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What is a karyotype? Why is a karyotype study performed? Preparing for analysis

13.06.2024

What is a karyotype test?

Karyotyping – as a method for identifying genetic disorders leading to infertility

Each spouse, when getting married, dreams that sooner or later the sonorous laughter of a baby will be heard in their home. However, not everyone is destined to know what it is to be parents. Infertile couples today (unfortunately!!!) are not uncommon. However, medical science is constantly developing and putting into practice new informative techniques that make it possible to identify the exact cause of the disease. AND karyotype studies- one of them.

Violations in the genetic material of one of the spouses (or both) often lead to infertility. However, such changes do not manifest themselves as any special clinical picture.

What is a human karyotype

All living organisms on earth differ not only in appearance. Each species is characterized by a specific set of chromosomes, unique to it, called a karyotype. There are 46 chromosomes in the human karyotype. 44 (or 22 pairs) are autosomes that are present in somatic cells and are the same in men and women. And 2 are sex chromosomes that determine the sex of a person. A woman has the same type of sex chromosomes (XX), while a man has different sex chromosomes (XY). Accordingly, the female karyotype is 46, XX; male karyotype – 46, XY.

The chromosome set contains all the genetic information about its owner. It remains unchanged throughout a person's life. The karyotype of the unborn child carries half the genetic information from the father and half from the mother.

In case of infertility, this is a necessary study!

Karyotyping is a necessary examination for infertility

Most often, a karyotype study is performed for infertility in cases where other causative factors have been excluded. But recently, more and more often, this study is prescribed as mandatory during a comprehensive examination, since a genetic defect can be combined with other reasons, and play, among others, a fatal role. After all, it is violations in the structure and number of chromosomes that often lead to the impossibility of conception and fetal defects.

Karyotype research belongs to the group of cytogenetic methods. There are 2 types of karyotyping:

  • prenatal – study of the chromosome set of the fetus;
  • studying the patient's genetic material.

Indications

The main indications for karyotype examination are:

  • a history of 2 or more spontaneous miscarriages;
  • infertility;
  • oligozoospermia;
  • non-obstructive form of azoospermia
  • primary (or secondary) amenorrhea;
  • frozen pregnancy;
  • cases of infant mortality in the first year of life or stillbirth of a child in the family;
  • the birth of a child with congenital combined defects;
  • delayed development of the baby (both physical and mental;
  • genetic diseases in parents and close relatives;
  • suspicion of a genetic pathology based on existing external signs (for example: a specific shape of the skull, fingers, anomalies of the external genitalia, eyes, nose, etc.);
  • examination of donors of genetic material.

What are the deviations from the norm?

Karyotype: deviations from the norm

The karyotype is laid down at the initial stages of the formation of the organism. And even then an incorrect karyotype may occur. In the case when malfunctions occur during the process of oogenesis or spermatogenesis, respectively, in a woman and in a man, education (during gametogenesis) in future parents, the genetic material of the zygote, which comes from the parents, is already damaged. And as soon as such a zygote begins to divide, all cells receive a “defective” karyotype.

Most often, embryos with an “incorrect” karyotype die in the early stages of pregnancy. This is due to the presence of various combined defects in them, in which further development is impossible. A woman has a miscarriage. In some cases (their share is 1.5-2%), the fetus still survives, and the pregnancy ends with the birth of a child with an abnormal karyotype. At the same time, already in the first hours of life, signs of congenital genetic abnormalities are determined, which leads to the need to study the karyotype of such a baby.

The main genetic abnormalities include:

  • Down's disease;
  • Patau syndrome;
  • Edwards syndrome;
  • Klinefelter's syndrome;
  • Shereshevsky-Turner syndrome;
  • cat cry syndrome;
  • polysomy on the X chromosome.

Karyotype disorders also include changes that directly affect the structure of chromosomes:

  • translocations are rearrangements that occur between different chromosomes and are characterized by the transfer of a fragment of one chromosome to another;
  • deletions – loss of a certain region of a chromosome;
  • inversion – rotation of a chromosome fragment by 180°;
  • duplication is the appearance of an additional copy of a certain section of a chromosome, which can be located directly behind the duplicated section, or in another place on the same chromosome, or in a completely different chromosome.

