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 th e three genes (LAMA3, LAMB3, LAMC2) encoding the laminin-5 chains cause jun ctional epidermolysis bullosa, a clinically and genetically heterogeneous b listering skin disease. Here, we describe a non-Herlitz. junctional epiderm olysis bullosa patient, compound heterozygote for two novel mutations affec ting the LAMC2 gene. The mutation in the paternal allele is a de novo splic e site mutation (522-1G -->A) that results in in-frame skipping of exon 4 a nd synthesis of a mutated gamma2 polypeptide (gamma2 Delta4) carrying a 33 amino acid deletion within the N-terminal domain V. The maternal mutation i s a one base pair insertion (3511insA) in the 3' terminal exon of LAMC2 res ulting in a frameshift and a premature termination codon. Mutation 3511insA is predicted to lead to the synthesis of a gamma2 polypeptide (gamma 2t) d isrupted in its alpha -helical C-terminal structure and truncated of the la st 25 amino acids. Keratinocytes isolated from the patient's skin showed a markedly decreased level of gamma2 chain mRNA and secreted scant amounts of laminin-5, which undergoes physiologic proteolytic processing. To investig ate the biologic function of the laminin-5 molecules synthesized by the pat ient, mutant gamma2 cDNAs were transiently expressed in gamma2-null keratin ocytes. Transfection of the gamma2 Delta4 cDNA resulted in restoration of l aminin-5 deposition onto the culture substrate, which demonstrates that the gamma2 polypeptides carrying a deletion in domain V, upstream of the gamma 2 proteolytic cleavage site, are assembled into native laminin-5 that is se creted and extracellularly processed. In contrast, transfection of a mutant cDNA expressing the gamma 2t chain failed to restore laminin-5 immunoreact ivity, which indicates that integrity of the Gamma-2 C-terminal amino acid sequences is required for laminin-5 assembly. These results correlate for t he first time a functional alteration in a laminin-5 domain with a mild jun ctional epidermolysis bullosa phenotype.