Aberrant splicing as potential modifier of the phenotype of junctional epidermolysis bullosa

Abstract

Background: A lack or dysfunction of the anchoring protein laminin-332 in the basement membrane leads to the skin blistering disorder junctional epidermolysis bullosa (JEB). The mutation c.628G>A in the gene LAMB3 encoding the laminin β3-chain is associated with generalized intermediate JEB; it may introduce an amino acid substitution (p.Glu210Lys) or disrupt splicing.

Objective: This retrospective study aimed at determining the effects of aberrant splicing on the JEB phenotype.

Methods: LAMB3 transcription was analysed in two siblings compound heterozygous for the LAMB3 mutations p.Glu210Lys and p.Arg635* with a diverging JEB phenotype from late childhood on. Laminin-332 levels in skin sections and in cultured keratinocytes were investigated by immunofluorescence staining. Real-time PCR was used to quantify LAMB3 expression in keratinocytes. RNA splice variants were identified by subcloning of a LAMB3 cDNA fraction and subsequent DNA sequencing. Structural models of laminin-332 helped to assess the impact of certain mutations on laminin-332 folding.

Results: Both siblings showed diminished LAMB3 expression. Laminin-332 was equally reduced in skin sections obtained during infancy but differed in keratinocytes isolated during adolescence. Although aberrant LAMB3 splicing with 26 variants was detected in both patients, splicing differed significantly: the full-length LAMB3 transcript harbouring the p.Glu210Lys mutation was found more often in the patient affected less severely (14/108 vs. 5/106 clones; P = 0.03). Structural modelling predicted that several deletions in LAMB3, but not the point mutation p.Glu210Lys, have an effect on laminin-332 folding and secretion.

Conclusions: Differential LAMB3 mRNA splicing in the patients may explain the disparate JEB phenotype. By elucidating the regulation of laminin-332 gene expression, these findings may contribute to the development of therapeutic strategies for JEB and might help to understand phenotype modification by splice-site mutations in other hereditary diseases.