A novel variant in tenascin-X may be associated with an Ehlers Danlos phenotype in patients with congenital adrenal hyperplasia. R. Morissette1,3, W. Chen2, Z. Xu3, J. Dreiling4, M. Quezado4, N. McDonnell3, D. Merke1,5 1) Clinical Center, The National Institutes of Health, Bethesda, MD, USA; 2) PreventionGenetics, Marshfield, WI, USA; 3) Laboratory of Clinical Investigation, The National Institute on Aging, The National Institutes of Health, Baltimore, MD; 4) Laboratory of Pathology, The National Cancer Institute, The National Institutes of Health, Bethesda, MD, USA; 5) The Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA.

   Background: Mutations in CYP21A2 result in congenital adrenal hyperplasia (CAH) due to 21-hydoxylase deficiency. Flanking CYP21A2 is TNXB, the gene encoding tenascin-X (TNX), an extracellular matrix glycoprotein that is highly expressed in connective tissue and regulates collagen fibrillogenesis and matrix maturation. TNX deficiency is a known cause of Ehlers Danlos syndrome (EDS), a connective tissue dysplasia. We previously reported that approximately 7% of CAH patients have haploinsufficiency for a contiguous deletion of CYP21A2 and TNXB resulting in CAH-X syndrome with characteristic clinical features of a connective tissue dysplasia, such as joint laxity, cardiac valvular abnormalities, and bifid uvula. In 10 of 330 patients, evidence of a CAH-X phenotype was found; however, a TNXB deletion was not identified. Therefore, we sought to determine the cause of the CAH-X phenotype in these CAH patients. Methods: Dermal fibroblasts and direct tissue from patients and controls were used for biochemical experiments. Sanger sequencing, western blotting, and immunohistochemistry are being used to investigate potential defects in TNX. Immunohistochemical analysis of elastin, fibrillin, and overall extracellular matrix organization in dermal tissue from patients and controls is ongoing. Results: We identified a potentially pathogenic variant c.12174C>G (p.Cys4058Trp) in TNXB in five CAH patients with an EDS phenotype. p.Cys4058Trp is in the fibrinogen, alpha/beta/gamma chain, and C-terminal globular domain of TNX. This variant currently is not found in the 1000 Genomes Project or dbSNP database. PolyPhen-2 software predicted that the variant is probably damaging. Software modeling showed that mutating this highly conserved cysteine disrupts a disulfide bond and likely results in protein misfolding. Western blot analysis in fibroblasts showed that this missense variant does not alter TNX expression compared to controls as expected. Immunohistochemical analysis is underway. Conclusion: A novel variant of a highly conserved cysteine in TNX is likely responsible for at least some of the connective tissue phenotypes found in patients with CAH. Patients with CAH due to 21-hydroxylase deficiency are at risk for also having a connective tissue dysplasia due to TNX deficiency. In addition to the known contiguous gene deletion, other types of TNXB genetic mutations may commonly occur in CAH patients.

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