BASICS OF MEDICAL BIOLOGY - 2012

Basics of Medical Genetics. Methods for Studying Human Heredity

Anthropogenetics (human genetics) and medical genetics are crucial Components of the modern theoretical training of future physicians. The Development of these fields took place almost simultaneously with the rise of classical Mendelian genetics. In 1865, the English physician F. Galton published his work "Inquiries into Human Faculty and Its Development", in which he proposed his biometric method for studying human heredity. A significant contribution was later made by Garrod, who established the hereditary nature of alkaptonuria (1902) and hypothesized the genetic origin of Metabolic Disorders. The first major victory of classical Mendelian genetics was the Discovery of the Inheritance of the ABO Blood group system (Bernstein, 1924).

The Selection/4.html">Development of Genetics was profoundly influenced by The Emergence of a new science in the 1950s — Molecular Genetics. In 1949, the pioneers Pauling and his colleagues identified the causes of Sickle-Cell Anemia as structural abnormalities in the Hemoglobin molecule. Further vital contributions were made by cytogeneticists (J. Tjio, A. Levan, 1956), who in the 1960s developed techniques for studying the Human Karyotype and examining Chromosomes.

The next stage in the evolution of these genetic fields was the Translation of genetic breakthroughs into public health practice. During the 1960s and 1970s, MEDICAL Genetic Counseling (MGC) experienced rapid growth, leading to the development and Structure/175.html">Implementation of Methods for counseling and preventing hereditary disorders (such as phenylketonuria, galactosemia, etc.), as well as prenatal diagnostic techniques for chromosomal and molecular diseases.

The application of novel methods in medical genetics elevated The Study of heredity to an unprecedented level, enabling researchers to investigate genetic mechanisms and the processes of their disruption at the CELLULAR AND MOLECULAR levels, and paving the way for decoding human chromosome maps and METABOLISM/28.html">The Genetic Code.

The Introduction of Cytogenetic Methods and somatic cell Hybridization opened up new possibilities for analyzing chromosomal DISEASES ASSOCIATED WITH aberrations in chromosome number and structure. The widespread adoption of biochemical and immunological techniques has led to the identification of over 3,000 hereditary disorders involving metabolic defects and abnormal biomolecule structures. DNA analysis methods (such as DNA profiling and Genetic Engineering) have made it possible to analyze genetic material at THE MOLECULAR LEVEL.

As a result, medicine has gained new opportunities to directly apply not only Diagnostics, but also the Prevention and Treatment of Hereditary disorders in clinical practice. S.M. Davidenkov, one of the founders of medical genetics in our country, was the first to apply genetic concepts in clinical settings, conducting analyses of numerous hereditary disorders and providing medical genetic counseling to affected families. To date, more than 5,000 Monogenic Disorders and 500 Chromosomal Disorders have been discovered, and alterations in hereditary material associated with several Multifactorial Diseases have been identified.

It is clear that the development of anthropogenetics and medical genetics has been driven by human practical needs, particularly the demands of medicine, and the necessity for the continued advancement of these fields is unquestionable. At the present stage, genetics possesses METHODS FOR STUDYING human heredity that can objectively determine the mode of inheritance of a trait, identify the level of biological Organization at which the disruption of genetic material occurred, and facilitate a comprehensive clinical and genetic examination of the patient.

The fundamental laws of heredity and Variability were discovered through the application of the hybridological method of genetic analysis (G. Mendel, 1865). However, this method cannot be applied to humans as an object of genetic research. First, artificial directional crossing is not feasible in humans. Second, the small number of offspring precludes The Use of statistical approaches. Third, the long generation interval (averaging 25 years) allows researchers to observe the succession of only 3 to 4 generations within a lifetime. Furthermore, human genetic studies are hindered by A large number of linkage groups, genotypic and phenotypic polymorphisms, and a significant environmental influence on the expressivity and penetrance of traits.

