Review Article The Fragile X Mental Retardation Syndrome 20 Years After the FMR1 Gene Discovery: an Expanding Universe of Knowledge *François Rousseau,1,2,3,4 Yves Labelle,2,3,4 Johanne Bussières,3 Carmen Lindsay3 Réseau de Médecine Génétique Appliquée, Fonds de Recherche en Santé du Québec; 2The APOGEE-Net/CanGèneTest Research and Knowledge Network (www.cangenetest.org); 3Unité de recherche en génétique humaine et moléculaire, Axe de recherche en évaluation des technologies et transfert des connaissances, Centre de recherche du CHUQ-Hôpital-SaintFrançois-d’Assise; 4Département de biologie moléculaire, biochimie médicale et pathologie, Faculté de Médecine, Université Laval, CRCHUQ-Hôpital St-François d’Assise, 10 rue de l’Espinay, Québec, Qc, Canada G1L 3L5. For correspondence: Dr François Rousseau, Francois.rousseau@mac.com 1 Abstract The fragile X mental retardation (FXMR) syndrome is one of the most frequent causes of mental retardation. Affected individuals display a wide range of additional characteristic features including behavioural and physical phenotypes, and the extent to which individuals are affected is highly variable. For these reasons, elucidation of the pathophysiology of this disease has been an important challenge to the scientiic community. 1991 marks the year of the discovery of both the FMR1 gene mutations involved in this disease, and of their dynamic nature. Although a mouse model for the disease has been available for 16 years and extensive research has been performed on the FMR1 protein (FMRP), we still understand little about how the disease develops, and no treatment has yet been shown to be effective. In this review, we summarise current knowledge on FXMR with an emphasis on the technical challenges of molecular diagnostics, on its prevalence and dynamics among populations, and on the potential of screening for FMR1 mutations. Introduction Approximately 2% of the general population is affected by mental retardation (MR)1,2 deined as ‘signiicant limitations in both intellectual functioning and adaptive behaviour as expressed in conceptual, social and practical adaptive skills’. Both environmental and genetic causes are involved, and the latter include chromosome number or structural anomalies, genomic disorders and monogenic diseases. The X chromosome is often involved in MR, explaining in part the higher incidence of MR in males compared to females.3 The FXMR syndrome is by far the most prevalent X-linked MR pathology, accounting for approximately 20% of all X-linked MR cases.4 FXMR is caused by the absence of a functional FMRP,5 most often as a result of transcriptional silencing of the corresponding FMR1 gene (i.e. the mRNA cannot be produced) on chromosome X.6,7 The transcriptional silencing occurs as a consequence of the expansion of the number of repeats of the trinucleotide CGG in the 5’ untranslated region (UTR) of the gene.6-9 An increase in the number of repeats results in hypermethylation of the repeat sequences and of the adjacent promoter region and silencing of the gene. In the normal population, there is a high degree of variation, or polymorphism, in the number of triplet repeats in the 5’ UTR of the FMR1 gene, ranging from 5 to 54 repeats with an average of 30 repeat units.10 Expansion of the trinucleotide repeats to between 55 and 200 repeats is termed a premutation state because the number of repeats in not suficient to cause MR,6 although this range of repeats may predispose to other disorders as discussed later. An expansion of more than 200 repeats, which is associated with abnormal methylation on the active X chromosome, is termed a full mutation and is associated with FXMR.6,11 FXMR was one of the irst diseases to be associated with trinucleotide repeat expansion, and to date at least 16 other neurological diseases have been found to be caused by this mechanism.12 Fragile X Phenotype Approximately 85% of males and 25–30% of females with a full FMR1 mutation have an IQ <70,13,14 with an average IQ of approximately 40 for males.15 Short-term memory for complex Clin Biochem Rev Vol 32 August 2011 I 135 Rousseau F et al. information, visuospatial skills and speech are the most commonly affected abilities. Severity of the deicits correlates with deiciency of the protein FMRP; individuals with only partial decrease in FMRP levels are more likely to present a borderline or normal IQ without learning disabilities.13,16,17 The lesser severity of these cases is likely due to a lower degree of FMR1 gene methylation than normally seen in FXMR which permits the production of at least some, albeit low, level of FMRP. Females are generally much less affected by the gene abnormality due to the X-linked nature of the mutation. The majority of females with the full mutation have a normal or borderline IQ,14 however most will have learning and/or psychological deicits.18,19 Physical characteristics of affected individuals include macrocephaly (long narrow face and prominent forehead, jaw and ears), macroorchidism (enlarged testicles) appearing prior to puberty, and a variety of physical features consistent with connective tissue abnormalities, including hyperextensible inger joints, lat feet, mitral valve prolapse, hypotonia, soft skin, and a high arched palate.20,21 At the neuroanatomical level, a consistent inding is the presence of abnormally long and dense dendritic spines,22,23 which is thought to be caused by defective dendritic spine development or maturation. Behavioural and psychological characteristics of FXMR individuals include shyness, social anxiety and avoidance, withdrawal, distractability, mood lability, depression, hyperactivity and irritability. The clinical heterogeneity of FXMR combined with the high incidence of the disease has prompted researchers to develop checklists that generate scores correlated with the likelihood of developing FXMR for a given patient. Many such checklists are currently proposed and new ones are published on a regular basis. They have been reviewed by van Karnebeek et al. in 2005,24 Johnson in 2008,25 and Guruju et al. in 2009.26 The overall conclusion of the latter reviews is that checklists are somewhat useful to identify individuals most likely to have FXMR, with clinical features showing the strongest predictive value being macroorchidism and macrocephaly. A close relationship between FXMR and autism has been documented. A mutation in the FMR1 gene is found in 2–7% of children diagnosed with autism, and the prevalence of autism in FXMR individuals is estimated to be 30%.27-29 Furthermore, individuals with FXMR but without classical signs of autism may display autistic-like features such as hand lapping and biting, gaze avoidance, tactile defensiveness, repetitive behaviours, hyperactivity and hypersensitivity to sensory stimuli.30,31 The neurobiological basis of the connection between FXMR and autism is unknown. Several proteins regulated by FMRP at the translational level are associated with autism, and FMRP deiciency affects synaptic plasticity and interferes with the γ-aminobutyric acid (GABA) and 136 I Clin Biochem Rev Vol 32 August 2011 glutamate systems, all of which are also involved in autism spectrum conditions.32 The link between FXMR and autism is intriguing in view of the fact that FXMR is associated with a speciic mutation and well-deined physical anomalies, while autism is diagnosed strictly on behavioural characteristics and is not currently correlated with speciic biological markers. Recently, several groups have identiied genes that appear to be associated with autism spectrum disorders,33-36 and these may eventually shed some light on the connection between FXMR and autism. Molecular Pathophysiology FXMR is caused by the absence of a functional FMRP, a protein located with polyribosomes and expressed in several tissues, albeit at different levels.37,38 As already mentioned, the most common mechanism for this deiciency is an expansion of a CGG-rich triplet repeat sequence in the irst exon (and coding for the 5’ UTR) of the FMR1 gene, and with expansion leading to its hypermethylation (Figure 1).7-9 Methylation is a common means of silencing genes, and in FXMR this hypermethylation induces a local chromatin condensation that prevents the binding of speciic transcription factors and the basal transcriptional machinery, leading to transcriptional silencing of the gene. Several transcription factors have been shown to regulate FMR1 expression: the upstream stimulatory factors 1 and 2, the nuclear respiratory factors 1 and 2, Sp1, and the cAMP-responsive element-binding protein (CREB).39,40 These factors bind to a 150 bp region adjacent to FMR1 transcription initiation site, which is in close proximity to the CGG repeat sequence. Approximately 250 bp upstream of the triplet repeat sequence, a CpG island is also hypermethylated in FXMR (Figure 1). Other modiications associated with hypermethylation of this region are reduced acetylation of lysine residues in histones H3 and H4,41 and a change in the methylation pattern of histone H3.42 The most likely mechanism of trinucleotide repeat expansion is slipped-strand mispairing during DNA replication.43 This might occur when Okazaki fragments formed by repetitive sequences are not anchored to unique sequences at their ends, thereby increasing the probability of slippage, which in turn would induce the addition of nucleotides to the template strand by repair enzymes to restore its complementarity to the newly synthesised strand, thus generating an expansion.44 While the latter mechanism could explain the accumulation of a small number of repeats, larger expansions would require a pause in the process of replication to allow the synthesis of a larger number of repeats. This could occur through the formation of secondary structures formed by the already present repeat sequences.45 On a larger scale, there is evidence that cis-acting sequences located approximately 50 kb proximal to the fragile X site may be involved in the expansion mechanism, and a Fragile X Syndrome: 20 Years On Figure 1. Structure of the human FMR1 gene. The schematic diagram at the very top of the igure represents the normal allele, and the schematic diagrams further below show the changes associated with the premutation and full mutation expansions