Samenvatting Les 3 'Repeat expansion disorders'

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