All of these specific characteristics led to the development of specialized methods for studying human genetics, while modern experimental techniques have made it possible to establish linkage groups for nearly every chromosome and to decipher the genetic code. The biometric approach (F. Galton, 1865), the capabilities of which have been greatly enhanced by modern computer technology, provides substantial assistance in studying the genetics of multifactorial traits.

The genealogical method (F. Galton, J. Adams, W. Weinberg) is based on tracing a specific trait across multiple generations while indicating the familial relationships among the pedigree members. Genealogy refers to the ancestral Lineage of an individual. The genealogical method is the most universal tool in human genetics. Technically, it consists of two stages: 1) constructing the pedigree, and 2) performing genealogical Analysis of the pedigree.

Pedigree construction begins with the proband. The proband is the individual whose pedigree is to be compiled and analyzed. Most commonly, this is a patient or a carrier of the Gene for the trait under investigation. Children of the same parental pair are called sibships (brothers and sisters). Usually, a pedigree is compiled for one or a few specific traits, as it is technically impossible to include all known traits at once. Pedigree construction utilizes standardized symbols proposed in 1931 by G. Just (see fig.).

Rules for constructing pedigrees: 1) the proband is indicated on the pedigree chart by an arrow; 2) individuals of the same generation occupy a single horizontal row or line; 3) generations are designated on the left by Roman numerals; the oldest generation is placed at the top of the pedigree and labeled with the numeral I, while the youngest is placed at the bottom; 4) all members of a single generation are arranged in birth order (from left to right) horizontally and designated by Arabic numerals.

To ensure the reliability of the analysis results, at least 3 to 4 generations should be included. The pedigree must encompass all family members: healthy individuals, affected individuals, stillbirths and miscarriages, those with physical defects, and the mentally impaired. The more reliable information available regarding the health status of the proband's relatives, the more informative the genealogical analysis will be. When referring to any family member, the generation number is stated first, followed by the individual's number within that generation, such as II-3 or III-7. A systematic Description of the data concerning the proband and their relatives (the legend) must be attached to the pedigree chart. The legend should reflect the following information: 1) the results of clinical and paraclinical examinations of the proband; 2) records of personal physical examinations of the proband's relatives; 3) a comparison of the findings from the proband's personal examination with data gathered from interviewing relatives; 4) written records regarding relatives residing in other locations.

Genealogical analysis of a pedigree makes it possible to determine: 1) The Nature of a trait (whether it is hereditary or non-hereditary); 2) the mode of inheritance; 3) the proband's zygosity (homozygous or heterozygous) regarding the trait under study; 4) the recurrence risk probability of the hereditary trait manifesting in offspring; 5) provided the pedigree includes the maximum possible number of relatives, it is also feasible to determine gene penetrance, analyze gene linkage, perform chromosome mapping, and study mutation rates and gene interaction mechanisms; 6) the lineage (paternal or maternal) through which the trait is transmitted.

The primary task of genealogical analysis is to determine whether the trait under investigation is hereditary or non-hereditary. If the trait (or disease) appears multiple times within a pedigree, its hereditary nature may be suspected. However, the possibility of phenocopies must be excluded. For instance, if the same teratogenic factor acted on a woman during all her pregnancies, she might give birth to children with identical anomalies. Another example is when identical occupational hazards cause similar pathologies in members of the same family. Once the hereditary nature of a trait (disease) has been established, the mode of inheritance must be determined: autosomal-dominant, autosomal-recessive, X-linked-dominant, X-linked-recessive, or Y-linked.

Autosomal-dominant inheritance (see fig.) is caused by the transmission across generations of a dominant gene localized in an autosome. This mode of inheritance is characterized by the following features:

1) given a sufficient number of offspring, the trait (disease) manifests in every generation (vertical transmission through the pedigree);

2) males and females inherit the trait with equal frequency;

3) both parents transmit the trait to their children to an equal degree;

4) one or both parents of an affected child are affected;

5) the probability of having an affected child in a family where one parent is heterozygous affected and the other is healthy (Aa × aa) is 50%.

