A comprehensive next-generation sequencing assay for the diagnosis of epidermolysis bullosa

Abstract

Background: Historically, diagnosis of epidermolysis bullosa has required skin biopsies for electron microscopy, direct immunofluorescence to determine which gene(s) to choose for genetic testing, or both. Methods: To avoid these invasive tests, we developed a high-throughput next-generation sequencing (NGS)-based diagnostic assay called EBSEQ that allows simultaneous detection of mutations in 21 genes with known roles in epidermolysis bullosa pathogenicity. Mutations are confirmed with traditional Sanger sequencing. Results: We present our EBSEQ assay and preliminary studies on the first 43 subjects tested. We identified 11 cases of epidermolysis bullosa simplex, five cases of junctional epidermolysis bullosa, 11 cases of dominant dystrophic epidermolysis bullosa, 15 cases of recessive dystrophic epidermolysis bullosa, and one case that remains without diagnosis. We also found an additional 52 variants of uncertain clinical significance in 17 of the 21 epidermolysis bullosa-associated genes tested. Three of the variants of uncertain clinical significance were also found in three other patients, for a total of 49 unique variants of uncertain clinical significance. We found the clinical sensitivity of the assay to be 75% to 98% and the analytical sensitivity to be 99% in identifying base substitutions and small deletions and duplications. Turnaround time was 3 to 6 weeks. Conclusions: EBSEQ is a sensitive, relatively rapid, minimally invasive, comprehensive genetic assay for the diagnosis of epidermolysis bullosa.