Structural chromosomal abnormalities

Question 1

what are the 2 types of translocation

Answer 1

reciprocal

robertsonian

Question 2

what does translocation mean

Answer 2

exchange of two segments between non-homologous chromosome

Question 3

what is the name give to the DNA repair mechanism whereby they rejoin the broken double stranded DNA

Answer 3

Non-homologous end joining (NHEJ)

Question 4

What happens when NHEJ goes wrong

Answer 4

the end joining occurs but onto different chromosomes creating derivative chromosomes
there is no net gain or loss

Question 5

How does translocation effect aligning of chromosomes during meiosis

Answer 5

Homologous chromosomes usually align as bivalent structures during metaphase 1, however since a translocation took place it means that it will align as a tetravalent structure. this is because the chromosomes segment must align with its complement homologous chromosome segment

Question 6

Result of unbalanced reciprocal translocation

Answer 6

-Many lead to miscarriage (hence why a woman with a high number of unexplained
miscarriages should be screened for a balanced translocation)
-Learning difficulties, physical disabilities
-Tend to be specific to each individual so exact risks and clinical features vary

Question 7

What is robertsonian translocation

Answer 7

• only occurs in acrocentric chromosome
• the p arms are chopped off and lost . Then the q arms are stuck together instead
• balanced carrier has 45 chromosomes and will be healthy because there is still a copy of both the q arm
• However if there are 46 chromosomes including robertonian then it will be unbalanced
• p arms encode rRNA
• trivalent alignment

Question 8

2 ways Trisomy 21 can arise and how will it look different on a karyotype

Answer 8

NDJ and Robertsonian
in NDJ there will be 3 chromosome present together
In robertsonian there will 2 chromosomes together with an extra on translocated chromosomes (e.g. chromosome 14)

Question 9

5 other structural chromosomal changes changes

Answer 9

• terminal deletion
• interstitial deletion
• inversion
• duplication
• ring chromosomes

Question 10

Deletions

Answer 10

-1 :7000 live births
-Deletion may be terminal or interstitial
-Causes a region of monosomy
-Haploinsufficiency Of some genes
-Contiguous gene syndronme (multiple, unrelated clinical features)
-Phenotype is specific for size and place on deletion
Gross deletions seen on metaphase spread on G-banded karyotype

Question 11

Microdeletions

Answer 11

• Many patients had no abnormality visible on rnetaphase spread
• High resolution banding, FISH and now CGH showed ‘micro’ deletions
• Only a few genes may be lost or gained
• Velocardiofacial (DiGeorge), 22q11
• Wolf-Hirschhorn, 4p16
• Williarns, 7q11
• Snith-Magenis, 17p11

Question 12

sources of sample for karyotyping for prenatal and postnatal

Answer 12

prenatal
--amniocentesis
-chorionic villus sampling
--cell-free fetal DNA 
postnatal
--blood
saliva

Question 13

Chromosome staining

Answer 13

• most common = G-banding

- G =Giemsa

Question 14

Why does bands form in chromosome staining

Answer 14

• chromatin
• 2 different sorts: euchromatin & heterochromatin
• Euchromatin GC-rich; loosely packed; genes active
• Heterochromatin = AT-rich; tightly packed; genes inactive
• Stain differently

Question 15

process of chromosomes staining work

Answer 15

-5ml venous blood taken
-add phytohemagglutinin and culture medium
-culture at 37 degree for 3 days
-add colchicine and hypotonic saline
-cells fixed
spread cell onto slide by dropping
-digest with trypsin and stain giemsa
-analyse metaphase spread
-karyotype

Question 16

what can you use g-banding to look for

Answer 16

aneuploidies
translocation
very large deletion

Question 17

what is FISH

Answer 17

-Fluorescent in situ hybridisation
-Hybridisation single stranded nucleic acid strand binds to a new single stranded nucleic acid
strand (DNA,’DNAor DNA/RNA)
-Cultured cells, metaphase spread
–Fluorescent probe
–Denature probe and target DNA
–Mix probe and target DNA
–Probe binds to target

Question 18

what is probe

Answer 18

• A single stranded DNA (or RNA) molecule
• Typically 20 — 1 OOO bases in length
• Labelled with a fluorescent or luminescent molecule (less cormnnly a radioactive
  isotope)
• In some techniques thousands or rfillions of probes are used simultaneously

Question 19

what can you use FISH to look for

Answer 19

aneuploidies
translocation
large deletion
– uses fluorescent probes for specific parts of the genome

Question 20

what is array CGH

Answer 20

--Array comparative 
genomic hybridisation 
-For detection of sub- 
microscopic 
chromosomal 
abnormalities 
-Patient DNA labelled 
Green 
-Control DNA labelled in red

Question 21

what can you use aCGH to look for

Answer 21

for microdeletions and microduplications

Question 22

what QF-PCR

Answer 22

• quantitative fluorescence polymerase chain reaction

- uses microsatellites

Question 23

what is microsatellite

Answer 23

-Short repeated sequences
-Number of repeats varies
between individuals
-Total length of microsatellite
sequence varies between
individuals
-Microsatellites: Length polymorphism
• Dinucleotide
• Trinucleotide
• Tetranucleotide
and so on..
-Short tandem repeat (STR) =Microsatellites=
simple sequence repeat (SSR)

Question 24

how to detect microsatellite

Answer 24

• Isolate DNA from individual
• Design primers specific to flanking sequences
• PCR amplification
• Gel electrophoresis
• PCR amplification of microsatellite region
• Genotype size of fragments on gel-based system
• Homozygotes = single product of specific size
• Heterozygotes = two different sized products

Question 25

PCR

Answer 25

-Exponential amplification of a DNA fragment of known sequence
-Components of the PCR reaction:
—Template — DNA to amplify
—primers — Short pieces Of SSDNA (15-30bp)
—polymerase — thermostable enzyme (Taq)
—Nucleotides — single base mixture (dNTPs)
—Buffer—To maintain pH
—MgC12 — Essential for polymerase activity
- PCR consists of incubating at three different temperatures
This results in three different processes happening:
Denaturation
Annealing
Extension

Question 26

QF-PCR

Answer 26

-Perform PCR using primers for microsatellite known to be on chromosome 21 (if testing for Down’s)
-Should be two copies Of microsatellite (one from one from father, like any
other autosornal locus, gene, whatever)
-If homozygous, there will be a single peak Of high signal
-If heterozygous, there will be two peaks of similar, lower signal