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Background: Recently, an exonic GGC repeat expansion (RE) was identified by long-read genome sequencing in the ZFHX3 gen, causing spinocerebellar ataxia type 4 (SCA4), a dominant form of ataxia with sensory neuropathy. However, the analysis of larger cohorts of patients remained demanding, resulting in a challenge to diagnose patients and leaving the question of anticipation in SCA4 unanswered. Objectives: We aimed to develop a GGC repeat test for clinical SCA4 screening and to apply this test to screen two large German SCA pedigrees and samples of unrelated patients collected over the last 25 years. Methods: We modulated a commercial GGC-RE kit (Bio-Techne AmplideX® Asuragen® PCR/CE FMR1 Reagents) with ZFHX3-specific primers and adapted PCR conditions. The test was applied to patients and 50 healthy controls to determine the exact repeat number. Clinical data were revised and correlated with the expanded allele sizes and an exploratory analysis of structural MRI was performed. Results: Repeat size, determined by our protocol for (GGC)n RE analysis shows a strong inverse correlation between repeat length and age at onset and anticipation in subsequent generations. The phenotype also appears to be more strongly expressed in carriers of longer RE. Clinical red flags were slowed saccades, sensory neuropathy and autonomic dysfunction. Conclusion: Our protocol enables cost-effective and robust screening for the causative SCA4 RE within ZFHX3. Furthermore, detailed clinical data of our patients gives a more precise view on SCA4, which seems to be more common among patients with ataxia than expected. [ABSTRACT FROM AUTHOR]

Trehalose is a naturally occurring sugar found in various food and pharmaceutical preparations with the ability to enhance cellular proteostasis and reduce the formation of toxic intracellular protein aggregates, making it a promising therapeutic candidate for various neurodegenerative disorders. Here, we explored the effectiveness of nutritional trehalose supplementation in ameliorating symptoms in a mouse model of Fragile X-associated tremor/ataxia syndrome (FXTAS), an incurable late onset manifestation of moderately expanded trinucleotide CGG repeat expansion mutations in the 5' untranslated region of the fragile X messenger ribonucleoprotein 1 gene (FMR1). An inducible mouse model of FXTAS expressing 90 CGG repeats in the brain had been previously developed, which faithfully captures hallmarks of the disorder, the formation of intracellular inclusions, and the disturbance of motor function. Taking advantage of the inducible nature of the model, we investigated the therapeutic potential of orally administered trehalose under two regimens, modelling disease prevention and disease treatment. Trehalose's effectiveness in combating protein aggregation is frequently attributed to its ability to induce autophagy. Accordingly, trehalose supplementation under the prevention regimen ameliorated the formation of intranuclear inclusions and improved the motor deficiencies resulting from the induced expression of 90 CGG repeats, but it failed to reverse the existing nuclear pathology as a treatment strategy. Given the favorable safety profile of trehalose, it is promising to further explore the potential of this agent for early stage FXTAS. [ABSTRACT FROM AUTHOR]

Expansion of a CGG repeat in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene on the X chromosome is the cause of Fragile X Syndrome (FXS). The repeat length of unaffected individuals varies between 5-40 repeats, whereas >200 repeats are observed in cases of FXS. The intermediate range between 55-200 repeats is considered the premutation range and is observed in roughly 1:300 females and 1:900 males in the general population. With the availability of large-scale whole genome sequence (WGS) data and the development of computational tools to detect repeat expansions, we systematically examined the role of FMR1 premutation alleles in autism spectrum disorder (ASD) susceptibility, assess the prevalence, and consider the allelic stability between parents and offspring. We analyzed the WGS data of 22,053 subjects, including 32 FXS positive controls, 1359 population controls, and 5467 ASD families. We observed no FMR1 full mutation range repeats among the ASD parent-offspring families but identified 180 family members with premutation range alleles, which represents a higher prevalence compared to the independent WGS control sample and previous reports in the literature. A sex-specific analysis between probands and unaffected siblings did not reveal a significant increase in the burden of premutation alleles in either males or females with ASD. PCR validation, however, suggests an overestimation of the frequency of FMR1 premutation range alleles through computational analysis of WGS data. Overall, we show the utility of large-scale repeat expansion screening in WGS data and conclude that there is no apparent evidence of FMR1 premutation alleles contributing to ASD susceptibility.

The pathological huntingtin (HTT) trinucleotide repeat underlying Huntington disease (HD) continues to expand throughout life. Repeat length correlates both with earlier age at onset (AaO) and faster progression, making slowing its expansion an attractive therapeutic approach. Genome-wide association studies have identified candidate variants associated with altered AaO and progression, with many found in DNA mismatch repair (MMR)-associated genes. We examine whether lowering expression of these genes affects the rate of repeat expansion in human ex vivo models using HD iPSCs and HD iPSC-derived striatal medium spiny neuron-enriched cultures. We have generated a stable CRISPR interference HD iPSC line in which we can specifically and efficiently lower gene expression from a donor carrying over 125 CAG repeats. Lowering expression of each member of the MMR complexes MutS (MSH2, MSH3, and MSH6), MutL (MLH1, PMS1, PMS2, and MLH3), and LIG1 resulted in characteristic MMR deficiencies. Reduced MSH2, MSH3, and MLH1 slowed repeat expansion to the largest degree, while lowering either PMS1, PMS2, or MLH3 slowed it to a lesser degree. These effects were recapitulated in iPSC-derived striatal cultures where MutL factor expression was lowered. CRISPRi-mediated lowering of key MMR factor expression to levels feasibly achievable by current therapeutic approaches was able to effectively slow the expansion of the HTT CAG tract. We highlight members of the MutL family as potential targets to slow pathogenic repeat expansion with the aim to delay onset and progression of HD and potentially other repeat expansion disorders exhibiting somatic instability. [Display omitted] The expanded trinucleotide repeat underlying Huntington disease (HD) continues to lengthen across the carrier's lifetime and correlates with disease onset. A core of DNA mismatch repair factors has been identified as onset modifiers, and we show that lowering expression of these factors slows repeat expansion in striatal neurons derived from HD iPSCs. [ABSTRACT FROM AUTHOR]

Expanded CAG repeats in coding regions of different genes are the most common cause of dominantly inherited spinocerebellar ataxias (SCAs). These repeats are unstable through the germline, and larger repeats lead to earlier onset. We measured somatic expansion in blood samples collected from 30 SCA1, 50 SCA2, 74 SCA3, and 30 SCA7 individuals over a mean interval of 8.5 years, along with postmortem tissues and fetal tissues from SCA1, SCA3, and SCA7 individuals to examine somatic expansion at different stages of life. We showed that somatic mosaicism in the blood increases over time. Expansion levels are significantly different among SCAs and correlate with CAG repeat lengths. The level of expansion is greater in individuals with SCA7 who manifest disease compared to that of those who do not yet display symptoms. Brain tissues from SCA individuals have larger expansions compared to the blood. The cerebellum has the lowest mosaicism among the studied brain regions, along with a high expression of ATXN s and DNA repair genes. This was the opposite in cortices, with the highest mosaicism and lower expression of ATXN s and DNA repair genes. Fetal cortices did not show repeat instability. This study shows that CAG repeats are increasingly unstable during life in the blood and the brain of SCA individuals, with gene- and tissue-specific patterns. Large repeated sequences can cause spinocerebellar ataxias (SCAs). They expand in size over time in the blood and the brain, and this instability varies between SCAs and brain regions. This research helps us understand how these genetic changes evolve throughout a person's life, providing insights for future therapeutic strategies. [ABSTRACT FROM AUTHOR]

Introduction: Spinocerebellar ataxia type 36 (SCA36) is caused by large GGCCTG repeat expansion in the NOP56 gene. The genetic diagnosis based on Southern blot is expensive and time-consuming. This study aimed to evaluate the reliability and effectiveness of whole exome sequencing (WES) for routine genetic diagnosis of suspected SCA36 patients. Methods: Pathogenic repeat expansions for SCAs including SCA36 were first analyzed based on WES data using ExpansionHunter in five probands from SCA families, then the results were confirmed by triplet repeat primed polymerase chain reaction (TP-PCR) and Southern blot. Results: GGCCTG repeat expansion in NOP56 was indicated in all five probands by WES, then it was found in 11 SCA patients and three asymptomatic individuals by TP-PCR. The sizes of GGCCTG repeat expansions were confirmed to be 1,390–1,556 by Southern blot. The mean age at onset of the patients was 51.0 ± 9.3 (ranging from 41 to 71), and they presented slowly progressive cerebellar ataxia, atrophy and fasciculation in tongue or limb muscles. Conclusion: The patients were clinically and genetically diagnosed as SCA36. This study proposed that WES could be a rapid, reliable, and cost-effective routine test for the preliminarily detection of SCA36 and other ataxia diseases. [ABSTRACT FROM AUTHOR]

The C9orf72 hexanucleotide repeat expansion (HRE) is a common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The inheritance is autosomal dominant, but a high proportion of subjects with the mutation are simplex cases. One possible explanation is de novo expansions of unstable intermediate-length alleles (IAs). Using haplotype sharing trees (HSTs) with the haplotype analysis tool kit (HAPTK), we derived majority-based ancestral haplotypes of HRE samples and discovered that IAs containing ≥18–20 repeats share large haplotypes in common with the HRE. Using HSTs of HRE and IA samples, we demonstrate that the longer IA haplotypes are largely indistinguishable from HRE haplotypes and that several ≥18–20 IA haplotypes share over 5 Mb (>600 markers) haplotypes in common with the HRE haplotypes. These analysis tools allow physical understanding of the haplotype blocks shared with the majority-based ancestral haplotype. Our results demonstrate that the haplotypes with longer IAs belong to the same pool of haplotypes as the HRE and suggest that longer IAs represent potential premutation alleles. Using haplotype sharing trees and majority-based ancestral haplotypes, we demonstrate that the C9orf72 hexanucleotide repeat expansion and alleles with ≥18–20 repeats share large haplotype blocks, indicating very recent shared ancestry. This was in sharp contrast to the shorter alleles, suggesting that expansions might be formed from the longer repeat alleles. [ABSTRACT FROM AUTHOR]

Spinocerebellar ataxia type 31 (SCA31) is an autosomal‐dominant neurodegenerative condition caused by a 2.5–3.8 kb‐long complex repeat containing (TGGAA/TTCCA)n in an intron shared by two genes called brain expressed, associated with Nedd4 (BEAN1) and thymidine kinase 2 (TK2) located in the human chromosome 16q22.1. Since BEAN1 and TK2 are transcribed in mutually opposite directions in human brains, two independently transcribed RNAs containing either (UGGAA)n or (UUCCA)n are likely to associate with the pathogenesis of SCA31. Recently, a minor TK2 mRNA isoform called TK2‐EXT was confirmed to be transcribed in human cerebellum, suggesting that (UUCCA)n is indeed expressed. The level of TK2 mRNA and TK2 protein expression levels was both preserved in SCA31 human cerebellum, suggesting that the expression of (UUCCA)n does not affect the expression of TK2, and hence, the function of TK2 seemed to be preserved. On the other hand, the other penta‐nucleotide RNA repeat (UGGAA)n, expressed through BEAN1 transcription, is likely to conform toxicity through forming abnormal RNA structures called RNA foci in the nucleus of expressing cells. In addition, three proteins TDP‐43, FUS, and hnRNPA2/B1 that commonly have a capacity to bind with (UGGAA)n reduced the number of RNA foci, and ameliorated the phenotype brought by (UGGAA)n in Drosophila. A small compound naphthyridine carbamate dimer that binds to (UGGAA)n dampened the (UGGAA)n toxicity in Drosophila, further supporting the idea that (UGGAA)n expressed by BEAN1 is pathogenic. Therefore, a plausible approach to treat SCA31 may be considered by administering agents with a capacity binding to (UGGAA)n. [ABSTRACT FROM AUTHOR]

A self-pollinated, annual, and nutrient-rich legume, Lens tomentosus has excellent economic value since seeds of L. tomentosus are used all over the world. Chloroplast genome provides a valuable resource for phylogenetic reconstruction and divergence between legume species because of its maternal inheritance and low rearrangement rate. L. tomentosus complete chloroplast genome sequence was revealed through next-generation sequencing in this study. The length of L. tomentosus chloroplast genome was found to be 123,548 bp. The annotated genome carries 107 genes, 77 of which were protein-coding, 4 rRNA, and 27 tRNA genes. While other legume species contain two inverted repeat regions, two Lens species, L. tomentosus, and L. culinaris, have only one which classifies them in the IRLC clade. In addition, 66 simple sequence repeats (SSRs) were found in the L. tomentosus chloroplast genome, and multiple sequence alignment analysis revealed SSR polymorphisms unique to L. tomentosus. As a result, certain genic regions, such as ycf1 and paf1 (ycf3), can be potential markers for cultivar identification in future studies. This study will provide genomic data resources to the lentil genetics as it sequenced the first complete chloroplast genome L. tomentosus and compared it with L. culinaris chloroplast genome. [ABSTRACT FROM AUTHOR]

A recessive Short Tandem Repeat expansion in RFC1 has been found to be associated with cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), and to be a frequent cause of late onset ataxia and sensory neuropathy. The usual procedure for sizing these expansions is based on Southern Blotting (SB), a time-consuming and a relatively imprecise technique. In this paper, we compare SB with Optical Genome Mapping (OGM), a method for detecting Structural Variants (SVs) based on the measurement of distances between fluorescently labelled probes, for the diagnosis of RFC1 CANVAS and disease spectrum. The two methods are applied to 17 CANVAS patients' blood samples and resulting sizes compared, showing a good agreement. Further, long-read sequencing is used for two patients to investigate the agreement of sizes with either SB or OGM. Our study concludes that OGM represents a viable alternative to SB, allowing for a simpler technique, a more precise sizing of the expansion and ability to expand analysis of SV in the entire genome as opposed to SB which is a locus specific method. [ABSTRACT FROM AUTHOR]

Abstract Familial adult myoclonic epilepsy (FAME) is an adult‐onset neurological disease characterized by cortical tremor, myoclonus, and seizures due to a pentanucleotide repeat expansion: a combination of pathogenic TTTCA expansion associated with a TTTTA repeat in introns of six different genes. Repeat‐primed PCR (RP‐PCR) is an inexpensive test for expansions at known loci. The analysis of the SAMD12 locus revealed that the repeats have different size, configuration, and composition. The TTTCA repeats can be very long (>1000 repeats) but also very short (14 being the shortest identified). Here, we report siblings of European descent with the clinical diagnosis of FAME yet a negative RP‐PCR test. Using short‐read genome sequencing, we identified the pentanucleotide expansion in intron 4 of SAMD12, which was confirmed by CRIPSR‐Cas9‐mediated enrichment and long‐read sequencing to be of (TTTTA)~879(TTTCA)3(TTTTA)7(TTTCA)7 configuration. Our finding is the first to associate the SAMD12 locus in European patients with FAME and currently represents the shortest identified TTTCA expansion. Our results suggest that the SAMD12 locus should be tested in patients with suspected FAME independent of ethnicity. Furthermore, RP‐PCR may miss the underlying mutation, and genome sequencing may be needed to confirm the pathogenic repeat.

