4.3.7.1 The scientific problem addressed by the
project. This project aims to solve the fundamental and socially significant
problem of studying the basics of hereditary predisposition to widespread
cancer (breast cancer, ovarian cancer, kidney cancer) and allergic diseases
(bronchial asthma, allergic rhinitis, atopic dermatitis), as well as to assess
the effectiveness of therapy to reveal the etiology and pathogenesis of these
diseases and optimize treatment. Oncological and allergic diseases are among
the widespread, severe and disabling diseases, in the case of cancer with a
high frequency of fatal outcome. Genetic and phenotypic heterogeneity,
insufficient study of molecular pathogenesis, late diagnostics, presence in a
large number of patients of heavy, difficult to treat forms of the considered
pathologies testify to the necessity to develop modern highly effective systems
for forecasting the risk of development of these diseases and detection at
early stages. In recent years, a number of large-scale genetic studies of these
diseases have been conducted, and significant progress in the study of
hereditary nature is associated with the study of large samples within the
framework of international consortiums and the active use of modern methods of
research: genome-wide association analyses (GWAS), whole exome, whole genome
and targeting sequencing with the use of NGS-technology (next generation
sequencing), genome-wide gene expression, epigenome-wide association study (EWAS),
etc. Along with genetic research, in recent years there has been an active
study of epigenetic mechanisms of cancer and allergic diseases, such as DNA
methylation, histone modification, differential activity of microRNA, which
allows to significantly expand understanding of the nature of interaction
between environmental and genetic factors. Despite the numerous genetic and
epigenetic studies of these pathologies, there are many unresolved issues that
require a comprehensive and multifaceted approach.Despite the success of
clinical pharmacology, the treatment of cancer and allergic diseases still
remains a challenge. The emergence of new drugs for the treatment of
oncological diseases - immune checkpoint inhibitors (ICI) - is a revolutionary
achievement in the field of oncology. In some patients, the effect after the
use of this group of drugs can persist for a long time, significantly
increasing the life expectancy of patients compared with previous treatment. At
present, a marker for assessing the efficacy of the ICI drugs is considered to
be high expression of PD-L1, however, this marker is not specific, sometimes
with a high level of its expression patients do not respond to treatment, and
at the same time, there are cases when the drug is effective for some patients
with low expression of PD-L1. However, there is no other marker that could
indicate the efficacy of ICI therapy to predict the risk of developing
resistance to therapy. In this regard, it is relevant to search for markers to
monitor the effectiveness of treatment of patients with kidney cancer with ICI
drugs based on a comprehensive genetic and epigenetic analysis of the tumor and
its microenvironment.The effectiveness of drug therapy, the optimal class and
mode of dosing of drugs in patients with allergic diseases (AZ) are also
largely determined by genetic and epigenetic factors, which should be taken
into account when determining the treatment tactics. Taking into account the
observed variability of patients' response to the therapy and the presence of
marked interpopulation differences in the frequency of polymorphic variants of
genes involved in the metabolism of drugs, it is relevant to conduct
pharmacogenetic studies of AZ taking into account the ethnicity of patients.
All the above points to the relevance, scientific significance and timeliness
of comprehensive study of these pathologies using an interdisciplinary
approach, the main advantage of which is to combine the deep fundamental
knowledge of specialists in the field of molecular genetics and bioinformatics
in order to obtain significant results from a fundamental and practical point
of view. Solution of tasks set in this project will provide an opportunity to
characterize the process of formation of genetic predisposition to oncology and
allergy in general, as well as to certain diseases, to identify highly specific
potential markers of development, course and effectiveness of therapy of these
diseases, will contribute to the development of approaches to timely prevention
and treatment. 4.3.7.2 Relevance of the problem, scientific significance of the
problem solution. Relevance and importance of planned scientific research is
dictated by the main strategy of modern medicine - its transfer to the level of
personalized medicine (PM), which is integrated medicine, which includes the
development of personalized treatment, testing for susceptibility to disease,
prevention, diagnosis and treatment of diseases, taking into account individual
genetic characteristics of patients. The necessary conditions for the
development and implementation of PM methods are the identification of genetic
and epigenetic mechanisms of disease pathogenesis. Revealing of molecular
mechanisms of pathogenesis of diseases, their concrete clinical phenotypes is a
basis for development of methods of early diagnostics and the newest methods of
treatment. Oncological diseases are often associated with mutations in key
genes. The identification of major events contributing to the development of
malignant neoplasms and the general mechanisms of their pathogenesis can
facilitate the development of new approaches to cancer diagnosis and treatment.
Analysis of the results of large-scale GWAS allows identifying completely new
risk loci common to several cancers. The identification of such "cross
locuses" can reveal common genetic factors underlying the formation of
malignant tumors and facilitate simultaneous risk assessment of a wide range of
cancer diseases. The data available to date in studies on predictive markers
for different tumours are often contradictory due to different research
approaches, ethnic variability, statistical errors and the small number of
observations, and therefore work in this area is highly promising and relevant.
Identification of cancer risk factors contributing to the development of
malignant neoplasms using data from full genome studies is being conducted
worldwide, and the ultimate goal of such studies is to develop effective
systems for predicting the risk of disease development and choosing the right
treatment tactics and strategy.Advances in cancer immunotherapy over the past
decade are considered important in the medical and biological sciences. The
development and progression of cancer causes a strong antitumor immune
response, through which the immune system can destroy malignant cells. Immune
checkpoint inhibitors are a group of modern immunoncological drugs whose
mechanism of action aims to restore normal antitumor immune response by
blocking the inhibitor receptors of T-lymphocytes, so-called key immune points
- the type 4 antigen associated with anticytotoxic T-lymphocyte (CTLA-4) and
the programmable cell death protein (PD-1), its ligands PD-L1 and PD-L2, and
the associated inhibitory signals allowing tumor cells to evade ("escape")
immunological surveillance. Immune regulation of control points is a subject of
intensive study worldwide, but the underlying mechanisms regulating the
expression of PD1 and PD-L1 have not been fully studied. The physiological role
of immune control points is to maintain independent tolerance and minimize the
degree and duration of inflammatory reactions. The introduction of therapeutic
antibodies (Nivolumab, Pembrolysumab, Avelumab) targeting the PD-1 / PD-L1
pathway into clinical practice has led to significant improvements in patient
treatment outcomes. Unfortunately, most patients have tumor insensitivity to
this class of drugs and further progression of the tumor process against the
background of the ongoing treatment. In the mechanism of antitumor immunity in
general and the mechanism of action of ICI there are many unclear questions.
The factors underlying sensitivity to control point inhibitors are currently
being intensively studied. The performance markers currently available are not
specific. Therefore, the search for markers that lead to resistance to ICI
drugs is very relevant and crucial in identifying patients who are less likely
to respond to treatment, which could allow for the choice of the most effective
treatment tactics and increase the economic impact of the drugs. According to
the World Allergy Organization, about 30-40% of the world population suffer
from various allergic diseases [http://www.worldallergy.org]. Bronchial asthma,
allergic rhinitis and atopic dermatitis (atopic triad) are three common,
interrelated allergic diseases that often transform from one to another and are
associated with each other. These epidemiological studies allow classifying
allergic diseases as allergies, assuming the presence of both common
(syntropic) genes apparently responsible for common pathogenesis
(predisposition to allergies in general) and genes specific to different groups
of diseases [Freudin M.B., 2010; Sun H.L., et al., 2012; Pinart M., et al.,
2014; Ziyab A., et al., 2014; Garcia-Aymerich J., et al., 2015; Gough H., et
al., 2015; Jacob L., et al., 2016].In recent years, a number of large-scale
genetic studies of AZ have been conducted, significant progress in the study of
hereditary nature of AZ is associated with the study of large samples within the
framework of international consortia and the active use of modern methods of
research: genome-wide association analyses (GWAS), whole exome, whole genome
and targeting sequencing with the use of NGS-technology (next generation
sequencing), genome-wide gene expression, epigenome-wide association study
(EWAS), etc. More than 300 genes were detected, the function of protein
products of which is closely related to the development of AZ [Holloway J.W. et
al., 2010; Meng J.-F. et al., 2010; Ortiz R.A. andBarnes K.S., 2015;
Willis-Owen S.A.G. et al., 2018]. More than 20 genome-wide linkage analysis of
BA, atopy, AR and AD have been carried out. Chromosomal regions closely bonded
to the development of AZ were identified, and positional-cloning genes for BA
and AD were identified [Zhang Y. et al., 2003; Marchet al., 2011; Ortiz R.A.
and Barnes K.S., 2015; Willis-Owen S.A.G. et al., 2018]. More than 90 GWAS of
BA and its various clinical phenotypes, 8 GWAS AR and 10 GWAS AD were
performed, which revealed more than 1000 polymorphic variants of genes
associated with AZ, performing various roles, including inflammatory cell
functions and cell activation (IL13, IL6R, DENND1B, LRRC32, IL2RB, IL1RL1),
barrier function of the epithelium (IL33, IL1RL1, C11orf30, TSLP, CDHR3), smooth
muscle contraction of the respiratory tract (PDE4D), apoptosis and cell
differentiation (GSDMB), etc. [www.genome.gwas.org]. Along with genetic
studies, epigenetic mechanisms of development of allergic diseases, such as DNA
methylation, histone modification, and differential activity of microRNA, have
been actively studied in recent years, which makes it possible to significantly
expand understanding of the nature of interaction between environmental and
genetic factors [Specjalski K., Jassem E., 2019]. A large number of AZ studies
have been devoted to the role of small or other non-coding RNAs. MicroRNA play
an important role in posttranscriptional regulation of gene expression by
binding to target RNA carriers (mRNA) for their suppression or degradation,
which leads to an overall reduction in gene expression [Carnino J. M., et al.,
2018]. MicroRNAs have an undoubted effect on the regulation of allergic
inflammation. The main set of microRNA involved in the pathogenesis of AZ
includes miR-196a2, miR-146a, miR-499, miR-148a, miR-148b, miR-152, miR-149,
miR-21, miR-223, miR-142-5p, miR-142-3p, miR-146b, miR-155, let-7, miR-193b and
miR-375. Some individual microRNAs can be used as biomarkers of allergic
diseases [Specjalski K., Jassem E., 2019]. In the last decade pharmacogenetic
AZ studies have been actively conducted all over the world, the relevance of
which is explained by the observed heterogeneity of patients' response to
therapy and the presence of patients with severe diseases resistant to therapy.
In a significant part of patients the control of symptoms is not effective
enough, which leads to the development of heavier forms and serious
complications. It has been established that genetic predisposition by 60-80%
determines individual variability in the effectiveness of treatment of patients
with a certain group of drugs [Isidoro-García M. et al., 2017; Farzan N. et
al., 2017]. Despite the numerous genetic and epigenetic studies of AZ, there
are still many unresolved issues, the study of which requires a comprehensive
and versatile approach. Clinical heterogeneity of this group of diseases with
similar etiopathogenesis indicates the need to identify risk factors
predisposing to the development of allergy in general, as well as certain
allergic diseases in individuals of different ethnic backgrounds, in order to
develop approaches to timely prevention and treatment. The search for new
genetic and epigenetic markers of the effectiveness of glucocorticosteroid and
antihistamine drugs for the development of new approaches to diagnosis and
treatment of these diseases is relevant.In connection with the above, it is
planned to conduct a comprehensive study of genetic and epigenetic mechanisms
for the development of a number of cancer and allergic diseases using modern
genome analysis technologies to develop effective approaches to diagnosis,
prevention and treatment of these diseases. 4.3.7.3 Specific challenge within
the problem to which the project is directed, its scope. This project is a
fundamental biomedical research aimed at uncovering the molecular mechanisms of
pathogenesis of complex oncological and allergic diseases in order to develop
new effective approaches to DNA diagnosis and pathogenetic treatment. In order
to achieve the goal of the study, the following objectives are set:1.Search for
common genetic markers of risk of breast cancer, ovarian cancer, as well as
specific markers of different forms of these diseases (breast cancer
estrogen-receptor positive (ER+) and breast cancer estrogen-receptor negative
(ER-), ovarian serous cancer) based on genome-wide studies. 2.Identification of
genetic and epigenetic risk markers for allergic diseases and the effectiveness
of their therapy with glucocorticosteroids and antihistamines. 3.Identification
of genetic and epigenetic markers of kidney cancer risk and efficacy of their
therapy with immune checkpoint inhibitors. 4.3.7.4 Scientific novelty of the
set task, justification of achievability of the task solution and possibility
to get the planned results.The scientific novelty of this study is to implement
a comprehensive approach to solving the fundamental and socially significant
problem of studying the molecular bases of hereditary predisposition to
widespread cancer (breast cancer, ovarian cancer and kidney cancer) and
allergic diseases (bronchial asthma, allergic rhinitis, atopic dermatitis), as
well as to find markers of efficacy / resistance of their treatment. The
project will search for markers of risk of widespread cancer and allergic
diseases based on analysis of genetic and epigenetic factors. In addition,
given the existing heterogeneity of patients' response to therapy and the
presence of patients resistant to therapy for severe cancers and allergic
diseases, special attention will be paid to the search for markers of efficacy
of therapy for these two groups of diseases.In particular, a transcriptome
analysis will be carried out for breast cancer of various forms and for ovarian
cancer to identify common and specific markers of risk for their development.
In addition, molecular genetic features of patients with metastatic kidney
cancer, who are treated with immune chekpoint inhibitors and differently
respond to the treatment, will be searched for using the most advanced
molecular genetic research methods. The project plans to evaluate genetic and
epigenetic markers and their impact on the effectiveness of therapy with ICI
drugs. In order to solve and achieve the set tasks, well characterized
representative samples of kidney cancer patients demonstrating an effective
response to ICI therapy and, conversely, resistant to the therapy will be used.
All patients will be examined and characterized by highly qualified specialists
from the Republican Clinical Oncology Dispensary and the Oncology and Urology Departments
of Bashkir State Medical University. Informed consent for participation in the
experiment will be obtained from each individual. The project will apply modern
high-tech methods of molecular genetic research using OpenArray technology on
QuantStudio™ 12K Flex. The study of genetic and epigenetic markers for the
development of allergic diseases and the effectiveness of response to therapy
with glucocorticosteroid and antihistamine drugs in individuals of Russian,
Tatar and Bashkir ethnicity has not been previously conducted in this project,
and therefore has a marked scientific novelty and practical importance for
understanding the pathogenesis of diseases and prescribing adequate
personalized therapy. To date, genetic and epigenetic studies of AZ have
revealed many polymorphic variants of genes that have a moderate impact on the
pathogenesis and efficiency of AZ therapy, but the data obtained in various
studies often contradict each other and do not explain the observed variability
of therapeutic response of AZ patients to drugs. In this project it is planned
molecular genetic study of AZ on different samples of patients, taking into
account phenotypes and endotypes of diseases, with clear criteria for assessing
the effectiveness of therapeutic response to drugs is appropriate. The project
will also study the role of polymorphism of biogenesis genes and precursors of
microRNA in the development of AZ and sensitivity to therapy in patients with
AZ, which is very relevant due to the fact that currently available data on the
importance of regulatory microRNA in the development of allergic inflammation
are few. One of the prerequisites for achieving the set objectives and
obtaining the planned results is a high level of professionalism of the team of
performers, who for many years have been studying genetic and epigenetic
factors of predisposition to the development of widespread cancer and allergic
diseases. Currently, the laboratory has a DNA and RNA bank of patients with
various oncological diseases (n=2000), allergic diseases (n=906) and a control
group (n=2000). The search for mutations in some genes, which are important
participants of the common signaling pathways controlling the genome integrity,
cell cycle, apoptosis and cell differentiation, causing hypersensitivity to
ionizing radiation, was carried out [Bermisheva M.A., 2006-2019; Gilyazova
I.R., 2008-2019; Klementova E.A., 2012-2019]. In Russians, Tatars and Bashkirs
living in the Republic of Bashkortostan, genetic markers of risk of development
of AZ (AD, BA and AR) by polymorphic variants of candidate genes have been
determined [Karunas A.S. et al., 2004, 2007; 2012, Fedorova Yu.Yu. et al.,
2009-2011; Gimalova G.F. et al., 2012-2017]. In the framework of the integrated
project of the Sixth EU Framework Program GABRIEL, a full-scale analysis of the
association of bronchial asthma in individuals of different ethnic origins was
carried out [Karunas A.S., 2011, 2015]. The pharmacogenetic study of BA studied
polymorphic variants of genes involved in the metabolism of beta-2-agonists,
glucocorticosteroids, and antileukotrienes [Fedorova Yu.; Karunas A.S., 2016,
2019].The team uses the most modern methods of research in the field of
physico-chemical, molecular and cell biology, which is facilitated by the
available unique expensive devices, equipment and availability of necessary
computer programs. All performers of the project have extensive experience in
carrying out relevant research in the framework of grants RFBR, FTP,
U.M.N.I.K., START, grants of the European Union, etc., research and practical
work in the development of test systems of monogenic and multifactor, including
human cancer. All works planned in the project correspond to the world level.
The expected results will be of priority nature, characterized by a high degree
of scientific novelty and prospects of implementation of the proposed
approaches to achieve the goal of the work and will make a significant
contribution to the genetics of cancer and pulmonology diseases. 4.3.7.5
Current state of research on this problem. Malignant neoplasms have become a
global problem of our time. Annually, more than 18 million new cancer cases and
about 9.6 million deaths are registered worldwide. Cancer incidence is
projected to increase by about 60% by 2040 [Globocan, 2018]. Oncopathology is
the second most important cause of death in the world and in Russia after
cardiovascular pathology [Kaprin et al., 2018]. Its morbidity is steadily
growing globally, which is determined by objective processes of population
ageing and the convergence of changing social and environmental factors. Thus,
in Russia the average annual growth rate of malignant neoplasm morbidity in
2008-2018 exceeded 1.7%. This has an impact on reduced life expectancy,
increased irreparable population losses and colossal economic damage. Today
there is no doubt that genetic and epigenetic disorders in the genome are the
trigger mechanism of malignant tumor formation and development. In the last
decade the genetic nature of oncological diseases has been actively studied all
over the world, including Russia, which is due both to the undoubted urgency of
studying risk factors for the development of this group of diseases and to the
emergence of new methods such as genome-wide association analysis (GWAS), whole
genome and targeting sequencing based on NGS technologies. Rare mutations with
high penetration can explain only a small number of family cases of breast
cancer (BC) or ovarian cancer (OC), they do not explain the causes of sporadic
forms of disease. However, there is considerable evidence of the genetic
contribution to risk even for sporadic breast cancer. Most researchers believe
that the genetic architecture of sporadic disease is polygenic, in which
susceptibility is the result of the combined effect of many low impact options.
Large-scale GWAS studies aim to find such options by working with large
patient/control samples.To date, more than 60 GWAS breast cancer has been
performed. As this number grows, the advantage of meta-analysis - combining
evidence from multiple studies - becomes apparent. The first large-scale
meta-analysis was conducted by Michailidou et al. (2013) in 2013 included 55342
cases of breast cancer and 54455 controls out of nine GWAS, new loci associated
with the risk of disease were identified. In another work involving more than
120000 people from 52 studies, another 15 loci of predisposition were
identified [Michailidou et al., 2015]. Specific loci have been identified to
explain the development of different subtypes of breast cancer. Many studies
have been devoted to studying genetic determinants of cancer taking into
account the status of estrogen receptor (ER), progesterone receptor (PR) and/or
HER2 expression. Seven SNPs were found to have a significantly greater effect
in patients with ER + than in patients with ER-, and only two SNPs remained
associated with ER, breast cancer, after adjustment for multiple testing
[Broeks et al., 2011; Figueroaet al., 2011]. Stevens et al. (2013) examined 65
variants associated with breast cancer and found that although 38 were
associated with both ER + and ER, the others were unique to only one subtype.
