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

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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.

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Acknowledgments

This work was supported by the W. Garfield Weston Foundation [ER, JR, MCT, LZ], The James Hunter ALS Initiative [LZ, JR], Ministero della Istruzione, dell’Università e della Ricerca Scientifica Italy [IR, LP], Argentine Research Council-CONICET [EIS], Ministry of health-IRCCS-RF-2010-2319722 [SS], Cassa di Risparmio Firenze 2012-0471 [SS], Cassa di Risparmio Pistoia e Pescia 2012-0159 [BN], and the NIHR UCLH Biomedical Research Centre and the MNDA [PF]. PMcK holds an Alzheimer Society of Canada Doctoral Award. PMcG holds a Milton Safenowicz Postodoctoral Fellowship from the American ALS Association.

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    Authors and Affiliations

    1. Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Krembil Discovery Tower, 60 Leonard Street, Toronto, ON, M5T 2S8, Canada

      Zhengrui Xi, Ming Zhang, Maria Carmela Tartaglia, Philip McGoldrick, Paul McKeever, Danielle Moreno, Christine Sato, Yan Liang, Janice Robertson & Ekaterina Rogaeva

    2. Regional Neurogenetic Centre, Azienda Sanitaria Provinciale Catanzaro, Lamezia Terme, CZ, Italy

      Amalia C. Bruni, Raffaele G. Maletta & Rosanna Colao

    3. Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK

      Pietro Fratta

    4. Neurogenetics Unit, National Hospital for Neurology and Neurosurgery, Queen Square, London, WC1N 3BG, UK

      James M. Polke, Mary G. Sweeney & Ese Mudanohwo

    5. Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), University of Florence, Florence, Italy

      Benedetta Nacmias & Sandro Sorbi

    6. Division of Neurology, Department of Medicine, University of Toronto, 1 King’s College Circle, Toronto, ON, M5S 1A8, Canada

      Maria Carmela Tartaglia & Ekaterina Rogaeva

    7. Neurology I, Rita Levi Montalcini Department of Neuroscience, University of Torino, Turin, Italy

      Innocenzo Rainero, Elisa Rubino & Lorenzo Pinessi

    8. Neurology Unit, Department of Pathophysiology and Transplantation, University of Milan, Centro Dino Ferrari, Fondazione Ca’ Granda, IRCCS Ospedale Maggiore Policlinico, Milan, Italy

      Daniela Galimberti

    9. Laboratorio de Biología Molecular, Instituto de Investigaciones Neurológicas Dr. Raúl Carrea (FLENI), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina

      Ezequiel I. Surace

    10. Sunnybrook Health Sciences Centre, 2075 Bayview Ave., Toronto, ON, M4N 3M5, Canada

      Julia Keith & Lorne Zinman

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    Corresponding author

    Correspondence to Ekaterina Rogaeva.

    Additional information

    Z. Xi and M. Zhang contributed equally to this work.

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    Xi, Z., Zhang, M., Bruni, A.C. et al. The C9orf72 repeat expansion itself is methylated in ALS and FTLD patients. Acta Neuropathol 129, 715–727 (2015). https://doi.org/10.1007/s00401-015-1401-8

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    • DOI: https://doi.org/10.1007/s00401-015-1401-8

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