Availability of mRNA Obtained from Peripheral Blood Mononuclear Cells for Testing Mutation Consequences in Dystrophic Epidermolysis Bullosa

Abstract

Dystrophic epidermolysis bullosa (DEB) is an inheritable blistering disease caused by mutations in COL7A1, which encodes type VII collagen. To address the issue of genotype-phenotype correlations in DEB, analyzing the consequences of COL7A1 mutations using mRNA is indispensable. Herein we established a novel method for testing the effect of mutations in DEB using COL7A1 mRNA extracted from peripheral blood mononuclear cells (PBMCs). We investigated the consequences of four COL7A1 mutations (c.6573 + 1G > C, c.6216 + 5G > T, c.7270C > T and c.2527C > T) in three Japanese individuals with recessive DEB. The novel method detected the consequences of two recurrent COL7A1 mutations (c.6573 + 1G > C, c.6216 + 5G > T) and a novel COL7A1 mutation (c.7270C > T) accurately. In addition, it detected aberrant splicing resulting from a COL7A1 mutation (c.2527C > T) which was previously reported as a nonsense mutation. Furthermore, we revealed that type VII collagen-expressing cells in PBMCs have similar cell surface markers as mesenchymal stem cells; they were CD105⁺, CD29⁺, CD45^-, and CD34^-, suggesting that a small number of mesenchymal stem cells or mesenchymal stromal cells are circulating in the peripheral blood, which enables us to detect COL7A1 mRNA in PBMCs. Taken together, our novel method for analyzing mutation consequences using mRNA obtained from PBMCs in DEB will significantly contribute to genetic diagnoses and novel therapies for DEB.

Keywords: dystrophic epidermolysis bullosa; mesenchymal stem cells; mesenchymal stromal cells; mutational analysis; type VII collagen: mRNA.

Figure 1

COL7A1 mutational analysis results. Genomic DNA and messenger RNA (mRNA) were obtained from patient peripheral blood mononuclear cells (PBMCs). Results of agarose gel electrophoresis for the products of reverse transcription polymerase chain reaction (RT-PCR) and results of direct sequencing analysis of genomic DNA and complementary DNA (cDNA) are presented. (a) Patient 1, (b) Patient 2, and (c) Patient 3.

Figure 2

Type VII collagen expression in cells isolated from PBMCs obtained using the Human Mesenchymal Stem Cell Enrichment Cocktail. (a) COL7A1 mRNA expression of cultured normal human epidermal keratinocyte (NHEK), normal human dermal fibroblast (NHDF), Peripheral blood mononuclear cell (PBMC), and human mesenchymal stem cell (hMSC) was analyzed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). (b) The isolated cells were cultured in mesenchymal stem cell culture medium for 14 days. Scale bar, 100 μm. (c) COL7A1 mRNA expression in isolated cells. Cells were treated with or without 1 nM of transforming growth factor- (TGF-β). Quantitative analysis of COL7A1 mRNA expression was performed using RT-qPCR.

Figure 3

Immunological characteristics of cells isolated from peripheral blood mononuclear cells (PBMCs) using the Human Mesenchymal Stem Cell Enrichment Cocktail. (a) Expression of lineage markers CD105 or CD45 was investigated using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Left lane, cultured human mesenchymal stem cells (hMSCs); middle lanes, cells isolated from PBMCs; right lane, PBMCs. (b) Immunophenotyping of cultured and isolated cells. Immunofluorescence staining for lineage markers CD105, CD29, CD45, and CD34 (green); type VII collagen (red); and nuclear component (blue). DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate. Scale bar, 100 μm.