C9orf72 Promoter Methylation Analysis

Methylation levels for the C9orf72 promoter in patients were determined as previously described (45 (link)). Briefly, genomic DNA from the cerebellum was extracted using the Qiagen DNeasy Blood and Tissue kit and subject to overnight digestion with HhaI and HaeIII (double-digested) or just HaeIII (mock) alone. A small aliquot of DNA was amplified using primers flanking the HhaI cutsite within the C9orf72 promoter region using 2x FastStart SYBR Green Master (Roche) on the ABI StepOnePlus machine. The difference in cycles to threshold amplification between double and mock digested DNA was calculated as methylation values. Spearman’s correlation was calculated using gene counts for each gene and methylation values for each C9orf72 mutation case. R package ‘lsr’ with ‘correlate’ function (with options corr.method =“spearman” and p.adjust.method=“fdr”) using the Spearman’s correlation and FDR adjusted p-values was used. Only correlations from genes with HUGO gene symbols were calculated and plotted against gene fold change for each gene that was calculated by DESeq2. This was done using both significantly differentially expressed genes linked to the C9orf72 mutation (DESeq2 FDR p-value < 0.05) and genome-wide using all expressed genes.