Parental origin of germ-line and somatic mutations in the retinoblastoma gene

Segregation analysis of polymorphic sites within the retinoblastoma (RB) gene and on chromosome 13, as well as the parental origin of the lost allele in the tumor, were analyzed in 24 families with RB patients. Four mutant alleles transmitted through the germ-line and seven de novo germ-line mutant alleles were identified in 11 patients with hereditary RB. Segregation analysis within the RB gene and on chromosome 13 was useful for DNA diagnosis of susceptibility to RB in relatives of hereditary patients, even if mutations were not identified. All seven de novo germ-line mutant alleles were paternally derived. The bias toward the paternal allele for de novo germ-line mutations of the RB gene was statistically significant. Seven paternal alleles and six maternal alleles were lost in 13 non-hereditary RB tumors with no bias in the parental origin of the somatic allele loss. These results suggest that the physical environment or a deficiency in DNA repair during spermatogenesis may be associated with significant risk factors for de novo germ-line mutations.     

Aneugenic effect of ionizing radiation in mammalian and human somatic cells

Aneuploidy is among the most serious impairments of hereditary material in somatic and germline cells of living organisms. Chromosome loss or the appearance of an extra homolog in the chromosome set can result in either cell death or the development of various neoplasms with high probability of malignancy. It was traditionally believed that ionizing radiation produces primarily a clastogenic effect. However, there is apparently an aneugenic component of radiation, with mechanism different from that of structural chromosome damage. The present review focuses on the evidence for the existence of the aneugenic effect of ionizing radiation in mammalian and human somatic cells.     

Chromosome 13 restriction fragment length polymorphisms

Summary The gene locus for hereditary retinoblastoma is on human chromosome 13, band q14. With this gene localization in mind, we cloned DNA fragments from this chromosome. Three of the fragments identify restriction fragment length polymorphisms. These three fragments are from the region 13q12–13q22, the chromosome region which contains the retinoblastoma locus. We expect that these restriction fragment length polymorphisms will be linked to the retinoblastoma locus, and that they will serve in certain retinoblastoma families as predictors of retinoblastoma gene carriers.They will also be useful in studies of other gene loci thought to be on chromosome 13.This research was supported by grants from the National Institutes of Health HD04807, CA29883, and EY04543, by a grant from Fight for Sight, Inc., New York City, and by the Anna Fuller Fund     

Erbliche Disposition zu Tumoren des Auges

Ocular neoplasms are associated with several hereditary cancer predisposition syndromes. Of these, hereditary retinoblastoma is the most important. Historically, hereditary retinoblastoma has been the prototype dominant cancer predisposition, and Knudson’s study of this tumour has resulted in the two-step mutation hypothesis, which has been seminal for developing a further understanding of hereditary cancers. Although ocular manifestations are of minor relevance for the prognosis in tumour syndromes other than retinoblastoma, they may be important for the recognition of the underlying cancer predisposition. There is no monogenic predisposition for the development of uveal melanoma, which is the most frequent malignant ocular neoplasm in humans. However, epidemiological data suggest that genetic factors may play a role. The study of somatic alterations in these tumours has shown that the loss of one chromosome 3 is closely associated with an adverse prognosis. Such a somatic genotype-phenotype association is highly exceptional among solid tumours.     

Repair of potentially lethal X-ray damage in fibroblasts derived from patients with hereditary and D-deletion retinoblastoma

We examined X-ray induced potentially lethal damage repair (PLDR) in density inhibited plateau phase cultures of six fibroblast strains derived from patients with hereditary retinoblastoma and two patients with D-deletion retinoblastoma and compared them to three normal controls. PLD was measured in hereditary retinoblastoma (7 Gy exposure) and normal cells (7 and 9 Gy exposure) after 24 h repair time. PLD survival curves were performed at 2-9 Gy on six retinoblastoma and three normal control cell strains. Thus, PLDR was compared at equitoxic survival levels as well as after exposure to equal doses of radiation. Some retinoblastoma strains showed normal PLDR whereas others were possibly deficient. Implications of PLDR for susceptibility to radiation-induced and spontaneous tumours in hereditary retinoblastoma patients are discussed.     

