Novel mutations in the LAMC2 gene in non-Herlitz junctional epidermolysis bullosa: Effects on laminin-5 assembly, secretion, and deposition

Laminin-5 is the major adhesion ligand of epithelial cells. Mutations in the three genes (LAMA3, LAMB3, LAMC2) encoding the laminin-5 chains cause junctional epidermolysis bullosa, a clinically and genetically heterogeneous blistering skin disease. Here, we describe a non-Herlitz junctional epidermolysis bullosa patient, compound heterozygote for two novel mutations affecting the LAMC2 gene. The mutation in the paternal allele is a de novo splice site mutation (522-1G→A) that results in in-frame skipping of exon 4 and synthesis of a mutated γ2 polypeptide (γ2Δ4) carrying a 33 amino acid deletion within the N-terminal domain V. The maternal mutation is a one base pair insertion (3511insA) in the 3′ terminal exon of LAMC2 resulting in a frameshift and a premature termination codon. Mutation 3511insA is predicted to lead to the synthesis of a γ2 polypeptide (γ2t) disrupted in its α-helical C-terminal structure and truncated of the last 25 amino acids. Keratinocytes isolated from the patient's skin showed a markedly decreased level of γ2 chain mRNA and secreted scant amounts of laminin-5, which undergoes physiologic proteolytic processing. To investigate the biologic function of the laminin-5 molecules synthesized by the patient, mutant γ2 cDNAs were transiently expressed in γ2-null keratinocytes. Transfection of the γ2Δ4 cDNA resulted in restoration of laminin-5 deposition onto the culture substrate, which demonstrates that the γ2 polypeptides carrying a deletion in domain V, upstream of the γ2 proteolytic cleavage site, are assembled into native laminin-5 that is secreted and extracellularly processed. In contrast, transfection of a mutant cDNA expressing the γ2t chain failed to restore laminin-5 immunoreactivity, which indicates that integrity of the γ2 C-terminal amino acid sequences is required for laminin-5 assembly. These results correlate for the first time a functional alteration in a laminin-5 domain with a mild junctional epidermolysis bullosa phenotype.