A methylation study of C9orf72 and the development of a novel diagnostic test
University of Toronto, Tanz Centre for Research in Neurodegenerative Diseases
It was recently recognized that two fatal neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), belong to the same clinico-pathological spectrum. One of the most significant discoveries in human genetics is the recent detection of the (G4C2)30-1600 repeats expansion in the C9orf72 gene, which is the most common cause of both FTLD and ALS. In addition, we reported the association of C9orf72 with Alzheimer’s disease and Parkinson’s disease, suggesting a role for C9orf72 in several common disorders of aging. However, the current technology cannot resolve the precise length of the expansion allele above ~50 repeats and only detects the presence/absence of the expansion (yes/no test). The first studies suggested that ~30 repeats could be pathological. However, our previous study raised concern about a reliable cutoff for the pathological repeat number, which is essential for both basic research and the utility of genetic screening in patient care. We propose to establish an assay reflecting the functional consequence of the repeat expansion and translate this knowledge into an empirical test that has predictive value for early disease diagnosis.
Relevance to the acceleration of therapeutics for neurodegenerative diseases of aging
Our project is novel in translational research and is related to several neurodegenerative diseases. We will study C9orf72 by applying well-established methods in the epigenetic field. In general, epigenetic mechanisms are a heritable and dynamic means of regulating numerous genomic functions, including gene expression, through modifications of DNA. There is increasing evidence that, in addition to DNA sequence and the environment, epigenetic modifications of DNA may contribute to complex neurodegenerative diseases. The use of an epigenetic concept is a novel approach for studying the consequences of the repeat expansion and will provide important knowledge to C9orf72-related pathogenesis.
We hope to understand whether the epigenetic profile could underlie the major molecular mechanism of C9orf72 pathogenesis and thus develop the diagnostic potential using this information. We will be able to use our results to establish a more reliable cutoff for pathological C9orf72 alleles and hence improve patient diagnosis. Importantly, it is possible that the pathological cutoff is disease-dependent (e.g. ALS vs. FTLD); and could be modulated by individual genetic background. Therefore, an epigenetic-based functional test supplementing the routine genetic test will be of major diagnostic value.
An expanded six-nucleotide-repeat in the C9orf72 gene is the most common cause of inherited Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Generally, ALS and FTLD patients have several hundred to several thousand repeats in their DNA, contrary to healthy people who have less than 30 repeats.
Since the repeats occur in noncoding DNA and should have no effect on the C9orf72 protein sequence and structure, the predicting the disease mechanism is difficult. So far, three possible mechanisms have been proposed: (1) deficiency of the gene product (C9orf72 protein); (2) a toxic repeat-containing RNA that sequesters RNA-binding proteins; and/or (3) an unconventional translation of the DNA repeats into dipeptide repeats that form toxic neuronal inclusions.
Our project was designed to investigate the first hypothesis. We had good reasons to suspect that methylation, a type of DNA modification, might turn off C9orf72 expression leading to a ~ 50% reduction in C9orf72 protein. First, methylation-driven gene silencing occurs in other disorders caused by repeats in DNA, including Friedreich’s ataxia, Fragile X mental retardation, and myotonic dystrophy. Second, the C9orf72 sequence contains two CpG islands that are prime targets for methylation (upstream and one downstream of the repeat itself). Third, the repeat is located in the regulatory region that could control C9orf72 gene expression.
To search for methylated sites in the C9orf72 gene, we collected DNA samples from normal controls and from different neurodegenerative disease patients (ALS, FTLD, Parkinson Disease and Alzheimer Disease) both with and without the C9orf72 expansion. Using two independent experimental methods, we identified that 26 sites (all upstream of the repeat), were methylated in many expansion carriers, but not in controls. When dividing people into groups of no methylation, low methylation (1 to 3 sites methylated), and high methylation (4 to 26 sites methylated), we found no methylation was the norm among controls and expansion-free patients (>91%), and the rest of them had only low methylation (2-9%). In patients with the expansion, up to 36% were highly methylated at the C9orf72 locus and 26-38% fell into the low methylation category. Importantly, we did not detect methylation for intermediate alleles (up to 43 repeats), bringing to question the current cutoff of 30-repeats for pathological alleles.
Methylation level showed no correlation with age of disease onset, but more methylation was associated with a faster disease course of ALS. In addition, a high methylation level occurs more frequently in familial ALS patients than in sporadic ALS patients. The high methylation level was also inherited along with the expansion in ALS families.
Although our results showed that methylation level at the C9orf72 locus would not predict whether a person gets ALS or FTLD, a trend of more ALS patients having high methylation was observed. Future studies with a larger sample size will help us to clarify this question.
Our ongoing studies will evaluate the methylation profile of the repeat itself to further understand the disease mechanism of C9orf72 in ALS and FTLD.