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• W2136896331 abstract "The G4C2 repeat expansion in C9orf72 is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We tested the hypothesis that the repeat expansion causes aberrant CpG methylation near the G4C2 repeat, which could be responsible for the downregulation of gene expression. We investigated the CpG methylation profile by two methods using genomic DNA from the blood of individuals with ALS (37 expansion carriers and 64 noncarriers), normal controls (n = 76), and family members of 7 ALS probands with the expansion. We report that hypermethylation of the CpG island 5′ of the G4C2 repeat is associated with the presence of the expansion (p < 0.0001). A higher degree of methylation was significantly correlated with a shorter disease duration (p < 0.01), associated with familial ALS (p = 0.009) and segregated with the expansion in 7 investigated families. Notably, we did not detect methylation for either normal or intermediate alleles (up to 43 repeats), bringing to question the current cutoff of 30 repeats for pathological alleles. Our study raises several important questions for the future investigation of large data sets, such as whether the degree of methylation corresponds to clinical presentation (ALS versus FTLD). The G4C2 repeat expansion in C9orf72 is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We tested the hypothesis that the repeat expansion causes aberrant CpG methylation near the G4C2 repeat, which could be responsible for the downregulation of gene expression. We investigated the CpG methylation profile by two methods using genomic DNA from the blood of individuals with ALS (37 expansion carriers and 64 noncarriers), normal controls (n = 76), and family members of 7 ALS probands with the expansion. We report that hypermethylation of the CpG island 5′ of the G4C2 repeat is associated with the presence of the expansion (p < 0.0001). A higher degree of methylation was significantly correlated with a shorter disease duration (p < 0.01), associated with familial ALS (p = 0.009) and segregated with the expansion in 7 investigated families. Notably, we did not detect methylation for either normal or intermediate alleles (up to 43 repeats), bringing to question the current cutoff of 30 repeats for pathological alleles. Our study raises several important questions for the future investigation of large data sets, such as whether the degree of methylation corresponds to clinical presentation (ALS versus FTLD). Amyotrophic lateral sclerosis (ALS [MIM 612069]) and frontotemporal lobar degeneration (FTLD [MIM 600274]) constitute a neurodegenerative syndrome, with individual cases presenting along a clinico-pathological spectrum. Individuals with FTLD exhibit primary dementia commonly characterized by early behavioral and/or language changes, whereas ALS is characterized by the degeneration of motor neurons responsible for voluntary movements. Both syndromes may occur within the same family or the same person. The most common known genetic cause of ALS and FTLD is the recently discovered noncoding hexanucleotide (G4C2)n > 30 repeat expansion in the chromosome 9 open reading frame 72 gene (C9orf72 [MIM 614260]),1DeJesus-Hernandez M. Mackenzie I.R. Boeve B.F. Boxer A.L. Baker M. Rutherford N.J. Nicholson A.M. Finch N.A. Flynn H. Adamson J. et al.Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.Neuron. 2011; 72: 245-256Abstract Full Text Full Text PDF PubMed Scopus (3382) Google Scholar, 2Renton A.E. Majounie E. Waite A. Simón-Sánchez J. Rollinson S. Gibbs J.R. Schymick J.C. Laaksovirta H. van Swieten J.C. Myllykangas L. et al.ITALSGEN ConsortiumA hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD.Neuron. 2011; 72: 257-268Abstract Full Text Full Text PDF PubMed Scopus (3081) Google Scholar which accounts for 24%–37% of familial and 6%–7% of sporadic cases in whites.3Rademakers R. C9orf72 repeat expansions in patients with ALS and FTD.Lancet Neurol. 