The Ataxia Magazine Issue 215 reported on a new paper on Friedreich’s ataxia (FA) from the team led by Professor Paola Giunti at the London Ataxia Centre, which has been recently published: https://www.mdpi.com/1422-0067/22/14/7507.
Genes are made of a chain of components, identified by a letter (A, T, C or G). In FA, the FXN gene has excessive copies of a series of repeated G-A-A letters. However, interruptions can occur in this continuous sequence of GAA repeats.
This study of 101 FA patients identified short interruptions at both ends of the GAA repeats. Small interruptions were found more commonly, and those towards the end of the GAA repeats were most frequent. Importantly, interruptions towards the end of the GAA repeat sequence were associated with later ages of disease onset.
The interruption towards the end of the GAA sequence is predicted to delay onset of symptoms by approximately nine years relative to those lacking interruptions. This study highlights the role of interruptions in modulating the FA condition and prognosis.
Dr Suran Nethisinghe is joint first author of the paper and a postdoctoral research associate working with Prof Paola Giunti at the London Ataxia Centre. Please read his full interview about the paper below.
An introduction to Dr Suran Nethisinghe
Question 1. How long have you been working on Ataxia research?
It is more than a decade I am working on the genetic ataxias. I have been mainly working on repeat disorders, and the role of interruptions in those repeats and how it affects disease severity. I also have been working on other ataxias such as ARSACS.
My PhD was in virology, in basic science. However, I wanted to work on research with a direct impact on patients. Therefore, when the highly translational project came up on interruptions in SCAs (spinocerebellar ataxias) with Prof Paola Giunti, I jumped at the chance. It was my first post-doc, and I have enjoyed ataxia research ever since.
Scientific phrases to clarify to understand the paper
Question 2. What is a GAA trinucleotide repeat in simple terms?
A GAA trinucleotide repeat is a stretch of DNA sequence where the letters in the DNA sequence are G-A-A. The trinucleotide repeat means that these 3 letters are repeated over and over. So it forms a long stretch of repeats that reduces the “production” of the relevant protein, frataxin. In FA patients it normally ranges from 70 to over a thousand.
Question 3. What is a repeat expansion in simple terms?
When DNA is repeated over and over in a long series it is unstable. It has the tendency to increase in length or expand, hence the term repeat expansion. The actual mechanism for repeat expansion is still being investigated but one theory suggests that, when the repetitive DNA comes to be replicated (e.g. when DNA is duplicated during cell division), the machinery that does the replication may slip or stutter as it goes along this repeat DNA sequence. The slippage, if it goes unchecked, will lead to the repeat becoming even longer. The longer the repeat stretch the more likely slippage or stuttering will occur and therefore the more unstable the repeat becomes.
These repeats are present in the general population but need to pass a certain threshold in size to cause the disease in an individual. The GAA repeat tract contains 5 to 68 repeats in the general population, whereas 66 to 1700 repeats can cause FA. The longer the repeat, the more severe the disease. As you can see, there is some overlap in the upper end of the number of repeats in the general population and the lower end of the disease-causing number of repeats (indeed an FA patient has been reported with 56 repeats) so this threshold is not a strict cut-off.
In FA, the repeats are present in a stretch of DNA that does not code for protein, but their expansion causes the production of the protein affected in FA, frataxin, to be turned off like a tap in a process called gene silencing. It is the lack of frataxin that causes the disease.
Question 4. What are ‘interruptions’ mentioned in your paper?
The repeat for FA has the sequence GAA, and this is repeated several times. For example, in an individual with FA they might have 600 copies of GAA all in a row. An interruption is a change in the sequence to disrupt the repeat. Often you will have a single change in the letters, e.g. GAG. Typically, it will be an insertion or removal of a single letter. Interruptions make the stretch impure, interrupting the sequence.
Previously, our team at the London Ataxia Centre along with our collaborators published a report showing that large interruptions in this continuous sequence of GAA repeats are rare in FA patients (https://www.frontiersin.org/articles/10.3389/fncel.2018.00443/full).
Question 5. How did you detect the ‘interruptions’?
In our previous study we used restriction enzymes as a tool to detect interruptions. Restriction enzymes cut DNA at a specific sequence and the enzyme called MboII can cut, or ‘digest’, DNA at a pure GAA repeat. Any changes in the pattern of DNA after this digestion, would suggest that interruptions are present. The study showed that these interruptions detected by MboII are rare. However, MboII is only sensitive enough to detect large-scale interruptions and cannot detect small interruptions.
With our current study, we used a technique called triplet repeat primed PCR (TP PCR). PCR allows you to amplify DNA based on a certain sequence, like photocopying a certain area of DNA. Triplet repeat primed means that we are amplifying using a specific sequence of repeat. So, we are using this method to amplify DNA based on the GAA repeat.
The advantage of this method over MboII is that it can detect small interruptions, however the sensitivity of this method is such that we can only detect interruptions about 100 repeats into the repeat at each end of the repeat region.
Question 6. What is does ‘the 5’ and 3’ ends of the GAA repeat tract’ mean in the paper?
This is just technical notation for the start (5’) or end (3’) of the GAA repeat region.
Findings and significance of the paper
Question 7. What were the key findings of your paper?
We were able to show that these small interruptions are often single letter changes in the GAA repeat and are more commonly present than large-scale interruptions that were previously described in our other study.
We used a cohort that comprised of mainly patients who were taking part in the EFACTS study at the London Ataxia Centre (http://www.e-facts.eu/) and collaborators. The EFACTS study involves participants having annual visits and researchers studying the change in the condition over time. I would like to thank these patients for their participation. Since FA is a relatively rare disorder, we would not have had the numbers to form this kind of study without their active participation. We are very grateful for the participation of patients in EFACTS and other studies that are ongoing. If anyone would like to take part in EFACTS visit: https://www.ataxia.org.uk/ataxia-research/taking-part-in-research/for-people-with-friedreichs-ataxia/.
We grouped the patients in our cohort into those that have interruptions towards the start, interruptions towards the end, interruptions at both ends, or those that do not have interruptions at either end of the repeat region.
An individual has two copies (also known as alleles) of the frataxin gene which contain GAA repeats. An individual inherits one copy (allele) of the frataxin gene containing a GAA repeat tract from each parent. The size of the GAA repeat on each allele (copy) may differ, with the smaller one being called GAA1 and the longer one being called GAA2. The longer the repeat, the more silencing of the gene, so less of the protein is produced. Therefore, the amount of protein you are actually getting is determined by the shorter allele, GAA1. Typically, we use this to determine disease severity, in terms of age of onset. An earlier age of onset is linked to increasing size of GAA1.
We found that individuals with interruptions towards the end of the repeat tract tend to have shorter repeat GAA1 repeat sizes and have later disease onset. This suggests that interruptions towards the end of the repeat is key in modulating the FA condition.
Another output of this study is how we mathematically modelled the age of disease onset from the GAA1 repeat size. In the paper, using our novel model, the interruption towards the end of the GAA sequence is predicted to delay onset of symptoms by approximately nine years relative to those lacking interruptions. This finding could possibly inform genetic counselling. However, although our study is useful, the cohort numbers in some of the sub-groups remain small and would benefit from more data (participating patients) to improve the model.
Question 8. What is the significance of this research for FA patients?
These results are exciting as interruptions will help in understanding how FA will unfold for individuals. This is the first step to possibly translating these findings into clinical practice. It will possibly help with grouping FA patients in clinical trials. We are grateful for the participation of patients in this study. After my PhD, I was eager to work in research that has the potential to have a real impact on patient care. I would encourage patient participation in ongoing studies as this will help unravel the pathological mechanism of the disease and possibly exploring new therapeutic options.
In addition, it is important to move forward in the understanding of the repeats. There is a lot to still be discovered about the role of interruptions. Why do interruptions towards the end of the GAA sequence have such an impact? How does a single interruption have such an impact? It seems remarkable and the mechanism behind this keeps me inspired to try and understand what interruptions are doing. It is a fascinating area.
Furthermore, we would like to understand in detail how the interruptions affect FA. New sequencing technologies are being developed that could shed light on this and it is something we are looking to pursue.
Date posted: 27/09/21