Fibroblasts from Retinoblastoma patients: Enhanced growth in fetal calf serum and a normal response to ionizing radiation

Retinoblastoma is a rare malignant eye tumor found almost exclusively in young children. In 30% of cases, the tumor is bilateral and is inherited as an autosomal dominant trait. In such patients, all of the cells in the body must carry the mutation predisposing to retinoblastoma. To search for the expression of the gene in cells outside the retina, we have studied several in vitro properties of skin fibroblasts from patients with bilateral retinoblastoma. Measurement in low concentrations of fetal calf serum of the initial growth rate and the plating efficiency show that fibroblasts from retinoblastoma donors grow significantly better than those from normal donors. However, we were unable to confirm the results of other investigators that fibroblasts from donors with bilateral retinoblastoma are unusually sensitive to ionizing radiation. In family studies, skin fibroblasts from normal siblings had the same radiation sensitivity as fibroblasts from sibling with retinoblastoma.     

Meiotic segregation analysis of RB1 alleles in retinoblastoma pedigrees by use of single-sperm typing

In hereditary retinoblastoma, different epidemiological studies have indicated a preferential paternal transmission of mutant retinoblastoma alleles to offspring, suggesting the occurrence of a meiotic drive. To investigate this mechanism, we analyzed sperm samples from six individuals from five unrelated families affected with hereditary retinoblastoma. Single-sperm typing techniques were performed for each sample by study of two informative short tandem repeats located either in or close to the retinoblastoma gene (RB1). The segregation probability of mutant RB1 alleles in sperm samples was assessed by use of the SPERMSEG program, which includes experimental parameters, recombination fractions between the markers, and segregation parameters. A total of 2,952 single sperm from the six donors were analyzed. We detected a significant segregation distortion in the data as a whole (P=.0099) and a significant heterogeneity in the segregation rate across donors (.0092). Further analysis shows that this result can be explained by segregation distortion in favor of the normal allele in one donor only and that it does not provide evidence of a significant segregation distortion in the other donors. The segregation distortion favoring the mutant RB1 allele does not seem to occur during spermatogenesis, and, thus, meiotic drive may result either from various mechanisms, including a fertilization advantage or a better mobility in sperm bearing a mutant RB1 gene, or from the existence of a defectively imprinted gene located on the human X chromosome.     

Brca2 (XRCC11) Deficiency Results in Radioresistant DNA Synthesis and a Higher Frequency of Spontaneous Deletions

We show here that the radiosensitive Chinese hamster cell mutant (V-C8) of group XRCC11 is defective in the breast cancer susceptibility gene Brca2. The very complex phenotype of V-C8 cells is complemented by a single human chromosome 13 providing the BRCA2 gene, as well as by the murine Brca2 gene. The Brca2 deficiency in V-C8 cells causes hypersensitivity to various DNA-damaging agents with an extreme sensitivity toward interstrand DNA cross-linking agents. Furthermore, V-C8 cells show radioresistant DNA synthesis after ionizing radiation, suggesting that Brca2 deficiency affects cell cycle checkpoint regulation. In addition, V-C8 cells display tremendous chromosomal instability and a high frequency of abnormal centrosomes. The mutation spectrum at the hprt locus showed that the majority of spontaneous mutations in V-C8 cells are deletions, in contrast to wild-type V79 cells. A mechanistic explanation for the genome instability phenotype of Brca2-deficient cells is provided by the observation that the nuclear localization of the central DNA repair protein in homologous recombination, Rad51, is reduced in V-C8 cells.     

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.     

Mutant spectra of irradiated CHO AL cells determined with multiple markers analyzed by flow cytometry

We have previously developed a sensitive and rapid mammalian cell mutation assay which is based on a Chinese hamster ovary cell line that stably incorporates human chromosome 11 (CHO A(L)) and uses flow cytometry to measure mutations in CD59. We now show that multiparameter flow cytometry may be used to simultaneously analyze irradiated CHO A(L) cells for mutations in five CD genes along chromosome 11 (CD59, CD44, CD90, CD98, CD151) and also a GPI-anchor gene. Using this approach, 19 different mutant clones derived from individual sorted mutant cells were analyzed to determine the mutant spectrum induced by ionizing radiation. All clones analyzed were negative for CD59 expression and PCR confirmed that at least CD59 exon 4 was also absent. As expected, ionizing radiation frequently caused large deletions along chromosome 11. This technology can readily be used to rapidly analyze the mutant yield as well as the spectrum of mutations caused by a variety of genotoxic agents and provide greater insight into the mechanisms of mutagenesis.     

