Autosomal Dominant Cutis Laxa with Severe Lung Disease: Synthesis and Matrix Deposition of Mutant Tropoelastin Zsolt Urban, Jimin Gao, F. Michael Pope,

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Autosomal Dominant Cutis Laxa with Severe Lung Disease: Synthesis and Matrix Deposition of Mutant Tropoelastin Zsolt Urban, Jimin Gao, F. Michael Pope, Elaine C. Davis Journal of Investigative Dermatology Volume 124, Issue 6, Pages 1193-1199 (June 2005) DOI: 10.1111/j.0022-202X.2005.23758.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Clinical characterization of family cutis laxa (CL)-1. (A) Prematurely redundant and sagging facial skin of the proband III.1 at age 40 y. (B) The pedigree of CL-1 indicates autosomal dominant inheritance. Hatched symbols indicate individuals (I.2, II.9, and III.11) who were reportedly affected but were not available for examination. Small diamonds (IV.2 and IV.3) indicate miscarriages; large diamonds denote multiple unaffected individuals within the same generation. (C) Posterioanterior (PA) chest X-ray (viewed in AP position) of III.1 shortly before lung transplantation at the age of 51 y. Hyperexpanded lung fields indicate emphysema. Bilateral basal shadows reveal lung fibrosis. Note the enlarged left pulmonary artery along the left heart border demonstrating pulmonary hypertension. (D) Inelastic skin and generalized CL in individual IV.4 at birth. Journal of Investigative Dermatology 2005 124, 1193-1199DOI: (10.1111/j.0022-202X.2005.23758.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Histological and electron microscopic evaluation of dermal elastic fibers in patient III.1. (A) Hematoxylin–eosin staining of a skin section shows metachromatic, fragmented, and clumped (arrows) elastic fibers. (B) Elastic van Giesson (EVG) staining of a skin section from III.1 indicates disorganization of elastic fibers in the upper (arrows) and deep (arrowheads) dermis. (C) EVG staining of a control skin section shows normal, candelabra-like organization of elastic fibers in the papillary dermis (arrows). Transmission electron microscopy of dermal elastic fibers (El) in III.1 (D) demonstrates diminished microfibrillar component (arrows) on the periphery of elastic fibers compared with a normal control (E). Collagen bundles (Col) appear normal. Magnification bars are 100 μm (A–C) and 2 μm (D–E), respectively. Journal of Investigative Dermatology 2005 124, 1193-1199DOI: (10.1111/j.0022-202X.2005.23758.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Abnormal tropoelastin (TE) and elastin mRNA in cutis laxa (CL)-1. (A) Skin fibroblasts from affected individuals III.1 and IV.4 were used to assess TE synthesis by metabolic labeling and immunoprecipitation. In addition to normal TE (68 kDa), a prominent 120 kDa protein (arrow) was detected in CL-1 samples only. Note that fibronectin (FN) binds directly to the immunoprecipitation matrix and thus is present without the use of a primary antibody (Kuusela et al, 1984). Molecular weight marker positions are shown in kilodaltons (kDa). (B) Total RNA was isolated from fibroblasts from CL patients III.1 and IV.4 and a normal control (CON) and was analyzed by RNA-blot analysis using human elastin (Olson et al, 1995) and β-actin cDNA as probes. In addition to the normal 3.5 kb elastin mRNA (elastin), fibroblasts from patients III.1 and IV.4 also contained a 5 kb elastin mRNA (arrow). Molecular weight marker positions are shown in kilobases (kb). (C) Total mRNA from III.1 and a normal control was analyzed by 3′ rapid amplification of cDNA ends (3′-RACE) using an upstream gene-specific primer complementary to exon 30. The mutation-specific 3′-RACE product (arrow) was sequenced to determine the structure of the 3′-end of the mutant mRNA. Molecular weight marker positions are shown in base pairs. Journal of Investigative Dermatology 2005 124, 1193-1199DOI: (10.1111/j.0022-202X.2005.23758.