2012; 11: 297-298Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar This discovery was confirmed in multiple data sets, including ours;4Gijselinck I. Van Langenhove T. van der Zee J. Sleegers K. Philtjens S. Kleinberger G. Janssens J. Bettens K. Van Cauwenberghe C. Pereson S. et al.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; 11: 54-65Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 5Sabatelli M. Conforti F.L. Zollino M. Mora G. Monsurrò M.R. Volanti P. Marinou K. Salvi F. Corbo M. Giannini F. et al.ITALSGEN ConsortiumC9ORF72 hexanucleotide repeat expansions in the Italian sporadic ALS population.Neurobiol. Aging. 2012; 33 (e15–e20): 1848Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 6Smith B.N. Newhouse S. Shatunov A. Vance C. Topp S. Johnson L. Miller J. Lee Y. Troakes C. Scott K.M. et al.The C9ORF72 expansion mutation is a common cause of ALS+/-FTD in Europe and has a single founder.Eur. J. Hum. Genet. 2013; 21: 102-108Crossref PubMed Scopus (175) Google Scholar, 7Xi Z. Zinman L. Grinberg Y. Moreno D. Sato C. Bilbao J.M. Ghani M. Hernández I. Ruiz A. Boada M. et al.Investigation of c9orf72 in 4 neurodegenerative disorders.Arch. Neurol. 2012; 69: 1583-1590Crossref PubMed Scopus (83) Google Scholar however, the disease mechanism related to the C9orf72 G4C2 repeat expansion remains unknown. There are several hypotheses about the functional consequences of the repeat expansion. The toxic gain-of-function model is related either to the sequestering of RNA binding proteins by RNA foci consisting of pre-mRNA containing the repeat expansion1DeJesus-Hernandez M. Mackenzie I.R. Boeve B.F. Boxer A.L. Baker M. Rutherford N.J. Nicholson A.M. Finch N.A. Flynn H. Adamson J. et al.Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.Neuron. 2011; 72: 245-256Abstract Full Text Full Text PDF PubMed Scopus (3382) Google Scholar or to the non-ATG-initiated translation from the repeat expansion in all reading frames leading to the aggregation of dipeptide-repeat proteins in neurons.8Ash P.E. Bieniek K.F. Gendron T.F. Caulfield T. Lin W.L. Dejesus-Hernandez M. van Blitterswijk M.M. Jansen-West K. Paul 3rd, J.W. Rademakers R. et al.Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS.Neuron. 2013; 77: 639-646Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar, 9Mori K. Weng S.M. Arzberger T. May S. Rentzsch K. Kremmer E. Schmid B. Kretzschmar H.A. Cruts M. Van Broeckhoven C. et al.The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS.Science. 2013; 339: 1335-1338Crossref PubMed Scopus (864) Google Scholar Another model is a loss-of-function mechanism (haploinsufficiency), supported by observations that the expansion leads to ∼50% reduction of C9orf72 mRNA in frontal cortex and lymphoblasts derived from heterozygous mutation carriers.1DeJesus-Hernandez M. Mackenzie I.R. Boeve B.F. Boxer A.L. Baker M. Rutherford N.J. Nicholson A.M. Finch N.A. Flynn H. Adamson J. et al.Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.Neuron. 2011; 72: 245-256Abstract Full Text Full Text PDF PubMed Scopus (3382) Google Scholar, 4Gijselinck I. Van Langenhove T. van der Zee J. Sleegers K. Philtjens S. Kleinberger G. Janssens J. Bettens K. Van Cauwenberghe C. Pereson S. et al.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; 11: 54-65Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar Finally, it is possible that all three mechanisms contribute to the disease. Here we tested the hypothesis that the expansion may cause aberrant CpG methylation of C9orf72 leading to downregulation of its mRNA, as was shown for different C9orf72 transcripts in a few published expansion carriers.1DeJesus-Hernandez M. Mackenzie I.R. Boeve B.F. Boxer A.L. Baker M. Rutherford N.J. Nicholson A.M. Finch N.A. Flynn H. Adamson J. et al.Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.Neuron. 2011; 72: 245-256Abstract Full Text Full Text PDF PubMed Scopus (3382) Google Scholar, 4Gijselinck I. Van Langenhove T. van der Zee J. Sleegers K. Philtjens S. Kleinberger G. Janssens J. Bettens K. Van Cauwenberghe C. Pereson S. et al.