Karyotype analysis

One of the methods of cytogenetic research aimed at studying chromosomes is karyotyping. The analysis has a number of indications for the conduct, as well as several types. 

Karyotype is a set of human chromosomes. It describes all the features of genes: size, quantity, shape. Normally, the genome consists of 46 chromosomes, of which 44 are autosomal, that is, they are responsible for hereditary traits (hair and eye color, ear shape and other). The last pair is the sex chromosomes that determine the karyotype: women 46XX and men 46XU.

The diagnostics process reveals any violations of the genome:

• Changes in the quantitative composition.
• Violation of the structure.
• Violation of quality.

As a rule, karyotyping is performed by a newborn to determine genetic anomalies. The analysis is also shown for couples who are planning a pregnancy. In this case, the study reveals a discrepancy between genes, which can lead to the birth of a child with hereditary pathologies.

Types of molecular karyotyping:

1. Target

Assigned to confirm various abnormalities and syndromes. It allows to determine the causes of pregnancy loss: frozen fetus, miscarriage, interruption according to medical indications. Defines the etiology of an additional set of chromosomes with triploidias. The analysis is performed on microarrays with 350 thousand markers, concentrated in clinically significant areas of chromosomes. The resolving power of this study is from 1 million bp.

2. Standard

Identifies violations in the genome of clinical significance. Diagnoses microdeletion syndromes and pathologies associated with autosomal dominant diseases. Determines the causes of chromosomal abnormalities in undifferentiated syndromes in patients with developmental anomalies, congenital malformations, delayed psychomotor development, autism.

It allows to reveal chromosomal abnormalities in the prenatal period. The technique determines aneuploidy, pathological microdeletions in the fetus. The study is conducted on a microarray with 750,000 high density markers, which cover all significant parts of the genome. The resolving power of the standard karyotype analysis is from 200,000 bp.

3. Expanded

Allows to establish the causes of chromosomal abnormalities in undifferentiated syndromes in children. Identifies pathogenic deletions, that is, the disappearance of chromosome regions and duplication - additional copies of genes. Diagnoses sites with loss of heterozygosity, causes of autosomal recessive pathologies.

Extended chromosome microarray analysis is performed using a high-density microarray that contains more than 2.6 million individual high-density markers. The resolving power of this study allows covering the entire genome and ranges from 50,000 bp. Thanks to this, all sections of the gene code are studied with the utmost precision, which makes it possible to reveal the smallest structural disturbances.

As a rule, the analysis for karyotype is carried out according to the purpose of the geneticist. Depending on the medical indications, one of the above types can be assigned. A standard study costs less, but it is rarely prescribed, because it does not reveal many chromosomal abnormalities. Target karyotyping is a more expensive analysis, so it is prescribed in the presence of clinical signs of syndromes and other anomalies. Extended diagnosis is the most expensive and most informative, as it allows to fully study all 23 sets of chromosomes.

Where to pass the analysis on karyotype?

Chromosomal micromatrix analysis is handed over as prescribed by a geneticist. The study is aimed at studying the genome of the patient and revealing any anomalies in its structure.

Chromosomes are DNA strands, their number and structure has its own specificity for each species. The human body contains 23 pairs of chromosomes. One pair determines the sex: in women 46XX chromosomes, and in men 46XY. The remaining genes are autosomes, that is, non-genes.

Features of karyotyping:

• The analysis is carried out 1 time, since the chromosome set does not change throughout life.
• Allows to establish the causes of reproductive problems in the spouses.
• Diagnoses multiple malformations in children.
• Identifies genetic abnormalities.

Karyotype is given in a specialized medical laboratory or in a genetic center. The study is conducted by a qualified doctor. As a rule, the tests are ready within 1-2 weeks. The results are deciphered by the geneticist.

Indications for the procedure of the karyotype analysis

The procedure of karyotyping is assigned to newborn babies for the detection of genetic abnormalities and hereditary pathologies, as well as for men and women at the stage of pregnancy planning. There are also a number of other indications for the analysis:

• Male and female infertility of unknown origin.
• Male infertility: severe and non-obstructive oligozoospermia, teratozoospermia.
• Spontaneous abortion: miscarriages, frozen fetus, premature birth.
• Primary amenorrhea.
• Cases of early death of newborns in anamnesis.
• Children with chromosomal abnormalities.
• Children with multiple congenital malformations.
• The parents' age is more than 35 years.
• Multiple unsuccessful attempts at IVF fertilization.
• Hereditary disease in one of the future parents.
• Hormonal disorders in women.
• Spermatogenesis of unknown etiology.
• Closely related marriages.
• Unfavorable ecological living environment.
• Prolonged contact with chemicals, irradiation.
• Harmful habits: smoking, alcohol, drugs, drug dependence.