respectively. Dark grey lines show the transcribed sequence, black boxes the exons, and the light grey box the CpG island. The ATG translation initiation codon and the 17 exons and alternative splicing junctions are shown. The size of arrows correlates with amount of RNA which is produced. The term Me indicates methylation of full mutations. Restriction sites and the site for the probe StB12.3 are also shown. Sources:11,186-188. SNP variant located in this region cosegregates with a subset of chromosome haplotypes at highest risk of expansion.46 One question still debated is whether the repeat expansion occurs before or after fertilisation of the ovum. Affected individuals show mosaicism in their somatic cells; that is, the number of FMR1 triplet repeat sequences varies in their somatic cells, having full mutations of different sizes and sometimes premutations in a subpopulation of cells. In addition, affected males show only premutations in their sperm.47 Such observations are consistent with a postzygotic expansion model; that is, occurring after fertilisation of the ovum. On the other hand, there is evidence consistent with a prezygotic model.48 An observation supporting the latter model is that while only premutations are observed in mature sperm cells, full mutations are observed in oocytes and foetal spermatogonia,49 with the presence of premutations in mature sperm, as FMRP is strongly expressed in testis, possibly due to a somatic selection process.38 It may be that both mechanisms occur. Not all FXMR patients show an expansion of the FMR1 triplet repeat sequence. In a small number of cases, deletions ranging from 355 bp to 13 Mb have been identiied.50-56 Point mutations have also been identiied in the FMR1 coding sequence, leading to a non-functional protein.57,58 In addition, a massively parallel DNA sequencing project has recently identiied more than one hundred FMR1 sequence variants in developmentally delayed male subjects,59 suggesting a role of FMRP in this phenotype. Population Genetics and Origins of Mutations While FXMR appears to be prevalent worldwide, founder effects have been described in a number of different ethnic groups,60 and there are speciic FXMR-associated haplotypes in populations known to have founder effects, such as the French-Canadian61 and Israeli populations.62 However there are also a limited number of FXMR-associated haplotypes in other populations not prone to founder effects, such as the French.63 These observations have supported the general belief that FMR1 expansions were initiated in a limited number of normal FMR1 alleles and that the initial early events occurred many generations ago (up to 90 generations) in a limited number of chromosomal backgrounds.64 Morton and Macpherson studied the population genetics of FMR1 and proposed that FMR1 mutations arose from a multistep mutational process that progressed from normal size alleles to relatively stable intermediate size alleles, then to unstable premutation alleles, and inally to full mutation alleles which are associated with FMR1 shutdown, lack of FMRP and presence of FXMR.65 Some observations suggest that the initial mutation mechanism (from normal to premutation allele) might differ from the process leading from premutation to full mutation.66 Different risk factors for instability have been proposed, including structure of the repeat (length of CGG arrays and their interruption by AGG repeats), haplotype background (cis-acting factors),46,61 trans-acting factors, and most importantly, the parental origin of transmission (full mutations being inherited only from a maternally-transmitted FMR1 allele of premutation or full mutation size). In a large study involving 13 laboratories in eight countries, Nolin et al. observed that the smallest premutation allele having expanded to a full mutation in one generation contained 59 repeats,67 although an even smaller sequence (56 triplet repeats) had expanded to a full mutation in a single generation according to another study.68 Therefore, for diagnostic purposes, we have conservatively deined the lower threshold of the premutation allele range as 55 repeats in this review. The study of mutational processes and their timing has yet to provide insight into the mechanisms responsible for these very peculiar dynamic mutations that cause the inactivation Clin Biochem Rev Vol 32 August 2011 I 137 Rousseau F et al. Figure 2. Structure of the human FMR1 protein (FMRP). The nuclear localisation sequence (NLS), hnRNP K-protein homology domains (KH1 and 2), nuclear export signal (NES), and Arg-Gly-Gly domain (RGG) are shown. Numbers indicate amino acids, and the regions of FMRP bound by several of its interacting partners are shown (see also Table 1). of the FMR1 gene and therefore FXMR. Human population genetics, the study of transmission of FMR1 arrays in animal models, as well as in vitro investigations using triplet repeat arrays will likely provide these much needed insights in the future. Gene and Protein Function(s) FMRP is widely expressed in various tissues, with the highest levels found in the brain and testis,69 which correlates with the phenotype observed in FXMR. The full-length protein has 632 amino acids and ive clearly characterised domains (Figure 2), including two hnRNP K-protein homology (KH) domains in its central region and an Arg-Gly-Gly (RGG) domain at its carboxy-terminal end. Extensive alternative splicing occurs at the 3’-end of the gene in exons 12, 14, 15 and 17 (Figure 1), potentially giving rise to at least 12 different isoforms. The most common splicing product generates the isoform lacking exon 12, whereas the isoform lacking exon 14 (which encodes the nuclear export signal) has the lowest expression level.70 The protein also contains a nuclear localisation signal (Figure 2), indicating that it may shuttle between the nucleus and cytoplasm, a property that has been conirmed by electron microscopic studies.71 In the brain, FMRP is exclusively expressed in differentiated neurons, mostly in the hippocampus and granular layer of the cerebellum.69,72 FMRP expression is also detected in synapses but not in axons.71 Besides interacting with itself, several other proteins interact with FMRP (Table 1), including the closely related FXR1 and FXR2 proteins. These proteins show 86% and 70% similarity to FMRP respectively, and possess the same domain organisation. All three proteins are associated with polyribosomes in an RNA-dependent manner.73 FMRP also interacts with non-RNA binding proteins such as the SUMO-conjugating enzyme UBC9, the Ran-binding protein 9, and nucleolin. While the actual physiological signiicance of these associations is currently unclear, a large amount of data clearly point to a role of FMRP in mRNA transport and 138 I Clin Biochem Rev Vol 32 August 2011 translation.74-78 Overexpression of FMRP both in vivo and in vitro suppresses translation.79-81 FMRP has been shown to bind to a signiicant percentage of brain mRNAs, with a preference for mRNAs containing a G-quartet structure or a U-rich sequence.82-86 The association of FMRP with polyribosomes is dependent upon the binding to the KH2 domain of a tertiary RNA structure called the kissing complex.87 FMRP can be phosphorylated, a modiication which apparently favours its association with stalled ribosomes.88 Therefore, dephosphorylation of FMRP may trigger the translation of FMRP-associated mRNAs. Furthermore, an elegant study by Miyashiro et al. has identiied several mRNAs that bind FMRP and show a change in abundance and/or distribution in the brains of fmr1 knockout (KO) mice.89 Conirming and extending these observations, Kao et al. used time-lapse imaging to demonstrate the role of FMRP in the transport and expression of CaMKIIα mRNA at the dendritic spines in hippocampal neuron primary cultures upon stimulation of group I metabotropic glutamate receptors.90 Although the exact role of FMRP is still largely unknown in other cell types, the evidence accumulated to date strongly suggests that one signiicant role of FMRP in neurons is to regulate synaptic plasticity via the transport and translation of speciic mRNAs at dendritic spines. FMRP also appears to play a role in brain development, as it suppresses the expression of the microtubule-associated protein 1B (MAP1B) mRNA during synaptogenesis in the mouse neonatal brain.91 The role of FMRP in microtubule regulation has recently been conirmed in Drosophila, where dFMRP was shown to regulate the axonal transport of mitochondria via microtubule network formation.92 Because of its role in such general regulatory systems as mRNA transport and expression and microtubule formation, a detailed understanding of the function of FMRP will likely require more sophisticated experimental techniques such as time-lapse imaging as mentioned above. As discussed later, FMRP has also been shown to be involved in the miRNA pathway suggesting new mechanisms of action. Clin Biochem Rev Vol 32 August 2011 I 139 Protein Name UniProtKB Accession Number Cellular Compartments, Molecular Functions and Biological Processes Experimental Evidence of Association with FMRP Fragile X mental retardation syndromerelated protein 1 P51114 Nucleolus; developmental protein; differentiation; myogenesis Co-puriication;201,202 pull-down;73 two-hybrid;73 co-immunoprecipitation191,202 Fragile X mental retardation syndromerelated protein 2 P51116 Cytoplasm; RNA binding; protein binding Far-western;73 co-puriication;201,202 coimmunoprecipitation;73,191,202 two-hybrid;73 pull-down73,201 Nuclear fragile X mental retardation-interacting protein 1 Q9UHK0 Nucleus; RNA processing Co-immunoprecipitation;190 two-hybrid;189,190 pull-down189,190 Nuclear fragile X mental retardation-interacting protein 2 Q7Z417 Cytoplasm; nucleus; RNA binding; protein binding Two-hybrid;189 co-immunoprecipitation;189 nuclear magnetic resonance;199 pull-down189 Cytoplasmic FMR1interacting protein 1 Q7L576 Cell junction; cell projection; cytoplasm; synapse; synaptosome; developmental protein; cell shape; differentiation; neurogenesis Two-hybrid;200 co-immunoprecipitation;200 pull-down200 Cytoplasmic FMR1interacting protein 2 Q96F07 Cell junction; cytoplasm; synapse; synaptosome; protein binding; apoptosis; cell adhesion Two-hybrid;189,200 pull-down200 Nucleolin P19338 Co-immunoprecipitation202 Ran-binding protein 9 Q96S59 Nuclease-sensitive element-binding protein 1 P67809 Cytoplasm; nucleus; RNA binding; nucleotide binding; protein C-terminus binding; telomeric DNA binding; angiogenesis Cytoplasm; nucleus; Ran GTPase binding; microtubule nucleation; protein complex assembly Cytoplasm; nucleus; repressor; transcription; transcription regulation; mRNA processing; mRNA splicing SUMO-conjugating enzyme UBC9 P63279 Nucleus; ligase; cell cycle; cell division; chromosome partition; Host-virus interaction; mitosis; Ubl conjugation pathway Two-hybrid193 Tudor domain-containing protein 3 Q9H7E2 Cytoplasm; nucleus; nucleic acid binding Pull-down;197 co-immunoprecipitation197 Kinesin-like protein KIF3C O14782 Microtubule; motor protein; ATP binding; kinesin complex Pull-down;195 two-hybrid;195 coimmunoprecipitation195 Poly(ADP-ribose) glycohydrolase Q86W56 Cytoplasm; nucleus; poly(ADP-ribose) glycohydrolase activity; carbohydrate metabolic process Co-immunoprecipitation196 Microspherule protein 1 Q96EZ8 Nucleus; nucleolus; protein binding; protein modiication process Two-hybrid;194 pull-down194 * STRING database, version 8.3, June 2010. Pull-down;198 co-immunoprecipitation;198 two-hybrid198 Co-immunoprecipitation192 Fragile X Syndrome: 20 Years On Table 1. FMRP interacting proteins.* Rousseau F et al. Animal Models An fmr1 KO mouse was produced in 1994, shortly after the discovery of the FMR1 gene in 1991.93 This model appeared adequate to study the pathology of the disease at the time as the KO mouse displayed macroorchidism, learning deicits, and hyperactivity. In addition, the increase in dendritic spine density and the abnormally long and immature spines observed in FXMR patients are also observed in this mouse model.94-96 Further study of the fmr1 KO mouse showed that a form of protein synthesis-dependent synaptic plasticity, termed long-term depression and triggered by the activation of metabotropic glutamate receptors, is selectively enhanced in the hippocampus of mice lacking FMRP.97 This is not only consistent with a role of FMRP in translation repression, but may also be related to the cognitive deicits observed in FXMR patients since long-term depression is believed to be involved in memory and learning. More recently, it was shown that synaptic functions were also affected in the amygdala of FMRP KO mice.98 This observation might be related to the emotional symptoms of the disease as the amygdala has a pivotal role in extracting the affective signiicance of sensory stimuli and mediating the formation of emotional memories.99 These studies have further validated the mouse FMRP KO model to gain insights into FXMR pathology. Several attempts have been made to rescue the phenotype of KO mice.100 The most successful approach consisted in using a yeast artiicial chromosome (YAC) containing the human FMR1 gene to produce a transgenic mouse that was then crossed with the KO mouse.101 The KO/YAC progeny showed normal testicular development, indicating functional rescue by the human protein. However, these mice showed only partial rescue of the behavioural phenotypes, suggesting that cell type speciicity and/or expression levels of the protein might be important factors in these phenotypes. Several additional attempts have been made to generate mouse models for the FMR1 expansion mutation. A transgenic mouse was generated with a (CGG)98 repeat sequence replacing the endogenous short CGG repeat mouse sequence in the FMR1 promoter, which showed only moderate instability upon both female and male transmission.102 In addition, mice with up to 230 CGG repeats were observed, and although this length is within the range of full human mutations, no abnormal methylation was detected.103 These results suggest that although the mouse may be an appropriate model to study FMRP deiciency, it is not yet adequate to study the expansion mechanism leading to abnormal methylation. Inactivation of the dFmr1 gene in Drosophila leads to enlarged synaptic terminals and defective coordinated behaviour in simple light tests. However, the lies are viable, display no gross anatomical anomalies, and appear normal in a range 140 I Clin Biochem Rev Vol 32 August 2011 of behavioural tests.104 Such a relatively mild phenotype is unexpected in view of the fact that the Drosophila genome does not encode the FXR1 and FXR2 homologues of FMR1 found in mammalian genomes.105 The zebraish, however, does possess all three genes, and FMRP loss-of-function studies in this model using morpholino antisense oligonucleotides show that FMRP is required for normal axonal branching in trigeminal ganglion neurons and normal cartilage formation in craniofacial development.106 The latter model therefore appears appropriate to study the role of FMRP in cartilage formation, a phenotype that has not yet been reported in the mouse model and which could be related to the connective tissue abnormalities and macrocephaly documented in FXMR patients. Involvement of microRNAs MicroRNAs (miRNAs) are a class of small (18–25 nt) noncoding RNAs that are involved in a wide range of basic biological processes including differentiation and apoptosis.107 They are generated from a precursor form (pre-miRNAs) by Dicer, a cytoplasmic RNAse III enzyme, and then integrated into a complex called RNA-Induced Silencing Complex (RISC), where they negatively regulate the expression of speciic mRNAs, either through cleavage of the mRNA or by translational repression.108,109 FXMR was one of the irst genetic disorders to be associated with miRNAs.110,111 Both mammalian FMRP and its Drosophila homologue dFMRP were found to interact with Dicer and with the Argonaute proteins that are the catalytic components of the RISC complex.111-113 In the adult mouse brain, FMRP interacts with both Dicer and Argonaute1 at postsynaptic densities.114 Considering that FMRP is an RNA-binding protein associated with both Dicer and the RISC complex, one of its proposed roles in the miRNA pathway might be to guide the miRNA produced by Dicer into the RISC complex. Several miRNAs have been shown to speciically interact with FMRP. In Drosophila, dFMRP is required for the proper processing of pre-miRNA-124a: dFMRP deiciency leads to a reduction of mature miRNA-124a levels and an increase in pre-miRNA-124a levels.115 The miRNA-124a is speciically expressed in the central nervous system and ectopic expression of miRNA-124a leads to decreased dendritic branching of dendritic arborisation sensory neurons in Drosophila.115 These observations are remarkably consistent with the inding of abnormally long dendritic spines in FXMR patients lacking FMRP.22,23 In the mouse brain, FMRP has been shown to interact with several miRNAs, most notably miRNA-125b and miRNA-132.116 Both of these miRNAs are involved in synaptic plasticity and dendritic spine morphology. The target of miRNA-125b is the mRNA encoding N-methyl-D-aspartate (NMDA) receptor subunit NR2A, which is also associated with FMRP Fragile X Syndrome: 20 Years On in the mouse brain.116 Taken together, these observations suggest that the miRNA pathway may play a highly signiicant role in the phenotype of FXMR patients. Molecular Diagnostics Identiication of the nature of FXMR mutations and their properties has revolutionised the diagnosis of FXMR which, before 1991, relied mainly on cytogenetic analysis for the typical fragile site in Xq27.3.6,7,11 Molecular diagnostic analysis of the FMR1 gene in fragile X families has shown a high degree of genetic homogeneity. Indeed, >95% of all FXMR cases are likely due to the same type of mutation – the expansion of a CGG-rich triplet repeat – in the 5’ UTR of the irst exon of the gene. These expansions of a triplet repeat sequence (comprised mainly of CGG repeats, sometimes interrupted by AGG repeats) thus constitute the vast majority of FXMR mutations, and therefore FXMR molecular diagnosis relies on detecting these expansions. Point mutations have also been described in association with the phenotype but they appear to be much rarer than triplet repeat expansions.57,58 Less frequently, FXMR can be due to a number of other types of mutations, and a recent survey by massively parallel sequencing of 963 development-delayed males without FMR1 CGG repeat expansions identiied 130 novel FMR1 sequence variants, including one missense mutation of a conserved residue, 3 promoter mutations affecting the in vitro level of transcription, and 10 non-coding variants of possible functional signiicance.59 These variants conirm, as expected, that as with other genes, the FMR1 gene might also be affected by mutations other than the typical triplet repeats. Guidelines for FXMR molecular diagnosis have been published and are summarised in Table 2. Some guidelines propose clinical best practices for indications for referral or testing, while others propose laboratory best practices for actually performing the molecular diagnostic testing, detecting mutations and reporting the results. FXMR molecular diagnosis is methodologically challenging for several reasons, including the heterogeneity of full mutations in somatic cells (including blood cells), mosaicism in expansion size and methylation, the presence of a normal X chromosome and X-inactivation in females, expansions that are refractory to PCR, and the incomplete/absent methylation found in certain prenatal samples.117 Molecular diagnosis of FXMR is thus much more complex than regular point mutation or deletion analysis. Due to the very high level of complexity, a laboratory with speciic expertise in FXMR testing and in all the atypical modes of presentation of FMR1 mutations is an important asset for high quality results. Indications for Molecular Diagnosis Reported indications for testing include molecular conirmation of diagnosis, carrier diagnosis and prenatal diagnosis. The clinical situations where it is good practice to suspect FXMR are highlighted in Table 3. The clinical presentation of the disease is highly variable and therefore the relevance of FMR1 testing relies strongly on the expertise of the geneticist evaluating each case. With respect to the rate of positive results among samples referred for FMR1 genotyping, due to the large array of non-speciic and rather frequent symptoms that can lead