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; 11: 54-65Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar Notably, the G4C2 repeat is mapped to the promoter region of the NM_018325.3 transcript, and the promoter region of two other C9orf72 transcripts (NM_001256054.1 and NM_145005.5) overlaps a CpG island 5′ of the repeat (Figure S1 available online). Cytosine (C) methylation of CpG dinucleotides is an important epigenetic modification10Klose R.J. Bird A.P. Genomic DNA methylation: the mark and its mediators.Trends Biochem. Sci. 2006; 31: 89-97Abstract Full Text Full Text PDF PubMed Scopus (1909) Google Scholar that could lead to gene expression silencing as a result of hypermethylation of CpG islands11Jaenisch R. Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals.Nat. Genet. 2003; 33: 245-254Crossref PubMed Scopus (4661) Google Scholar, 12Bird A. DNA methylation patterns and epigenetic memory.Genes Dev. 2002; 16: 6-21Crossref PubMed Scopus (5409) Google Scholar (defined as >200 bp regions with >50% CG content and >0.6 ratio of observed to expected number of CG-dinucleotides). Several genes with repeat expansions are known to have aberrant CpG methylation leading to downregulation of gene expression. For example, in individuals with Friedreich ataxia (MIM 229300), hypermethylation was detected upstream of the GAA expansion in intron 1 of FXN (MIM 606829), which leads to a deficit of FXN mRNA in blood, heart, and brain.13Al-Mahdawi S. Pinto R.M. Ismail O. Varshney D. Lymperi S. Sandi C. Trabzuni D. Pook M. The Friedreich ataxia GAA repeat expansion mutation induces comparable epigenetic changes in human and transgenic mouse brain and heart tissues.Hum. Mol. Genet. 2008; 17: 735-746Crossref PubMed Scopus (205) Google Scholar, 14Greene E. Mahishi L. Entezam A. Kumari D. Usdin K. Repeat-induced epigenetic changes in intron 1 of the frataxin gene and its consequences in Friedreich ataxia.Nucleic Acids Res. 2007; 35: 3383-3390Crossref PubMed Scopus (157) Google Scholar, 15Evans-Galea M.V. Carrodus N. Rowley S.M. Corben L.A. Tai G. Saffery R. Galati J.C. Wong N.C. Craig J.M. Lynch D.R. et al.FXN methylation predicts expression and clinical outcome in Friedreich ataxia.Ann. Neurol. 2012; 71: 487-497Crossref PubMed Scopus (93) Google Scholar DNA hypermethylation is also caused by a CGG expansion in the 5′ UTR of FMR1 (MIM 309550) responsible for fragile X mental retardation syndrome (MIM 300624).16Sutcliffe J.S. Nelson D.L. Zhang F. Pieretti M. Caskey C.T. Saxe D. Warren S.T. DNA methylation represses FMR-1 transcription in fragile X syndrome.Hum. Mol. Genet. 1992; 1: 397-400Crossref PubMed Scopus (570) Google Scholar, 17Pieretti M. Zhang F.P. Fu Y.H. Warren S.T. Oostra B.A. Caskey C.T. Nelson D.L. Absence of expression of the FMR-1 gene in fragile X syndrome.Cell. 1991; 66: 817-822Abstract Full Text PDF PubMed Scopus (1235) Google Scholar, 18Bell M.V. Hirst M.C. Nakahori Y. MacKinnon R.N. Roche A. Flint T.J. Jacobs P.A. Tommerup N. Tranebjaerg L. Froster-Iskenius U. et al.Physical mapping across the fragile X: hypermethylation and clinical expression of the fragile X syndrome.Cell. 1991; 64: 861-866Abstract Full Text PDF PubMed Scopus (299) Google Scholar Moreover, the CTG expansion in the 3′ UTR of DMPK (MIM 605377) causes hypermethylation19López Castel A. Nakamori M. Tomé S. Chitayat D. Gourdon G. Thornton C.A. Pearson C.E. Expanded CTG repeat demarcates a boundary for abnormal CpG methylation in myotonic dystrophy patient tissues.Hum. Mol. Genet. 2011; 20: 1-15Crossref PubMed Scopus (103) Google Scholar of the promoter of a downstream gene, reducing its transcription.20Klesert T.R. Otten A.D. Bird T.D. Tapscott S.J. Trinucleotide repeat expansion at the myotonic dystrophy locus reduces expression of DMAHP.Nat. Genet. 1997; 16: 402-406Crossref PubMed Scopus (220) Google Scholar, 21Thornton C.A. Wymer J.P. Simmons Z. McClain C. Moxley 3rd, R.T. Expansion of the myotonic dystrophy CTG repeat reduces expression of the flanking DMAHP gene.Nat. Genet. 