Molecular cytogenetic practical

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

4. hybridization PCR 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)

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. PRINS • = Primed in situ labeling Fluorescently labeled nucleotides

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

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. 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. 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. 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 4 1 4 8 1 8 Normal finding

33. t(11;21) Painting probes – detection of unbalanced translocation of 11 and 21 chromosomes

34. Patient 1

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

39. 180 kb ELN LIMK1 D7S613 CENTROMERE TELOMERE MOLECULAR CYTOGENETIC ANALYSIS OF 7q11.23 MICRODELETION • 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 aorticstenosis could lead to a heart attack already in childhood (sudden death of the child).

42. Good bye!