Eduard V. GENEROZOV, PhD in Biology, Associate Professor, Head of Laboratory
Andrey V. ZHELANKIN, PhD in Biology, Senior Research Scientist
Elvira A. BONDAREVA, PhD in Biology, Senior Research Scientist
Ildus I. AKHMETOV, Dr. in Biology, Research Scientist
Elena S. KOTOVA, PhD in Biology, Research Scientist
Nikolay A. KULEMIN, PhD in Biology, Research Scientist
Lubov O. SKORODUMOVA, PhD in Biology, Research Scientist
Irina V. FEDYUSHKINA, PhD in Biology, Research Scientist
Alexandra V. KANYGINA, Junior Research Scientist
Ekaterina A. SEMYONOVA, Junior Research Scientist
Elena I. SHAROVA, Junior Research Scientist
Konstantin A. BABALYAN, Junior Research Scientist
Elena S. ZAKHAROVA, PhD in Biology, Research Laboratory Assistant
Liliya N. YULMETOVA, Laboratory Assistant
Topics of scientific interest and lines of research
The Laboratory of Human Molecular Genetics was established in April, 2004 and is a part of the Department of Biomedicine and Genomics.
Research carried out in the laboratory is based on the use of complex, integrative approaches aimed at characterization and establishment of the role of genetic and epigenetic factors in pathogenesis of widespread socially significant diseases. In recent years, the research has been focused on genetic aspects of cardiovascular pathology, ophthalmologic diseases, oncogenetics, and genetics of muscle activity. New generation sequencing (NGS) methods based on Illumina and Oxford Nanopore technologies are actively used for whole-genome, whole-exome and targeted genomic analysis, transcriptional profiling, analysis of non-coding RNA, and assessment of individual variability of T-cell receptors. Studies are performed using high-density genotyping with DNA microarrays (Illumina), allowing the analysis of a fixed set of polymorphisms with a coverage density of up to 5 million polymorphic variants (SNPs) per sample, as well as DNA methylation analysis.
Bioinformatic methods of analysis are an integral part of modern genetic research. A wide range of bioinformatic methods is applied in the laboratory. The FRCC PCM computing cluster consists of 17 nodes (24-48 CPU cores, 64-256 Gb RAM), a high-performance server for resource-intensive tasks (128 CPU cores, 2 Tb RAM) and a 205 Tb storage system. Original bioinformatic approaches for processing and annotating NGS sequencing data are being developed. In recent years, new approaches to reference design have been implemented to obtain copy number variation (CNV) estimates from whole-exome and whole-genome sequencing data. One of the new directions of research is devoted to the analysis of neoantigenic characteristics of peptides for the tasks of personalized cancer immunotherapy.
More details about the scientific research performed in the laboratory can be found in the Current Projects section.
A set of equipment for the comprehensive genetic analysis is available:
- Low temperature and domestic refrigerators
- Desktop centrifuge with cooling and without
- Boxes for setting the amplification reactions
- Thermocycler DNA ENGINE TETRADTM (MJ Research)
- MALDI ToF mass spectrometer MICROFLEX (Bruker Daltoniks)
- Sequencer ABI Prism-3730 Genetic Analyzer (Applied Biosystems, USA; Hita-chi, Japan)
- Ion Personal Genome Machine® (PGM™) System (Applied Biosystems, USA)
- SOLiD 4 (Applied Biosystems, USA)
- GS FLX+ Sequensing System (Roche)
- TECAN Freedom Evo Workstation (Illumina, USA)
- iScan (Illumina, USA)
- Electrophoretic Equipment
Our researchers involved in muscle genetics studies have been the members of Athlome Project Consortium for the last 6 years and co-authored several publications with other Athlome members including:
- Genathlete study research groups (USA, leaders: Claude Bouchard, Tuomo Rankinen, Bernd Wolfarth);
- Myology and genetics research groups (Great Britain, Japan, Sweden. Heads: Cecilia Lindgren, Noriyuki Fuku, Ola Hansson;
- Epigenetic of muscle memory research group (Norwegian School of Sport Sciences). Leader: Adam P. Sharples.
