Nucleotide repeats in the C9ORF72 gene are the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Since their discovery, the hexanucleotides have presented a puzzle for scientists, since they occur in noncoding DNA and should have no effect on protein structure. Recently, two research groups reported that the repeats are, in fact, translated, suggesting that an aberrant dipeptide repeat protein may be at the heart of the disease. Now, researchers at the University of Toronto in Canada offer up a different explanation for repeat toxicity. In the June 6 American Journal of Human Genetics, senior author Ekaterina Rogaeva and colleagues posit that transcription-silencing methyl groups clog the C9ORF72 promoter, reduce transcription of the gene, and speed up disease progression. Methylation, which occurs at 26 different loci in C9ORF72, correlates with the presence of the hexanucleotide expansion, according to the research. The work supports the idea that ALS and FTLD result from reduced production of the C9ORF72 protein. The presence and concentration of methyl groups might prove useful as a biomarker for disease, the authors speculated. “Perhaps methylation may be more severe in ALS than in FTLD, or vice versa,” said Rogaeva.

The function of the C9ORF72 protein remains unknown, although sequence analyses have suggested a role in membrane trafficking (see ARF related news story on Zhang et al., 2012, and Levine et al., 2013). Scientists generally consider that 30 or more repeats in the gene cause ALS or FTLD (see ARF related news story on Renton et al., 2011, and Dejesus-Hernandez et al., 2011). Three possible disease mechanisms have been proposed: Improper transcription or translation leading to haploinsufficiency; a toxic RNA that sequesters RNA-binding proteins such as TDP-43 or FUS; or the abnormal translation of the repeats into small peptides that form neuronal inclusions (see Dejesus-Hernandez et al., 2011; Gijselinck et al., 2012; ARF related news story on Mori et al., 2013; and ARF related news story on Ash et al., 2013), Rogaeva, first author Zhengrui Xi, and colleagues investigated the haploinsufficiency hypothesis.

The researchers had good reason to suspect that methylation might turn off C9ORF72 expression. For one, methylation-driven gene silencing occurs in other repeat disorders, including Friedreich's ataxia (Evans-Galea et al., 2012), Fragile X mental retardation (Sutcliffe et al., 1992), and myotonic dystrophy (López Castel et al., 2011; Klesert et al., 1997; Thornton et al., 1997). In addition, the C9ORF72 sequence contains two CpG islands, cytosine- and guanine-rich regions that are prime targets for methylation, one upstream and one downstream of the repeat itself.

To hunt for methylated nucleotides in the C9ORF72 gene, the researchers collected DNA samples from 37 people with ALS due to the C9ORF72 expansion, 64 people with ALS but no expansion, and 76 normal control cases. They digested the DNA with restriction endonucleases that only cut unmethylated nucleotide motifs. The researchers identified two sites upstream of the repeat that were methylated in expansion carriers, but not in controls.

Since the restriction enzyme analysis was limited to specific endonuclease sites, the scientists then turned to a more general approach to identify other methylated bases. They treated the DNA with bisulfite to convert unmethylated cytosines to thymines, and then sequenced the DNA areas flanking the repeat region for cytosines that remained intact. They identified 26 different sites, all upstream of the repeat. Because the repeats interfere with DNA amplification and sequencing, the researchers were unable to analyze methylation sites within the expansion itself, but it is possible they are also modified, said Rogaeva.

How does methylation status relate to disease? Xi and colleagues divided people into no methylation, low methylation (one to three sites tagged), and high methylation (four to 26 sites methylated) groups. Among controls and expansion-free ALS patients, no methylation was the norm, although a few subjects had low methylation. Rogaeva had predicted expansion carriers would all have DNA with several methyl groups, but was surprised to see that 10 of them had no methylation, a dozen were in the low methylation group, and only 15 had highly methylated C9ORF72.

Does the level of methylation tell something about the person who carried it or their disease? The methylation numbers showed no correlation with age, age of disease onset, or the body part—such as the throat or limbs—where symptoms tend to start. However, more methylation associated with a faster disease course.

Might other disease characteristics depend on methylation? Rogaeva and colleagues will test if methylation correlates with repeat length or disease phenotype, such as whether a person gets ALS or FTLD. It would be interesting to look at the downstream consequences of C9 methylation, added Veronique Belzil of the Mayo Clinic in Jacksonville, Florida. Methyl groups on DNA often cause methylation and deacetylation of histones, which then more tightly wind up DNA, leading to gene silencing, she noted.

If methylation does correlate with repeat length, or distinguish ALS versus FTLD, it might be a good surrogate marker for diagnosis or prognosis, Rogaeva suggested. Methylation might also help scientists designate the proper cutoff for a safe number of repeats, she said. For now, researchers consider 30 or more repeats to be pathological. But Rogaeva found no hypermethylation in cases with up to 43 repeats. Belzil was cautious about using methylation as a marker, noting that some people with just over 30 repeats do get sick, and the methyl groups may not be able to differentiate borderline cases with 30-43 repeats.

What ties this DNA modification to the repeat expansion? Though the mechanism remains unclear, Rogaeva suspects that the expansion somehow leads to overmethylation. Belzil suggested that might create a haploinsufficiency that causes few problems in young people, but precipitates disease as they age. The current study does not discount other potential mechanisms for C9ORF72-based ALS and FTLD, Rogaeva added. A combination of toxic repeat peptides, a reduction in proper C9ORF72 translation, and perhaps other mechanisms could all contribute, she speculated.—Amber Dance


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News Citations

  1. C9ORF72 Function: Is the ALS Protein a Membrane Traffic Cop?
  2. Corrupt Code: DNA Repeats Are Common Cause for ALS and FTD
  3. RNA Twist: C9ORF72 Intron Expansion Makes Aggregating Protein
  4. Second Study Sees Intron in FTLD Gene Translated

Paper Citations

  1. . Discovery of Novel DENN Proteins: Implications for the Evolution of Eukaryotic Intracellular Membrane Structures and Human Disease. Front Genet. 2012;3:283. PubMed.
  2. . The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs. Bioinformatics. 2013 Feb 15;29(4):499-503. Epub 2013 Jan 16 PubMed.
  3. . A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron. 2011 Oct 20;72(2):257-68. Epub 2011 Sep 21 PubMed.
  4. . Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron. 2011 Oct 20;72(2):245-56. Epub 2011 Sep 21 PubMed.
  5. . A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study. Lancet Neurol. 2012 Jan;11(1):54-65. PubMed.
  6. . The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science. 2013 Mar 15;339(6125):1335-8. Epub 2013 Feb 7 PubMed.
  7. . Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron. 2013 Feb 20;77(4):639-46. PubMed.
  8. . DNA methylation represses FMR-1 transcription in fragile X syndrome. Hum Mol Genet. 1992 Sep;1(6):397-400. PubMed.
  9. . Expanded CTG repeat demarcates a boundary for abnormal CpG methylation in myotonic dystrophy patient tissues. Hum Mol Genet. 2011 Jan 1;20(1):1-15. PubMed.
  10. . Trinucleotide repeat expansion at the myotonic dystrophy locus reduces expression of DMAHP. Nat Genet. 1997 Aug;16(4):402-6. PubMed.
  11. . Expansion of the myotonic dystrophy CTG repeat reduces expression of the flanking DMAHP gene. Nat Genet. 1997 Aug;16(4):407-9. PubMed.

Further Reading


  1. . The C9ORF72 mutation brings more answers and more questions. Alzheimers Res Ther. 2013 Feb 18;5(1):7. PubMed.
  2. . C9orf72 G4C2 repeat expansions in Alzheimer's disease and mild cognitive impairment. Neurobiol Aging. 2013 Jun;34(6):1712.e1-7. PubMed.
  3. . The disease-associated r(GGGGCC)n repeat from the C9orf72 gene forms tract length-dependent uni- and multimolecular RNA G-quadruplex structures. J Biol Chem. 2013 Apr 5;288(14):9860-6. PubMed.
  4. . C9ORF72 hexanucleotide expansions of 20-22 repeats are associated with frontotemporal deterioration. Neurology. 2013 Jan 22;80(4):366-70. PubMed.
  5. . Investigation of C9orf72 repeat expansions in Parkinson's disease. Neurobiol Aging. 2013 Jun;34(6):1710.e7-9. PubMed.
  6. . Concurrence of multiple sclerosis and amyotrophic lateral sclerosis in patients with hexanucleotide repeat expansions of C9ORF72. J Neurol Neurosurg Psychiatry. 2013 Jan;84(1):79-87. PubMed.

Primary Papers

  1. . Hypermethylation of the CpG Island Near the G4C2 Repeat in ALS with a C9orf72 Expansion. Am J Hum Genet. 2013 May 22; PubMed.