Mr.Kulachat Sae - Jang

Ph.D student in Medical Technology

4436692 MTMT/D

Group 17

Assignment six

SKY and CGH

SKY and CGH Spectral Karyotyping (SKY) and Comparative Genomic Hybidization (CGH) are complimentary fluorescent molecular cytogenetic techniques. SKY permits the simultaneous visualization of all human or mouse chromosomes in a different color, facilitating the identification of chromosomal aberrations. CGH utilizes the hybridization of differentially labeled tumor and reference DNA to generate a map of DNA copy number changes in tumor genomes.

summary for case nomber UOK122

UOK122 is case of renal cell carcinoma at kidney in human and in this case is male sex and solid type of tumor.

Cytogenetic Summary.

Clone/ Cell : 1

Cell Count : 5

Ploidy : 3n+, Hypertriploid (70-80)

Spectral Karyotyping (SKY)

SKY is a molecular cytogenetic technique, which allows one to simultaneously visualize all human (or mouse) chromosomes in a different color. This facilitates karyotype analysis considerably.

Spectral Karyotyping in this case.

70-78<+4n>,XX,-Y,-Y,der(1)t(1;22)(p12;q11.2),der(3)t(3;5)(p12;q12)x2,- 4,del(4)(q12q31.1),-6,der(6)t(6;9)(p11.2;q22.1),+7,-8,i(8)(q10),-9,-9, i(11)(p10),del(11)(q14),del(12)(q24)x2,-13,-13,-14,-14,i(15)(q10),+16, +del(16)(q23q23.2),-17,i(17)(q10),-18,-18,-19,i(19)(q10),-21,-22,-22[c p5]

Chromosome specific probe pools (chromosome painting probes) are generated from flow-sorted chromosomes and amplified and fluorescently labeled by degenerate oligonucleotide-primed polymerase chain reaction. After in situ hybridization of these differentially labeled probe pools each metaphase chromosome can be recognized based on its unique spectral signature. Image acquisition is based on a combination of epifluorescence microscopy, CCD imaging, and Fourier spectroscopy. This allows the measurement of the entire emission spectrum with a single exposure at all image points.

In this case will use SKY for screening genomes for chromosomal aberrations in human disease and of human renal cell carcinoma at kidney. By making possible the unambiguous identification of even complex and hidden chromosomal abnormalities.

SKY images and analysis.

Chromosome 1 : 3 chromosomes is normal and abnormal fragment 1 present in 5 cell(s) that is 22qter to q11.2 and 1p12 to qter

Chromosome 2 : 4 chromosomes is normal and no abnormal fragment.

Chromosome 3 : 2 chromosomes is normal and abnormal fragment 1 present in 5 cell(s) 5qter to q13 and 3p12 to qter

Chromosome 4 : 2 chromosomes is normal and no abnormal fragment.

Chromosome 5 : 3 chromosomes is normal and no abnormal fragment.

Chromosome 6 : 2 chromosomes is normal and no abnormal fragment.

Chromosome 7 : 5 chromosomes is normal and no abnormal fragment.

Chromosome 8 : 2 chromosomes is normal and no abnormal fragment.

Chromosome 9 : 2 chromosomes is normal and no abnormal fragment.

Chromosome 10 : 4 chromosomes is normal and no abnormal fragment.

Chromosome 11 : 2 chromosomes is normal and abnormal fragment 1 present in 5 cell(s) 11pter to p10 and 11p10 to pter abnormal fragment 2 present in 3 cell(s) 11pter to q14

Chromosome 12 : 2 chromosomes is normal and abnormal fragment 1 present in 5 cell(s) 12pter to q24

Chromosome 13 : 2 chromosomes is normal and no abnormal fragment.

Chromosome 14 : 2 chromosomes is normal and no abnormal fragment.

Chromosome 15 : 3 chromosomes is normal and no abnormal fragment.

Chromosome 16 : 5 chromosomes is normal and no abnormal fragment.

Chromosome 17 : 2 chromosomes is normal and no abnormal fragment.

Chromosome 18 : 2 chromosomes is normal and no abnormal fragment.

Chromosome 19 : 2 chromosomes is normal and abnormal fragment 1 present in 5 cell(s)

Chromosome 20 : 4 chromosomes is normal and no abnormal fragment.

Chromosome 21 : 3 chromosomes is normal and no abnormal fragment.

Chromosome 22 : 2 chromosomes is normal and no abnormal fragment.

Chromosome X : 2 chromosomes is normal and no abnormal fragment.

Chromosome Y : no chromosomes is normal and no abnormal fragment.

Comparative genomic hybridization (CGH)

Comparative genomic hybridization (CGH) is a fluorescent molecular cytogenetic technique that identifies DNA gains, losses, and amplifications, mapping these variations to normal metaphase chromosomes. It is a powerful tool for screening chromosomal copy number changes in tumor genomes and has the advantage of analyzing entire genomes within a single experiment. It is particularly applicable to the study of tumors which do not yield sufficient metaphases for cytogenetic analysis and can be applied to fresh or frozen tissues, cell lines, and archival formalin-fixed paraffin-embedded samples.

Comparative genomic hybridization produces a map of DNA sequence copy number as a function of chromosomal location throughout the entire genome. Differentially labeled test DNA and normal reference DNA are hybridized simultaneously to normal chromosome spreads. The hybridization is detected with two different fluorochromes. Regions of gain or loss of DNA sequences, such as deletions, duplications, or amplifications, are seen as changes in the ratio of the intensities of the two fluorochromes along the target chromosomes.

CGH is based on quantitative two-color fluorescence in situ hybridization. Equal amounts of differentially labeled tumor genomic DNA and normal reference DNA are mixed together and hybridized under conditions of Cot-1 DNA suppression to normal metaphase spreads. The labeled probes are detected with two different fluorochromes, e.g., FITC for tumor DNA and TRITC for the normal DNA. The difference in fluorescence intensities along the chromosomes in the reference metaphase spread are a reflection of the copy number changes of corresponding sequences in the tumor DNA.

Found chromosomal aberrations in human solid tumors has identified non-random tumor and tumor-stage specific genetic changes.

CGH image and analysis.

Chromosome 1 : fragment loss and starts from 1pter to 1q12.

Chromosome 2 : No fragment descriptions.

Chromosome 3 : fragment loss and starts from 3pter to 3p14.

Chromosome 4 : fragment s loss and starts from 4q22 to 4q35.

Chromosome 5 : fragment loss and starts from 5pter to 5q11.2 and gain starts from 5q23 to 5q35

Chromosome 6 : fragment loss and starts from 6q12 to 6q27.

Chromosome 7 : fragment gain and starts from 7p22 to 7q36.

Chromosome 8 : fragment loss and starts from 8pter to 8q11.1.

Chromosome 9 : fragment loss and starts from 9pter to 9q34

Chromosome 10 : no fragment descriptions

Chromosome 11 : fragment loss and starts from 11q12 to 11q25.

Chromosome 12 : fragment loss and starts from 12q24 to 12qter.

Chromosome 13 : fragment loss and starts from 13pter to 13q34.

Chromosome 14 : fragment loss and starts from 14pter to 14q32.

Chromosome 15 : fragment gain and starts from 15q10 to 15qter.

Chromosome 16 : fragment gain and starts from 16pter to 16q24.

Chromosome 17 : fragment loss and starts from 17pter to 17q11.2.

Chromosome 18 : fragment loss and starts from 18pter to 18q23.

Chromosome 19 : fragment loss and starts from 19pter to 19q12.

Chromosome 20 : no fragment descriptions.

Chromosome 21 : fragment loss and starts from 21pter to 21q21.

Chromosome 22 : fragment loss and starts from 22pter to 22q13.

Chromosome X : no fragment descriptions.

Chromosome Y : fragment loss and starts from Ypter to Yq12.

Summary

CGH and SKY in tumors.

Fluorescence in situ hybridization techniques allow the visualization and localization of DNA target sequences on the chromosomal and cellular level and have evolved as exceedingly valuable tools in basic chromosome research and cytogenetic diagnostics. Recent advances in molecular cytogenetic approaches, namely comparative genomic hybridization and spectral karyotyping, now allow tumor genomes to be surveyed for chromosomal aberrations in a single experiment and permit identification of tumor-specific chromosomal aberrations with unprecedented accuracy. Comparative genomic hybridization utilizes the hybridization of differentially labeled tumor and reference DNA to generate a map of DNA copy number changes in tumor genomes. Comparative genomic hybridization is an ideal tool for analyzing chromosomal imbalances in archived tumor material and for examining possible correlations between these findings and tumor phenotypes. Spectral karyotyping is based on the simultaneous hybridization of differentially labeled chromosome painting probes (24 in human), followed by spectral imaging that allows the unique display of all human (and other species) chromosomes in different colors. Spectral karyotyping greatly facilitates the characterization of numerical and structural chromosomal aberrations, therefore improving karyotype analysis considerably. We review these new molecular cytogenetic concepts, describe applications of comparative genomic hybridization and spectral karyotyping for the visualization of chromosomal aberrations as they relate to human malignancies and animal models thereof, and provide evidence that fluorescence in situ hybridization has developed as a robust and reliable technique which justifies its translation to cytogenetic diagnostics.