Hepatitis C virus (HCV) genotyping (RNA determination) (genotypes 1a, 1c, 2, 3a/3b)

Competition “Bio/Mol/Text”-2020/2021

This work was published in the “Viruses and Microorganisms” category of the “Bio/Mol/Text” competition 2020/2021.

The general partner of the competition is the annual biotechnology conference BiotechClub, organized by the international innovative biotechnology company BIOCAD.

The sponsor of the competition is SkyGen: a leading distributor of life science products on the Russian market.

Competition sponsor: the largest supplier of equipment, reagents and consumables for biological research and production.

"Book" sponsor of the competition - "Alpina Non-Fiction"

History of the emergence of concepts

The works of Charles Darwin became the main impetus in the development of the science of heredity and variability. He was the first to put forward the hypothesis of the separation of cells in the body, from which another individual emerges as a result. Thus, Darwin began to develop the theory of pangenesis and his work as a result became the impetus for the development of the science of heredity and variability.

In 1865, G. Mendel managed to formulate the basic laws of genetics by conducting experiments with different varieties of peas: the law of uniformity of first-generation hybrids, the law of segregation, the law of independent inheritance of traits. The date of birth of genetics is attributed to 1900; its term was proposed by W. Batson in 1906. Based on an already known concept, in 1909. Wilhelm Johansen introduced the concept of "gene". Around the same period, he introduced the concept of “phenotype,” thereby emphasizing the hereditary characteristics of genetics.

Later, the German zoologist W. Hacker studied the relationship between the genotypes and phenotypes of organisms, which was later called phenogenetics.

In general, the history of the development of genetics has a rich past; it is divided into three stages:

Stage 1 (1900-1930). The period of classical genetics, Mendelism. Natural discrete heredity has been established. The chromosome theory and the theory of mutations were created.

Stage 2 (1930-1953) . Research was carried out in the field of molecular genetics and the provisions of classical genetics were revised. An integrated approach to research began to be used.

Stage 3 (1953 to present). Decoding the genetic code. The internal and external influences of the variability process are considered. Structural and systemic knowledge of the deep essence of the gene is carried out.

Why do we need to know about genetic variants of viruses?

News about coronavirus remained the most read throughout 2022, and the number of scientific publications about SARS-CoV-2 was almost five times higher than the number of articles on another “high-profile” topic - CRISPR/Cas systems: according to PubMed, there are already more than 60 thousand of them! And here it is more important to monitor not so much the dramatic statistics of infections and outcomes, but rather the potential and abilities of the pathogen. After all, viruses can mutate and thus generate new genetic variants [1].

Genetic variants of a virus are the genomes of a certain type (or strain) of a virus that differ from each other in nucleotide sequence, and these differences can lead to the emergence of new strains. “Genotype”, “subtype”, “genetic variant” are terms that reflect the degree of genomic differences (in descending order).

As knowledge on this topic accumulates, scientists’ concerns only grow stronger: mutations can affect both virulence and the severity of the disease, the development of drug resistance and the likelihood of re-infection [2].

SARS-CoV-2 was divided into 3 genotypes in the spring of 2022: A, B and C [1]. But now special attention is paid to some genetic variants of this virus and their geographic distribution (Fig. 1) [3]. It is known that even the smallest change in the genetic material of the pathogen can greatly affect the course or spread of the disease. What could the mutations in the SARS-CoV-2 genome have affected?

Figure 1a. Schematic representation of the evolution of SARS-CoV-2

[3]

Figure 1b. Map of the distribution of genetic variants of SARS-CoV-2

[3]

For example, a single mutation in the spike protein gene, which caused the replacement of its 614th amino acid, D614G , contributed to the spread of SARS-CoV-2 around the world [4], [5].

Another mutation, C14408T , in the sequence encoding the viral RNA-dependent RNA polymerase (RdRp), has the potential to influence the development of drug resistance [6], [7]. But the most striking case can be considered when in a patient one genetic variant of SARS-CoV-2 was replaced by another [8]. The fact is that our immune system essentially builds protection against a certain viral genotype. If the virus changes, the organism that encountered its previous variant may not recognize the new one.

However, we can calm down a little: the mutations of SARS-CoV-2, although they created a good fuss, have not yet shown clear clinical significance - unlike changes in a number of other viruses. They will be discussed further.

