Novel Mutations in the LAMC2 Gene in Non-Herlitz Junctional Epidermolysis Bullosa: Effects on Laminin-5 Assembly, Secretion, and Deposition Daniele Castiglia,

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Novel Mutations in the LAMC2 Gene in Non-Herlitz Junctional Epidermolysis Bullosa: Effects on Laminin-5 Assembly, Secretion, and Deposition Daniele Castiglia, Patrizia Posteraro, Mari Pinola, Giovanna Zambruno Journal of Investigative Dermatology Volume 117, Issue 3, Pages 731-739 (September 2001) DOI: 10.1046/j.0022-202x.2001.01453.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Immunofluorescence localization of laminin-5 in non-H JEB keratinocytes. With antibodies to laminin γ2 chain (a, b) and heterotrimeric laminin-5 (c, d), cytoplasmic staining as well as labeling of the extracellular matrix are visible in patient's keratinocytes (a, c). The intensity of the extracellular staining is strikingly reduced, however, compared to normal control (b, d). Bars: 7 µm (a, b); 10 µm (c, d). Journal of Investigative Dermatology 2001 117, 731-739DOI: (10.1046/j.0022-202x.2001.01453.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Northern blot and immunoprecipitation analysis of laminin-5. (A) Expression of LAMC2 gene was assessed by northern blot of total RNA purified from cultured patient and normal keratinocytes. Membranes were hybridized with a 32P-labeled LAMC2 specific probe and, for loading control, the GAPDH probe. The intensity of the signal for the γ2 chain mRNA appears markedly reduced in patient (Pt) compared to control keratinocytes (C). (B) Cultured keratinocytes from the non-H JEB patient (lanes 1, 3) and a healthy volunteer (lanes 2, 4) were labeled with [35S] methionine and cysteine, and conditioned media were immunoprecipitated with antibodies against laminin-5 β3 (lanes 3, 4) and γ2 chains (lanes 1, 2). The eluates were then analyzed on 6% SDS-PAGE gels under reducing conditions. Protein-bound radioactivity in conditioned media was counted, and equivalent amounts of radioactivity were immunoprecipitated from patient and control media. Highly reduced levels of unprocessed and processed γ2 chain are observed in patient's cells (1, 3) in comparison with normal control keratinocytes (2, 4). Journal of Investigative Dermatology 2001 117, 731-739DOI: (10.1046/j.0022-202x.2001.01453.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Identification and verification of the heterozygous mutations 3511insA and 522-1G→A in the proband LAMC2 gene. (A) Total genomic DNA was subjected to PCR using primers that amplify a 462 bp region comprising exon 23 of LAMC2 gene. In comparison with normal control DNA, nucleotide sequencing of patient's DNA reveals a single base insertion (A at position 3511), resulting in a frameshift and a PTC 110 bp downstream. The mutation is designated 3511insA. (B) Verification of mutation 3511insA was performed by ASO hybridization of PCR products from the parents, the proband, and a normal control (C). The wild-type (WT) oligomer hybridizes to all DNA samples, whereas the mutant (M) oligonucleotide hybridizes only to DNA from the proband and his mother, indicating that they are heterozygous for mutation 3511insA. (C) Total genomic DNA was subjected to PCR using primers that amplify a 205 bp region encompassing LAMC2 exon 4. Compared with the DNA of a normal control, nucleotide sequencing of non-H JEB patient's DNA reveals a G-to-A transition at the 3′ acceptor splice site of intron 3. The mutation is designated 522-1G→A. (D) ASO analysis performed on 205 bp amplimers corresponding to exon 4 with wild-type (WT) and mutated (M) oligomers confirms the heterozygous state of mutation 522-1G→A only in the proband's DNA (3), suggesting the de novo occurrence of the mutation in the paternal allele. Journal of Investigative Dermatology 2001 117, 731-739DOI: (10.1046/j.0022-202x.2001.01453.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Allele-specific analysis of the transcripts and cDNA sequencing. (A) RT-PCR amplification of total RNA obtained from patient's cultured keratinocytes using paternal (lane 1) or maternal (lane 2) specific antisense oligonucleotides that end at the C/A polymorphism detected at nt 858. Paternal-specific PCR products are represented by two fragments, of 376 and 279 bp. By contrast, a single band of 378 bp is derived from the maternal allele. (B) Sequencing of 279 bp fragment shown in lane 1A reveals an aberrant mRNA bearing the in-frame skipping of exon 4 (99 bp) (mutant cDNA 1), whereas sequencing of the 376 bp band derived from the paternal allele shows an out-of-frame transcript carrying the deletion of the first two bases of exon 4 (mutant cDNA 2). (C) In the middle is schematically represented the genomic structure of the region from exon 3 to exon 5 of the LAMC2 paternal allele carrying mutation 522-1G→A. The aberrantly spliced transcripts are represented above and below the mutated allele. Splicing events are drawn by dotted lines. The mutation and the cryptic splice site within exon 4 are indicated. (D) RT-PCR amplification followed by AspI restriction enzyme digestion was carried out using oligonucleotides spanning the region containing the 858 C/A polymorphism (nt 707–1145) on cDNA from a normal homozygous C/C individual (lane 1) and the heterozygous C/A patient (lane 2). Two digested bands of 286 and 153 bp are visible in the C/C homozygous subject. In the patient, the maternal transcripts correspond to the same 286 and 153 cleaved bands, whereas the paternal transcripts remain undigested and correspond to the 439 bp band. Note that the intensity of the two maternal bands is similar to that of the paternal one. The markers for molecular size (100 bp DNA ladder, from 100 to 2000 bp) are in lane M. Journal of Investigative Dermatology 2001 117, 731-739DOI: (10.1046/j.0022-202x.2001.01453.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Immunofluorescence staining of γ2-null LSV5 cells transiently expressing the wild-type and mutated γ2 chains. Forty-eight hours after transfection, cells were stained with antibodies to laminin γ2 chain (a, b, c) and to heterotrimeric laminin-5 (d, e, f). In LSV5 cells the γ2 chain and, consequently, laminin-5 molecules are absent (Miquel et al, 1996), but reappear after transfection with the wild-type γ2 cDNA resulting in strong intracytoplasmic and extracellular labeling (a, d). LSV5 cells transfected with plasmid pCγ2Δ4 to express the exon-4-deleted γ2 chain show an intense cytoplasmic labeling and a fainter extracellular staining with both antibodies used (b, e). LSV5 cells transfected with plasmid pCγ2t to express the C-terminal truncated γ2 chain show immunostaining with anti-γ2 antibody (c) but no reactivity with anti-laminin-5 antibody (f). Transfection experiments were repeated three times with similar results. Scale bar: 12 µm. Journal of Investigative Dermatology 2001 117, 731-739DOI: (10.1046/j.0022-202x.2001.01453.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effect of frameshift mutation 3511insA on protein sequence and secondary structure. (A) Alignment and conservation between the C-terminal sequences of wild-type murine γ1 (Swissprot accession No. P02468) and human γ2 chains. The mutant γ2 chain with the aberrant amino acid sequence generated by the frameshift mutation 3511insA and starting from residue 1132 is also shown. The ‘d’ positions in the heptad repeat of laminin long arm is indicated above the sequences. The γ1 sequences required for dimer and trimer assembly are boxed. The mutant chain terminates prematurely at residue 1168. New proline and glycine residues in the mutant sequence that are known to act as α-helix breakers are underlined. (B) Secondary structure prediction plots according toGarnier et al (1996). The normal sequence shows propensity for α-helix (WT γ2). By contrast, the mutant sequence shows a severe disruption of the α-helix acquiring a random coil profile (Mut γ2). Black, α-helix; gray, random coil. Journal of Investigative Dermatology 2001 117, 731-739DOI: (10.1046/j.0022-202x.2001.01453.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions