Incomplete Silencing of Full Mutation Alleles in Males with Fragile X Syndrome is Associated with Autistic Features

Background: Fragile X syndrome (FXS) is a common monogenic cause of intellectual disability with autism features. While it is caused by loss of the 1 product (FMRP), mosaicism for active and inactive alleles, including alleles termed premutation (PM: 55-199 CGGs), is not uncommon. Importantly, both PM and active full mutation (FM: ≥ 200 CGGs) alleles often express elevated levels of mRNA that are thought to be toxic. This study determined if complete mRNA silencing from FM alleles and/or levels of mRNA (if present) in blood are associated with intellectual functioning and autism features in FXS.

Methods: The study cohort included 98 participants (70.4% male) with FXS (FM-only and PM/FM mosaic) aged 1-43 years. A control group of 14 females were used to establish control mRNA reference range. Intellectual functioning and autism features were assessed using the Mullen Scales of Early Learning or an age-appropriate Wechsler Scale and the Autism Diagnostic Observation Schedule-2nd Edition (ADOS-2), respectively. mRNA was analysed in venous blood collected at the time of assessments, using the real-time PCR relative standard curve method.

Results: Females with FXS had significantly higher levels of mRNA ( < 0.001) than males. mRNA levels were positively associated with age ( < 0.001), but not with intellectual functioning and autistic features in females. FM-only males (aged < 19 years) expressing FM mRNA had significantly higher ADOS calibrated severity scores compared to FM-only males with completely silenced ( = 0.011). However, there were no significant differences between these subgroups on intellectual functioning. In contrast, decreased levels of mRNA were associated with decreased intellectual functioning in FXS males ( = 0.029), but not autism features, when combined with the PM/FM mosaic group.

Conclusion: Incomplete silencing of toxic FM RNA may be associated with autistic features, but not intellectual functioning in FXS males. While decreased levels of mRNA may be more predictive of intellectual functioning than autism features. If confirmed in future studies, these findings may have implications for patient stratification, outcome measure development, and design of clinical and pre-clinical trials in FXS.