Parental age and seasonal variation in the births of children with sporadic retinoblastoma: a mutation-epidemiologic study

Summary Statistical analysis of parental age data from 225 sporadic cases of bilateral retinoblastoma, plus ten sporadic cases of chromosome deletion or translocation involving 13q14 that was identified as of paternal origin, revealed no evidence of paternal or maternal age effect. Parental exposure to ionizing radiation or chemical mutagens, the effect of which is accumulated with advancing age, does not seem to play a major role in the production of germinal mutations at the responsible (RB) locus. Furthermore, analysis of variation in the month of birth of 753 children with sporadic unilateral retinoblastoma did not show any significant deviation from the controls or a cyclic trend. The occurrence of nonheritable retinoblastoma is not likely to be associated with certain viruses such as human adenovirus 12 whose activity varies markedly with season. These results, together with the fairly uniform pattern in the incidence of this tumor among different populations, suggest that most, if not all, cases of sporadic retinoblastoma are caused by some intrinsic biological mechanisms, and not by environmental mutagens that may vary with respect to time and place.     

Antimutagenic effects in humans.

The application of antimutagenicity studies to human somatic mutation is discussed, with emphasis on the potential for future studies. Five assay-gene combinations are now available for measuring human somatic mutation in lymphocytes and erythrocytes. Results with these combinations have defined the human background levels, and show clear responses of mutant frequency to a variety of mutagens. The testing of antimutagenic effects on background frequencies is feasible, but has not yet been done. The major uncertainty in such studies is the unknown age of mutant cells in the background, since only the newly forming mutants are potentially susceptible to most antimutagenic treatments. Intervention studies in the face of active mutagenicity and the use of other genotoxicity endpoints, such as chromosome aberrations, micronuclei and DNA adducts, are considered briefly.     

International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC working paper 5/1: Perspectives in mutation epidemiology. 1. Incidence and prevalence of genetic disease (excluding chromosomal aberrations) in human populations

Detailed knowledge of the birth frequency or the cumulative incidence over all ages of genetic diseases in human populations is a prerequisite for assessing the magnitude of possible genetic hazards caused by environmental mutagens. However, both theoretical and practical difficulties are involved in precisely measuring the total frequency of these diseases. Two sets of data from large-scale population studies, one from Northern Ireland and the other from British Columbia, are compared with each other and with the results from ad hoc surveys for individual monogenic disorders. With due allowance for differences in approach, examination indicates that the data from the large-scale population studies are inadequate. However, it could provide a crude estimate of the total frequency of genetic diseases and a fairly reliable estimate of the individual frequency of certain genetic disorders with early onset that are familiar and readily diagnosed. In addition to environmental mutagens, there are a number of factors associated with current human activity that may change the incidence of genetic diseases. In order to monitor the human population for environmental mutagens, the change in frequency of sporadic cases of those genetic diseases that arose from fresh mutation and that can be easily detected as early as possible should be followed closely. The mechanism of data collection currently being employed in some countries for childhood cancers, certain congenital malformations, and inborn errors of metabolism could be extended to include the so-called sentinel phenotypes. The rationale and feasibility of using retinoblastoma and Wilms' tumor (nephroblastoma) as examples of such population monitoring are described.     

Organ-specific mutations in Chinese hamsters induced by chemical carcinogens

Good qualitative correlations between mutation in bacteria and carcinogenic effects in laboratory animals have been shown by a number of workers. As a result, in vitro microbial mutation assays are now widely used in a preliminary screen for detecting possible mammalian mutagens and, by implication, possible carcinogens.In order to investigate the relationship between mutation and carcinogenesis in vivo, a method has been developed in which somatic mutation can be studied in specific organs of Chinese hamsters after acute dosing with chemical carcinogens. Using this approach, in which chemicals are subject to the full spectrum of mammalian metabolism and detoxification, changes in mutation frequency are studied in primary cultures of tissues obtained from hamsters dosed with the test chemical. Forward mutation is assayed using 8-azaguanine-resistance or ouabain-resistance as genetic markers.In preliminary experiments an increase in mutation at both loci was detected in cultures derived from lung tissue of hamsters after intraperitoneal dosing with the direct-acting mutagens and weak carcinogen, ethyl methanesulphonate, or with diethylnitrosamine, a carcinogen that requires metabolic activation before mutagenic activity can be demonstrated in vitro.Subsequent experiments have shown the induction of mutation in cells derived from the bladder of animals dosed with diethylnitrosamine or methyl nitrosourea, from the lungs of hamsters dosed with urethane, and in a variety of tissues from animals dosed with methanesulphonates, 2-acetylaminofluorene and 3-methylcholanthrene.     

Retinoblastoma

Retinoblastoma is a rare eye tumor of childhood that arises in the retina. It is the most common intraocular malignancy of infancy and childhood; with an incidence of 1/15,000–20,000 live births. The two most frequent symptoms revealing retinoblastoma are leukocoria and strabismus. Iris rubeosis, hypopyon, hyphema, buphthalmia, orbital cellulites and exophthalmia may also be observed. Sixty per cent of retinoblastomas are unilateral and most of these forms are not hereditary (median age at diagnosis two years). Retinoblastoma is bilateral in 40% of cases (median age at diagnosis one year). All bilateral and multifocal unilateral forms are hereditary. Hereditary retinoblastoma constitutes a cancer predisposition syndrome: a subject constitutionally carrying an RB1 gene mutation has a greater than 90% risk of developing retinoblastoma but is also at increased risk of developing other types of cancers. Diagnosis is made by fundoscopy. Ultrasound, magnetic resonance imaging (MRI) and computed tomography (CT) scans may contribute to diagnosis. Management of patients with retinoblastoma must take into account the various aspects of the disease: the visual risk, the possibly hereditary nature of the disease, the life-threatening risk. Enucleation is still often necessary in unilateral disease; the decision for adjuvant treatment is taken according to the histological risk factors. Conservative treatment for at least one eye is possible in most of the bilateral cases. It includes laser alone or combined with chemotherapy, cryotherapy and brachytherapy. The indication for external beam radiotherapy should be restricted to large ocular tumors and diffuse vitreous seeding because of the risk of late effects, including secondary sarcoma. Vital prognosis, related to retinoblastoma alone, is now excellent in patients with unilateral or bilateral forms of retinoblastoma. Long term follow-up and early counseling regarding the risk of second primary tumors and transmission should be offered to retinoblastoma patients.     

Mutation as a cause of genetic disease

Mutational changes can be conveniently classified into two sorts: those that appear to involve single genes and are generally referred to as gene mutations, and those that involve chromosomal segments containing many genes, or even whole chromosomes, and are referred to as chromosomal mutations. Both of these kinds of mutation occur in germ-cell lineages and contribute substantially to inherited disease, or pre-disposition to disease, and both also occur in somatic cells and contribute to acquired disease. The mutation rates for inherited disease ascribed to mutation in a single gene differ for different genes and are age-dependent. Moreover, a single disease entity, such as haemophilia B, may be the result of any one of a number of different alterations within the gene responsible for the disease. The mutation rate for inherited chromosomal mutation is also age-dependent, particularly so in the case of mutations involving alterations in chromosome number. Studies in experimental animals demonstrate that exposure to physical or chemical mutagens results in increasing the incidence of inherited gene and chromosomal mutations. However, such increases have not been unequivocally demonstrated in human populations exposed to known mutagens. Studies on mutation in human lymphoid or epithelial somatic cells clearly demonstrate an increased frequency in cells taken from people exposed to ionizing radiations or chemical mutagens or in cells exposed in vitro. The consequences of such mutations will depend upon their nature and the origins and functions of the cells in which they occur. Of particular importance are mutations influencing cell growth and proliferation, and both gene and chromosomal mutations are implicated as causal factors in the development of human cancers.     

Quantification of the paternal allele bias for new germline mutations in the retinoblastoma gene

New germline mutations in the human retinoblastoma gene are known to arise preferentially on paternally derived chromosomes, but the magnitude of that bias has not been measured. We evaluated 49 cases with a new germline mutation and found that in 40 cases (82%) the mutation arose on the paternally derived allele. We also evaluated 48 cases likely to have a somatic initial mutation; in this group the initial mutation arose on paternal or maternal chromosomes with approximately equal frequency. There was no statistically significant difference in the average age of fathers of children with new paternal germline mutations from the average age of fathers of children with new maternal germline mutations or somatic initial mutations. Combining the data with that from previous reports from other groups, the proportion of new germline mutations arising on a paternally derived allele is 85% (based on 72 cases; 95% confidence interval = 76–93%). This number can be useful in the genetic counseling of some families with retinoblastoma. Received: 18 December 1996 / Accepted: 30 April 1997     

Contribution of BRCA1 germline mutation in patients with sporadic breast cancer

Hereditary artifacts in BRCA1 gene have a significant contributory role in familial cases of breast cancer. However, its germline mutational penetrance in sporadic breast cancer cases with respect to Pakistani population has not yet been very well defined. This study was designed to assess the contributory role of germline mutations of this gene in sporadic cases of breast cancer. 150 cases of unilateral breast cancer patients, with no prior family history of breast cancer and no other disorders or diseases in general with age range 35–75 yrs, were included in this study.Mutational analysis for hot spots on Exon 2, 3 and 13 of BRCA1 was done by using Single Strand Conformational Polymorphism (SSCP). Sequence analysis revealed five variants (missense) and one novel splice site mutation at exon 13. No germline mutation was observed on the remaining exons with respect sporadic breast cancer cases in Pakistani population. A vast majority of breast cancer cases are sporadic; the present study may be helpful for designing a better genetic screening tool for germline BRCA mutations in sporadic breast cancer patients of Pakistani population. Further studies involving a screening of entire coding region of BRCA1 is required to explore the merits of genetic diagnosis and counseling in breast cancer patients.     

Analyses of spontaneous pink mutant events in the stamen hairs of Tradescantia clone BNL 4430 cultivated in a nutrient solution circulating growth chamber

In order to obtain more fundamental data on Tradescantia clone BNL 4430, one of the most suitable testers for environmental mutagens, the occurrences of spontaneous somatic pink mutations in the stamen hairs were scored for 52 weeks from 12 December 1998 to 10 December 1999, cultivating the young inflorescence-bearing shoots with roots in a nutrient solution circulating (NSC) growth chamber. The environmental conditions in the chamber were 22.0+/-0.5 degrees C during the 16h day with the light intensity of 7.5klx from white fluorescent tubes, and 20.0+/-0.5 degrees C at night. During the scoring period, 697,443 stamen hairs with an average cell number of 25.36 were observed and 2642 pink mutant events (PMEs) were detected. The overall spontaneous mutation frequency was 1.56+/-0.03 PMEs per 10(4) hair-cell divisions, and the frequency was significantly lower in May, July and August and significantly higher in November and December. By analyzing the sectoring patterns of 1856 PMEs (70.25% of PMEs detected), the most of 172 cases of multiple (two to five) pink sectors observed in the same hairs (scored as 232 PMEs for calculating mutation frequency) were found to be the results of events involving somatic recombinations occurred in single cells or cell lineages, rather than those of two or more independent somatic mutations occurred in different cells. This finding clearly shows the significance of somatic recombinations in producing such multiple sectors (382 sectors in total) which occupied 19.0% of the 2006 pink sectors in total analyzed. Somatic recombinations were considered to be playing a significant role also in producing single PMEs in the stamen hairs.     

Constitutional RB1-gene mutations in patients with isolated unilateral retinoblastoma.

In most patients with isolated unilateral retinoblastoma, tumor development is initiated by somatic inactivation of both alleles of the RB1 gene. However, some of these patients can transmit retinoblastoma predisposition to their offspring. To determine the frequency and nature of constitutional RB1-gene mutations in patients with isolated unilateral retinoblastoma, we analyzed DNA from peripheral blood and from tumor tissue. The analysis of tumors from 54 (71%) of 76 informative patients showed loss of constitutional heterozygosity (LOH) at intragenic loci. Three of 13 uninformative patients had constitutional deletions. For 39 randomly selected tumors, SSCP, hetero-duplex analysis, sequencing, and Southern blot analysis were used to identify mutations. Mutations were detected in 21 (91%) of 23 tumors with LOH. In 6 (38%) of 16 tumors without LOH, one mutation was detected, and in 9 (56%) of the tumors without LOH, both mutations were found. Thus, a total of 45 mutations were identified in tumors of 36 patients. Thirty-nine of the mutations-including 34 small mutations, 2 large structural alterations, and hypermethylation in 3 tumors-were not detected in the corresponding peripheral blood DNA. In 6 (17%) of the 36 patients, a mutation was detected in constitutional DNA, and 1 of these mutations is known to be associated with reduced expressivity. The presence of a constitutional mutation was not associated with an early age at treatment. In 1 patient, somatic mosaicism was demonstrated by molecular analysis of DNA and RNA from peripheral blood. In 2 patients without a detectable mutation in peripheral blood, mosaicism was suggested because 1 of the patients showed multifocal tumors and the other later developed bilateral retinoblastoma. In conclusion, our results emphasize that the manifestation and transmissibility of retinoblastoma depend on the nature of the first mutation, its time in development, and the number and types of cells that are affected.     

