Fragile X Syndrome Cases Research Articles

8. Optical genome mapping analysis of FMR1 expansions in fragile X syndrome

Fragile X syndrome (FXS) is associated with cognitive impairment and intellectual disability. Nearly all cases of FXS are associated with an expansion of a CGG triplet repeat in the FMR1 gene. Phenotype severity is often correlated with the expansion size; thus, accurate sizing of the CCG repeat array is crucial. The repetitive nature of these regions presents difficulties: 1. PCR is unable to traverse through long repeats; 2. sequencing has limited read lengths; 3. Southern blot may be inaccurate, time consuming, and expensive. Optical genome mapping (OGM) has the potential to address some of these shortcomings. OGM utilizes images of ultra-long DNA molecules, labeled at specific sequence motifs linearized in nanochannel arrays and can be used for structural and copy number variation calling and absence of heterozygosity analysis. Bionano Genomics has developed a targeted analysis workflow for analysis of FMR1. To evaluate the capability of measuring repeat arrays consistent with normal (<45 repeats), premutation (55-200 repeats), and full mutation (>200 repeats), we analyzed the FMR1 repeats in 75 samples. We also analyzed the repeat region on 20 phenotypically normal control subjects. We observed alleles consistent with annotation across the entire range of repeat counts. Analytical sensitivity was measured at 97% with 100% PPV for expansions >200 repeats. The largest expansion we detected was ∼1000 repeats. In the control samples, we measured repeat counts below the full mutation cutoff in all cases. Repeatability studies were carried out to show analytical consistency and to establish minimum molecule quality requirements. EnFocus™ analysis report provides pass/fail for QC metrics for input data quality as well as analytic measurement quality using internal control regions of the genome on each autosome. OGM performance for FMR1 repeat lengths analyzed here show a much higher dynamic range compared to PCR and higher precision compared to Southern blot.

High Throughput Small Molecule Screen for Reactivation of FMR1 in Fragile X Syndrome Human Neural Cells.

Fragile X syndrome (FXS) is the most common inherited cause of autism and intellectual disability. The majority of FXS cases are caused by transcriptional repression of the FMR1 gene due to epigenetic changes that are not recapitulated in current animal disease models. FXS patient induced pluripotent stem cell (iPSC)-derived gene edited reporter cell lines enable novel strategies to discover reactivators of FMR1 expression in human cells on a much larger scale than previously possible. Here, we describe the workflow using FXS iPSC-derived neural cell lines to conduct a massive, unbiased screen for small molecule activators of the FMR1 gene. The proof-of-principle methodology demonstrates the utility of human stem-cell-based methodology for the untargeted discovery of reactivators of the human FMR1 gene that can be applied to other diseases.

Clinical Characteristics of Fragile X Syndrome Patients in Japan.

Fragile X syndrome (FXS) is a well-known X-linked disorder clinically characterized by intellectual disability and autistic features. However, diagnosed Japanese FXS cases have been fewer than expected, and clinical features of Japanese FXS patients remain unknown. We evaluated the clinical features of Japanese FXS patients using the results of a questionnaire-based survey. We presented the characteristics of seven patients aged 6 to 20 years. Long face and large ears were observed in five of seven patients. Macrocephaly was observed in four of five patients. The meaningful word was first seen at a certain time point between 18 and 72 months (median = 60 months). Developmental quotient or intellectual quotient ranged between 20 and 48 (median = 29). Behavioral disorders were seen in all patients (autistic spectrum disorder in six patients, hyperactivity in five patients). Five patients were diagnosed by polymerase chain reaction analysis, and two patients were diagnosed by the cytogenetic study. All physicians ordered FXS genetic testing for suspicious cases because of clinical manifestations. In the present study, a long face, large ears, macrocephaly, autistic spectrum disorder, and hyperactivity were observed in almost cases, and these characteristics might be common features in Japanese FXS patients. Our finding indicated the importance of clinical manifestations to diagnosis FXS. However, the sample size of the present study is small, and these features are also seen to patients with other disorders. We consider that genetic testing for FXS should be performed on a wider range of intellectually disabled cases.

Deletion of the KH1 Domain of Fmr1 Leads to Transcriptional Alterations and Attentional Deficits in Rats.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene. It is a leading monogenic cause of autism spectrum disorder and inherited intellectual disability and is often comorbid with attention deficits. Most FXS cases are due to an expansion of CGG repeats leading to suppressed expression of fragile X mental retardation protein (FMRP), an RNA-binding protein involved in mRNA metabolism. We found that the previously published Fmr1 knockout rat model of FXS expresses an Fmr1 transcript with an in-frame deletion of exon 8, which encodes for the K-homology (KH) RNA-binding domain, KH1. Notably, 3 pathogenic missense mutations associated with FXS lie in the KH domains. We observed that the deletion of exon 8 in rats leads to attention deficits and to alterations in transcriptional profiles within the medial prefrontal cortex (mPFC), which map to 2 weighted gene coexpression network modules. These modules are conserved in human frontal cortex and enriched for known FMRP targets. Hub genes in these modules represent potential therapeutic targets for FXS. Taken together, these findings indicate that attentional testing might be a reliable cross-species tool for investigating FXS and identify dysregulated conserved gene networks in a relevant brain region.

Modeling Fragile X Syndrome in Drosophila.

Intellectual disability (ID) and autism are hallmarks of Fragile X Syndrome (FXS), a hereditary neurodevelopmental disorder. The gene responsible for FXS is Fragile X Mental Retardation gene 1 (FMR1) encoding the Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in RNA metabolism and modulating the expression level of many targets. Most cases of FXS are caused by silencing of FMR1 due to CGG expansions in the 5′-UTR of the gene. Humans also carry the FXR1 and FXR2 paralogs of FMR1 while flies have only one FMR1 gene, here called dFMR1, sharing the same level of sequence homology with all three human genes, but functionally most similar to FMR1. This enables a much easier approach for FMR1 genetic studies. Drosophila has been widely used to investigate FMR1 functions at genetic, cellular, and molecular levels since dFMR1 mutants have many phenotypes in common with the wide spectrum of FMR1 functions that underlay the disease. In this review, we present very recent Drosophila studies investigating FMRP functions at genetic, cellular, molecular, and electrophysiological levels in addition to research on pharmacological treatments in the fly model. These studies have the potential to aid the discovery of pharmacological therapies for FXS.

STEP inhibition reverses behavioral, electrophysiologic, and synaptic abnormalities in Fmr1 KO mice

Fragile X syndrome (FXS) is the leading cause of inherited intellectual disability, with additional symptoms including attention deficit and hyperactivity, anxiety, impulsivity, and repetitive movements or actions. The majority of FXS cases are attributed to a CGG expansion that leads to transcriptional silencing and diminished expression of fragile X mental retardation protein (FMRP). FMRP, an RNA binding protein, regulates the synthesis of dendritically-translated mRNAs by stalling ribosomal translation. Loss of FMRP leads to increased translation of some of these mRNAs, including the CNS-specific tyrosine phosphatase STEP (STriatal-Enriched protein tyrosine Phosphatase). Genetic reduction of STEP in Fmr1 KO mice have diminished audiogenic seizures and a reversal of social and non-social anxiety-related abnormalities. This study investigates whether a newly discovered STEP inhibitor (TC-2153) could attenuate the behavioral and synaptic abnormalities in Fmr1 KO mice. TC-2153 reversed audiogenic seizure incidences, reduced hyperactivity, normalized anxiety states, and increased sociability in Fmr1 KO mice. Moreover, TC-2153 reduced dendritic spine density and improved synaptic aberrations in Fmr1 KO neuronal cultures as well as in vivo. TC-2153 also reversed the mGluR-mediated exaggerated LTD in brain slices derived from Fmr1 KO mice. These studies suggest that STEP inhibition may have therapeutic benefit in FXS.

Capture-recapture methodology to study rare conditions using surveillance data for fragile X syndrome and muscular dystrophy

BackgroundRare conditions can be catastrophic for families and the implications for public health can be substantial. Our study compared basic surveillance through active medical record review with a linked administrative data file to assess the number of cases of two rare conditions, fragile X syndrome (FXS) and muscular dystrophy (MD) in a population.MethodsTwo methods of data collection were used to collect information from five counties comprising two standard metropolitan statistical areas of South Carolina. The passive system relied mostly on health claims data using ICD-9 CM diagnostic codes. The active system relied on a nurse abstracting records from a list of all licensed physicians with specialties in neurology, orthopedics, and genetics.ResultsThere were 141 FXS cases and 348 MD cases that met the case definitions using active surveillance. Additional cases were found for both conditions but they were determined to not be true cases. After linking the actively collected MD and FXS cases to passive datasets, we found that the estimated total numbers of cases were similar to using capture-recapture analysis; the positive predictive values for cases identified in the passive system were 56.6% for MD and 75.7% for FXS.ConclusionsApplying capture-recapture methods to passively collected surveillance data for rare health conditions produced an estimate of the number of true cases that was similar to that obtained through active data collection.

Incidence of Fragile X syndrome in Ireland.

Described as the commonest single gene cause of learning disability internationally, the incidence of Fragile X syndrome (FXS) has never previously been determined in Ireland. The aim of this work was to determine the observed incidence of FXS in the island of Ireland; the Republic of Ireland (ROI) and Northern Ireland (NI) separately and combined. Ascertainment was achieved for a cross-sectional study by a retrospective, clinical and laboratory database review of positive FXS cases, born in either ROI or NI, between years 2000-2009 inclusive. The observed incidence of FXS per 10,000 live births in the island of Ireland in males was 0.94 (95%CI: 0.75-1.13) or ∼1:10,600 and in females was 0.23 (95%CI: 0.14-0.32) or ∼1:43,000. Comparable testing rates for FXS are present in ROI and NI, with on average 1.48% (1.30% in ROI, 1.96% in NI) of live male births and 0.4% (0.35% in ROI, 0.55% in NI) of live female births undergoing analysis which is comparable to other centres internationally. This study demonstrates the observed incidence of FXS in the island of Ireland is (i) approximately half the estimated worldwide incidence in males and is not explained by low levels of testing, and (ii) approximately one quarter the estimated worldwide incidence in females which may be explained by low levels of testing. © 2017 Wiley Periodicals, Inc.

From FMRP Function to Potential Therapies for Fragile X Syndrome

Fragile X syndrome (FXS) is caused by mutations in the fragile X mental retardation 1 (FMR1) gene. Most FXS cases occur due to the expansion of the CGG trinucleotide repeats in the 5' un-translated region of FMR1, which leads to hypermethylation and in turn silences the expression of FMRP (fragile X mental retardation protein). Numerous studies have demonstrated that FMRP interacts with both coding and non-coding RNAs and represses protein synthesis at dendritic and synaptic locations. In the absence of FMRP, the basal protein translation is enhanced and not responsive to neuronal stimulation. The altered protein translation may contribute to functional abnormalities in certain aspects of synaptic plasticity and intracellular signaling triggered by Gq-coupled receptors. This review focuses on the current understanding of FMRP function and potential therapeutic strategies that are mainly based on the manipulation of FMRP targets and knowledge gained from FXS pathophysiology.

Intranuclear inclusions in a fragile X mosaic male

Lack of the fragile X mental retardation protein leads to Fragile X syndrome (FXS) while increased levels of FMR1 mRNA, as those observed in premutation carriers can lead to Fragile X- associated tremor ataxia syndrome (FXTAS). Until recently, FXTAS had been observed only in carriers of an FMR1 premutation (55–200 CGG repeats); however the disorder has now been described in individuals carriers of an intermediate allele (45–54 CGG repeats) as well as in a subject with a full mutation with mosaicism.Here, we report on molecular and clinical data of a male FMR1 mosaic individual with full and premutation alleles. Molecular analysis of FMR1 and FMRP expression in this subject is consistent with a FXS phenotype. We observed reduced expression of FMRP in both peripheral blood and brain leading to the FXS diagnosis. In addition, a dramatic 90% depletion of both FMR1 mRNA and FMRP levels was observed in the blood, as normally observed in FXS cases, and an even greater depletion in the brain. A clinical report of this patient, at age 71, described neurodegenerative signs of parkinsonism that were likely, in retrospect, part of a FXTAS scenario as post-mortem examination shows the presence of intranuclear inclusions, the hallmark pathology of FXTAS.The findings presented in this study indicate co-morbidity for both FXS and FXTAS in this individual carrying both full and premutation FMR1 alleles. In addition, based on symptoms and pathological and molecular evidence, this report suggests the need to redefine the diagnostic criteria of FXTAS.

Fragile X Syndrome in China

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability (ID), and the leading monogenic cause of autism spectrum disorders. The FXS cases in China were first reported in 1984, and effort has been made to improve the wellbeing of FXS patients. However, the general Chinese population is unfamiliar with FXS due to the limited public education. Even among those with medical training, there is a lack of awareness. Here our review aims to provide basic information on FXS, introduce the clinical aspects of FXS in China, and outline future research and policy recommendations that may in the future improve FXS genetic testing and counselor training in the Chinese health system. [N A J Med Sci. 2013;6(4):181-185. DOI: 10.7156/najms.2013.0604181]

Species-Dependent Posttranscriptional Regulation of NOS1 by FMRP in the Developing Cerebral Cortex

Fragile X syndrome (FXS), the leading monogenic cause of intellectual disability and autism, results from loss of function of the RNA-binding protein FMRP. Here, we show that FMRP regulates translation of neuronal nitric oxide synthase 1 (NOS1) in the developing human neocortex. Whereas NOS1 mRNA is widely expressed, NOS1 protein is transiently coexpressed with FMRP during early synaptogenesis in layer- and region-specific pyramidal neurons. These include midfetal layer 5 subcortically projecting neurons arranged into alternating columns in the prospective Broca's area and orofacial motor cortex. Human NOS1 translation is activated by FMRP via interactions with coding region binding motifs absent from mouse Nos1 mRNA, which is expressed in mouse pyramidal neurons, but not efficiently translated. Correspondingly, neocortical NOS1 protein levels are severely reduced in developing human FXS cases, but not FMRP-deficient mice. Thus, alterations in FMRP posttranscriptional regulation of NOS1 in developing neocortical circuits may contribute to cognitive dysfunction in FXS.