Most of the rare changes in the genome that may contribute significantly to
disease remain unexplored. Thanks to modern mass parallel sequencing
technologies, both common and rare variations can be identified. Over the past
few years, research using NGS technology has been conducted to study the
molecular characteristics of cancer, identify new genetic changes that
contribute to oncogenesis, cancer progression and metastasis, heterogeneity of
the tumor and its evolution. Impressive results have been obtained in the study
of ovarian cancer [Cancer Genome Atlas Research Network. Integrated genomic
analyses of ovarian carcinoma. Nature. 2011], colorectal cancer (Cancer Genome
Atlas Research Network. Comprehensive molecular characterization of human colon
and rectal cancer), breast cancer (Cancer Genome Atlas Research Network.
Comprehensive molecular portraits of human breast tumours), lung cancer, liver
cancer, kidney cancer, head and neck cancer, melanoma, acute myeloid leukemia,
etc. In the study of breast cancer, it is shown that there is a certain
specificity of a number of identified mutations with respect to certain
subtypes of breast cancer and disorders of specific pathological pathways. For
example, mutations in the MAP3K1 gene are common in Luminal A type of breast
cancer. Some mutations specify therapeutic possibilities of their use, for
example, mutant GATA3 can serve as a prognostic marker in case of application
of aromatase inhibitors. In Thompson E.'s work with co-authors (2012),
mutations were found in FANCC and BLM DNA repair genes as a result of exotic
sequencing in a sample of patients with hereditary disease. This method increases
the possibilities of ongoing research and allows to identify not only
pathogenic mutations in the already known genes responsible for the development
of breast cancer, but also find new options in the genes that are not yet
associated with the disease. Kidney cancer (renal cell cancer (RCC)) -
heterogeneous group of malignant tumors that develop from cells of proximal
twisted kidney tubules (renal parenchyma) [Matveev et al., 2013; Ljungberget
al., 2015]. Currently, there are 4 main histological types of RCC, each of
which is characterized by specific molecular and genetic disorders that
determine the potential for malignancy, metastasis, and sensitivity to drug
treatment. The most common is clear cell renal cell cancer (ccRCC),
representing up to 85% of all kidney tumors. CcRCC is characterized by
mutations in tumor growth suppressor genes located on the short shoulder of the
third chromosome, among which the greatest contribution to the disease is made
by changes in the genes of von Hippel-Lindau (VHL) and polybromo 1 (PBRM1)
[Matveev et al., 2013; Ljungberget al., 2015; Mikhailenko et al., 2016]. Less
common are chromophilic (papillary) (7-14%), chromophobic (4-10%), and cancer
of collective ducts (1-2%) [Bejanova et al., 2017].In Russia, among tumors of
urogenital system RCC ranks 2nd after prostate malignancies [Chissov et al.,
2017] and 1st-3rd in terms of growth rates [Davydov et al., 2014; Kaprin et
al., 2015; Chissov et al., 2017]. By the time the diagnosis is made, 25-30% of
patients are diagnosed with neglected forms of the disease with remote
metastases. Radical nephrectomy is considered to be the main method of renal
cell cancer treatment, at the same time there is a high resistance of kidney
cancer to chemotherapy and weak response to treatment with hormonal drugs, and
the effectiveness of cytokine therapy (interleukin 2, interferon alpha) does
not exceed 18-20% (Nosov et al., 2014). Radiation therapy for renal cancer is
practically ineffective.Since 2006, 11 drugs belonging to anti-VEGF groups
(inhibitors of vascular endothelial growth factor, angiogenesis inhibitors),
mTOR signal pathway inhibitors, and immune control point inhibitors have been
registered for systemic therapy of kidney cancer. Anti-VEGF drugs inhibit the
formation of blood vessels and thus prevent vascularization of the tumor,
causing it to "choke". Inhibitors of mTOR signaling pathway inhibit
cell growth and survival, while inhibitors of immune control points
"disarm" the tumor before natural antitumor immunity. At the heart of
the targeting therapy paradigm is knowledge of specific genomic disorders,
mutations that contribute to the survival and multiplication of malignant
cells. Detecting similar mutations that determine the sensitivity of a tumor to
immunotherapy could help to identify patients who have been shown to undergo
it. For example, it has been shown that in some cases such mutations have an
impact on the immune system. In patients with melanoma, against the background
of the BRAF V600E mutation, the secretion of a number of factors suppressing
the processes of activation of immune cells is observed, while suppressing the
BRAF signaling pathway, the secretion of these factors decreases. However, in
clinical studies of anti-PD1 drugs, no differences in their effectiveness
depending on the presence of mutations of BRAF were found. It is also known
that mutations in PTEN, ALK, LKB1 genes may lead to increased secretion of
PD-L1 by tumor cells, which makes their study as biomarkers of immunotherapy
effectiveness promising, however, at present no specific mutations associated
with the effectiveness of immunotherapy have been revealed [Simmonset al.,
2017].The majority of classical cytostatics and targeting preparations used
nowadays are characterized by a narrow spectrum of antitumor action and rapid
development of resistance. In contrast, modern immunotherapeutic preparations
allow achieving a clinical response in a wide range of malignant tumors. An
important advantage of immunotherapy is that preparations of this class do not
directly affect tumor cells, but restore the reactivity of the body's own
immune system, which should reduce the risk of resistance development. Despite
these advantages, the mechanisms through which the effect of immunotherapeutic
drugs is realized remain largely unknown. Renal cell cancer (RCC) is one of the
most immunosensitive tumors, as evidenced by the experience of non-specific
immunomodulators, such as drugs interleukin-2 or interferon-α. The
basis for the development of new immunotherapeutic preparations was the results
of research showing that tumor cells "escape" from immunological
surveillance use mechanisms, under physiological conditions necessary to
prevent the development of autoimmune aggression and damage to their own tissues
[Grünwaldet al., 2016; Leeet al., 2016; Schmidingeret al., 2018].This process
is regulated by cellular and molecular factors, among which T-cell inhibitor
receptors, the so-called immune control points, occupy a significant place
[Koshkinet al., 2016; Matarazaet al., 2016]. The most studied of them are
CTLA-4 (cytotoxic T-lymphocyteassociatedprotein 4) [Callahanet al., 2013;
Poprachet al., 2017; Sakamuriet al., 2017; Simmonset al., 2017] and PD-1
(rogrammedcelldeathpathway 1) [Ottet al., 2013; Rumyantsev et al., 2016; Rosset
al., 2017]. PD-1 is a membrane protein belonging to the CD28 / CTLA-4 family of
T-cell regulators. PD-1 plays an important role in suppressing T-cell activity
and preventing autoimmune reactions. When PD-1 interacts with its ligands,
apoptosis of cytotoxic lymphocytes in the tumor microenvironment is triggered,
while the activity of regulatory T-lymphocytes increases, which contributes to
the "escape" of the tumor from immunological surveillance [Pardollet
al., 2012; Patelet al., 2015; Rumyantsev et al., 2016; Kluchagina et al.,
2017]. The PD-1 / PD-L1 block strengthens antitumor immunity, reducing the
number and/or immunosuppressive activity of regulatory T-lymphocytes and
restoring the activity of effector T cells, as well as stimulating the proliferation
of memory B-cells. The mechanism of action of drugs affecting the PD-1
signaling pathway is related to blocking the interaction of its receptors and
their specific ligands, PD-L1 and PD-L2. It can be assumed that a necessary
condition for the implementation of the action of drugs of this group is the
use of malignant cells of this signaling pathway to escape the immune system
surveillance. In this case, the presence of expression of PD-1 ligands in the
tumor could allow identifying patients who would benefit from the use of drugs
suppressing the activity of this signaling pathway. PD-L2 expression is found
in some solid tumors, however, the role of PD-L2 expression in tumors on the
effectiveness of immunotherapeutic drugs application has not been studied so
far. Available data show that the presence of PD-L2 expression correlates with
PD-L1 expression and is rarely seen in isolation [Callahanet al., 2013].
MicroRNAs, in particular, networks of microRNAs which directly and indirectly
control the expression of immune control point receptors, are of particular
interest in connection with immunotherapy of oncopathology. MicroRNAs that
specifically inhibit only one control point are suitable for assessing whether
the patient will benefit from therapy and for assessing the response to the
immune control point blockade once therapy has begun. Regulatory-disturbing
microRNAs in autoimmune disease can be used to monitor and predict immune side
effects. Finally, microRNAs that target multiple immune control points are
ideal therapeutic targets because they mimic a blockade with multiple immune
control point inhibitors that have proven better than single antibody therapy.
One strategy for determining whether a patient will respond to control point
inhibitors may be the expression of microRNAs that control the level of immune
control points. For example, miR-124 microRNA levels have been found to be
significantly reduced in all classes and pathological types of gliom compared
to normal brain tissue, and have been identified as an important modulator of
STAT3 signal transmission [Sempereet al., 2010; Scariaet al., 2013]. It has
also been shown that increased regulation of miR-124 in glioma cancer stem
cells inhibits the STAT3 pathway. In addition, systemic treatment of miR-124
with mature microRNA (intra-tumor or intravenous infection) has demonstrated a
therapeutic effect against glioma in mouse glioblastoma models [Wiklundet al.,
2011; Westermannet al., 2014]. The obtained effects indicate that the action of
miR-124 depends on the difference between antitumor immune response mediated by
T cells [Wonget al., 2008].It is known that single-nucleotide polymorphic
variants (SNP) can influence the efficiency and toxicity of drugs [Evans WE,
Relling MV., 2004]. Since medications interact with malignant endothelial cells
and tumor microenvironment, it has been suggested that the patient's genetic
background plays an important role in kidney cancer therapy. In addition,
polymorphic variants can be analyzed in easily accessible biological samples
such as blood and, unlike somatic mutations, the SNP profile remains stable
throughout the patient's life. For these reasons, SNP are widely studied as
biomarkers of the response of kidney cancer to various types of drug
therapy.Genetic variability in various immune mediators is an important
determinant in susceptibility to a wide range of autoimmune disorders and
neoplasms, as well as in the progression and outcome of the disease.
Understanding of immunogenetic variability is necessary not only for
understanding heterogeneity of immune answers of antitumor system of an
organism, but also for providing rational therapy planning. Cytokines play a
crucial role in increasing and maintaining immune responses against many
pathogens, including tumors. Genetic variability of genes encoding
pro-inflammatory and anti-inflammatory cytokines and chemokines may lead to
changes in function or number of associated cytokines and, therefore, is
considered an important determinant of antitumor immunity. Thus, it has been
shown that the polymorphic variant in the promoter area of IL1B (c.-511C>T)
encoding the beta-interleukin-1 gene (mediator of inflammatory response,
proliferation, differentiation and apoptosis of cells) is associated with the
risk of cervical cancer in Korean, northern Indian, Chinese Han Chinese and
Egyptian populations [AkashM.Mehtaet al., 2017]. The study of polymorphic
variants of immune response genes has shown that they can influence the
regulatory role of microRNA. Thus, in the study of Wu D. with colleagues in
patients with colorectal cancer it was demonstrated that some alleles of
polymorphic variants in ICOS gene (inducible co-stimulatory molecule) can
influence the ability of microRNA-21-3p, microRNA-186-5p, microRNA-323b-5p, microRNA-1207-5p,
microRNA-1279, microRNA-2117 and microRNA-3692-3p to regulate the expression of
B7/CD28 molecules. It was suggested that rs7628626, rs13505, rs4404254,
rs1559931 and rs4675379, violating the regulatory role of microRNA in the
expression of B7/CD28 molecules, contribute to the appearance and progression
of colorectal cancer [Wu D et al., 2015]. When studying stomach cancer it was
shown that polymorphism in 3'UTR of B7-1 gene (SNP rs1599795 A> T) leads to
much higher risk of disease development. This polymorphic variant is able to
weaken the interaction between microRNA-361-5p and B7-1, at the same time
strengthening the binding of microRNA-132-3p/microRNA-212-3p and B7-1, which
leads to the reduction of B7-1 regulation and cancer development [Wu R. et al.,
2014].The polymorphic variant of rs4143815 in 3'-UTR region of PD-L1 gene, one
of the key molecules in ICTI therapy, promotes excessive expression and
increased risk of stomach adenocarcinoma as a result of disturbed interaction
between microRNA-570 and PD-L1 mRNA [Wang W. et al., 2013].In renal cancer a
relationship between polymorphic variants of lambda-3 interferon (IL28B) and
prognosis of the disease has been demonstrated. It was shown that carriers of
minor alleles had unfavorable response to VEGF therapy, worse prognosis,
increased expression of pAkt (Ser-473) and PD-L1 in the primary tumor. These
data suggest that the presence of SNP IFN-lambda3 minor alleles may be
clinically associated with increased tumor aerobic glycolysis, immunosuppression,
tumor resistance to therapy and poor survival in patients with renal cancer
[Nukui et al., 2018]. It was also noted that polymorphic variant of CTLA-4
rs231775 gene has potential as a prognostic biomarker. Patients with genotype
GG showed increased overall survival rate in comparison with patients with
genotype GA or AA [Liu X. et al., 2018]. According to a number of studies,
G-allele CTLA-4 rs231775 is associated with reduced CTLA-4 expression [Anjos S
et al., 2002; Sun T et al., 2008; Ligers A et al., 2001]. As a result of
reduced protein expression or reduced binding ability, G-allele may be
associated with reduced inhibition of activated T-cells. Polymorphic variants
in PD-1 and PD-L1 genes may also contribute to understanding differences in immunosuppressive
function. PD-1 rs10204525 has been reported to be associated with general
survival in patients with local regional stomach cancer [Sunakawa Y et al.,
2017] as well as in patients with resectable colorectal cancer [Yoon S et al.,
2016], which suggests that it may serve as a prognostic marker. It has been
reported that PD-L1 rs4143815 is significantly associated with the
deterioration of the overall survival in patients with non-small cell lung
cancer who underwent resection [Lee SY et al., 2017].The application of modern
immunotherapeutic preparations makes it possible to achieve a stable clinical
response in a part of patients with various malignant neoplasms. Despite
impressive results, the majority of patients have tumor insensitivity to drugs
of this class and further progression of the tumor process on the background of
the treatment. In this regard, the search for prognostic and predictive markers
of the effectiveness of immunotherapy, which will allow to identify a subgroup
of patients with immunosensitive tumors, is a fundamentally important direction
in the development of this type of drug therapy. Allergic diseases (AZ) are one
of the most common chronic diseases, based on the body's hypersensitivity to
certain environmental factors, which it considers potentially dangerous.
According to the World Allergy Organization, about 30-40% of the world's
population suffer from various AZs [http://www.worldallergy.org]. Due to high
prevalence, a significant decline in quality of life and disability of
patients, the study of these diseases is among the priority areas. Bronchial
asthma (BA), allergic rhinitis (AR) and atopic dermatitis (AD) are the three
common, interrelated AZs that often transform from one to another and are
associated with each other. The sequential nature of these diseases, from AD in
childhood to more severe allergies in the form of BA and AR in adults, is
commonly referred to in allergology as an "atopic march". AZ have a
complex multifactorial nature, heterogeneity of which is manifested by
different phenotypes of the disease in different people under the influence of
genetic and epigenetic factors of constantly changing environmental conditions.
These epidemiological studies allow us to classify AZ as a syntropy, assuming the
presence of both general genes responsible for the development of pathogenesis
and genes specific to different groups of diseases [Freudin M.B., 2010; Sun
H.L., et al., 2012; Ziyab A., et al., 2014; Garcia-Aymerich J., et al., 2015;
Bellanti J.A., 2019]. Their research allows us to classify AZ as a syntropy,
assuming that there are both general genes responsible for pathogenesis and
genes specific to different disease groups (Freudin M.B., 2010; Sun H.L., et
al., 2012; Ziyab A., et al., 2014; Garcia-Aymerich J., et al., 2015; Bellanti
J.A., 2019).Since the mid-1990s, there have been numerous attempts to identify
candidate genes that determine the hereditary component of AZ. More than 300
genes have been identified, whose protein product function is closely related
to AZ development [Holloway J.W. et al., 2010; Ortiz R.A. and Barnes K.S.,
2015; Willis-Owen S.A.G. et al., 2018]. More than 20 genome-wide linkage
studies of BA, atopy, AR and AD have been carried out. Chromosomal regions
closely bonded to the development of AZ were identified, and positional-cloning
genes for BA and AD were identified [Zhang Y. et al., 2003; March et al., 2011;
Ortiz R.A. and Barnes K.S., 2015; Willis-Owen S.A.G. et al., 2018]. Significant
progress in the study of hereditary nature of AZ is associated with the study
of large samples within the framework of international consortiums and the
active use of modern methods of research: GWAS analysis, whole exome, whole
genome and targeting sequencing with the use of NGS-technology, whole-genome
gene expression profiling, whole-epigenome analysis (EWAS), etc. In the
Catalogue of published research GWAS registered more than 90 GWAS BA, about
eight GWAS AR and ten GWAS AD [http://www.ebi.ac.uk/gwas/]. The most
statistically significant associations with AD, repeatedly confirmed in studies
of different groups of authors on different samples, showed polymorphic
variants of genes located in the regions 17q12-21 (GSDMB, ORMDL3, GSDMA, IKZF3,
ZPBP2), 6p21.32 (HLA-DQA1, HLA-DQ, HLA-DQB1, HLA-DQA2, HLA-DOA, HLA-DRA,
HLA-DRB5, HLA-DPB1, PBX2, NOTCH4, C6orf10, BTNL2), 9p24.1 (IL33), 2q12 (IL18R1,
IL1RL1, IL1RL2) and 5q22.1 (TSLP). Functioning of most of the polymorphic loci
associated with BA is associated with the immune response, Th2 cell differentiation,
inflammatory processes, barrier function of the epithelium, and others. (IL33,
IL1RL1, C11orf30, TSLP, CDHR3), inflammatory processes (IL13, IL6R, DENND1B,
LRRC32, IL2RB, IL1RL1) and others [Ortiz R.A. and Barnes K.S., 2015;
Willis-Owen S.A.G. et al., 2018; Ferreira M.A. et al., 2017; Zhu Z. et al.,
2018]. 8 GWAS allergic rhinitis were performed, in which more than 100
associated polymorphic variants were identified, many of which are also
associated with other allergic diseases HLA-DQB1, HLA-DQA1 (6p21).32), IL1RL1,
IL1RL1 (2q12.1), LRRC32, C11orf30 (11q13.5), GSDMB, ZPBP2 (17q12-21), IL33,
RANBP6 (9p24.1), IL13, RAD50 (5q31.1) et al. (http://www.ebi.ac.uk/gwas/, Waage
J. et al., 2018). In 10 atopic dermatitis GWAS performed so far, more than 130
associated polymorphic variants were found, among which also a part of loci
coincides with other allergic diseases, including high level associated
polymorphic variants of genes IL18R1 (2q12), HLA-B (6p21.32), FLG (1q21.3)
(http://www.ebi.ac.uk/gwas/). A number of results of whole exome sequencing
(WES) have been published in families with AD [DeWan A.T. et al., 2012], in
patients with AD of African-American origin [Margolis D.J. et al., 2014] and in
Ethiopian patients with ichthyosis and dermatitis [Taylan F. et al., 2015], in
families with AD [Won I. Heoet al., 2017; Luo W. et al., 2017]. The conducted
associative studies, including GWAS, showed the existence of a number of common
(syntropic) genetic risk factors for AZ, predisposing to the development of various
allergic diseases and combined allergies, and specific risk factors
predisposing to the development of certain diseases. For example, polymorphic
loci 11q13.5 (c11orf30-LRRC32) polymorphic loci initially identified in GWAS AD
[Esparza-Gordilloet al., 2009] were also associated with AR [Ramasamy A. et
al., 2011] and BA [Marenholzet al., 2011]. The genes IL13 and RAD50 (11q13.5)
are associated with the development of AD and AD [Moffatt M.F. et al., 2010,
Weidinger S. et al., 2013], and the genes GSDMB and ORDML3 (17q21) are
associated with AD and AR [Moffatt M.F. et al., 2007, 2010; Fuertes E. et al.,
2015].In the last decade pharmacogenetic AZ studies have been actively
conducted all over the world, the relevance of which is explained by the
observed heterogeneity of patients' response to therapy and the presence of
patients with severe diseases resistant to therapy. In a significant part of
patients the control of symptoms is not effective enough, which leads to the
development of heavier forms and serious complications. It was found that
genetic predisposition of 60-80% determines individual variability in the
effectiveness of therapy of patients by a certain group of drugs
[Isidoro-García M. et al., 2017; Farzan N. et al., 2017]. Pharmacogenetic studies
of AZ are carried out both with the help of polymorphic variants of candidate
genes, and with the use of GWAS and other modern approaches. Most of the work
is devoted to the study of genes that take part in metabolism of the main
groups of drugs used to treat allergic inflammation (glucocorticosteroids,
antihistamines, etc.) [Tantisira K.G. et al., 2004, Sharma S. et al.,
2005; Bryantseva O.N. et al. 2006; Klotsman M. et al., 2007; Zhdanova M.V. et
al., 2008; Telleria J.J. et al., 2008; Tantisira K.G. et al. 2009;
García-Martín E., 2009; Szczepankiewicz A. et al., 2010; Pietras T. et al.,
2011; Hizawa N., 2011; Simon T. et al., 2012; Tsartsali L. et al., 2012, Mougey
E. B. et al., 2013; Mironova J. A., et al. 2013; Panek M. et al. 2013; Mougey
E. et al., 2013; Danielewicz H., 2014; Kmyta V. et al., 2015; Awasthi S. et
al., 2015, Raje N. et al., 2015, Keskin O. et al., 2016; Slankard M. et al.
