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An Introduction to Cytogenetics


Dr. Eman Mosaad El-Hefny ElClinical Pathology Department South Egypt Cancer Institute


It i th study of chromosomal structure, is the t d f h l t t p pathology, function and behavior. gy,


- CHROMOSOMES g genetic material. material. carry most of the f h

- E h pair of choromosomes consists of Each i f h it f one p paternal and one maternal chromosome diploidy. The intact set is diploidy. passed to each daughter cell at every mitosis. mitosis.

CLASSIFICATION The normal human karyotype is made of 23 pairs (46) chromosomes: 46) chromosomes: · 22 pairs of autosomes: numbered from autosomes: 1 to 22 by order of length. length. · 1 pair of gonosomes ( i f (sex chromosomes): chromosomes): h ) XX in the female, XY in the male. , male.

Each chromosome has a centromere (CEN): (CEN):

It divides the chromosome into 2 arms: the arms: short arm (p arm) and the long arm (q arm). arm). e.g. t(14;18)(q22.3;p12) t(14;18)(q22. 12)

When the short arm is nearly as long as the long arm, the chromosome is said metacentric. metacentric. t t i If it i shorter, th chromosome i said is h t the h is id sub-metacentric. sub-metacentric. When it is very short, but still visible, the chromosome is said to be sub-telocentric. sub-telocentric. When extremely short, virtually invisible, the chromosome is said acrocentric. acrocentric.



CONSTITUTIONAL: CONSTITUTIONAL: All the tissues (the whole patient) hold the th same anomaly. Th chromosome anomaly. The h l error was already present in the embryo. yp embryo. y ACQUIRED: ACQUIRED: Q Only one organ is involved, the other tissues being normal. The patient has a normal. cancer of the affected organ. "Acquired organ. anomalies" herein refers to malignancies. malignancies.

HOMOGENEOUS: HOMOGENEOUS: When all the cells (studied) carry the anomaly. anomaly. e.g. 1: a constitutional anomaly h i tit ti l l having occurred in a parental gamete (e.g. + 21) p g ( (e. 21) will be found in each of the cells of the resulting child (homogeneous trisomy 21) 21).

MOSAIC: MOSAIC: When l Wh only some cells carry th anomaly ll the l whilst others are normal (or carry ( y another anomaly). anomaly). e.g. 1: A non-disjunction (e.g. + 21) non(e. 21) having occurred in the zygote after a few cell divisions: Only some of the embryo divisions: cells (and later, of the child's cells) will carry the anomaly (46, XY/47, XY, +21). 46, XY/47, 21)

NUMERICAL: NUMERICAL: If there is one (or more) chromosome(s) in excess (trisomy) (e.g. +21) (e. 21) or missing (monosomy). (monosomy). STRUCTURAL: STRUCTURAL: If structural changes occur within the chromosomes themselves, not necessarily accompanied by any numerical change. change. - The change is balanced, if there is no loss or gain of genetic material. i f i material. i l - Unbalanced, if there is deletion and/or , duplication of chromosome segment(s). segment(s).


1 ­ Meiotic non-disjunction: non-disjunction:

- N Non di j disjunction i fi t meiotic di i i ti in first i ti division p produces 4 unbalanced gametes. g gametes. -Non disjunction in second division produces 2 unbalanced and 2 normal d b l d d l g gametes. gametes. 2 - Fertilization anomaly. anomaly.

B - MOSAICISM: MOSAICISM: A mosaic individual is made of 2 (or more) cell populations characterised by difference (s) in the chromosomes. These chromosomes. cell populations however come from 1, populations, however, and only 1, zygote (When recording, a mosaic is denoted by a slash between the various clones observed, e.g. trisomy 21 presenting as a mosaic: 46, XY / 47, XY, mosaic: 46, 47, +21). 21)

II - STRUCTURAL ANOMALIES 1 - Reciprocal translocation - A m t al e change bet een terminal mutual exchange between segments from the arms of 2 chromosomes. chromosomes. Provided h h P id d that there i no l is loss or alteration l i at the points of exchange, the new arrangement is genetically balanced, and called a Balanced rearrangement. rearrangement. g - It is recorded as (e.g. t(9;22)(q34;q11)). (e. t(9 22)(q34; 11)).

Complex translocations: translocations: Three, or more b k and more th Th breaks d than two chromosomes can participate in p p exchange, leading to some very complicated rearrangements. rearrangements. The surviving, balanced forms are seen usually as cyclical translocations. The recent translocations. introduction of FISH-painting indicates FISHthat such complex translocations are much more frequent than we have realised. realised.

2 - Robertsonian translocation: -Fusion of 2 acrocentrics very close to the centromeres, most often in the p arms, giving rise to a dicentric chromosome. chromosome. The Th rearranged chromosome i l d th d h includes the long arms of the 2 acrocentrics, while g , most of the short arm material is lost. lost. - Recorded as [e.g. t(14q21q)]. [e. t(14q21q)] q)].

3 - Deletion: Deletion: - Loss of a segment, either interstitial or terminal, f t i l from a chromosome. I chromosome. Invariably, h i bl y, but not always, results in the loss of important genetic material. material.

- Deletion is therefore unbalanced rearrangement. rearrangement.


- Recorded as [e.g. del(5)(q14q34)]; [ [e. del(5)(q14q34)]; ( )] 2 breakpoints are recorded when the deletion is clearly interstitial. interstitial. - Only 1 breakpoint is recorded when the deletion seems terminal. terminal.

4 - Ring: Ring: - A centric ring involves the deletion (often small) of the ends of both arms (incl ding (including the telomeres) and rejoining of the median j g segment to itself in a circular structure. structure. - I an unbalanced rearrangement. Is b l d rearrangement. t - Recorded as (e.g. r(13)(p12q33) ). (e. r(13)(p12q33)

5 - Inversion: Inversion: - Inversion occurs when a segment of g chromosome breaks, and rejoining within the chromosome effectively inverts it. it. - Recorded [e.g. inv(9) (p11q13)]. [e. inv(9 (p11q13)].

5a - Paracentric inversion An inversion is termed paracentric when the segment involved lies wholly within one chromosome arm. arm. 5b - Pericentric inversion An inversion is said pericentric when the two break-points involved are sited on breakopposite sides of the centromere. centromere.

6 - Isochromosome: Isochromosome: - Loss of a complete arm, "replaced" by the d li ti th duplication of th f the other arm th ( q (equivalent to a monosomy y for one arm and trisomy for the other). other). - This is an unbalanced rearrangement. rearrangement. - Recorded as (e.g. i(17q) or i(17)(q10) (e. i(17q) i(17)(q10)

7 - Insertion: Insertion: An interstitial segment of a chromosome g is deleted and transferred to a new position i some other chromosome or iti in th h even somewhere else within the same chromosome. chromosome. Recorded as e.g. ins(2)(p13q31q34). ins(2)(p13q31q34)

8 - Duplication: Duplication: - Direct: A segment of chromosome is Direct: repeated, once or several times, the duplicated segment keeping the same orientation with respect to the centromere ("tandem duplication"). duplication"). - Inverted: Th d li t d segment t k Inverted: The duplicated I t d t takes the opposite orientation. pp orientation. -Recorded as e.g. dup(21)(P13q21). dup(21)(P13q21)

9 - Di t i : Dicentric: Dicentric - A chromosome with 2 centromeres: centromeres: Simplistically, it is the alternative rejoining mode of the reciprocal translocation, but it can originate by several other mechanisms. mechanisms. - Recorded as e.g. dic (13;21)(P20q35). 13;21)(P20q35)

10 - Complex Rearrangements: p g

Involving more than 2 chromosomes and/or more than 3 breakpoints. breakpoints.

11 - Marker: Marker: A non-recognisable, nonpersistent chromosome, recorded as mar. mar. - Variable sized often big element in a sized, big, cancer process. Since FISH-painting process. FISHtechniques have been developed. developed.

12 - Double minute; Homogeneously minute; staining region: region: -Double minute: recorded as DM. Appear as very small, usually paired dots. - Multiple copies indicates a much more i i i i co p e s u o . complex situation.


1- Culture and microscopic examination. examination 2- Banding Banding. 3- Karyotype and idiogram. idiogram


- Chromosomes in metaphase can be identified using certain staining techniques, so called banding. banding. - Cells are cultured and then stopped in Ce s e cu u ed d e s opped metaphase to maximize the number of suitable cells. Th are th spread on a it bl cells. They ll then d slide, stained with a suitable dye and , y visualized in the microscope. microscope.

A band is defined as that part of a chromosome which is clearly distinguishable from its adjacent segments b appearing d k t by i darker or brighter with one or more banding g g techniques. techniques. The chromosomes are visualized as consisting of a continuous series of bright and dark bands. bands.

The banding techniques fall into two principal groups: groups: 1) th those resulting i b d di t ib t d lti in bands distributed along the length of the whole g g chromosome, such as G-, Q- and Rbands. bands. 2) those that stain a restricted number of specific bands or structures. eg C-bands, structures. and nucleolus organizer regions NOR's regions, NOR s (at terminal regions of acrocentric chromosomes). chromosomes).

Karyotype y yp

-A systemized array of the chromosomes of a single cell prepared by photography. photography. - The banding pattern of each chromosome is specific and can be shown I th f the form of stylized ideal k f t li d id l karyotype t known as an idiogram. idiogram. g

Fluorescence In-Situ Hybridization In[FISH]

Fluorescence In-Situ Hybridization is a Inmethod used to identify specific parts of a chromosome. chromosome. For example, if you know the sequence of example, a certain gene, but you don't know on which chromosome the gene is located located, you can use FISH to identify the chromosome in question and the exact location of the gene. Or, if you suspect g gene. Or, y p that there has been a translocation in a chromosome, chromosome you can use a probe that spans the site of breakage/translocation. breakage/translocation.

Here s Here's how it works: works: - M k a probe complementary t th Make b l t to the known sequence. When making the sequence. q g probe, label it with a fluorescent marker, e.g. fluorescein fluorescein, by incorporating nucleotides that have the marker attached to them. Put the chromosomes them. on a microscope slide and denature them. them.

- Denature the probe and add it to the microscope slide, letting the probe hybridize to its complementary site. site. Wash ff the W h off th excess probe and l k at b d look t the chromosomes in a fluorescence microscope. microscope. The probe will show as one or more fluorescent signals in the microscope, depending on how many sites it can hybridize to. to.

- If there has been no translocation at that point, you will see one signal, since the probe hybridizes to one place on the chromosome. chromosome. - If, however, there has been a translocation, o translocation, you will see two signals ill t o signals, since the probe can hybridize to both ends of the translocation point. point.

To use FISH efficiently, y, you have to know what you're looking for, i.e. you usually suspect a particular defect defect, based on the appearance of certain chromosomes, etc. etc.

Multicolour Fluorescence In-Situ Hybridization In[M[M-FISH ]

- One of the most attractive features of FISH is the possibility of simultaneously visualizing multiple targets in different colours. colours. - M-FISH offers the possibility of p y including internal hybridization controls, and it f ilit t t l d facilitates th the comprehensive analysis of clinical p y samples that are short in supply and not easily retrievable. retrievable.

A novel approach has been developed to identify all human chromosomes within one M-FISH experiment. experiment. It is based on measurement of unique spectra for each of the 24 differentiall differentially labeled chromosomes paint probes p p permitting automated spectra-based spectrachromosome classification. classification.



Agarose Gel Electrophoresis Single band


Reverse transcriptase PCR (RT-PCR) (RT-

Southern Blotting

Microarray Assay

Clinical Applications 1. Inborn errors of metabolism: eg.

· · · · · · · · Duchenne muscular dystrophy Myotonic dystrophy Neurofibromatosis Friedreich's F i d i h' ataxia: i G-6-PD Hemophelia B, Sickle cell thalassemia, HB C disease Osteogenesis imperfecta Poly cystic kidney i i


Clinication Applications (cont.)

2. Sex chromosome anomalies:eg.

· · · · · · · · Klinefelter syndrome (47XXY) Kli f l d (47XXY) 47XXY). XXY). Turner syndrome (45X). y (45X). ) AML [eg. M3 t(15,17)] [ M3 t(15 17)] 15, ALL CLL CML [t(9,22) ph] [t(9 22) MDS Lymphoma

3. Hematological malignancies:

Clinication Applications (cont.)

4. Solid tumors: eg.

· · · · · · · · Familial polyposis. Familial breast cancer. Hereditary non-polyposis colon cancer. nonSmall ll l S ll cell lung cancer. Melanoma. Neuroblastoma. Retinoblastoma. Retinoblastoma Wilms' tumor


1. Multiple congenital anomalies. 2. Unexplained mental retardation. 3. Sexual ambiguity or abnormalities in sex development. 4. Infertility. 5. 5 Recurrent miscarriage miscarriage. 6. Unexplained stillbirth. 7. Malignancy and chromosome breakage syndromes.


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