What is cytogenetics?

It is a branch of genetic involving the study of the cell and the chromosomes in detail. The chromosomal studies include the grouping and numbering of chromosomes, studying their structures, their variations in numbers, and the related conditions. It involves the behavior of the chromosomes during the mitotic and the meiotic phases. Studying chromosomes involves various techniques. Conventional cytogenetic techniques include karyotyping and banding techniques. Molecular cytogenetic techniques involve modern tools to detect chromosomal aberrations. FISH, Array-CGH, and many others help in studying the chromosomes.

The human cytogenetics relates to the study of human chromosomes and the mechanisms and anomalies associated with the same. The plant and animal cytogenetics cover the study of the respective chromosomes and the conditions associated with the structural and numerical variations. The human chromosomes involve autosomes and sex chromosomes. The total complement of chromosomes includes 46 number of chromosomes. The study of every chromosome in detail includes revealing its structure, the banding pattern, the genes in every chromosome, the conditions associated with same, and overall functioning of the chromosome.

The human cytogenetics involves the study of human chromosomes and the anomalies associated with the same. Let us discuss the human chromosomes in detail.

Image 1: Chromosomes and cytogenetics

Human chromosomes:

The chromosomes occur in the nucleus. They consist of DNA compacted with histone proteins. The word chromatin indicates the strands of the chromosomal material in the interphase. It shows the presence of coiled and extended regions. There are two types of chromatin such as euchromatin and the heterochromatin. The euchromatin involves highly active DNA required for the transcription process. It stains lighter than the heterochromatin. Males possess X and Y chromosomes. The females possess two X chromosomes. The sizes and the shapes of the chromosomes vary as per the phase of the cell cycle. The size of the metaphase chromosome is 5mm. Chromosomes acquire different shapes during the anaphase such as the rods, V, J, T, or X shaped.

A metaphase chromosome reveals five main structural components such as the satellite, the telomere, the chromatids, the primary constriction, and the secondary constriction. The two symmetrical halves running parallel to one another indicate the chromatids. The ones adjacent to each other are known as sister chromatids. The primary constriction is also known as the centromere. It pins up the chromatids together. The centromere divides the chromosome into its respective arms. The short chromosomal arm is known as the p arm. The long chromosomal arm is known as the q arm. The secondary constriction or the nucleolar organizer region gets associated with the nucleolus and its formation. The position of the centromere is different for every chromosome.

Chromosome pair number	Group of the chromosome	Type of the chromosome based on its structure	Abnormalities associated with the structure or number
1	A	Metacentric	1p36 deletion syndrome
2	A	Submetacentric	2q37 deletion syndrome Cancers
3	A	Metacentric	3p deletion syndromes Microdeletion syndromes Cancers
4	B	Submetacentric	Cancers Wolf-Hirschhorn syndrome
5	B	Submetacentric	5q31.3 microdeletion syndrome  Cri-du-chat syndrome
6	C	Submetacentric	6q24-related transient neonatal diabetes mellitus  Cancers
7	C	Submetacentric	Russell-Silver syndrome Saethre-Chotzen syndrome Williams syndrome
8	C	Submetacentric	Recombinant 8 syndrome Trichorhinophalangeal syndrome type II
9	C	Submetacentric	Bladder cancer Chronic myeloid leukemia Kleefstra syndrome
10	C	Submetacentric	Cancers
11	C	Submetacentric	Jacobsen syndrome Neuroblastoma
12	C	Submetacentric	Pallister-Killian mosaic syndrome
13	D	Acrocentric	Retinoblastoma Trisomy 13
14	D	Acrocentric	FOXG1 syndrome Multiple myelomas Ring chromosome 14 syndrome
15	D	Acrocentric	Prader-Willi syndrome Angelman syndrome
16	E	Metacentric	16p11.2 deletion syndrome 16p11.2 duplication Rubinstein-Taybi syndrome
17	E	Submetacentric	17q12 deletion syndrome 17q12 duplication Acute promyelocytic leukemia Miller-Dieker syndrome Potocki-Lupski syndrome Smith-Magenis syndrome
18	E	Submetacentric	Tetrasomy 18p Trisomy 18
19	F	Metacentric	19p13.13 deletion syndrome
20	F	Metacentric	Alagille syndrome Cancers Ring chromosome 20 syndrome
21	G	Acrocentric	Down’s syndrome
22	G	Acrocentric	22q11.2 deletion syndrome 22q11.2 duplication 22q13.3 deletion syndrome Emanuel syndrome
X	-	Submetacentric	Klinefelter syndrome Triple X syndrome Turner syndrome X-linked acro gigantism
Y	-	Acrocentric	47, XYY syndrome 48, XXYY syndrome Y chromosome infertility

Karyotyping:

It helps in grouping the chromosomes from A to G. Human cells possess 22 pairs of autosomes (non-sex chromosomes). There are two sex chromosomes known as X and Y respectively. The procedure of karyotyping involves three main steps such as culturing the cells to get the chromosomes, banding or staining technique, and observation under the microscope followed by software analysis. After arranging the chromosomes in respective groups, they get analyzed for the presence or absence of the structural or numerical variations.

Image 2: Chromosome Banding

Chromosome banding:

The banding techniques help in identifying the dark and the light regions or chromosome bands. Various techniques exist in chromosome banding. The G-banding is the most commonly used technique. It involves treating the chromosomes with trypsin. The trypsin denatures the proteins present in the chromosome. The next step involves staining the chromosomes with Giemsa solution. The light and dark bands form due to this type of staining technique. Thus, it is possible to visualize them under the microscope. The Q banding method helps in staining the chromosomes with quinacrine mustard. The banding patterns mimic that of the G banding. The R-banding technique involves pre-heating of the chromosomes before staining with the Giemsa. It involves a reverse banding pattern as compared to the G banding. The Centromere and the secondary constriction regions get stained using C- banding.

Fluorescence in-situ hybridization:

The FISH technique utilizes a single-stranded DNA probe labeled with fluorescent labels. The single-stranded DNA probe gets annealed with the complementary target sequence on the chromosome. Hence, it detects the regions with similar sequences. Three main types of FISH probes include Centromeric probes, chromosome-specific unique sequence probes, and whole chromosome paint probes. The sequences found near the Centromeric regions show the presence of repetitive DNA. Also, the regions of centromere themselves show a large number of repetitive sequences. Thus, Centromeric probes help in identifying the sequences. The sub-microscopic deletions and duplications get identified using the chromosome-specific unique sequence probe. To visualize the entire chromosome, the cytogeneticists use whole chromosome paint probe. 

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Chromosome analysis

The chromosome analysis involves many conditions such as congenital malformations, mental retardation, repeated abortions, sex determination, and prenatal diagnosis. The procedure of chromosome analysis involves techniques such as karyotyping and FISH. A microscopic examination of chromosomes for detection of abnormalities or novel mutations involves chromosome analysis. It helps to detect monosomy, trisomy, infertility issues, repeated abortions, hereditary syndromes, and chromosomal aberrations. It helps in detecting the disorder or a disease. It helps in the genetic counseling and consultation. It is possible to diagnose hematological disorders as well. Modern FISH techniques detect cytogenetic abnormalities in haemato-lymphoid malignancies and hereditary cancers. Let us know the technique in detail.

Karyotyping:

It is a procedure to obtain karyotype of an individual. A karyotype is nothing but a chromosome complement of an individual. It depicts a photomicrograph of the metaphase chromosome arranged in a standard sequence. The review article describes the procedure in detail.

The human peripheral blood lymphocytes culturing and processing helps to obtain a karyotype. A sterile syringe is used to collect the sample. It involves the collection of 5ml of the nervous blood in a heparinized tube. Addition of heparin prevents blood clotting. The procedure primarily involves the addition of the heparinized whole blood sample into the culture medium consisting of RPMI-1640, fetal calf serum, phytohaemagglutinin, and antibiotics. The culture media and the fetal calf serum nourish the lymphocytes. Phytohaemagglutinin stimulates cell division in lymphocytes. Addition of the antibiotics prevents the infection. The culture vial involves incubation of 3 days at 37⁰C temperature.

The incubation of the culture vials leads to the division of the lymphocytic cells. The addition of a mitotic inhibitor (colchicine) at the end of the third day, helps a lot by preventing the spindles. It arrests the cells in metaphase. Visibility of the chromosomes is best during the metaphase. Then the culture vials are kept undisturbed for two hours. After 2 hours the lymphocytes are centrifuged. Then the cells undergo saline treatment. The saline creates a hypotonic treatment. The cells swell under the hypotonic environment. Followed by re-centrifugation, the procedure requires removal of the supernatant. After discarding the supernatant, the cells get fixed in a fixative such as Carnoy’s fixative. A careful dropping of few drops of the cell suspension on a clean, pre-chilled, grease free slide enables the chromosomes to get dispersed. The processing of the slides and staining them helps to visualize the chromosomes under a microscope. Karyotyping software helps to arrange the chromosomes in groups thereby generating a photomicrograph of chromosomes known as a karyotype.

Observation of the chromosomes:

A karyotype reflects the differences in the absolute sizes of the chromosomes. It helps to study the DNA duplication. The differences in the relative sizes of the chromosomes arise due to the interchange of the chromosomal segments. The chromosomal segments may interchange in unequal lengths. A karyotype helps to observe various types of chromosomal abnormalities known as deletions, duplications, translocations, and inversions. For example, a translocation arises due to differences in the Centromeric position. Grouping of the chromosomes helps in identifying the correct number of the chromosomes. Also, the karyotyping enables to study the number and the position of the satellites.

Giemsa banding:

It commonly helps in analyzing the chromosomes. Giemsa banding technique first involves treatment of the chromosomes with a denaturing agent known as trypsin. It helps in denaturing the proteins. A stain known as Giemsa helps to stain the slides. Staining with the Giemsa stain creates a unique banding pattern to the chromosomes. The pattern shows light and dark bands. A specific banding pattern helps to visualize the long and short arms of the chromosome. Giemsa stain is a complex stain specific to the phosphate groups of the DNA. The light and dark stripes appearing on the chromosomal arms depict the bands. They appear after staining the cell preparation.

The heterochromatin depicts a dark band. The euchromatin depicts a light band. Heterochromatin is rich in the repetitive DNA sequences. Other types of banding techniques include R-banding, C-banding, Q-banding, T-banding and silver staining.

Three main types of karyotyping include classical karyotyping, spectral karyotyping, and virtual karyotyping. The above method of karyotyping involves Giemsa banding belonging to the classical karyotyping. The spectral karyotyping helps to visualize all the chromosomes in different colors. This type of karyotyping is known as SKY technique. A digital karyotype helps to quantify the DNA copy number.

Image : Giemsa banding of chromosomes

Fluorescence in situ hybridization (FISH):

A metaphase chromosome consists of a DNA packed into it in a highly condensed form. The DNA sequence helps to detect the defects. The FISH technique uses a single-stranded DNA probe. It has a unique ability to anneal with the complementary DNA sequence on the chromosome. A DNA probe is a single-stranded sequence. It gets labeled radioactively. The DNA probe detects the DNA fragments with similar sequences. The hybridization of the DNA sequence with the probe sequence enables it to be visualized using autoradiography.

The FISH technique involves different types of probes. The Centromeric probes are nothing but the DNA sequences found in and around the centromere. They are specific to a particular chromosome and have a repetitive sequence. A chromosome-specific unique sequence probe identifies sub-microscopic deletions and duplications. A whole chromosome paint helps to visualize an entire chromosome. Examination of slides under a microscope detects the presence of a hybridized fluorescent signal. It also detects the absence of the chromosomal material if there is no signal. Not only metaphase chromosomes but also non-metaphase chromosomes such as interphase chromosomes hybridize with the fluorescent labeled probes.

Chromosome painting is the next version of FISH. In this technique, the hybridization probe involves a mixture of DNA molecules specific for different regions of a single chromosome.    