2016; Voropaev E.V. et al. 2018]. A number of epigenetic studies of AZ therapy
have been carried out, as a result of which differential methylated areas of
gene promoter areas associated with the effectiveness of therapy have been
identified; the role of modifications of histones and microRNA in the formation
of therapeutic resistance to certain groups of drugs has been shown [Li L.B.,
2010; McAlees J.W., et al., 2011; Fu A. et al., 2012; Solberg O.D., 2012;
Gaffin J. et al. 2014; Wu C., et al. 2014; Xiao C. et al., 2015; Zhu J. et al.,
2016; Huo X., 2016; Toraih E.A. et al. 2017; Zhang X. et al., 2017; Mironova
J.A. et al., 2017; Zhang S. et al., 2018; Yu B. et al., 2019].
Glucocorticosteroids (GCs) are considered the most effective anti-inflammatory
drugs for all allergopathologies [GINA, 2019]. The anti-inflammatory effect of
GCs is realized by their interaction with glucocorticoid receptors, which leads
to changes in expression of genes involved in inflammation [Farzan N. et al.,
2017]. According to the literature, the change in the efficacy of GCs therapy
in AZ patients is associated with polymorphic variants in the genes of
glucocorticoid receptor NR3C1, corticotropin-releasing hormone CRHR1
[Isidoro-García M., et al., 2017; Dai Y. Et al., 2018]. The role of polymorphic
variants of genes TBX21, WDR21A, eNOS, ORMDL3, HDAC1 and ADRB2 in the
development of AZ and the efficacy of treatment has been established in a
number of works [Farzan N. et al., 2017]. At GWAS efficacy of treatment of AD
patients new genes associated with changes in pharmacological response to GCs
were found - genes of glucocorticoid-induced transcript 1 of GLCCI1 [Tantisira
K.G. et al., 2011], transcription factor TFT [Tantisira K.G. et al., 2011],
2012], protein gene with ZNF432 domains [Wu A.C. et al., 2014], allantoicases
(ALLC) [Park T.J. et al.., 2014], a protein containing leucine rich repetitions
and F-box domain (FBXL7) [Park H.W. et al., 2014], myosin 1E (MYOE1) [Wang Y.
Et al., 2015], genes of apolipoprotein B APOBEC3B and APOBEC3C
[Hernandez-Pacheco N, et al. 2019], etc.]. Antihistamines are one of the most
frequently prescribed drugs to achieve more effective control of allergic
diseases, and in recent years, there has been an active study of the genes that
determine the effectiveness of therapeutic response of patients with AZ to this
group of drugs. Antihistamines have anti-edema, antispasmodic, anticholinergic,
antipruritic, antiserotonin and local anesthetic effects and can prevent
bronchospasm caused by histamine and histamine-like substances. Histamine is a
biogenic amine with a wide range of physiological functions of the body, the
level of which is influenced by key enzymes involved in its metabolism
[Lutsenko T.M., 2016]. In human organism histamine is formed from amino acid
L-histidine as a result of its decarboxylation by histidine decarboxylase,
which is encoded by HDC gene. Histamine is metabolized by oxidative
decomposition of amino acid 1, coded by AOC1 gene, and by methylation of
histamine-N-n-methyltransferase, coded by HNMT gene; histamine action is
realized through interaction with HRH1, HRH2, HRH3, and HRH4 transmembrane receptors
[Kucher A.N., Cherevko N.A., 2018]. A large number of functional studies of
histamine metabolism are conducted in patients with AD and other allergic
diseases. A much higher level of histamine was found in bronchi of patients
with AD [Lutsenko T.M., 2016, Raje N. et al, 2015]. A number of studies have
found that polymorphic loci of genes encoding proteins involved in histamine
metabolism play a role in the pathogenesis of BA and other allergic diseases,
determine therapeutic response of patients to treatment with antihistamines
[Sharma S. et al.., 2005; Kennedy M.J. et al., 2008; Garcia-Martin E. et al.,
2009; Szczepankiewicz A. et al., 2010; Jones B.L. andKearns G.L., 2011; Simon
T. et al., 2012; Raje N. et al., 2015]. GWAS has found associations of polymorphic
variants localized in or near genes of histamine metabolic pathway (PSAP, SCG3,
ADCYAP1) with bronchial asthma (www.genome.gwas.org). Active use of
genome-wide, epigenome-wide and other modern technologies in order to find new
markers of patients' sensitivity to the use of a particular group of drugs
allowed to bring the pharmacogenetic research of AZ to a qualitatively new
level. However, each of the identified polymorphic variants of susceptibility
genes to these diseases has only a small impact, and even the total effect of
multiple genes can not be the main cause of the disease, such a phenomenon is
defined as a phenomenon of "missed heredity". In order to better
understand the contribution of genetic factors to the formation of a trait, a
polygenic analysis should be performed with mandatory evaluation of intergenic
interactions, and the influence of epigenetic factors should be taken into
account [Cookson W., Moffat M., 2011; Ober C., Yao T.C., 2011; Lee J. et al.
2018]. At present, the influence of epigenetic processes on changes in gene
activity without changes in its coding sequence is actively studied, in
particular, in the study of pathogenesis of multifactor diseases. Epigenetic
mechanisms such as DNA methylation, histone modifications, and differential
activity of microRNA have been most studied, which allow to explain the
significant contribution of heredity to the formation of allergic diseases
[Menzella F. et al.,2016; Kabesh M., 2016]. A large number of AZ studies are
devoted to the role of small or other non-coding RNAs. MicroRNAs (miRNAs) are a
group of small single-chain coded non-coding RNAs (about 18-22 nucleotides).
MicroRNAs play an important role in posttranscriptional regulation of gene
expression by binding the bases to target RNA carriers for their suppression or
degradation, which leads to an overall decrease in the expression of the gene
broadcast from this miRNA [Carnino J. M., et al., 2018]. In recent years, the
significant role of microRNA has also been established in the development of
numerous immunological and inflammatory diseases, including allergic ones.
Rebane A., Akdis C.A. (2014) suggested that microRNAs contribute to the
development of AZ by participating in the pathway imbalance Th1/Th2,
contributing to chronic inflammation and tissue remodeling [Rebane A., Akdis
C.A., 2014]. Specific microRNA profiles were demonstrated at BA, AR and AD.
Quite a few studies indicate the role of microRNA in the development of
respiratory diseases, in particular, BA [Oglesby I.K. et al., 2010; Li L.J. et
al., 2011; Qin H.B. et al., 2012; Angulo M. et al., 2012]. Single-nucleotide
polymorphic variants in both microRNA genes and their targets are associated
with AZ. The polymorphic variant in pre-miR-146a gene associated with reduced
expression of mature miR-146a is associated with reduced risk of AZ in Chinese
and Mexican patients [Su X.W. et al., 2011; Jimenez-Morales S. et al., 2012].
The association with reduced risk of BA has been established with polymorphic
variants located in the miR-148/miR-152 binding site in the 3'UTR region of the
HLA HLAG class I gene, and with OOP in the ITGB3 binding site of the miR-124
gene [Tan Z. et al., 2007; Zhang Y. et al., 2013]. One of the approaches to
identifying microRNAs is to evaluate their level of expression, which is
differentiated between normal and affected tissue. The evaluation of microRNA
expression profiles in human tissue biopsy specimens and cellular models of
mice with BA allowed to reveal differential expression of ~ 10-20% of microRNA.
