Deletion of the Cytoplasmatic Domain of BP180/Collagen XVII Causes a Phenotype with Predominant Features of Epidermolysis Bullosa Simplex Marcel Huber,

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Deletion of the Cytoplasmatic Domain of BP180/Collagen XVII Causes a Phenotype with Predominant Features of Epidermolysis Bullosa Simplex Marcel Huber, Edgar Frenk, Daniel Hohl Journal of Investigative Dermatology Volume 118, Issue 1, Pages 185-192 (January 2002) DOI: 10.1046/j.0022-202x.2001.01617.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Clinical features of the patient. (A) Presence of grouped blisters on the buttock at birth. (B) Patient at the age of 7 mo showing blisters at different stages after trauma, which were healing without scarring. Journal of Investigative Dermatology 2002 118, 185-192DOI: (10.1046/j.0022-202x.2001.01617.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Immunofluorescence staining for the intracellular domain of BP180/collagen XVII is absent in the patient's skin. Frozen sections from control and patient skin were incubated with antibodies against the intracellular and extracellular BP180/collagen XVII domains. (A) Staining of normal control skin with antibodies to the extracellular NC16a domain of BP180/collagen XVII. (B) Strongly reduced staining of the patient's skin with the NC16a domain antibodies in one skin biopsy. The faint staining is localized in the roof of a small blister (the star denotes the blister cavity), indicating junctional blister formation. (C) Positive staining of the patient's skin with the NC16a antibodies in a second skin biopsy. In this specimen, the signal is observed on the floor of the blister, indicating intraepidermal splitting. (D) Staining of control skin with antibody 1A8c against the intracellular domain of BP180/collagen XVII. (E) Negative staining of patient's skin with antibody 1A8c. (F) Positive staining of patient's skin with antibody 1D1 against the extracellular domain of BP180/collagen XVII. Journal of Investigative Dermatology 2002 118, 185-192DOI: (10.1046/j.0022-202x.2001.01617.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Electron microscopy reveals abnormalities in hemidesmosome and keratin intermediate filament structures. Heterogeneous hemidesmosomes of varying size and structure were found in the proband's skin. Moreover, the keratin intermediate filament bundles do not extend into the hemidesmosomes. The other ultrastructural units of the basement membrane zone, lamina lucida, lamina densa, and the anchoring fibrils appeared normal. Bar: 0.25 µm. Journal of Investigative Dermatology 2002 118, 185-192DOI: (10.1046/j.0022-202x.2001.01617.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Northern blot analysis shows a large deletion in the 5′ part of the BP180/collagen XVII mRNA. Total RNA was isolated from keratinocytes cultured in high calcium medium established from the patient (lanes 1, 4) and from normal control individuals (lanes 2, 3, 5, 6). The northern blot was hybridized with a BP180/collagen XVII 5′ probe (nucleotides 97–636, GenBank M91669) in lanes 1–3, or with a BP180/collagen XVII 3′ probe (nucleotides 3555–4402, GenBank M91669) in lanes 4–6. As loading control glyceraldehyde-3-phosphate-dehydrogenase was used to ensure equal amounts of RNA. Journal of Investigative Dermatology 2002 118, 185-192DOI: (10.1046/j.0022-202x.2001.01617.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Immunoblot analysis reveals that patient keratinocytes produce only BP180/collagen XVII of 130 kDa molecular weight. Proteins extracted from cultured cells and culture medium were immunoblotted with the NC16a antibody. Lane 1: Both full-length BP180/collagen XVII (upper arrowhead) and the shed ectodomain (lower arrowhead) were present in cell extracts from normal keratinocytes (Co). Lane 2: The shed ectodomain was precipitated from the medium of normal keratinocytes. Lane 3: The cell extracts of the patient's keratinocytes (EB) contained mutant BP180/collagen XVII polypeptide of approximately 130 kDa (arrow) and the shed ectodomain (lower arrowhead). Lane 4: The soluble ectodomain was present in the medium of the patient's keratinocytes (lower arrowhead). Identical results were obtained when using antibodies against the carboxy-terminal region or against the Col15 domain (see Materials and Methods). On the right, the migration positions of molecular weight standards, from top to bottom 200 kDa, 111 kDa, 80 kDa, and 46 kDa, are shown. Journal of Investigative Dermatology 2002 118, 185-192DOI: (10.1046/j.0022-202x.2001.01617.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Pedigree and mutation analysis in the COL17A1 gene. (A) The pedigree of the family demonstrates that the mutation R1226X (black areas) and the genomic deletion (dotted areas) of the proband (1) occurred in a compound heterozygous manner, because the father (3) is the carrier of R1226X and the mother (2) of the deletion. (B) Sequence analysis of the patient's PCR product containing exon 51 showed a C-to-T transition at nucleotide position 3781, designated R1226X. A restriction endonuclease site for Taq1 is eliminated by the mutation. Accordingly, the 342 bp PCR product (upper band) of a normal control (C1) and the mother (2) were cleaved into a 185 bp (lower band) and a 157 bp fragment, whereas the proband (1), the father (3), and a previously characterized unrelated patient with the same mutation (C2) showed an additional uncleaved band of 342 bp. (C) The maternal mutation was a large genomic deletion, extending from intron 2 to intron 15. In normal controls, an identical 42 bp sequence was found within both introns. In the proband's DNA, this 42 bp sequence is present once and is flanked at the 5′ end by the sequence corresponding to normal intron 2 (underlined) and at the 3′ end by the sequence corresponding to normal intron 15 (doubly underlined). Journal of Investigative Dermatology 2002 118, 185-192DOI: (10.1046/j.0022-202x.2001.01617.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Southern blot analysis of the maternal deletion. Genomic DNA from the patient (lane 1) and a normal individual (lane 2) was digested with XhoI and hybridized with a BP180/collagen XVII exon 2 probe. The arrowhead marks the start of the electrophoresis. Arrows mark the position of the molecular weight markers of 21 kbp, 5.2 kbp, 4.3 kbp, 3.5 kbp, and 2 kbp (top to bottom). Journal of Investigative Dermatology 2002 118, 185-192DOI: (10.1046/j.0022-202x.2001.01617.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions