[Genetic safety of cellular therapy].

This paper presents the main results of the study on chromosome and genome variability of mesenchymal stem cell cultures from bone marrow and adipose tissue carried out in the Laboratory of Mutagenesis, Research Centre for Medical Genetics, over the last three years. Genome stability was assessed from DNA damage using the DNA comet assay, karyotyping and registration of aneuploidy by the FISH method. We found that DNA damage rate in MSC cultures from bone marrow was 3.9% and 3.8% at the early (2-5) passages and the late (10-15) passages respectively. The cultures were characterized by high dispersion of individual values. Karyotyping showed mosaicism in both types of MSC cultures at the early and late stages of cultivation. The fraction of abnormal cells in some cultures amounted to 80-90%. Evaluation of aneuploidy in interphase cells revealed 1.34% of aneuploid cells (on the average) per one "conventional" chromosome; their overall frequency in the genome amounted to 20-40%. The frequency of aneuploid cells was similar at the early and late passages. Cultures with clones of trisomic and monosomic cells were revealed. The probability of occurrence of abnormal cells may increase by virtue of de novo mutations in the culture and as a result of positive selection of the cells existing in the organism that exhibit a higher reproduction rate in culture. Based on the experimental data on mutational process, selection of mutant cells and clone formation, it is concluded that cytogenetic control of stem cells is necessary to ensure the safety of cellular therapy.

Statistical analysis of clone formation in cultures of human stem cells.

We performed a statistical analysis of clone formation from aneuploid cells (chromosomes 6, 8, 11, X) in cultures of bone marrow-derived human multipotent mesenchymal stromal cells by spontaneous level of aneuploidy at different terms of culturing (from 2 to 19 cell cycles). It was found that the duration of cell cycle increased from 65.6 h at passages 2-3 to 164.5 h at passage 12. The expected ratio of aneuploid cells was calculated using modeled 5, 10, 20 and 30% selective preference in reproduction. The size of samples for detecting 10, 25, and 50% increased level of aneuploidy was calculated. The presented principles for evaluation of aneuploid clone formation may be used to distinguish clones of any abnormal cells.

Spontaneous aneuploidy and clone formation in adipose tissue stem cells during different periods of culturing.

Cytogenetic analysis of 13 mesenchymal stem cell cultures isolated from normal human adipose tissue was carried out at different stages of culturing. The incidence of chromosomes 6, 8, 11, and X aneuploidy and polyploidy was studied by fluorescent in situ hybridization. During the early passages, monosomal cells were more often detected than trisomal ones. A clone with chromosome 6 monosomy was detected in three cultures during late passages.

Methodological guidelines for genetic safety testing of cell transplants.

A combination of karyotyping and aneuploidy analysis by interphase fluorescent in situ hybridization is a sensitive method for evaluation of genetic stability of stem cell cultures. The methodology and specific features of preparing and analyzing the cytogenetic preparations are described as exemplified by human multipotent mesenchymal stromal cells.

Aneuploidy of stem cells isolated from human adipose tissue.

The incidence of autosomes 6 and 8 aneuploidy in stem cell cultures derived from adipose tissue was evaluated at different stages of culturing. Monosomy was more incident than trisomy during the early passages. Distribution of cultures by the incidence of aneuploidy in different chromosomes was virtually the same. Clones with chromosome 6 monosomy were detected in two cultures during late passages.

[Chromosomal instability parameters in the population affected by nuclear explosions at the Semipalatinsk nuclear test site]. / Pokazateli nestabil'nosti khromosomnogo apparata u naseleniia, postradavshego v rezul'tate iadernykh vzryvov na Semipalatinskom poligone.

A population genetic survey of 149 persons who were born and have permanently lived in the contaminated zones of the Semipalatinsk region has been performed. A cytogenetic study has demonstrated that the frequency of aberrant cells is 1.7-3 times higher than control parameters. The total frequencies of chromosome aberrations are 3.43 +/- 0.48, 3.1 +/- 0.3, 1.8 +/- 0.2, and 1.15 +/- 0.17 aberrations per 100 cells in the populations of the extreme radiation risk (ERR), maximum radiation risk (MaxRR), minimum radiation risk (MinRR), and control zones, respectively. The high chromosome aberration rate in all three zones of radiation risk has been detected mainly due to radiation-induced chromosome markers, including paired fragments (1.2 +/- 0.2, 0.94 +/- 0.13, and 0.43 +/- 0.06 per 100 cells, respectively), dicentric and ring chromosomes (0.44 +/- 0.04, 0.45 +/- 0.07, and 0.11 +/- 0.02 per 100 cells, respectively), and stable chromosome aberrations (0.74 +/- 0.16, 0.8 +/- 0.1, and 0.63 +/- 0.13 per 100 cells, respectively). The qualitative spectra of the cytogenetic lesions observed in these groups indicate a mutagenic effect of ionizing radiation on chromosomes in the populations studied.

[Chromosomal abnormalities in lymphocytes from a patient with Werner's syndrome in intact culture and after treatment with halogenated analogs of thymidine]. / Khromosomnye anomalii u bol'nogo sindromom Vernera v intaktnoi kul'ture limfotsitov i posle vozdeistviia galogenovykh analogov timidina.

Four balanced chromosomal translocation, deletion of chromosome 15, and a break in chromosome 11 were detected in 100 G-banded metaphases of cultured lymphocytes of a patient with Werner's syndrome. We observed aneuploidy that included both trisomies and monosomies for various chromosomes. Halogenated analogs of thymidine in low doses increased significantly the incidence of chromosome aberrations accompanied by fragments. 5-Iododeoxyuridine induced lesions in centromeric regions of B-group chromosomes in 44.4% of all the cases of breaks. A hypothesis is proposed about the existence of a special mechanism for genetic control in changes in the cell nucleus and mitotic chromosome transformation. This mechanism can be manifested after the application of halogenated analogs of thymidine. The mutation involved in Werner's syndrome is presumably related to this mode of genetic control.

[Inheritance of Wolff-Parkinson-White syndrome and evolution of its clinical symptoms in families of patients in a prospective trial]. / Nasledovanie sindroma Vol'fa-Parkinsona-Uaita i évoliutsiia ego klinicheskikh proiavlenii v sem'iakh bol'nykh pri prospektivnom nabliudenii.

Medicogenetic examination was conducted in families of 46 patients (21 women and 25 men aged 16-74 years) with Wolff-Parkinson-White (WPW) syndrome. A total of 256 relatives were investigated (136 women and 120 men aged 2 to 85 years). As a result, the diagnosis of preexcitation syndrome and phenomenon was made initially in 75(29.3%) of the relatives: WPW syndrome, Clerc-Levy-Cristesco (CLC) syndrome, CLC phenomenon was made in 6(2.3%), 27(10.6%) and 42(16.4%) relatives, respectively. Additional conduction pathways in the families with WPW syndrome are inherited by the autosome-dominant type with penetrability 0.94(94%) and clinical polymorphism. Prospective observation of the families revealed evolution of the clinical symptoms (development of arrhythmia) in the relative with CLC or WPW phenomenon in unfavorable exo- and endogenic factors. WPW syndrome evolution in the patients ran with aggravation of arrhythmia though 12 patients showed improvement.

[Radio-ecological and genetic assessment of late consequences of Totsk nuclear explosion]. / Radioékologicheskaia i geneticheskaia otsenka otdalennykh posledstvii Totskogo iadernogo vzryva.

Radio-ecological and cytogenetic assessments were made in the vicinity of Totsk nuclear explosion in the Orenburg Region. Increased contents of radioactive isotopes Cs and Pu were detected in the soil. A comparative cytogenetic analysis indicated that the population continued to be exposed to radioactive agents.

[Molecular-genetic characteristics of the deleted region of chromosome 8q24.1 in Langer-Giedion and tricho-rhino-phalangeal type I syndromes]. / MOlekuliarno-geneticheskaia kharakteristika oblasti deletsii khromosomy 8q24.1 pri sindromakh Langer-Gidiona i trikho-rino-falangovom I tipa.

A molecular-genetic characterization of deletions in part of chromosome 8q24.1 was performed in patients with Langer-Giedion syndrome (six patients) and triho-rhino-phalangeal syndrome type I (three patients) by means of Southern blot hybridization analysis, restriction fragment length polymorphism and single-strand conformation polymorphism, analysis. Four families with multiple exostosis chondrodysplasia (MECD) also underwent the same analysis. Results of deletion mapping allowed determination of the probable region of localization of the proposed gene of MECD at D8S67 locus. By means of a polymorphic DNA probe obtained from the locus an additional hybridization signal was revealed only in patients with MECD. Other polymorphic DNA probes and microsatellite sequences confirmed the results of deletion mapping and detected haplotypes on the chromosomes with a mutation in the proposed MECD gene.

[Clinical and biological significance of chromosome disorders in acute leukemia in children]. / Klinicheskoe i biologicheskoe znachenie khromosomnykh narushenii pri ostrykh leikozakh u detei.

Chromosomal examination was made in the bone marrow and lymphocytes of the peripheral blood of 37 children with acute leukemia. Of them 25 had acute lymphoblastic leukemia (ALL), 12 had acute nonlymphoblastic leukemia (ANLL). NA karyotype was registered in 13 patients (5 with ALL, 9 with ANLL), AA karyotype in 17 children (12 with ALL, 5 with ANLL). In All the following chromosomal markers were identified: del(6)/(q22), del(7)(q32), del(11)(q23), 9p+, t(8; 14) (q24; q32). In ANLL there were: 46, XY, t(3; 8)/46, XY; 47, XY, +16, +(2; 14)--M1-form; 46, XX, t(8; 21); 46, XX, t(8; 21)/46, XX--M2-form; 47, XX, +mar/46, XX; 47, XY, +12--M4-form; 46, XX, del(16)/46, XX, 47, XY, +16; 48, XY, +5, +8--M5-form FAB.

[Molecular diagnosis of fragile X-chromosome syndrome (Martin-Bell syndrome) in patients of native populations]. / Molekuliarnaia diagnostika sindroma fragil'noi X-khromosomy (sindrom Martina-Bella) u bol'nykh otechestvennykh populiatsii.

63 families at-rist of Fragile X-syndrome (FraX) are subjected to Southern blot analysis with the DNA probes Ox1.9 and Ox0.55. Molecular studies have confirmed an initial clinical diagnosis of FraX in 26 families earlier studied cytogenetically and in 11 of 27 families with only some clinical traits of FraX syndrome in proband. Full mutation and premutation condition of FMR-1 gene was ascertained in 34 and rejected in 18 close relatives of probands with the proved FraX syndrome in 37 and families. Four different patterns of pathological alleles are detected at electrophoretograms of DNA samples restricted by endonuclease RcoRI and hybridized to the DNA probe Ox1.9. Prenatal diagnosis of FraX was carried out in two cases at the 1st and 2nd trimester of pregnancy. Perspective of broad application of molecular methods for early diagnostics and prophylactic of FraX syndrome are briefly discussed.

[Chromosome fragile sites in patients with multiple primary tumors and familial forms of breast cancer]. / Izuchenie khromosomnoi sait-lomkosti u bol'ynkh s pervichno-mnozhestvennymi opukholiami i semeinymi formami raka molochnoi zhelezy.

An analysis was carried out of chromosomal site-fragility in patients with primary multiple tumors and familial breast cancer. A possible correlation is discussed between fragile sites, breakpoints in chromosome rearrangements in cancer patients and the localization of oncogenes mapped in chromosomal regions involved in said rearrangements.

[Prenatal cytogenetic diagnosis using chromosomal preparations of the native biopsy samples of chorionic villi, cultured chorionic cells and amniocytes]. / Dorodovaia tsitogeneticheskaia diagnostika s ispol'zovaniem khromo- somnykh preparatov iz nativnogo biotata vorsinchatogo khoriona, kul'tiviruemykh kletok khoriona i amniotsitov.

Analysis of the results of prenatal cytogenetic diagnosis carried out in the first and second pregnancy trimesters in more than 300 women permitted comparing the efficacies of two methodologic approaches, diagnostic amniocentesis and chorion sampling , with due consideration for the methodologic errors typical of these methods and of the tested biologic material. Up to 5% of the diagnoses are erroneous if the diagnosis is based on chorion sampling data, whereas in amniocentesis the share of diagnostic errors is lower by an order. The authors have given a theoretical rationale for and tried a methodologic approach, involving the employment of the 'direct' chromosomal preparations from villous chorion biopsy specimens and the so-called 'maintained' cell culture technique, that permits obtaining chromosomal preparations of higher quality and, consequently, helps improve the accuracy of chromosomal diagnosis.

[Phenotype and "critical segments" of chromosomes during partial aneuploidy for chromosome 4 in man]. / Fenotip i "kriticheskie segmenty" khromosom pri chastichnykh aneuploidiiakh po khromosome 4 u cheloveka.

Computerized analysis of sparse matrix, based on the list of involved organs, body parts, extremities, function etc. (total item number about 600) was performed for different cytogenetically identified anomalies of human chromosome 4 (35 cases of 4p-, 32 cases of 4p+, 39 cases of 4q-, 39 cases of 4q+; both published and original data were used). For each of the four types of partial aneusomy, 4 specific enough groups of traits were revealed which had been found in 50% of respective patients, at least. Such "nuclei" of traits were highly similar to those given in comprehensive modern manuals. However, 4p- and 4q- could only be classified as strictly enough delineated chromosomal syndromes. The 4(p14-pter) region was found to be the most likely crucial segment for the Wolf-Hirschhorn syndrome.

[Del(1)(q22-q25) syndrome. Cytogenetics and phenotype]. / Sindrom del(1)(q22-q25). Tsitogenetika i fenotip.

A male infant is described with dysmorphology of the head and face, neck, extremities and genitalia, as well as growth and mental retardation and with the de novo interstitial deletion of the proximal segment of the long arm of chromosome 1-del (1) (q22-q25). Comparison of the phenotypic characteristics of this patient with those of previously described patients with similar deletion confirms the existence of the proximal 1q deletion syndrome.