Solve-RD is a pan-European rare disease (RD) research program that aims to identify disease-causing genetic variants in previously undiagnosed RD families. We utilized 10-fold coverage HiFi long-read sequencing (LRS) for detecting causative structural variants (SVs), single nucleotide variants (SNVs), insertion-deletions (InDels), and short tandem repeat (STR) expansions in extensively studied RD families without a clear molecular diagnosis. Our cohort includes 293 individuals from 114 genetically undiagnosed RD families selected by European Rare Disease network (ERN) experts. Of these, 21 families were affected by so-called 'unsolvable' syndromes for which genetic causes remain unknown, and 93 families with at least one individual affected by a rare neurological, neuromuscular, or epilepsy disorder without genetic diagnosis despite extensive prior testing. Clinical interpretation and orthogonal validation of variants in known disease genes yielded thirteen novel genetic diagnoses due to de novo and rare inherited SNVs, InDels, SVs, and STR expansions. In an additional four families, we identified a candidate disease-causing SV affecting several genes including a MCF2/FGF13 fusion and PSMA3 deletion. However, no common genetic cause was identified in any of the 'unsolvable' syndromes. Taken together, we found (likely) disease-causing genetic variants in 13.0% of previously unsolved families and additional candidate disease-causing SVs in another 4.3% of these families. In conclusion, our results demonstrate the added value of HiFi long-read genome sequencing in undiagnosed rare diseases.

The authors have declared no competing interest.

The Solve-RD project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 779257. This research is supported (not financially) by several ERNs: ERN on Intellectual disability, TeleHealth, Autism and Congenital Anomalies (ERN ITHACA)-Project ID No 101085231; ERN on Rare Neurological Diseases (ERN RND)-Project ID No 101155994; ERN for Neuromuscular Diseases (ERN Euro-NMD)-Project ID No 101156434; and ERN for Rare and Complex Epilepsies (ERN EpiCARE) - Project ID No 101156811. The ERNs are co-funded by the European Union within the framework of the Third Health Program. V.A.Y. and J.G. received funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) via the project NFDI 1/1 'GHGA - German Human Genome-Phenome Archive' (No 441914366). L.Sa. received funding from the Sigrid Juselius Foundation (Fellowship No 220540). RS received funding from the Bundesministerium fur Bildung und Forschung (BMBF) through funding for the TreatHSP network (grant 01GM2209A) and the National Institute of Neurological Disorders and Stroke (NINDS) under Award Number R01NS072248. H.H. was supported by the DFG (HE8803/1-1 to H.H.)

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:

Ethics committee/IRB of University of Tuebingen, Tuebingen, Germany, gave ethical approval for this work (ClinicalTrials.gov Nr.: NCT03491280; https://clinicaltrials.gov/study/NCT03491280). All individuals were recruited via the respective European Reference Networks. Inclusion criteria were a clinical rare disease diagnosis in at least one family member by one of the associated expert centres and a not conclusive exome or genome analysis at time of submission. We did not exclude anyone based on sex, gender, ethnicity, race, age or any other socially relevant groupings. Each patient entry was associated with its submitting investigator or clinician and linked to its corresponding European Reference Network. The responsibility of checking the data was suitable for submission to the RD-Connect GPAP and Solve-RD lay with the data submitter as required by their Code of Conduct (institution: Fundacio Centre de Regulacio Genomica) and Data Sharing Policy (institution: Solve-RD general assembly), respectively. In some cases, individuals had to be re-consented prior to data submission. This study adheres to the principles set out in the Declaration of Helsinki.

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).

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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

Long-read sequencing data for the complete study cohort have been deposited in EGA under controlled access (accession codes pending).