Copy number variants

Question 1

Why are some chromosomal abnormalities too small to detect with banding?

Answer 1

Can’t see them if they happen within a band. Can’t tell a slightly smaller band from a slightly larger band

Question 2

How do we detect small deletions and duplications that can’t be detected with karyotyping?

Answer 2

Comparative genomic hybridization - CGH arrays

Question 3

How do we do comparative genomic hybridization on a slide?

Answer 3

1. Extract genomic DNA from the patient and a healthy control
2. Differentially label
3. Hybridize to normal metaphase chromosomes on a slide
4. Use software to calculate fluorescence ratios between the samples
5. Yellow if there’s no difference in copy number, or fluoresce one colour or the other more if there was a copy number variant

Question 4

Why has array CGH essentially replaced CGH using metaphase chromosomes on a slide?

Answer 4

Easier to interpret and higher resolution

Question 5

How do we do array CGH?

Answer 5

1. Isolate genomic DNA from the patient and a healthy control
2. Differentially label
3. Hybridize to the array with printed oligonucleotides or clones on the chip
4. Detect fluorescence
5. Anything yellow is the same copy number, one colour or the other indicates duplication or deletion

Question 6

What is chromothripsis?

Answer 6

Chromosome shattering and extensive rearrangement in a single catastrophic event

Question 7

What are the 4 features of chromothripsis?

Answer 7

1. Breakpoints usually clustered in a limited area of a single chromosome/handful of chromosomes
2. Copy numbers oscillating
3. Haplotypes getting rearranged into random orientation and order
4. Some fragments are lost

i.e. rearrangements and deletions! Lots of them

Question 8

Would chromothripsis be constitutional?

Answer 8

No, event is extremely rare and would only happen in a single cell

Question 9

What is uniparental disomy?

Answer 9

A zygote where both copies of a chromosome came from the same parent

Question 10

What are the 4 mechanisms of uniparental disomy?

Answer 10

1. Gametic complementation
2. Monosomy rescue
3. Mitotic recombination
4. Trisomy rescue

Question 11

How does uniparental disomy result from gametic complementation?

Answer 11

2 abnormal gametes with complementary defects are used in a fertilization event. One gamete is missing a copy of that chromosome and the other has two copies of it. The result is a diploid that has both copies of that chromosome from the same parent

Question 12

How does uniparental disomy result from monosomy rescue?

Answer 12

Endoreduplication in a zygote made from one normal gamete fertilized by one lacking a chromosome

Question 13

How does uniparental disomy result from mitotic recombination?

Answer 13

Crossing over during mitosis, resulting in segmental UPD where only certain parts of the chromosome show UPD

Question 14

How does uniparental disomy result from trisomy rescue?

Answer 14

Loss of a chromosome in a zygote formed from a normal gamete fertilizing a diploid gamete. Can also result in a normal zygote depending on which copy of the extra chromosome gets lost

Question 15

Which ones of the mechanisms for the formation of uniparental disomy results in constitutional UPD? Which ones result in segmental UPD and mosaicism?

Answer 15

Constitutional: gametic complementation, monosomy rescue, trisomy rescue
Segmental: mitotic recombination

Question 16

What problems can arise from uniparental disomy?

Answer 16

Imprinted genes. If the affected chromosome has two copies of an imprinted gene, then there’s no active copy of that gene and results in developmental disorders

Question 17

How are genes imprinted?

Answer 17

Methylation and chromatin alterations, and other epigenetic modifications

Question 18

What happens to imprinting in germline cells?

Answer 18

Gets erased and reestablished during meiosis depending on the sex of the individual. All maternally imprinted genes will be imprinted in the germline of a female, and all paternally imprinted genes will be imprinted in the germline of a male