Collagen XII in normal and unclassified Ehlers-Danlos Syndrome skin fibroblasts

Ehlers-Danlos Syndrome (EDS) is an inheritable disorder that affects collagenous extracellular matrix. It is divided into 6-9 classifications based on phenotype and/or gene abnormality. Patients with the unclassified form of EDS present with generalized periodontitis in addition the more characteristic joint and skin abnormalities. Collagen type XII is a member of the Fibril Associated Collagens with Interrupted Triple-helices (FACIT) subfamily of collagens, and has a distribution that is coincident with the aforementioned clinical abnormalities. The type XII protein has previously been demonstrated, by Western blot, to have a higher electrophoretic mobility in patients with the unclassified form of Ehlers-Danlos Syndrome when compared to controls. Objectives: The purpose of this study was to evaluate the 5’ end of the cDNA to determine whether or not the apparent decrease in size of the type XII was due to an alternative splicing and/or translation start event. Methods: Tissue cultures of dermal fibroblasts from a patient with unclassified EDS and an age and sex-matched control were grown to confluence in alpha-modified MEM supplemented with 10% FCS, ascorbate and antibiotic/antimycotic. The cells were trypsinized and RNA extracted. The RNA was subjected to 5’ RACE utilizing a type XII-specific anti-sense primer, and the resulting cDNA’s were cloned into the TOPO® cloning vector. Colonies were picked, grown in small scale and mini-prepped to isolate DNA. Several clones from each group were sent for automated sequencing and the sequences compared. Results: Although there were variations in the size of the 5’ untranslated region, consistent with previous studies, there were no apparent differences in the open reading frames between the experimental and control groups in the region under investigation. Conclusions: This supports the hypothesis that the defect in the type XII collagen molecule in unclassified EDS does not reside within the 5’ coding region of type XII collagen.