Applications of Molecular Cytogenetics

1
Applications of MolecularCytogenetics

• 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
Examples
der 5
4
der 5
9
9
5
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
6
(No Transcript)
7
(No Transcript)
8
Specific Translocations in Hematopoeitic Cancers

• Distinct rearrangements in specific neoplasms
• Reproducibly involve same chomosomal segments
• Usually involve oncogene activation
• Leukemia two-gene fusion-gt 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(922)(q34q11) 98 (100) ABL/BCR
AML-M2 t(821)(q22q22) 18 (30) ETO/AML1
AML-M3 T(1517)(q22q11) 14 (98) PML/RARA
AML-M4 Inv(16)(p13q22) 6 (100) MYH11,CBFB
AML-M5 T(1011)(p1323) 11 (30) AF10,MLL
ALL-Pre B T(119)(q23p13) PBX1/TCF3
ALL-B T(814)(q24q32) MYC/IGH
ALL-T T(1114)(p13q11) RBTN1/TCRA
NHL-B T(814)(q24q32) MYC/IGH
NHL-T t(25)(p23q35) ALK/NPM
CML-B T(1114)(q13q32) CCND1/IGH
CML-T T(814)(q24q11) MYC/TCRA
MM T(1114)(q13q32) CCND1/IGH

10
Satellite (centromeric) probe on Xchromosome
45,X or 46,XY
Possible karyotype?
11
X- and Y-centromeric probes
Green X Red Y
46,XY
Determine probable karyotype.
12
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

13
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
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
antimongoloid slant of eyelids
hypertelorism
low set dysplastic ear
micromandibula
Inborn cardiac defect (e.g. tetralogy of Fallot),
thymic hypoplasia (or aplasia).
17
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.
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
low set ears
abnormal teeth
open mouth, thick lip
elfin face
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)

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