CNSD 3.4 ALS and non-coding DNA variants

What is the problem in ALS?

There is a missing heritability, whereas in 12 out of 100 cases a genetic cause can be identified.

What is a non-coding DNA variant?

A DNA sequence that does not encode for proteins.

How much of the human genome is non-coding?

• 4M variants per human genome

• 1% in protein-coding sequences and 99% not in protein coding DNA

What is the influence of non-coding DNA?

It can influence mRNA processing, chromatin interactions, altering transcription, altering proteins, etc.

What 3 functional elements are impacted by non-coding variants?

• Canonical/cryptic splice sites & mRNA splicing: alternative splicing will lead to different mRNAs that lead to different protein products

• 3’ UTR usage & mRNA stability: UTR determines how long the mRNA will stay in the cell, and will thus be translated until it will be broken down

• Promoters/enhancers & gene expression: the promotor is the primary source of gene regulation. DNA loops bind the enhancer to the promotor and then different transcription factors are recruited to start the transcription. This can be influenced by the fact that there might not be looping or because of a mutation in the binding site of the transcription factors, a mutation in the promotor, etc.

Why is the study of non-coding variants difficult?

There are a lot of them. 21 countries → >10.000 ALS genomes → 170M DNA variants. Moreover, a lot od non-coding functional elements are cell specific and it is hard to predict what a given non-coding variant does (not the right tools yet).

What is an example of a non-coding DNA variant in ALS?

C9ORF72

What does GWAS suggest?

That the majority of heritable disease risk in human populations is determined by non-coding variants.

What does the non-coding mutation C9ORF72 cause?

More repeats, and therefore also both loss- and gain of function of the gene.

• Loss of function: the number of proteins goes down

• Gain of function: RNA toxicity and DPR toxicity

What is meant by canonical and cryptic splice site variants in ALS?

• Canonical = what you expect too see in a normal person

• Cryptic = disease focused

What kind of mutations on the canonical splice sites in KIF5A by rare variants are responsible for ALS?

Mutations on the tail (cargo binding) of KIF5A.

What does the mutation in KIF5A cause?

Almost all patients had mutations that caused changes in the splicing. With normal splicing, exon 27 is in the RNA, but because of the mutation exon 27 is removed. This will change the form and thus the function of the protein. This is because you are missing part of your protein, wherefore interactions will change.

What is not reliable about the mutation in KIF5A?

The found splicing-altering variant is actually upstream of exon 27. It is thus actually too far way from the exon to determine if it actually causes different splicing. With the help of coloring, you can see if exon skipping takes place. You will then look at the amount of mScarlet still present.

What are challenges when researching splicing?

There are several factors that can influence splicing. For example, you might have to deal with exons that are not true exons. If these are present, it will lead to a disruption in the mRNA sequence.

What is UNC13A?

A cryptic site suppressed by TDP-43. In a lot of studies UNC13A was found to be associated with ALS, but follow-up studies proved that this was actually not the case.

What led to the belief that UNC13A was associated with ALS?

TDP-43 might play a role. TDP-43 causes inhibition of the splicing site, wherefore the splicing changes. Therefore, ALS patients will get more exons that look like exons, but do not belong in the mRNA.

What elements are important in gene expression?

• Promotors

• Enhancer

• Cell specificity

Promotors and enhancers may be cell specific.

What is meant by active DNA?

Open chromatin conformations that can be read.

What does ATACseq do?

It is a technology to map active DNA. It is a technique with which you can find the open areas in the chromatin and thus the active DNA. You can than perform a genetic analysis of the rare ATAC regions found in ALS.

So we find thus enhancer that seems protective and neuron-specific. What would you do next?

Look at which genes the enhancer regulates. You remove the enhancer and look at which gene expression goes down.

CFAP140 seems to be regulated by this enhancer. What do you do next?

Experimental design: iPSCs → neuronal differentation → motor neurons → knock down CFAP41 → phenotype amelioration

What are future directions to study ncDNA variants in ALS?

• Deeper single cell mapping of key functional elements

• AI to predict functional impact of non-coding variants: e.g. a model where you put sequencing data and it makes, based on the given data, new data which can predict is the area is open or closed

• Mapping other functional non-coding elements:

  5’ UTR: translational control, stability

  3’ UTR: translational control, subcellular localization, stability