Response to Eura et al.

Sequential development of parkinsonism in two patients with oculopharyngodistal type myopathy in GIPC1-related repeat expansion disorder

Background and PurposeCGG repeat expansion in the 5′ untranslated region (5′UTR) of the Notch 2 N-terminal-like C gene (NOTCH2NLC) has been associated with neuronal intranuclear inclusion disease (NIID) and oculopharyngodistal myopathy type 3 (OPDM3). Few OPDM3 patients have been reported. This report describes two OPDM3 patients with novel imaging findings who presented the typical features of NIID, and reviews all OPDM3 cases available in the literature.MethodsThe available clinical, imaging, and pathological information was reviewed and investigated. CGG repeat expansion in the 5'UTR of NOTCH2NLC was tested using the repeat-primed polymerase chain reaction (PCR), followed by the fluorescence amplicon-length PCR to determine the number of CGG repeats.ResultsOur two OPDM3 patients and most patients reported in the literature developed the typical clinical characteristics of NIID, including leukoencephalopathy, peripheral neuropathy, cognitive deterioration, pigmentary retinopathy, ataxia, tremor, acute encephalitis-like episodes, pigmentary retinopathy, miosis, and sensorineural hearing loss. In addition to typical imaging findings of NIID, our two patients exhibited diffusion weighted imaging (DWI) hyperintensities in the middle cerebellar peduncles, which have not been described previously. Muscle biopsies revealed rimmed vacuoles and p62-positive intranuclear inclusions in the myofibers in both patients. The skin biopsy performed in one patient detected typical eosinophilic intranuclear inclusions. Genetic analysis identified CGG repeat expansion in NOTCH2NLC as the causative mutation in the two patients.ConclusionsOur two patients with OPDM3 had clinical characteristics of NIID and exhibited DWI abnormality in the cerebellum. Our results indicate that OPDM3 is within the spectrum of NIID and that DWI hyperintensities in the cerebellum are helpful for diagnosing NIID or OPDM3.

Expansion of CGG repeats (CGGexp) in the 5’ untranslated region (5’UTR) of the FMR1 gene underlies the fragile X-associated conditions including tremor/ataxia syndrome (FXTAS), a late-onset neurodegenerative disease. One pathomechanism of FXTAS is the repeat-associated non-AUG-initiated (RAN) translation of CGG repeats of mutant FMR1 mRNA, resulting in production of FMRpolyG, a toxic protein containing long polyglycine tract. To identify novel modifiers of RAN translation we used an RNA-tagging system and mass spectrometry-based screening. It revealed proteins enriched on CGGexp-containing FMR1 RNA in cellulo, including a ribosomal protein RPS26, a component of the 40S subunit. We demonstrated that RPS26, together with its chaperone TSR2, modulates FMRpolyG production and its toxicity. We also found that the number of proteins produced via RPS26-sensitive translation was limited, and 5’UTRs of mRNAs encoding these proteins were guanosine and cytosine-rich. Moreover, the silencing of another component of the 40S subunit, the ribosomal protein RPS25, also induced repression of FMRpolyG biosynthesis. Results of this study suggest that the composition of the 40S subunit plays important role in noncanonical CGGexp-related RAN translation.

The CGG repeat expansion in RILPL1 is associated with oculopharyngodistal myopathy type 4

Repeat expansion of CGG in LRP12 has been identified as the causative variation of oculopharyngodistal myopathy (OPDM). However, to our knowledge, the clinicopathologic features of OPDM with CGG repeat expansion in LRP12 (hereafter referred to as OPDM_LRP12) remain unknown. To identify and characterize the clinicopathologic features of patients with OPDM_LRP12. This case series included 208 patients with a clinical or clinicopathologic diagnosis of oculopharyngeal muscular dystrophy (OPDM) from January 1, 1978, to December 31, 2020. Patients with GCN repeat expansions in PABPN1 were excluded from the study. Repeat expansions of CGG in LRP12 were screened by repeat primed polymerase chain reaction and/or Southern blot. Clinical information, muscle imaging data obtained by either computed tomography or magnetic resonance imaging, and muscle pathologic characteristics. Sixty-five Japanese patients with OPDM (40 men [62%]; mean [SD] age at onset, 41.0 [10.1] years) from 59 families with CGG repeat expansions in LRP12 were identified. This represents the most common OPDM subtype among all patients in Japan with genetically diagnosed OPDM. The expansions ranged from 85 to 289 repeats. A negative correlation was observed between the repeat size and the age at onset (r2 = 0.188, P = .001). The most common initial symptoms were ptosis and muscle weakness, present in 24 patients (37%). Limb muscle weakness was predominantly distal in 53 of 64 patients (83%), but 2 of 64 patients (3%) had predominantly proximal muscle weakness. Ptosis was observed in 62 of 64 patients (97%), and dysphagia or dysarthria was observed in 63 of 64 patients (98%). A total of 21 of 64 patients (33%) had asymmetric muscle weakness. Aspiration pneumonia was seen in 11 of 64 patients (17%), and 5 of 64 patients (8%) required mechanical ventilation. Seven of 64 patients (11%) developed cardiac abnormalities, and 5 of 64 patients (8%) developed neurologic abnormalities. Asymmetric muscle involvement was detected on computed tomography scans in 6 of 27 patients (22%) and on magnetic resonance imaging scans in 4 of 15 patients (27%), with the soleus and the medial head of the gastrocnemius being the worst affected. All 42 muscle biopsy samples showed rimmed vacuoles. Intranuclear tubulofilamentous inclusions were observed in only 1 of 5 patients. This study suggests that OPDM_LRP12 is the most frequent OPDM subtype in Japan and is characterized by oculopharyngeal weakness, distal myopathy that especially affects the soleus and gastrocnemius muscles, and rimmed vacuoles in muscle biopsy.

