TY - JOUR T1 - A unified analytic framework for prioritization of non-coding variants of uncertain significance in heritable breast and ovarian cancer JF - bioRxiv DO - 10.1101/031419 SP - 031419 AU - Eliseos J. Mucaki AU - Natasha G. Caminsky AU - Ami M. Perri AU - Ruipeng Lu AU - Alain Laederach AU - Matthew Halvorsen AU - Joan HM. Knoll AU - Peter K. Rogan Y1 - 2015/01/01 UR - http://biorxiv.org/content/early/2015/11/11/031419.abstract N2 - Background Sequencing of both healthy and disease singletons yields many novel and low frequency variants of uncertain significance (VUS). Complete gene and genome sequencing by next generation sequencing (NGS) significantly increases the number of VUS detected. While prior studies have emphasized protein coding variants, non-coding sequence variants have also been proven to significantly contribute to high penetrance disorders, such as hereditary breast and ovarian cancer (HBOC). We present a strategy for analyzing different functional classes of non-coding variants based on information theory (IT).Methods We captured and enriched for coding and non-coding variants in genes known to harbor mutations that increase HBOC risk. Custom oligonucleotide baits spanning the complete coding, non-coding, and intergenic regions 10 kb up- and downstream of ATM, BRCA1, BRCA2, CDH1, CHEK2, PALB2, and TP53 were synthesized for solution hybridization enrichment. Unique and divergent repetitive sequences were sequenced in 102 high-risk patients without identified mutations in BRCA1/2. Aside from protein coding changes, IT-based sequence analysis was used to identify and prioritize pathogenic non-coding variants that occurred within sequence elements predicted to be recognized by proteins or protein complexes involved in mRNA splicing, transcription, and untranslated region (UTR) binding and structure. This approach was supplemented by in silico and laboratory analysis of UTR structure.Results 15,311 unique variants were identified, of which 245 occurred in coding regions. With the unified IT-framework, 132 variants were identified and 87 functionally significant VUS were further prioritized. We also identified 4 stop-gain variants and 3 reading-frame altering exonic insertions/deletions (indels).Conclusions We have presented a strategy for complete gene sequence analysis followed by a unified framework for interpreting non-coding variants that may affect gene expression. This approach distills large numbers of variants detected by NGS to a limited set of variants prioritized as potential deleterious changes.ASSEDAAutomated Splice Site and Exon Definition AnalysisBICBreast Cancer Information Core DatabaseCASAVAConsensus Assessment of Sequencing and VariationCIS-BP-RNACatalog of Inferred Sequence Binding Preferences of RNA binding proteinsCRACComplex Reads Analysis and ClassificationDM2Domain Mapping of Disease MutationsENIGMAEvidence-based Network for the Interpretation of Germline Mutant AllelesExPASyExpert Protein Analysis SystemGATKGenome Analysis ToolkitHBOCHereditary Breast and Ovarian CancerHGMDHuman Gene Mutation DatabaseIARCInternational Agency for Research on CancerIGVIntegrative Genomics ViewerIndelInsertion/deletionITInformation theoryLOVDLeiden Open Variant DatabaseMGLMolecular Genetics LaboratoryMLPAMultiplex Ligation Probe AmplificationNGSNext-Generation SequencingPTBPolypyrimidine tract binding proteinPTTProtein Truncation TestPWMPosition Weight MatrixRBBSRNA-Binding protein Binding SiteRBPRNA-Binding ProteinRBPDBRNA-Binding Protein DataBaseRiIndividual informationRsequenceMean information contentSHAPESelective 2’-Hydroxyl Acylation analyzed by Primer ExtensionSNVSingle Nucleotide VariantSRFSplicing Regulatory FactorSRFBSSplicing Regulatory Factor Binding SiteSSSplice SiteTFTranscription FactorTFBSTranscription Factor Binding SiteUTRUntranslated RegionVCFVariant Call FileVUSVariants of Uncertain SignificanceΔRiChange in individual information.Patient Sample IDs are assigned in following mannernumber-number+letter (i.e. 1–1A). If a sample was repeated, the IDs are separated by a “.” (i.e. 1–1A.2–1A) ER -