Homozygous-Recessive Characteristics as a Biomarker of Predisposition for COVID-19.

Coronavirus disease (COVID-19), a new form of severe acute respiratory syndrome, has caused a global pandemic. The aim of this study was to analyze homozygous-recessive characteristics (HRC) in the group of COVID-19 patients, considering their gender, forms of the disease (mild and severe symptoms), risk factors: hypertension, diabetes mellitus type 2, hyperlipidemia, smoking habits, and the distribution of ABO blood group. Using the HRC test, we analyzed 20 HRCs in a sample of 321 individuals: 205 patients and 116 controls. The average HRC in patients was significantly higher than controls, as well as in patients with severe symptoms compared to patients with mild symptoms. The patients with higher HRC (cut-off ≤5.5) experienced a significantly increased risk of disease of 2.3 times (OR = 2.315, p < .0005). Our results indicate that the HRC test could be used as a screening in recognizing predisposition for COVID-19.

Morphogenetic Variability as Potential Biomarker of Neurogenic Lesion Degree in Children with Spina Bifida.

AIMS: In this study we analyzed the degree of genetic homozygosity among spina bifida patients with different degrees of neurogenic lesion (N = 82), as well as their clinical and neurological characteristics, compared to healthy control individuals (N = 100). METHODS: According to clinical and electromyographic findings, we separately assessed the type of neurogenic lesion (paresis or paralysis). Regarding the degree of neurogenic lesion, patients were classified into three groups: mild, moderate and severe. We analyzed six muscles. For assessing the degree of individual genetic homozygosity, we tested the presence and distribution of 15 homozygous recessive characteristics (HRC). RESULTS: The predominant type of neurogenic lesion was paresis. Every third evaluated muscle was affected in the group with mild neurogenic lesion, while more than half were affected in the group with severe neurogenic lesion. The average values of HRCs among different groups of patients and the control showed the population-genetic differences that exist among them (control HRC/15=3.0±0.2; mild HRC/15=3.6±0.2; moderate HRC/15=4.8±0.3; severe neurogenic lesion HRC/15=5.0±0.3). CONCLUSIONS: Spina bifida patients have a significant increase of recessive homozygosity and a decreased variability compared to the control group. As neurogenic lesions are more severe, more affected muscles are present, as well as the increase of individual recessive homozygosity.

Polyphenolic contents of Teucrium polium L. and Teucrium scordium L. associated with their protective effects against MMC-induced chromosomal damage in cultured human peripheral blood lymphocytes.

Teucrium species have been used in traditional medicine for treatment of different diseases. The aim of this study was to investigate polyphenolic contents by high-performance liquid chromatography (HPLC), and the genotoxic effect of methanolic extracts of Teucrium polium and Teucrium scordium using the cytokinesis-block micronucleus (CBMN) assay on human peripheral blood lymphocytes (PBLs) from healthy donors. The HPLC  analysis  showed that extracts consist of phenolic acid (gallic, vanillic, caefic, chlorogenic, p-coumaric, sinapic) and flavonoids (catechin, rutin, myricetin, luteolin, quercetin and apigenin). Cultures were treated with extracts of both plants separately and in combinations with mitomycin C (MMC). In separate treatments, both herbal extracts significantly induced micronucleus (MN) frequency only at the highest concentrations. All concentrations of T. scordium , except the lowest, and all concentrations of T. polium extracts in combined treatment with MMC significantly reduced the frequency of MN. The extract of T. polium did not significantly aefct the nuclear division index (NDI), whereas T. scordium in higher concentrations, separately and in combined treatment with MMC, significantly decreased the NDI value. Our results suggest that both herbal extracts in combination with MMC have antimutagenic (T. polium) and proapoptotic effects (T. scordium), which indicates their protective effects in PBLs.

High micronucleus frequency in peripheral blood lymphocytes of untreated cancer patients irrespective of gender, smoking and cancer sites.

Chromosomal instability could be one of primary causes for malignant cell transformation. The objective of the present study was to evaluate the spontaneous genetic damages in circulated lymphocytes of newly diagnosed cancer patients by using cytokinesis-block micronucleus (CBMN) assay, with respect to the factors that might affect micronucleus frequency (i.e. age, gender, smoking habits and cancer sites). Micronuclei (MN) are small nuclei that are originated from chromosome fragments or whole chromosomes. The analyzed samples included 44 untreated cancer patients (19 females and 25 males with mean age of 60.89 years) with different cancer sites (12 patients with breast cancer, 5 with uterine cancer and 27 with cancer of pharynx). Control group included 40 healthy donors (28 females and 12 males with mean age of 43.95 years). The mean baseline MN frequency was significantly higher (p < 0.001) in cancer patients (15.18 +/- 5.05 MN/1000 BN cells ranging from 4 to 27) than the baseline frequency in healthy controls (6.45 +/- 2.75 MN/1000 BN cells, ranging from 1 to 11). There was no gender difference in baseline MN frequency in cancer patients and healthy controls. Moreover, the MN frequency did not significantly differ among cancer sites, and between smokers and non-smokers in both patient and control samples. In conclusion, untreated cancer patients may be associated with an increase of chromosomal instability in peripheral blood lymphocytes, irrespective of gender, cigarette smoking and cancer sites.

Fifty years of Balkan endemic nephropathy: challenges of study using epidemiological method.

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial disease associated with urothelial cancer, which affects people living in the alluvial plains along the tributaries of the Danube River. Challenges of studying BEN using the epidemiological method are multiple. The natural history from exposure to occurrence of the disease may take many years. The early stages of BEN are not easily detectable clinically, as the disease is asymptomatic until a significant decline in function occurs, and even then symptoms are usually non-specific. The natural history of BEN is complex, possibly with multiple risk factors operating both at the stage of initiation of renal damage and in its progression. In BEN, genetic susceptibility is due to multiple genes of small effects, gene-gene interactions, and gene-environment interactions of complex nature that are difficult to assess with current study designs. BEN is now kidney disease of the old people, and many risk factors for disease such as smoking, alcohol consumption, obesity, and diabetes could contribute to the kidney damage. Evidence is presented that environmental rather than genetic factors play a decisive role in the etiopathogenesis of BEN. Aristolochic acid, described as a culprit of BEN in 1959, is confirmed in 2007 by the molecular biology methods. Mycotoxins and polycyclic aromatic hydrocarbons, leached from lignites and found in the vicinity of endemic settlements, deserve further investigation. Despite advances in understanding the epidemiology of BEN, more research is needed on the patterns of BEN over time and between places, and on identifying the contributions of modifiable risk factors in initiating and hastening progression of BEN in order to improve the scope for preventing BEN. Primary prevention is still at the beginning. Knowledge accumulated in the fifty years of BEN research and new data about prevention and treatment of chronic kidney disease reveal several effective methods in secondary and tertiary prevention of BEN. Genetic epidemiology could establish the relative size of the genetic effect in relation to other sources of variation in disease risk (i.e., environmental effects such as intrauterine environment, physical and chemical effects, as well as behavioral and social aspects). Public health authorities in the several countries having aristolochic acid nephropathy should take immediate measures for reducing dietary exposure of residents to Aristolochia.

Morphogenetic variability during selection of elite water polo players.

In this population-genetic study, we compared morphological and genetic variability of a control group of individuals with that of developing and elite water polo players from Serbia, using a test of determination of homozygously recessive characteristics in humans (HRC-test). Comparisons of the frequencies of ABO and Rh blood types were also made for the groups examined. The degree of genetic homozygosity showed not only statistically significant differences in the mean values obtained for the studied samples (control group 5.1 +/- 0.2; emerging water polo players 3.5 +/- 0.1; elite water polo players 1.9 +/- 0.3 HRCs, out of 20 analysed characteristics), but also differences in the type of distribution, as well as the presence of specific combinations of such traits. During identification of swimming talents, the degree of genetic homozygosity showed a significant decrease, from 4.8 +/- 0.3 in the group of pre-competitive young boys, to 2.7 +/- 0.3 in selected adult team members, to only 1.9 +/- 0.3 in elite water polo players. In the group of pre-competitive young individuals, all characteristics tested also had homo-recessive combinations, while in the group of elite water polo players only 8 of 20 traits were expressed as homozygously recessive. In the group of elite water polo players, who were World and European champions as well as Olympic champions, the frequencies of the A and B blood types were low, AB was absent, and the frequency of the O blood type was high at 72.2% (i.e., almost twice the expected percentage).

Treatment of pregnant women with a betamimetic and verapamil increases the micronuclei frequency in umbilical cord blood lymphocytes.

In prevention of preterm labor, betamimetics are used in gynecological practice mostly combined with antiarrhythmic verapamil because of their therapeutic cardiovascular side effects. The aim of this study was to investigate the influence of a betamimetic (ritodrine hydrochloride, fenoterol or hexoprenaline) and verapamil (administered to mothers) on the frequency of micronuclei (MN) in umbilical cord blood lymphocytes of neonates, using cytokinesis-block micronucleus test. The analyzed sample included 23 babies whose mothers received the therapy and 30 control babies whose mothers received no therapy. The average MN frequency was significantly higher in the neonates whose mothers received the therapy (8.13 +/- 2.69 MN/1000 BN cells), in comparison with the baseline frequency in untreated controls (3.30 +/- 2.63 MN/1000 BN cells), with probability p < 0.05. The highest MN frequency was found in neonates whose mothers received fenoterol and verapamil (2.8-fold i.e. 9.10 +/- 3.00 MN/1000 BN cells), while ritodrine hydrochloride and hexoprenaline combined with verapamil induced 2.3-fold and 2.2-fold higher MN values than in controls (7.50 +/- 3.33 MN/1000 BN cells and 7.29 +/- 0.95 MN/1000 BN cells). Multiple linear regression analysis showed that MN frequency was affected only by the maternal therapeutic treatment, while the neonates' sex, maternal age, cigarette smoking, and therapeutic doses did not affect the MN frequency in umbilical lymphocytes of neonates. We conclude that the treatment of pregnant women with a betamimetic and verapamil significantly increases the MN frequency in umbilical cord blood lymphocytes of neonates, regardless to therapeutic doses.

Micronuclei in cord blood lymphocytes as a biomarker of transplacental exposure to environmental pollutants.

Exposure to environmental pollutants can result in chromosomal instability, which can produce a wide variety of effects on human health. In the spring of 1999, extensive environmental pollution happened in Kragujevac (the city in the central Serbia) with damages of soil, water and air, caused by the air strikes on "Zastava" complex. Because we found significant increase of micronuclei in newborns born 12 months after this environmental pollution (in the beginning of 2000), the purpose of the present study was to follow the frequency of micronuclei in lymphocytes of newborns born seven years after pollution (in 2006). The frequencies of micronuclei were estimated in cord blood lymphocytes of 41 newborns (20 males and 21 females) by application of cytokinesis-block (CB) micronucleus test. The obtained results showed that the mean value of micronuclei was significantly decreased in newborns born in 2006 in comparison to the mean value of micronuclei in newborns born 12 months after contamination (4.73 +/- 3.38 micronuclei/1,000 CB cells vs 9.36 +/- 5.60 micronuclei/1,000 CB cells), with probability p < 0.001. Newborn's gender, mother's age (19-40 years) and maternal cigarette smoking (< 20 cigarettes per day) did not show any noticeable effects on micronuclei frequencies in the analyzed newborns. There was relationship between the micronuclei frequencies and the level of environmental pollution (F = 6.95, p = 0.000). Our results suggest that the environment profoundly influences genetic constitution of newborns, and that micronucleus assay in cord blood lymphocytes is an important method for evaluation of transplacental mutagens.

Allozyme polymorphism of Mdh and alpha-Gpdh in Ixodes ricinus populations: comparison of borreliae-infected and uninfected ticks.

Ixodes ricinus Linnaeus (Acari: Ixodidae) ticks are vectors of numerous infectious diseases in humans and animals. The allozyme variability of MDH and alpha-Gpdh was detected by native polyacrylamide gel electrophoresis in I. ricinus natural populations in three localities in Serbia. Four alleles of Mdh locus (MDH 1, MDH 2, MDH 3 and MDH X) and four alleles of alpha-Gpdh locus (VS, S, F and VF) were detected. Interpopulation differences in Mdh and alpha-Gpdh allele frequencies were statistically insignificant. Significant difference in alpha-Gpdh allele frequencies between males and females was recorded in the largest sample only. Differences in allele frequencies, detected between borreliae-infected and uninfected I. ricinus ticks, were close to the level of statistical significance, especially for alpha-Gpdh locus. Clear significant difference appeared in females when sexes were tested separatelly (P = 0.037). It is interesting that genotypes containing rarer alleles (MDH 1 and S) were infected in higher proportion in comparison to other genotypes. Our results point towards a possible role of Mdh and alpha-Gpdh loci in I. ricinus ticks in the determination of energy requirements for host seeking. Sex differences in alpha-Gpdh allele frequencies suggest that selective pressure, concerning efficiency of reserve materials utilisation, points to alpha-Gpdh rather than to Mdh locus.

Monitoring of lymphocyte micronuclei among newborns from Kragujevac in Central Serbia before and after environmental contamination.

The air strikes on "Zastava" complex in Kragujevac, in the spring of 1999, caused extensive environmental pollution with damage to soil, water and air. Since the main problem was the leakage of several tones of polychlorinated biphenyls (PCBs) as well as heavy metals Cr and Ni into the environment and groundwater, we decided to evaluate influence of the environmental contamination on eventual changes of genetic constitution of human body cells. The subjects analyzed were 36 phenotypically healthy newborn babies, who were born 12 months (n=22) and 18 months (n=14) after environmental contamination, and 25 newborns in 1998 as a control group. For the assessment of mutagenic effects of environmental pollutants in vivo, the cytokinesis-block micronucleus test was employed. The results show significant increase of micronuclei (MN) in lymphocytes from newborns born 12 months after contamination (9.36 +/- 5.60), in comparison to controls (5.53 +/- 3.02) and newborns born 18 months after contamination (6.14 +/- 3.57). Only 3 newborns (12%) out of 25 controls showed more than 1 MN/1000 binucleated cells in respect to 8/22 after 12 months (36%) and 2/14 after 18 months from contamination (14%). The mean number of binucleated cells with MN significantly varied in function of sex and environmental changes in newborns born 12 months after contamination (as demonstrated by the analysis of covariance F=9.61, p <0.003). After 18 months of contamination, environmental components had no noticeable effects on MN frequency (F=0.5, p >0.48). These results suggest that the exposure to environmental pollutants in utero affects genetic constitution of fetus and increases MN values in their body cells, which is detectable right after birth in peripheral blood lymphocytes.

Effect of various doses of gestogens on micronuclei frequency in human peripheral blood lymphocytes of pregnant women.

BACKGROUND: Since gestogens, in the form of hormonal substitution therapy, have been proposed to have a role in the prevention of threatened spontaneous abortions during the first three months of pregnancy, we decided to evaluate possible genotoxic effects of these preparations. METHODS: A total of 30 pregnant women, with a diagnosis of threatened spontaneous abortions, received the gestogen therapy in the first 3 months, and a sample of 30 pregnant women without indication for hormonal therapy were included as the control group. For investigation of mutagenic effects of gestogens in vivo the cytokinesis block (CB) micronucleus (MN) test was applied. RESULTS: Average MN frequency in the control group was 6.79 +/- 0.69 MN/1000 cells. The second analysed group included 12 patients with threatened spontaneous abortions, who received gestogen therapy in doses of 100-750 mg. Average MN frequency in these patients before therapy was 11.83 +/- 1.33 MN/1000 cells, and after therapy it was 16.50 +/- 1.32 MN/1000 cells (P < 0.001). The third analysed group comprised nine patients, who received gestogen therapy in doses of 750-2000 mg. Average MN frequency in these patients before therapy was 15.67 +/- 3.00 MN/1000 cells, and after therapy was 23.89 +/- 2.49 MN/1000 cells (P < 0.001). The fourth analysed sample comprised nine patients, treated with gestogen doses of 2000-8400 mg. The average MN frequency in these patients before therapy was 11.89 +/- 1.63 MN/1000 cells, and after therapy was 21.22 +/- 2.80 MN/1000 cells (P < 0.001). CONCLUSIONS: The increase of therapeutic gestogen doses was followed by an increase of average MN frequency. The greatest rise of MN frequency (1.8-fold) was observed in the group of patients who were treated with the highest gestogen doses (2000-8400 mg). The smallest increase (1.4-fold) of MN frequency was found in the group of patients whose therapeutic doses were the lowest (100-750 mg).

Viabilities of originally natural O-chromosomal inversion homo- and heterokaryotypes in Drosophila subobscura.

The influence of epistatic interactions of lethal and non-lethal genes upon viability of Drosophila inversion karyotypes is poorly known. In this paper we present comparative results for viabilities of 21 originally natural O-inversion homo- and 38 heterokaryotypes in a D. subobscura population. We observed strong heterotic effect in viability of O-lethal heterozygotes irrespective of different inversion backgrounds, which indicates a mechanism for protection of a great number of lethal genes. In O-non-lethal heterozygotes the heterotic effect in viability was absent irrespective of different inversion backgrounds. Our results showed a great extent of genetic load and high abundance of O-chromosomal arrangements in the population analyzed. It belongs to the set of central European populations. An epistasis of lethal genes present in O-inversion hetero- and not present in O-inversion homokaryotypes of moderate or low frequencies could be good example for co-adaptation of chromosomal inversions with regard to the genetic load. This represents a more efficient mechanism for limitation of genetic load than alternative mechanisms for protection of lethals. Except for lethal genes, possible epistatic interactions of mildly deleterious (subvital) genes, could also be responsible for limiting the extent of genetic load in natural D. subobscura populations. We demonstrated a non-random distribution of several combinations of viability classes among different O-inversion homo- and heterokaryotypes. As a consequence of that, the viabilities of the O-inversion homokaryotypes compared to heterokaryotypes were significantly higher at low frequencies than in moderate or high frequencies. This shows frequency-dependence as a mechanism of balancing selection for protection of O-chromosomal inversions in natural D. subobscura populations. In addition, the viabilities of the O-inversion homokaryotypes of lower frequency, compared to homokaryotypes of moderate or high frequency, were significantly higher. This again indicates the existence of supergene selection as another mechanism for protection of chromosomal inversions, as co-adapted complexes in natural D. subobscura populations.