Spontaneous chromosomal aberrations in Fanconi anaemia,ataxia telangiectasia fibroblast and Bloom's syndrome lymphoblastoid cell lines as detected by conventional cytogenetic analysis and fluorescence in situ hybridisation (FISH) technique

Several primary and transformed human cell lines derived from cancer prone patients are employed routinely for biochemical and DNA repair studies. Since transformation leads to some chromosomal instability a cytogenetic analysis of spontaneous chromosome aberrations in fibroblast cell lines derived from patients with Fanconi anaemia (FA), ataxia telangiectasia (AT), and in lymphoblastoid cell lines derived from patients with Bloom's syndrome (BS), was undertaken. Unstable aberrations were analysed in Giemsa stained preparations and the chromosome painting technique was used for evaluating the frequencies of stable aberrations (translocations). In addition, the frequency of sister-chromatid exchanges (SCEs) was determined in differentially stained metaphases. The SV40-transformed fibroblasts from these cell lines have higher frequencies of unstable aberrations than the primary fibroblasts. In the four lymphoblastoid cell lines derived from BS patients higher frequencies of spontaneously occurring chromosomal aberrations in comparison to normal TK6wt cells were also evident. The frequency of spontaneously occurring chromosome translocations was determined with fluorescence in situ hybridisation (FISH) and using DNA libraries specific for chromosomes 1, 2, 3, 4, 7, 8, 11, 14, 19, 20 and X. The translocation levels were found to be elevated for primary FA fibroblasts and lymphoblastoid cells derived from BS patients in comparison with control cell lines, hetero- and homozygote BS cell lines not differing in this respect. The SV40-transformed cell lines showed very high frequencies of translocations independent of their origin and almost every cell contained at least one translocation. In addition, clonal translocations were found in transformed control TK6wt and AT cell lines for chromosomes 20 and 14, respectively. The spontaneous frequencies of SCEs were similar in transformed fibroblasts derived from normal individuals and AT patients, whereas in SV40-transformed FA cells these were higher (4-fold). Among cell lines derived from BS patients, heterozygote lines behaved like control, whereas in homozygote cell lines very high frequencies of SCEs (about 12-fold) were evident.     

Effects of X-irradiation on cell-cycle progression, induction of chromosomal aberrations and cell killing in ataxia telangiectasia (AT) fibroblasts

Survival, cumulative labeling indices, chromosomal aberrations and cell-cycle distribution by flow microfluorometry (FMF) were studied in fibroblasts from normal and three ataxia telangiectasia (AT) families after X-irradiation during density-inhibition of growth and immediate release by subculture to low density. Homozygotic AT (proband) fibroblasts were very hypersensitive to cell killing by X-irradiation (D0 = 40-45 rad). Fibroblasts from AT heterozygotes (parents) were minimally hypersensitive, with D0's (100-110 rad) slightly lower than those for normal fibroblasts (D0 = 120-140 rad). There were three different response groups for a G1 phase block induced by 400 rad of X-rays: (1) minimal or no G1 block was observed in AT homozygote cell strains; (2) 10-20% of the cells were blocked in G1 in normal cell strains; and (3) 50% or more of the cells were blocked in AT heterozygote strains. FMF profiles and cumulative labeling indices showed that homozygotic AT cells irradiated in plateau phase moved into the S-phase following subculture with no additional delay over non-irradiated controls. Homozygotic AT cells showed not only a 4-5 times higher frequency of X-ray-induced chromosomal aberrations than normal strains, but approximately 30% of these were of the chromatid-type. There were no differences in the frequency or type of X-ray-induced chromosomal aberrations between normal and heterozygotic AT cells.     

Heterogeneity of chromosomal breakage levels in epithelial tissue of ataxia-telangiectasia homozygotes and heterozygotes

Summary The objective of this study was to obtain an estimate of the frequency distribution of spontaneous chromosomal breakage occurring in vivo in oral epithelia of 20 ataxiatelangiectasia patients (A-T homozygotes) and 26 parents (A-T obligate heterozygotes). Samples of exfoliated cells were obtained from each individual by swabbing the oral cavity and preparing air-dried slides. The percentage of exfoliated cells with micronuclei (MEC frequency) was used as an in vivo indicator for the amount of chromosomal breakage occurring in the tissue. As a population group, MEC frequencies of the A-T patients differed significantly from controls (mean for A-T patients, 1.51; for controls, 0.29; P<0.01). However, the values observed in individual patients ranged from MEC frequencies 10- to 12-fold above control values, to frequencies overlapping the upper values observed in the controls. Similarily, MEC frequencies observed among the A-T heterozygotes differed significantly from controls (mean for A-T heterozygotes, 1.02, mean for controls, 0.29; P<0.01). However, only 16 of the 26 individuals sampled had MEC frequencies >0.5%, the 90th percentile for controls (compared with 16 of the 20 A-T patients examined). Of the A-T patients 11 had been previously assigned to complementation groups on the basis of sensitivity to x-irradiation. Seven of the patients belonged to group A and had MEC frequencies ranging from 0.3% to 1.9% with the remaining patients belonging to group C with MEC frequencies of 0.2% to 0.9%. The data presented in this paper suggest that although levels of spontaneous breakage in epithelial tissues of A-T patients and A-T obligate heterozygotes are often significantly elevated, this is not the case in all individuals.     

High frequency of spontaneous translocations revealed by FISH in cells from patients with the cancer-prone syndromes ataxia telangiectasia and Nijmegen breakage syndrome.

The application of fluorescence in situ hybridization (FISH) using whole-chromosome paints (WCPs) is proving to be a very powerful technique for revealing chromosomal instability that, for the most part, has gone undetected by conventional cytogenetic analysis. We have analyzed the frequency of translocations in lymphocytes and lymphoblastoid cell lines from ataxia telangiectasia (AT) and Nijmegen breakage syndrome (NBS) homozygotes and heterozygotes using a three-color chromosome-painting technique (WCP 1, 2, 4). With this assay we were able to detect an increased frequency of spontaneous translocations in AT homozygotes (median, 18.47 +/- 10.82 translocations per 1,000 metaphase cells; 10 patients) and AT heterozygotes (median, 7.87 +/- 3.15 translocations per 1,000 cells; 7 patients), in comparison to controls (median, 2.26 +/- 1.75 translocations per 1,000 cells; 10 controls). Analysis of NBS homozygotes (median, 19.05 +/- 11.27 translocations per 1,000 cells; 5 patients) and NBS heterozygotes (median, 6.93 +/- 3.04 translocations per 1,000 cells; 6 patients) also showed an increased frequency of translocations in these patients compared to controls. The presence of such hitherto undetected chromosomal aberrations corroborate previous findings of spontaneous chromosomal instability in AT and NBS patients, as manifested by an increased rate of open breaks and rearrangements involving chromosomes 7 and 14. Moreover, we show that the degree of genomic instability in AT and NBS patients is even higher than previously established and that some AT and NBS heterozygotes evidence spontaneous chromosomal instability as well. These increased levels of nonspecific translocations could be an important risk factor for the development of malignancies in homozygotes and heterozygotes for ATM or NBS1 gene mutations.     

In vivo chromosomal instability in ataxia-telangiectasia homozygotes and heterozygotes

Summary The exfoliated cell micronucleus test was used to monitor in vivo chromosomal instability in a population comprised of five ataxia-telangiectasia (A-T) homozygotes and seven obligate heterozygotes (parents of A-T patients). This assay was previously validated as a procedure for quantifying non-invasively carcinogen-induced chromosomal aberrations occurring in vivo in epithelial tissues of both the oral cavity and the urinary bladder. The procedure involved taking airdried smears of three sites in the oral cavity of each examined individual. Desquamated urinary bladder cells were collected by centrifugation of freshly voided urine samples. Frequencies of exfoliated cells in these preparations were determined and compared with control values (individuals with no genetic chromosomal instability and no known carcinogene exposure) for these sites. Exforliated cell micronucleus (MEC) frequencies were elevated 5- to 14-fold in samples from the A-T homozygotes. This elevation in MEC frequency occurred for both the oral cavity and urinary bladder. Five out of the seven obligate A-T heterozygotes had an elevated MEC frequency in samples from the oral cavity. In addition, all examined urine samples from A-T heterozygotes contained an elevated percentage of micronucleated cells. These data suggest that this assay is suitable for in vivo monitoring of groups of individuals in which genetically produced chromosomal damage occurs. The possibility of A-T heterozygote detection with this simple procedure is of particular significance, since such individuals are believed to comprise up to 1% of the general population, and have been identified as being at elevated risk for cancer.     

No induction of interchromosomal effect in male meiosis of Chinese hamsters with reciprocal translocations

Chinese hamsters from five strains with reciprocal translocations, T(1;3)7Idr, T(1;3)8Idr, T(1;2)9Idr, T(7;9)16Idr, and T(1;5)17Idr, and a karyotypically normal strain, CHS/Idr, were used to look for an interchromosomal effect by chromosomal analysis of meiotic cells and one-cell embryos. The frequencies of nondisjunction at first meiosis in five normal (+/+) males, calculated by doubling the number of hyperhaploid cells, ranged from 0.43% to 1.33%, and there was no significant difference in frequency among individuals. On the other hand, the frequency of hyperhaploid cells in males heterozygous for each translocation ranged from 3.0% to 11.8%, and the frequency of hyperhaploid cells with an extra translocation-unrelated chromosome ranged from 0.2% to 0.4%, which is no different from that estimated from scoring of +/+ males at the second meiotic metaphase. In one-cell embryos from crosses between karyotypically normal females and male heterozygotes for T(1;2)9Idr and T(7;9)16Idr, 1.1% and 0.5% of embryos had an extra translocation-unrelated chromosome. Compared with the control, the frequency of meiotic nondisjunction showed no increase in male heterozygotes for the reciprocal translocations. Therefore, the results suggest that multivalents and rearranged chromosomes existing at first and second meiosis in male Chinese hamsters exert no influence on segregation of normal bivalents and chromosomes unrelated to the rearrangements.     

Chronic gamma-irradiation results in increased cell killing and chromosomal aberration with specific breakpoints in fibroblast cell strains derived from non-Hodgkin's lymphoma patients.

Cultured skin fibroblast cells from 6 patients with non-Hodgkin's lymphoma (NHL) and 2 clinically normal subjects were compared for cell survival and chromosomal aberration after chronic gamma-irradiation. Fibroblasts from an ataxia telangiectasia (AT) homozygote and an AT heterozygote were used as positive controls. Following irradiation, fibroblasts from all 6 NHL patients showed an increase in both cell death and chromosomal aberration (breaks and rearrangements) compared to the normal subjects. The difference in the frequency of chromosomal aberration between the normals and the NHL patients remained virtually unchanged over a period of 24-72 h post irradiation incubation of the cells. Cell cycle analysis by flow cytometry carried out in 1 normal and 1 NHL fibroblast cell strain showed that more cells representing the NHL patient were in G2/M phase compared to the normal at various times of cytogenetic analysis. While the AT homozygote appeared to be the most radiosensitive, the AT heterozygote showed a slightly higher incidence of cell death and chromosomal aberration than the normals. The cellular and chromosomal radiosensitivity of fibroblast cell lines from the NHL patients differed slightly from that of the AT heterozygote but clearly occupied an intermediate position between the AT homozygote and the normal subjects. Cells from 3 of the NHL patients showed radiation-induced specific chromosomal breaks involving chromosomes 1, 2, 6, 8, 10 and 11 which correspond to known fragile sites. Such breakpoints associated with increased radiosensitivity may be indicative of predisposition to malignancy in the patients studied.     

Estimating meiotic disjunction frequencies in chicken translocation heterozygotes based on embryonic mortality

Chickens heterozygous for a chromosomal translocation [MN t(1;4)] were intercrossed and the progeny were analyzed for their chromosome complement. A ratio of 1 homozygous translocation carrier to 4 heterozygous translocation carriers to 1 homozygous standard chromosome carrier was noted (n = 520), rather than the 1:2:1 ratio expected from Mendelian segregation. The excess of heterozygous carriers was apparently caused by union of complementary duplication/deficient gametes. Embryonic death occurred in 68% of fertile zygotes. This finding fits very closely to expectations if alternate and adjacent meiotic disjunctions occurred at equal frequencies. As alternate disjunction frequencies increase from 0.5 to 1.0 in inter se matings, the proportion of inviable zygotes among fertilized ova will decrease from 0.625 to 0 and the proportion of translocation heterozygotes among viable progeny will decrease from 0.667 to 0.5. In instances where alternate and adjacent disjunction occur at equal frequency, preferential recovery of translocation carriers will occur. This may contribute to chromosomal diversity within a species, and possibly lead to speciation.     

Radiation induced apoptosis in ataxia telangiectasia homozygote, heterozygote and normal cells

Recent reports suggest that the radiation-induced, p53-dependent, apoptotic response is aberrant in ataxia telangiectasia (AT) cells. We investigated the possibility that an aberrant apoptotic response to ionizing radiation may also be the characteristic of AT heterozygotes and may facilitate in discriminating AT heterozygotes from the general population. Log phase, Epstein Barr virus (EBV) transformed lymphoblastoid cell lines and primary lymphocytes from three AT families were irradiated and the apoptotic response at 30h post radiation was measured by flow cytometry using TUNEL and hypodiploid methods. Our results show that the apoptotic response of AT homozygote (ATM-/-), AT heterozygote (ATM+/-) and normal cells (ATM+/+) to ionizing radiation, measured by the hypodiploid and TUNEL methods using flow cytometry, is dose and time dependent. Furthermore, this response is paradoxical in that ATM (-/-) lymphoblastoid cells were characterized by a reduced post radiation apoptotic response compared to their normal counterparts. Heterozygote (ATM+/-) lymphoblastoid cells displayed an intermediate response to ionizing radiation. In contrast, primary, non-transformed AT cells exhibited the same apoptotic response as their normal counterparts. Our results thus indicate that pre-radiation, EBV-transformed, lymphoblastoid cell lines from individual families may be useful in discriminating ATM status, but patient-derived, primary AT homozygous, heterozygous and normal primary cultured lymphocytes cannot be discriminated by this assay.     

Association between synaptonemal complexes of sex and autosomal bivalents in male tx/ty mice as a possible cause of their sterility

Electron microscopic study of total preparations of synaptonemal complexes of spermatocytes I from sterile heterozygous male mice--t12/tw18; tw5/twPa-1; twPa-1/tw18 was performed. T/tw18 and C3H/N fertile heterozygotes were used in each variant as control. The cells are karyotyped in all experiments, as based on the measurements of the length of 19 SC autosomes and SC sex complex. All sterile compounds (spermatocytes) demonstrate high frequency of different types of associations (72%) between sex chromosomes and the autosome 17 carrying a chromosomal aberration in the region of the T-locus. The heterozygotes tx/ty used in our experiments show no disruption of chromosome synapsis, when even studied under electron microscope, though some atypical changes in the ultrastructure of chromosome axes and frequent atypical associations of the axes of XY-sex bivalents in sterile heterozygous animals exist.     

Louis-Bar syndrome: spontaneous and induced chromosomal aberrations in lymphocytes and micronuclei in lymphocytes,oral mucosa and hair root cells

Summary Louis-Bar (L-B) syndrome, also called ataxia-telangiectasia, is cytogenetically characterized by an increased frequency of spontaneous and induced chromosomal aberrations (CA) in cultured lymphocytes and skin fibroblasts. However, it is not yet clear whether the chromosomal instability is also present in uncultured cells. The spontaneous and bleomycin-induced CA in peripheral lymphocytes of 8 L-B patients were evaluated. The micronucleus test was also performed, for the first time in lymphocytes by the cytokinesis-block method, and in uncultured cells of the oral cavity and hair root. The spontaneous frequency of CA and micronuclei in lymphocytes was about 3 times higher in L-B patients than in controls, these two cytogenetic parameters being highly correlated. Moreover, the induction by bleomycin of CA was higher in patients than in controls. The micronuclei in buccal and hair root cells of patients were normal. It remains to be determined whether the different responses obtained with cultured and uncultured cells are the result of the different L-B gene expression of chromosomal instability or whether they arise because of a particular cell sensitivity to culture conditions. The spontaneous and induced CA in lymphocytes of heterozygotes cultured in the presence of L-B serum were studied to evaluate a possible increased sensitivity of heterozygotes to a possible diffusible clastogenic factor present in the plasma of L-B patients. We could not demonstrate the presence of any factor that enhances CA in normal subjects or in heterozygote carriers.     

Clastogenic effect of ethanol in chronic and abstinent alcoholics

Alcoholism is one of the main causes of damage for human health, being relevant to study the induction of chromosomal aberrations (CA) by ethanol, and to investigate the individual susceptibility to diseases caused by alcoholism. A cytogenetic study was performed in human peripheral blood lymphocytes of 29 heavy chronic alcoholics, 11 alcoholics in abstinence, and 10 controls. The values of the chromosomal aberrations, mitotic indexes (MI) and proliferation indexes (PI) were determined. A molecular cytogenetic study was also carried out using fluorescence in situ hybridization (FISH) method with DNA library probes for chromosomes 1, 3 and 6, in lymphocytes from chronic alcoholic individuals in comparison with a control group. The results showed that the CA frequencies for chronic alcoholics (5.15 CA/100 cells) and alcoholics in abstinence (3.87 CA/100 cells) were higher than those obtained for control individuals (1.72 CA/100 cells). The mean translocation frequencies (equivalent to the genome) were calculated for six chronic alcoholics (0.267 translocations/100 cells) and six alcoholics in abstinence (0.167 translocations/100 cells), whose values were significantly higher than those observed for six control individuals (0.067 translocations/100 cells). The CA frequencies were not statistically different when smoker and non-smoker alcoholics were compared, indicating that although the smoking habit had significantly increased (four-fold) the CA frequency in healthy control individuals, a lack of interaction effect was observed within the group of alcoholics when smokers and non-smokers were compared. The CA frequencies presented by alcoholics in abstinence were similar to those obtained for chronic alcoholics. Therefore, chronic ethanol intoxication can lead to chromosome damage and disturbances in the metabolism of endogenous and exogenous compounds, which may persist for a long time, and constitute a relevant factor of risk for the development of neoplasias.     

Clastogenic effect of ethanol in chronic and abstinent alcoholics

Alcoholism is one of the main causes of damage for human health, being relevant to study the induction of chromosomal aberrations (CA) by ethanol, and to investigate the individual susceptibility to diseases caused by alcoholism. A cytogenetic study was performed in human peripheral blood lymphocytes of 29 heavy chronic alcoholics, 11 alcoholics in abstinence, and 10 controls. The values of the chromosomal aberrations, mitotic indexes (MI) and proliferation indexes (PI) were determined. A molecular cytogenetic study was also carried out using fluorescence in situ hybridization (FISH) method with DNA library probes for chromosomes 1, 3 and 6, in lymphocytes from chronic alcoholic individuals in comparison with a control group. The results showed that the CA frequencies for chronic alcoholics (5.15 CA/100 cells) and alcoholics in abstinence (3.87 CA/100 cells) were higher than those obtained for control individuals (1.72 CA/100 cells). The mean translocation frequencies (equivalent to the genome) were calculated for six chronic alcoholics (0.267 translocations/100 cells) and six alcoholics in abstinence (0.167 translocations/100 cells), whose values were significantly higher than those observed for six control individuals (0.067 translocations/100 cells). The CA frequencies were not statistically different when smoker and non-smoker alcoholics were compared, indicating that although the smoking habit had significantly increased (four-fold) the CA frequency in healthy control individuals, a lack of interaction effect was observed within the group of alcoholics when smokers and non-smokers were compared. The CA frequencies presented by alcoholics in abstinence were similar to those obtained for chronic alcoholics. Therefore, chronic ethanol intoxication can lead to chromosome damage and disturbances in the metabolism of endogenous and exogenous compounds, which may persist for a long time, and constitute a relevant factor of risk for the development of neoplasias.     

Diagnosis of heterozygous states for adenine phosphoribosyltransferase deficiency based on detection of in vivo somatic mutants in blood T cells: application to screening of heterozygotes.

An accurate diagnosis of heterozygotes for autosomal recessive disorders with unknown mutations can be difficult. Using a unique phenomenon occurring in vivo, we designed a method for the diagnosis of heterozygotes for adenine phosphoribosyltransferase (APRT) deficiency which makes way for a qualitative distinction between normal and heterozygous subjects. We cultured peripheral blood mononuclear cells with 2,6-diaminopurine, an APRT-dependent cytotoxin, to search for in vivo mutational cells. Fifteen putative heterozygotes examined were found to possess such mutant cells at rather high frequencies; thus, a false negative diagnosis is unlikely. The analysis of genomic DNA in 82 resistant clones from two of the heterozygotes clarified that 64 (78%) had lost the germinally intact alleles. Thirteen members of APRT-deficient families were examined; eight proved to be heterozygotes. Among 425 individuals from two separate residential areas of Japan, two heterozygotes were found. The authenticity of the heterozygosity was validated by two separate methods for the two heterozygotes; hence, a false positive diagnosis can be ruled out. Our data showed a calculated heterozygote frequency of 0.47% (95% confidence limits; 0.05%-1.7%), a value compatible with that (1.2%) calculated from data concerning the incidence of 2,8-dihydroxyadenine urolithiasis. This novel genetic approach for identifying heterozygotes is now being tested to search for other enzyme deficiencies in humans.     

Spontaneous and induced chromosomal damage and mutations in Bloom Syndrome mice

Bloom Syndrome (BS) is characterized by both cancer and genomic instability, including chromosomal aberrations, sister chromosome exchanges, and mutations. Since BS heterozygotes are much more frequent than homozygotes, the issue of the sensitivity of heterozygotes to cancer is an important one. This and many other questions concerning the effects of BLM (the gene responsible for the BS) are more easily studied in mice than in humans. To gain insight into genomic instability associated with loss of function of BLM, which codes for a DNA helicase, we compared frequencies of micronuclei, somatic mutations, and loss of heterozygosity (LOH) in Blmtm3Brd homozygous, heterozygous, and wild-type mice carrying a cII transgenic reporter gene. It should be noted that the Blmtm3Brd is inserted into the endogenous locus with a partial duplication of the gene, so some function of the locus may be retained. The cII reporter gene was introduced from the Big Blue mouse by crossing them with Blmtm3Brd mice. All measurements were made on F2 mice from this cross. The reticulocytes of Blmtm3Brd homozygous mice had more micronuclei than heterozygous or wild-type mice (4.5, 2.7, and 2.5 per thousand, respectively; P < 0.01) but heterozygotes did not differ significantly from wild-type. Unlike spontaneous chromosome damage, spontaneous mutant frequencies did not differ significantly among homozygous, heterozygous, and wild-type mice (3.2 x 10(-5), 3.1 x 10(-5), and 3.1 x 10(-5), respectively; P > 0.05). Mutation measurements were also made on mice that had been treated with ethyl-nitrosourea (ENU) because Bloom Syndrome cells are sensitive to ethylating agents. The ENU-induced mutation frequency in Blmtm3Brd homozygous, heterozygous, and wild mice were 54 x 10(-5), 35 x 10(-5), and 25 x 10(-5) mutants/plaques, respectively. ENU induced more mutations in Blmtm3Brd homozygous mice than in wild-type mice (P < 0.01), but not significantly more in heterozygous mice (P = 0.06). Spontaneous LOH did not differ significantly among the genotypes, but ENU treatment induced much more LOH in Blmtm3Brd homozygous mice, as measured by means of the Dlb-1 test of Vomiero-Highton and Heddle. Hence, these Blmtm3Brd mice resemble Bloom Syndrome except that they have normal frequencies of spontaneous mutation. The fact that these mice have elevated rates of both cancer and chromosomal aberrations (as shown by more micronuclei and LOH) but normal rates of spontaneous mutation, shows the greater importance of chromosomal events than mutations in the origin of their cancers.     

Chromosomal control of chiasma distribution in house mice. Analysis of chiasma distribution in homo- and heterozygotes by inversion in chromosome 1

Examination of chiasma distribution in the chromosome 1 in male mice homo- and heterozygous for distal inversion In(1)12Rk and in normal mice was carried out. No differences in chiasma distribution was found between homozygotes for the inversion and homozygotes for normal chromosome 1. A drastic change in this trait was revealed in heterozygous animals. In heterozygotes, the telomeric segments of SC were asynapsed and unavailable for recombination. This leads to significant decrease in the frequency of bivalents bearing chiasmata in pretelomeric region. In turn, it produced chiasma redistribution in proximal noninverted portion of the bivalent 1. These results could be interpreted as evidence for chromosomal control of chiasma distribution pattern: the distance of certain part of the chromosome from telomere and interference (which also operates at the chromosomal level) are more important for determination of the chiasmata frequency in the given region, than its genetic content.     

Cytogenetic characterization of ataxia telangiectasia (AT) heterozygotes using lymphoblastoid cell lines and chronic γ-irradiation

Summary Lymphoblastoid cell lines (LCLs) derived from two patients identified as ataxia telangiectasia (AT), two obligate AT heterozygotes and two controls (healthy subjects with no known genetic disease or relationship to AT patients) were compared with respect to the induction of chromosomal breaks by acute and chronic -irradiation. Although there was a considerable increase in the frequency of chromosomal breaks per cell in the LCLs of AT patients resulting from acute irradiation, the small increase occurring in the LCLs of the AT heterozygotes made it difficult to distinguish them from the controls. Following chronic -irradiation, however, the frequency of chromosomal breaks per cell in the LCLs of the AT heterozygotes occupied a significantly distinct position from that of the controls. These observations suggested that the use of chronic irradiation may be a better choice in the cytogenetic characterization of AT heterozygotes.     

