Laboratory of Functional Genomics

Functional analysis of nucleotide sequence variants

Active use of NGS-approaches in DNA diagnosis of hereditary diseases has led not only to an increase in its effectiveness, but also to the emergence of a new problem, i.e. difficulties in the interpretation of the nucleotide sequence variants. At present, a geneticist applies family analysis, bioinformatics predictions, and population frequencies in determining pathogenicity found by sequencing of variants. At the same time, it is impossible to determine the status of many variants unambiguously. In such cases, it is possible to conduct functional analysis experiments. The laboratory of functional genomics develops various approaches to solve this problem, including the following: splicing studies, measurement of channel activity, study of protein localization, and analysis of protein-protein interactions. A unique experiment design is developed for each variant to determine the pathogenicity of the variant under test.

Study of the function of long non-coding human RNA

At present, about 2.5 thousand clinical trials of drugs for gene therapy are conducted worldwide. Several of them have already been approved for clinical use. Well-characterized protein-coding genes are the targets of the vast majority of such drugs. However, in recent years, there is an increasing number of researches on long non-coding RNAs (lncRNA) and their involvement in various cellular processes. Functions of only about two hundred of 16 thousand genes of human DNA are described. However, their possible contribution to development of about 200 different diseases has been shown; at that, in some cases, they are among key elements of pathogenesis. This makes long non-coding RNAs a promising target for gene therapy. The laboratory conducts research on the structure and function of long non-coding RNAs. For this purpose, a wide range of modern molecular methods, such as siRNA knockdown, determination of RNA partners of the transcript under test using RNA-pull-down, wound healing assay and MTT assay methods .

The potential siRNA in kidney transplantation

A critical shortage of donor organs is the main problem of transplantation. This is a common problem for all countries where transplantations are performed. Every year, the number of potential recipients on the waiting lists for organ transplantation is steadily increasing, while the number of donors remains approximately constant. Donors with brain death are the main source of donor organs, whose death is ascertained under controlled conditions, which allows to carry out explantation of organs under physiological conditions, with preserved blood circulation, but the number of such donors is not enough, and this situation will always be the same. The use of organs from donors with a sudden irreversible circulatory arrest is promising, but this is prevented by graft damage due to ischemia and subsequent reperfusion, as in this case, the explantation is performed urgently, in the absence of blood circulation.

The use of normothermal extracorporeal perfusion of organs of the deceased person with his modified blood is very promising, which is the most physiological way to restore the viability of transplants. However, the potential of therapeutic window of the effect of this method for improvement of long-term results of organ transplantation obtained by means of organ-preserving technologies is not fully represented. Approaches based on gene therapy, in particular, the use of small interfering RNA (siRNA) may be among the most effective and safe solutions. Small interfering RNAs have shown themselves to be highly effective, specific and safe inhibitors of unwanted gene expression. We suggest using them to suppress central molecular targets that activate pathogenetic signaling pathways such as apoptosis, necrosis, and/or necroptosis. Therefore, the use of siRNA in organ transplantation can significantly improve the quality of transplanted organ and increase its life time.

Facioscapulohumeral muscular dystrophy (Landouzy-Dejerine)

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic disease of autosomal recessive type characterized by variable number of copies of the repeated 3.3 kb units at 10q26. It is shown that in patients with this disease the number of repetitions is less than 11, whereas in a healthy person it can reach more than 100. At present, the molecular diagnosis of this disease is carried out only in foreign laboratories by Southern blot hybridization, which requires the use of radioactive DNA probes. Our laboratory has developed a new approach to the diagnosis of this disease that does not require the use of radioisotope compounds. The method is also simple in analysis and requires less DNA.

Over recent years, the molecular genetic study of the pathogenesis of this disease has advanced significantly. Key participants of molecular processes were found and characterized. Non-coding RNA capable of activating protein-coding genes that trigger the main cellular processes that lead to the development of the disease was the trigger of the pathogenetic process. At present, there are various postgenomic technologies that allow to suppress the expression of targeted genes with high specificity and efficiency. The laboratory is developing approaches based on these technologies to turn off the expression of non-coding RNA, which should lead to a therapeutic effect.

1. Marakhonov AV, Tabakov VY, Zernov NV, Dadali EL, Sharkova IV, Skoblov MY. Two novel COL6A3 mutations disrupt extracellular matrix formation and lead to myopathy from Ullrich congenital muscular dystrophy and Bethlem myopathy spectrum. Gene. 2018 Jun 9. pii: S0378-1119(18)30677-2.
2. Filatova A.Yu., Vasilyeva T.A., Skoblov M.Yu., Kadyshev V.V., Voskresenskaya A.A., Marakhonov A.V., Zinchenko R.A. Functional confirmation of pathogenicity in the PAX6 gene optional intron sequence. Medical Genetics. 2018, Volume 17, No. 4 (189), pp. 42-47.
3. Plotnikova O.M., Skoblov M.Yu. The effectiveness of programs predicting miRNA-mRNA interactions. Molecular Biology, 2018, Volume 52, No. 2, pp. 1-23.
4. Filatova A.Yu., Sparber P.A., Krivosheyeva I.A., Skoblov M. Yu.. Long non-coding RNAs are a promising target for the various diseases treatment. Bulletin of RSMU, 2017, 3, p. 5-17.
5. Zernov N, Skoblov M, Baranova A, Boyarsky K. Mutations in gonadotropin-releasing hormone signaling pathway in two nIHH patients with successful pregnancy outcomes. Reprod Biol Endocrinol. 2016 Aug 20;14(1):48. doi: 10.1186/s12958-016-0183-8.
6. Zernov NV, Skoblov MY, Marakhonov AV, Shimomura Y, Vasilyeva TA, Konovalov FA, Abrukova AV, Zinchenko RA. Autosomal Recessive Hypotrichosis with Woolly Hair Caused by а Mutation in the Keratin 25 Gene Expressed in Hair Follicles. J Invest Dermatol. 2016 Jun;136(6):1097-105. doi: 10.1016/j.jid.2016.01.037.
7. Skoblov M.Yu., Skobeeva V.A., Baranova A.V. Mechanisms of transgenerational epigenetic inheritance and their significance for human biology. Genetics. 2016, Volume 52, No. 3, p. 1-10.
8. Filatova A.Yu., Skoblov M.Yu. Use of siRNA in the cardiovascular diseases therapy. "Clinical and experimental surgery. Academician B.V. Petrovsky Journal" 2014. - No. 4. - P. 12–19.
9. Andrey Marakhonov, Nataliya Sadovskaya, Ivan Antonov, Ancha Baranova, Mikhail Skoblov. Analysis of discordant Affymetrix probesets casts serious doubt on idea of microarray data reutilization. BMC Genomics. 2014, 15 (Suppl 12): S8).
10. Glebova K, Reznik ON, Reznik AO, Mehta R, Galkin A, Baranova A, Skoblov M. siRNA Technology in Kidney Transplantation: Current Status and Future Potential. BioDrugs. 2014 Aug; 28(4):345-61.
11. Skoblov M, Marakhonov A, Marakasova E, Guskova A, Chandhoke V, Birerdinc A, Baranova A. Protein partners of KCTD proteins provide insights about their functional roles in cell differentiation and vertebrate development. Bioessays. 2013 Jul;35(7):586-96. doi: 10.1002/bies.201300002.
12. Gurvich OL, Skoblov M. Real-time PCR and multiplex approaches. Methods Mol Biol. 2011;784:1-13.
13. Birerdinc A, Nohelty E, Marakhonov A, Manyam G, Panov I, Coon S, Nikitin E, Skoblov M, Chandhoke V, Baranova A. Pro-apoptotic and antiproliferative activity of human KCNRG, a putative tumor suppressor in 13q14 region. Tumour Biol. 2010 Jan; 31(1):33-45.