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Case Reports
. 2009 Nov;85(5):593-605.
doi: 10.1016/j.ajhg.2009.09.013. Epub 2009 Oct 15.

Mutations in LTBP4 cause a syndrome of impaired pulmonary, gastrointestinal, genitourinary, musculoskeletal, and dermal development

Zsolt Urban  1 , Vishwanathan Hucthagowder, Nura Schürmann, Vesna Todorovic, Lior Zilberberg, Jiwon Choi, Carla Sens, Chester W Brown, Robin D Clark, Kristen E Holland, Michael Marble, Lynn Y Sakai, Branka Dabovic, Daniel B Rifkin, Elaine C Davis
Affiliations
Case Reports

Mutations in LTBP4 cause a syndrome of impaired pulmonary, gastrointestinal, genitourinary, musculoskeletal, and dermal development

Zsolt Urban et al. Am J Hum Genet. 2009 Nov.

Abstract

We report recessive mutations in the gene for the latent transforming growth factor-beta binding protein 4 (LTBP4) in four unrelated patients with a human syndrome disrupting pulmonary, gastrointestinal, urinary, musculoskeletal, craniofacial, and dermal development. All patients had severe respiratory distress, with cystic and atelectatic changes in the lungs complicated by tracheomalacia and diaphragmatic hernia. Three of the four patients died of respiratory failure. Cardiovascular lesions were mild, limited to pulmonary artery stenosis and patent foramen ovale. Gastrointestinal malformations included diverticulosis, enlargement, tortuosity, and stenosis at various levels of the intestinal tract. The urinary tract was affected by diverticulosis and hydronephrosis. Joint laxity and low muscle tone contributed to musculoskeletal problems compounded by postnatal growth delay. Craniofacial features included microretrognathia, flat midface, receding forehead, and wide fontanelles. All patients had cutis laxa. Four of the five identified LTBP4 mutations led to premature termination of translation and destabilization of the LTBP4 mRNA. Impaired synthesis and lack of deposition of LTBP4 into the extracellular matrix (ECM) caused increased transforming growth factor-beta (TGF-beta) activity in cultured fibroblasts and defective elastic fiber assembly in all tissues affected by the disease. These molecular defects were associated with blocked alveolarization and airway collapse in the lung. Our results show that coupling of TGF-beta signaling and ECM assembly is essential for proper development and is achieved in multiple human organ systems by multifunctional proteins such as LTBP4.

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Figures

Figure 1
Figure 1
Clinical Manifestations of Patients with LTBP4 Mutations (A–E) Characteristics of patient 1 at 1 mo of age included (A and B) flattened midface with a wide nasal bridge, long philtrum, micrognathia, swollen eyelids, receding forehead and dolichocephaly, (C) redundant skin on the trunk and (D) extremities, and (E) widely spaced first and second toes with transverse plantar creases. (F) X-ray imaging demonstrated dilated intestines, laterally displaced liver, compressed chest, and inguinal hernia. (G) Upper gastrointestinal imaging highlighted esophageal tortuosity. (H) Contrast imaging of the bladder showed severe diverticulosis. (I–K) Clinical pictures of patient 2 at 2 mo of age showed (I) craniofacial features similar to patient 1, as well as (J and K) cutis laxa. (L) CT image at 3 mo showing hyperinflation of the anterior and atelectasis of the posterior lung. (M) X-ray image showing bell-shaped chest, atelectasis of the right lung, and hyperinflation of the left lung. (N) Severe bladder diverticulosis. (O and P) Clinical photographs of Patient 3 taken at 3 yrs 1 mo illustrate cutis laxa with premature aged appearance throughout the body, including (O) the legs and (P) the abdomen. The patient also had a gastrostomy tube and tracheostomy. (Q) Chest CT image of parasternal diaphragmatic hernia with cystic changes and atelectasis of the lung at 3 yrs 6 mo of age. (R) Upper gastrointestinal contrast image showing stomach diverticula at 3 yrs 6 mo of age. (S) Urinary tract contrast imaging showing bladder diverticula at 2 yrs 4 mo of age.
Figure 2
Figure 2
Mutations Found in LTBP4 (A) The parents of patient 1 were second cousins. (B) DNA sequencing showed that patient 1 (V.1) was homozygous for mutation p.Q1185fsX1211, whereas his unaffected father (Fa) and mother (Mo) were both heterozygous. (C) The parents of patient 2 were unaffected and unrelated. (D) Patient 2 (II.1) was compound heterozygous for mutation p.P264fsX300 inherited from his mother (Mo) and mutation p.C857X inherited from his father (Fa). (E) Patient 3 was an offspring of first-cousin parents. (F) Homozygous mutation p.C274G (arrowhead) in patient 3 (IV.1) compared to normal sequence in a control individual (Co). (G) The pedigree of patient 4. (H) Patient 4 (II.1) was compound heterozygous for mutations p.C857X and p.P1376fsX1403. Neither of these mutations was found in his mother (Mo), suggesting that at least one of the mutations was de novo. No DNA sample was available from the father. (I) The cysteine residue replaced by the mutation p.C274G found in patient 3 is highly conserved in all vertebrates. (J) Schematic representation of the domain structure of LTBP4, showing the location of the mutations.
Figure 3
Figure 3
Functional Consequences of LTBP4 Mutations In Vitro (A) Quantitative RT-PCR analysis showed severely reduced expression of LTBP4 in skin fibroblasts from three patients (1, 2, and 4) in comparison to three controls, triplicates each. Results were normalized to β-actin (ACTB) and expressed as group averages ± standard errors of means. The expression of TGF-β1 (TGFB1) was not altered significantly (NS) in patients. (B) Patient 1 (P1) expressed low levels of full-length (FL) LTBP4 in comparison to control (Co) fibroblasts. Cycloheximide (CHX) treatment (+) partially rescued the expression of the FL isoform. Del 28,29 shows the exon 28, 29 skipping product, HD indicates a heteroduplex. (C) Immunoblotting confirmed severely reduced LTBP4 levels in conditioned media of fibroblasts from patient 1 (P1). LTBP1 secretion was normal in both control (Co) and patient fibroblasts. (D) Dual immunostaining in patient 1 (P1) shows an abundant fibrillin-1 (FBN1, green) network but very little staining for LTBP4 (red). Control cells (Co) show strong colocalization of FBN1 and LTBP4 (yellow, orange). LTBP1 (green) and FBN1 (red) were strongly colocalized in both patient and control cells. Nuclei were counterstained with DAPI (blue). Magnification bars represent 50 μm. (E) Measurement of TGF-β signaling by coculturing of reporter cells with fibroblasts from patients 1, 2, and 4 (P, triplicates each) or two controls (Co, six replicates each). Negative controls (None, six replicates) were reporter cells only. The presence of TGF-β receptor inhibitor (TGFBR Inh) is shown by a + sign and absence by a − sign. Luminescence units were converted to TGF-β concentration with the use of a TGF-β1 standard curve. Significant difference is shown by a p value (t test) and error bars show standard errors of means.
Figure 4
Figure 4
Impaired Elastic Fiber and Tissue Architecture in Patients with LTBP4 Mutations (A) Hart's elastin stain of the skin from a 9-mo-old control (Co) shows robust, strongly stained horizontal elastic fibers in the deep dermis (arrowhead) and fine candelabra-like vertical fibers in the papillary dermis (arrow). (B and C) Skin sections from (B) patient 1 (P1) and (C) patient 2 (P2) lack elastic fibers in the papillary dermis and have fragmented, diffuse fibers in the deep dermis. (D) Lung sections from patient 2 showed dramatically enlarged (> 200 μm) saccular air spaces and rarefied elastic fibers confined mostly to the saccular wall (black arrowheads) or to arrested septal primordia (blue arrowheads). Capillaries were enlarged and had thickened walls (arrows). (E) Other areas of the lung contained collapsed air spaces (arrowhead) and airways filled with proteinaceous exudates (arrows). (F–J) Bronchi had (F) discontinuities (arrowheads) in the elastic sheet of the lamina propria and (G) fragmented elastic fibers in the submucosa. Aortic sections from patient 2 showed overall preserved vessel wall structure, but (H) discontinuities in elastic lamellae (arrowheads), (I) cystic degeneration of the media (arrowheads), and (J) fragmentation of the internal elastic lamina (arrowheads) were observed. (K) Electron microscopy of dermal elastic fibers from a 7-mo-old control (Co) showed robust insoluble elastin core (El) surrounded by a fine layer of microfibrils (Mf). (L) In patient 1 (2 mo old), dermal elastic fibers had a diminished elastin core (El), with abundant microfibrils (Mf) and globular elastin aggregates at the periphery of the fiber (Gl). (M) Ultrastructural abnormalities of pulmonary elastic fibers in Ltbp4S−/− mice were identical to those of patient 1. Magnification bars represent 20 μm (A–C, G, J); 200 μm (D, E, H, I); 100 μm (F); 0.5 μm (L); and 0.2 μm (K, M).
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