GESTALT-Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing

National Institute On Aging

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Abstract

1- GESTALT donors' recruitment and enrollment is still ongoing. We are seeing participants out to year 8. 2-Transcriptome: RNA sequencing of immune cells was completed for approximately 1,400 samples. Cell-specific gene expression analysis was performed for six immune cell types from 50 donors, with the findings published in 2022. Age-associated changes in gene expression are currently being analyzed, and a manuscript is in preparation. In a separate study, coding and long noncoding RNAs were identified in cultured primary skin fibroblasts from 82 donors. The results of this study were published in Aging Cell. 3- Proteome: Proteomic analysis of 13 immune cell types from approximately 1,000 samples across 99 GESTALT donors has been completed, and a manuscript is currently in preparation. The proteomes of cultured primary skin fibroblasts from GESTALT donors were previously published in Aging Cell. Additionally, multiple papers have been published on serum and plasma proteomics. 4- Epigenetics a. Methylome: DNA methylation data were obtained from 99 donors. Cell-specific and age-associated methylation analyses were published in two separate papers. Additionally, we investigated DNA methylation and gene expression changes during the differentiation of naïve to memory lymphocytes (B cells, CD4⁺ T cells, and CD8⁺ T cells). These studies were complemented by activation assays, and a manuscript is currently in preparation. b. Chromatin Accessibility: Age-associated changes in chromatin accessibility were studied in monocytes and B cells (naïve and memory). Open chromatin profiling in monocytes was conducted on samples from 21 donors (young: 20–55 years, n = 12; old: 60–85 years, n = 9). Principal component analysis (PCA) of differentially open chromatin (DOC) regions revealed three distinct clusters: exclusively old (n = 5), exclusively young (n = 7), and a mixed group (n = 4 old + 5 young). The heterogeneous nature of the healthy older population was marked by significant enrichment of DOCs containing NF-κB and ETS transcription factor motifs. Genes associated with these DOCs were enriched in pro-inflammatory pathways, including NF-κB and NOD-like receptor signaling, indicating a diverse “inflamm-aging” phenotype. Further, intracellular cytokine assays of monocytes from the five older donors revealed a distinct cytokine expression profile. Pro-inflammatory cytokines such as IL-6 and IL-1β were expressed at baseline, whereas TNF-α was not detected, either at baseline or after endotoxin (LPS) stimulation. Ongoing integrative analyses are examining chromatin accessibility variations alongside DNA methylation, transcriptomic data, and serum cytokine levels. A manuscript is currently in preparation. In B cells, chromatin accessibility studies highlighted features distinguishing naïve and memory lymphocytes. Notably, OCT motifs were enriched in memory B cell-specific open chromatin regions, while ETS motifs were prominent in naïve B cells. A manuscript detailing chromatin accessibility and differentiation features of lymphocytes is also in preparation.