CGG Repeat Expansion Research Articles

Fragile X Syndrome: Scientific Background and Screening Technologies

Fragile X is the most common inherited cause of mental retardation with a prevalence of 1 in 4000 for males and 1 in 5000 to 8000 for females. The American College of Medical Genetics and Genomics has recommended diagnostic testing for fragile X in symptomatic persons, women with ovarian dysfunction, and persons with tremor/ataxia syndrome. Although medical and scientific professionals do not currently recommend screening nonsymptomatic populations, improvements in current treatment approaches and ongoing clinical trials have generated growing interest in screening for fragile X. Here, we briefly review the relevant molecular basis of fragile X and fragile X testing and compare three different molecular technologies available for fragile X screening in both males and females. These technologic approaches include destabilizing the CGG-repeat region with betaine and using chimeric CGG-targeted PCR primers, using heat pulses to destabilize C-G bonds in the PCR extension step, and using melting curve analysis to differentiate expanded CGG repeats from normals. The first two-step method performed with high sensitivity and specificity. The second method provided agarose gel images that allow identification of males with expanded CGG repeats and females with expanded CGG-repeat bands which are sometimes faint. The third melting curve analysis method would require controls in each run to correct for shifting optimal cutoff values.

Reversibility of neuropathology and motor deficits in an inducible mouse model for FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder affecting carriers of the fragile X-premutation, who have an expanded CGG repeat in the 5'-UTR of the FMR1 gene. FXTAS is characterized by progressive development of intention tremor, ataxia, parkinsonism and neuropsychological problems. The disease is thought to be caused by a toxic RNA gain-of-function mechanism, and the major hallmark of the disease is ubiquitin-positive intranuclear inclusions in neurons and astrocytes. We have developed a new transgenic mouse model in which we can induce expression of an expanded repeat in the brain upon doxycycline (dox) exposure (i.e. Tet-On mice). This Tet-On model makes use of the PrP-rtTA driver and allows us to study disease progression and possibilities of reversibility. In these mice, 8 weeks of dox exposure was sufficient to induce the formation of ubiquitin-positive intranuclear inclusions, which also stain positive for the RAN translation product FMRpolyG. Formation of these inclusions is reversible after stopping expression of the expanded CGG RNA at an early developmental stage. Furthermore, we observed a deficit in the compensatory eye movements of mice with inclusions, a functional phenotype that could be reduced by stopping expression of the expanded CGG RNA early in the disease development. Taken together, this study shows, for the first time, the potential of disease reversibility and suggests that early intervention might be beneficial for FXTAS patients.

Improving preimplantation genetic diagnosis for Fragile X syndrome: two new powerful single-round multiplex indirect and direct tests.

Fragile X syndrome (FraX) is caused by the expansion of an unstable CGG repeat located in the Fragile X mental retardation 1 gene (FMR1) gene. Preimplantation genetic diagnosis (PGD) can be proposed to couples at risk of transmitting the disease, that is, when the female carries a premutation or a full mutation. We describe two new single-cell, single-round multiplex PCR for indirect and direct diagnosis of FraX on biopsied embryos. These tests include five unpublished, highly heterozygous simple sequence repeats, and the co-amplification of non-expanded CGG repeats for the direct test. Heterozygosity of the new markers ranged from 69 to 81%. The mean rate of non-informative marker included in the tests was low (26% and 23% for the new indirect and direct tests, respectively). This strategy allows offering a PGD for FraX to 96% of couples requesting it in our centre. A conclusive genotype was obtained in all cells with a rate of cells presenting an allele dropout ranging from 17% for the indirect test to 26% for the direct test. The new indirect test was applied for eight PGD cycles: 32 embryos were analysed, 9 were transferred and 3 healthy babies were born. By multiplexing these highly informative markers, robustness of the diagnosis is improved and the loss of potentially healthy embryos (because they are non-diagnosed or misdiagnosed) is limited. This may increase the chances of success of couples requesting a PGD for FraX, in particular, when premature ovarian insufficiency in premutated women leads to a reduced number of embryos available for analysis.

