Review. Advances in clinical and molecular understanding of the FMR1 premutation and fragile X-associated tremor/ataxia syndrome

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1 Advances in clinical and molecular understanding of the FMR1 premutation and fragile X-associated tremor/ataxia syndrome Randi Hagerman, Paul Hagerman Lancet Neurol 2013; 12: Department of Pediatrics and the MIND Institute (R Hagerman MD) and Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, CA, USA (P Hagerman MD) Correspondence to: Dr Paul Hagerman, Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, CA 95616, USA Fragile X syndrome, the most common heritable form of cognitive impairment, is caused by epigenetic silencing of the fragile X (FMR1) gene owing to large expansions (>200 repeats) of a non-coding CGG-repeat element. Smaller, so-called premutation expansions ( repeats) can cause a family of neurodevelopmental phenotypes (attention deficit hyperactivity disorder, autism spectrum disorder, seizure disorder) and neurodegenerative (fragile X-associated tremor/ataxia syndrome [FXTAS]) phenotypes through an entirely distinct molecular mechanism involving increased FMR1 mrna production and toxicity. Results of basic cellular, animal, and human studies have helped to elucidate the underlying RNA toxicity mechanism, while clinical research is providing a more nuanced picture of the range of clinical manifestations. Advances of knowledge on both mechanistic and clinical fronts are driving new approaches to targeted treatment, but two important necessities are emerging: to define the extent to which the mechanisms contributing to FXTAS also contribute to other neurodegenerative and medical disorders, and to redefine FXTAS in view of its differing presentations and associated features. Introduction Many neurologists are unaware of the stark distinction between the neurodevelopmental disorder fragile X syndrome and the neurodegenerative disorder fragile X-associated tremor/ataxia syndrome (FXTAS). These mechanistically and clinically distinct syndromes are often conflated because both arise from mutated forms of the fragile X mental retardation 1 (FMR1) gene. Although both disorders are caused by expansions of a trinucleotide (CGG) repeat element in a non-coding portion of the gene, fragile X syndrome is caused by expansions of greater than 200 CGG repeats, whereupon the gene undergoes methylation-coupled silencing. The consequent absence of FMRP (FMR1 protein), which is important for proper synapse develop ment and function, causes intellectual disability and an autism spectrum disorder in most males, and similar, albeit less severe, intellectual and behavioural disorders in females. By contrast, FXTAS is clinically manifest almost exclusively among carriers of premutation repeat expansions ( CGG repeats), in whom the FMR1 gene remains fully active; the gene generally expresses increased levels of its mrna, 1 which is thought to result in CGG-repeat-induced toxicity and the adult-onset neuro degenerative disorder. 2 5 Thus, although fragile X syndrome and FXTAS are caused by CGG-repeat expansions within the same gene, they affect different groups of individuals (ie, possessing full mutation vs premutation alleles), are clinically apparent at opposite ends of the age range, and arise through different pathogenic mechanisms (gene silencing and RNA toxicity, respectively). In this Review, we focus on advances in understanding of the clinical manifestations among carriers of premutation alleles, which include but are not limited to those with FXTAS. Manifestations in premutation carriers Before the discovery of the FMR1 gene in 1991, and for some years after, carriers of premutation alleles were thought to be clinically unaffected, but female carriers of premutation alleles often had children with the genetic disorder fragile X syndrome. With the discovery of the FMR1 gene, the basis of this propensity was recognised as being due to instability of the CGG repeat, with substantial conversion from premutation to full mutation expansions occurring during matrilineal transmission. However, shortly before discovery of the gene, Cronister and colleagues 6 reported that incidence of early ovarian failure (before the age of 40 years) was much higher in female carriers (about 20%) than in the general population (about 1%). Fragile X-associated primary ovarian insufficiency is now known to be the most common cause of early menopause. 7 Additional clinical manifestations among carriers of premutation alleles were identified throughout the 1990s. Psychiatric problems, including depression and anxiety, were recognised during this period, 8 although these clinical features were initially thought to be associated with the stress of raising children with fragile X syndrome. Results of more extensive controlled studies 9 13 using validated psychiatric clinical interviews, such as the structured clinical interview for DSM-IV disorders (SCID), 14 have established that depression, anxiety, or both, occur in about 40% of carriers. We now know that carriers are intrinsically more vulnerable to depression and anxiety, because these problems can occur before having children affected by fragile X syndrome. 9,13 However, this intrinsic vulnerability is also sensitive to the stress of raising a child with the syndrome or caring for a parent with FXTAS The discovery of FXTAS in ,4 brought a new dimension to premutation involvement. FXTAS is a progressive neurodegenerative disorder involving core features of intention tremor and gait ataxia, with associated white matter disease and global brain atrophy. 2,4,5,18 21 The identification of FXTAS helped to validate earlier reports of nebulous emotional or neuropsychiatric symptoms in premutation carriers, by anchoring the severe end of a spectrum of clinical features experienced by carriers of premutation alleles Vol 12 August 2013

2 The premutation allele is common in the general population, with estimates of prevalence dependent on the population assessed. In Israel, roughly one in females is a carrier. 22 In Spain, estimates of carrier prevalence (one in 130 females and one in 250 males; n=5235; CGG repeat range 10 77; modal value 30) used newborn bloodspots collected previously as part of the national newborn screening programme, which were provided to the investigators as consecutive, de-identified samples, thus largely avoiding sample bias. 23 In a Wisconsin study, 24 the corresponding numbers were one in 151 females and one in 468 males (n=6747; CGG repeat range 9 135; modal value 31); this population-based sample (saliva-derived DNA) was restricted to high-school graduates, thus introducing a possible bias towards reduced prevalence. In a more recent bloodspot screening study in California, 25 one in 209 females and one in 430 males were found to be carriers (n=14 207; CGG repeat range 6 44; modal value 30). The Californian study was subject to bias associated with consent procedures for newborn screening, although a subset of the sample was tested anonymously after the identity of the source of the newborn bloodspot was hidden. 25 Neurodevelopmental problems associated with the premutation Accompanying the growing awareness of clinical symptoms in adult carriers of premutation alleles is an increasing appreciation of neurodevelopmental problems, which are more common in boys with the premutation who might have a higher rate of attention deficit hyperactivity disorder (ADHD), shyness, social deficits, autism spectrum disorder, and (occasionally) intellectual disability The prevalence of developmental or behavioural problems, including autism spectrum disorder or ADHD, is higher in boys who present clinically with a behavioural problem than in other family members identified with cascade testing (autism spectrum disorders 8%; ADHD 38%). 26,27,30 Why some carriers, but not others, will have some form of neurodevelopmental involvement is not known; nor is it possible to predict, on the basis of the presence or absence of neurodevelopmental features, whether an individual will eventually develop FXTAS. Although the severity of amygdala dysfunction in young adult men and the age of onset of FXTAS 31,32 both correlate with the number of CGG repeats in the premutation range, penetrance remains incomplete. This observation suggests that additional genetic, epigenetic, and environmental factors affect disease development. Premutation carriers with more than 100 CGG repeats are more likely than those with fewer repeats to have mild deficits in FMRP, developmental problems, 28 and manifest altered amygdala function. 11 Deficits in FMRP lead to upregulation of the mglur5 system 33 and downregulation of the GABA A system, 34 resulting in an excitatory inhibitory imbalance that is observed in the premutation mouse-model neurons. 35 A transcranial magnetic stimulation study 36 showed that asymptomatic adult women with the premutation have reduced GABA A -mediated intracortical and afferent inhibition from the cerebellum to the primary motor cortex compared with age-matched controls. These GABA A deficits might predispose carriers to the ADHD symptoms shown by about 30% of boys with the premutation. 26,27 The GABA A deficits can also lead to an increased frequency of burst-type firing in networks of cultured premutation mouse neurons, 35 which is probably the basis for the high rate of seizures (8 13%) in carriers. 27,37,38 Moreover, the presence of seizures is significantly associated with the development of autism spectrum disorder in boys with the premutation. 27 The association between seizures and autism spectrum disorders is also found in other neurodevelopmental disorders, including tuberous sclerosis and neurofibromatosis. 39 These data suggest that early treat ment of seizures is important in premutation carriers and in other disorders associated with autism or autism spectrum disorders. Clinical and radiological features of FXTAS The original descriptions of FXTAS emphasise a presentation of intention tremor, gait ataxia, or both, with an onset typically between the ages of 60 and 65 years. 3,4,40 Intention tremor and ataxia are generally deemed to be the core features of FXTAS and are the major clinical diagnostic criteria (table 1). 4,41 The diagnostic criteria also include memory problems and executive function deficits. Additionally, Brunberg and colleagues 18 recognised that hyperintensity of the middle cerebellar peduncle (MCP) was a distinctive feature of the white matter disease detected by T2 FLAIR MRI in those with FXTAS. Because this MCP finding was highly specific (albeit not unique) to FXTAS, it was designated as a major criterion for diagnosis of definite FXTAS. The MCP sign is found in roughly 60% of men with FXTAS, 5,19 and those with FXTAS and the MCP sign are likely to have more severe cognitive deficits and a longer history of symptoms than those without the MCP sign. 5 The MCP sign is observed in only 13% of women with FXTAS. 19 Other common neuroimaging signs of FXTAS include white matter hyperintensities in the pons, insula, splenium of the corpus callosum, and periventricular region. 2,21,42,43 Additionally, thinning of the corpus callosum, 2 cerebellar atrophy, 44 and white matter abnormalities, as assessed by diffusion tensor imaging, particularly involving frontocerebellar tracts, can be seen in those with FXTAS and in adult asymptomatic premutation carriers who do not have FXTAS These findings suggest that the RNA toxicity initiates brain changes well before the onset of FXTAS. Although neuropathy was recognised early as a common clinical problem in patients with FXTAS, 4 it was not included in the initial diagnostic criteria. 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3 Observation/description Examination and degree Molecular Major CGG repeats (premutation) Radiological Major MRI white matter lesions in middle cerebellar peduncle Minor MRI white matter lesions in cerebral white matter Minor Moderate-to-severe generalised atrophy Clinical Major Intention tremor Major Gait ataxia Minor Parkinsonism Minor Moderate-to-severe short-term memory deficit Minor Executive function deficit Neuropathological Major FXTAS inclusions Diagnostic category Presence of expanded CGG repeat (molecular), and Definite Presence of one major radiological sign plus (i) one major clinical symptom or (ii) the presence of FXTAS inclusions Probable Presence of one major radiological sign and one minor clinical symptom, or two major clinical symptoms Possible Presence of one minor radiological sign and one major clinical symptom Table 1: Current diagnostic criteria for FXTAS 4,41 Sub sequently, neuropathy was established as a common early symptom in carriers who later developed FXTAS, and was observed by electromyography in 81% (13 of 16) of patients with FXTAS. 2 Poston and colleagues 51 noted a generalised reflex myoclonus in a 60-year-old patient with FXTAS, and showed that tapping deep tendons in the arms or legs with a reflex hammer resulted in single jerks of the neck, trunk, abdomen, and arms. This same reflex myoclonus occurred when stroking the palm or soles with a key. 51 In our experience, this finding is common with advanced cases of FXTAS, particularly with frontal release signs such as a snout reflex. Parkinsonism is a minor diagnostic criterion of FXTAS, 4 and has been subsequently documented in 64% (14 of 22) of individuals with FXTAS. 2 SPECT studies of the nigrostriatal system, specifically dopamine transporter density, have shown deficits with significantly reduced uptake of ¹²³I FP-CIT in the putamen bilaterally in patients with FXTAS These findings show not only cerebellar problems, but also basal ganglia dysfunction in FXTAS, although dopaminergic dysfunction is not always present in those with FXTAS. 55 Typically, the age of onset of FXTAS is in the early 60s, and it presents with onset of tremor. 40 However, findings from diffusion tensor imaging suggest that asymptomatic changes in the brain associated with the pathological changes of FXTAS exist well before the onset of tremor or ataxia Typical early symptoms include neuropathy, erectile dysfunction, or other autonomic symptoms, such as episodes of hypotension; however, cognitive decline is occasionally the initial symptom. 56 Juncos and colleagues 57 reported that essential tremor can last for years, or sometimes decades, before the onset of ataxia, which marks a stage of more rapid decline. Subsequently, on average, falls begin 6 years after initial symptoms, use of a cane after 15 years, and death after 21 years. 40 Leehey and colleagues 40 reported that lifespan after diagnosis of FXTAS ranged from 5 to 25 years. Results of studies have shown several new symptoms associated with FXTAS (panel). Olfactory dysfunction has not previously been reported, but was recorded in 80% of patients with definite FXTAS. 57 As individuals progress from the possible to the definite stages of FXTAS, the symptoms in the panel become more common. In late stage FXTAS, for example, problems with swallowing, urinary and bowel incontinence, sedation in the day, and muscle weakness are more common. 40,57,72,73 Neuropsychological deficits Some FXTAS-associated cognitive problems, including memory and executive function deficits, are often present at the time an individual shows tremor and ataxia; 5,74 77 however, many men with FXTAS have a history of attention problems in childhood that might have led to a diagnosis of ADHD. 26,27,78 Women with the premutation also have an increased rate of ADHD before onset of FXTAS. 79 Sevin and colleagues 57 surveyed all known carriers older than 50 years in France and identified many with cognitive decline without obvious motor deficits. Grigsby and colleagues 76 also reported executive function deficits in a subgroup of older (aged years, mean 59) carriers who did not have tremor or ataxia. Hunter and colleagues 80 found no evidence of executive function deficits in carriers who are younger than 50 years. However, their conclusion contrasts with those of other reports, 76,78,81 in which such deficits were found to be increased in premutation carriers without FXTAS, including some younger than 50 years. The difference in these findings is connected with the different measures used to detect executive function deficits. The Behavior Dyscontrol Scale is very sensitive to these deficits in carriers, but the Hunter study 80 did not use this measure. Yang and colleagues 77 showed that the executive function deficits in patients with FXTAS correlated with changes in event-related potential, particularly in P3 with reduced amplitude and latency on an auditory so-called oddball paradigm. The P3 component of the event-related potential is known to be associated with selective attention, attention orienting to salient or unexpected events, and with working memory. Smaller P3 amplitudes correlated with larger CGG repeats and higher FMR1 mrna levels in those with Vol 12 August 2013

4 FXTAS. The executive function and visual spatial processing deficits deteriorated over time, leading to a broader cognitive decline, although Brega and colleagues 82 have documented that the executive function deficits mediate the other secondary cognitive changes in learning, memory, and visual spatial processing. Neuroimaging studies have corroborated the eventrelated potential findings with volumetric studies, showing significant atrophy in several frontal brain regions, including the dorsomedial and dorsolateral prefrontal cortex, the orbitofrontal and anterior cingulate cortex, and the medial parietal (precuneus) and superior parietal cortex. 44 Executive function deficits are also documented in a functional MRI study 83 of a verbal working memory task in carriers. The researchers reported reduced activation in core working memory areas, right ventral inferior frontal cortex, and left premotor or dorsal inferior frontal cortex for both premutation carriers with and without FXTAS compared with controls. Seritan and colleagues 84 showed that 42% of people with late-stage FXTAS develop significant cognitive decline and meet diagnostic criteria of dementia. Dementia is far less common in women with FXTAS, 84 although in a neuropathology study of eight women with the premutation, four were diagnosed with dementia before death, and almost all had some neuropathological features of Alzheimer s disease at autopsy. 85 All eight women had intranuclear inclusions, although the diagnosis of FXTAS was not made before death in about half. Pathogenesis of premutation disorders and FXTAS Triggering events Evidence strongly suggests the existence of a triggering event involving sequestration of one or more proteins by the expanded CGG repeat in the FMR1 mrna 5 UTR (figure 1) By analogy with protein sequestration in myotonic dystrophy, 89,90 the so-called RNA toxicity in FXTAS is thought to involve the sequestration of one or more RNA binding proteins, thus reducing their capacity to carry out their normal functions. Candidate proteins include HNRNPA1B2, 91,92 PURA, 93,94 KHDRBS1, 95 and DGCR8. 96,97 Evidence exists for all of these proteins for in-vitro association with CGG-repeat-containing RNA, as well as in-vivo evidence of a functional role in mediating effects of the CGG-repeat RNA. However, compelling functional linkage to FXTAS neuro degeneration does not exist for any of these candidates, except perhaps DGCR8, underscoring the need for further research. Although strong evidence exists for the role of PURA in mediating neurodegeneration in drosophila, PURA has not been detected in the intranuclear inclusions in murine models, 98 and whether it is present in human inclusions is inconclusive. 91,98 By contrast with the situation with PURA, evidence is growing that HNRNPA1B2 has a role in mediating at least some aspects of neurodegeneration, not only in drosophila, 92,99,100 but also in cultured rat neurons, 101 where the RNA binding protein is thought to assist mrna dendritic transport. Although several candidate protein mediators of the CGG-repeat mrna toxicity exist, more work is needed in mammalian systems to clarify their respective roles, if any, in the pathogenesis of premutationassociated disorders, including FXTAS. Finally, although most evidence favours the CGG-repeat-mediated, protein-sequestration model, other models for RNA toxicity have been proposed. Such models include the possibility that the CGG-repeat-containing RNA initiates a conformational transition in one or more proteins that have prion-like domains, 86 analogous to amyloid plaque formation in Alzheimer s disease, or that repeat-associated non-atg translation is occurring, yielding altered polypeptide products that could themselves lead to aggregation and neuronal toxicity Such a mechanism Panel: Established and expanded features of FXTAS Autonomic problems Constipation/irritable bowel syndrome 58,59 Erectile dysfunction 60 Problems swallowing 40 Gastrointestinal reflux 40 Orthostatic hypotension 40 Hypertension* 49,61 Urinary urgency and incontinence 40,62 Cardiac arrhythmia 62 Dizzy spells or vertigo 62 Sensory Olfactory dysfunction* 58 Hearing loss* 5 Neuropathy* 4,5,48,50,63 Sleep problems Insomnia* 64 Sleep apnoea* 65 Daytime sleeping 65 Motor symptoms Tremor* ; ataxia* 2 5 Muscle weakness 40 Parkinsonism* 4,52,54,55 Psychiatric Depression*; anxiety* 11,66 Irritability 67 Chronic pain Fibromyalgia* 2,68 70 Neuropathic pain* 49 Immune-mediated disorder Hypothyroidism* 49,68,69,71 Fibromyalgia* 49,68 70 *Features that occur in a greater number of those with FXTAS compared with age-matched controls. Established features. Vol 12 August

5 CGG-repeat RNA binding protein(s) Reduced free RNA binding proteins Increasing CGG-repeat length ~ ~ ~~ FMRP Reduced FMRP Functional deficiency of CGG-repeat RNA binding protein(s) FMRP deficiency Figure 1: Schematic diagram of the sequestration model for RNA toxicity in the fragile X premutation disorders, including fragile X-associated tremor/ataxia syndrome One or more RNA-binding proteins bind to the CGG-repeat RNA in a length-dependent fashion, such that little binding occurs in the normal CGG-repeat range. Excess binding or sequestration of those proteins leads to a functional insufficiency for their normal function(s). Reduced FMRP concentrations might contribute to the premutation phenotypes for the larger premutation alleles. has been proposed for expanded hexanucleotide (GGGGCC)-repeat alleles of the C90RF72 gene, a genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. Todd and colleagues 104 have shown the presence of RAN translation products from FMR1 mrna involving initiation of protein synthesis upstream of the CGG repeat and a shift in codon frame (CGG to GGC), resulting in a peptide that contains a polyglycine repeat sequence and additional downstream aminoacid sequence. The relative importance of this mechanism to FXTAS neurodegeneration needs further study. However, a premutation mouse model 105 that presumably does not support repeatassociated non-atg translation 104 shows significant Purkinje cell dropout, as is the case in FXTAS in human beings. Features of cellular dysregulation Although there are many potential downstream pathways that could mediate cellular dysregulation, as suggested by the previously mentioned protein candidates, two general processes are emerging as core mediators of cellular dysregulation and dysfunction: specifically, altered mitochondrial function and altered calcium (Ca² + ) regulation 35 (figure 2). Evidence for mitochondrial dysfunction exists for both cultured fibroblasts from adult premutation carriers with and without FXTAS, and from post-mortem brain tissue from patients with FXTAS. Several general features are apparent, 106 including reduced oxidative phosphorylation capacity, decreased mitochondrial levels, and increased precursor-to-mature ratios of several nuclear-encoded subunits of electron transport complexes, indicative of a defect in mitochondrial protein import. This mitochondrial functional deficiency could explain the increased oxidative stress in CNS tissue. 107 Qurashi and colleagues 109 recorded an accumulation of transcripts from stress response genes in the drosophila model of the premutation, suggesting a cellular response to oxidative stress, mitigation of an inflammatory response, or both; such inflammatory or immune-mediated processes co-occur in individuals with FXTAS at a higher rate than expected in the general population. 49,68,70,85,110,111 Kaplan and colleagues 108 showed that hippocampal neurons from premutation knock-in mice (about 170 CGG repeats) have reduced mitochondria with decreased mitochondrial mobility and increased rates of basal oxygen consumption and proton leak. Importantly, these hippocampal neurons were obtained from neonatal mice, which do not manifest any overt features of neurodegeneration. Mitochondrial dysfunction could be a very early feature of the incipient processes that might eventually lead to FXTAS in adulthood. Also evident in the neonatal period are abnormalities of neuronal morphology and network activity (mouse hippocampal neurons), as well as altered Ca² + regulation. 35,112 Remarkably, the burst-type firing observed with the premutation mouse neurons on multi-electrode arrays, which is perhaps associated with the heightened seizure activity in childhood for premutation carriers, is completely reversed with mglur5 antagonists or the GABA A receptor positive modulator allopregnanolone. 35 These observations suggest that overlapping downstream pathways exist between premutation and full mutation disorders, even if the initial triggering events (RNA toxicity, FMRP deficiency) are distinct. Additional factors leading to neurodegeneration and FXTAS One of the puzzling aspects of the neurodegenerative process leading to FXTAS is why the syndrome only affects some individuals who have a CGG repeat of a particular size. This issue is of particular importance because essentially all mice that carry comparably sized CGG repeats show early developmental neuronal cell dysfunction and later inclusion formation, but they do not have the debilitating neurodegenerative process that a proportion of humans who are premutation carriers have. Part of the answer to this paradox probably involves additional genetic or environmental factors, such as smoking, 7 environmental toxins, 113 toxicity from general anaesthesia, 114 and chemotherapy, 115 which would be expected to aggravate any underlying neuropathology, moving an intrinsically subclinical neuronal dysfunction to an overt neurodegenerative process (figure 3). Much more work is needed in this area, because understanding of these added stressors could lead to improved preventive measures Vol 12 August 2013

6 Although RNA toxicity seems to be the dominant mechanism for pathogenesis in FXTAS, several alternative models have been proposed. First, the RNA might trigger a conformational transition in one or more proteins that harbour prion-like domains, leading in turn to a propagating protein aggregate along the lines of amyloid plaque formation in Alzheimer s disease. 86,116 In this regard, King and colleagues 116 noted that HNRNPA2B1, which is found in the intranuclear inclusions of FXTAS, 18,31,91,92 ranks among RNA recognition motif proteins as having a high potential to form prion-like protein aggregates. By analogy with the prionogenic proteins TDP-43 and FUS, which form aggregates in the degenerating motor neurons in amyotrophic lateral sclerosis, King and colleagues 116 proposed that the expanded CGG repeat in the FMR1 mrna might trigger such an aggregation of HNRNPA2B1. However, by contrast with the exclusively intranuclear inclusions of FXTAS, all the prionogenic proteins listed by King and colleagues 116 form cytoplasmic aggregates, 86 which have not been observed in patients with FXTAS. Moreover, in a drosophila model of FXTAS that shows neuronal degeneration and nuclear and cytoplasmic inclusions, overexpression of HNRNPA2B1 actually suppresses the neurodegenerative phenotype. 92 A second issue linked to the pathogenic mechanism in FXTAS is what part, if any, is played by reduced FMRP concentrations in carriers with repeat numbers in the upper premutation range. Low FMRP concentrations cannot be the driving factor in premutation-associated disease, because FXTAS and fragile X-associated primary ovarian insufficiency are not seen in people with fully methylated alleles in the full-mutation range (>200 repeats), in whom FMRP concentrations are notably reduced or absent. Moreover, Hashem and colleagues 117 showed that CGG expression as RNA is necessary and sufficient to produce the intranuclear inclusions characteristic of FXTAS and the associated neuronal cell death, under conditions where FMRP concentrations are not different from normal wild-type concentrations. Notwithstanding the fact that FMRP concentrations are unlikely to lead to the neuro degenerative phenotype, lowered concentrations could still contribute to the severity of premutation-associated disease, particularly in cognitive or behavioural dysfunction. Neuropathology One of the hallmarks of FXTAS is the presence of intranuclear inclusions in neurons and astrocytes throughout the CNS of affected individuals. 18,118 The inclusions are tau and synuclein negative, but do contain FMR1 mrna 119 and several additional proteins that, when sequestered, are expected to exacerbate cellular dysregulation and thus potentiate FXTAS. 97 Although women and men with FXTAS have similar numbers of neuronal inclusions, women seem to have far fewer astrocytic inclusions for unknown reasons. 85 CGG-repeat number and inclusion load 31 are each strongly positively correlated with an earlier onset of the neurodegenerative disorder. 32,120 Surprisingly, in view of the presumed CNS basis of the disorder, results of studies have shown the presence of the intranuclear inclusions throughout the peripheral nervous system, including the paraspinal ganglia, pericardiac ganglia, and myenteric plexus. 121,122 Intranuclear inclusions are also observed in the adrenal gland (cortex and medulla), pituitary, islets of Langerhans, thyroid, heart, and testicular Leydig cells. 61,121,122 The presence of intranuclear inclusions throughout the peripheral nervous system and in several non-nervous tissues raises two important points about FXTAS pathogenesis: first, the broad distribution indicates that the mechanisms leading to FXTAS and, presumably, the broader range of premutation phenotypes are functioning outside of the CNS. For example, autonomic dysfunction (eg, orthostatic hypotension, impotence), commonly occurring before 100 μv 100 μv 2 s 2 s Ca 2+ Figure 2: Stylised neuron with single synapse represented, showing cellular dysregulation in fragile X-associated tremor/ataxia syndrome CGG-repeat expansion leads to downstream effects that include reduced mitochondrial function, altered calcium regulation, and increased, synchronous firing of neurons in mouse hippocampal neuronal networks. N=nucleus. M=mitochondria. ER=endoplasmic reticulum. M M Ca 2+? N ER N ER Vol 12 August

7 Neuronal and glial cell dysfunction Additional (genetic) factors Environmental (toxic) insults Non-degenerative Progressive CNS dysfunction Additional medical problems (eg, illness, surgery) Degenerative FXTAS Figure 3: Schematic representation of the progression of CNS dysfunction Dysfunction primarily driven by the premutation CGG-repeat expansion, but also modulated by second-gene effects and various environmental exposures (eg, untreated hypertension or hypothyroidism, smoking or use of other agents that promote oxidative damage, major illness or, anecdotally, surgery requiring general anaesthesia). FXTAS=fragile X-associated tremor/ataxia syndrome. the onset of tremor or balance problems, might indicate inclusions in pericardial ganglia and testicular Leydig cells, respectively. 60 Similarly, inclusions in the cardiac tissue and pericardial ganglia probably indicate a disease process that results in cardiac arrhythmias. 122 Moreover, fragile X-associated primary ovarian insufficiency could arise as a direct consequence of dysfunction in crucial peripheral tissues (eg, hypothalamic-pituitary-adrenal axis). 122 Second, the presence of inclusions indicates that the core elements of cellular dysregulation are not limited to the CNS, and therefore could be studied in samples and cultured cells from those tissues (eg, fibroblasts). 123 Neuropathology reports for patients with FXTAS have helped to increase understanding of the co-occurrence of other disorders, including Lewy body dementia, Alzheimer s disease, and multiple sclerosis. 18,85,111,123,124 In particular, the added cell stress associated with a second CNS disorder would be expected to exacerbate the cellular dysregulation that potentiates FXTAS, by contributing to mitochondrial dysfunction (figure 2) Investigation of common or overlapping neuropathological mechanisms between FXTAS and other CNS disorders will increase our understanding of the variability of disease expression and progression in both FXTAS and the other disorders. Expanded spectrum of clinical features associated with the premutation Our understanding of the core and associated features of FXTAS has expanded to include a spectrum of neurological, psychological, endocrine, and immunerelated characteristics that occur with increased frequency in patients with FXTAS (panel). Other symptoms commonly seen in those with FXTAS have not been assessed in comparison with age-matched controls. Moreover, carriers without clinical evidence of FXTAS could also have some of these features, although less frequently than those with FXTAS. 49 An emerging view of the disorder is therefore that it is one end of a spectrum of clinical involvement that spans the entire age range. The core neurological features originally described in FXTAS occur in roughly 40% of male carriers older than 50 years, 126 and about 16% of female carriers older than 50 years, 12,49,69 although penetrance has been less well documented in women. 52,71,125,127 Penetrance increases with age in both men and women. Women with FXTAS have less white matter disease and brain atrophy on MRI and less dementia in late-stage FXTAS than do men. 19,84 One important caveat to these differences is that they were generally ascertained through families with known fragile X syndrome probands, which biases the allele size-distribution towards larger expansions, and hence greater propensity for clinical involvement in premutation carriers. 128 Who will develop FXTAS or other forms of premutation clinical involvement is probably modulated by factors such as environmental toxins, chemotherapy, drug and alcohol abuse, and surgery, or background genetic predisposition (figure 3). 113,115 Chemotherapy can exacerbate white matter disease and FXTAS symptoms, 115 and women who smoke develop fragile X-associated primary ovarian insufficiency earlier than non-smokers, 7 probably because of additional oxidative stress. On the basis of our observations, FXTAS symptoms seem to progress more rapidly in people addicted to alcohol or drugs, in some instances because of addiction to opiates that were originally used to control neuropathic pain or fibromyalgia; illicit drugs have been shown to exacerbate white matter disease, and even use of cannabis (street-obtained) for pain control can exacerbate it. 133 In our experience, the onset of FXTAS often occurs after prolonged general surgery in ageing premutation carriers. Some types of general inhalant anaesthesia are intrinsically toxic to neurons, 114 and might therefore exacerbate the underlying neuronal pathology associated with FXTAS. Immune-mediated disorders are more common in premutation carriers than controls, particularly hypothyroidism and fibromyalgia. 49,68,69,71 In a study of 55 women carriers with FXTAS, 72 3% had at least one immune-mediated disorder compared with 46 5% of women carriers without FXTAS and 31 5% of age-matched controls. 68 The estimated odds ratio (OR) for immune-mediated disorders was 2 6 (95% CI , p=0 015) for women with FXTAS compared with premutation carriers without FXTAS; the ORs of an immune-mediated disorder in carriers with FXTAS (5 5, , p<0 001) or without FXTAS (2 1, , p=0 034) were also signi ficantly higher than for controls. Features of an immune-mediated disorder are often present before the development of FXTAS, although how the preceding immune-mediated disorder might influence the sub sequent development of FXTAS is unclear. Un treated hypothyroidism can exacerbate cognitive deficits; therefore, routine screening for hypothyroidism is recommended by the primary health-care provider in ageing women with the Vol 12 August 2013

