Transcriptomic profiling of unmethylated full mutation carriers implicates TET3 in FMR1 CGG repeat expansion methylation dynamics in fragile X syndrome

TL;DR

This study explores why some people with a genetic mutation linked to fragile X syndrome do not develop the expected gene silencing, suggesting a role for the TET3 gene in DNA methylation.

Contribution

The study identifies TET3 as a potential regulator of methylation dynamics in fragile X syndrome, based on transcriptomic and genomic analyses of rare unmethylated cases.

Findings

TET3 is significantly downregulated in unmethylated full mutation carriers compared to controls and FXS patients.

Genomic analysis of TET3 in unmethylated carriers reveals rare variants, though no clear functional variants were identified.

TET3 is proposed as a candidate factor for the lack of methylation in the FMR1 locus in some individuals.

Abstract

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by the expansion of a CGG repeat in the 5’UTR of the FMR1 (fragile X messenger ribonucleoprotein 1) gene. Healthy individuals possess a repeat 30–55 CGG units in length. Once the CGG repeat exceeds 200 copies it triggers methylation at the locus. This methylation covers the FMR1 promoter region and silences expression of the gene and the production of FMRP (fragile X messenger ribonucleoprotein). The loss of FMRP is responsible for a number of pathologies including neurodevelopmental delay and autism spectrum disorder. Methylation of the expanded repeat in the FMR1 locus is the causal factor for FXS, however it is not known why the expanded repeat triggers this epigenetic change or how exactly DNA methylation is established. Intriguingly, genetic engineering of expanded CGG repeats of over 300 copies in the FMR1 locus in mice remains unmethylated. Also in humans, in very rare cases, individuals can have an FMR1 CGG expansion > 200 copies but the locus remains unmethylated. These unmethylated full mutation (UFM) individuals give us a rare opportunity to investigate the mechanism of FMR1 promoter methylation.

Fibroblasts were obtained from a healthy control, an FXS patient and two unmethylated full expansion carriers. RNA was extracted and comparative transcriptomic analysis was performed on all samples. Whole genome sequencing was carried out on DNA from the two UFM carriers and the results analysed to investigate DNA variants that could explain the observed differences in gene expression.

Our analyses focused on genes involved in epigenetic modification. We show that Tet methylcytosine dioxygenase 3 (TET3), a gene involved in DNA methylation, is significantly downregulated in UFM carriers compared to healthy controls or FXS patient derived cells. Genomic analyses reveal a number of rare variants present in the TET3 locus in UFM carriers when compared to the reference genome. However, no clear modifying TET3 variants were identified.

Our results suggest that TET3 is a candidate factor responsible for the lack of methylation of the expanded FMR1 locus. Further analyses are needed to further elucidate this relationship, however given its potential to directly interact with CGG repeats and its ambiguous role in 5-hydroxy-methylation of CG containing sequences, TET3 is a strong candidate for further exploration.

The online version contains supplementary material available at 10.1186/s11689-025-09609-5.

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