A Complex of Pyrosequencing-Based Methods for Detection of Somatic Mutations in Codons 600 and 601 of the BRAF gene.

The aim of the study is to develop methods for the differentiation of mutations in the BRAF codon 600 and to increase the sensitivity of the K601E mutation detection. Materials and Methods: The nucleotide sequence of the BRAF codons 592-602 was identified using the PyroMark Q24 genetic analysis system. The mutations search in codon 600 was conducted using the 600-S primer in line with the following order of adding nucleotides: GCTGTCАTCTGCTAGCTAGAC (corresponding to nucleotides 1799-1786). The K601E mutation was detected using the 601-S primer in line with the following order of nucleotide addition: GCTACTCACTGTAG (corresponding to nucleotides 1801-1793). The analytical characteristics of the proposed methods for somatic mutations' detection were determined using dilutions of plasmid DNA samples containing the BRAF gene region without mutations or with one of the following mutations: V600E, V600R, V600K, V600M, and K601E. Validation was performed on 132 samples of biological material obtained from the thyroid nodules. Results: The developed methods allow to determine 2% of the V600E or V600M mutations, 1% of the V600K and V600R mutations, and 3% of the K601E mutations in samples with high DNA concentration; it is also possible to confidently detect at least 5% of the mutant allele for all mutations in low concentration samples (less than 500 copies/PCR). During biological material testing, 53 samples with the V600E mutation were detected; the proportion of the mutant allele was 4.9-50.0%. Conclusion: A complex of methods for determination of the nucleotide sequence of the BRAF codons 592-601 and the algorithm for testing samples and analyzing mutations in the BRAF codons 600-601 was developed. The method provides sufficient sensitivity to detect frequent mutations in codons 600 and 601 and allows them to be precisely differentiated.

Associations of Toll-like Receptor Gene Polymorphisms with NETosis Activity as Prognostic Criteria for the Severity of Pneumonia.

The aim of the study was to determine the molecular genetic prognostic criteria for the severity of the course pneumonia based on the analysis of the association of genetic polymorphism in toll-like receptors with the severity of NETosis. MATERIALS AND METHODS: The study included 38 patients with the main diagnosis of community-acquired pneumonia with a severe course. All the patients underwent standard clinical laboratory examinations, computed tomography of the thoracic organs, microbiological examination of blood and tracheobronchial aspirate. The level of neutrophilic extracellular traps (NETs) in blood smears was determined on the 1st-2nd and 5th-7th days of hospitalization. Genotyping of rs5743551 (TLR1), rs5743708 (TLR2), and rs4986790 (TLR4) polymorphic loci was performed by pyrosequencing. RESULTS: The level of NETs on the 1st day of admission was statistically significantly lower in heterozygous and homozygous carriers of rs4986790 (TLR4) polymorphism (AG and GG genotypes) compared with patients with the wild-type genotype (AA genotype) (p<0.05). When comparing the number of NETs with genotypes for rs5743708 (TLR2) and rs5743551 (TLR1) polymorphisms, no statistically significant correlation was found (p>0.05). The study of the NET level in dynamics demonstrated a decrease in the NETosis activity of neutrophils during the first week of hospitalization (p<0.05). The presence of the G allele in the patient's genotype for rs5743551 (TLR1) polymorphism increases the risk of a poor outcome of the disease (p<0.0001) (OR=20.3; 95% CI (4.3-135.0)). CONCLUSION: The obtained data suggest that level of NETs is a marker of the activity of neutrophils which are closely related to the studied genetic polymorphisms, and affects the prognosis of the pneumonia outcome.

[A comparative analysis of allele frequencies of rs1801133 and rs1801131 of MTHFR in patients with stroke and healthy people from the Moscow region]. / Sravnitel'nyi analiz chastot allel'nykh variantov rs1801133 i rs1801131 gena MTHFR v vyborke patsientov, perenesshikh ishemicheskii insul't, i u zdorovogo naseleniia v Moskovskom regione.

AIM: To study genetic characteristics of the population of the Moscow region and analyze the association of rs1801133 and rs1801131 of MTHFR with the risk of ischemic stroke (IS). MATERIAL AND METHODS: A sample of 170 and 115 patients with atherothrombotic and cardioembolic subtypes of IS and 360 residents of the Moscow region without IS were examined. MTHFR alleles were determined by a multiplex real-time polymerase chain reaction. RESULTS AND CONCLUSION: No association between the frequencies of MTHFR alleles and the risk of ischemic stroke was found. The comparison of allele frequencies with those in Caucasian populations published in the dbSNP (NCBI) and 1000 Genomes Project databases revealed significant differences for rs1801133 from the EUR 1000 Genomes Project. The allele frequency data for MTHFR could increase the accuracy and reliability of the individual risk calculation for multifactorial diseases in the Russian population.

[Complex assessment of the contribution of genetic factors to the risk of ischemic stroke]. / Kompleksnaia otsenka vklada geneticheskikh faktorov v razvitie ishemicheskogo insul'ta.

AIM: To develop a method of the complex assessment of genetic risk for ischemic stroke (IS) and evaluate its effectiveness. MATERIAL AND METHODS: Genotyping of 182 patients with atherothrombotic and cardioembolic subtypes of IS and 360 healthy individuals of 48 single nucleotide polymorphic loci (SNP) associated with the risk of II and its subtypes was performed. RESULTS AND CONCLUSION: In each group of SNPs, composite indicators of genetic risk of IS in groups of patients and healthy controls were identified. Differences between the calculated values of the genetic risk in these groups were significant (p <0,05). The quality of the binary classification validated by ROC-analysis confirmed the predictive potential of the proposed method of risk calculation for determining the genetic predisposition to the development of IS.

[Characterization of retrotransposon Bov-B LINE reverse transcriptase gene sequences in parthenogenetic lizards Darevskia unisexualis and bisexual species D. nairensis and D. valentini].

Cloning and sequencing of the partial reverse transcriptase gene (750 bp) of the Bov-B LINE retrotransposon have been held in parthenogenetic lizards Darevskia unisexualis and its assumed parental bisexual species D. nairensis and D.valentini. It was shown that the percentage of transcriptionally active copies of this gene, which does not contain a stop codon, is almost the same in the three species and is about 75%. The intragenomic divergence level of these sequences is low and was found to be 2.6% in D. unisexualis, 1.9% in D. nairensis, and 1.6% in D. valentini. The phylogenetic analysis shows the distribution of copies of D. unisexualis in each of the two clusters of RT sequences characteristic of D. nairensis and D. valentini. This result supports the view of the hybrid origin of D. unisexualis and does not exclude intraspecific hybridization between D. nairensis and D. valentini.

[Intra- and interspecific polymorphism of (AAT)n in microsatellite locus du47D in parthenogenetic species of the genus Darevskia].

The molecular structure of the allelic variants of (AAT)n of the Du47D microsatellite locus was determined in parthenogenetic lizards Darevskia dahli, D. armeniaca, and D. rostombekovi. Comparative analysis of these alleles showed that they were characterized by perfect structure of microsatellite cluster, and were different in the number of (AAT) monomeric units, as well as in the combinations of species-specific substitutions and deletions in the microsatellite flanking regions. Molecular structure of microsatellite cluster, species-specific single nucleotide polymorphism (SNP), and different representation of alleles Du47 in the samples of parthenogenetic species examined point to the origin of the alleles from different bisexual species, which is consistent with the hybrid nature of unisexual species of the genus Darevskia. In addition, these data reflect different combination patterns of interspecific hybridization events with the participation of the same bisexual species upon the formation of hybrid genomes of parthenogenetic species. Possible application of the allelic variants of microsatellite loci of parthenogenetic lizards as the genetic markers for the analysis of the genomes of parthenogenetic species in the light of evolution, ecology, and parthenogenetic type of reproduction in vertebrates is discussed.

[Molecular nature of allelic polymorphism of highly variable microsatellite locus Du161(arm) in unisexual lizard Darevskia armeniaca (Lacertidae)].

In the genome of unisexual (parthenogenetic) lizard Darevskia armeniaca, highly variable locus Du 161 (arm) was discovered. Analysis of allelic polymorphism was carried out using locus-specific PCR of the lizard DNA specimens from 13 isolated Armenian populations (N = 138). In the sample examined, a total of 12 Du 161(arm) alleles were identified, and their differences at the level of primary DNA structure were determined. Sequence analysis of the Du 161 (arm) alleles showed that their microsatellite clusters contained repeats of one type (GATA repeats). Allelic Du 161 (arm) variants differed in the number of GATA monomers in microsatellite, point mutations of transition and transversion types, located at fixed distances from microsatellite cluster, and by single nucleotide insertions, as well as by longer insertions located within and outside of the microsatellite cluster. Moreover, point mutations formed different combinations (haplotypes), typical of certain alleles. These combinations can be used for the analysis of the origin and inheritance of these alleles in D. armeniaca, as well as for investigation of their interspecific variation in the representatives of the genus Darevskia.

[Molecular structure of the allelic variants of (AAT)n microsatellite locus Du47D in the parthenogenetic species Darevskia unisexualis and bisexual parental species D. valentini and D. raddei].

Microsatellite repeats are one of the most widespread elements of the eukaryotic genome, but are poorly studied in species with clonal reproduction. PCR analysis and DNA sequencing were used to study the molecular structure of the allelic variants of microsatellite locus Du47D in the parthenogenetic species Darevskia unisexualis and its evolutionary ancestors, bisexual species D. raddei and D. valentini, of the genus Darevskia (Lacerta saxicola complex). Sequencing showed that the allelic variants of the D. unisexualis Du47D locus and the alleles of its D. raddei and D. valentini orthologs have a perfect microsatellite cluster structure, differ in number of ATT monomeric units, and have certain species-specific combinations of nucleotide substitutions, deletions, and insertions in the microsatellite-flanking DNA sequences. The Du47D alleles that the parthenogenetic species inherited from D. valentini or from D. raddei were identified.

[Thermodynamic characterization of di-, tri-, and tetranucleotide loci in parthenogenetic lizards Darevskia unisexualis].

Allelic polymorphism of three microsatellite loci from the genome of parthenogenetic lizard Darevskia unisexualis was characterized using analysis of free energy (Gibbs energy) of the DNA/DNA duplex formation within the stepwise mutational model. It was demonstrated that the number of microsatellite cluster monomericic units would change to decrease the mean free energy of the locus. In addition, based on the analysis of nucleotide composition, the GC content of each locus was evaluated, and belonging of the loci examined to certain isochore families was suggested.

[The polymorphism and structural peculiarities of two noncoding regions of liver fluke Fasciola hepatica (Plathelminthes: Trematoda) mitochondrial genome].

Structural characteristics and polymorphism of long (LNR) and short (SNR) mitochondrial non-coding regions of the liver fluke Fasciola hepatica were studied. The flukes were obtained from several populations of Russia and Belarus. The amplification of LNR yielded a set of 10 fragments with the length of neighbouring ones differing in one tandem repeat (85 bp; published earlier for Australian fluke). LNR amplification fragments of different length were cloned and sequenced. Comparison of the LNR sequences of Australian and Belarussian flukes revealed 3 nucleotide substitutions and one point heteroplasmy of the first nucleotides in the imperfect repeat and four adjacent perfect repeats. Positions of the three mutations coincide in perfect and imperfect repeats and the frequency of mutations is 4-4.7% while the frequency of heteroplasmic sites varies from 0.1 to 1.2%. It was shown that the presence of mutations and the heteroplasmy of one site can change the structure and stability of the putative secondary structures of the perfect and imperfect re- peats. The amplification of SNR of F. hepatica from several populations yielded fragments which differed from the published SNR sequence of Australian F. hepatica in the single transversion. Both non-coding regions have several conservative and potentially regulatory sequences. Probable cause of heteroplasmy and concerted origin of substitutions in different repeats are discussed.

[Molecular genetic characteristic of dinucleotide loci in parthenogenetic lizards Darevskia unisexualis].

In the present study, the first molecular genetic investigation of dinucleotide (GT)n microsatellite loci in parthenogenetic lizards Darevskia unisexualis was performed. New polymorphic locus, Du214, (GeneBank ac no. EU252542) was identified and characterized in detail. It was demonstrated that allele of this locus differed in the size and structure of microsatellite locus, as well as in point mutations, the combinations of which enabled the isolation of stabile fixed double nucleotide substitutions A-A (alleles 2 and 4) and G-T (alleles 1, 3, 5, and 6). Double nucleotide substitutions described were also identified in the orthlogous loci of the parental species genomes, D. raddei (G-T) and D. valentine (A-A). Based on the analysis of allele distribution pattern at this locus in all populations of parthenospecies D. unisexualis, mathematic model was elaborated and realized. Using this model, frequencies of allelic variants for all populations of the species of interest were calculated and population genetic structure of D. unisexualis was characterized. Genetic contribution of each population to the species gene pool was determined. The data obtained demonstrated that microsatellite variation was one of the factors of clonal and genetic diversity of a parthenospecies.

[Molecular structure of allelic variants of microsatellite loci Du281 and Du47 in unisexual and bisexual lizard species of the genus Darevskia].

Experimental data on the molecular structure and variability of microsatellite loci in unisexual and bisexual lizard species of the genus Darevskia were analyzed. The allelic variants of Du281 and Du47 were found to differ in the number of monomers, the structure of microsatellite clusters, and point mutations in these clusters and flanking DNA. Interspecific comparison of alleles of these loci revealed both variable regions in the microsatellite clusters and allele-specific evolutionarily conserved nucleotide groups. In general, the results of comparative structural analysis of allelic variants testify to a high genetic similarity of the unisexual and bisexual lizard species studied and reveals the characteristic features of their interspecies variability.

Molecular characterization of allelic variants of (GATA)n microsatellite loci in parthenogenetic lizards Darevskia unisexualis (Lacertidae).

Populations of parthenogenetic lizards of the genus Darevskia consist of genetically identical animals, and represent a unique model for studying the molecular mechanisms underlying the variability and evolution of hypervariable DNA repeats. As unisexual lineages, parthenogenetic lizards are characterized by some level of genetic diversity at microsatellite loci. We cloned and sequenced a number of (GATA)n microsatellite loci of Darevskia unisexualis. PCR products from these loci were also sequenced and the degree of intraspecific polymorphism was assessed. Among the five (GATA)n loci analysed, two (Du215 and Du281) were polymorphic. Cross-species analysis of Du215 and Du281 indicate that the priming sites at the D. unisexualis loci are conserved in the bisexual parental species, D. raddei and D. valentini. Sequencing the PCR products amplified from Du215 and Du281 and from monomorphic Du323 showed that allelic differences at the polymorphic loci are caused by microsatellite mutations and by point mutations in the flanking regions. The haplotypes identified among the allelic variants of Du281 and among its orthologues in the parental species provide new evidence of the cross-species origin of D. unisexualis. To our knowledge, these data are the first to characterize the nucleotide sequences of allelic variants at microsatellite loci within parthenogenetic vertebrate animals.

[Finding of Bov-B LINE retroelement in parthenogenetic and bisexual lizard species of the genus Darevskia (Lacertidae)].

The Bov-B LINE retrotransposon was first discovered in Ruminantia and was long considered to be specific for this order. Later, this mobile element was described in snakes and some lizard species. Analysis of phylogenetic relationships of Bov-B LINE elements from different ruminants, snakes, and lizard species led to the suggestion on horizontal transfer of this retrotransposon from Squamata to Ruminantia. In the Squamata group, Bov-B LINE element was found in all snakes and some lizard species examined. The element was not detected in the genomes of some species of the genera Lacerta and Podarcis. In the present study, using PCR amplification and sequencing of PCR products, Bov-B LINE element was identified in the genomes of parthenogenetic and bisexual species of the genus Darevskia (Lacertidae), as well as in such species as Lacerta agilis and Zootoca vivipara, where this retrotransposon had not been not detected before.

[Study of allelic polymorphism of (GATA)n-containing loci in parthenogenetic lizards Darevskia unisexualis (Lacertidae)]. / Izuchenie allel'nogo polimorfizma (GATA)n-soderzhashchikh lokusov partenogeneticheskikh iashcherits Darevskia unisexualis (sem. Lacertida).

The genesis of mini- and microsatellite loci, which is under extensive study in humans and some other bisexual species, have been virtually overlooked in species with clonal mode of reproduction. Earlier, using multilocus DNA fingerprinting, we have examined variability of some mini- and microsatellite DNA markers in parthenogenetic lizards from the genus Darevskia. In particular, mutant (GATA)n-restrictive DNA fragments were found in Darevskia unisexualis. In the present study, we examined intraspecific polymorphism of three cloned loci of D. unisexualis--Du323, Du215, and Du281--containing (GATA)7GAT(GATA)2, GAT(GATA)9, and (GATA)10TA(GATA) microsatellite clusters, respectively. Different levels of intrapopulation and interpopulation variability of these loci were found. Locus Du281 showed the highest polymorphism--six allelic variants (in the sample of 68 DNA specimens). Three alleles were found for locus Du215. The Du325 locus was electrophoretically invariant. The primers chosen for loci Du323, Du215, and Du281 were also used for PCR analysis of homologous loci in two presumptive parental bisexual species, D. valentini and D. nairensis. The PCR products of the corresponding loci of the parental species had approximately the same size (approximately 200 bp) as their counterparts in D. unisexualis, but the polymorphism levels of the paternal, maternal, and hybrid species were shown to be somewhat different. These data on the structure of the D. unisexualis loci provide a possibility to study genetic diversity in the parthenogenetic species D. unisexualis and other related unisexual and bisexual species of this genus, which can provide new information on the origin of parthenogenetic species and on the phylogenetic relationships in the genus Darevskia. These data can also be used for resolving problems of marking the lizard genome, which is still poorly studied.

Instability of (GATA)n microsatellite loci in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae).

Mini- and microsatellites, comprising tandemly repeated short nucleotide sequences, are abundant dispersed repetitive elements that are ubiquitous in eukaryotic genomes. In humans and other bisexual species hypervariable mini- and microsatellite loci provide highly informative systems for monitoring of germline and somatic instability. However, little is known about the mechanisms by which these loci mutate in species that lack effective genetic recombination. Here, multilocus DNA fingerprinting was used to study M13 minisatellite and (GATA)n microsatellite instability in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae). DNA fingerprinting of 25 parthenogenetic families, from six isolated populations in Armenia (comprising a total of 84 siblings), using the oligonucleotide (GATA)4 as a hybridization probe, revealed mutant fingerprinting phenotypes in 13 siblings that differed from their mothers in several restriction DNA fragments. In three families (8 siblings), the mutations were present in the germline. Moreover, the mutant fingerprint phenotypes detected in siblings were also present in population DNA samples. No intrafamily variations in DNA fingerprint patterns were observed with the M13 minisatellite probe. Estimates of the mutation rate for (GATA)n microsatellite loci in D. unisexualis showed that it was as high as that seen in some bisexual species, reaching 15% per sibling or 0.95% per microsatellite band. Furthermore, in one case, a somatic (GATA)n microsatellite mutation was observed in an adult lizard. These findings directly demonstrate that mutations in (GATA)n microsatellite loci comprise an important source of genetic variation in parthenogenetic populations of D. unisexualis.

[Superinstability (TCT/TCC)n of microsatellite DNA in parthenogenetic lizards Darevskia unisexualis (family Lacertidae)]. / Supernestabil'nost' (TCC/TCC)n mikrosatellitnykh DNK u partenogeneticheskikh iashcherits Darevskia unisexualis (sem. Laceridae).

Using multilocus DNA fingerprinting, we have examined variability of (TCT)n microsatellite and M13 minisatellite DNA repeats in populations, families, and tissues of Caucasian parthenogenetic rock lizards Darevskia unisexualis (Lacertidae). It has been shown for the first time that population and family DNA samples of D. unisexualis (75 samples in total) have individually specific DNA fingerprinting patterns of (TCT)n fragments. Analysis of inheritance of (TCT)n microsatellites in 46 first-generation progeny in 17 parthenogenetic D. unisexualis families revealed their extremely high instability. Mutant TCT fingerprint phenotypes were found in virtually each animal of the progeny. Moreover, varying fragments in the progeny and their original variants in the mothers were shown to simultaneously contain (TCT)n and (TCC)n polypyrimidine clusters. At the same time, no variability of (TCT)n fragments has been detected in the tissues and organs of mature parthenogenetic lizards and in the analogous tissues of the two-week-old progeny of this year. This suggests the absence of somatic mosaicism and methylation of the corresponding loci in the samples. Along with the hyperinstability of (TCT/TCC)n polypyrimidine clusters, we have shown that the population and family DNA fingerprinting patterns of M13 minisatellites were invariable and monomorphic in the same DNA samples of D. unisexualis. Our results indicate that mutations at loci containing polypyrimidine microsatellites significantly contribute to the total genomic variability of parthenogenetic lizards D. unisexualis.