1. Cytogenetic and molecular cytogenetic analysis in clinical genetics
5th year
Clinical genetics

2. Eduard Kočárek eduard.kocarek@lfmotol.cuni.cz
Phone (office):
Phone (Plzenska):
Secretary of the Institute of biology and medical genetics
phone – 502

3. Basic cytogenetic examinations
Interphase cells
Barr body (sex chromatin)
Metaphase cells – staining of chromosomes
Solid staining
G-banding
R-banding
C-banding
Q-banding
Ag-NOR

4. Molecular cytogenetic examinations
Identification of chromosomal abnormalities by means of molecular biological methods
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 etc.)
Gene chips, resp. array CGH 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. FISH (fluorescent in situ hybridization)
Locus specific probes
Centromeric probe
target DNA
denaturation
hybridization
probe
M-FISH
Whole chromosome painting probes

6. Quantitative fluorescent PCR QF-PCR
Primer 1
In case of informative polymorphism each peak represents one locus on one chromosome.
denaturation, annealing
Primer 2
PCR
Capillary electrophoresis

7. QF-PCR – normal finding

8. QF-PCR – identification of trisomy

9. MLPA Multilocus ligase-dependent probe amplification P
„stuffer“ sequence
Hybridization sequence
Target DNA P

10. MLPA

11. MLPA – computer evaluation

12. MLPA result – deletions in the dystrophine gene

13. DNA attached to the membrane
MAPH
Multilocus amplifiable probe hybridization
DNA attached to the membrane
Mixture of probes
DNA sample
hybridization
Probe 1
Probe 2
primers
primers
Capillary electrophoresis
amplification of attached probes

14. Indications of cytogenetic and molecular cytogenetic examinations

15. Basic cytogenetic chromosomal analysis
Patient
Basic cytogenetic chromosomal analysis
Molecular cytogenetic analysis (mostly FISH)
Molecular biological analysis

16. Indications for postnatal 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

17. What to do before indication of postnatal 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)

18. Tissue samples for postnatal chromosomal analysis
Peripheral blood
Fibroblasts from skin biopsy
Epithelial cells from buccal smear (only in rare cases for Barr body identification or FISH)
Bone marrow (hemoblastosis)
Solid tumor
Autopsy material (in case of the death of a patient)

19. How to take a blood sample for chromosomal analysis?
Disinfect the skin with alcohol (96% ethanol)
Use tube with heparin or lithium-heparin to prevent blood clotting.
(The EDTA tube is used only for the DNA isolation.)

20. 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

21. Indication Result INDICATION Various samples RESULT Metaphase cells
Using molecular cytogenetic analysis the aberration was discovered in other family members
in vitro cultivation is possible
In vitro cultivation
is impossible
Metaphase cells
(cultivated lymphocytes, amniocytes etc.)
Interphase cells (e.g. tumor cells, tissues taken from autopsy material or histological sections)
Various samples
Chromosomal analysis
Negative finding, detailed examination is necessary
(e.g. microdeletions)
Chromosomal abnormality remains unclear
(e.g. marker chromosomes, rearrangements including 3 and more chromosomes, mosaics)
Chromosomal aberration is found and described
(e.g. aneuploidies, simple structural rearrangements)
Molecular cytogenetic analysis
RESULT
Result

22. Basic cytogenetic chromosomal analysis
Patient
Basic cytogenetic chromosomal analysis
Molecular cytogenetic analysis (mostly FISH)
Molecular biological analysis

23. In which cases we have to indicate detailed molecular biological analysis?
Neither chromosomal nor molecular cytogenetic analysis led to relevant conclusion
Suspicion to monogenic disorder (e.g. fragile X syndrome)
Some cases of microdeletion syndromes could be associated with very short deletions or spot mutations that can't be identified by means of common molecular cytogenetic methods.
Identification of uniparental disomy, or other imprinting defects (especially in Prader-Willi/Angelman syndromes).

24. Patient 1

25. 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.

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

27. 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 could be examined by 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?

28. Microdeletion should be confirmed by the FISH analysis
Patient 1 - karyotype
Normal finding:
46,XY
Microdeletion should be confirmed by the FISH analysis

29. 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)

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

31. 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).

32. Thank you for your attention and good bye!