DNA methylation and proteomic profiling of postmortem brain tissue reveals epigenetic dysregulation and inflammatory signaling in fragile X-associated tremor/ataxia syndrome

Background

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by a CGG repeat expansion in the premutation range of FMR1 . The epigenetic landscape of the FXTAS brain remains largely uncharacterized. We performed genome-wide DNA methylation profiling of postmortem prefrontal cortex and targeted proteomic profiling to investigate molecular alterations associated with FXTAS and evaluate convergence on inflammatory signaling.

Methods

DNA methylation was profiled in postmortem prefrontal cortex from 27 male FXTAS cases and 29 male controls using Illumina MethylationEPIC arrays. Surrogate variable analysis was used to account for latent sources of variation. Differentially methylated positions (DMPs) were defined as well as differentially methylated regions (DMRs). Exploratory functional analyses and comparison with published FXTAS transcriptomic and neurodegenerative-disease methylation datasets. Targeted proteomic profiling using the Olink Inflammation panel was performed in 9 male FXTAS cases and 12 male controls.

Results

We identified 98 DMPs which mapped to 70 genes. Notable DMP-associated genes included CYP2E1 , related to oxidative stress; TRAF3 , BATF , RCOR1 , and MSI2 , with roles in immune and NF-κB-related signaling; and genes associated with myelination ( LINGO1 ), synaptic function ( SYT3 ), and zinc homeostasis ( SLC39A4 ). TRAF3 , a negative regulator of NF-κB and type I interferon signaling, contained one of the most significant DMPs. Exploratory Reactome analysis highlighted biological themes related to inflammatory resolution, axonal growth inhibition, zinc homeostasis, and CYP2E1 metabolism, although no pathway remained significant after multiple-testing correction. Transcription-factor motif enrichment and protein–protein interaction analyses identified no significant higher-order regulatory or interaction modules. Seven DMP-associated genes overlapped with differentially expressed genes from an independent FXTAS mouse transcriptomic study, whereas no gene-level overlap was detected with the Alzheimer’s, Parkinson’s, or Huntington’s disease methylation signatures examined. Olink profiling identified 60 significantly altered inflammation-related proteins, 59 of which showed lower abundance in FXTAS, including TLR3, IRAK1, IRAK4, IKBKG/NEMO, and C1QA.

Conclusion

These findings identify previously unrecognized DNA methylation alterations in FXTAS prefrontal cortex involving genes related to oxidative stress, myelination, zinc homeostasis, and innate immune/NF-κB signaling. Convergence between methylation changes in immune-regulatory genes, including TRAF3 , and a predominantly reduced inflammatory protein signature provides complementary evidence for altered inflammatory signaling in FXTAS.