Unravelling the clinical spectrum and the role of repeat length in C9ORF72 repeat expansions

Since the discovery of the C9orf72 repeat expansion as the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis, it has increasingly been associated with a wider spectrum of phenotypes, including other types of dementia, movement disorders, psychiatric symptoms and slowly progressive FTD. Prompt recognition of patients with C9orf72-associated diseases is essential in light of upcoming clinical trials. The striking clinical heterogeneity associated with C9orf72 repeat expansions remains largely unexplained. In contrast to other repeat expansion disorders, evidence for an effect of repeat length on phenotype is inconclusive. Patients with C9orf72-associated diseases typically have very long repeat expansions, containing hundreds to thousands of GGGGCC-repeats, but smaller expansions might also have clinical significance. The exact threshold at which repeat expansions lead to neurodegeneration is unknown, and discordant cut-offs between laboratories pose a challenge for genetic counselling. Accurate and large-scale measurement of repeat expansions has been severely hindered by technical difficulties in sizing long expansions and by variable repeat lengths across and within tissues. Novel long-read sequencing approaches have produced promising results and open up avenues to further investigate this enthralling repeat expansion, elucidating whether its length, purity, and methylation pattern might modulate clinical features of C9orf72-related diseases.

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Identification of GGC repeat expansion in the NOTCH2NLC gene in amyotrophic lateral sclerosis

Neurology ◽

10.1212/wnl.0000000000010945 ◽

2020 ◽

Vol 95 (24) ◽

pp. e3394-e3405 ◽

Cited By ~ 1

Author(s):

Yanchun Yuan ◽

Zhen Liu ◽

Xuan Hou ◽

Wanzhen Li ◽

Jie Ni ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Mainland China ◽

Repeat Expansion ◽

Fisher Exact Test ◽

Healthy Controls ◽

Exact Test ◽

Repeat Expansions ◽

Neuronal Intranuclear Inclusion ◽

Electrophysiologic Studies ◽

Lateral Sclerosis

ObjectiveTo determine whether the GGC repeats in the NOTCH2NLC gene contribute to amyotrophic lateral sclerosis (ALS).MethodsIn this study, 545 patients with ALS and 1,305 healthy controls from mainland China were recruited. Several pathogenic mutations in known ALS-causative genes (including C9ORF72 and ATXN2) and polynucleotide repeat expansions in NOP56 and AR genes were excluded. Repeat-primed PCR and GC-rich PCR were performed to determine the GGC repeat size in NOTCH2NLC. Systematic and targeted clinical evaluations and investigations, including skin biopsy and dynamic electrophysiologic studies, were conducted in the genetically affected patients.ResultsGGC repeat expansion was observed in 4 patients (numbers of repeats 44, 54, 96, and 143), accounting for ≈0.73% (4 of 545) of all patients with ALS. A comparison with 1,305 healthy controls revealed that GGC repeat expansion in NOTCH2NLC was associated with ALS (Fisher exact test, 4 of 545 vs 0 of 1,305, p = 0.007). Compared to patients with the neuronal intranuclear inclusion disease (NIID) muscle weakness–dominant subtype, patients with ALS phenotype carrying the abnormal repeat expansion tended to have a severe phenotype and rapid deterioration.ConclusionOur results suggest that ALS is a specific phenotype of NIID or that GGC expansion in NOTCH2NLC is a factor that modifies ALS. These findings may help clarify the pathogenic mechanism of ALS and may expand the known clinical spectrum of NIID.

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Expanded GGGGCC repeat transcription is mediated by the PAF1 complex in C9orf72-associated FTD.

10.1101/319376 ◽

2018 ◽

Cited By ~ 2

Author(s):

Lindsey D. Goodman ◽

Mercedes Prudencio ◽

Luis F. Martinez-Ramirez ◽

Matthews Lan ◽

Michael Parisi ◽

...

Keyword(s):

Frontal Cortex ◽

Large Scale ◽

Transcriptional Regulator ◽

Repeat Expansion ◽

Transcript Levels ◽

Associated Diseases ◽

Paf1 Complex ◽

Rna Levels

Expression of an expanded (G4C2)30+ repeat found in C9orf72 is the most prominent mutation in familial FTD and ALS. An unbiased RNAi-based, large-scale screen in (G4C2)49-expressing Drosophila identified the CDC73/PAF1 complex (PAF1C) as a novel suppressor of (G4C2)49-toxicity. Downregulation of PAF1C, an activator of elongating RNAPII, caused suppression by reducing (G4C2)49-RNA levels. Remarkably, only PAF1C components Paf1 and Leo1 were selective for transcription of a long repeat expansion; transcript levels produced from shorter and longer repeat-containing transgenes were similarly affected by other components and Spt4, a previously identified transcriptional regulator of G4C2-repeats. Congruent with our fly data, PAF1 and LEO1 were upregulated in the frontal cortex of C9+ FTD patients and their expression correlated to expression of repeat-containing C9orf72. Surprisingly, this affect was specific to C9+ FTD versus C9+ ALS. This is the first evidence that PAF1C is playing a role in C9orf72-associated FTD. Further, PAF1C may affect other repeat-associated diseases.

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Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease

10.1101/176651 ◽

2018 ◽

Cited By ~ 1

Author(s):

Mark T. W. Ebbert ◽

Stefan Farrugia ◽

Jonathon Sens ◽

Karen Jansen-West ◽

Tania F. Gendron ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Repeat Expansion ◽

Whole Genome ◽

Short Read ◽

Short Read Sequencing ◽

Sequencing Technologies ◽

Long Read ◽

Repeat Expansions ◽

Targeted Approach

AbstractBackground: Many neurodegenerative diseases are caused by nucleotide repeat expansions, but most expansions, like the C9orf72 ‘GGGGCC’ (G4C2) repeat that causes approximately 5-7% of all amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases, are too long to sequence using short-read sequencing technologies. It is unclear whether long-read sequencing technologies can traverse these long, challenging repeat expansions. Here, we demonstrate that two long-read sequencing technologies, Pacific Biosciences’ (PacBio) and Oxford Nanopore Technologies’ (ONT), can sequence through disease-causing repeats cloned into plasmids, including the FTD/ALS-causing G4C2 repeat expansion. We also report the first long-read sequencing data characterizing the C9orf72 G4C2 repeat expansion at the nucleotide level in two symptomatic expansion carriers using PacBio whole-genome sequencing and a no-amplification (No-Amp) targeted approach based on CRISPR/Cas9.Results: Both the PacBio and ONT platforms successfully sequenced through the repeat expansions in plasmids. Throughput on the MinlON was a challenge for whole-genome sequencing; we were unable to attain reads covering the human C9orf72 repeat expansion using 15 flow cells. We obtained 8x coverage across the C9orf72 locus using the PacBio Sequel, accurately reporting the unexpanded allele at eight repeats, and reading through the entire expansion with 1324 repeats (7941 nucleotides). Using the No-Amp targeted approach, we attained >800x coverage and were able to identify the unexpanded allele, closely estimate expansion size, and assess nucleotide content in a single experiment. We estimate the individual’s repeat region was >99% G4C2 content, though we cannot rule out small interruptions.Conclusions: Our findings indicate that long-read sequencing is well suited to characterizing known repeat expansions, and for discovering new disease-causing, disease-modifying, or risk-modifying repeat expansions that have gone undetected with conventional short-read sequencing. The PacBio No-Amp targeted approach may have future potential in clinical and genetic counseling environments. Larger and deeper long-read sequencing studies in C9orf72 expansion carriers will be important to determine heterogeneity and whether the repeats are interrupted by non-G4C2 content, potentially mitigating or modifying disease course or age of onset, as interruptions are known to do in other repeat-expansion disorders. These results have broad implications across all diseases where the genetic etiology remains unclear.

