CHAPTER 1. BACKGROUND AND SIGNIFICANCE
1.1 Ehlers-Danlos Syndrome Type IV.
1.1.1 EDS: A Connective Tissue Disorder. Ehlers-Danlos Syndrome (EDS) is a connective tissue disorder in which mutations in a protein found within the tissue matrix, called collagen, has been identified as the primary cause of the disease. EDS Type IV (EDS IV) is the most severe form of EDS and is a dominantly heritable connective tissue disorder in which the gene for type three collagen (COL3 A1) is often mutated. Patients with the disorder commonly exhibit fragility in the skin, blood vessels, and viscera, with loose and hyperextensibile joints and skin (Figure 1). These observations classically define the disorder, yet current opinion suggests that collagen mutations may cause more common diseases related to modest phenotypes that are difficult to diagnose or associate with a malfunction in collagen biosynthesis. Patients with EDS often experience abnormal rupturing of internal arteries and hollow organs that sometimes leads to sudden death. The rupturing of internal organs is characteristic of tissue that is weak, and it is suspected that the destabilization of the extra cellular matrix (ECM) by malformed COL3 (COL3A1) is the cause (Thakker-Varia, Anderson et al. 1995; Meldon, Brady et al. 1996; Schwarze, Schievink et al. 2001).
Here we describe the investigation of fibroblasts harvested from a subgroup of EDSIV patients that have collagen point mutations at sites known to be important during the biosynthesis of collagen, called splice-site mutations. Our investigation is focused primarily on the biological process of splicing in an attempt to understand why, under defined temperature ranges, the product of splicing is altered. It has been reported that cells carrying this kind of mutation can be rescued at lower temperatures and it is hypothesized that the availability of protein components of the splicing machinery is responsible for this phenomenon (Wu, Kuivaniemi et al. 1993). Microarray technology was employed to assess the levels and changes in gene expression of fibroblasts grown in culture at 31°, 37°, and 42° C. The study was performed with fibroblasts from a subgroup of EDS patients that have mutations in their COL3A1 gene at intron/exon boundaries, the region that contains splice- control sequences and are sensitive to temperature change. Disruption of normal splicing leads to disruption of the patient's connective tissue and inevitably to their disease phenotype. All patients in this study have point mutations in sequences involved in splice-site selection, and some show temperature-dependent splicing patterns. By investigating the pattern of gene expression from these patient cells in conjunction with temperature change, it may be possible to extend what is known about temperature sensitivity to splice-site selection, and possibly provide greater insight into the nature of the EDS disorder.