Regional-specific calibration enables application of bioinformatic evidence for clinical classification of 5’ cis-regulatory variants in Mendelian disease

To date, clinical genetic testing and approaches to classify genetic variants in Mendelian disease genes have focused heavily on exonic coding and intronic gene regions. This multi-step study was undertaken to provide an evidence base for selecting and applying bioinformatic approaches for use in clinical classification of 5’ cis-regulatory region variants. Curated datasets of rare clinically reported disease-causing 5’ cis-regulatory region variants, and variants from matched genomic regions in population controls, were used to calibrate six bioinformatic tools as predictors of variant pathogenicity. Likelihood ratio estimates were aligned to code weights following ClinGen recommendations for application of the American College of Medical Genetics (ACMG)/American Society of Molecular Pathology (AMP) classification scheme. Considering code assignment across all reference dataset variants, performance was best for CADD (81.2%) and REMM (81.5%). Optimized thresholds provided moderate evidence towards pathogenicity (CADD, REMM), and moderate (CADD) or supporting (REMM) evidence against pathogenicity. Both sensitivity and specificity of prediction were improved when further categorizing variants based on location in an EPDnew-defined promoter region. Combining predictions (CADD, REMM, and location in a promoter region) increased specificity at the expense of sensitivity. Importantly, the optimal CADD thresholds for assigning ACMG/AMP codes PP3 (≥10) and BP4 (≤8) were vastly different to recommendations for protein-coding variants (PP3 ≥ 25.3; BP4 ≤22.7); CADD <22.7 would incorrectly assign BP4 for >90% of reported disease-causing cis-regulatory region variants. Our results demonstrate the need to consider a tiered approach and tailored score thresholds to optimize bioinformatic impact prediction for clinical classification of cis-regulatory region variants. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement RV, MM, ALD and ABS were supported by NHMRC Funding (APP177524). The work of A.L.D. was also supported in part by National Institutes of Health grant R01 CA264971. ### 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: All the data used for this study was openly available before the commencement of the study, and were originally located by the following citations. Biggs H, Parthasarathy P, Gavryushkina A, Gardner PP. ncVarDB: a manually curated database for pathogenic non-coding variants and benign controls. Database (Oxford). 2020;2020. Landrum MJ, Lee JM, Benson M, Brown GR, Chao C, Chitipiralla S, et al. ClinVar: improving access to variant interpretations and supporting evidence. Nucleic Acids Res. 2018;46(D1):D1062-D7. Caron B, Luo Y, Rausell A. NCBoost classifies pathogenic non-coding variants in Mendelian diseases through supervised learning on purifying selection signals in humans. Genome Biol. 2019;20(1):32. Smedley D, Schubach M, Jacobsen JOB, Kohler S, Zemojtel T, Spielmann M, et al. A Whole-Genome Analysis Framework for Effective Identification of Pathogenic Regulatory Variants in Mendelian Disease. Am J Hum Genet. 2016;99(3):595-606. 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 and scripts are available either in the supplemental information or at .