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Project titleRole of genes encoding the family of chaperones Hero, in the development and course of ischemic stroke.

Keywordsischemic stroke; Hero proteins; heat shock proteins; chaperones; genetic variants; gene expression; genome editing; gene knockout; ischemia of the brain; gene-gene interactions; gene networks; gene-environmental interactions.

Annotation
Despite significant efforts by the global medical community in the prevention and treatment of ischemic stroke (IS), this disease remains the third leading cause of death worldwide and is the leading cause of disability, cognitive decline and dementia. In ischemia of the nervous tissue, hyperproduction of reactive oxygen species, calcium overload and accumulation of hydrogen ions mediate the transition of hypoxic stress to chemical stress. Such a violation of homeostasis leads to the proteotoxic effect of accumulated metabolites and denaturation of intracellular proteins. It is the critical accumulation of damaged enzymes and structural proteins that causes irreversible changes that lead to necrosis or programmed cell death of neurons. To reduce the structural and functional consequences of hypoxic and other negative influences, a special class of proteins, chaperones, functions in the cells of all studied organisms. The main function of chaperones is to restore the native tertiary and quaternary structure of damaged proteins. In March 2020, scientists from Japan discovered a new class of chaperones, Hero proteins, encoded by genes: C9ORF16, C11ORF58, BEX3, SERBP1, SERF2, and C19ORF53. Due to their unusual heat resistance and unstructured nature, this class of proteins has been called heat-resistant obscure - heat-resistant unknowns, Hero. Their main mechanism of action is to physically shield other proteins. Such a function is provided by a flexible structure and a high negative or positive charge of the molecules (Tsuboyama et al, 2020). Of particular interest is the demonstrated physicochemical stability of Hero proteins, which have a very specific structure and withstand extreme conditions. At the moment, it is already known that Hero have the ability to stabilize various proteins both in vitro and in cells; prevent the accumulation of abnormal proteins in cells; exhibit pronounced protein-stabilizing and antiaggregatory effects, for example, prevent pathological aggregation of TDP-43; suppress neurotoxicity; reduce proteotoxic stress. Taking into account the fact that the Hero family of proteins was discovered quite recently, the genetic aspects of their involvement in susceptibility to human diseases are practically not studied and taking into account the important role of chaperones in the molecular mechanisms of the development and course of ischemic stroke, this line of research seems to be extremely relevant. Within the framework of the project, for the first time in the world, it is planned to conduct a comprehensive molecular analysis of the involvement of genes encoding Hero proteins in the formation of a predisposition and the course of IS. In addition, to assess how actively cells “recruit” Hero proteins during ischemia, it is planned to assess the level of expression of Hero genes in different post-stroke periods in the saliva of patients with IS (saliva will be investigated as the most relevant marker of gene expression in the brain). Based on the results obtained, the most clinically significant Hero genes will be selected and their role in the physiology and ischemic resistance of the brain will be studied in transgenic animal models. To do this, it is planned to create mice with knockout or overexpression (in case of lethal knockout) of one of the genes encoding the Hero protein. This line will be analyzed for the transcriptome of the brain tissue. Also, transgenic animals will be subjected to modeling of ischemic brain damage, followed by a comprehensive study of ischemic resistance: functional tests to assess neurological deficits, analysis of the histological picture of the brain, analysis of the expression of genes reflecting the functional state of the brain.

Expected results
Within the framework of this project, for the first time in the world, a molecular genetic study of the role of Hero proteins in relation to the risk of development and the severity of ischemic stroke (IS) will be carried out. To implement the project, a systematic approach will be used, including bioinformatics analysis, genotyping, transcriptome analysis and phenotyping of animals with knockout genes encoding Hero. On a sample of at least 1000 patients with ischemic stroke / 1000 healthy individuals, the role of polymorphism of genes of the Hero family in the formation of a predisposition to the development of ischemic stroke and its subtypes will be investigated. For the first time, functional studies of genes encoding Hero proteins (analysis of gene expression) will be carried out both in healthy people and in different types of ischemic stroke. Conducting functional studies at different periods of stroke (acute, acute) will provide more complete information on the molecular mechanisms of the involvement of the studied genes in the pathogenesis of IS and its subtypes. For the first time, transgenic animals with knockout or overexpression of clinically significant Hero genes will be created and characterized. For the first time in transgenic animal models, the role of Hero in ischemic brain damage will be studied. From a practical point of view, this study will not only allow conducting a comprehensive analysis of the role of a new class of chaperones in the development of AI, but will also open up the potential for new methods of AI therapy, including those based on the use of the recombinant Hero proteins (due to their special physicochemical properties, recombinant Heroes should have high bioavailability and stability, which allows them to be positioned as a potentially promising class of pharmacological drugs for neuroprotection).