Fragile X syndrome is the leading cause of inherited mental impairment and is associated with expansions of CGG repeats within the FMR1 gene. To detect expanded CGG repeats, we developed a dual-mode single-molecule fluorescence assay that allows acquisition of two parallel, independent measures of repeat number based on (1) the number of Cy3-labeled probes bound to the repeat region and (2) the physical length of the electric field-linearized repeat region, obtained from the relative position of a single Cy5 dye near the end of the repeat region. Using target strands derived from cell-line DNA with defined numbers of CGG repeats, we show that this assay can rapidly and simultaneously measure the repeats of a collection of individual sample strands within a single field of view. With a low occurrence of false positives, the assay differentiated normal CGG repeat lengths (CGG( N ), N = 23) and expanded CGG repeat lengths (CGG( N ), N = 118), representing a premutation disease state. Further, mixtures of these DNAs gave results that correlated with their relative populations. This strategy may be useful for identifying heterozygosity or for screening collections of individuals, and it is readily adaptable for screening other repeat disorders.

Repeat expansion disorders (REDs) encompass over 50 inherited neurological disorders and are characterized by the expansion of short tandem nucleotide repeats beyond a specific repeat length. Particularly intriguing among these are multiple fragile X-associated disorders (FXds), which arise from an expansion of CGG repeats in the 5' untranslated region of the FMR1 gene. Despite arising from repeat expansions in the same gene, the clinical manifestations of FXds vary widely, encompassing developmental delays, parkinsonism, dementia, and an increased risk of infertility. FXds also exhibit molecular mechanisms observed in other REDs, that is, gene- and protein-loss-of-function and RNA- and protein-gain-of-function. The heterogeneity of phenotypes and pathomechanisms in FXds results from the different lengths of the CGG tract. As the number of repeats increases, the structures formed by RNA and DNA fragments containing CGG repeats change significantly, contributing to the diversity of FXd phenotypes and mechanisms. In this review, we discuss the role of RNA and DNA structures formed by expanded CGG repeats in driving FXd pathogenesis and how the genetic instability of CGG repeats is mediated by the complex interplay between transcription, DNA replication, and repair. We also discuss therapeutic strategies, including small molecules, antisense oligonucleotides, and CRISPR-Cas systems, that target toxic RNA and DNA involved in the development of FXds.

Two novel intragenic variants in the FMR1 gene in patients with suspect clinical diagnosis of Fragile X syndrome and no CGG repeat expansion

ObjectivesAutism spectrum disorder (ASD) is a major neurodevelopmental disorder characterized by persistent deficits in social communication along with restricted, repetitive patterns of behaviour and interests. FMR1 gene, which causes Fragile X syndrome (FXS), is the most common single-gene contributor to ASD. Variations in CGG repeat length within the 5' untranslated region of FMR1 are central to the etiology of FXS. Given the established connection between FXS and ASD, this study investigated whether the trinucleotide repeat region of FMR1 is associated with ASD in Bangladeshi children.MethodsSeventy-one children participated in the study, including 39 with ASD and 32 age-matched controls. The FMR1 region was amplified using polymerase chain reaction and subsequently sequenced.ResultsThere was no statistically significant difference in the number of CGG repeats between patients with ASD and controls (p>0.01), with the most common repeat number being 27 in both groups. Interspersion of the two AGG trinucleotides among the CGG repeats was the most common pattern found in the study participants, with frequencies of 56.67% and 50.00% in the ASD and the control groups, respectively.ConclusionThis study provides preliminary evidence that CGG repeat expansion in the FMR1 gene is unlikely to represent a major genetic contributor to ASD in Bangladeshi children. However, given the limited sample size, further investigations with a larger cohort are required to confirm these findings.

BackgroundFragile X syndrome (FXS) is the most common inherited form of intellectual disability caused by a CGG repeat expansion in the 5′ untranslated region of the FMR1 gene. When the number of repeats exceeds 200, the gene becomes hypermethylated and is transcriptionally silenced, resulting in FXS. Other allelic forms of the gene that are studied because of their instability or phenotypic consequence include intermediate alleles (45–54 CGG repeats) and premutation alleles (55–200 repeats). Normal alleles are classified as having <45 CGG repeats. Population screening studies have been conducted among American and Australian populations; however, large population‐based studies have not been completed in China.Methods and ResultsIn this work we present FXS screening results from 10,145 women of childbearing age from China. We first created and tested a standard panel that was comprised of normal, intermediate, premutation, and full mutation samples, and we performed the screening after confirming the consistency of genotyping results among laboratories.ConclusionBased on our findings, we have determined the intermediate and premutation carrier prevalence of 1/130 and 1/634, respectively, among Chinese women.

Fragile X syndrome (FXS) is characterized by intellectual impairment caused by CGG repeat expansion in the FMR1 gene. When repeats exceed 200, they induce DNA methylation of the promoter and the repeat region, resulting in transcriptional silencing of the FMR1 gene and the subsequent loss of FMRP protein. In the past decade or so, research has focused on the role of FMRP as an RNA-binding protein involved in translation inhibition in the brain in FXS model mice, particularly by slowing or stalling ribosome translocation on mRNA. More recent advances have shown that FMRP has a profound role in RNA splicing, at least in some cases by modulating the translation of splicing factor mRNAs. In a surprise, the human FMR1 gene is transcribed in most cases even with a full CGG expansion. However, much of the FMR1 that is produced is misspliced, which can be corrected by splice-switching antisense oligonucleotide (ASO) administration. Other recent findings suggest that inhibition of multiple kinases can demethylate the FMR1 gene and induce the formation of an R-loop in the CGG repeat region, leading to contraction of the repeat and FMRP restoration. These insights are paving the way for possible future therapeutic approaches for this disorder. We highlight the importance of FMRP restoration by ASO-mediated splice switching or CGG repeat modulation as key advances that may lead to successful treatments for FXS.

Hypomethylation of an Expanded FMR1 Allele Is Not Associated with a Global DNA Methylation Defect

This study determined whether CGG repeat length moderates the relationship between age and performance on selective measures of executive function in premutation carriers (PM) who are asymptomatic for a recently described late-onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). Forty PM men aged 18-69 years with a family history of fragile X syndrome underwent neuropsychological tests of inhibition and working memory. We examined only men who are asymptomatic for FXTAS. Multiple regression analyses were conducted to examine the moderating role of CGG repeat length on the relation between age and performance on inhibition and working memory tasks. With increasing age and only in men with an FMR1 expansion in the upper premutation range (>100 CGG repeats) was there an association between age and poorer task performance on selective executive function measures involving inhibition (p < 0.05) and executive working memory (p < 0.01). Men in the lower premutation range (<100 CGG repeats) were relatively risk-free from any cognitive aging effects associated with CGG repeat expansions. We conclude that neural networks in the prefrontal cortex may be highly susceptible to age-related neurotoxic effects in the upper size range of the FMR1 premutation. Future longitudinal studies will be needed to determine whether specific executive markers may serve to distinguish those at greatest risk for severe cognitive decline or dementia associated with FXTAS.

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.

Premutation in the FMR1 gene occur in the general population with an estimated prevalence 1 in 130-260 females and 1 in 250-810 males. Carriers of premutation are at risk of development of spectrum of neurological, psychiatric and immunological disorders in adulthood. Fragile X-associated disease caused by dynamic mutation (expansion of CGG repeats) can be divided into three disorders: FXS - Fragile X syndrome, FXPOI - Fragile X-associated primary ovarian insufficiency, FXTAS -Fragile X-associated tremor/ataxia syndrome, which can be present in few generations of one family. Immuno-mediated disorders are more common in premutation carriers as compared to control group, especially hypothyroidism and fibromyalgia. Although FMR1-associated conditions are not curable, timely diagnosis through genetic testing is important as it can lead to implementation of treatment strategies and behavioral interventions considered to improve symptoms. Knowledge of expanded allele status for females helps them to make more informed reproductive decisions.