Clinical Diagnostics Flashcards
What are some examples of chromosomal abnormalities
Aneuploidy due to non-disjunction
Translocations
Duplications/Insertions
Microdeletions
What are the three cytogenetic studies
Karyotype
FISH
Array-CGH
Karyotype Vs Array CGH for large chromosomal abnormalities
Array-CGH cannot reveal balanced translocations cos no gain or loss
How does non-disjunction cause aneuploidy
Meiosis I - paired chromosomal fail to separate
Meiosis II - sister chromatids fail to separate
What are the clinical features of Down Syndrome
ANS
What are the clinical features of Patau’s and Edward’s syndrome
18 Patau’s - cleft lip, extra fingers, mid part of brain fused
18 Edward’s - clenched hand, rocker bottom feet
What are translocations
Two different chromosomes sustain single break and incorrectly joined
Results in exchange of material between non-homologues chromosomes
What are the types of translocation
Balanced - no deleterious phenotype unless it affects regulation of a gene
Unbalanced = too much or little of a chromosome
Reciprocal - when reciprocal parent gamete undergoes meiotic division, it cannot line up properly = exchange of material between non-homologous chromosomes
Robertsonian - acrocentric chromosomes involved, they may fuse
When having a baby this may lead to a loss or a trisomy
What is DiGeorge Syndrome
22q11 microdeletion
How is can DiGeorge Syndrome be diagnosed using clinical diagnostics
Can’t use FISH as it is too small of a deletion, and only if you’re certain of the diagnosis
Array-CGH used instead
What is fragile X syndrome
Fragile X syndrome (FXS) is a genetic disorder characterized by mild-to-moderate intellectual disability.
What kind of disorder is fragile X - (dominant, recessive etc.)
X-linked dominant affecting X chromosome at Q27.3 FMR1 coding for FMRP
Caused by expansion in 5’ UTR CAG repeat
Why is it difficult to test for fragile X syndrome
Most tests require PCR but as it is a CAG repeat it is GC rich thus poorly sequenced (high Tm)
There may be artefacts, misalignment etc.
How is fragile X syndrome tested
Historically- microscope
Now - southern blotting to size by length
Triplet PCR
What is southern blotting
Separation based on length
Describe the process of southern blotting
Probe hybridises near the repeat, with ECORI restriction sites on either side of the repeat
Restriction enzymes digest the DNA, and this is then run on agarose gel
Following this is the transfer to nitrocellulose membrane using alkaline buffer
DNA is hybridised, X ray film applied and then radiation is applied to develop the film
What is triplet primed PCR
Used for CAG repeat expansions - method depends on the disorder tested
For fragile X
Forward (2)
Fluorescent probe binding upstream of the repeat
Repeat probe binding randomly to the repeat regions
Reverse - binds the downstream flanking region only, or that region AND some of the repeat regions
This results in amplification of the repeat at different lengths creating a ladder
The larger the region the wider the ladder due to more possible binding sites for the primers
Output in electropherogram
This uses capillary electrophoresis to separate by fragment size, and fluorescence analysis
Describe the triplet primed PCR
Forward (2)
Fluorescent probe binding upstream of the repeat
Repeat probe binding randomly to the repeat regions
Reverse - binds the downstream flanking region only, or that region AND some of the repeat regions
This results in amplification of the repeat at different lengths creating a ladder
The larger the region the wider the ladder due to more possible binding sites for the primers
Uses capillary electrophoresis and fluorescence analysis visualised in an electropherogram
What are the complications with triplet primed PCR
AGG interruption within CAG regions leads to decreased peak height
What are the pros of triplet primed PCR + electropherogram
Can be applied to many repeat expansion disorders
Less laborious, no radioactivity and uses modern lab equipment
Little DNA requires - high sample throughput
ingle cell testing - pre implantation test of embryo
What are the cons of triplet primed PCR + electropherogram
Methylation cannot be detected
Cannot accurately measure expansions larger than those in mutation zone
Problem between premutation and full mutation expansion differentiation
Why cant WGS be used for fragile X syndrome
Due to PCR problems
