Variant Identification in Ichthyosis Patients

Pathogenic variants of ichthyosis patients were identified previously using Sanger sequencing or gene panel sequence analysis. For this study, genomic DNA was extracted from patient fibroblasts and hiPSCs with Genomic DNA Minikit (Invitrogen, Carlsbad, CA, USA), following the kit instructions. Primers for TGM1 exon 5, PNPLA1 exon 5 and ERCC2 exon 5 were designed with Primer 3 [46 (link)] (available upon request). PCR was performed using standard protocols. Products were purified and prepared for sequencing reaction with BigDye^® Terminator v1.1 Kit (Applied Biosystems, Foster City, CA, USA) and run in a Sequence Analyser 3130XL (Applied Biosystems, Foster City, CA, USA). Sequences were analyzed with SeqmanPro software (DNASTAR Inc., Madison, WI, USA). Variant pathogenicity prediction was performed by in silico tools MutationTaster [47 (link)], SIFT [48 (link)], Polyphen-2 [49 (link)] and Human Splicing Finder 3.0 [50 (link)]. Variant novelty was accessed by searching ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and GnomAD v2.1.1 (https://gnomad.broadinstitute.org/).