Chromosome painting has proven useful for the detection of chromosomal rearrangements, although the presence of cells containing clonal aberrations can have an effect on the outcome of cytogenetic analyses (e.g. aberration frequency and chromosomal distribution studies). Cells with clonal chromosomal changes have been found in studies of both radiation-exposed Chernobyl cleanup workers ("liquidators") and healthy unexposed human subjects. We have used a simple statistical method to aid in the identification of individuals from distinct Chernobyl radiation-exposed and unexposed control populations who may possess cells containing clonal rearrangements. A chi2 value determined from the observed number of aberrations and the expected number based on chromosome length that corresponds to a probability less than 0.005 appears to be an indicator of clonality. These selected individuals can be analyzed further for clonality, thereby sparing detailed examination of the entire population. Here we present an analysis of individuals possessing clonal aberrations to assess the influence of clonality on the results of cytogenetic studies. Our results show that the subtraction of clonal events from the chi2 calculation for the "outliers" results in nearly all of these values losing their statistically significant deviation from proportionality. These adjustments can also be made to prevent the overestimation of frequencies of chromosome aberrations for biodosimetry. The frequency of clonal aberrations appears to increase as a function of age in control subjects, whereas an age effect was not evident in Chernobyl liquidators. This suggests that spontaneous and radiation-induced clonal expansion are occurring in control subjects and liquidators, respectively.
To investigate within the framework of a multilaboratory study the suitability of FISH chromosome painting to measure so-called stable translocations in peripheral lymphocytes of Mayak nuclear-industrial workers (from the Southern Urals) and their use for retrospective biodosimetry.
Chromosime analyses were carried out from 69 workers who had received protracted occupational radiation exposures (0.012-6.065 Gy) up to approximately 40 years before blood sampling. Twenty-one unexposed people living in the same area were controls. A multicolour FISH-painting protocol with the target chromosomes 1, 4 and 8 simultaneously with a pancentromeric probe was used to score potentially transmissible chromosome-type aberrations (reciprocal translocations 2B and related 'one-way' patterns I-III according to the S&S classification).
Individual biodosimetry estimates were obtained in terms of these potentially long-term surviving aberration types based on the linear component of a low dose-rate gamma-ray calibration curve produced using identical staining and scoring protocols. For comparison, the workers personal and total background doses were converted to red bone marrow doses. The estimated doses were mainly lower than would be predicted by the calibration curve, particularly at accumulated higher dose levels.
Owing to the limited life-time of circulating T-lymphocytes, the long-term persistence of translocations in vivo requires the assumption of a clonal repopulation of these naturally senescing cells from the haemopoietic stem cell compartments. Obviously such a replacement cannot be fully achieved, leading to a temporal decline even of the yield of transmissible aberrations types. Assuming further a highly selective capacity of stem cells against any type of chromosomal damage and the fact that one must rely on partial genome findings, the potential of FISH chromosome painting for retrospective dose reconstruction is probably limited to a decade or so after high-level protracted radiation exposure.
A critical survey of all published measurements made so far aimed at retrospective biological dosimetry using fluorescence in situ hybridisation (FISH) techniques on some workers at the Mayak reprocessing plant and on members of the Techa River cohort is given. Each individual has a recorded dose derived from personal monitoring measurements, usually external gamma-rays for Mayak workers or from reconstruction techniques, usually internally derived for the Techa River cohort. From the person's age, which affects the control level, and the stated dose, an expected number of translocations is calculated for each individual and comparisons made to the observed numbers of translocations. From this, an assessment of how well FISH studies can help to validate existing estimates of dose is made. This varies from study to study. Good agreement is generally obtained for the Techa River cohort and lower doses of the Mayak cohort. Rather poorer agreement applies to the more highly exposed Mayak workers. Some of the discrepancy could be because the FISH painting technique was new and was applied to populations before a proper investigation on how to use it for retrospective biological dosimetry had taken place. In addition, too few cells were generally scored per individual so that statistical uncertainties were large.
The etiology of mental retardation (MR), often presenting as developmental delay in childhood, is unknown in approximately one-half of cases. G-banding is the standard method for investigating those suspected of having a chromosomal etiology; however, detection of structural abnormalities is limited by the size and pattern of the G-bands involved. Rearrangements involving subtelomeric regions have been shown to cause MR and this has generated interest in investigating the prevalence of these rearrangements using telomere-specific probes. In addition, because cryptic interchromosomal rearrangements may not be small or confined to chromosomal ends, spectral karyotyping (SKY) using chromosome-specific painting probes may be of value. We report here a study using these two FISH-based techniques in 50 children with idiopathic MR or developmental delay and normal GTG-banded karyotypes. Our objective was to assess the prevalence of cryptic rearrangements in this population using subtelomeric FISH and SKY. Three rearrangements were detected by subtelomeric FISH: a derivative 5 from a maternal t(5;21); a recombinant 11 from a paternal pericentric inversion; and a 2q deletion that was also present in the mother. Only the derivative 5 was detected by SKY. SKY did not detect any interstitial interchromosomal rearrangement. The prevalence of clinically significant cryptic rearrangements by subtelomeric FISH and SKY was thus 4% (95% confidence interval 0.5-13.7) and 2% (95% CI 0.05-10.7), respectively. This study supports the view that G-banding does not detect all clinically significant chromosomal abnormalities and that subtelomeric FISH and SKY can detect some of these abnormalities.
The incidence of papillary thyroid carcinoma (PTC) increases significantly after exposure of the head and neck region to ionizing radiation, yet we know neither the steps involved in malignant transformation of thyroid epithelium nor the specific carcinogenic mode of action of radiation. Such increased tumor frequency became most evident in children after the 1986 nuclear accident in Chernobyl, Ukraine. In the eight years following the accident, the average incidence of childhood PTCs (chPTC) increased 70-fold in Belarus, 200-fold in Gomel, 10-fold in the Ukraine and 50-fold in Tschnigov, Kiev, Rovno, Shitomyr and Tscherkassy compared to the rate of about 1 tumor incidence per 106 children per year prior to 1986 (Likhtarev et al., 1995; Sobolev et al., 1997; Jacob et al., 1998). To study the etiology of radiation-induced thyroid cancer, we formed an international consortium to investigate chromosomal changes and altered gene expression in cases of post-Chernobyl chPTC. Our approach is based on karyotyping of primary cultures established from chPTC specimens, establishment of cell lines and studies of genotype-phenotype relationships through high resolution chromosome analysis, DNA/cDNA micro-array studies, and mouse xenografts that test for tumorigenicity. Here, we report the application of fluorescence in situ hybridization (FISH)-based techniques for the molecular cytogenetic characterization of a highly tumorigenic chPTC cell line, S48TK, and its subclones. Using chromosome 9 rearrangements as an example, we describe a new approach termed 'BAC-FISH' to rapidly delineate chromosomal breakpoints, an important step towards a better understanding of the formation of translocations and their functional consequences.
Speculation has long surrounded the question of whether past exposure to ionizing radiation leaves a unique permanent signature in the genome. Intrachromosomal rearrangements or deletions are produced much more efficiently by densely ionizing radiation than by chemical mutagens, x-rays, or endogenous aging processes. Until recently, such stable intrachromosomal aberrations have been very hard to detect, but a new chromosome band painting technique has made their detection practical. We report the detection and quantification of stable intrachromosomal aberrations in lymphocytes of healthy former nuclear-weapons workers who were exposed to plutonium many years ago. Even many years after occupational exposure, more than half the blood cells of the healthy plutonium workers contain large (>6 Mb) intrachromosomal rearrangements. The yield of these aberrations was highly correlated with plutonium dose to the bone marrow. The control groups contained very few such intrachromosomal aberrations. Quantification of this large-scale chromosomal damage in human populations exposed many years earlier will lead to new insights into the mechanisms and risks of cytogenetic damage.