Jennifer West

Overexpression of Transforming Growth Factor-β Is Associated With Increased Hyaluronan Content and Impairment of Repair in Marfan Syndrome Aortic Aneurysm

Background— Marfan syndrome (MFS), a condition caused by fibrillin-1 gene mutation is associated with aortic aneurysm that shows elastic lamellae disruption, accumulation of glycosaminoglycans, and vascular smooth muscle cell (VSMC) apoptosis with minimal inflammatory response. We examined aneurysm tissue and cultured cells for expression of transforming growth factor-beta1 to -beta3 (TGFβ1 to 3), hyaluronan content, apoptosis, markers of cell migration, and infiltration of vascular progenitor cells (CD34). Methods and Results— MFS aortic aneurysm (6 males, 5 females; age 8 to 78 years) and normal aorta (5 males, 3 females; age 22 to 56 years) were used. Immunohistochemistry showed increased expression of TGFβ1 to 3, hyaluronan, and CD34-positive microcapillaries in MFS aneurysm compared with control. There was increased expression of TGFβ1 to 3 and hyaluronan in MFS cultured VSMCs, adventitial fibroblasts (AF), and skin fibroblasts (SF). Apoptosis was increased in MFS (VSMC: mean cell loss in MFS 29%, n of subjects=5, versus control 8%, n=3, P<0.05; AF: 28%, n=5 versus 7%, n=5, P<0.05; SF: 29%, n=3 versus 4%, n=3, not significant). In MFS, there was a 2-fold increase in adventitial microcapillaries containing CD34-positive cells compared with control tissue. Scratch wound assay showed absence of CD44, MT1-MMP, and β-3 integrin at the leading edge of migration in MFS indicating altered directional migration. Western blot showed increased expression of TGFβ1 to 3 in MFS but no change in expression of CD44, MT1-MMP, or β-3 integrin compared with controls. Conclusions— There was overexpression of TGF-β in MFS associated with altered hyaluronan synthesis, increased apoptosis, impaired progenitor cell recruitment, and abnormal directional migration. These factors limit tissue repair and are likely to contribute to aneurysm development.

Angiotensin-converting enzyme and regulation of blood pressure in a large Australian family

1. Animal studies have implicated the angiotensin‐converting enzyme (ACE) gene as an inherited risk factor contributing towards elevation of blood pressure.

An integrated approach to management of Marfan syndrome caused by an FBN1 exon 18 mutation in an Australian Aboriginal family

To the Editor: Marfan syndrome (MFS) is a severe autosomal dominant trait affecting the cardiovascular system, eyes, skeleton, dura, lungs, skin, and integument (1), caused by mutations of the fibrillin 1 gene (FBN1) on chromosome 15 (2). Death frequently results from dissection or rupture of the ascending aorta. Patients may have subluxation of the lens, usually, bilaterally. Skeletal manifestations include long arms and legs, long fingers, scoliosis, and pectus deformities. Other complications include lumbosacral dural ectasia and susceptibility to pneumothorax, herniae, and striae. The prevalence of MFS may be as much as one in 3–5000 (1). The penetrance of MFS appears high, but there is extensive phenotypic variability among affected individuals, even within families (2, 3). We have studied a large family of European and Australian Aboriginal origin. Sixty-two individuals at risk of MFS have been examined clinically since 1993. As shown in Fig. 1a, 18 family members had no features of MFS. Seventeen people independently met the criteria for a diagnosis of MFS (1), and 13 met these criteria when family history was taken into account, including two (IV3 and IV8) without lens subluxation. Three children (V18, V29, and V33) had lens subluxation but no other signs of MFS. Several individuals had involvement of one or two systems. None of these could be given a diagnosis of MFS. Patients could not be assessed for the major criterion lumbosacral dural ectasia (4, 5), because of the lack of imaging facilities in the remote area where they were examined. A positive result for this test would permit a diagnosis of MFS in five individuals, three with subluxated lenses (V18, V29, and V33), one with skeletal and ocular involvement (V1), and one with a dilated aorta and skeletal involvement (V4). In V3, the presence of dural ectasia would be suggestive but not conclusive. Immunohistochemical staining of fibrillin (6) in fibroblast cultures (established from punch biopsies of forearm or abdomen skin) of four affected family members (IV3, IV6, IV7, and IV21) revealed significant abnormalities (Fig. 1b). In control fibroblasts, most of the fibrillin was located in the extracellular area. The fibres were long, smooth, and fine in appearance. In the four affected family members examined, most of the fibrillin was found within the cells. The limited amount of fibrillin in the extracellular matrix was disorganized and appeared clumped rather than fibrous. DNA was extracted from blood or cheek cells (7). Polymerase chain reaction amplification of DNA was performed according to the protocol of Research Genetics (Huntsville, AL). Microsatellites were scored independently by two observers blind to the diagnosis. A LOD score (8) of 3.21 at y of 0 for the FBN1 microsatellite marker (9) and 6.36 at y of 0 for marker CYP-19 (10) indicated that the disease was closely linked to or at the FBN1 gene. As shown in Table 1, affected family members were found to carry the FBN1 2 allele and the CYP-19 178 bp allele. Twenty-one exons of the FBN1 gene were examined for DNA variants by single-strand conformation (SSC) analysis (11, 12). A variant was found using DNA from exon 18 of IV20 and was present in all tested affected individuals but not in healthy relatives, unrelated spouses, or 34 unrelated MFS patients (Table 1). Exon 18 was amplified with unlabeled primers to obtain material for direct automated sequencing using a kit (Applied Biosystems Inc., Foster City, CA). Sequencing was performed with both the sense and antisense amplification primers. Exon 18 DNA of IV20 showed a transition that replaced adenine with guanine at nucleotide position 2262. This mutation converts tyrosine to cysteine at position 754 in the amino acid sequence (Y754C). Six other family members, who met the revised diagnostic Clin Genet 2004: 65: 66–69 Copyright # Blackwell Munksgaard 2004 Printed inDenmark. All rights reserved CLINICALGENETICS

Recent developments in the diagnosis of Marfan syndrome and related disorders

Marfan syndrome is a multisystem disorder of connective tissue that is inherited in an autosomal dominant fashion, and results from mutation of the FBN1 gene on human chromosome 15. There are a number of conditions of the connective tissue with a similar phenotype that can be confused with Marfan syndrome. Modifications of the diagnostic criteria have recently been published, facilitating the differentiation of Marfan syndrome from these conditions. It is still difficult to use modern genetic testing for diagnosis because Marfan syndrome can be caused by many different mutations in FBN1, a large gene with 65 coding segments, while mutations in other genes can cause overlapping phenotypes. Several clinical trials of drug therapy, including the antihypertensive drug losartan, are in progress.

Association analysis of six candidate genes in a sample of Australian hypertensive patients.

1. Essential hypertension is characterized by increased vascular resistance due to narrowing of the small arterioles. This may be influenced by vasoactive substances, cell growth and vascular remodelling.