RNA translation is tightly controlled in eukaryotic cells to regulate gene expression and maintain proteome homeostasis. RNA binding proteins, translation factors, and cell signaling pathways all modulate the translation process. Defective translation is involved in multiple neurological diseases including amyotrophic lateral sclerosis (ALS). ALS is a progressive neurodegenerative disorder and poses a major public health challenge worldwide. Over the past few years, tremendous advances have been made in the understanding of the genetics and pathogenesis of ALS. Dysfunction of RNA metabolisms, including RNA translation, has been closely associated with ALS. Here, we first introduce the general mechanisms of translational regulation under physiological and stress conditions and review well-known examples of translation defects in neurodegenerative diseases. We then focus on ALS-linked genes and discuss the recent progress on how translation is affected by various mutant genes and the repeat expansion-mediated non-canonical translation in ALS. [ABSTRACT FROM AUTHOR]

Background: More than 50 loci are associated with spinocerebellar ataxia (SCA), and the most frequent subtypes share nucleotide repeats expansion, especially CAG expansion. Objective: The objective of this study was to confirm a novel SCA subtype caused by CAG expansion. Methods: We performed long‐read whole‐genome sequencing combined with linkage analysis in a five‐generation Chinese family, and the finding was validated in another pedigree. The three‐dimensional structure and function of THAP11 mutant protein were predicted. Polyglutamine (polyQ) toxicity of THAP11 gene with CAG expansion was assessed in skin fibroblasts of patients, human embryonic kidney 293 and Neuro‐2a cells. Results: We identified THAP11 as the novel causative SCA gene with CAG repeats ranging from 45 to 100 in patients with ataxia and from 20 to 38 in healthy control subjects. Among the patients, the number of CAA interruptions within CAG repeats was decreased to 3 (up to 5–6 in controls), whereas the number of 3′ pure CAG repeats was up to 32 to 87 (4–16 in controls), suggesting that the toxicity of polyQ protein was length dependent on the pure CAG repeats. Intracellular aggregates were observed in cultured skin fibroblasts from patients. THAP11 polyQ protein was more intensely distributed in the cytoplasm of cultured skin fibroblasts from patients, which was replicated with in vitro cultured neuro‐2a transfected with 54 or 100 CAG repeats. Conclusions: This study identified a novel SCA subtype caused by intragenic CAG repeat expansion in THAP11 with intracellular aggregation of THAP11 polyQ protein. Our findings extended the spectrum of polyQ diseases and offered a new perspective in understanding polyQ‐mediated toxic aggregation. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

There is growing recognition that epivariations, most often recognized as promoter hypermethylation events that lead to gene silencing, are associated with a number of human diseases. However, little information exists on the prevalence and distribution of rare epigenetic variation in the human population. In order to address this, we performed a survey of methylation profiles from 23,116 individuals using the Illumina 450k array. Using a robust outlier approach, we identified 4,452 unique autosomal epivariations, including potentially inactivating promoter methylation events at 384 genes linked to human disease. For example, we observed promoter hypermethylation of BRCA1 and LDLR at population frequencies of ∼1 in 3,000 and ∼1 in 6,000, respectively, suggesting that epivariations may underlie a fraction of human disease which would be missed by purely sequence-based approaches. Using expression data, we confirmed that many epivariations are associated with outlier gene expression. Analysis of variation data and monozygous twin pairs suggests that approximately two-thirds of epivariations segregate in the population secondary to underlying sequence mutations, while one-third are likely sporadic events that occur post-zygotically. We identified 25 loci where rare hypermethylation coincided with the presence of an unstable CGG tandem repeat, validated the presence of CGG expansions at several loci, and identified the putative molecular defect underlying most of the known folate-sensitive fragile sites in the genome. Our study provides a catalog of rare epigenetic changes in the human genome, gives insight into the underlying origins and consequences of epivariations, and identifies many hypermethylated CGG repeat expansions.

Familial adult myoclonus epilepsy (FAME) results from the same pathogenic TTTTA/TTTCA pentanucleotide repeat expansion in six distinct genes encoding proteins with different subcellular localizations and very different functions, which poses the issue of what causes the neurobiological disturbances that lead to the clinical phenotype. Postmortem and electrophysiological studies have pointed to cortical hyperexcitability as well as dysfunction and neurodegeneration of both the cortex and cerebellum of FAME subjects. FAME expansions, contrary to the same expansion in DAB1 causing spinocerebellar ataxia type 37, seem to have no or limited impact on their recipient gene expression, which suggests a pathophysiological mechanism independent of the gene and its function. Current hypotheses include toxicity of the RNA molecules carrying UUUCA repeats, or toxicity of polypeptides encoded by the repeats, a mechanism known as repeat‐associated non‐AUG translation. The analysis of postmortem brains of FAME1 expansion (in SAMD12) carriers has revealed the presence of RNA foci that could be formed by the aggregation of RNA molecules with abnormal UUUCA repeats, but evidence is still lacking for other FAME subtypes. Even when the expansion is located in a gene ubiquitously expressed, expression of repeats remains undetectable in peripheral tissues (blood, skin). Therefore, the development of appropriate cellular models (induced pluripotent stem cell‐derived neurons) or the study of affected tissues in patients is required to elucidate how FAME repeat expansions located in unrelated genes lead to disease. [ABSTRACT FROM AUTHOR]

Familial adult myoclonic epilepsy (FAME) is an adult‐onset neurological disease characterized by cortical tremor, myoclonus, and seizures due to a pentanucleotide repeat expansion: a combination of pathogenic TTTCA expansion associated with a TTTTA repeat in introns of six different genes. Repeat‐primed PCR (RP‐PCR) is an inexpensive test for expansions at known loci. The analysis of the SAMD12 locus revealed that the repeats have different size, configuration, and composition. The TTTCA repeats can be very long (>1000 repeats) but also very short (14 being the shortest identified). Here, we report siblings of European descent with the clinical diagnosis of FAME yet a negative RP‐PCR test. Using short‐read genome sequencing, we identified the pentanucleotide expansion in intron 4 of SAMD12, which was confirmed by CRIPSR‐Cas9‐mediated enrichment and long‐read sequencing to be of (TTTTA)~879(TTTCA)3(TTTTA)7(TTTCA)7 configuration. Our finding is the first to associate the SAMD12 locus in European patients with FAME and currently represents the shortest identified TTTCA expansion. Our results suggest that the SAMD12 locus should be tested in patients with suspected FAME independent of ethnicity. Furthermore, RP‐PCR may miss the underlying mutation, and genome sequencing may be needed to confirm the pathogenic repeat. [ABSTRACT FROM AUTHOR]

Hexanucleotide GGGGCC repeat expansion in C9ORF72 is the most prevalent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One pathogenic mechanism is the aberrant accumulation of dipeptide repeat (DPR) proteins produced by the unconventional translation of expanded RNA repeats. Here, we performed genome-wide CRISPR-Cas9 screens for modifiers of DPR protein production in human cells. We found that DDX3X, an RNA helicase, suppresses the repeat-associated non-AUG translation of GGGGCC repeats. DDX3X directly binds to (GGGGCC)n RNAs but not antisense (CCCCGG)n RNAs. Its helicase activity is essential for the translation repression. Reduction of DDX3X increases DPR levels in C9ORF72-ALS/FTD patient cells and enhances (GGGGCC)n-mediated toxicity in Drosophila. Elevating DDX3X expression is sufficient to decrease DPR levels, rescue nucleocytoplasmic transport abnormalities, and improve survival of patient iPSC-differentiated neurons. This work identifies genetic modifiers of DPR protein production and provides potential therapeutic targets for C9ORF72-ALS/FTD.

