Roberts syndrome: phenotypic variation, cytogenetic definition and heterozygote detection.

Five cases of Roberts syndrome (RS) in four nuclear families are reported and the wide range of phenotypic variation among them is described. This is in contrast with the remarkable uniformity of the cytogenetic findings. Indirect immunofluorescence with seric antibodies from patients with CREST, revealed that the centromeric structures are normal in RS thus confirming J. German's assumption that the chromatid repulsion is confined to the heterochromatin. The authors quantified the phenomenon of centromeric heterochromatin separation (as occasionally revealed by C-bands in normal subjects) in obligate heterozygotes and possible heterozygotes for RS. The results are indicative of the possibility to screen for heterozygotes. The nosology of RS and related syndromes is discussed in view of the cytogenetic findings and the natural history of the disease.     

Studies of mitotic and centromeric abnormalities in Roberts syndrome: Implications for a defect in the mitotic mechanism

Roberts syndrome is an inherited human condition that is of particular interest because separation of centromeres and constitutive heterochromatin is observed in metaphase chromosomes. In this study we investigated the frequency of other cytological abnormalities in three Roberts syndrome patients. Our findings when taken with previous cytological reports emphasize that there are other features that are equally characteristic of Roberts syndrome: (1) aneuploidy with random chromosome loss and (2) micronuclei and/or nuclear lobulations of 8%–24% of interphase cells. We observed abnormal chromosome movement involving one or all the chromosomes during anaphase. Evidence is presented suggesting that aneuploidy, micronuclei and abnormal nuclear morphology are a direct result of lagging chromosomes. The cytological features documented for Roberts syndrome indicate that this is a human mitotic mutant.by T.C. Hsu     

Mapping of a single locus capable of complementing the defective heterochromatin phenotype of Roberts syndrome cells

Roberts syndrome (RS) is a developmental disorder characterized by tetraphocomelia and a broad spectrum of additional clinical features. Most patients with RS exhibit characteristic cytogenetic phenotypes, which include an abnormal appearance of pericentromeric heterochromatin on metaphase chromosomes, referred to as "heterochromatic repulsion." In the present study, we use complementation of this abnormal cytogenetic phenotype as a means to identify a specific region of the normal human genome capable of rendering phenotypic correction. We screened the entire human genome, using a transient chromosome-transfer assay, and demonstrated complementation exclusively after the transfer of proximal chromosome 8p, a result subsequently confirmed by stable microcell-mediated chromosome transfer. Additionally, homozygosity mapping was used to refine the interval of this complementing locus to 8p21. The results are consistent with the notion that the single gene defect responsible for heterochromatic splaying and developmental abnormalities maps to chromosome 8p21.     

Sensitivity of Bloom syndrome fibroblasts to mitomycin C

Lymphocytes and fibroblasts from people with Bloom syndrome, an autosomal recessive disorder associated with a predisposition to a wide variety of cancers, are known to be hypersensitive to ethylating agents as measured by sister chromatid exchange induction. Recently, hypersensitivity to cell killing by mitomycin C has also been reported in Bloom syndrome fibroblasts from three donors. We report here results which confirm the hypersensitivity of Bloom syndrome fibroblasts as measured by cell killing but show that they have a normal sensitivity to mitomycin C as measured by sister chromatid exchange induction. These results are discussed in terms of their relevance to the diversity of response of Bloom syndrome cells to mutagens, and the nature of the primary defect in Bloom syndrome.     

Chromatid repulsion associated with Roberts/SC phocomelia syndrome is reduced in malignant cells and not expressed in interspecies somatic-cell hybrids.

Different cell types from a female patient with Roberts/SC phocomelia syndrome were evaluated quantitatively for the presence of repulsion of heterochromatin and satellite regions of mitotic chromosomes. Whereas EBV-transformed lymphoblasts from an established cell line revealed these phenomena at frequencies equal to those in PHA-stimulated lymphocytes and cultured skin fibroblasts, aneuploid cells from a metastatic melanoma displayed them at 50% lower frequency. Cocultivation of the patient's fibroblasts with either an immortal Chinese hamster cell line or with a human male fibroblast strain carrying a t(4;6)(p14;q21) translocation showed that the phenomenon was not corrected or induced by a diffusible factor or by cell-to-cell contact. In each experiment, only the patient's metaphase spreads revealed chromatid repulsion. In fusion hybrids between the patient's fibroblasts and an established Chinese hamster cell line, the human chromosomes behaved perfectly normally, suggesting that the gene product which is missing or mutant in Roberts/SC phocomelia syndrome is supplied by the Chinese hamster genome.     