Low incidence of deletion of the esterase D locus in retinoblastoma patients

Esterase D was quantitatively measured in the red blood cells from three patients from three separate kindreds who had abnormalities of chromosome 13. The esterase D activity was proportional to the number of copies of the q14 region of chromosome 13 present. These findings confirm published data localizing the esterase D gene to chromosome band 13q14, a region which is important in the etiology of retinoblastoma. Fifty-one additional retinoblastoma patients not known to have any chromosomal defect also underwent esterase D determination. In none of these patients did the esterase D measurement detect a 13q14 deletion. The normal esterase D levels in this series of 51 retinoblastoma patients suggest that deletion of an esterase D locus is infrequent in retinoblastoma patients. It must be noted that patients who are mosaics, with a 13q14 deletion in only a fraction of all somatic cells, could possibly have normal red blood cell esterase D levels. Further study is necessary to determine if esterase D determination of all retinoblastoma patients is a worthwhile clinical tool.     

Functional complementation of the radiation-sensitive mutant M10 cell line by human chromosome 5

The mouse lymphoma (L5178Y) cell mutant M10 is defective in rejoining DNA double-strand breaks and is hypersensitive to ionizing radiation. The introduction of human chromosome 5 into M10 cells by microcell mediated chromosome transfer complemented the ionizing-radiation hypersensitivity defect of this cell line. The presence of chromosome 5 in the microcell hybrids was shown using PCR with chromosome-specific primers and fluorescence in situ hybridization. From this data we conclude that the gene that corrects the radiation hypersensitivity of M10 cells is located on chromosome 5 and tentatively assigned to the 5q14 to 5pter region. We designate this gene XRCC4L.     

X-radiation-induced chromosome breakage in retinoblastoma lymphocytes

We have examined the spontaneous and X-radiation-induced chromosomal damage in normal humans and in patients with retinoblastoma using the BudR-Giemsa technique in lymphocytes cultured for 48 h. 9 sporadic unilateral non-hereditary cases, 11 hereditary cases (8 bilateral sporadic and 3 unilateral hereditary cases) and 20 healthy individuals were studied simultaneously. No difference in the spontaneous frequency of chromatid and chromosome aberrations was observed between patients and controls. After treatment with 150 rad the frequency of chromosome exchange aberrations was higher in unilateral hereditary cases than the controls (42.0% +/- 5.3 and 22.3% +/- 2.6 respectively; p = 0.05). In bilateral sporadic retinoblastoma 2 different groups were observed. A hypersensitive group showed a significant increment in radiation-induced chromosomal exchange aberrations over the control group (46.2% +/- 5.4 and 24.2% +/- 2.1 respectively; p = 0.01). The other group had a chromosomal exchange frequency similar to normal individuals (26.5% +/- 2.0 and 24.2% +/- 0.4 respectively; p = 0.10). Sporadic unilateral non-hereditary retinoblastoma had an exchange chromosomal aberration frequency similar to control individuals (26.1% +/- 2.8 and 24.6% +/- 2.7 respectively; p greater than 0.10). These results suggest that: There is no relationship between spontaneous chromosome fragility and retinoblastoma. Sporadic unilateral non-hereditary retinoblastoma has normal chromosome sensitivity to X-irradiation. Some hereditary cases of retinoblastoma are sensitive to X-rays while others behave like normals. A mutation or a submicroscopic deletion at a DNA repair locus which is independent of the retinoblastoma gene may cause this radiosensitivity.     

Survival of 60Co-irradiated herpes simplex virus in 15 human diploid fibroblast cell strains

The present study was designed to determine the extent to which herpes simplex virus (HSV) may be utilized to study the repair of DNA damaged by ionizing radiation. We investigated the survival of 60Co-irradiated HSV in cell strains derived from 2 normal controls and 13 patients with a broad range of diseases associated with possible DNA repair deficiencies. Irradiation was performed under two conditions to vary the type of damage incurred by the virus. HSV survival was greatly enhanced when the virus was irradiated in such a way that the indirect effects of ionizing radiation were minimized. We found no correlation between cellular hypersensitivity to ionizing radiation and survival of irradiated HSV. Reduced levels of virus survival were found in only 1 cell strain. When cells were treated with ionizing radiation or UV light prior to infection, no enhancement of virus survival was observed.     

Mutability of the liver cells in the progeny of irradiated male rats

The descendants of the first generation of radiation exposed rat males exhibited an increased sensitivity of liver cells to the mutagenic effect of ionizing radiation as was determined by the incidence of chromosome aberrations. It is assumed that the stability of the hereditary apparatus of these animal cells is diminished due to the presence therein of radiation induced injuries of mutation and epigenomic origin at a heterozygous condition.     

Studies on the radiosensitivity of cells from patients with basal cell naevus syndrome.

No difference in survival was observed between cultured cells from basal cell naevus syndrome (BCNS) patients and normal controls following exposure of fibroblasts to ionizing radiation. Potential lethal damage repair in BCNS cells, measured by holding experiments, was also no different from normal. G0-irradiated lymphocytes from BCNS patients were found to have a significantly higher level of X-ray-induced chromosome aberrations compared with normals. This increase is, however, small, and, taken together with the survival data, suggests that increased cell killing as a measure of the unusual clinical radiosensitivity is not the major effect of the BCNS gene.     

Deletion of chromosome region 13q14 is transmissible and does not always predispose to retinoblastoma

Summary During routine screening of retinoblastoma patients for esterase D activity in red blood cell lysates a patient was identified with only 50% of normal enzyme activity. Chromosome analysis showed that this patient had a small deletion within chromosome region 13q14. Parental studies showed that, whereas the father had normal enzyme levels, the mother had esterase D levels which were also 50% of normal and a similar small 13q14 deletion. Ophthalmological examination failed to demonstrate any retinal abnormality in either parent. Thus wer present the first case not only of the direct transmission of a 13q14 deletion within a family but also of an individual in whom the deletion has not predisposed to tumour formation.     

Modeling viability of mammalian cells exposed to ionizing radiation with different linear energy transfer

A mathematical model of the survival of mammalian cells exposed to ionizing radiation is proposed, which takes into account nonuniform radiosensitivity of different regions of DNA. The model is based on the assumption that the double-strand breaks of the anchor DNA, induced either directly by irradiation or by enzymes, play a key role in the lethal radiation effect. The model survival curves are in good agreement with experimental curves measured in different laboratories.     

Measuring the spectrum of mutation induced by nitrogen ions and protons in the human-hamster hybrid cell line A(L)C

Astronauts can be exposed to charged particles, including protons, alpha particles and heavier ions, during space flights. Therefore, studying the biological effectiveness of these sparsely and densely ionizing radiations is important to understanding the potential health effects for astronauts. We evaluated the mutagenic effectiveness of sparsely ionizing 55 MeV protons and densely ionizing 32 MeV/nucleon nitrogen ions using cells of two human-hamster cell lines, A(L) and A(L)C. We have previously characterized a spectrum of mutations, including megabase deletions, in human chromosome 11, the sole human chromosome in the human-hamster hybrid cell lines A(L)C and A(L). CD59(-) mutants have lost expression of a human cell surface antigen encoded by the CD59 gene located at 11p13. Deletion of genes located on the tip of the short arm of 11 (11p15.5) is lethal to the A(L) hybrid, so that CD59 mutants that lose the entire chromosome 11 die and escape detection. In contrast, deletion of the 11p15.5 region is not lethal in the hybrid A(L)C, allowing for the detection of chromosome loss or other chromosomal mutations involving 11p15.5. The 55 MeV protons and 32 MeV/nucleon nitrogen ions were each about 10 times more mutagenic per unit dose at the CD59 locus in A(L)C cells than in A(L) cells. In the case of nitrogen ions, the mutations observed in A(L)C cells were predominantly due to chromosome loss events or 11p deletions, often containing a breakpoint in the pericentromeric region. The increase in the CD59(-) mutant fraction for A(L)C cells exposed to protons was associated with either translocation of portions of 11q onto a hamster chromosome, or discontinuous or "skipping" mutations. We demonstrate here that A(L)C cells are a powerful tool that will aid in the understanding of the mutagenic effects of different types of ionizing radiation.