Figure 2: The six-step phenotypic-driven strategy applied for variant filtration and interpretation.
(1) Targeted analysis of the WES data, focusing on a list of genes associated with the phenotype of the patients indicated by the clinical features according to HPO (Human Phenotype Ontology) (https://hpo.jax.org/app/) and/or OMIM (Online Mendelian Inheritance in Man) (https://www.omim.org/) and/or in-house in-silico gene lists, which were supplemented with genes from literature searches and established protein-protein interaction networks; (2) Exclusion of variants with Minor Allele Frequency (MAF) >1% according to the gnomAD database (https://gnomad.broadinstitute.org/) and/or the 1000 Genomes Project database (http://www.1000genomes.org/), (3) Subsequent filtering of remaining variants based on their category and gene/genomic localization, initially discarding synonymous variants, intronic variants, variants in intergenic regions and, 3′ and 5′ UTR regions. (At this step, synonymous variants were kept on a separately list for further investigation if appropriate); (4) The resulting variant list (including rare, nonsense, missense, splice site, and frameshift INDELs), was subsequently filtered according to the in-silico pathogenicity prediction, phenotype correlation, mode of inheritance and whether variants had already been recorded and classified using the VarSome database (https://varsome.com/). Human Splicing Finder (https://www.genomnis.com/access-hsf) was used to assess suspected splice site variants along with a concomitant review of the literature; (5) variant classification according to the American College of Medical Genetics and Genomics (ACMG) recommendations. The five categories comprise pathogenic, likely pathogenic, Variant of Uncertain Significance (VUS), likely benign and benign variants; (6) Finally, a multidisciplinary team of molecular biologists, laboratory geneticists and clinical geneticists, evaluated the variant(s) most relevant to the patient’s phenotype and decided on the variants to be disclosed in the final report.