October 31, 2011 (Montreal, Quebec) — A hexanucleotide repeat expansion in the C9ORF72 gene has been identified as the cause of chromosome 9–linked amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD), accounting for a substantial proportion of both familial ALS and FTD in affected patients. FTD is the second most common form of dementia in patients younger than 65 years of age.
The results of the study were discussed here at the 12th International Congress of Human Genetics and the 61st American Society of Human Genetics Annual Meeting, and were published October 20 in Neuron.
A consortium of investigators undertook parallel, next-generation, deep resequencing of the chromosome 9p21 region in DNA that had been enriched for chromosome 9, said Bryan Traynor, MD, chief, neuromuscular diseases research group, laboratory of neurogenetics, National Institute of Aging, National Institutes of Health, in Bethesda, Maryland, at a press conference. The DNA was obtained from a person affected with familial ALS.
Analysis of sequence data from this DNA eventually revealed 8 novel variants in a 232 kb block in the target region of chromosome 9. "When I looked at those 8 variants, I noticed that 6 of them lay within a 30 base-pair window, which is strange," Dr. Traynor explained. Examination of individual sequence reads within this region showed that the computer algorithm had lined them incorrectly.
"However, when I lined the sequences manually, it became clear to me that this case carried more hexanucleotide repeats than a standard human reference genome. That was our Eureka moment — we knew there was something going on here," Dr. Traynor said. Subsequent screening of case and control samples for the presence of this GGGGCC hexanucleotide repeat with repeat-primed polymerase chain reaction (PCR) confirmed that the expanded hexanucleotide repeat was present in every single affected case in those families, he added.
Personal Search
Dr. Traynor's personal search for the genetic cause of ALS actually began in Finland, where the population is homogeneous and thus conducive to genome-wide association studies (GWAS). Finland also has the highest incidence of ALS in the world. Previous studies have implicated a specific locus on chromosome 9 that was clearly important for ALS, although characterization of the specific genetic defect remained elusive. "When we did GWAS of ALS in Finland, we found a very high association signal on chromosome 9 within this previously defined locus on an interval that was about 7.2 million base pairs," Dr. Traynor explained.
They whittled this down to about 232,000 base pairs. Even though the region contained only 3 genes, "we knew it was going to be very difficult to find," he added. Dr. Traynor and colleagues, therefore, formed a consortium with other laboratories that had ALS families linked to chromosome 9. These labs provided the National Institutes of Health with all of their DNA samples, so they could then be exposed to infinite sequencing and computation.
Having subsequently identified the expanded hexanucleotide repeat, investigators went back to their original Finnish cohort in which repeat-primed PCR analysis showed that the same mutation on the C9OFR72 gene was expanded in 28.1% of Finnish ALS cases and in only 0.4% of controls. "We knew that whatever the genetic lesion was, when we found it, it would be present with a high frequency in the Finnish. Lo and behold, it was, accounting for nearly 50% of familial cases in Finland and about 20% of sporadic cases," Dr. Traynor said.
What investigators were not expecting to find was the same mutation occurring at a high frequency in "outbred" European populations. Having screened a cohort of 268 familial ALS probands in Germany, Italy, and North America, investigators found that 38.1% of these probands carried the same hexanucleotide repeat expansion in C9OFR72.
Although a number of other genes cause a certain proportion of familial ALS, "we went from understanding about one quarter of familial ALS to understanding two thirds of familial ALS in outbred European populations; in the Finnish population, it's even higher," Dr. Traynor observed. Dr. Traynor also noted that it has become clear that ALS and FTD are part of the same spectrum.
They tested the hypothesis that the hexanucleotide repeat expansion underlies a proportion of FTD cases as well. Having measured its occurrence in a cohort of 75 Finnish FTD cases, repeat-primed PCR analysis showed that the percentage of FTD cases carrying this mutation was comparable to that of the Finnish ALS cohort. As Dr. Traynor stressed, the function of C9OFR72 is not known.
They speculate, however, that this large expansion generates toxic RNA species within the nucleus, where they end up amalgamating into a ball. "This ball soaks up other RNA binding proteins and mucks up transcription; if you do not have transcription, the cell is dead," he noted.
Lucie Bruijn, PhD, chief scientist, The ALS Association, Washington, DC, told Medscape Medical News that they are "extremely excited" about these findings.
"This is a huge breakthrough in the field of ALS. We will probably learn something about FTD as well, so it is a major change in the field," she said. Dr. Bruijn pointed out that the same genetic abnormality was identified by a separate group of researchers, led by Rosa Rademakers, PhD, from the Mayo Clinic, Jacksonville, Florida, further solidifying the validity of the findings (Neuron. 2011;72:245-256).
As Dr. Bruijn observed, it is extremely interesting that the hexanucleotide repeat is found in the noncoding region of a gene on chromosome 9p21, "so it sits in DNA where protein is not going to be read out." As the study by Rademakers et al showed, this implicated area of DNA normally repeats only 2 to 23 times in healthy individuals; in ALS and FTD patients, it repeats between 700 to 1600 times.
This likely leads to a major dysregulation of RNA metabolism, noted Dr. Bruijn, preventing proteins from carrying out normal function in the cell.
Dr. Traynor and Dr. Bruijn have disclosed no relevant financial relationships.
Neuron. 2011;72:257-268. Abstract
12th International Congress of Human Genetics (ICHG) and the 61st American Society of Human Genetics (ASHG) Annual Meeting. Presented October 20, 2011.
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