THE FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

ANALYSIS IN PRADER-WILLI SYNDROME

Authors: Monica Stoian, Maria Puiu, Valerica Belengeanu

Department of Medical Genetics, University of Medicine and Pharmacy “Victor Babes” Timisoara, Romania

Fluorescence in situ hybridization (FISH) technique is an important analysis not only in cytogenetic research but also in routine clinical diagnostics for Prader-Willi Syndrome.

Prader-Willi syndrome (PWS) is a multisystemic disorder caused by the loss of expression of paternally transcribed genes within chromosome 15q11-q13.

• 70% of PWS patients have the deletion of the 15q11.2-q13 region on the paternal chromosome

• 20-30% of the PWS patients have maternal uniparental disomy

• 2-5% patients display an imprinting defect • occasionally, deletions occur from chromosomal

translocation

Objective

Perform FISH analysis for the patients included in the study in order to determine whether the cause of the Prader-Willi Syndrome is the microdeletion of 15q11-q13 of the paternal chromosome.

The analysis were supported by the CNMP Grant 42113, 2008-2011.

Material and method

• Peripheral blood on heparin was taken from 5 patients (2 boys and 3 girls) with parents consent

• 72 hours cultured lymphocytes were harvested according to standard procedure and slides were made.

• Conventional banding using trypsin and GTG staining was performed

• On the best slides FISH analysis was performed

Results Cytogenetic analysis

• Conventional cytogenetic analysis was performed, as it is an analysis that should be included in testing for deletion, as occasionally the deletion is a result of a chromosomal translocation.

• Using high resolution banding, cytogenetic analysis might point to a deletion.

• It is a cheap and affordable analysis in all genetic laboratories in Romania

All patients investigated showed a normal karyotype, without translocations.

FISH analysis

FISH analysis was performed using Vysis- Abbott: Vysis® Prader-Willi/Angelman Region Probes according to manufacturers protocol and adapted from the Cytogenetic Laboratory at Ulleval University Hospital, Oslo, Norway protocol.

FISH • is a molecular-cytogenetic technique used

to detect and localize the presence or absence of specific DNA sequences on chromosomes.

• uses fluorescent probes that bind to only those parts of the chromosome with which they show a high degree of sequence similarity (they are designed for).

Fluorescence microscope is needed to find out whether the fluorescent probe bound to the chromosomes.

Probe Name Probe Location Fluorophore

Vysis LSI (D15Z1) 15p11.2 SpectrumGreen

Vysis LSI SNRPN 15q11-13 SpectrumOrange

Vysis LSI PML 15q22 SpectrumOrange

Probes

Probe Name Probe Location Fluorophore

Vysis LSI (D15Z1) 15p11.2 SpectrumGreen

Vysis LSI D15S11 15p11-q13 SpectrumOrange

Probes

Other available probes from Vysis- Abbott:

• Prader-Willi/Angelman Region Probe - LSI D15S10 SpectrumOrange/CEP 15 (D15Z1) SpectrumGreen/PML SpectrumOrange

• Prader-Willi/Angelman Region Probe - LSI GABRB3 SpectrumOrange/CEP 15 (D15Z1) SpectrumGreen

Cytocell also has a designed probe: Prader-Willi/Angelman (SNRPN) Region Probe

• SNRPN/Imprinting Centre: Red Fluorophore;15q Subtelomere Specific Probe (clone 154P1): Green Fluorophore

Technique

• Freshly made slides were kept in the freezer until probe was available.

• Slides were place the in the 37º C water bath in 2X SSC solution for 30 minutes

• 3 series of dehydration in alcohol solutions 70%, 80%, 100% at room temperature each for 2 minutes

• The slides were air dried

Probe preparation

• 7µl hybridization buffer (LSI [locus specific] /wcp [whole chromosome paint])

• 1µl probe

• 2µl purified water

Denaturation of the slides

• The slides were placed in denaturation solution for 2 minutes in 73º C water bath

• 3 series of dehydration in alcohol solutions 70%, 80%, 100% ice-cold each for 2 minutes

Denaturation of the probe for 5 min at 73-75ºC (water bath or PCR machine)

• The slides were placed on a slide warmer (40-50 ºC) and the denaturated 10µl probe was apply.

• The area was covered with 22x22 mm cover glass and sealed with a cow-gum.

Hybridization• The slides were place in a humidified

box in 37 ºC overnight.

Slide washing• The slides were immersed in

0.4XSSC/0.3% NP40 at 73±1ºC and wash for 2 minutes

• The slides were wash for 30 sec-1 minute in 2XSSC/0.1% NP40 at room temperature

- 10 µl of DAPI II counterstain was applied on hybridization area and covered with cover-glass

Slide examination

• Slides were examined using Carl-Zeiss AxioImager M1 microscope with fluorescence and MetaSystems Isis software.

• At least 30 metaphases were evaluated for each patient.

• 5-10 images were captured for each patient.

SNRPN

SNRPN

SNRPN

SNRPN

PWSR

PWSR

SNRPN

SNRPN

SNRPN

SNRPN

Discussions and conclusions

• All result came out negative for the microdeletion 15q11-q13;

• This is in contrast to the data from literature: 70% of PWS patients have the deletion;

• FISH analysis should also be performed in order to correctly diagnose the molecular class for the PWS patients (besides DNA methylation analysis, DNA polymorphism analysis and mutation analysis for the imprinting center).

Thank you!