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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