Karyotyping of children is carried out in such cases:

• Congenital malformations.
• Mental retardation.
• Delay in psychomotor development.
• Microanomalia and delayed psycho-speech development.
• Sexual anomalies.
• Violation or delay of sexual development.
• Growth retardation.
• Prognosis of the child's health.

Diagnosis is recommended for all spouses at the stage of pregnancy planning. Also, the analysis can be carried out during pregnancy, that is, prenatal chromosomal research.

How does the karyotype look?

The totality of signs of a complete set of chromosomes is a karyotype. For the systematization of chromosome analyzes, the International Cytogenetic Nomenclature is used, which is based on differential staining of the genome for a detailed description of the entire strand of DNA.

The study reveals:

• Trisomy - in the pair there is a third extra chromosome.
• Monosomy - one pair lacks one chromosome.
• Inversion - the turn of the genome.
• Translocation is movement of sites.
• Deletion is the loss of the site.
• Duplication - doubling the fragment.

The results of the analysis are recorded using this system:

1. The total number of chromosomes and the set of genitalia are 46, XX; 46, XY.
2. Extra and missing chromosomes are indicated, for example, 47, XY, + 21; 46, XY-18.
3. The short arm of the genome is denoted by the symbol - p, and the long - q.
4. The translocation is t, and the deletion is del, for example 46, XX, del (6) (p12.3)

The ready analysis for karyotype is as follows:

• 46, XX - the norm or rate the woman.
• 46, XY is the norm of a man.
• 45, X - Shereshevsky-Turner syndrome.
• 47 XXY - Klinefelter's syndrome.
• 47, XXX - trisomy on the X chromosome.
• 47, XX (XY), +21 - Down's syndrome.
• 47, XY (XX), + 18 - Edwards syndrome.
• 47, XX (XY), + 13 - the Patau syndrome.

Cytogenetic research reveals various anomalies in the structure of DNA strands. The analysis also diagnoses predispositions to many diseases: endocrine pathologies, hypertension, joint damage, myocardial infarction and others.

[1], [2], [3], [4], [5], [6], [7], [8]

Preparation

Blood cells are used for the analysis on karyotype, therefore it is very important to prepare correctly for diagnosis.

Preparation for chromosome research begins 2 weeks before it is taken and consists of excluding the influence on the body of such factors:

• Acute and chronic diseases.
• Reception of medicines.
• Drinking alcohol and drugs, smoking.

For the analysis, venous blood is used in 4 ml. Blood sampling is carried out on an empty stomach.

[9], [10], [11], [12], [13], [14], [15], [16]

Technique of the karyotype analysis

The human genome can not be seen with the naked eye, the chromosomes are visible only under a microscope at certain phases of cell division. To determine the karyotype, single-nucleated leukocytes, skin fibroblasts or bone marrow cells are used. For the study, cells are suitable in the metaphase of mitosis. The biological fluid is placed in a test tube with lithium and heparin. Blood is cultured for 72 hours.

Then the culture is enriched with special substances, which stop cell division on the necessary phase for diagnosis. From the culture do drugs on the glass, which are subject to investigation. Additional information on the state of the genome is obtained by its coloration. Each chromosome has striation, which is clearly visible after staining.

In a classical chromosomal study, the staining is carried out with various dyes and their mixtures. The dye binds to different parts of the genome differently, making the staining uneven. Due to this, a complex of transverse marks is formed, which reflect the linear heterogeneity of the chromosome.

Basic staining methods:

• Q - gives images with high detail. This method was called the staining of Caspersson with acrichine-mustard with diagnostics under a fluorescent microscope. It is used to analyze the genetic sex, identify translocations between X and Y, Y and autosomes, and also for screening of mosaicism with Y chromosomes.
• G is a modified Romanovsky-Giemsa method. Has a higher sensitivity in comparison with Q. It is used as a standard method of cytogenetic analysis. Identifies small aberrations, marker chromosomes.
• R - is used to detect homologous G and Q negative regions. The genome is treated with acridine orange dye.
• C - analyzes centromeric regions of chromosomes with constitutive heterochromatin and the veritable distal part of Y.
• T - used to analyze telomeric DNA strand regions.

The colored and fixed cells are photographed under a microscope. From the resulting set of photographs form a numbered set of pairs of autosomes, that is, a systematized karyotype. The image of DNA strands is oriented vertically, the numbering depends on the size, with a pair of sex chromosomes covering the set.

Blood preparations are analyzed under a microscope of 20-100 metaphase plates to detect quantitative and structural aberrations.

• Quantitative aberrations are changes in the number of genes. Similar is observed with Down's syndrome, when there is an extra 21 chromosomes.
• Structural aberrations are a change in the chromosomes themselves. This can be a fall of the genome, the transfer of one part to another, a rotation of 180 degrees and more.

The technique of karyotyping is a laborious process. The study is conducted by highly qualified specialists. To diagnose a single person's genome it can take a whole working day.

Analysis of karyotype of spouses

When marrying, many couples face the problem of conception. Cytogenetic analysis is shown to solve reproductive problems. Karyotyping of spouses allows to reveal anomalies in the structure of the genome, which interfere with having children or disturbing the process of gestation. Change karyotype is impossible, but thanks to the diagnosis, you can determine the true causes of infertility and abortion, find ways to solve them.

Chromosomal micromatrix analysis is performed to detect abnormalities in the structure of the structure and the number of DNA strands that can cause hereditary diseases in a future child or infertility of the spouses. There are international standards for analyzing future parents:

• Chromosomal pathologies in the genus, in the family.
• Miscarriage of a pregnancy in an anamnesis.
• The age of the pregnant is older than 35 years.
• Prolonged mutagenic effects on the body.

To date, such methods of karyotyping are used:

1. Analysis of chromosomes in blood cells.

It allows to identify cases of infertility, when the chance to have a child is significantly reduced or completely absent from one of the spouses. The survey also determines the risk of genome instability. To treat abnormalities, patients can be prescribed antioxidants and immunomodulators, which reduce failure of conception.

Venous blood is taken for the study. From the biological fluid, lymphocytes are isolated, which are stimulated in a test tube, treated with a special substance, stained and studied. For example, with Klinefelter's syndrome, which manifests itself as male infertility, there is an extra chromosome 47 XX in the karyotype. Also, structural changes in the genome can be identified: inversion, deletion, translocation.

2. Prenatal examination.

Defines chromosome pathologies of the fetus in the early stages of pregnancy. Such a study is necessary for the diagnosis of genetic diseases or malformations that lead to fetal death in utero.

To conduct research, such methods can be used:

• Non-invasive - safe for the mother and fetus. Diagnosis is carried out with the help of a child's ultrasound and a detailed biochemical analysis of a woman's blood.
• Invasive - biopsy of the chorion, cordocentesis, placentocentesis, amniocentesis. For the analysis, cells of the placenta or chorion, amniotic fluid or blood from the umbilical cord are collected. Despite the high accuracy of diagnosis, invasive procedures have an increased risk of complications, therefore, they are performed only on strict medical indications: fetal pathology diagnosed during pregnancy, maternal child over 35, parents with chromosomal abnormalities, and biochemical blood markers.

For cytogenetic research, not only blood, but also ejaculate can be used. This method is called Tunel and allows to determine one of the most common causes of male infertility under the condition of a normal karyotype - fragmentation of sperm DNA.

If there are gene mutations or chromosomal aberrations in one of the spouses, the doctor tells about possible risks and the probability of a child with deviations. Since the gene pathologies are incurable, the further decision of the couple is taken independently: use donor material (sperm, egg), risk giving birth or staying without children.

If the deviations in the genome are detected in the processes of gestation, both in women and in embryos, doctors recommend that such pregnancies be interrupted. This is due to the increased risk of a baby's birth with serious, and in some cases inconsistent with life, deviations. Conducting the analysis and deciphering their results is done by a geneticist.

[17], [18], [19], [20], [21]

Blood test for karyotype

Most often, karyotyping is performed on the analysis of venous blood by culturing its cells. But for carrying out cytogenetic research, another biological material can be used:

• Cells from amniotic fluid.
• Placenta.
• Cells of embryos.
• Abortive material.
• Bone marrow.

What material will be taken for diagnosis depends on the cause and task of the analysis. An approximate algorithm for blood testing:

• A small volume of liquid for 72 hours is placed in a nutrient medium at a temperature of 37 ° C.
• Since the chromosomes are visible at the stage of the metaphase of cell division, a reagent is added to the biological medium, which stops the fission process in the required phase.
• The cell culture is stained, fixed and analyzed under a microscope.

Blood analysis for karyotype provides a high-precision detection of any anomalies in the structure of DNA strands: intrachromosomal and interchromosomal rearrangements, changes in the order of location of fragments of the genome, and others. The main purpose of diagnosis is to identify genetic diseases.