The distribution of repressive histone modifications on silenced FMR1 alleles provides clues to the mechanism of gene silencing in fragile X syndrome

Fragile X syndrome (FXS) is the most common heritable cause of intellectual disability and the most common known cause of autism. Most cases of FXS result from the expansion of a CGG·CCG repeat in the 5' UTR of the FMR1 gene that leads to gene silencing. It has previously been shown that silenced alleles are associated with histone H3 dimethylated at lysine 9 (H3K9Me2) and H3 trimethylated at lysine 27 (H3K27Me3), modified histones typical of developmentally repressed genes. We show here that these alleles are also associated with elevated levels of histone H3 trimethylated at lysine 9 (H3K9Me3) and histone H4 trimethylated at lysine 20 (H4K20Me3). All four of these modified histones are present on exon 1 of silenced alleles at levels comparable to that seen on pericentric heterochromatin. The two groups of histone modifications show a different distribution on fragile X alleles: H3K9Me2 and H3K27Me3 have a broad distribution, whereas H3K9Me3 and H4K20Me3 have a more focal distribution with the highest level of these marks being present in the vicinity of the repeat. This suggests that the trigger for gene silencing may be local to the repeat itself and perhaps involves a mechanism similar to that involved in the formation of pericentric heterochromatin.

Parkinsonism and cognitive decline in a fragile X mosaic male

Parkinsonism and cognitive decline in a fragile X mosaic male

Fragile X and autism: Intertwined at the molecular level leading to targeted treatments.

Fragile X syndrome (FXS) is caused by an expanded CGG repeat (> 200 repeats) in the 5' untranslated portion of the fragile mental retardation 1 gene (FMR1), leading to deficiency or absence of the FMR1 protein (FMRP). FMRP is an RNA carrier protein that controls the translation of several other genes that regulate synaptic development and plasticity. Autism occurs in approximately 30% of FXS cases, and pervasive developmental disorder, not otherwise specified (PDD-NOS) occurs in an additional 30% of cases. Premutation repeat expansions (55 to 200 CGG repeats) may also give rise to autism spectrum disorders (ASD), including both autism and PDD-NOS, through a different molecular mechanism that involves a direct toxic effect of the expanded CGG repeat FMR1 mRNA. RNA toxicity can also lead to aging effects including tremor, ataxia and cognitive decline, termed fragile X-associated tremor ataxia syndrome (FXTAS), in premutation carriers in late life. In studies of mice bearing premutation expansions, there is evidence of early postnatal neuronal cell toxicity, presenting as reduced cell longevity, decreased dendritic arborization and altered synaptic morphology. There is also evidence of mitochondrial dysfunction in premutation carriers. Many of the problems with cellular dysregulation in both premutation and full mutation neurons also parallel the cellular abnormalities that have been documented in autism without fragile X mutations. Research regarding dysregulation of neurotransmitter systems in FXS, including the metabotropic glutamate receptor (mGluR)1/5 pathway and γ aminobutyric acid (GABA)A pathways, have led to new targeted treatments for FXS. Preliminary evidence suggests that these new targeted treatments will also be beneficial in non-fragile X forms of autism.

Implication of screening for FMR1 and FMR2 gene mutation in individuals with nonspecific mental retardation in Taiwan.

Fragile X syndrome (FXS) is the most common form of familial mental retardation (MR), attributable to (CGG)n expansion in the FMR1 gene. FRAXE is less frequent, associated with a similar mutation of the FMR2 gene. This study attempted to ascertain the prevalence of both disorders in Taiwan, as well as to develop a method to effectively find carriers. A total of 321 patients with nonspecific MR were screened for the FMR1 and FMR2 mutation. Four of 206 boys and men (1.9%) and 1 in 115 girls and women (0.9%) were identified as having FXS. All four FXS boys or men could be identified by Southern blot analysis, as well as by a simple nonradioactive polymerase chain reaction analysis. None of the 206 boys or men had FMR2 full mutation. This confirmed the low incidence of FRAXE in Chinese. FXS appears to be more prevalent among patients with mild MR, because 4 of the 5 patients with FXS were from the 115 with mild MR (3.48%) and only 1 was from the other 206 with severe MR (0.49%). All five FXS cases were maternally inherited. Other family members were resistant to further searching for carriers. It is worth noting that none of these mothers had a discernible premarital family history of MR. Thus the negative family history could not preclude the possibility that a woman was a carrier. To identify female carriers of childbearing age, beyond the scope of family history, is thus worthy of further exploration. Screening men for carriers using this inexpensive method is probably feasible, even though normal transmitting men have no immediate risk of producing a child with the disease. Female carriers can then be effectively identified from these normal transmitting men and can take all preventive measures.