Neurological disorders caused by novel non-coding repeat expansions: clinical features and differential diagnosis.

Nucleotide repeat expansions in the human genome are a well-known cause of neurological disease. In the past decade, advances in DNA sequencing technologies have led to a better understanding of the role of non-coding DNA, that is, the DNA that is not transcribed into proteins. These techniques have also enabled the identification of pathogenic non-coding repeat expansions that cause neurological disorders. Mounting evidence shows that adult patients with familial or sporadic presentations of epilepsy, cognitive dysfunction, myopathy, neuropathy, ataxia, or movement disorders can be carriers of non-coding repeat expansions. The description of the clinical, epidemiological, and molecular features of these recently identified non-coding repeat expansion disorders should guide clinicians in the diagnosis and management of these patients, and help in the genetic counselling for patients and their families.

Somatic and intergenerational G4C2 hexanucleotide repeat instability in a human C9orf72 knock-in mouse model.

Expansion of a G4C2 repeat in the C9orf72 gene is associated with familial Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). To investigate the underlying mechanisms of repeat instability, which occurs both somatically and intergenerationally, we created a novel mouse model of familial ALS/FTD that harbors 96 copies of G4C2 repeats at a humanized C9orf72 locus. In mouse embryonic stem cells, we observed two modes of repeat expansion. First, we noted minor increases in repeat length per expansion event, which was dependent on a mismatch repair pathway protein Msh2. Second, we found major increases in repeat length per event when a DNA double- or single-strand break (DSB/SSB) was artificially introduced proximal to the repeats, and which was dependent on the homology-directed repair (HDR) pathway. In mice, the first mode primarily drove somatic repeat expansion. Major changes in repeat length, including expansion, were observed when SSB was introduced in one-cell embryos, or intergenerationally without DSB/SSB introduction if G4C2 repeats exceeded 400 copies, although spontaneous HDR-mediated expansion has yet to be identified. These findings provide a novel strategy to model repeat expansion in a non-human genome and offer insights into the mechanism behind C9orf72 G4C2 repeat instability.

A genome-wide spectrum of tandem repeat expansions in 338,963 humans.

The Genome Aggregation Database (gnomAD), widely recognized as the gold-standard reference map of human genetic variation, has largely overlooked tandem repeat (TR) expansions, despite the fact that TRs constitute ∼6% of our genome and are linked to over 50 human diseases. Here, we introduce the TR-gnomAD (https://wlcb.oit.uci.edu/TRgnomAD), a biobank-scale reference of 0.86 million TRs derived from 338,963 whole-genome sequencing (WGS) samples of diverse ancestries (39.5% non-European samples). TR-gnomAD offers critical insights into ancestry-specific disease prevalence using disparities in TR unit number frequencies among ancestries. Moreover, TR-gnomAD is able to differentiate between common, presumably benign TR expansions, which are prevalent in TR-gnomAD, from those potentially pathogenic TR expansions, which are found more frequently in disease groups than within TR-gnomAD. Together, TR-gnomAD is an invaluable resource for researchers and physicians to interpret TR expansions in individuals with genetic diseases.

Glucose hypometabolism prompts RAN translation and exacerbates C9orf72-related ALS/FTD phenotypes.

The most prevalent genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia is a (GGGGCC)n nucleotide repeat expansion (NRE) occurring in the first intron of the C9orf72 gene (C9). Brain glucose hypometabolism is consistently observed in C9-NRE carriers, even at pre-symptomatic stages, but its role in disease pathogenesis is unknown. Here, we show alterations in glucose metabolic pathways and ATP levels in the brains of asymptomatic C9-BAC mice. We find that, through activation of the GCN2 kinase, glucose hypometabolism drives the production of dipeptide repeat proteins (DPRs), impairs the survival of C9 patient-derived neurons, and triggers motor dysfunction in C9-BAC mice. We also show that one of the arginine-rich DPRs (PR) could directly contribute to glucose metabolism and metabolic stress by inhibiting glucose uptake in neurons. Our findings provide a potential mechanistic link between energy imbalances and C9-ALS/FTD pathogenesis and suggest a feedforward loop model with potential opportunities for therapeutic intervention.

Investigating Repeat Expansions in NIPA1, NOP56, and NOTCH2NLC Genes: A Closer Look at Amyotrophic Lateral Sclerosis Patients from Southern Italy.

The discovery of hexanucleotide repeats expansion (RE) in Chromosome 9 Open Reading frame 72 (C9orf72) as the major genetic cause of amyotrophic lateral sclerosis (ALS) and the association between intermediate repeats in Ataxin-2 (ATXN2) with the disorder suggest that repetitive sequences in the human genome play a significant role in ALS pathophysiology. Investigating the frequency of repeat expansions in ALS in different populations and ethnic groups is therefore of great importance. Based on these premises, this study aimed to define the frequency of REs in the NIPA1, NOP56, and NOTCH2NLC genes and the possible associations between phenotypes and the size of REs in the Italian population. Using repeat-primed-PCR and PCR-fragment analyses, we screened 302 El-Escorial-diagnosed ALS patients and compared the RE distribution to 167 age-, gender-, and ethnicity-matched healthy controls. While the REs distribution was similar between the ALS and control groups, a moderate association was observed between longer RE lengths and clinical features such as age at onset, gender, site of onset, and family history. In conclusion, this is the first study to screen ALS patients from southern Italy for REs in NIPA1, NOP56, and NOTCH2NLC genes, contributing to our understanding of ALS genetics. Our results highlighted that the extremely rare pathogenic REs in these genes do not allow an association with the disease.

Precise editing of pathogenic nucleotide repeat expansions in iPSCs using paired prime editor.

Nucleotide repeat expansion disorders, a group of genetic diseases characterized by the expansion of specific DNA sequences, pose significant challenges to treatment and therapy development. Here, we present a precise and programmable method called prime editor-mediated correction of nucleotide repeat expansion (PE-CORE) for correcting pathogenic nucleotide repeat expansion. PE-CORE leverages a prime editor and paired pegRNAs to achieve targeted correction of repeat sequences. We demonstrate the effectiveness of PE-CORE in HEK293T cells and patient-derived induced pluripotent stem cells (iPSCs). Specifically, we focus on spinal and bulbar muscular atrophy and spinocerebellar ataxia type, two diseases associated with nucleotide repeat expansion. Our results demonstrate the successful correction of pathogenic expansions in iPSCs and subsequent differentiation into motor neurons. Specifically, we detect distinct downshifts in the size of both the mRNA and protein, confirming the functional correction of the iPSC-derived motor neurons. These findings highlight PE-CORE as a precision tool for addressing the intricate challenges of nucleotide repeat expansion disorders, paving the way for targeted therapies and potential clinical applications.

SUMO protease FUG1, histone reader AL3 and chromodomain protein LHP1 are integral to repeat expansion-induced gene silencing in Arabidopsis thaliana.

Epigenetic gene silencing induced by expanded repeats can cause diverse phenotypes ranging from severe growth defects in plants to genetic diseases such as Friedreich's ataxia in humans. The molecular mechanisms underlying repeat expansion-induced epigenetic silencing remain largely unknown. Using a plant model with a temperature-sensitive phenotype, we have previously shown that expanded repeats can induce small RNAs, which in turn can lead to epigenetic silencing through the RNA-dependent DNA methylation pathway. Here, using a genetic suppressor screen and yeast two-hybrid assays, we identified novel components required for epigenetic silencing caused by expanded repeats. We show that FOURTH ULP GENE CLASS 1 (FUG1)-an uncharacterized SUMO protease with no known role in gene silencing-is required for epigenetic silencing caused by expanded repeats. In addition, we demonstrate that FUG1 physically interacts with ALFIN-LIKE 3 (AL3)-a histone reader that is known to bind to active histone mark H3K4me2/3. Loss of function of AL3 abolishes epigenetic silencing caused by expanded repeats. AL3 physically interacts with the chromodomain protein LIKE HETEROCHROMATIN 1 (LHP1)-known to be associated with the spread of the repressive histone mark H3K27me3 to cause repeat expansion-induced epigenetic silencing. Loss of any of these components suppresses repeat expansion-associated phenotypes coupled with an increase in IIL1 expression with the reversal of gene silencing and associated change in epigenetic marks. Our findings suggest that the FUG1-AL3-LHP1 module is essential to confer repeat expansion-associated epigenetic silencing and highlight the importance of post-translational modifiers and histone readers in epigenetic silencing.

Reversal of C9orf72 mutation-induced transcriptional dysregulation and pathology in cultured human neurons by allele-specific excision.

Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the C9orf72 locus in a patient-derived induced pluripotent stem cell (iPSC) line and a non-diseased wild-type (WT) line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 frontotemporal dementia/amyotrophic lateral sclerosis in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding single nucleotide polymorphism in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using digital droplet PCR (ddPCR), we determined that transcription from the mutant allele is upregulated at least 10-fold, and that sense transcription is independently regulated from each allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat poly-GP expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production, and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all dipeptide repeat proteins, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing.

Abundant transcriptomic alterations in the human cerebellum of patients with a C9orf72 repeat expansion.

The most prominent genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) is a repeat expansion in the gene C9orf72. Importantly, the transcriptomic consequences of the C9orf72 repeat expansion remain largely unclear. Here, we used short-read RNA sequencing (RNAseq) to profile the cerebellar transcriptome, detecting alterations in patients with a C9orf72 repeat expansion. We focused on the cerebellum, since key C9orf72-related pathologies are abundant in this neuroanatomical region, yet TDP-43 pathology and neuronal loss are minimal. Consistent with previous work, we showed a reduction in the expression of the C9orf72 gene and an elevation in homeobox genes, when comparing patients with the expansion to both patients without the C9orf72 repeat expansion and control subjects. Interestingly, we identified more than 1000 alternative splicing events, including 4 in genes previously associated with ALS and/or FTLD. We also found an increase of cryptic splicing in C9orf72 patients compared to patients without the expansion and controls. Furthermore, we demonstrated that the expression level of select RNA-binding proteins is associated with cryptic splice junction inclusion. Overall, this study explores the presence of widespread transcriptomic changes in the cerebellum, a region not confounded by severe neurodegeneration, in post-mortem tissue from C9orf72 patients.

DDX3X overexpression decreases dipeptide repeat proteins in a mouse model of C9ORF72-ALS/FTD.

Hexanucleotide repeat expansion in C9ORF72 (C9) is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One of the proposed pathogenic mechanisms is the neurotoxicity arising from dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG (RAN) translation. Therefore, reducing DPR levels emerges as a potential therapeutic strategy for C9ORF72-ALS/FTD. We previously identified an RNA helicase, DEAD-box helicase 3 X-linked (DDX3X), modulates RAN translation. DDX3X overexpression decreases poly-GP accumulation in C9ORF72-ALS/FTD patient-derived induced pluripotent stem cell (iPSC)-differentiated neurons (iPSNs) and reduces the glutamate-induced neurotoxicity. In this study, we examined the in vivo efficacy of DDX3X overexpression using a mouse model. We expressed exogenous DDX3X or GFP in the central nervous system (CNS) of the C9-500 ALS/FTD BAC transgenic or non-transgenic control mice using adeno-associated virus serotype 9 (AAV9). The DPR levels were significantly reduced in the brains of DDX3X-expressing C9-BAC mice compared to the GFP control even twelve months after virus delivery. Additionally, p62 aggregation was also decreased. No neuronal loss or neuroinflammatory response were detected in the DDX3X overexpressing C9-BAC mice. This work demonstrates that DDX3X overexpression effectively reduces DPR levels in vivo without provoking neuroinflammation or neurotoxicity, suggesting the potential of increasing DDX3X expression as a therapeutic strategy for C9ORF72-ALS/FTD.

RFC1 and FGF14 Repeat Expansions in Serbian Patients with Cerebellar Ataxia.

BACKGROUND: The newly discovered intronic repeat expansions in the genes encoding replication factor C subunit 1 (RFC1) and fibroblast growth factor 14 (FGF14) frequently cause late-onset cerebellar ataxia. OBJECTIVES: To investigate the presence of RFC1 and FGF14 pathogenic repeat expansions in Serbian patients with adult-onset cerebellar ataxia. METHODS: The study included 167 unrelated patients with sporadic or familial cerebellar ataxia. The RFC1 repeat expansion analysis was performed by duplex PCR and Sanger sequencing, while the FGF14 repeat expansion was tested for by long-range PCR, repeat-primed PCR, and Sanger sequencing. RESULTS: We identified pathogenic repeat expansions in RFC1 in seven patients (7/167; 4.2%) with late-onset sporadic ataxia with neuropathy and chronic cough. Two patients also had bilateral vestibulopathy. Repeat expansions in FGF14 were found in nine unrelated patients (9/167; 5.4%) with ataxia, less than half of whom presented with neuropathy and two-thirds with global brain atrophy. Tremor and episodic features were the most frequent additional characteristics in carriers of uninterrupted FGF14 repeat expansions. Among the 122 sporadic cases, 12 (9.8%) carried an expansion in either RFC1 or FGF14, comparable to 4/45 (8.9%) among the patients with a positive family history. CONCLUSIONS: Pathogenic repeat expansions in RFC1 and FGF14 are relatively frequent causes of adult-onset cerebellar ataxia, especially among sporadic patients, indicating that family history should not be considered when prioritizing ataxia patients for testing of RFC1 or FGF14 repeat expansions.

Sequence composition changes in short tandem repeats: heterogeneity, detection, mechanisms and clinical implications.

Short tandem repeats (STRs) are a class of repetitive elements, composed of tandem arrays of 1-6 base pair sequence motifs, that comprise a substantial fraction of the human genome. STR expansions can cause a wide range of neurological and neuromuscular conditions, known as repeat expansion disorders, whose age of onset, severity, penetrance and/or clinical phenotype are influenced by the length of the repeats and their sequence composition. The presence of non-canonical motifs, depending on the type, frequency and position within the repeat tract, can alter clinical outcomes by modifying somatic and intergenerational repeat stability, gene expression and mutant transcript-mediated and/or protein-mediated toxicities. Here, we review the diverse structural conformations of repeat expansions, technological advances for the characterization of changes in sequence composition, their clinical correlations and the impact on disease mechanisms.

Pathogenic CANVAS (AAGGG)n repeats stall DNA replication due to the formation of alternative DNA structures.

CANVAS is a recently characterized repeat expansion disease, most commonly caused by homozygous expansions of an intronic (A2G3)n repeat in the RFC1 gene. There are a multitude of repeat motifs found in the human population at this locus, some of which are pathogenic and others benign. In this study, we conducted structure-functional analyses of the pathogenic (A2G3)n and nonpathogenic (A4G)n repeats. We found that the pathogenic, but not the nonpathogenic, repeat presents a potent, orientation-dependent impediment to DNA polymerization in vitro. The pattern of the polymerization blockage is consistent with triplex or quadruplex formation in the presence of magnesium or potassium ions, respectively. Chemical probing of both repeats in vitro reveals triplex H-DNA formation by only the pathogenic repeat. Consistently, bioinformatic analysis of S1-END-seq data from human cell lines shows preferential H-DNA formation genome-wide by (A2G3)n motifs over (A4G)n motifs. Finally, the pathogenic, but not the nonpathogenic, repeat stalls replication fork progression in yeast and human cells. We hypothesize that the CANVAS-causing (A2G3)n repeat represents a challenge to genome stability by folding into alternative DNA structures that stall DNA replication.

RFC1 repeat expansions in downbeat nystagmus syndromes: frequency and phenotypic profile.

OBJECTIVES: The cause of downbeat nystagmus (DBN) remains unknown in a substantial number of patients ("idiopathic"), although intronic GAA expansions in FGF14 have recently been shown to account for almost 50% of yet idiopathic cases. Here, we hypothesized that biallelic RFC1 expansions may also represent a recurrent cause of DBN syndrome. METHODS: We genotyped the RFC1 repeat and performed in-depth phenotyping in 203 patients with DBN, including 65 patients with idiopathic DBN, 102 patients carrying an FGF14 GAA expansion, and 36 patients with presumed secondary DBN. RESULTS: Biallelic RFC1 AAGGG expansions were identified in 15/65 patients with idiopathic DBN (23%). None of the 102 GAA-FGF14-positive patients, but 2/36 (6%) of patients with presumed secondary DBN carried biallelic RFC1 expansions. The DBN syndrome in RFC1-positive patients was characterized by additional cerebellar impairment in 100% (15/15), bilateral vestibulopathy (BVP) in 100% (15/15), and polyneuropathy in 80% (12/15) of cases. Compared to GAA-FGF14-positive and genetically unexplained patients, RFC1-positive patients had significantly more frequent neuropathic features on examination and BVP. Furthermore, vestibular function, as measured by the video head impulse test, was significantly more impaired in RFC1-positive patients. DISCUSSION: Biallelic RFC1 expansions are a common monogenic cause of DBN syndrome.

Ribosomal quality control factors inhibit repeat-associated non-AUG translation from GC-rich repeats.

A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1 and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter assays and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation-suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders.

PolyGR and polyPR knock-in mice reveal a conserved neuroprotective extracellular matrix signature in C9orf72 ALS/FTD neurons.

Dipeptide repeat proteins are a major pathogenic feature of C9orf72 amyotrophic lateral sclerosis (C9ALS)/frontotemporal dementia (FTD) pathology, but their physiological impact has yet to be fully determined. Here we generated C9orf72 dipeptide repeat knock-in mouse models characterized by expression of 400 codon-optimized polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice recapitulate key features of C9ALS/FTD, including cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. TGF-ß1 was one of the top predicted regulators of this ECM signature and polyGR expression in human induced pluripotent stem cell neurons was sufficient to induce TGF-ß1 followed by COL6A1. Knockdown of TGF-ß1 or COL6A1 orthologues in polyGR model Drosophila exacerbated neurodegeneration, while expression of TGF-ß1 or COL6A1 in induced pluripotent stem cell-derived motor neurons of patients with C9ALS/FTD protected against glutamate-induced cell death. Altogether, our findings reveal a neuroprotective and conserved ECM signature in C9ALS/FTD.

eIF5 stimulates the CUG initiation of RAN translation of poly-GA dipeptide repeat protein (DPR) in C9orf72 FTLD/ALS.

Tandem GGGGCC repeat expansion in C9orf72 is a genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Transcribed repeats are translated into dipeptide repeat proteins via repeat-associated non-AUG (RAN) translation. However, the regulatory mechanism of RAN translation remains unclear. Here, we reveal a GTPase-activating protein, eukaryotic initiation factor 5 (eIF5), which allosterically facilitates the conversion of eIF2-bound GTP into GDP upon start codon recognition, as a novel modifier of C9orf72 RAN translation. Compared to global translation, eIF5, but not its inactive mutants, preferentially stimulates poly-GA RAN translation. RAN translation is increased during integrated stress response, but the stimulatory effect of eIF5 on poly-GA RAN translation was additive to the increase of RAN translation during integrated stress response, with no further increase in phosphorylated eIF2α. Moreover, an alteration of the CUG near cognate codon to CCG or AUG in the poly-GA reading frame abolished the stimulatory effects, indicating that eIF5 primarily acts through the CUG-dependent initiation. Lastly, in a Drosophila model of C9orf72 FTLD/ALS that expresses GGGGCC repeats in the eye, knockdown of endogenous eIF5 by two independent RNAi strains significantly reduced poly-GA expressions, confirming in vivo effect of eIF5 on poly-GA RAN translation. Together, eIF5 stimulates the CUG initiation of poly-GA RAN translation in cellular and Drosophila disease models of C9orf72 FTLD/ALS.

Resting-state fMRI functional connectome of C9orf72 mutation status.

OBJECTIVE: The resting-state functional connectome has not been extensively investigated in amyotrophic lateral sclerosis (ALS) spectrum disease, in particular in relationship with patients' genetic status. METHODS: Here we studied the network-to-network connectivity of 19 ALS patients carrying the C9orf72 hexanucleotide repeat expansion (C9orf72+), 19 ALS patients not affected by C9orf72 mutation (C9orf72-), and 19 ALS-mimic patients (ALSm) well-matched for demographic and clinical variables. RESULTS: When compared with ALSm, we observed greater connectivity of the default mode and frontoparietal networks with the visual network for C9orf72+ patients (P = 0.001). Moreover, the whole-connectome showed greater node degree (P < 0.001), while sensorimotor cortices resulted isolated in C9orf72+. INTERPRETATION: Our results suggest a crucial involvement of extra-motor functions in ALS spectrum disease. In particular, alterations of the visual cortex may have a pathogenic role in C9orf72-related ALS. The prominent feature of these patients would be increased visual system connectivity with the networks responsible of the functional balance between internal and external attention.

Role of the repeat expansion size in predicting age of onset and severity in RFC1 disease.

RFC1 disease, caused by biallelic repeat expansion in RFC1, is clinically heterogeneous in terms of age of onset, disease progression and phenotype. We investigated the role of the repeat size in influencing clinical variables in RFC1 disease. We also assessed the presence and role of meiotic and somatic instability of the repeat. In this study, we identified 553 patients carrying biallelic RFC1 expansions and measured the repeat expansion size in 392 cases. Pearson's coefficient was calculated to assess the correlation between the repeat size and age at disease onset. A Cox model with robust cluster standard errors was adopted to describe the effect of repeat size on age at disease onset, on age at onset of each individual symptoms, and on disease progression. A quasi-Poisson regression model was used to analyse the relationship between phenotype and repeat size. We performed multivariate linear regression to assess the association of the repeat size with the degree of cerebellar atrophy. Meiotic stability was assessed by Southern blotting on first-degree relatives of 27 probands. Finally, somatic instability was investigated by optical genome mapping on cerebellar and frontal cortex and unaffected peripheral tissue from four post-mortem cases. A larger repeat size of both smaller and larger allele was associated with an earlier age at neurological onset [smaller allele hazard ratio (HR) = 2.06, P < 0.001; larger allele HR = 1.53, P < 0.001] and with a higher hazard of developing disabling symptoms, such as dysarthria or dysphagia (smaller allele HR = 3.40, P < 0.001; larger allele HR = 1.71, P = 0.002) or loss of independent walking (smaller allele HR = 2.78, P < 0.001; larger allele HR = 1.60; P < 0.001) earlier in disease course. Patients with more complex phenotypes carried larger expansions [smaller allele: complex neuropathy rate ratio (RR) = 1.30, P = 0.003; cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) RR = 1.34, P < 0.001; larger allele: complex neuropathy RR = 1.33, P = 0.008; CANVAS RR = 1.31, P = 0.009]. Furthermore, larger repeat expansions in the smaller allele were associated with more pronounced cerebellar vermis atrophy (lobules I-V ß = -1.06, P < 0.001; lobules VI-VII ß = -0.34, P = 0.005). The repeat did not show significant instability during vertical transmission and across different tissues and brain regions. RFC1 repeat size, particularly of the smaller allele, is one of the determinants of variability in RFC1 disease and represents a key prognostic factor to predict disease onset, phenotype and severity. Assessing the repeat size is warranted as part of the diagnostic test for RFC1 expansion.

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.