Aneurysm and dissection are major diseases of the aorta and are often asymptomatic until a life-threatening event like ischemic organ damage or rupture occurs. Marfan Syndrome (MFS) is a diverse yet clinically recognized subgroup of people at risk for aneurysm, including dissecting aneurysm, and constitutes a significant fraction (estimated at 5–7.5% 12) of all individuals with ascending and thoracic aortic aneurysmal disease. MFS incidence is estimated to be 1 in 5–10,000 3. Our long-term goal is to develop an assay that will identify people at risk for aneurysm before the disease process has reached an advanced state. This report is a small step in that direction.

In this study, we focus on individuals diagnosed with Marfan syndrome. The prevalence of MFS combined with its clinical recognition makes it an excellent model system for studies on aneurysmal disease. MFS is an autosomal dominant heritable disorder caused by mutations in the fibrillin-1 (FBN1) gene 45, with more than 500 unique mutations identified 6. FBN1 mutations show a high degree of penetrance but considerable inter- and intra-familial variability in their phenotype 3. The variable penetrance suggests that environmental factors and/or disease modifying genes also contribute to the phenotype. Neonatal MFS correlates to mutations within exons 24–32 and MFS defined by mutations in exons 59–65 carry a reduced risk of aortic pathology. Large-scale comparisons between MFS individuals with premature termination mutations and cysteine substitutions in FBN1 revealed significant differences in ocular, skeletal and hypermobility features but no difference in the frequency of ascending aortic aneurysm 78. Apart from these observations, determining the nature of the mutation (a time and labor intensive process) does not improve prediction of the severity of the disease, the risk of aneurysm development or of its progression 79. These limited genotype-phenotype correlations suggest that genes other than FBN1 may significantly influence the phenotype, and their identification may lead to a more informative test of risk.

Fibroblasts are not smooth muscle cells. However, in culture they display a stable phenotype with stress fibers composed of cytoplasmic actins and a splice variant of cellular fibronectin 10. The increased mechanical stress on dermal fibroblasts seeded at low density produces a cell culture population consisting of 70–80% myofibroblasts. The term "myofibroblast" was proposed over 30 years ago to describe the fibroblasts that appeared in granulation tissue at the sight of open wounds 11. Recently, it has been recognized that Thy-1 surface expression defines a subpopulation of fibroblasts capable of differentiating into myofibroblasts 12. We can detect Thy-1 expression in both affected and unaffected skin fibroblasts by array and have confirmed that observation by quantitative real time polymerase chain reaction (qRT-PCR, data not shown). Thus, the skin cultures we used were "myofibroblast" like.

In the last several years, use of DNA microarrays to analyze gene expression has emerged as a promising technology for disease classification and prognosis and for identification of genes that could be potential causes, bio-markers or drug targets 13141415161718. However, there are limits to the sensitivity of microarrays for detecting genes expressed at low levels as well as additional confounding problems associated with arrays 1920212223. Consequently, in common with most recent studies 2024, we go beyond mere classification by independently validating expression levels using quantitative qRT-PCR and validating the results in a second population.

In the present study, we used total RNA in oligo dT primed cDNA reactions to identify an expression phenotype associated with the MFS genotype in cultured skin fibroblasts. Our results show a clearly recognizable expression phenotype in cultured fibroblasts. We of course do not expect exactly the same expression phenotype in aortic smooth muscle cells, but we do expect some overlap in the perturbed pathways, as they share the same root cause. Some of the identified genes, including elastin and several collagens, are obvious targets for roles in the development and maintenance of the extracellular matrix environment and cell-matrix contacts. Additional genes validated by qRT-PCR analysis, including the vitamin D receptor, programmed cell death-10 and the LIM domain only 7, may represent genes that will provide new insight into the disease process. Our ability to detect a MFS expression phenotype in cultured fibroblasts provides both a simple method for large-scale screening and a basis for mechanistic studies of the genes identified as differentially expressed.

An overview of our experimental design is shown in Figure 1. In brief, we analyzed total RNA from 36 subjects by spotted membrane DNA macroarray. We used Research Genetics spotted cDNA membrane arrays to analyze gene expression in fibroblast cultures, 17 with characterized mutations in the FBN1 gene (7 missense, all cysteine substitutions, 9 nonsense and 1 multi-exon deletion; 14 of 17 had an aortic phenotype) and 19 unaffected controls (details in Table 1). We selected differentially expressed genes between MFS and unaffected samples based on estimated False Discovery Rate (FDR)25. We calculated the 4132 p-values for the two-sample t-tests associated with the expression differences between affected and control for each gene on the array. At a q-value (estimated FDR) threshold of 0.001, 283 genes (265 with identifiable unique Unigene ID numbers) out of a total of 4132 are selected as differentially expressed in MFS vs. unaffected. The top ranking genes based on ratio are listed in Table 2 [see Additional File 1 for the entire list]. At the same q-value threshold, we found 175 differentially expressed genes (165 with unique Unigene ID) between missense and unaffected samples, and 111 differentially expressed genes (106 with unique Unigene ID) between nonsense and unaffected samples. The q-values are only approximate, since their validity depends on various assumptions such as normality of the expression levels within groups. To globally assess the reliability of these test results while making fewer statistical assumptions, we performed a permutation test (see Methods) to determine if the large number of significant genes we found could easily have arisen by chance. Of 50,000 random permutations for MFS and unaffected samples, no permutations have more than 283 differentially expressed genes, making the array results highly statistically significant, with an empirical p-value smaller than 0.00002. Furthermore, the only permutations exhibiting a substantial number of significant genes were those permutations that happened to separate most of the MFS subjects from most of the unaffected controls. Over forty five thousand permutations showed no significant genes, and the average number called significant was only 0.45, making it very likely that our estimated false discovery rate of 0.001 is conservative. [See Additional File 1 for the complete 4132-gene dataset.]