Annotation of the results obtained in 2022
As part of the project implementation in 2022, the selection of genes and polymorphisms for molecular genetic studies was carried out. In total, 31 SNPs in 6 Hero genes (BEX3, C11ORF58, C19ORF53, C9ORF16, SERBP1, SERF2), as well as 59 SNPs in 30 genes of heat shock proteins, co-chaperones, and chaperone regulators were selected for the study (BAG1, BAG2, BAG3, BAG4, BAG5, DNAJA1, DNAJA3, DNAJB1, DNAJB2, DNAJC7, HSBP1, HSF1, HSF2, HSP90AA1, HSPA12B, HSPA14, HSPA1A, HSPA1B, HSPA1L, HSPA2, HSPA4, HSPA5, HSPA6, HSPA8, HSPA9, HSPB8, HSPD1, ST13, STIP1, STUB1). When selecting DNA markers, the haplotype structure of genes, the potential functional significance of SNPs, and the frequency of the minor allele in European populations (0.05 or more) were taken into account. The functional significance of the SNPs selected for the study was assessed bioinformatically using a range of tools: FuncPred (SNPinfo Web Server, https://snpinfo.niehs.nih.gov/snpinfo/snpfunc.html), HaploReg (v4.1) (http://archive .broadinstitute.org/mammals/haploreg/haploreg.php), rSNPBase (http://rsnp.psych.ac.cn/index.do); RegulomeDB (version 2.0)(https://regulomedb.org/regulome-search/), atSNP (http://atsnp.biostat.wisc.edu/search), QTLbase (http://mulinlab.org/qtlbase/search .html), GTex Portal (https://gtexportal.org/home/), Blood eQTL browser (https://www.eqtlgen.org/). Genotyping of 49 genetic variants was carried out on a complete sample of patients/controls (at least 2000 samples for each locus). Genotyping of 41 genetic variants was carried out in the amount of 1290 samples. Two approaches were used for genotyping: MALDI-TOF mass spectrometry using the MassArray Analyzer 4 platform (Agena Bioscience) and real-time PCR with allele discrimination using TaqMan probes. Data from molecular genetic studies performed during 2022 were used to conduct preliminary genetic and statistical calculations. To compare the frequencies of minor alleles of the studied SNPs of Central Russian residents with populations (it was performed for SNPs genotyped in the full sample), we used data published as part of the 1000 Genomes Project, Phase 3 (http://www.ensembl.org) and registered in European populations (GBR, IBS, TSI, FIN, CEU). It was found that the frequencies of minor alleles in most of the studied SNPs correspond to European populations. Differences (between 3 or more out of 5 analyzed European populations) were found only for 3 SNPs: rs12566098 SERBP1, rs12561767 SERBP1, rs196336 BAG3. The analysis of associations of genotypes with the risk of IS was carried out by the logistic regression analysis. A log-additive model was used to interpret the results of the analysis of associations of SNPs with IS. As a result of the analysis of associations of SNPs in the general group of patients/controls, links have been found with 11 SNPs, 7 of which are localized in the Hero genes (rs12566098 SERBP1, rs10766342 C11ORF58, rs11024032 C11ORF58, rs11826990 C11ORF58, rs3203295 C11ORF58, rs10832676 C11ORF58, rs4757429 C11ORF58), 4 in the genes of heat shock proteins and their coregulators rs10892958 HSPA8, rs11682567 HSPD1, rs2227956 HSPA1L and rs7749960 BAG2 . For SNPs genotyped in the full sample (49 SNPs), in addition to the analysis of associations in the general groups, an analysis of associations depending on gender, smoking status, age of manifestation of IS, and IS localization was carried out. In men, associations with 5 genetic variants were found: 2 SNPs of the Hero genes (rs12566098 SERBP1 and rs10832676 C11ORF58) and 3 SNPs of the heat shock protein genes rs17155992 HSPA14, rs1136141 HSPA8, rs10892958 HSPA8. In women, associations were established exclusively with polymorphic loci of the Hero genes rs12566098 SERBP1, rs11826990 C11ORF58, rs4644832 SERF2, rs2901077 C19ORF53, rs2900262 C9ORF16 (5 genetic variants in total). An analysis of the associations of the studied DNA markers depending on the smoking status revealed that in non-smoking individuals, 3 SNPs in the Hero genes (rs12566098 SERBP1, rs1058074 SERBP1, rs4644832 SERF2) and 1 SNP in the heat shock protein gene of the A family (Hsp70) rs1136141 HSPA8 are associated with the IS risk. In smoking individuals, the development of IS is associated with rs753856 HSPA6, rs556439 HSF2, rs10766342 C11ORF58, rs7928675 C11ORF58, rs11826990 C11ORF58, rs3203295 C11ORF58, rs10832676 C11ORF58, rs1136141 HSPA8, rs10892958 HSPA8, rs7155973 HSP90AA1. Analysis of the associations of the studied SNPs with biological and clinical parameters in patients with IS revealed a relationship between rs11666524 C19ORF53, rs556439 HSF2C and body mass index; rs4655707, rs12566098, rs6702742, rs1058074, rs12561767 SERBP1 - with the area of the lesion in stroke; rs196336, rs196329 BAG3 - with the level of total cholesterol; rs1846936, rs3802963 C11ORF58 - with the level of triglycerides; rs13161158 HSPA4 - with prothrombin time; rs7155973 HSP90AA1, rs1136141 HSPA8, rs13161158 HSPA4 – with international normalized ratio; rs4655707 SERBP1, rs2277947 C19ORF53, rs6702742 SERBP1, rs11024030, rs11024032, rs11826990, rs3203295, rs4757429 C11ORF58, rs12561767 SERBP1 - with the activated partial thromboplastin time. Using the MB-MDR method, 12 most significant two-order models of epistatic interactions associated with the development of IS were identified. All the best two- order models of gene-gene interactions included exclusively polymorphic variants of the Hero genes - C11ORF58 and C19ORF53. Using the MB-MDR method, 24 most significant three-order models of gene-gene interactions associated with the development of IS were identified. The significance of all models was confirmed using a permutation test. All the best three-order models of gene-gene interactions were based on the interactions of polymorphic loci of the Hero genes - rs2277947 C19ORF53 and rs11666524 C19ORF53, which were included in all the best three-order models of gene-gene interactions. The best two-order models of gene-environment interactions included both polymorphic loci of the Hero genes (SERBP1, C11ORF58) and other genes encoding heat shock proteins and their regulators (HSPA8, HSPA6, HSF2, HSPA1B, BAG1, BAG3, HSPA12B, HSPA9, HSPA1A , HSPA14, DNAJB1). The basis of the most significant 3-order models of gene-environment interactions, in addition to smoking, included polymorphic loci of the Hero genes (C19ORF53, C11ORF58, SERBP1, SERBP1), as well as genes of the HSP families and their co-regulators (HSPA8, HSPA12B, HSPA6, HSF2, DNAJB1 ). The functional annotation of the SNPs of the Hero genes showed their high regulatory potential: binding to expression quantitative trait loci, DNA methylation quantitative trait loci (cis-eQTL, cis-mQTL-effects), influence on histone modifications, influence on DNA binding to transcription factors that are jointly involved in biological processes pathogenetically significant for IS. In the first year of the project, 1 article "C9orf16 (BBLN) gene, encoding a member of Hero proteins, is a novel marker in ischemic stroke risk" was published in the journal "Research Results in Biomedicine" indexed by Scopus. The scientific results achieved within the framework of this project were presented in the report "Genes, encoding heat-resistant obscure (Hero) proteins: new players in ischemic stroke pathogenesis" at the conference BGRS/SB-2022 (Novosibirsk, 2022) (https://bgrssb.icgbio.ru/2022/ru/2022/07/01/genes-encoding-heat-resistant-obscure-hero-proteins-new-players-in-ischemic-stroke-pathogenesis/).

Publications

1. Kobzeva K. A., Shilenok I. V., Belykh A. E., Gurtovoy D. E., Bobyleva L. A., Krapiva A. B., Stetskaya T. A., Bykanova M. A., Mezhenskaya A. A., Lysikova E. A., Freidin M. B., Bushueva O. Yu. C9orf16 (BBLN) gene, encoding a member of Hero proteins, is a novel marker in ischemic stroke risk Research Results in Biomedicine., 2022;8(3):278-292 (year - 2022) https://doi.org/10.18413/2658-6533-2022-8-3-0-2

2. Bushueva O., Soldatov V. , Belykh A. , Kobzeva K. , Soldatovа M. , Gurtovoy D. , Shilenok I. , Deykin A. Genes, encoding heat-resistant obscure (Hero) proteins: new players in ischemic stroke pathogenesis Abstracts / Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences. – Novosibirsk: ICG SB RAS, 2022. – 1148 p., The Thirteenth International Multiconference (04–08 July 2022, Novosibirsk, Russia); Bioinformatics of Genome Regulation and Structure/Systems Biology (BGRS/SB-2022). – 2022. – P. 398-398. (year - 2022) https://doi.org/10.18699/BGRS/SB-2022-000

Annotation of the results obtained in 2023
At the 2nd stage of the project, in the general group of patients/controls, associations of ischemic stroke (IS) with 8 SNPs in chaperone genes and GWAS genes were established: BAG2, HSPA1L, PITX2, CASZ1, AC016251.1, ATXN2, TWIST1. Analysis of associations depending on gender revealed that in men, 9 genetic variants in the genes DNAJA2, BAG2, HSPA1B, HSPA1L, PITX2, ZFHX3, AC016251.1, ATXN2 are associated with IS. In women, connections have been established with 5 genetic variants: in the PITX2, CASZ1, PRDM16, AC016251.1, ATXN2 genes. In smokers, 3 SNPs are associated with the development of IS: in the BAG2, MMP3, AC016251.1 genes. In non-smoking individuals, 5 SNPs in the genes HSPA1L, PITX2, AC016251.1, ATXN2, TWIST1 are associated with the development of IS. In individuals with low consumption of fresh vegetables and fruits, 9 SNPs are associated with the development of IS: in the genes DNAJB1, HSBP1, HSPA4, FGF5, PITX2, LINC02577, ZFHX3, AC016251.1, ZCCHC14. In individuals with a normal level of consumption of fresh vegetables and fruits, 5 SNPs are associated with the development of IS: in the genes BAG2, HSP90AA1, DENND5A, CASZ1, AC016251.1. Analysis of the associations of SNPs studied at the 2nd stage of the project with clinical and laboratory parameters revealed that polymorphic variants of the HSPA4, HSPA5, HSPA1L, BAG3 genes are associated with body mass index in patients with IS; SNPs of the genes DENND5A, FURIN, CDK6, CENPQ, HSPB8 – with age of manifestation of IS; SNPs of the BAG3, STUB1, HSPD1, HSPA1L, DNAJB1 genes - with the number of strokes; rs12682626 BAG4 – with the area of the lesion in stroke; SNPs HSPA14, PRDM16, BAG5 – with the level of total cholesterol in patients with IS; SNPs PRDM16, LINC02577 – with triglyceride levels; SNPs FGF5, MMP3, ATXN2, HSPD1, STUB1; HSPA1B – with prothrombin time; SNPs HSPA14, LINC02577, AC016251.1, SH3PXD2A, HSPD1 – with international normalized ratio; SNPs DENND5A, BAG5, STIP1 – with an indicator of activated partial thromboplastin time. Using the MB-MDR method (the analysis included genotyping data obtained at stages 1 and 2 of the project), 2- and 3-locus models of intergenic interactions associated with an increased risk of IS were established. The best two-locus G×G models were formed exclusively from polymorphic variants of the Hero genes (primarily C19orf53 and C11orf58); Moreover, the most significant three-locus G×G models were SNPs of Hero genes in interaction with GWAS loci. Also, the MB-MDR method established 2- and 3-level models of gene-environment interactions associated with the risk of