Dit document omvat mijn samenvatting (info slides + notities) van Les 3 van het vak Neurogenetics gegeven door Rosa Rademakers. Ik heb dit voor elke les, buiten les 10. Daarvan heb ik wel notities bij de slides. Je mag me hiervoor ook altijd contacteren :))
Class 3
Thing about last week
- We can ignore the figure
- Loss of TDP-43 induce cryptic exons
o If you have the normal transcript you see 4 thick bands
o When you knock down the TDP43 you see loss of these 4 bands
What are tandem repeat variants?
Human genetic variation
- SNPs: 3,8 millions compared to the reference genome
o EJect single nucleotide
- Indels: 800 000
- Structural variants: 25 to 30 000 variants
o AJect a large part of your genome
o Some of these can be identified with short read sequencing
Tandem repeats
- Perfectly repeated or interruption
- DNA motif can change entirely during repeat expansion
- Depending on the length of the motif diJerent names are used
o Short tandem repeats
o Larger than 10 n is a VNTR
o Then you have even larger repeats
- Many tandem repeats are unstable
o Expension or contraction of tandem repeats à longer and shorter
o Longer repeats are less stable and more likely to expand
o DNA replication: strand that is synthetized can dissociate …
o In bottom case: one unit looped out
- Show variation in length
o Across the generation the repeats will become longer or shorter
- Some of the repeats that are unstable can be pathogenic
o Pink = individuals that will develop a disease
o Gap between black and pink à not enough observation
o CANVAS repeats has a long zone that is tolerated, amother one can have
an shorter zone that is tolerated
o Permissive haplotype
o Premutation allele: not pathogenic, but your oJspring is at high risk
- Anticipation
o Through heightened awareness within family à further expansion of
repeat à oJspring will be more aJected
o Number = allele length à across generations it will become longer
- Timeline of discoveries
o Linkage locus was first used to identify a locus
o Then next generation sequencing for example
, Lab methods for tandem repeat analysis
Southern blot
- Restriction site that will be cleaved
- Fragment of 1 kb and the other fragment will become longer through expension
PCR
- Fluorescent primers
- Problems of false negatives because of CG rich or too long
Repeat-primed PCR
- You will get a ladder-like pattern
- You don’t know the size
Short-read sequencing
- Become a problem when the reads become longer then the read length
Long-read sequencing
- Direct observation of the length of repeat
- Some targeted methods where you use Cas9 or selective sequencing
- Databases for structural variants from long reads are currently under
development
- Quite common to be above 60 units
RNA - fish
- Fluorescent probes that will hybridise to RNA
- Wash away excess probes
- Macroscopy to visualize
Disease mechanisms
Location of pathogenic expensions matters
- Location of repeat is important: can be In intron, UTR … -> will determine the
pathogenicity
- Why do have coding expansions have trinucleotide motif and intronic expansions
can have diJerent lengths?
o More severe if it is a out of frame expansion
- Why are there no pathogenic intergenic expansions
o Transcription is important
- All these things can happen at the same time, we don’t know which one is
responsible for the phenotype
Protein-coding repeats
- More of CG -> more glutamine -> longer protein à protein will misfold à will
aggregate à neurodegeneration
o Normal protein function can be lossed
RNA repeats can aggregate
- Intteruptions aJect structure, will lead to pathological consequences
Repetitive RNA may form RNA foci
- Function will be toxic and the RNA binding proteins will have a loss of function
- Primary gain of function à secondary, downstream you will have loss of function
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