Preparation for karyotyping

The test should not be taken on an empty stomach. 3-4 weeks before the procedure, you should avoid taking antibacterial drugs.

How is karyotyping performed?

Carrying out karyotyping

The patient's blood is drawn, from which lymphocytes are subsequently isolated. Biological material is analyzed at the stage when the cell enters the process of division. To do this, cells are placed in a test tube and stimulated to trigger the mechanisms of mitosis. After a few days, when the chromosomes can be examined, the process is stopped (certain substances are used for this).

The cells are placed on a glass slide, stained with special dyes, and viewed under a light microscope. This research technique allows us to examine the structural features of chromosomes, their shape and size, the presence of heterogeneous zones and locations of constrictions.

The image obtained in the microscope is recorded using a camera (several times to obtain a more accurate picture). Next, the images are studied and analyzed.

To obtain the most reliable result, the karyotype of several cells (11 or 13) is examined.

The results obtained are studied by geneticists. If any abnormalities are detected in the karyotype of both porters (or one), the specialist determines whether it is the cause of infertility. If the answer is positive, in order to minimize the effect of this factor and get rid of the diagnosis of infertility, an individual scheme of further actions is drawn up for this couple.

If the risk of congenital anomalies in the unborn baby remains high, it is recommended to conduct a study of the genetic material of the fetus at the onset of pregnancy. This procedure is carried out in the early stages, is highly informative and allows you to prevent the birth of a child with complex, severe and even incompatible defects.

There are several types of prenatal diagnostics:

Non-invasive– includes an ultrasound of the fetus and a biochemical blood test for the presence of specific markers in it. All these methods are safe, but do not make it possible to “see” the child’s karyotype.

Invasive– involves the collection of fetal genetic material (eg, amniotic fluid, umbilical cord blood) through penetration into the uterus. This technique allows you to study the karyotype and identify genetic diseases of the unborn child. Due to the risk of complications, invasive intervention is performed only when indicated.

The human karyotype consists of 46 chromosomes. The very definition of a karyotype implies not only an analysis of the number of chromosomes, but also a description of their structure. The fact is that in different species of living organisms the number of chromosomes can coincide, but their structure never completely coincides. Thus, the karyotype (including that of humans) is species-specific, i.e., unique for each type of living organism, which allows them to be distinguished from others.

On the other hand, some individuals of the same species may have slight deviations from the normal karyotype, i.e., have an abnormal karyotype. In humans, karyotypes with chromosomes 47 and 45 are often found.

The 46 chromosomes that make up the human karyotype are present in almost every somatic (non-reproductive) cell of the body and represent 23 pairs of homologous chromosomes. More precisely, 22 pairs of autosomes and one pair of sex chromosomes. Moreover, in women the sex chromosomes are homologous (XX), but in men they are not (XY).

Thus, karyotype is a diploid (2n) set of chromosomes. (The exception is karyotypes of haploid (n) organisms.) Half of the chromosomes of the karyotype are inherited by the organism from the mother, the other from the father.

It is necessary to distinguish between the concepts of karyotype, genotype and genome. Mostly under karyotype understand the structural features of the complete set of chromosomes of an individual or species. Genotype- this is the totality of all the genes of an individual, which also involves the analysis of a diploid set of chromosomes, but at the gene level (analysis of the totality of genes of the organism), and not at the level of chromosome structure. Under genome often understand the totality of hereditary material of a haploid set of chromosomes (in the case of diploid eukaryotes). A genome is a set of genes that “describes” the species characteristics of an organism. For example, all people have genes that determine the development of eyes, arms, legs, a complex brain, etc. Such general features of the structure and functioning of individuals of a species are determined by the genome. But people differ from each other in eye color, temperament, body length, etc. To analyze such variations within the same genome, the concept of genotype is used.

The correct number of chromosomes in a human karyotype was first determined in the 50s of the 20th century. At this time, it was only possible to measure the length of the chromosomes themselves and the length of their arms (p - short arm, q - long). Based on this data, scientists classified the chromosomes.

Later (in the 60s, early 70s) a method of differentially staining chromosomes with different dyes was invented. The use of certain dyes led to cross-striations of chromosomes (the appearance of many alternating stripes on them). Moreover, for each pair of homologous chromosomes, the bands had exclusively their own characteristics (number, thickness), but were always the same, regardless of the type of cells and individuals of the species.