to fragile X testing, there is a low pick-up rate; usually <1% of male samples referred for testing will have a full mutation (Clinical Molecular Genetics Society, Practice guidelines, 2005; Table 2). If a more pronounced degree of MR is used as a testing criterion (as opposed to borderline-mild MR), or with the use of clinical checklists, this yield can increase to 4% as recently reported in a systematic review.24 In females, the yield is lower, and as low as 0.3%. The latter review recommends that molecular investigations for FXMR be performed in all boys with MR. In this review, the two checklist items that seemed to be the most useful in children were the presence of a family history of MR and absence of microcephaly. The authors also suggest that studies in girls should not be performed routinely and only in the presence of positive clues such as a family history of MR. In our recent experience, between January 2000 and August 2010, our laboratory received a total of 5892 samples for FMR1 testing. Of these, the number of samples with a positive result was 150 (2.6%), comprised of 94 (1.6%) premutations, 39 (0.67%) full mutations, 11 (0.19%) size mosaics (premutation + full mutation) and 6 (0.1%) methylation mosaics. Fortyseven samples were of quality or quantity insuficient for analysis. The median age at diagnosis of a full mutation was 4 years for males (n=38) and 17 years for females (n=18). The median age of identiication of premutation female carriers was 32 years (n=78), i.e. well into the reproductive period of their lives, and clearly not early enough to offer them timely genetic counselling considering their high risk of having a child affected by FXMR. A very large series of 119,232 FXMR testing requests in the US was published in 2007 by Strom and colleagues.118 The overall rate of positive results was 1.3%, with 1.4% of males and 0.61% of females displaying full mutation genotypes, while 1.7% of tested females carried a premutation. The same series included 307 prenatal FXMR tests which revealed that the risk of expansion to a full mutation was 5% between 50 and 75 triplets, 30% between 76 and 100 triplets and 100% for >100 triplets. Clin Biochem Rev Vol 32 August 2011 I 141 Guidelines Description Reference Reference for Superseded Versions Carrier Screening for Fragile X Syndrome. American Congress of Obstetricians and Gynecologists (ACOG), Committee Opinion No. 469, October 2010. Recommendations regarding testing for fragile X syndrome. 203 226 Signiicance of tri-nucleotide repeats. A clariication of the American College of Medical Genetics guidelines. 208 Practice guideline of the Society of Obstetricians and Gynecologists of Canada and the Canadien College of Medical Geneticists, Committee Opinion No. 216. American College of Medical Genetics (ACMG): Technical standards and guidelines for Fragile X testing: a revision. European Molecular Genetics Quality Network (EMQN): Draft best practice guidelines for molecular analysis in Fragile X syndrome. Recommendations regarding screening for fragile X in the obstetrical and gynaecological population in Canada. 206 Educational resource for clinical laboratory geneticists. 227 Standards for the performance and interpretation of genetic tests and reporting. 228 ACMG Practice Guideline: Diagnostic and carrier testing. Recommendations and general guidelines to aid clinicians in making referrals for diagnostic and carrier testing for fragile X syndrome. 214 Clinical Molecular Genetics Society (CMGS): Practice guideline for molecular diagnosis of Fragile X syndrome. Recommendations regarding referral categories and molecular diagnosis of fragile X syndrome. 229 Genetic counseling for Fragile X syndrome: Updated recommendations of the National Society of Genetic Counselors Review of the molecular genetics of fragile X syndrome, clinical phenotype, indications for genetic testing and interpretation of results, risks of transmission, family planning options, psychosocial issues, and references for professional and patient resources. 210 Consensus Conference of ACMG: Guidelines regarding the evaluation of patients with mental retardation. Guidelines for fragile X testing in patients with mental retardation. 207 American Academy of Pediatrics (AAP) Committee on Genetics: Health supervision for children with Fragile X syndrome. Guidelines for paediatricians caring for children with molecular conirmation of fragile X and advice for pregnant women with a prenatal diagnosis of fragile X syndrome. 205 Guidelines for genetic counsellors. Development of an approach to inform relatives in fragile X families about genetic risk. 211 Guidelines for the diagnosis of Fragile X syndrome. Presentation of strategies that can be applied for postnatal and prenatal diagnosis of fragile X syndrome. It contains different probes and enzymes sourced from numerous publications. 213 209 204 212 Rousseau F et al. 142 I Clin Biochem Rev Vol 32 August 2011 Table 2. Guidelines for testing for Fragile X syndrome. Fragile X Syndrome: 20 Years On Table 3. Indications for referring for fragile X mental retardation testing. Development delay Learning/behavioural dificulties Speech delay Autistic features Asperger syndrome Attention deicit disorder/attention deicit hyperactivity disorder (ADD/ADHD) Social dysfunction Poor eye contact Challenging behaviour Large head Hand lapping/biting Dysmorphic facies Carrier detection Prenatal diagnosis Premature ovarian failure (especially if familial) A recent study on the timeliness of diagnosis for FXMR in the US concluded that despite patient advocacy, recommendations for prompt referrals, and increased exposure to FXMR in the literature, there was no change in age at diagnosis between 2001 and 2007, suggesting that earlier identiication will remain a challenge unless screening is envisaged.119 The high prevalence of the disease and of premutation carriers in the general population has prompted discussions on the relevance of establishing screening programs for FXMR (see below). Recent data on transmission of FMR1 premutations in the general population suggest that a signiicant proportion of such alleles are at risk of expansion to a full mutation.120-122 This is discussed in more detail in the section on screening for FXMR. These studies on transmission suggest that FMR1 premutations from the general population (i.e. from those without a family history) have a similar risk of expansion with respect to their size as in fragile X families, thus these data would not support the need to wait for further research to evaluate whether such alleles are stable or not in unaffected families. One other condition that is part of the differential diagnosis of FXMR is the MR syndrome associated with dynamic mutations in the FRAXE gene called FMR2.123 FRAXE resembles FXMR but is rarer and has been much less studied than FXMR. Although a thorough discussion of this syndrome is beyond the topic of the present review, it is worth mentioning that these mutations can be detected using the same Southern blots as are used in investigations of FXMR (EcoRI + EagI), albeit using a different probe (OxE18). They also have a presentation similar to FMR1 mutations, i.e. with the same normal size range, premutation size range and full mutation sizes associated with abnormal methylation on the active X chromosome.123 The phenotype is believed to be less severe than FXMR. FMR1 Molecular Diagnostic Methods Although no systematic review has been published recently on the methods used for FMR1 genotyping, it appears that Southern blotting is still the most frequently used method, with the support of PCR-based methods to discriminate between small premutations (ca. 55–65 triplets) and large intermediate size alleles (45–54 triplets) (European Molecular Genetics Quality Network (EMQN), Table 2). Many other tools, mostly PCR-based, have been proposed (see below), some of which are also FDA-approved. There is, however, a lack of large studies comparing head-to-head the analytical and clinical performances of the various FMR1 genotyping methods available for all types of the more challenging FMR1 genotypes. The Gold Standard – Southern Blotting Southern blotting, a technique where genomic DNA is digested with speciic restriction enzymes and hybridised to a gene-speciic probe, has the advantage of being able, in a single experiment, to determine both the size and methylation status of FMR1 mutations (Figure 3). However, the method is labour-intensive and can take several days to yield results. It is also not always possible to clearly discriminate small premutations (55–65 triplets) from large intermediate size alleles (45–54 repeats). In such cases, a PCR-based assay is frequently used, since it can amplify small mutations and be loaded on a high resolution gel electrophoresis platform (capillary or traditional). A good 20 cm EcoRI-EagI Southern blot will usually have a resolution of 45 bp (or 15 triplet repeats) in the range of the 2.8 kb band, which provides excellent sensitivity even for small premutations of 55 triplet repeats, in the presence of a normal control (30 triplet repeats) in an adjacent position. Southern blotting using unampliied genomic DNA also has the advantage of providing a reliable estimate of the size distribution of FMR1 alleles in the sample, although it cannot precisely determine the size of mutations. The routine use of Southern blotting has been challenged,117 (EMQN, Table 2) and this type of analysis should be considered of very high complexity, requiring considerable experience in the laboratory in order to detect irregular types of mutations such as very heterogeneous smears, or mosaicism that includes both normal size alleles and full mutations (see below). Of note, the ‘irregular’ genotypes are also challenging for PCR-based methods. Nevertheless, Southern blotting is still considered by the vast majority of referral laboratories as the gold standard of FMR1 molecular diagnostic methods,124 Clin Biochem Rev Vol 32 August 2011 I 143 Rousseau F et al. Figure 3. Different FMR1 genotypes detected on EcoRI+EagI Southern blots of genomic DNA using probe StB12.3 (A to J). Fine size-analysis by PCR of the triplet array (K). Normal males typically show a unique 2.8 kb band (arrow in A3,4,10,13,14,E4,H1,3), and occasionally an additional fainter band resulting from partial EcoRI digest (A9,11) or EagI digest (A20). Normal females typically show two bands representing the active (2.8 kb arrow) and inactive (5.2 kb arrow head) X chromosomes which are very useful as controls for normal size unmethylated and methylated alleles (A2,7,8,12,B1,3,C1,D1,3,E1,F2,G1,I1,4,J3). Premutationcarrier males will have no band at 2.8 kb but one migrating more slowly (A16). Premutation-carrier females will typically show four distinct bands, two in the unmethylated range (2.8 to ~3.4 kb) and two in the methylated range (5.2 to ~5.8 kb) (A21). The two methylated bands may be close enough to sometimes show a merged signal in overexposed blots (A1,15,17,18,19,22,I3), or with small premutations (J2). Full mutations can appear as one or more homogeneous bands (A5,D2,F1,I2) or as a heterogeneous pattern that can be a faint smear (J1,4). In males, typical full mutations migrate above the 5.2 kb size marker, are associated with the absence of signal below this size, and may be homogeneous (A5) or heterogeneous (upper part of E3 and H2). In females, full mutations are accompanied by the signal of the normal FMR1 allele present in each cell, that is randomly divided between the active and inactive X-chromosomes. Full mutations in females (A6,F1,G2,J1) can be easily missed if they are very heterogeneous and the blot is not exposed suficiently. In women, if close to 100% of the cells analysed have their mutated FMR1 allele on either the active or inactive X-chromosome (I2), then the pattern observed will have only two bands. Small premutations may also be missed in this context (not shown) if a normal female sample is not next to the sample being tested (we tend to group females together on a Southern blot). More complex patterns are observed with so-called mosaic patterns. In males, it is possible to observe size-mosaics with a premutation in some cells and a full mutation in others (B2,E3,H2) or, more rarely, with a normal allele in some cells and a full mutation in others (D2). In females, size-mosaics (premutation + full mutation) will show extra bands (above 5.2 kb) compared to those expected from a premutation-carrier (C2). Finally, methylation-mosaic patterns are characterised by the presence of mutations of full-mutation size (i.e. >~600 bp more than the 2.8kb or 5.2kb bands) both in the methylated (above 5.2kb) and unmethylated (2.8 to 5.1) ranges (I5, J4). These can be observed in males, females and also in chorionic villi prenatal samples (where methylation is incomplete, I5, J4). Note that J4 is a chorionic villi sample of a female foetus with a very heterogeneous and unmethylated large (i.e. full) mutation. Panel K shows PCR and high-resolution acrylamide gel analysis of FMR1 alleles in the range between normal alleles and premutations, which are more dificult to resolve on Southern blot. The number of repeats in the expansion is shown at the right of the panel. and can be used to detect all types of mutations and complex infrequent patterns such as those discussed below. In terms of analytical sensitivity and speciicity, a high quality Southern blot analysis (without signiicant background and with strong signal and appropriate quality control) analysing both size and methylation status of the FXMR locus is believed to have 144 I Clin Biochem Rev Vol 32 August 2011 >98% sensitivity and close to 100% speciicity for the FMR1 dynamic mutations causing this disease.11 PCR-based FMR1 Genotyping Methods Due to its technical simplicity and rapidity, PCR is a preferred method for molecular diagnostics in general. However, Fragile X Syndrome: 20 Years On because FMR1 mutations are very GC-rich (most of the time showing very long stretches of >200 virtually pure CGG repeats), they are very dificult to amplify using PCR. Further, in the presence of mutations of highly heterogeneous sizes, PCR will favour ampliication of shorter alleles, and thus produce a biased, unreliable assessment of the size distribution of mutations. Considerable efforts have been invested in developing PCR methods able to compete with the sensitivity and speciicity of Southern blotting, and even to determine the methylation status of FMR1 mutations (see below). These methods do not seem to have been widely adopted for the moment (EMQN, Table 2), although important efforts in marketing PCR assays have led to the commercialisation of a number of diagnostic kits (e.g. by Quest Diagnostics, Celera Genomics, Abbott and Assuragen). A number of laboratories will use a PCR-based assay as a irst-pass and perform a Southern blot analysis only for samples with an observed or suspected allele >~50 repeats.118 Reliable estimates of the sensitivity of these approaches for detecting each type of FMR1 abnormal genotype are yet to be published. Thus there is no reliable estimate of the proportion of full mutations (especially mosaic cases) that could be missed by PCR-based approaches used as a front line test. Complexity in FMR1 Genotyping Although triplet repeat expansion in the 5’ UTR of the FMR1 gene is the main type of FXMR mutation which is expected in most cases of FXMR, the expansions can take many forms in any given individual (Figure 3), and there are speciic types of mutation patterns that the laboratory must seek and keep in mind in order to minimise the false negative rate of the assay used. Guidelines have recommended that clinical laboratories performing FMR1 genotyping be very well acquainted with the assay they are using and with all the various mutation patterns that can be observed, even if these are observed rarely (EMQN, Table 2). Complexity in FMR1 genotyping includes the following phenomena: somatic heterogeneity (mosaicism) in the size of the CGGrich triplet expansions; presence of abnormal methylation of the FMR1 gene; somatic heterogeneity (mosaicism) in the methylation pattern; incomplete methylation patterns in prenatal samples; interference by the normal X-chromosome in women; dificulty in PCR assays (as mentioned above); and sensitivity of both Southern blot and PCR analyses, especially in samples from females. Mosaicism in Mutation Size FMR1 full mutations are notoriously heterogeneous in nature and have been described as such since their identiication in 1991.6,11 It seems that a signiicant proportion of carriers of full mutations show some degree of mosaicism.14,125 Given that FMR1 full mutations are always inherited from the mother (from either a premutation or a full mutation), the zygote is believed to inherit an expanded FMR1 allele. Thus, the mutation heterogeneity observed in tissues of individuals with full mutations is thought to be due to somatic instability.126 It has been reported that ethidium bromide used in Southern Blot gels may, by causing sheering of DNA, artifactually increase the apparent heterogeneity of FMR1 mutations.127 However, the heterogeneity is still observed even when staining techniques other than ethidium bromide are used (unpublished data), and thus this possibility remains unconirmed. Even in a male with a full mutation genotype, different cells may harbour different mutation sizes, ranging from a normal allele with 20–30 repeats up to very large expansions of >700 triplets.11,17,125 This, of course, poses a challenge for genotyping methods, and especially for those that rely on PCR of the expanded sequences, which are likely to generate more amplicons from the smaller size alleles than from the very large ones. Although Southern blotting is more powerful for detection of mosaicism, the cost of the procedure is higher than PCR-based testing for routine diagnosis, although this does depend somewhat on the costing model. For example, in the Quebec public health care setting, the cost of one Southern blot-based test can be as low as US$75 compared to US$450– 750 in the US (The National Fragile X Foundation). In such a context, clinical laboratories may elect to use Southern blotting as their standard test to detect FMR1 triplet repeat expansions. Full mutations rarely show a homogeneous somatic pattern, and on most occasions will be detected as smears on a Southern blot.11 Premutations are more stable and appear as single bands on a Southern blot. However, the reverse is not true, as implied previously; that is, a signiicant proportion of individuals with full mutations will also display premutations in some cells (about 20% of samples with a full mutation in our population as detected by Southern blotting). The proportion of cells showing premutations can be as high as 50% with the rest harbouring a full mutation. One of the most challenging situations is when a signiicant proportion of cells harbour a normal size allele in a male while the rest have a very heterogeneous set of full mutations (Figure 3). The other challenging situation for Southern blotting is when a woman carrying a premutation has all of her premutated alleles on the inactive X chromosome, producing an apparently normal band pattern with only two bands (normal allele on the active X and premutated allele on the inactive X). In those cases, if the premutation is small, it may be dificult to distinguish from a normal allele on the Southern blot using EcoRI/EagI or HindIII/EagI restriction enzymes. Clin Biochem Rev Vol 32 August 2011 I 145 Rousseau F et al. Methylation Mosaicism In addition to size heterogeneity, full mutations will occasionally show heterogeneity in the methylation pattern with large unmethylated expansions in a fraction of the genomic fragments analysed.14,17 These are termed ‘methylation mosaics’ (as opposed to size mosaics) and they represent about 10% of samples with a full mutation in our population as detected by Southern blotting. However, due to the fact that all the alleles are >200 triplet repeats, they are still usually readily distinguished from premutations or normal alleles if the method distinguishes methylated from unmethylated alleles. It has been reported that when a signiicant proportion of the alleles in the analysed leukocytes is unmethylated, even for large alleles, the phenotype may be much less severe.17,128-130 Prenatal Diagnosis Prenatal diagnosis of FXMR using cytogenetics was in place prior to discovery of the gene but had important limitations in terms of