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. 2013 Jan;34(1):111-21.
doi: 10.1002/humu.22165. Epub 2012 Aug 13.

Comprehensive clinical and molecular analysis of 12 families with type 1 recessive cutis laxa

Bert Callewaert  1 , Chi-Ting Su, Tim Van Damme, Philip Vlummens, Fransiska Malfait, Olivier Vanakker, Bianca Schulz, Meghan Mac Neal, Elaine C Davis, Joseph G H Lee, Aicha Salhi, Sheila Unger, Ketil Heimdal, Salome De Almeida, Uwe Kornak, Harald Gaspar, Jean-Luc Bresson, Katrina Prescott, Maria E Gosendi, Sahar Mansour, Gérald E Piérard, Suneeta Madan-Khetarpal, Frank C Sciurba, Sofie Symoens, Paul J Coucke, Lionel Van Maldergem, Zsolt Urban, Anne De Paepe
Affiliations

Comprehensive clinical and molecular analysis of 12 families with type 1 recessive cutis laxa

Bert Callewaert et al. Hum Mutat. 2013 Jan.

Abstract

Autosomal recessive cutis laxa type I (ARCL type I) is characterized by generalized cutis laxa with pulmonary emphysema and/or vascular complications. Rarely, mutations can be identified in FBLN4 or FBLN5. Recently, LTBP4 mutations have been implicated in a similar phenotype. Studying FBLN4, FBLN5, and LTBP4 in 12 families with ARCL type I, we found bi-allelic FBLN5 mutations in two probands, whereas nine probands harbored biallelic mutations in LTBP4. FBLN5 and LTBP4 mutations cause a very similar phenotype associated with severe pulmonary emphysema, in the absence of vascular tortuosity or aneurysms. Gastrointestinal and genitourinary tract involvement seems to be more severe in patients with LTBP4 mutations. Functional studies showed that most premature termination mutations in LTBP4 result in severely reduced mRNA and protein levels. This correlated with increased transforming growth factor-beta (TGFβ) activity. However, one mutation, c.4127dupC, escaped nonsense-mediated decay. The corresponding mutant protein (p.Arg1377Alafs(*) 27) showed reduced colocalization with fibronectin, leading to an abnormal morphology of microfibrils in fibroblast cultures, while retaining normal TGFβ activity. We conclude that LTBP4 mutations cause disease through both loss of function and gain of function mechanisms.

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Conflict of interest statement

The authors have no conflict of interest to declare.

Figures

Figure 1
Figure 1. Schematic representation of Fibulin-5 (panel A) and LTBP4 (panel B), with all identified mutations to date
An amino acid scale is shown above and below the schematic representation of the fibulin-5 and LTBP4 proteins, respectively. Mutations identified in this study are depicted in bold. Previously reported mutations are shown in italic and marked with (1) (Claus, et al., 2008); (2) (Elahi, et al., 2006; Loeys, et al., 2002); (3) (Nascimento, et al., 2010); or * (Urban, et al., 2009). A: Fibulin-5. Yellow, Signal peptide, Blue, calcium binding epidermal growth factor – like domain (cbEGF-like) (the first cbEGF-like domain includes an insertion); Red, Fibulin specific globular domain. The 4th EGF-like domain is shown in detail. The disulfide bond between C217 and C230 is indicated by a dashed line. B: The long form of LTBP4. Green, 4-Cys domain; red, 8-Cys (TB) domain; purple, hybrid domain; blue, calcium binding epidermal growth factor-like domain; yellow, EGF-like domain.
Figure 2
Figure 2. Expression analysis of LTBP4 at the mRNA and protein levels
A: Relative expression of LTBP4 mRNA (normalized to expression of household genes YWHAZ, GAPDH, and HPRT1) on total RNA samples extracted from patient skin fibroblasts (I:IV-6, C:II-2, E:II-1). Premature termination mutations resulted in greatly reduced levels of LTBP4 mRNA in C:II-1, E:II-1, and a more modest reduction in I:IV-6. Bars indicate 95% confidence interval. B: Sequencing of a RT-PCR product from fibroblast I:IV-6 shows the presence of mutation c. 4127dupC (p.Arg1377Alafs*27) compared to a control. C: Schematic representation of the truncated LTBP4 protein predicted based on the presence of the mutation p.Arg1377Alafs*27 compared to the full length (long form) of LTBP4 protein. D: Representative image of immunoblots of LTBP4 and fibrillin-1 (FBN1) in conditioned media from control and mutant fibroblasts. LTBP4 was probed using an antibody raised against the amino-terminal half of LTBP4 (N-half) and an antibody against the carboxy-terminus (C-term). LTBP4 is detected as a long form (~175kDa), a short form (~150 kD) and a proteolytic fragment (~120kDa). Fibrillin-1 appears as a single 350 kD protein. C1: control 1; C2: control 2. Patients: C:II-2, E:II-1 and I:IV-6. Severely reduced expression of LTBP4 was noted in patient C:II-2 and E:II-1. LTBP4 was detectable in the media of I:IV-6 using the anti-N-half antibody but not using the anti-C-term antibody.
Figure 3
Figure 3. Immunostaining for LTBP4 and fibrillin-1
Control and mutant fibroblasts (I:IV-6, C:II-2 and E:II-1) were stained for LTBP4 (green) and fibrillin-1 (FBN1; red). Nuclei were counterstained in blue. Fibroblasts from patient I:IV-6 show abnormal morphology of fibrillin-1 microfibrils and altered localization of LTBP4 to fibrillin-1. Fibroblasts from patients C:II-2 and E:II-1 lack LTBP4 staining in the extracellular matrix. Magnification bars: 50 µm.
Figure 4
Figure 4. The distribution of fibronectin and LTBP4 in fibroblast extracellular matrix
Green color, red color and yellow color indicate fibronectin (FN), LTBP4 and colocalization, respectively. Nuclei were counterstained in blue. Control cells show patchy colocalization of LTBP4 with fibronectin. Cells from I:IV-6 deposit largely distinct fibronectin and LTBP4-containing networks. Cells from C:II-2 and E:II-I show no matrix staining for LTBP4. Magnification bars: 50µm.
Figure 5
Figure 5. Immunofluorescence staining for LTBP1 and fibrillin-1
Control and patient (I:IV-6, C:II-2 and E:II-1) fibroblasts were stained for LTBP1 (red) and fibrillin-1 (FBN1; green). Nuclei were counterstained in blue. The same patchy colocalization pattern is observed between LTBP1 and fibrillin-1 in both control and mutant cells. However, microfibril bundles are thicker and wavier in fibroblasts form I:IV-6. Magnification bars: 100 µm.
Figure 6
Figure 6. Electron microscopic findings in LTBP4 and FBLN5 related cutis laxa
In a control skin biopsy, electron microscopy of the elastic fiber consists of an elastin core (e) surrounded by microfibrils (mf), which form physical continuity and the same directionality as peripheral processes of the elastin core. In the patient (C:II-2), the elastin core (e) consists of globular deposits, which are poorly connected to each other and to peripheral microfibrils (mf). This abnormal elastic fiber morphology is similar to a previously published patient with a homozygous LTBP4 mutation (LTBP4−/−) and a knockout mouse for the short form of ltbp4 (Ltbp4S−/−). Images (LTBP4−/−) and (Ltbp4S−/−) were taken from (Urban, et al., 2009). Electron microscopic abnormalities of elastic fibers in a patient with a homozygous FBLN5 mutations (FBLN5−/−) showed small rounded elastin deposits poorly connected to microfibrils. Image (FBLN5−/−) was taken from (Hu, et al., 2006). Magnification bars: Control, C:II-2, LTBP4−/−: 500 nm; LTBP4S−/−: 200 nm; FBLN5−/− 1000 nm.
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