8 premutation with or without neurological symptoms. 49 Since the observation of inclusions within peripheral neurons, autonomic dysfunction as an associated feature of FXTAS has been more readily interpreted as a consequence of involvement of the peripheral nervous system. 121,122 The need for a pacemaker is not uncommon in those with FXTAS, partly because of the associated bradycardia, which is a consequence of the involvement of pericardial ganglia or cardiac conduction system. However, pacemakers can interfere with confirmation of FXTAS, because they are a contraindication for MRI, which provides a major diagnostic criterion for FXTAS (table 1). 3 Moreover, gastrointestinal symptoms (eg, constipation) could be connected to peripheral disease within the myenteric plexus. Hypertension was initially reported in 60% of women with FXTAS 49 and has subsequently been found to be significantly increased in men with FXTAS (OR 3 22; 95% CI , p=0 0003) relative to controls. 61 Male carriers without FXTAS did not show a significant increase in hypertension compared with controls (OR 1 61, , p=0 164). The Framingham Heart Study 134 reported associated white matter findings connected to hypertension. This study found that an increased systolic blood pressure was linearly associated with decreased regional fractional anisotropy and increased mean diffusivity, especially in the anterior corpus callosum, the inferior fronto-occipital fasciculi, and the fibres projecting from the thalamus to the superior frontal gyrus. Hypertension was also strongly associated with reduced grey matter volumes. Therefore, to avoid exacerbation of white matter disease in individuals with the premutation, hypertension should be aggressively diagnosed, treated, and monitored. FXTAS-associated psychiatric problems often begin before the emergence of tremor and ataxia in patients who develop FXTAS. 9,10,13,66 The increased lifetime prevalence of mood disorders (65%) and of anxiety disorders (52%) in individuals with FXTAS is greater than in those without the disorder. 10,66 Treatment of FXTAS Targeted treatments for FXTAS that can reverse the features of this disorder have not been established. However, several symptom-directed treatments exist that can help those with FXTAS 58, 72, 135 and family members who are affected by the level of care needed for these patients. 136 Irritability and outburst behaviour is common in people with FXTAS; 136 family caregivers often become depressed and need treatment and counselling. 136,137 Treatment of depression is of particular importance because the condition is common in FXTAS, and untreated depression in elderly people can lead to further cognitive decline. 58,72,138 Several anecdotal reports describe partial responses for the symptomatic treatment of intention tremor in FXTAS, including the use of β blockers, primidone, or topiramate. 2,58,72 Deep brain stimulation has also been reported to be beneficial for severe intention tremor of FXTAS in two reports, 139,140 but this method also exacerbated ataxia in two of three cases in the report by Hagerman and colleagues. 140 Botulinum toxin A injections have also been reported to help treatment of tremor in FXTAS. 72 Existing treatments for ataxia are not generally effective, although case reports have reported some benefit from use of riluzole, varenicline, amantadine, or buspirone. 72,141 Physical therapy might also improve strength and stability in those with ataxia, although a controlled study has not been done. 58 Interventions for cognitive decline in FXTAS have included memantine 142 and anticholinesterase inhibitors, 72 with case reports describing positive results. Venlafaxine has helped with attention and executive function deficits, and improved depression in several cases, but a controlled trial has not been undertaken. 73,143 Treatment for the underlying RNA toxicity of FXTAS needs further investigation. The observation that allopregnanolone normalises network activity in neurons cultured from premutation mice, 35 coupled with the observed GABA A deficit in patients with FXTAS, 36 suggests that a GABA A agonist might be beneficial to those with FXTAS. Moreover, at the cellular level an mglur5 antagonist similarly improved neuronal network activity, 35 indicating an excitatory inhibitory imbalance reminiscent of fragile X syndrome. Both GABA agonists and mglur antagonists are being studied in individuals with fragile X syndrome. 143,144 In view of the significant oxidative stress in cells harbouring premutation alleles, 35,106,107,112 antioxidants could be of some benefit as an adjunct to the treatment of FXTAS; however, no controlled trials have been completed for antioxidants or other specific treatments or interventions for FXTAS or, more broadly, for any form of premutation manifestations. Urgent additional treatment studies are needed. One potential approach to target directly the underlying pathogenic mechanisms that give rise to FXTAS would be to use synthetic oligonucleotides to degrade the expanded CGG-repeat mrna, thus eliminating the RNA trigger. Since the site of action of the CGG-repeatcontaining mrna is likely to be intranuclear, oligonucleotides that operate within the nucleus would be needed. One encouraging example of this approach involved the use of a synthetic so-called gapmer oligonucleotide for targeted knockdown, both in culture and in an in-vivo mouse model involving direct intramuscular delivery, of the CAG-repeat DMPK RNA associated with myotonic dystrophy part Although this type of approach has great potential for treatment of FXTAS, substantial barriers remain. In particular, because FXTAS is principally a CNS disorder, delivery of the oligonucleotides across the blood brain barrier would be needed. Vol 12 August

9 Reconceptualisation and redefinition of FXTAS Because the initial definition of FXTAS focused on the core features of progressive tremor and ataxia, and the associated cognitive decline, premutation-associated features that are not manifestly progressive (eg, emotional, behavioural disturbances) should not be regarded as part of the FXTAS diagnosis, even though they might involve the same underlying pathogenic mechanisms as the neurodegenerative phenotype. Furthermore, symptoms of intermittent numbness and tingling, immune-mediated disorders, or fragile X-associated primary ovarian insufficiency, which are very common in carriers with and without FXTAS, 49 do not presage FXTAS. In view of the nearly universal presence of neuronal dysfunction in neonatal premutation mice, we propose that a state of cellular dysfunction (eg, altered mitochondrial function, disturbed calcium regulation) exists in carriers that potentiates adult-onset clinical involvement and eventual neurodegeneration, but that might not be sufficient for an overt phenotype in the absence of one or more additional genetic or environmental factors (figure 3). Thus, symptoms of FXTAS could be precipitated by trauma, severe illness (cancer and chemotherapy 115 ), prolonged surgery with general anaesthesia or exposure to toxins, 113 genetic predisposition to mitochondrial dysfunction, 106,107 or the coexistence of another disease such as multiple sclerosis 49,110,111 or Alzheimer s disease. 85 In this regard, Cornish and colleagues 146 identified a set of neuropsychological deficits, including executive function deficits and increasing impulsivity, in older carriers that might portend FXTAS. Moreover, Yang and colleagues 77 reported on a subgroup of individuals with the pre mutation who had increasing dysfunction seen with diffusion tensor imaging studies involving fronto-cerebellar circuits, which might also identify those with the highest risk of FXTAS. However, longitudinal studies are still needed for both of these investigations to assess the ability of these approaches to predict who will develop FXTAS a crucial undertaking to enable early treatment for those who will eventually develop the neuro degenerative disorder. Finally, although FXTAS has been framed exclusively as a premutation-associated disorder, the neurodegenerative features of FXTAS have been observed in a small number of individuals with an unmethylated fullmutation allele, 147 presumably as a consequence of continued production of raised levels of FMR1 mrna, 148 and in some with copy numbers in the grey zone (45 54 CGG repeats). 73,149 Additionally, small intra nuclear inclusions associated with FXTAS have been reported in three males with full mutation alleles. 59 Loesch and colleagues 147 described a 65-year-old man with a full mutation (multiple alleles between 247 and 480 repeats) that was completely unmethylated, with an FMR1 mrna concentration that was 3 5 times higher than the mean control level elevation of FMR1 mrna. This individual was a truck driver with a borderline IQ (an IQ of 70 80, indicating low intelligence) who developed alcoholism aged 54 years and subsequently had memory deficits, type 2 diabetes, stomach cancer surgery, worsening of tremor and ataxia, and eventually dementia. His MRI had classic signs for FXTAS, including the MCP sign and white matter involvement of the pons, corpus callosum, and periventricular area. The diagnostic criteria for FXTAS need to be updated, and should include the possibility that individuals with CGG-repeat expansions that lie outside of the premutation range can develop the disorder. The broader issue of how to define clinical features that are included or excluded from the criteria also needs to be revisited. Conclusions and future research directions FXTAS represents the severe end of premutationassociated disorders, which include fragile X-associated primary ovarian insufficiency, developmental delays, autism spectrum disorder, ADHD, anxiety, depression, and various medical and neurological problems, such as migraine, fibromyalgia, neuropathy, sleep apnoea, hypertension, and hypothyroidism. These problems are associated with RNA toxicity caused by elevated concentrations of FMR1 RNA in premutation carriers. Future research directions should focus on variable penetrance of clinical involvement, and the environmental and genetic factors that can precipitate or prevent clinical manifestations. Except for a controlled trial of memantine that is currently underway (NCT ), FXTAS-directed treatment approaches have not been investigated. Future directions for treatment should be driven by molecular advances regarding the triggering mechanism of FXTAS, and by targeted treatments that have the potential to halt or reverse the neuro degeneration of FXTAS. Prophylactic interventions that protect the brain from white matter changes and from oxidative stress should be studied. Because the premutation is common in the general population and most carriers will have at least one premutation-associated disorder, we recommend that clinicians test for the premutation (via DNA testing) when one of the premutation disorders is present, particularly when symptoms of tremor, ataxia, or primary ovarian insufficiency are observed. Genetic counselling for the family is recommended if a carrier is identified. 136 Search strategy and selection criteria We searched PubMed for articles published in English from January, 1990, to April, 2013, using the following terms: "fragile X", "FXTAS", "primary ovarian failure", "premutation", "RNA toxicity", "neurodegeneration", "FMR1", "fibromyalgia", "CGG repeat", "neurotoxicity", and "neuropathology". Articles were selected on the basis of relevance; when many papers addressed the same topic one or more were cited as representative of the issue Vol 12 August 2013

10 Contributors Both authors contributed equally to all aspects of the production of this manuscript. PH generated the figures with input from RH. Conflicts of interest RH has received funding from Novartis, Roche, Seaside Therapeutics, Curemark, and Forest for treatment studies in fragile X syndrome or autism. RH has also consulted with Novartis regarding treatment studies in fragile X syndrome. PH is an uncompensated collaborator with Pacific Biosciences regarding new FMR1 sequencing strategies. PH holds patents for FMR1 genotyping and protein tests, and has applied for a joint patent with Pacific Biosciences on sequencing methodology for CGG-repeat determination. Acknowledgments This work was supported by National Institutes of Health grants R01HD036071, R01AG032115, UL1DE019583, R01AG03119, and a National Center for Research Resources grant UL1RR We thank Elisabeth Makhoul and Michele Ono for editorial assistance in manuscript preparation. 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