Mutations in the tail domain of the neurofilament heavy chain gene increase the risk of amyotrophic lateral sclerosis

Objective Genetic variation in the neurofilament heavy chain gene ( NEFH ) has been convincingly linked to the pathogenesis of multiple neurodegenerative diseases, however, the relationship between NEFH mutations and ALS susceptibility has not been robustly explored. We therefore wanted to determine if genetic variants in NEFH modify ALS risk. Methods We performed fixed and random effects model meta-analysis of published case-control studies reporting NEFH variant frequencies using next-generation sequencing, microarray or PCR-based approaches. Comprehensive screening and rare variant burden analysis of NEFH variation in the Project MinE ALS whole-genome sequencing data set was also conducted. Results We identified 12 case-control studies that reported NEFH variant frequencies, for a total of 9,496 samples (4,527 ALS cases and 4,969 controls). Fixed effects meta-analysis found that rare (MAF<1%) missense variants in the tail domain of NEFH increase ALS risk (OR 4.56, 95% CI 2.13-9.72, p<0.0001). A total of 591 rare NEFH variants, mostly novel (78.2%), were found in the Project MinE dataset (8,903 samples: 6,469 cases and 2,434 controls). Burden analysis showed ultra-rare (MAF <0.1%) pathogenic missense variants in the tail domain are associated with ALS (OR 1.94, 95% CI 0.86-4.37, Madsen-Browning p=0.039), replicating and confirming the meta-analysis finding. High-frequency rare (MAF 0.1-1%) tail in-frame deletions also confer susceptibility to ALS (OR 1.18, 95% CI 0.67-2.07, SKAT-O p=0.03), which supports previous findings. Interpretation This study shows that NEFH tail domain variants are a risk factor of ALS and supports the inclusion of missense and in-frame deletion NEFH variants in ALS genetic screening panels. ### Competing Interest Statement JHV reports to have sponsored research agreements with Biogen and Astra Zeneca. AAC reports consultancies or advisory boards for Amylyx, Apellis, Biogen, Brainstorm, Cytokinetics, GenieUs, GSK, Lilly, Mitsubishi Tanabe Pharma, Novartis, OrionPharma, Quralis, Sano, Sanofi, and Wave Pharmaceuticals. The other authors have nothing to report. ### Funding Statement The authors are supported by South London and Maudsley NHS Foundation Trust; MND Scotland; Motor Neurone Disease Association; National Institute for Health Research; Darby Rimmer MND Foundation; Spastic Paraplegia Foundation and Rosetrees Trust. HM is supported by GlaxoSmithKline and the KCL funded centre for Doctoral Training (CDT) in Data-Driven Health. AAK is funded by ALS Association Milton Safenowitz Research Fellowship (grant number22-PDF-609.DOI :10.52546/pc.gr.150909.), The Motor Neurone Disease Association (MNDA) Fellowship (Al Khleifat/Oct21/975-799), The Darby Rimmer Foundation, and The NIHR Maudsley Biomedical Research Centre. AI is funded by the Motor Neurone Disease Association and The NIHR Maudsley Biomedical Research Centre. AAC is an NIHR Senior Investigator (NIHR202421) and has received support from an EU Joint Programme - Neurodegenerative Disease Research (JPND) project. The work is supported through the following funding organisations under the aegis of JPND - [www.jpnd.eu][1] (United Kingdom, Medical Research Council (MR/L501529/1; MR/R024804/1) and Economic and Social Research Council (ES/L008238/1)) and through the Motor Neurone Disease Association, My Name'5 Doddie Foundation, and Alan Davidson Foundation. This study represents independent research part funded by the National Institute for Health Research (NIHR) Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King's College London. Project MinE Belgium was supported by a grant from IWT (no 140935), the ALS Liga België, the National Lottery of Belgium and the KU Leuven Opening the Future Fund. PVD holds a senior clinical investigatorship of FWO-Vlaanderen (G077121N) and is supported by the E. von Behring Chair for Neuromuscular and Neurodegenerative Disorders, the ALS Liga België; and the KU Leuven funds "Een Hart voor ALS", "Laeversfonds voor ALS Onderzoek" and the "Valéry Perrier Race against ALS Fund". ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Genetic data generated from these cohorts can be downloaded upon request from the Project MinE Data Browser (). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. [1]: https://www.jpnd.eu