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

Daniele Castiglia, Patrizia Posteraro, Flavia Spirito, Mari Pinola, Corrado Angelo, Pietro Puddu, Guerrino Meneguzzi, Giovanna Zambruno

Research output: Contribution to journalArticle

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 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.

Original languageEnglish
Pages (from-to)731-739
Number of pages9
JournalJournal of Investigative Dermatology
Volume117
Issue number3
DOIs
Publication statusPublished - 2001

Fingerprint

Junctional Epidermolysis Bullosa
Genes
Mutation
Complementary DNA
Keratinocytes
Amino Acids
Peptides
Transfection
Exons
Skin
kalinin
Nonsense Codon
Heterozygote
Skin Diseases
Base Pairing
Restoration
Amino Acid Sequence
Adhesion
Epithelial Cells
Alleles

Keywords

  • Inherited blistering skin diseases
  • Laminin γ2 chain
  • Molecular genetics

ASJC Scopus subject areas

  • Dermatology

Cite this

Novel mutations in the LAMC2 gene in non-Herlitz junctional epidermolysis bullosa : Effects on laminin-5 assembly, secretion, and deposition. / Castiglia, Daniele; Posteraro, Patrizia; Spirito, Flavia; Pinola, Mari; Angelo, Corrado; Puddu, Pietro; Meneguzzi, Guerrino; Zambruno, Giovanna.

In: Journal of Investigative Dermatology, Vol. 117, No. 3, 2001, p. 731-739.

Research output: Contribution to journalArticle

Castiglia, Daniele ; Posteraro, Patrizia ; Spirito, Flavia ; Pinola, Mari ; Angelo, Corrado ; Puddu, Pietro ; Meneguzzi, Guerrino ; Zambruno, Giovanna. / Novel mutations in the LAMC2 gene in non-Herlitz junctional epidermolysis bullosa : Effects on laminin-5 assembly, secretion, and deposition. In: Journal of Investigative Dermatology. 2001 ; Vol. 117, No. 3. pp. 731-739.
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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 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.",
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AU - Zambruno, Giovanna

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