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ИННОВАЦИОННЫЕ ТЕХНОЛОГИИ В ПЕДИАТРИИ И ДЕТСКОЙ ХИРУРГИИ
Silva M.L.M.4, de Jesus Marques-Salles T.1 'Pediatric Oncohematology Center, Hospital Oswaldo Cruz/ Pos Graduation Course of the Faculty of Medical Sciences, University of Pernambuco, Recife/PE, Brazil; 2Biologic Sciences Institute, Pernambuco University, Recife/PE, Brazil;
3Jena University Hospital, Institute of Human Genetics, Jena, Germany;
"Cytogenetic Department, National Center for Bone Marrow Transplant (CEMO-INCA), National Cancer Institute, Rio de Janeiro/RJ, Brazil
Fanconi Anemia (FA) is an inherited disorder with congenital and developmental abnormalities, bone marrow failure and an extreme risk to develop myelodys-plastic syndrome or acute myeloid leukemia. Cytogenetic surveillance of bone marrow is an important part of the clinical management of FA as cytogenetic aberrations can help to estimate the moment of malignant trans-fomation. Here, we characterized bone marrow features and implications of chromosomal aberrations in four cases of FA. The molecular karyotyping revealed different acquired abnormalities in three of the four cases, e.g. a high complex and rare karyotype. A fourth patient was also included as an asymptomatic carrier with duplication of chromosome 1. These features showed that FA provokes serious chromosomes abnormalities which in most cases evolve to hematological malignances with fatal outcome. Due to the appearance of rare and complex chromosomal abnormalities, molecular cytogenetics studies are important to determine the real significance of chromosomal aberrations in malignancies derived from FA.
ALPHA-FETOPROTEIN AND ITS VALUE FOR PREDICTING PREGNANCY OUTCOMES - A RE-EVALUATION
Darouich A.A.1, Liehr T.1, Weise A.1, Schleußner E.2, Kiehntopf M.3, Schreyer I.1'4
'Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Jena, Germany; 2Jena University Hospital, Friedrich Schiller University, Placenta-Labor, Department of Obstetrics, Jena, Germany;
3Jena University Hospital, Friedrich Schiller University, Institute of Clinical Chemistry and Laboratory Diagnostics, Jena, Germany; "Jena University Hospital, Zentrum für ambulante Medizin, Jena, Germany
A retrospective study based on 3,119 singleton and 56 twin pregnancies is presented. The standard levels of am-nion fluid derived alpha-fetoprotein level (AF-AFP) between 12th and 36th week of gestation were determined. Additionally, acetylcholinesterase (AChE) test results for 63 cases, ultrasonography results for 32 cases and abnormal karyotypic findings for 100 cases were available for selected cases. According to the present data the AF-AFP test is reliable and provides expected test results
in terms of population studies. However, individual AF-AFP test results can be subject to high individual variations. In this study AF-AFP multiple of medians (MoM) over 1.7 were indicative for neuronal tube defects and/or omphalocele in only 6.3% of the cases, while such AF-AFP values were hints on severe sonographic signs in 62% of the cases. Also, altered AF-AFP concentrations were present in 82% of cytogenetically abnormal cases. Overall, even though predicative value of the AF-AFP-test is matter of discussion it continues to be widely applied in invasive prenatal diagnostics. This study indicates that it only can be applied reliably in combination with other tests like banding cytogenetics, ultrasonography and all embedded in well-established genetic counseling.
MOLECULAR CHARACTERIZATION OF KMT2A FUSION PARTNER GENES IN 13 CASES OF PEDIATRIC LEUKEMIA WITH COMPLEX OR CRYPTIC KARYOTYPES
Garcia D.R.N.12, de Souza M.T.2-3, de Figueiredo A.F.2-3, Othman M.A.K.5, Abdelhay E.3, de Matos R.R.C.23, Meyer C.6, Marschalek R.6, Land M.G.P.14, Liehr T.5, Ribeiro R.C.7, Silva M.L.M.123 1Clinical Medicine Postgraduate Program, College of Medicine, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil;
Cytogenetics Department, Bone Marrow Transplantation Unit, National Cancer Institute, Rio de Janeiro, Brazil; 3Oncology Post Graduation Program, National Cancer Institute, Rio de Janeiro, Brazil; "Martagao Gesteira Institute of Pediatrics and Child Development, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil;
'Institute of Human Genetics, Jena University Hospital, Jena, Germany;
6Institute of Pharmaceutical Biology, Diagnostic Center of Acute Leukemia, Goethe-University of Frankfurt, Frankfurt/Main, Germany;
'Department of Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States
In pediatric acute leukemias, reciprocal chromosomal translocations frequently cause gene fusions involving the lysine (K)-specific methyltransferase 2A gene (KMT2A, also known as MLL). Specific KMT2A fusion partners are associated with the disease phenotype (lym-phoblastic vs. myeloid), and the type of KMT2A rearrangement also has prognostic implications. However, the KMT2A partner gene cannot always be identified by banding karyotyping. We sought to identify such partner genes in 13 cases of childhood leukemia with uninformative karyotypes by combining molecular techniques, including multicolor banding FISH, reverse-transcriptase PCR, and long-distance inverse PCR. Of the KMT2A fusion partner genes, MLLT3 was present in five patients, all with acute lymphoblastic leukemia, MLLT1 in two patients, and MLLT10, MLLT4, MLLT11, and AFF1
РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 4, 2016 ROSSIYSKIY VESTNIK PERINATOLOGY IPEDIATRII, 4, 2016
Раздел 6. Наследственные заболевания и врожденные пороки развития
in one patient each. Reciprocal reading by long-distance inverse PCR also disclosed KMT2A fusions with PITPNA in one patient, with LOC100132273 in another patient, and with DNA sequences not compatible with any gene in three patients. The most common KMT2A breakpoint region was intron/exon 9 (3/8 patients), followed by intron/exon 11 and 10. Finally, multicolor banding revealed breakpoints in other chromosomes whose biological and prognostic implications remain to be determined. We conclude that the combination of molecular techniques used in this study can efficiently identify KMT2A fusion partners in complex pediatric acute leukemia karyotypes.
COMPARATIVE ANALYSIS OF INDIVIDUAL CHROMOSOME INVOLVEMENT IN MICRONUCLEI INDUCED BY MITOMYCIN C AND BLEOMYCIN IN HUMAN LEUKOCYTES
Hovhannisyan G.1, Aroutiounian R.1, Babayan N.1,2, Harutyunyan T.1, Liehr T.3
'Department of Genetics and Cytology, Faculty of Biology, Yerevan State University, Yerevan, Armenia; 2Institute of Molecular Biology, National Academy of Sciences, Yerevan, Armenia;
3Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Jena, Germany
Micronucleus (MN) assay is a well standardized approach for evaluation of clastogenic/aneugenic effects of mutagens. Fluorescence in situ hybridization (FISH) is successfully used to characterize the chromosomal content of MN. However, the relationships between nuclear positioning, length, and gene density of individual chromosomes and their involvement in MN induced by different mutagens have not been clearly defined. Chromosomal content of MN was characterized in human leukocytes treated with mitomycin C (MMC) and bleomycin (BLM) by FISH using centromeric (cep) and whole-chromosome painting (wcp) probes. Involvement of chromosomes 8, 15 and 20 in MMC-induced and chromosomes 1, 9 and 16 in BLM-induced MN was studied, and correlated with chromosome size, gene density and interphase position. The results obtained were analyzed together with previous own data on the frequencies of inclusion of chromosomes 3, 4, 6, 7, 9, 16, 17, 18, and X in MMC-induced MN. It could be shown that MMC- and BLM-induced MN could contain material derived from all chromosomes investigated. Involvement of whole chromosomes 8, 15 and 20 in MMC-induced MN negatively correlated with gene density; however, analysis together with earlier studied chromosomes did not confirm this correlation. Inclusion of chromosomes 8, 15 and 20 in MMC-induced MN does not depend on their size and interphase position; the same result was found for the twelve overall analyzed chromosomes. In BLM-treated cells significant correlation between frequencies of involvement of chromosomes 1, 9 and 16 in MN and their size was found.
Our results clearly revealed that BLM differs from MMC with respect to the distribution of induced chromosome damage and MN formation. Thus, DNA-damaging agents with diverse mechanism of action induce qualitatively different MN with regard to their chromosomal composition. Also this study demonstrates the utility of combined sequential application of cep and wcp probes for efficient detection of MN chromosomal content in terms of centric and acentric fragments.
CONSTITUTIONAL GENOME AND CHROMOSOME INSTABILITIES IN THE AUTISTIC BRAIN
lourov I. Y.1-2-3, Vorsanova S.G.1-2-4, Liehr T.5, Zelenova M.A.1-2-4, Kurinnaia O.S.1-2-4, Vasin K.S.1-2-4, Kolotii A.D.1-2, Korostelev S.A.6, Yurov Y.B.1-2-4 'Mental Health Research Center, Moscow, Russian Federation;
2Research and Clinical Institute for Pediatrics named after Y. E. Veltishev at the Pirogov Russian National Research Medical University, Ministry of Health, Moscow, Russian Federation;
3Department of Medical Genetics, Russian Medical Academy of Postgraduate Education, Ministry of Health, Moscow, Russian Federation;
"Moscow State University of Psychology and Education,
Moscow, Russian Federation;
5Institute of Human Genetics, Jena, Germany;
6FSBI «Research Centre for Medical Genetics», Moscow,
Russian Federation
Introduction. Chromosome and genome instabilities confined to the human brain are associated with neurological and psychiatric diseases. However, the autistic brain has not been studied in context of genomic copy number variations. Here, we have evaluated copy number variations (CNV) and chromosomal rearrangements in the autistic brain.
Materials and Methods. SNP/oligonucleotide micro-array and FISH were used to evaluate 22 post-mortem brain samples of 12 patients with autism (cerebellum and cerebral cortex). Additionally, an original bioinformat-ics technology to interpret and prioritize CNV was used.
Results. Chromosomal mosaicism for additional chromosome X in a male chromosome complement and mosaic isodicentric chromosome 15 were detected in 2 unrelated samples. This data were confirmed by FISH. A sample exhibited duplication at 20q13.2q13.33 (~10 Mb). Constitutional chromosome instability mimic chromothripsis manifesting as non-mosaic and mosaic multiple CNV (from 7000 to 32100 and from 22 to 61, respectively) was detected in 3 cases. Mean CNV amount in the cerebellum was higher than in the cortex (287.8 vs. 259.7). Finally, all the samples of the autistic brain exhibited an increased background level of chromosome X an-euploidy as to control brain samples addressed previously.
Conclusions. We show that the autistic brain is hallmarked by constitutional genome (chromosome) instabil-
РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 4, 2016 ROSSIYSKIY VESTNIK PERINATOLOGY IPEDIATRII, 4, 2016
