The shared ancestry between the C9orf72 hexanucleotide repeat expansion and intermediate-length alleles using haplotype sharing trees and HAPTK.

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.

Advances in the Structure of GGGGCC Repeat RNA Sequence and Its Interaction with Small Molecules and Protein Partners.

The aberrant expansion of GGGGCC hexanucleotide repeats within the first intron of the C9orf72 gene represent the predominant genetic etiology underlying amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). The transcribed r(GGGGCC)n RNA repeats form RNA foci, which recruit RNA binding proteins and impede their normal cellular functions, ultimately resulting in fatal neurodegenerative disorders. Furthermore, the non-canonical translation of the r(GGGGCC)n sequence can generate dipeptide repeats, which have been postulated as pathological causes. Comprehensive structural analyses of r(GGGGCC)n have unveiled its polymorphic nature, exhibiting the propensity to adopt dimeric, hairpin, or G-quadruplex conformations, all of which possess the capacity to interact with RNA binding proteins. Small molecules capable of binding to r(GGGGCC)n have been discovered and proposed as potential lead compounds for the treatment of ALS and FTD. Some of these molecules function in preventing RNA-protein interactions or impeding the phase transition of r(GGGGCC)n. In this review, we present a comprehensive summary of the recent advancements in the structural characterization of r(GGGGCC)n, its propensity to form RNA foci, and its interactions with small molecules and proteins. Specifically, we emphasize the structural diversity of r(GGGGCC)n and its influence on partner binding. Given the crucial role of r(GGGGCC)n in the pathogenesis of ALS and FTD, the primary objective of this review is to facilitate the development of therapeutic interventions targeting r(GGGGCC)n RNA.

Supramolecular Polymorphism of (G4C2)n Repeats Associated with ALS and FTD.

Guanine-rich DNA sequences self-assemble into highly stable fourfold structures known as DNA-quadruplexes (or G-quadruplexes). G-quadruplexes have furthermore the tendency to associate into one-dimensional supramolecular aggregates termed G-wires. We studied the formation of G-wires in solutions of the sequences d(G4C2)n with n = 1, 2, and 4. The d(G4C2)n repeats, which are associated with some fatal neurological disorders, especially amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), represent a challenging research topic due to their extensive structural polymorphism. We used dynamic light scattering (DLS) to measure translational diffusion coefficients and consequently resolve the length of the larger aggregates formed in solution. We found that all three sequences assemble into longer structures than previously reported. The d(G4C2) formed extremely long G-wires with lengths beyond 80 nm. The d(G4C2)2 formed a relatively short stacked dimeric quadruplex, while d(G4C2)4 formed multimers corresponding to seven stacked intramolecular quadruplexes. Profound differences between the multimerization properties of the investigated sequences were also confirmed by the AFM imaging of surface films. We propose that π-π stacking of the basic G-quadruplex units plays a vital role in the multimerization mechanism, which might be relevant for transformation from the regular medium-length to disease-related long d(G4C2)n repeats.

Recognition of expanded GGGGCC hexanucleotide repeat by synthetic ligand through interhelical binding.

An expanded GGGGCC hexanucleotide (G4C2) repeat within the non-coding region of C9ORF72 gene has been identified as the most common genetic cause of FTD/ALS kindred, and synthetic ligand targeting this pathological expansion sequence holds a promising approach for the disease interference. We here describe the naphthyridine carbamate tetramer, p-NCTB, as a binding ligand to hairpin G4C2 repeat. p-NCTB simultaneously recognizes two distal CGGG/CGGG sites in G4C2 repeat DNA and RNA leading to the formation of the interhelical (inter- and intrastrand) binding complexes. The intrastrand binding was predominant when p-NCTB bound to long repeat sequence that accommodates multiple binding sites by folding into hairpins, while the interstrand binding was exclusive for short repeat sequence. The binding of p-NCTB showed repeat-length selectivity: the longer repeat sequence is a better target for p-NCTB. p-NCTB demonstrated inhibition of transcription against G4C2 repeat template in vitro in a repeat length-dependent manner.

ALS and FTD linked GGGGCC-repeat containing DNA oligonucleotide folds into two distinct G-quadruplexes.

BACKGROUND: The most common genetic cause of neurological disorders ALS and FTD is a largely increased number of GGGGCC repeats in C9orf72 gene. Non-canonical structures including G-quadruplexes adopted by expanded repeats are hypothesized to be crucial in pathogenesis. Recently, we have shown that structural polymorphism of oligonucleotide d(G4C2)3G4 is reduced by dG to 8Br-dG substitution. High-resolution structure of one of the two major G-quadruplexes adopts antiparallel topology comprising of four G-quartets. Herein, we describe the topology of the second major G-quadruplex structure and influence of folding conditions on relative populations of the two folds. METHODS: Influence of folding conditions was explored by 1H 1D NMR. Determination of topology was achieved by 2D NMR complemented with PAGE and CD. UV melting experiment was used to explore thermal stability of structures. RESULTS: Two structures adopted by oligonucleotide d(G4C2)3GGBrGG denoted AQU and NAN coexist in solution and ratio of their populations is determined by pH and rate of cooling when folding from thermally denatured state in the presence of K+ ions. CONCLUSIONS: AQU is kinetically favored and forms by folding at low pH, while NAN is favored thermodynamically and at neutral pH. AQU and NAN share similar antiparallel topology with four G-quartets and three edgewise loops, however they exhibit distinct structural and dynamic properties. GENERAL SIGNIFICANCE: Novel G-quadruplex topology adds insight into diverse polymorphism of DNA sequences comprising potentially pathological GGGGCC repeat. Relative populations of the two structures and their dependence on folding conditions contribute to understanding of factors that govern G-quadruplex folding. This article is part of a Special Issue entitled "Gquadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Distribution of the C9orf72 hexanucleotide repeat expansion in healthy subjects: a multicenter study promoted by the Italian IRCCS network of neuroscience and neurorehabilitation.

Introduction: High repeat expansion (HRE) alleles in C9orf72 have been linked to both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); ranges for intermediate allelic expansions have not been defined yet, and clinical interpretation of molecular data lacks a defined genotype-phenotype association. In this study, we provide results from a large multicenter epidemiological study reporting the distribution of C9orf72 repeats in healthy elderly from the Italian population. Methods: A total of 967 samples were collected from neurologically evaluated healthy individuals over 70 years of age in the 13 institutes participating in the RIN (IRCCS Network of Neuroscience and Neurorehabilitation) based in Italy. All samples were genotyped using the AmplideXPCR/CE C9orf72 Kit (Asuragen, Inc.), using standardized protocols that have been validated through blind proficiency testing. Results: All samples carried hexanucleotide G4C2 expansion alleles in the normal range. All samples were characterized by alleles with less than 25 repeats. In particular, 93.7% of samples showed a number of repeats ≤10, 99.9% ≤20 repeats, and 100% ≤25 repeats. Conclusion: This study describes the distribution of hexanucleotide G4C2 expansion alleles in an Italian healthy population, providing a definition of alleles associated with the neurological healthy phenotype. Moreover, this study provides an effective model of federation between institutes, highlighting the importance of sharing genomic data and standardizing analysis techniques, promoting translational research. Data derived from the study may improve genetic counseling and future studies on ALS/FTD.

Molecular conformations and dynamics of nucleotide repeats associated with neurodegenerative diseases: double helices and CAG hairpin loops.

Pathogenic DNA secondary structures have been identified as a common and causative factor for expansion in trinucleotide, hexanucleotide, and other simple sequence repeats. These expansions underlie about fifty neurological and neuromuscular disorders known as "anticipation diseases". Cell toxicity and death have been linked to the pathogenic conformations and functional changes of the RNA transcripts, of DNA itself and, when trinucleotides are present in exons, of the translated proteins. We review some of our results for the conformations and dynamics of pathogenic structures for both RNA and DNA, which include mismatched homoduplexes formed by trinucleotide repeats CAG and GAC; CCG and CGG; CTG(CUG) and GTC(GUC); the dynamics of DNA CAG hairpins; mismatched homoduplexes formed by hexanucleotide repeats (GGGGCC) and (GGCCCC); and G-quadruplexes formed by (GGGGCC) and (GGGCCT). We also discuss the dynamics of strand slippage in DNA hairpins formed by CAG repeats as observed with single-molecule Fluorescence Resonance Energy Transfer. This review focuses on the rich behavior exhibited by the mismatches associated with these simple sequence repeat noncanonical structures.

Knock in of a hexanucleotide repeat expansion in the C9orf72 gene induces ALS in rats.

BACKGROUND: The GGGGCC (G4C2) repeat expansion in the human open reading frame 72 on chromosome 9, C9orf72, is the most common cause of amyotrophic lateral sclerosis (ALS). Studies in transgenic mouse models have linked the pathogenic mechanism of G4C2 repeat expansion to RNA foci or the accumulation of unnatural dipeptide repeats in neurons. However, only one of the existing transgenic mouse lines developed typical ALS. METHODS: C9orf72 knockin rats were generated by knockin of 80 G4C2 repeats with human flanking fragments within exon1a and exon1b at the rat C9orf72 locus. Protein expression was detected by western blot. Motor coordination and grip force were measured using a Rotarod test and a grip strength test. Neurodegeneration was assessed by Nissl staining with cresyl violet. RESULTS: C9orf72 haploinsufficiency reduced C9orf72 protein expression 40% in the cerebrum, cerebellum and spinal cords from knockin rats (P < .05). The knockin (KI) rats developed motor deficits from 4 months of age. Their falling latencies and grip force were decreased by 67% (P < .01) and 44% (P < .01), respectively, at 12 months of age compared to wild-type (WT) mice. The knockin of the hexanucleotide repeat expansion (HRE) caused a 47% loss of motor neurons in the spinal cord (P < .001) and 25% (5/20) of female KI rats developed hind limb paralysis at 13 to 24 months. CONCLUSION: Motor defects in KI rats may result from neurotoxicity caused by HRE and the resulting reduction in C9orf72 protein due to haploinsufficiency. These KI rats could be a useful model for investigating the contributions of loss-of-function to neurotoxicity in C9orf72-related ALS.

Comprehensive genotyping of the C9orf72 hexanucleotide repeat region in 2095 ALS samples from the NINDS collection using a two-mode, long-read PCR assay.

OBJECTIVE: Expansion of the G4C2 repeat tract in the C9orf72 gene is linked to frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Here, we provide comprehensive genotyping of the C9orf72 repeat region for the National Institute of Neurological Disorders and Stroke (NINDS) ALS collection (n = 2095), using a novel bimodal PCR assay capable of amplifying nearly 100% GC-rich sequences. METHODS: A single-tube 3-primer PCR assay mode, resolved using capillary electrophoresis, was used for sizing up to 145 repeats with single-repeat accuracy, for detecting expansions irrespective of their overall size, and for flagging confounding 3' sequence variations (SVs). A modified two-primer PCR mode, resolved via agarose gel electrophoresis, provided further size information for hyper-expanded samples (>145 repeats) up to ∼5.8 kb amplicons (∼950 G4C2 repeats). RESULTS: Within the evaluated cohort, 177 (8.4%) samples were expanded, with 175 (99%) samples being hyper-expanded. 3'-SVs were identified in 64 (3.1%) samples, and were most common in expanded alleles. Genotypes of all 606 (29%) homozygous samples were confirmed using an orthogonal PCR assay. CONCLUSION: This study and PCR method may improve and standardize molecular characterization of the C9orf72 locus, and have the potential to inform phenotype-genotype correlations and therapeutic development in ALS/FTD.

Purα Repaired Expanded Hexanucleotide GGGGCC Repeat Noncoding RNA-Caused Neuronal Toxicity in Neuro-2a Cells.

Expanded hexanucleotide GGGGCC repeat in a noncoding region of C9ORF72 is the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). However, its molecular pathogenesis remains unclear. In our previous study, the expanded GGGGCC repeats have been shown to be sufficient to cause neurodegeneration. In order to investigate the further role of expanded GGGGCC repeats in the neuron, the normal r(GGGGCC)3 and mutant-type expanded r(GGGGCC)30 expression vectors were transfected into Neuro-2a cells. Cell proliferation, dendrite development, and the proteins' levels of microtubule-associated protein-2 (MAP2) and cyclin-dependent kinase-5 (CDK5) were used to evaluate the cell toxicity of GGGGCC repeats on Neuro-2a cells. The results were shown that expression of expanded GGGGCC repeats caused neuronal cell toxicity in Neuro-2a cells, enhanced the expression of pMAP2 and pCDK5. Moreover, overexpression of Purα repaired expanded GGGGCC repeat-inducing neuronal toxicity in Neuro-2a cells and reduced the expression of pMAP2 and pCDK5. In all, our findings suggested that the expanded GGGGCC repeats might cause neurodegeneration through destroyed neuron cells. And the GGGGCC repeat-induced neuronal cell toxicity was inhibited by upregulation of Purα. We inferred that Purα inhibits expanded GGGGCC repeat-inducing neurodegeneration, which might reveal a novel mechanism of neurodegenerative diseases ALS and FTD.

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

BACKGROUND: 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 MinION 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 8× 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 > 800× 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.

All in the Family: Repeats and ALS/FTD.

In 2011, an intronic (G4C2)•(G2C4) expansion was shown to cause the most common forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This discovery linked ALS with a clinically distinct form of dementia and a larger group of microsatellite repeat diseases, and catalyzed basic and translational research.

C9orf72 hexanucleotide repeat expansions and Ataxin 2 intermediate length repeat expansions in Indian patients with amyotrophic lateral sclerosis.

Repeat expansions in the chromosome 9 open reading frame 72 (C9orf72) gene have been recognized as a major contributor to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia in the Caucasian population. Intermediate length repeat expansions of CAG (polyQ) repeat in the ATXN2 gene have also been reported to increase the risk of developing ALS in North America and Europe. We screened 131 ALS patients and 127 healthy controls from India for C9orf72 and ATXN2 repeat expansions. We found pathogenic hexanucleotide expansions in 3 of the 127 sporadic ALS patients, in 1 of the 4 familial ALS patients, and in none of the healthy controls. In addition, our findings suggest that the 10 base-pair deletion that masks detection of C9orf72 repeat expansion does not explain the low frequency of this repeat expansion among Indian ALS patients. Intermediate length polyQ expansions (27Qs-32Qs) in the ATXN2 gene were detected in 6 of the 127 sporadic ALS patients and 2 of the 127 of the healthy controls. Long ATXN2 polyQ repeats (≥33Qs) were not present in any of the ALS patients or controls. Our findings highlight the need for large-scale multicenter studies on Indian ALS patients to better understand the underlying genetic causes.

Amyotrophic Lateral Sclerosis with Frontotemporal Dementia in the Presence of C9orf72 Repeat Expansion-A Case Report.

Amyotrophic lateral sclerosis and frontotemporal dementia are significant neurodegenerative illnesses with possible genetic predispositions. The C9orf72 gene and the GGGGCC repeat expansions of it are reported to have a causative role in the expression of these conditions. We report a case of a patient with autosomal dominant amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) in the presence of C9orf72 repeat expansion. We believe our case further supports the theory that the presence of C9orf72 repeat expansion in patients with a family history of amyotrophic lateral sclerosis and/or frontotemporal dementia significantly increases their risk of developing either or both diseases. The development of antisense oligonucleotides that might target GGGGCC RNA sequences theoretically may have a therapeutic role in mitigating the clinical expression of these illnesses.

Identification of a novel loss-of-function C9orf72 splice site mutation in a patient with amyotrophic lateral sclerosis.

Abnormal expansion of a hexanucleotide GGGGCC repeat in the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia in Caucasians. However, the underlying pathologic mechanisms remain controversial, and both loss-of-function and gain-of-function models have been proposed. To gain further insight into these mechanisms, we performed mutation analysis of C9orf72 in 276 Han Chinese patients with ALS. We identified GGGGCC expansions in 2 cases of sporadic ALS with 38 and 63 repeats, as well as a novel splice site mutation (c.601-2A>G) in a third case. These genetic alterations were not detected in 332 control patients without neurological disease. Intriguingly, functional analysis revealed that the splice site mutation disrupted the reading frame, creating a premature stop codon (p.I201fsX235). Decreased levels of the mutant messenger RNA were detected in patient cells, suggesting that it may undergo nonsense-mediated messenger RNA decay. Taken together, these results demonstrate that C9orf72 mutation is infrequently associated with ALS in Han Chinese patients and suggest that a splice site mutation in C9orf72 may lead to loss of function due to haploinsufficiency of the resulting protein.

GGGGCC microsatellite RNA is neuritically localized, induces branching defects, and perturbs transport granule function.

Microsatellite expansions are the leading cause of numerous neurodegenerative disorders. Here we demonstrate that GGGGCC and CAG microsatellite repeat RNAs associated with C9orf72 in amyotrophic lateral sclerosis/frontotemporal dementia and with polyglutamine diseases, respectively, localize to neuritic granules that undergo active transport into distal neuritic segments. In cultured mammalian spinal cord neurons, the presence of neuritic GGGGCC repeat RNA correlates with neuronal branching defects, and the repeat RNA localizes to granules that label with fragile X mental retardation protein (FMRP), a transport granule component. Using a Drosophila GGGGCC expansion disease model, we characterize dendritic branching defects that are modulated by FMRP and Orb2. The human orthologs of these modifiers are misregulated in induced pluripotent stem cell-differentiated neurons (iPSNs) from GGGGCC expansion carriers. These data suggest that expanded repeat RNAs interact with the messenger RNA transport and translation machinery, causing transport granule dysfunction. This could be a novel mechanism contributing to the neuronal defects associated with C9orf72 and other microsatellite expansion diseases.