Cytogenetics

Question 1

Karotyping

Answer 1

numerical and large structural abberations

scans whole genome

requires mitotic cells

days to weeks

Question 2

FISH

Answer 2

metaphase and interphase

targeted cytogenetics

structural abnormalities (aneusomies) < 5Mb, microdeletions, microduplications

cryptic rearrangements

requres knowledge of targets

Question 3

Array CGH

Answer 3

scan whole genome for copy number changes and small rearrangements

Question 4

DNA sequencing

Answer 4

now good for detailed views of known gene target, soon useful for detailed scan of whole genome

Question 5

cytogenetic techniques

Answer 5

karotype

FISH

Question 6

molecular genetic techniques

Answer 6

array

sequencing

Question 7

DNA resolution

Answer 7

Question 8

Karotype Procedure

Answer 8

suspension cultures: blood, bone marrow

monolayer cultures: amniotic fluid, chorionic villi, products of conception, solid tumors

need proliferating cells that live well in a dish

specimens are minced and disaggregated by treatment w collagenase

plated as monolayer cultures

growth

metaplase cells are harvested, swolloe, dropped on glass slides and burst

chromosomes are stained and banded using Giemsa and trypsin (GTG)

Question 9

structrue of chromosome

Answer 9

dark band - heterochromatin: AT rich, tissues specific genes expressed

Euchromatin: GC-rich, housekeeping genes

Question 10

acrocentric

Answer 10

“p” short arm is super short - it’s hard to see it but still present

satellite

Question 11

submetacentric

Answer 11

arm length is unequal

Question 12

metacentric

Answer 12

2 arms equal in length, balanced translocation = fusion of 2 acrocentric chromosomes

Question 13

advantages of karotype

Answer 13

numerical abnormalities - too many too few

trisomy 21, 13, 18, Monosomy

large quantitative structural abnormalities (deletions, duplications, amplifications, big translocations)

large qualititave structural abnormalities (translocations, inversions, insertions)

Question 14

disadvantages of karyotype

Answer 14

requires actively dividing cells

requires time (growth of cells)

resolution varies with tissues

contamination w normal cells (mother and chid, tumor and non tumor)

best for big changes over 5 Mb

usually cannot detect single gene rearrangements (tumors)

Question 15

FISH procedure

Answer 15

1. denature dsDNA into single strands
2. add fluoro labled single strand probe (always DNA!) w sequence complentary to chromosomal region of interest
3. re-anneal DNA to allow probe binding to target
4. wash unbound proble
5. evaluate by fluoro microscope - nucleus is counterstained

Question 16

Metaphase vs Interphase FISH

Answer 16

Metaphase:

requires cell culture

chromosome morphology retained = hybridization signals can be localized

like classic karyotype - stopped them in metaphase,

use knowledge of what chromosome you are looking at - can see how many chromosomes there are (numerical abnormalities!) and molecular abnormalities that are too small to see on karyotye

very good for prenatal! can see number AND microdeletions

Interphase:

fixed tissues

chromosome morphology unavailable

signalys can’t be mapped reliably to chromosomes (FNA, single cells, formalin-fixed paraffin sections)

cells are out of M phase - did not proliferatue - previously fixed tissue, don’t see chromosomes, ony fish signals!

done on a lot of cancer tissues when asking specific questions - don’t really care about karyotype

Question 17

Break-apart probe

Answer 17

gene specific FISH:

1 gene - 2 probes for 2 diff regions close to each other

normal: both signals together/overlapping
abnormal: signals apart

rearrangement w differnet partners forms fusion protein

Question 18

Fusion probe

Answer 18

Gene specific FISH

2 genes - 2 probes - one for each gene

normal: both signals apart
abnormal: fused/overlapping

fusion protein is pathologic

Question 19

disadvantages of FISH

Answer 19

have to know target!

need clinical suspicion

limited number of colors

limited resolution - can’t see small inertions/deletions (have to be able to detect w the eye - if probe is very small can’t see it

Question 20

aCGH procedure

Answer 20

array + competitive + genomic + hybridization

slide w dots: tons of probes! chunks of DNA printed w areas of interest

pt DNA and controlled DNA - different colors - compete!

if 1:1 yellow - balance!

if more pt dna for region - gain!

if more control dna for a region - loss!

5-15 probes for everything so no mistakes

Question 21

when aCGH is the right test

Answer 21

looking for small DNA gains/losses that would not be detected by karyotype and can’t target w FISH bc unknown target

developmental delay, autism, growth, recurrent pregnancy loss/unexplained infertility

first tear w many unexplained abnormalities

unbalanced changes!

high resolution, sensitivity, specificity

molecular karyotype

Question 22

aCGH disadvantages

Answer 22

does not detect balanced translocations, small insertions, deletions

if only a few cells affected - may not see!

mosiacism - array will even out all dna

chimerism - fusion of more than one fertilized zygote in embryonic development

Question 23

balanced rearrangement

Answer 23

no DNA loss (almost) or gain

array: balanced signal - same amt DNA

reciprocal, inversions

Question 24

unbalanced rearrangements

Answer 24

DNA loss or gain

Array shows

unbalanced translocations

deletions

duplications

amplifications

Question 25

Robertsonian Transloaction

Answer 25

whole arm exchange of acrocentric chromosomes - most involve non homologous (13 and 14)

in short arm recombination - bicentric chromosome w 2 long arms and most of short arm material lost

1 centromere is inactive

chromosome is stable

balanced carriers = parent - 45 chromosome

2/3 child is effected w monosomy/trisomy

Question 26

when sequencing?

Answer 26

mutations, small insertions/deletions

clinical testing:

targeted therapy for tumors

constitutional defects: hearing loss, cardiomyopathy, syndromes (risk for next preg>

genotyping - hla testing paternity forensics

pharmacogenetics - drug metabolism