Characterization of DNA hypermethylation in the cerebellum of c9FTD/ALS patients

Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, Florida 32224, USA.
Brain research (Impact Factor: 2.84). 10/2014; 1584. DOI: 10.1016/j.brainres.2014.02.015


A significant portion of patients suffering from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two diseases commonly seen in comorbidity, carry an expanded noncoding hexanucleotide repeat in the C9orf72 gene, a condition collectively referred to as c9FTD/ALS. Repeat expansions, also present in other neurodegenerative diseases, have been shown to alter epigenetic mechanisms and consequently lead to decreased gene expression, while also leading to toxic RNA gain-of-function. As expression of multiple C9orf72 transcript variants is known to be reduced in c9FTD/ALS cases, our group and others have sought to uncover the mechanisms causing this reduction. We recently demonstrated that histones H3 and H4 undergo trimethylation at lysines 9 (H3K9), 27 (H3K27), 79 (H3K79), and 20 (H4K20) in all pathogenic repeat carrier brain samples, confirming the role of altered histone methylation in disease. It was also reported that about 40% of c9ALS cases show hypermethylation of the CpG island located at the 5′ end of the repeat expansion in blood, frontal cortex, and spinal cord. To determine whether the same CpG island is hypermethylated in the cerebella of cases in whom aberrant histone methylation has been identified, we bisulfitemodified the extracted DNA and PCR-amplified 26 CpG sites within the C9orf72 promoter region. Among the ten c9FTD/ALS (4 c9ALS, 6 c9FTD), nine FTD/ALS, and eight disease control samples evaluated, only one c9FTD sample was found to be hypermethylated within the C9orf72 promoter region. This study is the first to report cerebellar hypermethylation in c9FTD/ALS, and the first to identify a c9FTD patient with aberrant DNA methylation. Future studies will need to evaluate hypermethylation of the C9orf72 promoter in a larger cohort of c9FTD patients, and to assess whether DNA methylation variation across brain regions reflects disease phenotype.

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Available from: Melissa Erin Murray, Feb 18, 2014
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    • "The C9orf72 protein product remains poorly characterized but structurally resembles Differentially Expressed in Normal and Neoplastic cells (DENN) proteins, which are involved in membrane trafficking processes (Zhang et al., 2012). Alleles with expansions larger than 30 repeats are enriched with repressive epigenetic marks within the gene promoter including histone 3 lysine 9 trimethylation (H3K9me3) and cytosine methylation (5mC) (Belzil et al., 2014). Such heterochromatinization corresponds to reduced transcription rates, leading to the hypothesis that haploinsufficiency may contribute to the pathology of C9orf72- related ALS and FTD (Gendron et al., 2014). "
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    ABSTRACT: Among several genetic mutations known to cause amyotrophic lateral sclerosis (ALS), a hexanucleotide repeat expansion in the C9orf72 gene is the most common. In approximately 30% of C9orf72-ALS cases, 5-methylcytosine (5mC) levels within the C9orf72 promoter are increased, resulting in a modestly attenuated phenotype. The developmental timing of C9orf72 promoter hypermethylation and the reason why it occurs in only a subset of patients remain unknown. In order to model the acquisition of C9orf72 hypermethylation and examine the potential role of 5-hydroxymethylcytosine (5hmC), we generated induced pluripotent stem cells (iPSCs) from an ALS patient with C9orf72 promoter hypermethylation. Our data show that 5mC levels are reduced by reprogramming and then re-acquired upon neuronal specification, while 5hmC levels increase following reprogramming and are highest in iPSCs and motor neurons. We confirmed the presence of 5hmC within the C9orf72 promoter in post-mortem brain tissues of hypermethylated patients. These findings show that iPSCs are a valuable model system for examining epigenetic perturbations caused by the C9orf72 mutation and reveal a potential role for cytosine demethylation.
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    ABSTRACT: The G4C2-repeat expansion in C9orf72 is a common cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). C9orf72 transcription is reduced in expansion carriers implicating haploinsufficiency as one of the disease mechanisms. Indeed, our recent ALS study revealed that the expansion was associated with hypermethylation of the CpG-island (5′of the repeat) in DNA samples obtained from different tissues (blood, brain and spinal cord). However, the link between FTLD and methylation of the CpG-island is unknown. Hence, we investigated the methylation profile of the same CpG-island by bisulfite sequencing of DNA obtained from blood of 34 FTLD expansion carriers, 166 FTLD non-carriers and 103 controls. Methylation level was significantly higher in FTLD expansion carriers than non-carriers (P = 7.8E−13). Our results were confirmed by two methods (HhaI-assay and sequencing of cloned bisulfite PCR products). Hypermethylation occurred only in carriers of an allele with >50 repeats, and was not detected in non-carriers or individuals with an intermediate allele (22–43 repeats). As expected, the position/number of methylated CpGs was concordant between the sense and anti-sense DNA strand, suggesting that it is a stable epigenetic modification. Analysis of the combined ALS and FTLD datasets (82 expansion carriers) revealed that the degree of methylation of the entire CpG-island or contribution of specific CpGs (n = 26) is similar in both syndromes, with a trend towards a higher proportion of ALS patients with a high methylation level (P = 0.09). In conclusion, we demonstrated that hypermethylation of the CpG-island 5′of the G4C2-repeat is expansion-specific, but not syndrome-specific (ALS versus FTLD).
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    ABSTRACT: The expanded GGGGCC hexanucleotide repeat in the non-coding region of the C9orf72 gene on chromosome 9p21 has been discovered as the cause of approximately 20–50% of familial and up to 5–20% of sporadic amyotrophic lateral sclerosis (ALS) cases, making this the most common known genetic mutation of ALS to date. At the same time, it represents the most common genetic mutation in frontotemporal dementia (FTD; 10–30%). Because of the high prevalence of mutant C9orf72, pre-clinical efforts in identifying therapeutic targets and developing novel therapeutics for this mutation are highly pursued in the hope of providing a desperately needed disease-modifying treatment for ALS patients, as well as other patient populations affected by the C9orf72 mutation. The current lack of effective treatments for ALS is partially due to the lack of appropriate biomarkers that aide in assessing drug efficacy during clinical trials independent of clinical outcome measures, such as increased survival. In this review we will summarize the opportunities for biomarker development specifically targeted to the newly discovered C9orf72 repeat expansion. While drugs are being developed for this mutation, it will be crucial to provide a reliable biomarker to accompany the clinical development of these novel therapeutic interventions to maximize the chances of a successful clinical trial. This article is part of a Special Issue entitled ALS complex pathogenesis. This article is part of a Special Issue entitled ALS complex pathogenesis.
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