Why are genotypes determined?

In order to find out which genotype of hepatitis C is the most dangerous, first of all you should clarify what a genotype is . The term in question refers to types of hepatoviruses, the main difference of which is a certain gene set.

They are indicated by Arabic numerals. They have additional quasi-types (subtypes), which are marked with Latin letters. An example of a pathogen type and quasi-type designation is gen 1a.

Regardless of which genotype is the most dangerous for hepatitis C, many of them have a very unstable set of genes. For this reason, the pathogen mutates extremely quickly , resulting in resistance to even the most powerful antiviral drugs.

Thus, determining the type of pathogen is then necessary to determine the appropriate therapy for each specific case. Moreover, which is the most dangerous genotype for hepatitis C can be found out from statistical data obtained on the basis of official studies.

Hepatitis viruses and company

Hepatitis

In 2022, the Nobel Prize in Physiology or Medicine was awarded for the discovery of the causative agent of one of the hepatitises, the hepatitis C virus (HCV) [9]. The disease can occur without visible symptoms, but the virus severely affects the liver. In extreme cases, cirrhosis or even liver cancer develops [10]. Fortunately, there are already effective treatments for hepatitis C: according to Nobel laureate Harvey Alter, direct-acting antiviral drugs can cure 95–98% of patients [9]. So what's the catch?

The hepatitis C virus has seven genotypes (Table 1) [10], and in order to choose the optimal course of treatment, it is necessary to first determine the genotype of the pathogen that infected a particular patient. Despite the fact that pangenotypic (universal, independent of the genetic variant of the virus) treatment regimens have already appeared, they are more difficult to reach patients, including because of their cost [11].

Interestingly, HCV genotypes 1 and 4 are usually less responsive to therapy and require longer treatment than genotypes 2 and 3 [12].
Table 1. HCV genotypes and subtypes [13]

At the same time, due to high genetic variability, that is, large differences between viral genotypes (up to 30–35%), it is not possible to create an effective vaccine against HCV. Compounding the problem, testing vaccine prototypes requires small animal models, and it is difficult to produce mice that can both develop hepatitis C and mimic the human immune response to infection. Finally, to study the virus in vitro, it is necessary to produce infectious HCV particles, and cell cultures capable of this have only recently appeared [14].

Sequencing of the viral genome showed that a recombinant variant of HCV (RF2k/1b), genetically similar to genotypes 1 and 2, is circulating in Russia. Due to such masking, detection of this genotype is difficult, and therefore treatment can be less effective [15].

So, in order to effectively cope with hepatitis C, you need to determine the genotype of its causative agent. It is equally important to know the genotype of the virus that causes another hepatitis - hepatitis B. Yes, there is a vaccine against HBV [16]. But! There is reason to believe that HBV genotypes, despite their relatively small variability (about 8% of the genome), have different effects on the severity of the disease [17], [18].

It is worth considering, however, that the results of studies of the relationship of viral genotypes with the characteristics of the course of the disease are very contradictory. Most often, this is due to the insufficient size of the studied samples, which, in turn, is due to the difficulties of mass genotyping. Because of this, for example, in Taiwan, the development of hepatocellular carcinoma is associated with HBV genotype B, and in Japan and China - with HBV genotype C [19]. However, some authors conclude that the HBV genotype does not have much clinical significance at all [20].

Although there are no general recommendations for HBV genotyping yet, a number of scientists are calling for it to be made mandatory, since pan-genotypic therapy for hepatitis B has not been developed, and the effectiveness of drugs sometimes varies depending on the genotype.

Detection of the virus genotype can be a good help for doctors in at least two directions: in determining the likelihood of developing a particular form of the disease in a particular patient and in personalizing treatment. However, there are very few such studies; one might even say that this field has not yet been plowed at all.

Dengue

We have already noted that when developing vaccines, it is necessary to know and take into account the diversity of virus genotypes. In this regard, striking examples include approaches to the production of vaccines against influenza and a less common disease, dengue fever, the causative agent of which we will now talk about [21].

Figure 2. Geographical distribution of dengue endemic areas in 2011

[21]

The dengue virus has four serotypes, that is, four groups of viruses with a common antigenic structure. This pathogen has more genotypes, which makes it more difficult to track them. Alas, knowledge of the serotype is sometimes not enough for high-quality vaccination. For example, the low effectiveness of one of the vaccines against serotype 2 in clinical trials can be explained by the large variability of its constituent genotypes [22].

