CFM 2: Molecular Diagnostics and Cytogenetics

• Karyotyping
• FISH
• Array CGH
• DNA sequencing

• Telocentric: centromere on end
• Acrocentric: p-arm is so short it is hard to see
• Submetacentric: centromere not quite in the middle
• Metacentric: centromere right in the middle

Cells that proliferate in a culture (e.g. amniotic fluid, chorionic villi, umbilical blood, preimplantation blastomeres, blood, fibroblasts, hematologic or solid tumors)

• Pros: good for looking at numerical abnormalities (trisomy, monosomy); large quantitative structural abnormalities (deletions, duplications, amplifications); large qualitative structural abnormalities (translocations, inversions, insertions)
• Cons: Requires actively dividing cells, requires time to grow the cells, resolution varies with tissues, low resolution (best for changes >5Mb), contamination with normal cells

• Identification of complex or cryptic rearrangements
• Identification of segmental aneusomies
• Identification of marker or ring chromosome origin
• Aneuploidy (extra or missing copies of single chromosomes)

• Take a cell
• Denature dsDNA into ssDNA strands
• Add fluorescence labeled ss probes to bind to complementary sequences on chromosomal region of interest
• Re-anneal DNA to allow probe to bind
• Wash off unbound probe
• Evaluate by fluorescence microscope (nucleus counterstained with DAPI)

• Metaphase FISH: FISH done on karyotyped cells, so you can see which chromosome the problem happens on; requires cell culture
• Interphase FISH: cannot tell which chromosome the genetic change happens on

Use centromeric FISH probe specific to chromosome 21 centromere and see how many chromosome 21s you have

• 2 probes for 2 different regions of a single gene close to each other
• Normal - both signals are together and overlapping
• Abnormal - the signals are apart

• 2 genes
• 2 probes, 1 for each gene
• Normal - both signals are apart
• Abnormal - signals are fused/overlapping

• You have to know something about your target
• Clinical suspicion is crucial
• Limited number of colors
• Limited resolution; cannot visualize small insertions/deletions because you need to find them by eye

subtelomeric FISH probes

• Array competitive genomic hybridization
• Patient DNA probed one color; control DNA probed another
• Mix pt and control DNA in equal amounts
• Add to an array with probes for specific regions of genome
• If there is a gain in pt's DNA, it will be more the pt's color than the control's
• If there is a loss in pt's DNA, it will be more control's color than pt's

Looking for small DNA gains/losses that would not be detected by karyotype and we cannot target them with FISH (unknown target)

• Does not detect balanced translocations
• Does not detect small insertions/deletions
• Detection limit
• Mosaicism (presence of two or more populations of cells with different genotypes in one individual who has developed from a single fertilized egg)
• Chimerism (two or more genotypes occur from fusion of more than one fertilized zygote in early stages of embryonic development)

• Whole arm exchange in acrocentric chromosomes (most often non-homologous chromosomes) that leads to viable fetus
• Long arms from acrocentric chromosomes fuse to form one chromosome with all long arms, and one with all short arms (which gets lost)

NextGen sequencing