TERRA,CpG methylation,and telomere heterochromatin: Lessons from ICF syndrome cells

Self-reinforcing negative feedback loops are commonly observed in biological systems. RNA-mediated negative feedback loops have been described in the formation of heterochromatin at centromeres in fission yeast and the inactive X chromosome in mammalian cells. The telomere repeat-containing RNA (TERRA) has also been implicated in the formation of telomeric heterochromatin through a self-reinforcing negative feedback loop. In cells derived from human ICF syndrome, TERRA levels are abnormally elevated and telomeres are abnormally shortened. We now show that telomere heterochromatin is also abnormal in ICF cells. We propose that ICF cells fail to reinforce the TERRA-dependent negative feedback loop as a result of the inability to establish heterochromatin at subtelomeres. This failure is likely due to the lack of DNMT3b and DNA methylation, which is a genetic lesion associated with ICF syndrome. Failure of this feedback mechanism leads to catastrophic telomere dysfunction and chromosome instability.     

On the localization of mitomycin C-induced aberrations in normal human and Fanconi's anaemia cells

This paper summarizes the results of a series of experiments with primary cultures of normal human fibroblasts and lymphocytes designed to investigate chromatid aberration 'break-point' localization after a 1-h pulse of mitomycin C. For discontinuities and interchanges, 60-70% of the inferred 'break-points' were localized to defined paracentric heterochromatin and the centromeric regions (i.e. approximately 21% by length of the normal karyotype), irrespective of 'dose', aberration frequency, sample time or cycle sub-phase as determined by replication banding. Chromatid intrachanges are non-(or negatively) localized because of an inescapable scoring bias. SCE in fibroblasts show no such localization. Cells from a number of Fanconi's anaemia subjects were examined. In poorly growing cultures, localization was as high as in normal cells but in vigorous cultures localization was reduced to approximately 30%. It is suggested that the enhanced aberration sensitivity of this syndrome could arise because non-localized aberrations, usually eliminated before division in normal cells, are allowed to reach mitosis in FA cells.     

Comparison of HepG2 feeder cells generated by exposure to gamma-rays, X-rays, UV-C light or mitomycin C for ability to activate 7,12-dimethylbenz[a]anthracene in a cell-mediated Chinese hamster V79/HGPRT mutation assay

The cell-mediated Chinese hamster V79/HGPRT mutagenicity assay is an established in vitro testing method. Although gamma-irradiated human HepG2 hepatoma cells have been used recently for chemical activation, an alternative is now needed due to scheduled retirement of the available gamma-source. X-irradiation, 254 nm UV-C light and mitomycin C were examined as possible HepG2 mitotic inhibitors, and treated cells compared for activation of 7, 12-dimethylbenz[a]anthracene (DMBA). In colony-forming assays, V79 and HepG2 cells differed in sensitivity to DMBA, with V79 survival declining sharply between 1-2.5 microM (LD50=1.75 microM) while HepG2 survival decreased gradually, beginning at 0.01 microM DMBA (LD50=0.045 microM). When HepG2 feeder cells generated by each method were included in V79/HGPRT mutation assays, activation of 1 microM DMBA was found to vary according to the mitotic inhibitor used, with mutation frequencies decreasing in the order 4000 rads gamma-rays>25 microg/ml mitomycin C>4000 rads X-rays>25 J/m2 UV-C light. Only assays containing gamma-irradiated HepG2 cells generated an increase (2-3-fold) in mutation frequency when DMBA exposure was extended from 24 to 48 h. The effect of HepG2 preincubation with either Aroclor 1254 or DMBA on feeder cell activation of DMBA was also assessed using concentrations of Aroclor 1254 (10 microg/ml) or DMBA (1.0 microM) which were found to produce optimum induction of ethoxyresorufin-O-deethylase (EROD) activity (3.1-fold and 2-fold increases, respectively). Compared to results obtained with uninduced HepG2 cells, assays incorporating HepG2 cells activated by either Aroclor 1254 or DMBA produced slightly increased V79/HGPRT mutation frequencies after 24 h of exposure to mutagen; however, a 48 h incubation with mutagen in the presence of HepG2 preincubated with either Aroclor 1254 or DMBA resulted in higher mutation frequencies regardless of the mitotic inhibitor treatment. EROD activity was also induced 1.4-fold following exposure of HepG2 cells to mitomycin C alone. Although gamma-irradiation remains the treatment of choice for producing metabolically active HepG2 feeder cells, comparison of the alternatives tested suggests that mitomycin C would be a convenient and suitable replacement.     

