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CGG repeat expansions within the premutation range (55–200) of the FMR1 gene can lead to Fragile X-associated tremor/ataxia syndrome and Fragile X-associated neuropsychiatric disorders. These CGG repeats are translated into a toxic polyglycine-containing protein, FMRpolyG. Pathology of Fragile X-associated tremor/ataxia syndrome and Fragile X-associated neuropsychiatric disorders comprises FMRpolyG- and p62-positive intranuclear inclusions. Diagnosing a FMR1-premutation carrier remains challenging, as the clinical features overlap with other neurodegenerative diseases. Here, we describe two male cases with Fragile X-associated neuropsychiatric disorders-related symptoms and mild movement disturbances and novel pathological features that can attribute to the variable phenotype. Macroscopically, both donors did not show characteristic white matter lesions on MRI; however, vascular infarcts in cortical- and sub-cortical regions were identified. Immunohistochemistry analyses revealed a high number of FMRpolyG intranuclear inclusions throughout the brain, which were also positive for p62. Importantly, we identified a novel pathological vascular phenotype with inclusions present in pericytes and endothelial cells. Although these results need to be confirmed in more cases, we propose that these vascular lesions in the brain could contribute to the complex symptomology of FMR1-premutation carriers. Overall, our report suggests that Fragile X-associated tremor/ataxia syndrome and Fragile X-associated neuropsychiatric disorders may present diverse clinical involvements resembling other types of dementia, and in the absence of genetic testing, FMRpolyG can be used post-mortem to identify premutation carriers.

STUDY QUESTION Does repeat-associated non-AUG (RAN) translation play a role in fragile X-associated primary ovarian insufficiency (FXPOI), leading to the presence of polyglycine containing protein (FMRpolyG)-positive inclusions in ovarian tissue? SUMMARY ANSWER Ovaries of a woman with FXPOI and of an Fmr1 premutation (PM) mouse model (exCGG-KI) contain intranuclear inclusions that stain positive for both FMRpolyG and ubiquitin. WHAT IS KNOWN ALREADY Women who carry the FMR1 PM are at 20-fold increased risk to develop primary ovarian insufficiency (FXPOI). A toxic RNA gain-of-function has been suggested as the underlying mechanism since the PM results in increased levels of mRNA containing an expanded repeat, but reduced protein levels of fragile X mental retardation protein (FMRP). Recently, RAN translation has been shown to occur from FMR1 mRNA that contains PM repeat expansions, leading to FMRpolyG inclusions in brain and non-CNS tissues of fragile X-associated tremor/ataxia syndrome (FXTAS) patients. STUDY DESIGN, SIZE, DURATION Ovaries of a woman with FXPOI and women without PM (controls), and ovaries from wild-type and exCGG-KI mice were analyzed by immunohistochemistry for the presence of inclusions that stained for ubiquitin and FMRpolyG . The ovaries from wild-type and exCGG-KI mice were further characterized for the number of follicles, Fmr1 mRNA levels and FMRP protein expression. The presence of inclusions was also analyzed in pituitaries of a man with FXTAS and the exCGG-KI mice. PARTICIPANTS/MATERIALS, SETTING, METHODS Human ovaries from a woman with FXPOI and two control subjects and pituitaries from a man with FXTAS and a control subjects were fixed in 4% formalin. Ovaries and pituitaries of wild-type and exCGG mice were fixed in Bouin's fluid or 4% paraformaldehyde. Immunohistochemistry was performed on the human and mouse samples using FMRpolyG, ubiquitin and Fmrp antibodies. Fmr1 mRNA and protein expression were determined in mouse ovaries by quantitative RT-PCR and Western blot analysis. Follicle numbers in mouse ovaries were determined in serial sections by microscopy. MAIN RESULTS AND THE ROLE OF CHANCE FMRpolyG-positive inclusions were present in ovarian stromal cells of a woman with FXPOI but not in the ovaries of control subjects. The FMRpolyG-positive inclusions colocalized with ubiquitin-positive inclusions. Similar inclusions were also observed in the pituitary of a man with FXTAS but not in control subjects. Similarly, ovaries of 40-week-old exCGG-KI mice, but not wild-type mice, contained numerous inclusions in the stromal cells that stained for both FMRpolyG- and ubiquitin, while the ovaries of 20-week-old exCGG-KI contained fewer inclusions. At 40 weeks ovarian Fmr1 mRNA expression was increased by 5-fold in exCGG-KI mice compared with wild-type mice, while Fmrp expression was reduced by 2-fold. With respect to ovarian function in exCGG-KI mice: (i) although the number of healthy growing follicles did not differ between wild-type and exCGG-KI mice, the number of atretic large antral follicles was increased by nearly 9-fold in 40-week old exCGG-KI mice (P < 0.001); (ii) at 40 weeks of age only 50% of exCGG-KI mice had recent ovulations compared with 89% in wild-type mice (P = 0.07) and (iii) those exCGG-KI mice with recent ovulations tended to have a reduced number of fresh corpora lutea (4.8 ± 1.74 versus 8.50 ± 0.98, exCGG-KI versus wild-type mice, respectively, P = 0.07). LIMITATIONS, REASONS FOR CAUTION Although FMRpolyG-positive inclusions were detected in ovaries of both a woman with FXPOI and a mouse model of the FMR1 PM, we only analyzed one ovary from a FXPOI subject. Caution is needed to extrapolate these results to all women with the FMR1 PM. Furthermore, the functional consequence of FMRpolyG-positive inclusions in the ovaries for reproduction remains to be determined. WIDER IMPLICATIONS OF THE FINDINGS Our results suggest that a dysfunctional hypothalamic-pituitary-gonadal-axis may contribute to FXPOI in FMR1 PM carriers. STUDY FUNDING/COMPETING INTERESTS This study was supported by grants from NFXF, ZonMW, the Netherlands Brain Foundation and NIH. The authors have no conflict of interest to declare.