References:
[1] Medical genetics, G.P. Pal
[2] Molecular Cytogenetics: Protocols and Applications, Yao-Shan Fan  
[3] Human Chromosomes: Structure, Behavior, and Effects, Eeva Therman, Millard Susman
[4] The AGT Cytogenetics Laboratory Manual, Marilyn S. Arsham, Margaret J. Barch, Helen J. Lawce  

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A review on human chromosomes

An Introduction to chromosomes:

The ability of the chromosomes in getting stained reveals the true nature of the chromosome (derived from the Greek work word. The word chroma indicates color). These stainable bodies appear like threads under a microscope. They contain the genetic material, mainly the DNA coiled around the proteins. Human chromosomes are visible with when the cell is undergoing mitotic or meiotic cell division. There are total 46 chromosomes in each human cell.

·    Autosomes: There are total 44 autosomes or 22 pairs. Each pair consists of homologous chromosomes. In a pair, one chromosome comes from the father and the other chromosome comes from the mother.

·        Sex Chromosomes: There are two different types of sex chromosomes, X and Y respectively.

The average size of the human metaphase chromosome is 5 millimeters. Chromosomes tightly coil and get condensed during metaphase. Chromosomes appear in different shapes during each phase of the cell cycle. They appear thread-like during interphase. During metaphase, chromosomes look like rod-shaped. They look like V, J or rod-shaped during anaphase. Von Hartz coined the term chromosome. The scientists who first discovered the structure of chromosomes were Schleiden, Virchow, and Bütschli. Walter Sutton and Theodor Boveri independently developed the chromosome theory of inheritance in 1902.

An interphase nucleus contains strands of a material called chromatin. There are two regions in the chromatin, mainly coiled and extended regions.

·    Heterochromatin: It is the dark staining area of the chromatin. There are two types of heterochromatin. Constitutive heterochromatin contains repetitive sequences. It is present near the centromere. Constitutive heterochromatin never expresses itself. There is one more type of heterochromatin, known as facultative heterochromatin, which expresses itself.

·        Euchromatin: It is the light staining area of the chromatin.

The chief constituent of chromatin is DNA, the blueprint of life. At the time of cell division, chromatin strands coil into compact structures, so that they easily fit into the cell nucleus. Chromosomes appear as thick rods only during the cell division. They uncoil and form chromatin at the end of cell division.

Image: Human chromosomes revealed through karyotyping

Structure of the chromosome:

Metaphase chromosome appears clearly under the microscope. Following are the principal point to be discussed:

·        Chromatids: Each metaphase chromosome consists of two symmetrical halves parallel to each other. They are called chromatids. These chromatids are present in the form of chromonema during prophase. There are two types of chromatids mainly, sister and non-sister chromatids. Two chromatids are present on a single chromosome. Thus they are called sister chromatids. The concept of dyad describes a pair of sister chromatids. These structures join with the help of a centromere.  Non-sister chromatids are either of the two chromatids of a chromosome pair.

·        Centromere: A centromere is a light staining constricted area to which both the chromatids are attached. A centromere divides the chromatids into short and long arms respectively. The short arm is known as “p” arm. The long arm is known as “q” arm. Centromere produces a primary constriction. It is the position of the centromere. It is different for different chromosomes. Secondary constriction is also known as nucleolar organizer region. It is involved in the formation of the nucleolus. Human centromere consists of several hundred kilobases of repetitive DNA. Centromeres are the sites where spindle fibers are attached. Thus, centromere helps in the movement of the chromosome.

·     Satellite: A satellite is a region that is attached to the chromosome by a thread of chromatin. It is present at the distal region of the arm of the chromosome.

·        Telomere: A special DNA-protein complex is present at the ends of the chromosomes. This complex is known as a telomere, with tandem repeats of TTAGGG-3’ sequences between 3-20 kilobases in length. Telomeres are not genes since they do not code for any functional molecule. Telomeres provide structural stability to the chromosomes by sealing their ends. Telomeres protect the chromosomes from damage. They also protect the chromosome from fusing into a ring or binding to other DNA.

Classification of chromosomes:

1.     Classification based on the position of the centromere:  

·     Metacentric: The two arms are almost equal in their lengths. The location of the centromere is at the center of the chromosome.

·   Submetacentric: The two arms of the submetacentric chromosomes are unequal in length. The location of the centromere is slightly away from the center.

·   Acrocentric: One arm of an acrocentric chromosome is short. Whereas, the other arm is long.

·      Telocentric: A telocentric chromosome has only one arm.

2.     Standard classification: It is also known as Denver classification. It classifies chromosomes into seven groups, depending on the length of the chromosomes.

·  Group A: This group consists of pairs of chromosomes 1, 2 and 3. Chromosome 1 is the largest human chromosome. It represents 8% of the total DNA content.  Chromosome 2 is the second one. The third chromosome represents 6.5 % of the total DNA content.

·       Group B: This group consists of pairs of chromosome 4 and 5.

·    Group C: This group consists of pairs of chromosome 6, 7, 8, 9, 10, 11 and 12.

·        Group D: This group consists of pairs of chromosomes 13, 14 and 15.

·        Group E: This group consists of pairs of chromosomes 16, 17 and 18.

·        Group F: This group consists of pairs of chromosomes 19 and 20.

·        Group G: This group consists of pairs of chromosomes 21 and 22.

·  Sex Chromosomes: There are two types of sex chromosomes. X chromosome and Y chromosome are called sex chromosomes.

3.     Paris nomenclature: According to this method, the long and short arms of the chromosomes have specific regions that get stained. These regions are further stained using banding techniques. Such techniques may not only help to identify specific chromosomes, but also find out the location within the chromosome. Banding techniques may help to detect minor structural abnormalities.

What is sex chromatin?

The nucleus in the interphase is in the resting phase. An interphase nucleus shows a dark stain chromatin mass attached on one side of the nuclear membrane. Sex chromatin is also known as the Barr body. It is observed only in females. However, the chromatin determination using the Barr body is not as accurate as the Karyotyping technique.

What are chromosomal aberrations?

Chromosomal mutations or aberrations are variation in the normal chromosome structure or chromosome number.

A deletion is a chromosomal mutation in which a part of a chromosome is missing. Chromosomal breaks result in deletions. Sometimes an entire chromosome may get deleted. Duplication may lead to doubling of a chromosomal segment. Excision of a chromosomal segment follows reinsertion leading to an inversion. A translocation is a chromosomal mutation in which a chromosome segment gets positioned in a different location in the genome.

Chromosome Analysis:

Chromosome analysis indicates a proper diagnosis of many clinical conditions. It is a microscopic analysis of chromosomes in the dividing cells. Chromosomal analysis can detect chromosome number and structure.

Uses of chromosome analysis are as follows:

·        Detection of congenital malformations, mental retardation, and repeated abortions.

·        Prenatal diagnosis

·        Diagnosis of malignancies

Karyotyping:

Karyotyping is a test to evaluate the number and the structure of the chromosomes. In this procedure, the metaphase chromosomes are obtained and photographed. The procedure of karyotyping is specialized. Peripheral blood lymphocytes, bone marrow cells or amniotic fluid samples are collected and analyzed for chromosomes. A photo-micrograph reveals chromosomes scattered randomly. These chromosomes are arranged into groups, using the software. A karyotype can be sued to detect chromosomal abnormalities.

Chromosome Banding:

Analysis of chromosome becomes precise with the help of banding techniques. There are four types of banding techniques such as G-banding, Q-banding, R-banding, and C-banding. A unique pattern of light and dark bands are obtained using the G-banding technique.

Fluorescence in-situ hybridization (FISH):

FISH is a new diagnostic technique that involves a single-stranded probe annealing to its complementary sequence. FISH can be used to detect minute chromosomal aberrations, malignancies, and study of chromosomes.

References:
[1] Medical genetics, G.P. Pal
[2] Human Genetics, 3/e, Gangane
[3] Vogel and Motulsky's Human Genetics: Problems and Approaches, Friedrich Vogel, Gunter Vogel, Arno G. Motulsky
[4] Biology for the IB Diploma: Standard and Higher Level, Andrew Allott
[5] Principles of Medical Genetics, Thomas D. Gelehrter

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