Based on the differential coloring method, schematic maps were developed ( karyograms, idiograms) human karyotype, on which each chromosome from the haploid set (or two homologous chromosomes from the diploid set) was assigned a number, and the striation of the chromosomes was drawn. Autosomes were numbered in descending order of size (the largest chromosome was numbered 1, the smallest was numbered 22). The sex chromosomes had the number 23. In addition, the chromosomes were combined into groups.

The human karyotype contains all three types of chromosomes: metacentric(equal arms: p = q), submetacentric(p acrocentric (basically there is only a q arm).

The arm of a chromosome is its region from the centromere (primary constriction) to the telomere (located at the end). In the idiograms of the human karyotype (as well as many domestic and laboratory living organisms), each arm of each chromosome has its own standard-approved band numbering (and two levels of numbering are used: groups are numbered, and individual bands in each group are numbered). The numbering goes from the centromere to the telomeres. Currently, scientists have been able to determine the localization of certain genes in a number of bands.

In addition to karyograms, a special karyotype recording standard is used. In the case of humans, normal karyotypes are written as 46, XX (for a woman) and 46, XY (for a man). In the case of genomic (not to be confused with gene) extra or missing autosomes are indicated using the chromosome number and the “+” or “-” sign; sex chromosomes are indicated explicitly. For example:

  • 47, XX, 21+ (woman with an additional 21 chromosome),
  • 47, XXY (male with an extra X chromosome).

Karyotype anomalies may concern not only the number of chromosomes, but also changes in their structure (chromosomal mutations). Any part of a chromosome can turn over (inversion), be removed (deletion), transferred to another chromosome (translocation), etc. For such cases, there is also a separate recording standard. For example:

  • 46, XY, 5p- (deletion of the entire short arm of chromosome 5 occurred),
  • 46, XX, inv (3)(q1.1-1.4) (in the long arm of the 3rd chromosome there was an inversion of the section starting with number 1.1 and ending with number 1.4).

Karyotype , a set of characteristics of the chromosome set characteristic of each biological species. These signs include:

  • number,
  • size and shape of chromosomes,
  • position on the chromosomes of the primary constriction (centromere),
  • the presence of secondary constrictions,
  • alternation of heterochromatic and euchromatic regions, etc.

The karyotype serves as a “passport” of the species, reliably distinguishing it from the karyotypes of other species. Constancy of all signs The species karyotype is ensured by the precise processes of chromosome distribution among daughter cells in mitosis and meiosis (these processes can be disrupted by chromosomal mutations).

Karyotype is the complete set of chromosomes in human cells. The normal chromosome content in human somatic (non-embryonic) cells is 46 chromosomes, organized into 23 pairs. Each pair consists of one chromosome received from the mother and one received from the father.

Appearance of chromosomes changes significantly during the cell cycle: during interphase, chromosomes are localized in the nucleus, as a rule, despiralized and difficult to observe, therefore, cells in one of the stages of their division are used to determine the karyotype - metaphase of mitosis.

Chromosomes in a light microscope at the metaphase stage are DNA molecules, packaged using special proteins in dense supercoiled rod-shaped structures. Thus, a large number of chromosomes are packed into a small volume and placed in a relatively small volume of the cell nucleus. The arrangement of chromosomes visible in the microscope is photographed and assembled from several photographs. systematic karyotype- a numbered set of chromosome pairs of homologous chromosomes. In this case, the chromosome images are oriented vertically, with short arms up, and their numbering is carried out in descending order of size. A pair of sex chromosomes (X and Y in a man, X and X in a woman) is placed at the very end of the image of the chromosome set.

When studying a karyotype, which is usually carried out at the metaphase stage of the cell cycle, the following is used:

  • microphotography,
  • special methods for coloring chromosomes and other methods.

To obtain a classic karyotype, chromosomes are stained with various dyes or their mixtures: due to differences in the binding of the dye to different parts of the chromosomes, staining occurs unevenly and characteristic banded structure(a complex of transverse marks), reflecting the linear heterogeneity of the chromosome and specific for homologous pairs of chromosomes and their sections (with the exception of polymorphic regions, various allelic variants of genes are localized). The first chromosome staining method to produce such highly detailed images was developed by the Swedish cytologist Kaspersson (Q-staining). Other dyes are also used, such techniques are collectively called differential staining of chromosomes.

