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

Abstract

A significant number 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 bisulfite-modified 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.

Keywords: Amyotrophic lateral sclerosis; C9orf72; DNA methylation; Epigenetics; Frontotemporal dementia; Repeat expansion.

Figure 1

The C9orf72 promoter is hypermethylated in one c9FTD patient. (A) Schematic representation showing the three known transcript variants of C9orf72; V1: NM_145005.5, V2: NM_018325.3, V3: NM_001256054.1. Red triangles mark the repeat location. Red brackets mark the amplified region after the first PCR run. The two CpG islands located at the 5' and 3' end of the repeat region are shown in green. (B) Chromatograms showing sequence amplification of the CpG island located at the 5' end of the repeat region after bisulfite treatment of the DNA isolated from cerebellum. The top panel shows the reference sequence. The middle panel shows a section of the sequence obtained from the c9FTD patient hypermethylated at the C9orf72 promoter region (c9FTD/ALS sample # 8, see Table 1). The last panel shows a section of the sequence obtained from a c9FTD patient in whom no methylated sites were identified (c9FTD/ALS sample #4, see Table 1). Blue line over thymines in the reference sequence marks sites of bisulfite conversion from cytosines. CpG sites are highlighted in orange. (C) Bar graph representing the number of samples showing hypermethylation, low methylation, or no methylation of the CpG island located at the 5' end of the repeat region in cerebellum of each group.