Associations of cold receptor TRPM8 gene single nucleotide polymorphism with blood lipids and anthropometric parameters in Russian population.

We analyzed associations of single nucleotide polymorphisms rsl13004520 (R247T), rs11562975 (L250L), rs7593557 (S419N), rs11563208 (I1016I), and rs11563071 (V1058V) of the cold receptor TRPM8 (2q37.1) gene with blood plasma lipids and anthropometric parameters in Russian population (randomly chosen residents of Novosibirsk: 507 women and 459 men, mean age 57 years). The studied polymorphisms are localized in regions encoding NH2-terminal (R247T, L250L, S419N) and COOH-terminal (I1016I, V1058V) cytoplasmic domains of the channel. We showed association of single nucleotide polymorphism V1058V with the levels of total cholesterol and LDL and HDL cholesterol, and association of I1016I polymorphism with triglyceride content. Polymorphisms L250L and S419N correlated with anthropometric parameters (body mass index and waist and hip circumferences).

[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.

[Evaluation of association between 9 genetic polymorphism and myocardial infarction in the Siberian population].

AIM: to evaluate association between genetic polymorphism (SNPs) and myocardial infarction (identified in recent GWAS) as markers of high risk of myocardial infarction (MI) in Siberian population. Patients were divided into 2 groups - MI patients and control group (ratio 1:2) and presented the sapmle of population of Novosibirsk (9400 patients, 45-69 years) within international project HAPIEE (Health, Alcohol and Psychosocial factors In Eastern Europe). 200 patients with MI (129 men, 71 women) were included. Control group - individuals without MI (420) matched for age and sex. Genomic DNA was extracted from venous blood by phenol-chloroform extraction. Gene polymorphism of genes tested by real-time PCR according to protocol (probes TaqMan, Applied Biosystems, USA) with the use of ABI 7900HT. The following SNPs were studied: rs28711149, rs499818, rs619203, rs10757278 and rs1333049 (hr. 9), rs1376251, rs2549513, rs4804611, rs17465637. The association of SNP and MI was confirmed for 4 of 9 studied SNPs: rs1333049 (hr. 9), rs10757278 (hr. 9), rs499818 (hr. 6), rs619203 gene ROS1. Heart rate was associated with rs1333049 and rs10757278. Glucose level was associated with rs619203, rs28711149 and rs1376251. Total cholesterol and atherogenic index was associated with rs28711149. For the first time in Russian population the associations of GWAS with myocardial infarction SNPs was detected for rs619203, rs499818, rs1333049 and rs10757278. These genetic markers can be used for assessing the risk of myocardial infarction in Russian population.

Association of cold receptor TRPM8 gene polymorphism with blood lipid indices and anthropometric parameters in Shorians.

We analyzed single nucleotide polymorphisms of the cold receptor TRPM8 gene as genetic markers of blood serum lipid indices in Shorians. Associations were found between rs11562975 (L250L) TRPM8 gene mononucleotide polymorphism with total cholesterol and LDL cholesterol and between rs28901637 (P249P) and HDL cholesterol. No associations of P249P and L250L with triglyceride level were found. L250L polymorphism was associated with anthropometric parameters characterizing lipid metabolism (hip and waist circumferences). The TRPM8 gene is likely to be involved in the regulation of lipid metabolism.

[HFE gene polymorphism in the population of Northern Asia].

Human HFE gene haplotype analysis with reference to IVS2(+4)t/c, IVS4(-44)t/c, IVS5(-47)a/g polymorphic sites was performed in different North Asian ethnic groups. Of the eight possible intronic haplotypes, TTG, TTA, CTA and CCA were identified. High frequency of the CCA haplotype appears to be a characteristic feature of all Asian native populations. Potential functional importance of IVS4(-44)t/c polymorphism is demonstrated. Patients presenting with iron overload syndrome are shown to have low frequency of IVS4(-44)c.

[Relationship of single-nucleotide polymorphism rs11562975 in thermo-sensitive ion channel TRPM8 gene with human sensitivity to cold and menthol].

The examination of people belonging to the Russian ethnic group revealed that 20.3% of subjects had heterozygous genotype, containing the C-allele in single nucleotide polymorphism rs11562975, located in exon 7 of the gene encoding the temperature-sensitive ion channel TRPM8. Functional differences, associated with sensitivity to cold and menthol were identified between subjects with different genotypes of the polymorphism rs11562975 (GG and GC). Subjects with heterozygous genotype GC were characterized by increased sensitivity to cold and reduced sensitivity to menthol, agonist of the ion channel TRPM8, compared with subjects with homozygous genotype GG.