developing IS. The best 2-level G×E models were formed due to smoking and predominantly polymorphic loci of chaperone genes, incl. encoding heat shock proteins and their regulators. The best 3-level G×E models were formed due to smoking, chaperone genes, and polymorphic variants of the Hero genes. Analysis of the interaction network of genes associated with the development of IS revealed their physical interactions, co-expression, common pathogenetic pathways, as well as interactions through common protein domains, bioinformatically predicted interactions. We found 10 biological processes (GO) that were overrepresented for the IS-associated set of genes; These GOs are involved primarily in processes associated with the regulation of proteostasis (protein folding, cell response to misfolded proteins, refolding, 'de novo' folding, response to topologically incorrect proteins), as well as processes involved in the regulation of "heat shock" playing significant role in cell adaptation under stress conditions. A comprehensive functional annotation of SNPs was carried out, the associations of which with the risk of developing IS were established at stages 1 and 2 of the project. 35 SNPs affect DNA binding to TFs that are jointly involved in the regulation of neuronal apoptosis, cellular response to hypoxia and oxidative stress, vascular endothelial proliferation, cellular response to inflammatory factors, as well as neuronal differentiation, neurogenesis, and vasculogenesis. 26 IS-associated SNPs via cis-eQTL influence the expression of an additional 150 genes in brain tissue, blood vessels, and blood cells that are jointly involved in protein refolding of the cellular heat shock response. 22 SNPs are associated with cis-mQTL-mediated effects on the level of methylation of CpG sites in brain tissues. 17 SNPs are characterized by pronounced effects on histone modifications in blood cells and brain tissues. A comparative analysis of the expression of Hero genes in samples isolated from peripheral blood revealed differences between controls and patients in the acute phase of stroke in the expression level of 2 genes: C19orf53 and SERBP1. The level of expression of Hero genes in ischemic and non-ischemic human brain tissue is characterized by variability, but the differences do not reach the level of statistical significance. As a result of a series of experiments to create a mouse with a knockout in the D8Ertd738e gene (ortholog of C19orf53) using the CRISP/CAS9 method, five primary transgenic mice were obtained in which various mutations of the target gene were found, including frameshift mutations, extended deletions, and insertions. Generation F1 was obtained. A molecular genetic analysis of the F1 generation is being carried out to determine the variants of the vertically transmitted mutations. In 2023, 9 patents for inventions were received; 4 Wos/Scopus articles were published, of which 1 article was published in Q1; 1 article (Scopus) was accepted for publication. Links to articles published in 2023: https://pubmed.ncbi.nlm.nih.gov/37240062/ https://pubmed.ncbi.nlm.nih.gov/37372351/ https://pubmed.ncbi.nlm.nih.gov/37252629/ http://iramn.ru/journals/bbm/2023/10/7717/

Publications

1. Belykh A.E., Soldatov V.O., Stetskaya T.A., Kobzeva K.A., Soldatova M.O., Polonikov A.V., Deykin A.V., Churnosov M.I., Freidin M.B., Bushueva O.Y. Polymorphism of SERF2, the gene encoding a heat-resistant obscure (Hero) protein with chaperone activity, is a novel link in ischemic stroke IBRO Neuroscience Reports, 2023. – Vol. 14. – P. 453-461 (year - 2023) https://doi.org/10.1016/j.ibneur.2023.05.004

