[A study of genetic variants in patients with ischemic stroke in human rat gene orthologues]. / Izuchenie geneticheskikh variantov u patsientov s ishemicheskim insul'tom v chelovecheskikh ortologakh krysinykh genov.

OBJECTIVE: To study the associations of nine genetic variants with the risk and dynamics of recovery (outcome) of ischemic stroke (IS) using the developed protocol for the search for genomic markers based on a bioinformatic approach to the study of single nucleotide polymorphisms (SNPs) in human orthologues of rat genes differentially expressed under conditions of induced cerebral ischemia. MATERIAL AND METHODS: We identified and analyzed nine SNPs in 553 Russians (331 IS patients and 222 controls). The National Institutes of Health Stroke Scale (NIHSS) was used to assess stroke severity. Functional recovery after stroke was assessed using the modified Rankin scale (mRS). The principles of selection of polymorphic markers analyzed in the study were determined according to the protocol developed by us earlier. Selected SNP tags were genotyped using real-time polymerase chain reaction (PCR) TaqMan. RESULTS: The relationship of SNP with both the risk of IS and the dynamics of its recovery was investigated. SNP rs66782529 (LGALS3) was associated with negative IS outcomes (p=0.048). SNPs rs62278647 and rs2316710 (PTX3) were significantly associated with IS risk (p=0.000029 and p=0.0025, respectively). The associations for rs62278647 and rs2316710 were found only in females, suggesting a gender-related PTX3 polymorphism. CONCLUSION: This study not only reveals some new genetic links to IS and its consequences, but also shows how the study of gene variations in a rat model of cerebral ischemia can be useful in the search for genetic markers of this disease in humans.

[The Peptide Drug ACTH(4-7)PGP (Semax) Suppresses mRNA Transcripts Encoding Proinflammatory Mediators Induced by Reversible Ischemia of the Rat Brain].

Due to its nootropic, neuroprotective, and immunomodulatory effects, the peptide Semax is utilized in the treatment of ischemic stroke. Our earlier RNA-Seq analysis of the transcriptome in an ischemic model of transient occlusion of the middle cerebral artery showed an increase in the mRNA levels of many proinflammatory genes, and the suppression of their induction by Semax. However, for many relevant genes, including Il1a, Il1b, Il6 and Tnfa, the levels of their expression were too low for detailed quantitative evaluation. Here we utilize qRT-PCR to analyze the effects of the Semax peptide on the expression of weakly expressed mRNAs encoding several proinflammatory mediators, and show that exposure to Semax leads to a statistically significant decrease in the Il1a, Il1b, Il6, Ccl3, and Cxcl2 mRNAs, which compensates for the increase in the transcription of these genes induced by ischemia-reperfusion. We conclude that the observed protective effect of Semax in the model of stroke may be due to its anti-inflammatory effects. We also discuss the limitations of the RNA-Seq when applied to quantifying less abundant transcripts as compared to the real-time RT-PCR method.

Neuroprotective Effects of Peptides in the Brain: Transcriptome Approach.

The importance of studying the action mechanisms of drugs based on natural regulatory peptides is commonly recognized. Particular attention is paid to the peptide drugs that contribute to the restoration of brain functions after acute cerebrovascular accidents (stroke), which for many years continues to be one of the main problems and threats to human health. However, molecular genetic changes in the brain in response to ischemia, as well as the mechanisms of protective effects of peptides, have not been sufficiently studied. This limits the use of neuroprotective peptides and makes it difficult to develop new, more efficient drugs with targeted action on brain functions. Transcriptome analysis is a promising approach for studying the mechanisms of the damaging effects of cerebral ischemia and neuroprotective action of peptide drugs. Beside investigating the role of mRNAs in protein synthesis, the development of new neuroprotection strategies requires studying the involvement of regulatory RNAs in ischemia. Of greatest interest are microRNAs (miRNAs) and circular RNAs (circRNAs), which are expressed predominantly in the brain. CircRNAs can interact with miRNAs and diminish their activity, thereby inhibiting miRNA-mediated repression of mRNAs. It has become apparent that analysis of the circRNA/miRNA/mRNA system is essential for deciphering the mechanisms of brain damage and repair. Here, we present the results of studies on the ischemia-induced changes in the activity of genes and peptide-mediated alterations in the transcriptome profiles in experimental ischemia and formulate the basic principles of peptide regulation in the ischemia-induced damage.

[The association of hemostasis system genes with the development of ischemic stroke in patients under the age of 50 years]. / Assotsiatsiia genov sistemy gemostaza s riskom razvitiia ishemicheskogo insul'ta u patsientov v vozraste do 50 let.

AIM: To study an influence of polymorphic variants of hemostasis system genes on the risk of ischemic stroke (IS) in patients of the Slavic population under the age of 50 years. MATERIAL AND METHODS: Ninety-two patients (19 women and 73 men), aged 18-50 years, were examined. The diagnosis of stroke was confirmed by neuroimaging (CT or MRI) in all patients. Polymorphic alleles of GP1BA, F2, F5 were studied by a real-time polymerase chain reaction using the TaqMan technology. RESULTS AND CONCLUSION: An analysis of the GP1BA -5T/C polymorphism showed that it was associated with IS in young men, lacunar stroke and stroke due to thrombosis of the brachiocephalic arteries. This association was not found in young women. The F5 G1691A polymorphism was associated with lacunar stroke. The F2 G20210A polymorphism was associated with stroke due to thrombosis of the brachiocephalic arteries.

[Effects of ischemia on the expression of neurotrophins and their receptors in rat brain structures outside the lesion site, including on the opposite hemisphere].

Neurotrophins stimulate the regeneration of neural tissue after lesions. It is also known that the sources of neurogenesis and cerebral function recovery are predominantly located in subcortical brain structures. The effects of ischemia on the expression of genes that encode neurotrophins (Bdnf, Ngf, Nt-3) and their receptors (TrkB, TrkA, TrkC, p75) in brain structures outside the lesion site were studied 3, 24, and 72 h after irreversible unilateral occlusion of the middle cerebral artery in rats. Changes in the mRNA expression of these genes were assessed by relative quantification using real-time RT-PCR. Sham surgery was found to stimulate the expression of genes that encode neurotrophins (Bdnf, Ngf) and their receptor (p75). It has been shown that ischemia influenced the expression of neurotrophins (Bdnf, Ngf, Nt-3) and their receptors (TrkB, TrkA, TrkC, p75) in brain structures outside the lesion focus, including the contralateral hemisphere. The downregulation of Bdnf and TrkB transcripts and Ngf and TrkA upregulation in the contralateral cortex on the first day of ischemia obviously reflected stress response. On day 3, Nt-3 transcription increased in all investigated structures outside the lesion focus. In the contralateral hemisphere, relative levels of TrkA and TrkC mRNA expression increased, while p75 expression decreased. Presumably, the observed changes in gene transcription serve to facilitate neuroplasticity and neural tissue regeneration.

ITPR1 gene p.Val1553Met mutation in Russian family with mild Spinocerebellar ataxia.

BACKGROUND: Spinocerebellar ataxias (SСAs) are a highly heterogeneous group of inherited neurological disorders. The symptoms of ataxia vary in individual patients and even within the same SCA subtype. A study of a four-generation family with autosomal dominant (AD) non-progressive SCA with mild symptoms was conducted. The genotyping of this family revealed no frequent pathogenic mutations. So the objective of this study was to identify the genetic causes of the disease in this family with the technology of whole-exome sequencing (WES). METHODS AND RESULTS: WES, candidate variant analysis with further Sanger sequencing, mRNA secondary structure prediction, and RSCU analysis were performed; a heterozygous missense mutation in ITPR1 was identified. CONCLUSION: Our study confirms the fact that ITPR1 gene plays a certain role in the pathogenesis of SCAs, and, therefore, we suggest that c.4657G>A p.Val1553Met) is a disease-causing mutation in the family studied.

PECULIARITIES OF THE STRUCTURE AND EXPRESSION OF HUMAN SPHINGOMYELIN SYNTHASE 1 GENE (SGMS1).

The article is a review of the results of the study of the structural and functional organization of the human sphingomyelin synthase 1 gene (SGMS1) in Human Molecular Genetics Department of Institute of Molecular Genetics RAS. SGMS1 gene encodes an essential enzyme which is involved in the synthesis of sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide, wich determines its participation in the regulation of intracellular vesicular transport, cholesterol metabolism, cell proliferation, apoptosis and other significant processes. Our research has shown that the SGMS1 gene is located on the chromosome 10, has a size of 320 kb and contains more than 20 exons. A detailed study of the SGMS1 gene's structure allowed us to identify the variety of its transcripts. mRNA isoforms with different fragments of 5R untranslated region (5R UTR) and encoding the full length protein, as well as transcripts resulting from alternative combinations of exons and containing the coding region of the gene and 3R UTR have been discovered. We have found new transcripts among the products of SGMS1 gene ­ circular RNAs, which mostly contained sequences of multi-exon 5R UTR of the gene. They are conservative and predominantly expressed in the brain. Circular RNAs of SGMS1 gene had a large number of binding sites for a microRNA that may determine the functional significance of these molecules. The review describes the latest information about the structural and functional organization of the human gene SGMS1 as well as the features of its expression.

[POSSIBLE ROLE OF TRANSTHYRETIN IN THE BIOLOGICAL MECHANISM OF THE REGULATORY PEPTIDE NEUROPROTECTION.]

The peptide preparation Semax has been effectively used for therapy of ischemic stroke. However, the mechanisms of its action are insufficiently understood and actively studied. The full-genome analysis of the transcriptome implemented in our recent work dem- onstrated that under conditions of focal ischemia of rat brain the Semax modified the profile of the transcription activity of many genes. In this case, the difference in the transcription levels of the gene encoding the protein transthyretin (Ttr) expression in rats under the pathological conditions of ischemia and in the presence of Semax was very high. High similarity between the effects of Ttr and coupled molecular systems with the Semax effects in ischemic stroke allowed us to suggest that the neuroprotection mechanisms of Semax (and, possibly, of other neuroprotection mechanisms of Semax) could be mediated by Ttr. In this review, we discussed the role of Ttr in CNS and its possible role in the neuroprotection mechanism of Semax.

[Alternative promoters localised in SGMS1 gene introns take part in regulation of its expression in human tissues].

Sphingomyelin synthase 1 (SMS1) is an enzyme of vital importance which is responsible for the synthesis of sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide in eukaryotic cells. Previously we have investigated in detail the structure of SGMS1 human gene and identified a lot of its transcripts. We revealed the isoforms of mRNA differing in the 5'-UTR and coding the full-length protein, and also the transcripts arising from alternative combination of the exons localized in the coding region of the gene and 3'-UTR. From the results of computer analysis it follows that the synthesis of transcripts differing in the 5'-UTR is enabled by the different promoters of SGMS1 gene. It has been found in the present work by the method of real-time PCR that the content of five alternative transcripts of this gene, differing in the 5'-UTR, is substantially dissimilar among human tissues. In all the investigated tissues those transcripts are presented most prominently whose synthesis takes place under the control of the distal promoter including exon 1. In lesser extent are presented the transcripts including 5'-end exons whose synthesis is enabled by the promoters localized in introns of this gene. The differential level of content of SGMS1 gene transcripts, differing in the 5'-UTR, indicates that the use of the alternative promoters is tissue-specific and apparently strictly regulated. The structural organization of 5'-UTR variants of SGMS1 transcripts, directed by alternative promoters, is substantially different; this can provide regulation of the gene functioning on post-transcriptional level.

[Analysis of mutations in patients with suspected autosomal dominant form of the Parkinson disease].

The Parkinson disease (PD) is a severe neurological disorder. Diverse genetic systems and environmental factors are involved in the pathogenesis of this disease. However, despite extensive research into the disease, its causes are not fully elucidated, and the exact spectrum of genes and mutations involved in the development of hereditary forms of PD has not been fully clarified yet. The present work is devoted to the analysis of mutations that lead to the development of monogenic forms of PD in patients with suspected autosomal dominant form of PD using Multiplex Ligation-dependent Probe Amplification (MLPA). We have identified several mutations (G2019S in LRRK2, heterozygous deletions of 2-3, 3-4 exons and heterozygous duplication of 2-4 exons in PARK2, deletion of 3 exon in PARK7) that lead to the development of PD in only 7 people out of 70 (18.4%), which suggests the need for further search of new mutations, for example, using exome sequencing. In the future it will help to develop the molecular genetic tests for early preclinical diagnosis and risk evaluation of the development of PD, and to understand better the causes and mechanisms of this disease.

[Expression of sphingomyelin synthase 1 (SGMS1) gene varies in human lung and oesophagus cancer].

The investigation of molecular mechanisms contributing to cancer progression is the burning problem ofcurrent research. Considerable attention has been given to the study of gene expression in cancer cells. Sphingomyelin synthase 1 gene (SGMS1) is one of the genes whose expression can be altered in cancer. SMS1 enzyme, encoded by this gene, catalyzes the synthesis of sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide. SMS1 may maintain the balance between cell death and survival by regulating the formation of the pro-apoptotic mediator ceramide and anti-apoptotic mediator diacylglycerol. In addition, the changes in sphingomyelin level and sphingomyelin synthase activity have been observed in cancers of many tissues. However the peculiarities of SGMS1 gene transcription have been insufficiently explored. In this work the expression of transcripts of SGMS1 has been investigated by the method of Real Time PCR in matched pairs of samples of human lung and oesophagus cancer and adjacent tissues without pathology. A significant decrease in SMS1 transcripts expression has been found in samples of human lung cancer. At the same time, in the samples of human oesophagus cancer and adjacent tissue, expression of SMS1 transcripts varies insignificantly: it is increased in 7 and decreased in 5 of 15 samples. The obtained results indicate that SGMS1 gene is differently expressed in cancers of different genesis.

Molecular mechanisms of pathogenesis of Parkinson's disease.

Parkinson's disease is a complex disease characterized by a progressive degeneration of nigrostriatal dopaminergic neurons. The development of this condition is defined by the interaction between the genetic constitution of an organism and environmental factors. Analysis of the genes associated with development of monogenic forms of disease has allowed pointing out several mechanisms involved in Parkinson's disease pathogenesis such as the ubiquitin-proteasome degradation, differentiation of dopaminergic neurons, mitochondrial dysfunction, oxidative damage, and others. In this review, a variety of data which throw light on molecular mechanisms underlying pathogenesis of Parkinson's disease will be considered.

Genetic polymorphisms and the efficacy and toxicity of cisplatin-based chemotherapy in ovarian cancer patients.

Platinum drugs are among the most active and widely used agents in the treatment of different cancers. However, the great individual variability in both outcome and toxicity of platinum chemotherapy requires the identification of genetic markers that can be used to screen patients before treatment. In this study, 21 polymorphisms in 10 genes, the protein activities of which may be addressed in different aspects of cisplatin metabolism, were tested for correlations with efficacy and toxicity of cisplatin-cyclophosphamide regimen in 104 ovarian cancer patients. The glutathione S-transferase P1 (GSTP1) Ile105Val polymorphism was strongly associated with progression-free survival (chi(2)=12.12, P=0.002). The allelic status of the GSTA1 -69 C>T polymorphism correlated with the overall survival: patients with T/T genotype survived longer than C/C carriers (P=0.044). Thrombocytopenia, anemia and neuropathy were less frequent among patients with the GSTM1-null or GSTM3 intron 6 AGG/AGG genotypes. Severe neutropenia was associated with the TP53 72 Pro/Pro, XPD 312 Asp/Asn and XRCC1 399 Arg/Arg genotypes. A higher risk of nephrotoxicity was noted for patients with the heterozygous ERCC1 19007 T/C and 8092 C/A genotypes. No correlations were found between genotypes and complete tumor responses.

A common leucine-rich repeat kinase 2 gene mutation in familial and sporadic Parkinson's disease in Russia.

A PARK8 form of Parkinson's disease (PD) is caused by a novel gene, leucine-rich repeat kinase 2 (LRRK2), and a single mutation G2019S was found in a proportion of LRRK2-associated cases of diverse ethnic origins. We performed the LRRK2 G2019S mutation analysis in 304 Russian patients with PD, including 291 sporadic and 13 autosomal dominant cases. The frequency of the LRRK2 G2019S was 0.7% amongst the sporadic patients (2/291) and 7.7% amongst familial PD (1/13). The mutation was also found in three unaffected relatives and absent in 700 control chromosomes. One patient carrying the LRRK2 G2019S was found earlier to have an additional mutation, a heterozygous duplication of exon 5 of the parkin gene. All patients carrying the LRRK2 G2019S exhibited typical levodopa-responsive parkinsonism, and severe levodopa-induced dyskinesia was observed in the patient carrying the LRRK2 and parkin mutations. There was notable variability in ages of the disease onset in G2019S carriers not explained by APOE genotypes. Two subsets of G2019S-positive patients had different PARK8 haplotypes suggesting that the LRRK2 G2019S in Russian patients had arisen independently on different chromosomes. Identification of common LRRK2 mutations in some PD patients without an overt family history has notable implications for genetic counseling.

Polymorphisms at locus D1S80 and other hypervariable regions in the analysis of Eastern European ethnic group relationships.

BACKGROUND: It has been hypothesized that, whereas many loci are used to generate phylogenetic relationships, the utilization of those that yield the most information could increase the accuracy of any multilocus phylogenetic reconstruction. Among these is the D1S80 hypervariable minisatellite region, which has been shown to be highly polymorphic globally, and it was of interest to compare the nearest neighbours and distant populations of Eastern Europe using the D1S80 polymorphism. AIM: The study evaluated the capacity of the D1S80 locus to discriminate between populations from different ethnic groups in Russia and the Republic of Belarus, revealing the polymorphism parameters of the populations studied. SUBJECTS AND METHODS: Hypervariable D1S80 minisatellite polymorphism was studied in 15 populations, belonging to six distinct ethnic groups from the Russian Federation (Russians, Komis, Maris, Udmurts, Kalmyks, and Yakuts) and the Republic of Belarus (Byelorussians). The data were analysed with other results reported for D1S80 polymorphism among Eastern Europeans, and were analysed together with those previously reported for Eastern European populations for the 3'ApoB, DMPK, DRPLA, and SCA1 hypervariable loci. Genetic diversity analysis was carried out using multidimensional scaling (MDS) of Nei's genetic distances. RESULTS: The Eastern Slavonic populations (Russians, Ukrainians, and Byelorussians) are closely associated, and outermost from populations of Asian origin (Kalmyks and Yakuts). The populations that inhabit the Volga-Ural region (Udmurt, Komi, Mari, and Bashkir ethnic groups) revealed intermediate characteristics. CONCLUSION: The clustering of populations demonstrated here using D1S80 alone coincides with the analysis of five hypervariable region (HVR) loci, and is consistent with linguistic, geographic, and ethnohistorical data. These results are in agreement with most studies of mtDNA, Y-chromosomal, and autosomal DNA diversity in Eastern Europe. The D1S80 locus is convenient for population analyses, and may be used as part of a set of similar markers, which should allow the easy resolution of small differences in population structures.

Variability of the 3'APOB minisatellite locus in Eastern Slavonic populations.

OBJECTIVE: To describe and compare the 3' apolipoprotein (Apo) B minisatellite allele frequency distributions of Eastern Slavonic populations and their Uralic, Altaic, and Caucasian speaking neighbors. METHODS: Healthy individuals of 10 populations among Russians, Byelorussians, Komis and Bashkirs were studied for variable number tandem repeats (VNTRs) in the 3'ApoB minisatellite region. Data were analyzed with other results reported for this polymorphism in eastern Europeans and Siberians. RESULTS: Allele frequency spectra in Eastern Slavonic, Northern Caucasian and Finno-Ugric speaking populations are bimodal with the main peak in alleles 34-36 and a secondary mode around allele 48, whereas Altaic speaking populations have a unimodal allele frequency distribution with a peak of around 34-36 VNTRs. Population relationships were revealed using both multidimensional scaling analysis (based on Nei's genetic distance estimate) and testing for genetic heterogeneity. Eastern Slavonic populations (Russians, Ukrainians, Byelorussians) were most closely related to each other and formed a separate tight clusterwhen plotted. Testing for genetic heterogeneity among the Eastern Slavonic ethnic groups revealed maximum diversity among Byelorussians, followed by Russians, then Ukrainians. The 3'ApoB minisatellite variability reveals little heterogeneityamong the Eastern Slavonic ethnic groups, whereas there wassignificant heterogeneity for Northern Caucasian and Altaic speakers. CONCLUSION: For this 3'ApoB polymorphism the Eastern Slavonic populations, despite their wide geographical distribution, appear to be much more homogenous than other ethnic groups of the region. Multidimensional scaling analysis of these data allowed for differentiation between individual populations from an ethnic group even if there is little heterogeneity.

p53 polymorphisms in Russia and Belarus: correlation of the 2-1-1 haplotype frequency with longitude.

Four different polymorphisms in the human p53 gene (a 16-bp duplication in intron 3, and three RFLPs: for Bsh1236I at codon 72, for MspI in intron 6 and for BamHI in the 3' flanking region) and extended haplotypes were studied in nine geographically diverse populations from Russia and Belarus. The Yakuts differed from all other populations, as they had a significantly higher frequency of the BamHI A1 allele. Most populations did not differ significantly from each other in the frequency of the Bsh1236I polymorphism. The 16-bp duplication A1 allele and MspI A2 allele frequencies were significantly higher in the Yakut and Khant populations. Linkage disequilibrium values (D') between BamHI and other polymorphic sites were not significant in many cases; for this reason we have used the 16 bp-Bsh1236I-MspI haplotype frequencies only. Of eight possible haplotypes, five were observed in the populations investigated. Haplotype 1-2-2 was the most frequent in all populations. The next most common haplotype, 1-1-2, was present at very similar frequencies among the Byelorussians and Russians from Smolensk, but was more frequent in other populations. The frequency of haplotype 2-1-1 showed a nearly continuous decrease from West to East (from 17.857% among the Byelorussians to 0.685% in the Yakuts from the Verkhoyansk) and correlated with longitude (Spearman's r = -0.8667, P = 0.0025), which may be due to natural selection and adaptation. The relationships among populations were evaluated by means of Nei's D(A) distances for the 16 bp-Bsh1236I-MspI haplotype frequencies. Based on the multidimensional scaling analysis a correlation between p53 haplotype frequencies and ethnicity is supposed.

Connection between p53 gene Bam HI RFLP polymorphism with the volume of brain infarction in patients with carotid atherothrombotic ischemic stroke.

PURPOSE: The aim of our investigation was to study the connection between p53 gene Bam HI RFLP polymorphism and the brain infarction volume in patients with atherothrombotic ischemic stroke that could highlight certain genetic aspects of the individual sensibility of brain tissue to acute ischemia. MATERIALS AND METHODS: Diallelic Bam HI RFLP polymorphism in 5' flanking region p53 gene was studied in 96 patients with carotid atherothrombotic stroke from Moscow population. Magnetic resonance imaging was conducted on day 7 after the stroke onset. The manual morphometry and "Osiris" morphometric hardware (by the Hospital of the University of Geneva) were used for assessment of the infarction volume. RESULTS: The predominance of small-size infarctions (< 40 cm3) was revealed in patients with (-/-) Bam HI RFLP p53 genotype versus patients with (-/+) (X2 = 19.7; P < 0.001) and (+/+) (X2 = 12.288; P < 0.001) genotypes. According to the Bayesian's statistics, in patients with (-/-) p53 Bam HI genotype the development of a small-size infarction in atherothrombotic ischemic stroke can be prognosticated with probability more than 65%. CONCLUSIONS: A significant association between p53 gene Bam HI RFLP polymorphism and the infarction volume was found in patients with carotid atherothrombotic stroke from Moscow population. These results additionally confirm that apoptosis plays an important role in the formation of ischemic brain lesion and that drugs with anti-apoptotic properties may prove beneficial in stroke patients.