Chromosome fragility at FRAXA in human cleavage stage embryos at risk for fragile X syndrome

Fragile X syndrome (FXS), the most common inherited intellectual disability syndrome, is caused by expansion and hypermethylation of the CGG repeat in the 5' UTR of the FMR1 gene. This expanded repeat, also known as the rare fragile site FRAXA, causes X chromosome fragility in cultured cells from patients but only when induced by perturbing pyrimidine synthesis. We performed preimplantation genetic diagnosis (PGD) on 595 blastomeres biopsied from 442 cleavage stage embryos at risk for FXS using short tandem repeat (STR) markers. In six blastomeres, from five embryos an incomplete haplotype was observed with loss of all alleles telomeric to the CGG repeat. In all five embryos, the incomplete haplotype corresponded to the haplotype carrying the CGG repeat expansion. Subsequent analysis of additional blastomeres from three embryos by array comparative genomic hybridization (aCGH) confirmed the presence of a terminal deletion with a breakpoint close to the CGG repeat in two blastomeres from one embryo. A blastomere from another embryo showed the complementary duplication. We conclude that a CGG repeat expansion at FRAXA causes X chromosome fragility in early human IVF embryos at risk for FXS.

Validation of a Commercially Available Screening Tool for the Rapid Identification of CGG Trinucleotide Repeat Expansions in FMR1

Recently developed PCR-based methods for fragile X syndrome testing are often regarded as screening tools because of a reduced reliance on Southern blot analysis. However, existing PCR methods rely essentially on capillary electrophoresis for the analysis of amplicons. These methods not only require an expensive capillary electrophoresis instrument but also involve post-PCR processing steps. Here, we evaluated a commercially available PCR-based assay that uses melt curve analysis as a screening tool for the rapid detection of CGG repeat expansions in the fragile X mental retardation 1 (FMR1) gene. On the basis of testing with well-characterized DNA samples, the assay gave a detection limit of 10 ng per reaction and an analytic specificity beyond 150 ng per reaction. Furthermore, the melt temperatures critical for result interpretation were found to be closely linked to the CGG expansion lengths with great consistency (CV < 0.55%). The clinical performance of the assay was established with 528 blinded and previously analyzed clinical samples, yielding results of 100% sensitivity (95% CI, 91.0%-100%) and 99.6% specificity (95% CI, 98.5%-99.9%) in detecting expansions >55 CGG repeats in FMR1. This new approach eliminates post-PCR handling for all non-expanded samples, and exemplifies a truly efficient screening procedure.

Characterization and Early Detection of Balance Deficits in Fragile X Premutation Carriers With and Without Fragile X-Associated Tremor/Ataxia Syndrome (FXTAS)

Fragile X-associated tremor/ataxia syndrome (FXTAS) results from a "premutation" size 55-200 CGG repeat expansion in the fragile X mental retardation 1 (FMR1) gene. Core motor features include cerebellar gait ataxia and kinetic tremor, resulting in progressive mobility disability. There are no published studies characterizing balance deficits in FMR1 premutation carriers with and without FXTAS using a battery of quantitative measures to test the sensory integration underlying postural control, automatic postural reflexes, and dynamic postural stability limits. Computerized dynamic posturography (CDP) and two performance-based balance measures were administered in 44 premutation carriers, 21 with FXTAS and 23 without FXTAS, and 42 healthy controls to compare balance and functional mobility between these groups. Relationships between FMR1 molecular variables, age, and sex and CDP scores were explored. FXTAS subjects demonstrated significantly lower scores on the sensory organization test (with greatest reductions in the vestibular control of balance), longer response latencies to balance perturbations, and reduced stability limits compared to controls. Premutation carriers without FXTAS also demonstrated significantly delayed response latencies and disrupted sensory weighting for balance control. Advancing age, male sex, increased CGG repeat size, and reduced X activation of the normal allele in premutation carrier women predicted balance dysfunction. These postural control deficits in carriers with and without FXTAS implicate dysfunctional cerebellar neural networks and may provide valuable outcome markers for tailored rehabilitative interventions. Our findings suggest that CDP may provide sensitive measures for early detection of postural control impairments in at-risk carriers and better characterize balance dysfunction and progression in FXTAS.

