V. Alifirova

NMDA receptor genotypes associated with the vulnerability to develop dyskinesia

Dyskinesias are involuntary muscle movements that occur spontaneously in Huntingtons disease (HD) and after long-term treatments for Parkinsons disease (levodopa-induced dyskinesia; LID) or for schizophrenia (tardive dyskinesia, TD). Previous studies suggested that dyskinesias in these three conditions originate from different neuronal pathways that converge on overstimulation of the motor cortex. We hypothesized that the same variants of the N-methyl-D-aspartate receptor gene that were previously associated with the age of dyskinesia onset in HD were also associated with the vulnerability for TD and not LID. Genotyping patients with LID and TD revealed, however, that these two variants were dose-dependently associated with susceptibility to LID, but not TD. This suggested that LID, TD and HD might arise from the same neuronal pathways, but TD results from a different mechanism.

Association of SNPs of CD40 Gene with Multiple Sclerosis in Russians

Multiple sclerosis (MS) is a serious, incurable neurological disease. In 2009, the ANZgene studies detected the suggestive association of located upstream of CD40 gene in chromosome 20q13 (p = 1.3×10−7). Identification of the causal variant(s) in the CD40 locus leads to a better understanding of the mechanism underlying the development of autoimmune pathologies. We determined the genotypes of rs6074022, rs1883832, rs1535045, and rs11086996 in patients with MS (n = 1684) and in the control group (n = 879). Two SNPs were significantly associated with MS: rs6074022 (additive model C allele OR = 1.27, 95% CI = [1.12–1.45], p = 3×10−4) and rs1883832 (additive model T allele OR = 1.20, 95% CI = [1.05–1.38], p = 7×10−3). In the meta-analysis of our results and the results of four previous studies, we obtain the association p-value of 2.34×10−12, which confirmed the association between MS and rs6074022 at a genome-wide significant level. Next, we demonstrated that the model including rs6074022 only sufficiently described the association. From our analysis, we can speculate that the association between rs1883832 and MS was induced by LD, whereas rs6074022 was a marker in stronger LD with the functional variant or was the functional variant itself. Our results indicated that the functional variants were located in the upstream region of the gene CD40 and were in higher LD with rs6074022 than LD with rs1883832.

Replicative association analysis of genetic markers of cognitive traits with Alzheimer’s disease in the Russian population

A replicative association analysis of Alzheimer’s disease (AD) was carried out for 15 genetic markers that have been associated with cognitive disorders in genome-wide association studies. In the Russian population, AD was associated with CSMD1 rs2616984 (OR = 1.50, 95% CI 1.07–2.09, p-value = 0.018) and, potentially, with NOTCH4 rs313296 (OR = 1.53, 95% CI 0.98–2.39, p-value = 0.06) and NRIP1 rs2229741 (OR = 1.35, 95% CI 0.99–1.85, p-value = 0.061). Combinations of epistatically interacting genes (CSMD1 and NRIP1; NOTCH4, CSMD1, and NRIP1; and TLR4, CSMD1, and NRIP1) were identified, along with their genotype combinations that showed a significant association with AD and the highest predictive values. Possible molecular mechanisms of the gene involvement in AD pathogenesis are discussed. A bioinformatics analysis of the biological processes, molecular functions, and protein-protein interactions for the AD genes indicated that the genes may play a modulating or modifying role, acting together in various regulatory and signaling pathways involved in AD.

A rare variant in the sortilin-related receptor 1 gene is associated with declined cognitive functions in the elderly

AIM To estimate the association of rs11218343 in the sortilin-related receptor 1 (SORL1) gene with cognitive performance in the elderly and with Alzheimers disease (AD) in the Russian population. MATERIAL AND METHODS A sample included 586 elderly people (mean age 70.9±5.7 years) without AD diagnosis and 100 patients with late-onset AD (mean age 72.1±7.8 years) from the Tomsk population. SORL1 rs11218343 was genotyped using PCR and MALDI-TOF mass spectrometry. Cognitive performance in the sample of elderly without AD was assessed by Montreal Cognitive Assessment (MoCA) test. RESULTS Allele frequencies of the SORL1 polymorphism were not significantly different between the elderly without AD and AD patients. However mean MoCA score in the carriers of the rare allele (19.00±6.61) was significantly lower than in homozygotes for the common variant (22.25±3.89) (F=4.97; p=0.026). CONCLUSION The rare variant in SORL1 gene previously associated with AD in genome-wide association studies and meta-analyses was associated with lower total МоСА scores in the random sample of elderly people that suggests declined cognitive functions in the carriers of this variant in elderly.

Predictive genetic model for levodopa-induced dyskinesia in patients with Parkinson's disease

Predictive genetic model for levodopa-induced dyskinesia in patients with Parkinsons disease S.A. Ivanova(1), V.M. Alifirova(2), M.B. Freidin(3), I.V. Pozhidaev(4), O.Y. Fedorenko(4), N.A. Bokhan(4), I.A. Zhukova(5), N.G. Zhukova(5), B. Wilffert(6), A.J.M. Loonen(6) (1)Mental Health Research Institute, Molecular Genetics and Biochemistry Laboratory, Tomsk, Russia (2)Siberian State Medical University, Department of Neurology and Neurosurgery, Tomk, Russia (3)Tomsk National Research Medical Center of the Russian Academy of Sciences, Research Institute of Medical Genetics, Tomsk, Russia (4)Tomsk National Research Medical Center of the Russian Academy of Sciences, Mental Health Research Institute, Tomsk, Russia (5)Siberian State Medical University, Department of Neurology and Neurosurgery, Tomsk, Russia (6)University of Groningen, Groningen Research Institute of Pharmacy, Groningen, The Netherlands Parkinsons disease (PD), a common neurodegenerative disorder caused by the loss of the dopaminergic input to the basal ganglia, is commonly treated with levodopa (L-DOPA). The use of this drug, however, is severely limited by adverse effects. Levodopa-induced dyskinesia (LID) is one of these and characterized by involuntary muscle movements that occur as a consequence of chronic levodopa treatment. LID is a substantial barrier to effective symptomatic management of PD as up to 45% of L-DOPA users develop LID within 5 years [1]. Clinical heterogeneity of LID suggests a significant role of endogenous factors in determining their prevalence. Some evidences suggest a relationship between LID and specific genetic variants, such as polymorphisms in the genes controlling enzymes responsible for drug and monoamine metabolism, neurotransmitter receptors and proteins involved in oxidative stress or antioxidant function [2–4]. Objective: To investigate a contribution of polymorphic variants of neurotransmitter receptors and cytochrome genes in the development of LID in PD patients. Methods: A total of 212 PD patients who received L-DOPA therapy were studied. Dyskinesia was assessed by using the Abnormal Involuntary Movement Scale (AIMS). DNA extraction and genotyping were conducted according to standard protocols and blind to the clinical status of the subjects. Genotyping was carried out for 72 SNPs of DRD1, DRD2, DRD2/ANKK1, DRD3, DRD4, HTR2C, HTR3A, HTR3B, HTR6, HTR2A, HTR1A, HTR1B, CYP1A2*1F, CYP2D6*3, CYP2D6*4, CYP2C19*3, CYP2C19*17, CYP2C19*2, and GSTP1 genes using MassARRAY® Analyzer 4 (Agena Bioscience™) and the set SEQUENOM Consumables iPLEX Gold 384. Discriminant analysis and receiver operating curve (ROC)-analysis were carried out to build a genetic predictive model for dyskinesia. Results: Group of PD patients consists of 149 females and 83 males (age ranging from 40 to 86 years, average age 68.7 ± 7.6 years). The mean age of onset is 60.04 ± 9.46 years, average disease duration is 9.79 ± 5.57 years. Dyskinesia was reported in 57 (26.9%) patients. The best discriminant model was obtained with the following predictors: rs11721264, rs165774, rs3758653, rs4245147, rs6313, rs1364043, rs2734849, rs324035, rs6311, rs11246226 and rs4244285. These polymorphisms are localized in the following genes: DRD3 (rs11721264, rs324035), DRD4 (rs3758653, rs11246226), DRD2 (rs4245147, rs2734849), HTR2A (rs6313, rs6311), HTR1A (rs1364043). The discriminant model using this set of SNPs gives the error of classification about 13% and the AUC 0.795. Depending on the anticipated frequency of LID, positive and negative predictor values varied between 0.745–0.834 and 0.864–0.916, respectively. We hypothesized in our previous studies that the pathological basis of LID might be degeneration of indirect pathway medium spiny neurons [5]. These indirect pathway medium spiny neurons carry type 2 family dopamine receptors (DRD2, DRD3, DRD4), and HTR2A receptors. Moreover, dopamine release may be promoted by inhibiting serotonergic neurotransmission. Hence, the current findings are well in line with this hypothesis. Conclusion: The resulting panel of 11 SNPs provides a sufficiently high accuracy of LID prediction. The use of this panel in a prospective study will clarify the prospects for its application in clinical practice for predicting risk of LID in patients with PD. References [1] Rascol, O., Brooks, D.J., Korczyn, A.D., et al., 2000. Study Group: A five–year study of the incidence of dyskinesia in patients with early Parkinsons disease who were treated with ropinirole or L–DOPA. N Engl J Med 342, 1484–1491. [2] Ivanova, S.A., Fedorenko, O.Yu., Freidin, M.B., et al., 2016. Dissimilar mechanistic background of peripheral and orofacial hyperkinesia in patients with Parkinsons disease and levodopa-induced dyskinesia. Physiology and Pharmacology 19, 216–221. [3] Kaplan, N., Vituri, A., Korczyn, A.D., et al., 2014. Sequence variants in SLC6A3, DRD2, and BDNF genes and time to levodopa-induced dyskinesias in Parkinsons disease. J Mol Neurosci 53 (2), 183–188. [4] Loonen, A.J.M., Ivanova, S.A., 2016. Role of 5-HT2C receptors in dyskinesia. International Journal of Pharmacy and Pharmaceutical Sciences 8 (1), 5–10. [5] Loonen, A.J., Ivanova, S.A., 2013. New insights into the mechanism of drug-induced dyskinesia. CNS Spectr 18, 15–20. Keywords: Genetics / Molecular genetics Parkinsons disease levodopa-induced dyskinesia

Dopamine receptors genes polymorphisms in Parkinson patients with levodopa-induced dyskinesia

Dopamine receptors genes polymorphisms in Parkinson patients with levodopa-induced dyskinesia I. Pozhidaev(1), V.M. Alifirova(2), M.B. Freidin(3), I.A. Zhukova(2), O.Y. Fedorenko(1), D.Z. Osmanova(1), Y.S. Mironova(2), B. Wilffert(4), S.A. Ivanova(1), A.J.M. Loonen(5) (1)Mental Health Research Institute, Molecular Genetics and Biochemistry, Tomsk, Russia (2)Siberian State Medical University, Neurology and Neurosurgery, Tomsk, Russia (3)Research Institute for Medical Genetics, Laboratory of Population Genetics, Tomsk, Russia (4)Groningen Research Institute of Pharmacy, Pharmacotherapy and Clinical Pharmacology, Groningen, The Netherlands (5)Groningen Research Institute of Pharmacy, Pharmacotherapy in Psychiatric Patients, Groningen, The Netherlands Introduction: Long-term levodopa treatment of Parkinsons disease (PD) is frequently complicated by spontaneously occurring involuntary muscle movements called levodopa-induced dyskinesia (LID). LID are a substantial barrier to effective symptomatic management of Parkinsons disease (PD), as up to 45% of L-DOPA users develop LID within 5 years [1]. The exact pathological mechanism of this complication has not yet been elucidated. A lot of studies nowadays which approved complex genetic nature of LID. And these genes are involved not only for oxidative stress, but in drug metabolism too [2–4]. Objective: This study aimed to investigate a possible contribution of polymorphic variants of DRD1, DRD2, DRD2/ANKK1, DRD3, DRD4 genes in the development of LID in PD patients. Methods: 212 patients with Parkinsons disease on levodopa therapy were investigated. Dyskinesia was measured by using Abnormal Involuntary Movement Scale (AIMS). DNA extraction and fluorogenic 5′-exonuclease TaqMan genotyping assays were conducted according to standard protocols and blind to clinical status of the subjects. Genotyping was carried out on 28 SNPs of dopamine receptors (rs4532, rs936461, rs6275, rs1801028, rs4245147, rs134655, rs6277, rs1076560, rs2283265, rs179997, rs6279, rs1076562, rs2734842, rs2734849, rs11721264, rs167770, rs3773678, rs963468, rs7633291, rs2134655, rs9817063, rs324035, rs1800828, rs167771, rs6280, rs1587756, rs3758653, rs11246226) on the MassARRAY® Analyzer 4 (Agena Bioscience™) using the set SEQUENOM Consumables iPLEX Gold 384. SPSS software was used for statistical analysis. Statistical significance of the association testing was established using permutations. P-value <0.05 after permutations was considered statistically significant. Results: Patients in our cohort demonstrated typical PD demographics, with a mean age of onset of 60.04 ± 9.46 years, a mean disease duration of 9.79 ± 5.57 years. Dyskinesia was reported in 57 (26.9%) patients. The distribution of genotypes of studied genes corresponded to the Hardy-Weinberg equilibrium. We found that 5 polymorphisms (rs4245147, rs6275, rs2734842, rs6279, rs1076562) are significantly associated with LID. All these polymorphisms are located in DRD2 gene. In logistic regression models adjusted for the covariates, such as age, gender and duration of disease only one of the studied markers was associated with LID (rs4245147). Odds ratio for carriers of the genotype TT is 1.73 [95% CI: 1.12–2.70], which indicates the predisposing effect of this genotype on the development of dyskinesia. Polymorphisms in the dopamine receptors genes play significant role in the therapy response to L-DOPA as well as in various of its adverse effects. We hypothesized that single nucleotide polymorphisms in DR genes may result in a clinical phenotype contributing to an increased risk of LID. This appears to be especially true for rs4245147 of the DRD2 gene. Hence, this gene polymorphism is a good candidate for studying (genetic) biomarkers predicting the risk of developing this movement disorder. Conclusion: Rs4245147 polymorphism of the DRD2 gene is a putative component of a set of biomarkers predicting the vulnerability to develop dyskinesia. References [1] Rascol, O., Brooks, D.J., Korczyn, A.D., et al., 2000. Study Group: A five–year study of the incidence of dyskinesia in patients with early Parkinsons disease who were treated with ropinirole or L–DOPA. N Engl J Med 342, 1484–1491. [2] Ivanova, S.A., Fedorenko, O.Y., Freidin, M.B., et al., 2016. Dissimilar mechanistic background of peripheral and orofacial hyperkinesia in patients with Parkinsons disease and levodopa-induced dyskinesia. Physiology and Pharmacology 19, 216–221. [3] Kaplan, N., Vituri, A., Korczyn, A.D., et al., 2014. Sequence variants in SLC6A3, DRD2, and BDNF genes and time to levodopa-induced dyskinesias in Parkinsons disease. J Mol Neurosci 53 (2), 183–1880. [4] Rieck, M., Schumacher-Schuh, A.F., Altmann, V., et al., 2012. DRD2 haplotype is associated with dyskinesia induced by levodopa therapy in Parkinsons disease patients. Pharmacogenomics 13 (15), 1701–1710. Keywords: Receptors Dopamine Genetics / Molecular genetics

Replicative Analysis Of 30 Snps In Russian Patients With Alzheimer’s Disease

Background Alzheimer’s disease is a highly heritable genetically heterogeneous disorder with 60%–80% of risk attributed to genetic factors. Two forms of the disease are known as Early-onset familial Alzheimers disease and Late-onset sporadic Alzheimers disease. Although scientists know how brain cells of persons with Alzheimers disease are affected, and additionally understand some of the genetic explanations of the disease, the precise cause of this disease is still unclear. There are over 90 Genome-Wide Association Studies for Alzheimers disease. However, only a few associations were replicated in independent data. The aim of this study was to analyze associations of 30 SNPs reported in GWAS with Alzheimer’s disease in Russian population of Siberian region. Methods 108 patients with AD and 285 healthy controls, matched to the patients by age, gender, and ethnicity were included in this study. 30 SNPs were genotyped by MALDI-TOF mass-spectrometry using MassARRAY Analyzer 4 (Sequenom). Allele-specific ORs and associated p-values were calculated. Results We found three significant associations of SNPs with AD in Russian patients of Siberian region: rs17594526 at TCF4 gene (OR = 1.77, p=0.003), rs11064768 at CCDC60 gene (OR = 2.15, p= 0.02) and rs12922317 at SNX29 gene (OR = 1.47, p= 0.02). These genetic markers were previously reported in GWAS associated with schizophrenia. Discussion Only the TCF4 gene has known functional importance for cognitive dysfunctions of schizophrenia and Alzheimers disease. As a transcription factor, the TCF4 gene regulates the expression of other genes involved in cell differentiation, survival, and neurodevelopment. Genetic markers of CCDC60 and SNX29 are associated with Alzheimer’s disease but their role in pathogenesis of the disease is not clear. This work was supported by the Russian Science Foundation (project # 16-14-00020).

[The association of the DRD3 gene with Parkinson's disease].

OBJECTIVE To investigate the association between dopamine receptor DRD3 gene tag single nucleotide polymorphisms (SNPs) and the risk of Parkinsons disease (PD). MATERIAL AND METHODS One hundred and forty-three patients with PD and 96 healthy individuals from the Russian population were examined. Ten tag SNPs (rs963468, rs2134655, rs9817063, rs324035, rs11721264, rs1800828, rs3773678, rs167770, rs167771, rs7633291) within DRD3 have been genotyped. RESULTS AND CONCLUSION Associations between 4 polymorphisms (rs11721264, rs3773678, rs167771, rs324035) and PD have been found. Our study confirms the involvement of polymorphic features of dopamine receptors genes in the pathophysiology in PD.

Polymorphisms of DRD2, DRD3, DRD4 and HTR2C genes in levodopa-induced dyskinesias in Parkinson's disease

Levodopa-induced dyskinesias (LID) are involuntary muscle movements that occur as a consequence of chronic levodopa (L-DOPA) treatment. LID are a substantial barrier to effective symptomatic management of Parkinsons disease (PD), up to 45% of L-DOPA users develop LID within 5 years [1]. Clinical heterogeneity of LID suggests a significant role of endogenous factors in determining their prevalence. Some evidence suggest a relationship between LID and specific genetic changes, such as changes in the genes controlling enzymes responsible for drug metabolism, neurotransmitter receptors, as well as proteins involved in oxidative stress or antioxidant function [2]. Our previous results showed that the susceptibility to LID was associated with two NMDA receptor (GRIN2A) variants [3]. Objective: To investigate contribution of polymorphic variants of HTR2C serotonin receptor gene and DRD2, DRD3 and DRD4 dopamine receptors genes in the development of LID in PD patients. Methods: 143 patients with Parkinsons disease were examined. Dyskinesias were estimated with use of Abnormal Involuntary Movement Scale (AIMS). Orofaciolingual LID (LIDof) and limb-truncal LID (LIDlt) were assessed with AIMS items 1-4 and 5-7, respectively. DNA extraction and fluorogenic 5- exonuclease TaqMan genotyping assays were conducted according to standard protocols and blind to the clinical status of the subjects. Genotyping was carried out on 23 polymorphic variants of DRD2, DRD3, DRD4 and HTR2C genes (rs6275, rs1800497, rs1799732, rs71653615, rs11721264, rs167770, rs3773678, rs963468, rs7633291, rs2134655, rs9817063, rs324035, rs1800828, rs167771, rs3758653, rs6318, rs5946189, rs569959, rs17326429, rs4911871, rs3813929, rs1801412, rs12858300). The softwares “R” and SPSS were used for statistical analysis. The Hardy-Weinberg equilibrium (HWE) of genotypic frequencies was tested by the chi-square test. For the SNPs in the X-chromosomal HTR2C gene, deviation from HWE was not calculated. Results: Associations of 5 polymorphisms with levodopainduced dyskinesias in patients with PD were revealed. Polymorphisms associated with orofaciolingual LID virtually did not overlap with polymorphisms associated with limb-truncal LID, indicating the different genetic basis of these phenotypes. Moreover, LIDof and LIDlt are associated with different genes polymorphisms. LIDof is associated with DRD4 (rs3758653) and HTR2C (rs4911871, rs5946189) genes polymorphisms whereas LIDlt is associated with only one of studied polymorphism. Polymorphisms of DRD3 gene (rs2134655, rs963468) are associated with severity of dyskinesia: rs2134655 - with orofaciolingual LID; rs963468 - with limb-truncal LID, but not with the fact of the presence of dyskinesia itself. Conclusions: Polymorphisms in the genes coding for neurotransmitter receptors play significant roles in the therapy response to L-DOPA as well as in various adverse effects. We hypothesised that single nucleotide polymorphisms in specific genes, particularly those coding for dopamine and serotonin receptors, may result in a clinical phenotype contributing to an increased risk of LID. Thus, the polymorphisms of genes possessing protective or predisposing effects in development of levodopa induced dyskinesia in PD have been revealed that would allow predicting risk of development of movement disorders.

Correlation of concentrations of glutamate in serum samples and disability in multiple sclerosis

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that has been implicated in several biological processes in multiple tissues and cell types and acts as a ligand for five G-protein coupled receptors, generally referred to as S1P1–S1P5. FTY720 is a sphingosine analogue that, after phosphorylation by sphingosine kinases, binds to four of the five known S1P receptors. As an immunomodulatory drug, FTY720 is known to prevent lymphocyte egress through abrogation of S1P signaling via receptor internalization. Its clinical efficacy has been established in multiple sclerosis. In addition, an emerging body of experimental evidence points to potential direct effects on neurons, astrocytes, and glial cells. The effects of FTY720 on cells of the peripheral nerve, however, have not been investigated in vivo so far. To study the effects of FTY720 on peripheral nerve regeneration, we performed sciatic nerve crush in C57Bl/6 mice. Two weeks post-crush, we assessed sciatic nerve functionality by electrophysiology and walking track analysis, by histology the degree of myelination and axonal integrity. To better understand potentially relevantmechanisms we studied cAMP, a crucial factor for axonal regeneration, as well as oxidative stress, Clinical as well as electrophysiological measures indicated a significant improvement of axonal regeneration in FTY720-treated mice compared to control animals. Histology revealed a significantly increased thickness of myelin sheaths in crushed nerves of FTY720-treatedmice. In these nerves, cAMP levels were found to be increased, whereas dinitrophenyl-labeling of free protein carbonyls and visualization using HRP-DAB pointed to lower oxidative stress in FTY720-treated animals. Our data suggest that FTY720 may exhibit direct effects within the peripheral nervous system in vivo propagating peripheral nerve regeneration.