The group of miRNAs with changed level of expression in pulmonary tissue
included let-7c, miR-21, miR-29, miR-135, miR-142, miR-146, miR-150, miR-155,
miR-181, miR-193, miR-223, miR-365, miR-375, miR-452 and miR-615 [Sonkoly E.G.
et al, 2010; Garbacki N. et al., 2011; Lu S. et al., 2012; Lu T.X. et al.,
2012; Vennegaard M.T. et al., 2012; Lacedonia D. et al., 2017]. Allergic
rhinitis has been found to be characterized by typical microRNA expression
profiles. Among numerous candidates for miR-181a AR biomarkers miR-181a seems
to be especially promising. Serum level of this microRNA has been significantly
reduced in AR patients and its expression level negatively correlates with the
disease severity, Th2 cytokines (IL-4, IL-5) and positively correlates with Th1
cytokines (IL-12, interferon γ) [Liu W. et al., 2016b]. The combination of
miR-126-5p, miR-19a-5p and miR-26a-5p can confirm the diagnosis of AR with a
sensitivity of 90% and specificity of 70% (Jia M. et al., 2018). There is less
literature data on the role of microRNA in AP development. Most studies confirm
increased expression of miRNAs (miR-21, miR-146a, miR-203) in AD patients as
well as in patients with AD and AR [Wu G. et al., 2015]. Other microRNAs, such
as miR-720, are expressed exclusively in AD patients, probably because of their
role in organ-specific functions, such as regulation of keratinocyte cell cycle
[Chikh A. et al., 2011]. The serum expression level of miR-483-5p in children
with AD was also higher, but only in children with other associated atopic
diseases [Lv Y. et al., 2014]. The significant influence of microRNA on the
formation of therapeutic response has been established. On the BA mouse model
it was found that miR-146a expression is suppressed during dexamethasone treatment
and correlates with the total number of inflammatory cells [Feng M.J. et al.,
2012]. Differential expression of 26 microRNA in response to dexamethasone
treatment was revealed, in particular, a significant increase of expression of
mmu-miR-144-3p and mmu-miR-451a in response to the use of GCs in mice with BA
was shown [Plank M.W. et al., 2015]. A number of analyses of the microRNA
expression profile in AD patients were carried out, which resulted in the
detection of differential expression of 9 microRNA in response to the use of
GCS [Solberg O.D.,2012], and a cluster of microRNA associated with a low need
for GCS [Zhang S. et al., 2018]. Significantly higher levels of miR-21 and
miR-126 expression were found [Wu C., et al. 2014], miR-1165-3p [Wu C., et al.
2018] in patients with BA who do not use IGCS compared to the treatment group.
Significant increase of miR-181b-5p level in plasma of patients with BA after
IGCS treatment during 4 weeks was shown [Huo X., 2016]. Several studies of the
role of microRNA in the sensitivity of therapeutic response to beta-2 agonists
have been conducted. The genotype rs3746444*GG of polymorphic variant of
miR-499 binding site was associated with much lower bronchodilator response
after inhalation of beta-2 agonist with salbutamol in patients with BA from
Egypt [Toraih E..A. et al. 2017], the miR-16 expression level targeted by the
ADRB2 gene is proposed as a biomarker predicting the efficacy of the response
to salmeterol therapy [Yu B. et al.., 2019].Thus, microRNA have an undoubted
influence on the regulation of allergic inflammation. The main set of miRNAs
involved in the pathogenesis of AZ includes the highly-expressed miR-21,
miR-223, miR-146a, miR-142-5p, miR-142-3p, miR-146b, miR-155 and low let-7,
miR-193b and miR-375. Some individual microRNAs can be used as biomarkers of
allergic diseases. Another aspect is possible therapeutic applications. The use
of microRNA as biomarkers should be studied with an emphasis on possible
identification of endotypes and phenotypes of allergic diseases. Given the
complex relationship between microRNA and target genes regulated by multiple
microRNAs, this area requires further study.The main feature of epigenetic
mechanisms is their reversibility, which determines the prospects of their use
for the development of innovative treatments for BA and other allergic
diseases. At present, it is the study of epigenetic factors that determine the
risk of allergic diseases and the sensitivity of patients to the recommended
therapy that is extremely relevant. Thus, despite the numerous genetic and
epigenetic studies of oncological and allergic diseases, there are many
unresolved issues, the study of which requires a comprehensive and multifaceted
approach.The analysis of published literature data confirms the compliance of
the planned research to the world level and the lack of direct analogues of the
work. The expected results of the study are characterized by a high degree of
novelty and practicality of the proposed approaches to achieve the goal of work.
4.3.7.6. Proposed methods and approaches, overall work plan for the duration of
the project. Approaches to the project tasks aimed at studying cancer and
allergic diseases are determined by the latest scientific achievements in the
field of genetics, molecular biology, oncology, based on the current knowledge
on the etiopathogenesis of renal cancer, breast cancer, ovarian cancer,
bronchial asthma, atopic dermatitis and allergic rhinitis. Experimental work
will be carried out using advanced genome analysis technologies, adequate
selection of the object of study. The analysis will be carried out in
comparison groups "case-control" with the evaluation of the
association of diseases with individual polymorphic variants and mutations of
genes.The present study will include patients with light cell kidney cancer
(300 people), who are treated with immune control point inhibitors and react
differently to the treatment. In addition, healthy individuals (300) whose
primary relatives did not have cancer will also be included in the study as a
comparison group. The criteria for including patients in the study will be the
histologically verified diagnosis of clear cell renal cell carcinoma, the
presence of metastases and the prescription of ICI drugs. Criteria for exclusion
- kidney carcinomas of other histological types, the presence of other
malignant tumors in the family history, urinary tract infections, urolithiasis.
Collection of biological samples of patients with kidney cancer will be made by
employees of the department of urology of Bashkiria State Medical University
clinic. The informed consent will be received from each participant of
research. All samples will be characterized by histological type of tumor,
TNM-classification and stage of disease.Breast cancer and ovarian cancer
research will be carried out on a formed group of patients with breast cancer
(800 people), patients with ovarian cancer (250 people) and healthy women (800
people). Biological material was collected at the Clinical Oncological Dispensary
of the Ministry of Health of the Republic of Bashkortostan (Ufa) for the period
2007-2014. The participants in the study gave their informed consent to
conducting molecular genetic studies. Clinical and laboratory methods of
research were used in the collection and processing of material. In the course
of the study, data on the ethnicity of patients were specified by means of a
survey and determination of the nationality of parents in three generations.
The average age of a manifestation of breast cancer in patients was 52 years.
During the survey, it was established that 10% of the breast cancer patients
had a burdened family history. About 90 per cent of women with breast cancer in
our sample belong to the three main ethnic groups living in the Republic of
Bashkortostan - Russian, Bashkir and Tatar - 47 per cent, 13 per cent and 26
per cent, respectively. The age of patients with RH varied from 17 to 80 years
(52.2 ± 7.4 years). In 8.8 per cent of cases of women with nuclear age, family
cases of breast cancer were detected. In terms of ethnic composition, the group
of ovarian cancer patients is not homogeneous and is represented by Russians
-49%, Tatars - 28%, Ukrainians - 6%, Bashkirs - 6%, and others.The work will
use a representative sample of allergy patients (BA, AR and AD), aged 2 to 67
years (900 people). The examined patients are patients of the children's
department of the Clinic of the Federal State Educational Institution of Higher
Education "Bashkir State Medical University" of the Russian Ministry
of Health, pulmonology and allergology departments of the State Budgetary
Institution of the Republican Children's Clinical Hospital "City Clinical
Hospital No. 21" in Ufa, and allergology departments of the Republican
Children's Clinical Hospital in Ufa. The diagnosis of diseases was established
by qualified physicians in accordance with the criteria of programme documents
for the diagnosis, treatment and prevention of diseases. The sample of patients
will be divided into groups depending on the presence and combination of
different AZ: 1. general sample of AZ patients; 2. Patients with clinical
manifestations of only BA (without AR and AD); 3. AR patients (without BA and
AD); 4. AR patients (without BA and AR); 5. Patients with combined clinical manifestations
of BA, AR and AD; 6. Patients with BA and AD (without AR); 7. Patients with BA
and AR (without AD); 8. Patients with AR and AD (without AD). The criteria for
inclusion of patients into the study group is the established diagnosis and
therapy with glucocorticosteroids and antihistamines for at least 3 months. As
a control group will be investigated, consisting of 589 practically healthy
persons from 2 to 66 years old, with no manifestations of AZ and burdened
hereditary atopic diseases. All participants of the study or their parents (in
children's age individuals) gave their informed consent for conducting
molecular genetic studies. DNA isolation of patients and individuals of the
control group is performed by phenol-chloroform extraction. The quality of DNA
preparations is estimated by the ratio of optical densities A260/A280 with the
use of spectrophotometer NanoDrop 1000 (Thermo Fisher Scientific, USA).The
search for genes responsible for the development of various forms of breast
cancer and nuclear radiation will be based on genomic analysis (analysis of
nucleotide substitutions, structural variations in the coding part of the
genome, meta-analyses of GWAS and TWAS) using NGS technology (next generation
sequencing). In particular, NGS and NG will perform meta-analysis of gene
expression data (TWAS) and GWAS meta-analysis with consideration of different
disease types (ER + and ER-) in order to identify common and specific genes
involved in disease development.To understand whether polymorphic sites in the
genes of microRNA biogenesis, microRNA precursors or sites linking them to the
target are factors involved in the formation of individual predispositions to
the development and course of allergic and cancer diseases, the biological role
of microRNA in the pathogenesis of these diseases will be studied. A study of ~
30 polymorphic variants of biogenesis genes and precursors of microRNA will be
carried out, for which there was found a connection with AZ according to the
data of foreign authors (miR-196a2, miR-146a, miR-499 (Trinh H.K.T. et al.,
2017); miR-148a, miR-148b, miR-152 (HLA-G, Tan Z. et al.), 2007); miR-149 (Hu
D. et al., 2017), etc.), as well as selection and subsequent study of
polymorphic variants in microRNA gene binding sites, the targets of which,
according to the databases, are mRNAs of the genes studied in the project.
Based on the study of literature data and work with bioinformatic resources,
selection of ~ 30 polymorphic loci located in the genes of microRNA,
participants of biosynthesis and processing of microRNA or sites of binding
microRNA to the target, which are involved in the pathogenesis of cancer
diseases, will also be carried out. Selection of polymorphic variants in
microRNA genes will be carried out using the database of the National Center
for Biotechnology Information (https://www.ncbi.nlm.nih.gov/), Ensembl Genome
Browser (www.ensembl.org), polymorphism of microRNA binding sites
(http://compbio.uthsc.edu/miRSNP/miRSNP_detail_all.php). Selection and
genotyping of ~ 30 polymorphic loci selected based on the results of previous
work will be carried out to identify genetic markers for the development and
effectiveness of allergic disease therapy, as well as AZ-associated data from
candidate genes and full-scale genome studies carried out in various world
populations, including genes, involved in the response to AZ therapy with
glucocorticosteroids (GR, CRHR1, GLCCI1, T (TFT), ZNF432, ALLC, FBXL7, CMTR1,
MAGI2) and antihistamines (AOC1, HNMT, MAOB, ALDH7A1, HRH1, HRH2, HRH3, HRH4),
etc.)., in patients with BA, AR, AD and combined allergopathology. To determine
the genetic markers of risk and effectiveness of renal cancer therapy,
genotyping of ~ 30 polymorphic variants of genes of pro- and anti-inflammatory
cytokines (TNFA, IL1B, IL10, etc.), chemokines (CXCL12 (SDF-1)), receptors,
components of antigen processing apparatus (ERAP1, LMP7, TAP1, etc.) will be
performed.), microRNA (miR-146a, miR-196a2, miR-499, etc.) and other
polymorphic loci, which will be selected during the formation of panels for
genotyping based on a thorough analysis of data from literature sources and
bioinformatics databases. Single Nucleotide Polymorphisms (SNP) analysis will
be performed by genotyping followed by association analysis using the OpenArray®Real-Time
PCR System (QuantStudio™ 12K Flex Real-Time PCR System, Applied Biosystems,
USA) and real-time fluorescence detection PCR (FLASH/RTAS). The OpenArray
Real-Time PCR System is a unique platform for performing reactions in nanoliter
volumes. The 3 μl reaction mixture (TaqMan® OpenArray® Genotyping Master Mix,
Applied Biosystems, USA) is mixed with 3 μl DNA samples (required concentration
50 ng) in a 384-well plate. The Quant Studio™12K FlexReal-Time PCR allele
discrimination method involves the use of metal plates (array) into which the
resulting mixture is then transferred. This study involves the use of arrays
designed to simultaneously detect 60 single-nucleotide polymorphic variants in
a DNA sample. Previously, each well of the slide contains VIC and FAM-labeled
oligonucleotide probes with the possibility of simultaneous analysis of 60
polymorphic variants of genes. The signal level from each well, the
fluorescence image evaluation is done after the real-time PCR is completed
using the QuantStudio™ 12K Flex Instrument touch screen system. The obtained
DNA sample genotyping results are analyzed using QuantStudio™ 12K
FlexSoftware.Statistical processing of the study results will be performed
using STATISTICA v.6.0 [StatSoft], BIOSTAT [Glanz S.C., 1999], SNPStats
(http://bioinfo.iconcologia.net/snpstats/start.htm), PLINK 1.07
(http://pngu.mgh.harvard.edu/~purcell/plink/ index.shtml) and Microsoft Excel
software.The degree of associations will be evaluated in odds ratio (OR)
values. When comparing the frequencies of genotypes and alleles in pairs in
groups of patients and healthy individuals, the χ2 (P) criterion will be used
for 2x2 Yets-adjusted conjugation tables for continuity. Differences in
statistical criterion p<0.05 will be evaluated as significant. Correction
for multiple testing will be performed using FDR (False Discovery Rate)
(Benjiamini &Hochberg) estimation method provided by PLINK 1.07 package.
Intergenic interactions will be analyzed based on the results of modeling using
MDR (Multifactor-Dimensionality Reduction) [Ritchie et al, 2001] and GMDR
(Generalized Multifactor-Dimensionality Reduction) programs [Lou et al, 2007;
Chen et al, 2011].Overall work plan for the entire duration of the
project:1.Perform a search of genetic markers of breast and ovarian cancer risk
as well as specific markers of different types of these diseases on the basis
of full-scale genomic research data. 2. Carry out research of 60 polymorphic
loci located in genes of microRNA precursors, participants of biosynthesis and
processing of microRNA or sites of microRNA binding with targets, in groups of
patients with cancer, allergic diseases and in corresponding control groups.3.
Conduct a study of 60 polymorphic loci associated with cancer and allergic
diseases according to candidate and full genome studies, as well as metabolism
of drugs used for the treatment of this pathology, in groups of patients with
cancer and allergic diseases and in appropriate control groups. 4. To carry out
the analysis of association of gene-genotyped polymorphic loci with the
development and course of oncological and allergic diseases taking into account
the ethnicity of individuals, the degree of severity of diseases, the
effectiveness of the therapy; analysis of intergenic interactions. 5. Identify
genetic and epigenetic markers of risk for the development and effectiveness of
cancer and allergy therapy. 6. Present project results at Russian and foreign
conferences, publish articles in peer-reviewed journals.Thus, in the framework
of this project we offer for the first time to conduct a comprehensive study of
the contribution of genetic and epigenetic factors in the development, course
and effectiveness of cancer and allergy therapy in individuals of different
ethnic backgrounds. 4.3.7.7. The team of performers has a scientific backlog
for the project (in this paragraph, a textual description of the backlog is
filled out, and the placement of other supporting information is described in
paragraph 4.3.19).For many years, the Laboratory of Human Molecular Genetics
has been collaborating with the Oncology Department of the Bashkir State
Medical University and the Republican Clinical Oncology Health Center of the
Ministry of Health of the Republic of Bashkorostan and is studying the
molecular genetic basis for the development of a number of oncological
diseases. Currently, the laboratory has a DNA bank for patients with various
oncological diseases, including cancer of the breast, ovaries, kidney (n =
2000) and the control group (n=2000). With the informed consent of the same
patient, blood and paired tissue samples are taken (normal / tumor). All
patients whose DNA is available in the laboratory collection are well
characterized clinically by oncologists, histologists. In the laboratory,
patients with various oncological diseases are searching for mutations in genes
that are important participants in the general signaling pathways that control
the integrity of the genome, the cell cycle, apoptosis, and cell
differentiation.As a result of molecular genetic studies of breast and ovarian
cancer, interesting results have been obtained. Thus, the analysis of the
spectrum and frequency of mutations in the BRCA1, BRCA2, CHEK2, NBN, ATM,
PALB2, BLM genes in patients with breast and ovarian cancer from the Republic
of Bashkortostan showed that the 5382insC mutation in the BRCA1 gene is most
common among the studied mutations associated with high risk of developing
malignant neoplasms in women of the Republic of Bashkortostan. Mutations in the
CHEK2 gene (dele9.10 (5kb)) and in the PALB2 gene (c.172_175delTTTG)
[Bermisheva, 2014] were also detected in patients with ovarian cancer. The
study of new candidate genes associated with cancer. For example, it was found
that the ERCC4 * p.Arg799Trp mutation is not associated with a high risk of
developing breast cancer and ovarian cancer in women in our region (Bermisheva,
2020). It was also demonstrated that the WRN * p.R1406X variant is not
pathogenic for the development of ovarian and breast cancer [Bermisheva, 2019].
It has been proven that the common c.1047-2A> G variant of the CDK12 gene in
the Tatars and Bashkirs population is not clinically significant in hereditary
breast cancer [Bogdanova, 2019]. An interesting direction in the research of
oncological diseases was the study of the role of miRNAs in the development of
tumors. Analysis of polymorphic variants in the DROSHA / RNASEN, DGCR8, DICER1,
XPO5, RAN, PIWIL1 / HIWI, AGO1 / EIF2C1, AGO2, GEMIN4, GEMIN3 / DDX20, DDX5
genes involved in microRNA processing in a sample of 778 patients (417 patients
with breast cancer and 361 healthy women), showed that the polymorphic loci
rs11060845 and rs10773771 in the PIWIL1 gene, rs3809142/RAN, rs10719/DROSHA,
rs1640299/DGCR8, rs563002/DDX20, rs595055/AGO34 gland cancer and rs595055/AGO4
and rs595055/AGO4 are associated with women of Russian ethnicity [Bermisheva,
2018].As a result of studies conducted within the framework of the consortium
for the study of breast cancer and cancer (ВСАС, ОСАС), the key mechanisms
underlying these diseases have been identified. New polymorphic variants have
been identified associated with the risk of developing tumors of the breast,
ovaries, as well as genetic loci specific for breast subtypes. Most variants
are located not in genes, but in those regions that regulate gene activity
(Mavaddat N, 2019, Michailidou K, 2017, Milne R, 2017, Phelan C, 2017).The
study of the molecular genetic basis for the development of clear cell renal
cancer has been conducted in the laboratory since 2010. During this period, a
search was made for changes in the nucleotide sequence in the von Hippel-Lindau
gene (VHL), polybromo-1 (PBRM1) in patients with clear cell renal cancer
(ccRCC). Mutations in the VHL gene were detected in 21.9% (23/105) of cases of
primary kidney tumors, in the PBRM1 gene in 18% of cases. For the first time
eleven somatic mutations were identified that were not previously described in
the literature and were not registered in the databases [Gilyazova IR, 2013].