[22], [23], [24], [25], [26], [27]

Genetic analysis of karyotype

Cytogenetic diagnosis aimed at studying the size, number and shape of chromosomes is genetic karyotyping. The analysis has such indications for carrying out:

• Identification of birth defects.
• Risk of a child with hereditary pathologies.
• Suspicion of infertility.
• Violation of spermogram.
• Unintention of pregnancy.
• Drawing up a plan for the treatment of certain types of neoplastic tumors.

Also, genetic analysis for karyotype is included in the list of mandatory for spouses who plan to have children.

Most often, the study reveals such pathologies:

1. Aneuplodia is a change in the number of chromosomes both in the direction of increasing and decreasing. Violation of balance leads to miscarriages, the birth of infants with severe congenital pathologies. The mosaic form of aneuploidy causes Down syndrome, Edwards syndrome and other very incompatible diseases.
2. Rebuild karyotype - if the changes are balanced, then the chromosome set is not broken, but simply differently ordered. With unbalanced changes there is a threat of gene mutations, which is especially dangerous for future generations.
3. Translocation is an unusual structure of DNA strands, that is, the replacement of one fragment of the genome by another. In most cases, it is inherited.
4. Violation of sexual differentiation is an extremely rare chromosomal disorder, which is not always manifested by external symptoms. Non-compliance with phenotypic sex can be one of the causes of infertility.

Analysis for karyotype is performed in genetic laboratories, qualified doctors of genetics.

Karyotype analysis with aberrations

Aberrations are disorders in the structure of chromosomes, caused by their discontinuities and redistribution with loss or duplication of genetic material. Karyotyping with aberrations is a study aimed at detecting any changes in the structure of the genome.

Types of aberrations:

• Quantitative - a violation of the number of chromosomes.
• Structural - a violation of the structure of the genome.
• Regular - are determined in most or all cells of the body.
• Irregular - arise due to the impact on the body of various adverse factors (viruses, radiation, chemical effects).

The analysis determines the karyotype, its features, signs of the impact of various negative factors. Chromosomal investigation with aberrations is carried out in such cases:

• Infertility in marriage.
• Spontaneous miscarriages.
• Cases of stillbirth in the anamnesis.
• Early infant mortality.
• Frozen pregnancy.
• Congenital malformations.
• Violation of sexual differentiation.
• Suspicions of chromosomal pathology.
• Delayed mental, physical development.
• Examination before IVF, ICSI and other reproductive procedures.

Unlike classical karyotyping, this analysis takes more time to hold and costs more.

Karyotype analysis for a child

According to medical statistics, congenital pathologies play a significant role in the causes of infant mortality. For the timely detection of genetic abnormalities and hereditary diseases, the child is analyzed for karyotype.

• Most often, children are diagnosed with trisomy - Down's syndrome. This pathology occurs in 1 out of 750 babies and manifests itself in various kinds of deviations in both physical and intellectual development.
• In second place in the prevalence of Klinefelter's syndrome. It manifests itself as a delay in sexual development in adolescence and occurs in 1 out of 600 newborn males.
• Another genetic pathology diagnosed in 1 out of 2,500 female children is the Shereshevsky-Turner syndrome. In childhood, this disease makes itself felt by increased pigmentation of the skin, swelling of the feet, hands and shins. During puberty, there is a lack of menstruation, the hairline under the armpits and on the pubic, also the mammary glands are not developed,

Karyotyping is necessary not only for toddlers with visible deviations, as it allows to suspect genetic problems and begin their correction. The analysis is handed over in the medical-genetic center. Depending on the age of the child, the blood can be taken from the heel or from the vein. If necessary, a geneticist may require an analysis of the karyotype and parents.

Analysis of the newborn karyotype

Neonatal screening is the first analysis performed by newborns. The study is conducted in the maternity house for 3-4 days of life, for premature babies on day 7. Early karyotyping allows revealing genetic abnormalities and disturbances in the structure of DNA before the appearance of visible pathological symptoms.

For early diagnosis, use blood from the heel of the baby. Cytogenetic research is aimed at identifying such common pathologies among children as:

• Phenylketonuria is a hereditary disease characterized by a decrease in activity or the absence of an enzyme that cleaves the amino acid phenylalanine. When progressing leads to disruption in the brain and mental retardation.
• Cystic fibrosis - affects the glands that produce secrets, digestive juices, sweat, saliva, mucus. Causes disruption in the functioning of the lungs and organs of the digestive tract. The disease is inherited.
• Congenital hypothyroidism is a lesion of the thyroid gland with insufficient production of its hormones. It leads to a delay in physical and mental development.
• Adrenogenital syndrome is a pathological condition in which the adrenal cortex produces an inadequate amount of hormones. Because of this, the development of genital organs is disrupted.
• Galactesymia is a pathology in which the transformation of galactose into glucose is disturbed. Treatment consists of the rejection of dairy products. Without timely diagnosis, it can cause blindness and death.

