NEW YORK (GenomeWeb News) – A new study is adding heft to the notion that cytoskeletal pathway alterations can spur amyotrophic lateral sclerosis, or ALS, commonly known as Lou Gehrig's Disease.
An international effort spearhead by investigators at the University of Massachusetts used exome sequencing to look for genetic glitches behind an inherited form of ALS, which accounts for around 10 percent of ALS cases.
As they reported online yesterday in Nature, the researchers found multiple ALS-associated mutations in the PFN1 gene when they pored over protein-coding sequences for individuals from two extended families with familial ALS. And mutations in PFN1 seemed to explain an estimated 1 to 2 percent of the familial ALS cases tested for the discovery and follow-up phases of the study.
PFN1 codes for a profilin protein that interacts with actin and helps it grow from single subunits to filaments, making it the latest in a string of cytoskeleton-related genes to be implicated in ALS.
"We know of at least three other [risk] genes that seem to affect cytoskeletal pathways — and probably more than that if you include [motor neuron] diseases related to ALS," University of Massachusetts neurology researcher John Landers, the study's senior author, told GenomeWeb Daily News.
Though more functional analyses of normal and mutant PFN1 are needed, the team's cell line experiments so far are consistent with an actin-related role for PFN1 in helping to build the axon structures that electrical signals traverse as messages move between nerve cells.
Together with findings from past studies, such experiments suggest that axon malfunction stemming from impaired cytoskeletal scaffold function could be one cause of the progressive motor neuron death characterizing ALS.
Past genetic studies have identified several genes that are altered in ALS, Landers explained, including some involved in cytoskeletal function or RNA processing. Even so, with almost half of all familial ALS cases stemming from indeterminate genetic factors, he and his colleagues suspected that they might find new genetic contributors to ALS by focusing on families affected by the progressive, ultimately fatal, neurological disease.
"We were looking to find additional ALS genes to see if there's a common thread between all these different genes," Landers said.
For the first phase of the study, the team did exome sequencing on two individuals per family, focusing on individuals with ALS from the furthest branches of each family tree.
Within each of the two large ALS families included in the study, affected individuals had been tested and found to be negative for mutations in known ALS-associated genes, researchers explained.
Using Nimblegen SeqCap EZ exome arrays, they captured targeted protein-coding sequencing for each individual and then sequenced the exomes to a depth of more than 150 times, on average, with the Illumina GAII or HiSeq2000. After tossing out variants that turned up in dbSNP, National Heart, Lung, and Blood Institute Exome Sequencing Project data, or 1000 Genomes Project data, the team tested the suspicious variants that remained by Sanger sequencing, searching for alterations that segregated with disease in each family.
In both families, individuals with ALS carried mutations to the chromosome 17 gene PFN1 that appear to be inherited in a dominant manner, though the nature of the mutation detected differed between the two families.
PFN1 mutations turned up when the team did targeted sequencing of the gene in hundreds more individuals from familial ALS, too. Overall, seven of 274 familial cases tested carried alterations in the gene.
When they sequenced the gene in 816 non-familial, sporadic ALS cases, the investigators found two individuals with PFN1 mutations. These appeared to fall at the less pathogenic and lower penetrance end of the PFN1 alteration spectrum, Landers noted.
In a series of follow-up experiments, meanwhile, researchers showed that changes to PFN1 can, indeed, impact profilin's function in the cytoskeleton. For example, in primary motor neuron cell lines containing some mutant forms of the gene, profilin protein clumped together rather than spreading out in the cell's cytoplasm as it normally does. And some of these mutant profilin aggregates contained a protein encoded by another ALS related gene: TDP-43.
Consistent with the observation that mutations in PFN1 typically turn up near sites coding for actin-binding sites, the researchers reported, profilin mutations in cells also coincided with impaired actin protein binding and the production of stubbier-than-usual axons.
"In healthy neurons, PFN1 acts almost like a railroad tie for fibrous filaments called actin, which make up the axon," Landers explained in a statement. "PFN1 helps bind these filaments to each other, promoting outgrowth of the axon. … Here we show that mutant PFN1 may contribute to ALS pathogeneses by accumulating in these aggregates and altering the actin dynamics in a way that inhibits axon outgrowth."
These latest revelations not only add to the mounting evidence that cytoskeletal malfunction might be at play in ALS and other motor neuron disorders, the study's authors noted, but they also point to the cytoskeleton pathway as one in a growing list of potential treatment targets.
"There is growing evidence that ALS may be caused by a variety of cellular defects, and that it is a not a disorder with a single origin," according to Amelie Gubitz, who directs the National Institute of Neurological Disorders and Stroke's neurodegeneration research program.
Gubitz was not directly involved in the study, though NINDS did provide some of the funding for the effort.
"Whether and where these disease pathways converge is an active area of research with important implications for therapy development," she said in a statement.
For their part, Landers said he and his colleagues are continuing to search for new ALS risk genes using exome sequencing and other approaches.