Chromatin changes caused by expansion of CGG repeats in fmr1 gene

Fragile X syndrome is an inherited neurodegenerative disease with a frequency of 1/4000–1/6000. It is the main cause of inheritable mental retardation. The progression of the disorder is caused by CGG repeat expansion in 5′ UTR of fmr1 (fragile X mental retardation 1) gene. The normal allele contains ≤ 54 repeats. Alleles containing 55–200 repeats induce fragile X-associated disorders: fragile X-associated tremor/ataxia syndrome and fragile-X associated primary ovarian insufficiency. Alleles containing ≤ 200 repeats induce fragile X syndrome. The absence of FMRP protein is the main reason for the progression of the syndrome. FMRP is RNA-binding protein responsible for neuronal differentiation. In case of increasing CGG triplets number the expression of fmr1 gene is repressed. Results of CGG expansion are DNA methylation, histone methylation and deacetylation. Repression transcription factors bind this chromatin and lead to the progression of the disorder. In this review, we discuss how heterochromatinization is induced by CGG repeat expansion in the promoter region of fmr1 gene.

A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function.

Loss of function of the FMR1 gene leads to fragile X syndrome (FXS), the most common form of intellectual disability. The loss of FMR1 function is usually caused by epigenetic silencing of the FMR1 promoter leading to expansion and subsequent methylation of a CGG repeat in the 5′ untranslated region. Very few coding sequence variations have been experimentally characterized and shown to be causal to the disease. Here, we describe a novel FMR1 mutation and reveal an unexpected nuclear export function for the C-terminus of FMRP. We screened a cohort of patients with typical FXS symptoms who tested negative for CGG repeat expansion in the FMR1 locus. In one patient, we identified a guanine insertion in FMR1 exon 15. This mutation alters the open reading frame creating a short novel C-terminal sequence, followed by a stop codon. We find that this novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells. We also reveal an evolutionarily conserved nuclear export function associated with the endogenous C-terminus of FMRP. In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes.

Fragile X-associated tremor/ataxia syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that affects some but not all carriers of small, noncoding CGG-repeat expansions (55-200 repeats; premutation) within the fragile X gene (FMR1). Principal features of FXTAS include intention tremor, cerebellar ataxia, Parkinsonism, memory and executive function deficits, autonomic dysfunction, brain atrophy with white matter disease, and cognitive decline. Although FXTAS was originally considered to be confined to the premutation range, rare individuals with a gray zone (45-54 repeats) or an unmethylated full mutation (>200 repeats) allele have now been described, the constant feature of the disorder remaining the requirement for FMR1 expression, in contradistinction to the gene silencing mechanism of fragile X syndrome. Although transcriptional activity is required for FXTAS pathogenesis, the specific trigger(s) for FXTAS pathogenesis remains elusive, highlighting the need for more research in this area. This need is underscored by recent neuroimaging findings of changes in the central nervous system that consistently appear well before the onset of clinical symptoms, thus creating an opportunity to delay or prevent the appearance of FXTAS.

Single-Nucleotide Mutations in FMR1 Reveal Novel Functions and Regulatory Mechanisms of the Fragile X Syndrome Protein FMRP.

Fragile X syndrome is a monogenic disorder and a common cause of intellectual disability. Despite nearly 25 years of research on FMR1, the gene underlying the syndrome, very few pathological mutations other than the typical CGG-repeat expansion have been reported. This is in contrast to other X-linked, monogenic, intellectual disability disorders, such as Rett syndrome, where many point mutations have been validated as causative of the disorder. As technology has improved and significantly driven down the cost of sequencing, allowing for whole genes to be sequenced with relative ease, in-depth sequencing studies on FMR1 have recently been performed. These studies have led to the identification of novel variants in FMR1, where some of which have been functionally evaluated and are likely pathogenic. In this review, we discuss recently identified FMR1 variants, the ways these novel variants cause dysfunction, and how they reveal new regulatory mechanisms and functionalities of the gene.

Aspectos clínicos, moleculares y farmacológicos en los trastornos asociados a gen 1 del retraso mental del X frágil

BackgroundFragile X syndrome, the most common inherited cause of intellectual disability, is associated with a broad spectrum of disorders across different generations of a single family. This study reviews the clinical manifestations of fragile X-associated disorders as well as the spectrum of mutations of the fragile X mental retardation 1 gene (FMR1) and the neurobiology of the fragile X mental retardation protein (FMRP), and also provides an overview of the potential therapeutic targets and genetic counselling. DevelopmentThis disorder is caused by expansion of the CGG repeat (>200 repeats) in the 5 prime untranslated region of FMR1, resulting in a deficit or absence of FMRP. FMRP is an RNA-binding protein that regulates the translation of several genes that are important in synaptic plasticity and dendritic maturation. It is believed that CGG repeat expansions in the premutation range (55 to 200 repeats) elicit an increase in mRNA levels of FMR1, which may cause neuronal toxicity. These changes manifest clinically as developmental problems such as autism and learning disabilities as well as neurodegenerative diseases including fragile X-associated tremor/ataxia syndrome (FXTAS). ConclusionsAdvances in identifying the molecular basis of fragile X syndrome may help us understand the causes of neuropsychiatric disorders, and they will probably contribute to development of new and specific treatments.

Distribution of fragile X mental retardation 1 CGG repeat and flanking haplotypes in a large Chinese population.

Fragile X syndrome is mainly caused by a CGG repeat expansion within the 5′ UTR of the fragile X mental retardation 1 (FMR1) gene. Previous analyses of the FMR1 CGG repeat patterns and flanking haplotypes in Caucasians and African Americans have identified several factors that may influence repeat instability. However, the CGG repeat patterns and distribution for FRAXAC2 have not yet been investigated in mainland Chinese. We surveyed the CGG repeat lengths in 1113 Han Chinese (534 males and 579 females), and the CGG repeat patterns of 534 males were determined by sequence analysis. We also explored the flanking haplotypes (DXS548-FRAXAC1-FRAXAC2) in 566 unaffected and 28 unrelated fragile X Chinese males. The most frequent alleles for DXS548 and FRAXAC1 were identical between our Chinese population and other Asian populations. We identified several low-abundance alleles for DXS548 and FRAXAC1 not found in previous studies in mainland Chinese and Taiwanese cohorts. The most frequent allele was (CGG)29 followed by (CGG)30, and the most frequent patterns were 9 + 9 + 9, 10 + 9 + 9, and 9 + 9 + 6 + 9, similar to those in Singaporeans. We identified only one premutation female carrier with 89 CGG repeats in the 1113 Han Chinese. A few associations between the CGG repeat patterns and flanking haplotypes were determined in this study. In general, the Chinese population had a smaller number of alleles and lower expected heterozygosity for all three STR markers and FRAXA locus when compared with Caucasians and African Americans. We identified a novel haplotype 7-3-5 + that is significantly associated with the full mutation.

Peripheral neuropathy and parkinsonism: a large clinical and pathogenic spectrum.

Peripheral neuropathy (PN) has been reported in idiopathic and hereditary forms of parkinsonism, but the pathogenic mechanisms are unclear and likely heterogeneous. Levodopa-induced vitamin B12 deficiency has been discussed as a causal factor of PN in idiopathic Parkinson's disease, but peripheral nervous system involvement might also be a consequence of the underlying neurodegenerative process. Occurrence of PN with parkinsonism has been associated with a panel of mitochondrial cytopathies, more frequently related to a nuclear gene defect and mainly polymerase gamma (POLG1) gene. Parkin (PARK2) gene mutations are responsible for juvenile parkinsonism, and possible peripheral nervous system involvement has been reported. Rarely, an association of parkinsonism with PN may be encountered in other neurodegenerative diseases such as fragile X-associated tremor and ataxia syndrome related to premutation CGG repeat expansion in the fragile X mental retardation (FMR1) gene, Machado-Joseph disease related to an abnormal CAG repeat expansion in ataxin-3 (ATXN3) gene, Kufor-Rakeb syndrome caused by mutations in ATP13A2 gene, or in hereditary systemic disorders such as Gaucher disease due to mutations in the β-glucocerebrosidase (GBA) gene and Chediak-Higashi syndrome due to LYST gene mutations. This article reviews conditions in which PN may coexist with parkinsonism.

FMR1 gene mutation screening by TP-PCR in patients with premature ovarian failure and fragile-X

CGG repeat expansion in the FMR1 gene is associated with fragile X syndrome, fragile X-associated tremor/ ataxia syndrome and fragile X-associated primary ovarian insufficiency. In this study, FMR1 gene mutation screening was carried out in 50 patients. Among them, 12(%24) were POF and 19 (%38) were Fragile-X. We also examined the parents of the Fragile-X patients. DNA was extracted from blood with kit procedure. To examine expansion of the fragile-X CGG repeat, TP-PCR assay was performed and all amplicons were evaluated on an ABI3130XL Genetic Analyzer System by Fragman analysis. The data were analyzed by Gene Mapper Program. As a result of this study, the patients were identified with the fragile-X whose FMR1 gene CGG alleles have been observed in normal range. However, in patients who were referred with premature ovarian failure, pre-mutation frequency was observed as 6.6%. Only limited study in Turkish population reported frequency of pre-mutation carrier in POF and Fragile-X. Detection of pre-mutation carrier is important for next generation to have healthy siblings. We emphasize that TP-PCR technique is clear, reliable, sensitive, easy and fast method to detect pre-mutation. However, full mutations have to be examined by the technique of Southern blot in the diagnosis of fragile-X.

Fragile X mental retardation protein expression in Alzheimer's disease.

The FMR1 protein product, FMRP, is an mRNA binding protein associated with translational inhibition of target transcripts. One FMRP target is the amyloid precursor protein (APP) mRNA, and APP levels are elevated in Fmr1 KO mice. Given that elevated APP protein expression can elicit Alzheimer’s disease (AD) in patients and model systems, we evaluated whether FMRP expression might be altered in Alzheimer’s autopsy brain samples and mouse models compared to controls. In a double transgenic mouse model of AD (APP/PS1), we found no difference in FMRP expression in aged AD model mice compared to littermate controls. FMRP expression was also similar in AD and control patient frontal cortex and cerebellum samples. Fragile X-associated tremor/ataxia syndrome (FXTAS) is an age-related neurodegenerative disorder caused by expanded CGG repeats in the 5′ untranslated region of the FMR1 gene. Patients experience cognitive impairment and dementia in addition to motor symptoms. In parallel studies, we measured FMRP expression in cortex and cerebellum from three FXTAS patients and found reduced expression compared to both controls and Alzheimer’s patient brains, consistent with animal models. We also find increased APP levels in cerebellar, but not cortical, samples of FXTAS patients compared to controls. Taken together, these data suggest that a decrease in FMRP expression is unlikely to be a primary contributor to Alzheimer’s disease pathogenesis.

Cis-acting DNA sequence at a replication origin promotes repeat expansion to fragile X full mutation.

Fragile X syndrome (FXS) is caused by CGG repeat expansion that leads to FMR1 silencing. Women with a premutation allele are at risk of having a full mutation child with FXS. To investigate the mechanism of repeat expansion, we examined the relationship between a single-nucleotide polymorphism (SNP) variant that is linked to repeat expansion in haplogroup D and a replication origin located ∼53 kb upstream of the repeats. This origin is absent in FXS human embryonic stem cells (hESCs), which have the SNP variant C, but present in the nonaffected hESCs, which have a T variant. The SNP maps directly within the replication origin. Interestingly, premutation hESCs have a replication origin and the T variant similar to nonaffected hESCs. These results suggest that a T/C SNP located at a replication origin could contribute to the inactivation of this replication origin in FXS hESCs, leading to altered replication fork progression through the repeats, which could result in repeat expansion to the FXS full mutation.

A CGG-repeat expansion mutation in ZNF713 causes FRA7A: association with autistic spectrum disorder in two families.

We report de novo occurrence of the 7p11.2 folate-sensitive fragile site FRA7A in a male with an autistic spectrum disorder (ASD) due to a CGG-repeat expansion mutation (∼450 repeats) in a 5' intron of ZNF713. This expanded allele showed hypermethylation of the adjacent CpG island with reduced ZNF713 expression observed in a proband-derived lymphoblastoid cell line (LCL). His unaffected mother carried an unmethylated premutation (85 repeats). This CGG-repeat showed length polymorphism in control samples (five to 22 repeats). In a second unrelated family, three siblings with ASD and their unaffected father were found to carry FRA7A premutations, which were partially or mosaically methylated. In one of the affected siblings, mitotic instability of the premutation was observed. ZNF713 expression in LCLs in this family was increased in three of these four premutation carriers. A firm link cannot yet be established between ASD and the repeat expansion mutation but plausible pathogenic mechanisms are discussed.

Point mutation frequency in the FMR1 gene as revealed by fragile X syndrome screening

Fragile X syndrome (FXS) is a common cause of intellectual disability, developmental delay and autism spectrum disorders. This syndrome is due to a functional loss of the FMR1 gene product FMRP, and, in most cases, it is caused by CGG repeat expansion in the FMR1 promoter. Yet, also other FMR1 mutations may cause a FXS-like phenotype. Since standard molecular testing does not include the analysis of the FMR1 coding region, the prevalence of point mutations causing FXS is not well known. Here, high resolution melting (HRM) was used to screen for FMR1 gene mutations in 508 males with clinical signs of mental retardation and developmental delay, but without CGG and GCC repeat expansions in the FMR1 gene and AFF2 genes, respectively. Sequence variations were identified by HRM analysis and verified by direct DNA sequencing. Two novel missense mutations (p.Gly482Ser in one patient and p.Arg534His in two unrelated patients), one intronic and two 3′-untranslated region (UTR) variations were identified in the FMR1 gene. Missense mutations in the FMR1 gene might account for a considerable proportion of cases in male patients with FXS-related symptoms, such as those linked to mental retardation and developmental delay.

Induced expression of expanded CGG RNA causes mitochondrial dysfunction in vivo

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder affecting carriers of premutation forms of the FMR1 gene, resulting in a progressive development of tremor, ataxia and neuropsychological problems. The disease is caused by an expanded CGG repeat in the FMR1 gene, leading to an RNA gain-of-function toxicity mechanism. In order to study the pathogenesis of FXTAS, new inducible transgenic mouse models have been developed that expresses either 11CGGs or 90CGGs at the RNA level under control of a Tet-On promoter. When bred to an hnRNP-rtTA driver line, doxycycline (dox) induced expression of the transgene could be found in almost all tissues. Dox exposure resulted in loss of weight and death within 5 d for the 90CGG RNA expressing mice. Immunohistochemical examination of tissues of these mice revealed steatosis and apoptosis in the liver. Decreased expression of GPX1 and increased expression of cytochrome C is found. These effects were not seen in mice expressing a normal sized 11CGG repeat. In conclusion, we were able to show in vivo that expression of an expanded CGG-repeat rather than overexpression of a normal CGG-repeat causes pathology. In addition, we have shown that expanded CGG RNA expression can cause mitochondrial dysfunction by regulating expression levels of several markers. Although FTXAS patients do not display liver abnormalities, our findings contribute to understanding of the molecular mechanisms underlying toxicity of CGG repeat RNA expression in an animal model. In addition, the dox inducible mouse lines offer new opportunities to study therapeutic interventions for FXTAS.

Fragile x premutation.

Whereas full mutation CGG-repeat expansions (>200 repeats) of the fragile X gene (FMR1) give rise to the neurodevelopmental disorder, fragile X syndrome (FXS); smaller, ‘premutation’ expansions (55 to 200 repeats) are now gaining increasing recognition as the basis for a spectrum of clinical involvement, from neurodevelopmental problems; to mid-adult disorders, such as primary ovarian insufficiency and mood and psychiatric disorders; to the late-adult-onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). The premutation disorders are thought to arise through a molecular mechanism involving toxicity of the elevated levels of expanded CGG-repeat mRNA (‘RNA toxicity’), a process that is entirely distinct from the FMR1 protein-deficiency that gives rise to FXS. However, despite the importance of the spectrum of clinical disorders associated with the premutation, and a high prevalence rate (1 in 130 to 250 females and 1 in 250 to 810 males), relatively little attention has been paid to these disorders and there is a general lack of awareness among clinicians as to the distinction between the premutation disorders and FXS. To address this lack of awareness, an international conference on the premutation was held in Perugia, Italy, in June 2013. The conference covered the expanding range of clinical involvement, refinements of the assessments and tools for characterizing such involvement, and the rapidly expanding understanding of the pathogenic molecular and cellular mechanisms that give rise to the spectrum of involvement among premutation carriers. All of these advances support ongoing efforts to develop new targeted treatments for the premutation disorders. As an outgrowth of the meeting, papers were solicited from the conference attendees such that groups of scientists and clinicians would develop works that broadly covered the topics of the meeting. The following papers represent that effort.