It must be taken into account that with autosomal-dominant inheritance, a trait may skip a generation. This occurs when manifestations of a condition do not appear immediately, but rather at a specific age, such as in Huntington's disease (average age of onset is 38–40 years). In the event of death at an earlier age, no data regarding the potential disease of this family member will be available, but the probability of the disease appearing in descendants remains. Reduced expressivity of the mutant gene can lead to mild or masked forms of the disease, and suppression by an epistatic gene is also possible.

Class="center">Autosomal-Dominant Inheritance

Autosomal Recessive Inheritance is caused by the transmission of a recessive gene located on an autosome across generations. Typical features of this type include:

1) even with a sufficient number of offspring, the trait (disease) does not manifest in every generation;

2) siblings (brothers and sisters) are predominantly affected (horizontal inheritance) rather than parents and children, as is the case in Autosomal dominant inheritance;

3) males and females inherit the trait with equal frequency;

4) parents of an affected child are usually phenotypically healthy (heterozygous carriers of the mutant gene).

5) the probability of having affected children increases in consanguineous marriages;

6) only recessive homozygotes (aa) are affected, while heterozygotes (Aa) are healthy but act as carriers of the pathological gene;

7) the probability of having affected children from healthy heterozygous parents (Aa x Aa) is 25%.

Fig. 24. Standard symbols for pedigree construction.

Autosomal Recessive Inheritance

Fig. 25. Autosomal dominant inheritance (polydactyly).

X-linked dominant inheritance (fig.) is caused by the localization of a dominant gene in the region of the X chromosome that lacks a homolog on the Y chromosome. This type is characterized by:

Fig. 26. Autosomal recessive inheritance (phenylketonuria).

Fig. 27. X-linked dominant inheritance (vitamin D-resistant Rickets).

1) an affected father (X^AY) will have all daughters affected, as they receive the paternal X chromosome with the dominant gene; all sons will be born healthy, as they inherit the maternal X chromosome;

2) if the mother is homozygous for the abnormal gene (X^AX^A), she will transmit the trait (disease) to all daughters and all sons;

3) if the mother is heterozygous, she will transmit the trait (disease) to half of her daughters (X^AX^a) and half of her sons (X^aY); the probability of having healthy children is 50% regardless of sex.

4) the trait manifests equally in both males and females.

Conditions inherited in an X-linked dominant manner include: vitamin D-resistant rickets and brown tooth enamel.

Fig. 28. X-linked recessive inheritance pattern (hemophilia).

X-linked recessive inheritance (fig.) is caused by the localization of a recessive trait gene in the region of the X chromosome that lacks a homolog on the Y chromosome. Typical features of this inheritance pattern are:

1) the trait is observed in the pedigree horizontally and vertically, often skipping generations; affected individuals include sibs, cousins, and maternal relatives (the proband's uncles).

2) predominantly males are affected;

2) absence of trait (disease) transmission from father to son;

3) a carrier female (X^AX^a) transmits the disease to half of her sons, all daughters are healthy, but half of them are carriers;

4) all daughters of an affected father (X^aY) will be phenotypically healthy yet heterozygous carriers;

5) if the mother is homozygous (X^aX^a) for the given gene, all sons will be affected.

6) the parents of an affected child are typically phenotypically healthy, though maternal relatives may be affected (females being heterozygous carriers of the mutant gene).

7) the probability of trait manifestation in offspring is 25% (when the mother is heterozygous; from a hemizygous father, the trait is passed to daughters, but remains phenotypically unexpressed in them).

Conditions inherited in an X-linked recessive manner include hemophilia, color blindness, Duchenne muscular dystrophy, and Lesch-Nyhan syndrome. Pedigrees with X-linked recessive inheritance are characterized by a predominance of affected males.

