Purpose: To identify genetic etiologies and genotype/phenotype associations for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs). Methods: We coupled phenotyping with exome or genome sequencing of 467 pedigrees with genetically unsolved oCCDDs, integrating analyses of pedigrees, human and animal model phenotypes, and de novo variants to identify rare candidate single nucleotide variants, insertion/deletions, and structural variants disrupting protein-coding regions. Prioritized variants were classified for pathogenicity and evaluated for genotype/phenotype correlations. Results: Analyses elucidated phenotypic subgroups, identified pathogenic/likely pathogenic variant(s) in 43/467 probands (9.2%), and prioritized variants of uncertain significance in 70/467 additional probands (15.0%). These included known and novel variants in established oCCDD genes, genes associated with syndromes that sometimes include oCCDDs (e.g., MYH10, KIF21B, TGFBR2, TUBB6), genes that fit the syndromic component of the phenotype but had no prior oCCDD association (e.g., CDK13, TGFB2), genes with no reported association with oCCDDs or the syndromic phenotypes (e.g., TUBA4A, KIF5C, CTNNA1, KLB, FGF21), and genes associated with oCCDD phenocopies that had resulted in misdiagnoses. Conclusion: This study suggests that unsolved oCCDDs are clinically and genetically heterogeneous disorders often overlapping other Mendelian conditions and nominates many candidates for future replication and functional studies.

D.G.M. is a paid adviser to GlaxoSmithKline; Insitro; Variant Bio and Overtone Therapeutics and has received research support from AbbVie; Astellas; Biogen; BioMarin; Eisai; Merck; Microsoft; Pfizer and Sanofi-Genzyme; none of these activities are related to the work presented here. M.E.T. is provided with research reagents and/or resources from Microsoft; Illumina; Pacific Biosciences; and Ionis Pharmaceuticals; none of these are related to the work presented here. A.O.D.L. is on the scientific advisory board for Congenica Inc. S.A.D.G. is an employee and stockholder of Regeneron Pharmaceutical.

This study was funded in part by NEI R01EY027421 and NHLBI X01HL132377 (E.C.E). Sequencing and analysis were provided by the Broad Institute Center for Mendelian Genomics (Broad CMG) and were funded by: the National Human Genome Research Institute (NHGRI) grant UM1HG008900 (with additional support from the National Eye Institute, and the National Heart, Lung and Blood Institute). Analysis was also supported by NHGRI grants U01HG011755 and R01HG009141, NIMH grant MH115957, along with grant 2022-309464 from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation. J.A.J. was supported by T32GM007748, 5T32NS007473, 5T32EY007145, and the Harvard Medical School William Randolph Hearst Fund. G.L. was supported by the Fonds de recherche en Sante du Quebec (FRQS) fellowship. E.G. and A.S.L. were supported by the Manton Center for Orphan Disease Research funding. P.M.M.R. was supported by R01 EY027421-02S1 and R01 EY027421-04S1. M.C.W. was supported by NEI 5K08EY027850 and BCH Ophthalmology Foundation Faculty Discovery Award. H.B. was supported by K99/R00DE026824. D.G.M. acknowledges a National Health and Medical Research Council investigator grant (#2009982). M.C.W., S.M., and D.G.H. receive research support from Childrens Hospital Ophthalmology Foundation, Inc., Boston, MA. E.C.E. is a Howard Hughes Medical Institute Investigator.

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 Boston Childrens Hospital gave ethical approval for this work. This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Boston Childrens Hospital (BCH), Boston, MA (protocol 05-03-036R). Data were collected in accordance with the ethical guidelines of BCH.

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.

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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.

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Exome/genome sequencing data are accessible under dbGaP accession numbers phs001247.v1.p1 and phs001272.v2.p1. SNVs, indels, and SVs that were newly ACMG/AMP/ClinGen-classified by our study were submitted to ClinVar (Accession IDs: SUB14307097, SUB14310205, SUB14279226). A subset of variants were previously interpreted by the ACMG/AMP criteria by our team and submitted to ClinVar under separate accession IDs (pedigree ENG_1580, SCV001445961.1; pedigree 42, SCV001445941.1; pedigree ENG_CMO, SCV003761257.1; pedigree 71, SCV002507051.1 and SCV000693896.1; pedigree 144, SCV001430799.1; pedigree 38, SCV001449530.1). A variant in pedigree 13 was previously interpreted by the Undiagnosed Diseases Network for the same individual as we report in our cohort (SCV000837689.1). All other data produced in the present work are contained in the manuscript.