We have detected small numbers of intranuclear inclusions in granule cell and astrocytic cell populations in the dentate gyrus of the hippocampus of 3 older adult cases with FXS. We were also able to identify the presence of intranuclear inclusions in the frontal cortex of each case with FXS. Although the presence of such inclusions is generally considered to be restricted to the premutation range (wherein the FMR1
gene is active and producing elevated levels of expanded CGG-repeat containing FMR1
mRNA) it had remained a formal possibility that either residual transcriptional activity of unmethylated full mutation alleles (methylation mosaicism) (34
) or the existence of small numbers of transcriptionally-active premutation alleles (size mosaicism) could give rise to occasional inclusion-bearing cells in FXS (). Indeed, mice with unmethylated, full mutation alleles do possess small numbers of intranuclear inclusions (42
). Interestingly, the Fmr1
gene remains fully unmethylated above 200 CGG repeats in CGG knock-in mice (42
). The present study provides the first report of intranuclear inclusions in full mutation carriers with FXS, supporting the idea that even low expression levels of premutation- or full-mutation length FMR1
mRNA are sufficient to promote intranuclear inclusion formation. In the CGG knock-in mouse model, the existence of inclusions for alleles with more than 200 CGG repeats is consistent with an RNA-based mechanism for cellular toxicity and intranuclear inclusion formation (25
), since the Fmr1
gene retains transcriptional activity (42
). Importantly, the mouse model suggests that the premutation- and full-mutation length alleles themselves, and not necessarily elevated Fmr1
expression levels, are sufficient to result in inclusion formation, albeit at relatively low levels (42
The RNA-based model of inclusion formation raises the expectation that there is at least residual transcriptional activity in the 3 FXS cases. Indeed, measurement of bulk FMR1 mRNA yielded low but detectable levels of FMR1 mRNA in the frontal cortex of each of the FXS cases studied. Using immunofluorescent staining we also detected very low levels of FMRP immunoreactivity as a measure of gene activity in the same brain regions where the FMR1 mRNA levels were quantified (i.e. frontal cortex). The residual FMR1 mRNA and FMRP protein expression do not permit definition of the type of mosaicism giving rise to residual expression and inclusion formation; however, Southern blot analysis of these 3 cases suggests the presence of both size-and methylation-mosaicism in at least one of them, i.e. Case 3.
The absence of ubiquitin-positive intranuclear inclusions in cells negative for FMRP reactivity is important because it supports the models proposing that the expanded (premutation or full mutation) FMR1 mRNA is critical for inclusion formation. Based on a semiquantitative analysis it appears that within the population of FMRP-reactive cells, the numbers of intranuclear inclusions are similar to those previously reported in FXTAS cases (i.e. 10%–20%). This finding is important because the relatively low number of inclusions in the hippocampi of these FXS cases most likely reflects the relative sparseness of FMRP reactivity seen in the cortex.
It should be noted that the presence of rare intranuclear inclusions in a predominately full mutation background in the context of FXS does not imply that such intranuclear inclusions are indicators of any clinically important neurodegenerative process similar to that seen in FXTAS. Rather, the presence of intranuclear inclusions in the context of FMR1
gene expression (albeit at a reduced level) most likely reflects cellular dysfunction within the small number of cells that harbor the unmethylated full mutation- and/or premutation-sized alleles. An important consideration for future studies of individuals with the full mutation and FXS is whether continued transcription of unmethylated full mutation alleles (34
) could result in a distinct, but related, neurodegenerative process to FXTAS, or whether such unmethylated alleles contribute to susceptibility or generation of a completely separate neurodegenerative disorder unrelated to FXTAS. Although movement disorders have been reported in 38.6% of aging individuals (males; n = 44) with FXS (44
), at present there is no evidence that such additional neurological diseases are related to mosaicism of the FMR1
gene. Moreover, no pathological anatomical features have been reported in FXS cases that would suggest features indicative of a movement disorder (44
). Importantly, Parkinsonism was reported in 9% of the males (n = 44) in the study of Utari et al (44
). For the current study it is likely that Case 1 had parkinsonian features (37
), but these were unrelated to the methylation mosaicism on the FMR1
Furthermore, it should be emphasized that none of the 3 FXS cases demonstrated FXTAS symptomatology, including peripheral neuropathy or intention tremor; nor did they have Bergmann gliosis or white matter hyperintensity in the middle cerebellar peduncles. The lack of FXTAS features, in conjunction with the clear presence of FXS symptoms, strongly suggests these FXS cases did not manifest neurodegenerative features similar to those of FXTAS. Further clinical-molecular studies of aging patients with the full mutation, both with and without mosaicism, are needed in order to assess the importance and the functional role of rare intranuclear inclusion formation in patients with FXS. The fact that inclusions, although few, can be found in some cases of FXS despite extremely low levels of full mutation FMR1 mRNA would suggest that elevated FMR1 mRNA levels are not directly responsible for inclusion formation; rather, even low levels of FMR1 mRNA may be sufficient to support the formation of inclusions.