Skin Expression of Metalloproteinases and Tissue Inhibitor of Metalloproteinases in Sibling Patients with Recessive Dystrophic Epidermolysis and Intrafamilial Phenotypic Variation  Sylvie Igondjo Tchen, Sabah Ghomrasseni, Sylvie Séguier, Frédérick Gaultier, Sylvie Fraitag, Gaston Godeau  Journal of Investigative Dermatology  Volume 121, Issue 2, Pages 273-279 (August 2003) DOI: 10.1046/j.1523-1747.2003.12325.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Immunostaining of collagen type VII. RDEB affected sibling brothers with the same mutations in COL7A1: demonstrative intrafamilial phenotypic variability. Journal of Investigative Dermatology 2003 121, 273-279DOI: (10.1046/j.1523-1747.2003.12325.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Zymography. Gelatinolytic activities detected on gelatin zymograms. Lane a, control healthy donor no. 1; lane b, control healthy donor no. 2; lane c, patient no. 1 (patient M) with RDEB; lane d, patient no. 2 (patient T) with RDEB; lane e, patient no. 3 (patient F) with RDEB; lane f, control healthy donor no. 1+EDTA; lane g, control healthy donor no. 1+Pefablock. With controls 1 and 2 (lanes a, b) two lysis bands appear at 92kDa (progelatinase B) and at 72kDa (progelatinase A). With two affected patients (patient M, lane c; patient F, lane e) three lysis bands appear clearly at 92kDa, 72kDa, and 66kDa (activated gelatinase A), and a lysis band at the limit of detection appears at 88kDa (activated gelatinase B). Only with patient T (lane d) were four intense lysis bands detected on the gelatin zymogram at 92kDa, 88kDa, 72kDa, and 66kDa. Lane f, gelatinolytic activities were suppressed by adding EDTA; lane g, gelatinolytic activities were unaffected by Pefablock. Journal of Investigative Dermatology 2003 121, 273-279DOI: (10.1046/j.1523-1747.2003.12325.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Quantification of gelatinases from RDEB skin of patients M, T, and F and control skin 1 and 2 from healthy donors after zymographic analysis. Ordinate: gelatinase activity, gel U per mg of tissue weight, where U=surface of the lysis band (in pixels)×gray levels×1000. Abscissa: control 1 and control 2, healthy donors; RDEB M, T, F, patients M, T F, respectively, with RDEB; Unaff, unaffected skin; Aff, affected skin; 92kDa, pro-MMP-9; 88kDa, activated MMP-9; 72kDa, pro-MMP-2; 66kDa, activated MMP-2. With control 1 and control 2 low gelatinolytic activities were detected at 92kDa (pro-MMP-9) and 72kDa (pro-MMP-2). With the three patients (M, T, and F), the active forms of MMP-2 (66kDa) and MMP-9 (88kDa), which were never detected with controls, were observed and quantified by image analysis. Journal of Investigative Dermatology 2003 121, 273-279DOI: (10.1046/j.1523-1747.2003.12325.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Quantification of MMP and TIMP after dot blotting analysis. (a) Quantification of MMP-1; (b) quantification of TIMP-1; (c) quantification of MMP-2; (d) quantification of TIMP-2; (e) quantification of MMP-3; (f) quantification of MMP-9. Quantification was performed after dot blotting analysis. Quantities of MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 appear on the ordinate as DQ=surface of the spot on dot blotting (in pixels)×(255 gray level). On abscissa: control 1 and control 2, healthy donors; RDEB M, T, F, patients M, T, F, respectively, with RDEB; Unaff, unaffected skin; Aff, affected skin. Low amounts of MMP-1, MMP-2, MMP-3, MMP-9, and TIMP-2 were detected in controls 1 and 2. With the three affected patient increased amounts of MMP-1, MMP-2, MMP-3, and MMP-9 were detected compared with controls. Concerning TIMP-1 (b) low amounts were detected in the unaffected and affected skin of patient F (the most affected) compared with the two controls and the other two affected patients. For this patient an increased amount of MMP-1 (a) was detected in his unaffected skin compared with the two controls and the other two affected patients. Journal of Investigative Dermatology 2003 121, 273-279DOI: (10.1046/j.1523-1747.2003.12325.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Western blotting: Immunodetection of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2. Cont, control healthy donor no. 1; Unaff, unaffected skin; Aff, affected skin. The amounts of MMP-2, MMP-3, and MMP-9 were increased for lesioned (affected) or unlesioned (unaffected) skin of patient F compared with the other two RDEB patients (patients T and M) and control donor 1. For patient F the amount of MMP-1 from the unaffected skin exceeded that detected for patients M and T and for control donor 1, and the amounts of TIMP-1 detected from affected and unaffected skin were decreased by comparison with those detected for patients M and T, and for control donor 1. Concerning TIMP-2 a slight increase was observed for patient F (affected and unaffected skins) compared with control donor 1 and with the other two RDEB patients M and T. Journal of Investigative Dermatology 2003 121, 273-279DOI: (10.1046/j.1523-1747.2003.12325.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions