The C9orf72 repeat expansion itself is methylated in ALS and FTLD patients

Abstract

The most common cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) is a G4C2-repeat expansion in C9orf72. However, the lower limit for pathological repeats has not been established and expansions with different sizes could have different pathological consequences. One of the implicated disease mechanisms is haploinsufficiency. Previously, we identified expansion-specific hypermethylation at the 5' CpG-island near the G4C2-repeat, but only in a fraction of carriers (up to 36 %). Here, we tested the hypothesis that the G4C2-repeat itself could be the main site of methylation. To evaluate (G4C2)n -methylation, we developed a novel assay, which was validated by an independent methylation-sensitive restriction enzyme assay. Notably, both assays are qualitative but not quantitative. Blood DNA was available for 270 unrelated individuals, including 71 expansion carriers. In addition, we investigated blood DNA from family members of 16 probands, and 38 DNA samples from multiple tissues of 10 expansion carriers. Finally, we tested DNA from different tissues of an ALS patient carrying a somatically unstable 90-repeat. We demonstrated that the G4C2-expansion is generally methylated in unrelated carriers of alleles >50 repeats (97 %), while small (<22 repeats) or intermediate (22-90 repeats) alleles were completely unmethylated. The presence of (G4C2)n -methylation does not separate the C9orf72-phenotypes (ALS vs. ALS/FTLD vs. FTLD), but has the potential to predict large vs. intermediate repeat length. Our results suggest that (G4C2)n -methylation might sometimes spread to the 5'-upstream region, but not vice versa. It is stable over time, since (G4C2)n -methylation was detected in carriers with a wide range of ages (24-74 years). It was identified in both blood and brain tissues for the same individual, implying its potential use as a biomarker. Furthermore, our findings may open up new perspectives for studying disease mechanisms, such as determining whether methylated and unmethylated repeats have the same ability to form a G-quadruplex configuration.

Development of Therapeutics for C9ORF72 ALS/FTD-Related Disorders

“…In fact, all three transcriptional variants of C9ORF72 seem to be reduced in patients' tissues as a possible consequence of the promoter's hypermethylation of CpG islands and as a result of the HRE's hypermethylation itself in the expanded allele [57][58][59], thus leading to a potentially deleterious condition of haploinsufficiency. The role of epigenetic processes has already been studied in other repeat expansion disorders, such as Fragile X-associated Tremor/ Ataxia Syndrome (FXTAS), in which the overexpression of FMR1 mRNA can be reversed by histone acetyltransferase (HAT) inhibitors [60].…”

Elevated Global DNA Methylation Is Not Exclusive to Amyotrophic Lateral Sclerosis and Is Also Observed in Spinocerebellar Ataxia Types 1 and 2

“…More recent studies have reported an increase in 5-mC levels detectable in blood samples of sALS patients [16], and some have suggested abnormalities in the regulatory machinery which takes part in the maintenance of DNA methylation [17, 18]. Alongside holistic methylation studies, others have focused on the methylation status of the C9orf72 gene promoter, shown to be hypermethylated in expansion carrier ALS and FTD patients [19-22]. Such aberrant methylation has been suggested to be a disease modifier acting through a loss-of-function [23] rather than a toxic gain-of-function mechanism [12, 24].…”

Cross-Sectional Survey of Relevant Literatures as to the Current Proposed Disease Mechanisms and Treatments of Amyotrophic Lateral Sclerosis (ALS)

“…70 The increasing evidence for the presence of this sequence in FALS cases combined with the late age of onset for symptoms has led some studies to evaluate the possibility that this irregularity in RNA processing caused by the hexanucleotide expansion may be initially corrected by conventional cellular repair mechanisms only to eventually overwhelm the system in later life. 79 This expansion itself may not be inherently irregular but instead trigger pathogenicity in differentially methylated states 80,81 and has also demonstrated a concordance with abnormal localization of TDP-43, a hallmark of ALS neuronal pathology. 68 Murine models with induced GGGGCC sequences have also been constructed, evidencing a gain of function relationship theorized elsewhere.…”