Structure of C9ORF72 repeat identified by MND Association funded researchers

New exciting findings announced today provide the first insight into the structure and function of a repeated six letter genetic sequence in an MND gene called C9ORF72.

Understanding the function of C9ORF72, and how it could go wrong to cause MND, could assist researchers in the future to identify potential treatments that target the disease.

The finding was identified by University College London researchers including Dr Adrian Isaacs and Dr Pietro Fratta. Dr Fratta is a recipient of a Medical Research Council/MND Associaiton’s Lady Edith Wolfson Clinical Research Fellowship.

Their findings were published in the reputable scientific journal Scientific Reports on 21 December 2012.

C9ORF72 – the plot thickens

In 2011, MND Association funded researchers discovered that a repeated six-letter code within a gene called C9ORF72 can cause MND and a related condition called fronto-temporal dementia (FTD) for approximately 40% of cases with a family history of MND and/or FTD. Having a family history of MND is rare and affects 5-10% of people with MND.

In people without MND, this six-letter code (GGGGCC) is repeated up to 30 times. In C9ORF72 MND or FTD, this sequence can be excessively repeated between 700 and 1,400 times.

Since this pivotal discovery, researchers have started their journey to search for answers to find out more about C9ORF72 and how it can cause MND.

This study aimed to identify whether the six-letter code normally forms a specific structure when in its copy (RNA) form. Forming a structure normally means that something has a particular role. If this seemingly innocent piece of repetitive code does form a structure, then it could mean that excessively repeating it could cause problems by being over active, or by stopping other functions.

Dr Adrian Isaacs
Dr Adrian Isaacs

Dr Isaacs who led the study explains, Nothing is currently known about how the mistake in C9ORF72 kills motor neurones. The mistake in C9ORF72 is similar to mistakes that cause some other neurological diseases.”

“In these diseases the mistake leads to the formation of toxic aggregates of RNA –RNA is a copy of DNA that is made when a gene is switched on and is important for the generation of proteins.”

 Dr Issacs and colleagues used advanced analytical chemistry to identify the structure that the repeated six-letter code (GGGGCC) forms and to suggest its potential role.

RNA G-Quadruplex, glorified Battenberg cake

This shape, and structure that has been identified for the repeated six letter code in the copy of C9ORF72 is called an ‘RNA G-quadruplex’.

In real life terms, an RNA G-quadruplex would look –with some artistic license granted – like a Battenberg cake.

RNA G-quadruplex looks like a Battenberg cake

The four coloured sponge squares would be the individual letters of the code – all being the ‘GGGG’ part of the sequence running along the length of the structure and forming four ‘slices’.

Each line of four Gs (coloured length of sponge), is stuck together to another line of four Gs in the structure by strong hydrogen bonds (the jam!).  This forms the four-square pattern that makes up each ‘slice’. Each line of four Gs is also attached to its phosphate backbone, which is the outermost section of the structure (the marzipan).

The only addition to the Battenberg that’s missing to create an RNA G-quadruplex would be a metal ball, or ion sitting in the middle of each of the four slices.

What does it do?

Having a structure means that the repeated six-letter code is of importance to find out whether it has a function. Having a function would then mean that the genetic expansion could have a detrimental effect on its usual role in the cell.

The forming of these Battenberg cake-like structures means that it could perform a specific role in the body. To date, quadruplexes have been identified as having a number of roles in the body, including editing copies of genes to create functional proteins.

Dr Pietro Fratta
Dr Pietro Fratta

Dr Pietro Fratta explains how this structure could play a role in C9ORF72 MND, “One possibility is that the RNA G-quadruplexes accumulate in motor neurones and then different proteins within the cell somehow bind to this structure and get stuck. As a result the motor neurones malfunction and perhaps even ultimately die.”

Commenting on these findings, MND Association’s Director of Research Development Dr Brian Dickie said “The UCL scientists have opened up an exciting new avenue of research.”

“At the moment we know very little about whether, or how, these RNA structures may be linked to MND, but evidence from other diseases indicates that they are biologically active and therefore likely to be important to the function and health of nerve cells.”

Following this finding, the next steps for researchers will be to determine the function of the G-quadruplex in nerve cells, and the effects of the excessive repeat in MND has on the function of these quadruplexes.

Paper reference: Fratta P. et al. C9orf72 hexanucleotide repeat associated with amyotrophic lateral sclerosis and frontotemporal dementia forms RNA G-quadruplexes. Scientific Reports 2012 DOI: 10.1038/srep01016

Read our news release on this story.

Chromosome 9 finally reveals its secrets

It’s taken a huge international collaboration, including 3 MND Association-funded scientists, to discover a genetic mistake that appears to cause almost 40% of cases of familial (inherited) MND – that’s nearly twice as many as are caused by mutations in the SOD1 gene and more than three times as many as are caused by TDP-43 and FUS combined. Yet despite the fact that it’s relatively common, the rogue gene proved especially difficult to find.

Digging for genes

Our genetic code is arranged into 23 pairs of subunits called chromosomes. Earlier work had homed in on an area on chromosome 9 that appeared to be significantly associated with both MND and the related neurodegenerative disease frontotemporal dementia (FTD), but nobody could drill down as far as the problem gene itself. As a result, chromosome 9 became something of an ‘archaeological dig site’ for MND researchers, with several groups using cutting edge techniques to try and excavate the elusive causative gene that they knew was lurking somewhere in the short arm of this chromosome. The successful international team, which included almost 60 scientists at 37 institutes, finally discovered the exact location and nature of the aberrant genetic code by looking in the most unlikely of places – in the stretches of DNA that do not actually provide any instructions for building proteins, otherwise known as non-coding DNA.

What did the researchers unearth?

The research team studied DNA samples from a Welsh family affected by inherited MND and FTD that was already known to be associated with chromosome 9, as well as samples from a similar Dutch family and a large number of Finnish inherited and non-inherited MND cases. In among the non-coding DNA in a chromosome 9 gene called C9ORF72, the researchers found a 6-letter genetic ‘word’ which, in healthy individuals, is consecutively repeated up to about 20 times. However, in the Welsh and Dutch families and a large proportion of the Finnish familial cases, the 6-letter word was repeated as many as 250 times. This phenomenon is known as a ‘repeat expansion’. The researchers went on to check for this repeat expansion in familial MND cases from North America, Germany and Italy, and found it cropped up in 38% of them. They even found it in a much smaller proportion of sporadic cases from Finland, suggesting that it could be an important risk factor in at least some people with the  non-inherited form of the disease.

What does the discovery mean for MND research?

Despite the fact that the repeat expansion does not directly affect the instructions for building a protein, there is good reason to believe that it can still lead to significant neuronal damage. At the moment it is not fully understood how this happens, but one possibility is that it leads to the production of excessive and consequently toxic quantities of RNA, the molecule that provides the cell with a more usable copy of DNA. Disruption to RNA processing has already been implicated as a disease mechanism in MND – this is the pathway through which faulty TDP-43 and FUS are thought to exert their effects – so C9ORF72 may provide scientists with another piece of the RNA jigsaw.

The effect of the repeat expansion is clearly open to influence. Among those people with the repeat expansion, some experienced only FTD, others showed only muscle weakness, and some had both MND and FTD.  The reasons for this variation in symptoms will be just one area that scientists will now want to look into. This overlap between MND and FTD is something that researchers are very keen to understand, and the C9ORF72 discovery may be the key to solving this puzzle. They will also want to better understand how the repeat expansion causes damage, and that will include trying to find out what C9ORF72 actually does – at the moment this is unknown. (Maybe it’ll get a more interesting name along the way!) Building on the new finding in this way could help move us closer to an effective treatment.

For now, a more tangible consequence of the discovery could be a genetic test for people already diagnosed with familial MND who want to understand more about the basis of their disease. Such a test will take a little time to develop but should become available in the UK in the next few months. When it does, it will be accessible to genetics labs across the country. Anyone interested should speak to their doctor or specialist nurse.  

Dead heat

Just as archaeologists might question whether a newly discovered artefact is the real thing, so scientists need double-checking when they claim to have made a new discovery. Fortunately, a second team hit upon C9ORF72 at exactly the same time, and their results will be published alongside the work described here, in the journal ‘Neuron’. The race to the ‘Lost Ark’ of chromosome 9 ended in a tie, but has provided the research community with a major piece of the MND puzzle on which to build future discoveries.

Article: Renton A, Majounie E, Waite A et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked amyotrophic lateral sclerosis-frontotemporal dementia. Neuron (2011).

Read our press release on the C9ORF72 story.