Theme:

Modern methods of diagnosis

for hereditary diseases
 Purpose:

Study modern methods of

diagnostics for
hereditary diseases.
1. Genealogical method. Rules for building
pedigrees. Genetic analysis of pedigrees.
2. Cytogenetic method
3.Biochemical method.
4.Molecular genetic method.
1.
 Genealogical method
(from the Greek. genealogia
– pedigree) is based on a
graphical representation of the
pedigree scheme and the study
of the nature of inheritance of a
certain trait or disease.
• For the first time the genealogical
method was proposed an English
scientist By Francis Galton in
1865.

Francis Galton
• Galton was trying to solve
the problem of giftedness,
analyzing the pedigrees of
outstanding figures in
science, sports, military
Affairs, art, etc. using the
genealogical method.
• As a result, the scientist we
were able to analyze the
pedigrees of 300 families,
numbering up to 1000
outstanding people.
 Main task
• Establishing the hereditary nature of the disease
• Establishing the type of disease inheritance
• Determining the number of people who need research
to identify heterozygous carriers of a mutant gene
• The establishment of heterogeneity of hereditary
diseases
• Use in medical and genetic consultations
• The establishment of penetrantnost
• Evaluating the intensity of gene expression
• The clinical and genealogical
method conditionally
includes
3 stages:
1. Collecting information
about your family.
2. Build a family tree.
3. The pedigree analysis.
• Collecting family information
begins with proband's.
• Proband – the person for
whom the pedigree is
compiled.
• Most often, this is a patient
with the studied disease or a
carrier of the studied trait.
• Then proband's other relatives
are examined, primarily his
parents and grandparents. sibs
of DM.
• Sibs of DM – siblings, i.e.
children from the same family.
• Second stage –
building a pedigree
• For analysis and visual
presentation of the collected
information, use graphic
representation of the pedigree.
• To do this, use standard
symbols accepted around the
world.
Standard characters that are used when creating a pedigree:
• Third stage - the pedigree
analysis

Purpose of pedigree analysis

– the establishment of
genetic patterns.
1. establishing the hereditary character
of the trait.
2. defining the inheritance type.

 autosomal dominant,
 autosomal recessive
 linked to sex chromosomes
Cytogenetic method
By the term «cytogenetics»
understand the field of science
that studies the structure and
function of chromosomes.
The cytogenetic method allows:
•Study the karyotype of an organism;
•To study the types and causes of chromosomal mutations;
•To diagnose chromosome diseases;
•Determine the genetic sex of the body in a phenotypic disorder.

The cytogenetic method includes:

• the metaphase plate method.
• method of sex chromatin;

1) Method of determination sex chromatin it is used to study the number of

sex chromosomes in the interphase nuclei. This is an indirect determination
of the number of chromosomes.
Determination of sexual chromatin is used for diagnostics of diseases caused
by violation of the number of X-chromosomes, extra-diagnostics of gender
even on the remnants of the fabric. For example: in a woman with karyotype
45, X0 (Shereshevsky-Turner syndrome, monosomy-X) and normally in men
XU cell nuclei do not contain sex chromatin. With the syndrome trisomy-X in
a woman forms two blocks, in a man with karyotype 47 (XH) – one block
chromatin's, with karyotype 48, XXXX – two.
•

The relationship between the number of X-chromosomes (I), the number of

Barr cells in the cells of the oral mucosa (II), and “drumstick” in the nuclei of
white blood cells (ІІІ).
In 1949, M. Barr and C. Bertram found a small brightly colored body in the nuclei of
cat neurons. Later, scientists proved that it is contained only in the nuclei of female
cells. Males don't have it. This little body was called Barr's body. Similar bodies
have been found in organisms with sex chromosomes XX.
•

Sex chromatin (Barr's body). Shown

with an arrow.
Taurus Of Barra (sex chromatin) – it's a helical X-chromosome that is inactivated
in embryogenesis before the development of the gonads. Normally, in women, 20-
60 % of cells in the nucleus contain a single sex chromatin body. The number of
lumps of sexual chromatin is always one less than the number of X-chromosomes.
Most often, sex chromatin is determined in epithelial cells of the cheek mucosa
(buccal scraping), as well as in neutrophils in the form of a nucleus outgrowth
(drumstick).
2) Method metaphase plate (karyotyping) allows you to study the number and
structure of chromosomes. It is used to diagnose a variety of inherited
diseases, study chromosomal abnormalities in cells.
3)
The method consists of the following steps:

Getting chromosomes
• In order to prepare a metaphase plate most often, peripheral blood cells
(lymphocytes) are taken. The fraction of lymphocytes is obtained as a result
of blood centrifugation. Then, to stimulate mitosis, phytohemaglutinin (a
nutrient medium) is added, and to stop mitosis – colchicine (destroys the
threads of the dividing spindle). After that, the cells are treated with a
hypotonic solution. Cell membranes are torn, and chromosomes freely lie at a
certain distance from each other (metaphase plates). Culture is fixed and
preparations are prepared.
•
Staining of chromosomes
• The drug is colored with dyes depending on the tasks of the study:
– General – for counting the number of chromosomes;
– differential: R, G – for determining
homologous chromosomes, Q, C – for determining
aberration, the origin of chromosomes.
• Cover with a protective glass, examine under a microscope (or make
microphotographs).
Chromosome analysis
• They study chromosomes: length, shape, location of the centromere,
etc.
• Make up a karyogram.
Karyogram – this is the order of each pair of chromosomes in terms of
their size: from the larger to the smaller, the sex chromosomes are
taken out separately.
•
•
Method metaphase plate (karyotyping)

Preparation of cytogenetic preparations by culturing peripheral

blood lymphocytes
 Fluorescence in situ
hybridization, (FISH) -
метод
• FISH-method allows you to identify individual
chromosomes and their individual sections on the
screen metaphase plates (chromosomes in a state
of maximum condensation and visualization);
• In interphase cores (non-condensed chromosomes,
without a clear morphological structure) based on
the features of their molecular and genetic structure.
• The method includes the use of specially prepared
(fluorescent)
DNA samples for detecting genetic defects at the
chromosomal level and performing molecular
cytogenetic diagnostics using modern fluorescence
microscopy.
•
 The method of fluorescent hybridization in situ it was developed
thanks to the success of human molecular genetics and is completely
new, convenient and safe.
 FISH-method it does not require a high level of cytogenetics, allows
you to identify the most complex chromosomal abnormalities, and the
use of modern multimedia equipment further simplifies the diagnosis
process.
 Limits of use method FISH incredibly wide: from the localization of a
specific gene to the transcription complex rearrangements across
multiple chromosomes.
 It allows you to identify the origin of aberrant chromosomes, as well
as microchromosomal abnormalities which are not available for
differential staining methods.
 Significant advantage FISH the method is the use of interphase nuclei
for the diagnosis of pathologies.
 Depending on the goals, you can use different variants of fluorescent
hybridization, which allows you to achieve the desired result as
reliably as possible.
 Another important feature is that a single cell (metaphase plate or
interphase core) can be repeatedly labeled by washing after each
application of the probe, which increases the number of detected
sequences tenfold in the work with just one cell.
 Application FISH method
Molecular cytogenetic diagnostics using FISH the
method is used in various sections of medical
genetics:
 to determine numerical and structural chromosomal
abnormalities in clinical cytogenetics;
 for identification of marker (mini-, extra)
chromosomes;
 for determination of aneuploidies in non-dividing
prenatal and postnatal interphase cells;
 to determine the various specific and nonspecific
chromosomal abnormalities in oncologenesis;
 for the analysis of chromosomal abnormalities in
clinical cytogenetics (isochromosomes, balanced
chromosomal abnormalities in various clinical
pictures of the syndrome, etc.).
•
Stages FISH - method
The essence of the
method consists in the
preparation of short
DNA sequences, called
transcription factors.
probes, which are
complementary to the
DNA sequences that
represent the object of
study.
Probes hybridize
(bind) with
complementary DNA
sites and are labeled
by the fact that they
are fluorescent label,
they allow you to see
the localization of
genes of interest in the
DNA or chromosome
structure.
Biochemical method
• Biochemical method founded by A. Garrod in 1902.
This method allows us to study the
phenotypic effect of a gene when changing
the structure of an enzymatic protein.
Changing the sequence or number of
nucleotides in a gene leads to a violation of
the DNA code, and therefore to a violation
of the structure of protein molecules. The
consequence of this is a decrease in the
activity of the enzyme or its absence, the
accumulation of unusual metabolic
products, which leads to pathology.

Biochemical methods diagnose metabolic diseases, establish the

heterozygosity of parents. In recent years, special programs have been
developed and used for mass research in various countries.
 Biochemical method
• Phase one – screening-program screening. For this stage, a small number of
simple, accessible methods are usually used (Express methods): chemical or
microbiological tests. This is how a risk group is identified.

• In phase a clarification is performed (confirmation of the diagnosis or

deviation for a false-positive reaction at the first stage). For this purpose,
precise chromatographic, mass-spectrometric, and other methods for
determining enzymes, amino acids, and so on are used.
•
Molecular genetic
method.
DNA diagnostics — this is one of the most
modern high-tech research methods.
DNA diagnostics
combines several
research methods, the
most common of which
is the method of
PCR (polymerase chain
reaction).
• Polymerase chain reaction (PCR, PCR it was invented in 1983
by an American scientist named Kary Mullis.
• The principle of the method is to double (amplify) the DNA
region bounded by primers, using the enzyme DNA
polymerase.
• For each subsequent amplification cycle, both the original
DNA site and newly synthesized fragments (amplifiers) are
doubled.
• As a result, the number of fragments increases exponentially
(a chain reaction). After 30 to 40 cycles, their number exceeds
several billion, which makes it possible to detect them by
various methods.
• Amplification (lat. amplificatio - gain, magnification), in
molecular biology - increasing the number of copies DNA. In
the cell, amplification occurs as a result of replications In
artificial conditions, increasing the number of copies of DNA is
achieved using polymerase chain reaction.
• Primer — a short oligo-or polynucleotide sequence with a
free z'ON-group, complementary to a single-stranded DNA or
RNA; from its 3’-end, the DNA polymerase begins to build up
the polydeoxyribonucleotide chain.
 The stages of research and
development
• 1. Isolation of nucleic acids. At the first stage, all the DNA is extracted (for DNA-containing
microorganisms) or RNA (for the method NASBA or RNA-containing viruses) from the test
material.
• 2. Actual PCR or amplification. The solution containing a mixture of nucleotides, a PCR
buffer, a polymerase and primers is added to the DNA extracted at the first stage. PCR is
performed as follows: first, the reaction mixture is heated to a temperature of 90-94° C,
thereby causing DNA denaturation, then the temperature is lowered to 50-70°C C depending
on the nucleotide sequence of the primers, so that the annealing takes place in strictly
complementary sites, and finally, the temperature optimal for the operation of the DNA
polymerase is set in the mixture. When these cycles are repeated, the number of copies of
the DNA region located between the landing sites of primers increases exponentially.
• 3. Records of the results. Accumulated amplification products (a large number of DNA copies
between the primer landing sites) can be detected by gel electrophoresis. In addition, when
using fluorescently labeled probes, it is possible to take into account the results of changes in
fluorescence relative to negative control.
•
• NASBA (Nucleic Acids Sequence-Based Amplification)- unlike conventional PCR, the target for
NASBA (Nuclear Acids Sequence-Based Amplification) is RNA molecules of microbial
ribosomes, which gives a number of advantages
 Components of the reaction
•
• To perform PCR in the simplest case, the following components are required:
• The DNA matrix, which contains the portion of DNA that is required to amplify.
• Two primer's, complementary opposite ends of different chains of the required
DNA fragment.
• Thermostable DNA polymerase — enzyme, which catalyzes the DNA
polymerization reaction. Polymerase for use in PCR must remain active at high
temperature for a long time, so they use enzymes isolated from thermophiles —
Thermus aquaticus (Taq-polymerase), Pyrococcus furiosus (Pfu polymerase),
Pyrococcus woesei (Pwo-polymerase) and others.
• Deoxyribonucleoside triphosphates (dATP, dGTP, dCTP, dTTP).
• Mg2+ ions required for polymerase activity.
• Buffer solution, which provides the necessary reaction conditions — pH, the ionic
strength of the solution. Contains salts, bovine serum albumin.
•
• In order to identify and reproduce in the existing sample exactly the DNA
sequence that is needed, you need to know the nucleotide sequences of
its ends, in order to create complementary short (18-30 nucleotides)
fragments - primers. In the figure, these known ends of the sequence are
marked in blue, and black is the fragment of DNA that is needed.
•
 First-stage - denaturation

The double-stranded DNA matrix is heated to 94-96 °C (or 98 °C if

a particularly thermally stable polymerase is used) for 0.5—2 min.to
allow the DNA strands to separate.
 Second stage -
annealing.

The temperature is lowered, and the primers connect When the chains have separated,
the temperature is lowered so that the primers can communicate with a single-stranded
matrix with complementary parts of the DNA.This stage is called annealing. The
annealing temperature depends on the composition of primers and is usually selected by
4-5°C is below their melting point. Stage time — 0.5—2 min. The wrong choice of
annealing temperature leads either to poor binding of primers to the matrix (at an inflated
temperature), or to binding in the wrong place and the appearance of non-specific
products (at an undervalued temperature).
The main element PCR "this is a multiple heat cycle in which a DNA sample is exposed
to three different temperatures.
 Third stage - elongation

Commonly used Taq and Pfu

polymerases are most active
at 72 °C

Polymerase is an enzyme that can complete the second chain of DNA. In order to start
doing this, it needs a piece where the DNA is already double-stranded, and this is the
place where the DNA interacts with the primer. The polymerase always completes the
chain from the 5' to the 3'end (these names are related to the orientation of the
deoxyribose sugar in the chain; in ordinary double-stranded DNA, the chains are
directed opposite to each other:
5'_____________3'
3'_____________5'
 • Now there are four DNA strands that are too long in the solution. Two - the ones
that were, and two built on primers. But if you repeat the process again, you will
get the following picture:
•

If the original DNA chains could be considered conditionally infinite in both directions,
then the strands obtained in the first cycle are infinite in one direction, and on the second
they are limited by the primer. When such chains interact with the second primer, they
will produce pieces that are bounded on both sides. In total, several dozen cycles are
carried out in PCR, so the absolute majority of the reaction product will be short
necessary sequences, with which it will be possible to perform various manipulations, in
the simplest case - to accelerate on the chromatograph and compare the resulting strips
with those that would have turned out if there was ureaplasm or someone else.
 Scheme of doubling of DNA fragments in PCR (Andy
Vierstraete, 2001)
Scheme of doubling of DNA fragments in PCR

For the amplification process, the structure of primers must be identical (complementary) a
section of the original DNA. If this does not happen (there is no specific DNA), then there i
no doubling of the DNA. If there is not a single DNA molecule in the solution with a site tha
is complementary to the added primers, then the PCR reaction will not go, even though a
million other DNA molecules will be floating in the solution. This is the reason for the high
specificity of the PCR method.
 Advantages of the PCR method
1. Versatility. Using PCR, you can determine the DNA in any biological samples. Moreover, this
applies equally to both microbial and human DNA.
2. High specificity. Specificity is determined by the fact that the PCR determines a unique gene
site that is characteristic only for this pathogen. To increase specificity, it is possible to
identify several different genes of a single microbe. For example, to determine Ureaplasma
urealyticum, both the 16S-RNA gene and the urease gene can be detected. And to identify
Chlamydia trachomatis, in addition to determining chromosomal DNA and cryptic plasmid
DNA, it became possible to detect ribosomal RNA (NASBA). This significantly increases the
reliability of the study.
3. High sensitivity. The polymerase chain reaction can detect single copies of DNA. On average,
the sensitivity threshold of most modern test systems ranges from 10 to 100 copies of DNA.
This significantly exceeds the sensitivity of cultural research methods.
4. Small volume of biological material. The analysis is possible in a minimal sample volume (up to
several microliters), which is extremely important in Pediatrics, neonatology, neurology, and
forensic medicine.
5. The possibility to diagnose not only acute but latent infections.Especially effective is the PCR
method for diagnosing difficult to cultivate, uncultivated and persistent forms of
microorganisms, which are often encountered in latent and chronic infections.
 Method of specific restriction of DNA (blot
hybridization)
• Transfer method DNA from agarose gel to nitrocellulose or
nylon membranes was called by Southern southern
blotting (southern — South)).
• Transfer method RNA from agarose gel to nitrocellulose or
nylon membranes was called Northern blotting (nothern —
northern).
•
• Transfer method protein from polyacrylamide gel to
nitrocellulose or nylon membranes (Burnette, 1981) was
named Western blotting (western — Eastern).
•
• In the described types of blotting, electrophoresis and vacuum are
also used to organize the flow of DNA, RNA and proteins through
the gel to the membrane instead of capillary forces.