sensitivity, especially for female foetuses. In the prenatal context, DNA-based molecular assays can be performed on chorionic villi or cultured amniocytes, and are much more reliable.11 As mentioned earlier, the risk of expansion of a premutation into a full mutation in a single generation depends on the size of the premutation, and varies between 5–10% for small premutation alleles (<75 triplets) and 100% for premutations of ≥100 triplets. These igures are used in genetic counselling with at-risk couples to weigh their decision with respect to invasive prenatal diagnosis. Chorionic villus samples will frequently present with an unestablished methylation pattern where the expected signal of the methylated inactive normal X-chromosome is incomplete or absent.11,130 In those cases, full FMR1 mutations are also not necessarily methylated, and the laboratory cannot therefore obtain information about an important criterion for full mutations: abnormal methylation. In such cases where methylation is not well established and the size of FMR1 expansion is in the large premutation or small full mutation range, it may be necessary to recommend the sampling, culture and analysis of amniocytes because methylation is established by the time amniocentesis is usually performed. New Developments in Laboratory Tests The limitations and challenges posed by current molecular diagnostic methods for FXMR have led to the development of many alternative approaches, with the aim of simplifying or automating the identiication of either FXMR cases (inactivated FMR1 gene lacking FMRP), or unaffected carriers (premutations). These novel approaches are numerous and we will present only an overview here with the intent of showing possible alternative avenues for FXMR diagnosis or screening in the future. Note however that many of the new tests have 146 I Clin Biochem Rev Vol 32 August 2011 not been thoroughly evaluated and compared with the current gold standard method, and as such have not yet been adopted by many clinical laboratories. This is particularly so in the context of samples showing challenging mutation proiles (such as mosaics with a normal allele and a full mutation, or premutation and full mutation mosaics), and their diagnostic performance in such cases remains unclear. Immunocytochemistry The availability of well-characterised anti-FMRP antibodies has raised the possibility of using immunohistochemistry assays of blood smears (or hair roots), with the diagnosis of FXMR based on the proportion of FMRP-positive cells.131-134 More recently, a quantitative assay for FMRP has been proposed,135 but FMRP levels in whole blood are below the sensitivity of the assay, so it remains a research tool, for example for molecular biology experiments where larger quantities of FMRP may be encountered. Better PCR Assays and PCR/MS Considerable efforts have also been placed in developing PCR assays with a better yield for FMR1 alleles with a larger number of triplet repeats.136,137 PCR assays have also been combined with novel means of detection such as mass spectrometry.138 PCR Combined with Capillary Southern Blotting New methodological algorithms have been proposed such as PCR followed by capillary Southern blotting.139 Triplet-Primed PCR Methods showing high promise due to their sensitivity for large expansions involve triplet-primed PCR assays. Different variations of this approach have been proposed.140-142 However, these methods do not allow determination of FMR1 methylation patterns. Methylation-Speciic PCR Methylation-speciic PCR methods for methylated FMR1 alleles such as bisulite-based PCR have been proposed.121,143-147 One such method has been used on >36,000 de-identiied newborn samples isolated from dried blood spots. It appears to have high sensitivity and speciicity for males with full FMR1 mutations that are abnormally methylated. MALDI-TOF Mass Spectrometry Recently, a matrix-assisted laser desorption/ionisation-time of light mass spectrometry (MALDI-TOF MS) assay was described that identiied two so-called ‘fragile X-related epigenetic elements’ showing a high degree of correlation with the methylation status of the FMR1 CpG island as measured by classical diagnostic assays.148 The authors propose that the Fragile X Syndrome: 20 Years On high throughput, low cost and speciicity of this MS assay for methylated FMR1 alleles make it an interesting tool for FXMR diagnosis and population screening. Pre-implantation Diagnostic Methods For pre-implantation diagnosis, single-cell assays need to be developed and implemented. PCR-based assays of this type have been proposed.149,150 Reference Materials Traceability of biological analyses requires the availability of reference materials with well-deined properties.151 There are different categories of reference materials, with various levels of validation with respect to properties such as stability, homogeneity and commutability (i.e. validity on several different diagnostic technical platforms). For FXMR, reference materials such as the fragile X (FMR1) control produced by the National Institute of Standards and Technology (NIST; SRM 2399) are available. Another reference material consists of a panel of 5 genomic DNA samples endorsed by the European Society of Human Genetics and approved as an International Standard by the Expert Committee on Biological Standardization at the WHO.152 Validation of FMR1 genotypes in reference materials requires large-scale characterisation of candidate materials.153 Genotype-Phenotype Correlations Genotype-phenotype correlation studies have been published since the cloning of the gene.14,154-156 The disorder associated with an FMR1 full mutation is FXMR. As described above, FXMR can be observed at different levels of severity, especially regarding the degree of intellectual disability, which is recognised as an IQ of 40–70 in males, whereas females are usually less severely affected; even so, they are not unaffected and female carriers of premutations and full mutations have been reported to suffer more frequently from social phobia than a control group of mothers of autistic children.154 Study of FMR1 mutation characteristics and palmar and inger epidermal ridge measures have shown that the size of FMR1 mutations is correlated with the thumb ridge count (in males) and breadth (males and females), but not with FMRP levels.156 A small proportion of males with a full mutation genotype are so-called ‘high-functioning males’.129,130,157 They present with a full mutation genotype but have IQs >70 and can manage simple everyday tasks. This is in contrast with classical FXMR males who show a more signiicant degree of MR. It appears that this high-functioning capacity is frequently associated with the presence of a lower degree of methylation than might be expected for full mutations and therefore associated with some residual expression of the FMRP protein. But in general, it has not been possible thus far to ind any markers that are highly predictive of MR for individuals with a full mutation. FMR1 Premutation Disorders Since the discovery of the association between the FMR1 gene and FXMR, two syndromes speciic to FMR1 premutations in the absence of FXMR-related phenotypes have been described, namely fragile X-related premature ovarian insuficiency (FXPOI) and fragile X tremor and ataxia syndrome (FXTAS). As these syndromes appear later in life than FXMR, their existence introduces an element of predictive testing into any prenatal or postnatal testing for FXMR in fragile X families. Fragile X-Related Premature Ovarian Insuficiency FXPOI is deined as menopause occurring prior to the age of 40 years,158 while ‘early menopause’ is deined as menopause occurring prior to the age of 45 years. Female carriers of the FMR1 premutation (55–200 CGG repeats) are at increased risk for FXPOI, with a relative risk of 21 compared to the general population,159 as well as early menopause and ovarian dysfunction and decreased fertility in general.160 Conversely, amongst women with idiopathic sporadic premature ovarian failure syndrome, or its rarer familial form, approximately 2–14% are estimated to carry a FMR1 premutation.159 Carriers of the full mutation do not appear to be at risk for these conditions. FXPOI has not been observed in carriers of alleles smaller than 59 triplet repeats. Fragile X-Associated Tremor and Ataxia Syndrome FXTAS is a late onset neurodegenerative disorder, usually affecting males over 50 years of age.161-163 The clinical presentation of FXTAS is heterogeneous, and usually in the seventh decade with intention tremor, cerebellar gait ataxia, and frontal executive dysfunction sometimes associated with some speciic radiologic and neuropathological signs.163,164 Females comprise only a small part of the FXTAS population and their symptoms tend to be less severe and can be different from those of men.163 FXTAS affects the neurological system and progresses at variable rates among affected individuals. The pathogenic basis of FXTAS is, paradoxically, postulated to be overexpression of the expanded CGG repeat of the FMR1 gene, rather than gene silencing,165 but the precise mechanism remains unclear. All individuals with FXTAS are carriers of FMR1 gene premutations. The syndrome has not been observed in carriers of alleles smaller than 59 triplet repeats. Other FMR1 Premutation-Associated Syndromes It has been reported that small expansions of the FMR1 gene (including alleles between 40 and 54 triplets as well as premutations) were enriched in patients with idiopathic Parkinson’s disease and other causes of parkinsonism, and thus Clin Biochem Rev Vol 32 August 2011 I 147 Rousseau F et al. might be associated with parkinsonism.166,167 These indings have not been reproduced consistently.168,169 Conirmation of this association therefore awaits further studies. Treatment Several therapeutic avenues for FXMR have been tested on small series of patients, with unconvincing results.170,171 These include folic acid supplementation, dextroamphetamine, methylphenidate, and L-acetylcarnitine. A recent systematic review of pharmacological therapeutic approaches for FXMR concluded that there is no robust evidence to support recommendations for pharmacological treatments in patients with FXMR in general, or in those with