1997; 16: 407-409Crossref PubMed Scopus (195) Google Scholar, 22Korade-Mirnics Z. Tarleton J. Servidei S. Casey R.R. Gennarelli M. Pegoraro E. Angelini C. Hoffman E.P. Myotonic dystrophy: tissue-specific effect of somatic CTG expansions on allele-specific DMAHP/SIX5 expression.Hum. Mol. Genet. 1999; 8: 1017-1023Crossref PubMed Scopus (40) Google Scholar To gain new insight into the C9orf72 disease mechanism, we investigated the CpG methylation profile around the G4C2 repeat by two methods using genomic DNA collected from our well-characterized Canadian ALS cohort and normal controls. Informed consent was obtained from all participants in accordance with the ethical review board. Study participants were diagnosed with ALS at the ALS Clinic of Sunnybrook Health Sciences Centre in Toronto according to established criteria.23Clinical and neuropathological criteria for frontotemporal dementia. The Lund and Manchester Groups.J. Neurol. Neurosurg. Psychiatry. 1994; 57: 416-418Crossref PubMed Scopus (1842) Google Scholar, 24Brooks B.R. Miller R.G. Swash M. Munsat T.L. World Federation of Neurology Research Group on Motor Neuron DiseasesEl Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis.Amyotroph. Lateral Scler. Other Motor Neuron Disord. 2000; 1: 293-299Crossref PubMed Scopus (4002) Google Scholar, 25Hughes A.J. Daniel S.E. Lees A.J. Improved accuracy of clinical diagnosis of Lewy body Parkinson’s disease.Neurology. 2001; 57: 1497-1499Crossref PubMed Scopus (626) Google Scholar Expansion carriers were defined by the previously suggested 30-repeat cutoff.1DeJesus-Hernandez M. Mackenzie I.R. Boeve B.F. Boxer A.L. Baker M. Rutherford N.J. Nicholson A.M. Finch N.A. Flynn H. Adamson J. et al.Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.Neuron. 2011; 72: 245-256Abstract Full Text Full Text PDF PubMed Scopus (3382) Google Scholar, 2Renton A.E. Majounie E. Waite A. Simón-Sánchez J. Rollinson S. Gibbs J.R. Schymick J.C. Laaksovirta H. van Swieten J.C. Myllykangas L. et al.ITALSGEN ConsortiumA hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD.Neuron. 2011; 72: 257-268Abstract Full Text Full Text PDF PubMed Scopus (3081) Google Scholar DNA from blood was available for 37 unrelated ALS expansion carriers (mainly identified in our previous study7Xi Z. Zinman L. Grinberg Y. Moreno D. Sato C. Bilbao J.M. Ghani M. Hernández I. Ruiz A. Boada M. et al.Investigation of c9orf72 in 4 neurodegenerative disorders.Arch. Neurol. 2012; 69: 1583-1590Crossref PubMed Scopus (83) Google Scholar) and 140 noncarriers (64 ALS cases and 76 controls >65 years old). Sample characteristics are presented in Table 1. The clinical parameters between ALS expansion carriers and noncarriers are similar, except that carriers have an earlier age of onset (p = 0.04) and higher percentage of familial cases (p = 2 × 10−6), which is a known association.4Gijselinck I. Van Langenhove T. van der Zee J. Sleegers K. Philtjens S. Kleinberger G. Janssens J. Bettens K. Van Cauwenberghe C. Pereson S. et al.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; 11: 54-65Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar In addition, we investigated blood DNA from available family members of seven ALS probands with the expansion (including four simplex cases) and three previously reported7Xi Z. Zinman L. Grinberg Y. Moreno D. Sato C. Bilbao J.M. Ghani M. Hernández I. Ruiz A. Boada M. et al.Investigation of c9orf72 in 4 neurodegenerative disorders.Arch. Neurol. 2012; 69: 1583-1590Crossref PubMed Scopus (83) Google Scholar carriers of intermediate alleles (24, 32, 39 repeats) diagnosed with Parkinson disease (MIM 168600). DNA was also extracted from blood, frontal cortex, and cervical spinal cord for seven autopsy-confirmed expansion carriers, as well as from frontal cortex of 14 controls.Table 1Sample CharacteristicsBloodBrain (Frontal Cortex)ALS ExpALS NonexpControlALS ExpControlNumber of samples376476714Age at sample collection60.5 ± 9.566.5 ± 9.373.0 ± 7.656.7 ± 7.878.1 ± 7.7Age at onset58.6 ± 9.463.7 ± 11.4–55.3 ± 8.1–Female (frequency)15 (0.41)25 (0.39)48 (0.63)35Family history ( frequency)18 (0.49)5 (0.08)–5–DiagnosisALS ( frequency)32 (0.86)54 (0.84)–4–ALS-FTD ( frequency)5 (0.14)10 (0.16)–3–Range of G4C2 repeatsSmall allele2–112–82–82–102–5Larger allele35, exp2–222–19exp2–12Abbreviation is as follows: exp, expansion. Open table in a new tab Abbreviation is as follows: exp, expansion. To assess the level of methylation we developed two assays. The basis of the methylation-sensitive restriction enzyme assay was adapted from previous reports26Allen R.C. Zoghbi H.Y. Moseley A.B. Rosenblatt H.M. Belmont J.W. Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation.Am. J. Hum. Genet. 1992; 51: 1229-1239PubMed Google Scholar, 27Rogaev E.I. Lukiw W.J. Lavrushina O. Rogaeva E.A. St George-Hyslop P.H. The upstream promoter of the beta-amyloid precursor protein gene (APP) shows differential patterns of methylation in human brain.Genomics. 1994; 22: 340-347Crossref PubMed Scopus (48) Google Scholar (the experimental principle is shown in Figure S2). In brief, 100 ng of DNA was digested overnight at 37°C with 1 U methylation-sensitive restriction enzyme (HhaI, HpaII, or HpyCH4IV, New England Biolabs), followed by 10 min inactivation at 95°C. The enzyme digests unmethylated DNA, and therefore the following PCR would produce an amplification product only for methylated DNA. As an internal control, a duplicate reaction for each DNA sample was prepared without the enzyme. The digested and undigested DNA was amplified in parallel (Tm = 60.5°C) (Table S1). PCR products (1.5 μl) were resolved on a 1.5% agarose gel. To quantify the band intensity, black/white inverted gel images were analyzed by the Gel Analysis tool in Image J, and the observed methylation (OM) ratio was calculated based on the band intensities (PCR from digested DNA ÷ PCR from undigested DNA). The direct bisulfite sequencing assay was adapted from previous reports.19López Castel A. Nakamori M. Tomé S. Chitayat D. Gourdon G. Thornton C.A. Pearson C.E. Expanded CTG repeat demarcates a boundary for abnormal CpG methylation in myotonic dystrophy patient tissues.Hum. Mol. Genet. 2011; 20: 1-15Crossref PubMed Scopus (103) Google Scholar, 28Parrish R.R. Day J.J. Lubin F.D. Direct bisulfite sequencing for examination of DNA methylation with gene and nucleotide resolution from brain tissues.Curr. Protoc. Neurosci. 2012; Chapter 7 (Unit 7, 24)PubMed Google Scholar, 29Jiang M. Zhang Y. Fei J. Chang X. Fan W. Qian X. Zhang T. Lu D. Rapid quantification of DNA methylation by measuring relative peak heights in direct bisulfite-PCR sequencing traces.Lab. Invest. 2010; 90: 282-290Crossref PubMed Scopus (83) Google Scholar It is based on the conversion of unmethylated C to T by bisulfite treatment, while methylated C remain unchanged. Bisulfite treatment was performed with 100 ng of DNA by the Imprint DNA Modification kit (Sigma). The investigated region is shown in Figure S3. After conversion, the region 5′ of the G4C2 repeat was amplified by a seminested PCR (Table S1). Bisulfite-treated DNA (2 μl) was amplified by FastStart PCR Master Mix (Roche) by touchdown PCR (for primers BSP_1F, BSP_1R: Tm from 68°C to 58°C at a rate of −1°C/cycle and fixed at 58°C for 30 cycles; for primers BSP_2F, BSP_2R: Tm from 67°C to 57°C at a rate of −1°C/cycle and fixed at 57°C for 20 cycles; 3 min extension for each cycle). Methylation was detected by direct inspection of sequencing chromatograms and an integrated tool within Mutation Surveyor version 4.0 (Softgenetics). Only samples with >95% conversion rate of non-CpG C were included in the analyses. To avoid quantification errors caused by relative peak height measurements at each CpG, broad categories were used to score methylation level: unmethylated CpG (T peak) or methylated CpG (T/C double peaks). Of note, “C peak only” (indicating 100% methylation) was not observed in any sample. For each sample we obtained the total number of methylated CpG sites detected in the inspected region. A linear regression analysis was performed between the OM ratio (HhaI assay) and number of methylated CpG sites (bisulfite sequencing assay) to calculate the correlation between the assays. A nonlinear regression