Molecular Mechanisms in Pentanucleotide Repeat Diseases

The number of neurodegenerative diseases resulting from repeat expansion has increased extraordinarily in recent years. In several of these pathologies, the repeat can be transcribed in RNA from both DNA strands producing, at least, one toxic RNA repeat that causes neurodegeneration by a complex mechanism. Recently, seven diseases have been found caused by a novel intronic pentanucleotide repeat in distinct genes encoding proteins highly expressed in the cerebellum. These disorders are clinically heterogeneous being characterized by impaired motor function, resulting from ataxia or epilepsy. The role that apparently normal proteins from these mutant genes play in these pathologies is not known. However, recent advances in previously known spinocerebellar ataxias originated by abnormal non-coding pentanucleotide repeats point to a gain of a toxic function by the pathogenic repeat-containing RNA that abnormally forms nuclear foci with RNA-binding proteins. In cells, RNA foci have been shown to be formed by phase separation. Moreover, the field of repeat expansions has lately achieved an extraordinary progress with the discovery that RNA repeats, polyglutamine, and polyalanine proteins are crucial for the formation of nuclear membraneless organelles by phase separation, which is perturbed when they are expanded. This review will cover the amazing advances on repeat diseases.

Multi-site Technical Performance and Concordance of Optical Genome Mapping: Constitutional Postnatal Study for SV, CNV, and Repeat Array Analysis

Background The standard of care (SOC) cytogenetic testing methods, such as chromosomal microarray (CMA) and Fragile-X syndrome (FXS) testing, have been employed for the detection of copy number variations (CNVs), and tandem repeat expansions/contractions that contribute towards a sizable portion of genetic abnormalities in constitutional disorders. However, CMA is unable to detect balanced structural variations (SVs) or determine the precise location or orientation of copy number gains. Karyotyping, albeit with lower resolution, has been used for the detection of balanced SVs. Other molecular methods such as PCR and Southern blotting, either simultaneously or in a tiered fashion have been used for FXS testing, adding time, cost, and complexity to reach an accurate diagnosis in affected individuals. Optical genome mapping (OGM), innovative technology in the cytogenomics arena enables a direct, high-resolution view of ultra-long DNA molecules (>150 kbp), which are then assembled de novo to detect germline SVs ranging from 500 bp insertions and deletions to complex chromosomal rearrangements. The purpose of this study was to evaluate the performance of OGM in comparison to the current SOC methods and assess the intra- and inter-site reproducibility of the OGM technique. We report the largest retrospective dataset to date on OGM performed at five laboratories (multi-site) to assess the robustness, QC performance, and analytical validation (multi-operator, and multi-instrument) in detecting SVs and CNVs associated with constitutional disorders compared to SOC technologies. Methods This multi-center IRB-approved, double-blinded, study includes a total of 331 independent flow cells run (including replicates), representing 202 unique retrospective samples, including but not limited to pediatric-onset neurodevelopmental disorders. This study included affected individuals with either a known genetic abnormality or no known genetic diagnosis. Control samples (n=42) were also included. Briefly, OGM was performed on either peripheral blood samples or cell lines using the Saphyr system. The OGM assay results were compared to the human reference genome (GRCh38) to detect different types of SVs (CNV, insertions, inversions, translocations). A unique coverage-based CNV calling algorithm was also used to complement the SV calls. Analysis of heterozygous SVs was performed to assess the absence of heterozygosity (AOH) regions in the genome. For specific clinical indications of FSHD1 and FXS, the EnFocus FXS and FSHD1 tools were used to generate the region-specific reports. OGM data was analyzed and visualized using Access software (version 1.7), where the SVs were filtered using an OGM specific internal control database. The samples were analyzed by laboratory analysts at each site in a blinded fashion using ACMG guidelines for SV interpretation and further reviewed by expert geneticists to assess concordance with SOC testing results. Results Of the first 331 samples run between five sites, 99.1% of sample runs were completed successfully. Of the 331 datasets, 219 were assessed for concordance by the time of this publication; these were samples that harbored known variants, of which 214/219 were detected by OGM resulting in a concordance of 97.7% compared to SOC testing. 47 samples were also run in intra- and inter-site replicate and showed 100% concordance for pathogenic CNVs and SVs and 100% concordance for pathogenic FMR1 repeat expansions. Conclusion The results from this study demonstrate the potential of OGM as an alternative to existing SOC methods in detecting SVs of clinical significance in constitutional postnatal genetic disorders. The outstanding technical performance of OGM across multiple sites demonstrates the robustness and reproducibility of the OGM technique as a rapid cytogenomics testing tool. Notably, OGM detected all classes of SVs in a single assay, which allows for a faster result in cases with diverse and heterogeneous clinical presentations. OGM demonstrated 100% concordance for pathogenic variants previously identified including FMR1 repeat expansions (full mutation range), pathogenic D4Z4 repeat contractions (FSHD1 cases), aneuploidies, interstitial deletions, interstitial duplications, intragenic deletions, balanced translocations, and inversions. Based on our large dataset and high technical performance we recommend OGM as an alternative to the existing SOC tests for the rapid detection and diagnosis of postnatal constitutional disorders.

Genome-wide tandem repeat expansions contribute to schizophrenia risk

Tandem repeat expansions (TREs) can cause neurological diseases but their impact in schizophrenia is unclear. Here we analyzed genome sequences of adults with schizophrenia and found that they have a higher burden of TREs that are near exons and rare in the general population, compared with non-psychiatric controls. These TREs are disproportionately found at loci known to be associated with schizophrenia from genome-wide association studies, in individuals with clinically-relevant genetic variants at other schizophrenia loci, and in families where multiple individuals have schizophrenia. Our findings support the involvement of genome-wide rare TREs in the polygenic nature of schizophrenia.

Aurintricarboxylic Acid Decreases RNA Toxicity in a C. elegans Model of Repeat Expansions

Pathologic expansions of DNA nucleotide tandem repeats may generate toxic RNA that triggers disease phenotypes. RNA toxicity is the hallmark of multiple expansion repeat disorders, including myotonic dystrophy type 1 (DM1). To date, there are no available disease-modifying therapies for DM1. Our aim was to use drug repositioning to ameliorate the phenotype of affected individuals in a nematode model of DM1. As the RNA interference pathway plays a key role in mediating RNA toxicity, we investigated the effect of aurintricarboxylic acid. We demonstrated that by perturbing the RNA interference machinery using aurintricarboxylic acid, we could annihilate the RNA toxicity and ameliorate the phenotype. As our approach targets a universal disease mechanism, it is potentially relevant for more expansion repeat disorders.

Comparison of TCF4 repeat expansion length in corneal endothelium and leukocytes of patients with Fuchs endothelial corneal dystrophy

Expansion of CTG trinucleotide repeats (TNR) in the transcription factor 4 (TCF4) gene is highly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Due to limitations in the availability of DNA from diseased corneal endothelium, sizing of CTG repeats in FECD patients has typically been determined using DNA samples isolated from peripheral blood leukocytes. However, it is non-feasible to extract enough DNA from surgically isolated FECD corneal endothelial tissue to determine repeat length based on current technology. To circumvent this issue, total RNA was isolated from FECD corneal endothelium and sequenced using long-read sequencing. Southern blotting of DNA samples isolated from primary cultures of corneal endothelium from these same affected individuals was also assessed. Both long read sequencing and Southern blot analysis showed significantly longer CTG TNR expansion (>1000 repeats) in the corneal endothelium from FECD patients than those characterized in leukocytes from the same individuals (<90 repeats). Our findings suggest that the TCF4 CTG repeat expansions in the FECD corneal endothelium are much longer than those found in leukocytes.

Familial adult myoclonic epilepsy (FAME): clinical features, molecular characteristics, pathophysiological aspects and diagnostic work-up

Familial adult myoclonic epilepsy (FAME) is a rare autosomal dominant disorder characterized by myoclonus and seizures. The genetic variant underlying FAME is an intronic repeat expansion composed of two different pentamers: an expanded TTTTA, which is the motif originally present at the locus, and an insertion of TTTCA repeats, which is usually located at the 3′ end and likely corresponds to the pathogenic part of the expansion. This repeat expansion has been identified so far in six genes located on different chromosomes, which remarkably encode proteins with distinct cellular localizations and functions. Although the exact pathophysiological mechanisms remain to be clarified, it is likely that FAME repeat expansions lead to disease independently of the gene where they occur. We herein review the clinical and molecular characteristics of this singular genetic disorder, which interestingly shares clinical features with other more common neurological disorders whose etiology remains mainly unsolved.

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS): from clinical diagnosis towards genetic testing

The cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is a late-onset and recessively inherited ataxia. For many years, CANVAS has been diagnosed based on the clinical phenotype. Only recently, a large biallelic pentanucleotide repeat expansion in the replication factor C subunit 1 (RFC1) gene has been identified as the underlying genetic cause for the large majority of CANVAS cases. Subsequently, other phenotypes such as ataxia with chronic cough, incomplete CANVAS and MSA-C-like phenotypes have been associated with biallelic RFC1 repeat expansions. Because of this heterogeneity it has been suggested to change the name of the disease to “RFC1 disease”. Chronic cough is characteristic and can precede neurological symptoms by years or decades. In the neurological examination signs of cerebellar, sensory, and vestibular ataxia are frequently observed. Nerve conduction studies usually show absent or markedly reduced sensory nerve action potentials. On brain MRI cerebellar degeneration and spinal cord alterations are common. In later disease stages more widespread neurodegeneration with additional involvement of the brainstem and basal ganglia is possible. As yet, the exact incidence of RFC1-associated neurological diseases remains uncertain although first studies suggest that RFC1-related ataxia is common. Moreover, the pathophysiological mechanisms caused by the large biallelic pentanucleotide repeat expansions in RFC1 remain elusive. Future molecular and genetic research as well as natural history studies are highly desirable to pave the way towards personalized treatment approaches.