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Topic_4.doc
Петрашенко Вікторія Олександрівна
2014
Topic 4: “СITOGENETIC METHODS OF RESEARCH IN CLINIC”
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Topic 4: “СITOGENETIC METHODS OF RESEARCH IN CLINIC”
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Topic 4: “СITOGENETIC METHODS OF RESEARCH IN CLINIC”
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Topic 4: “СITOGENETIC METHODS
OF RESEARCH IN CLINIC”
1. The general aim - to know the main principles of citogenetic methods and their using in practical
medicine
2. Student must know:
The structure and kinds of chromosomes.
Methods of taking material for citogenetic investigation.
Methods of painting of chromosomes.
Region of using of citogenetic methods.
3. Student must be able:
To
To
To
To
treat karyogram in normal and pathological conditions.
explain the mechanism of separation of somatic and germ cells.
explain methods for preparations of mitotic chromosomes.
differentiate methods of chromosome painting.
4. Plan of conducting of studies
Introduction
Classroom
Control and correction of initial level of knowledges
Computer class
Essence and types of citogenetic method
Classroom
Methods of painting of chromosomes, their essence
Classroom
Demonstration of karyograms
Classroom
Educational control and correction of level of knowledges
Classroom
Conclusion
Classroom
5 min
10 min
25 min
20 min
10 min
5 min
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Chromosome
A chromosome is an organized structure of DNA and protein that is found in cells. It is a single piece of
coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes
also contain DNA-bound proteins, which serve to package the DNA and control its functions.
Fig. 1. Chromosome structure
In a eukaryotic cell, the chromosomes in the nucleus forms a condensed structure with proteins called
as chromatin. The proteins in association with DNA are Histones. Histones are the proteins rich in
positively charged amino acids like lysine and argignine. These positively charged amino acids in histone
binds tightly to the negatively charged phosphate groups of DNA.
Chromosome packaging: Packaging structure of eukaryotic chromosomal DNA is called as
nucleosome which gives negative super coiling. Generally 146 ton200 nucleotide base pairs are wrapped
by histone octamer. Histone octamer contains two cpies core histones viz: H2A, H2B, H3 and H4.
Types of Chromosomes:
♦ Autosomes. Autosomes are structures that contain the hereditary information. They do not
contain information related to reproduction and sex determination. They are identical in both sexes, i.e.,
male and female species of humans. There are 46 (2n) chromosomes in humans. Of these 46
chromosomes, there are 44 pairs of autosomes and contain information related to the phenotypic
characters.
♦ Allosomes/ Heterosomes. The allosomes are sex chromosomes that are different from
autosomes in form, behavior and size. There are a pair of allosomes in humans. The X chromosomes are
present in the ovum and either the X or Y chromosome can be present in the sperm. These
chromosomes help in determination of sex of the progeny. If the offspring receives X chromosome from
the mother as well as father, it results in a female child (XX). If the offspring receives one X and one Y
chromosome from the parents, it results in a male child (XY). In simple words, it is the donation of X or Y
chromosome by the father that helps in determination of the sex of the child.
Apart from these two categories, chromosomes can further be divided according to the location of the
centromeres.
♦ Chromosome Types: Based on Centromere Position
Chromosomes are divided into four types based on the centromere position. These four types are as
follows:
⟿ Metacentric Chromosome
The metacentric chromosome has its centromere centrally located between the two arms. This gives
the chromosome a typical 'V' shape that is seen during the anaphase. The arms of this chromosome are
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roughly equal in length. In certain cells, fusion of two acrocentric chromosomes leads to formation of
metacentric chromosome.
⟿ Submetacentric Chromosome
The arms of the submetacentric chromosome are said to be unequal in length. This is because the
kinetochore is present in the sub median position. This gives rise to the 'L' shape of the submetacentric
chromosome.
⟿ Telocentric Chromosome
Also known as the monarchial type of chromosomes, they have a centromere that is located towards
the end of the chromosome. Thus, telocentric chromosomes have a 'rod' shaped appearance. In some
cases, the telomeres extend from both the chromosome ends. The telocentric chromosome is not
present in humans.
⟿ Subtelocentric Chromosome
Chromosomes that have a centromere that is located closer to the end than the center, are called
subtelocentric chromosomes.
⟿ Acrocentric Chromosome
The location of the centromere in the acrocentric chromosome is subterminal. This causes the short
arm of the chromosome to become really short making it very difficult to observe.
⟿ Holocentric Chromosome
In holocentric chromosomes, the centromere runs through the entire length of the chromosome.
These chromosomes are very common in cells belonging to organisms in the animal and plant kingdom.
Cell Division.
There are two types of cell division: mitosis and meiosis. Mitosis is the type of cell division that occurs
in most cells of the body. It is during mitosis, specifically the prophase stage of mitosis, that
chromosomes are visible and easy to identify for karyotyping. In mitosis, two genetically identical
daughter cells are produced from a single parent cell. Before cell division, DNA replication has occurred
so that there is a doubled amount of DNA and the chromosomes contain two identical sister chromatids.
Mitosis is divided into stages. Prophase is characterized by spiraling of the chromosome threads into
coils to form microscopically identifiable chromosomes; the nuclear membrane and the nucleolus
disappear and the mitotic spindle forms. In metaphase, the chromosomes condense and are clearly
visible as distinct structures. The centromeres of the chromosomes attach to the microtubules of the
mitotic spindle and the chromosomes align at the middle of the cell along the spindle. Anaphase is
characterized by division of the chromosomes along their longitudinal axis to form two daughter
chromatids and migration of each chromatid of the pair to opposite poles of the cell. Telophase, which
completes mitosis, is characterized by reconstitution of the nuclear membrane and nucleolus,
duplication of the centrioles, and cytoplasmic cleavage to form the two daughter cells.
Meiosis is the form of cell division that occurs to produce germ cells or gametes (sperm and egg). A
diploid cell (with two sets or 46 chromosomes) divides to form haploid cells (with one set or 23
chromosomes). Meiosis is divided into two parts: meiosis I and meiosis II. DNA replication occurs before
meiosis I. In male meiosis, the germ cell begins division with two times the normal cellular amount of
DNA. In meiosis I, each daughter cell gets one of the duplicated chromosomes of each pair. At the
beginning of meiosis II, each cell contains 23 chromosomes, each with a duplicated pair of chromatids. In
meiosis II, the duplicated pair separate and each daughter cell ends up with one of each of the 23
chromosomes, that is, there are four daughter cells, each with a haploid (half the normal number) set of
chromosomes. In female meiosis, rather than going through cell divisions during meiosis I, one diploid
set of chromosomes condenses and forms a polar body, and during meiosis II, one of the haploid sets of
chromosomes condenses and forms the second polar body, resulting in one egg with a haploid (half the
normal number) set of chromosomes and two polar bodies that contain three sets of chromosomes.
There is exchange between chromosomes (crossing over of chromosome segments) during meiosis,
leading to new alignment and combination of genes. Two common errors of cell division occur during
meiosis that result in abnormal numbers of chromosomes and chromosomal anomalies. The first is
nondisjunction, in which two chromosomes fail to separate and thus migrate together into one of the new
cells, producing one cell with two copies of the chromosome and one cell with no copy. The second is
anaphase lag, in which a chromatid is lost because it fails to move quickly enough during anaphase to
become incorporated into one of the new daughter cells.
Karyotype
In general, the karyotype is the characteristic chromosome complement of a eukaryote species.. The
preparation and study of karyotypes is part of cytogenetics.
Each animal has characteristic number of their chromosomes; these are generally present in diploid
that is 2n form. Karyotype is the complete set of chromosome present in cells of an organism. Karyotype
of human has 22 pairs of homologous chromosomes and one pair of sex chromosome. Precisely males
have 22 pairs of homologous chromosome, one X and one Y chromosome where as females have 22
pairs of homologous chromosome and a pair of X chromosomes.
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Aberrations in chromosome numbers leads to genetic disorders. Euchromatin is the active
chromosome where DNA transcribes and translates to give proteins. Heterochromatin is the inactive
form of chromosome and functions to maintain the chromatin structure.
Methodology.
Chromosome studies can be obtained from any dividing nucleated cell. The techniques for
visualization require condensation of chromatin material that occurs at cell division. Cytogenetic studies
are usually performed on blood lymphocytes, but cytogenetic studies of fibroblasts must be considered if
there is a suspicion of mosaicism. Chromosome studies for prenatal diagnosis are performed with cells
obtained from amniotic fluid, chorionic villi tissue, fetal blood, or in preimplantation prenatal diagnosis by
analysis of a blastomere.
Karyotyping refers to the systematic arrangement from a photograph or by a computer of previously
stained and banded chromosomes of a single cell by pairs. The cells are cultured, arrested in mitosis
during metaphase, and then fixed and stained. If finer details are necessary, prophase chromosomes
may be examined. Because prophase chromosomes are longer and less condensed, they show 600–
1,200 bands, compared with metaphase chromosomes, in which only 400–600 bands are usually visible.
Trypsin-Giemsa staining gives G banding. Quinacrine gives the Q (fluorescent) banding. Special stains are
used to demonstrate centromeres.
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Fig.2. FISHing for abnormalities
Incorporation of FISH (also known as molecular cytogenetics) has greatly increased the sensitivity of
chromosome studies. The process of FISH involves hybridization of cloned fluorochrome-labeled DNA
fragments (probes) to target chromosomal sequences. Probe DNA is denatured in the presence of Cot-1
DNA that binds highly and moderately repetitive genomic sequences to allow for select hybridization of
the unique sequence DNA to the target. Probe detection is accomplished by UV-light excitement of a
fluorochrome, such as fluorescein-5-thiocyanate (FITC) or rhodamine that is either directly attached to
the probe DNA or attached to a hapten (biotin or digoxygenin)-labeled probe.
Using FISH technology, subtle aberrations in chromosome content can be readily assessed and linked
with clinical disorders. FISH has not only been used to detect submicroscopic alterations in metaphase
cells, it may also be performed on interphase nuclei. Importantly, FISH probes have been mapped to
chromosomal locations and molecular sequence maps, allowing for the direct connection of data being
generated from the human genome project to clinical phenotypes caused by cytogenetic abnormalities.
These links provide clues to the molecular basis of disease and the genes involved.
Comparative genomic hybridization is a molecular cytogenetic technique that allows simultaneous
enumeration of every chromosome. It involves the isolation of test DNA from a single cell (multiplied by
polymerase chain reaction) or multiple cells from the test individual and comparison with DNA from a
normal reference individual. The test DNA is labeled in a different way (green fluorochrome) from the
reference DNA (red fluorochrome). The test and reference DNA are simultaneously hybridized to normal
chromosomes. The fluorescent images reveal ratios of green to red and can be used to identify extra or
missing chromosomal material.
Chromosomal anomalies occur in 0.4% of live births. They are an important cause of mental
retardation and congenital anomalies. Chromosomal anomalies are present in much higher frequencies
among spontaneous abortions and stillbirths. The phenotypic anomalies that result from chromosomal
aberrations are mainly due to imbalance of genetic information. Chromosomal anomalies include
abnormalities of chromosome number and structure.
Fig.3. Karyotype (a) and partial results of a subtelomeric probe panel (ToTelVysion,
Vysis, Inc., Downers Grove, IL) analysis (b) for a female patient with developmental delay
and autism. The probe cocktail shown contains probes for chromosomal regions 2p
(green), 2q (red), X centromere (blue) and Xq/Yq (yellow). This patient had a subtle
deletion of the subtelomeric region on chromosome 2q (arrows)
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Fig.4. FISH with a probe for TUPLE1 on a metaphase cell from a patient with Di
syndrome revealing a deletion of 22q11.2 (a), and on an interphase cell from a patient
with Dup(22)(q11.2q11.2) syndrome/
FISHing got more interesting with the ability to simultaneously visualize all 24 human chromosome
pairs with different colors. Spectral karyotyping (SKY, Figure 4) or Multiplex-FISH (M-FISH) techniques
incorporate combinatorial or ratio-based fluorochrome labeling schemes to produce 24 colors, one for
each chromosome. To accomplish this, whole chromosome probes, composed of unique and moderately
repetitive sequences from an entire chromosome, are labeled with specific combinations of fluorescent
dyes. The probes are generated from DNA from a particular chromosome that is isolated from the rest of
the genome by flow sorting, creation of somatic cell hybrids containing a single human chromosome or
area of a chromosome, or microdissection of chromosomes and subsequent amplification of the
dissected DNA sequences via the PCR. Hybridization of the collection of whole chromosome probes to
metaphase cells produces continuous fluorescent signals on each of the chromosomes. For SKY, the
spectral characteristics of each pixel are scored by an inferometer; whereas for M-FISH, images are
collected through a series of excitation and emission filters and computer software is used to classify
each chromosomal segment. The computer then applies a distinct pseudocolor for each chromosome
allowing the cytogeneticist to view a karyotype with each chromosome “painted” a different color.
This type of analysis has been especially useful for defining complex rearrangements, such as those
seen in neoplastic disorders and solid tumors. For example, in a study of 30 children with acute
lymphoblastic leukemia whose leukemic blast cells lacked chromosomal abnormalities detectable by
conventional cytogenetics or whose blast cells had multiple chromosomal abnormalities that could not
be completely characterized by G-banding analysis, SKY identified three cryptic translocations in the
previously normal category and was also successful in defining the nature of the chromosomal
abnormalities in 4 of the 10 patients with marker and derivative chromosomes. SKY and M-FISH have
also been used to determine the origin of de novo duplications and marker chromosomes in pre- and
postnatal cases.
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Fig.5. Schematic of cytogenetic analysis using spectral karyotyping (SKY). (a) Flowsorted human chromosomes are combinatorially labeled with at least one, and as many
as five, fluorochrome combinations to create a unique spectral color for each
chromosome pair. (b) The SKY probe mixture is hybridized to a metaphase chromosome
preparation. (c) Detection of the hybridized sample. (d) Visualization of the SKY
hybridization using a Spectracube connected to an epifluorescence microscope. (e)
Spectral classification assigns a discrete color to all pixels, which is converted to a
display image that is based on the fluorescence intensities of all of the chromosomepainting probes: (1) classification colors are assigned to the chromosomes; (2) this
measurement forms the basis for automated chromosome identification.
Examples of questions.
5. What is the karyotype?
a) is the characteristic chromosome complement of a eukaryote species;
b) a condensed structure with proteins;
c) the proteins rich in positively charged amino acids like lysine and argignine;
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d) an organized structure of DNA and protein that is found in cells.
4. What is the nucleosome?
a) is the inactive form of chromosome and functions to maintain the chromatin structure;
b) a condensed structure with proteins;
c) an organized structure of DNA and protein that is found in cells;
d) packaging structure of eukaryotic chromosomal DNA.
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