- Molecular aspects of strength training research group (Manchester Metropolitan University, Liverpool John Moores University). Leaders: Alun Williams and Robert Erskine.
Ophthalmogenetic projects are developed in close cooperation with the Eye Microsurgery Center under the Russian Ministry of Health. The research is carried out in close cooperation with the Russian Ministry of Health, the Helmholtz Research Center for Eye Microsurgery and the Ufa Research Institute of Eye Diseases of the Academy of Sciences of the Republic of Bashkortostan. Collaborative research with the clinical departments of N.I. Pirogov Russian National Research Medical University is carried out.
PhD. theses have been prepared and defended over the past few years as part of the ongoing projects:
Alexandra Vladimirovna Belodedova for the degree of PhD in Medical Sciences in the specialties "ophthalmology" and "genetics" on the topic: "Diagnosis and surgical treatment of keratoconus based on in-depth study of genetic aspects of the disease and femto-assisted keratoplasty", https://www.mntk.ru/specialists/dissovet/zashita2020/belodedova/
Borisov Oleg Vitalievich for the degree of PhD in Biological Sciences in Mathematical Biology, Bioinformatics on "Investigation of the contribution of genetic factors in the variability of morphofunctional characteristics of human muscle fibers using a combination of bioinformatic methods of full genomic associative analysis," https://mipt.ru/education/post-graduate/soiskateli-biologicheskienauki.php.
Every year, students from the Molecular and Translational Medicine Department of MIPT and students from the Biology Department of M.V. Lomonosov Moscow State University perform master's and bachelor's theses in the laboratory:
Anton D. Voronov, MIPT, bachelor’s degreee in Applied Mathematics and Physics: Search for genetic variants associated with primary corneal endothelial dystrophy in patients without expansion of trinucleotide repeats in TCF4 gene,'" supervised by Skorodumova L.O., Sharova E.I.
Liliya N. Yulmetova, MIPT, master's degree in Bioinformatics: Identification of new genetic loci associated with Fuchs corneal endothelial dystrophy", supervised by Skorodumova L.O., Sharova E.I.
Alisa A. Shichanina, MIPT, bachelor's degree in Innovative Pharmacology: Search for candidate variants of keratoconus in the rs2721051 linkage disequilibrium block", supervised by Skorodumova L.O.
Diana Latypova, MIPT, bachelor’s degree in Bioinformatics: Molecular and genetic characterization of chemically induced tumors of the large intestine as a model of colorectal cancer in BALB/cJ mice, supervised by Kanygina A.V., Sharova E.I.
Tatiana Tsedilina, master's degree program in Biology Department of MU: Analysis of possible functional connection of LOXHD1 and AGBL1 genes with Fuchs corneal dystrophy, supervised by Sharova E.I.
One of the important directions of research of the laboratory is ophthalmogenetics, in particular, genetics of Fuchs corneal endothelium dystrophy (FECD) and keratoconus.
In the Laboratory of Molecular Human Genetics, the prevalence of 10 markers associated with FECD was analyzed for the first time in Russia (Skorodumova L.O. et al., 2018).
Samples from patients with FECD and control group participants were collected in S.N. Fedorov Eye Microsurgery Center. The frequencies of the marker alleles rs613872, rs17595731, and expansion of trinucleotide repeats CTG18.1 in the TCF4 gene were 78%, 14%, and 72%, respectively. It was shown that expansion of CTG18.1 trinucleotide repeats in the TCF4 gene is common in a significant proportion of Russian patients with FECD and study of its involvement in FECD pathogenesis is relevant in Russia.
Within the framework of the RFF grant of the Presidential Research Project Program implemented by leading scientists, including young scientists 2017-2019 (the pathogenetic role of expansion of trinucleotide repeats in the TCF4 gene in the development of FECD was investigated, samples of corneal endothelium of patients with DF and donors were collected. They were used to obtain transcriptomes of corneal endothelial samples (Nikitina A.S. et al., 2019). Analysis of differentially expressed genes between FECD and control groups revealed the activation of signaling pathways modeling extracellular matrix, phagocytosis and immune response.
Another research is dedicated to the genetic predisposition to keratoconus. Markers associated with keratoconus were selected that could be most relevant for genotyping in the Russian population (Skorodumova L.O. et al., 2019). As part of the RFBR grant "The role of genetic and autoimmune factors in the pathogenesis of keratoconus" 2017-2020, a DNA sample bank of keratoconus patients and cataract patients (control sample) was collected together with the S.N. Fedorov Eye Microsurgery Institute and the Ufa Research Institute of Eye Diseases. Genotyping of variants associated with keratoconus, rs1536482 near COL5A1 gene, rs2721051 near FOXO1 gene and rs1324183 between MPDZ and NF1B genes was carried out for the first time in Russia. A significant association with keratoconus was found for all three variants. We also searched for rare variants in the COL5A1 gene promoter in patients with keratoconus who were carriers of at least one rs1536482 allele. Rare variants in the COL5A1 promoter were found to have no significant role in keratoconus susceptibility associated with rs1536482 (Skorodumova L.O. et al., 2021). These results substantiated the current search for potential causal variants in the boundaries of loci that are cohesively inherited with rs1536482 and rs2721051 variants.
As part of the work on the role of autoimmune mechanisms in the pathogenesis of keratoconus, the repertoire of T cells in corneal samples and blood mononuclei of patients with keratoconus (Skorodumova L.O. et al., 2020) was analyzed. In general, the possibility to obtain libraries of transcripts encoding T-cell receptors showed the presence of these cells in corneal samples, which has not been reported before.
Research support of high-performance sports medicine is one of the research areas pursued by FMBA of Russia. As part of this topic, the laboratory has for many years conducted research to analyze the impact of genetic factors on human physical abilities. Information about the genetic basis of the physiological characteristics of individuals with unique physiological characteristics, which is essential for professional athletes, is of fundamental value, since it reveals the general mechanisms of adaptation of the body to intense physical exertion. The results of such research are also of practical importance: they are applied to sport selection, correction of training process parameters, as well as diagnostics and treatment of patients who are not professional athletes. Since 2011, with the participation of laboratory specialists, large-scale work has been carried out to genotype large samples of professional athletes in the Russian Federation, with a total population exceeding 2000 people. A series of associative studies were performed on the obtained data; the results were presented in 37 scientific publications.
In the last 5 years, the research interest of the laboratory in this area has been focused on studying the role of genetic factors in individual variability of muscle fiber composition, their influence on the development of strength and speed characteristics of skeletal muscles, regulation of fatigue and recovery. The key feature of skeletal muscle is plasticity, i.e. the ability to change its functional characteristics in response to a change in contractile activity. The study of molecular and genetic mechanisms underlying this plasticity is an urgent task both from the point of view of normal physiology (development and maintenance of performance, improvement of sports results), and from the point of view of understanding the pathogenesis of many diseases.
Studies in this area have been supported by the Russian Science Foundation. For example, in 2017-2019, a work was successfully carried out under grant No. 17-15-01436 of the Russian Science Foundation "Comprehensive analysis of the contribution of genetic, epigenetic, and environmental factors to individual variability in the composition of human muscle fibers." At present, a work is being carried out under the Russian Science Foundation project No. 21-15-00362 "Investigation of the molecular-genetic mechanisms of morphofunctional changes of human muscle fibers during high-intensity physical activity". A characteristic feature of these projects is the study of skeletal muscle biopsy material obtained from volunteers during the experiment with controlled physical exercise. Using a comprehensive methodological approach including genotyping, transcriptional profiling, and epigenetic analysis, we expect to be able to characterize new patterns in the molecular genetic mechanisms of muscle fiber phenotype regulation.
Endogenous and exogenous factors of obesity
One of the directions of research of the laboratory is the study of endogenous and exogenous risk factors for obesity in the adult population of Moscow (supervised by E.A. Bondareva, Ph.D. in Biological Sciences). This work was supported by the Russian Foundation for Basic Research, Grant № 20-09-00276 "Fatigenic" environment of the megapolis.
The "obesogenic" environment is an environment that contributes to high caloric intake and a sedentary lifestyle. Factors forming the "globesogenic" environment are under the close attention of scientific community in the light of their influence on the global epidemic of obesity of modern mankind ("globesity" - global obesity). The aim of the project is to investigate the fundamental problem of genotype-medium interactions by studying the interaction of human genetic background with a complex of factors of the "fatogenic" environment of the metropolis and their influence on the amount of fat and its topography in the adult population of Moscow. The novelty of the study lies in the interdisciplinary approach to the problem of obesity (total, latent and visceral) in the adult population of the metropolis, which assesses the complex of endogenous and exogenous factors characteristic of the big city environment provoking excessive fat deposition or, conversely, protecting against it. Upon completion of the project, unique data will be obtained, which will enable an assessment of the impact of the "fattygenic" environment of the metropolis on the formation of the morphological status in different sex and age groups of the adult population of Moscow against the background of a complex of endogenous factors.
We will analyze the relationship between the polymorphisms in genes associated with fat accumulation, brown and beige adipose tissue activity and body composition and somatotype parameters depending on the gender and age of the subjects, as well as against the background of various lifestyle modifications and dietary features and in the absence of them.
Immuno-oncology and immunogenetics
In recent years, the laboratory has been actively developing research in the field of immunological studies. Experimental and bioinformatic approaches have been implemented in the laboratory, allowing the typing of major and minor alleles of macaque A and B MHC genes using Illumina sequencing platform technology. In addition, the technology for sequencing full-length MHC transcripts on the MinIon platform is being developed.
A set of works was implemented as part of the state assignment "Targeted Vaccine". Approaches were developed and implemented both to identify candidate tumor neoantigens in postoperative material and to validate their real antigenicity for the immune system of a patient with colorectal cancer. The approaches have been tested in vitro as well as in animal models using cell lines.
MicroRNAs regulate gene expression at the post-transcriptional level and are typically detected in the extracellular fraction of human biological fluids, which makes them promising diagnostic and prognostic biomarkers of various pathologies. The study of circulating microRNA profiles as biomarkers of human diseases became one of the research areas of the laboratory after the state assignment "Development and implementation of transcriptomics methods in clinical practice" in 2017 (cipher: miRNA-risk). In collaboration with I.M. Sechenov First Moscow State Medical University (Sechenov University), studies of circulating plasma miRNAs as potential biomarkers of paroxysmal atrial fibrillation as well as various forms of acute coronary syndrome were conducted on samples of healthy volunteers and patients of the university clinical hospital. The results of these studies led to the publication of two articles in IJMS and Biomolecules in 2020 and 2021. In 2021, the project #21-75-00114, "Influence of pre-analytical factors on the profiles of circulating extracellular microRNAs for diagnostics of cardiovascular diseases" led by Andrey Zhelankin received funding from the Russian Science Foundation as part of the Young Scientists Research Initiative project. Implementation of the grant involves a comprehensive assessment of differences in circulating plasma microRNA profiles due to the use of different types of anticoagulants (ACD, sodium citrate, CTAD and EDTA) for blood sampling. This will identify specific microRNA profiles for which changing blood collection tubes may be critical in the study of biomarkers of cardiovascular disease. The laboratory has expertise in isolation of circulating microRNAs from plasma and serum, microRNA detection by real-time PCR, and microRNA sequencing on the Illumina platform.
Research & Development “Development of high-throughput method for genetic polymorphisms analysis for fundamental studies of socially significant diseases in humans” (2009-2011) in the frames of Federal Target Program "Research and Scientific-Pedagogical Personnel of Innovative Russia in 2009-2013.
Research & Development “Characterization of pharmacogenomic markers, causing individual, genetically determined sensitivity/resistance to the drugs used in the treatment of cancer” 2012. funded by the Ministry of Health Care and Social Development of Russian Federation
1. Gordeeva V, Sharova E, Arapidi G. Progress in Methods for Copy Number Variation Profiling. Int J Mol Sci. 2022 Feb 15;23(4):2143. https://doi.org/10.3390/ijms23042143
2. Skorodumova L.O., Belodedova A.V., Sharova E.I., Zakharova E.S., Iulmetova L.N., Bikbov M.M., Usubov E.L., Antonova O.P., Selezneva O.V., Levchenko A., Fedorenko O.Y., Ivanova S.A., Gainetdinov R.R., Malyugin B.E. Rare single nucleotide variants in COL5A1 promoter do not play a major role in keratoconus susceptibility associated with rs1536482. BMC ophthalmology // 2021;21(1):1-9. https://doi.org/10.1186/s12886-021-02128-6
3. Guilherme JPLF, Semenova EA, Borisov OV, Larin AK, Moreland E, Generozov EV, Ahmetov II. Genomic predictors of testosterone levels are associated with muscle fiber size and strength. Eur J Appl Physiol. 2022 Feb;122(2):415-423. doi: 10.1007/s00421-021-04851-w. Epub 2021 Nov 18. DOI: https://link.springer.com/article/10.1007/s00421-021-04851-w
4. Zhelankin AV, Stonogina DA, Vasiliev SV, Babalyan KA, Sharova EI, Doludin YV, Shchekochikhin DY, Generozov EV, Akselrod AS. Circulating Extracellular miRNA Analysis in Patients with Stable CAD and Acute Coronary Syndromes. Biomolecules. 2021 Jun 29;11(7):962. doi: 10.3390/biom11070962. https://doi.org/10.3390/biom11070962
5. Gordeeva V, Sharova E, Babalyan K, Sultanov R, Govorun VM, Arapidi G. Benchmarking germline CNV calling tools from exome sequencing data. Sci Rep. 2021 Jul 13;11(1):14416. https://doi.org/10.1038/s41598-021-93878-2
6. Skorodumova L., Kanygina A., Belodedova A., Sharova E., Malyugin B. Shift of T-cell repertoire in cornea of keratoconus patients // European Journal of Human Genetics. - 2020. - Vol. 28. - Suppl. 1. - P. 196-197. https://doi.org/10.1038/s41431-020-00739-z
7. Turner DC, Gorski PP, Maasar MF, Seaborne RA, Baumert P, Brown AD, Kitchen MO, Erskine RM, Dos-Remedios I, Voisin S, Eynon N, Sultanov RI, Borisov OV, Larin AK, Semenova EA, Popov DV, Generozov EV, Stewart CE, Drust B, Owens DJ, Ahmetov II, Sharples AP. DNA methylation across the genome in aged human skeletal muscle tissue and muscle-derived cells: the role of HOX genes and physical activity. Sci Rep. 2020 Sep 21;10(1):15360. https://doi.org/10.1038/s41598-020-72730-z
8. Zhelankin AV, Vasiliev SV, Stonogina DA, Babalyan KA, Sharova EI, Doludin YV, Shchekochikhin DY, Generozov EV, Akselrod AS. Elevated Plasma Levels of Circulating Extracellular miR-320a-3p in Patients with Paroxysmal Atrial Fibrillation. Int J Mol Sci. 2020 May 15;21(10):3485. https://doi.org/10.3390/ijms21103485
9. Kusić D, Connolly J, Kainulainen H, Semenova EA, Borisov OV, Larin AK, Popov DV, Generozov EV, Ahmetov II, Britton SL, Koch LG, Burniston JG. Striated muscle-specific serine/threonine-protein kinase beta segregates with high versus low responsiveness to endurance exercise training. Physiol Genomics. 2020 Jan 1;52(1):35-46. https://doi.org/10.1152/physiolgenomics.00103.2019
10. Moreland E, Borisov OV, Semenova EA, Larin AK, Andryushchenko ON, Andryushchenko LB, Generozov EV, Williams AG, Ahmetov II. Polygenic Profile of Elite Strength Athletes. J Strength Cond Res. 2020 Dec 3. https://doi.org/10.1519/jsc.0000000000003901
11. Kotova ES, Savochkina YA, Doludin YV, Vasilyev AO, Prilepskay EA, Potoldykova NV, Babalyan KA, Kanygina AV, Morozov AO, Govorov AV, Enikeev DV, Kostryukova ES, Ilina EN, Govorun VM, Pushkar DY, Sharova EI. Identification of Clinically Significant Prostate Cancer by Combined PCA3 and AMACR mRNA Detection in Urine Samples. Res Rep Urol. 2020 Sep 17;12:403-413. doi: 10.2147/RRU.S262310. eCollection 2020. https://doi.org/10.2147/rru.s262310
12. Seaborne RA, Hughes DC, Turner DC, Owens DJ, Baehr LM, Gorski P, Semenova EA, Borisov OV, Larin AK, Popov DV, Generozov EV, Sutherland H, Ahmetov II, Jarvis JC, Bodine SC, Sharples AP. UBR5 is a novel E3 ubiquitin ligase involved in skeletal muscle hypertrophy and recovery from atrophy. J Physiol. 2019 Jul;597(14):3727-3749. doi: 10.1113/JP278073. Epub 2019 Jun 11. https://doi.org/10.1113/jp278073
13. Nikitina A.S., Belodedova A.V., Malyugin B.E., Sharova E.I., Kostryukova E.S., Larin A.K., Veselovsky V.A., Antonova O.P., Skorodumova L.O. Dataset on transcriptome profiling of corneal endothelium from patients with Fuchs endothelial corneal dystrophy // Data in brief. - 2019. - Vol. 25. – P. 104047. https://doi.org/10.1016/j.dib.2019.104047
14. Skorodumova L.O., Belodedova A.V., Sharova E.I., Malyugin B.E. Search for genetic markers for precise diagnostics of keratoconus // Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry. – 2019. – Vol. 13. – №. 3. – P. 214-227. https://doi.org/10.1134/S1990750819030090
15. Skorodumova L.O., Belodedova A.V., Antonova O.P., Sharova E.I., Akopian T.A., Selezneva O.V., Kostryukova E.S., Malyugin B.E. CTG18.1 Expansion is the Best Classifier of Late-Onset Fuchs' Corneal Dystrophy Among 10 Biomarkers in a Cohort From the European Part of Russia // Invest Ophthalmol Vis Sci. – 2018. – Т. 59. – №11. – С. 4748–4754. https://doi.org/10.1167/iovs.18-24590
16. Kanygina AV, Sharova EI, Sultanov RI, Schelygin YA, Doludin YV, Kostryukova ES, Generozov EV. Targeted gene sequencing panels: applicability for neoantigen profiling of colon and rectal adenocarcinoma. Biomed Khim. 2018 Nov;64(6):517-524. https://doi.org/10.18097/pbmc20186406517
17. Babalyan K, Sultanov R, Generozov E, Sharova E, Kostryukova E, Larin A, Kanygina A, Govorun V, Arapidi G. LogLoss-BERAF: An ensemble-based machine learning model for constructing highly accurate diagnostic sets of methylation sites accounting for heterogeneity in prostate cancer. PLoS One. 2018 Nov 2;13(11):e0204371. https://doi.org/10.1371/journal.pone.0204371
Links to online resources
http://vesicles.niifhm.ru/ - online tool for calculating centrifugation parameters for separating particles with different masses. A detailed description is provided in our publication:
Livshits MA, Khomyakova E, Evtushenko EG, Lazarev VN, Kulemin NA, Semina SE, Generozov EV, Govorun VM. Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol. Sci Rep. 2015 Nov 30;5:17319. https://doi.org/10.1038/srep17319