Acta Neuropathologica

, Volume 134, Issue 5, pp 715–728 | Cite as

Conserved DNA methylation combined with differential frontal cortex and cerebellar expression distinguishes C9orf72-associated and sporadic ALS, and implicates SERPINA1 in disease

  • Mark T. W. Ebbert
  • Christian A. Ross
  • Luc J. Pregent
  • Rebecca J. Lank
  • Cheng Zhang
  • Rebecca B. Katzman
  • Karen Jansen-West
  • Yuping Song
  • Edroaldo Lummertz da Rocha
  • Carla Palmucci
  • Pamela Desaro
  • Amelia E. Robertson
  • Ana M. Caputo
  • Dennis W. Dickson
  • Kevin B. Boylan
  • Rosa Rademakers
  • Tamas Ordog
  • Hu LiEmail author
  • Veronique V. BelzilEmail author
Original Paper


We previously found C9orf72-associated (c9ALS) and sporadic amyotrophic lateral sclerosis (sALS) brain transcriptomes comprise thousands of defects, among which, some are likely key contributors to ALS pathogenesis. We have now generated complementary methylome data and combine these two data sets to perform a comprehensive “multi-omic” analysis to clarify the molecular mechanisms initiating RNA misregulation in ALS. We found that c9ALS and sALS patients have generally distinct but overlapping methylome profiles, and that the c9ALS- and sALS-affected genes and pathways have similar biological functions, indicating conserved pathobiology in disease. Our results strongly implicate SERPINA1 in both C9orf72 repeat expansion carriers and non-carriers, where expression levels are greatly increased in both patient groups across the frontal cortex and cerebellum. SERPINA1 expression is particularly pronounced in C9orf72 repeat expansion carriers for both brain regions, where SERPINA1 levels are strictly down regulated across most human tissues, including the brain, except liver and blood, and are not measurable in E18 mouse brain. The altered biological networks we identified contain critical molecular players known to contribute to ALS pathology, which also interact with SERPINA1. Our comprehensive combined methylation and transcription study identifies new genes and highlights that direct genetic and epigenetic changes contribute to c9ALS and sALS pathogenesis.


Amyotrophic lateral sclerosis C9orf72 DNA methylation Epigenetic modification SERPINA1 Transcriptome regulation 



We are extremely grateful to all individuals who agreed to donate their brains to research. This study was supported by the National Institutes of Health/National Institute on Aging [AG16574-17 J PILOT (V.V.B.)]; National Institutes of Health/National Institute of Neurological Disorders and Stroke [R21NS074121 (K.B.B.), P01NS084974 (D.W.D., K.B.B., and R.R.]; Mayo Clinic Center for Individualized Medicine (V.V.B., K.B.B., and H.L.); ALS Association (K.B.B.), Donors Cure Foundation (H.L.), and the Robert Packard Center for ALS Research at Johns Hopkins. V.V.B. is recipient of the Career Transition Award from ALS Canada and Brain Canada, the Milton Safenowitz Post-Doctoral Fellowship from the Amyotrophic Lateral Sclerosis Association, the Post-Doctoral Fellowship from the Canadian Institutes of Health Research, the Career Development Award for Young Investigators in Neurosciences from the Siragusa Foundation, and the Research Fellowship from the Robert and Clarice Smith & Abigail Van Buren Alzheimer’s Disease Research Foundation. M.T.W.E received the PhRMA Foundation Research Starter grant.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

401_2017_1760_MOESM1_ESM.pdf (808 kb)
Supplementary material 1 (PDF 808 kb)


  1. 1.
    Akalin A, Kormaksson M, Li S, Garrett-Bakelman FE, Figueroa ME, Melnick A et al (2012) methylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles. Genome Biol 13:R87. doi: 10.1186/gb-2012-13-10-r87 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Amaral PP, Dinger ME, Mercer TR, Mattick JS (2008) The eukaryotic genome as an RNA machine. Science 319:1787–1789. doi: 10.1126/science.1155472 CrossRefPubMedGoogle Scholar
  3. 3.
    Belzil VV, Bauer PO, Gendron TF, Murray ME, Dickson D, Petrucelli L (2014) Characterization of DNA hypermethylation in the cerebellum of c9FTD/ALS patients. Brain Res 1584:15–21. doi: 10.1016/j.brainres.2014.02.015 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Belzil VV, Bauer PO, Prudencio M, Gendron TF, Stetler CT, Yan IK et al (2013) Reduced C9orf72 gene expression in c9FTD/ALS is caused by histone trimethylation, an epigenetic event detectable in blood. Acta Neuropathol 126:895–905. doi: 10.1007/s00401-013-1199-1 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Belzil VV, Gendron TF, Petrucelli L (2012) RNA-mediated toxicity in neurodegenerative disease. Mol Cell Neurosci 56C:406–419. doi: 10.1016/j.mcn.2012.12.006 Google Scholar
  6. 6.
    Belzil VV, Katzman RB, Petrucelli L (2016) ALS and FTD: an epigenetic perspective. Acta Neuropathol. doi: 10.1007/s00401-016-1587-4 PubMedPubMedCentralGoogle Scholar
  7. 7.
    Byrne S, Heverin M, Elamin M, Bede P, Lynch C, Kenna K et al (2013) Aggregation of neurologic and neuropsychiatric disease in amyotrophic lateral sclerosis kindreds: a population-based case-control cohort study of familial and sporadic amyotrophic lateral sclerosis. Ann Neurol 74:699–708. doi: 10.1002/ana.23969 CrossRefPubMedGoogle Scholar
  8. 8.
    Byrne S, Walsh C, Lynch C, Bede P, Elamin M, Kenna K et al (2011) Rate of familial amyotrophic lateral sclerosis: a systematic review and meta-analysis. J Neurol Neurosurg Psychiatry 82:623–627. doi: 10.1136/jnnp.2010.224501 CrossRefPubMedGoogle Scholar
  9. 9.
    Cox DW (2001) Alpha-1-antitrypsin. In: Scriver CRB, Sly AL, Valle D (eds) The metabolic and molecular bases of inherited disease, vol IV, 8th edn. McGraw-Hill, New York, pp 5559–5584Google Scholar
  10. 10.
    Donnelly CJ, Zhang PW, Pham JT, Haeusler AR, Mistry NA, Vidensky S et al (2013) RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention. Neuron 80:415–428. doi: 10.1016/j.neuron.2013.10.015 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Fratta P, Mizielinska S, Nicoll AJ, Zloh M, Fisher EM, Parkinson G et al (2012) C9orf72 hexanucleotide repeat associated with amyotrophic lateral sclerosis and frontotemporal dementia forms RNA G-quadruplexes. Sci Rep 2:1016. doi: 10.1038/srep01016 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Gendron TF, van Blitterswijk M, Bieniek KF, Daughrity LM, Jiang J, Rush BK et al (2015) Cerebellar c9RAN proteins associate with clinical and neuropathological characteristics of C9ORF72 repeat expansion carriers. Acta Neuropathol 130:559–573. doi: 10.1007/s00401-015-1474-4 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Gibson SB, Figueroa KP, Bromberg MB, Pulst SM, Cannon-Albright L (2014) Familial clustering of ALS in a population-based resource. Neurology 82:17–22. doi: 10.1212/01.wnl.0000438219.39061.da CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Guerreiro R, Bras J, Hardy J (2015) SnapShot: genetics of ALS and FTD. Cell 160(798):e791. doi: 10.1016/j.cell.2015.01.052 Google Scholar
  15. 15.
    Haeusler AR, Donnelly CJ, Periz G, Simko EA, Shaw PG, Kim MS et al (2014) C9orf72 nucleotide repeat structures initiate molecular cascades of disease. Nature 507:195–200. doi: 10.1038/nature13124 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Holoch D, Moazed D (2015) RNA-mediated epigenetic regulation of gene expression. Nat Rev Genet 16:71–84. doi: 10.1038/nrg3863 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Jung J, Bonini N (2007) CREB-binding protein modulates repeat instability in a Drosophila model for polyQ disease. Science 315:1857–1859. doi: 10.1126/science.1139517 CrossRefPubMedGoogle Scholar
  18. 18.
    Kalsheker NA (1996) Alpha 1-antichymotrypsin. Int J Biochem Cell Biol 28:961–964CrossRefPubMedGoogle Scholar
  19. 19.
    Kamboh MI, Minster RL, Kenney M, Ozturk A, Desai PP, Kammerer CM et al (2006) Alpha-1-antichymotrypsin (ACT or SERPINA3) polymorphism may affect age-at-onset and disease duration of Alzheimer’s disease. Neurobiol Aging 27:1435–1439. doi: 10.1016/j.neurobiolaging.2005.07.015 CrossRefPubMedGoogle Scholar
  20. 20.
    Krueger F, Andrews SR (2011) Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27:1571–1572. doi: 10.1093/bioinformatics/btr167 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Lam L, Chin L, Halder RC, Sagong B, Famenini S, Sayre J et al (2016) Epigenetic changes in T-cell and monocyte signatures and production of neurotoxic cytokines in ALS patients. FASEB J. doi: 10.1096/fj.201600259RR PubMedPubMedCentralGoogle Scholar
  22. 22.
    Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. doi: 10.1038/nmeth.1923 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Liu EY, Russ J, Wu K, Neal D, Suh E, McNally AG et al (2014) C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD. Acta Neuropathol 128:525–541. doi: 10.1007/s00401-014-1286-y CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Mattick JS, Amaral PP, Dinger ME, Mercer TR, Mehler MF (2009) RNA regulation of epigenetic processes. BioEssays 31:51–59. doi: 10.1002/bies.080099 CrossRefPubMedGoogle Scholar
  25. 25.
    Morahan JM, Yu B, Trent RJ, Pamphlett R (2009) A genome-wide analysis of brain DNA methylation identifies new candidate genes for sporadic amyotrophic lateral sclerosis. Amyotroph Lateral Scler 10:418–429. doi: 10.3109/17482960802635397 CrossRefPubMedGoogle Scholar
  26. 26.
    Morris KV, Mattick JS (2014) The rise of regulatory RNA. Nat Rev Genet 15:423–437. doi: 10.1038/nrg3722 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Nagase T, Nakayama M, Nakajima D, Kikuno R, Ohara O (2001) Prediction of the coding sequences of unidentified human genes. XX. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res 8:85–95CrossRefPubMedGoogle Scholar
  28. 28.
    Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, Chou TT et al (2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314:130–133CrossRefPubMedGoogle Scholar
  29. 29.
    Pace JM, Corrado M, Missero C, Byers PH (2003) Identification, characterization and expression analysis of a new fibrillar collagen gene, COL27A1. Matrix Biol 22:3–14CrossRefPubMedGoogle Scholar
  30. 30.
    Pennacchio LA, Bickmore W, Dean A, Nobrega MA, Bejerano G (2013) Enhancers: five essential questions. Nat Rev Genet 14:288–295. doi: 10.1038/nrg3458 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Polymenidou M, Lagier-Tourenne C, Hutt KR, Bennett CF, Cleveland DW, Yeo GW (2012) Misregulated RNA processing in amyotrophic lateral sclerosis. Brain Res 1462:3–15. doi: 10.1016/j.brainres.2012.02.059 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Prell T, Grosskreutz J (2013) The involvement of the cerebellum in amyotrophic lateral sclerosis. Amyotroph Lateral Scler Frontotemporal Degener 14:507–515. doi: 10.3109/21678421.2013.812661 CrossRefPubMedGoogle Scholar
  33. 33.
    Prudencio M, Belzil VV, Batra R, Ross CA, Gendron TF, Pregent LJ et al (2015) Distinct brain transcriptome profiles in C9orf72-associated and sporadic ALS. Nat Neurosci 18:1175–1182. doi: 10.1038/nn.4065 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Rademakers R, van Blitterswijk M (2013) Motor neuron disease in 2012: novel causal genes and disease modifiers. Nat Rev Neurol 9:63–64. doi: 10.1038/nrneurol.2012.276 CrossRefPubMedGoogle Scholar
  35. 35.
    Renoux AJ, Todd PK (2012) Neurodegeneration the RNA way. Prog Neurobiol 97:173–189. doi: 10.1016/j.pneurobio.2011.10.006 CrossRefPubMedGoogle Scholar
  36. 36.
    Renton AE, Chio A, Traynor BJ (2014) State of play in amyotrophic lateral sclerosis genetics. Nat Neurosci 17:17–23. doi: 10.1038/nn.3584 CrossRefPubMedGoogle Scholar
  37. 37.
    Spitz F (2016) Gene regulation at a distance: from remote enhancers to 3D regulatory ensembles. Semin Cell Dev Biol. doi: 10.1016/j.semcdb.2016.06.017 PubMedGoogle Scholar
  38. 38.
    Su Z, Zhang Y, Gendron TF, Bauer PO, Chew J, Yang WY et al (2014) Discovery of a biomarker and lead small molecules to target r(GGGGCC)-associated defects in c9FTD/ALS. Neuron 83:1043–1050. doi: 10.1016/j.neuron.2014.07.041 CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Tan RH, Kril JJ, McGinley C, Hassani M, Masuda-Suzukake M, Hasegawa M et al (2016) Cerebellar neuronal loss in amyotrophic lateral sclerosis cases with ATXN2 intermediate repeat expansions. Ann Neurol 79:295–305. doi: 10.1002/ana.24565 CrossRefPubMedGoogle Scholar
  40. 40.
    Therrien M, Dion PA, Rouleau GA (2016) ALS: recent Developments from Genetics Studies. Curr Neurol Neurosci Rep 16:59. doi: 10.1007/s11910-016-0658-1 CrossRefPubMedGoogle Scholar
  41. 41.
    Wang YC, Liu HC, Liu TY, Hong CJ, Tsai SJ (2001) Genetic association analysis of alpha-1-antichymotrypsin polymorphism in Parkinson’s disease. Eur Neurol 45:254–256. doi: 10.1159/000052138
  42. 42.
    Xi Z, Rainero I, Rubino E, Pinessi L, Bruni AC, Maletta RG et al (2014) Hypermethylation of the CpG-island near the C9orf72 G4C2-repeat expansion in FTLD patients. Hum Mol Genet. doi: 10.1093/hmg/ddu279 PubMedGoogle Scholar
  43. 43.
    Xi Z, Yunusova Y, van Blitterswijk M, Dib S, Ghani M, Moreno D et al (2014) Identical twins with the C9orf72 repeat expansion are discordant for ALS. Neurology 83:1476–1478. doi: 10.1212/WNL.0000000000000886 CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Xi Z, Zhang M, Bruni AC, Maletta RG, Colao R, Fratta P et al (2015) The C9orf72 repeat expansion itself is methylated in ALS and FTLD patients. Acta Neuropathol 129:715–727. doi: 10.1007/s00401-015-1401-8 CrossRefPubMedGoogle Scholar
  45. 45.
    Xi Z, Zinman L, Moreno D, Schymick J, Liang Y, Sato C et al (2013) Hypermethylation of the CpG island near the G4C2 repeat in ALS with a C9orf72 expansion. Am J Hum Genet 92:981–989. doi: 10.1016/j.ajhg.2013.04.017 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Zeng Q, Subramaniam VN, Wong SH, Tang BL, Parton RG, Rea S et al (1998) A novel synaptobrevin/VAMP homologous protein (VAMP5) is increased during in vitro myogenesis and present in the plasma membrane. Mol Biol Cell 9:2423–2437CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Zhang K, Donnelly CJ, Haeusler AR, Grima JC, Machamer JB, Steinwald P et al (2015) The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature 525:56–61. doi: 10.1038/nature14973 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Mark T. W. Ebbert
    • 1
  • Christian A. Ross
    • 2
  • Luc J. Pregent
    • 1
  • Rebecca J. Lank
    • 1
  • Cheng Zhang
    • 2
  • Rebecca B. Katzman
    • 1
  • Karen Jansen-West
    • 1
  • Yuping Song
    • 1
  • Edroaldo Lummertz da Rocha
    • 2
    • 6
  • Carla Palmucci
    • 3
  • Pamela Desaro
    • 3
  • Amelia E. Robertson
    • 3
  • Ana M. Caputo
    • 3
  • Dennis W. Dickson
    • 1
  • Kevin B. Boylan
    • 3
  • Rosa Rademakers
    • 1
  • Tamas Ordog
    • 4
    • 5
  • Hu Li
    • 2
    Email author
  • Veronique V. Belzil
    • 1
    Email author
  1. 1.Department of NeuroscienceMayo ClinicJacksonvilleUSA
  2. 2.Department of Molecular Pharmacology and Experimental TherapeuticsMayo ClinicRochesterUSA
  3. 3.Department of NeurologyMayo ClinicJacksonvilleUSA
  4. 4.Epigenomics Program, Center for Individualized MedicineMayo ClinicRochesterUSA
  5. 5.Department of Physiology and Medical EngineeringMayo ClinicRochesterUSA
  6. 6.Stem Cell Transplantation Program, Department of Pediatric Hematology and OncologyBoston Children’s Hospital and Dana-Farber Cancer InstituteBostonUSA

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