1. Molecular cytogenetic practical
FISH method

2. Two kinds of cytogenetic examination:
Basic chromosomal analysis
staining methods (solid staining, G-banding etc.)
Based on analysis of metaphase chromosomes
Molecular cytogenetic analysis
Identification of chromosomal abnormalities using molecular biological methods

3. Basic chromosomal analysis
Patient
Basic chromosomal analysis
Family of the patient
Molecular cytogenetic analysis
Molecular biological analysis

4. Molecular cytogenetic examinations
In most of cases interphase cells could be used for analysis (with exception of whole chromosome painting probes and M-FISH)
Examples of methods:
in situ hybridization and its modifications (CGH, M-FISH, fiber FISH atd.)
Gene chips, resp. array CGH, DNA microarray etc.
PRINS, PCR in situ
quantitative fluorescent PCR, real time PCR
methods based on amplification of probe attached to target sequence (MLPA, MAPH)
hybridization
PCR

5. Written test 20 questions 20 minutes
In some multiple-choice questions more than one answer could be correct.

6. Hybridization
target DNA
denaturation
hybridization
probe

7. Hybridization Molecular (on isolated DNA)
In situ (on biological structures – i.e. interphase nuclei, metaphase chromosomes, cells or tissues)

8. Probe
A part of DNA (or RNA) that is complementary to certain sequence on target DNA (i.e. DNA of the patient)
Plasmid, phage DNA, cosmid (or combination of phage and plasmid DNA), YAC
PCR-product (amplification of certain segment of chromosomal DNA)

9. Labeling of probes Radioactive Enzymatic Fluorescent
Fluorescent in situ hybridization (FISH)

10. FISH

11. Types of probes Centromeric (satellite) probes Locus specific probes
Whole chromosome painting probes

12. 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
Analysis of low-frequency mosaic
Diagnosis of submicroscopic (cryptic) chromosomal rearrangements
Microdeletion syndromes
Amplification of oncogenes and microdeletion of tumor-suppressor genes in malignancies

13. Fluorescently labeled nucleotides
PRINS
= Primed in situ labeling
Fluorescently labeled nucleotides

14. Quantitative fluorescent PCR QF-PCR
In case of informative polymorphism each peak represents one locus in one chromosome.
denaturation, annealing
PCR
Capillary electrophoresis

15. QF-PCR – normal finding

16. QF-PCR – identification of trisomy

17. Presentations
mFISH
CGH

18. FISH gallery and practical tasks

19. Satellite (centromeric) probe – chromosome 7

20. Satellite (centromeric) probe on X–chromosome
45,X or 46,XY
Possible karyotype?

21. X- and Y-centromeric probes
Green = X
Red = Y
46,XY
Determine probable karyotype.

22. Hybridization of interphase nuclei with X-centromeric probe
What will be the most possible chromosomal finding (or findings)?
Mosaic karyotype
45,X/46,XX
46,XY/46,XX
47,XXY/46,XY
45,X/47,XXY

23. Marker is a derivative X chromosome; Possible karyotype: 47,XX,der(X)
X-centromeric probe – identification of small supernumerary chromosome (marker chromosome) X
How you would conclude this FISH finding? X
Marker is a derivative X chromosome;
Possible karyotype: 47,XX,der(X)
mar

24. Hybridization with a telomeric probe

25. Lokus specific probe – detection of SRY region

26. Combination of locus-specific probe with a centromeric one
Green = X-centromere
Red = SRY
Normal male - 46,XY;
SRY is not deleted.
Possible karyotype?

27. Locus specific probe – examination of chromosome 22q11.2 microdeletion
Red signals = HIRA region on 22q11.2
Green signals = control probe on ARSA region (subtelomeric part of 22q)
Is it possible to confirm microdeletion?

28. Microdeletion confirmed (loss of one red signal)
Deleted chromosome – red signal absent
Red signal –TUPLE1 (HIRA) locus
Green signal –ARSA locus (control probe)
normal chromosome – red signal on HIRA locus is present
Microdeletion 22q11.2 is associated with DiGeorge syndrome or velocardiocacial syndrome.

29. „antimongoloid“ slant of eyelids
DiGeorge syndrome
„antimongoloid“ slant of eyelids
hypertelorism
low set dysplastic ear
micromandibula
Inborn cardiac defect (e.g. tetralogy of Fallot), thymic hypoplasia (or aplasia).

30. Tetralogy of Fallot
Left normal heart, right heart of the patient with the tetralogy of Fallot
Tetralogy of Fallot – combination of 4 different inborn cardiac defects

31. Amplification confirmed
Locus specific probes – examination of oncogene HER2/NEU amplification (red signals)
Normal finding
Amplification confirmed

32. Painting probes – examination of chromosomes 1, 4 and 8
Normal finding

33. Painting probes – detection of unbalanced translocation of 11 and 21 chromosomes

34. Patient 1

35. See the photo of the patient and note abnormal phenotypic features.
2-years old boy with mental retardation
Inborn cardiac defect – supravalvular aortic stenosis.
See the photo of the patient and note abnormal phenotypic features.

36. Patient 1 (boy, 2 years) irides stellatae hypertelorism low set ears
abnormal teeth
open mouth, thick lip
„elfin face“

37. Patient 1
Phenotypic features and inborn defects are typical for Williams-Beuren syndrome
This syndrome is caused by microdeletion of the long arm of the chromosome 7 (sub-band 7q11.23).
In 95% of patients this microdeletion is identified by means of the FISH method.
Before the molecular cytogenetic analysis basic cytogenetic examination is recommended.
Which type of probe you would use for FISH analysis of microdeletion of the chromosome 7?

38. Microdeletion should be confirmed by the FISH analysis
Patient 1 - karyotype
Normal finding:
46,XY
Microdeletion should be confirmed by the FISH analysis
Which kind of probe you would use for FISH analysis of microdeletion of the chromosome 7?

39. MOLECULAR CYTOGENETIC ANALYSIS OF 7q11.23 MICRODELETION
ELN
LIMK1
D7S613
CENTROMERE
TELOMERE
180 kb
LOCUS SPECIFIC PROBE FOR THE CRITICAL REGION ELN/LIMK/D7S613
(labeled with the Spectrum Orange, red signal)
CONTROL PROBE D7S522
(labeled with the Spectrum Green, green signal)

40. Patient 1 – conclusion of the molecular cytogenetic examination
Microdeletion of 7q11.23 chromosome confirmed.
Diagnosis: Williams-Beuren syndrome

41. Prognosis of patients with the Williams-Beuren syndrome
Neonatal hypercalcemia
Mild to moderate mental retardation
Supravalvular aortic stenosis could lead to a heart attack already in childhood (sudden death of the child).

42. Good bye!