Meiotic studies of male common shrews (Sorex araneus L.) from a hybrid zone between chromosome races

Thirty-three adult male common shrews (Sorex araneus L.) were collected from a hybrid zone between two chromosomal races that differed in Robertsonian metacentrics. Anaphase I nondisjunction frequencies were estimated on the basis of metaphase II counts. RIV and CV complex heterozygotes (four-element rings and five-element chains at meiosis I, respectively) had substantially higher nondisjunction rates than homozygotes and simple Robertsonian heterozygotes. However, at least in the case of RIV-forming hybrids, increased nondisjunction frequency did not result from malsegregation of the heterozygous complex. Extra elements found in hyperploid spreads were most frequently acrocentrics, that could not originate from a fully metacentric multivalent. Complex heterozygotes were also characterized by higher frequencies of univalents observed at diakinesis I. However, univalents did not originate from complex configurations, which were regularly formed with usually one chiasma per chromosome arm. Hence, we suppose that the presence of multivalents in the cell affects pairing and segregation of other elements at meiosis I.     

Influence of ataxia telangiectasia gene dosage on bleomycin-induced chromosome breakage and inhibition of replication in human lymphoblastoid cell lines

Long-term lymphoblastoid cell lines, obtained by E-B virus transformation of peripheral blood lymphocytes, retain many of the features of hypersensitivity to environmental agents found in primary cultures and fibroblast strains from patients with genetic diseases. Primary lymphocyte cultures from patients with ataxia telangiectasia, a cancer-prone genetic disease, have increased sensitivity to chromosomal damage induced by the radio-mimetic drug, bleomycin. In order to study the expression of ataxia telangiectasia gene dosage in lymphoblastoid cell lines, we examined chromosomal aberrations in lines containing two, one, or no alleles for ataxia telangiectasia. These were derived from ataxia telangiectasia homozygotes, from ataxia telangiectasia obligate heterozygotes, and from presumably normal donors, respectively. Chromosome preparations were made 46 h after a 2 h exposure to bleomycin and scored for chromosome breakage, for the relative rate of cell replication as assessed by sister chromatid differentiation patterns, and for the frequency of sister chromatid exchanges. Baseline frequencies of chromosome breakage and sister chromatid exchanges, and baseline rates of cell replication were similar in all nine lymphoblastoid cell lines. Following treatment with 25 or 250 mU/ml bleomycin, all the lymphoblastoid cell lines showed increased chromosome breakage and decreased cell replication. The lymphoblastoid cell lines from the ataxia telangiectasia homozygotes had significantly increased chromosome breakage and decreased rate of cell replication after either bleomycin dose in comparison with the normal or with the ataxia telangiectasia heterozygous lines. Sister chromatid exchange frequencies were not altered by bleomycin exposure.     

Associative overdominance: Evaluating the effects of inbreeding and linkage disequilibrium

Expressions are obtained for the expected phenotypic values of homozygous and heterozygous genotypes for a neutral marker locus linked to a locus segregating for a recessive deleterious gene. The phenotypic values are functions of the allele frequencies at the marker locus, the inbreeding coefficient and the degree of association of the deleterious gene with the marker alleles. The analysis is extended to more than two alleles at the marker locus. Either linkage disequilibrium or inbreeding alone can produce an apparent superiority of heterozygotes for the marker locus (unless specified otherwise, the terms ‘homozygote’ and ‘heterozygote’ will refer to the marker locus). The effect of linkage disequilibrium on the difference between the heterozygote and homozygote values can be positive (associative overdominance) or negative (associative underdominance), depending on the frequencies of the marker alleles and the degree of their association with the deleterious gene. Inbreeding has always a positive effect. In general, the expected value of a homozygote is a positive function of its allele frequency. When the various homozygous genotypes are combined into one class and the various heterozygous genotypes into another, the phenotypic difference of the two classes is a function of the evenness of the allelic frequency distribution. Inbreeding is a more likely explanation of associative overdominance if the frequency of the deleterious gene is low, but its effect on the character high. Conversely, linkage disequilibrium is more likely if the frequency is high and the effect low. The degrees of association between marker alleles and the deleterious gene can, in principle, be estimated from the observed phenotypic scores and used to calculate expected multi-locus genotype scores. This could provide the basis for statistical tests of the associative overdominance hypothesis as an explanation of observed correlations between multi-locus heterozygosity and phenotypic traits.