[Polymorphism in the human 2'-5'-oligoadenylate synthetase genes (OAS), associated with predisposition to severe forms of tick-borne encephalitis, in populations from North Eurasia].

2'-5'-oligoadenylate synthetases are a family of interferon-induced enzymes which play an important role in the antiviral defense in mammals. In human genome three genes encoding functional synthetases (OAS1, OAS2 and OAS3) form a cluster. Previously we found that particular genotypes and/or alleles of five single nucleotide polymorphisms (SNPs) located within OAS2 and OAS3 genes are associated with predisposition to severe forms of tick-borne encephalitis (TBE) in Russian population. In current study we investigated the distribution of three of that SNPs (OAS3rs2285932 (C/T Ile438Ile), OAS3rs2072136 (G/A, Ser567Ser) and OAS2 rs15895 (G/A, Trp720Ter relative to p71 isoform)) in seven populations from North Eurasia: Caucasians (Russians and Germans (from Altai region)), Central Asian Mongoloids (Altaians, Khakasses, Tuvinians and Shorians) and Arctic Mongoloids (Chukchi). Differences between populations in genotype, allele and haplotype frequencies and in linkage disequilibrium structure for these SNPs were detected. We found that these frequencies correlate with the ethnicity of the populations and with their supposed differential exposure to TBE virus. Particularly, the lowest frequencies of G/G genotype for OAS3 gene rs2072136 SNP (that according to our previously obtained data is associated with predisposition to severe forms of TBE) were found in Altaians, Khakasses, Tuvinians and Shorians who may highly contact with TBE virus in places of their habitation. Thus, data obtained allow to suppose that TBE virus might act as a selection factor for particular OAS genes variants in Central Asian Mongoloids.

[Analysis of polymorphism of three positions of promoter region of TNF- gene in patients with ischemic heart disease, unstable angina and myocardial infarction].

We studied polymorphism of three positions of promoter region of TNF alpha gene in patients with ischemic heart disease (IHD), development of unstable angina (UA) and acute myocardial infarction (MI). Analysis of frequencies of genotypes in position A308 showed that heterozygotness in patients with cardiovascular diseases significantly decreased. Lowering of risk of development of MI in carriers of heterozygous variant of the gene in position A308 was shown. In the group of patients with Q wave MI frequency of A308 AA genotype rose.

[Analysis of the gene polymorphism of matrix metalloproteinase-2 and -9 in patients with coronary heart disease].

AIM: To analyze the gene polymorphisms of matrix metalloproteinase-2 and -9 in patients with coronary heart disease (CHD). MATERIALS AND METHODS: The influence of single nucleotide polymorphism in the promoter region of the gene of matrix metalloproteinase (MMP)-2 at position -1306 and that of MMP-9 at position -1562 on the development of complications was studied in 181patients with atherosclerosis by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of amplification products. RESULTS: A significant genotype increase with enhanced MMP-9 gene transcriptional activity was found in the patients aged 55 years inclusively who had been diagnosed as having myocardial infarction as compared with an older age group and, on the contrary, a rise in the MMP-2 genotype with a high promoter activity in the older age group. CONCLUSION: The findings suggest that the functional polymorphism in the promoter region of the MMP-2 and MMP-9 genes plays a certain role in the development of acute coronary events.

Ethnicity-specific distribution of TRPM8 gene variants in Eurasian populations: signs of selection.

The TRPM8 gene encodes the ion channel, which is a cold receptor in afferent neurons of the mammalian somatosensory system. We studied the frequency of haplotype distribution from six SNPs in the TRPM8 gene in Eurasian human populations, including Russians, Kazakhs and Chukchi. Four of the six SNPs are located in exon 7 (rs13004520, rs28901637, rs11562975, rs17868387), rs7593557 is in exon 11. These exons encode parts of the N-terminus, which is necessary for channel functioning in the plasma membrane of neurons. The rs11563071 is in exon 23 encoding part of the C-terminus. The primary difference in population distribution of haplotypes determines the SNP from exon 11 which leads to Ser419Asn substitution in protein. The most pronounced differences in the patterns of diversity and frequencies of haplotypes were observed between Chukchi and Russians. The frequency of major H1 haplotype encompassing the 419Ser gene variant differs in examined populations; 0.738 (Russians), 0.507 (Kazakhs) and 0.337 (Chukchi), p < 0.001. The TRPM8 gene variants encoding 419Asn and carrying the minor alleles of rs28901637 (P249P) and rs11562975 (L250L) in exon 7 are characteristic of Asian populations. The frequency of all 419Asn variants in Chukchi is comparable to that in Africans, however, the minor allele frequencies of rs28901637, rs11562975 in Africans is low. Apparently in the process of human colonization of Eurasia, minor alleles of these SNPs diverged depending on rs7593557 structure in exon 11. We analyzed sequences of five TRPM8 mRNA isoforms extracted by researchers from different tissues. Sequence analysis demonstrates that they are transcribed from major H1 variant of the TRPM8 gene but contain different translation start codons, which are generated by alternative splicing from pro-mRNA.

