Fluorescent in situ Hybridization FISH Principles and Applications

Fluorescent in situ Hybridization (FISH) Principles and Applications Biotechnology Students Training Course By Dr. Basim Ayesh 1

2 Advantages and Disadvantages of conventional cytogenetic technique n 1. 2. n 1. Advantages Enable the entire genome to be viewed at one time. 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. Disadvantages Detect major structural abnormalities (one band = 6 mb of DNA ~ 150 genes ). 1. Labor intensive and highly dependent upon operator experience and skills.

3 Molecular cytogenetics: Fluorescence in situ Hybridization (FISH) n n 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: q q n 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

4 FISH Identifies chromosomal abnormalities

5 Indications for chromosomal analysis n n n n 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

6 What to do before indication of chromosomal analysis? n Exclude possibility of non-genetic affection q q q n 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)

7 In which conditions we have to indicate FISH analysis? n The material doesn't contain metaphase chromosomes q q n n n 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 q q Microdeletion syndromes Amplification of oncogenes and microdeletion of tumor-suppressor genes in malignancies

8 Tissue samples for FISH analysis n Peripheral blood n Fibroblasts from skin biopsy n Epithelial cells from buccal smear n Bone marrow (hemoblastosis) n Solid tumor biobsies

9 How to take a blood for FISH analysis? n Disinfect the skin with alcohol (70% ethanol) n Use tube with heparin or lithiumheparin to prevent blood clotting (metaphase preparations). n EDTA tube may also be used (Interphase preparations).

10 FISH Procedure n n n n 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

11 TYPES OF FISH PROBES

12 Locus specific probes n n 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 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.

13 Green: Chromosome 13 Red: Chromosome 21 Normal Down syndrome

Buccal Cells from Swab

15 Dual fusion probes n n 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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17 Dual color break-apart probes n n 0. 6– 1. 5 Mb Lowers background as well as identifies translocation events Designed to bind to the intact chromosome flanking the translocation breakpoint. When a translocation occurs, the two probes separate

18 Centromeric probes (CEPs) n n 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. q This combination of CEP and dual color probes comprises a tricolor probe

19 Telomeric probes n n 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.

20 Advantages of Interphase FISH n Interphase cells for FISH do not require culturing of the cells and stimulating division to get metaphase spreads q q n 200– 500 cells can be analyzed microscopically using FISH q n interphase FISH is faster than methods using metaphase cells valuable for analysis of cells that do not divide well in culture, including fixed cells. the sensitivity of detection is higher than that of metaphase procedures, which commonly examine 20 spreads. Monitor recurrent or residual disease in BMT pt.

21 Metaphase FISH n n n 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

22 Spectral karyotyping (SKY) and multiple fluoeescent hybridization (M-FISH) n n 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.

23 SKY Advantages: n n Mapping of chromosomal breakpoints. Detection of subtle translocations. Identification of marker chromosomes, homogeneously staining regions, and double minute chromosomes. Characterization of complex rearrangements. Disadvantages: n n n Very expensive equipments. The technique is labor intensive. Dose not detect structural rearrangements within single chromosome. Low resolution (up to 15 mb ). Specific, not a screening method. a

24 limitations of FISH n n 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 q q n n n 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

25 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 Cryptic Rearrangements of telomeres various Not detected ~100% 1 p 36 deletion 1 p 36. 3 Few ~99% >95% Wolf-Hirschhorn 4 p 16. 3 Most ~99% >95% Cri-du-chat 5 p 15. 2 Most ~99% >95% Williams-Beuren 7 q 11. 2 Almost none ~99% Not detected Prader-Willi 15 q 11 -q 13 Unreliable ~70% Not detected Angelman 15 q 11 -q 13 Unreliable ~70% Not detected Miller-Dieker lissencephaly 17 p 13. 3 Few >90% Smith-Magenis 17 p 11. 2 Some >95% Not detected Velocardiofacial/Di. Georage 1 22 q 11. 2 Rarely >95% Not detected Some detected

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28 Trisomy 21 n n n 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 21 q 22. 3 region appears to contain the gene(s) responsible for the congenital heart disease observed in Down syndrome.

29 Trisomy 13 n n n 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 13 q 14 -13 q 32 with variable expression, gene interactions, or interchromosomal effects.

30 Trisomy 18 n n 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.

31 X-Y related syndromes n Turner syndrome: q n Metafemales or triple-X females: q n Inherit three X chromosomes; their genotype is XXX or more rarely XXXX or XXXXX. Klinefelter syndrome: q n Occurs when females inherit only one X chromosome; their genotype is X 0. Males inherit one or more extra X chromosomes; their genotype is XXY or more rarely XXXY, XXXXY, or XY/XXY mosaic. XYY syndrome: q Males inherit an extra Y chromosome; their genotype is XYY.

32 n Poseidon™ Repeat Free™ Chromosome 13/21, X/Y/18 specific DNA Probes Vial 1 q Critical region 1 (red): n q Critical region 2 (green): n n The 21 q specific DNA probe is direct-labeled with Platinum. Bright 550. The 13 q 14 specific DNA probe is direct-labeled with Platinum. Bright 495. Vial 2 q Critical region 3 (blue): n q Critical region 4 (green): n q The 18 SE DNA probe is direct-labeled with Platinum. Bright 415. The X SE DNA probe is direct-labeled with Platinum. Bright 495. Critical region 5 (red): n The Y SE DNA probe is direct-labeled with Platinum. Bright 550.

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34 Intended use n n 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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36 Chromosome 13/21 specific probe n A dual-color assay to detect gains of chromosome 21 and 13. q Trisomy 21 (3 R 2 G) n n q Trisomy 13 (2 R 3 G) n n q 3 red signal at the 21 q 22 region 2 green signals for chromosome 13 3 green signals at the 13 q 14 region 2 red signals for chromosome 21 Normal 13, 21 (2 R 2 G) n 2 single color red (R) and green (G)

37 Chromosome X/Y/18 specific probe n n n n n 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