title: Novel mutations in the LAMC2 gene in non-Herlitz junctional epidermolysis bullosa: effects on laminin-5 assembly, secretion, and deposition.
abstract: 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 gamma2 polypeptide (gamma2Delta4) 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 gamma2 polypeptide (gamma2t) disrupted in its alpha-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 gamma2 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 gamma2 cDNAs were transiently expressed in gamma2-null keratinocytes. Transfection of the gamma2Delta4 cDNA resulted in restoration of laminin-5 deposition onto the culture substrate, which demonstrates that the gamma2 polypeptides carrying a deletion in domain V, upstream of the gamma2 proteolytic cleavage site, are assembled into native laminin-5 that is secreted and extracellularly processed. In contrast, transfection of a mutant cDNA expressing the gamma2t chain failed to restore laminin-5 immunoreactivity, which indicates that integrity of the gamma2 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.
authors.affiliation: Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata, IRCCS, Rome, Italy. d.castiglia@idi.it
authors.forename: D
authors.initials: D
authors.lastname: Castiglia
authors.forename: P
authors.initials: P
authors.lastname: Posteraro
authors.forename: F
authors.initials: F
authors.lastname: Spirito
authors.forename: M
authors.initials: M
authors.lastname: Pinola
authors.forename: C
authors.initials: C
authors.lastname: Angelo
authors.forename: P
authors.initials: P
authors.lastname: Puddu
authors.forename: G
authors.initials: G
authors.lastname: Meneguzzi
authors.forename: G
authors.initials: G
authors.lastname: Zambruno
mesh_terms: D000595:Amino Acid Sequence; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002648:Child; D004820:Epidermolysis Bullosa / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007797:Laminin / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D000097794:Kalinin
chemical_list: D015815:Cell Adhesion Molecules; D007797:Laminin; D000097794:Kalinin; C400567:LAMC2 protein, human