Myotonic dystrophy (DM) is the most common muscular dystrophy in adults. Dominantly inherited CTG and CCTG repeat expansions in DMPK and CNBP genes cause DM type 1 (DM1) and 2 (DM2), respectively. These genetic defects lead to the abnormal splicing of different mRNA transcripts, which are thought to be responsible for the multiorgan involvement of these diseases. In ours and others' experience, cancer frequency in patients with DM appears to be higher than in the general population or non-DM muscular dystrophy cohorts. There are no specific guidelines regarding malignancy screening in these patients, and the general consensus is that they should undergo the same cancer screening as the general population. Here, we review the main studies that investigated cancer risk (and cancer type) in DM cohorts and those that researched potential molecular mechanisms accounting for DM carcinogenesis. We propose some evaluations to be considered as malignancy screening in patients with DM, and we discuss DM susceptibility to general anesthesia and sedatives, which are often needed for the management of cancer. This review underscores the importance of monitoring the adherence of patients with DM to malignancy screenings and the need to design studies that determine whether they would benefit from a more intensified cancer screening than the general population. [ABSTRACT FROM AUTHOR]

Genetics are fundamental to understanding the pathophysiology of neurological disease, including movement disorders. Genetic testing in clinical practice has changed dramatically over the last few decades. While the likelihood of establishing an etiological diagnosis is greater now with increased access to testing and more advanced technologies, clinicians face challenges when deciding whether to test, then selecting the appropriate test, and ultimately interpreting and sharing the results with patients and families. In this review, we use a case-based approach to cover core aspects of genetic testing for the neurologist, namely, genetic testing in Parkinson's disease, interpretation of inconclusive genetic test reports, and genetic testing for repeat expansion disorders using Huntington disease as a prototype. [ABSTRACT FROM AUTHOR]

Hexanucleotide repeats expansion in the C9orf72 gene is the most common cause of familial and sporadic amyotrophic lateral sclerosis (ALS) cases in Europe. In this study we aimed to determine the size and distribution of C9orf72 alleles, and investigate the possible association of the repeat size with several clinical parameters in ALS patients from Serbia. Patients were recruited from 2011-2021 and analysed using fragment length analysis and Southern blot. Out of 383 ALS patients, we have detected 31 (8.09%) patients with repeat expansion. In the total ALS cohort, clinical overlap with frontotemporal dementia (FTD) was registered in 17 (4.44%) patients, and among them, 5 (29.41%) were expansion carriers. There was no difference in the age of onset, age at the examination or disease duration, gender, and the frequency of spinal and bulbar onset between patients with and without C9orf72 expansion. The presence of positive family history (34.48% vs. 15.65%) and FTD (16.13% vs. 3.41%) was more frequent in expansion-positive vs. expansion-negative patients. In expansion-positive patients, significantly higher values of the largest detected repeat were found in patients with ALS in the family, and in expansion-negative, a higher median value of the smaller allele was noted in patients with a positive family history of ALS, dementia, and both in comparison to the rest of the group. A correlation of the repeat size was not found with the age of onset in both patients with and without the expansion. This is the first detailed study of C9orf72 sizing in ALS patients from Serbia. Our results emphasize the need for C9orf72 genetic screening in ALS patients with/without FTD. [ABSTRACT FROM AUTHOR]

IntroductionNeuromuscular disorders (NMDs) have a heterogeneous etiology. A genetic diagnosis is key to personalized healthcare and access to targeted treatment for the affected individuals.MethodsIn this study, 861 patients with NMDs were analyzed with genome sequencing and comprehensive variant calling including single nucleotide variants, small insertions/deletions (SNVs/INDELs), and structural variants (SVs) in a panel of 895 NMD genes, as well as short tandem repeat expansions (STRs) at 28 loci. In addition, for unsolved cases with an unspecific clinical presentation, the analysis of a panel with OMIM disease genes was added.ResultsIn the cohort, 27% (232/861) of the patients harbored pathogenic variants, of which STRs and SVs accounted for one-third of the patients (71/232). The variants were found in 107 different NMD genes. Furthermore, 18 pediatric patients harbored pathogenic variants in non-NMD genes.DiscussionOur results highlight that for children with unspecific hypotonia, a genome-wide analysis rather than a disease-based gene panel should be considered as a diagnostic approach. More importantly, our results clearly show that it is crucial to include STR- and SV-analyses in the diagnostics of patients with neuromuscular disorders.

A recessive Short Tandem Repeat expansion in RFC1 has been found to be associated with cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), and to be a frequent cause of late onset ataxia and sensory neuropathy. The usual procedure for sizing these expansions is based on Southern Blotting (SB), a time-consuming and a relatively imprecise technique. In this paper, we compare SB with Optical Genome Mapping (OGM), a method for detecting Structural Variants (SVs) based on the measurement of distances between fluorescently labelled probes, for the diagnosis of RFC1 CANVAS and disease spectrum. The two methods are applied to 17 CANVAS patients’ blood samples and resulting sizes compared, showing a good agreement. Further, long-read sequencing is used for two patients to investigate the agreement of sizes with either SB or OGM. Our study concludes that OGM represents a viable alternative to SB, allowing for a simpler technique, a more precise sizing of the expansion and ability to expand analysis of SV in the entire genome as opposed to SB which is a locus specific method.

Background: Coding and noncoding repeat expansions are an important cause of neurodegenerative diseases. Objective: This study determined the clinical and genetic features of a large German family that has been followed for almost 2 decades with an autosomal dominantly inherited spinocerebellar ataxia (SCA) and independent co‐occurrence of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Methods: We carried out clinical examinations and telephone interviews, reviewed medical records, and performed magnetic resonance imaging and positron emission tomography scans of all available family members. Comprehensive genetic investigations included linkage analysis, short‐read genome sequencing, long‐read sequencing, repeat‐primed polymerase chain reaction, and Southern blotting. Results: The family comprises 118 members across seven generations, 30 of whom were definitely and five possibly affected. In this family, two different pathogenic mutations were found, a heterozygous repeat expansion in C9ORF72 in four patients with ALS/FTD and a heterozygous repeat expansion in DAB1 in at least nine patients with SCA, leading to a diagnosis of DAB1‐related ataxia (ATX‐DAB1; SCA37). One patient was affected by ALS and SCA and carried both repeat expansions. The repeat in DAB1 had the same configuration but was larger than those previously described ([ATTTT]≈75[ATTTC]≈40‐100[ATTTT]≈415). Clinical features in patients with SCA included spinocerebellar symptoms, sometimes accompanied by additional ophthalmoplegia, vertical nystagmus, tremor, sensory deficits, and dystonia. After several decades, some of these patients suffered from cognitive decline and one from additional nonprogressive lower motor neuron affection. Conclusion: We demonstrate genetic and clinical findings during an 18‐year period in a unique family carrying two different pathogenic repeat expansions, providing novel insights into their genotypic and phenotypic spectrums. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

