A Homozygous Nonsense Mutation in Type XVII Collagen Gene (COL17A1) Uncovers an Alternatively Spliced mRNA Accounting for an Unusually Mild Form of Non-Herlitz.

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A Homozygous Nonsense Mutation in Type XVII Collagen Gene (COL17A1) Uncovers an Alternatively Spliced mRNA Accounting for an Unusually Mild Form of Non-Herlitz Junctional Epidermolysis Bullosa Laura Ruzzi, Patrizia Posteraro, Giovanna Zambruno, Daniele Castiglia, Marina D'Alessio Journal of Investigative Dermatology Volume 116, Issue 1, Pages 182-187 (January 2001) DOI: 10.1046/j.1523-1747.2001.00229.x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Identification and verification of the homozygous mutation R795X in the probands' COL17A1 gene and inheritance in the families. (a) Sequence analysis of the patients' PCR products spanning exons 26–34. Compared with normal cDNA (upper panel), a C→T transition at nucleotide position 2488 is evident in patients' cDNA (middle panel). In addition, a second transcript carrying the in-frame skipping of exon 33 is present in patients' cells (lower panel). The 2488C→T transition converts the codon for arginine CGA with the signal for premature termination of translation TGA at amino acid position 795. The mutation is therefore designated R795X. Base substitution is underlined. (b) Total genomic DNA from all affected individuals was subjected to PCR using primers that amplify a 247 bp fragment encompassing exon 33. Compared with a normal control (upper panel), direct nucleotide sequencing of the non-Herliz JEB patients confirms the presence of the 2488C→T transition at the homozygous state (lower panel). (c) Pedigrees of the non-Herlitz JEB families and verification of mutation 2488C→T with HaeIII restriction endonuclease digestion. Genomic DNA amplification of the region containing the mutation, using sense primer 5′-CTTGTTCTAATCACATCA-3′ and antisense primer 5′ CCAGTGAAAAGAAAGAAA 3′, results in the synthesis of a 95 bp PCR product. The amplification conditions were 95°C for 8 min; 94°C, 45 s; 48°C, 45 s; 72°C, 45 s (30 cycles); and extension at 72°C for 10 min. Whereas the HaeIII digestion of the PCR product yields two bands, 57 and 38 bp in size, in a normal control (lane C), in the presence of the 2488C→T substitution the 95 bp fragment is not digested. Patients A-V.1, A-V.2, A-V.5, B-IV.1, C-II.1, and D-II.1 are all homozygous for the mutation, whereas the healthy mother of A-V.1 and A-V.2 and the son of A-V.5 are heterozygous. The corresponding PCR product from the healthy individual A-V.4 is normally digested. Journal of Investigative Dermatology 2001 116, 182-187DOI: (10.1046/j.1523-1747.2001.00229.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Amplification of exon 33 alternatively processed transcripts in patients' and normal keratinocytes. Two percent agarose gel fractionation of RT-PCRs of total RNA extracted from cultured keratinocytes of four unrelated healthy donors (lanes 2–5), the heterozygous healthy carrier A-VI.1 (lane 6), patient A-V.5 (lane 7), 2342delG GABEB cells (lane 10), and from normal human lymphocytes (lane 1). Lane 8 corresponds to blank (no template added to the PCR). Lane 9 shows PCR amplification performed on a cloned wild-type cDNA fragment spanning exons 26–34 as positive (a)/negative (b) control. Whereas with primers Pr 7L/Pr 7R encompassing exons 26–34 an additional 358 bp band is detected only in the patient and, to a minor degree, in the healthy carrier (a), the use of a primer pair Pr 7L/Pr 32:34R, the latter spanning the junction between exons 32 and 34, evidences a 338 bp product, corresponding to the exon 33-deleted transcript, also in normal human keratinocytes and in 2342delG GABEB cells (b). Identity of the 338 bp fragment shown in panel B was checked by direct sequencing. (c) Amplification of GAPDH served as cDNA quality control. A 100 bp ladder (Boehringer Mannheim) was used as molecular size marker. Journal of Investigative Dermatology 2001 116, 182-187DOI: (10.1046/j.1523-1747.2001.00229.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Immunoblot detection of residual type XVII collagen in patients' keratinocytes. Protein extracts of normal and patients' keratinocytes were separated by SDS-PAGE, transferred to nitrocellulose filters, and stained for type XVII collagen (BP180) expression using MoAb 1A8C. Lane 1, extract from normal cells (four times diluted); lane 2, extract from patient A-V.5 cells; lane 3, extract from patient D-II.1 cells; lane 4, extract from 2342delG GABEB cells. Journal of Investigative Dermatology 2001 116, 182-187DOI: (10.1046/j.1523-1747.2001.00229.x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions