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.