In addition, the team of authors conducted an analysis of allelic deletions of
von Hippel-Lindau tumor suppressor genes (VHL), the family Ras-associated
protein of the family 1 (RASSF1) and the fragile histidine triad (FHIT) gene
using STR markers in 110 paired DNA samples from tumor tissue of the kidney and
normal renal parenchyma. The frequency of heterozygosity loss at the two
studied loci for the VHL gene was 32.7% of cases of informative samples, for
the RASSF1 and FHIT gene, 32 and 30% of cases of informative samples,
respectively. Moreover, we have identified associations of allelic deletions in
the FHIT gene with tumor size [Kutlyeva et al, 2012]. The loss of
heterozygosity in the FHIT gene is associated with minimal tumor size and the
initial stage of the disease, which indicates early inactivation of the FHIT
gene. An analysis was also performed of aberrant methylation of CpG islands of
the promoter region of the VHL gene. Aberrant methylation is determined only in
samples of tumor tissue and is not detected in normal renal parenchyma. The
frequency of aberrant methylation was 3.8% in primary kidney tumors in patients
with ccRCC. A similar analysis was performed for the gene of the
cyclin-dependent kinase inhibitor CDKN2A encoding the p16 protein. Aberrant
methylation is determined only in samples of tumor tissue and is not detected
in normal renal parenchyma. The frequency of promoter methylation of this gene
in primary tumors was 20.9%. In our study, biallelic inactivation of the VHL
gene was detected in 30.4% (7/23) cases. The results of our analysis of
biallelic inactivation of the VHL gene indicate its important role in the onset
and development of clear cell renal cancer [I. Gilyazova, 2012; Kutlyeva L.,
2012].In addition, to date, analysis of the expression of 758 mature miRNAs using
OpenArray technology and the QuantStudio 12K FlexReal-Time PCR System has been
performed, increased expression of miRNA-210 and miRNA-642 has been detected in
kidney tumor samples compared to normal tissue. To assess the involvement of
methylation of CpG islands that overlap the promoter regions of miRNA genes in
the pathogenesis of kidney cancer, methylation analysis of 22 miRNA genes in
tumor tissue of the kidney and normal renal parenchyma of patients with clear
cell renal cell cancer using EpiTectMethyl II PCR Array technology (Qiagen) was
performed. The analysis revealed two genes and two miRNA clusters with
statistically significant changes in methylation levels: let-7g, miRNA-301a,
miRNA-23b / -24-1 / -27b cluster and miRNA-30c-1 / -30e cluster. Subsequent
analysis of differential methylation miRNA gene expression established a
statistically significant increase in miRNA-301a, miRNA-23b expression in
kidney tumors in patients with kidney cancer compared with normal renal
parenchyma. A statistically significantly lower level of let-7g expression was
found in kidney tumors compared with adjacent normal tissue [Klimentova et al.,
2019].Analysis of the expression of the miRNA-200 family (microRNA-200a,
microRNA-200b, microRNA-200c, microRNA-141 and microRNA-429) in metastatic
kidney cancer showed a significant decrease in the expression of miRNA-200c in
kidney tumors compared to normal tissue [Gilyazova et al ., 2019].In addition,
the laboratory team is studying the molecular genetic basis of other oncological
diseases. Since 2010, the group of authors has been part of the international
consortium for the study of breast cancer (BCAC) and ovarian cancer (OCAC). In
a joint study with the BCAC consortium, potential candidate genes MAP3K1,
SLC4A7, STXBP4, TGFb1, PIK3CA, LSP1 and others associated with the disease were
identified [Milne R, 2011; Ghoussaini M, 2012; Dörk T. et al., 2019], new loci
associated with different histological types of epithelial ovarian cancer were
identified [Phelan C. et al., 2017].Studies of allergic diseases (bronchial
asthma, allergic rhinitis, atopic dermatitis) are carried out by the team of
the Laboratory of Human Molecular Genetics in collaboration with the Department
of Pediatric Diseases and the Department of Propaedeutics of Internal Diseases
of the Bashkir State Medical University (BSMU) since 1999. Representative DNA
samples from patients with BA, AR, AD (about 900 people) and the control group
of healthy individuals (600 people), corresponding to each other by gender, age
and ethnically accessories. A partial sample of patients with familial cases of
bronchial asthma was recruited. A partially formed bank of RNA isolated from
the peripheral blood of patients with BA, AR and healthy individuals (20
people, respectively). Russians, Tatars and Bashkirs living in the Republic of
Bashkortostan have identified genetic risk markers for the development of
atopic dermatitis, bronchial asthma and allergic rhinitis by polymorphic
variants of cytokine genes (IL4, IL4RA, IL9, IL10, IL13, CCL11, TNFA) and
xenobiotic detoxification genes ( CYP1A1, CYP2C9, CYP2C19, CYP2D6, GSTM1,
GSTT1, NAT2) [Karunas A.S. et al., 2004, 2007; 2012; Khusnutdinova E.K.,
Karunas A.S., 2007; Ramazanova N.N. et al., 2007, Fedorova Yu.Yu. et al., 2009,
2010, 2011]. In all ethnic groups, a similarity of risk markers for the
development of upper and lower respiratory tract allergic diseases — allergic
rhinitis, bronchial asthma, and their combined manifestation was found. It was
revealed that the most important risk marker for the development of atopic
dermatitis and other allergic diseases against the background of AD are
mutations in the filaggrin FLG gene [Karunas A.S. et al., 2012]. In the study
of 64 polymorphic loci of image-recognizing receptor genes, an association of
blood pressure with polymorphic gene variants of Toll-like receptors (TLR1,
TLR6 and TLR10) was established [Gimalova et al., 2014]. Within the framework
of the integrated project of the Sixth Framework Program of the European Union
GABRIEL, a genome-wide analysis of the association of 610,000 polymorphic loci
scattered throughout the genome with the development of bronchial asthma in
individuals of Russian, Tatar and Bashkir ethnicity was carried out. The
polymorphic loci associated with the development of AD with a genome-wide
significance level (p <4.79x10-7) and localized in the region 17q12-q21 were
identified [Karunas AS, 2011]. For the first time, a pronounced association of
AD with polymorphic loci of the beta1,4-galactosyltransferase 1 gene of B4GALT1
(in the region of 9p13) and the IGFBP3 gene encoding protein 3 binding to
insulin-like growth factors (in the region of 7p12.3) was found [Karunas AS,
Khusnutdinova E. K., 2013]. In a genome-wide analysis of the association and a
replicative study on an independent sample of individuals conducted in separate
ethnic groups, ethnic-specific markers of the risk of developing AD were
identified in Russians (MUC19 (12q12) and KIAA1671 (22q11.23), Tatars (RAPGEF4
(2q31.1), rs12719740 (rs12719740 ( 15q26.3) and rs10435941 (9q21.2)) and the
Bashkirs (GRIA1 (5q33.2), rs12719740 (15q26.3), DACT2 (6q27)) [Karunas A.S.,
Khusnutdinova E.K., 2013; Karunas A.S. et al., 2015].As part of a
pharmacogenetic study of asthma was made an analysis of polymorphic variants of
the beta2-adrenoreceptor gene ADRB2, the glucocorticosteroid receptor GR, the
corticotropin releasing hormone CRHR1 receptor and TBX21 transcription factor
in patients with bronchial asthma and healthy individuals living in the
Republic of Bashkortostan. Markers of an increased risk of developing AD, its
severe form, uncontrolled and partially-controlled course of the disease with a
decrease in spirography parameters (FVC, OFV1, MOS25, MOS50, MOS75) [Murzina
RR, 2015; Fedorova Yu.Yu., Karunas A.S. et al., 2016]. In the study of
polymorphic variants of genes involved in the metabolism of widely used
anti-asthma drugs - glucocorticosteroids (GLCCI1, T (TFT), ZNF432, ALLC, FBXL7,
CMTR1, MAGI2) and antileukotriene drugs (ALOX5, ALOX5AP, LTA4H, LTLC4 gene
variants GLCCl1, FBXL7, MAGI2, T (TFT), ALLC, LTA4H, ALOX5AP and LTC4S in the
development and course of bronchial asthma during therapy with
glucocorticosteroid and antileukotrienic drugs [Fedorova Yu.Yu., Karunas AS et
al., 2019].The research results were published in peer-reviewed Russian and
foreign journals, reported at Russian and international conferences. Two
patents for predicting the risk of developing AD have been developed. (Karunas
A.S., Shalukhina A.R., Zagidullin S.Z., Islamgulov D.V., Khusnutdinova E.K. A
method for predicting the risk of developing bronchial asthma. Patent for the
invention No. 2324937. Priority of the invention November 27, 2006;
Khusnutdinova E.K., Karunas A.S., Fedorova Yu.Yu. Nurgalieva A.Kh., Yunusbaev
BB. A method for predicting the risk of developing bronchial asthma Patent for
invention No. 2510508. Priority of invention 13.07.2012).At present, a unique
complex of instruments and equipment has been assembled at the IBG UFRC RAS,
which allows solving the most complex research problems and tasks. The
Institute has the following modern equipment necessary to carry out the work
planned in this project: automatic DNA sequencer model ABI Prism 3500
(AppliedBiosystems, USA); NanoDrop 1000 spectrophotometer (ThermoScientific, USA),
MastercyclerClassic amplifiers (Eppendorf, Germany), T100 (Bio-Rad, USA), Milli
Q Academic ultrapure water production system (Millipore, USA), Multifuge 1S-R
refrigerated centrifuge (ThermoScientificHeraus) , 5415R refrigerated
microcentrifuge (Eppendorf, Germany), MiniSpinPlus microcentrifuge (Eppendorf,
Germany), personal vortex V-1 plus (Biosan, Latvia), Elf-4, Elf-8 power sources
(DNA-Technology, Russia) and PowerPackBasic (BioRad, USA), electrophoresis
chambers Mini-ProteanTetraCell (BioRad, USA), VE-20, SE-1 and SE2 (Helikon,
Russia), laminar boxes BAVnp-01- “Laminar-S” -1.2 (Mias, Ros this), electronic
scales OHAUSAdventurer (China), autoclave MelagEuroklav 23 VS (Germany),
solid-state thermostats “Gnom” (DNA technology, Russia) and “Termite” (DNA
technology, Russia), magnetic stirrer MSH-300 (Biosan, Latvia), thermostat
TS-40 (Russia), programmable rotator mixer (Multi RS-60), low-temperature
refrigerators -85C models MDF190, MDF-192 and MDF 32UVL (Sanyo, Japan),
SpeedVac concentrator (Savant, USA), GelDoc XR gel documentation systems
(BioRad, USA) and DocPrint 001.FDC (VilberLourmat, France), a local area
network of computing and processing computers.The project team has extensive
experience in the field of molecular genetic studies of oncological diseases
and has all the methods of molecular biological analysis, including the
experience of microRNA expression using OpenArray technology using the
QuantStudio 12K FlexReal-Time PCR System, experience in exome sequencing with
subsequent bioinformatics data processing. High qualification of team members
is confirmed by the availability of publications in leading scientific
journals. 4.3.7.8. Detailed work plan for the first year of the project. 1. To
search for genetic markers of the risk of breast and ovarian cancer developing,
as well as specific markers of various types of these diseases based on data
from genome-wide studies. 2. Form a panel containing ~ 60 polymorphic loci
located in the genes of miRNA precursors, participants in miRNA biosynthesis
and processing, or miRNA binding sites with the target; 3. Genotyping
polymorphic variants using the QuantStudio ™ 12K Flex system
(AppliedBiosystems) in patients with cancer and allergic diseases.