If, according to the results of the analysis of the karyotype in the newborn, any deviations or anomalies are detected, then a set of additional studies is performed to clarify the diagnosis. Such early diagnosis will help to identify any problems in the child's body in a timely manner and begin treatment.

[28], [29], [30], [31], [32], [33], [34]

How much analysis is done for the karyotype?

The duration of chromosome research takes from 10 to 21 days. When the results are ready depends on the type of analysis, that is, with aberrations or classical karyotyping.

The ready analysis for karyotype contains such information:

• The number of chromosomes.
• Is there any change in the structure of the chromosomes.
• Are there any violations in the order of the genome.

Deciphering the results and their interpretation is the responsibility of the geneticist. If any abnormalities are detected, the doctor gives medical advice for further diagnosis or guidance regarding treatment.

Normal performance

Normal karyotypes for humans are 46, XX or 46, XY. As a rule, their change occurs in the early stages of the development of the body:

• Most often, the disorder occurs during the period of gametogenesis (pre-embryonic development), when the parental sex cells produce the kyotype of the zygote. The further development of such a zygote leads to the fact that all cells of the embryo contain an abnormal genome.
• Violation can occur in the early stages of dividing the zygote. In this case, the embryo contains several cellular clones with different karyotypes. That is, mosaicism develops-the multiplicity of karyotypes of the whole organism and its organs

Changes in the genome are manifested by various pathologies and vices. Consider the common karyotype anomalies:

• 47, XXY; 48, XXXY - Klinefelter's syndrome, polysomy on the X-chromosome in men.
• 45X0; 45X0 / 46XX; 45, X / 46, XY; 46, X iso (Xq) - Shereshevsky-Turner syndrome, X chromosome monosomy, mosaicism.
• 47, XXX; 48, XXXX; 49, XXXXX - polysomy on the X chromosome, trisomy.
• 47, XX, + 18; 47, ХY, + 18 - Edwards syndrome, trisomy on the 18 chromosome.
• 46, XX, 5p- - catnip scream syndrome, deletion of short arm 5 pairs of genome.
• 47, XX, + 21; 47, XY, + 21 - Down's disease, trisomy on 21 chromosomes.
• 47, XX, + 13; 47, ХY, + 13 - Patau syndrome, trisomy on chromosome 13.

Cytogenetic research is aimed at determining the state of DNA strands, identifying defects and anomalies. Any deviation from normal indices is an occasion for a complex examination of the body.

[35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46]

The device for analysis

To decipher the karyotype, the sequencing method is used. This technique was developed in 1970 and is based on determining the sequence of amino acids in DNA. Sequencing machines use interactive cyclic enzymatic reactions with further processing and comparison of the results obtained.

Basic functions of sequencers:

• Primary complete study of unknown genomes, exoms, transcripts.
• Karyotyping.
• Paleogenetics.
• Metagenomics and microbial diversity.
• Resequencing and mapping.
• DNA methylation analysis.
• Analysis of transcripts.

At the first stage, the apparatus creates a library of random sequences of DNA strands. Then creates amplicon by PCR, which are used as samples. At the final stage, the primary structure of all fragments is determined.

Sequencers of the latest generation are fully automated and widely used for genomic analysis, minimizing the production of erroneous results due to the human factor.

Interpretation of results of the analysis on karyotype

Interpretation of the results of cytogenetic research is done by a geneticist. As a rule, the analysis is ready in 1-2 weeks and can look like this:

• 46XX (XY), are grouped into 22 pairs and 1 pair of sex. The genome has a normal size and structure. Anomalies not revealed.
• The genome is broken, more than 46 chromosomes are detected. The shapes and sizes of one / several chromosomes are abnormal. The pairs of the genome are broken / incorrectly grouped.

With regard to pathological abnormalities in the karyotype, they distinguish such common disorders:

• Trisomy is an extra somatic chromosome. Down Syndrome, Edwards Syndrome.
• Monosomy is the loss of one chromosome.
• Deletion is the absence of a genome site. -46, xx, 5p-cat's scream syndrome.
• Translocation is the transfer of one section of the genome to another.
• Duplication is the duplication of a fragment.
• Inversion - rotation of a fragment of a chromosome.

Based on the results of the analysis on karyotype, the doctor makes a conclusion about the state of the genotype and the degree of genetic risk. At the slightest changes in the structure of DNA strands, a set of additional studies is assigned. Identified aberrations may not be manifested, but increase the risk of the birth of children with genetic abnormalities.