Deletions within COL7A1 Exons Distant from Consensus Splice Sites Alter Splicing and Produce Shortened Polypeptides in Dominant Dystrophic Epidermolysis.

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Deletions within COL7A1 Exons Distant from Consensus Splice Sites Alter Splicing and Produce Shortened Polypeptides in Dominant Dystrophic Epidermolysis Bullosa Anavaj Sakuntabhai, Nadja Hammami-Hauasli, Christine Bodemer, Ariane Rochat, Catherine Prost, Yann Barrandon, Yves de Prost, Mark Lathrop, Fenella Wojnarowska, Leena Bruckner-Tuderman, Alain Hovnanian The American Journal of Human Genetics Volume 63, Issue 3, Pages 737-748 (September 1998) DOI: 10.1086/302029 Copyright © 1998 The American Society of Human Genetics Terms and Conditions

Figure 1 Clinical presentation of patients and of patient 1's daughter. a, Patient 1 at age 32 years, showing dystrophic finger nails with minimal scarring. b, Patient 1's daughter, showing congenital absence of skin of the posterior aspect of the right leg and heel. c, Patient 2 at age 10 years, showing blistering and atrophic scarring on the pretibial area and knees, with numerous and hypertrophic milia. The American Journal of Human Genetics 1998 63, 737-748DOI: (10.1086/302029) Copyright © 1998 The American Society of Human Genetics Terms and Conditions

Figure 2 Genomic amplification of exons 73 and 87 from COL7A1, and RT-PCR amplification of corresponding regions in patients 1 and 2. a, Family 1, in which amplification of exon 73 from genomic DNA showed an extra band (258 bp) migrating faster than the band of normal size (286 bp), in both patient 1 (lanes P1) and her affected daughter (lane Da). Sequencing of this lower band showed a 28-bp deletion (6081del28) in exon 73, located 72 bp upstream of the donor splice sites of intron 73. In patient 1, RT-PCR amplification of the COL7A1 cDNA region from exons 71–76 showed a smaller band (458 bp) than would be expected from the deletion. Sequence analysis of this band confirmed 6081del28 and revealed abnormal splicing of the last 62 nucleotides of exon 73. b, Family 2, in which genomic amplification of exons 86 and 87 showed a smaller band (377 bp) in addition to the band of normal size (404 bp) in both patient 2 (lanes P2) and her affected father (lane Fa2). Sequencing of the lower band showed a 27-bp deletion in exon 87, located 27 bp upstream of the donor splice site of intron 87. In patient 2, RT-PCR amplification of the COL7A1 cDNA region encompassing exons 85–89 showed a band of reduced intensity and smaller size (184 bp) than would be predicted from the deletion. Sequencing of this band (184 bp) revealed skipping of exon 87. The reduction in intensity of this band may be due to variable degradation of the mutated transcript, despite the fact that the reading frame is maintained. Bands of abnormal size are denoted by an asterisk (*). Numbering of the nucleotides refers to the cDNA sequence published by Christiano et al. (1994b) (Genbank accession number L02870). The American Journal of Human Genetics 1998 63, 737-748DOI: (10.1086/302029) Copyright © 1998 The American Society of Human Genetics Terms and Conditions

Figure 3 Schematic representation of the genomic deletions of exons 73 and 87 and associated abnormal splicing. Exons and parts of exons present in the mRNA are shown as blank boxes, genomic deletions of exons are shown as black boxes, and the exons or parts of the exons that are removed by abnormal splicing are shown as boxes with hatching. a, Patient 1. The 28-bp genomic deletion in exon 73 leads to abnormal splicing of the last 62 bp of this exon after the activation of a cryptic GT donor splice site (underlined). The 10-bp sequence (nucleotides 6109–6118) encoding the new amino acids E2028, S2029, and L2030 between the 3′ end of the genomic deletion and the cryptic GT donor splice is shown as in gray shaded. b, Patient 2. The 27-bp genomic deletion of exon 87 produces skipping of the entire exon 87. These splicing abnormalities occurred despite the fact that the consensus donor splice-site sequences of intron 73 and 87 remain intact. The American Journal of Human Genetics 1998 63, 737-748DOI: (10.1086/302029) Copyright © 1998 The American Society of Human Genetics Terms and Conditions

Figure 4 Indirect IF staining of control and patient's keratinocytes, with antibodies to type VII collagen. Staining of normal human keratinocytes (a and c) exhibits a weak granular intracellular IF, whereas staining of keratinocytes of patients 1 (b) and (d) clearly show increased immune reactivity, indicating intracellular accumulation of type VII procollagen. The American Journal of Human Genetics 1998 63, 737-748DOI: (10.1086/302029) Copyright © 1998 The American Society of Human Genetics Terms and Conditions

Figure 5 Immunoblotting of type VII collagen from control and patient's keratinocyte extracts. a,Intensity of 320-kD type VII procollagen immunoblot band, which was stronger in keratinocyte extracts of patients 1 (lane Pat 1) and 2 (lane Pat 2) than in those of controls (lanes Co). b, Semiquantitative immunoblot-based assay (König and Bruckner-Tuderman 1992), demonstrating that the relative intensity of type VII procollagen bands in the control, patient 1, and patient 2 was 1:1.7:2.3, indicating that the DDEB keratinocytes accumulated type VII procollagen. The American Journal of Human Genetics 1998 63, 737-748DOI: (10.1086/302029) Copyright © 1998 The American Society of Human Genetics Terms and Conditions

Figure 6 Pepsin digestion of type VII procollagen, and immunoblotting with antibodies to the carboxy-terminus of type VII collagen. Normal and DDEB keratinocyte extracts were digested with pepsin, to produce smaller triple-helical type VII procollagen fragments. In untreated extracts of the control and patient 1 and 2, the 320-kD type VII procollagen band was present (lanes −Pepsin). A limited digestion with pepsin (lanes +Pepsin) resulted in the loss of the intact procollagen and in appearance of the full-length triple helix and its carboxy-terminal half, the P1 fragment (Burgeson 1993). The amino-terminal half of the triple helix, the P2 fragment, was not recognized by the antibodies used here. In contrast to what was seen in the control with a distinct P1 fragment, P1 derived from mutant type VII procollagen of patient 1 appeared as a diffuse and broadened band, and pepsin digestion of mutant type VII procollagen of patient 2 resulted in a double P1 band, indicating that the mutant type VII collagen molecules were hybrid trimers of normal length and shortened polypeptides. In the case of patient 1, pepsin treatment did not result in complete digestion of the procollagen molecules but produced abnormal high–molecular-weight cleavage products, suggesting that inclusion of mutant polypeptides in the trimeric molecules had led to altered proteinase sensitivity. The American Journal of Human Genetics 1998 63, 737-748DOI: (10.1086/302029) Copyright © 1998 The American Society of Human Genetics Terms and Conditions

Figure 7 Electron microscopy of tissue of (a) the control, which shows AFs (arrows) of normal length and ultrastructure that insert into the lamina densa, (b) skin from patient 1, which shows hypoplastic AFs reduced in number, and (c) patient 2, which shows tissue separation (asterisk [*]) below the lamina densa and rare and poorly formed AFs. (Scale bar 0.5 μm; final magnification 26,400×) The American Journal of Human Genetics 1998 63, 737-748DOI: (10.1086/302029) Copyright © 1998 The American Society of Human Genetics Terms and Conditions

Figure 8 Proposed mechanisms for genomic deletions 6081del28 and 6847del27. a, Patient 1 (6081del28). Formation of a hairpin loop by intrastrand pairing of inverted repeats 1-4 and 2-3 is shown. The presence of stretches of G and C (underlined) located immediately upstream of the loop would have favored slippage of the DNA polymerase and replication across the base of the loop, causing the loop to be removed from the intervening sequence. Alternatively, excision of the loop may have occurred during DNA replication. b, Patient 2 (6847del27). The presence of an 8-bp direct repeat (CCTGGCCC [underlined]) may have favored accidental nucleotide pairing and deletion of the intervening 27-bp sequence during DNA replication. The American Journal of Human Genetics 1998 63, 737-748DOI: (10.1086/302029) Copyright © 1998 The American Society of Human Genetics Terms and Conditions