In Silico Prediction of Effect of Missense Mutations

Eight software programs were used to predict the effects of missense variants: two online in silico prediction tools (CADD v.1.3 and PolyPhen-2) and six complementary tools (Grantham Difference, SIFT v.4.0.3, SpliceSiteFinder-like, MaxEntScan, NNSplice v.0.9, and Human Splicing Finder v.2.4.1) embedded in Alamut visual software v.2.9.0 (Interactive Biosoftware). The presence or absence of the variants was checked in population databases, including the Genome Aggregation Database (gnomAD), 1000 Genomes, and the Exome Variant Server (EVS) as well as in disease databases: the Leiden Open Variation Database (LOVD), ClinVar, and the Human Gene Mutation Database (HGMD) (last accessed May 2017). Evolutionary conservation for human neurofibromin GenBank: {"type":"entrez-protein","attrs":{"text":"NP_000258.1","term_id":"4557793","term_text":"NP_000258.1"}}NP_000258.1 residues 804–950 was evaluated using Clustal software v.2.0.12. The palindromic sequences and quadruplex forming G-Rich sequences (QGRS) were identified by Palindrome search and QGRS Mapper, respectively.

Interpretation of variant pathogenicity was performed based on the American College of Medical Genetics and Genomics (ACMG) recommendations.⁴³