There is an opinion that if the effectiveness of dengue vaccines in new clinical trials remains low, when creating vaccine preparations it will be necessary to build on the results of genotyping [21]. This is also necessary to exclude a possible antibody-dependent increase in infection if the vaccine does not protect against all serotypes/genotypes that an individual organism may encounter.

Note

Antibody-dependent enhancement (ADE) is a phenomenon in which the binding of a virus to suboptimal neutralizing or non-neutralizing antibodies promotes its penetration into immune cells and subsequent replication in them. Because of ADE, untested vaccines can be very dangerous: when faced with infection, instead of providing protection, some of them can worsen the course of the disease [23].

To summarize, knowledge of virus genotypes is essential for tracking the spread of diseases around the world, for treating them, and for obtaining effective vaccines. Unfortunately, so far genotype-specific control of infections is greatly limited, among other things, by the lack of information about viral strains.

But how does this strain diversity appear in the first place?

Territorial distribution

Hepatitis C is common in all WHO regions, but the burden of the disease varies by region. An estimated 12 million people have hepatitis C in both the WHO Eastern Mediterranean Region and the WHO European Region, respectively. In the South-East Asia and Western Pacific regions, approximately 10 million people each suffer from chronic hepatitis C, while in the African Region and the Americas Region, the number of people living with chronic hepatitis C is estimated to be 9 and 5 million, respectively.

Viral cunning: mechanisms of genome modification

Viruses are able to change their properties inside host cells and, as a result, become more infectious and more dangerous. However, don't be afraid. Typically, viral evolution proceeds in the direction of decreasing lethality, since it is much easier for a virus that does not kill its host and causes almost no symptoms to spread throughout the population. Although the genomes of viruses change with a fairly high frequency, in fact, mutations more often turn out to be neutral, not entailing noticeable consequences for the virus and its host. Very few of them actually contribute to further evolution [24].

The ability of viruses to change is based on mutations and/or the exchange of genetic material between different viruses, the results of which are fixed or not fixed by the pressure of natural selection.

A mutation is a change in the nucleotide sequence in a certain part of the genome, often leading to changes in the structure and/or functions of an organism or virus. Mutations can occur due to errors in the enzymes that create copies of genomes, or under the influence of the environment.

When two related viruses infect the same cell, hybrid viruses containing altered genetic material compared to the parent forms can be formed. This process is called recombination or reassortment if viruses have a segmented genome (it is packaged into a particle with separate segments such as mini-chromosomes) [25].

For reference

Before the introduction of the term “reassortment” into scientific use, recombination was the name given to any exchange of genes, including those processes that are now competently called reassortment [25].

Reassortment is very similar to the birth of children from parents: when two different virions enter the same cell and begin to reproduce, they can exchange parts of their genomes (Fig. 3). The result will be a new variation (strain) of the virus, which may differ significantly from its ancestors [25]. Such viruses sometimes become unrecognizable to the immune system and spread, causing epidemics and pandemics. This scenario is typical, for example, for influenza viruses. Some researchers blame viral reassortment for the Spanish flu pandemic [26].

Figure 3. Reassortment of viruses, which leads to the emergence of a strain with altered genetic material

[27]

Thanks to mutations and the exchange of genetic material, the virus can even change its host. So SARS-CoV-1 passed from bats to the civet family (musangs and civets), and then to people. In the early 2000s, this virus caused an epidemic that affected 29 countries [28].

New genotypes can also arise during laboratory passages, when several animals are sequentially infected with the virus, isolating from them a new pure culture of the pathogen [29]. This procedure is needed, in particular, for testing antiviral drugs and vaccines.

The properties of the virus change stepwise during passages. In the first passages, virions are mainly found that have changed one genetic trait. But with an increase in the number of passages, changes in many genetic characteristics are observed in the vast majority of viral particles.

In addition to recombination and mutations, virus variability can be caused by the influence of the host (host-controlled variation). Such modifications do not affect the genetic material of the virus and are found, for example, in DNA-containing bacteriophages, influenza and Sendai viruses. A cell can influence the nature of the viral components synthesized in it. Or the viral particle may contain host proteins and lipids. Therefore, when the host cell changes, the structure of the virion shell also changes the antigens with which the body recognizes the virus.

