structural chromosomal abnormalities Flashcards
describe structural abnormalities
translocations= reciprocal, robertsonian
inversion
deletion
duplication
rings
isochromsomes
describe translocation
exchange of two segments between non-homologous chromosomes
reciprocal or robertsonian
non homologous end joining
No net gain or loss of genetic material
Involve any chromosome and any size fragment
Common: 1 in 930 people
Plus copy of normal 1 and normal 22
Carrier of balanced translocation
results 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
robertsonian translocation
Two acrocentric chromosomes join near centromere with the loss of p arms
Balanced carrier has 45 chromosomes
If 46 chromosomes present including Robertsonian then must be unbalanced
p arms encode rRNA (multiple copies so not deleterious to lose some)
Robertsonian translocations 13;14 and 14;21 relatively common. 21;21 translocation leads to 100% risk of Down syndrome in fetus
outcomes of translocations
Very difficult to predict
Only have approximate probability of producing possible gametes
Some unbalanced outcomes may lead to spontaneous abortion of conceptus so early that not seen as problem
Some unbalanced outcomes may lead to miscarriage later on and present clinically
Some may result in live-born baby with various problems
deletions
1:7000 live births
Deletion may be terminal or interstitial
Causes a region of monosomy
Haploinsufficiency of some genes
Contiguous gene syndrome (multiple, unrelated clinical features)
Phenotype is specific for size and place on deletion
Gross deletions seen on metaphase spread on G-banded karyotype
micro deletions
Many patients had no abnormality visible on metaphase 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
Williams, 7q11
Smith-Magenis, 17p11
source of sample
Prenatal Amniocentesis Chorionic villus sampling Cell-free fetal DNA Postnatal Blood Saliva
chromosome staining
Most common = G-banding
G = Giemsa
Why bands?
Chromatin
2 different sorts: euchromatin & heterochromatin
Euchromatin = GC-rich; loosely packed; genes active
Heterochromatin = AT-rich; tightly packed; genes inactive
Stain differently
summary of G banding
How does karyotype of patient differ from expected?
Uses a chemical stain
Uses metaphase chromosomes
Takes several days at least
Looks for aneuploidies, translocations & very large deletions
FISH
Fluorescent in situ hybridisation Hybridisation = single stranded nucleic acid strand binds to a new single stranded nucleic acid strand (DNA/DNA or DNA/RNA) Cultured cells, metaphase spread Fluorescent probe Denature probe and target DNA Mix probe and target DNA Probe binds to target
what is a probe
single stranded DNA (or RNA) molecule
Typically 20 – 1000 bases in length
Labelled with a fluorescent or luminescent molecule (less commonly a radioactive isotope)
In some techniques thousands or millions of probes are used simultaneously
summary of FISH
How does karyotype of patient differ from expected?
Uses fluorescent probes for SPECIFIC parts of genome
Uses metaphase chromosomes
Takes several days at least
Looks for aneuploidies, translocations & large deletions
Traditional FISH
array CGH:
Array comparative genomic hybridisation
For detection of sub-microscopic chromosomal abnormalities
Patient DNA labelled Green
Control DNA labelled Red
summary of aCGH
How many copies of a particular genomic region does the patient have?
Uses fluorescent probes to differentiate between patient and control
Uses extracted DNA
Looks for microdeletions and microduplications
QF-PCR
Quantitative fluorescence polymerase chain reaction
Trisomies 13, 18 and 21
Uses microsatellites
micro satellites
Short repeated sequences
Number of repeats varies between individuals
Total length of microsatellite sequence varies between individuals
detecting microsatellitles
Isolate DNA from individual
Design primers specific to flanking sequences
PCR amplification
Gel electrophoresis
detecting micro satellites 2
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
MgCl2 – Essential for polymerase activity
PCR 2
PCR consists of incubating at three different temperatures
This results in three different processes happening:
Denaturation
Annealing
Extension
PCR exponential amplification
The PCR quickly reaches an exponential amplification phase…but remember it doesn’t go on forever!
The components of the PCR reaction are not unlimited so at some point the reaction will slow down and stop
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 mother, one from father, like any other autosomal 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
summary of QF-PCR
How many copies of a chromosome does the patient have?
Uses fluorescent probes for SPECIFIC microsatellite markers on SPECIFIC chromosomes
Uses extracted DNA
Quick (~48hrs)
Looks for aneuploidies
Need to know what you’re looking for
non-invasive pre-natal testing and NGS
Cell free fetal DNA Maternal blood sample Trisomy testing Next-generation sequencing “High chance” indicator for invasive test
summary of NIPT and NGS
Uses extracted DNA from mother
Looks for aneuploidies
Utilises next generation sequencing
Screening not diagnostic
