Fragile X syndrome: An overview and update of the FMR1 gene

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1 Received: 8 May 2017 Revised: 9 June 2017 Accepted: 10 June 2017 DOI: /cge REVIEW Fragile X syndrome: An overview and update of the FMR1 gene M. Mila 1,2 M.I. Alvarez-Mora 1,2 I. Madrigal 1,2 L. Rodriguez-Revenga 1,2 1 Biochemistry and Molecular Genetics Department, Hospital Clinic, Institut d'investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain 2 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Madrid, Spain Correspondence Montserrat Milà, Department of Biochemistry and Molecular Genetics, Hospital Clinic, Institut d'investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, C/Villarroel 170, Barcelona, Spain. mmila@clinic.cat Funding information Instituto de Salud Carlos III (ISCIII), Grant/ Award number: PI12/00879; Fondo Europeo de Desarrollo Regional (FEDER); Generalitat de Catalunya, Grant/Award number: 2014 SGR603. Fragile X syndrome (FXS) is the most common cause of inherited intellectual disability and the leading form of the monogenic cause of autism. Fragile X mental retardation type 1 (FMR1) gene premutation is the first single-gene cause of primary ovarian failure (Fragile X-associated primary ovarian insufficiency [FXPOI]) and one of the most common causes of ataxia (fragile X-associated tremor/ataxia syndrome [FXTAS]), multiple additional phenotypes such as fibromyalgia, hypothyroidism, migraine headaches, sleep disturbances, sleep apnea, restless legs syndrome, central pain syndrome, neuropathy and neuropsychiatric alterations has been described. Clinical involvement in men and women carrying the FMR1 premutation currently constitutes a real health problem in the society that should be taken into account. It is important to highlight that while in FXS there is aloss-of-function ofthefmr1 gene, in premutation associated disorders there is a gain of FMR1 mrna function. To date, the tremendous progress achieved in the understanding of the pathophysiology of FXS, has led to the development of several targeted therapies aimed at preventing or improving the neurological manifestations of the disease. This review is an update of the diseases associated with the FMR1 gene. KEYWORDS FMR1 expansion, fragile X syndrome, FXPOI, FXTAS, genetic counseling 1 FRAGILE X SYNDROME: AN OVERVIEW AND UPDATE OF THE FMR1 GENE In 1943, Martin-Bell described for the first time a familial syndrome of intellectual disability (ID) affecting men, which also showed dysmorphic features and macroorchidism. 1 Subsequently, in 1969 Lubs associated a cytogenetic marker with this syndrome, showing fragility at the terminal end of the long arm of the X chromosome (Xq27.3) in a percentage of the metaphases, leading to the name fragile X syndrome (FXS). 2 In 1991, 3 groups working independently cloned the fragile X mental retardation type 1 (FMR1) gene. 3 5 These authors also described for the first time a special mutation called dynamic mutation, which consisted of a CGG triplet expansion that increases along the generations. During the following decade, geneticists tested a large number of patients for this mutation, increasing their knowledge and describing new human diseases caused by dynamic mutations. In 1999, the study of fragile X pedigrees revealed a higher incidence of primary ovarian insufficiency related to the FMR1 gene, and this condition was named premature ovarian failure (POF) and then later fragile X premature ovarian insufficiency (FXPOI). 6 This term encompasses a continuum of severity in ovarian dysfunction ranging from normal menses and normal hormonal levels, although reduced fertility, to the most severe form of this condition in which folliclestimulating hormone is elevated, menses are abnormal or absent, and fertility is drastically reduced. 7 After further studies, Hagerman et al described a neurological disease related to the FMR1 premutation that was named fragile X-associated tremor/ataxia syndrome (FXTAS). 8 Finally, since 2007 the spectrum of the clinical phenotype associated with the FMR1 premutation has continuously widened. This review is an update of the diseases associated with the FMR1 gene. 2 FRAGILE X SYNDROME FXS (#MIM300624; ORPHA 908) is the most common cause of inherited ID (1%-2% of all ID) and the leading form of the monogenic cause of autism and autism spectrum disorders (ASD) (Figure 1). The 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Clinical Genetics. 2018;93: wileyonlinelibrary.com/journal/cge 197

2 198 MILA ET AL. premutation alleles that exhibit CGG repeat lengths between 55 to 200 CGGs. In this situation, the FMR1 gene is also transcribed and translated, and the CpG island is non-methylated. However, premutation carriers have normal or slightly reduced synthesis of FMRP and increased levels of mrna (2- to 8-fold more than normal alleles), and these individuals are asymptomatic for FXS. Premutation carriers have a risk of having affected offspring as the number of CGGs is unstable and tends to increase with each cellular division. Moreover, people carrying premutation alleles are at risk of developing some disorders characteristic of this status such as: FXPOI, FXTAS and emotional disorders, among others. In Western populations the frequency of premutation alleles in females ranges from 1 in 151 to 1 in 259, although there is evidence of ethnic and racial variability. In Israel, for example, the premutation frequency is around 1 in 113 and in Taiwan 1 in In males the rate ranges from 1 in 468 to 1 in ,18 FIGURE 1 Two siblings, boy and girl, carrying fragile X mental retardation type 1 (FMR1) gene full mutation affected with fragile X syndrome FMR1 gene is inherited as a X-linked dominant trait with a reduced penetrance of 80% in males and 30% to 50% in females The hallmark physical characteristics of FXS include post-pubertal macroorchism, a long face, hyperextensible joints, and prominent ears. The incidence is variable depending on the population; although 1 in 4000 males and 1 in 7000 females are well accepted worldwide. 9 The FXS results from the inactivation of the FMR1 gene. While clinical manifestations of classic FXS have not varied since the first descriptions of the syndrome, the implication of the FMR1 gene in autism and ASD has gained great relevance in later years. Approximately 30% to 50% of FXS patients meet full Diagnostic and Statistical Manual of Mental Disorders-4th edition-text Revision (DSM-IV-TR) criteria for AS, with 60% to 74% fulfilling criteria for ASD. Over 90% of individuals with FXS display some form of atypical behavior which are characteristics of autism including difficulties in social interaction, 10 and approximately 2% to 5% of all the diagnoses of ASD are due to mutations within the FMR1 gene of the FMR1 gene. 11 In most cases the molecular basis of the syndrome is a dynamic mutation: an expansion CGG trinucleotide repeat located in the untranslated region of the first exon of the FMR1 gene and hypermethylation of the CGG repeats and the adjacent CpG island. 3 5 The FMR1 gene spans 17 exons and 40 kb of genomic DNA. It transcribes an mrna of 3.9 kb. The translated protein is called fragile X Mental retardation protein (FMRP), which is necessary from early stages of development and throughout life. 12 In the general population the number of CGG repeats is polymorphic, presenting from 6 to 44 CGGs, and the adjacent CpG island, which acts as a promoter, is non-methylated. Alleles harboring 6 to 44 CGGs are considered normal and remain stable upon transmission. A second class of alleles within the upper normal range contains 45 to 54 CGGs. This range, known as the gray zone, corresponds to intermediate alleles (IAs) which can be stable or slightly unstable, if there are no AGG interruptions, being transmitted to subsequent generations with the possibility of expanding to a premutated allele. 13 The frequency of IAs in the general population varies from 1 in 35 to 1 in The next class is Finally, when the CGG number is greater than 200 repeats, these alleles are in the full mutation range. The CpG island and the repeats themselves are methylated, and this methylation switches off the gene, blocking transcription and no FMRP protein is produced. Male individuals with the full mutation are always affected with FXS, while only 30% to 50% of females carrying the full mutation are affected due to the X-inactivation phenomena induced by the presence of 2 X chromosomes. The clinical manifestations of FXS may vary according to the presence of a mosaicism, different methylation levels of the full mutated allele or by X-inactivation leading to differential FMRP expression within tissues. More than 25% of patients show a premutation-full mutation mosaicism caused by the somatic instability of the full mutation in early embryogenesis. Methylation mosaicism can also be observed in large expansions with incomplete or null methylation. These mosaicisms allow the expression of some FMRP and, in some cases, have been associated with milder ID in males or full mutated males who display normal phenotypes referred to as high-functioning males. 19 In up to 98% of cases, FXS is due to a CGG repeat expansion which is always inherited, but any other FMR1 mutation leading to a loss-of-function of the gene may also cause FXS or an FXS-like phenotype. Point mutations or deletions affecting the FMR1 gene are responsible for up to 1% to 2% of FXS cases. These mutations can be de novo or inherited from a carrier mother. 20 It has been suggested that patients with a clinical FXS-like phenotype but not carrying the full mutation should be screened for FMR1 mutations. 3 FRAGILE X MENTAL RETARDATION PROTEIN FMRP is the protein codified by the FMR1 gene; it is the major protein regulator of the translation of many RNAs involved in synaptic plasticity. 21 FMRP is a selective RNA-binding protein that inhibits the translation of its RNA targets, and although it is mostly expressed in brain and spermatogonia, it is ubiquitously expressed from the early stages of development through postnatal life. Many genes bound to FMRP participate in modeling synaptic plasticity, thus, cognitive impairment is caused by the absence of the FMRP in neurons. The localization of the FMRP is largely cytoplasmatic, being associated

3 MILA ET AL. 199 with the polyribosomes attached to the endoplasmatic reticulum and with free ribosomes at the bases of dendrites and within dendritic spines. 22 FMRP plays a fundamental role in the synapses and the normal development of dendrites. In healthy neurons, FMRP modulates the local translation of numerous synaptic proteins; the synthesis of which is required for the maintenance and regulation of long-lasting changes in synaptic strength. FMR1 gene transcription shows alternative splicing in exons 12, 14, 15 and 17 resulting in the expression of multiple FMR1 mrna isoforms and potentially of FMRP. 23 The distribution of these isoforms differs according to the different brain regions except for the hippocampus and the olfactory bulb at least in mice brains. 24 FMRP binds to approximately 4% of mrnas in the mammal brain, regulating protein synthesis, and the lack of FMRP implies excess basal translation (for further review see Reference 25). Approximately one-third of all RNAs encoding pre- and post-synaptic proteins are targets of FMRP. This role as a transcription factor may explain the phenotypic complexity of the FXS and its variable expression. 4 DIAGNOSIS OF FXS Diagnosis of FXS is based on the determination of the precise CGG number and the methylation status of the CpG island. Diagnosis has considerably improved in the last years with the advances in technology. Although Southern blot is still considered the gold standard of DNA methodology for the diagnosis of FXS, this technique is being gradually replaced by the triplet repeat-primed polymerase chain reaction (TP-PCR) approach. This PCR provides information about the exact CGG number, AGG interruptions, and discriminates between normal, intermediate, premutation and full mutation alleles as well as homozygous females from women with an allele in the normal range and another allele in the premutation or full mutation range. 26 A methylation-specific PCR, TP-MS-PCR, has been developed, and this technique is able to evaluate the methylation status, being even more sensitive than Southern blot analysis. 27 Currently the best method for routine prenatal and postnatal diagnosis consists in a combination of TP-PCR and TP-MS-PCR using DNA extracted from any tissue. The sensitivity and specificity of these 2 PCRs combined are greater than 99%. An indirect linkage approach with microsatellite markers within and surrounding the FMR1 gene is still used for pre-implantation or pre-conceptional genetic diagnosis because of the difficulty in amplifying CGG expansions in only 1 cell. On the other hand, an immunocytochemistry method using an antibody against FMRP is mainly used in cases in which sample collection is difficult or in large-scale screening of a male population. The tissues of choice are blood or hair roots, but this technique has limitations, with the most important drawbacks being found in females and premutation carriers. Recently some new methods have been developed. 28,29 Nevertheless, in individuals without FMRP suspected of having FXS, a cascade DNA family study is always required before a definitive diagnosis is made. Several best practice guidelines for the molecular genetic testing and diagnosis of FXS and other FMR1-associated disorders have been published. 30 Currently FMR1 mutation testing is recommended for: (1) individuals of either sex with ID, developmental delay or autism, (2) individuals who have a family history of FXS, (3) women with a family history of FMR1-associated disorders, including FXPOI, women with reproductive or fertility problems associated with elevated levels of follicle stimulating hormone, (4) individuals with late onset tremor or cerebellar ataxia of unknown origin, and (5) potential gamete donors given the relatively high prevalence of premutations in the general population. 5 CLINICAL INVOLVEMENT IN FMR1 PREMUTATION CARRIERS Since 1999 our understanding of FMR1-associated disorders has dramatically expanded from the initial belief that premutation carriers were clinically unaffected to that of an increasing spectrum of clinical involvement. FXPOI and FXTAS are 2 well-established FMR1- associated disorders that affect approximately 20% of female premutation carriers and half of older premutation adult men, respectively. The reproductive system of permutated women is commonly impaired. The reproductive capacity of a woman depends principally on 2 factors, the ovarian follicular reserve and the quality of the oocytes within these follicles. Premutation women present a continuum of diminished ovarian reserve that gives rise to menstrual cycle irregularities, decreased fertility and hormone fluctuations. 7 Overall, female carriers present menopause approximately 5 years earlier than controls 31 and are also at risk of developing FXPOI as well as all the clinical manifestations of chronic hypoestrogenism such as impaired bone health, increased cardiovascular risk, among others. Nevertheless, these women present spontaneous ovulations during menopause with the risk of having an affected child. In addition, in the last years an increasingly broad spectrum of clinical manifestations including psychiatric disorders (eg, anxiety and depression), enhanced stress, vertigo, olfactory dysfunction, hearing loss, chronic pain syndromes (eg, fibromyalgia and chronic migraine), hypothyroidism, hypertension and sleep apnea have been described especially in women 32,33 (Table 1). Although women generally have less severe clinical manifestations associated with FXTAS, they present reproductive/ovarian, immune-mediated and psychiatric disorders at a higher frequency than the observed in the general population. 32 In parallel with the wider phenotypic spectrum described among adult FMR1 premutation carriers there is a growing awareness of neurodevelopmental problems in childhood. Although this aspect of clinical involvement is still poorly recognized, higher rates of attention deficit hyperactivity disorder (ADHD), shyness, social deficits, ASD and, less commonly and controversially, ID in infants with the premutation, have been described. 34 Special consideration should be given to IAs, as some studies also report behavioral and developmental symptoms in male and female children harboring these alleles Nevertheless, a definitive association between the presence of IAs and clinical manifestations remains unclear The clinical phenotypes associated with premutation alleles described to date all have incomplete penetrance in common which may be due to a combination of genetic backgrounds and

4 200 MILA ET AL. TABLE 1 Clinical manifestation associated to FMR1 premutation (PM) carriers a Cohort studied a Association to FXTAS disease Association to CGG repeat length Immune-mediated disorders Fibromyalgia PM females Probably in FXTAS/possibly in no-fxtas Possibly Thyroid disease PM females Probably in FXTAS/possibly in no-fxtas Possibly Neurodevelopmental phenotypes Working memory deficiencies PM males Definitively in FXTAS/probably in no-fxtas Possibly Language problems PM females Definitively in FXTAS/probably in no-fxtas Not associated Spatiotemporal processing impairment Young-adult PM carriers Definitively in FXTAS/probably in no-fxtas Possibly Arithmetic weaknesses PM females Definitively in FXTAS/probably in no-fxtas Possibly Developmental delay PM carriers Definitively in FXTAS/probably in no-fxtas Possibly Neurocognitive and psychiatric involvement Anxiety disorders PM females Possibly in FXTAS/possibly in no-fxtas Not associated Mood disorders PM carriers Probably in FXTAS/probably in no-fxtas Possibly Autism spectrum disorder PM children and adults Probably in FXTAS/possibly in no-fxtas Not associated ADHD PM females Definitively in FXTAS/probably in no-fxtas Probably Other clinical manifestations Migraine PM carriers Possibly in FXTAS/possibly in no-fxtas Not associated Hypertension PM carriers Possibly in FXTAS/possibly in no-fxtas Possibly Peripheral neuropathy PM carriers Definitively in FXTAS/probably in no-fxtas Evidenced Abbreviations: ADHD, attention deficit hyperactivity disorder; FMR1, fragile X mental retardation type 1; FXTAS, fragile X-associated tremor/ataxia syndrome. a Modified from Wheeler et al. 32 environmental factors that increase the likelihood of clinical involvement. Few genetic risk factors have been described as modulators of disease risk. Hunter et al 42 reported that the rs singlenucleotide polymorphism (SNP) of the CRHR1 gene, which is associated with differential cortisol activation, may contribute to depression and anxiety symptoms in premutation female carriers. Similarly, Silva et al 43 found an association between the presence of allele APOE4 and the risk of developing FXTAS, and Alvarez-Mora et al 44 with the phenomena of X inactivation. Lozano et al 45 suggested the influence of a second (genetic) hit (a copy number variant) on the appearance and severity of neurological problems associated with the FMR1 premutation allele. Finally, the progression of FXTAS has been shown to be more rapid in patients that have been exposed to environmental toxins such as chronic use of addictive substances (opiates, alcohol, and cocaine) or chemotherapy. 33 Overall, the advances in describing and characterizing FMR1- associated disorders will provide a more precise scenario of the clinical involvement of these alleles. 6 MOLECULAR BASIS OF FMR1 PREMUTATION It is important to highlight that the molecular mechanisms of the classical FXS and premutation-associated disorders are completely different. While in FXS there is a loss-of-function of the FMR1 gene, in premutation-associated disorders there is a gain of FMR1 mrna function. Four molecular mechanisms related to the pathologies associated with the FMR1 premutation have been proposed. First, elevated levels of the expanded FMR1 mrna lead to a RNA-gain-of function toxicity, in which the excess of the expanded CGG mrna itself is toxic to the cell. This toxicity is mediated by the sequestration of RNA-binding proteins that partially impair their normal function in the cell (for further review see Reference 46). Increased expression of the premutation allele is thought to be the main cause of clinical manifestations in premutation carriers because the expanded FMR1 mrna and the sequestered proteins form aggregates leading to the formation of intranuclear inclusions present in several tissues. These intranuclear inclusions represent the neuropathological hallmarks of FXTAS, which contain more than 20 different proteins as well as FMR1 mrna but not FMRP. 47 The molecular mechanism of the pathogenesis of FXPOI and FXTAS is considered to be the same. The precise molecular mechanisms of FXPOI, are unknown. However, it has been proposed that follicular impairment can occur at various stages of follicular development. Insights from a knock-in mouse model showed that the increase of mrna levels was sufficient to reduce the number of follicles leading to reduced fertility. 48 On the other hand some authors have found an accelerated loss of follicles although with a normal development of the primordial follicle pool, suggesting an intrinsic problem of the ovary. 49 Likewise, gene expression profiling in blood from FMR1 premutation female carriers with and without FXPOI showed that signaling mechanisms necessary for the maintenance of the survival and activation of the primordial follicle as well as oocyte maturation are down-regulated in FXPOI women. 50 Second, repeat-associated non-atg (RAN) translation, which has also been recognized in other neurodegenerative disorders caused by repeat expansions, 51 has been proposed as a putative mechanism triggering the pathogenesis of premutation alleles. It has been shown that repetitive RNA motifs can support the initiation of translation in

5 MILA ET AL. 201 the absence of an AUG start codon resulting in polypeptides that contribute to neuronal toxicity. Todd et al 52 were the first group to demonstrate the presence of a poly-glycine peptide, named FMRPolyG, which is toxic to cells and is detectable in intranuclear inclusions of post-mortem FXTAS brain samples. On the other hand, Buijsen et al 53 reported the presence of intranuclear inclusions in ovaries of premutation carrier with FXPOI that stained positive for both FMRpolyG and ubiquitin suggesting that this mechanism also plays a role in FXPOI. Recently, Sellier et al 54 suggested that the expression of FMRpolyG is pathogenic per se, while the sole expression of CGG RNA is not. These authors suggest that the synthesis of FMRpolyG by RAN translation alters the nuclear lamina architecture and drives pathogenesis in FXTAS. The description of an antisense transcript produced from FMR1 together with several long non-coding RNA (lncrnas) generated from the FMR1 gene locus that are differentially expressed among premutation carriers has also been proposed as a third molecular mechanism triggering FMR1-associated disorders Expression levels of some of these lncrnas (ie, FMR4 and FMR6) have been found to be significantly altered in post-mortem brain tissue samples of FMR1 premuation carriers. 56,57 Moreover, it has been demonstrated that they may play a key role in cell survival, promoting mechanisms of both cell proliferation and apoptosis. 56 In addition, it has been proposed that FMR6 may play a role in the pathogenesis of FXPOI. Transcription levels of FMR4 and FMR6 have been measured in granulosa cells detecting a significant non-linear association between the number of CGG repeats and the levels of FMR6, but not FMR4, with the highest levels of FMR6 being found in carriers of 80 to 120 CGG repeats. 58 These 3 models are exclusively based on post-transcriptional mechanisms, but emerging evidence highlight the involvement of cotranscriptional processes, as a fourth molecular mechanism that might also be involved in the pathogenesis of FXTAS. Loomis et al 59 reported the formation of stable RNA-DNA hybrids, known as R-loops, during transcription through the expanded CGG repeat track. It has been suggested that increased transcriptional activity associated with premutation alleles may lead to DNA damage at the FMR1 locus resulting in the activation of DNA damage response. 33 This mechanism is based on the presence of the γh2ax protein in the intranuclear inclusions of FXTAS patients which accumulates by excessive R-loop formation leading to the subsequent activation of DNA damage response. 59 Initially, the role of low FMRP levels was discarded in FMR1- associated disorders, as full mutated individuals with no FMRP do not develop FXTAS and FXPOI. However, currently it is suggested that the slightly reduced FMRP levels detected among FMR1 premutation carriers might explain some neurodevelopmental problems observed in children with the premutation. In addition RNA toxicity is also associated with amygdala dysfunction, resulting in cognitive deficits, anxiety, ASD, social avoidance, and aggressive behavior GENETIC COUNSELING In FXS, as in any other X-linked disorders, the probability of a son or daughter inheriting the mutated chromosome from a mother carrier is 50%, while all the daughters and none of the sons of a carrier father will receive the mutation. However, the affectation of the child depends on the dynamic mutation process, that is, if the mutation remains in the premutation range or progresses to a full mutation. Variables found to influence the stability of FMR1 include gender, CGG repeat number, and the presence of AGG interruptions. The expansion from a premutation allele to a full mutation is almost exclusively through female meioses, and thus, all daughters from a male with a premutation will inherit the premutation and will not manifest FXS. Nowadays, it is known that the expansion risk of a FMR1 allele depends both on CGG repeat size and the presence of AGG interruptions. The biological function of these interruptions seems to stabilize the gene during transmission and decreases the risk of DNA polymerase slippage during DNA replication. 61 In the general population, almost 95% of alleles have 1 or 2 AGG interruptions, with the most common allele pattern being of 2 AGG repeats at positions 10 or 11 and 20 or 21. In contrast, premutation alleles contain no or few AGGs at the 5 0 end, and they contain long stretches of uninterrupted CGGs. Presumably, these alleles with no AGG interruptions confer increased risk for unstable transmissions. Therefore, knowledge of the distribution of these interruptions in the IAs or premutation alleles is important for genetic counseling (Table 2). Premutation alleles are highly unstable and may expand to a higher CGG repeat size or even to a full mutation in only 2 generation. The smallest known allele that has expanded to full mutation in a single generation harbored 56 CGG. 62 Recently IA have gained relevance because these alleles may exhibit intergenerational instability associated with the number of AGGs. 13 Counselors must have a solid understanding of the FMR1 gene and the different FMR1-associated disorders (Figure 2). All individuals identified as carriers of intermediate or premutation alleles should be referred for genetic counseling to discuss possible future risks of FMR1-associated disorders. In prenatal diagnosis, this is of particular relevance, as this leads to many ethical considerations (for further discussion see Reference 63). Prenatal identification of female fetuses with the FMR1 full mutation also poses a significant challenge, as there is no way to predict phenotypic severity. This uncertainty generates anxiety for TABLE 2 Risk of expansion of FMR1 alleles considering maternal CGG repeat size and/or the effect of AGG interruption a CGG repeats AGG interruptions Not considering AGGs 0% 1% 2% > Not detected Abbreviation: FMR1, fragile X mental retardation type 1. Not detected a Data was obtained from previous reports: 918 maternal transmissions from premutation carriers <90 CGGs 80 and 171 maternal transmissions from premutation carriers with 90 CGGs. 81

6 202 MILA ET AL. I 1 2 FXTAS II FXPOI N III N N FXS ASD A+FXS FXS N N.. Normal allele Premutation Full Mutation FIGURE 2 Pedigree of a fragile X syndrome family showing different fragile X mental retardation type 1 (FMR1)- associated disorders. FXTAS, fragile X tremor-ataxia syndrome; FXPOI, Fragile X-associated primary ovarian insufficiency; ASD, autistic spectrum disorder; A, autism; FXS, fragile X syndrome parents. On the basis of these observations, pre-test and post-test genetic counseling is really important and should be always offered. 8 THERAPEUTIC APPROACHES IN FXS To date, the tremendous progress achieved in the understanding of the pathophysiology of FXS has evolved with the elucidation of many molecular and cellular processes that are compromised in the FXS. This has led to the development of several targeted therapies aimed at preventing or improving the neurological manifestations of the disease. Despite these advances, an effective targeted therapy currently remains elusive, and, to date, no drug has been approved for the treatment of FXS. Nevertheless, several promising therapeutic strategies are currently being assessed in FXS clinical studies. Based on the knowledge acquired over time, 2 direct approaches are currently being investigated for the treatment of FXS: (1) reactivation of the FMR1 gene, and (2) compensation of the lack of FMRP, acting on the pathways in which it is involved. Restoring FMR1 gene activity and therefore, FMRP expression, is based on the concept that the epigenetic changes that block transcription are potentially reversible. The idea is to convert a non-functional methylated full mutation to a functional unmethylated full mutation. The description of individuals with normal intelligence with a completely or partially unmethylated full mutation that expresses FMRP, together with the fact that the CGG-repeat in the 5 0 untranslated region (5 0 UTR) does not influence the open reading frame of a mutated FMR1 gene, provide the proof-of-principal for this approach. 64 There are several studies that report that FMR1 gene silencing could, to some extent, be reversed in FXS patient cells. Compounds that target repressive chromatin modifications by either blocking DNA methyltransferases (5-azacytidine [5-azaC] or 5-azadC) or by affecting the histone acetylation status (valproic acid [VPA] or L-acetylcarnitine [LAC]) have been tested. 65 Although the use of 5-azaC or 5-azadC has achieved a significant reactivation of FMR1 gene expression in FXS cellular models, 66 the reactivation was modest and transient. Furthermore, the use of these compounds in therapeutic strategies has 2 major objections. First, their lack of specificity that could lead to potential unforeseeable damaging consequences in long-term treatment. Second, in order to be effective, the cells must divide, which is generally not the case with FXS target cells. On the other hand, VPA, an antiepileptic drug, and LAC, have been tested in clinical trials showing effectiveness in ameliorating ADHD symptoms, 67 although the in vitro reactivation effect on the FMR1 gene was shown to be minimal. 68 CRISPR/Cas9 genome editing technology has recently been used to excise the expanded CGG repeat from the full mutation allele in FXS cells, achieving FMR1 reactivation in most of the cell lines studied. 69 The successful results achieved in these preliminary studies open a promising new cell therapy line for future treatment of FXS. In FXS, the lack of FMRP causes dysregulation and usually overexpression of many FMRP RNA targets, which can cause imbalances in neurotransmission and deficits in synaptic plasticity. In fact, examination of brains of FMR1 knock-out mice revealed altered dendritic spine morphology and density, evidencing the critical functions of FMRP in synaptic connections. 70 The absence of FMRP observed in the brain of FMR1 knock-out mice, also causes hyperactivation of metabotropic glutamate receptor (mglur) signaling, which leads to increased protein synthesis and defects in synaptic plasticity including enhanced long-term depression (LTD) of transmission. This observation led to the mglur theory of FXS, which proposes that excessive signaling through mglurs contributes to the psychiatric and neurological aspects of the FXS phenotype. 71 FMRP also regulates translation pathways activated by other receptors such as muscarinic (M1) acetylcholine receptors and Gq-linked receptors, including dopamine D1 receptors. 72 Activation of these receptors induces FMRP dephosphorylation, which results in a loss of FMRP repressor function and, hence, to the synthesis of new synaptic proteins. Some of the FMRP-regulated proteins are likely responsible for increased α-amino- 3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) receptor