So, the formation of new viral genotypes is a completely common phenomenon based on standard evolutionary processes. However, how can we detect these genotypes?

Which genotype of hepatitis C is the most dangerous?

Any HCV infection carries a certain danger to the body of its carrier. But, still, which genotype is the most dangerous for hepatitis C?

The opinions of scientists and hepatologists differ. Some consider gen 1 due to the extreme viral load and difficulties in carrying out therapeutic measures. Other researchers are of the opinion that the third gene form due to the large number of complications associated with this variant of infection.

Regardless of which is the most dangerous genotype for hepatitis C presented above, any such disease requires urgent treatment . Otherwise, severe complications of the disease may occur even with relatively loyal gen 2 HCV.

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What is the most dangerous genotype for hepatitis C?

How are virus genotypes determined?

Now that we know how viral genotypes arise and how the course, spread and treatment of the disease can depend on the genetic variant of the pathogen, let's move on to discussing laboratory methods. They allow us to determine which genotype of virus caused the disease in a particular patient. Let's consider methods for detecting genotypes using the example of the HCV virus. Currently, laboratory tests used in the diagnosis and treatment of hepatitis C include serological tests for the detection of antibodies to HCV, molecular tests for the detection and quantification of HCV RNA, as well as methods for genotyping the pathogen [30].

When initially diagnosing hepatitis C, symptoms and levels of liver enzymes (especially alanine aminotransferase, ALT) in the blood are taken into account. In patients with suspected hepatitis C, antibodies to HCV are detected by enzyme-linked immunosorbent assay (EIA) [31] or chemiluminescent immunoassay (CIA). In populations at low risk of infection with this virus, a negative EIA or CIA result is sufficient to rule out infection with hepatitis C. But it is important to remember that in patients with HIV or end-stage renal disease, serological tests for HCV antibodies may give false-negative results. If the test reveals antibodies or the possibility of a false negative result is accepted, the RNA of the virus itself should be looked for in the patient's body.

Determining HCV infection status, treatment decisions, and monitoring response depend on three important factors: detection of HCV RNA, its quantity, and viral genotype. There are several commercial test systems available to evaluate all these parameters. They vary in detection range, sensitivity, specificity, cost, and difficulty of use.

Detection of hepatitis C virus RNA is based on the principle of nucleic acid amplification [32], and a combination of types of polymerase chain reaction (PCR) is used: qualitative PCR, transcription-mediated amplification (TMA) and quantitative real-time PCR.

HCV genotyping is necessary to prescribe the optimal treatment regimen. The genotype of this virus can be determined by several methods that target the viral protein genes (E1, NS4 and NS5) and the 5′ untranslated region (5′-UTR) of the HCV genome. These include simple PCR, hybridization with genotype-specific probes, and real-time PCR. However, to more accurately determine the viral genotype, next generation sequencing (NGS) methods should be used [33].

Mechanisms of transmission

Hepatitis C virus is a blood-borne virus. The most common routes of transmission are:

• reuse or insufficient sterilization of medical equipment, in particular syringes and needles, in health care settings;
• transfusion of unscreened blood and blood products;
• sharing injection equipment when injecting drugs.

HCV can also be transmitted from an infected mother to her child, and through sexual contact that involves contact with blood (for example, people with multiple sex partners or men who have sex with men). However, these mechanisms of transmission are less common.

Hepatitis C is not spread through breast milk, food, water, or casual contact such as hugging, kissing, or sharing food or drink with an infected person.

WHO estimates that there will be 1.5 million new cases of HCV infection worldwide in 2022.

Our project HaploSense

As we have already understood, determining genotypes is difficult, since there are practically no quick and accessible tests. Our team of undergraduate and graduate students called Moscow 2022 decided to change this situation.

At the international iGEM competition this year, we presented a project for a detector of hepatitis C virus genotypes. The detection is based on the CRISPR-Cas system, with which many readers are already familiar [34], [35], and the operating principle is reminiscent of the popular SHERLOCK (Specific High) detection methods sensitivity Enzymatic Reporter unLOCKing) and DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) [35]. But there are also important differences, which we will now talk about.

In our test system, we used the smallest Cas protein discovered so far, CasX (or Cas12e) from Deltaproteobacteria: it consists of 980 amino acid residues compared to 1300 for Cas9 [36]. Like Cas13a and Cas12a, small CasX has collateral nuclease activity (it can nonspecifically cut nucleotide sequences that it encounters after binding to a target): it allows it to cut oligonucleotides with fluorescent tags after the protein has attached to the viral genome. But HCV is an RNA virus, so in order for CasX to bind to it, it is necessary to carry out an additional reaction: using reverse transcription to obtain a complementary DNA sequence.

So, in our system there are four main stages (Fig. 4):

1. HUDSON. The sample is heated to 95 °C, which neutralizes both the virus and RNases [37].
2. RT-LAMP. Reverse transcription and isothermal amplification of the genetic material of the virus are carried out - already in the form of DNA [38]. Thanks to the use of LAMP (loop-mediated isothermal amplification), expensive PCR devices are not needed for testing; you only need a simple thermostat to maintain a constant temperature.
3. Detection. CasX binds to target DNA and, through collateral activity, cuts short single-stranded DNA labeled with the fluorophore FAM and biotin at opposite ends.
4. Visualization. The resulting mixture is applied to test strips with two binding zones: one is a control zone, containing streptavidin and interacting with any oligonucleotides labeled with biotin (both cut and whole), the second is a binding zone of cut oligonucleotides without biotin, but with FAM. Thus, when the strip is placed in a fluorimeter, two lines will mean that the patient is infected with HCV of a certain genotype.

So far, our test system involves the detection of only one genetic variant of HCV - recombinant RF2k/1b, common in Russia. In 2022, access to the laboratory was closed for us, so the system still has to be assembled and tested. If all laboratory tests are successful, we plan to expand the specificity of the system to all genotypes of the virus, without increasing the number of devices.

Figure 4. HCV genotype detection scheme using HaploSense

This seemingly complex system will make it possible to determine the genotype of the hepatitis C pathogen faster, outside a specially equipped laboratory and with no less accuracy than PCR. We hope that one day this approach will help make testing widespread and accessible.

Treatment

Primary HCV infection does not always require treatment, since in some patients the immune system itself successfully copes with the virus. However, treatment is indicated for those patients in whom hepatitis C becomes chronic. The goal of therapy is complete cure of hepatitis C.

The 2022 WHO guideline update recommends pangenotypic direct-acting antiviral (DAA)-based therapy. DAAs can cure most HCV-infected people; however, the course of treatment is short (usually from 12 to 24 weeks), and its duration depends on the absence or presence of liver cirrhosis.

WHO recommends treatment for all persons with chronic HCV infection aged 12 years and older. In many high- and upper-middle-income countries, the cost of treatment with pangenotypic DAAs remains high. However, in many countries (mainly low- and lower-middle-income countries), prices have fallen sharply with the advent of generic drugs.

Access to HCV treatment is improving but remains too limited. In 2022, of the 58 million HCV carriers worldwide, about 21% (15.2 million people) knew their diagnosis; Moreover, by the end of 2019, about 62% (9.4 million) of diagnosed carriers of chronic HCV infection had completed a course of DAA treatment. Much more needs to be done to achieve the 2030 goal of treating 80% of those infected with HCV worldwide.

More about iGEM

In 2022, a team of students from the Faculty of Biology of Moscow State University presented their development at the most prestigious international competition in synthetic biology - The International Genetically Engineered Machines competition (iGEM). The name translates from English as “International Competition of Genetically Engineered Machines.” This competition was established in 2003 at the Massachusetts Institute of Technology (Boston, USA). Schoolchildren, undergraduates and graduate students from all over the world and with different academic backgrounds, from journalists and economists to IT specialists and biologists, gather in teams, work on a pressing problem throughout the year and present their projects at the autumn, final Giant Jamboree conference. The iGEM competition is unique in that it creates an open community of people who share their work with each other and the world. The competition is also distinguished by the complexity and diversity of the projects: the emphasis is on the application of engineering principles and approaches, the use of modeling methods, discussion and elaboration of the project with potential users and experts from different fields (science, business, law, public administration, etc.). Educational activities, popularization of science, building bridges between scientists and society in order to discuss current problems - all this is also part of the team’s tasks. This year, due to the pandemic, the competition was held online. The core of our team consisted of students from the Faculty of Biology of Moscow State University, who gained invaluable experience and won a gold medal. However, we will talk more about iGEM next time [39].

Basic concepts of genetics

In addition to these two concepts, the basic concepts of genetics include others:

1. Heredity is the transfer of characteristics from one living organism to another;
2. Variability is the process of the offspring acquiring distinctive characteristics during individual development;
3. Allelic gene – a gene that occupies identical loci in homologous chromosomes;
4. Heterozygote is an individual that carries a gene of a different molecular structure;
5. Homozygote is an individual that carries a gene with the same molecular structure.

Teamwork

At the end of March 2022, the Moscow 2022 team assembled a new lineup to participate in the iGEM competition (Fig. 5). Then, for the first time, strangers met each other with a common desire - to create a project in the field of synthetic biology that could change something in the world.

The idea for our project appeared only a month and a half later, at the beginning of May. iGEM ​​offers complete thematic freedom: you can make bread from yeast on Mars, purify water with bacteria, synthesize natural hair dye, or develop disease monitoring systems. The choice of topic for our project was greatly influenced by the team leader, Alexey Konstantinovich Shaitan. His laboratory uses CRISPR-Cas systems, and on their basis it is possible to create disease detectors. The COVID-19 pandemic was just beginning, but it was already clear that SARS-CoV-2 needed to be detected quickly, on a large scale and with high accuracy. This is how we created our project - with the goal of detecting coronavirus genotypes.

A very important requirement of the competition is the relevance of the development. In other words, it was important to understand that people need our project and will use it. This makes iGEM projects very different from the routine work of scientific laboratories. That is why we met with experts, wrote letters to the chief epidemiologist of the Ministry of Health, Nikolai Ivanovich Briko, and posted posts on social networks. The feedback completely changed our project: HaploSense refocused on detecting hepatitis C virus genotypes.

On the other hand, the detector itself was important - how it would work, look, whether we could assemble it at all. Here our sponsor, the BIOCAD company, came to our aid. The company's experts advised us on the idea of ​​the project and its feasibility in such a short time. They told us how to prepare patents and bring a product to market. And these are far from simple processes.

As a result, we analyzed a mountain of relevant literature, were able to come up with a complete system based on CRISPR-Cas technologies, and carried out its modeling and bioinformatics analysis.

It must be said that iGEM organizers help teams along the entire journey: they organize seminars, introduce specialists, and create convenient platforms. And they especially support interaction between teams. For six months we communicated and met with motivated guys from America, Estonia, India, France, Germany and Russia (this year two teams from our country participated in the competition). After such meetings, we realized that we could make a good, high-quality project, even as students.

Figure 5. iGEM Moscow 2020 team

The pandemic, of course, played a role in the work of our team: we worked remotely and met each other offline only four times. We really became united when the competition deadlines approached. We met the deadline thanks to the well-coordinated work of the team. It would have been impossible for one person to cope with: in addition to developing the project idea, modeling and conducting experiments, we had to create a website describing the project, two videos about the team and work, fill out a safety form, write a review on the topic, and that’s not all. That is why the iGEM project is primarily a team effort.

The results of the competition are summed up in the fall, in early November, at the Giant Jamboree - a huge conference where teams, professors, lecturers and judges gather to discuss projects, note their strengths and weaknesses, form new collaborations and just have a good time. This is the final of iGEM: the projects have already been prepared, the competition criteria have been met. It would seem that you can exhale... But here the teams meet with judges - researchers who will evaluate projects and review them in detail. We, of course, also communicated with the judges (online this year): on Sunday, at 17:00, we were asked why we used LAMP, how many test strips we would use, and much more... And then our team waited a week , full of synthetic biology lectures and networking with peers from around the world, as well as synthetic biology quizzes and an awards ceremony. As a result, we won a gold medal and created a good project, which we will continue to develop and, we hope, bring to the status of a full-fledged test system suitable for ordinary clinics.

We encourage all young researchers to take part in iGEM. This competition has become the best event of 2022 for us. It is possible to create a project from scratch, you just need the desire!

What is a phenotype?

A phenotype is a collection of characteristics that an individual has at a particular stage of development.

“The phenotype is formed on the basis of the genotype”

The set of internal and external characteristics that an organism acquires during its development forms a phenotype. Each organism has its own individual internal and external features: the nature of metabolism, the degree of functioning of the body. This constitutes its phenotype.

Accidents of development

The variability of developmental phenomena depends on many reasons. Heredity tends to reduce developmental variability, whereas conditions not associated with heredity tend to increase it. Some developmental researchers identify four types of random factors that influence developmental variability:

• accidents in the selection of parental pairs, the genes of which make up the genotype of the individual;
• randomness of epigenetic (that is, external to the genotype) processes within individual ontogenesis;
• the randomness of the maternal environment in which the individual develops;
• the randomness of the non-maternal environment in which the individual develops.

Although these are random events, they all have an element of heredity. The genotype is inherited from the parents, and the offspring and parents have common genes that influence the course of individual development. Epigenetic processes within the body represent the influence of other cells or their products on the activity of the genotype of a given cell. Since all cells in the body have the same genotype, it is natural that epigenetic influences are associated with heredity. However, epigenetic processes are stochastic, open to the influence of environmental factors of the organism and, therefore, to any historical accidents.

The maternal environment of mammals is a very important element of the external environment. Mothers provide the intrauterine and postnatal (baby care and education) environment for the child. It is clear that these conditions are influenced by the mother's genotype. Partially, the mother's genes are shared with the offspring, so the maternal environment can be inherited. The maternal environment is also sensitive to historical contingencies.

Nonmaternal environmental effects also influence developmental variability. This includes factors that are chosen by the individual himself or shaped by the people around him, including relatives with whom he shares genes. Therefore, these environmental effects, to some extent, are also influenced not only by random environmental events, but also by genes, and are also inherited (genotype-environmental covariation).

Thus, in accordance with the above classification, in all the described elements of the environment external to a given individual, there are mechanisms for inheritance, both genetic and non-genetic (various traditions, etc.).

Naturally, non-heritable factors also influence development. These are those features of the environment that are not associated with changes caused by the developing individual himself or his related environment. They can be either random or natural. Regular changes include cyclical changes (change of day and night, change of seasons, etc.), widespread influences (gravity) or predictable factors (temperature, pressure). Non-heritable factors are also present in the maternal and other social environment (quality of maternal nutrition, maternal stress level, number and gender of siblings, etc.). Randomly or systematically changing environmental events contribute to variability in development.

All events external to genes that take place during the process of ontogenesis, together with genetic factors, create the background against which development takes place. Due to the impact of a huge variety of regular and random events in ontogenesis, developing systems can organize and reorganize. Genes make development possible, but other components that influence the development of the system are no less important participants in the development process.

At the beginning of the presentation, defining the concept of phenotype, we emphasized that the phenotype is the result of the interaction of the genotype and the environment, however, in the light of what has been said about the process of individual development, we must make some clarification in this formulation and, along with environmental factors, mention accidents of development that cannot be reduced to purely environmental influences. If we tried to graphically depict the dependence of the phenotype on various factors, then we would need at least a four-dimensional space in which, in addition to the axes for genotype and environment, there would also have to be an axis for the accidents of development.

Genotype, genome and gene pool

The term “genome” was proposed by the German biologist G. Winkler 10 years later than the concept of “genotype” was formulated.

Genome also refers to a collection of genes, but unlike a genotype, a gene is considered as a nucleotide sequence of DNA in haploid cells (with a single set of chromosomes), and not as an allele (an alternative form of the same gene) in a diploid set of chromosomes.

In order not to get lost in the jungle of genetics, it is important to understand the main thing:

The genome, being the hereditary material of an organism, is stored in several (sometimes in one) chromosomes, the number of which is unique for a particular species.

The genome size (based on the number of genes it contains) varies widely, reaching several tens of thousands of units. The fewest genomes were found in the simplest viruses (several hundred), the largest number were found in representatives of the plant world (for example, there are more than 46,000 of them in rice).

According to recent studies, the human genome consists of 46 chromosomes (23 pairs), which contain about 22-25 thousand genes.

Individuals from different populations can interbreed and produce offspring. The gene pool of a species is made up of the gene pool of populations.

The characteristic features of the gene pool include the following distinctive qualities:

1. Under constant environmental conditions, the gene pool remains unchanged.
2. When external conditions change, individuals whose genes possess traits useful for survival gain an advantage. It is these individuals that will pass on the most valuable qualities to their offspring during sexual reproduction.
3. The genes responsible for the most “advantageous” traits accumulate from generation to generation, creating the basis for changes in the gene pool.
4. The variability of the gene pool as a mechanism of natural selection is directional and helps to improve the adaptive functions of the body.

The evolutionary changes occurring in the population can be traced in the following examples. In harsh climatic conditions (extreme cold, heat), the proportion of genotypes that increase the thermal insulation of organisms increases.

In other cases, the preservation of a population largely depends on genes encoding the color of the animal (in order to improve camouflage), or the synthesis of protective enzymes (liquids, gases), or behavior patterns, etc.

All this makes the population (or species as a whole) more resistant to the external environment, and therefore ensures its survival.

It happens that some genes (in parents or their descendants) turn out to be damaged (mutated), but this has practically no effect on the genotype, since mutations caused by such genes are unstable and are almost never repeated in inheritance. It follows that the human genome is absolutely stable.

How can environment interact with genotype during development?

It is clear that the result of development - the phenotype - depends on the joint action of genes and the environment. Genes and traits are linked through a complex network of developmental pathways. All individual differences that differential psychologists and psychogeneticists are concerned with are the result of the developmental circumstances of specific individuals in specific environments. Often individuals brought up in apparently different environments have much in common. Conversely, siblings raised in the same family, seemingly under similar circumstances, due to subtle differences in the conditions of upbringing and development, will actually experience very different influences from both the physical and social environment.

Thus, the process of interaction with the environment is complex and ambiguous. Note also that psychologists and other researchers often use the term “interaction” in a statistical sense when examining the interaction of individual factors in the production of any measurable effect. Let us emphasize that the statistical interaction of factors and the interaction of genes and environment in individual development are completely different things. They should not be confused.

For us, the formulation is quite familiar, which states that the manifestation of a phenotype is the result of the interaction of the genotype with the environment during development. However, if you think about this statement, it does not seem so obvious. After all, interaction presupposes that its participants come into contact and come into contact. In fact, our genotype, that is, the genetic apparatus, is hidden deep inside the cell and is separated from the external environment not only by the integument of the body, but also by the cellular and nuclear membranes. How can the external environment interact with genetic structures?

It is clear that genes and the surrounding world are not in direct contact. The organism as a whole interacts with the external environment; genes interact with various biochemical substances inside the cell. But various cellular substances can be influenced by the outside world. Let's consider what is known about these processes today's science. To do this, we will again have to turn to molecular genetics and consider in more detail how genes function, since in the previous presentation we only stated that the main function of a gene is to encode the information necessary for the synthesis of a specific protein.

The relationship between phenotype and genotype

The genotype of an organism determines its phenotype. All living organisms have DNA, which provides instructions for the production of molecules, cells, tissues and organs. DNA contains the genetic code, which is also responsible for directing all cellular functions, including mitosis, DNA replication, protein synthesis, and molecular transport.

An organism's phenotype (physical traits and behavior) is determined by its inherited genes. Genes are specific sections of DNA that encode the structure of proteins and determine various characteristics. Each gene is located on a chromosome and can exist in more than one form. These different forms are called alleles, which are located in specific locations on specific chromosomes. Alleles are passed from parents to offspring through sexual reproduction.

Diploid organisms inherit two alleles for each gene; one allele from each parent. The interactions between alleles determine the phenotype of an organism. If an organism inherits two identical alleles for a particular trait, it is homozygous for that trait. Homozygous individuals express one phenotype for a given trait. If an organism inherits two different alleles for a particular trait, it is heterozygous for that trait. Heterozygous individuals can express more than one phenotype for a given trait.

Complete, incomplete and co-dominance

Traits can be dominant or recessive. In complete dominance inheritance patterns, the phenotype of the dominant trait completely masks the phenotype of the recessive trait. There are also cases where the relationship between different alleles does not show complete dominance. In incomplete dominance, the dominant allele does not completely mask the other allele. This results in a phenotype that is a mixture of the phenotypes observed in both alleles. With codominance, both alleles are fully expressed. This results in a phenotype in which both traits are observed independently of each other.