Fanconi anaemia cells are not uniformly deficient in unhooking of DNA interstrand crosslinks,induced by mitomycin C or 8-methoxypsoralen plus UVA

Summary Fanconi anaemia (FA) cells are extremely sensitive to crosslinking agents, e. g. mitomycin C, but only moderately sensitive to trimethylpsoralen plus UVA. Evidence has been reported suggesting that there is a deficient DNA crosslink repair mechanism in FA cells, but others failed to confirm this conclusion using other methods and other crosslinking agents. We reinvestigated the mitomycin C and 8-methoxypsoralen crosslink repair in FA cells with a high sensitivity to mitomycin C. Although an essentially similar methodology was used to that previously described, no difference between the control and FA cell strains was observed, neither for mitomycin C- nor for 8-methoxypsoralen-induced crosslinks.     

Chinese hamster cell mutant, V-C8, a model for analysis of Brca2 function

The previously described Chinese hamster cell mutant V-C8 that is defective in Brca2 shows a very complex phenotype, including increased sensitivity towards a wide variety of DNA damaging agents, chromosomal instability, abnormal centrosomes and impaired formation of Rad51 foci in response to DNA damage. Here, we demonstrate that V-C8 cells display biallelic nonsense mutations in Brca2, one in exon 15 and the other in exon 16, both resulting in truncated Brca2 proteins. We generated several independent mitomycin C (MMC)-resistant clones from V-C8 cells that had acquired an additional mutation leading to the restoration of the open reading frame of one of the Brca2 alleles. In two of these revertants, V-C8-Rev 1 and V-C8-Rev 6, the reversions lead to the wild-type Brca2 sequence. The V-C8 revertants did not gain the entire wild-type phenotype and still show a 2.5-fold increased sensitivity to mitomycin C (MMC), higher levels of spontaneous and MMC-induced chromosomal aberrations, as well as abnormal centrosomes when compared to wild-type cells. Our results suggest that Brca2 heterozygosity in hamster cells primarily gives rise to sensitivity to DNA cross-linking agents, especially chromosomal instability, a feature that might also be displayed in BRCA2 heterozygous mutation carriers.     

The induction of mitotic gene conversion by chemical and physical mutagens as a function of culture age in the yeast, Saccharomyces cerevisiae

Summary Cultures of yeast progressing from the exponential to the stationary phase of growth show increased resistance to the lethal effects of the chemical mutagens nitrous acid, ethyl methane sulphonate and mitomycin C and increased sensitivity to the lethal effects of UV light. Induced mitotic intragenic recombination produced by gene conversion also shows variation in its response to the growth phase after mutagen treatment. Higher frequencies of recombination per surviving cell were found after nitrous acid and ethyl methane sulphonate treatment of stationary phase cells wherease identical frequencies were produced by UV and mitomycin C treatment in both growth phases.The results were consistent with the hypothesis that the more nitrous acid and ethyl methane sulphonate resistant stationary phase cells were more active in postreplication repair. The sensitivity of exponential phase cells to nitrous acid and ethyl methane sulphonate may result from both increased mutagen uptake and reduced postreplication repair activity. In contrast, irrespective of growth phase all cells surviving UV and mitomycin C treatment appear to have undergone identical levels of post-replication repair.     

Electrochemical mutagen screening using microbial chip

Electrochemical microbial chip for mutagen screening were microfabricated and characterized by scanning electrochemical microscopy (SECM). Salmonella typhimurium TA1535 with a plasmid pSK1002 carrying a umuC'-'lacZ fusion gene was used for the whole cell mutagen sensor. The TA1535/pSK1002 cells were exposed to mutagen solutions containing 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamido (AF-2), mitomycin C (MMC) or 2-aminoanthracene (2-AA) and embedded in a microcavity (5nl) on a glass substrate using collagen gel. The beta-galactosidase expression on the microbial chip was electrochemically monitored using p-aminophenyl-beta-d-galactopyranoside (PAPG) as the enzymatic substrate. This system has several advantages compared with the conventional umu test: drastic reduction of the sample volume, less time-consuming for beta-galactosidase detection (free from substrate reaction time) and lower detection limit for the three mutagens (AF-2, MMC, 2-AA). Finally, a multi-sample assay was carried out using the microbial array chip with four microcavities.     

Expression cloning of multiple human cDNAs that complement the phenotypic defects of ataxia-telangiectasia group D fibroblasts.

Ataxia-telangiectasia (A-T) is an inherited human disease of unknown etiology associated with neurologic degeneration, immune dysfunction, cancer risk, and genetic instability. A-T cells are sensitive to ionizing radiation and radiomimetic drugs, offering the possibility of cloning A-T genes by phenotypic complementation. We have used this sensitivity to isolate the first human cDNAs reported to complement A-T cells in culture. Complementation group D A-T fibroblasts were transfected with an episomal vector-based human cDNA library, approximately 610,000 resultant transformants were treated with the radiomimetic drug streptonigrin-resistant, and nine unrelated cDNAs were recovered from 29 surviving streptonigrin-resistant clones. Five cDNAs were mapped, but none localized to 11q23, the site of A-T complementation group A and C loci. Four of the mapped cDNAs conferred mutagen resistance to A-T D fibroblasts on secondary transfection. One cDNA was identified as a fragment of dek, a gene involved in acute myeloid leukemia. The dek cDNA fragment and pCAT4.5, a 4.5-kb cDNA that mapped to 17p11, independently complemented three different phenotypic abnormalities of A-T D fibroblasts (mutagen sensitivity, hyper-recombination, and radio-resistant DNA synthesis). The pCAT4.5 cDNA did not complement the mutagen sensitivity of an A-T group C fibroblast line, suggesting that it represents a candidate disease gene for group D A-T. Our results indicate that phenotypic complementation alone is insufficient evidence to prove that a candidate cDNA is an A-T disease gene. The complementing cDNAs may represent previously uncharacterized genes that function in the same pathway as does the A-T gene product(s) in the regulation of cellular responses to DNA damage.     

Chromosomal instability and cellular hypersensitivity to X-radiation of cultured fibroblasts derived from porokeratosis patients'' skin

Porokeratosis is a rare genetic skin disorder known to be associated with a propensity to develop skin cancer. To further elucidate the previously reported cytogenetic and cellular abnormalities, we studied karyotypic changes and the sensitivity to X-ray irradiation of cultured fibroblasts derived from skin lesions and normal-appearing skin of 3 patients with porokeratosis. Cultured fibroblasts from normal-appearing skin of 9 controls were similarly examined. Porokeratosis subjects had a greater number of cells with chromosomal abnormalities than controls. Two porokeratosis strains which were derived from the normal-appearing skin of a patient had a noticeable clone of abnormal cells. Porokeratosis fibroblasts were hypersensitive to the lethal effects of X-radiation. This hypersensitivity was common to both the lesion-derived strains and the ones derived from normal-appearing skin. The 2 strains with clonal abnormal cells were also similarly hypersensitive to X-radiation. These results suggest that chromosomal instability is strongly related to porokeratosis and that X-ray hypersensitivity is an inherent abnormality in cultured fibroblasts of porokeratosis patients.     

The Cellular Phenotype of Roberts Syndrome Fibroblasts as Revealed by Ectopic Expression of ESCO2

Cohesion between sister chromatids is essential for faithful chromosome segregation. In budding yeast, the acetyltransferase Eco1/Ctf7 establishes cohesion during DNA replication in S phase and in response to DNA double strand breaks in G2/M phase. In humans two Eco1 orthologs exist: ESCO1 and ESCO2. Both proteins are required for proper sister chromatid cohesion, but their exact function is unclear at present. Since ESCO2 has been identified as the gene defective in the rare autosomal recessive cohesinopathy Roberts syndrome (RBS), cells from RBS patients can be used to elucidate the role of ESCO2. We investigated for the first time RBS cells in comparison to isogenic controls that stably express V5- or GFP-tagged ESCO2. We show that the sister chromatid cohesion defect in the transfected cell lines is rescued and suggest that ESCO2 is regulated by proteasomal degradation in a cell cycle-dependent manner. In comparison to the corrected cells RBS cells were hypersensitive to the DNA-damaging agents mitomycin C, camptothecin and etoposide, while no particular sensitivity to UV, ionizing radiation, hydroxyurea or aphidicolin was found. The cohesion defect of RBS cells and their hypersensitivity to DNA-damaging agents were not corrected by a patient-derived ESCO2 acetyltransferase mutant (W539G), indicating that the acetyltransferase activity of ESCO2 is essential for its function. In contrast to a previous study on cells from patients with Cornelia de Lange syndrome, another cohesinopathy, RBS cells failed to exhibit excessive chromosome aberrations after irradiation in G2 phase of the cell cycle. Our results point at an S phase-specific role for ESCO2 in the maintenance of genome stability.     

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.     

Abnormalities in the cell-division cycle in Roberts syndrome fibroblasts: a cellular basis for the phenotypic characteristics?

