After variant detection, successive filtering steps are applied to prioritize candidate variants. Functional validation integrates multiomics and experimental approaches to assess the biological impact of prioritized variants. (A) Transcriptomic validation by RNA sequencing (RNA-seq) can reveal molecular consequences of candidate variants at the transcript level. Abnormal expression of the affected gene may indicate altered transcriptional regulation or mRNA stability. Monoallelic expression suggests allelic imbalance, often due to nonsense-mediated decay or promoter methylation. Aberrant splicing may include exon skipping (canonical exons omitted), intron retention (intronic sequences retained due to splice site disruption), pseudoexon inclusion (cryptic intronic sequences aberrantly incorporated), or shortened or extended exons, resulting from alternative splice donor or acceptor usage. (B) Epigenomic methods assess DNA and chromatin modifications that influence gene expression. Bisulfite sequencing provides base-resolution maps of cytosine methylation. Assay for transposase-accessible chromatin sequencing (ATAC-seq) identifies regions of open chromatin, marking regulatory regions, such as promoters and enhancers. Chromatin immunoprecipitation sequencing (ChIP-seq) maps histone modifications or transcription factor binding. (C) Proteomic validation: Proteomic analyses evaluate the downstream impact of variants on protein abundance and function. Mass spectrometry can be used in qualitative modes to identify protein isoforms or posttranslational modifications and in quantitative modes to measure differential protein levels across samples. Complementary affinity-based platforms, such as Olink or SomaScan, provide targeted, high-throughput quantification using specific molecular binders. (D) In vivo and cellular validation: Functional assessment of variant pathogenicity often employs patient-derived samples, such as fibroblasts or induced pluripotent stem cells, which can be differentiated into disease-relevant cell types (e.g., neurons). CRISPR-based genome editing enables introduction or correction of variants to test causality, and rescue experiments — where reexpression of the wild-type allele restores normal function — provide strong evidence of pathogenicity.