an additional diagnosis of attention deicit hyperactivity disorder or autism.171 Much hope has been placed in a better understanding of the dysfunctional cellular mechanisms involved in FXMR to generate novel therapeutic avenues.172 Work on neuronal plasticity of FMR1-rescued KO mice has prompted some researchers to suggest that the neurological and cognitive deicits of individuals born with an inactive FMR1 gene may be partly reversible.173 There is also a need for further studies on the effectiveness of behavioural and social interventions for patients affected with FXMR.170 New compounds are being tested for treating FXMR based on knowledge derived from pathophysiology studies as well as from experiments on mouse models of FXMR. Some are in phase II trials, such as a single-isomer version of baclofen, an already approved muscle relaxant. There are other drug candidates as well.171 High quality clinical controlled trials will be needed to provide strong evidence of beneit and lead to recommendations for FXMR treatment. Also, reports on such clinical trials should follow the Consolidated Standards of Reporting Trials (CONSORT) recommendations.174 Finally, the future development of FXMR treatments of demonstrable effectiveness will be an essential component of any potential strategy for neonatal population screening for FXMR. Population Prevalence and Screening Mutation Prevalence Given the high prevalence of FXMR in many different countries and populations, researchers have sought to determine the prevalence of FXMR among children from a variety of backgrounds including those with MR, but also in those with special education needs (SEN), as well as the asymptomatic general population. Tables 4 and 5 present the largest studies (with >1000 individuals) published thus far for these different population types. When considering males with SEN only, the combined prevalence of full mutations from the three studies (Table 4) was 30/7380 (or 1/246 (0.4%), 95% CI: 1/181–1/383),175-177 148 I Clin Biochem Rev Vol 32 August 2011 which is more than 17-fold higher (Chi square (1df) = 206, p<0.00001) than that reported by large studies of unselected newborns from the general population (see next paragraph). Frequency of full mutations in males with SEN is about seven-fold lower than the high frequency of full mutations in probands from families with no family history of FXMR referred for FMR1 genotyping as reported by a large national study in France by Biancalana et al. (606/20,816 or 1/34; 95% CI: 1/31.8–1/37.3).178 The prevalence of full mutations in the general population, as well as that of premutations in women of childbearing age, have been the subjects of several studies. Table 5 summarises the large studies (>1000 population-based unselected samples) that have been published thus far. Eight studies (Table 5, studies 1-4, 9, 12, 15, 17 ) provide estimates of the incidence of full mutations in consecutive newborns from the general population. The incidence of FMR1 full mutations was 23 in the equivalent of a total of 98,088 consecutive newborns, representing an incidence of 1/4264 in newborns (95% CI: 1/3027–1/7211). Five studies (Table 5, studies 3, 5, 8, 11, 17) provide an estimate of the prevalence of FMR1 premutation carriers amongst pregnant women yielding an overall estimate of 294/62,695 (or 1/213, 95% CI: 1/191–1/241). One of the studies, from Israel, showed a much higher prevalence.120 If this study is removed from the combined results, then the prevalence of premutation carrier females is 63/26,212 (or 1/416, 95% CI: 1/333–1/552). Finally, three studies (Table 5, studies 13, 15, 18) have examined unrelated and unselected men or women from the general population to estimate the prevalence of FMR1 mutations. As some methods used, such as PCR, may not be suitable to detect full mutations with a high degree of sensitivity, we will only include the results for premutations hereafter. This data can be merged with the prevalence of FMR1 premutations in pregnant women to compute an allele frequency of FMR1 premutations per X-chromosome tested. Globally, there were 475 premutations out of 185,878 independent X-chromosomes tested, or an allele frequency of 1/391 (95% CI: 1/359–1/430). If the studies conducted in Israel are removed, the igures are lower, with a premutation allele frequency of 117/84,244 (or 1/720, 95% CI: 1/609–1/879). Given the relatively high prevalence of FMR1 premutations in the general population, one of the main questions was the degree of instability of premutation size alleles identiied by screening the general population compared to that observed in fragile X families. A few studies have taken a direct approach to this question by screening for mutations in pregnant women and testing the FMR1 genotype in their offspring.62,120,122,179 Careful analysis of the exact number of transmissions of FMR1 alleles of 55 or more triplets, as Study Region Sample Size and Characteristics Full Mutations Mosaic Premutation Prevalence and/or 95% CI 1176 Tasmania, Australia a) 1,253 children (M) with SEN 0 0 0 b) 578 normal newborns (M) 0 0 0 Premutation 0 (CI: 0-1/417) for SEN 0 (CI: 0-1/192) for normal newborns US a) 1,618 (M) and 694 (F) Caucasian children with SEN 0 (M), 4 (F) NA 0 (M), 2 (F)† b) 777 (M) and 326 (F) AfricanAmerican children with SEN 3 (M), 0 (F) 0 (M), 0 (F)† c) Other ethnicities: 155 (M), 69 (F) 0 (M), 0 (F) 0 (M), 0 (F)† a) 3,732 subjects with SEN selected from 135,798 (M) from the general poulation aged 5-18 y. 20, including 2 methylation mosaics 3 2* b) 2,932 mothers of SEN subjects from a) above. 0 0 1* 2175 3177 England Premutation M: 0/2,471 (CI: 0-1/1,783) in SEN and general population F: 1/531 (CI: 1/3,058-1/132) in SEN and general population Full mutation M: 1/353 (CI: 1/805-1/164) in SEN and 1/3,623 (CI: 1/6,024-1/2,212) in general population F: 0/1,061 (CI: 0-1/765) in SEN population Full mutation: 1/162 (CI: 1/243-1/108) Premutation (in control X chromosomes): 1/2,932 (CI: 1/16,954-1/517) Clin Biochem Rev Vol 32 August 2011 I 149 4178 France a) 14,867 (M) and 5,949 (F) with MR and referred for FXMR testing. 417 (M), 60 (F) NA NA Full mutation: M: 1/36 (CI: 1/39-1/32) F: 1/99 (CI: 1/127-1/77) 5215 Canada 912 (M) and 638 (F) DD subjects 2,073 unselected subjects of both genders NA NA 5 (M), 1 (F) ¶ 1¶ Cyprus 314 (M) and 236 (F) DD subjects 112 unaffected subjects (betathalassemia samples) Full mutation NA Premutation DD M: 1/182 (1/430-1/78) DD F: 1/638 (1/3,984-1/112) Unselected (Canada only): 1/2,073 (1/12,090-1/366) 0 (M), 1 (F) ¶ 7¶ SEN = special educational needs; DD = developmental disabilities; CI = 95% conidence interval; NA = not applicable or no data. Premutation deinition used: * 61-200 CCG, † 61-199 CCG, ¶ 55-200 CCG. Fragile X Syndrome: 20 Years On Table 4. Prevalence of FMR1 mutations in children with special educational needs, mentally retarded probands and individuals with developmental disabilities. Study Gender Region and/or Ethnicity Sample Source Sample Size Full Mutations Mosaics Premutations Prevalence (95% CI) 1219 Male Castile and Leon, Spain Consecutive newborns 5,267 2 NA 21† Premutation 1/251 (1/164-1/385) Full mutation 1/2,633 (1/714-1/10,000) 2121 Male Georgia, US Consecutive newborns 36,124 4 NA NA Caucasian M: 1/4,063 (1/10,477-1/1,580) African-American M: 1/5,490 (1/20,017-1/1,506) Hispanic M: 1/5,396 (1/30,567-1/953) Caucasian (45%) African American (30%) Hispanic (15%) Other (8%) 2 1 0 Estimate for the general population of 36,124 (M) 1/5,161 M (1/10,653-1/2,500) Female Quebec City, Canada Newborns, (M and F) Mainly FrenchCanadian 4224 Male 5120 Female 3122 Foetuses (gender not speciied) 21,411 0 24,449** 2 (M) North Carolina, US Screening test on newborns offered after delivery 1,459 2 NA 2† Full mutation: 1/730 (1/4,500-1/129) Israel Pregnant women, no known family history of FXMR, or suspected potential carriers 40,079 (36,483 with no family history + 3,596 potential carriers) 5 (1 with no family history + 3 in potential carriers) 1 (potential carrier) 255¶ (231with no family history + 24 potential carriers) Carrier frequencies for 36,483 F with no family history: 1/157 Premutation: 1/158 Full mutation: 1/36,000 370 30 NA Prenatal diagnosis offered to women carrying a premutation or a full mutation NA Premutation F: 1/549 (1/417-1/800). For 24,449 newborns, prevalence of fragile X syndrome is 1/12,225 (upper CI: 1/4,638) Estimated full mutation: M: 1/6,209 (upper CI: 1/1,718) F: upper CI 1/3,390 Matched mother-newborn sample pairs for unselected deliveries 39§ 23§ 163¶ Carrier frequency for 3,596 potential carriers: 1/128 Premutation: 1/150 Full mutation: 1/899 Rousseau F et al. 150 I Clin Biochem Rev Vol 32 August 2011 Table 5. Prevalence of FMR1 mutations in the general population. Male California, US Postnatal and prenatal testing Male Worldwide Female 333 x 862 59,525 364 1008 x Foetuses: 307 Foetuses: 15 (M), 7 (F) Foetuses: 17 (M), 16 (F) Meta-analysis of studies involving individuals with MD and individuals from the general population MD: 3,265 NA Female 7222* NA 59,707 NA POP: 50,576 MD: 0-5% NA NA POP: 186 (0.4%) Clin Biochem Rev Vol 32 August 2011 I 151 8217 Female US Pregnant women and women wishing to be pregnant, with no known fragile X history 2,292 NA NA F: 6† Premutation 1/382 (1/836-1/175) 9225 Male Taiwan Consecutive newborns 10,046 1 NA M: 6† Premutation 1/1,674 (1/3,656-1/767) Full mutation: 1/10,046 (1/1,773-1/57,250) 10230 Male US Economic evaluation combining data from 15 studies NA NA NA Female Full mutation M: 1/3,847 (1/2,263-1/12,830) F: 1/2,364 (1/1,195-1/115,473) Premutation M: 1/809 (1/531-1/1,698) F: 1/104 (1/96-1/112) 11221 Female Taiwan Pregnant women tested randomly 1,002 0 NA 0 0/1,002 (0-1/61) 12223 Male Catalonia, Spain Consecutive newborns 4,932 2 NA 4†† Premutation 1/1,233 (1/4,525-1/482) Full mutation 1/2,469 (1/20,408-1/684) 13218 Male Mostly FrenchCanadian from Quebec City, Canada Consecutive individuals who were submitted to a blood test 10,572 NA NA 13§§ Premutation 1/813 (1/527-1/1,781) Fragile X Syndrome: 20 Years On 6118 Gender Region and/or Ethnicity Sample Source Sample Size Full Mutations Mosaics Premutations Prevalence (95% CI) 14216 Male Caucasians Compilation of studies including selected ‘at risk’ or clinically diagnosed groups, and