The development of a novel biomarker for ALS and FTD based on epigenetic studies of C9orf72
University of Toronto, Tanz Centre for Research in Neurodegenerative Diseases, Sunnybrook Health Sciences Centre
Frontotemporal Dementia (FTD) is characterized by behavioral/language changes, while Amyotrophic Lateral Sclerosis (ALS) is result of the degeneration of motor neurons leading to paralysis. The most common mutation for both syndromes is a repeat-expansion in C9orf72, usually ranging from hundreds to thousands of repeats. Currently it is unclear whether expansion alleles with different sizes have the same pathological consequence, and the lower-limit for pathological repeat number has not been determined. One of the disease mechanisms is related to low level of C9orf72 in mutation carriers. With the support of 2012 Rapid Response Grant, we demonstrated that the methylation level of the region nearby of the pathological repeat was significantly higher in ALS and FTD expansion carriers vs. non-carriers. Intriguingly, we detected a high methylation level in only 36% of mutation carriers; however down-regulation of C9orf72 expression was reported in all of the evaluated carriers. Therefore, additional DNA regions could be subject to methylation. In the current proposal, we aim to develop a novel assay to directly evaluate the DNA methylation profile of the repeat itself and study potential links between DNA methylation and histone methylation that could also be involved in gene silencing. After establishing the new assay, we will use it to screen our DNA samples obtained from ~600 controls, ~400 ALS cases and ~500 FTD cases.
Relevance to the acceleration of therapeutics for neurodegenerative diseases of aging
The mutation in C9orf72 is accounting for up to 37% familial and 7% sporadic cases in Caucasians. No other gene discovered to date has been shown to cause neurodegenerative disease in such a high number of patients. The unifying objective of this program is to uncover properties that can be exploited for the generation of rational strategies for differential diagnostics for C9orf72-related disease that circumvents the technical difficulty in measuring the length of the expansion. For instance, we hope to explain a wide clinical diversity of C9orf72-related phenotypes ranging from pure ALS to mixed ALS/FTD to pure FTD with highly variable age of onset (20s-80s years) and rate of disease progression (a few months to decades).
We will determine if the methylation level of the repeat differentiates between disease status (ALS vs. FTD) and other clinical parameters (e.g. age of onset or disease duration). Also, we will assess if the methylation level is reflecting the repeat length. For example, we will compare carriers of intermediate alleles (20-50 repeats) with carriers of small alleles (<20 repeats) or large alleles (>50 repeats). If we find that investigated markers correlate with disease status, sub-phenotypes or disease severity, we will be able to establish criteria for pathological C9orf72 alleles and therefore greatly improve patient diagnosis and genetic counseling.
There is a class of mutations that can be described as “stuttering DNA” with long tracks of repeats. The repeat expansion in the C9orf72 gene is the most common genetic cause of ALS and FTD. The size of the expansions is very variable between different individuals (30-8000 repeats), and whether these alleles have the same pathological significance is unknown. Understanding the mechanistic basis of the toxicity behind mutant C9orf72 is vital to the development of effective therapies and diagnostics. Expansion carriers produce about half the normal amount of C9orf72 protein. Because the addition of methyl groups to a DNA region could silence gene expression, we investigated if such modifications explain the reduction in C9orf72 protein. We hypothesize that the methylation suppresses the production of toxic RNAs and peptides, which also leads to collateral damage (suppression of the normal C9orf72 protein).
Evaluating methylation within the repeats was not an easy task. During the grant period we successfully developed a novel method to analyze the methylation status of the repeat expansion, and demonstrated that methylation is a direct consequence of the expansion. When we examined the data from multiple blood and brain samples, a clear pattern emerged. For DNA with up to 90 repeats, no methylation was present, but once this region extends beyond 90 repeats, it always picks up methyl groups. This pattern held true regardless of a person’s diagnosis of ALS or FTD. Notably, the final interpretation of our results will be the subject of ongoing investigation.
Our assay has qualities of a biomarker based on the three following arguments. First, it could predict large vs. small/intermediate alleles: methylation was detected only for large expansions, but not for normal (2-22 repeats) or intermediate alleles (up to 90 repeats). Second, the methylation was identified in both blood and brain tissue of the same individual. Third, the methylation is stable over time, since it was detected in carriers with a wide range of ages (24–74 years); and it is present in both patients and currently unaffected (presymptomatic) individuals.
Importantly, our assay is useful for establishing a more accurate cut-off for pathological number of repeats. We concluded that small expansions (<90 repeats) might be considered ‘‘pre-mutations’’ to reflect their propensity to expand in the next generation and/or within different tissues of the same individual, including brain. For instance, we reported a Canadian family in which 70-repeats from the father (unaffected at age 89 years) expanded during parent-offspring transmission and started the first generation affected by ALS (four children carry a 1750-repeat allele). Hence, our study supports the hypothesis of multiple origins for the expansion; and follow-up studies might help explain the unusually high frequency of C9orf72-patients without family history of ALS or FTD. Likely, these cases represent the first generation in which the mutation jumped to the pathological range.
Notably, our assay revealed that the epigenetic profile has been reset in immortalized skin cells (fibroblasts). To further investigate DNA methylation at the C9orf72 locus in fibroblasts and embryonic stem cell lines, we established international collaboration with teams in the UK and Israel. Such studies are important because cell models are used for investigating the ALS/FTD disease mechanism.
We generated data that contributed to 17 presentations and 14 pear-review publications, including 5 manuscripts in preparation that will likely be published in 2016.