Albertine Nijenhuis

A very mild form of non-Herlitz junctional epidermolysis bullosa: BP180 rescue by outsplicing of mutated exon 30 coding for the COL15 domain

Abstract: Mutations in the gene COL17A1 cause non‐Herlitz junctional epidermolysis bullosa. Here, we describe a patient who, despite two heterozygous mutations in COL17A1, has an extremely mild form of the disease missing most of the characteristic clinical features. DNA analysis revealed a frame‐shift mutation 3432delT and a nonsense mutation 2356C→T (Q751X). cDNA analysis showed that the deleterious effect of the latter mutation was skirted by deleting the premature termination codon containing exon 30. In this way, the reading frame was restored, resulting in a 36 nucleotides shorter mRNA transcript. Immunoblot analysis showed expression of the 180‐kDa bullous pemphigoid antigen (BP180) with a slightly higher SDS‐PAGE mobility, in line with the deletion of 12 amino acids from the COL15 domain. Immunofluorescence of skin sections showed diminished, but correctly localised expression of BP180, and this, in concert with the mild clinical phenotype, suggests that this COL15 mutated BP180 is still partly functional.

Two type XVII collagen (BP180) mRNA transcripts in human keratinocytes: A long and a short form

We have analysed BP180 mRNA expression in normal human keratinocytes. Here we report the presence in normal keratinocytes of two COL17A1 transcripts which differ by 0.6 kb in length. Both mRNAs hybridized on Northern blot with probes directed to sequences encoding intracellular and extracellular fragments of BP180. By blast homology search alignments we extended the 3′ untranslated region (3′UTR) of the known BP180 mRNA sequence by 877 bases to completion. Three of 20 cDNAs identified by blast searches contained a 610 bp deletion in this new 3′UTR sequence. Northern blot analysis with a probe complementary to this deleted sequence showed binding only to the larger mRNA. The deletion of 610 nucleotides in the smaller mRNA was verified by reverse transcription–PCR and sequencing. Genomic PCR showed the new sequence to be an extension of exon 56 of the COL17A1 gene which suggests that the second mRNA is generated by differential splicing. In normal keratinocytes the level of the smaller transcript was 5–15% of that of the larger transcript whereas in a squamous cell carcinoma cell line this ratio was reversed, the smaller mRNA being three times more abundant than the larger mRNA. The biological significance of this newly identified transcript in protein synthesis and tissue expression or in cell differentiation, proliferation or adhesion is as yet unknown.

Keratinocyte Binding Assay Identifies Anti-Desmosomal Pemphigus Antibodies Where Other Tests Are Negative

The serological diagnosis of pemphigus relies on the detection of IgG autoantibodies directed against the epithelial cell surface by indirect immunofluorescence (IIF) on monkey esophagus and against desmoglein 1 (Dsg1) and Dsg3 by ELISA. Although being highly sensitive and specific tools, discrepancies can occur. It is not uncommon that sera testing positive by ELISA give a negative result by IIF and vice versa. This brings diagnostic challenges wherein pemphigus has to be ascertained or ruled out, especially when no biopsy is available. We utilized the ability of anti-Dsg3 and anti-Dsg1 IgG to bind in specific desmosomal patterns to living cells to investigate these discrepancies between IIF and ELISA. Living cultured primary normal human keratinocytes were grown under differentiating conditions to induce adequate expression of Dsg1 and Dsg3, incubated with patient serum for 1 h, and then stained to visualize bound IgG. We investigated two different groups; sera from patients with a positive direct immunofluorescence (DIF) and inconsistent serological findings (n = 43) and sera with positive ELISA or IIF but with negative DIF (n = 60). As positive controls we used 50 sera from patients who fulfilled all diagnostics criteria, and 10 sera from normal human subjects served as negative controls. In the DIF positive group, IgG from 39 of the 43 sera bound to the cells in a desmosomal pattern while in the DIF negative group none of the 60 sera bound to the cells. This shows that for pemphigus patients, ELISA and IIF can be negative while anti-desmosomal antibodies are present and vice versa that ELISA and IIF can be positive in non-pemphigus cases. In absence of a biopsy for DIF, such findings may lead to misdiagnosis.