consecutive individuals Several thousand individuals tested, results extrapolated to the general Caucasian population 1/3,7171/8,918 (M) NA 1/1,000†§ NA Healthy preconceptional or pregnant women with no familial history of mental retardation 14,334 3 Foetuses of carriers 177 5 Women referred for fragile X screening 9,660 0 Foetuses of fragile X carriers 74 5 Pregnant women with no family history of FXMR 1,477 0 0 18¶¶ Foetuses of carriers with a premutation or full mutation 24 1 1 1¶¶ Consecutive individuals who were submitted to a blood test 10,624 NA NA 41## Female 1562 Female Various ethnic groups from Israel Foetuses (gender not speciied) 16220 Female Tel Aviv, Israel Foetuses (gender not speciied) 17179 Female Kuopio Hospital, Finland Foetuses (gender not speciied) 1861 Female Quebec City, Canada Mainly FrenchCanadian 1/246 to 1/468†§ NA 127# Premutation: 1/113 85# NA 85¶¶ Premutation carriers 50 CGG and more: 1/114 55 CGG and more: 1/159 39 ¶¶ (including 4 with expansion) NA Premutation 1/259 (1/373-1/198) M = male; F = female; FXMR = fragile X mental retardation syndrome; MD = movement disorders; POP = general population; CI = 95% conidence interval; NA = not applicable or no data. * Meta-analysis of 14 genetic screens, including 5 for the population data only. ** Not all tested, only tested if mother >50 repeats. Premutation deinition used: † 55-200 CGG; § 55 CGG or more; ¶ 55-199 CGG; x 56-200 CGG; †† 53-200 CGG; §§ 54-200 CGG; ¶¶ 50 CGG or more; †§ 61-200 CGG; # 51-200 CGG; ## 55-230 CGG. Rousseau F et al. 152 I Clin Biochem Rev Vol 32 August 2011 Study Fragile X Syndrome: 20 Years On identiied by screening of the general population, reveals that 26 out of 248 such alleles expanded to a full mutation when transmitted by a carrier woman. This corresponds to a rate of expansion to a full mutation of 10.4% (95% CI: 6.7–14.3%). As the mean size of premutations identiied in the general population is lower than in fragile X families, this suggests that their expansion rate is similar with respect to their size. Thus, one important conclusion from the integration of these studies is that women carriers of FMR1 premutations, even without a history of FXMR, apparently have a signiicant risk of transmitting an expanded full mutation to their offspring. Thus, providing genetic counselling to pregnant women carriers of an FMR1 premutation might justify prenatal or postnatal screening for FXMR. Population Screening With a strong evidence-base supporting the high incidence of FXMR (1/4200 in newborns), the high frequency of premutation carrier women (~1/360), and the availability of highly sensitive and speciic testing methods to detect FMR1 mutation carriers, it has been proposed that the option of adding fragile X testing to prenatal screening for Down’s syndrome seems reasonable. Other alternatives to fragile X screening that have been considered include postnatal screening including newborn screening, but this would call for the availability of an eficient therapeutic approach which does not yet exist. Three different health technology assessment reports on fragile X screening have been published in the past 10 years. The irst report, by Murray et al., recommended that limited paediatric screening for FXMR and cascade screening in affected families was valuable and should continue.180 Prenatal screening and preventing an affected birth was estimated to cost less than one-seventieth of the estimated lifetime cost of care of an affected individual (US$13,000 vs US$1 million (1992)). However, it was also recommended that more research be carried out before considering any active screening program at the NHS, including the feasibility of antenatal screening. In 2001, the second report, by Pembery et al., underscored the absence of reliable estimates from population-based data of the prevalence of premutations as well as the unreliable estimates of the risk of expansion to a full mutation for such alleles in the lower premutation size range, especially those found in the general population.181 Simulations showed that systematic case inding and cascade screening in families can only reach half of the premutation carriers. The costs of FXMR have social as well as inancial components, and families support the concept of screening. Finally, this report called for more research on the risks for women in the general population carrying FMR1 alleles between 55 and 65 repeats to transmit a full mutation to their offspring. A third report, by Song et al. in 2003, also reviewed the literature and modelled fragile X screening scenarios.182 The authors concluded that both prenatal screening and cascade screening were feasible and acceptable according to the empirical evidence available. Thorough simulations that compared cascade screening with prenatal screening showed that population-based prenatal screening was more effective, but more expensive than active cascade screening. On the other hand, active cascade screening of affected families was more eficient, cheaper, but less effective than population-based prenatal screening. The projected costs of a prenatal screening program would be between 12 and 20 times that of cascade screening, and the incremental cost per FXMR birth avoided would be 30fold for prenatal screening (£284,779) compared to active cascade screening of affected families (£8494). It was thus suggested that both strategies be evaluated in large-scale trials with the possibility of inding approaches for combining both strategies simultaneously or sequentially. In 2009, an update of a Cochrane systematic review speciically addressing preconception and antenatal screening for FXMR was published by Kornman et al.183 These authors searched for randomised clinical trials comparing women tested without a family history of FXMR with women tested only in the presence of a positive family history or other undiagnosed mental illness or impairment. No such trials were found, which led the authors to conclude that trials were needed in order to weigh the beneits and risks of screening women for FMR1 mutated alleles, compared with the current practice of active cascade testing and testing women with an increased risk perceived as a history of FXMR or MR. Addition of FMR1 carrier testing to the existing Down’s syndrome prenatal screening programs was deemed attractive due to the programs already in place and because the FMR1 screening assay in the mother would be highly sensitive to and speciic for the risk of an affected pregnancy. Nevertheless, such FMR1 testing would still be expensive considering the molecular testing of maternal samples and the more complex information to provide to women candidates for the screening, compared with the explanations on Down’s syndrome, a better known and less complex disease. A systematic review of population-based screening for FXMR was published in 2010 by Hill et al.184 It concluded that targeted counselling as well as educational strategies will be essential for women from the general population who are offered screening, and that future studies of screening for FXMR should also explore the psychological aspects of such testing, and not only the uptake of testing and mutation frequency. The American College of Medical Genetics and the American Congress of Obstetricians and Gynecologists are currently Clin Biochem Rev Vol 32 August 2011 I 153 Rousseau F et al. working on recommendations for population-based carrier screening and for newborn screening. It will be interesting to see the formulation of these recommendations, but in the absence of well designed clinical trials comparing the various strategies to the current practice, it will be dificult for these organisations to propose a speciic approach with a strong evidence base to support their conclusions and recommendations. The existence of speciic diseases associated with FMR1 premutations, and perhaps the even more frequent intermediate size alleles (40–54 repeats), brings a new level of complexity and challenge to any populationbased screening program that would have the capacity to detect such alleles, as it is likely that carriers of such alleles would need counselling.185 Conclusion Since the identiication of the dynamic mutations and the FMR1 gene associated with the disease in 1991, our knowledge of FXMR has improved on many fronts. Understanding the nature and functions of FMRP and the effects of its absence on neuronal function as well as on cognitive development is paving the way towards potential therapeutic interventions. Determination of the premutation and full mutation prevalence in the general population, and of the instability of such mutations outside of families affected with the disease, combined with the development of cheaper screening methods for FMR1 mutations, is slowly building the case for more systematic approaches to identify cases early enough to allow for genetic counselling, and in the longer term, to initiate early, eficient therapeutic interventions when they become available. Ever since the beginning, the FMR1 gene has brought many surprises to the scientiic and medical communities. There are still many individuals and families that are struggling with the serious conditions provoked by the dysfunctions caused by mutations of this gene. It is thus quite encouraging that more and more efforts and energy are invested not only in solving the many riddles of the FMR1 gene and associated syndromes, but also, and most especially, in developing and validating therapeutic interventions that will ease the burden of all individuals and families affected by these common mutations. Acknowledgements: The authors wish to thank the reviewers and editors for their constructive comments and suggestions for this review. Dr François Rousseau holds a Fonds de Recherche en Santé du Québec/Québec Ministry of Health and Social Services/Centre Universitaire de Québec Research Chair in health technology assessment and evidence-based laboratory medicine. The work having led to this review was in part supported by the APOGÉENet/CanGèneTest Research and Knowledge Network in 154 I Clin Biochem Rev Vol 32 August 2011 Genetic Health Services, funded by the Canadian Institutes for Health Research (www.cangenetest.org). 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