The C9orf72 repeat size correlates with onset age of disease, DNA methylation and transcriptional downregulation of the promoter

Abstract

Pathological expansion of a G4C2 repeat, located in the 5' regulatory region of C9orf72, is the most common genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). C9orf72 patients have highly variable onset ages suggesting the presence of modifying factors and/or anticipation. We studied 72 Belgian index patients with FTLD, FTLD–ALS or ALS and 61 relatives with a C9orf72 repeat expansion. We assessed the effect of G4C2 expansion size on onset age, the role of anticipation and the effect of repeat size on methylation and C9orf72 promoter activity. G4C2 expansion sizes varied in blood between 45 and over 2100 repeat units with short expansions (45–78 units) present in 5.6% of 72 index patients with an expansion. Short expansions co-segregated with disease in two families. The subject with a short expansion in blood but an indication of mosaicism in brain showed the same pathology as those with a long expansion. Further, we provided evidence for an association of G4C2 expansion size with onset age (P<0.05) most likely explained by an association of methylation state of the 5' flanking CpG island and expansion size in blood (P<0.0001) and brain (P<0.05). In several informative C9orf72 parent–child transmissions, we identified earlier onset ages, increasing expansion sizes and/or increasing methylation states (P=0.0034) of the 5' CpG island, reminiscent of disease anticipation. Also, intermediate repeats (7–24 units) showed a slightly higher methylation degree (P<0.0001) and a decrease of C9orf72 promoter activity (P<0.0001) compared with normal short repeats (2–6 units). Decrease of transcriptional activity was even more prominent in the presence of small deletions flanking G4C2 (P<0.0001). Here we showed that increased methylation of CpGs in the C9orf72 promoter may explain how an increasing G4C2 size lead to loss-of-function without excluding repeat length-dependent toxic gain-of-function. These data provide insights into disease mechanisms and have important implications for diagnostic counseling and potential therapeutic approaches.

Detection of long repeat expansions from PCR-free whole-genome sequence data

“…1). Because the expanded repeat is not present in the reference, these paired IRRs may not align to the C9orf72 repeat locus and could either not align at all or misalign to a different locus in the genome (Church et al 2015;Gijselinck et al 2016). To identify unaligned or misaligned IRRs, we tested every (MAPQ = 0) read in all 182 expanded ALS samples of the first cohort identified by the first round of RP-PCR as having the C9orf72 repeat expansion.…”

“…Because high-throughput WGS technologies are currently limited to ∼150 base pair (bp) read lengths, variantcalling methods that rely on reads aligned to the reference are subsequently limited to repeat lengths less than 150 bases (Narzisi and Schatz 2015). Many pathogenic repeat expansions have repeats spanning hundreds to thousands of base pairs (Dürr et al 1996;Gatchel and Zoghbi 2005;Kronquist et al 2008;Gijselinck et al 2016), so it has been assumed that short-read sequencing technologies may not be able to identify pathogenic repeat expansions (Loomis et al 2013;Ashley 2016).…”

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

“…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]. …”

Development of Therapeutics for C9ORF72 ALS/FTD-Related Disorders

“…This discovery has opened the path for progress in the knowledge and treatment of these devastating diseases. C9ORF72 HRE-linked pathogenesis has been suggested to be elicited by means of loss-offunction and toxic gain-of-function mechanisms that comprise the following: (1) transcribed sense GGGGCCexp or antisense (CCCCGGexp) RNAs that sequester proteins, therefore interfering with their physiological function [8]; (2) sense or antisense expanded RNAs that are translated via repeatassociated non-AUG initiated (RAN) translation to generate toxic dipeptide repeat proteins (DPRs) [10][11][12][13]; or (3) haploinsufficiency that originates from diminished transcription of the C9ORF72 exonic sequence [14,15] (Fig. 1).…”