¹Tohoku University Graduate School of Medicine, Department of Developmental Neuroscience, Sendai, Japan

Background: Fragile X syndrome (FXS) is a neurodevelopmental disorder (NDD) caused by CGG expansion in the FMR1 gene, leading to transcriptional silencing of the gene and subsequent loss of Fragile X mental retardation protein (FMRP), an RNA-binding protein essential for cortical neurogenesis during brain development (Casingal et al., 2020; Saffary and Xie, 2011). Notably, female premutation carriers, particularly those with a paternally inherited premutation, exhibit an increased incidence of premature ovarian failure (Hundscheid et al., 2000), suggesting a paternal effect. However, how the absence of FMRP influences genomic imprinting in the brain remains unclear.

Aims & Objectives: We aim to investigate the potential transgenerational effects originating from FXS fathers. Using a mouse model of FXS (Fmr1 knockout; Fmr1-KO), we sought to elucidate novel mechanisms by which paternal, neurodevelopmental disorder–associated influences on offspring, with a particular focus on imprinting regulation during brain development.

Method: To comprehensively characterize FMRP-associated transcriptomic changes, RNA sequencing was performed using telencephalic tissues from wild-type (WT) and Fmr1 KO fathers (patFmr1-KO). Genes whose expression was specifically reduced in patFmr1-KO were selected for further analysis. To investigate transcriptional regulation, DNA methylation was assessed by nanopore sequencing combined with PCR-based targeted profiling.

Results: A specific paternally expressed gene (PEG) showed a marked reduction in expression in the embryonic telencephalon of patFmr1-KO mice regardless of their genotypes. This decrease was further validated by quantitative real-time PCR in embryonic cortices. In situ hybridization revealed disrupted regional expression of the PEG in the patFmr1-KO telencephalon. DNA methylation analysis demonstrated significantly increased CpG methylation around the PEG promoter in patFmr1-KO embryonic brains compared with WT paternal controls. Allele-specific analysis indicated that this hypermethylation occurred selectively on the paternal allele. Although similar yet weak hypermethylation was detected in peripheral tissues such as lung and heart, the most pronounced effect was observed in the brain.

Discussion & Conclusions: Our findings suggest that the absence of FMRP in the father can transmit epigenetic alterations to the offspring, leading to hypermethylation of the PEG promoter and consequent transcriptional silencing during embryonic brain development. These results support a role for FMRP in regulating imprinting in a paternal-origin-dependent manner and may have potential implications for the problem of missing heritability in NDDs. Ongoing work is examining whether patFmr1-KOoffspring display behavioral phenotypes reminiscent of NDDs.