Nenad Bukvic

Sex chromosome loss, micronuclei, sister chromatid exchange and aging: a study including 16 centenarians

In the present study we analysed the possible effect of age, sex and smoking on the mean values of micronucleus (MN) and sister chromatid exchange (SCE) frequencies on peripheral blood obtained from 38 subjects ranging in age from 16 to 63 years and 16 centenarians. The mean number of binucleated cells with micronuclei varied in function of age and sex (as demonstrated by the analysis of covariance (F=13.13; P<0.001), particularly evident was the increment observed in women with increasing age (interaction age/sex: F=5.53; P<0.05). Smoking habits had no effects on MN frequency (F=0.36; P>0.05). Sex (F=4.18; P<0.05) and smoking habits (F=14.64; P<0.001) influenced significantly SCE per cell frequencies, but age had no effects on them (F=2.45; P>0.05). The age-associated increase of sex chromosome loss was studied using fluorescence in situ hybridisation (FISH) on interphase nuclei. The loss of Y signals was observed in approximately 10% of interphase cells from the centenarians males, that is six times more often than in the younger control men (approximately 1.6%). The frequency of X signal loss (approximately 1.7%) in young women was similar to that observed in male controls of the same age but the incidence of the X chromosome aneuploidy in centenarian females was appreciably higher (approximately 22%) than that found for the Y chromosome in males. These results were correlated with the data on MN formation and a positive correlation between the percentage of aneuploid cells (FISH) and MN values was observed (r=0.50; P<0.05).

Sister chromatid exchange (SCE) and micronucleus (MN) frequencies in lymphocytes of gasoline station attendants

Peripheral blood lymphocytes from 22 men with low average exposure (229 micrograms/m3 = 0.72 ppm) to benzene and 19 control men were investigated for Sister Chromatid Exchange (SCE) frequency. The majority of the men (21 exposed, 19 controls) were also investigated using the micronucleus assay (MN). The exposed subjects were employed at 10 different gas stations in or near the city (Bari/South Italy). SCE frequencies were significantly related with age and smoking habits, on the contrary no relation was observed between SCE and length of employment (SCE = 7.41 + 0.03.age (*) + 0.0001.length of employment (n.s.) + 0.03.cigarette consumption (*); F = 4.87; p < 0.01; (*) significant; (n.s.) non-significant). MN frequencies were significantly increased in relation with length of employment; but no relation was observed when age and smoking habits were taken into consideration (regression model: MN = 18.03 + 0.006.age (n.s.) + 0.32.length of employment (*) - 0.1.cigarette consumption (n.s.); F = 4.138; p < 0.05).

A homozygous frameshift mutation in the ESCO2 gene: Evidence of intertissue and interindividual variation in Nmd efficiency

Roberts syndrome (RS) is a rare disorder characterized by tetraphocomelia and several other clinical features. Cells from RS patients exhibit characteristic premature separation of heterochromatic region of many chromosomes and abnormalities in cell cycle. Mutations in the ESCO2 gene have recently been identified in 20 RS families. We performed mutational analysis of the ESCO2 gene in two fetuses diagnosed with RS and their normal parents. In both fetuses, we identified homozygosity for the c. 745_746delGT mutation, while the non‐consanguineous parents were both heterozygous for the same mutation. Considering the position of the mutation identified, we carried out qualitative and quantitative real‐time ESCO2 cDNA analysis on RNA isolated from CVS‐stromal cells in one fetus, amniocytes in the second fetus, and lymphocytes from the heterozygous parents. The results of this analysis showed that despite the presence of a premature termination codon (PTC) 112 nucleotides upstream of the next exon3–exon4 junction, the mutant ESCO2 mRNA was present in both fetuses, albeit at low levels, indicating a partial resistance to nonsense mediated decay (NMD). Interestingly, when cells derived from the two fetuses were treated with an inhibitor of translation, they revealed the presence of tissue and individual variability in NMD efficiency, despite the identical mutational status. The existence of such a variation in the NMD efficiency could explain the broad intrafamilial and interfamilial variability in the clinical presentation of RS patients, and in other genetic diseases where nonsense mutations are responsible for most of the mutation load. Moreover, considering that a mutated full length mRNA was produced in both fetuses, we used Western blot analysis to demonstrate the absence of the ESCO2‐truncated protein in cells derived from both fetuses and in a lymphoblastoid cell line derived from the parents. J. Cell. Physiol. 209: 67–73, 2006.

An unusual dicentric Y chromosome with a functional centromere with no detectable alpha-satellite

We describe an unusual marker chromosome Y. This marker is present in 5% of the lymphocytes of a dysgenetic woman showing a mosaic karyotype 45,X/46,XY/ 47,XY+mar. Q-banding revealed that the marker was morphologically identical to the Y chromosome of the patient but presented the primary constriction in the heterochromatic region. C-banding confirmed that the heterochromatic region was C-positive; furthermore, it showed two spots in the euchromatic region in a position corresponding to that of the centromere in the normal Y Fluorescence in situ hybridization with the centromere-specific probe pDP 97 and the pancentromeric alpha-satellite probe α27α30 failed to detect any signal at the primary constriction site. To improve the characterization of the marker chromosome, hybridization was performed using pDP 105, a probe located on the short arm of the Y chromosome, together with chromosome-Y- specific paint-hybridizing to the single sequence spanning the Y short arm. In both cases, positive signals telomeric to the inactive centromere were observed. Possible mechanisms resulting in the formation of the marker chromosome are discussed.

Cytogenetic studies in coke oven workers

Chromosome aberrations, micronuclei, and sister chromatid exchanges (SCE) were evaluated in cultured lymphocytes of coke oven workers of an Italian steel industry plant, occupationally exposed to polycyclic aromatic hydrocarbons, and in a group of unexposed controls from a non-oven plant in the same area. No differences were found between exposed and controls for rates of total abnormal metaphases (including and excluding gaps), chromatid-type and chromosome-type aberrations, cells with 2 or more breaks, and for micronuclei. On the contrary, SCE were significantly increased in the exposed versus the controls, but, when smoking habits were considered, the increase was significant only for smokers.

Acute radiodermatitis from accidental overexposure to X-rays

Approximately 2 weeks after accidental overexposure to X-ray radiation, a worker developed acute radiodermatitis on fingers of both hands. Exposure simulation indicated that total ionizing radiation absorbed by his fingers amounted to about 20 Gy. After 2 years, acute radiodermatitis evolved to chronicity of lesions with presence of atrophic skin, teleangiectasia, alopecia, and dyskeratosis on three right-hand fingers. Cytogenetic dosimetry of peripheral blood lymphocytes, performed 2 months after acute radiation, showed an increase of micronuclei (7% vs. 1 +/- 0.4% according to laboratory reference data). The increase was ascribed to the high dose of ionizing radiation absorbed by circulating lymphocytes in the vessels of overexposed tissues. The cytogenetic examination was repeated 27 months after acute irradiation; it was found that the percentage of micronuclei had been restored to within reference levels. The possibility of using cytogenetic dosimetry, following acute partial exposure to X-rays, not just as an indicator of previous exposure, but also as an indicator of the absorbed radiation dose is examined. Lastly, the possible stochastic effects that may set in on the skin of the affected fingers and the need for periodically monitoring the evolution of chronic skin lesions, are discussed.

Trisomy 13 mosaicism in a phenotypically normal child: Description of cytogenetic and clinical findings from early pregnancy beyond 2 years of age†‡

The clinical outcomeof mosaic trisomy 13 detected at prenatal diagnosis may vary from a clinically serious entity similar to the Patau syndrome to physical and mental normality [Delatycki and Gardner, 1997]. This range presumably reflects the proportion and tissue distribution of the trisomic cells. Since most of the pregnancies in which a fetus with mosaic trisomy 13 was diagnosed were terminated, there is a lack of follow-up data [Delatycki and Gardner, 1997; Eubanks et al., 1998; Wallerstein et al., 2000] especially for cases in which a high percentage (>50%) of trisomic cells was observed. Thus, genetic counselling is particularly difficult [Delatycki et al., 1998]. Herein, we describe a patient with trisomy 13 mosaicism detected at amniocentesis resulting in a normal live birth. We report on the clinical history and cytogenetic findings in different tissue sources obtained during prenatal life, at birth, and up to age 2 years. On follow-up the child showed mild growth delay in body, weight, and head size. A female fetus with trisomy 13 mosaicism was detected prenatally by amniocentesis performed at 16 weeks of gestation because of advanced maternal age (43 years). Twenty-seven of 34 clones scored (80%) showed trisomy 13. Microsatellite analysis indicated that the additional chromosome was of maternal origin, but we were unable to establish if the error occurred in the second meiotic division or in a post-zygotic mitosis. As ultrasonography did not reveal fetal abnormalities; the couple decided to continue the pregnancy, requesting further studies for confirmation. Karyotyping of cord blood at 19 weeks of gestation showed 10% trisomic cells of 100 cells scored. A chorionic villus sample (CVS), obtained at the same time as cordocentesis, showed that all the 11 cells analyzed from the short-term culture (cytotrophoblast cells) as well as the 13 cells scored from 10 foci from long-term cultures (mesenchymal cells) were 47,XX,þ13. The pregnancy was normal and delivery was at 38 weeks of gestation with birth weight of 2,270 g (3rd centile), birth length of 47 cm (>10th centile), and head circumference of 31 cm ( 3rd centile). The baby showed large angiomas on the forehead and in the occipital region and smaller angiomas on the left hand. ECG and echocardiography showed no heart defects; brain ultrasonography was normal; and the ophthalmologic examination did not indicate eye or retinal defects. Kidney ultrasonography showed a slight right pyelectasia. Chromosome re-examination performed on cord blood at delivery confirmed the previous result, that is, 10% trisomic cells of 107 cells scored. At 6 months, the baby weighed 5,000 g (<3rd centile) with a head circumference of 40.5 cm ( 3rd centile) and was 60 cm (<3rd centile) tall. Hearing screening by the BOEL-Test performed at 9 months of age was negative. Neurologic and psycomotor

