Cytogenetic and molecular cytogenetic analysis in clinical genetics

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Cytogenetic and molecular cytogenetic analysis
in clinical genetics

• 5th year
• Clinical genetics

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Eduard Kocárek

• eduard.kocarek_at_lfmotol.cuni.cz
• Phone (office) 224 435 980
• Secretary of the Institute of biology and medical
  genetics
• phone [phone]

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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

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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)

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FISH (fluorescent in situ hybridization)
Locus specific probes
Centromeric probe
target DNA
denaturation
hybridization
probe
M-FISH
Whole chromosome painting probes
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PRINS

• Primed in situ labeling

Fluorescently labeled nucleotides
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Quantitative fluorescent PCRQF-PCR
Primer 1
In case of informative polymorphism each peak
represents one locus on one chromosome.
denaturation, annealing
Primer 2
PCR
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QF-PCR normal finding
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QF-PCR identification of trisomy
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MLPA
Multilocus ligase-dependent probe amplification
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MLPA
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MLPA computer evaluation
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MLPA result deletions in the dystrophine gene
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MAPH
Multilocus amplifiable probe hybridization
DNA attached to the membrane
Mixture of probes
DNA sample
hybridization
Probe 1
Probe 2
Capillary electrophoresis
amplification of attached probes
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Indications of cytogenetic and molecular
cytogenetic examinations
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Patient
Basic cytogenetic chromosomal analysis
Molecular cytogenetic analysis (mostly FISH)
Molecular biological analysis
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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

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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)

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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)

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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.)

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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

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Indication
INDICATION
Result
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Patient
Basic cytogenetic chromosomal analysis
Molecular cytogenetic analysis (mostly FISH)
Molecular biological analysis
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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).

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Patient 1
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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.
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Patient 1 (boy, 2 years)
irides stellatae
hypertelorism
low set ears
abnormal teeth
open mouth, thick lip
elfin face
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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?
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Patient 1 - karyotype
Normal finding 46,XY
Microdeletion should be confirmed by the FISH
analysis
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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)

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Patient 1 conclusion of the molecular
cytogenetic examination

• Microdeletion of 7q11.23 chromosome confirmed.
• Diagnosis Williams-Beuren syndrome

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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).

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Thank you and good bye!