Moderation of Phenotypic Severity in Dystrophic and Junctional Forms of Epidermolysis Bullosa Through In-Frame Skipping of Exons Containing Non-Sense or Frameshift Mutations

Abstract

Non-sense mutations on both alleles of either the type VII collagen gene (COL7A1) or the genes encoding laminin 5 (LAMA3, LAMB3, or LAMC2) usually result in clinically severe forms of recessive dystrophic or junctional epidermolysis bullosa, respectively. In this study we assessed two unrelated families whose mutations in genomic DNA predicted severe recessive dystrophic epidermolysis bullosa or junctional epidermolysis bullosa phenotypes but in whom the manifestations were milder than expected. The recessive dystrophic epidermolysis bullosa patients had a homozygous single base-pair frameshift mutation in exon 19 of COL7A1 (2470insG). Clinically, there was generalized blistering but only mild scarring. Skin biopsy revealed positive type VII collagen immunoreactivity and recognizable anchoring fibrils. The junctional epidermolysis bullosa patients were compound heterozygotes for a frameshift/non-sense combination of mutations in exons 3 and 17 of LAMB3 (29insC/Q834X). These patients did not have the lethal form of junctional epidermolysis bullosa but, as adults, displayed the milder generalized atrophic benign epidermolysis bullosa variant. There was undetectable laminin 5 staining at the dermal-epidermal junction using an antibody to the beta3 chain, but faintly positive alpha3 and gamma2 chain labeling, and there was variable hypoplasia of hemidesmosomes. To explain the milder recessive dystrophic epidermolysis bullosa and junctional epidermolysis bullosa phenotypes in these families, reverse transcription-polymerase chain reaction, using RNA extracted from frozen skin, was able to provide evidence for some rescue of mutant mRNA transcripts with restoration of the open- reading frame. In the recessive dystrophic epidermolysis bullosa patients, transcripts containing in-frame skipping of exon 19 of COL7A1 in the cDNA were detected, and in the junctional epidermolysis bullosa patients transcripts with in-frame skipping of exon 17 of LAMB3 were identified. The truncated proteins encoded by these transcripts are expected to lack certain critical domains involved in cell-matrix attachment, but may still be able to contribute to adhesion thereby moderating the severity of the skin blistering. This study shows the limitations in predicting phenotype in epidermolysis bullosa solely based on mutation analysis of genomic DNA and emphasizes the importance of immunohistochemistry, electron microscopy, and mRNA assessment as parallel investigations.

Epidermolysis Bullosa Simplex in Israel

“…Also, we cannot exclude compensatory mechanisms, such as the progressive development of exon skipping as shown in other forms of EB. 41 In contrast with the varying expression of the dominant mutations, all EBS cases due to homozygous nonsense mutations reported in this series were associated with a severe phenotype as previously reported in the literature. 15,[42][43][44] Thus, it seems that in these cases a severe course can be predicted with confidence during genetic counseling.…”

Antisense-mediated exon skipping: A versatile tool with therapeutic and research applications

“…Truncating mutations in the COL7A1 gene are associated with dystrophic epidermolysis bullosa, a disease characterized by severe blistering of the skin (Uitto et al 1995;Fine et al 2000). In contrast, mutations leading to inframe exon skipping result in milder cases, suggesting that reading frame restoration might have therapeutic potential for this disease (McGrath et al 1999). Using mutation-specific AONs targeting exon 70 resulted primarily in the skipping of the mutated exon, while the normal exon was included in the mRNA.…”

Genética Molecular das Epidermólises Bolhosas

“…47 Outra aplicação importante da genética molecular ocorre no diagnóstico pré-natal (DPN), 2,48 examinando-se o DNA fetal obtido do córion e não a pele fetal. O DPN feito a partir das lesões necessita da obtenção de fragmento da pele, o qual deve ser representativo da enfermidade, para evitar resultado falso-negativo, devendo-se esperar até a décima oitava ou vigésima semana.…”