Nces (NCATS). CReATe is funded through collaboration between NCATS and the National Institute of Neurological Disorders and Stroke (NINDS). This work was also supported, in part, by a grant from the NIH R01 NS088689 (R.B and C.M.). Availability of data and materials All data generated in this study are published in this article and its supplementary information files. Authors’ contributions RE, CM and ZZ designed the study. RE, GTC and NSA performed experiments and data analysis. TFG performed DPR assessment. MB, CW and RHB contributed expertise and intellectual feedback. RE and ZZ wrote the paper and all authors approved the manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval All experimental procedures involving transgenic mice were performed in accordance with the guidelines of Institutional Animal Care and Use Committee of the University of Massachusetts Medical School. Induced pluripotent stem cells were derived from the blood of consenting individuals within our C9ORF2-ALS patient population under IRB approved protocols at the University of Miami Miller School of Medicine.Conclusions We have determined that epigenetic perturbations seen in C9-ALS patients are also observed in C9-BAC mice and that R-loops are unlikely to be initiators of DNA methylation at the expanded C9ORF72 locus. Since many repeat expansion mutations, including PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28045099 C9-ALS, have an epigenetic component that is directly linked to pathology, our observations are of significant importance for understanding the mechanism of disease and are of consequence for therapeutic development efforts utilizing C9-BAC mouse models. Additional filesAdditional file 1: Figure S1. Relative quantification (RQ) values of mouse beta-actin, GAPDH and 18S endogenous controls in C9-BAC mouse cortex PD173074 dose across different age groups are shown (A). Absolute copy number of human C9ORF72 transcripts per microliter in C9-BAC mice as determined by digital droplet PCR (B), one-way ANOVA (p < 0.001) and Bonferroni's multiple comparison test was performed between neonatal (0wks) and the remaining age groups, significance is indicated by p < 0.05 * and p < 0.01 **. (JPEG 238 kb) Additional file 2: Figure S2. Copy number variation analysis for human C9ORF72 transgene in C9-BAC mouse brain cortex with hypermethylated (me+), unmethylated (me-) promoter and wild-type mouse (WT). (JPEG 142 kb) Additional file 3: Figure S3. Linear regression analysis of the mean C9ORF72 promoter methylation percentile (as determined by bisulfite pyrosequencing) and glycine-proline dipeptide abundance (A). R square and p values for individual CpG dinucleotides are indicated (B). Quantitative PCR assessment of C9ORF72 expression in hypermethylated C9-BAC mice indicated by open shapes (17wks square, 30wks triangle, 36wks circle) and their age group counterparts (C), error bars represent SEM. (JPEG 530 kb)Publisher's NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details 1 Department of Psychiatry Behavioral Sciences, Center for Therapeutic Innovation, University of Miami Miller School of Medicine, 1501 NW 10th Ave, BRB-413, Miami, FL 33136, USA. 2Department of Neurology, University of Massachusetts Medical School, Worchester, MA 016555, USA. 3Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224, USA. 4Department of Neurology, University of Miami.
