Molecular cytogenetic practical

FISH method

Two kinds of cytogenetic examination:

• Basic chromosomal analysis– staining methods (solid staining, G-banding

etc.)

– Based on analysis of metaphase chromosomes

• Molecular cytogenetic analysis– Identification of chromosomal abnormalities

using molecular biological methods

Patient

Basic chromosomal analysis

Molecular cytogenetic analysis

Family of the patient

Molecular biological analysis

Molecular cytogenetic examinations

• In most of cases 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 atd.)– Gene chips, resp. array CGH, DNA microarray etc.– PRINS, PCR in situ– quantitative fluorescent PCR, real time PCR– methods based on amplification of probe attached to

target sequence (MLPA, MAPH)

hybr

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nP

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Written test

• 20 questions

• 20 minutes

• In some multiple-choice questions

more than one answer could be correct.

Hybridization

target DNA

probe

denaturation

hybridization

Hybridization

• Molecular (on isolated DNA)

• In situ (on biological structures – i.e.

interphase nuclei, metaphase

chromosomes, cells or tissues)

Probe

• A part of DNA (or RNA) that is complementary to certain sequence on target DNA (i.e. DNA of the patient)

• Plasmid, phage DNA, cosmid (or combination of phage and plasmid DNA), YAC

• PCR-product (amplification of certain segment of chromosomal DNA)

Labeling of probes

• Radioactive

• Enzymatic

• Fluorescent

– Fluorescent in situ hybridization (FISH)

FISH

Types of probes

Centromeric (satellite) probes

Locus specific probes

Whole chromosome painting probes

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

PRINS• = Primed in situ labeling

Fluorescently labeled nucleotides

Quantitative fluorescent PCRQF-PCR

Capillary electrophoresis

denaturation, annealing

PCR

In case of informative

polymorphism each peak represents one locus in one

chromosome.

QF-PCR – normal finding

QF-PCR – identification of trisomy

Presentations

• mFISH

• CGH

FISH gallery and practical tasks

Satellite (centromeric) probe – chromosome 7

Satellite (centromeric) probe on X–chromosome

45,X or 46,XYPossible karyotype?

X- and Y-centromeric probes

46,XYDetermine probable karyotype.

Green = X

Red = Y

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)?

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

Hybridization with a telomeric probe

Lokus specific probe – detection of SRY region

Combination of locus-specific probe with a centromeric one

Green = X-centromere

Red = SRY

Normal male - 46,XY; SRY is not deleted.

Possible karyotype?

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?

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 or velocardiocacial syndrome.

DiGeorge syndrome

hypertelorism

micromandibulalow set dysplastic

ear

„antimongoloid“ slant of eyelids

Inborn cardiac defect (e.g. tetralogy of Fallot), thymic hypoplasia (or aplasia).

Tetralogy of Fallot

Left normal heart, right heart of the patient with the tetralogy of Fallot

Tetralogy of Fallot – combination of 4 different

inborn cardiac defects

Locus specific probes – examination of oncogene HER2/NEU amplification (red signals)

Normal finding

Amplification confirmed

Painting probes – examination of chromosomes 1, 4 and 8

Normal finding

1

1

4

4

88

Painting probes – detection of unbalanced translocation of 11 and

21 chromosomes

t(11;21)

Patient 1

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.

Patient 1 (boy, 2 years)

irides stellataehypertelorism

open mouth, thick lip

abnormal teeth

„elfin face“

low set ears

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 is identified by means of 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?

Patient 1 - karyotypeNormal finding:

46,XY

Microdeletion should be

confirmed by the FISH analysisWhich kind of probe you would use for

FISH analysis of microdeletion of the chromosome 7?

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

Patient 1 – conclusion of the molecular cytogenetic examination

• Microdeletion of

7q11.23 chromosome

confirmed.

• Diagnosis: Williams-

Beuren syndrome

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).

Good bye!