For years, researchers have been unable to find the most common genetic cause of ALS, but now its treatment is gaining ground.
By Elie Dolgin
Mark Price’s family has a long history of neurological illness. His sister and uncle died of amyotrophic lateral sclerosis (ALS) and his mother and aunt suffered from dementia. However, it wasn’t until he himself started speaking out of turn in 2010, shortly before his daughter Sharon’s wedding, that he realised there could be a genetic cause underlying the family’s tragic medical history.
During the year, Price was diagnosed with ALS and Sharon wondered if she – or her future children – would be next. I gave up my plans for a while and said to myself, ‘I can’t start having children until this is completely cleared up’,” recalls Sharon, who was 26 at the time. Initially, Sharon Price’s doctors were unable to identify any deficiencies in the ALS-related genes known at the time. Then, in September 2011, it was revealed that two teams of scientists had found a new gene linked to ALS, one that could explain up to 40% of familial cases and 10% of ‘sporadic’ cases. What’s more, this gene was the cause of around 30% of hereditary cases of a disease known as frontotemporal degeneration (FTD). Finally, it explained why this neurodegenerative disease often affects members of families who also suffer from motor neurone disease (ALS), such as Price’s family.
Mark Price in a family photo with his daughters in 1988. In 2011, he was diagnosed with ALS and found to have a defective C9ORF72 gene.
The genetic culprit is called C9ORF72 – after its location on chromosome 9 in a region known as the open reading frame (ORF) 72, which has an unusual nucleotide sequence pattern. In some people with ALS or FTD, a short piece of DNA in a non-coding part of C9ORF72 is repeated hundreds or even thousands of times; in healthy people, the same sequence – GGGGCC – is repeated less than two dozen times.
In early 2012, Price was tested for theC9ORF72 repetitive expansion. The test came back positive and he passed away a year later. And while Sharon and her two sisters mourned their father, they also had to face up to the fact that they each had a 50% chance of carrying the genetic abnormality. They now had to decide whether or not to be tested.
House hunting
The story of C9ORF72 begins with the German psychiatrist Anton von Braumühl, who first made the link between ALS and FTD in 1932. However, it was not until the mid-2000s, when the genes of several generations of large families affected by the two diseases were studied, that researchers began to focus on the short arm of chromosome 9 as the host of the gene in question. By 2010, they had reduced the study area to a strand of 232,000 nucleotides – tiny by genomic standards. However, none of the four genes in this region contained protein-modifying mutations that could explain the link between the diseases.
“It’s as if we knew which street to look down but didn’t know the exact house,” explains Ammar Al-Chalabi, neurologist and clinical geneticist at King’s College London.
The hunt was on for the gene responsible. At least five European and North American research teams set out to find it. Many thought they would be off the hook, but C9ORF72 turned out to be “very devious”, explains Ekaterina Rogaeva, a molecular geneticist at the University of Toronto in Canada. “It’s not exactly an easy field to work in”.
A group led by Rosa Rademakers, a neurogeneticist at the Mayo Clinic in Jacksonville, Florida, studied three generations of the same family whose 10 members had ALS, FTD or both. They didn’t know exactly what to look for in the genomes of these patients, “so we looked for anything that looked suspicious”, said Rademakers. So we looked for anything that looked suspicious,” explained Rademakers. That included the GGGGCC-rich piece of C9ORF72.
She and her colleagues used polymerase chain reactions (PKR) to extend this region, and identified an unusual inheritance pattern: for each family member with a neurodegenerative disease, the PKR test indicated two identical copies of C9ORF72, whereas they should have had different variants.
It was a mystery to Rademakers, until she realised that the genetic defect was greater than the upper limit that PKR could read. So she and her colleagues turned to a more sensitive technique called repeat primer PKR and observed a large expansion of repeats – but only in affected family members. None of the healthy family members, nor a thousand healthy control cases, showed this repeat expansion.
The researchers tested 696 other people with ALS or FTD to ensure that this recurrence was not unique to the family they had studied. They found the C9ORF72 mutation in 59 unrelated people, including 22 with no known family history of neurodegenerative disease. Further experiments revealed that the GGGGCC strand was repeated at least 700 times.
Rademakers remembers his excitement at this discovery, which could not be without consequences. Around the same time, an international consortium led by Bryan Traynor, a neurologist and geneticist at the US National Institute on Aging in Bethesda, Maryland, made the same discovery. Traynor got wind of the expansion of repeats because of technical shortcomings in another method of DNA analysis – next-generation sequencing. “It was exciting to sit in front of the computer and find out what was really going on,” he says.
The two teams published their results simultaneously in September 2011 in Neuron, beating the other groups. “They were indeed faster than us”, says Al-Chalabi, “but we were actually happy with that”.
Thanks to in vitro fertilisation, Sharon Stone (left) and Jodie Price, Price’s daughters, were able to avoid passing on the defective gene to their children. Photo: Sarah Keayes/The Photo Pitch
Exciting times
This discovery had an immediate impact. The frequency of the C9ORF72 disorder in patients “made it clear to everyone seriously interested in ALS that we had to work on this subject”, explains Pamela Shaw, a neurologist at the University of Sheffield in the UK.
Brian Dickie, Director of Research and Development at the Motor Neurone Disease Association in Northampton, UK, remembers flying from London to a meeting in the US in September. It was five days after the publication of the Rademakers and Traynor treatises. Several ALS researchers and clinicians were on board, and one of them had copies of the manuscripts with him. “During the flight, we passed them around,” says Dickie. “It was very exciting.
Several drug manufacturers were interested in this discovery. “It was hard to ignore the discovery of C9ORF72,” explains Brian Zambrowicz, head of functional genomics at Regeneron Pharmaceuticals, a Tarrytown, New York-based company founded to tackle neurodegenerative diseases, but which broadened its strategy 20 years ago after its first drug candidate to help people with ALS failed. According to Zambrowicz, the discovery of C9ORF72 prompted the company to refocus on ALS therapies, starting with the creation of a C9orf72 mouse model.
Ionis Pharmaceuticals, which specialises in RNA-based antisense therapies capable of eliminating disease-causing genes, also reacted very quickly. “The day the treaties were published, we immediately drew up a plan”, recalls Frank Bennett. He is Senior Vice President of Research at Ionis, based in Carlsbad, California. Within two years, Frank Bennett and his academic collaborators had demonstrated that an antisense drug could reduce aberrant levels of C9ORF72 mRNA in cell cultures. Just over two years later, they had proof-of-concept data for mouse models. A key Ionis drug candidate is now undergoing preclinical toxicology studies, with human testing expected to begin early next year.
According to Lucie Bruijn, head of science at the ALS Association in Washington DC, the speed of these developments can be explained in part by the influx of researchers keen to track down the mechanisms by whichC9ORF72 interference leads to disease. The repeated expansion was reminiscent of those found in other neurodegenerative disorders, notably Huntington’s disease, myotonic dystrophy and spinocerebellar ataxia. In addition, there was a genetic overlap with FTD. Historically, researchers studying these brain diseases worked in isolation, but after the discovery of C9ORF72, they joined forces to pursue a common goal.
“Suddenly, a lot of clinicians and scientists became interested in ALS,” says Bruijn. “This gave a huge boost to the field”. The idea of why GGGCC mutations might cause ALS or FTD had little to do with the expansion of the repeat, but rather with the normal protein C9ORF72. Rademakers found that levels of the normal protein were reduced in people with the genetic disorder. Although little is known about the role of this protein, it is thought to be involved in the transport of molecules within cells. Rademakers’ observation suggested that lower levels of normal C9ORF72 could be responsible for pathological brain responses.
Initial studies seemed to refute this hypothesis. Mice whose neurons expressed little or no C9orf72 protein showed no behaviour likely to indicate a neurodegenerative disease, and their brains showed no molecular characteristics typical of ALS or FTD. More recently, however, several teams have identified immune deficiencies in mice lacking C9orf72 in all tissues. Taken together, these results indicate that lower levels of active C9orf72 per se do not cause neuronal degeneration, although altered immune responses may exacerbate the severity or progression of the disease. “This could be a contributing factor”, says neuroscientist Jeroen Pasterkamp from the University Medical Centre Utrecht in the Netherlands, “but in combination with other mechanisms”.
Work to do
The most obvious alternative mechanism is RNA toxicity. Other diseases caused by expansions of non-coding repeats can be explained by aggregates of aberrant RNA in the nucleus which bind to ‘house proteins’, normally responsible for the proper functioning of the cell, but which have now seized up. Based on this hypothesis, molecular neuroscientist Adrian Isaacs and his colleagues at University College London created transgenic fruit flies to check whether these aggregates were pathogenic. And they were in for a surprise.
Flies with more than 100 GGGGCC repeats showed signs of C9ORF72-mediated neurodegeneration – but only if the RNA containing the repeats could be translated into a protein, and not if the RNA was entangled with translation stop signals. In other words, RNA aggregates as such were not sufficient to induce the disease. The malformed proteins seemed to be the real culprits. “I was convinced it would be RNA toxicity”, said Isaacs, “but the fly data told a different story”.
Proteins resulting from GGGGCC expansion are produced by an unusual process that does not require a start signal and that can occur even for repeated sequences located in non-coding genetic regions. Laura Ranum, a neurogeneticist at the University of Florida College of Medicine in Gainesville, was the first to describe this phenomenon in 2010, using tissues from people with spinocerebellar ataxia and myotonic dystrophy, as well as mouse models of these diseases.
According to Ranum, his findings were largely ignored by the research community. Many doubted the reality of the mechanism. However, then came the Symposium on RNA Binding Proteins in Neurological Diseases in November 2011 in Arlington, Virginia, where Rademakers and Traynor spoke about C9ORF72 and Ranum spoke about the unusual shape of protein translation. The link was quickly established.
Dieter Edbauer sat in the audience and listened to Ranum. He remembers getting out his laptop to see what kind of proteins the expansion of C9ORF72 could create. As the repeat is six nucleotides long – and protein synthesis depends on a triplet code – Edbauer realised that C9ORF72 could produce a handful of different proteins, each containing two amino acids that repeat ad infinitum. He typed in each of these potential dipeptide repeat proteins. “I looked left and right to see if anyone saw what I was doing,” recalls Edbauer, who is a molecular neuroscientist at the German Centre for Neurodegenerative Diseases in Munich. “I thought everyone had the same idea, but it turned out not to be the case”.
Fifteen months later, in February 2013, Edbauer and colleagues reported that these proteins accumulated throughout the brains of people with C9ORF72. A few days later, Rademakers and colleagues at the Mayo Clinic, led by molecular neuroscientist Leonard Petrucelli, published similar findings. Ranum did the same before the end of the year.
Since then, there has been increasing evidence to support the idea that at least some of the repetitive proteins are “unequivocally toxic and malignant”, says Paul Taylor, a molecular geneticist at St Jude Children’s Research Hospital in Memphis, Tennessee. It appears that these proteins cause neurodegeneration by stopping the trafficking of molecular cargo between the nucleus and cytoplasm in brain cells. “The essential disorder of C9ORF72 is linked to this nuclear transport”, explains Jeffrey Rothstein, a neurologist at the Johns Hopkins University School of Medicine in Baltimore, Maryland.
Pointing the finger of blame
Some researchers are now prepared to point the finger at these problematic proteins as the sole culprits when it comes to C9ORF72-mediated diseases. “To put it bluntly, toxic polydipeptides don’t contribute to disease, they underlie it,” says Steven McKnight, a biochemist at the University of Texas Southwestern Medical Center at Dallas. McKnight believes that the RNA aggregates and the reduction in normal C9ORF72 protein levels are “rather insignificant”.
However, most researchers remain sceptical. “For these simple model systems, the evidence that the protein is toxic is overwhelming,” says Aaron Gitler, a molecular neuroscientist at Stanford University School of Medicine in California, but, he adds, “in the context of human disease, it could always be a combination of factors and I have to keep my options open.”
The debate about the mechanisms of the disease is not purely theoretical: it is at the root of drug development. Some companies, including Neurimmune in Zurich, Switzerland, and Voyager Therapeutics in Cambridge, Massachusetts, are focusing solely on blocking repetitive proteins or trying to prevent their manufacture, while others, such as Karyopharm Therapeutics in Newton, Massachusetts, are hoping to inhibit deficiencies in nuclear transport without directly targeting the products of the C9ORF72 gene.
However, some therapeutic strategies, such as antisense, do not depend on the exact nature of the mechanism. Because antisense drugs can stop the production of RNA and protein, it doesn’t matter which is the causative factor in brain cells,” explains Paul Bolno, CEO of Wave Life Sciences in Cambridge, Massachusetts, which is preparing to test an antisense therapy targeting C9ORF72 on patients next year. And because levels of the repeat proteins can be detected in cerebrospinal fluid, it’s easy to assess whether or not the drug is working. “You have a measurable biomarker,” says Bolno.
Given the speed at which research by drug manufacturers is progressing, it is entirely possible that an effective therapy for C9ORF72-mediated diseases will be available by the time Mark Price’s relatives begin to show symptoms of neurodegeneration. His youngest daughter Haley is encouraged by this prospect. “Hats off to the scientific community,” she says. On the other hand, she worries that policymakers are not doing enough to support the preventive health measures available today to avoid C9ORF72-related illnesses.
Keen to expand the family, Haley and her sisters also had themselves tested for C9ORF72 shortly after their father tested positive. “Unfortunately”, says eldest sister Jodie, “there was bad news for everyone”. Since then, each sister has undergone in vitro fertilisation several times. During the process, they also had the embryos checked for the C9ORF72 disorder before implanting them. Emotionally, physically and financially, it was hard for all of them. The cost alone, they estimated, was around €100,000. But in the end, it was “confirmation that science works wonders and that we could get rid of this family curse”, says Haley.
Sharon’s son Jack recently celebrated his third birthday, Jodie will give birth to a daughter in mid-November and Haley has two frozen embryos ready to be used after her wedding on 9 December.
Translation: Bart De Becker
Source: Nature Outlook