7 MILA ET AL. 203 internalization, leading to a reduction in long-term potentiation (LTP) throughout the brain. 73 Overall, excessive constitutive activation of mglur-mediated dendritic protein synthesis is responsible for an imbalance in excitatory and inhibitory neurotransmission in FXS. Recent targeted clinical trials in FXS have attempted to rectify this excitatory/inhibitory imbalance believed to contribute to the pathophysiology of FXS. The use of mglur blockers and γ-aminobutyric acid (GABA) agonists has shown to be efficacious in rescuing synaptic deficits in pre-clinical studies of mouse and fly models. 74,75 In this scenario, mglur5 negative modulators (including fenobam, AFQ056, STX107 and RO ) have been tested in several clinical trials on FXS, although they were abandoned due to their lack of efficacy. 76 Since then, the focus has moved away from the mglur antagonists and shifted to the GABA system and to other receptors (dopamine, serotonin, or tropomyosin receptor kinase B) that have reduced function in FXS. In order to correct the deficits in GABAergic signaling, several GABAergic compounds have been tested (ie, riluzole, acamprostae, ganaxolone, and arbaclofen; Table 3). In these studies GABA-A agonists are directed at compensating for GABA-A subunit deficiencies, while GABA-B agonists act pre-synaptically to block glutamate release. Positive effects have been obtained for some of the clinical trials based on these compounds, leading to phase II and phase III developments. Other targeted treatment strategies have been designed and directed at either: (1) reducing the excessive activity of individual proteins normally regulated by FMRP (eg, increased protein levels of matrix metalloproteinase 9 [MMP-9] or striatal-enriched protein tyrosine phosphatase), (2) increasing the expression and activation of surface AMPA receptors; (3) using micrornas (mirnas) to block excessive translation of mrnas normally regulated by FMRP; and (4) correcting abnormal channel activities normally directly regulated by FMRP 77 (Table 3). Some of these strategies have shown benefits in early-phase or open-label trials in FXS. For example, the administration of lithium, which is thought to rescue glycogen synthase kinase-3 (GSK-3) activity, showed promising effects on behavior and cognition in FXS mice, 78 and resulted in improvement in abnormal extracellular signal-regulated kinase (ERK) phosphorylation rates in in vivo patient-derived cell lines. Administration of minocycline, which has an inhibitory effect on MMP- 9, has also shown positive effects with mild global clinical improvement in children with FXS. 65 Finally, promising pre-clinical results have been obtained with the administration of lovastatin, cannabinoids, omega-3 and other neutraceuticals. 79 Despite all target-based treatments tested, at present the treatment of FXS is supportive and depends on the symptoms presented, with therapy and educational strategies designed to treat co-occurring medical and behavioral symptoms. Supportive therapies involve a combination of applied behavioral analysis, medications, occupational therapy, physical therapy and speech-language therapy (PubMed Health Information). Drugs approved for indications other than FXS are currently being administered. For example, LAC and VPA have been used to treat ADHD symptoms in FXS, melationin has been tested to improve insomnia, and aripiprazole (an antipsychotic) is currently prescribed to reduce irritability. 76 Given the global developmental delay that most individuals with FXS present, it is necessary to provide supportive therapy during the early stages of growth in order to maximize the potential benefits. Furthermore an individualized behavioral, therapeutic and educational intervention plan is needed to achieve the best results. 77 It is important to highlight that all the clinical trials have so far been tested based on evidence obtained from different FXS animal models, basically mouse and fly. New therapeutic approaches should be tested in a human cellular model. Indeed, Fragile X Syndrome-induced pluripotent stem (FXS-iPS) cells represent a potentially useful cellular model for testing new drugs. Furthermore, considering the large number of mrnas targeted by FMRP and its widespread expression, most of the clinical trials with a single drug performed up to now have been inconclusive. Future trials should be aimed at correcting the multiple pathways known to be disrupted by the lack of FMRP. TABLE 3 Fragile X syndrome and targeted treatments a Drug mechanism Target Compound Modulating excitatory neurotransmission mglur5 antagonists Fenobam Mavoglutant/AFQ056 Basimglurant/RO STX107 N-methyl-D-aspartate (NMDA) Memantine receptor antagonist Modulator of AMPA signaling Cx516 Modulating inhibitory neurotransmission Targeting GABA receptors Arbaclofen Ganaxolone Acamprosate Riluzone Metadoxine Modulating intracellular signaling pathways Inhibition of GSK-3 Lithium Inhibition of ERK pathway Lovastatin Modulating specific targets of FMRP Inhibition of MMP-9 Minocycline Abbreviations: AMPA, α-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate; GABA, γ-amino-butyric acid; FMRP, fragile X Mental retardation protein; GSK-3, glycogen synthase kinase-3; MMP-9, matrix metalloproteinase 9. a Modified from Davenport et al 76 and van Karnebeek et al. 82

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