Acta Neuropathologica

, Volume 129, Issue 5, pp 715–727 | Cite as

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

  • Zhengrui Xi
  • Ming Zhang
  • Amalia C. Bruni
  • Raffaele G. Maletta
  • Rosanna Colao
  • Pietro Fratta
  • James M. Polke
  • Mary G. Sweeney
  • Ese Mudanohwo
  • Benedetta Nacmias
  • Sandro Sorbi
  • Maria Carmela Tartaglia
  • Innocenzo Rainero
  • Elisa Rubino
  • Lorenzo Pinessi
  • Daniela Galimberti
  • Ezequiel I. Surace
  • Philip McGoldrick
  • Paul McKeever
  • Danielle Moreno
  • Christine Sato
  • Yan Liang
  • Julia Keith
  • Lorne Zinman
  • Janice Robertson
  • Ekaterina Rogaeva
Original Paper

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.

Keywords

ALS FTLD C9orf72 G4C2-repeat Methylation 

Supplementary material

401_2015_1401_MOESM1_ESM.docx (5.9 mb)
Supplementary material 1 (DOCX 6066 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Zhengrui Xi
    • 1
  • Ming Zhang
    • 1
  • Amalia C. Bruni
    • 2
  • Raffaele G. Maletta
    • 2
  • Rosanna Colao
    • 2
  • Pietro Fratta
    • 3
  • James M. Polke
    • 4
  • Mary G. Sweeney
    • 4
  • Ese Mudanohwo
    • 4
  • Benedetta Nacmias
    • 5
  • Sandro Sorbi
    • 5
  • Maria Carmela Tartaglia
    • 1
    • 6
  • Innocenzo Rainero
    • 7
  • Elisa Rubino
    • 7
  • Lorenzo Pinessi
    • 7
  • Daniela Galimberti
    • 8
  • Ezequiel I. Surace
    • 9
  • Philip McGoldrick
    • 1
  • Paul McKeever
    • 1
  • Danielle Moreno
    • 1
  • Christine Sato
    • 1
  • Yan Liang
    • 1
  • Julia Keith
    • 10
  • Lorne Zinman
    • 10
  • Janice Robertson
    • 1
  • Ekaterina Rogaeva
    • 1
    • 6
  1. 1.Tanz Centre for Research in Neurodegenerative DiseasesUniversity of TorontoTorontoCanada
  2. 2.Regional Neurogenetic Centre, Azienda Sanitaria Provinciale CatanzaroLamezia TermeItaly
  3. 3.Department of Neurodegenerative DiseaseUCL Institute of NeurologyLondonUK
  4. 4.Neurogenetics UnitNational Hospital for Neurology and NeurosurgeryLondonUK
  5. 5.Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA)University of FlorenceFlorenceItaly
  6. 6.Division of Neurology, Department of MedicineUniversity of TorontoTorontoCanada
  7. 7.Neurology I, Rita Levi Montalcini Department of NeuroscienceUniversity of TorinoTurinItaly
  8. 8.Neurology Unit, Department of Pathophysiology and TransplantationUniversity of Milan, Centro Dino Ferrari, Fondazione Ca’ Granda, IRCCS Ospedale Maggiore PoliclinicoMilanItaly
  9. 9.Laboratorio de Biología Molecular, Instituto de Investigaciones Neurológicas Dr. Raúl Carrea (FLENI)Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)Buenos AiresArgentina
  10. 10.Sunnybrook Health Sciences CentreTorontoCanada

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