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Tau Accumulation via Reduced BAG3-mediated Autophagy Is Required for GGGGCC Repeat Expansion-Induced Neurodegeneration

10.1101/727008 ◽

2019 ◽

Author(s):

Xue Wen ◽

Ping An ◽

Hexuan Li ◽

Zijian Zhou ◽

Yimin Sun ◽

...

Keyword(s):

Tau Protein ◽

Neurodegenerative Disorders ◽

Motor Performance ◽

Molecular Mechanisms ◽

Repeat Expansion ◽

Cag Repeats ◽

Reduced Activity ◽

Lateral Sclerosis

SUMMARYExpansions of trinucleotide or hexanucleotide repeats lead to several neurodegenerative disorders including Huntington disease (HD, caused by the expanded CAG repeats (CAGr) in the HTT gene) and amyotrophic lateral sclerosis (ALS, could be caused by the expanded GGGGCC repeats (G4C2r) in the C9ORF72 gene), of which the molecular mechanisms remain unclear. Here we demonstrate that loss of the Drosophila orthologue of tau protein (dtau) significantly rescued in vivo neurodegeneration, motor performance impairments, and shortened life-span in Drosophila models expressing mutant HTT protein with expanded CAGr or the expanded G4C2r. Importantly, expression of human tau (htau4R) restored the disease-relevant phenotypes that were mitigated by the loss of dtau, suggesting a conserved role of tau in neurodegeneration. We further discovered that G4C2r expression increased dtau accumulation, possibly due to reduced activity of BAG3-mediated autophagy. Our study reveals a conserved role of tau in G4C2r-induced neurotoxicity in Drosophila models, providing mechanistic insights and potential therapeutic targets.

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The diversity, structure and function of heritable adaptive immunity sequences in the Aedes aegypti genome

10.1101/127498 ◽

2017 ◽

Cited By ~ 1

Author(s):

Zachary J. Whitfield ◽

Patrick T. Dolan ◽

Mark Kunitomi ◽

Michel Tassetto ◽

Matthew G. Seetin ◽

...

Keyword(s):

Aedes Aegypti ◽

Single Molecule ◽

Large Scale ◽

Insect Immunity ◽

Genomic Context ◽

Long Read ◽

Aegypti Genome ◽

Endogenous Viral Elements ◽

And Function

AbstractThe Aedes aegypti mosquito is a major vector for arboviruses including dengue, chikungunya and Zika virus. Combating the spread of these viruses requires a more complete understanding of the mosquito immune system. Recent studies have implicated genomic endogenous viral elements (EVEs) derived from non-retroviral RNA viruses in insect immunity. Because these elements are inserted into repetitive regions of the mosquito genome, their large-scale structure and organization with respect to other genomic elements has been difficult to resolve with short-read sequencing. To better define the origin, diversity and biological role of EVEs, we employed single-molecule, real-time sequencing technology to generate a high quality, long-read assembly of the Ae. aegypti-derived Aag2 cell line genome. We leverage the quality and contiguity of this assembly to characterize the diversity and genomic context of EVEs in the genome of this important model system. We find that EVEs in the Aag2 genome are acquired through recombination by LTR retrotransposons, and organize into larger loci (>50kbp) characterized by high LTR density. These EVE containing loci are associated with increased transcription factor binding sight density and increased production of anti-genomic piRNAs. We also detected piRNA processing corresponding to on-going viral infection. This global view of EVEs and piRNA responses demonstrates the ubiquity and diversity of these heritable elements that define small-RNA mediated antiviral immunity in mosquitoes.

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Enhanced insulin signalling ameliorates C9orf72 hexanucleotide repeat expansion toxicity in Drosophila

eLife ◽

10.7554/elife.58565 ◽

2021 ◽

Vol 10 ◽

Author(s):

Magda L Atilano ◽

Sebastian Grönke ◽

Teresa Niccoli ◽

Liam Kempthorne ◽

Oliver Hahn ◽

...

Keyword(s):

Mammalian Cells ◽

Insulin Signalling ◽

Repeat Expansion ◽

Adult Brain ◽

Systemic Injection ◽

Hexanucleotide Repeat ◽

Repeat Expansions ◽

Hexanucleotide Repeat Expansion ◽

Lateral Sclerosis ◽

Dipeptide Repeat

G4C2 repeat expansions within the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeats undergo repeat-associated non-ATG translation to generate toxic dipeptide repeat proteins. Here, we show that insulin/IGF signalling is reduced in fly models of C9orf72 repeat expansion using RNA sequencing of adult brain. We further demonstrate that activation of insulin/IGF signalling can mitigate multiple neurodegenerative phenotypes in flies expressing either expanded G4C2 repeats or the toxic dipeptide repeat protein poly-GR. Levels of poly-GR are reduced when components of the insulin/IGF signalling pathway are genetically activated in the diseased flies, suggesting a mechanism of rescue. Modulating insulin signalling in mammalian cells also lowers poly-GR levels. Remarkably, systemic injection of insulin improves the survival of flies expressing G4C2 repeats. Overall, our data suggest that modulation of insulin/IGF signalling could be an effective therapeutic approach against C9orf72 ALS/FTD.

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C9orf72-generated poly-GR and poly-PR do not directly interfere with nucleocytoplasmic transport

Scientific Reports ◽

10.1038/s41598-019-52035-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 14

Author(s):

Joni Vanneste ◽

Thomas Vercruysse ◽

Steven Boeynaems ◽

Adria Sicart ◽

Philip Van Damme ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Motor Neurons ◽

Hela Cells ◽

Nuclear Import ◽

Nucleocytoplasmic Transport ◽

Repeat Expansions ◽

Import And Export ◽

Lateral Sclerosis ◽

Dipeptide Repeat

Abstract Repeat expansions in the C9orf72 gene cause amyotrophic lateral sclerosis and frontotemporal dementia characterized by dipeptide-repeat protein (DPR) inclusions. The toxicity associated with two of these DPRs, poly-GR and poly-PR, has been associated with nucleocytoplasmic transport. To investigate the causal role of poly-GR or poly-PR on active nucleocytoplasmic transport, we measured nuclear import and export in poly-GR or poly-PR expressing Hela cells, neuronal-like SH-SY5Y cells and iPSC-derived motor neurons. Our data strongly indicate that poly-GR and poly-PR do not directly impede active nucleocytoplasmic transport.

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The role of prefrontal cortex in cognitive control and executive function

Neuropsychopharmacology ◽

10.1038/s41386-021-01132-0 ◽

2021 ◽

Author(s):

Naomi P. Friedman ◽

Trevor W. Robbins

Keyword(s):

Prefrontal Cortex ◽

Executive Function ◽

Cognitive Control ◽

Large Scale ◽

Psychiatric Symptoms ◽

General Factor ◽

Genetic Studies ◽

Working Memory Updating ◽

Goal Directed Behavior

AbstractConcepts of cognitive control (CC) and executive function (EF) are defined in terms of their relationships with goal-directed behavior versus habits and controlled versus automatic processing, and related to the functions of the prefrontal cortex (PFC) and related regions and networks. A psychometric approach shows unity and diversity in CC constructs, with 3 components in the most commonly studied constructs: general or common CC and components specific to mental set shifting and working memory updating. These constructs are considered against the cellular and systems neurobiology of PFC and what is known of its functional neuroanatomical or network organization based on lesioning, neurochemical, and neuroimaging approaches across species. CC is also considered in the context of motivation, as “cool” and “hot” forms. Its Common CC component is shown to be distinct from general intelligence (g) and closely related to response inhibition. Impairments in CC are considered as possible causes of psychiatric symptoms and consequences of disorders. The relationships of CC with the general factor of psychopathology (p) and dimensional constructs such as impulsivity in large scale developmental and adult populations are considered, as well as implications for genetic studies and RDoC approaches to psychiatric classification.

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ATXN1 repeat expansions confer risk for amyotrophic lateral sclerosis and contribute to TDP-43 mislocalization

Brain Communications ◽

10.1093/braincomms/fcaa064 ◽

2020 ◽

Vol 2 (2) ◽

Cited By ~ 3

Author(s):

Gijs H P Tazelaar ◽

Steven Boeynaems ◽

Mathias De Decker ◽

Joke J F A van Vugt ◽

Lindy Kool ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Large Scale ◽

Amyotrophic Lateral Sclerosis Patient ◽

International Study ◽

Spinocerebellar Ataxia Type ◽

Published Data ◽

Polyglutamine Expansions ◽

Similar Disease ◽

Repeat Expansions ◽

Lateral Sclerosis

Abstract Increasingly, repeat expansions are being identified as part of the complex genetic architecture of amyotrophic lateral sclerosis. To date, several repeat expansions have been genetically associated with the disease: intronic repeat expansions in C9orf72, polyglutamine expansions in ATXN2 and polyalanine expansions in NIPA1. Together with previously published data, the identification of an amyotrophic lateral sclerosis patient with a family history of spinocerebellar ataxia type 1, caused by polyglutamine expansions in ATXN1, suggested a similar disease association for the repeat expansion in ATXN1. We, therefore, performed a large-scale international study in 11 700 individuals, in which we showed a significant association between intermediate ATXN1 repeat expansions and amyotrophic lateral sclerosis (P = 3.33 × 10−7). Subsequent functional experiments have shown that ATXN1 reduces the nucleocytoplasmic ratio of TDP-43 and enhances amyotrophic lateral sclerosis phenotypes in Drosophila, further emphasizing the role of polyglutamine repeat expansions in the pathophysiology of amyotrophic lateral sclerosis.

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An update on the neurological short tandem repeat expansion disorders and the emergence of long-read sequencing diagnostics

Acta Neuropathologica Communications ◽

10.1186/s40478-021-01201-x ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Sanjog R. Chintalaphani ◽

Sandy S. Pineda ◽

Ira W. Deveson ◽

Kishore R. Kumar

Keyword(s):

Tandem Repeat ◽

Short Tandem Repeat ◽

Fragile X ◽

Cost Effective ◽

Repeat Expansion ◽

Main Body ◽

Cgg Repeat ◽

Long Read ◽

Repeat Expansions ◽

Short Tandem

Abstract Background Short tandem repeat (STR) expansion disorders are an important cause of human neurological disease. They have an established role in more than 40 different phenotypes including the myotonic dystrophies, Fragile X syndrome, Huntington’s disease, the hereditary cerebellar ataxias, amyotrophic lateral sclerosis and frontotemporal dementia. Main body STR expansions are difficult to detect and may explain unsolved diseases, as highlighted by recent findings including: the discovery of a biallelic intronic ‘AAGGG’ repeat in RFC1 as the cause of cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS); and the finding of ‘CGG’ repeat expansions in NOTCH2NLC as the cause of neuronal intranuclear inclusion disease and a range of clinical phenotypes. However, established laboratory techniques for diagnosis of repeat expansions (repeat-primed PCR and Southern blot) are cumbersome, low-throughput and poorly suited to parallel analysis of multiple gene regions. While next generation sequencing (NGS) has been increasingly used, established short-read NGS platforms (e.g., Illumina) are unable to genotype large and/or complex repeat expansions. Long-read sequencing platforms recently developed by Oxford Nanopore Technology and Pacific Biosciences promise to overcome these limitations to deliver enhanced diagnosis of repeat expansion disorders in a rapid and cost-effective fashion. Conclusion We anticipate that long-read sequencing will rapidly transform the detection of short tandem repeat expansion disorders for both clinical diagnosis and gene discovery.

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