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460 SHORT COMMUNICATION A Silent COL17A1 Variant Alters Splicing and Causes Junctional Epidermolysis Bullosa Julia HOFFMANN 1 , Federica CASETTI 1 , Antonia REIMER 1,2 , Juna LEPPERT 1 , Gabriele GRÜNINGER 1 and Cristina HAS 1 * 1 Department of Dermatology, and 2 Berta-Ottenstein-Programme, Faculty of Medicine, Medical Center – University of Freiburg, Hauptstrasse 7, DE-79104 Freiburg, Germany. *E-mail: [email protected] Accepted Jan 23, 2019; E-published Jan 23, 2019 Type XVII collagen is a type II transmembrane protein present in specialized adhesion structures, the hemi- desmosomes, which anchor the intermediate filaments to the cell membrane and to the underlying basement membrane. Mutations in COL17A1, the gene encoding type XVII collagen cause a spectrum of disorders, being associated with junctional epidermolysis bul- losa (EB, MIM 226650), amelogenesis imperfecta and epithelial recurrent erosion dystrophy (MIM 122400) (1). The phenotype depends on the type and dosage of the pathogenic variants. Biallelic COL17A1 mutations, which lead to premature termination codons, diminish the adhesive capacity of the hemidesmosomes and lead to generalized skin blistering, extensive wounds, enamel hypoplasia, nail dystrophy and irrevers­ible hair loss in patients with junctional EB generalized intermediate (1). Genotype–phenotype correlations show that residual amounts of type XVII collagen resulting from in-frame splicing errors, or from the substitution of specific key amino acids, lead to milder clinical manifestations in localized or late-onset junctional EB (2–6). METHODS COL17A1 mutation analysis was performed in a cohort of 68 patients with junctional EB. After obtaining informed consent, EDTA-blood samples and skin biopsies were taken from the index cases, and if possible from parents. The study was approved by the ethics committee of the University of Freiburg, and conducted ac- cording to the principles of the Declaration of Helsinki. Mutational analysis of the coding region and exon-intron boundaries of the COL17A1 gene was performed either by Sanger sequencing or by a next-generation sequencing (NGS) multigene panel containing genes known to be associated with EB (7, 8). Immunofluorescence mapping was done as reported, with a panel of antibodies to adhesion proteins of the dermal–epidermal junction to determine the subtype of EB (9). For type XVII collagen detection, the poly- clonal serum NC16A (10) and the monoclonal antibody NC16A3 (Abcam, Cambridge, UK) were employed. Keratinocytes were isolated from the skin sample of case 1 using standard methods, and cultured in keratinocyte growth medium (Invitrogen, KGM, MA, USA), supplemented with epidermal growth factor and bovine pituitary extract. Total RNA was isola- ted from subconfluent keratinocyte cultures using the RNAeasy ® FFPE kit (QIAGEN, Hilden, Germany), reverse transcribed into cDNA (Fermentas, St Leon-Rot, Germany) and PCR amplified with primers spanning COL17A1 exon 46 (Table SI 1 ). Amplicons were cloned into the TopoTA vector, and DNA isolated from single clones submitted to Sanger sequencing. Immunoblot analysis was https://www.medicaljournals.se/acta/content/abstract/10.2340/00015555-3133 1 doi: 10.2340/00015555-3133 Acta Derm Venereol 2019; 99: 460–461 performed with keratinocyte lysate of case 1 and from a normal control and detected with the affinity purified NC16A polyclonal sera. In silico predictions of the consequences of the variant were performed with Mutation Taster and Human Splicing Finder (http://www.umd.be/HSF3/). RESULTS AND DISCUSSION Using a diagnostic approach combining mutation analysis and immunofluorescence mapping, biallelic COL17A1 pathogenic variants were identified in 64 of 68 junctional EB cases, while in 4 cases, a single COL17A1 heterozygous pathogenic allele was identified (97.06% detection rate). Careful re-evaluation revealed in all 4 patients the same heterozygous variant of uncertain sig- nificance (gnomAD), c.3198C>T, p.Ser1066Ser (Table SI 1 , Fig. S1a 1 ). Analysis of the DNA from the parents confirmed segregation of the variants in the families and the compound heterozygous state in the index cases. According to ExAC, dbSNP, HGMD professional and 1000Genomes, the variant c.3198C>T, rs369035370, was found in heterozygous state in 2 individuals and has a minor allele frequency of <0.01. In silico predictions indicate that c.3198T alters splicing by the following putative mechanisms: (i) by marginal increase in the activity of the exonic cryptic donor splice site GTTgtgagt (underlined, Mutation Taster: from 0.2185 to 0.2356; Human Splicing Finder: 2.74% increase), (ii) by forma- tion of a new donor splice site TGAgttact (underlined, Human Splicing Finder: 69.34% increase), and (iii) by alteration of an exonic splicing enhancer (ESE) site, agctac (Human Splicing Finder) (Fig. S1b 1 ). To verify these predictions and address the patho- genicity of this variant of uncertain significance, total RNA was isolated from keratinocytes of case 1 (com- pound heterozygous for p.Gly803* and p.Ser1066Ser). COL17A1 mRNA levels were strongly reduced in the keratinocytes of case 1 compared with control cells (Fig. S1c 1 ). Sequencing revealed that the allele carry- ing the mutation c.2407G>T, p.Gly803* was partially degraded due to mRNA decay (Fig. S1c 1 ). Sequencing of cloned amplicons identified the normal nucleotide C, the variant T, or an abnormal sequence at position c.3198 (Fig. S1d 1 ). The abnormal sequence consisted in the usage of an upstream donor splice site within exon 46 (prediction (i)), leading to removal of 16 nu- cleotides c.3193_3208del, frame shift and formation of a premature termination codon, p.Val1065Leufs*35 in the NC5 (non-collagenous 5) domain of type XVII This is an open access article under the CC BY-NC license. www.medicaljournals.se/acta Journal Compilation © 2019 Acta Dermato-Venereologica.