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.

der 5 Examples

der 5 SpectrumGold WCP 5 + SpectrumRed WCP der 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 CMLt(9;22)(q34;q11)98% (100%)ABL/BCR AML-M2t(8;21)(q22;q22)18% (30%)ETO/AML1 AML-M3T(15;17)(q22;q11)14% (98%)PML/RARA AML-M4Inv(16)(p13q22)6% (100%)MYH11,CBFB AML-M5T(10;11)(p13;23)11% (30%)AF10,MLL ALL-Pre BT(1;19)(q23;p13)PBX1/TCF3 ALL-BT(8;14)(q24;q32)MYC/IGH ALL-TT(11;14)(p13;q11)RBTN1/TCRA NHL-BT(8;14)(q24;q32)MYC/IGH NHL-Tt(2;5)(p23;q35)ALK/NPM CML-BT(11;14)(q13;q32)CCND1/IGH CML-TT(8;14)(q24;q11)MYC/TCRA MMT(11;14)(q13;q32)CCND1/IGH

Satellite (centromeric) probe on X–chromosome 45,X or 46,XY Possible karyotype?

X- and Y-centromeric probes 46,XY Determine 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

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?

Case 1: DiGeorge syndrome hypertelorism micromandibula low set dysplastic ear „antimongoloid“ slant of eyelids Inborn cardiac defect (e.g. tetralogy of Fallot), thymic hypoplasia (or aplasia).

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.

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) irides stellatae hypertelorism open mouth, thick lip abnormal teeth „elfin face“ low set ears

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  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) ELNLIMK1D7S613 CENTROMERETELOMERE 180 kb

Case 2 – conclusion of the molecular cytogenetic examination Microdeletion of 7q11.23 chromosome confirmed. Diagnosis: Williams- Beuren 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