Background: X‐linked dystonia‐parkinsonism (XDP) is a neurodegenerative disorder caused by the intronic insertion of a SINE‐VNTR‐Alu (SVA) retrotransposon carrying an (AGAGGG)n repeat expansion in the TAF1 gene. The molecular mechanisms by which this mutation causes neurodegeneration remain elusive. Objectives: We investigated whether (AGAGGG)n repeats undergo repeat‐associated non‐AUG (RAN) translation, a pathogenic mechanism common among repeat expansion diseases. Methods: XDP‐specific RAN translation reporter plasmids were generated, transfected in HEK293 cells, and putative dipeptide repeat proteins (DPRs) were detected by Western blotting. Immunocytochemistry was performed in COS‐7 cells to determine the subcellular localization of one DPR. Results: We detected putative DPRs from two reading frames, supporting the translation of poly‐(Glu‐Gly) and poly‐(Arg‐Glu) species. XDP RAN translation initiates within the (AGAGGG)n sequence and poly‐(Glu‐Gly) DPRs formed nuclear inclusions in transfected cells. Conclusions: In summary, our work provides the first in‐vitro proof of principle that the XDP‐linked (AGAGGG)n repeat expansions can undergo RAN translation. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society [ABSTRACT FROM AUTHOR]

Recently, an intronic biallelic (AAGGG)n repeat expansion in RFC1 was shown to be a cause of CANVAS and adult-onset ataxia in multiple populations. As the prevalence of the RFC1 repeat expansion in Dutch cases was unknown, we retrospectively tested 9 putative CANVAS cases and two independent cohorts (A and B) of 395 and 222 adult-onset ataxia cases, respectively, using the previously published protocol and, for the first time optical genome mapping to determine the size of the expanded RFC1 repeat. We identified the biallelic (AAGGG)n repeat expansion in 5/9 (55%) putative CANVAS patients and in 10/617 (1.6%; cohorts A + B) adult-onset ataxia patients. In addition to the AAGGG repeat motif, we observed a putative GAAGG repeat motif in the repeat expansion with unknown significance in two adult-onset ataxia patients. All the expanded (AAGGG)n repeats identified were in the range of 800–1299 repeat units. The intronic biallelic RFC1 repeat expansion thus explains a number of the Dutch adult-onset ataxia cases that display the main clinical features of CANVAS, and particularly when ataxia is combined with neuropathy. The yield of screening for RFC1 expansions in unselected cohorts is relatively low. To increase the current diagnostic yield in ataxia patients, we suggest adding RFC1 screening to the genetic diagnostic workflow by using advanced techniques that attain long fragments. [ABSTRACT FROM AUTHOR]

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is an inherited late-onset neurological disease caused by bi-allelic AAGGG pentanucleotide expansions within intron 2 of RFC1. Despite extensive studies, the pathophysiological mechanism of these intronic expansions remains elusive. We screened by clinical exome sequencing two unrelated patients presenting with late-onset ataxia. A repeat-primer polymerase chain reaction was used for RFC1 AAGGG intronic expansion identification. RFC1 mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction. We identified the first two CANVAS affected patients who are compound heterozygous for RFC1 truncating variants (p.Arg388* and c.575delA, respectively) and a pathological AAGGG expansion. RFC1 expression studies in whole blood showed a significant reduction of RFC1 mRNA for both patients compared to three patients with bi-allelic RFC1 expansions. In conclusion, this observation provides clues that suggest bi-allelic RFC1 conditional loss-of-function as the cause of the disease. [ABSTRACT FROM AUTHOR]

Myotonic dystrophy (DM) is the most common form of muscular dystrophy in adults, caused by unstable genomic expansions of CTG or CCTG repeats. Mutant RNA transcripts containing expanded repeats form ribonuclear foci and cause a toxic gain‐of‐function by perturbing splicing factors in the nucleus, resulting in misregulation of alternative pre‐mRNA splicing, known as "spliceopathy." The misregulated splicing is thought to be responsible for multisystemic symptoms in DM. Although no curative treatment exists, recent advances in basic and translational research provide clues on therapeutic interventions for DM. Here, the RNA‐mediated molecular pathomechamism and therapeutic approaches targeting the toxic RNA with antisense oligonucleotides and small molecules are reviewed. [ABSTRACT FROM AUTHOR]

Introduction: The biallelic repeat expansion (AAGGG)exp in RFC1 causes cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). Recently, cognitive impairment has been reported in patients with CANVAS and a broader neurodegenerative process associated with RFC1 has been suggested. Furthermore, rare cases of multiple system atrophy, Parkinson's disease, amyotrophic lateral sclerosis or CANVAS with features of dementia with Lewy bodies have been found.Objective: We hypothesized that the biallelic (AAGGG)exp is associated with neurodegeneration manifested as cognitive symptoms and that atypical RFC1 disease may be found among patients with cognitive disorder.Methods: Clinical data on nine patients with biallelic (AAGGG)exp were reviewed and 564 patients with Alzheimer's disease or frontotemporal dementia (FTD) were investigated for biallelic RFC1 (AAGGG)exp.Results: Five patients with biallelic (AAGGG)exp were found with a cognitive impairment and in four of them the phenotype resembled FTD. However, biallelic (AAGGG)exp was not detected among patients with Alzheimer's disease or FTD.Conclusion: Cognitive impairment is a feature in patients with the biallelic (AAGGG)exp, but the pathogenic expansion seems to be rare in patients with dementia. Studies on patients with diverse phenotypes would be useful to further explore the involvement of RFC1 in neuronal degeneration and to identify atypical phenotypes, which should be taken into account in clinical practice. [ABSTRACT FROM AUTHOR]

Expansion of a CGG repeat in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene on the X chromosome is the cause of Fragile X Syndrome (FXS). The repeat length of unaffected individuals varies between 5–40 repeats, whereas >200 repeats are observed in cases of FXS. The intermediate range between 55–200 repeats is considered the premutation range and is observed in roughly 1:300 females and 1:900 males in the general population. With the availability of large-scale whole genome sequence (WGS) data and the development of computational tools to detect repeat expansions, we systematically examined the role of FMR1 premutation alleles in autism spectrum disorder (ASD) susceptibility, assess the prevalence, and consider the allelic stability between parents and offspring. We analyzed the WGS data of 22,053 subjects, including 32 FXS positive controls, 1359 population controls, and 5467 ASD families. We observed no FMR1 full mutation range repeats among the ASD parent-offspring families but identified 180 family members with premutation range alleles, which represents a higher prevalence compared to the independent WGS control sample and previous reports in the literature. A sex-specific analysis between probands and unaffected siblings did not reveal a significant increase in the burden of premutation alleles in either males or females with ASD. PCR validation, however, suggests an overestimation of the frequency of FMR1 premutation range alleles through computational analysis of WGS data. Overall, we show the utility of large-scale repeat expansion screening in WGS data and conclude that there is no apparent evidence of FMR1 premutation alleles contributing to ASD susceptibility.

Abstract Background The genetics of cerebellar ataxia is complex. Hundreds of causative genes have been identified, but only a few cause more than single cases. The spectrum of ataxia-causing genes differs considerably between populations. The aim of the study was to investigate the molecular epidemiology of ataxia in the Finnish population. Patients and methods All patients in hospital database were reviewed for the diagnosis of unspecified ataxia. Acquired ataxias and nongenetic ataxias such as those related to infection, trauma or stroke were excluded. Sixty patients with sporadic ataxia with unknown etiology and 36 patients with familial ataxia of unknown etiology were recruited in the study. Repeat expansions in the SCA genes (ATXN1, 2, 3, 7, 8/OS, CACNA1A, TBP), FXN, and RFC1 were determined. Point mutations in POLG, SPG7 and in mitochondrial DNA (mtDNA) were investigated. In addition, DNA from 8 patients was exome sequenced. Results A genetic cause of ataxia was found in 33 patients (34.4%). Seven patients had a dominantly inherited repeat expansion in ATXN8/OS. Ten patients had mitochondrial ataxia resulting from mutations in nuclear mitochondrial genes POLG or RARS2, or from a point mutation m.8561C > G or a single deletion in mtDNA. Interestingly, five patients were biallelic for the recently identified pathogenic repeat expansion in RFC1. All the five patients presented with the phenotype of cerebellar ataxia, neuropathy, and vestibular areflexia (CANVAS). Moreover, screening of 54 patients with Charcot-Marie-Tooth neuropathy revealed four additional patients with biallelic repeat expansion in RFC1, but none of them had cerebellar symptoms. Conclusions Expansion in ATXN8/OS results in the majority of dominant ataxias in Finland, while mutations in RFC1 and POLG are the most common cause of recessive ataxias. Our results suggest that analysis of RFC1 should be included in the routine diagnostics of idiopathic ataxia and Charcot-Marie-Tooth polyneuropathy.

Expansion of hexanucleotide repeats (G4C2) in the non-coding region of the C9orf72 gene is the most known genetic cause of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and the combined ALS/FTD phenotype. Besides ALS and FTD, G4C2 repeat expansions were detected in other neurological disorders with variable frequency. These include, among others, two forms of atypical Parkinsonism, multiple system atrophy (MSA) and progressive supranuclear palsy (PSP). This study aimed to assess the potential role of C9orf72 repeat expansions among Serbian patients diagnosed with MSA and PSP. Genomic DNA of 44 MSA patients, 73 PSP patients, and 96 controls was extracted from peripheral blood, and normal C9orf72 alleles were analyzed by standard quantitative fluorescence polymerase chain reaction (QF-PCR) and fragment analysis. Subsequently, for all samples presenting a single allele, repeat-primed PCR was performed with two different sets of primers to avoid a false-negative result. Thirty repeats were used as a pathogenic cut-off and 20-29 repeats for the intermediate alleles. No pathological C9orf72 expansions were detected in the MSA and PSP patients nor the control subjects. In the MSA group, the most common was the allele with 2 repeats, and the largest repeat number was 14. Among PSP patients, the most common allele also had 2 repeats, while the largest detected repeat size within the normal range was 17. Also, we identified one PSP patient that had an intermediate size allele (25 repeats). We did not find correlation between the number of repeats and disease onset, age at the time of examination, or disease duration in MSA or PSP patients. Regarding family history, in PSP the sum of both allele repeats numbers was higher in patients with positive family history than in sporadic cases. The results presented in this study are the first systematic assessment of C9orf72 allele sizes among patients diagnosed with MSA and PSP in the Serbian population. Although the potential role of intermediate C9orf72 repeats in neurodegenerative disorders is still to be elucidated, our results support the current knowledge that C9orf72 repeat expansions are not associated with MSA and PSP. [ABSTRACT FROM AUTHOR]

Eukaryotic translation is tightly regulated to ensure that protein production occurs at the right time and place. Recent studies on abnormal repeat proteins, especially in age-dependent neurodegenerative diseases caused by nucleotide repeat expansion, have highlighted or identified two forms of unconventional translation initiation: usage of AUG-like sites (near cognates) or repeat-associated non-AUG (RAN) translation. We discuss how repeat proteins may differ due to not just unconventional initiation, but also ribosomal frameshifting and/or imperfect repeat DNA replication, expansion, and repair, and we highlight how research on translation of repeats may uncover insights into the biology of translation and its contribution to disease.

Spinocerebellar ataxia type 10 (SCA10) is a rare autosomal dominant ataxia caused by a large expansion of the (ATTCT)n repeat in ATXN10. SCA10 was described in Native American and Asian individuals which prompted a search for an expanded haplotype to confirm a common ancestral origin for the expansion event. All patients with SCA10 expansions in our cohort share a single haplotype defined at the 5'-end by the minor allele of rs41524547, located ~35 kb upstream of the SCA10 expansion. Intriguingly, rs41524547 is located within the miRNA gene, MIR4762, within its DROSHA cleavage site and just outside the seed sequence for mir4792-5p. The world-wide frequency of rs41524547-G is less than 5% and found almost exclusively in the Americas and East Asia-a geographic distribution that mirrors reported SCA10 cases. We identified rs41524547-G(+) DNA from the 1000 Genomes/International Genome Sample Resource and our own general population samples and identified SCA10 repeat expansions in up to 25% of these samples. The reduced penetrance of these SCA10 expansions may be explained by a young (pre-onset) age at sample collection, a small repeat size, purity of repeat units, or the disruption of miR4762-5p function. We conclude that rs41524547-G is the most robust at-risk SNP allele for SCA10, is useful for screening of SCA10 expansions in population genetics studies and provides the most compelling evidence to date for a single, prehistoric origin of SCA10 expansions sometime prior to or during the migration of individuals across the Bering Land Bridge into the Americas., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Expansion of an intronic (GGGGCC)n repeat within the C9ORF72 gene is the most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (C9-FTD/ALS), characterized with aberrant repeat RNA foci and noncanonical translation-produced dipeptide repeat (DPR) protein inclusions. Here, we elucidate that the (GGGGCC)n repeat RNA co-localizes with nuclear speckles and alters their phase separation properties and granule dynamics. Moreover, the essential nuclear speckle scaffold protein SRRM2 is sequestered into the poly-GR cytoplasmic inclusions in the C9-FTD/ALS mouse model and patient postmortem tissues, exacerbating the nuclear speckle dysfunction. Impaired nuclear speckle integrity induces global exon skipping and intron retention in human iPSC-derived neurons and causes neuronal toxicity. Similar alternative splicing changes can be found in C9-FTD/ALS patient postmortem tissues. This work identified novel molecular mechanisms of global RNA splicing defects caused by impaired nuclear speckle function in C9-FTD/ALS and revealed novel potential biomarkers or therapeutic targets., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Abstract Background Screening for short tandem repeat (STR) expansions in next-generation sequencing data can enable diagnosis, optimal clinical management/treatment, and accurate genetic counseling of patients with repeat expansion disorders. We aimed to develop an efficient computational workflow for reliable detection of STR expansions in next-generation sequencing data and demonstrate its clinical utility. Methods We characterized the performance of eight STR analysis methods (lobSTR, HipSTR, RepeatSeq, ExpansionHunter, TREDPARSE, GangSTR, STRetch, and exSTRa) on next-generation sequencing datasets of samples with known disease-causing full-mutation STR expansions and genomes simulated to harbor repeat expansions at selected loci and optimized their sensitivity. We then used a machine learning decision tree classifier to identify an optimal combination of methods for full-mutation detection. In Burrows-Wheeler Aligner (BWA)-aligned genomes, the ensemble approach of using ExpansionHunter, STRetch, and exSTRa performed the best (precision = 82%, recall = 100%, F1-score = 90%). We applied this pipeline to screen 301 families of children with suspected genetic disorders. Results We identified 10 individuals with full-mutations in the AR, ATXN1, ATXN8, DMPK, FXN, or HTT disease STR locus in the analyzed families. Additional candidates identified in our analysis include two probands with borderline ATXN2 expansions between the established repeat size range for reduced-penetrance and full-penetrance full-mutation and seven individuals with FMR1 CGG repeats in the intermediate/premutation repeat size range. In 67 probands with a prior negative clinical PCR test for the FMR1, FXN, or DMPK disease STR locus, or the spinocerebellar ataxia disease STR panel, our pipeline did not falsely identify aberrant expansion. We performed clinical PCR tests on seven (out of 10) full-mutation samples identified by our pipeline and confirmed the expansion status in all, showing absolute concordance between our bioinformatics and molecular findings. Conclusions We have successfully demonstrated the application of a well-optimized bioinformatics pipeline that promotes the utility of genome-wide sequencing as a first-tier screening test to detect expansions of known disease STRs. Interrogating clinical next-generation sequencing data for pathogenic STR expansions using our ensemble pipeline can improve diagnostic yield and enhance clinical outcomes for patients with repeat expansion disorders.

Myotonic dystrophy type 2 (DM2) is caused by CCTG repeat expansions in the CNBP gene, comprising 75 to >11,000 units and featuring extensive mosaicism, making it challenging to sequence fully expanded alleles. To overcome these limitations, we used PCR-free Cas9-mediated nanopore sequencing to characterize CNBP repeat expansions at the single-nucleotide level in nine DM2 patients. The length of normal and expanded alleles can be assessed precisely using this strategy, agreeing with traditional methods, and revealing the degree of mosaicism. We also sequenced an entire ~50 kbp expansion, which has not been achieved previously for DM2 or any other repeat-expansion disorders. Our approach precisely counted the repeats and identified the repeat pattern for both short interrupted and uninterrupted alleles. Interestingly, in the expanded alleles, only two DM2 samples featured the expected pure CCTG repeat pattern, while the other seven presented also TCTG blocks at the 3′ end, which have not been reported before in DM2 patients, but confirmed hereby with orthogonal methods. The demonstrated approach simultaneously determines repeat length, structure/motif, and the extent of somatic mosaicism, promising to improve the molecular diagnosis of DM2 and achieve more accurate genotype–phenotype correlations for the better stratification of DM2 patients in clinical trials.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized primarily by progressive loss of motor neurons. Although ALS occurs worldwide and the frequency and spectrum of identifiable genetic causes of disease varies across populations, very few studies have included African subjects. In addition to a hexanucleotide repeat expansion (RE) in C9orf72, the most common genetic cause of ALS in Europeans, REs in ATXN2, NIPA1 and ATXN1 have shown variable associations with ALS in Europeans. Intermediate range expansions in some of these genes (e.g. ATXN2) have been reported as potential risk factors, or phenotypic modifiers, of ALS. Pathogenic expansions in NOP56 cause spinocerebellar ataxia-36, which can present with prominent motor neuron degeneration. Here we compare REs in these genes in a cohort of Africans with ALS and population controls using whole genome sequencing data. Targeting genotyping of short tandem repeats at known loci within ATXN2, NIPA1, ATXN1 and NOP56 was performed using ExpansionHunter software in 105 Southern African (SA) patients with ALS. African population controls were from an in-house SA population control database (n = 25), the SA Human Genome Program (n = 24), the Simons Genome Diversity Project (n = 39) and the Illumina Polaris Diversity Cohort (IPDC) dataset (n = 50). We found intermediate RE alleles in ATXN2 (27–33 repeats) and ATXN1 (33–35 repeats), and NIPA1 long alleles (≥8 repeats) were rare in Africans, and not associated with ALS (p > 0.17). NOP56 showed no expanded alleles in either ALS or controls. We also compared the differences in allele distributions between the African and n = 50 European controls (from the IPDC). There was a statistical significant difference in the distribution of the REs in the ATXN1 between African and European controls (Chi-test p 8) in Europeans vs. Africans (Fisher’s p = 0.016). The distribution of RE alleles in ATXN2 and NOP56 were similar amongst African and European controls. In conclusion, repeat expansions in ATXN2, NIPA1 and ATXN1, which showed associations with ALS in Europeans, were not replicated in Southern Africans with ALS.

Trinucleotide repeat expansions fold into long, stable hairpins and cause a variety of incurable RNA gain-of-function diseases such as Huntington's disease, the myotonic dystrophies, and spinocerebellar ataxias. One approach for treating these diseases is to bind small molecules to these structured RNAs. Both Huntington's disease-like 2 (HDL2) and myotonic dystrophy type 1 (DM1) are caused by a r(CUG) repeat expansion, or r(CUG)exp. The RNA folds into a hairpin structure with a periodic array of 1 × 1 nucleotide UU loops (5′C U G/3′G U C; where the underlined nucleotides indicate the Us in the internal loop) that sequester various RNA-binding proteins (RBPs) and hence the source of its gain-of-function. Here, we report nuclear magnetic resonance (NMR)-refined structures of single 5′C U G/3′G U C motifs in complex with three different small molecules, a di-guandinobenzoate (1), a derivative of 1 where the guanidino groups have been exchanged for imidazole (2), and a quinoline with improved drug-like properties (3). These structures were determined using NMR spectroscopy and simulated annealing with restrained molecular dynamics (MD). Compounds 1 , 2 , and 3 formed stacking and hydrogen bonding interactions with the 5′C U G/3′G U C motif. Compound 3 also formed van der Waals interactions with the internal loop. The global structure of each RNA-small molecule complexes retains an A-form conformation, while the internal loops are still dynamic but to a lesser extent compared to the unbound form. These results aid our understanding of ligand-RNA interactions and enable structure-based design of small molecules with improved binding affinity for and biological activity against r(CUG)exp. As the first ever reported structures of a r(CUG) repeat bound to ligands, these structures can enable virtual screening campaigns combined with machine learning assisted de novo design. [ABSTRACT FROM AUTHOR]

Abstract Background Physiological disturbances in cortical network excitability and plasticity are established and widespread in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, including those harbouring the C9ORF72 repeat expansion (C9ORF72 RE ) mutation – the most common genetic impairment causal to ALS and FTD. Noting that perturbations in cortical function are evidenced pre-symptomatically, and that the cortex is associated with widespread pathology, cortical dysfunction is thought to be an early driver of neurodegenerative disease progression. However, our understanding of how altered network function manifests at the cellular and molecular level is not clear. Methods To address this we have generated cortical neurons from patient-derived iPSCs harbouring C9ORF72 RE mutations, as well as from their isogenic expansion-corrected controls. We have established a model of network activity in these neurons using multi-electrode array electrophysiology. We have then mechanistically examined the physiological processes underpinning network dysfunction using a combination of patch-clamp electrophysiology, immunocytochemistry, pharmacology and transcriptomic profiling. Results We find that C9ORF72 RE causes elevated network burst activity, associated with enhanced synaptic input, yet lower burst duration, attributable to impaired pre-synaptic vesicle dynamics. We also show that the C9ORF72 RE is associated with impaired synaptic plasticity. Moreover, RNA-seq analysis revealed dysregulated molecular pathways impacting on synaptic function. All molecular, cellular and network deficits are rescued by CRISPR/Cas9 correction of C9ORF72 RE. Our study provides a mechanistic view of the early dysregulated processes that underpin cortical network dysfunction in ALS-FTD. Conclusion These findings suggest synaptic pathophysiology is widespread in ALS-FTD and has an early and fundamental role in driving altered network function that is thought to contribute to neurodegenerative processes in these patients. The overall importance is the identification of previously unidentified defects in pre and postsynaptic compartments affecting synaptic plasticity, synaptic vesicle stores, and network propagation, which directly impact upon cortical function.

There are more than 30 inherited human disorders connected with repeat expansion (myotonic dystrophy type I, Huntington’s disease, Fragile X syndrome). Fragile X syndrome is the most common reason for inherited intellectual disability in the human population. The ways of the expansion development remain unclear. An important feature of expanded repeats is the ability to form stable alternative DNA secondary structures. There are hypotheses about the nature of repeat instability. It is proposed that these DNA secondary structures can block various stages of DNA metabolism processes, such as replication, repair and recombination and it is considered as the source of repeat instability. However, none of the hypotheses is fully conf irmed or is the only valid one. Here, an experimental system for studying (CGG)n repeat expansion associated with transcription and TCR­-NER is proposed. It is noteworthy that the aberrations of transcription are a poorly studied mechanism of (CGG)n instability. However, the proposed systems take into account the contribution of other processes of DNA metabolism and, therefore, the developed systems are universal and applicable for various studies. Transgenic cell lines carrying a repeat of normal or premutant length under the control of an inducible promoter were established and a method for repeat instability quantif ication was developed. One type of the cell lines contains an exogenous repeat integrated into the genome by the Sleeping Beauty transposon; in another cell line, the vector is maintained as an episome due to the SV40 origin of replication. These experimental systems can serve for f inding the causes of instability and the development of therapeutic agents. In addition, a criterion was developed for the quantif ication of exogenous (CGG)n repeat instability in the transgenic cell lines’ genome.

Hexanucleotide repeat expansion in C9ORF72 (C9) is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One of the proposed pathogenic mechanisms is the neurotoxicity arising from dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG (RAN) translation. Therefore, reducing DPR levels emerges as a potential therapeutic strategy for C9ORF72-ALS/FTD. We previously identified an RNA helicase, DEAD-box helicase 3 X-linked (DDX3X), modulates RAN translation. DDX3X overexpression decreases poly-GP accumulation in C9ORF72-ALS/FTD patient-derived induced pluripotent stem cell (iPSC)-differentiated neurons (iPSNs) and reduces the glutamate-induced neurotoxicity. In this study, we examined the in vivo efficacy of DDX3X overexpression using a mouse model. We expressed exogenous DDX3X or GFP in the central nervous system (CNS) of the C9–500 ALS/FTD BAC transgenic or non-transgenic control mice using adeno-associated virus serotype 9 (AAV9). The DPR levels were significantly reduced in the brains of DDX3X-expressing C9-BAC mice compared to the GFP control even twelve months after virus delivery. Additionally, p62 aggregation was also decreased. No neuronal loss or neuroinflammatory response were detected in the DDX3X overexpressing C9-BAC mice. This work demonstrates that DDX3X overexpression effectively reduces DPR levels in vivo without provoking neuroinflammation or neurotoxicity, suggesting the potential of increasing DDX3X expression as a therapeutic strategy for C9ORF72 - ALS/FTD. • AAV9-mediated DDX3X overexpression reduces DPR levels in C9-BAC mouse brain. • DDX3X overexpression decreases p62 inclusion in C9-BAC mice. • DDX3X overexpression does not cause neuroinflammation or neurotoxicity. [ABSTRACT FROM AUTHOR]

Myotonic dystrophy (DM) is the most common muscular dystrophy in adults. Dominantly inherited CTG and CCTG repeat expansions in DMPK and CNBP genes cause DM type 1 (DM1) and 2 (DM2), respectively. These genetic defects lead to the abnormal splicing of different mRNA transcripts, which are thought to be responsible for the multiorgan involvement of these diseases. In ours and others’ experience, cancer frequency in patients with DM appears to be higher than in the general population or non-DM muscular dystrophy cohorts. There are no specific guidelines regarding malignancy screening in these patients, and the general consensus is that they should undergo the same cancer screening as the general population. Here, we review the main studies that investigated cancer risk (and cancer type) in DM cohorts and those that researched potential molecular mechanisms accounting for DM carcinogenesis. We propose some evaluations to be considered as malignancy screening in patients with DM, and we discuss DM susceptibility to general anesthesia and sedatives, which are often needed for the management of cancer. This review underscores the importance of monitoring the adherence of patients with DM to malignancy screenings and the need to design studies that determine whether they would benefit from a more intensified cancer screening than the general population.

Myotonic dystrophy type 1 (DM1) is a dominantly inherited disorder due to a toxic gain of function of RNA transcripts containing expanded CUG repeats (CUGexp). Patients with DM1 present with multisystemic symptoms, such as muscle wasting, cognitive impairment, cataract, frontal baldness, and endocrine defects, which resemble accelerated aging. Although the involvement of cellular senescence, a critical component of aging, was suggested in studies of DM1 patient-derived cells, the detailed mechanism of cellular senescence caused by CUGexp RNA remains unelucidated. Here, we developed a DM1 cell model that conditionally expressed CUGexp RNA in human primary cells so that we could perform a detailed assessment that eliminated the variability in primary cells from different origins. Our DM1 model cells demonstrated that CUGexp RNA expression induced cellular senescence by a telomere-independent mechanism. Furthermore, the toxic RNA expression caused mitochondrial dysfunction, excessive reactive oxygen species production, and DNA damage and response, resulting in the senescence-associated increase of cell cycle inhibitors p21 and p16 and secreted mediators insulin-like growth factor binding protein 3 (IGFBP3) and plasminogen activator inhibitor-1 (PAI-1). This study provides unequivocal evidence of the induction of premature senescence by CUGexp RNA in our DM1 model cells. [ABSTRACT FROM AUTHOR]

Background: The plastid genomes of the green algal order Chlamydomonadales tend to expand their non-coding regions, but this phenomenon is poorly understood. Here we shed new light on organellar genome evolution in Chlamydomonadales by studying a previously unknown non-photosynthetic lineage. We established cultures of two new Polytoma-like flagellates, defined their basic characteristics and phylogenetic position, and obtained complete organellar genome sequences and a transcriptome assembly for one of them. Results: We discovered a novel deeply diverged chlamydomonadalean lineage that has no close photosynthetic relatives and represents an independent case of photosynthesis loss. To accommodate these organisms, we establish the new genus Leontynka, with two species (L. pallida and L. elongata) distinguishable through both their morphological and molecular characteristics. Notable features of the colourless plastid of L. pallida deduced from the plastid genome (plastome) sequence and transcriptome assembly include the retention of ATP synthase, thylakoid-associated proteins, the carotenoid biosynthesis pathway, and a plastoquinone-based electron transport chain, the latter two modules having an obvious functional link to the eyespot present in Leontynka. Most strikingly, the ~362 kbp plastome of L. pallida is by far the largest among the non-photosynthetic eukaryotes investigated to date due to an extreme proliferation of sequence repeats. These repeats are also present in coding sequences, with one repeat type found in the exons of 11 out of 34 protein-coding genes, with up to 36 copies per gene, thus affecting the encoded proteins. The mitochondrial genome of L. pallida is likewise exceptionally large, with its >104 kbp surpassed only by the mitogenome of Haematococcus lacustris among all members of Chlamydomonadales hitherto studied. It is also bloated with repeats, though entirely different from those in the L. pallida plastome, which contrasts with the situation in H. lacustris where both the organellar genomes have accumulated related repeats. Furthermore, the L. pallida mitogenome exhibits an extremely high GC content in both coding and non-coding regions and, strikingly, a high number of predicted G-quadruplexes. Conclusions: With its unprecedented combination of plastid and mitochondrial genome characteristics, Leontynka pushes the frontiers of organellar genome diversity and is an interesting model for studying organellar genome evolution. [ABSTRACT FROM AUTHOR]