Y-linked inheritance (holandric inheritance) is determined by the localization of a gene in the region of the Y chromosome that lacks a homolog on the X chromosome. With this pattern, the trait is transmitted exclusively through the male line from generation to generation—from father to all his sons, since a father passes his Y chromosome only to his sons. Traits inherited in a Y-linked manner include hypertrichosis of the pinna ("hairy ears") and ichthyosis (excessive Skin keratinization—"fish scale disease").

Fig. 29. Pedigree showing Y-linked trait inheritance.

Twin Method

This method was proposed in 1875 by F. Galton. The Essence of the twin method lies in comparing studied traits across different groups of twins, based on the similarities or differences in their genotypes and the environments in which they were raised. Twins are defined as offspring of the same mother who developed concurrently and were born in the same delivery. They are classified as monozygotic (MZ) or dizygotic (DZ). Monozygotic (identical) twins develop from a single zygote as a result of its division into 2, 3, or more embryos; they possess identical genotypes (100% shared genes), are always of the same sex, and bear a strong resemblance to each other. Dizygotic (fraternal, non-identical) twins develop during a single Pregnancy from separate zygotes (from two or more ova fertilized by different spermatozoa); they can be of the same sex or opposite sexes; share approximately 50% of their genes; and resemble each other no more than siblings born at different times. The incidence of twin births varies across countries, averaging roughly 1 in 86–88 deliveries. Monozygotic twins are born less frequently than dizygotic ones. On average, there is one pair of MZ twins for every 2–3 pairs of DZ twins. Dizygotic twins are more commonly born to women who have undergone gonadotropic hormone therapy, as well as to women aged 40–45. A predisposition to having twins is inherited maternally.

The twin method is most commonly used to: 1) assess the relative roles of HEREDITY AND ENVIRONMENT in the development of a trait (disease); 2) establish the hereditary nature of a trait; 3) determine Gene expressivity and penetrance; 4) evaluate the effectiveness of external factors on the Organism, such as therapeutic agents, upbringing, and education.

When applying this method, comparisons are made between: 1) monozygotic and dizygotic twins; 2) co-twins of monozygotic pairs; 3) twin study data and The population as a whole.

A number of methods have been proposed to determine twin zygosity (mono- or dizygosity): 1) the polysymptomatic method—studying phenotypic symptoms (Hair, eye, and skin pigmentation, hair form and scalp features, shape of the Nose, Lips, and auricles, fingerprint patterns); 2) portrait identification; 3) the questionnaire method (a questionnaire comprising 12 questions for the twins and 4 for the parents); 4) immunological methods. The only method capable of determining zygosity with 100% certainty is skin graft transplantation. Good engraftment indicates monozygosity, while rejection indicates dizygosity. Modern techniques for determining zygosity rely on molecular genetics.

To determine The Role of genotype versus environmental conditions in trait development, researchers compare the concordance (or discordance) of co-twins for specific traits. Concordance refers to the similarity between twins regarding the studied trait (expressed as a percentage). A twin pair is considered concordant if the trait manifests in both partners, and discordant if the trait is present in only one of them. The higher the concordance, the greater the role heredity plays in trait development. Traits such as eye and hair color, and Blood Groups, are entirely determined by the genotype.

Concordance (in %) for selected human traits in twins (MZ, DZ)

Analyzing trait inheritance in dizygotic twins allows researchers to explore an alternative scenario—The Influence of identical environmental conditions on the phenotypic expression of traits across different genotypes.

To determine the influence of genotype on trait expression, K. Holzinger's formula is employed, where H represents the heritability coefficient, k(MZ) is the pairwise correlation coefficient for identical twins (% concordance), and k(DZ) is the pairwise correlation coefficient for fraternal twins (% concordance).

When H equals one, the trait is entirely determined by the genotype; when H equals zero, environmental influences play the decisive role. An H coefficient of 0.5 indicates an equal impact of heredity and environment on the determination and development of a trait.

The Influence of the environment is denoted by the letter E, therefore

Н + Е = 1, Н = 1 - Е, а Е = 1 - Н

The Dermatoglyphic method (F. Galton, 1892)

This is a human genetics method based on the study of skin relief on the fingers, palms, and plantar surfaces of the feet (from Greek derma — skin, glyphē — to carve). Unlike other PARTS OF THE body, this area features epidermal ridges that form complex patterns. Dermatoglyphic patterns are genetically determined, possess an individual character just like the human genotype, and remain unchanged throughout life. There are no two people on Earth with identical fingerprint patterns (except for monozygotic twins). Dermatoglyphic studies are crucial for determining twin zygosity, diagnosing certain Hereditary diseases, in forensic medicine, and for personal identification in criminalistics. One of the branches of dermatoglyphics is dactyloscopy (the study of patterns on the fingertips). Other branches include palmoscopy (the study of palm patterns) and plantoscopy (the study of sole dermatoglyphics).

Dactyloscopy is a branch of dermatoglyphics that studies fingerprint patterns. The ridges on the fingers correspond to dermal papillae, which is why they are called papillary lines (from Latin papilla — nipple/pimple). The furrows between the papillae form grooves. The complete formation of tactile pattern details is finished by the sixth month of life and remains unchanged until death. There are three MAIN TYPES OF papillary patterns: 1) whorls W; 2) loops L: radial Lr and ulnar Lu; and 3) arches A (see figure)

Papillary lines of different flows never intersect, but they can converge, forming triradii (deltas). Ridge count is the number of papillary lines between the delta and the center of the pattern. Normally, it is 135 for women and 151 for men, averaging 15–20 per individual finger. In Turner syndrome (45, X0), the ridge count is high (180), whereas in Klinefelter syndrome (47, XXY), it is low (up to 50).

Fig. 30. Variants of fingerprint patterns:

a) prints; b) schematic arrangement of ridge lines and triradii: 1 — concentric pattern; 2 — loop; 3 — arch.

Palmoscopy. The relief of the palm is highly complex, comprising a series of fields, pads, and palmar creases. The central palmar fossa is surrounded by six elevations — pads. Near the Base of the thumb lies the thenar, near the opposite edge of the palm lies the hypothenar, and opposite the interdigital spaces are four interdigital pads. At the bases of digits II, III, IV, and V are digital triradii — points where three directions of papillary lines converge, designated by the Latin letters a, b, c, d. Near the wrist crease separating the HAND FROM THE forearm is the main (axial) palmar triradius. Drawing lines from triradii a and d to t forms the atd angle of the palm; normally it does not exceed 57°, but it changes in chromosomal disorders.

Fig. 31. The atd angle in normal conditions and in Chromosomal diseases:

1 — Patau syndrome; 2 — Down syndrome; 3 — Turner syndrome; 4 — normal; 5 — Klinefelter syndrome.

Pattern frequency varies across different populations. Calculating the value of triradii provides insight into pattern intensity, which increases as arches decrease and loops increase. Individual features of skin patterns are genetically determined. This has been proven by genetic research, in particular through twin studies involving monozygotic and dizygotic twins. The concordance coefficient for monozygotic twins is 0.8–0.9, whereas in dizygotic twins, concordance does not exceed 0.3–0.5.

Plantoscopy studies the patterns on the SOLE OF THE FOOT. Alterations in typical arch patterns have been observed in certain chromosomal diseases.

Extensive studies on dermatoglyphic features were conducted by T.D. Gladkova (1966), and on the hereditary determination of skin patterns by I.S. Huseva (1970, 1980). Based on these works, it was concluded that the quantitative parameters of skin ridge relief are programmed by a polygenic system comprising a small number of additively acting genes.

