Using computational approaches to enhance the interpretation of missense variants in the PAX6 gene

Purpose The PAX6 gene encodes a highly-conserved transcription factor involved in eye development. Heterozygous loss-of-function variants in PAX6 can cause a range of ophthalmic disorders including aniridia. A key molecular diagnostic challenge is that many PAX6 missense changes are presently classified as variants of uncertain significance. While computational tools can be used to assess the effect of genetic alterations, the accuracy of their predictions varies. Here, we evaluated and optimised the performance of computational prediction tools in relation to PAX6 missense variants. Methods Through inspection of publicly available resources (including HGMD, ClinVar, LOVD and gnomAD), we identified 241 PAX6 missense variants that were used for model training and evaluation. The performance of ten commonly-used computational tools was assessed and a threshold optimization approach was utilized to determine optimal cut-off values. Validation studies were subsequently undertaken using PAX6 variants from a local database. Results AlphaMissense, SIFT4G and REVEL emerged as the best-performing predictors; the optimized thresholds of these tools were 0.967, 0.025, and 0.772, respectively. Combining the prediction from these top-three tools resulted in lower performance compared to using AlphaMissense alone. Conclusion Tailoring the use of computational tools by employing optimized thresholds specific to PAX6 can enhance algorithmic performance. Our findings have implications for PAX6 variant interpretation in clinical settings. ### Competing Interest Statement E.B. is a paid consultant and equity holder of Oxford Nanopore, a paid consultant to Dovetail, and a non-executive director of Genomics England, a limited company wholly owned by the UK Department of Health and Social Care. All other authors declare no conflict of interests. ### Funding Statement The Wellcome Trust (224643/Z/21/Z, Clinical Research Career Development Fellowship to P.I.S.; 200990/Z/16/Z, Transforming Genetic Medicine Initiative to G.C.B.); the UK National Institute for Health Research (NIHR) Clinical Lecturer Programme (CL-2017-06-001652 to P.I.S.); the NIHR Manchester Biomedical Research Centre (NIHR203308); Retina UK and Fight for Sight (GR586, RP Genome Project - UK Inherited Retinal Disease Consortium to G.C.B.); the Indonesia Endowment Fund for Education (Lembaga Pengelola Dana Pendidikan (LPDP) scholarship to N.S.A.). ### Author Declarations 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 All data produced in the present work are included in the manuscript and the supplementary files * Acc : accuracy ACMG/AMP : the American College of Medical Genetics and Genomics and the Association for Molecular Pathology AUC : area under the ROC curve dbNSFP : database of non-synonymous functional prediction DM : disease-causing mutation DM? : likely disease-causing mutation with questionable pathogenicity gnomAD : Genome Aggregation Database GRCh38 : genome reference consortium human build 38 HD : homeodomain HGMD : Human Gene Mutation Database HGNC : HUGO Gene Nomenclature Committee LOVD : Leiden Open Variation Database MCC : Matthews correlation coefficient MCGM : Manchester Centre for Genomic Medicine OMIM : Online Mendelian Inheritance in Man PD : paired domain PAX 6 : paired box 6 PPV : positive predictive value ROC : receiver operating characteristic Sn : sensitivity Sp : specificity VUS : variant of uncertain significance