Cycloheximide Facilitates the Identification of Aberrant Transcripts Resulting from a Novel Splice-Site Mutation in COL17A1 in a Patient with Generalized.

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Cycloheximide Facilitates the Identification of Aberrant Transcripts Resulting from a Novel Splice-Site Mutation in COL17A1 in a Patient with Generalized Atrophic Benign Epidermolysis Bullosa Thomas N. Darling, Carole Yee, Brian Koh, John A. McGrath, Johann W. Bauer, Jouni Uitto, Helmut Hintner, Kim B. Yancey Journal of Investigative Dermatology Volume 110, Issue 2, Pages 165-169 (February 1998) DOI: 10.1046/j.1523-1747.1998.00103.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 GABEB keratinocytes show undetectable type XVII collagen and decreased COL17A1 mRNA. (A) Cellular extracts from biosynthetically radiolabeled GABEB keratinocytes were immunoprecipitated using preimmune rabbit serum (lane 1), rabbit anti-sera to type XVII collagen (lane 2), or rabbit anti-sera to bullous pemphigoid antigen 1 (BPAG1) (lane 3). Lane 2 shows no evidence of type XVII collagen or a truncated protein. A band of ≈230 kDa, corresponding to BPAG1, is visible in lane 3. (B) Northern blot study of total RNA extracted from cultured GABEB or normal keratinocytes preincubated for 2.5 h in the presence (+) or absence (–) of 10 μg cycloheximide (CHX) per ml. COL17A1 mRNA is decreased in GABEB keratinocytes as compared with normal human foreskin. In GABEB keratinocytes, cycloheximide increases COL17A1 mRNA. Equivalent loading of the RNA is shown by the similar relative intensity of the bands using a probe for β-actin. Journal of Investigative Dermatology 1998 110, 165-169DOI: (10.1046/j.1523-1747.1998.00103.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Identification of a maternally inherited mutation at the 3' acceptor splice site of exon 32. (A) CSGE of exon 32 with its flanking intronic regions demonstrates a heteroduplex band (→) in addition to a homoduplex band using DNA from the mother and the proband, but only a homoduplex band in the father and control individual. (B) Nucleotide sequencing of exon 32 shows the consensus AG at the acceptor splice site adjacent to exon 32 in the normal, but an AT in the mutant allele. (Note: The different nucleotide numbers in these two chromatograms relate to the first nucleotide counted by an automated sequencer in analysis of reactions performed on different days and do not represent corresponding sites in the two sequences.) (C Verification of the mutation by the introduction of a new Nla III site in the mutant. The PCR product from the father or unrelated control is cleaved into bands of 310 and 37 bp. The PCR product from the mother or the proband is cleaved into bands of 310, 182, 128, and 37 bp, as expected for individuals with one normal and one mutant allele. Journal of Investigative Dermatology 1998 110, 165-169DOI: (10.1046/j.1523-1747.1998.00103.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 The acceptor splice-site mutation causes abnormal processing of COL17A1 pre-mRNA, demonstrated in patient keratinocytes preincubated in the presence of cycloheximide. Primers spanning the mutation were used in RT-PCR studies performed using RNA from patient or normal keratinocytes cultured in the presence (+) or absence (–) of cycloheximide. For a control, reverse transcriptase was omitted (No RT), using RNA from patient keratinocytes preincubated with cycloheximide. RNA from both patient and normal keratinocytes yields the normal transcript of 544 bp. An additional, abnormally large RT-PCR product of about 800 bp is visualized using RNA from patient keratinocytes preincubated with cycloheximide. Identical results were obtained in four separate RT-PCR experiments. Journal of Investigative Dermatology 1998 110, 165-169DOI: (10.1046/j.1523-1747.1998.00103.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Normal and aberrant splicing of COL17A1 exon 32 in patient keratinocytes. Sequencing of RT-PCR products revealed four mutant transcripts, three produced from splicing at cryptic splice sites and one from skipping of exon 32. The different patterns of splicing are shown by the diagonal lines connecting regions spliced together. The restriction enzymes used to confirm the identities of the normal transcript as well as mutant transcripts 3 and 4 are shown at the top of the figure (→ indicate cleavage sites). Journal of Investigative Dermatology 1998 110, 165-169DOI: (10.1046/j.1523-1747.1998.00103.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions