[The relationship between the relative length of leukocyte telomeres and mtDNA copy number and acute coronary syndrome in a 15-year follow-up.]

OBJECTIVE: to study the association of relative leukocyte DNA telomere length with death from natural causes during a 15-year follow-up in a middle-aged and elderly Siberian population. Study of the association of the relative length of leukocyte telomeres (LTL) with fatal outcomes during a 15-year follow-up of a random population sample formed in 2003-2005 (n=9 360, 45-69 years old, Novosibirsk, HAPIEE project). The main group included the persons died from natural causes (except external) without a previous history of CVD and cancer (n=609); controls were stratified by sex and age (n=799). The analysis of relative LTL at baseline was performed using quantitative real-time PCR. We estimated the odds ratio of all-cause death per 1 decile shortening of LTD as a continuous variable in a multivariable-adjusted logistic regression. The carriers of shorter telomere carriers had an increased risk of death from natural causes over the next 15 years (OR=1,37, 95% CI 1,31-1,44) per decile of LTL decrease, regardless of other factors. The risk coefficients were similar for death from CVD (1,39), cancer (1,42), and other non-external causes (1,51). In studied middle-aged and elderly Siberian (Caucasoid) population cohort the LTL was an independent inverse predictor of the 15-year risk of death from natural causes.

[The relationship between the relative length of leukocyte telomeres and mtDNA copy number and acute coronary syndrome in a 15-year follow-up.]

We studied the relationship between the leucocyte telomere length (LTL) and the copy number of mitochondrial DNA (CNmtDNA) and the development of acute coronary syndrome during 15 years of follow-up. A random population sample was examined at baseline in 2003-2005 (n=9 360, men and women 45-69 years old, Novosibirsk, the HAPIEE project) and followed-up for 15 years. In the frame of nested case-control design, we selected cases - incident myocardial infarction/acute coronary syndrome (MI/ACS) among those free from baseline CVD (n=256) and sex- and age-stratified control among those free from baseline CVD and cancer and alive by the end of follow-up (n=799). The relative LTL and CNmtDNA were assessed using quantitative real-time PCR. Results. The carriers of shorter telomeres had increased 15-year risk of MI/ACS with adjusted OR=1,87 (95% CI 1,70-2,06) per 1 LTL decile independent of other factors. Fewer CNmtDNA was associated with increased risk of MI/ACS with adjusted OR=1,19 (95% CI 1,12-1,26) per 1 CNmtDNA decile. The identified associations were confirmed in tertile analysis and in stepwise analysis with continuous variables of both biomarkers. All associations persisted after adjusting for gender, age, and traditional CVD risk factors. Conclusion. The LTL and CNmtDNA were independent predictors of the 15-year risk of MI/ACS in the middle- and elderly Siberian (Caucasoid) population cohort. These findings highlight the need for further research to elucidate the mechanisms by which LTL and mtDNA copy number may affect human health.

Development of a Purified Viral Preparation for Studies of COVID-19 (SARS-CoV-2) Biology.

Different methods for producing bulk quantities of concentrated and purified SARS-CoV-2 for the use as antigens and for the research into COVID-19 biology were tested.

[Mitochondrial DNA copy number of leucocytes as ageing marker and risk factors for age-related diseases in human.]

We used quantitative real-time PCR method to analyse mtDNA copy number in a random subsample (n=996; 358 men aged 66,31±7,24 years; 468 women aged 67,62±7,1 years) selected from a population cohort (n=9 630) examined at baseline in international project HAPIEE in Novosibirsk, Russia, in 2003-2005. The participants were re-examined after 12 years in 2015-2017. The average relative number of mtDNA copies in peripheral blood leukocytes was greater in women than in men, independently of age and smoking (p=0,001). mtDNA copy number was inversely correlated with age both in men (p=0,005) and women (p<0,001). In age adjusted analysis, mtDNA copy number was inversely associated with waist, hip and heart rate in both sexes. In addition, mtDNA copy number in women was inversely associated with triglycerides and glucose, aterogenity index and positively with HDL cholesterol. In men, mtDNA copy number was positively associated with physical activity. The age-adjusted mean of mtDNA copy number among male never-smokers was greater than in smokers (p=0,003), and the mean mtDNA copy number was lower in women with diabetes than in women without diabetes (p=0,005). In both sexes, subjects with baseline history of hypertension had lower mtDNA copy number after 12-year follow-up than those without hypertension (p=0,05). This broadly supports the hypothesis that mtDNA copy number may act as biomarker of ageing.

Association of Level of Proprotein Convertase Subtilisin/Kexin Type 9 with Intima-Media Thickness in Patients with Familial Hypercholesterolemia.

We studied association of PCSK9 protein with the carotid artery intima-media thickness in patients with familial hypercholesterolemia (N=53; age 49.9±6.9 years) treated with statins. Blood level of PCSK9 protein was measured by ELISA; ultrasonography of the carotid arteries with measurement of the thickness of the intima-media complex of the common carotid arteries in the distal segment for 10 mm from the bifurcation on the far wall of the vessel was performed in on-line mode. The mean values were calculated for both sides, the maximum mean value was included in the analysis. It was shown that PCSK9 levels positively correlate with carotid artery intima-media thickness in patients with familial hypercholesterolemia.

[Frequency, spectrum, and functional significance of TP53 mutations in patients with diffuse large B-cell lymphoma].

A comparative analysis of oncogene mutations shows that variations in their frequency, spectrum, and hot-spot locations depends on the type of tumor and the ethnic origin of the population studied. The current version of the IARC TP53 Mutation Database lacks information about the frequency and spectrum of TP53 mutations in patients with DLBCL in Russia. The aim of this study was to assess the frequency and functional significance of TP53 mutations in patients with DLBCL in Novosibirsk. The TP53 coding sequence and the adjacent intron regions were analyzed by direct sequencing in the tumor material from 74 patients with DLBCL. Mutations of the TP53 coding sequence were found in 18 (24.3%) patients. These data are consistent with the frequency of TP53  mutations observed in other studies. The spectrum of nucleotide substitutions found in DLBCL specimens corresponded to that described in the IARC TP53 Mutation Database. According to bioinformatic data and to reported experiments in vitro, most of the mutations detected result in the production of functionally inactive p53. Our results show that DLBCL progression is accompanied by the functional selection for mutations in TP53 exons 5-8.

Haplotype analysis of the HFE gene among populations of Northern Eurasia, in patients with metabolic disorders or stomach cancer, and in long-lived people.

BACKGROUND: Previously, it was shown that the HFE gene (associated with human hereditary hemochromatosis) has several haplotypes of intronic polymorphisms. Some haplotype frequencies are race specific and hence can be used in phylogenetic analysis. We assumed that analysis of Caucasoid patients-living now in Western Siberia and having diseases associated with dietary habits and metabolic rate-will allow us to understand the processes of possible selection during settling of the northern part of Asia. RESULTS: Haplotype analysis of Northern Eurasian native and recently settled ethnic groups was performed on polymorphisms rs1799945, rs1800730, rs1800562, rs2071303, rs1800708, rs1572982, rs2794719, rs807209, and rs2032451 of this gene. The CCA haplotype of the rs2071303, rs1800708, and rs1572982 was found to be associated with HLA-A2 (39 %) in Asian populations. Haplotype analysis for the rs1799945, rs1800730, rs1800562, rs2071303, rs1800708, and rs1572982 was performed on Russian patients with some metabolic disorders or stomach cancer and among long-lived people. Decreased frequencies of the TTA haplotype (T in rs2071303, T in rs1800708, and A in rs1572982) were observed in the groups of patients with diseases associated with overweight (fatty liver disease, type 2 diabetes mellitus, or metabolic syndrome + arterial hypertension) as compared with the control sample. We detected significant differences in this haplotype's frequency between the patients with type 2 diabetes mellitus and Russian adolescents, elderly citizens, and long-lived people (χ(2) P value = 0.003, 0.010, and 0.015, respectively). CONCLUSIONS: No significant differences in frequencies of the alleles with mutations in coding regions of the HFE gene (C282Y, H63D, and S65C) were detected between the analyzed patients (with stomach cancer, metabolic syndrome, fatty liver disease, or type 2 diabetes mellitus) and the control Caucasoid sample. Monophyletic origin of H63D (rs1799945) was confirmed in Caucasoids and Northern Asians. The reasons for a sharp increase in the frequency of CCA haplotype of HFE in the Asian race remain unclear.

[Gene Polymorphism of the c-fms, ITGB3,CCR2, and DBH genes in the populations of old believers of the Tyumen oblast and Russian residents of Novosibirsk].

Old Believers of the Tyumen oblast have been studied compared with a control sample of Russian residents of the city of Novosibirsk. The former are a unique subpopulation, which has been relatively isolated from the rest of Russians in central and northern regions of Russia due to religious reasons since the middle of the 17th century. Polymorphisms in the genes for glycoprotein ITGB3, dopamine-ß-hydroxylase (DBH), and chemokine receptor CCR2 and two mutations in the c-fms gene have been analyzed. The populations are only similar in the c-fms indel. The frequencies of the rare alleles of CCR2, ITGB3, and 3'UTR of c-fms in the Old Believers are lower than in the sample of Novosibirsk Russians, and the rare allele of DBH is more frequent. A significant negative correlation is observed between DBH and CCR2 (r =-0.88; df = 4; P < 0.023). Apparently, these differences are related to the long-term isolation of Old Believers. This assumption is consistent with the fact that the levels of heterozygosity for most loci in Old Believers are lower than in Novosibirsk Russians.

[The Dynamics of the Composition of mtDNA Haplotypes of the Ancient Population of the Altai Mountains from the Early Bronze Age (3rd Millennium BC) to the Iron Age (2nd-1st Centuries BC)].

The mtDNA polymorphism in representatives of various archaeological cultures of the Developed Bronze Age, Early Scythian, and Hunnish-Sarmatian periods was analyzed (N = 34). It detected the dominance of Western-Eurasian haplotypes (70.6%) in mtDNA samples from the representatives of the ancient population of the Early Bronze Age--Iron Age on the territory of Altai Mountains. Since the 8th to the 7th centuries BC, a sharp increase was revealed in the Eastern-Eurasian haplogroups A, D, C, andZ (43.75%) as compared to previous cultures (16.7%). The presence of haplotype 223-242-290-319 of haplogroup A8 in Dolgans, Itelmens, Evens, Koryaks, and Yakuts indicates the possible long-term presence of its carriers in areas inhabited by these populations. The prevalence of Western-Eurasian haplotypes is observed not only in the Altai Mountains but also in Central Asia (Kazakhstan) and the South of the Krasnoyarsk Krai. All of the three studied samples from the Western-Eurasian haplogroups were revealed to contain U, H, T, and HV. The ubiquitous presence of haplotypes of haplogroup H and some haplogroups of cluster U (U5al, U4, U2e, and K) in the vast territory from the Yenisei River basin to the Atlantic Ocean may indicate the direction of human settlement, which most likely occurred in the Paleolithic Period from Central Asia.

[Association of decreased external pulmonary function with cognitive impairments].

AIM: To examine the association between the basic indicators of external respiratory function (ERF) and cognitive impairments (CIs). MATERIALS AND METHODS: The materials of a population-based cross-sectional study were used in the investigation; a representative sample consisted of 3818 men and women aged 45-69 years. Forced expiratory volume in the first second (FEV1), the ratio of FEV1 to forced vital capacity (FVC) were determined to assess ERF. CIs were evaluated using the 10-word test described by A.R. Luria, Cog-sum indices (the sum of correctly reproduced words with 3 attempts), and Cog-mean=Cog-sum/3. RESULTS: In patients with an FEV1/FVC <70%, the probability of a Cog-sum of <20 scores was twice higher than in those with an FEV1/FVC ≥70%; in patients with an FEV1 <80%, the probability of a Cog-sum of <20 scores was also 2 times higher than in those with an FEV1 ≥80%. Multiple regression analysis revealed no significant associations between Cog-sum index and smoking index, body mass index, and blood pressure. Cog-sum was inversely related to age and directly related to FEV1 and female sex. Binary logistic regression analysis showed that gender, age, and FEV1 were statistically significant variables. The relative risk of a Cog-sum of <20 that was 1.7 times higher in the men than in the women, 1.8 times higher in the patients with an FEV1 <80% than in those with an FEV1 ≥80% (model 1), 1.7 times higher in the patients with an FEV1/FVC <70% than in those with FEV1/FVC ≥70% (model 2) increased with age. CONCLUSION: The decrease in the basic indicators of ERF (FEV1, FEV1/FVC) is an independent predictor of CIs.

[Length telomere leukocytes as ageing markers and risk factors for age-related diseases in humans].

The purpose of the research was studying of leukocyte telomere length association with age, sex, risk factors for age-related diseases in Russian people of pre-retirement and retirement age. By quantitative real-time PCR method we studied the leukocyte telomere length in 398 men (56,3±7,2 years) and 365 women (56,6±7,1 years) selected from a population sample of 45-69 year-old residents of the Oktyabrsky and Kirovsky districts of Novosibirsk (9 400 people). The selection was formed in the course of work on the international project HAPIEE. As a result, an inverse correlation of telomere length with age (r=-0,159, р<0,001), with the ratio waist / hips (r=-0,107, p=0,003) was found out. The average length of telomeres in women significantly more than in men, p=0,031.The correlation of telomere length in males with weight (r=0,140, p=0,005), waist size (r=0,111, p=0,027) was found out. In women, there is an inverse correlation of telomere length with a waist size (r=-0,127, p=0,015), the ratio of waist / hips (r=-0,141, p=0,007). The length of telomeres is an inverse correlation with correlation with quantity of the cigarettes smoked (r=-0,121, р=0,024). The length of telomeres leukocytes correlates with age, smoking, and a number of phenotypical signs. In men with the family anamnesis burdened by malignancies leucocytes telomere length was found to be greater than in men without such anamnesis.

[Association of Some Homozygous Genotypes of Genes Regulating Inflammation, Destruction and Angiogenesis With Laboratory Markers of Atherosclerosis Course in Men With Stable Effort Angina].

Great number of factors stimulating or inhibiting production of proteins in inflammatory process influence serum levels of markers of inflammation. A number of homozygous genotypes of inflammation, destruction, and angiogenesis genes have been found to be associated with basic clinical-laboratory indices of inflammation and atherosclerotic process. The revealed genetic markers can be used as complimentary markers of prognosis of the disease course.

[MODERN METHODS OF PHYSICAL-CHEMICAL RESEARCH IN GASTROENTEROLOGY PRACTICE: THE EXPERIENCE OF INTERACTION].

UNLABELLED: The aim of this work was to assess the potential of some physical and chemical methods for studying erythrocytes and blood serum in gastroenterological practice by the example of colorectal cancer (CC). MATERIALS AND METHODS: A total of 26 persons with various stages of colorectal cancer and 16 healthy (control group) were examined. Parameters of erythrocytes and blood serum were investigated by light microscopy, dielectrophoresis in a non-uniform alternating electric field (DEF in NUAEF), terahertz spectroscopy, ellipsometry, Raman-spectroscopy. RESULTS: Polymorphism of erythrocytes, rigidity, viscosity, indexes of aggregation and destruction were significantly higher in patients with CC and polarizability, amplitude of erythrocyte deformation in NUAEF being lower than those in the controls. The study of erythrocytes by terahertz spectroscopy revealed the low levels of amplitude transmittance over the whole frequency range in CC patients compared to the controls. The increasing of refractive index, degree of heterogeneity of thin films obtained from the serum in CC patients were observed in considering the ellipsometric parameters. We found a significant increasing of the concentration of antigens to CD24 at the early stage of the disease. The areas of some peaks in Raman spectra were significantly lower in patients with CC compared to the healthy ones, it is possible due to a carotin deficiency. Most of the studied parameters were correlated with the stage of the disease. A set of optical methods for studying blood serum compared with those of histology and radiological methods of diagnosis showed their high sensitivity and specificity, positive and negative predictive value (80 % and above). CONCLUSION: The obtained results of the pilot study demonstrate the prospects of using physical and chemical methods of research of erythrocytes and blood serum for early diagnosis, stage of disease and monitoring the effectiveness of treatment of CC.

[Linkage disequilibrium and haplotypes of rs1042522, rs1625895 and rs17878362 gene TP53 markers in patients with diffuse large B-cell lymphoma].

Association of rs1042522, rs17878362 and rs1625895 markers with non-Hodgkin's lymphoma risk is not well studied. Large number of non-Hodgkin's lymphoma entities, as well as a small effect of each of the polymorphisms on the lymphomas risk, leads to the analyses of these markers in each of histological subtypes of lymphoma and to study the polymorphisms in the haplotype groups. The goal of this work was to analyze the frequency, haplotypes and linkage disequilibrium of rs1042522, rs1625895 and rs17878362 in the patients with diffuse large B-cell lymphoma and in control group. The differences in the structure of LD between rs17878362, rs1042522 and rs1625895 TP53 gene in the population of the Siberian region were shown. Haplotype approach wasmore informative in the analyses of association of the gene TP53 polymorphisms and the diffuse large B-cell lymphomas risk in case-control study than the study of each polymorphism. The association of haplotype wArgG with diffuse large B-cell lymphoma risk, and the protective effect of haplotypes wProG or dupProG were identified. The difference in the effects of the haplotype TP53 was noted depending on the homozygous or heterozygous diplotype.

[Genetic predictors of myocardial infarction in subjects of young age].

AIM OF THE STUDY: to investigate associations of single nucleotide polymorphisms (SNP) rs499818 (6p24.1), rs619203 of ROS1 gene (6q22), rs10757278 rs1333049 (9p21.3), rs2549513 (16q23.1), rs4804611 of ZNF627 gene (19p13.2) with myocardial infarction in subjects of young age. The group of patients with MI (n=99) aged less than 45 years and the control group (n=111) did not differ significantly by sex (=0,617), age (=0.291), arterial hypertension (=0.766), diabetes mellitus (=0.395), hypercholestolemia (=0.696), excessive body mass and obesity (=0.361), abdominal obesity (=0.831) and history of smoking (=0.400). There was significant difference between groups by burdened heredity (<0.001). Genomic DNA was obtained from venous blood by phenol-chloroform extraction. Genetic testing was performed by real time polymerase chain reaction using 7900HT Fast Real-Time PCR System according to manufacturers protocol. We found significant association between rs1333049 and rs10757278 and myocardial infarction (MI). Odds ratio (OR) of development of MI in carriers of risk allele rs1333049 was 2.4 (95% confidence interval [CI] 1.24 to 4.65), in carriers of G rs10757278 allele - 2.00 (95%CI 1.05 to 3.80). Association of risk alleles rs 1333049 and G rs10757278 with MI remained significant after adjustment for burdened family history (OR 4.25, 95%CI 1.39 to 12.99, and OR 3.04, 95%CI 1.09 to 8.52, respectively). Presence in the genotype of both risk alleles rs1333049 and G rs10757278 was associated with OR of MI development 2.40 (95%CI 1.20 to 4.82) which was not different from that associated with carriage of allele rs1333049 only. Possibly in our population both SNPs belong to one linkage block and correspondingly it is sufficient to genotype one SNP. No significant associations with MI were found for variants rs4804611, rs2549513, rs499818, rs619203. SNPs rs1333049 and rs10757278 of 9p21.3 locus are predictors of MI in young individuals not dependent on both traditional risk factors and presence of burdened family history.

[Intronic polymorphisms of аntionkogene TP53 in patients with indolent variants of Non-Hodgkin's lymphomas].

The frequency of distribution of alleles and genotypes of single nucleotide substitution G13494A in intron 6 and duplications dup16bp in intron 3 of the gene TP53 in 56 patients with indolent non-Hodgkin's lymphoma and variants in the controls was studied. The increase of the frequency of G-allele and G/G genotype of intron 6 of the gene TP53 in lymphoma patients compared with controls (91 and 84 % vs. 79 (p<0,01) and 63 % (p<0,01), respectively) was determined. It was found that individuals carrying the rare A-allele (i.e., having A/G or A/A genotype), had the risk of disease 3,23 times (OR=3,23; [95 % CI 1,50; 6,92], p<0,05) higher than the general population. No significant differences in the distribution of alleles and genotypes of dup16bp intron 3 of the TP53 gene between cases and controls were identified. The results indicate that the oligonucleotide G13494A substitution in intron 6 proapoptotic gene TP53 in older individuals may have a modulating effect on the risk of indolent lymphoma.

Association of polymorphism harbored by tumor necrosis factor alpha gene and sex of calf with lactation performance in cattle.

In a majority of mammals, male infants have heavier body mass and grow faster than female infants. Accordingly, male offspring nursing requires a much greater maternal energy contribution to lactation. It is possible that the maternal-fetal immunoendocrine dialog plays an important role in female preparation for lactation during pregnancy. Immune system genes are an integral part of gene regulatory networks in lactation and tumor necrosis factor alpha (TNFα) is a proinflammatory cytokine that also plays an important role in normal mammary gland development. The aim of this study was to evaluate the influence of the sex of calf and/or the -824A/G polymorphism in the promoter region of TNFα gene on milk performance traits in Black Pied cattle over the course of lactation. We also studied the allele frequency differences of -824A/G variants across several cattle breeds, which were bred in different climatic conditions. The G allele frequency decreased gradually over the course of lactation events in the Black Pied dairy cattle because of a higher culling rate of cows with the G/G genotype (p<0.001). In contrast to the genotypes A/A and A/G, cows with G/G genotype showed significant variability of milk and milk fat yield subject to sex of delivered calf. Milk yield and milk fat yield were significantly higher in the case of birth of a bull calf than with a heifer calf (p<0.03). The G allele frequency varies from 48% to 58% in Grey Ukrainian and Black Pied cattle to 77% in aboriginal Yakut cattle. Our results suggest that the TNFα -824A/G gene polymorphism may have an influence on the reproductive efforts of cows over the course of lactation events depending on the sex of progeny. Allocation of resources according to sex of the calf allows optimizing the energy cost of lactation. This may be a probable reason for high G allele frequency in Yakut cattle breeding in extreme environmental conditions. Similarly, the dramatic fall in milk production after birth of a heifer calf increases the probability of culling for the cows with the G/G genotype in animal husbandry.

[Prevalence of mutations BRCA1 5382insC, and CHEK2 1100delC in the population of Siberian region].

Frequencies of the 538insC mutation in the BRCA1 gene and the 1100delC mutation in the CHEK2 gene were compared in the group of breast cancer patients and the large-scale sample, consisting of 7920 DNA specimens from healthy residents of the city of Novosibirsk. Higher frequencies of these mutations in the patient group compared to the control sample (1.95 versus 0.25% for BRCA1 5382insC, and 1.78 versus 0.40% for CHEK2 1100delC) were observed, pointing to their association with susceptibility to breast cancer (OR = = 7.86, 95% CI 3.51-17.30 and OR =4.46, 95% C1 2.04-9.49, respectively).

[Risk of gastric cancer dependent on serological markers of atrophic gastritis: cohort study].

In a prospective study the risk of subsequent gastric cancer (GC) was assessed in persons aged 45-69 over 5 years after the initial testing with a set of serological tests (pepsinogen I, pepsinogen II, gastrin-17, antibodies to Helicobacter pylori). The presence of gastric atrophy markers was a significant predictor of GC in the forthcoming years. Non-invasive techniques may be used in the formation of high-risk groups, followed by GC active surveillance.

[CCR5, CCR2, apoe, p53, ITGB3 and HFE gene polymorphism in Western Siberia long-livers].

In order to estimate the distribution of some polymorphisms for the CCR5, CCR2, apoE, p53, ITGB3, and HFE genes in Russian long-livers from Western Siberia, a sample of 271 individuals (range 90-105 years) was examined. It was demonstrated that carriage of the delta32 polymorphism for the CCR5 gene, V64/polymorphism for the CCR2 gene, e2/e3/e4 for the apoE gene, L33P for the ITGB3 gene, as well as H63D and S65C polymorphisms for the HFE gene does not influence on predisposition to the longevity; carriage of the 282 Y allele for the HFE gene negatively influences on the longevity; carriage of the heterozygous genotype for the R72P polymorphism for the p53 gene correlates with the longevity of elderly people.