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.

A Novel Phenotype of Junctional Epidermolysis Bullosa with Transient Skin Fragility and Predominant Ocular Involvement Responsive to Human Amniotic Membrane Eyedrops

“…We hypothesize that wild-type LM332 released by basal cells in vivo is stable and accumulates at the BMZ, thus contributing to restore skin resilience soon after birth. In addition, splicing modulation favoring the wild-type/full length transcript might occur in postnatal life and contribute to increase wild-type/full size LM332 amount in the skin and thus to rescue skin fragility, as already described for different leaky splicing mutations in LAMB3 and ITGB4 [ 31 , 32 ]. Finally, the frequency of the wild-type spliced transcripts observed in primary keratinocytes in vitro may not exactly reflect the in vivo situation, and splicing efficiency may vary in different tissue contexts [ 33 ].…”

Splicing of the cake can affect the severity of epidermolysis bullosa

“…In addition to these mechanisms, in some families with recessive forms of EB, the children who all have the same two mutations, or the ‘genotype’, can on occasion have vastly different phenotypes. Sometimes, one of them improves with age whilst the other does not, as reported in this issue by Mittwollen et al 1 . of two siblings, a male and a female, with junctional EB (JEB) caused by LAMB3 gene mutations.…”

“…In the family reported by Mittwollen et al ., both siblings had one ‘ugly’ hotspot PTC mutation in LAMB3 , p.Arg635X, whilst the second mutation was supposed to be a relatively ‘good’ one, c.628G>A, predicting an amino acid substitution, p.Glu210Lys, and a generalized intermediate JEB phenotype 1,3 . However, this mutation resides at the end of exon 7 at the exon 7/intron 7 border, and can therefore affect splicing, being in fact a ‘bad’ mutation.…”

Clinical Perspectives of Gene-Targeted Therapies for Epidermolysis Bullosa

“…Additionally, recent reports indicate that modulation of regulatory splicing elements could be exploited to favorably alter mutation-based splicing patterns. Manipulation of crucial interactions of the spliceosome, e.g., by drug compounds or small molecules, may thus hold therapeutic potential via augmentation of (functional) full-length protein variants from splice mutations [ 78 , 79 ].…”