Roberts-SC phocomelia syndrome (RS) is a recessively inherited developmental disorder characterized by profound pre- and postnatal growth reduction, symmetrical limb reductions of varying severity, and craniofacial abnormalities. Many patients with RS exhibit a striking chromosomal abnormality involving the heterochromatic, C-banding regions of most chromosomes. Dermal fibroblast strains from three such patients were used to investigate in vitro cellular growth characteristics. Plating efficiency, colony-forming ability, and cell density at confluence in RS were compared with dermal fibroblast strains from pediatric patients without RS and fetal lung fibroblast strains. Time-lapse cinematography was used to study mitotic duration and cytokinesis in RS and various control fibroblast strains. Clearly, cell from affected individuals had deficiencies that led to a multitude of abnormalities at the cellular level. These included: abnormal mitosis and cytokinesis, reduced cell growth, atypical cell morphology, and altered chromosomal morphology at peri- and paracentromeric and nucleolar-organizing regions. These findings suggest that the basis for at least some of the phenotypic abnormalities characteristic of this trait may reside in the reduced growth rates of the cells during the course of development. This could account for the reduced pre- and postnatal growth rates as well as for the developmental abnormalities, since deficiencies of cells in developing anlagen could well lead to alterations in developmental patterns.     

The deficiency of PIP2 5-phosphatase in Lowe syndrome affects actin polymerization

Lowe syndrome is a rare X-linked disorder characterized by bilateral congenital cataracts, renal Fanconi syndrome, and mental retardation. Lowe syndrome results from mutations in the OCRL1 gene, which encodes a phosphatidylinositol 4,5 bisphosphate 5-phosphatase located in the trans-Golgi network. As a first step in identifying the link between ocrl1 deficiency and the clinical disorder, we have identified a reproducible cellular abnormality of the actin cytoskeleton in fibroblasts from patients with Lowe syndrome. The cellular abnormality is characterized by a decrease in long actin stress fibers, enhanced sensitivity to actin depolymerizing agents, and an increase in punctate F-actin staining in a distinctly anomalous distribution in the center of the cell. We also demonstrate an abnormal distribution of two actin-binding proteins, gelsolin and alpha-actinin, proteins regulated by both PIP(2) and Ca(+2) that would be expected to be altered in Lowe cells. Actin polymerization plays a key role in the formation, maintenance, and proper function of tight junctions and adherens junctions, which have been demonstrated to be critical in renal proximal tubule function, and in the differentiation of the lens. These findings point to a general mechanism to explain how this PIP(2) 5-phosphatase deficiency might produce the Lowe syndrome phenotype.     

The miRNA aberrant expression dependence on DNA methylation in HeLa cells treated with mitomycin C

The dependence of expression of miRNAs and their precursors (pre-miRNAs) on the DNA methylation level in HeLa cells 8 days after mitomycin C treatment was studied. A massive parallel DNA sequencing method was applied to analyze miRNA expression. 5-Azacytidine (DNA methylation inhibitor) was added to the medium 6 days after mutagenic agent exposure. The results indicated that the change in expression for some mature miRNAs (39 of 61) was accompanied by the change in the expression of their pre-miRNAs, while there were no significant changes in the expression of pre-miRNA for other mature miRNAs (22 of 61). The aberrant expression was maintained by 8 of 61 mature miRNAs and 6 of 55 pre-miRNAs in the induced HeLa cells after 5-azacytidine treatment. In addition, the expression of more than 90% of miRNAs, which indicated a significant change in expression after mitomycin C treatment, does not depend or depends slightly on the DNA methylation level in HeLa cells without mitomycin C treatment. The results suggest that mitomycin C induces aberrant DNA methylation which affects maintenance of changes in the miRNA expression in cell generations after mutagen treatment.     

Cohesinopathies: One ring, many obligations

Over 75 years ago, two human genetic disorders were initially described and named for their founding physicians: Cornelia de Lange (CdLS) and Roberts syndrome (RBS)/SC Phocomelia (SC). In the past 4 years, genetic studies of patients have revealed the primary genes involved in these disorders are the essential, evolutionarily conserved components of the cohesin pathway. This pathway serves to facilitate cohesion between replicated sister chromatids, thereby enabling proper chromosome segregation. As a result of these findings, these disorders now represent a novel class of human genetic disorders known as cohesinopathies. Over 60% of CdLS patients examined have de novo mutations in either: SCC2/NIPBL, SMC1, or SMC3, whereas the causative gene in Roberts syndrome and SC Phocomelia has been identified as ESCO2. Now modern genetic, biochemical, and cell biological approaches may be applied to determine the underlying mechanism of these genetic disorders.