Novel variants impairing Sp1 transcription factor binding in the COL7A1 promoter cause mild cases of recessive dystrophic epidermolysis bullosa

Abstract

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and most often severe genodermatosis characterized by recurrent blistering and erosions of the skin and mucous membranes after minor trauma, leading to major local and systemic complications. RDEB is caused by loss-of-function mutations in COL7A1 encoding type VII collagen (C7), the main component of anchoring fibrils which form attachment structures stabilizing the cutaneous basement membrane zone. Most of the previously reported COL7A1 mutations are located in the coding or intronic regions. We describe 6 patients with localized or intermediate RDEB for whom one recessive pathogenic variant in the coding region and a second variant in the COL7A1 promoter were identified. These substitutions, three of which are novel, are localized in two Sp1 binding sites of the promoter region. DNA pull-down assay showed a drastic reduction of Sp1 binding consistent with a dramatic decrease in COL7A1 transcript and almost undetectable C7 protein levels. Our results reveal that mutations in the COL7A1 promoter on the background of a null allele can underlie localized or intermediate RDEB. They further emphasize the functional importance of Sp1 motifs in the proximal COL7A1 promoter which should be carefully investigated for regulatory mutations in the case of RDEB with only one pathogenic variant identified in the coding or intronic regions.

Fig. 1. Clinical and molecular characterization of the probands.

a Pedigree of the probands (The number inside the symbol corresponds to the number of siblings). Previously described mutations are depicted in green, new mutations reported in this study are in red. b Clinical presentations of the probands. c Immunofluorescence staining of patient 1 with LH7.2 monoclonal antibody showing drastically reduced and discontinuous C7 staining along the dermo-epidermal junction in comparison with strong and continuous staining in the control. d Electron microscopy of patient 1 showing numerous anchoring fibrils (red arrows) beneath the lamina densa in an uncleaved area. e Electron microscopy of patient 1 showing absence of anchoring fibrils beneath the lamina densa in a cleaved area (The light grey area corresponds to the blister cavity). Bar = 500 nm. (§This mutation is a previously described splicing mutation which inhibits normal splicing of exon 3 producing 3 abnormal transcripts with premature stop codons).

Fig. 2. Molecular characterization of individuals 1, 3 and 4.1 with recessive dystrophic epidermolysis bullosa.

a, b GeneScan RT-PCR analysis showing the presence of a small amount of wild-type transcript in the fibroblasts of patients 1, 3 and 4.1. (For figure b, the PCR products from patients 3 and 4.1 were less diluted than the control to show the presence of the wt transcript) (c) Quantitative RT-PCR analysis of COL7A1 expression relative to PGK expression using primers amplifying exon 112 to exon 114 in COL7A1. COL7A1 transcript levels are strongly reduced in fibroblasts of patients 1, 3 and 4.1. Experiments were replicated three times.

Fig. 3. Western-Blot analysis and functional analysis of novel promoter variants.

a Western-blot analysis show very low amount of wild-type C7 protein in fibroblasts from RDEB patients 1, 3 and 4.1. Data are representative of three independent experiments. b, c Pull-down assay of Sp1 binding to a WT or mutated sequence of the COL7A1 promoter. HeLa Nuclear extracts (NE) were mixed with biotinylated DNA probes. The labeled DNA probes were precipitated with streptavidin-agarose beads, and precipitates were subjected to SDS-PAGE, followed by immunoblotting with anti-Sp1 antibody. Data are representative of three independent experiments. (Lane Sp1: Sp1 consensus sequence used; Lane NE: Nuclear extracts with no oligonucleotides are deposited on the gel to verify the presence of Sp1 in the nuclear extracts). b The mutated sequences c.-185T and c.-185A lead to reduced binding of the transcription factor Sp1 compared with the WT sequence (c.-185C). c The mutated sequence c.-215T shows decreased Sp1 binding compared to the WT sequence c.-215C. d Schematic representation of the COL7A1 promoter with transcription factor binding sites and corresponding transcription factors indicated. Schematic based on Kon and Naso [20, 21]. Previously described mutations are depicted in green, new mutations reported in this study are in red.