DNA methylation across the genome in aged human skeletal muscle tissue and muscle-derived cells: the role of HOX genes and physical activity.

Skeletal muscle tissue demonstrates global hypermethylation with age. However, methylome changes across the time-course of differentiation in aged human muscle derived cells, and larger coverage arrays in aged muscle tissue have not been undertaken. Using 850K DNA methylation arrays we compared the methylomes of young (27 ± 4.4 years) and aged (83 ± 4 years) human skeletal muscle and that of young/aged heterogenous muscle-derived human primary cells (HDMCs) over several time points of differentiation (0, 72 h, 7, 10 days). Aged muscle tissue was hypermethylated compared with young tissue, enriched for; pathways-in-cancer (including; focal adhesion, MAPK signaling, PI3K-Akt-mTOR signaling, p53 signaling, Jak-STAT signaling, TGF-beta and notch signaling), rap1-signaling, axon-guidance and hippo-signalling. Aged cells also demonstrated a hypermethylated profile in pathways; axon-guidance, adherens-junction and calcium-signaling, particularly at later timepoints of myotube formation, corresponding with reduced morphological differentiation and reductions in MyoD/Myogenin gene expression compared with young cells. While young cells showed little alterations in DNA methylation during differentiation, aged cells demonstrated extensive and significantly altered DNA methylation, particularly at 7 days of differentiation and most notably in focal adhesion and PI3K-AKT signalling pathways. While the methylomes were vastly different between muscle tissue and HDMCs, we identified a small number of CpG sites showing a hypermethylated state with age, in both muscle tissue and cells on genes KIF15, DYRK2, FHL2, MRPS33, ABCA17P. Most notably, differential methylation analysis of chromosomal regions identified three locations containing enrichment of 6-8 CpGs in the HOX family of genes altered with age. With HOXD10, HOXD9, HOXD8, HOXA3, HOXC9, HOXB1, HOXB3, HOXC-AS2 and HOXC10 all hypermethylated in aged tissue. In aged cells the same HOX genes (and additionally HOXC-AS3) displayed the most variable methylation at 7 days of differentiation versus young cells, with HOXD8, HOXC9, HOXB1 and HOXC-AS3 hypermethylated and HOXC10 and HOXC-AS2 hypomethylated. We also determined that there was an inverse relationship between DNA methylation and gene expression for HOXB1, HOXA3 and HOXC-AS3. Finally, increased physical activity in young adults was associated with oppositely regulating HOXB1 and HOXA3 methylation compared with age. Overall, we demonstrate that a considerable number of HOX genes are differentially epigenetically regulated in aged human skeletal muscle and HDMCs and increased physical activity may help prevent age-related epigenetic changes in these HOX genes.

Parkinson's Disease-Associated Changes in the Expression of Neurotrophic Factors and their Receptors upon Neuronal Differentiation of Human Induced Pluripotent Stem Cells.

Parkinson's disease (PD) is a neurodegenerative pathology resulting from the degeneration of dopaminergic (DA) neurons in the substantia nigra (SN). Neurotrophic factors (NTFs) and their receptors are key regulators of the survival, differentiation, and development of neurons. However, the role of these factors in the pathogenesis of PD is still unclear. Here, we analyzed the expression of NTFs and their receptors in human induced pluripotent stem cells (iPSCs) derived from the fibroblasts of patients with PD and healthy donors (HDs). Four PD-derived iPSC lines with different mutations and three cell lines from HDs at different stages of neuronal differentiation were used for RT-qPCR analysis and ELISA. We found that the mRNA levels of most analyzed genes were altered in PD-derived cells compared with those in HD-derived cells at all stages. Importantly, irrespective of PD-associated mutations, the mRNA levels of the BDNF and GDNF genes were mostly increased or unchanged in predominantly DA terminally differentiated neurons (TDNs) compared with those in HD-derived cells. Strikingly, in contrast to BDNF and GDNF mRNA levels, BDNF and GDNF protein levels were lower in almost all PD-derived TDNs than in HD-derived cells, thus indicating the dysregulation of NTF expression at the post-transcriptional level. We suggest that this dysregulation is one of the important signs of PD development.

[Targeted gene sequencing panels: applicability for neoantigen profiling of colon and rectal adenocarcinoma]. / Vozmozhnosti primeneniia targetnykh panelei dlia opredeleniia neoantigennogo profilia obraztsov adenokartsinomy tolstoi i priamoi kishki.

Cancer immunotherapy represents a promising and rapidly developing approach for the treatment of oncological diseases. Among the methods of personalized adjuvant immunotherapy, neoantigenic peptide-based drugs have demonstrated substantial efficiency. These drugs are designed to target mutant proteins arising from somatic alterations in the genome of tumor cells and thus stimulate immune response against tumor tissues. The methods of individual screening for potentially immunogenic mutations are mostly based on next-generation exome sequencing of tumor samples, which is a complex and costly procedure for clinical application. Targeted gene sequencing panels limited to a certain set of genes represent a reasonable alternative to WES. Targeted sequencing is also more efficient when there is a low amount of the sample DNA available. We have estimated the potential efficiency of targeted oncological panels in terms of somatic neoantigen profiling in colorectal cancer (colon and rectal adenocarcinoma). The clinical practice of identification of frequent somatic variants does not provide enough data for designing an efficient personalized drug when applied to low and medium mutated cancers such as colorectal cancer. Our analysis of 11 commercially available panels containing different number of genes has shown that neither the larger size of a panel nor its initial customization for colorectal cancer provides a significantly better estimation of an individual somatic mutation profile. The optimal approach is to use the general-purpose medium-sized cancer panels (2300-11200 amplicons and/or 150-600 genes). These panels allow to detect a sufficient number of immunogenic epitopes (>3) per patient for over 30-50% of patients.