Genet 302 lec 16 & 17
What is the purpose of comparative genomic hybridization (CGH)?
To detect unknown deletions, duplications, monosomies, and trisomies.
What is compared in CGH?
Reference DNA (normal diploid) is compared to test DNA (patient sample).
What technologies have been used historically for CGH?
Southern blots, PCR, and DNA microarrays.
What is the modern method for CGH?
Array comparative genomic hybridization (aCGH) using DNA microarrays.
Who developed two-channel aCGH and when?
Barrett et al., 2004.
In two-channel aCGH, how is reference DNA labeled?
Green.
In two-channel aCGH, how is test DNA labeled?
Red.
What is Step 1 of a two-channel aCGH experiment?
Label reference DNA green and test DNA red.
What is Step 2 of a two-channel aCGH experiment?
Add both labeled DNAs to the microarray slide.
What happens if equal amounts of reference and test DNA bind to a spot?
The spot appears yellow (red + green).
What instrument is used to scan aCGH slides?
A high-resolution microarray scanner (e.g., Agilent scanner).
What is calculated after scanning in aCGH?
The test/reference ratio (T/R).
What does a T/R ratio of 1 indicate?
Equal copy number in test and reference.
What does a T/R ratio less than 1 indicate?
Deletion in the test sample.
What does a T/R ratio greater than 1 indicate?
Duplication in the test sample.
Why is Log2(T/R) used instead of raw ratios?
Log base 2 elegantly represents fold changes and makes deletions and duplications symmetrical.
What is Log2(1)?
0.
What is Log2(0.5)?
-1 (heterozygous deletion).
What is Log2(1.5)?
Approximately +0.58 (heterozygous duplication).
In aCGH statistical coloring, what does blue indicate?
No significant difference.
What does green indicate on the graph?
Significantly below the line (deletion).
What does red indicate on the graph?
Significantly above the line (duplication).
What must you be able to do with Log2(T/R) graphs for the exam?
Draw and interpret them.
How does one-channel aCGH differ from two-channel aCGH?
All DNA is labeled one color and compared computationally to a reference dataset.
Which company is associated with one-channel aCGH?
Affymetrix.
In autosomal interpretation, where should most points cluster in a normal individual?
Around Log2(T/R) = 0.
In sex chromosome analysis with reference 46,XX and test 46,XY, what happens on the X chromosome?
Log2(T/R) values shift downward because males have one X instead of two.
If points on a chromosome are above the line across the entire chromosome, what does that indicate?
An extra chromosome (trisomy).
If points are below the line across an entire chromosome, what does that indicate?
Missing chromosome (monosomy).
In the chromosome number example (GM03576), what was the total chromosome number?
48.
If Log2(T/R) is strongly negative across a region of chromosome 18, what does that indicate?
Deletion.
If Log2(T/R) is moderately negative (~ -1), what type of deletion is most likely?
Heterozygous deletion.
Why is a homozygous deletion often non-viable?
Complete loss of essential genes is typically lethal.
If a region on chromosome 18 shows positive Log2(T/R) values, what does that indicate?
Duplication.
What must you be able to do for the exam from this lecture?
Draw aCGH results if given a genotype and determine genotype if shown aCGH results.
What is non-homologous end joining (NHEJ)?
A DNA repair mechanism that binds broken DNA ends and rejoins them without needing sequence homology.
How can NHEJ lead to chromosome rearrangements?
Incorrect rejoining of broken DNA ends can create deletions, duplications, inversions, or translocations.
What is a balanced rearrangement?
A structural chromosome change where all genetic material is still present but rearranged.
What is an unbalanced rearrangement?
A structural change where genetic material is missing or duplicated.
Which rearrangements are typically unbalanced: deletion, duplication, inversion, translocation?
Deletion and duplication.
Which rearrangements are typically balanced?
Inversion and translocation.
In the lecture example, what was unusual about the patient?
Unaffected parents had a daughter with an unknown genetic condition.
What did G-banding show in the patient?
A complex but apparently balanced rearrangement involving chromosomes 2, 3, and 5.
What was the karyotype shown by G-banding?
46,XX,t(2;3;5)(q21.3;q12;q13.3).
Why was the rearrangement suspected to be problematic despite appearing balanced?
The child had significant health issues.
What technique revealed the rearrangement was not truly balanced?
Array CGH (aCGH).
What abnormality did aCGH detect?
An interstitial deletion on chromosome 2.
What was the deletion described as?
del(2)(q21q21).
How can deletion size be estimated using aCGH?
By counting affected probes and multiplying by probe spacing (e.g., 1 probe per Mb).
If one probe represents 1 Mb and about six probes are missing, what is the deletion size?
Approximately 6 Mb.
What technique was used to confirm the deletion?
FISH (fluorescence in situ hybridization).
How many FISH probes were used in the confirmation?
Three adjacent BAC clones on chromosome 2.
What was the probe color pattern used in FISH?
Green – red – green.
What FISH result indicated a deletion?
Missing red signal on one chromosome 2.
Which gene was removed by the deletion?
ZEB2.
What is the cytogenetic location of ZEB2?
2q22.3.
What genetic condition is associated with deletion of ZEB2?
Mowat-Wilson syndrome.
What is the OMIM number for ZEB2?
605802.
What is the inheritance pattern of Mowat-Wilson syndrome?
Autosomal dominant.
Why can a rearrangement appear balanced by G-banding but still be pathogenic?
Small deletions at breakpoints may not be visible by G-banding.
What is the key exam skill from this lecture?
Integrating pedigree charts, G-banding, aCGH, FISH, PCR, and DNA sequencing to determine genotype and phenotype.