Biotechnology Students Training Course Biotechnology Students Training Course

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Advantages and Disadvantages of conventional cytogenetic technique

Advantages1. Enable the entire genome to be viewed at one time.

2. Suitable when a specific anomaly is suspected ( e.g. Philadelphia in CML ) and as a general diagnostic tool to detect additional chr. Abnormalities commonly seen in disease progression of CML.

Disadvantages1. Detect major structural abnormalities

(one band = 6mb of DNA ~ 150 genes ).

1. Labor intensive and highly dependent upon operator experience and skills.

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Molecular cytogenetics:Fluorescence in situ Hybridization (FISH) A process which distinctly paints and detects

RNA as well as DNA Structures, numbers and location in place in the cell or in situ.

FISH may be used with: Morphologically preserved chromosome preparations

(Metaphase). Fixed cells or tissue sections (Interphase)

Aids in gene mapping, toxicological studies, analysis of chromosome structural aberrations, and ploidy determination

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FISH Identifies chromosomal abnormalities

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Indications for chromosomal analysis

Suspicion to concrete chromosomal abnormality (concrete syndrome)

Multiple congenital anomalies or developmental delay

Mental retardation Gonadal dysgenesis Infertility Miscarriages Delivery of dead fetus or death of a newborn child Occurrence of certain malignancies

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What to do before indication of chromosomal analysis?

Exclude possibility of non-genetic affection Influence of teratogens or prenatal infections Complications during the birth (asphyxia, injuries of

the newborn child) Postnatal non genetic influence (injuries,

infections)

Exclude monogenic or multifactorial disorder (= without abnormal chromosomal finding)

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In which conditions we have to indicate FISH analysis?

The material doesn't contain metaphase chromosomes Unsuccessful cultivation It isn't possible to cultivate the tissue from patient (preimplantation

analysis, rapid prenatal examinations, examinations of solid tumors or autopsy material)

Analysis of complicated chromosomal rearrangements Identification of marker chromosomes Diagnosis of submicroscopic (cryptic) chromosomal

rearrangements Microdeletion syndromes Amplification of oncogenes and microdeletion of tumor-suppressor

genes in malignancies

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Tissue samples for FISH analysis

Peripheral blood

Fibroblasts from skin biopsy

Epithelial cells from buccal smear

Bone marrow (hemoblastosis)

Solid tumor biobsies

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How to take a blood for FISH analysis?

Disinfect the skin with alcohol

(70% ethanol)

Use tube with heparin or lithium-

heparin to prevent blood clotting

(metaphase preparations).

EDTA tube may also be used

(Interphase preparations).

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FISH Procedure Slides preparation from cultured (Metaphase) or

uncultured (Interphase) cells and fixed sing standard cytogenetic prosedure.

Fixed cells are exposed to a probe (60-200–kb fragment of DNA attached covalently to a fluorescent molecule).

Denature the chromosomes Denature the probe Hybridization: The probe will hybridize or bind to its

complementary sequences in the cellular DNA Fluorescence staining The bound probe can be visualized under a fluorescent

microscope in the nucleus of the cell

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Locus specific probes

A deletion or duplication of the region that is hybridized to the probe will result in a nucleus with only one signal or more than two signals, respectively.

Multiple probes spanning large regions are used to detect regional deletions.

Probes are designed to be specific to a particular chromosome or chromosomal regions

A probe to any unique region on a chromosome should yield an image of two signals per nucleus

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NormalNormal Down syndromeDown syndrome

GreenGreen: Chromosome 13: Chromosome 13RedRed: Chromosome 21: Chromosome 21

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Buccal Cells from Swab

Dual fusion probes

Sometimes called dual-colored probes

0.8–1.5 Mb in size Designed to bind to regions

spanning the breakpoint of both translocation partners.

A translocation will be observed as a signal from both the translocation junction and the reciprocal of the translocation junction; e.g., t(9;22) and t(22;9)

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Dual color break-apart probes

Designed to bind to the intact chromosome flanking the translocation breakpoint.

When a translocation occurs, the two probes separate

0.6–1.5 Mb Lowers background as well as identifies translocation

events

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Centromeric probes (CEPs)

Designed to hybridize to highly repetitive alpha satellite sequences surrounding centromeres.

These probes detect aneuploidy of any chromosome.

Combinations of centromeric probes and region-specific probes are often used to confirm deletions or amplifications in specific chromosomes.

Addition of a CEP to dual color probes serves as a control for amplification or loss of one of the chromosomes involved in the translocation. This combination of CEP and dual color probes

comprises a tricolor probe

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Telomeric probes

DNA probes specific to the telomeres of all human chromosomes.

Useful for the detection of chromosome structural abnormalities such as cryptic translocations or small deletions that are not easily visualized by standard karyotyping.

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Advantages of Interphase FISH Interphase cells for FISH do not require culturing

of the cells and stimulating division to get metaphase spreads interphase FISH is faster than methods using

metaphase cells valuable for analysis of cells that do not divide well in

culture, including fixed cells. 200–500 cells can be analyzed microscopically

using FISH the sensitivity of detection is higher than that of

metaphase procedures, which commonly examine 20 spreads.

Monitor recurrent or residual disease in BMT pt.

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Metaphase FISH Uses fluorescent probes that bind

to metaphase chromosomal regions or to whole chromosomes.

Whole chromosome paints: Probes that cover the entire chromosome, are valuable for detecting small rearrangements that are not apparent by regular chromosome banding.

Telomeric and centromeric probes are also applied to metaphase chromosomes to detect aneuploidy and structural abnormalities

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Spectral karyotyping (SKY) and multiple fluoeescent hybridization (M-FISH) By mixing combinations of

five fluors and using special imaging software, can distinguish all 23 chromosomes by chromosome specific colors.

This type of analysis can be used to detect abnormalities that affect multiple chromosomes as is sometimes found in cancer cells or immortalized cell lines.

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SKY

Advantages: Mapping of chromosomal breakpoints. Detection of subtle translocations. Identification of marker chromosomes, homogeneously

staining regions,and double minute chromosomes. Characterization of complex rearrangements.Disadvantages: Very expensive equipments. The technique is labor intensive. Dose not detect structural rearrangements within a

single chromosome. Low resolution (up to 15 mb ). Specific, not a screening method.

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limitations of FISH

The inability to identify chromosomal changes other than those at the specific binding region of the probe.

Preparation of the sample is critical in interphase FISH analysis to permeabilize the cells for optimal probe target interaction to maintain cell morphology.

Cannot detect small mutations. Miss Uniparental disomy. Miss Inversions. Probes are not yet commercially available for all

chromosomal regions Relativelly expensive

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Diagnostic Potential For Karyotype and FISH For Selected Disorders

Condition Locus studied

Karyotype Disease specific FISH

Telomere FISH

Aneuploidy various ~100% Not detected Detected by karyotype

Large deletions, large dupllications, translocation of large segments

various ~100% Not detected Detected by karyotype

CrypticRearrangements of telomeres

various Notdetected

Not detected ~100%

1p36 deletion 1p36.3 Few ~99% >95%

Wolf-Hirschhorn 4p16.3 Most ~99% >95%

Cri-du-chat 5p15.2 Most ~99% >95%

Williams-Beuren 7q11.2 Almost none

~99% Not detected

Prader-Willi 15q11-q13

Unreliable ~70% Not detected

Angelman 15q11-q13

Unreliable ~70% Not detected

Miller-Dieker lissencephaly 17p13.3 Few >90% Some detected

Smith-Magenis 17p11.2 Some >95% Not detected

Velocardiofacial/DiGeorage 1 22q11.2 Rarely >95% Not detected

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Trisomy 21

One of the most common chromosomal abnormalities in live born children

Causes Down syndrome, a particular combination of phenotypic features that includes mental retardation and characteristic facies (distinctive facial expressions associated with the condition).

Molecular analysis has revealed that the 21q22.1-q22.3 region appears to contain the gene(s) responsible for the congenital heart disease observed in Down syndrome.

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Trisomy 13

Also called Patau syndrome Is a chromosomal condition that is associated

with severe mental retardation and certain physical abnormalities.

The critical region has been reported to include 13q14-13q32 with variable expression, gene interactions, or interchromosomal effects.

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Trisomy 18

Causing Edwards syndrome is the second most common autosomal trisomy after trisomy 21.

Characterized by severe psychomotor and growth retardation, microcephaly, microphthalmia, malformed ears, micrognathia or retrognathia, microstomia, distinctively clenched fingers, and other congenital malformations.

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X-Y related syndromes

Turner syndrome: Occurs when females inherit only one X chromosome; their

genotype is X0.

Metafemales or triple-X females: Inherit three X chromosomes; their genotype is XXX or more rarely

XXXX or XXXXX.

Klinefelter syndrome: Males inherit one or more extra X chromosomes; their genotype is

XXY or more rarely XXXY, XXXXY, or XY/XXY mosaic.

XYY syndrome: Males inherit an extra Y chromosome; their genotype is XYY.

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Poseidon™ Repeat Free™ Chromosome 13/21, X/Y/18 specific DNA Probes Vial 1

Critical region 1 (red): The 21q specific DNA probe is direct-labeled with PlatinumBright550.

Critical region 2 (green): The 13q14 specific DNA probe is direct-labeled with

PlatinumBright495.

Vial 2 Critical region 3 (blue):

The 18 SE DNA probe is direct-labeled with PlatinumBright415.

Critical region 4 (green): The X SE DNA probe is direct-labeled with PlatinumBright495.

Critical region 5 (red): The Y SE DNA probe is direct-labeled with PlatinumBright550.

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Intended use

The probes are optimized to detect copy numbers of chromosome 21, 13, 18, X and Y

To be used with cultured or uncultured amniotic or WBCs slide preparations.

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Chromosome 13/21 specific probe

A dual-color assay to detect gains of chromosome 21 and 13. Trisomy 21 (3R2G)

3 red signal at the 21q22 region 2 green signals for chromosome 13

Trisomy 13 (2R3G) 3 green signals at the 13q14 region 2 red signals for chromosome 21

Normal 13, 21 (2R2G) 2 single color red (R) and green (G)

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Chromosome X/Y/18 specific probe

A triple-colour assay to detect gains or losses of chromosome X, Y and or 18.

Turner syndrome one green signal only at Xcen. Meta-Females (or Triple-X females) 3 or more green

signals at Xcen. Klinefelter 2 or more green and 1 red signal. XYY males one green and two red signals. normal X 2 single green signals Normal male one green and one red signal Trisomy 18 3 blue signals at 18 cen. normal chromosome 18 2 single blue signals

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