Types of differential staining of chromosomes

  • G-staining is a modified Romanovsky-Giemsa staining. The sensitivity is higher than that of Q-staining, therefore it is used as a standard method for cytogenetic analysis. Used to detect small aberrations and marker chromosomes (segmented differently than normal homologous chromosomes).
  • Q-staining - Kaspersson staining with quinine mustard with examination under a fluorescent microscope. Most often used to study Y chromosomes (quick determination of genetic sex, detection of translocations between the X and Y chromosomes or between the Y chromosome and autosomes, screening for mosaicism involving Y chromosomes).
  • R-staining - acridine orange and similar dyes are used, and areas of chromosomes that are insensitive to G-staining are stained. Used to identify details of homologous G- or Q-negative regions of sister chromatids, or homologous chromosomes.
  • C-staining - used to analyze the centromeric regions of chromosomes containing constitutive heterochromatin and the variable distal part of the Y chromosome.
  • T-staining - used to analyze telomeric regions of chromosomes.

Recently, a technique called so-called spectral karyotyping (fluorescent hybridization FISH), which consists of staining chromosomes with a set of fluorescent dyes that bind to specific regions of chromosomes. As a result of such staining, homologous pairs of chromosomes acquire identical spectral characteristics, which not only greatly facilitates the identification of such pairs, but also facilitates the detection of interchromosomal translocations, that is, movements of sections between chromosomes - translocated sections have a spectrum that differs from the spectrum of the rest of the chromosome.

The results are presented as karyograms(systematized arrangement of chromosomes cut out from a micrograph) or ideograms– a schematic representation of chromosomes arranged in a row as their length decreases.

Comparative analysis of karyotypes is used in karyosystematics to determine the evolutionary paths of karyotypes, to determine the origin of domestic animals and cultivated plants, to identify chromosomal abnormalities leading to hereditary diseases, etc.

Karyotyping is a method of cytogenetic research and consists of studying human chromosomes.

In the process of studying the chromosome set (karyotype), changes in the quantitative composition are determined and violations of the structures (quality) of chromosomes are identified.

Karyotyping is carried out once in a lifetime and allows you to determine the genome of a married man and woman, identify mismatches between the spouses’ chromosomes, which can cause the birth of a child with a developmental defect or a severe genetic disease, and also allows you to establish the reason why it is impossible for a given person to have children. married couple.

A karyotype is a set of human chromosomes with a complete description of all their characteristics (size, number, shape, etc.). Each person's genome normally consists of 46 chromosomes (23 pairs). 44 chromosomes are autosomal and are responsible for the transmission of hereditary characteristics in the family (hair color, ear structure, visual acuity, and so on). The last, 23rd pair is represented by sex chromosomes, which determine the karyotype of a woman 46XX and a man 46XY.

Indications for karyotyping

Ideally, all spouses wishing to become parents should undergo karyotyping, even if there are no indications for the analysis.

Many hereditary diseases that great-grandparents suffered from may not manifest themselves in humans, and karyotyping will help identify the pathological chromosome and calculate the risk of having a child with the pathology.

Mandatory indications for the procedure include:

  • the age of the future parents (35 years and older, even if only one of the spouses answers this item);
  • infertility of unknown origin;
  • repeated and unsuccessful attempts at artificial insemination (IVF);
  • the presence of a hereditary disease in one of the spouses;
  • hormonal balance disorders in women;
  • violation of sperm formation (spermatogenesis) with an unknown cause;
  • unfavorable ecological environment;
  • contact with chemicals and radiation exposure;
  • exposure to harmful factors on a woman, especially in the recent past: smoking, alcohol, drugs, taking medications;
  • the presence of spontaneous termination of pregnancy (miscarriages, premature births, missed pregnancies);
  • consanguineous marriages;
  • the presence of a child/children with chromosomal pathologies or congenital malformations.

The procedure for studying the karyotypes of spouses must be carried out at the stage of pregnancy planning. But the possibility of karyotyping is not excluded if a woman is pregnant. Then karyotyping is carried out not only of the spouses, but also of the unborn child (prenatal karyotyping).

Preparing for analysis

Since blood cells are used to determine the karyotype, it is necessary to exclude the influence of various factors that complicate their growth, which makes the analysis uninformative.

Approximately 2 weeks before donating blood for karyotyping analysis, the following factors should be prevented or avoided:

  • the presence of acute diseases or exacerbation of chronic ones;
  • taking medications, especially antibiotics;
  • drinking alcohol and smoking.

Mechanism

Preference is given to venous blood, which is taken from both spouses. Lymphocytes that are in the mitosis (division) phase are eliminated from the venous blood. Within three days, cell growth and reproduction are analyzed, for which lymphocytes are treated with a mitogen, which stimulates mitosis. During the division process, the researcher can observe the chromosomes, but the process of mitosis is stopped by special treatment. Then special preparations of chromosomes are prepared on glass.

To better reveal the structure of chromosomes, they are stained. Each chromosome has its own individual striations, which becomes clearly visible after staining. Then the stained smears are analyzed, during which the total number of chromosomes and the structure of each are determined. In this case, the striation of paired chromosomes is compared, and the result obtained is compared with the norms of the cytogenetic patterns of chromosomes.

The analysis usually requires no more than 12-15 lymphocytes; this number of cells makes it possible to identify quantitative and qualitative chromosome mismatches, and, consequently, a hereditary disease.

What does karyotyping reveal?

The interpretation of the karyotyping analysis is carried out by a geneticist. The analysis normally looks like 46XX or 46XY. But if any genetic pathology is detected, for example, the identification of a third extra chromosome 21 in a woman, then the result will look like 46XX21+.

What allows you to determine the analysis of the chromosome set:

  • trisomy - the third extra chromosome in a pair (for example, Down syndrome);
  • monosomy - one chromosome is missing in a pair;
  • deletion – loss of a section of a chromosome;
  • duplication – doubling of any fragment of a chromosome;
  • inversion – reversal of a chromosome section;
  • translocation – movement of sections (castling) of a chromosome.

For example, the discovery of a deletion in the Y chromosome is often the cause of impaired spermatogenesis and, consequently, male infertility. It is also known that deletions are the cause of some congenital pathologies in the fetus.

For the convenience of displaying the analysis result on paper when a change in the chromosome structure is detected, the long arm is written with the Latin letter q, and the short arm t. For example, if a woman loses a fragment of the short arm of chromosome 5, the result of the analysis will look like this: 46ХХ5t, which means “cry of the cat” syndrome (a genetic disorder characterized by the characteristic crying of a child and other congenital disorders).

In addition, karyotyping allows you to assess the state of genes. Using this research method it is possible to identify:

  • gene mutations that affect thrombus formation, which disrupts blood flow in small vessels during the formation of the placenta or implantation and can cause miscarriage/infertility;
  • gene mutation of the Y chromosome (in this case it is necessary to use donor sperm);
  • mutations of genes responsible for detoxification (low ability of the body to disinfect surrounding toxic factors);
  • a gene mutation in the cystic fibrosis gene helps to exclude the possibility of this disease in a child.

In addition, karyotyping helps diagnose genetic predisposition to many diseases, for example, myocardial infarction, diabetes mellitus, hypertension, joint pathology, etc.

What to do in case of deviations

If gene mutations or chromosomal aberrations are detected in one of the spouses at the stage of pregnancy planning, a geneticist explains to the couple the likelihood of having a sick child and the possible risks.

As you know, chromosomal and gene pathology is incurable, so the further decision falls on the shoulders of the future parents (use donor sperm or egg, risk having a child, or remain without children).

If chromosomal abnormalities are detected during pregnancy, especially in the embryo, the woman is offered to terminate the pregnancy. Doctors have no right to insist on termination of pregnancy.

For some chromosomal abnormalities (for example, the risk of having a child with a pathology is not high), a geneticist can prescribe a course of certain vitamins that reduce the likelihood of having a sick child.

A karyotype can be defined as a set of chromosomes of somatic cells, including structural features of chromosomes.

In multicellular organisms, all somatic cells contain the same set of chromosomes, that is, they have the same karyotype. In diploid organisms, the karyotype is the diploid set of chromosomes of the cell. The concept of karyotype is used not so much in relation to an individual, but in relation to a species. In this case they say that karyotype is species specific

, that is, each type of organism has its own special karyotype. And although the number of chromosomes in different species may be the same, they always have some differences in their structure.

Although the karyotype is primarily a species characteristic, it can vary somewhat among individuals of the same species. The most obvious difference is the unequal sex chromosomes in female and male organisms. In addition, various mutations may occur, leading to karyotype abnormalities. The number of chromosomes and the level of organization of the species do not correlate with each other.

Karyotypes of diploid (somatic) cells consist of pairs of homologous chromosomes. Homologous chromosomes are identical in shape and gene composition (but not in alleles). In each pair, one chromosome comes to the body from the mother, the other is from the father.

Karyotype study

Cell karyotypes are examined at the metaphase stage of mitosis. During this period of cell division, chromosomes are maximally spiralized and clearly visible under a microscope. In addition, metaphase chromosomes consist of two chromatids (sister) connected at the centromere.

The section of chromatid between the centromere and telomere (located at the end on each side) is called the arm. Each chromatid has two arms. The short arm is denoted by p, the long arm by q. There are metacentric chromosomes (arms are approximately equal), submetacentric (one arm is clearly longer than the other), acrocentric (actually only the q arm is observed).

When analyzing a karyotype, chromosomes are identified not only by their size, but also by the ratio of their arms. In all organisms of the same species, normal karyotypes for these characteristics (chromosome size, arm ratio) are the same.

Cytogenetic analysis involves the identification of all chromosomes of the karyotype. In this case, the cytological preparation is subjected to differential staining using special dyes that specifically bind to different sections of DNA. As a result, chromosomes acquire a specific striation pattern, which allows them to be identified.

Differential coloring method was discovered in the 60s of the 20th century and made it possible to fully analyze the karyotypes of organisms.

The karyotype is usually represented as an idiogram(a kind of scheme), where each pair of chromosomes has its own number, and chromosomes of the same morphological type are combined into groups. Within a group, chromosomes are arranged in size from largest to smallest. Thus, each pair of homologous chromosomes of the karyotype on the idiogram has its own number. Often only one chromosome of a pair of homologues is depicted.

For humans and many laboratory and farm animals, chromosome striation schemes have been developed for each staining method.

Chromosomal markers are bands that appear when stained. The stripes are grouped into districts. Both bands and regions are numbered from centromere to telomere. Some bands may indicate genes located on them.

Recording karyotypes

The karyotype record carries a certain characteristic of it. First, the total number of chromosomes is indicated, then the set of sex chromosomes. If there are mutations, genomic ones are indicated first, then chromosomal ones. The most common ones are: + (extra chromosome), del (deletion), dup (duplication), inv (inversion), t (translocation), rob (Robertsonian translocation).

Examples of recording karyotypes:

48, XY - normal karyotype of a male chimpanzee;

44, XX, del (5)(p2) - karyotype of a female rabbit in which division of the second section of the short (p) arm of the fifth chromosome occurred.

Human karyotype

The human karyotype consists of 46 chromosomes, which was precisely determined in 1956.

Before the discovery of differential coloration, chromosomes were classified by their total length and their centromeric index, which is the ratio of the length of the short arm of a chromosome to its total length. Metacentric, submetacentric and acrocentric chromosomes were found in the human karyotype. Sex chromosomes have also been identified.

Later, the use of differential staining methods made it possible to identify all chromosomes in a human karyotype. In the 1970s, rules (standards) for their description and designation were developed. Thus, autosomes were divided into groups designated by letters, each of which included chromosomes with a specific number: A (1-3), B (4, 5), C (6-12), D (13-15), E (16- 18), F (19, 20), G (21, 22). Sex chromosomes are the 23rd pair.

A normal human karyotype is written as follows:

46, XX - for women,

46, XY - for a man.

Examples of human karyotypes with abnormalities:

47, XX, 21+ - a woman with an extra 21st chromosome;

45, XY, rob (13, 21) - a man who has a Robertsonian translocation of the 13th and 21st chromosomes.