia (B-ALL). Low resolution banding cytogenetics at the time of diagnosis revealed a normal karyotype. However, retrospective application of fluorescence in situ hybridization-(FISH-) banding, locus specific FISH-probes, as well as multiplex ligation-dependent probe amplification and high resolution array-comparative genomic hybridization revealed previously cryptic aberrations. Overall a karyotype 46,XX,del(9)(p21.3p21.3),der(14)(pter->q32.33::q32.33->q13::q32.33->qter) was determined. The patient was treated according to PALG 5-ALL7-3 protocol and achieved complete remission. These findings indicate that a favorable prognosis is linked to these aberrations under the mentioned treatment. Supported in parts by the DAAD.
HIGH RATES OF SUBMICROSCOPIC ABERRATIONS IN KARYOTYPICALLY NORMAL ACUTE LYM-PHOBLASTIC LEUKEMIA
Othman M.A.K.1, Melo J.B.23, Carreira I.M.2'3, Rincic M.14, Glaser A.1, Grygalewicz B.5, Gruhn B.6, Wilhelm K.16, Rittscher K.1, Meyer B.7, Macedo S.M.L.8~9, de Jesus Marques S.T.10, Liehr T.1
Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Jena, Germany 2Laboratory of Cytogenetics and Genomics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal 3CIMAGO, Centro de Investigagao em Meio Ambiente, Geneticae Oncobiologia, Coimbra, Portugal 4Croatian Institute of Brain Research, Zagreb, Croatia 5Cytogenetic Laboratory, Maria Sklodowska-Curie Memorial Cancer Centre and Institute, Warsaw, Poland •Department of Pediatrics (Oncology and Hematology), Jena University Hospital, Friedrich Schiller University, Jena, Germany
7ZytoVision GmbH, Bremerhaven, Germany Cytogenetics Department, Bone Marrow Transplantation Unit, National Cancer Institute, Rio de Janeiro, RJ, Brazil
9Post Graduation Program in Oncology, National Cancer Institute (INCA), Rio de Janeiro, Brazil 10Pediatric Oncohematology Center, Hospital Oswaldo Cruz/ Pos Graduation Course of the Faculty of Medical Sciences, University of Pernambuco, Recife, PE, Brazil
Acute lymphoblastic leukemia (ALL) is not a single uniform disease. It consists of several subgroups with different cytogenetic and molecular genetic aberrations, clinical presentations and outcomes. Banding cytogenetics plays a pivotal role in the detection of recurrent chromosomal rearrangements and is the starting point of genetic analysis in ALL, still. Nowadays, molecular (cyto)genetic tools provide substantially to identify previously non-detectable, so-called cryptic chromosomal aberrations in ALL. However, ALL according to banding cytogenetics with normal karyotype — in short cytogenetically normal ALL (CN-ALL) — represent up to ~50% of all new diagnosed ALL cases. The overall goal of this study was to identify and characterize the rate of cryptic alterations in CN-ALL and to rule out if one single routine approach may be sufficient to detect most of the cryptic alterations present. 61 ALL patients with CN-ALL were introduced in this study. All of them underwent high resolution fluorescence in situ hybridization (FISH) analysis. Also DNA could be extracted from 34 ALL samples. These DNA-samples were studied using a commercially available MLPA (multiplex ligation-dependent probe amplification) probe set directed against 37 loci in hematological malignancies and/or array-comparative genomic hybridization (aCGH). Chromosomal aberrations were detected in 21 of 61 samples (~34%) applying FISH approaches: structural abnormalities were present in 15 cases and even numerical ones were identified in 6 cases. Applying molecular approaches copy number alterations (CNAs) were detected in 27/34 samples. Overall, 126 CNAs were identified and only 34 of them were detectable by MLPA (~27%). Loss of CNs was identified in ~80% while gain of CNs was present in ~20% of the 126 CNAs. A maximum of 13 aberrations was detected per case; however, only one aberration per case was found in 8 of all in detail studied 34 cases. Of special interest among the detected CNAs are the following new findings: del(15)(q26.1q26.1) including CHD2 gene was found in 20% of the studied ALL cases,
РОССИЙСКИЙ ВЕСТНИКПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 4, 2015
