Download Applications of Molecular Cytogenetics

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
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.
Examples
der 5
der 5
9
9
SpectrumGold
WCP 5 +
SpectrumRed
WCP 9
der 5
9
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
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
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
Satellite (centromeric) probe
on X–chromosome
Possible karyotype?
45,X or 46,XY
X- and Y-centromeric probes
Green = X
Red = Y
Determine probable karyotype.
46,XY
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
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;
mar
Possible karyotype:
47,XX,der(X)
Combination of locus-specific probe
with a centromeric one
Green = X-centromere
Red = SRY
Normal male - 46,XY;
Possible karyotype?
SRY is not deleted.
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?
Case 1: DiGeorge syndrome
„antimongoloid“
slant of eyelids
low set dysplastic
ear
hypertelorism
micromandibula
Inborn cardiac defect (e.g. tetralogy of Fallot),
thymic hypoplasia (or aplasia).
Microdeletion confirmed (loss of
one red signal)
Deleted chromosome –
red signal absent
Red signal –
TUPLE1 (HIRA)
locus
normal
chromosome –
red signal on
HIRA locus is
present
Green signal –
ARSA locus
(control probe)
Microdeletion 22q11.2 is associated with
DiGeorge syndrome.
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.
Case 2 (boy, 2 years)
hypertelorism
irides
stellatae
low set ears
abnormal teeth
open mouth,
thick lip
„elfin face“
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?
Case 2 - karyotype
Normal
finding:
46,XY
Microdeletion
should be
confirmed by
the FISH
analysis
MOLECULAR CYTOGENETIC ANALYSIS
OF 7q11.23 MICRODELETION
180 kb
ELN
CENTROMERE
 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)
LIMK1
D7S613
TELOMERE
Case 2 – conclusion of the
molecular cytogenetic examination
• Microdeletion of
7q11.23 chromosome
confirmed.
• Diagnosis: WilliamsBeuren syndrome
FISH
• Powerful adjunct to conventional
cytogenetic analysis
• Utilizes metaphases and non-mitotic
interphase nuclei
• Can be applied to fixed archived tumour
material
• Accurate, specific
FISH
• FISH - a process which vividly
paints chromosomes or portions of
chromosomes with fluorescent
molecules
• Human M-phase spread using DAPI
stain
FISH
• Identifies chromosomal
abnormalities
• Aids in gene mapping, toxicological
studies, analysis of chromosome
structural aberrations, and ploidy
determination
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
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
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
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)
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
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
FISH and Telomeres
• Application in cytogenetics - can
detect submicroscopic deletions
and cryptic translocations of genes
associated with unexplained mental
retardation and miscarriages