Nimblegen seqcap target enrichment

Following informed consent, patient genomic DNA was extracted from peripheral blood and, in two cases with EN (n. 17 and 26 in Table 1), also from the whole biopsy obtained from lesional skin. Mutations were identified through the targeted next generation sequencing (NGS) approach using a customized ichthyosis gene panel (Nextera Rapid Capture Custom Enrichment Kit, Illumina, San Diego, CA, USA; NimbleGen SeqCap Target Enrichment, Roche, Madison, WI, USA) and MiSeq or NextSeq550 sequencing platforms (Illumina). Identified variants were evaluated by the web-based tool VarSome [30 (link)] and validated by Sanger sequencing.

Two children affected with EBS-KLHL24 were studied (PT-1 and PT-2). PT-1 has been described (Case 1, (22 (link))), while PT-2 is a previously unreported 7-year-old female child, born to healthy non consanguineous healthy parents. Specifically, EBS-KLHL24 biopsies were taken from perilesional unblistered skin following skin rubbing, which results in basal keratinocyte vacuolization and microblistering, only detectable at microscopy analysis. Patient skin biopsies and blood samples were obtained after informed consent, with the approval of the Ethics Committees of participating Institutions and in conformity with the Helsinki guidelines. Normal human keratinocytes (NHKs) were obtained from skin or foreskin of age-matched healthy subjects undergoing surgery.
Skin biopsies were used for immunofluorescence antigen mapping and keratinocyte cultures as described (77 (link)). PT-2 genomic DNA was extracted from peripheral blood using QIAsymphony DSP DNA Mini Kit (Qiagen, Hilden, Germany), and sequence variants were identified through Next Generation Sequencing (NGS) approach (NimbleGenSeqCap Target Enrichment—Roche, Madison, WI, USA; Twist Human Core Exome Kit—Twist Bioscience, San Francisco, CA, USA) and NextSeq550 sequencing platform (Illumina, San Diego, CA, USA). Variant validation and segregation analysis were performed by Sanger sequencing.