In what one expert has called “the biggest advance in understanding amyotrophic lateral sclerosis (ALS) since the discovery of the SOD1 [superoxide dismutase 1] gene,” two research groups have announced the discovery of a new gene that underlies a large minority of familial cases of both ALS and frontotemporal dementia (FTD).
The gene, called chromosome 9 open reading frame 72 (C9ORF72), becomes the most common genetic cause of both ALS and FTD. The nature of the mutation suggests that a toxic build-up of RNA causes this form of the disease, potentially linking it to other recently discovered ALS genes, though not to SOD1, which has been up until now the most common gene for ALS.
Both studies were published in the Sept. 21 online edition of Neuron.
The hunt for the gene has been under way for the past five years, since several linkage studies and two whole-genome screens turned up an ALS “hot spot” on chromosome 9. Bryan Traynor, MD, led one of those studies, and also led one of the groups announcing the discovery of the new gene.
“We are pretty confident at this stage that the mutation accounts for about 40 percent of familial ALS cases of European descent,” said Dr. Traynor, investigator and chief of the Neuromuscular Diseases Research Group in the Laboratory of Neurogenetics at the National Institute of Aging. “It means we have gone from understanding only about a quarter of familial ALS, to about two thirds of familial ALS. SOD1 mutations cause 15 percent of familial ALS cases. We are talking about multiples of that.”
A smaller but nonetheless significant proportion of sporadic ALS cases are also explained by the mutation, he said.
The mutation is an expanded hexanucleotide repeat in a noncoding region of C9ORF72, a gene whose function is not known. The normal gene has between 2 and 20 GGGGCC units, while the mutant version has several hundred or more.
Rosa Rademakers, PhD, associate professor in the department of neuroscience at the Mayo Clinic in Jacksonville, FL, who led the second group of investigators, explained that the repeat is transcribed into RNA, which in the normal gene is edited out and degraded. But the expanded repeat folds up on itself, forming aggregates. This may be toxic to neurons and other cells in the brain and spinal cord, although future studies will be needed to confirm this hypothesis, she said.
After finding the chromosome 9 hot spot, Dr. Traynor set his sights on ALS patients in Finland, which has the world's highest incidence of the disease. That anomaly, he reasoned, suggested a common genetic cause, and one that might stand out more clearly there than in other, more heterogeneous populations. “The genetic homogeneity gives you the extra power you need,” he said.
Initial sequencing of a small number of Finnish ALS cases revealed the hexanucleotide repeat within C9ORF72. With that clue, his group undertook detailed sequencing in 402 ALS cases and 478 controls. They found the expanded repeat in almost 30 percent of patients, and fewer than 1 percent of controls. The expansion was present in 46 percent of familial patients, and 21 percent of sporadic patients. In a set of 268 familial patients and 262 controls of non-Finnish European ancestry, the expansion was found in 38 percent of patients, and no controls.
At the same time, Dr. Rademakers was studying a large family with branches in California, Minnesota, and British Columbia, with multiple members affected by ALS, FTD, or both. Analysis of their DNA indicated a linkage to chromosome 9. “When the same region emerged in the genome-wide association studies, we realized this region might have importance to a larger group of patients,” she said.
The size of the family allowed her to narrow down the region to one containing only five genes. Sequencing of each led to the expanded hexanucleotide repeat. She found the same mutation in about 23 percent of familial ALS patients unrelated to the family, and in about 4 percent of sporadic ALS patients. The same expansion was found in about 12 percent of familial and 3 percent of sporadic FTD patients.
“It has been clear for many years that there was likely to be some genetic link between ALS and FTD,” Dr. Rademakers said, since up to half of all ALS patients have some form of cognitive impairment, and half of FTD patients have some form of motor neuron impairment. “This is the first gene that brings them together on the genetic level.”
In examining brain and spinal cord pathology, Dr. Rademakers' group found that 25 percent of the cells in the frontal cortex and spinal cord of affected patients contained aggregates of expanded-repeat RNA, versus only 1 percent of cells in patients not carrying the expanded repeat.
While the length of the expanded repeat varies enormously among patients, there are not yet any data linking the length of the repeat with onset, progression, or clinical manifestation of either ALS or FTD.
The effect of the mutation on expression of the C9ORF72 protein is still unclear, with results differing between the two groups. Nothing is known about the protein, and its sequence doesn't suggest a relationship with any known proteins. “We do not know whether the protein is important for the disease,” Dr. Rademakers said, but both she and Dr. Traynor suspect the RNA aggregates are more likely to be the key to understanding the mutation's effect in the disease. Such aggregates have been increasingly linked to other neurodegenerative diseases, including myotonic dystrophy. “Is it a dual mechanism, with the protein involved as well? That still has to be determined,” she said.
Kurt Fischbeck, PhD, chief of the NINDS Neurogenetics Branch, called the discovery a “major advance,” and the most important since the discovery of SOD1 in 1993. “The field has focused on SOD for a long time, and this opens up a whole new territory, which may lead to new ideas about therapy.”
The discovery is interesting especially for what it suggests about possible mechanisms in ALS, he said, as it links the disorder to other repeat expansion diseases, especially myotonic dystrophy. The RNA aggregates in that disease have been shown to deplete the neuron's stock of an RNA-binding protein, accounting for many of the manifestations of myotonic dystrophy.
The discovery also further highlights the puzzling differences between SOD1-caused ALS and other forms of the disease. ALS caused by mutations in TAR DNA-binding protein 43 (TDP43), fused in sarcoma (FUS), and now C9ORF72 all appear to cause one or another problem in RNA metabolism, while there is no apparent RNA involvement in the SOD1 version. Both TDP43 and FUS are RNA-binding proteins, while SOD1 is not. Both C9ORF72 and TDP43 mutations, but not SOD1 mutations, lead to TDP43-containing protein aggregates as well; aggregates containing FUS protein occur in disease caused by FUS mutations. Dementia is rare in SOD1 ALS, but more common in other forms. “It is hard to put together,” Dr. Fischbeck said, but SOD1 “has got to connect up somehow.”