Zsolt Urban

Mutations in a gene encoding an ABC transporter cause pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by calcification of elastic fibres in skin, arteries and retina that results in dermal lesions with associated laxity and loss of elasticity, arterial insufficiency and retinal haemorrhages leading to macular degeneration. PXE is usually found as a sporadic disorder, but examples of both autosomal recessive and autosomal dominant forms of PXE have been observed. Partial manifestations of the PXE phenotype have also been described in presumed carriers in PXE families. Linkage of both dominant and recessive forms of PXE to a 5-cM domain on chromosome 16p13.1 has been reported (refs 8,9). We have refined this locus to an 820-kb region containing 6 candidate genes. Here we report the exclusion of five of these genes and the identification of the first mutations responsible for the development of PXE in a gene encoding a protein associated with multidrug resistance (ABCC6).

Impaired glycosylation and cutis laxa caused by mutations in the vesicular H + -ATPase subunit ATP6V0A2

We identified loss-of-function mutations in ATP6V0A2, encoding the a2 subunit of the V-type H+ ATPase, in several families with autosomal recessive cutis laxa type II or wrinkly skin syndrome. The mutations result in abnormal glycosylation of serum proteins (CDG-II) and cause an impairment of Golgi trafficking in fibroblasts from affected individuals. These results indicate that the a2 subunit of the proton pump has an important role in Golgi function.

Fibulin-4: A Novel Gene for an Autosomal Recessive Cutis Laxa Syndrome

Cutis laxa is a condition characterized by redundant, pendulous, and inelastic skin. We identified a patient with recessive inheritance of a missense mutation (169G-->A; E57K) in the Fibulin-4 gene. She had multiple bone fractures at birth and was diagnosed with cutis laxa, vascular tortuosity, ascending aortic aneurysm, developmental emphysema, inguinal and diaphragmatic hernia, joint laxity, and pectus excavatum by age 2 years. Her skin showed markedly underdeveloped elastic fibers, and the extracellular matrix laid down by her skin fibroblasts contained dramatically reduced amounts of fibulin-4. We conclude that fibulin-4 is necessary for elastic fiber formation and connective tissue development.

Genetic disorders of the elastic fiber system

Over the last decade, a considerable amount of new information has emerged describing the protein components of elastic fibers. It is now evident that elastic fibers are complex extracellular matrix polymers, composed of at least 19 different proteins that comprise both the microfibrillar and the amorphous components of elastic fibers. Mutations in three of the genes encoding the most abundant of these elastic fiber proteins result in a broad spectrum of elastic tissue phenotypes, ranging from skeletal and skin abnormalities to vascular and ocular defects. The following disorders will be discussed in this review: supravalvular aortic stenosis; Williams-Beuren syndrome; cutis laxa; Marfan syndrome; ectopia lentis; familial thoracic aortic aneurysms and dissections; MASS syndrome; isolated skeletal features of Marfan syndrome; Shprintzen-Goldberg syndrome; and congenital contractural arachnodactyly.

Connection between Elastin Haploinsufficiency and Increased Cell Proliferation in Patients with Supravalvular Aortic Stenosis and Williams-Beuren Syndrome

To elucidate the pathomechanism leading to obstructive vascular disease in patients with elastin deficiency, we compared both elastogenesis and proliferation rate of cultured aortic smooth-muscle cells (SMCs) and skin fibroblasts from five healthy control subjects, four patients with isolated supravalvular aortic stenosis (SVAS), and five patients with Williams-Beuren syndrome (WBS). Mutations were determined in each patient with SVAS and in each patient with WBS. Three mutations found in patients with SVAS were shown to result in null alleles. RNA blot hybridization, immunostaining, and metabolic labeling experiments demonstrated that SVAS cells and WBS cells have reduced elastin mRNA levels and that they consequently deposit low amounts of insoluble elastin. Although SVAS cells laid down approximately 50% of the elastin made by normal cells, WBS cells deposited only 15% of the elastin made by normal cells. The observed difference in elastin-gene expression was not caused by a difference in the stability of elastin mRNA in SVAS cells compared with WBS cells, but it did indicate that gene-interaction effects may contribute to the complex phenotype observed in patients with WBS. Abnormally low levels of elastin deposition in SVAS cells and in WBS cells were found to coincide with an increase in proliferation rate, which could be reversed by addition of exogenous insoluble elastin. We conclude that insoluble elastin is an important regulator of cellular proliferation. Thus, the reduced net deposition of insoluble elastin in arterial walls of patients with either SVAS or WBS leads to the increased proliferation of arterial SMCs. This results in the formation of multilayer thickening of the tunica media of large arteries and, consequently, in the development of hyperplastic intimal lesions leading to segmental arterial occlusion.

Mechanisms and treatment of cardiovascular disease in Williams-Beuren syndrome

Williams-Beuren syndrome (WBS) is a microdeletion disorder caused by heterozygous loss of approximately 1.5-Mb pairs of DNA from chromosome 7. Patients with WBS have a characteristic constellation of medical and cognitive findings, with a hallmark feature of generalized arteriopathy presenting as stenoses of elastic arteries and hypertension. Human and mouse studies establish that defects in the elastin gene, leading to elastin haploinsufficiency, underlie the arteriopathy. In this review we describe potential links between elastin expression and arteriopathy, possible explanations for disease variability, and current treatment options and their limitations, and we propose several new directions for the development of nonsurgical preventative therapies based on insights from elastin biology.

Aortic aneurysmal disease and cutis laxa caused by defects in the elastin gene

Background: Cutis laxa is an acquired or inherited condition characterized by redundant, pendulous and inelastic skin. Autosomal dominant cutis laxa has been described as a benign disease with minor systemic involvement. Objective: To report a family with autosomal dominant cutis laxa and a young girl with sporadic cutis laxa, both with variable expression of an aortic aneurysmal phenotype ranging from mild dilatation to severe aneurysm or aortic rupture. Methods and results: Histological evaluation of aortic aneurysmal specimens indicated classical hallmarks of medial degeneration, paucity of elastic fibres, and an absence of inflammatory or atherosclerotic lesions. Electron microscopy showed extracellular elastin deposits lacking microfibrillar elements. Direct sequencing of genomic amplimers detected defects in exon 30 of the elastin gene in affected individuals, but did not in 121 normal controls. The expression of mutant elastin mRNA forms was demonstrated by reverse transcriptase polymerase chain reaction analysis of cutis laxa fibroblasts. These mRNAs coded for multiple mutant tropoelastins, including C-terminally truncated and extended forms as well as for molecules lacking the constitutive exon 30. Conclusions: ELN mutations may cause severe aortic disease in patients with cutis laxa. Thus regular cardiac monitoring is necessary in this disease to avert fatal aortic rupture.

Mutations in LTBP4 Cause a Syndrome of Impaired Pulmonary, Gastrointestinal, Genitourinary, Musculoskeletal, and Dermal Development

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.

Loss-of-function mutations in ATP6V0A2 impair vesicular trafficking, tropoelastin secretion and cell survival

Autosomal recessive cutis laxa type 2 (ARCL2), a syndrome of growth and developmental delay and redundant, inelastic skin, is caused by mutations in the a2 subunit of the vesicular ATPase H+-pump (ATP6V0A2). The goal of this study was to define the disease mechanisms that lead to connective tissue lesions in ARCL2. In a new cohort of 17 patients, DNA sequencing of ATP6V0A2 detected either homozygous or compound heterozygous mutations. Considerable allelic and phenotypic heterogeneity was observed, with a missense mutation of a moderately conserved residue p.P87L leading to unusually mild disease. Abnormal N- and/or mucin type O-glycosylation was observed in all patients tested. Premature stop codon mutations led to decreased ATP6V0A2 mRNA levels by destabilizing the mutant mRNA via the nonsense-mediated decay pathway. Loss of ATP6V0A2 either by siRNA knockdown or in ARCL2 cells resulted in distended Golgi cisternae, accumulation of abnormal lysosomes and multivesicular bodies. Immunostaining of ARCL2 cells showed the accumulation of tropoelastin (TE) in the Golgi and in large, abnormal intracellular and extracellular aggregates. Pulse-chase studies confirmed impaired secretion and increased intracellular retention of TE, and insoluble elastin assays showed significantly reduced extracellular deposition of mature elastin. Fibrillin-1 microfibril assembly and secreted lysyl oxidase activity were normal in ARCL2 cells. TUNEL staining demonstrated increased rates of apoptosis in ARCL2 cell cultures. We conclude that loss-of-function mutations in ATP6V0A2 lead to TE aggregation in the Golgi, impaired clearance of TE aggregates and increased apoptosis of elastogenic cells.

Cutis laxa: A review

Cutis laxa is a rare disorder of elastic tissue resulting in loose, redundant, hypoelastic skin. Both acquired and inherited forms exist, some of which have significant systemic manifestations. Here, we review the various forms of cutis laxa, with focus on the inherited forms. Recent molecular studies have provided many new insights into the causes of cutis laxa and revealed greater genetic heterogeneity than previously appreciated.