analysis was used to evaluate the correlation between methylation level and disease duration. The goodness of fit measure (R2) was used to quantify these correlations. The p value was calculated by ANOVA to determine the significance of R2. Pearson’s or Spearman’s correlation coefficients were used to measure the correlation between independent variables: repeat size (<50 repeats), age, and methylation level. The nonparametric Mann-Whitney U test was used to compare continuous variables between two groups. The two-sided Pearson χ2 test or Fisher’s exact test (when expected value <5) were used to compare categorical variables. All analyses were performed with SPSS (v.20). By using the UCSC database, we detected two predicted CpG islands immediately flanking the G4C2 repeat (Figure S1), which contain restriction sites for three methylation-sensitive enzymes (HhaI, HpaII, and HpyCH4IV). To assess the sensitivity of these enzymes for methylation detection, we used a premixed calibration standard DNA (EpigenDx) consisting of a low-methylated DNA (<5% methylation), a high-methylated DNA (>85% methylation), and a mixture series of both. The region 5′ of the G4C2 repeat (fragment #1) has two HhaI, one HpaII, and three HpyCH4IV sites (Figure S4A). Analysis of fragment #1 revealed that the HhaI assay is the most sensitive, because it is able to detect methylation in a mixture containing only 5% high-methylated DNA, whereas the other two enzymes start to detect methylation from 25% to 50% (Figure S4B). No methylation was observed for the DNA of a randomly selected nonexpansion carrier (Figure S4B). By measuring band intensity (four replicates), we obtained a standard curve with R2 = 0.96, indicating the reliability of the quantification by the HhaI assay (Figure S4C). Therefore, the following case-control study was conducted with the HhaI assay. The same calibration DNA standards were used to assess the efficiency of the bisulfite sequencing assay. All non-CpG C were successfully converted to T (Figure S4D). As expected, only a few methylated C were detected in the low-methylated standard, and the assay was able to detect methylation of all CpG sites, even in the mixture with only 5% high-methylated DNA. CpG islands on both sides of the G4C2 repeat were analyzed by the HhaI assay (Figure 1A). The region 5′ (but not 3′) of the repeat revealed evidence of hypermethylation in expansion carriers (Figure 1B). The bisulfite sequencing assay also revealed evidence of hypermethylation at the 5′ region (as presented below) but not the 3′ region, which was evaluated in the pilot ALS cohort of 32 expansion carriers and 16 noncarriers (data not shown). A similar methylation pattern was reported for Friedreich ataxia15Evans-Galea M.V. Carrodus N. Rowley S.M. Corben L.A. Tai G. Saffery R. Galati J.C. Wong N.C. Craig J.M. Lynch D.R. et al.FXN methylation predicts expression and clinical outcome in Friedreich ataxia.Ann. Neurol. 2012; 71: 487-497Crossref PubMed Scopus (93) Google Scholar and myotonic dystrophy.19López Castel A. Nakamori M. Tomé S. Chitayat D. Gourdon G. Thornton C.A. Pearson C.E. Expanded CTG repeat demarcates a boundary for abnormal CpG methylation in myotonic dystrophy patient tissues.Hum. Mol. Genet. 2011; 20: 1-15Crossref PubMed Scopus (103) Google Scholar It was suggested that an expanded repeat could act as a barrier inhibiting the spread of methylation downstream;15Evans-Galea M.V. Carrodus N. Rowley S.M. Corben L.A. Tai G. Saffery R. Galati J.C. Wong N.C. Craig J.M. Lynch D.R. et al.FXN methylation predicts expression and clinical outcome in Friedreich ataxia.Ann. Neurol. 2012; 71: 487-497Crossref PubMed Scopus (93) Google Scholar however, the underlying mechanism is not well understood. Notably, the G4C2 repeat expansion itself could also be considered part of the CpG island and may be methylated as well, but currently available technologies do not allow the large expansion to be amplified by PCR or thoroughly sequenced. Next, methylation of the CpG island 5′ of the G4C2 repeat was assessed by both assays in the entire data set of 177 unrelated individuals. The OM ratio of the HhaI assay was significantly higher in the group of 37 ALS expansion carriers (mean ± SD = 0.52 ± 0.40) versus 64 ALS noncarriers (mean ± SD = 0.17 ± 0.09, Mann-Whitney U test p < 0.0001) or versus 76 controls (mean ± SD = 0.13 ± 0.07, Mann-Whitney U test p < 0.0001). Almost all samples without an expansion allele (138 out of 140) had an OM ratio below 0.3, which represents the methylation level of the low-methylated DNA standard (<5% methylation) in the HhaI assay standard curve. In contrast, 25 of 37 ALS expansion carriers had a ratio >0.3. A representative gel image of 12 samples from each group is shown in Figure 1D. The bisulfite sequencing assay revealed the methylation status 5′ of the G4C2 repeat beyond the two CpG sites recognized by the HhaI assay. A total of 26 CpG sites were assessed (Figure S3). A representative sequence chromatogram is shown in Figure 2A. In the nonexpansion group, 97% of samples (n = 136) were unmethylated (number of methylated CpG sites = 0). In contrast, 73% of expansion carriers (n = 27) had a number of methylated CpG sites ≥1, and methylation was detected at each of the 26 CpG sites (Figure S5A). We categorized the different methylation levels based on the maximum number of methylated CpG observed in controls: 0, no methylation; 1–3, low methylation; and 4–26, high methylation (Table 2). In agreement with the HhaI assay, a significant difference in methylation level between the expansion and nonexpansion groups was observed (p < 0.0001). No difference was found between the two nonexpansion groups (control versus ALS, p = 0.625).Table 2Methylation Level of Blood DNA from ALS Expansion Carriers, ALS Nonexpansion Carriers, and ControlsMethylation Level (Number of Methylated CpG)ALS ExpALS NonexpControlNFrequencyNFrequencyNFrequencyNo methylation (0)100.27630.98730.96Low methylation (1–3)120.3210.0230.04High methylation (4–26)150.4100.0000.00Total376476pa2-sided Pearson χ2 test was used, df = 2. Fisher’s exact test was used (when expected value < 5).: compared to ALS exp group<0.0001<0.0001pa2-sided Pearson χ2 test was used, df = 2. Fisher’s exact test was used (when expected value < 5).: compared to ALS nonexp group0.625Abbreviation is as follows: exp, expansion.a 2-sided Pearson χ2 test was used, df = 2. Fisher’s exact test was used (when expected value < 5). Open table in a new tab Abbreviation is as follows: exp, expansion. A scatter plot of the OM ratio against the number of methylated CpG sites revealed a significant correlation between the two assays (p < 0.0001) (Figure 2B). Bisulfite sequencing analysis of the 26 CpG sites revealed that the two CpG sites recognized by the HhaI assay (CpG2 and CpG8) are among the most frequently methylated sites in expansion carriers (70% and 30%, respectively), which is the basis of the strong correlation between the assays (Figure S5A). Notably, neither assay identified methylation in carriers with intermediate alleles (24, 32, 39, or 43 repeats; Figure 1C). Individuals without an expansion had a wide range of normal alleles (2–22 repeats) (Table 1), all of which had a low methylation level (methylated CpG sites < 3). Finally, the number of methylated CpG sites in expansion carriers and the size of their normal allele (2–11 repeats) were not correlated (Spearman’s correlation coefficient = 0.258, p = 0.124), indicating that the normal allele does not contribute to the methylation profile of expansion carriers. The level of methylation 5′ of the G4C2 repeat did not correlate with age at time of examination (range: 39–80 years old; Spearman’s correlation coefficient = −0.015, p = 0.931) or age of onset (range: 37–78 years old; Spearman’s correlation coefficient = 0.041, p = 0.814). However, it was inversely associated with disease duration (n = 24; OM: R2 = 0.388, p = 0.001; number of methylated CpG sites: R2 = 0.303, p = 0.005). Moreover, methylation was also significantly associated with familial ALS" @default.
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