Randi J. Hagerman

Elevated Levels of FMR1 mRNA in Carrier Males: A New Mechanism of Involvement in the Fragile-X Syndrome

Fragile-X syndrome is a trinucleotide-repeat-expansion disorder in which the clinical phenotype is believed to result from transcriptional silencing of the fragile-X mental retardation 1 (FMR1) gene as the number of CGG repeats exceeds approximately 200. For premutation alleles ( approximately 55-200 repeats), no abnormalities in FMR1-gene expression have been described, despite growing evidence of clinical involvement in premutation carriers. To address this (apparent) paradox, we have determined, for 16 carrier males (55-192 repeats), the relative levels of leukocyte FMR1 mRNA, by use of automated fluorescence-detection reverse transcriptase-PCR, and the percent of lymphocytes that are immunoreactive for FMR1 protein (FMRP). For some alleles with>100 repeats, there was a reduction in the number of FMRP-positive cells. Unexpectedly, FMR1 mRNA levels were elevated at least fivefold within this same range. No significant increase in FMR1 mRNA stability was observed in a lymphoblastoid cell line (160 repeats) derived from one of the carrier males, suggesting that the increased message levels are due to an increased rate of transcription. Current results support a mechanism of involvement in premutation carriers, in which reduced translational efficiency is at least partially compensated through increased transcriptional activity. Thus, diminished translational efficiency may be important throughout much of the premutation range, with a mechanistic switch occurring in the full-mutation range as the FMR1 gene is silenced.

Fragile X premutation tremor/ataxia syndrome: molecular, clinical, and neuroimaging correlates.

We present a series of 26 patients, all >50 years of age, who are carriers of the fragile X premutation and are affected by a multisystem, progressive neurological disorder. The two main clinical features of this new syndrome are cerebellar ataxia and/or intention tremor, which were chosen as clinical inclusion criteria for this series. Other documented symptoms were short-term memory loss, executive function deficits, cognitive decline, parkinsonism, peripheral neuropathy, lower limb proximal muscle weakness, and autonomic dysfunction. Symmetrical regions of increased T2 signal intensity in the middle cerebellar peduncles and adjacent cerebellar white matter are thought to be highly sensitive for this neurologic condition, and their presence is the radiological inclusion criterion for this series. Molecular findings include elevated mRNA and low-normal or mildly decreased levels of fragile X mental retardation 1 protein. The clinical presentation of these patients, coupled with a specific lesion visible on magnetic resonance imaging and with neuropathological findings, affords a more complete delineation of this fragile X premutation-associated tremor/ataxia syndrome and distinguishes it from other movement disorders.

The fragile-X premutation: a maturing perspective.

Carriers of premutation alleles (55-200 CGG repeats) of the fragile-X mental retardation 1 (FMR1) gene are often regarded as being clinically uninvolved. However, it is now apparent that such individuals can present with one (or more) of three distinct clinical disorders: mild cognitive and/or behavioral deficits on the fragile-X spectrum; premature ovarian failure; and a newly described, neurodegenerative disorder of older adult carriers, fragile-X-associated tremor/ataxia syndrome (FXTAS). Awareness of these clinical presentations is important for proper diagnosis and therapeutic intervention, not only among families with known cases of fragile-X syndrome but also more broadly for adults with tremor, gait ataxia, and parkinsonism who are seen in movement-disorders clinics.

The behavioral phenotype in fragile X: symptoms of autism in very young children with fragile X syndrome, idiopathic autism, and other developmental disorders.

This study was designed to explore the behavioral phenotype of autism in a group of young children with fragile X syndrome (FXS). Twenty-four children with FXS, ages 21 to 48 months, were compared with two well-matched groups: 27 children with autism (AD) and 23 children with other developmental delays (DD), on two standardized autism instruments, as well as on measures of development and adaptive behavior. Two FXS subgroups emerged. One subgroup (n = 16) did not meet study criteria for autism. Their profiles on the autism instruments and the developmental instruments were virtually identical to the other DD group. The other FXS subgroup (n = 8, or 33% of the total FXS group) met study criteria for autism. Their profiles on the autism instruments were virtually identical to the group with autism. The finding of two FXS subgroups raises a hypothesis of additional genetic influences in the FXS autism group, warranting further genetic studies.

Advances in the Treatment of Fragile X Syndrome

The FMR1 mutations can cause a variety of disabilities, including cognitive deficits, attention-deficit/hyperactivity disorder, autism, and other socioemotional problems, in individuals with the full mutation form (fragile X syndrome) and distinct difficulties, including primary ovarian insufficiency, neuropathy and the fragile X-associated tremor/ataxia syndrome, in some older premutation carriers. Therefore, multigenerational family involvement is commonly encountered when a proband is identified with a FMR1 mutation. Studies of metabotropic glutamate receptor 5 pathway antagonists in animal models of fragile X syndrome have demonstrated benefits in reducing seizures, improving behavior, and enhancing cognition. Trials of metabotropic glutamate receptor 5 antagonists are beginning with individuals with fragile X syndrome. Targeted treatments, medical and behavioral interventions, genetic counseling, and family supports are reviewed here.

A pilot open-label single-dose trial of fenobam in adults with fragile X syndrome

Objective: A pilot open label, single dose trial of fenobam, an mGluR5 antagonist, was conducted to provide an initial evaluation of safety and pharmacokinetics in adult males and females with fragile X syndrome (FXS). Methods: Twelve subjects, recruited from two fragile X clinics, received a single oral dose of 50–150 mg of fenobam. Blood for pharmacokinetic testing, vital signs and side effect screening was obtained at baseline and numerous time points for 6 h after dosing. Outcome measures included prepulse inhibition (PPI) and a continuous performance test (CPT) obtained before and after dosing to explore the effects of fenobam on core phenotypic measures of sensory gating, attention and inhibition. Results: There were no significant adverse reactions to fenobam administration. Pharmacokinetic analysis showed that fenobam concentrations were dose dependent but variable, with mean (SEM) peak values of 39.7 (18.4) ng/ml at 180 min after the 150 mg dose. PPI met a response criterion of an improvement of at least 20% over baseline in 6 of 12 individuals (4/6 males and 2/6 females). The CPT did not display improvement with treatment due to ceiling effects. Conclusions: Clinically significant adverse effects were not identified in this study of single dose fenobam across the range of dosages utilised. The positive effects seen in animal models of FXS treated with fenobam or other mGluR5 antagonists, the apparent lack of clinically significant adverse effects, and the potential beneficial clinical effects seen in this pilot trial support further study of the compound in adults with FXS.

Epigenetic Modification of the FMR1 Gene in Fragile X Syndrome Is Associated with Differential Response to the mGluR5 Antagonist AFQ056

An antagonist for the metabotropic glutamate receptor may improve symptoms in patients with fragile X syndrome whose FMR1 promoters are fully methylated. A Methylation Marker for Fragile X Syndrome Through the practice of meditation, students of Eastern philosophies are taught to turn down the noise to find the silence within. But for patients suffering from fragile X syndrome, it is the silence within that turns up the noise. In this disorder, a defect in the fragile X mental retardation 1 gene (FMR1) silences its expression, which gives rise to myriad molecular changes, most notably a turning up of signaling through the metabotropic glutamate receptor mGluR5. This noisy signaling pathway contributes to the cognitive deficits and differences that first become apparent in patients during childhood, and currently these symptoms are treatable only with supportive behavioral measures. But in mice and fruit flies that carry the same genetic defects as patients and also show enhanced glutamate receptor signaling and behavioral problems, administration of an mGluR5 antagonist improves the symptoms. Jacquemont et al. have now treated a group of 30 fragile X patients with such an antagonist. Not all subjects showed improvement, but an analysis of those who did revealed that the promoter of the FMR1 gene in drug-responsive patients is fully methylated, a sign that gene expression is completely silenced. This molecular aberration might serve as a signature that defines fragile X patients who could benefit from treatment with mGluR5 antagonists. In individuals with fragile X syndrome, the FMR1 gene can contain as many as several thousand extra repeats of the triplet base pairs CGG, a distortion that is accompanied by extra methylation at the gene’s promoter and thus impaired transcription. Because the number of triplet repeats differs widely from person to person—and even from generation to generation—there is a broad variation among patients in the structure of the gene and its methylation pattern. So when the authors tested the effects of a newly described mGluR5 inhibitor on fragile X patients, they assayed the methylation status of the FMR1 promoter, as well as running a large battery of behavioral tests designed to detect stereotypic behavior, hyperactivity, and inappropriate speech. In this clinical trial, the mGluR5 antagonist had no effect on the behaviors measured by these primary tests, but administration of the drug did correlate with differences observed in a secondary collection of tests, when the drug-treated patient group was compared with subjects who were given a placebo treatment. In a subsequent exploratory analysis, the authors found that each member of the subgroup of patients who harbored fully methylated FMR1 promoters showed improvement by the primary behavioral measures, exhibiting a boost in performance 19 or 20 days after treatment was started. The patient group with partially methylated promoters showed no such changes. This correlation between response to treatment and methylation status of the FMR1 promoter provides the basis for a larger study, appropriately designed to test whether methylation can serve as a predictor of a positive antagonist response in a population of patients with fragile X syndrome. It also offers hope that inhibition of the metabotropic glutamate system—believed to underlie many of the characteristic behaviors associated with fragile X—may be accomplished routinely, at least in patients in which the silence within lies in the FMR1 promoter. Fragile X syndrome (FXS) is an X-linked condition associated with intellectual disability and behavioral problems. It is caused by expansion of a CGG repeat in the 5′ untranslated region of the fragile X mental retardation 1 (FMR1) gene. This mutation is associated with hypermethylation at the FMR1 promoter and resultant transcriptional silencing. FMR1 silencing has many consequences, including up-regulation of metabotropic glutamate receptor 5 (mGluR5)–mediated signaling. mGluR5 receptor antagonists have shown promise in preclinical FXS models and in one small open-label study of FXS. We examined whether a receptor subtype–selective inhibitor of mGluR5, AFQ056, improves the behavioral symptoms of FXS in a randomized, double-blind, two-treatment, two-period, crossover study of 30 male FXS patients aged 18 to 35 years. We detected no significant effects of treatment on the primary outcome measure, the Aberrant Behavior Checklist–Community Edition (ABC-C) score, at day 19 or 20 of treatment. In an exploratory analysis, however, seven patients with full FMR1 promoter methylation and no detectable FMR1 messenger RNA improved, as measured with the ABC-C, significantly more after AFQ056 treatment than with placebo (P < 0.001). We detected no response in 18 patients with partial promoter methylation. Twenty-four patients experienced an adverse event, which was mostly mild to moderately severe fatigue or headache. If confirmed in larger and longer-term studies, these results suggest that blockade of the mGluR5 receptor in patients with full methylation at the FMR1 promoter may show improvement in the behavioral attributes of FXS.

Electrodermal responses to sensory stimuli in individuals with fragile X syndrome: a preliminary report.

The fragile X mutation and fragile X syndrome are associated with hyperarousal, hyperactivity, aggression, and anxiety. These may be related to strong reactions to auditory, tactile, visual, and olfactory stimuli [Hagerman, 1996b; Hagerman and Cronister, 1996]. However, almost no data exist describing hyperarousal and sensory sensitivity in individuals with the fragile X mutation. This study establishes a reliable laboratory paradigm for examining reactions to sensory stimuli. We found the pattern of electrodermal responses (EDRs) to stimulation in one sensory modality predicted the pattern of EDRs in four other sensory systems. In addition, the EDR pattern of individuals with the fragile X mutation was related to their FMR-protein expression. Finally, EDRs in individuals with fragile X syndrome were significantly different from those of normal controls, demonstrating greater magnitude, more responses per stimulation, responses on a greater proportion of trials, and lower rates of habituation. The findings support the theory that individuals with fragile X syndrome have a physiologically based enhancement of reactions to sensations. Because electrodermal activity indexes sympathetic nervous system activity, the data suggest that the over-arousal to sensation may involve the sympathetic system.

Fragile-X–Associated Tremor/Ataxia Syndrome (FXTAS) in Females with the FMR1 Premutation

We describe five female carriers of the FMR1 premutation who presented with symptoms of tremor and ataxia and who received a diagnosis of definite or probable fragile-X-associated tremor/ataxia syndrome (FXTAS). Unlike their male counterparts with FXTAS, none of the women had dementia. Females had not been reported in previous studies of FXTAS, suggesting that they may be relatively protected from this disorder. Brain tissue was available from one of the five subjects, a women who died at age 85 years; microscopic examination revealed intranuclear neuronal and astrocytic inclusions, in accord with the findings previously reported in males with FXTAS. The work-up of families with the FMR1 mutation should include questions regarding neurological symptoms in both older male and female carriers, with the expectation that females may also manifest the symptoms of FXTAS, although more subtly and less often than their male counterparts.

Autism Profiles of Males With Fragile X Syndrome.

Autism, which is common in individuals with fragile X syndrome, is often difficult to diagnose. We compared the diagnostic classifications of two measures for autism diagnosis, the ADOS and the ADI-R, in addition to the DSM-IV-TR in 63 males with this syndrome. Overall, 30% of the subjects met criteria for autistic disorder and 30% met criteria for PDD-NOS. The classifications on the ADOS and DSM-IV-TR were most similar, whereas the ADI-R classified subjects as autistic much more frequently. We further investigated the relationship of both FMRP and FMRI mRNA to symptoms of autism in this cohort and found no significant relationship between the measures of autism and molecular features, including FMRP, FMRI mRNA, and CGG repeat number.