Dosage effects of human ribosomal genes (rDNA) in health and disease.

Human ribosomal RNA genes encoding a pre-transcript of the three major ribosomal RNA (18S, 5.8S, and 28S rRNA) are tandemly repeated in human genome. Their total copy number varies from 250 to 670 per diploid genome with a mean of approximately 420 copies, but only a fraction of them is transcriptionally active. The functional consequences of human ribosomal RNA gene dosage are not widely known and often assumed to be negligible. Here, we review the facts of rRNA gene dosage effects on normal growth and aging, stress resistance of healthy individuals, and survivability of patients with chromosomal abnormalities, as well as on the risk and severity of some multifactorial diseases with proven genetic predisposition. An original hypothesis that rRNA gene dosage can be a modulating factor involved in the pathogenesis of schizophrenia and rheumatoid arthritis is put forward.

[Oxidative stress, rRNA genes, and antioxidant enzymes in pathogenesis of schizophrenia and autism: modeling and clinical advices].

Ribosomal genes (RG), or genes for rRNA, are represented by multiple tandem repeats in eukaryotic genomes, and just a part of them is transcriptionally active. The quantity of active copies is a stable genome feature which determines the cell's capability for rapid synthesis of proteins, necessary to cope with stress conditions. Low number of active RG copies leads to reduced stress resistance and elevated risk of multifactorial disorders (MFD). Oxidative stress (OS) in the brain cells is believed to be involved in the pathogenesis of infantile autism (IA) and schizophrenia, i.e., MFDs with a manifested genetic predisposition. With autism, OS markers are found almost in every research, whilst with schizophrenia, the OS data are contradictory. Earlier, in a sample of patients with schizophrenia, we have found significantly higher quantity of active RG copies than at the average in healthy population. Here we have estimated the number of active RG copies in a sample of patients with IA (n = 51) and revealed significantly lower mean value than in healthy population. A novel mathematical model of the dynamic pattern of OS has been proposed. The model is realized as an ordinary differential equation system, supposing induction of antioxidant protection enzymes being mediated by reactive oxygen species (ROS), with the subsequent decrease of ROS content in a cell. The rate of synthesis of antioxidant protection enzymes is limited by the ribosome synthesis rate which depends on the number of active RG copies. Analysis of the model showed that the system always approaches a single stable equilibrium point along a damped oscillation trajectory, which in some degree resembles the dynamics of 'predator-prey' interaction in Lotka-Volterra model. The stationary ROS level inversely depends on the number of active RG copies. Our study explains the inconsistency of clinical data of OS in schizophrenia and suggests a novel criterion for discriminative cytogenetic diagnostics of schizophrenia and IA, as well as allows to assume that antioxidant therapy should be effective only for children with low number of active RG copies.

[Cluster size polymorphism of active human ribosomal genes and simulation of the conditions of its stability through generations].

Based on selective silver nitrate staining of active ribosomal gene (AcRG) clusters in nucleolus organizer regions (NORs) of human metaphase chromosomes, a technique was developed earlier to estimate the AcRG dosage in individual genomes as a sum of arbitrary units (0-3) ascribed to the silver precipitate (AgNOR) on ten NORs. The AcRG dosage was considered to be an additive quantitative trait determined by five polymorphic autosomal loci (with for allelic forms for each locus). A database was created to contain the data on AcRG cluster variants for more than 1000 individual human genomes. In this study, the population frequencies of AcRG cluster variants were determined. The results agreed with the hypothesis that stabilizing selection acts at the zygotic and/or early embryogenetic stage to restrain the AcRG genomic dosage (copy number) within a range from 14.9 to 23.7 arbitrary units (the cell is unviable when the trait is beyond this range). The average zygotic losses due to selection were estimated at 9.1-9.9% for a real population. A computer model where the AcRG dosage of a progeny results from a random combination of the AgNORs of the five acrocentric chromosome pairs of the parents was developed and used to simulate the formation of a certain AcRG genomic dosage through generations in a human panmictic population with nonoverlapping generations. A combination of stabilizing selection by total AcRG copy number and a certain spontaneous mutation rate (the probability of changes in the cluster size of a NOR as a result of unequal crossingover in meiotic prophase) was shown to be a sufficient condition for the restrain of equilibrium population frequencies of AgNOR size variants in a human panmictic population. Using the model, the most probable spontaneous mutation frequency was predicted to be (2.1-2.3) x 10(-2) per NOR per generation for human AgNORs. The predicted frequency was within the 95% confidence interval of the experimental rate, which was determined by studying the inheritance of AgNOR variants in real families.

Abundance of ribosomal RNA gene copies in the genomes of schizophrenia patients.

OBJECTIVE: The ribosome is a critical component of the translation machinery. The key component of ribosomes is ribosomal RNA (rRNA). Dysregulation of rRNA biogenesis has been implicated in some human diseases. One of the factors affecting rRNA biogenesis is the ribosomal RNA genes (rDNA) copy number in the genome. The aim of this study was to examine the rDNA copy number (CN) variation in the genomes of patients with schizophrenia (SZ) compared to healthy controls (HC). METHODS: We evaluated rDNA CN in leukocytes of 179 subjects with SZ (108 male/71 female) in comparison with 122 HC (60 male/62 female) using two techniques: qPCR and nonradioactive quantitative hybridization (NQH), which is based on the use of biotinylated rDNA probes. RESULTS: rDNA CN (NQH) and rDNA CN (qPCR) was higher in SZ patients than in controls (median 542 vs 384, p=10-25 and median 498 vs 370, p=10-12). NQH was experimentally proved to be less sensitive to severe DNA damage than qPCR. The more DNA damage, the higher the ratio R=CN (NQH)/CN (qPCR). 15% of the SZ patients had significantly higher rDNA damage degree than the HC. CONCLUSION: Genomes of some SZ patients contain more ribosomal genes than those of HC. The elevated ribosomal genes copy number in human genome can be one of the genetic factors of schizophrenia development. This hypothesis requires further experimental studies to be corroborated or disproved.

Effects of the copy number of ribosomal genes (genes for rRNA) on viability of subjects with chromosomal abnormalities.

The number of active ribosomal genes (AcRG) was evaluated in 172 carriers of chromosomal abnormalities (CA) such as Down's syndrome (DS), Robertsonian translocations (RT), Klinefelter's and Turner's syndromes, trisomy Ð¥, disomy Y, and various structural CA. In controls (n=318), AcRG dosage varied from 119 to 190 copies with a mean of 151 copies per diploid genome. In CA carriers, except for DS newborns, AcRG dosage was not beyond these limits. As shown previously, only within these limits cellular homeostasis and organism's viability can be supported, while genomes beyond these limits are eliminated by embryonic loss. About 10% of embryos with DS and 50% of embryos with RT die/are aborted exclusively due to a surplus (DS) or a shortage (RT) of AcRG. AcRG dosage also affects the CA carrier's viability after birth, as demonstrated by comparing newborn and aged (10-40 y.o.) DS patients. Sampling range of AcRG dosage becomes considerably narrower with age: DS newborns ranged from 139 to 194 RG copies (σ2=3.59), while aged DS patients varied from 152 to 190 copies (σ2=1.55) with the same mean. Each CA group showed peculiarities in AcRG dosage distribution. We found that carriers of numerical abnormalities of gonosomes (sex chromosomes) concentrate within the area of medium, most adaptive dosages, whilst carriers of structural CA can only survive with relatively high AcRG number. Our article is the first ever to report an association of CA viability with the genomic number of AcRG.

Quantification of cell-free DNA in blood plasma and DNA damage degree in lymphocytes to evaluate dysregulation of apoptosis in schizophrenia patients.

Oxidative DNA damage has been proposed as one of the causes of schizophrenia (SZ), and post mortem data indicate a dysregulation of apoptosis in SZ patients. To evaluate apoptosis in vivo we quantified the concentration of plasma cell-free DNA (cfDNA index, determined using fluorescence), the levels of 8-oxodG in cfDNA (immunoassay) and lymphocytes (FL1-8-oxodG index, flow cytometry) of male patients with acute psychotic disorders: paranoid SZ (total N = 58), schizophreniform (N = 11) and alcohol-induced (N = 14) psychotic disorder, and 30 healthy males. CfDNA in SZ (N = 58) does not change compared with controls. In SZ patients. Elevated levels of 8-oxodG were found in cfDNA (N = 58) and lymphocytes (n = 45). The main sources of cfDNA are dying cells with oxidized DNA. Thus, the cfDNA/FL1-8-oxodG ratio shows the level of apoptosis in damaged cells. Two subgroups were identified among the SZ patients (n = 45). For SZ-1 (31%) and SZ-2 (69%) median values of cfDNA/FL1-8-oxodG index are related as 1:6 (p < 0.0000001). For the patients with other psychotic disorders and healthy controls, cfDNA/FL1-8-oxodG values were within the range of the values in SZ-2. Thus, apoptosis is impaired in approximately one-third of SZ patients. This leads to an increase in the number of cells with damaged DNA in the patient's body tissues and may be a contributing cause of acute psychotic disorder.

[The maternal effect in infantile autism: elevated DNA damage degree in patients and their mothers]. / Materinskii éffekt pri detskom autizme: povyshennyi uroven' povrezhdenii DNK u patsientov i ikh materei.

Infantile autism is a common disorder of mental development, which is characterized by impairments in the communicative, cognitive and speech spheres and obsessional stereotyped behaviour. Although in most cases, pathogenic factors remain unclear, infantile autism has a significant hereditary component, however, its etiology is also under the influence of environmental factors, including the condition of the mother's body during pregnancy ("maternal effect"). Oxidative stress is assumed to play a key role in the pathogenesis of infantile autism. It is known that oxidative stress has a prominent genotoxic effect, which is realized through inducing single and double strand breaks of the nuclear DNA. We evaluated the degree of DNA damage in patients with infantile autism and their mothers using DNA comet assay. The comet tail moment and DNA per cent ratio in the tail were assessed for each individual. The two parameters appeared to be strongly correlated (r=0.90). Mean and median values of both parameters were considerably higher in the sample of autistic children, than in age-matching healthy controls. Interestingly, these parameters were also elevated in healthy mothers of autistic children, with no difference from the values in the group of autistic children. The control group of healthy women of reproductive age, who had no children with autism, differed by the DNA comet tail moment from the group of mothers of autistic children, but did not differ significantly from the control group of healthy children. The results suggest that there are genotoxic factors in mentally healthy mothers of autistic children, which can determine the pathological process in the foeti via environmental "maternal effect" during gestation.

Functionalized Fullerene Increases NF-κB Activity and Blocks Genotoxic Effect of Oxidative Stress in Serum-Starving Human Embryo Lung Diploid Fibroblasts.

The influence of a water-soluble [60] fullerene derivative containing five residues of 3-phenylpropionic acid and a chlorine addend appended to the carbon cage (F-828) on serum-starving human embryo lung diploid fibroblasts (HELFs) was studied. Serum deprivation evokes oxidative stress in HELFs. Cultivation of serum-starving HELFs in the presence of 0.1-1 µM F-828 significantly decreases the level of free radicals, inhibits autophagy, and represses expression of NOX4 and NRF2 proteins. The activity of NF-κB substantially grows up in contrast to the suppressed NRF2 activity. In the presence of 0.2-0.25 µM F-828, the DSB rate and apoptosis level dramatically decrease. The maximum increase of proliferative activity of the HELFs and maximum activity of NF-κB are observed at these concentration values. Conclusion. Under the conditions of oxidative stress evoked by serum deprivation the water-soluble fullerene derivative F-828 used in concentrations of 0.1 to 1 µM strongly stimulates the NF-κB activity and represses the NRF2 activity in HELFs.

Toxic and DNA damaging effects of a functionalized fullerene in human embryonic lung fibroblasts.

Water-soluble fullerenes have been studied as potential nanovectors and therapeutic agents, but their possible toxicity is of concern. We have studied the effects of F-828, a soluble fullerene [C60] derivative, on diploid human embryonic lung fibroblasts (HELFs) in vitro. F-828 causes complex time-dependent changes in ROS levels. Inhibition of Nox4 activity by plumbagin blocks F-828-dependent ROS elevation. F-828 induces DNA breaks, as measured by the comet assay and γH2AX expression, and the activities of the transcription factors NF-kB and p53 increase. F-828 concentrations>25µM are cytotoxic; cell death occurs by necrosis. Expression levels of TGF-ß, RHOA, RHOC, ROCK1, and SMAD2 increase following exposure to F-828. Our results raise the possibility that fullerene F-828 may induce pulmonary fibrosis in vivo.