17 ?ethynylestradiol and norgestrel in combination induce micronucleus increases and aneuploidy in human lymphocyte and fibroblast cultures

Oral contraceptives are highly efficient and easily administered drugs; however, it must not be forgotten that they are composed of chemical substances which can be classified as potential carcinogens. Testing of a substance for genotoxicity represents a reliable approach both to evaluate the genetic hazard and to obtain information on its possible tumorigenic (cancerogenic) properties. The present study was undertaken to evaluate through carefully planned and controlled investigations the in vitro cytogenetic effects of oral contraceptives (ethynilestradiol and norgestrel mixed in the proportion 1:5) using three different concentrations, with two different durations of treatment (48 and 72 h), on two types of human cells (lymphocytes and fibroblasts) and a series of short-term test procedures: sister chromatid exchange (SCE), micronucleus test (MN), and chromosome aberrations (CA). In addition, the FISH procedure and in vitro anaphase and metaphase preparation analyses were performed. In contrast to CA and SCE frequencies, the frequency of MN in treated blood lymphocytes showed higher values by comparison with the controls, although the difference was statistically significant only for the lowest concentration (P = 0. 016). When using pancentromeric alphoid probes, the FISH procedure gave positive signals in more than 85% of micronuclei, clearly indicating that MN may contain whole chromosomes rather than acentric fragments. Unlike the lymphocytes, the fibroblasts showed dose-dependent effects, although those treated with the highest hormone concentrations showed an increased number of highly damaged cells (cytoplasmatic vacuolization, nuclear fragmentation, etc.), a decreased number of anaphase cells, a large number of which were abnormal, and a reduction of mitotic index. In conclusion, our data confirm that hormones do not induce structural chromosome aberrations in lymphocytes and indicate that ethynilestradiol and norgestrel have an aneugenic effect on fibroblast and lymphocyte cultures; FISH analysis on micronuclei from lymphocyte cultures and anaphase preparations from fibroblast cultures support this hypothesis. Teratogenesis Carcinog. Mutagen. 20:147-159, 2000.

Influence of Some Detoxification Enzyme Polymorphisms on Cytogenetic Biomarkers Between Individuals Exposed to Very Low Doses of 1,3-butadiene

Objective: To evaluate the variation of some biomarkers related to the level of enzymatic activity dependent on the different polymorphisms. Methods: We studied 27 butadiene-exposed workers and 37 controls using different biomarkers of the genotoxic effect. The genotypes were determined using polymerase chain reaction and restriction fragment length polymorphism-polymerase chain reaction techniques; the subjects were assigned to a specific group based on the microsomal epoxide hydrolase (mEH) activity predicted by their genotype (low, intermediate, high). Results: The studied biomarkers were not able to discriminate between exposed and control individuals, but sister chromatid exchange (SCE) and high frequency cells were influenced by smoking habits. Smokers having fast microsomal epoxide hydrolase activity showed higher SCE frequency (7.61) respect to those presenting intermediate (5.86) or slow (6.65) enzymatic activity. Conclusions: On the basis of these results, can we suppose the existence of an “intermediate genotype” advantage (at least for induction of SCE)?

Autism and Hypoplastic Corpus Callosum in a Case of Monocentric Marker Chromosome 15

An 8-year-old boy was diagnosed with autism, along with development delay, seizures, and hypoplastic corpus callosum. His karyotype was 47, XY, +mar.ish (15) (D15Z1+, SNRPN+, GABRB3+, PML-(de novo?). The supernumerary marker chromosome 15 with euchromatin was monosatellited and monocentric. Although autism, seizures, and mental and developmental retardation are not rare in association with a dicentric, bisatellited supernumerary marker chromosome 15, the present case is novel for a monocentric, monosatellited supernumerary marker chromosome 15 and the additional feature of hypoplastic corpus callosum. The present case provides support for the hypotheses that additional copies of different segments of proximal 15q are related to autism and to malformations of corpus callosum.