aaplications of molecular cytogenetics.ppt

Sharing an important presentation

1. Applications of Molecular
Cytogenetics
Dr Mohammed Alqahtani
CSLT(CG), CLSp(CG), RT,MBA, Ph.D
Genomic Medicine Unit Founder & Director
Center of Excellence in Genomic Medicine Research
Founder & Director

2. High Resolution Banding and FISH
Control Signals
Region-Specific Signal
The chromosome banding technique performed 20 years ago missed the
small deletion. High resolution banding developed more recently can
elucidate the abnormality. Fluorescence in situ Hybridization (FISH) is a
powerful technique in that it can reveal submicroscopic abnormalities
even in non-dividing cells.

3. der 5
Examples

4. der 5
SpectrumGold
WCP 5 +
SpectrumRed
WCP 9
9
5
9
9
der 5

5. bcr/abl Translocation Probe hybridized to an interphase
cell. Note the presence of one orange-pink signal, one
green signal and one yellow signal (fusion signal)
indicates the fusion of the bcr and abl oncogenes

8. Specific Translocations in
Hematopoeitic Cancers
•Distinct rearrangements in specific neoplasms
•Reproducibly involve same chomosomal segments
•Usually involve oncogene activation
–Leukemia: two-gene fusion-> chimeric protein
–Lymphoma: transcriptional control units are misplaced
leading to aberrant gene expression
•Presence can be used to:
–Classify & monitor the cancer
–Predict prognosis & therapeutic response

9. Disease
Ch. Abnormality
Overall frequency/
(Frequency in Sub) Involved genes
CML t(9;22)(q34;q11) 98% (100%) ABL/BCR
AML-M2 t(8;21)(q22;q22) 18% (30%) ETO/AML1
AML-M3 T(15;17)(q22;q11) 14% (98%) PML/RARA
AML-M4 Inv(16)(p13q22) 6% (100%) MYH11,CBFB
AML-M5 T(10;11)(p13;23) 11% (30%) AF10,MLL
ALL-Pre B T(1;19)(q23;p13) PBX1/TCF3
ALL-B T(8;14)(q24;q32) MYC/IGH
ALL-T T(11;14)(p13;q11) RBTN1/TCRA
NHL-B T(8;14)(q24;q32) MYC/IGH
NHL-T t(2;5)(p23;q35) ALK/NPM
CML-B T(11;14)(q13;q32) CCND1/IGH
CML-T T(8;14)(q24;q11) MYC/TCRA
MM T(11;14)(q13;q32) CCND1/IGH

10. Satellite (centromeric) probe
on X–chromosome
45,X or 46,XYPossible karyotype?

11. X- and Y-centromeric probes
46,XYDetermine probable karyotype.
Green = X
Red = Y

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

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

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

15. 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?

16. Case 1: DiGeorge syndrome
hypertelorism
micromandibula
low set dysplastic
ear
„antimongoloid“
slant of eyelids
Inborn cardiac defect (e.g. tetralogy of Fallot),
thymic hypoplasia (or aplasia).

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

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

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

20. Case 2
•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?

21. Case 2 - karyotype
Normal
finding:
46,XY
Microdeletion
should be
confirmed by
the FISH
analysis

22. 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)
ELN LIMK1 D7S613
CENTROMERE TELOMERE
180
 kb

23. Case 2 – conclusion of the
molecular cytogenetic examination
•Microdeletion of
7q11.23 chromosome
confirmed.
•Diagnosis: Williams-
Beuren syndrome

24. FISH
•Powerful adjunct to conventional
cytogenetic analysis
• Utilizes metaphases and non-mitotic
interphase nuclei
• Can be applied to fixed archived tumour
material
• Accurate, specific

25. FISH
•FISH - a process which vividly
paints chromosomes or portions of
chromosomes with fluorescent
molecules
•Human M-phase spread using DAPI
stain

26. FISH
•Identifies chromosomal
abnormalities
•Aids in gene mapping, toxicological
studies, analysis of chromosome
structural aberrations, and ploidy
determination

27. FISH
•Used to identify the presence and
location of a region of DNA or
RNA within morphologically
–preserved chromosome
preparations,
–fixed cells or
–tissue sections

28. FISH
•This means you can view a
segment or entire chromosome
with your own eyes
•Was often used during M phase
but is now used on I phase
chromosomes as well

29. FISH
•Advantage: less labor-intensive
method for confirming the
presence of a DNA segment
within an entire genome than
other conventional methods like
Southern blotting

30. FISH Uses
•Detection of high concentrations of
base pairs
•Eg: Mouse metaphase preparation
stained with DAPI (a non-specific
DNA binding dye with high affinity for
A-T bonds)

31. FISH and Telomeres
•Telomeric probes define the terminal
boundaries of chromosomes (5’ and
3’ ends)
•Used in research of chromosomal
rearrangements and deletions
related to cell aging or other genetic
abnormalities

32. FISH and Telomeres
•Special telomeric probes specific to
individual chromosomes have been
designed
•Probe is based on the TTAGGG
repeat present on all human
telomeres

33. FISH and Telomeres
•Application in cytogenetics - can
detect submicroscopic deletions
and cryptic translocations of genes
associated with unexplained mental
retardation and miscarriages