[Genetics of atrial fibrillation].

We carried out examination of 103 probands with atrial fibrillation (AF) and 301 their 1st, 2nd, and 3rd degree relatives (main group). In addition we examined 82 probands without clinical electrocardiographic signs of heart disease and 163 their 1st and 2nd degree relatives (control group). We found accumulation of AF in families of probands with this pathology. Segregation analysis of idiopathic forms of AF allowed to reveal autosomal dominant type of inheritance of this pathology. Heterozygous variant of Ser49Gly of betai-adrenoreceptor gene can be considered as one of genetic predictors of development of how primary and secondary AF.

[The spectrum of mutations in the low-density lipoprotein receptor gene in the Russian population].

The spectrum of mutations in the low-density lipoprotein (LDL) receptor gene was studied in a sample of hypercholesterolemia patients of Caucasoid origin from the population of Russia. The examined patients were 45 to 49-years-old and had the highest level of total serum cholesterol in this age group. Seven previously non-described mutations have been revealed in exon 9 (R410G; M412V) and in exon 12 (Y/Y576; N/N591; L605V; L605R; A612G). Twelve previously described mutations have been identified in exons 2 (C/C27), 5 (C261F; E240X), 6 (E288K), 8 (A391T), 9 (E418G; L432R; D433E), 11 (G/G549; E558K; L/L568), and 12 (G592E). Only one of these mutations was previously described in Russia in a clinical sample of patients with familial hypercholesterolemia. The spectrum of LDL receptor gene mutations in the population sample of patients with hypercholesterolemia significantly differs from the mutation spectrum in patients with familial hypercholesterolemia (clinical samples). Sequencing of the LDL receptor gene is a highly efficient method for identifying the markers of hypercholesterolemia predisposition in a population.

Transcription Regulatory Regions Database (TRRD): its status in 2002.

Transcription Regulatory Regions Database (TRRD) is an informational resource containing an integrated description of the gene transcription regulation. An entry of the database corresponds to a gene and contains the data on localization and functions of the transcription regulatory regions as well as gene expression patterns. TRRD contains only experimental data that are inputted into the database through annotating scientific publication. TRRD release 6.0 comprises the information on 1167 genes, 5537 transcription factor binding sites, 1714 regulatory regions, 14 locus control regions and 5335 expression patterns obtained through annotating 3898 scientific papers. This information is arranged in seven databases: TRRDGENES (general gene description), TRRDLCR (locus control regions); TRRDUNITS (regulatory regions: promoters, enhancers, silencers, etc.), TRRDSITES (transcription factor binding sites), TRRDFACTORS (transcription factors), TRRDEXP (expression patterns) and TRRDBIB (experimental publications). Sequence Retrieval System (SRS) is used as a basic tool for navigating and searching TRRD and integrating it with external informational and software resources. The visualization tool, TRRD Viewer, provides the information representation in a form of maps of gene regulatory regions. The option allowing nucleotide sequences to be searched for according to their homology using BLAST is also included. TRRD is available at http://www.bionet.nsc.ru/trrd/.

[Polymorphism of the gene of angiotensin-converting enzyme and apolipoprotein E gene in long-livers of Novosibirsk city]. / Polimorfizm gena angiotensin-prevrashchaiushchego fermenta i gena apolipoproteina E u dolgozhitelei goroda Novosibirska.

The levels of polymorphism of genes of angiotensin converting enzyme (ACE) and apolipoprotein E (Apo E) were studied in elderly and long-living people in Novosibirsk. The results of the study in the investigated group (97 subjects) were compared with polymorphism of these genes in Novosibirsk population group aged 25-64 who were investigated in MONICA Project survey and had DNA data base formed. Frequency of D/D genotype among senile and long-living men was 5.9%. It is 5 times lower than in men 55-64 years of age (p = 0.04). Similar decrease of this gene frequency was also found in women of the same age. In men older than 83 years of age 4 times lowering of 3/4 genotype of Apo E gene and 2 times increasing of frequency of 2/3 genotype were revealed when comprising frequency of these genotypes in people of middle age. In subjects of senile age and long-livers of both sexes genotype 4/4 was not revealed. Lipid levels were more favorable in women with genotype 2/3 of Apo E gene (comparatively lower mean level of total cholesterol and higher level of HDL cholesterol) if compared with genotypes 3/3 and 3/4.

[Unprimed synthesis of poly (d(A-T)), catalyzed by a preparation of Escherichia coli DNA polymerase I]. / Issledovanie prirody bezmatrichnogo sinteza d(A-T)-sopolimera, katalizirchemogo preparatami DNK-polimerazy I Escherichia coli.

The initial events of the de novo synthesis of poly[d(A-T)], catalyzed by preparations of E. coli DNA-polymerase I, were investigated. The data provide evidence that deoxynucleoside diphosphate: oligonucleotide deoxynucleotidyl transferase (dNDP-transferase), the enzyme which is able to catalyze unprimed polymerization of dNDP, participates in the process of initiation. This conclusion is based on the following data: 1) preincubation of E. coli DNA-polymerase I preparation with dADP and dTDT abolishes a lag-period in the poly[d(A-T)] synthesis; 2) dithiothreitol and N-ethylmaleinide, inhibitors of dNDP-transferase, inhibit de novo synthesis of [d(A-T)]-copolymer by preparations of E. coli DNA-polymerase I but do not effect primed synthesis ensured by this enzyme. High concentration of the substrate have similar effect. Using two-dimentional thin-layer chromatography and microcolumn chromatography on TEAE-cellulose we have shown that preliminary incubation of DNA-polymerase I preparations with dADP and dTDP results in the synthesis of short oligonucleotides (from di- to decanucleotides). Hydrolysis of these oligonucleotides with dilute sulfuric acid demonstrates that among the reaction products prevail oligoadenylates and oligothymidylates, but an appreciable amounts of heterooligomers including oligo[d(A-T)] were revealed as well. The model of so called de novo synthesis of regular polynucleotides is proposed, according to which dNDP-transferase, an accompanying enzyme in the preparations of DNA-polymerase I E. coli, is carrying out the synthesis of short oligonucleotides which form template-primer complexes repeatedly replicated by the DNA-polymerase I E. coli.

[Affinity modification of Escherichia coli Dna-polymerase I by 4-(N-2-chloroethyl-N-methylamino)-benzyl-gamma-amides of dTTP and dATP]. / Affinnaia modifikatsiia DNK-polimerazy I iz Escherichia coli 4-(N-2-chloroéthyl-N-methylamino)-benzyl-gamma-amidami dTTP i dATP.

The interaction of dTTP and dATP gamma-4-(N-2-chloroethyl-N-methylamino) benzylamidates with E. coli DNA-polymerase I were studied. dTTP and dATP gamma-4-(N-2-hydroxyethyl-N-methylamino) benzylamidates act as competitive inhibitors of DNA-polymerase I. Ki values for gamma-analogues of dTTP and dATP have been determined. Reactive dTTP and dATP derivatives are shown to be affinity reagent for this enzyme.

[Features of the structure and evolution of complex tandemly organized Bsp-repeats in the fox genome. II. Tissue-specific and recombinant BamHI-dimer sites]. / Osobennosti struktury i évoliutsii slozhnykh tandemno organizovannykh Bsp-povtorob genoma lisitsy. II. Tkanespetsificheskie i rekombinatsionnye saity BamHI-dimera.

The complex structure of the clustered Bsp-repeats in fox genome seems to have evolved throughout a long period of time as a result of multiplication, recombination and divergence events. The sequence of the subrepeat (SR) approximately 245 b.p long is the basic substructure for the hierarchically arranged Bam HI-repeat 1468 b.p. long. The monomer consists of 3 SRs with a 43-59% homology. A dimer is composed of 2 monomers with a 93% homology. Amplification of the Bsp-repeats during evolution seems to have occurred at least twice: first--on the SR ancestral form level, second--on the monomer level. Despite profound divergence, there are still conservative regions in SRs with sequences homologous to known functional sites in eukaryotes. However qualitative and quantitative composition of most functional motifs is stringently individual in every SR. The performed analysis revealed that throughout evolution SRs acquired significant amount of motifs homologous to promoter and enhancer regions in tissue-specific genes and virus regulatory regions. Functional motifs in separate SRs are being differently grouped. Most inducible motifs are located in the III and II subrepeats, putative promoters--in the II one; elements participating both in transcriptional and replicational processes--mainly in the I subrepeat. A few ensembles of functional motifs remotely resemble extended regulatory regions of some tissue-specific genes. The monomers are potentially capable of ensuring diverse aspects of transcriptional regulation. As a whole, motifs of the 3 SRs are potentially capable of regulating the RNA synthesis periodicity with respect to the cellular cycle, activation and repression of genetical material in response to signals from the environment (AP-1, AP-2, AP-4, T-antigen, etc) and temporal ("octamers") etc. Apart from the BamHI-dimer, a few homologues fragments were isolated from fox genome and sequenced. Some of them were rearranged with respect to the BamHI-dimer. Inversion locally alters the composition of motifs and the sequence acquires new functional potential. Thus, the analysis of the emergence and development of Bsp-repeat structural variations allows us to consider repetitive DNA sequences as an ideal material in constructing multiprofile regulatory sequences.

[Modular evolution of Bsp-repeats: formation of subrepeats and monomers preceded the divergence of four species of Canidae]. / Blochnaia évoliutsiia Bsp-povtorov: formirovanie subpovtorov i monomerov predshestvovalo raskhozhdeniiu chetyrekh vidov Canidae.

Determination of two nucleotide sequences of satellite-like DNA (Bsp-repeats) segments from the raccoon-like dog genome and analysis of these and three other homologous sequences from distantly related genomes of Canidae (silver fox, domestic dog, and gray fox) show that all these DNA fragments consist of five types of subrepeats (A, B, C, D, and E) and two types of monomers-ACB and ACED. Raccoon-like dog repeated segment of 1620 bp is composed of both type of monomers: ACBACED. Different monomers of the same type could be distinguished by subtypes of subrepeats. Building of a cladogram of 18 subrepeats and analysis of the structures of monomers and higher-order repeated units allowed us to reveal probable evolution events which have determined the hierarchical order in Bsp-repeat organization. Approximate periods of time for these evolution events were calculated from the number of substitutions per branch. Five main types of subrepeats and two types of monomers have been generated and propagated through several steps before divergence of the main phylogenetic trees of Canidae. Combinatorial variability appears to play a fundamental role in forming the hierarchical layers in Bsp-repeats structure.

[Features of the structure and evolution of complex, tandemly organized Bsp-repeats in the fox genome. I. Structure and internal organization of the BamHI-dimer]. / Osobennosti struktury i évoliutsii slozhnykh, tandemno organizovannykh, Bsp-povtorov genoma lisitsy. I. STruktura i vnutrenniaia organizatsiia BamHI-dimera.

A 1468 b.p. DNA BamHI-fragment homologous to the Bsp-repeat was isolated from the fox genome and sequenced. This fragment is an hierarchically arranged dimer. Its 734 b.p.-monomers consist of three subrepeats (SR), each 245 b.p. long, abundant with overlapping imperfect tandem repeats which in turn are rich in short direct related repeats (each 4-7 b.p. in size). The latters are mainly composed of AG, TG dinucleotides and their complements CT, CA. All subrepeats in the BamHI-dimer are flanked by motifs homologous to Jeffreys' sites. At certain points the sites are doubled. The above data allow to assume that the Bsp-repeat complex structure is likely to have developed throughout long multi-step evolution of relatively simple DNA sequences which had emerged de novo. Single substitutions, small inserts and deletions, multiple duplication and recombination events seem to have most contributed to the evolution of the Bsp-repeats. Single substitutions in SRs with respect to the consensus are not equally distributed along their length. A wave-like pattern of this distribution is the evidence for non-random character of mutations accumulation. A correspondence was noted between conservative regions in SR and the presence therein of functional motifs homologous to the binding sites of already known regulatory proteins.

[Reverse transcription of heterologous RNA by RNA-dependent DNA polymerase from Escherichia coli]. / Obratnaia transkriptsiia geteropolimernoi RNK s pomoshch'iu RNK-zavisimoi DNK-polimerazy Escherichia coli.

The appropriate conditions for the reverse transcription of rabbit globin mRNA by E. coli RNA-dependent DNA polymerase has been studied. By reducing the ionic strength, increasing the incubation temperature from 37 to 43 degrees and predenaturing the template it was possible to increase the cDNA size. The molar ratio of Mn2+ and dNTP optimal for cDNA synthesis is approximately 1.5-1.7:1. The increase of dNTP concentration from 0.05 to 0.4 mM each, under conditions of Mn2+ deficiency, results in the decrease of the cDNA size to 4S, obviously, the inhibitory effect of dNTP is not complexed with Mn2+. The synthesis of cDNA is inhibited also by the excess of Mn2+. Hybridization of cDNA with globin mRNA protects the former from S1-nuclease. Optimization of the conditions for reverse transcription of heterologous RNa by E. coli RNA-dependent DNA polymerase led to the increase of the cDNA length up to approximately 550 nucleotides which is about 70% of the RNA template length.