title: 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.
authors.affiliation: Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Krembil Discovery Tower, 60 Leonard Street, Toronto, ON, M5T 2S8, Canada.
authors.forename: Zhengrui
authors.initials: Z
authors.lastname: Xi
authors.forename: Ming
authors.initials: M
authors.lastname: Zhang
authors.forename: Amalia C
authors.initials: AC
authors.lastname: Bruni
authors.forename: Raffaele G
authors.initials: RG
authors.lastname: Maletta
authors.forename: Rosanna
authors.initials: R
authors.lastname: Colao
authors.forename: Pietro
authors.initials: P
authors.lastname: Fratta
authors.forename: James M
authors.initials: JM
authors.lastname: Polke
authors.forename: Mary G
authors.initials: MG
authors.lastname: Sweeney
authors.forename: Ese
authors.initials: E
authors.lastname: Mudanohwo
authors.forename: Benedetta
authors.initials: B
authors.lastname: Nacmias
authors.forename: Sandro
authors.initials: S
authors.lastname: Sorbi
authors.forename: Maria Carmela
authors.initials: MC
authors.lastname: Tartaglia
authors.forename: Innocenzo
authors.initials: I
authors.lastname: Rainero
authors.forename: Elisa
authors.initials: E
authors.lastname: Rubino
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Pinessi
authors.forename: Daniela
authors.initials: D
authors.lastname: Galimberti
authors.forename: Ezequiel I
authors.initials: EI
authors.lastname: Surace
authors.forename: Philip
authors.initials: P
authors.lastname: McGoldrick
authors.forename: Paul
authors.initials: P
authors.lastname: McKeever
authors.forename: Danielle
authors.initials: D
authors.lastname: Moreno
authors.forename: Christine
authors.initials: C
authors.lastname: Sato
authors.forename: Yan
authors.initials: Y
authors.lastname: Liang
authors.forename: Julia
authors.initials: J
authors.lastname: Keith
authors.forename: Lorne
authors.initials: L
authors.lastname: Zinman
authors.forename: Janice
authors.initials: J
authors.lastname: Robertson
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Rogaeva
mesh_terms: D000368:Aged; D000483:Alleles; D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics*; D000073885:C9orf72 Protein; D018899:CpG Islands; D019175:DNA Methylation; D042622:DNA Repeat Expansion; D005260:Female; D057174:Frontotemporal Lobar Degeneration / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D008297:Male; D008875:Middle Aged; D011506:Proteins / Q000235:genetics*; D015183:Restriction Mapping / Q000379:methods; D017422:Sequence Analysis, DNA
chemical_list: D000073885:C9orf72 Protein; C568651:C9orf72 protein, human; D011506:Proteins