Experimental work on induced mutations

The detection of changes in mutation rate in human populations remains extremely difficult. Thus estimation of genetic hazards of mutagens to man depends on extrapolation from experimental systems. Germ cells of animals show complex variations in sensitivity to mutagenic effects. Some agents predominantly affect stem cells or other immature germ cells, whereas others mainly affect later germ cell stages. Dose-response relations also vary both with the agent and with the stage or sex of germ cell treated. In man, in addition to single-gene defects and chromosome anomalies, conditions of complex or uncertain inheritance, such as congenital malformations, are clinically important. Genetic theory leaves unclear whether the incidence of these would be affected by a change in mutation rate. Recent research has shown that in mice the incidence of malformations is increased by exposure of the parents to mutagens, but the effect is small. Chromosomal non-disjunction is also clinically important. Again, recent research shows that its frequency can be changed by mutagens, but the effects vary with germ-cell stage. Thus, further research is needed to elucidate the relative contributions of different environmental mutagens to human genetic disease.     

Moving forward in determining the causes of mutations: the features of plants that make them suitable for assessing the impact of environmental factors and cell age

Currently, the types of factors that impact the mutation rate is a controversial issue. The marked attention towards identifying the factors that impact the genomic mutation rate is justified because mutations are the source of genetic variation underlying evolution and because many mutations have deleterious effects and can cause diseases. Although data showing correlations between germ cell division number and mutation rates (from epidemiological studies and molecular evolutionary rate analyses) have suggested that most mutations in animals are replication errors, this notion is highly debated and inconsistencies in the correlations suggest that other, replication-independent factors, could play an important role. Likely candidates include environmental parameters and cell age, but these issues have proved to be difficult to study using animals and in vitro systems, and consequently, very few or no data currently exist. The specific features of plants that make them powerful model systems for revealing the influence of the environment (natural environmental factors) and cell age on the spontaneous genomic mutation rate are discussed here. Overall, the evidence suggests that plants could be key biological systems for advancing our knowledge about how and why heritable mutations arise.     

Detection of mutations of the RB1 gene in retinoblastoma patients by using exon-by-exon PCR-SSCP analysis.

Most sporadic cases of retinoblastoma, malignant eye tumor of children, may require the identification of a mutation of the retinoblastoma gene (RB1 gene) for precise genetic counseling. We established a mutation detection system of and screened for the RB1 gene mutation in 24 patients with retinoblastoma--12 bilateral patients and 12 unilateral patients. Mutation analysis was performed by PCR-mediated SSCP analysis in the entire coding region and promoter region, as an initial screening method, followed by direct genomic sequencing. Possible oncogenic mutations were identified in 14 (58%) of 24 tumors, of which 6 were single base substitutions, 4 were small deletions, 3 were small insertions, and 1 was a complex alteration due to deletion-insertion. A constitutional somatic mosaicism was suggested in one bilateral patient. A majority (57%) of mutations were found in E1A binding domains, and all were presumed to truncate the normal gene products. The mutation analysis presented here may provide a basis for the screening system of RB1 gene mutations in retinoblastoma patients.     

A novel technique based on a PNA hybridization probe and FRET principle for quantification of mutant genotype in fibrous dysplasia/McCune-Albright syndrome

Somatic mutations are present in various proportions in numerous developmental pathologies. Somatic activating missense mutations of the GNAS gene encoding the Gs(alpha) protein have previously been shown to be the cause of fibrous dysplasia of bone (FD)/McCune-Albright syndrome (MAS). Because in MAS patients, tissues as diverse as melanocytes, gonads and bone are affected, it is generally accepted that the GNAS mutation in this disease must have occurred early in development. Interestingly, it has been shown that the development of an active FD lesion may require both normal and mutant cells. Studies of the somatic mosaic states of FD/MAS and many other somatic diseases need an accurate method to determine the ratio of mutant to normal cells in a given tissue. A new method for quantification of the mutant:normal ratio of cells using a PNA hybridization probe-based FRET technique was developed. This novel technique, with a linear sensitivity of 2.5% mutant alleles, was used to detect the percentage mutant cells in a number of tissue and cell culture samples derived from FD/MAS lesions and could easily be adapted for the quantification of mutations in a large spectrum of diseases including cancer.     

International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC working paper 5/5. Perspectives in mutation epidemiology. 5. Modern medical practice versus environmental mutagens: their possible dysgenic impact

We do not know how many mutations are being produced in human populations by exposure to environmental mutagens. If these mutagens caused a persistent rise in mutation rates, then ultimately there would be a proportional increase in the frequency of a variety of genetic diseases, including those that are difficult to treat and that require life-long care of affected individuals. In contrast, modern medical practices are relaxing selection pressure selectively with respect to disease, leading to a gradual increase in the frequency of certain genetic and partly genetic diseases that can be effectively treated. The pattern of this increase would differ from condition to condition, depending upon the mode of inheritance and the extent to which selection is relaxed; except for some special cases, the anticipated increase would generally be slow. Additional economic burdens on future society and families imposed by relaxed selection would mainly involve expenditures for relatively inexpensive treatments, and not those for expensive life-long care. Moreover, individuals treated successfully can be expected to contribute productively to society. With education and counseling for those who survive serious dominant and X-linked disorders, and with the development of accurate, inexpensive prenatal diagnosis, the presumed dysgenic effects of relaxed selection could be balanced.     

Analyses of spontaneous pink mutant events in the stamen hairs of Tradescantia clone BNL 4430 cultivated in a nutrient solution circulating growth chamber

In order to obtain more fundamental data on Tradescantia clone BNL 4430, one of the most suitable testers for environmental mutagens, the occurrences of spontaneous somatic pink mutations in the stamen hairs were scored for 52 weeks from 12 December 1998 to 10 December 1999, cultivating the young inflorescence-bearing shoots with roots in a nutrient solution circulating (NSC) growth chamber. The environmental conditions in the chamber were 22.0±0.5°C during the 16 h day with the light intensity of 7.5 klx from white fluorescent tubes, and 20.0±0.5°C at night. During the scoring period, 697,443 stamen hairs with an average cell number of 25.36 were observed and 2642 pink mutant events (PMEs) were detected. The overall spontaneous mutation frequency was 1.56±0.03 PMEs per 10⁴ hair-cell divisions, and the frequency was significantly lower in May, July and August and significantly higher in November and December. By analyzing the sectoring patterns of 1856 PMEs (70.25% of PMEs detected), the most of 172 cases of multiple (two to five) pink sectors observed in the same hairs (scored as 232 PMEs for calculating mutation frequency) were found to be the results of events involving somatic recombinations occurred in single cells or cell lineages, rather than those of two or more independent somatic mutations occurred in different cells. This finding clearly shows the significance of somatic recombinations in producing such multiple sectors (382 sectors in total) which occupied 19.0% of the 2006 pink sectors in total analyzed. Somatic recombinations were considered to be playing a significant role also in producing single PMEs in the stamen hairs.     