2. Irina Shilenok, Ksenia Kobzeva, Tatiana Stetskaya, Maxim Freidin, Maria Soldatova, Alexey Deykin, Vladislav Soldatov, Mikhail Churnosov, Alexey Polonikov, Olga Bushueva SERPINE1 mRNA Binding Protein 1 Is Associated with Ischemic Stroke Risk: A Comprehensive Molecular-Genetic and Bioinformatics Analysis of SERBP1 SNPs International Journal of Molecular Sciences, 2023. – V. 24. – №. 10. – P. 8716 (year - 2023) https://doi.org/10.3390/ijms24108716

3. Ksenia A Kobzeva, Maria O Soldatova, Tatiana A Stetskaya, Vladislav O Soldatov, Alexey V Deykin , Maxim B Freidin, Marina A Bykanova, Mikhail I Churnosov, Alexey V Polonikov, Olga Y Bushueva Association between HSPA8 Gene Variants and Ischemic Stroke: A Pilot Study Providing Additional Evidence for the Role of Heat Shock Proteins in Disease Pathogenesis Genes, 2023. – V. 14. – №. 6. – P. 1171. (year - 2023) https://doi.org/10.3390/genes14061171

4. T.A. Stetskaya, A.B. Krapiva, K.A. Kobzeva, D.E. Gurtovoy, G.V. Komkova, A.V. Polonikov, O.Y. Bushueva ПОЛИМОРФИЗМ ГЕНОВ АДАПТЕРНЫХ БЕЛКОВ ST13, STIP1 И РИСК ИШЕМИЧЕСКОГО ИНСУЛЬТА: ПИЛОТНОЕ ИССЛЕДОВАНИЕ Бюллетень экспериментальной биологии и медицины, 2023. – Т. 176. – №. 10. – С. 477-481. (year - 2023) https://doi.org/10.47056/0365-9615-2023-176-10-477-481

5. Tatiana A. Stetskaya, Ksenia A. Kobzeva, Sergey M. Zaytsev, Irina V. Shilenok, Galina V. Komkova, Natalia V. Goryainova, Olga Yu. Bushueva HSPD1 gene polymorphism is associated with an increased risk of ischemic stroke in smokers RESEARCH RESULTS IN BIOMEDICINE, - (year - 2024)

6. - Набор праймеров и зондов для генотипирования полиморфного локуса rs12566098 (C>G) гена SERBP1 у человека методом ПЦР в режиме "реального времени" -, №2807808 (year - )

7. - Способ генотипирования полиморфного локуса rs10104 (A>G) гена C19orf53 у человека методом ПЦР в режиме «реального времени» с применением аллель-специфических флуоресцентных зондов -, №2808842 (year - )

8. - Способ генотипирования полиморфного локуса rs346157 (A>G) гена C19orf53 у человека методом ПЦР в режиме «реального времени» с применением аллель-специфических флуоресцентных зондов -, №2808841 (year - )

9. - Способ генотипирования полиморфного локуса rs346158 (Т>С) гена C19orf53 у человека методом ПЦР в режиме «реального времени» с применением аллель-специфических флуоресцентных зондов -, №2808839 (year - )

10. - Способ генотипирования полиморфного локуса rs8107914 (С>Т) гена C19orf53 у человека методом ПЦР в режиме «реального времени» с применением аллель-специфических флуоресцентных зондов -, №2808846 (year - )

11. - Способ прогнозирования риска развития ишемического инсульта у курящих женщин популяции Центральной России на основе генотипирования полиморфизма rs1136141 (G>A) гена HSPA8 -, №2806497 (year - )

12. - Способ генотипирования полиморфного локуса rs6702742 (A>G) гена SERBP1 у человека методом ПЦР в режиме «реального времени» с применением аллель-специфических флуоресцентных зондов -, №2806911 (year - )

13. - Способ генотипирования полиморфного локуса rs12561767 (G>A) гена SERBP1 у человека методом ПЦР в режиме «реального времени» с применением аллель-специфических флуоресцентных зондов -, №2803522 (year - )

14. - Способ прогнозирования риска развития ишемического инсульта у курильщиков популяции Центральной России на основе генотипирования полиморфизма rs11682567 (T>G) гена HSPD1 -, №2805392 (year - )