Modern methods of diagnosis for hereditary diseases

The information-didactic block

Hereditary pathology is present in all areas of clinical medicine and accounts for a significant part
of the overall morbidity and mortality. According to current data, genetic factors account for 20-30% of
infant mortality, 40-50% of spontaneous abortions and miscarriages, 50% of cases of congenital deafness,
70% of cases of congenital blindness, and 80% of cases of mental retardation. In General, the health of
the population is determined by genetic factors by 18-20 %. According to who data for 1998, the
frequency of monogenic diseases in the human population is 1%, chromosomal diseases - 1%, and HPV -
3 %. Multifactorial diseases account for 92-93% of all chronic non-communicable diseases. At the same
time, 10-12 new hereditary diseases are described annually. If in 1966 in the first edition of Mac Cusick
"Mendelian inheritance in man" (MIM) consisted of about 1,500 inherited diseases and traits, the
electronic version of the catalog of 2000 includes more than 10,500 titles.
Despite the success achieved, it is still too early to talk about effective radical treatment of
hereditary diseases. Usually this is a dietary, symptomatic, replacement, corrective hormone or surgical
treatment. In this regard, the problem of early diagnosis and prevention is of great importance. The
effectiveness of prevention depends on the timely detection of sick or suspected sick people by General
practitioners and their referral to medical and genetic institutions.
Currently, clinical genealogical, cytogenetic, molecular genetic, and biochemical methods are
used to diagnose hereditary diseases.
The clinical and genealogical method is one of the old methods of studying human genetics.
Proposed by Galton in the late XIX th century. Allows you to determine the hereditary nature and type of
inheritance of the trait, its penetrance, and calculate the risk of having a sick child in the family. The
essence of this method is to identify family ties and trace the sign (disease) among relatives in a number
of generations.
It consists of the following stages:
1. Collecting information;
2. Pedigree;
3. Genealogical analysis.
The collection of information begins with the person seeking advice (proband). At this stage, the task is
reduced to the most complete collection of information about each family member by interviewing,
personal examination and, if necessary, special laboratory research.
The pedigree is drawn up in the form of a graphic diagram using special characters (Fig. 1). Generations
in the pedigree are indicated by Roman numerals from top to bottom, members of one generation - from
left to right in Arabic numerals. Brothers and sisters (siblings) are arranged in birth order. Thus, each
member of the pedigree has its own code, for example: I-2, II-5.

Figure: 1 Symbols of pedigree

Genealogical analysis is conducted for the purpose of:
1 - establishing the hereditary nature of the trait
2 - determination of the type of trait inheritance.
There are several main types of inheritance, which are respectively manifested in pedigrees
Autosomal dominant type (AD):
the disease occurs in every generation (vertical inheritance)
the ratio of sick and healthy is approaching 1:1;
equal occurrence of the trait in men and women;
in sick children, one of the parents is sick;
for healthy parents, all children are healthy;
two sick parents give birth to children with a more severe form of the disease;
if the penetration is incomplete, a generation slip is possible.
AD diseases are CHARACTERIZED by a wide variety of expressiveness of signs and terms of disease
manifestation, even within the same family. In most cases, these are pathological conditions that do not
cause serious damage to health and reproductive function - Marfan syndrome, polydactyly,
brachydactyly, etc. In homozygotes, the disease often manifests itself in a more severe form (sometimes
fatal).
Autosomal recessive type (AR):
the trait is shown in a generation (generation skip) and in several members of the generation at once
(horizontal inheritance);
inherited regardless of gender;
healthy parents may have sick children (25 %);
inheritance from an uncle (or aunt) to nephews - in the course of a chess knight.
AR diseases make up a large part of the genetic load of the human population. They appear only in
homozygotes (AA). Heterozygotes (AA) are clinically healthy, but they carry the abnormal gene and can
pass it on to children. The lack of children in modern families makes it difficult to establish recessive
diseases. Most AR diseases significantly reduce the viability of the body and are characterized by high
mortality (phenylketonuria, galactosemia, Wilson-Konovalov disease and other forms of fermentopathies,
oligophrenia, etc.).
 X-linked-dominant type (X D):
vertical and horizontal inheritance;
sick women are 2 times more common than men;
on average, the disease is less severe in women than in men;
sick men are born only from sick women, sick men only have sick daughters (Kris-cross-inheritance).
HD diseases are very variable in expression in women (vitamin-resistant rickets, enamel hypoplasia).

X-linked-recessive type (XR):

horizontal inheritance, generation skip;
only men get sick more often;
the disease is transmitted from grandfather to grandson through the daughter (carrier).
With this type of inheritance, women are more often heterozygous and phenotypically normal
(hemophilia, color blindness)

Y-linked type (Y) - holandric inheritance – the trait is only seen in men (syndactyly, hypertrichosis).
Cytoplasmic inheritance is characteristic of genes localized in the DNA of plastids and
mitochondria. Various mutations of mitochondrial genes that cause hereditary diseases in humans are
described. About 100 such diseases are known: Leber's optic nerve atrophy, oncocytoma (a benign
tumor), mitochondrial myopathy, delayed cardiopathy (a serious disease of young and middle age). The
main symptoms of mitochondrial diseases are associated with damage to the nervous system,
characterized by an early onset. The main diagnostic test is the phenomenon of mitochondrial heteroplasia
- the adhesion of mitochondria and their uneven distribution in the cytoplasm.
Inheritance of mitochondrial diseases is characterized by the following features:
- the disease is transmitted only from the mother;
- both sexes are ill;
- sick fathers don't pass the disease on to their children.
Polygenic inheritance - criteria for polygenic inheritance were first generalized by K. Carter in 1969 and
supplemented by F. Vogel and A. Motulski in 1989. They are diseases of hereditary predisposition that
depend on environmental conditions. The concordance of monozygotic twins for polygenic diseases is 4
times higher than the concordance of dizygotic twins. Polygenic diseases are characterized by:
1. Segregation in families, although there is no clear inheritance model.
2. The dependence of disease risk from:
- degree of kinship with the patient the proband, the higher the degree of relationship, the higher the risk
of disease;
- number of sick relatives:
- if both parents are healthy, the risk is 5-10 %;
- if one parent is ill, the risk is 10-20 %;
- both parents are ill - the risk is up to 40 %;
- from the rare sex - the more often the disease manifests in one sex, the higher the risk for the rare sex;
- from the heritability of the disease - the more genes affect the disease, the higher the risk.

3. calculating the risk of giving birth to a sick child.

To do this, you need to know:
1 - Homo - or heterozygous proband;
2 - trait penetrance.
Penetrance depends on the genetic environment and environmental factors. It can be calculated by
pedigree and using a formula (by proband concordance).
Calculation example:
In an AA x AA marriage, 50% of children will have the pathological a allele. If the penetrance of this trait
is 0.25, then the phenotypic trait will appear only in 12.5 % of children
Cytogenetic methods are a group of methods based on the microscopic study of human
chromosomes. These methods are used in the diagnosis of chromosomal diseases. The most common of
them is karyotyping - a routine method of studying the karyotype, which allows you to identify
violations in the number and structure of chromosomes. Using the method of differential staining, it is
possible to study the character of chromosomal aberrations and their localization by the characteristic
staining along the length of the chromosome. Basically, chromosomes are studied at the metaphase stage
of mitosis. For a more accurate analysis, chromosomes can be studied at the prometaphase stage, when
they are not yet fully spiralized and the length of the chromosome can distinguish not only regions and
segments, but also subsegments. This allows you to detect micro-rearrangements of chromosomes.
Thanks to the success of human molecular genetics, a fundamentally new method for studying
chromosomes has been developed - the fluorescent hybridization method (FISH method). For this
purpose, DNA probes are used-single – stranded DNA fragments with a known nucleotide composition (a
copy of the desired gene), which are complementary to the corresponding section of the chromosome. If
such a DNA probe is marked with a fluorescent label, then under the microscope the luminous point will
indicate the location of this probe on the chromosome. This method can be used to localize a gene and
decipher complex rearrangements between several chromosomes, which is not available for other
cytogenetic methods. It is used to diagnose chromosomal diseases associated with complex changes in the
structure of chromosomes.
Express methods are fast, cheap, and easy - to-perform preliminary diagnostic methods. At the
same time, easily accessible materials (blood, urine) are used in small quantities. Rapid detection of X -
and Y-chromatin allows you to determine the sex and changes in the number of sex chromosomes. Most
often, it is performed by scraping the cells of the cheek mucosa (buccal epithelium) and examining
colored preparations under a microscope. This method allows you to determine the number of X
chromosomes in the karyotype by the number of Barr bodies (one more than the number of X
chromosomes). The Barr body is an inactivated X-chromosome, and is found in 5-60% of cells of normal
women and 2-5% of cells of men in the form of an oval body under the nuclear membrane.
For detecting y-chromatin smears stained with a 0.005 % solution of acryxiniprit and viewed in a
fluorescent microscope. In this case, the Y-chromosome gives a bright green glow, which allows you to
determine the number of Y-chromosomes in the karyotype.
In blood smears, sexual X-chromatin can be determined by counting neutrophilic cells,where the
inactivated X-chromosome looks like an outgrowth of the nucleus in the form of a drumstick (drumstick
method).).
Molecular genetic methods are a large and diverse group of methods that can detect various
variations in the structure of DNA, up to the decoding of the primary sequence of nucleotide. Main stages
and variants of molecular genetic methods:
1. To obtain samples of DNA (or RNA) is the starting point of all methods. The source of DNA can
be any nucleated cells: peripheral blood leukocytes, chorion, amniotic cells, fibroblast cultures,
scraping of the cheek mucosa, several hair follicles, etc. This requires a very small amount of
material. The resulting DNA is suitable for all methods and can be stored frozen for a long time.
More often, to successfully diagnose a disease or heterozygous carrier, it is enough to have only a
small fragment of DNA. The desired fragment can be multiplied (amplified) in large quantities
using polymerase chain reaction (PCR). PCR is a method of DNA amplification in vitro. To do
this, you need to find out the nucleotide sequence of this fragment, and synthesize two
oligonucleotide primers (priming). The amplification process includes 3 stages: temperature
denaturation of DNA (separation of double-stranded DNA into single-stranded molecules)
joining primers to complementary sequences of single-stranded molecules (annealing) synthesis
of polynucleotide chains on single-stranded molecules within the boundaries of attached primers
using polymerase. Repeated repetition of this cycle allows you to multiply a certain DNA
sequence a million or more times.
2. Restriction (cutting) DNA fragments are a necessary step in molecular diagnostics. This
process is carried out by restrictases belonging to the group of bacterial endonucleases. In
human genetics, several dozen different types of restrictases are used, which cut double-
stranded DNA in strictly defined sections of the nucleotide sequence, with a length of 4-6 base
pairs. Different restrictases recognize different sequences, i.e. they have different restriction
sites. This results in a strictly defined number of DNA fragments of different lengths, typical
for each type of restriction. The method of restriction fragment length polymorphism (RDRP)
is widely used to identify the degree of DNA affinity of different organisms.
1. electrophoresis of DNA fragments - ensures the distribution of fragments on the surface of agarose or
polyacrylamide gel, depending on their size. DNA fragments move in a gel placed in a constant electric
field from the negative pole to the positive one at a speed that depends on the size of the fragment. The
length of each fragment can be determined by comparing the distance traveled by the fragment with the
distance traveled by a standard DNA sample with known dimensions.
2. Visualization (detection) of fragments in the gel is either the final stage of diagnosis, or a necessary
element of further analysis. The gel is treated with ethidium bromide, which binds to DNA. When the
GEL is UV-irradiated, a red glow is detected on its surface. Other options for coloring fragments that
allow automatic registration of results have also been developed.
3. Identification of DNA fragments - the selection of specific (desired) fragments from the gel is
currently performed more often using Southern blot hybridization. The desired fragment is identified
Using a special probe that has a known sequence of nucleotides and binds complementarily to this
fragment. This method consists of the following steps:
• the gel is placed in an alkali solution, in which the hydrogen bonds between the chains are destroyed and
single-stranded DNA fragments are obtained
• to visualize the desired fragments, a specific probe is used - a synthetic single-stranded oligonucleotide
of 16-30 bases, labeled with a radioactive or fluorescent label or antibodies. This probe is complementary
to the DNA fragment we need.
Thanks to the development of molecular genetic methods, it is now possible to diagnose almost any
disease associated with a violation in the structure of genetic material. These technologies have been
widely used in connection with human genome research. Most of the techniques are automated, and in the
future, they may become routine methods of decoding the genome of each person.
Biochemical methods are a group of methods that allow you to detect a defect in the metabolism
of a substance by determining it in the blood, urine, and other liquid media of the body. With their help,
more than 1000 congenital abnormalities of metabolism are described, which are expressed in the form of
accumulation or Vice versa deficiency of some products. The role of biochemical methods in detecting
heterozygous carriers of a pathological gene (load method) is particularly significant. These methods are
also used in screening programs for large-scale population studies to initially identify people with some of
the most common diseases.
Biochemical Express methods are easy and fast methods for diagnosing certain metabolic diseases. For
example, the addition of iron chloride in the urine of newborns causes a green color - which is a sign of
the presence of phenylpyruvic acid in the urine.
The Guthrie microbiological inhibitor test was originally proposed for the diagnosis of biochemical
disorders in newborns. Blood drops are taken from the newborn's heel onto filter paper discs and placed
on a culture of microorganisms grown in a certain nutrient medium. Accelerating the growth of these
microorganisms allows you to diagnose the presence of certain amino acids and carbohydrates in the
baby's blood above the norm.
All the methods listed above are also used in prenatal and early preclinical diagnostics of hereditary
diseases for the prevention of hereditary pathology.