Title: Novel Premature Termination Codon Mutations in the Laminin γ2-Chain Gene (LAMC2) in Herlitz Junctional Epidermolysis Bullosa
Abstract: To the Editor: Herlitz junctional epidermolysis bullosa (H-JEB), a severe blistering disorder affecting the skin and a variety of extracutaneous epithelia, leads to premature demise of the affected individuals during early postnatal period (Fine et al., 1991Fine J.-D. Bauer E.A. Briggaman R.A. et al.Revised clinical and laboratory criteria for subtypes of inherited epidermolysis bullosa: a consensus report by the Subcommittee on Diagnosis and Classification of the National Epidermolysis Bullosa Registry.J Am Acad Dermatol. 1991; 24: 119-135Abstract Full Text PDF PubMed Scopus (441) Google Scholar). A pathogenetic ultrastructural feature of the skin of affected individuals is abnormalities in hemidesmosome/anchoring filament complexes, associated with tissue separation within the lamina lucida at the dermal–epidermal basement membrane zone (Tidman and Eady, 1986Tidman M.J. Eady RaJ Hemidesmosome heterogeneity in junctional epidermolysis bullosa revealed by morphometric analysis.J Invest Dermatol. 1986; 86: 51-56Crossref PubMed Scopus (116) Google Scholar). A characteristic immunofluorescence finding is absent expression of laminin 5, a member of the laminin family of proteins, which consists of three polypeptides, the α3, β3, and γ2 chains (Aumailley and Krieg, 1996Aumailley M. Krieg T. Laminins: a family of diverse multifunctional molecules of basement membrane.J Invest Dermatol. 1996; 106: 209-214Crossref PubMed Scopus (115) Google Scholar). Since all three chains are required for stable assembly of trimeric laminin 5 molecules, the corresponding genes, LAMA3, LAMB3, and LAMC2, serve as potential candidate genes for mutations in H-JEB. In fact, a number of distinct mutations in these three genes have been demonstrated in patients with H-JEB, all mutations resulting in premature termination codons. Interestingly, however, the majority of the mutations (≈80%) reside in the LAMB3 genes, with only a few of them having been reported in the LAMA3 or LAMC2 genes (Pulkkinen et al., 1999Pulkkinen L. Uitto J. Christiano A.M. The molecular basis of the junctional forms of epidermolysis bullosa.in: Fine J.-D. Bauer E.A. Mcguire J. Moshell A. Epidermolysis Bullosa. Clinical, Epidemiologic, and Laboratory Advances, and the Findings of the National Epidermolysis Bullosa Registry. The Johns Hopkins University Press, Baltimore1999Google Scholar). In this study, we have delineated the molecular basis of skin blistering in a Japanese patient with H-JEB, and we now report two novel LAMC2 mutations. The proband, a 2 mo old female, was the first child of unrelated, clinically normal parents of Japanese origin (Figure 1a). The patient had a history of blistering of the skin and oral mucous membranes since birth. Biopsy, performed at the age of 2 mo, revealed negative expression for laminin 5, as determined by the monoclonal antibody GB3 (Verrando et al., 1991Verrando P. Blanchet-Bardon C. Pisani A. et al.Monoclonal antibody GB3 defines a widespread defect of several basement membranes and a keratinocyte dysfunction in patients with lethal junctional epidermolysis bullosa.Lab Invest. 1991; 64: 85-92PubMed Google Scholar), and staining with a monoclonal antibody 19-DEJ-1 was also negative (Fine et al., 1989Fine J.-D. Horiguchi Y. Couchman J.R. 19-DEJ-1, a hemisdesmosome-anchoring filament complex-associated monoclonal antibody: definition of a new skin basement membrane antigenic defect in junctional and dystrophic epidermolysis bullosa.Arch Dermatol. 1989; 125: 520-523Crossref PubMed Scopus (43) Google Scholar). Indirect immunofluorescence with antibodies recognizing type IV and type VII collagen showed positive staining on the floor of the dermal–epidermal blister, whereas staining for α6 and β4 integrins was positive in the roof of the blister, indicating the tissue separation at the level of lamina lucida. Transmission electron microscopy of normal appearing skin revealed hypoplastic hemidesmosomes. These findings are diagnostic of H-JEB, and the child died from complications of the disease at the age of 5 mo. For mutation analysis, DNA was isolated from the peripheral blood of the proband and her parents. The mutation detection was initiated by polymerase chain reaction (PCR) amplification of LAMB3 sequences, followed by heteroduplex scanning and automated nucleotide sequencing, using an established strategy (Pulkkinen et al., 1995Pulkkinen L. McGrath J.A. Christiano A.M. Uitto J. Detection of sequence variants in the gene encoding the β;3 chain of laminin 5 (LAMB3) by heteroduplex analysis of PCR amplified segments.Hum Mutation. 1995; 6: 77-84Crossref PubMed Scopus (54) Google Scholar). This approach did not reveal any putative pathogenetic mutations in LAMB3. Next, a similar mutation strategy was applied to search for mutations in the LAMC2 gene (Pulkkinen et al., 1997Pulkkinen L. McGrath J.A. Airenne T. et al.Detection of novel LAMC2 mutations in Herlitz junctional epidermolysis bullosa.Mol Med. 1997; 3: 124-135Crossref PubMed Scopus (22) Google Scholar). Examination of the proband’s PCR products corresponding to exon 5 disclosed a C-to-T transition at nucleotide position 556, as compared with the normal sequence (Figure 1b). This nucleotide substitution resulted in the change of a codon for glutamine to a stop codon (CAG→TAG), and the mutation was designated as Q186X. This same mutation was also found in the mother’s but not in the father’s DNA. Subsequent analysis of PCR products corresponding to exon 8 revealed a C-to-T transition at nucleotide position 1045 (Figure 1c). This nucleotide change resulted in substitution of a codon for arginine by a stop codon (CGA→TGA), a mutation designated as R349X. This mutation was also present in the father’s but not in the mother’s DNA. Thus, the proband was a compound heterozygote for two-stop codon mutations in the LAMC2 gene, Q186X/R349X, which were inherited from the mother and father, respectively. Both mutations predict truncated γ2-chain polypeptides that would be shortened by ≈85% and 70% of their length, respectively. In addition, premature termination codons have previously been shown to cause a marked reduction in the corresponding mRNA transcript level due to nonsense mediated mRNA decay (Cui et al., 1995Cui Y. Hagan K.W. Zhang S. Peltz S.W. Identification and characterization of genes that are required for the accelerated degradation of mRNAs containing a premature translational termination codon.Genes and Develop. 1995; 9: 423-436Crossref PubMed Scopus (220) Google Scholar). Thus, the severely truncated polypeptides, if synthesized at all, would be unable to contribute to the synthesis of functional laminin 5 molecules, thus explaining entirely negative immunofluorescence with the antibody GB3 that specifically recognizes a γ2-chain epitope only when the laminin 5 molecule is in native trimeric conformation (Matsui et al., 1995Matsui C. Nelson C.F. Hernandez G.T. Herron G.S. Bauer E.A. Hoeffler W.K. γ2 chain of laminin-5 is recognized by monoclonal antibody GB3.J Invest Dermatol. 1995; 105: 648-652Crossref PubMed Scopus (52) Google Scholar) Prior to this study, LAMC2 mutations have been reported in seven cases with H-JEB and in each case, with one exception, the proband is homozygous for the mutations (Pulkkinen et al., 1999Pulkkinen L. Uitto J. Christiano A.M. The molecular basis of the junctional forms of epidermolysis bullosa.in: Fine J.-D. Bauer E.A. Mcguire J. Moshell A. Epidermolysis Bullosa. Clinical, Epidemiologic, and Laboratory Advances, and the Findings of the National Epidermolysis Bullosa Registry. The Johns Hopkins University Press, Baltimore1999Google Scholar). In general, the mutations have been family specific, but a homozygous R95X/R95X has been reported in two unrelated patients (Abderdam et al., 1994Abderdam D. Galliano M.-F. Vailly J. et al.Herlitz’s junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for the γ2 subunit of nicein/kalinin (laminin-5).Nature Genetics. 1994; 6: 229-304Google Scholar;Pulkkinen et al., 1997Pulkkinen L. McGrath J.A. Airenne T. et al.Detection of novel LAMC2 mutations in Herlitz junctional epidermolysis bullosa.Mol Med. 1997; 3: 124-135Crossref PubMed Scopus (22) Google Scholar). These observations have implications for prenatal testing in pregnancies at risk for recurrence of H-JEB. Specifically, all three laminin genes, LAMA3, LAMB3, and LAMC2, can be candidates for mutations. Since these three genes have been mapped to distinct loci on chromosomes 18q11.2, 1q32, and 1q25–31, respectively, any subsequent prenatal testing has to be based on specific mutations, and cannot utilize linkage analysis. In summary, we have described two novel LAMC2 mutations in a patient with classic H-JEB. These mutations add to the expanding database on laminin 5 mutations in junctional forms of EB.