Cellular studies on retinoblastoma

Retinoblastoma may be hereditary or non-hereditary. The hereditary form involves either a predisposing gene transmissible as an autosomal dominant or a deletion at chromosome 13q14. An abnormal cellular response to ionizing radiation was suggested by the occurrence of secondary neoplasms within the field of therapeutic radiation in hereditary retinoblastoma patients. Hereditary retinoblastoma patients also show a predisposition to second neoplasms not related to therapy. In vitro studies on the radiation response of cells from retinoblastoma patients have generated conflicting results. Some laboratories, including our own, find that survival following ionizing irradiation of fibroblasts is within the normal range, other laboratories find an abnormal decrease in cell survival. X-ray-induced chromosome damage in G0-irradiated lymphocytes was slightly elevated compared to control subjects. Recent studies using chromosome 13 genetic markers suggest that retinoblastoma tumour cells are homo- or hemi-zygous for the mutant retinoblastoma gene. It seems unlikely that the mutant gene causes sensitivity to ionizing radiation but any tendency to chromosomal rearrangement in a gene carrier would increase the probability of tumour development.     

Differential sensitivity of peripheral blood lymphocytes of untreated leprosy patients to mitomycin C

The effects of a bifunctional alkylating agent mitomycin C (MMC), an effective inducer of chromosome aberrations and sister-chromatid exchanges (SCEs), have been studied in untreated leprosy patients. This was done to study the mutagen sensitivity of the leprosy patients. The frequency of chromosomal aberrations induced by MMC (conc. 0.01 microgram/ml) was 2.5% in controls, 3.6% in paucibacillary (PB), and 6.8% in multibacillary (MB) patients. The difference in the frequency of MMC-induced chromosome aberrations between the 3 groups studied was highly significant (p less than 0.01). Cultures grown with MMC showed the frequency of SCEs/cell to be 12.70 +/- 1.19 in controls, 19.97 +/- 3.51 in PB, and 29.66 +/- 5.92 in MB patients. The differences in the frequency of MMC-induced SCEs between the 3 groups were found to be highly significant (p less than 0.01). The enhanced frequencies of spontaneous and MMC-induced chromosome aberrations and SCEs observed in PB and MB patients indicate a clear differential mutagen sensitivity between PB and MB patients who are known to have different immunological status and thereby differ in the severity of the disease.     

Constitutive heterochromatin polymorphism and chromosome damage in viral hepatitis

The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCEs) were scored in relation to constitutive heterochromatin in 100 patients with viral hepatitis B, 100 patients with viral hepatitis A and 100 age- and sex-matched normal controls. 23.4%, 15% and 4% of the cells showed chromosomal aberrations in patients with hepatitis B, hepatitis A and normal controls respectively. Non-random involvement of chromosomal aberrations were also noted in chromosome 1 of patients with hepatitis B and A as compared to normal controls. The frequencies of SCEs (mean +/- S.D.) were found to be 10.40 +/- 2.83 in hepatitis B and 8.70 +/- 2.34 in hepatitis A. These values were significantly higher than the SCE frequency (mean +/- S.D.) of 5.88 +/- 2.25 observed in normal controls (P less than 0.001). The intra-chromosomal distribution of SCEs revealed a relatively increased incidence of SCEs in chromosome 1 of patients with hepatitis B and A as compared to normal controls. Analysis of constitutive heterochromatin polymorphism showed chromosome 1 qh+ to be the most frequent variant in patients with hepatitis B and A as compared to normal controls. The increased involvement of C-band variant 1 qh+ in patients with hepatitis B and A as compared to normal controls may indicate that extra heterochromatin offers additional sites for viral integration.     

Constitutional mosaic t(2;7)(q33;p22) and other rearrangements in a girl with Wilms' tumor

A 2-year-old girl with sporadic unilateral Wilms' tumor (WT) not associated with aniridia was found to have, besides other chromosome abnormalities, a t(2;7)(q33;p22) in 6% of her lymphocytes. A comparison with 7 previous WT cases without aniridia in whom diverse chromosomal aberrations were present, reveals a wide heterogeneity and lead us to tentatively classify such changes as causal, secondary, and casual.     

Bilateral Retinoblastoma: A Dominantly Inherited Affection

Ten survivors of sporadic bilateral retinoblastoma had 14 offspring, of whom eight were affected, seven of them in both eyes. Other reports from the literature raise the total of similar unselected cases to 19 survivors with a total of 39 offspring, of whom 17 were affected in both eyes and three in one eye.The high incidence of the bilateral affection in dominantly inherited retinoblastoma—as recorded in the literature—and in the offspring of survivors from sporadic bilateral retinoblastoma, as reported in the present study, establish all cases of bilateral retinoblastoma as a dominant disorder either in transmission or as a new mutation. This disorder, though fully or almost fully penetrant, is not always fully expressed. A small proportion, probably about 5 to 10% of all cases of the much more common sporadic unilateral affection, are in fact incompletely expressed germinal mutations for bilateral retinoblastoma. There is some evidence that histological appearances may distinguish these potentially transmissible unilateral tumours from the mass of unilateral retinoblastoma which have no genetic significance.     

Hereditary retinoblastoma: Penetrance,expressivity and age of onset

Summary Analyses of family data published in Japan concerning 29 kindreds with 2 or more cases of retinoblastoma revealed that, in the children who received the gene from a carrier parent, both penetrance and expressivity increase with increasing degree of expressivity in the parent. The estimate for the average degree of penetrance varies from 0.7 to 0.9, depending upon the method employed. The value will be increased with increasing number of survivors of hereditary retinoblastoma. Data were presented to indicate that the hereditary form of retinoblastoma onset tends to be later in unilateral than in bilateral affection, though in the non-hereditary form onset will be further delayed. No effect of birth order was detected. It was suggested that suppressor genes at other loci play a significant role in manifestation of the major dominant gene for retinoblastoma. Implications of these findings were discussed.This work, contribution number 1093 from the National Institute of Genetics, was supported by grants-in-aid from the Ministry of Education and the Ministry of Health and Welfare.     

Sister chromatid exchanges and chromosome aberrations in fibroblasts from patients with retinoblastoma

Summary The frequencies of sister chromatid exchanges (SCEs) and chromosome breaks were investigated in five diploid fibroblast strains derived from three patients with deletion 13[del(13)] retinoblastoma, one patient with a hereditary form of retinoblastoma, and one trisomy 13. The fibroblasts with del(13)(q14q22) showed slightly increased SCEs (at a P level of 5–10%), but the others, including del(13)(q12q14), the hereditary form of retinoblastoma, and trisomy 13, did not have increased SCEs as compared to normal controls. No increase in chromosome breaks was found in these fibroblasts. The results suggest that retinoblastoma is not associated with spontaneous increased chromosomal instability.     

Cytogenetic studies of blood lymphocytes of cosmonauts after long-ter, space flights

An analysis was performed of unstable chromosomal aberrations in peripheral blood of 36 cosmonauts after long-term space missions on "Mir" orbital station. 25 cosmonauts were examined before their flights to score spontaneous yields of cytogenetical damage. In all cases the doses absorbed by crews during space flights did not exceed permissible levels of irradiation, adopted for cosmonauts. The frequencies of chromosomal-type aberrations after space missions were found to increase significantly compared to the pre-flight levels. The yields of dicentrics and centric rings on the average were as high as 0.12 +/- 0.02 and 0.47 +/- 0.06% before and after the 1st flight, 0.18 +/- 0.05 and 0.71 +/- 0.11% before and after the 2nd flight respectively. During the inter-flight periods, usually lasted 1.5-2 years, the yields of chromosome damage lowered, but did not reach their spontaneous values. After each next flight the yields of chromosome aberrations increased again. The cytogenetical damage detected in cosmonauts' peripheral blood lymphocytes after chronic action of low doses of space radiation points out a possible increase in risks of stochastic effects in distant future for crews after long-term space missions.     

Aneuploidies, chromosome aberrations and dominant gene mutations detected in 113,913 consecutive newborn children in Mexico

Data on 113,913 liveborn children from a hospital in Guadalajara, Jalisco (Mexico), were analysed for birth defects (BD); mutation rates were calculated for sporadic aneuploidy, chromosome aberrations and dominant gene mutations. The results showed a general incidence of 13.92 BD cases per 1000 liveborns, of which 1.64% were chromosomal abnormalities, 1.50% were aneuploid, 0.14% were structural chromosome aberrations and 3.23% were dominant gene mutations. The mutation rates were 8.20 x 10(-4) chromosomal abnormalities, 7.5 x 10(-4) aneuploidies, 7.0 x 10(-5) chromosome aberrations and 1.61 x 10(-3) dominant gene mutations/gamete/generation, respectively. The lethality rate was 15.32% of the liveborns with BD. The described findings estimate the incidence of new human mutants detected at birth in a sample of the Mexican population. They show that the rate for some aneuploidies are similar to those found in other populations previously reported in the literature but the rates of chromosome and dominant gene mutations were different.     

Detection of submicroscopic deletions and a DNA polymorphism at the retinoblastoma locus

Summary DNA samples from 60 unrelated patients with retinoblastoma were screened by Southern blot hybridization using two probes that are closely linked to the retinoblastoma locus within human chromosome band 13q14. Seven of 44 patients with bilateral or multifocal unilateral retinoblastoma and one patient with unifocal unilateral retinoblastoma were found to have a heterozygous deletion for the anonymous DNA sequence H3-8. Three of the eight deletions did not include the esterase D locus and were undetectable by conventional cytogenetic analysis. The findings are compatible with the deletions being the cause of retinoblastoma in these cases and provide a basis for DNA diagnosis in nearly 20% of patients with bilateral and multifocal unilateral retinoblastoma. The H3-8 probe also detects a restriction fragment length polymorphism that is a useful genetic marker in some families.     

Spontaneous and induced chromosome instability in patients with fragile X syndrome

Spontaneous and degranol- and dimatiph-induced chromosomal instability in the lymphocyte culture of patients with fra-X syndrome was investigated. The cultures contained TC 199 and 5% FC serum. It was found that the frequency of spontaneous chromosomal aberrations (CA) was 7.3% in cells from patients with fra(X), 3.9% in patients with MR of unknown origin, and 1.3% in normal individuals. Spontaneous break-points in the patients with fra(X) were localized in 1p, 2q, 3p, 6q, 7q, 16 q more often than in normal individuals. No significant difference was found in SCEs. The cells of patients with fra(X) were not sensitive to the induction of CA by degranol. It was found that chromosomal telomeric changes (CTC) were mutagen-independent, remaining at the spontaneous level: in the patients with fra(X) CTC were 10.5% (9.5% fra-Xq27, and 1% autosomal telomeric changes); in normal individuals CTC were 0.1%.     

Mutation Spectrum of RB1 Gene in Unilateral Retinoblastoma Cases from Tunisia and Correlations with Clinical Features

Retinoblastoma, an embryonic neoplasm of retinal origin, is the most common primary intraocular malignancy in children. Somatic inactivation of both alleles of the RB1 tumor suppressor gene in a retinal progenitor cell through diverse mechanisms including genetic and epigenetic modifications, is the crucial event in initiation of tumorigenesis in most cases of isolated unilateral retinoblastoma. We analyzed DNA from tumor tissue and from peripheral blood to determine the RB1 mutation status and seek correlations with clinical features of 37 unrelated cases of Tunisian origin with sporadic retinoblastoma. All cases were unilateral except one who presented with bilateral disease, in whom no germline coding sequence alteration was identified. A multi-step mutation scanning protocol identified bi-allelic inactivation of RB1 gene in 30 (81%) of the samples tested. A total of 7 novel mutations were identified. There were three tumors without any detectable mutation while a subset contained multiple mutations in RB1 gene. The latter group included tumors collected after treatment with chemotherapy. There were seven individuals with germline mutations and all presented with advanced stage of tumor. There was no difference in age of onset of RB based on the germline mutation status. Thus 20% of the individuals with sporadic unilateral RB in this series carried germline mutations and indicate the importance of genetic testing all children with sporadic retinoblastoma. These findings help to characterize the spectrum of mutations present in the Tunisian population and can improve genetic diagnosis of retinoblastoma.     

The effects of exposure to different clastogens on the pattern of chromosomal aberrations detected by FISH whole chromosome painting in occupationally exposed individuals

The pattern of chromosomal aberrations (CA) was studied by fluorescence in situ hybridization (FISH) technique (whole chromosomes #1 and #4 painting) in workers occupationally exposed to any of the four following conditions: acrylonitrile (ACN), ethyl benzene (EB), carcinogenic polycyclic aromatic hydrocarbons (c-PAHs), and irradiation in nuclear power plants (NPP), respectively. Decrease in the relative frequency of translocations was observed in EB group, and an increase in reciprocal translocations in ACN and NPP-exposed groups. An increase in a relative number of insertions was registered under all four conditions (significant at ACN, EB, c-PAHs, quasisignificant at NPP-exposed groups). Significant differences in the percentage of lymphocytes with aberrations on chromosome #1 (58.8+/-32.7%, versus 73.8+/-33.6% in the controls, P < 0.05), and chromosome #4 (47.0+/-34.1%, versus 29.4+/-32.2%, P < 0.01) were found in workers exposed to ACN. Similarly, a decrease in the proportion of cells with aberration on chromosome #1 (61.0+/-24.0%, versus 73.8+/-33.6%, P < 0.05) and an increase on chromosome #4 (45.6+/-24.6%, versus 29.4+/-32.2%, P < 0.05) were observed in workers exposed to EB. Frequency of aberrant cells (%AB.C.) as well as genomic frequency of translocations (F(G)/100) increased with age (P < 0.001). Aging also increased the percentage of translocations and reciprocal translocations (P < 0.05), but decreased the relative number of acentric fragments (P < 0.01). Smoking led to significantly increased F(G)/100 (P < 0.05), but did not affect the pattern of chromosomal aberrations. Our results seem to indicate that different carcinogens may induce a different pattern of chromosomal aberrations.     

Retinoblastoma mutation rate in New Zealand and support for the two-hit model

Summary Age-specific incidence rates for 96 New Zealand patients with sporadic retinoblastoma peaked earlier for bilateral patients than for unilateral patients. The cumulative log survival until diagnosis for bilateral and unilateral patients followed linear and quadratic curves respectively, and supported the two-hit hypothesis for retinoblastoma. The germ cell mutation rate for retinoblastoma, assuming a single major gene, was calculated to be in the order of 9.3×10^-6 to 10.9×10^-6 for the New Zealand population.     

Effects of bleomycin on Down's syndrome lymphocytes in culture

Blood lymphocytes from 3 Down's syndrome (DS) and 3 age- and sex-matched normal probands were studied for the induction of chromosomal aberrations and sister-chromatid exchange (SCEs). Treatment with bleomycin (30 and 60 ng) at the initiation of culture showed a dose-dependent increase in the incidence of dicentric and ring chromosome aberrations. In contrast, the cells which were treated for the last 24 h in culture with bleomycin did not show an increase in chromosome-type aberrations. The proportion of metaphases in M1, M2, and M3 in cultures was not different between DS and normal cells. Sister-chromatid exchange frequency did not show significant changes between DS and normal individuals.     

Segregation analysis in a man heterozygous for a pericentric inversion of chromosome 7 (p13;q36) by sperm chromosome studies.

We have analyzed 140 sperm chromosome complements from a subfertile man heterozygous for an inv(7)(p13;q36). Seventy-five percent of the chromosome complements were not recombinant: 37.9% contained the normal chromosome 7, and 37.1% contained the inverted chromosome 7. Twenty-five percent of the 140 were recombinant: 7.1% carried a recombinant chromosome 7 with a duplication p and deletion q, 17.1% carried a recombinant chromosome 7 with a duplication q and deletion p, and 0.7% carried both recombinant chromosomes. The frequency of structural chromosomal aberrations unrelated to the inversion was 11.4%, and the frequency of aneuploidy was 2.9%. Both frequencies were not significantly different from those in control donors. Two sperm complements with a second independent, contiguous inversion involving one of the original breakpoints (q36) were observed (1.4%). The risk of producing chromosomally abnormal offspring or spontaneous abortions would be 34.3%. The proportion of X-bearing and Y-bearing sperm was 46.8% and 53.2%, respectively, not significantly different from the expected 1:1 ratio.     

Cytogenetic studies on the effect of feeding mice with stored wheat grains treated with malathion.

The cytogenetic effect of malathion residues in wheat grains stored for different periods of time (4, 12, 24 weeks) was evaluated in Swiss mice. The studies included: (1) chromosomal aberrations analysis in bone-marrow and spermatocyte cells; (2) chromosomal aberrations and sister chromatid exchange (SCE) analysis in spleen cell culture from mice fed with stored wheat grains. The tested doses were 8.36 (applied dose), 25.08 and 41.80 mg malathion kg(-1) wheat grains. The results demonstrated that the cytogenetic effect induced in different mouse tissues by malathion residues was dose-dependent and increased with increasing of both feeding and storage periods.Feeding mice with wheat grains stored for 4 weeks had a non-significant effect with respect to the induction of chromosomal aberrations or SCEs. Significant chromosome damage and increase of SCEs were observed in mice fed with wheat grains stored for 12 weeks. The maximum effect was recorded in mice fed for 12 weeks with the grains treated with the highest tested dose and stored for 24 weeks. However, mitomycin C i.p.-injected in mice at 1 mg kg(-1) body weight (b.w.) (positive control) induced a higher effect. The percentage of chromosome aberrations reached 13.60+/-0.98, 13.60+/-0.77 and 11.73+/-0.98 (P<0.01) in bone-marrow, cultured spleen cells and spermatocytes, respectively. The significant increase of abnormalities in spermatocytes was seen for univalent formation only, predominantly of the sex chromosomes. The frequency of SCEs was 10.76+/-0.62 per cell (P<0.01) in cultured spleen cells compared with 5.46+/-0.45 per cell for control and 14.66+/-0.54 per cell for the positive control.The obtained results indicate that malathion residues in stored wheat grains have potential genotoxic effect in mice under the conditions tested.     

Chromosomal aberration and sister-chromatid exchange frequencies in peripheral blood lymphocytes of a large human population sample

In order to assess the potential of cytogenetic determinations on peripheral blood lymphocytes as a means of monitoring human populations subject to low level occupational and environmental exposures to chemical mutagens and carcinogens, accurate baseline data are required. Accordingly, we have determined mean frequencies of chromosomal aberrations and of sister-chromatid exchanges, their variances, and the sources of this variance in a cohort of 353 healthy employees of the Brookhaven National Laboratory. A detailed protocol was adopted for blood sampling, lymphocyte culture, cytogenetic preparation and scoring in order to minimize variation from these potential sources. Scoring was divided between the Oak Ridge and the Brookhaven groups with duplicate scoring sufficient to evaluate and minimize the effect of any differences between laboratories or between individual scorers. In all, the data include 71,950 cells scored for chromosomal aberrations and 16,898 cells scored for sister-chromatid exchanges. The mean unadjusted frequency of sister-chromatid exchanges was 8.29 +/- 0.08/cell. As reported in other studies, cigarette smoking very significantly influenced sister-chromatid exchange frequencies; in our study the mean for smokers was 9.0 +/- 0.2, while that for non-smokers was 8.1 +/- 0.1/cell. The mean frequency was statistically higher in females than in males, regardless of smoking status. On the other hand, age of the subject did not significantly influence sister-chromatid exchange frequencies. Curiously, the subject's total white cell count did influence sister-chromatid exchange frequency. No other source of variation was found. The frequencies of chromosomal aberrations of all types were determined. The frequency of the most common unequivocal chromatid type, the chromatid deletion, was 0.81 +/- 0.05%, that of the most common unequivocal chromosome type, the dicentric, was 0.16 +/- 0.02%. No statistically significant influence was found of age or sex, nor of any other parameter tested, on the frequency of any chromosomal aberration type, with the single exception of long acentric fragments, often "supernumerary", believed to represent X chromosomes precociously separated at the centromere. Such fragments were significantly more frequent in samples from females than those from males, and showed a significant positive regression on age.     

Karyotypes of spontaneous human abortuses and several aspects of their phenotypic presentation]

In all, 172 human spontaneous abortuses were examined: 75 cases from the 1st trimester of pregnancy (all were karyotyped), and 97 ones--from 2nd and 3rd trimesters (59 cases were karyotyped). 31 cases of chromosomal anomalies were revealed. The incidence of chromosomal anomalies among the 1st and 2--3rd trimesters of spontaneous abortuses was 40.0+/-7.5 and 1.0+1.0%, resp. The general incidence of chromosomal anomalies among this series of spontaneous abortuses was equal to 18.0+/-2.9%. The question of phenotypical manifestations of chromosomal aberrations in spontaneous abortuses is discussed. The influence of gene structure of aberrant chromosomes on phenotypes of abortuses is supposed.     

Cytogenetic investigations in a family with ataxia telangiectasia

Summary Cytogenetic findings on a family with ataxia telangiectasia (A-T) in which three of four sibs were affected are described. The affected individuals had approximately twice the level of spontaneous chromosome breakage of a normla control, while the parents and the normal sib had no significant increase. Lymphocytes from all three A-T homozygotes showed specific stable chromosomal rearrangements involving chromosomes 7 and 14. All of these abnormalities involved breakage at the usual four sites associated with A-T (7p14, 7q35, 14q12, and 14q32). Two rearrangements detected in the eldest and most severely affected patient were clones, one of which [t(14;14)(p11;q12)] is not commonly found in A-T cells. No chromosomal rearrangements were encountered in lymphocytes from the control, the parents, or the normal sib. Lymphocytes from the A-T patients also were found to be 7–11 times more sensitive to the induction of chromatid aberrations by X-irradiation than control cells. Lymphocytes from the parents and normal sib showed a moderately increased frequency of X-ray induced aberrations compared with that of the control.