Structural chromosomal abnormalities Flashcards
what are the 2 types of translocation
reciprocal
robertsonian
what does translocation mean
exchange of two segments between non-homologous chromosome
what is the name give to the DNA repair mechanism whereby they rejoin the broken double stranded DNA
Non-homologous end joining (NHEJ)
What happens when NHEJ goes wrong
the end joining occurs but onto different chromosomes creating derivative chromosomes
there is no net gain or loss
How does translocation effect aligning of chromosomes during meiosis
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
Result of unbalanced reciprocal translocation
-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
What is robertsonian translocation
- 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
2 ways Trisomy 21 can arise and how will it look different on a karyotype
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)
5 other structural chromosomal changes changes
- terminal deletion
- interstitial deletion
- inversion
- duplication
- ring chromosomes
Deletions
-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
Microdeletions
- 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
sources of sample for karyotyping for prenatal and postnatal
prenatal --amniocentesis -chorionic villus sampling --cell-free fetal DNA postnatal --blood saliva
Chromosome staining
- most common = G-banding
- G =Giemsa
Why does bands form in chromosome staining
- chromatin
- 2 different sorts: euchromatin & heterochromatin
- Euchromatin GC-rich; loosely packed; genes active
- Heterochromatin = AT-rich; tightly packed; genes inactive
- Stain differently
process of chromosomes staining work
-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
what can you use g-banding to look for
aneuploidies
translocation
very large deletion
what is FISH
-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
what is probe
- 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
what can you use FISH to look for
aneuploidies
translocation
large deletion
– uses fluorescent probes for specific parts of the genome
what is array CGH
--Array comparative genomic hybridisation -For detection of sub- microscopic chromosomal abnormalities -Patient DNA labelled Green -Control DNA labelled in red
what can you use aCGH to look for
for microdeletions and microduplications
what QF-PCR
- quantitative fluorescence polymerase chain reaction
- uses microsatellites
what is microsatellite
-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)
how to detect microsatellite
- 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
PCR
-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
QF-PCR
-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
