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Bioinformatics-Based Identification of Expanded Repeats: A Non-reference Intronic Pentamer Expansion in RFC1 Causes CANVAS.

ABSTRACT: Genomic technologies such as next-generation sequencing (NGS) are revolutionizing molecular diagnostics and clinical medicine. However, these approaches have proven inefficient at identifying pathogenic repeat expansions. Here, we apply a collection of bioinformatics tools that can be utilized to identify either known or novel expanded repeat sequences in NGS data. We performed genetic studies of a cohort of 35 individuals from 22 families with a clinical diagnosis of cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS). Analysis of whole-genome sequence (WGS) data with five independent algorithms identified a recessively inherited intronic repeat expansion [(AAGGG)exp] in the gene encoding Replication Factor C1 (RFC1). This motif, not reported in the reference sequence, localized to an Alu element and replaced the reference (AAAAG)11 short tandem repeat. Genetic analyses confirmed the pathogenic expansion in 18 of 22 CANVAS-affected families and identified a core ancestral haplotype, estimated to have arisen in Europe more than twenty-five thousand years ago. WGS of the four RFC1-negative CANVAS-affected families identified plausible variants in three, with genomic re-diagnosis of SCA3, spastic ataxia of the Charlevoix-Saguenay type, and SCA45. This study identified the genetic basis of CANVAS and demonstrated that these improved bioinformatics tools increase the diagnostic utility of WGS to determine the genetic basis of a heterogeneous group of clinically overlapping neurogenetic disorders.

SUBMITTER: Rafehi H

PROVIDER: S-EPMC6612533 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Bioinformatics-Based Identification of Expanded Repeats: A Non-reference Intronic Pentamer Expansion in RFC1 Causes CANVAS.

Rafehi Haloom H Szmulewicz David J DJ Bennett Mark F MF Sobreira Nara L M NLM Pope Kate K Smith Katherine R KR Gillies Greta G Diakumis Peter P Dolzhenko Egor E Eberle Michael A MA Barcina María García MG Breen David P DP Chancellor Andrew M AM Cremer Phillip D PD Delatycki Martin B MB Fogel Brent L BL Hackett Anna A Halmagyi G Michael GM Kapetanovic Solange S Lang Anthony A Mossman Stuart S Mu Weiyi W Patrikios Peter P Perlman Susan L SL Rosemergy Ian I Storey Elsdon E Watson Shaun R D SRD Wilson Michael A MA Zee David S DS Valle David D Amor David J DJ Bahlo Melanie M Lockhart Paul J PJ

American journal of human genetics 20190620 1

Genomic technologies such as next-generation sequencing (NGS) are revolutionizing molecular diagnostics and clinical medicine. However, these approaches have proven inefficient at identifying pathogenic repeat expansions. Here, we apply a collection of bioinformatics tools that can be utilized to identify either known or novel expanded repeat sequences in NGS data. We performed genetic studies of a cohort of 35 individuals from 22 families with a clinical diagnosis of cerebellar ataxia with neur ...[more]

PMID: 31230722

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Project description:In eukaryote genomes, the polyadenylation site marks termination of mature RNA transcripts by a poly-adenine tail. The polyadenylation site is recognized by a dynamic protein complex, among which the poly-adenine-binding protein nuclear1 plays a key role. Reduced poly-adenine-binding protein nuclear1 levels are found in aged muscles and are even lower in oculopharyngeal muscular dystrophy patients. Oculopharyngeal muscular dystrophy is a rare, late onset autosomal dominant myopathy, and is caused by an alanine expansion mutation in poly-adenine-binding protein nuclear1. Mutant poly-adenine-binding protein nuclear1 forms insoluble nuclear aggregates leading to depletion of functional poly-adenine-binding protein nuclear1 levels. In oculopharyngeal muscular dystrophy models, increased utilization of proximal polyadenylation sites has been observed in tandem 3'-untranslated regions, and most often cause gene upregulation. However, global alterations in expression profiles canonly partly be explained by polyadenylation site switches within the most distal 3'-untranslated region. Most poly-adenine signals are found at the distal 3'-untranslated region, but a significant part is also found in internal gene regions, like introns, exons, and internal 3'-untranslated regions. Here, we investigated poly-adenine-binding protein nuclear1's role in polyadenylation site utilization in internal gene regions. In the quadriceps muscle of oculopharyngeal muscular dystrophy mice expressing expPABPN1 we found significant polyadenylation site switches between gene regions in 17% of genes with polyadenylation site in multiple regions (N?=?574; 5% False Discovery Rate). Polyadenylation site switches between gene regions were associated with differences in transcript expression levels and alterations in open reading frames. Transcripts ending at internal polyadenylation site were confirmed in tibialis anterior muscles from the same mice and in mouse muscle cell cultures overexpressing expPABPN1. The polyadenylation site switches were associated with nuclear accumulation of full-length transcripts. Our results provide further insights into the diverse roles of poly-adenine-binding protein nuclear1 in the post-transcriptional control of muscle gene expression and its relevance for oculopharyngeal muscular dystrophy pathology and muscle aging.

Project description:BackgroundNeuronal intranuclear inclusion disease (NIID) is a rare neurodegenerative disorder characterized by widespread intranuclear inclusions in the nervous system as well as multiple visceral organs. In 2019, expanded GGC repeats within the 5' untranslated region of the NOTCH2NLC gene was identified as the causative factor. NIID is a heterogeneous disorder with variable clinical manifestations including cognitive impairment, cerebellar ataxia, parkinsonism, paroxysmal symptoms, autonomic dysfunction, and muscle weakness. Although NIID primarily affects the central and peripheral nervous systems, growing evidence suggests potential cardiac abnormalities in NIID. However, the link between expanded GGC repeats within NOTCH2NLC and cardiac dysfunction remains uncertain.ResultsIn this study, we utilized two transgenic mouse models, expressing NOTCH2NLC-(GGC)98 ubiquitously or specifically in cardiomyocytes, and identified p62 (also known as sequestosome 1, SQSTM1)-positive intranuclear NOTCH2NLC-polyG inclusions in cardiomyocytes in two mouse models. We observed that both models exhibited cardiac-related pathological and echocardiographic changes, albeit exhibiting varying degrees of severity. Transcriptomic analysis revealed shared downregulation of genes related to ion channels and mitochondria in both models, with the cardiomyocyte-specific mice showing a more pronounced downregulation of mitochondria and energy metabolism-related pathways. Further investigations revealed decreased expression of mitochondria-related genes and electron transport chain activity. At last, we conducted a retrospective review of cardiac-related examination results from NIID patients at our hospital and also identified some cardiac abnormalities in NIID patients.ConclusionsOur study provided the first in vivo evidence linking GGC repeat expansions within NOTCH2NLC to cardiac abnormalities and highlighted the contribution of mitochondrial dysfunction in the development of cardiac abnormalities.

Dataset Information

Bioinformatics-Based Identification of Expanded Repeats: A Non-reference Intronic Pentamer Expansion in RFC1 Causes CANVAS.

Publications

Bioinformatics-Based Identification of Expanded Repeats: A Non-reference Intronic Pentamer Expansion in RFC1 Causes CANVAS.

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