1. Régen DROUIN, Geneticist
Fluorescence in situ hybridization (FISH) and the various types of FISH.
Régen DROUIN, Geneticist
MD, PhD, FACMG, FCCMG
Department of Medical Genetics, CHUS
& Department de Pediatrics,
Université de Sherbrooke, Sherbrooke, Quebec, Canada

2. Cytogenetics: - Chromosome Cytogenetics - Interphase Cytogenetics
- Conventional Cytogenetics
- Molecular Cytogenetics
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

3. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

4. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

5. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

6. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

7. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

8. Molecular Cytogenetic Techniques available:
FISH (Fluorescence In Situ Hybridization)
& variants: Q-FISH, express FISH, etc.
- PRINS (PRimed IN Situ labeling)
M-FISH (Multicolor-FISH) or SKY
(spectral Karyotype)
Band-FISH
- CGH (Comparative Genomic Hybridization)
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

9. Applications of Molecular cytogenetics
 Chromosome Identification
 Aneuploidy Detection
 Centromere Analysis
 Identification of Marker Chromosome
 Whole Chromosome Analysis (chromosome painting)
 Analysis of chromosome translocation
Detection of unique sequence (single-copy sequence)
Microdeletion investigation
Analysis of gene amplification
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

10. FISH Fluorescence in situ hybridization
Hybridation in situ avec visualisation en fluorescence
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

11. GCAATCGCCAATTATTCCAGGACTGGG CGTTAGCGGTTAATAAGCTCCTGACCC
Double-strand DNA
Denatured DNA
Single-strand DNA
Hybridization

12. Fluorescence In Situ Hybridization and Molecular Cytogenetics
1. Introduction
History:
ISH: John (1969), radioisotope probes hybridized to cell preparations and using autoradiography to detect the hybridization of the probes.
FISH: Pinkel (1986), Immunofluorescence technique - safety, rapidity, low background… Afterwards, so many techniques derived from FISH have been developed, e.g. CGH, SKY, M-FISH, fiber- FISH, PRINS…
Application: Gene mapping, detecting chromosome and gene changes…
Metaphase chromosomes---from all types of cytogenetic preparations;
Interphase cells---from above plus cytomorphological preparations;
Tissue sections---from tissue biopsy slides
Principles: See Fig.
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

13. Fluorescence: 2. Fluorescence and fluorescence microscope
Electromagnetic spectrum: UV (<400 nm, invisible) violet, blue, green,
yellow, orange and red ( nm, visible) infrared (>700 nm, invisible).
Energy increase with the wave length decrease.
Fluorescence:
Fluorescence,
Long wave light
UV or short
wave light
Some fluorochromes (dyes)
FITC (Fluorescein isothiocyanate)
Rhodamine
Texas red
DAPI (4’6-diamidino-2-phenylindole)
PI (Propidium Iodide)
heat
electron
Excitation (absorb energy)
Illumination (release)
Fig.: The principle of fluorochromes
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

14. Specimen on microscope slide
Fluorescence microscope:
Light source: High-pressure mercury vapor lamps, tungsten-halogen lamps, or xenon lamps.
Eye or Camera
Filters: 1. Exciting filter, to let a certain
wave length of light pass so that can excite the given fluorochrome
carried on sample.
2. Barrier filter, to allow the visible light pass so that the fluorescence can be seen by eyes or the image can be captured.
Transmitted and epi-illumination
Special requirements: No auto-fluorescence in any part of light path except for samples
Specimen on microscope slide

15. 3. Probes---a specific DNA fragment, usually 1 to 100 kb
length, complementary to the chromosome site
that we are interested in.
Probe Labeling: a). Indirect labeling, need antibodies to complete FISH procedure Haptens---Biotin-dUTP, digoxigenin-dUTP,
b). Direct labeling, the probe directly labeled with fluorochromes such as SpectralGreen and SpectralOrange One-step hybridization.
Labeling techniques: a). Nick translation
b). Random priming
c). PCR (Polymerase chain reaction)
DNase I makes nicks
DNA Polymerase adds dNTP, labeled dUTP at 3’ and remove dNTP at 5’ 5‘ 3‘ 5‘ 3‘
+※++※+
++※+++ 3‘ 5‘ 3‘
+※++※ 5‘
Fig.: Principle of Nick Translation
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

16. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

17. 4. Hybridization a). Denaturing of DNA probes
b). Denaturing of DNA template (chromosome)
c). Annealing (renature, hybridization)
d). Post-hybridization wash, stringency control
Factors
Level
Stringency
Results
(if inappropriate)
Temperature
High
Low
Low efficiency
High background
Concentration of salt
(SSC) solution
Concentration of formamide solution
e). Counterstaining
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

18. 4. Hybridization a). Denaturing of DNA probes
b). Denaturing of DNA template (chromosome)
c). Annealing (renature, hybridization)
d). Post-hybridization wash, stringency control
e). Counterstaining
Probe color(s)
Counterstain should be used
FITC (Green)
PI (Red)
Rhodamine or Texas Red (Red)
DAPI (Blue)
Mixed probe with multicolor
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

19. FISH targets : - Metaphase Chromosomes - Interphase Nuclei
- Fixed Tissues
- Cells in culture
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

20. DNA Condensation and fiber-FISH
1 400nm
Condensed section of chromosome
Extended section of chromosome
Chromatin fiber of packed nucleosomes
‘String-of-beads’
form of chromatin
DNA double helix
700nm
300nm
30nm
10nm
2 nm
DNA Condensation and fiber-FISH

21. A good FISH method should have:
- An extremely high specificity (extremely low background)
- A good sensitivity (good hybridization efficiency)
- Unambiguous recognition of the hybridization signal
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

22. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

23. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

24. Genetic diseases identified using molecular cytogenetics
Prader-Willi Syndrome
Angelman Syndrome
Miller-Dieker Syndrome
Williams Syndrome de Williams
DiGeorge and velo-cardio-facial Syndromes
Wolf-Hirschhorn Syndrome
Smith-Magenis Syndrome
Kallmann Syndrome
etc...
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

25. Di George
normal
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

26. Di George
normal
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

27. Deletion of one Di George locus
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

28. Prader-Willi Syndrome
(del.15q11-q13 pat ou DUP mat)
 Mental Retardation and behavior problems
 Deglutition problems and hypotonic newborn
 Bulimia presented by older children (obesity)
 Hypogonadism and incomplete puberty
 Acromicry
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

29. Angelman Syndrome (del.15q11-q13 mat ou DUP pat)
 Severe Mental Retardation
 Episodes of uncontrolled laughing
 Characteristic Facial Dysmorphism (low jaw and protruding tongue)
 Special behavior with disorganized movements (ataxic gait)
 Convulsions
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

30. PWS 15q22 control PML SNRPN 15q11q13
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

31. PWS del (15) (q11q13) 15q22 SNRPN control 15q11q13 PML
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

32. DEFINITION OF CRYPTIC CHROMOSOME REARRANGEMENTS
These are chromosomal anomalies not visible using standard high resolution cytogenetic technique (  550 bands per haploid genome). These anomalies are detectable only when using molecular cytogenetic techniques.
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

33. Pedigree Normal Deceased MDS ? Normal Current pregnancy (MDS)
Miller-Dieker Syndrome
Spontaneous Abortion
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada
Deceased MDS

34. Father
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

35. Father
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

36. Father of the propositus
FISH (Oncor)
MDS Probes 17p13.3 et RARA CR 17q21.1 16 17 17
Father of the propositus
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

37. PRimed IN Situ labeling
PRINS
PRimed IN Situ labeling
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

38. Incomplete Replication
Telomere
Simple DNA sequence (T2AG3) tandemly repeated, of variable length, located at the extremities of the chromosomes.
Telomeres are essential elements that protect the extremities of the chromosomes from degradation and ligation.
Shortening
Elongation
Equilibrium
Incomplete Replication
Addition of repetitions T2AG3 by the telomerase
Nuclease Activity
Senescence

39. Telomeres Specialized structures made of DNA and PROTEINS
Repeated DNA sequence: 2 à 15 kb
TTAGGG
AATCCC
Maintain the chromosome stability
Around 30 to 120 bp are lost per somatic cell division
Too short : cellular senescence and genetic instability
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

40. Measurement of telomeres
Average length of telomeres :
Measurement of terminal restriction fragments.
Digestion using restriction enzymes of purified DNA
Visualization and measurements of telomeric fragments by Southern blot
Cleavage of telomeres at variable distance
No information individual telomeres
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

41. Measurement of telomeres
Length of individual telomeres :
Quantitative FISH (Q-FISH)
Hybridization telomeric PNA probes
Measurements of the signal intensity
Length Profil of individual telomeres
Variation of hybridization efficiency ???
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

44. 1. Measurement of 1q telomere
100kb/29 m
100kb/19.5 m
100kb/14.1 m
2.9 m
2.1 m
1.5 m

47. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

49. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

50. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

51. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

52. A B C

53. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

54. The multi-color Karyotype
This is a technique that allows simultaneous identification and analysis of all chromosomes by attributing to each pair of autosomes, to the X and Y chromosomes, a specific and distinct color.
There are 2 methods:
Multiplex-Fluorescent In Situ Hybridization (M-FISH)
(Speicher et al., 1996)
SKY (Spectral Karyotyping)
(Schröck et al., 1996)
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

55. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

56. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

57. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

58. M-FISH
Sequential Acquisition of images corresponding to each fluorochrome, using 5 optic filters specific for the different fluorochromes.
FITC
TexasRed
DEAC 15 9 X
SpectrumOrange
Cy 5
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

59. SKY
The main part of the system: an optic head composed of an interferometer that measures the fluorescence spectrum and a CCD camera for the imaging.
The emission spectrum of the fluorescence can be simultaneously recorded for each point of the image.
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

60. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

61. Cell lines from a colorectal adenocarcinoma3 12 19 17 X
del(X)
ins(3;12) 4
del(4) 8
iso(13q)
der(18)
der(19)t(17;19)
der(8)
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

62. der(1)t(1;5) der(2)t(1;2) der(3)t(X;3) der(5)t(5;13) der(6)t(6;14)9 15 12 7
der(1)t(1;5)
B6-15
B6-1, B6-15
der(2)t(1;2)
der(3)t(X;3)
HT29, C7-1, C7-15
der(5)t(5;13)
der(6)t(6;14)
der(6)t(X;6;9)
ins(X;9)
HT29
C7-15
der(9)t(X;6;9)
der(12)t(7;12)
HT29, C7-1
der(13)t(5;13)
der(14)t(6;14)
C7-1, C7-15
dup(19p)

63. Representative Karyotype of the cell line
del(3p)
del(4)
der(5)t(5;13)
del(Xp)
der(6)t(6;14)
del(7p)
der(8)
ins(9;X)
iso(13q)
der(13)t(5;13)
der(14)t(6;14)
der(18)
dup(19p)
der(19)t(17;19) 13 14 15 16 17 18 19 20 21 22

65. m-FISH Karyotype
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

66. m-FISH Karyotype (False Colors)
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

67. Chromosome Painting 19 5 ? Painting 19 red & 5 green 8 m-FISH
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

68. inv(2)(q23;q34)
der(2)t(2;12)(q35;q12)
der(1)t(1;9)(q21;q12)
del(3)(q10)
i(3)(q10)
der(5)t(5;20)(q15;p12)
der(7)t(7;13)(q31;q12)
der(7)t(inv7;14)[(q21;q34)(q22;q24)]
der(9)t(1;9)(q21;q12)
der(10)t(3;10;12)(3qter3q21::12?::10p1510q26::10q2410qter)
der(9)t(8;9)(q12;p13)
i(12)(p11)
der(19)t(5;8;19)(19pter19q13.1::8?::19q13.219q13.3::5q345qter)
der(20)t(5;20)(q15;p12)
der(14)t(6;14)(q11.1;p11.1)
der(19)t(8;19)(?;p11)
del(18)(?)
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

69. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

70. Comparative Genomic Hybridization protocols Preparation of Metaphases
Preparation of Genomic DNA
Preparation of Metaphases
DNA labeling
Preparations
Hybridization
DNA detection
Image Capture
Image Treatment
Image Analysis

71. Genomic DNA of the tumor Metaphase Preparations
Normal Genomic DNA
Genomic DNA of the tumor
Hybridization
Metaphase Preparations
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

72. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

73. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

74. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

75. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

76. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

77. Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada

78. Team of Dr Régen DROUIN Cytogenetics Molecular Genetics Walid DRIDI
Macoura GADJI
Kada KRABCHI
Josée LAVOIE
Sandrine LACOSTE
Stéphane OUELLET
Patrick ROCHETTE
François VIGNEAULT
Éric BOUCHARD
Marc BRONSARD
Nathalie BASTIEN
Mélissa FERLAND
Isabelle PARADIS
Ju YAN
Department of Medical Genetics, CHUS & Department of Pediatrics, Université de Sherbrooke, Sherbrooke, Quebec, Canada