Somatic Cell Hybridization Methods

Somatic Cells contain the full Complement of Genetic information. This makes it possible to investigate numerous questions in human genetics that cannot be studied using a whole organism. Human somatic cells are obtained from various Organs (skin, Bone Marrow, Blood Cells, embryonic Tissues). Connective Tissue cells and blood lymphocytes are most commonly used.

In the 1960s, techniques were developed that allowed cells to be cultured under artificial conditions and cellular processes to be studied experimentally. This facilitated the development of somatic cell genetics (SCG) methods. These methods are used to study heredity and variability in somatic cell cultures, which compensates for the impossibility of applying genealogical and hybridological analysis to humans.

When cells of two different types fuse (under the influence of the Sendai parainfluenza virus, which is inactivated yet retains its ability to induce Cell Fusion), cells are formed that contain the nuclei of both parental cells within a common Cytoplasm — heterokaryons. Most heterokaryons perish, but those containing only two nuclei often continue their development and multiply by division. Following mitosis and subsequent cytokinesis, two mononucleated cells are formed from the binucleated heterokaryon. Each of these represents a synkaryon — a true hybrid cell possessing the chromosomes of both parental cells (see figure).

Somatic cell hybridization is performed broadly not only between different species, but also across types: human × mouse, human × mosquito, fly × chicken, etc. Depending on the purpose of the analysis, studies are conducted on heterokaryotic or synkaryotic cells. Synkaryrons are true hybrid cells because they feature a combination of two genomes. For example, human-mouse hybrid cells have 43 pairs of chromosomes: 23 from the human and 20 from the mouse. This method is used for human chromosome mapping.

Somatic cell genetics methods make it possible to:

1. study gene linkage and their chromosomal localization;

2. establish the primary action of genes and their interaction;

3. identify the Genetic heterogeneity of hereditary pathologies;

4. diagnose hereditary pathologies during the prenatal period.

Molecular and genetic methods

Sequencing (derived from the English word 'sequence') refers to a set of methods used to study The nucleotide sequence of DNA, reverse Transcription of DNA, and the Amplification (cloning) of individual DNA fragments by inserting them into bacterial Plasmids.

Such methods make it possible to:

1. study genetic material (gene sequences in DNA) and pinpoint the localization of disorders at the molecular level (Gene Mutations);

2. determine the nucleotide sequence of DNA and genes;

3. amplify structural genes (cloning) by introducing them into a bacterial cell;

4. recombine DNA molecules to produce essential substances (genetic engineering) based on human genes;

5. determine the exact localization of gene mutations using DNA probes.

Further advancements will enable the decoding of nucleotide sequences in both structural and regulatory regions of The Human Genome, as well as the future treatment of Genetic Disorders at the molecular level.

Fig. 32. Scheme of somatic cell hybridization resulting in The formation of syncaryons (heterokaryons).

Immunogenetic method

This method investigates genetic patterns using immunological reactions (antigen-antibody interaction) resulting in complex formation.

The immunogenetic method allows for the study of:

- the genetic determination and polymorphism of immune systems;

- the genetics of IMMUNOGLOBULINS and The Complement System;

- the genetics of the histocompatibility complex, transplantation Antigens, and immunoregulation factors;

- the genetics of the human HLA system (human leukocyte-associated Histocompatibility Antigens) and the genetic determination of Disease resistance associated with this system;

- the polymorphism of erythrocyte antigens and the genetics of blood groups.

The research utilizes:

- biological fluids (blood, saliva, CEREBROSPINAL FLUID) and tissues;

- cell cultures (HLA, Endocrine glands, bone marrow, leukocytes).

To identify the respective genes, their genetic markers (determinants), namely antigens, are determined.

Immunogenetic methods are essential for addressing issues in Organ and tissue transplantation, diagnosing hereditary pathologies of monogenic and multifactorial diseases, detecting genetic susceptibility and resistance to diseases, mapping Human chromosomes, and establishing correlations between immunological markers and diseases.