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 A tandem duplication in patients III.1 and IV.4. (A) Domain structure encoded by the normal elastin message is compared with that of the mutant mRNA detected in the cutis laxa (CL)-1 family. TE consists of alternating hydrophobic domains (open) and cross-linking domains (diagonally hatched), flanked by an N-terminal signal peptide and a C-terminal cysteine-containing, charged peptide (horizontally hatched). Exons 23 and 32 (asterisks) are normally subject to high-frequency alternative splicing. In the mutant mRNA, a 452 bp segment of intron 10 is present (shaded) that generates a missense peptide sequence encoded by the first 63 nucleotides. A portion of this unique sequence (underlined) was used to raise a mutant-specific polyclonal rabbit antibody. Note that rather than being a simple tandem duplication, the mutant mRNA is a product of a complex rearrangement, a duplication of the region encoded by exons 9—33; a third copy of exons 9–10 and intron 10 is added to the end of the mRNA. (B) PCR amplification of the breakpoint region from genomic DNA of a normal control (CON) and of CL patients III.1 and IV.4 using a sense primer complementary to intron 32 and an antisense primer complementary to intron 9. Size standard positions are shown in kilobase pairs. Panel C. Sequence analysis of the breakpoint-specific genomic PCR product identified a chimeric intron consisting of 155 bp of intron 33 and 95 bp of intron 8 (double arrows). The locations of exons 33 and 9 (boxes) and of the amplification primers (arrows) are indicated. (D) Southern blot analysis of the duplication breakpoint region. Genomic DNA samples from a normal control (CON) and from CL patient III.1 were digested with restriction enzymes BamHI, EcoRI, and HindIII. DNA fragments were subjected to Southern blot analysis using a breakpoint-specific PCR product (shown in panel B) as a probe. Size standard positions are shown on the left in kilobases. In addition to fragments identical to the control, duplication-specific 6, 5, and 5 kb fragments were detected in BamHI, EcoRI, and HindIII digests, respectively (arrowheads). Journal of Investigative Dermatology 2005 124, 1193-1199DOI: (10.1111/j.0022-202X.2005.23758.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Partial secretion of mutant tropoelastin (TE) in dermal fibroblasts. TE was immunoprecipitated from cell lysates and conditioned media from cultured skin fibroblasts. The protein was detected by western blotting using a different anti-TE antibody. The larger, mutant protein was detected in the lysate and media (arrows), indicating that the mutant protein was present in the cell and secreted into the medium. The presence of the mutant protein apparently did not affect the synthesis and secretion of the normal TE. Standard sizes and the calculated size of TE are shown in kilodaltons to the left. Journal of Investigative Dermatology 2005 124, 1193-1199DOI: (10.1111/j.0022-202X.2005.23758.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Immunofluorescence localization of the mutant tropoelastin (TE) in dermal fibroblasts. Subconfluent cultures control (A) and cutis laxa (CL) patient III.1 (B) fibroblasts were fixed and permeabilized for intracellular localization of the mutant TE protein using the intron 10 antibody. Whereas only background staining was seen in the control cells (A), a prominent, punctate, peri-nuclear staining was seen in the patient fibroblasts (B). Post-confluent fibroblast cultures from control (C) and CL-1 patient III.1 (D) were fixed and stained with the intron 10 antibody (specific for the mutant protein). Controls were negative (C), whereas the extracellular matrix of CL cells was positive for the mutant protein (D). Nuclei of control cells were counterstained to demonstrate the presence of the cell layer because staining with the intron 10 antibody was negative. Post-confluent fibroblast cultures from control (E) and CL-1 patient III.1 (F) were fixed and stained with an anti-TE antibody (recognizing both normal and mutant proteins). The abundance and intensity of elastic fibers were similar in both patient and control cultures. To indicate cell density, nuclei were also stained. Journal of Investigative Dermatology 2005 124, 1193-1199DOI: (10.1111/j.0022-202X.2005.23758.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions