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Abstract Numerous neurological disorders are linked to sequences rich in guanine repeats found in introns, exons, and regulatory regions of genes. These sequences have been observed to form stable G-quadruplex (GQ) structures both in vitro and in vitro. Cerebellar Ataxia with Neuropathy and Vestibular Areflexia Syndrome (CANVAS), a slowly progressive neurodegenerative disorder, is associated with the biallelic expansion of (AAGGG)n pathogenic repeats in the second intron of the RFC1 gene. Though these G-rich pathogenic repeats in other neurological diseases are associated with protein loss of function, RNA gain of function, and/or protein gain of function, not much is known about the pathological mechanism associated with CANVAS. Herein, we report the formation of stable GQ conformations in the CANVAS-associated repeats i.e., r(AAGGG)n, where ‘r’ stands for RNA. These GQs are critical regulators in neurological disorders leading to RNA foci formation and RNA binding protein sequestration. They also alter other causative processes like intron retention, which leads us to hypothesize a toxic Proteinopathy mechanism in CANVAS. Various biophysical and biomolecular assays characterized the interactions of three aggregation-prone RNA-binding proteins (RBPs): heterogeneous nuclear ribonucleoprotein H1/F (hnRNP H1/F), and DGCR8 with different pathogenic repeats [(AAGGG)9] in vitro, further affirming the hypothesis. The biophysical observations are further supported by molecular dynamics analysis and cell-based studies, putting us a step closer to elucidating the pathological mechanism(s) in CANVAS neuropathy, paving the way for the development of innovative therapeutic interventions.

Abstract Expansions of tandem repeats (TRs) cause approximately 60 monogenic diseases. We expect that the discovery of additional pathogenic repeat expansions will narrow the diagnostic gap in many diseases. A growing number of TR expansions are being identified, and interpreting them is a challenge. We present RExPRT (Repeat EXpansion Pathogenicity pRediction Tool), a machine learning tool for distinguishing pathogenic from benign TR expansions. Our results demonstrate that an ensemble approach classifies TRs with an average precision of 93% and recall of 83%. RExPRT’s high precision will be valuable in large-scale discovery studies, which require prioritization of candidate loci for follow-up studies.

Expansions of short tandem repeats (STRs) are associated with approximately 50 human neurodegenerative diseases. These pathogenic STRs are prone to form non-B DNA structure, which has been considered as one of the causative factors for repeat expansions. Minidumbbell (MDB) is a relatively new type of non-B DNA structure formed by pyrimidine-rich STRs. An MDB is composed of two tetraloops or pentaloops, exhibiting a highly compact conformation with extensive loop-loop interactions. The MDB structures have been found to form in CCTG tetranucleotide repeats associated with myotonic dystrophy type 2, ATTCT pentanucleotide repeats associated with spinocerebellar ataxia type 10, and the recently discovered ATTTT/ATTTC repeats associated with spinocerebellar ataxia type 37 and familial adult myoclonic epilepsy. In this review, we first introduce the structures and conformational dynamics of MDBs with a focus on the high-resolution structural information determined by nuclear magnetic resonance spectroscopy. Then we discuss the effects of sequence context, chemical environment, and nucleobase modification on the structure and thermostability of MDBs. Finally, we provide perspectives on further explorations of sequence criteria and biological functions of MDBs.

Abstract Background Expansions of short tandem repeats are the cause of many neurogenetic disorders including familial amyotrophic lateral sclerosis, Huntington disease, and many others. Multiple methods have been recently developed that can identify repeat expansions in whole genome or exome sequencing data. Despite the widely recognized need for visual assessment of variant calls in clinical settings, current computational tools lack the ability to produce such visualizations for repeat expansions. Expanded repeats are difficult to visualize because they correspond to large insertions relative to the reference genome and involve many misaligning and ambiguously aligning reads. Results We implemented REViewer, a computational method for visualization of sequencing data in genomic regions containing long repeat expansions and FlipBook, a companion image viewer designed for manual curation of large collections of REViewer images. To generate a read pileup, REViewer reconstructs local haplotype sequences and distributes reads to these haplotypes in a way that is most consistent with the fragment lengths and evenness of read coverage. To create appropriate training materials for onboarding new users, we performed a concordance study involving 12 scientists involved in short tandem repeat research. We used the results of this study to create a user guide that describes the basic principles of using REViewer as well as a guide to the typical features of read pileups that correspond to low confidence repeat genotype calls. Additionally, we demonstrated that REViewer can be used to annotate clinically relevant repeat interruptions by comparing visual assessment results of 44 FMR1 repeat alleles with the results of triplet repeat primed PCR. For 38 of these alleles, the results of visual assessment were consistent with triplet repeat primed PCR. Conclusions Read pileup plots generated by REViewer offer an intuitive way to visualize sequencing data in regions containing long repeat expansions. Laboratories can use REViewer and FlipBook to assess the quality of repeat genotype calls as well as to visually detect interruptions or other imperfections in the repeat sequence and the surrounding flanking regions. REViewer and FlipBook are available under open-source licenses at https://github.com/illumina/REViewer and https://github.com/broadinstitute/flipbook respectively.

Expanded tandem repeat DNAs are associated with various unusual chromosomal lesions, despiralizations, multi-branched inter-chromosomal associations, and fragile sites. Fragile sites cytogenetically manifest as localized gaps or discontinuities in chromosome structure and are an important genetic, biological, and health-related phenomena. Common fragile sites (∼230), present in most individuals, are induced by aphidicolin and can be associated with cancer; of the 27 molecularly-mapped common sites, none are associated with a particular DNA sequence motif. Rare fragile sites (≳ 40 known), ≤ 5% of the population (may be as few as a single individual), can be associated with neurodevelopmental disease. All 10 molecularly-mapped folate-sensitive fragile sites, the largest category of rare fragile sites, are caused by gene-specific CGG/CCG tandem repeat expansions that are aberrantly CpG methylated and include FRAXA, FRAXE, FRAXF, FRA2A, FRA7A, FRA10A, FRA11A, FRA11B, FRA12A, and FRA16A. The minisatellite-associated rare fragile sites, FRA10B, FRA16B, can be induced by AT-rich DNA-ligands or nucleotide analogs. Despiralized lesions and multi-branched inter-chromosomal associations at the heterochromatic satellite repeats of chromosomes 1, 9, 16 are inducible by de-methylating agents like 5-azadeoxycytidine and can spontaneously arise in patients with ICF syndrome (Immunodeficiency Centromeric instability and Facial anomalies) with mutations in genes regulating DNA methylation. ICF individuals have hypomethylated satellites I-III, alpha-satellites, and subtelomeric repeats. Ribosomal repeats and subtelomeric D4Z4 megasatellites/macrosatellites, are associated with chromosome location, fragility, and disease. Telomere repeats can also assume fragile sites. Dietary deficiencies of folate or vitamin B12, or drug insults are associated with megaloblastic and/or pernicious anemia, that display chromosomes with fragile sites. The recent discovery of many new tandem repeat expansion loci, with varied repeat motifs, where motif lengths can range from mono-nucleotides to megabase units, could be the molecular cause of new fragile sites, or other chromosomal lesions. This review focuses on repeat-associated fragility, covering their induction, cytogenetics, epigenetics, cell type specificity, genetic instability (repeat instability, micronuclei, deletions/rearrangements, and sister chromatid exchange), unusual heritability, disease association, and penetrance. Understanding tandem repeat-associated chromosomal fragile sites provides insight to chromosome structure, genome packaging, genetic instability, and disease.

Abstract Repeat expansions are responsible for over 40 monogenic disorders, and undoubtedly more pathogenic repeat expansions remain to be discovered. Existing methods for detecting repeat expansions in short-read sequencing data require predefined repeat catalogs. Recent discoveries emphasize the need for methods that do not require pre-specified candidate repeats. To address this need, we introduce ExpansionHunter Denovo, an efficient catalog-free method for genome-wide repeat expansion detection. Analysis of real and simulated data shows that our method can identify large expansions of 41 out of 44 pathogenic repeats, including nine recently reported non-reference repeat expansions not discoverable via existing methods.

Abstract Background Spinocerebellar ataxia (SCA) subtypes are often caused by expansions in non-coding regions of genes like SCA8, SCA10, SCA12 and SCA36. Other ataxias are known to be associated with repeat expansions such as fragile X-associated tremor ataxia syndrome (FXTAS) or expansions in the C9orf72 gene. When no mutation has been identified in the aforementioned genes next-generation sequencing (NGS)-based diagnostics may also be applied. In order to define an optimal diagnostic strategy, more information about the frequency and phenotypic characteristics of rare repeat expansion disorders associated with ataxia should be at hand. Methods We analyzed a consecutive cohort of 440 German unrelated patients with symptoms of cerebellar ataxia, dysarthria and other unspecific symptoms who were referred to our center for SCA diagnostics. They showed alleles in the normal range for the most common SCA subtypes SCA1-3, SCA6, SCA7 and SCA17. These patients were screened for expansions causing SCA8, SCA10, SCA12, SCA36 and FXTAS as well as for the pathogenic hexanucleotide repeat in the C9orf72 gene. Results Expanded repeats for SCA10, SCA12 or SCA36 were not identified in the analyzed patients. Five patients showed expanded SCA8 CTA/CTG alleles with 92-129 repeats. One 51-year-old male with unclear dementia symptoms was diagnosed with a large GGGGCC repeat expansion in C9orf72. The analysis of the fragile X mental retardation 1 gene (FMR1) revealed one patient with a premutation (>50 CGG repeats) and seven patients with alleles in the grey zone (41 to 54 CGG repeats). Conclusions Altogether five patients showed 92 or more SCA8 CTA/CTG combined repeats. Our results support the assumption that smaller FMR1 gene expansions could be associated with the risk of developing neurological signs. The results do not support genetic testing for C9orf72 expansion in ataxia patients.