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Background: The gene C9orf72 harbors a non-coding hexanucleotide repeat expansion known to cause amyotrophic lateral sclerosis and frontotemporal dementia. While previous studies have estimated the length of this repeat expansion in multiple tissues, technological limitations have impeded researchers from exploring additional features, such as methylation levels., Methods: We aimed to characterize C9orf72 repeat expansions using a targeted, amplification-free long-read sequencing method. Our primary goal was to determine the presence and subsequent quantification of observed methylation in the C9orf72 repeat expansion. In addition, we measured the repeat length and purity of the expansion. To do this, we sequenced DNA extracted from blood for 27 individuals with an expanded C9orf72 repeat., Results: For these individuals, we obtained a total of 7,765 on-target reads, including 1,612 fully covering the expanded allele. Our in-depth analysis revealed that the expansion itself is methylated, with great variability in total methylation levels observed, as represented by the proportion of methylated CpGs (13 to 66%). Interestingly, we demonstrated that the expanded allele is more highly methylated than the wild-type allele (P-Value = 2.76E-05) and that increased methylation levels are observed in longer repeat expansions (P-Value = 1.18E-04). Furthermore, methylation levels correlate with age at collection (P-Value = 3.25E-04) as well as age at disease onset (P-Value = 0.020). Additionally, we detected repeat lengths up to 4,088 repeats (~ 25 kb) and found that the expansion contains few interruptions in the blood., Conclusions: Taken together, our study demonstrates robust ability to quantify methylation of the expanded C9orf72 repeat, capturing differences between individuals harboring this expansion and revealing clinical associations., Competing Interests: Declarations. Ethics approval and consent to participate: All subjects agreed to be in the study, and biological specimens were obtained after informed consent with approval from the Mayo Clinic Institutional Review Board (IRB). Consent for publication: Not applicable. Competing interests: MDJ and RR hold a patent on methods to screen for the C9orf72 hexanucleotide repeat expansion., (© 2024. The Author(s).)

Amyotrophic lateral sclerosis is a heterogeneous, fatal neurodegenerative disease, characterized by motor neuron loss and in 50% of cases also by cognitive and/or behavioral changes. Mendelian forms of ALS comprise approximately 10-15% of cases. The majority is however considered sporadic, but also with a high contribution of genetic risk factors. To explore the contribution of somatic mutations and/or epigenetic changes to disease risk, we performed whole genome sequencing and methylation analyses using samples from multiple tissues on a cohort of 26 monozygotic twins discordant for ALS, followed by in-depth validation and replication experiments. The results of these analyses implicate several mechanisms in ALS pathophysiology, which include a role for de novo mutations, defects in DNA damage repair and accelerated aging., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Expansions of short tandem repeats are the cause of many neurogenetic disorders including familial amyotrophic lateral sclerosis, Huntington disease, and many others. Multiple methods have been recently developed that can identify repeat expansions in whole genome or exome sequencing data. Despite the widely recognized need for visual assessment of variant calls in clinical settings, current computational tools lack the ability to produce such visualizations for repeat expansions. Expanded repeats are difficult to visualize because they correspond to large insertions relative to the reference genome and involve many misaligning and ambiguously aligning reads., Results: We implemented REViewer, a computational method for visualization of sequencing data in genomic regions containing long repeat expansions and FlipBook, a companion image viewer designed for manual curation of large collections of REViewer images. To generate a read pileup, REViewer reconstructs local haplotype sequences and distributes reads to these haplotypes in a way that is most consistent with the fragment lengths and evenness of read coverage. To create appropriate training materials for onboarding new users, we performed a concordance study involving 12 scientists involved in short tandem repeat research. We used the results of this study to create a user guide that describes the basic principles of using REViewer as well as a guide to the typical features of read pileups that correspond to low confidence repeat genotype calls. Additionally, we demonstrated that REViewer can be used to annotate clinically relevant repeat interruptions by comparing visual assessment results of 44 FMR1 repeat alleles with the results of triplet repeat primed PCR. For 38 of these alleles, the results of visual assessment were consistent with triplet repeat primed PCR., Conclusions: Read pileup plots generated by REViewer offer an intuitive way to visualize sequencing data in regions containing long repeat expansions. Laboratories can use REViewer and FlipBook to assess the quality of repeat genotype calls as well as to visually detect interruptions or other imperfections in the repeat sequence and the surrounding flanking regions. REViewer and FlipBook are available under open-source licenses at https://github.com/illumina/REViewer and https://github.com/broadinstitute/flipbook respectively., (© 2022. The Author(s).)

We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40-64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington's disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered., Competing Interests: Declaration of Interests S.P.-B., A.B.S., J.A.H., H.R.M., and B.J.T. hold US, EU, and Canadian patents on the clinical testing and therapeutic intervention for the hexanucleotide repeat expansion of C9 or f72. S.W.S. serves on the scientific advisory council of the Lewy Body Dementia Association and is an editorial board member for the Journal of Parkinson’s Disease. B.J.T. is an editorial board member for JAMA Neurology, JNNP, and Neurobiology of Aging. V.S. is on the journal editorial boards of Amyotrophic Lateral Sclerosis, European Neurology, American Journal of Neurodegenerative Diseases, and Frontiers in Neurology. He has also received compensation for consulting services and speaking activities from AveXis, Cytokinetics, Italfarmaco, and Zambon. J.B.R. is an editor for Brain and has received compensation for consulting services from Asceneuron, Biogen, UCB, Astex, and SV Health. J.E.L. is a member of the scientific advisory board for Cerevel Therapeutics and a consultant and provides expert testimony for Perkins Coie., (Published by Elsevier Inc.)

C9orf72 hexanucleotide repeat expansions are the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Genetic testing for C9orf72 expansions in patients with ALS and/or FTD and their relatives has become increasingly available since hexanucleotide repeat expansions were first reported in 2011. The repeat number is highly variable and the threshold at which repeat size leads to neurodegeneration remains unknown. We present the case of an ALS patient who underwent genetic testing through our Motor Neurone Disease Clinic. We highlight current limitations to analysing and interpreting C9orf72 expansion test results and describe how this resulted in discordant reports of pathogenicity between testing laboratories that confounded the genetic counselling process. We conclude that patients with ALS or FTD and their at-risk family members, need to be adequately counselled about the limitations of current knowledge to ensure they are making informed decisions about genetic testing for C9orf72 . Greater collaboration between clinicians, testing laboratories and researchers is required to ensure risks to patients and their families are minimised.

Sporadic amyotrophic lateral sclerosis (ALS) is considered to be a complex disease with multiple genetic risk factors contributing to the pathogenesis. Identification of genetic risk factors that co-occur frequently could provide relevant insight into underlying mechanisms of motor neuron degeneration. To dissect the genetic architecture of sporadic ALS, we undertook a large sequencing study in 755 apparently sporadic ALS cases and 959 controls, analyzing 10 ALS genes: SOD1, C9orf72, TARDBP, FUS, ANG, CHMP2B, ATXN2, NIPA1, SMN1, and UNC13A. We observed sporadic cases with multiple genetic risk variants in 4.1% compared with 1.3% in controls. The overall difference was not in excess of what is to be expected by chance (binomial test, p = 0.59). We did, however, observe a higher frequency than expected of C9orf72 repeat carriers with co-occurring susceptibility variants (ATXN2, NIPA1, and SMN1; p = 0.001), which is mainly because of the co-occurrence of NIPA1 repeats in 15% of C9orf72 repeat carriers (p = 0.006)., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A non-coding hexanucleotide repeat expansion in the C9ORF72 gene is the most common mutation associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To investigate the pathological role of C9ORF72 in these diseases, we generated a line of mice carrying a bacterial artificial chromosome containing exons 1 to 6 of the human C9ORF72 gene with approximately 500 repeats of the GGGGCC motif. The mice showed no overt behavioral phenotype but recapitulated distinctive histopathological features of C9ORF72 ALS/FTD, including sense and antisense intranuclear RNA foci and poly(glycine-proline) dipeptide repeat proteins. Finally, using an artificial microRNA that targets human C9ORF72 in cultures of primary cortical neurons from the C9BAC mice, we have attenuated expression of the C9BAC transgene and the poly(GP) dipeptides. The C9ORF72 BAC transgenic mice will be a valuable tool in the study of ALS/FTD pathobiology and therapy., (Copyright © 2015 Elsevier Inc. All rights reserved.)