Micronuclei frequency in children exposed to environmental mutagens: a review

Cytogenetic monitoring has been traditionally used for the surveillance of populations exposed to genotoxic agents. In recent years sensitivity problems emerged in surveys of populations exposed to low levels of mutagens, and therefore alternative approaches have been explored. Biomonitoring studies in children are a promising field, since because of evident differences in the uptake, metabolism, distribution and excretion of mutagens this population seems to be more susceptible than adults. Further, the effect of major confounders such as cigarettes smoking, occupation, life-style, and dietary factors plays a minor role. Among cytogenetic assays, the micronucleus assay (MN) has several advantages and is increasingly used. A review was then carried out to synthesize the published data on the occurrence of MN in children and adolescents (age range 0-18 years), and to assess the impact of genotoxic exposure on MN frequency. Overall, 20 papers from international literature and 8 Russian papers were included. An effect of age was found within this age range, while the influence of gender on MN frequency was irrelevant. These results were confirmed by the re-analysis of data for 448 children selected from the HUMN database. An effect of chronic and infectious diseases on MN levels has been reported by various authors. Most studies describing the effect of exposure to genotoxic agents (ionizing radiation, chemicals, drugs, environmental tobacco smoke) found an increase of MN in exposed children. The limited number of published papers indicates that the conduct of properly designed studies on the effect of environmental pollutants in children may be difficult. This review confirmed the usefulness of MN assay in biomonitoring studies conducted in children, revealing that in many circumstances investigating children increases the sensitivity of the study, even with low dose exposures.     

Mutagenicity testing and risk estimation with mammals

Mammalian test systems are currently used for mutagenicity screening. The necessity and the limitations of standardizing these methods are discussed for the dominant-lethal assay. In addition to the refinement of standard methods, the development of new systems in mammals is emphasized. One promising approach is the detection of presumed somatic mutations. Another new development takes advantage of electrophoretic methods for detecting induced structural alterations of gene products. Mammalian experiments will be essential for the assessment of risks from chemical mutagens. The development of standards for the controlled use of chemical mutagens should be guided by the experience accumulated in radiation genetics. Two methods, the measurement of specific-locus mutation rates in mice and the direct determination fo the phenotypic damage of dominant genes affecting the skeleton of mice, are recommended for the assessment of the hazard of chemical mutagens.     

Effect of low dose irradiation on the reaction of blood lymphocytes of individuals with the somatic diseases

The reaction of blood lymphocytes on adaptive irradiation in vitro (0.05 Gy) and challenge irradiation (1.0 Gy) 5 h after has been studied among 320 children 3-16 years old by micronuclei test with cytochalasin B cytokinetic block. The adaptive response or the hypersensitivity phenomenon (HS) has been determined by the calculation of the number of binucleated cells with micronuclei (MN) in 1000-2000 binucleated cells. For each individual by the chi2 criteria significant differences between the frequencies of damaged cells by irradiation in challenge dose and by combined action of adaptive and challenge doses have been detected. On the base of the results obtained the connection between AR or HS and the somatic diseases of different type and the disturbancies in systems of humoral and cell immunity has been studied. It was shown that in the row of allergic diseases and tuberculous infection among the significant part of the children (approximately 50%) the HS phenomenon was registered. The connection between the HS phenomenon and inflammatory processes (without allergic components), the logopedic disturbancies wasn't observed. It was shown in the group of HS children the deviation in the cell immunity system is registered. Significant deviations in HS group in the system of the humoral immunity is observed. So by the HS phenomenon significant number of the children have the somatic diseases and the deviation in the systems of cell and humoral immunity.     

Evaluation of a mutation test using S49 mouse lymphoma cells and monitoring simultaneously the induction of dexamethasone resistance, 6-thioguanine resistance and ouabain resistance

A test for the detection of chemically induced mutants in S49 mouse lymphoma cells is described. These cells can be plated in parallel in several selective media; the induced frequencies of dexamethasone-resistant, 6-thioguanine-resistant and ouabain-resistant mutants were compared. The first two selection systems permit the detection of all kinds of mutation that result in alteration or partial or complete loss of the gene product concerned, whereas ouabain-resistant mutants can only be induced with strong point mutagens in these cells. Dexamethasone resistance is the marker induced at the highest frequency among these three. Data obtained from this selection system are therefore the most amenable to statistical analysis. Dexamethasone resistance is expressed within a short time after mutagenesis (3 days), and because S49 cells do not display metabolic co-operation, large numbers of cells can be screened. A metabolizing system in vitro with rat-liver homogenate may be included in tests of indirectly acting mutagens. These features make the S49 mutation test system using dexamethasone resistance as the main marker and other markers as internal controls an attractive tool in mutation testing in somatic cells in vitro.