Lecture 2: Diagnosis Techniques for Studying numerical + structural alterations to the human genome

**What are the 2 categories of cytogenetic testing?

Constitutional: Genetic changes are present in majority of all cells in the body (generally present from conception)

Cancer: genetic changes confined to a specific population of abnormal tumor or blood cells

*****What are the Tissue sources for CONSTITUTIONAL cytogenetic testing in pre vs. postnatal situations

• hint post (2) pre (4)

POST

• Peripheral blood lymphocytes

• Skin Fibroblasts

PRE

• Amniotic fluid

• Chorionic villi

• Product of conception

• Fetal tissue

*****What are the Tissue sources for CANCER cytogenetic testing

• hint (2)

1. bone marrow aspirate

2. Solid tumor - tissue

Overview of the study of the genome:

• cytogenetics vs. Molecular vs. Molecular cytogenetics/cytogenomics

• History of the 3 techniques

Cytogenetics: the microscopic study of whole chromosomes, large structural changes, and chromosome number (low resolution)

Molecular genetics: is the study of DNA, genes, and nucleotide sequences at the molecular level (very high resolution)

Molecular cytogenetics and cytogenomics: bridge the gap between traditional microscopic chromosome analysis and high-throughput molecular DNA technology

3 techniques (in chronological order): Banding, FISH, Microarray

*****Which of the 3 Techniques (banding, FISH, Microarray) is used on Cytogenetics?

G Banding

***What is Colcemid in the process of chromosome preparation from cultured cells?

Inhibits the Mitotic spindle formations

***What is the Application of G-banding

• what technique is used to view G banding

KARYOTYPING

• Seen at the MICROSCOPE

Example of a Diagnosis we can see from a karyotype

What is a Phildelphia chromosome?

Tiny chr 22

What is the difference between the chr. from a blood vs. cancer sample visually in a Karyotype?

SIZE

• Blood = BIGGER

• Cancer = SMALLER

Male, 27 y

Clinical indication: INFERTILITY

• What is the only thing we can tell from this karyotype?

ALL we can say is the Y is kinda short

*****Which of the 3 Techniques (banding, FISH, Microarray) is used in Molecular genetics??

FISH

• Fluorescence in-situ Hybridization

Unlike karyotype it is a TARGETED test

***How does fish work/ what are the principles/ basic steps

1. Design DNA probes

2. Label the probes

3. Denature probes + Target DNA

4. Hybridize the single strand probs and DNA

5. Visualization (probes will bind to area of interest they are designed to bind to)

****When performing FISH the Target DNA can be obtained from what 2 stages of the cell cycle?

1. METAPHASE chromosomes (more condensed)

2. INTERPHASE nuclei (less condensed)

****What are the 4 most commonly used classes of FISH PROBES

• postnatal/prenatal

1. Locus specific (Y)

2. Centromere specific (X)

3. Sub telomeric region (6)

4. Whole chromosome paints

*****FISH has a Targeted approach

• What things does it target

• What Abnormalities is this method typically used to DETECT?

Targets GENE/REGIONS of a chromosomes

Detects MICROdeletions/MICROduplications

****Going back to the example of the 27yo male with fertility (SHORT Y chr. from karyotype) a FISH was used

• What can be interpreted from the FISH results

• Normal on left. Patient on Right

• WHY did he have INFERTILITY

He has a ISOCHROMOSOME of Y

• Mistake in centromere division that caused the loss of both the long arms on his Y chromosome

The deleted region contained lots of genes related to fertility

****what is an ISOCHROMOSOME?

An abnormal chromosome where the two arms are mirror images of each other

*****What is CBFB break apart rearrangement probe

• HOW DOES IT WORK

FISH assays to detect structural changes, breaks, or rearrangements in the CBFB gene located on chromosome 16q22

How:

• 2 colored probes are used in the 16q22 region

• each overlaps with a section of the CBFB gene

• In NORMAL cases the 2 probes would be seen together when visualized

• But in this case there was an inversion causing there to be a separation between the red and green probes

*****What is FISH Scoring

• how does it work

• WHAT DOES IT DETECT

QUANTITATIVE FISH test

• count fluorescent signals of a specific probed gene under a microscope to determine if specific gene mutations, amplifications, deletions, or chromosomal abnormalities are present

Determines COPY NUMBER STATUS

• can compare copy numbers between a control gene and a target gene

*****What can a MULTICOLOUR FISH detect from this Karyotype?

• B-ALL = B cell acute lymphoblastic leukemia

REARRANGEMENT STATUS

• paint each chromosome its own color = easily tell which pieces of which chromosome moved where

****RECAP: What is the APPLICATION (aka what can it detect) of each of the Common classes of FISH Probes?

• Locus/ gene specific

• Chromosome specific CENTROMERE probes

• Chromosome specific PAINTING PROBES (whole chr + arm-specific)

• Multi color WHOLE CHR. painting probes

Diff between Chromosome specific Painting probe vs. Multicolor whole chr. painting probe =

• Chromosome specific = stains ONE specific chromosome (eg. chr 16)

• Whole chr = stains ALL chromosomes in the genome

***There is a 23yo male with Developmental delays + seizures

• which Genetic technique does the presence of developmental delays indicate for?

• WHY?

MICROARRAY

• Not cell by cell anymore but a more gene based technique

Microscopic and submicroscopic chromosomal imbalances, known as copy number variations (CNVs) or microdeletions/microduplications (such as 22q11.2 deletion syndrome), frequently underlie neurodevelopmental abnormalities

23yo male with Developmental delays + seizures

• What can we tell from this karyotype?

Nothing really, it looks normal at this resolution (low resolution)

*****Which of the 3 Techniques (banding, FISH, Microarray) is used on CYTOGENOMICS??

Array-Comparative Genomic Hybridization (aCGH/ Array CGH)

• aka chromosomal microarray or Microarray

VERY HIGH RESOLUTION

******ACGH are DNA based techniques. What is the SOURCE/SAMPLE taken to run ACGH?

Peripheral Blood samples in EDTA tubes

***What does it mean by ACGH are GENOME SCREENS?

a comprehensive laboratory test that evaluates a person's entire set of chromosomes at once to find missing or extra pieces of DNA

*****What do ACGH allow for the detection of?

SMALL chromosomal IMBALANCES (missing or extra genetic info) that cannot be detected by routine karyotype

• CNVs

*****What are CNVs?

Copy Number Variations

• microdeletions or Microduplications of segments of the genome, ranging from 50 bp to several mega bases

***What is a SPOTTED DNA microarray?

Laboratory technique where pre-synthesized DNA probes (oligonucleotides) are mechanically printed/”spotted” onto solid supports like glass slides using robotic pins

• pins dip into wells containing DNA then deposits each probe at designated locations on the array surface

****HOW do ACGH’s work? what are the basic steps

****What do the COLOURS

• yellow, red and green mean on a ACGH?

Yellow = control + patient = same

Red = more CONTROL DNA in a spot = Deletion in patient

Green = more PATIENT DNA in a spot = Duplication in patient

What does this result indicate about the Patient genome

CNV GAIN

What does this result indicate about the Patient genome

CNV LOSS

***How to interpret ACGH: What are the 5 International/National Guideline outcomes?

1. Pathogenic

2. Likely pathogenic

3. Uncertain clinical significance: VUS

4. Likely benign (no clinically significant abnormality detected + not reported)

5. Benign (no clinically significant abnormality detected + not reported)

*******How to interpret ACGH: For which of the 5 International/National Guideline outcomes are Family studies recommended?

First 3

1. Pathogenic

2. Likely pathogenic

3. Uncertain clinical significance: VUS

*****What are 6 Things to look for when INTERPRETING how serious the outcome of a ACGH is?

1. Gene content (OMIM/Refseq) + known syndromes- phenotypic correlations

   1. If there is gene sequence in the region dup/del = more likely dangerous

2. Dosage sensitive gene/region

3. DGV (database of genomic variance) + Local databases

   1. How often does this dup/del occur? if super often = likely benign

4. Size

5. Repetitive regions

6. Peer reviewed publications (amount of correlation/ # of papers)

7. Others

THERE WILL ALWAYS BE EXCEPTIONS

******What are 2 limitations of a ACGH/microarray?

1. Does not allow detection of balanced rearrangements (translocations, inversions) + low level mosaicism

2. Does not allow detection of regions consisting of highly repetitive sequences (Pericentromeric, heterochromatic regions, p-arms of acrocentric)

**What are the 3 possible outcomes of a microarray?

1. No abnormality: depends on level of resolution + area covered by test

2. CNV detected

3. Incidental findings: a potentially unrelated genetic risk

****What should be the next step if a CNV is detected?

Family studies

****What are 2 examples of possible Incidental findings of ACGH

• Cancer susceptibility locus

• Loci associated with adult onset

****Patient Earlier: 23yo male with Developmental delays + seizures

Could not identify what was wrong from karyotype = Ran ACGH

• What is a DUPLICATION of Chromosome 8q24.3 associated with?

• Neurodevelopmental disorders

• Epilepsy

*****Now that we know the specific area we are looking for that is causing the clinical indications we can run a FISH

• Patient Earlier: 23yo male with Developmental delays + seizures

Duplication of 8 is located on chr 14

and likely inherited from the parents as they have normal chr 8 and normal other chr 14

• Mother karyotype also normal but fish shows der chr 14

******Cytogenetic analysis RECAP: G banding, FISH, Microarray

• Can it detect microdel/dup

• Can it detect balanced rearrangements

• Resolution

• Genome screen vs. Targeted

• Does it study SINGLE or MULTIPLE cells