7 - Satellite cells from donors with spastic cerebral palsy exhibit altered DNA methylation patterns

Poster Number: 7
Publication Number: 7.109

Karyn Robinson, NemoursAlfred I. duPont Hospital for Children, Wilmington, DE, United States; Stephanie K. Lee, NemoursAlfred I. duPont Hospital for Children, Wilmington, DE, United States; Robert Akins, NemoursAlfred I. duPont Hospital for Children, Wilmington, DE, United States

Background: Individuals with spastic cerebral palsy (CP) have impaired longitudinal skeletal muscle growth and contractures that may contribute to impaired motor control, muscle atrophy, and increased need for clinical care or surgery. Muscle stem cells (satellite cells) are responsible for skeletal muscle growth and repair, and studies have demonstrated there is a decreased number of satellite cells in CP and that these cells exhibit inhibited myotube formation in vitro; however, the mechanisms responsible for these differences are unclear. Previous studies have established that individuals with CP have distinguishing DNA methylation patterns in blood cells and that alterations in DNA methylation patterns can remain stable over time.

Objective: We investigated whole genome DNA methylation pattern differences in satellite cell-derived myoblasts and myotubes from participants with spastic CP compared to controls.

Design/Methods: Surgical explants of skeletal muscle were obtained from subjects undergoing orthopedic surgery at the Nemours Children’s Hospital in Delaware following IRB approved informed consent/assent. Six subjects with spastic CP (average age=15.3 ± 2.7) and 6 control subjects (average age 13.9 ± 1.7) met inclusionary criteria. Satellite cells were isolated, validated, cultured, and differentiated. Nucleic acids were isolated from both proliferating satellite cell-derived myoblasts (SCDMBs) and differentiating satellite cell-derived myotubes (SCDMTs). DNA libraries were prepared from methyl-sensitive restriction endonuclease fragmented genomic DNA using HpaII, and next generation sequencing was performed on an Illumina ×10 platform.

Results: After initial filtering, 1.5 million CpG sites were included in the SCDMB analysis and 1.8 million in the SCDMT analysis. Significant differential DNA methylation between the CP and control cohorts was found at 544 CpG sites in SCDMBs and 1880 CpG sites in SCDMTs (FDR-corrected p-value < 0.05). Of these, 80 CpG sites were significantly different across both cell conditions with 36 significantly hypermethylated and 44 significantly hypomethylated in the CP compared to the control cohorts. The chromosomal localization of the significant CpG sites showed enrichment on chromosomes 12, 13, 14, and 18.Conclusion(s): Satellite cells derived from subjects with CP exhibited fundamental differences in DNA methylation patterns compared to controls. The particular enrichment of significant CpGs on chromosomes 12, 13, 14, and 18 could reveal potential regions to target for future functional and validation studies.