Recently, large-scale case-control analyses have been prioritized in the study of ALS. Yet the same effort has not been put forward to investigate additive moderate phenotypic effects of genetic variants in genes driving ALS risk, despite case-level evidence suggesting a potential oligogenic risk model. Considering its direct clinical and therapeutic implications, a large-scale robust investigation of oligogenicity in ALS is greatly needed. Here, we leveraged the Project MinE ALS Sequencing Consortium genome sequencing datasets of individuals with ALS (n = 6711) and controls (n = 2391) to identify signals of association between oligogenicity in known ALS genes (n=26) and disease risk, as well as clinical outcomes. Applying regression models to a discovery and replication cohort, we observed that the risk imparted from carrying rare variants in multiple known ALS genes was significant and was greater than the risk associated with carrying only a single rare variant, both in the presence and absence of variants in the most well-established ALS genes, such as C9orf72. However, in contrast to risk, the relationships between oligogenicity and ALS clinical outcomes, such as age of onset and survival, might not follow the same pattern as we did not observe any associations. Our findings represent the first large-scale, case-control assessment of oligogenic associations in ALS to date and confirm that oligogenic events involving known ALS risk genes are indeed relevant for the risk of disease in approximately 6% of ALS but not necessarily for disease onset and survival. This must be considered in genetic counselling and testing by ensuring the use of comprehensive gene panels even when a potential pathogenic variant has already been identified. Moreover, in the age of stratified medication and gene therapy, it supports the need of a complete genetic profile for the correct choice of therapy in all ALS patients.

The authors have declared no competing interest.

This is an EU Joint Programme Neurodegenerative Disease Research (JPND) project. The project is supported through the following funding organizations under the aegis of JPND http://www.neurodegenerationresearch.eu/ [United Kingdom, Medical Research Council (MR/L501529/1 and MR/R024804/1) and Economic and Social Research Council (ES/L008238/1)]. AAC is a NIHR Senior Investigator. AAC receives salary support from the National Institute for Health and Care Research (NIHR) Dementia Biomedical Research Unit at South London and Maudsley NHS Foundation Trust and King's College London. The work leading up to this publication was funded by the European Community's Health Seventh Framework Program (FP7/2007 2013; grant agreement number 259867) and Horizon 2020 Program (H2020-PHC-2014-two-stage; grant agreement number 633413). This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 Research and Innovation Programme (grant agreement no. 772376 EScORIAL. This study represents independent research part funded by the NIHR Maudsley Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King's College London. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, King's College London, or the Department of Health and Social Care. AAD is supported by the Canadian Institute of Health Research Banting Postdoctoral Fellowship Program. AI is funded by South London and Maudsley NHS Foundation Trust, MND Scotland, Motor Neurone Disease Association, National Institute for Health and Care Research, Spastic Paraplegia Foundation, Rosetrees Trust, Darby Rimmer MND Foundation, the Medical Research Council (UKRI) and Alzheimer's Research UK. SMKF is supported by grants from ALS Canada, Brain Canada, the Michael J. Fox Foundation, and the Montreal Neurological Institute Hospital. Project MinE Belgium was supported by a grant from IWT (n 140935), the ALS Liga Belgie, the National Lottery of Belgium and the KU Leuven Opening the Future Fund. AAK is funded by the ALS Association Milton Safenowitz Research Fellowship, The Motor Neurone Disease Association (MNDA) Fellowship, The Darby Rimmer Foundation, and The NIHR Maudsley Biomedical Research Centre.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

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, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

Individual whole-genome sequencing data are available and can be requested through Project MinE (https://www.projectmine.com/research/data-sharing/). A data access committee controls access to raw data, ensuring a FAIR data setup (https://www.datafairport.org). Details on the frequencies and gene burden test results are available on the ProjectMinE databrowser (http://databrowser.projectmine.com).