261 Assessment of readthrough of premature termination codons in xeroderma pigmentosum group C patients by real-time PCR and immunohistochemistry

Abstract

Xeroderma pigmentosum (XP) is a rare autosomal recessive DNA repair disorder caused by mutations in 8 genes, XP-A to XP-G, and Variant with severe sun sensitivity and10,000-fold increased risk of skin cancer. XP-C is the most common type in the U.S. About 12% of XP-C patients have premature termination codon (PTC) mutations that can lead to XPC protein elongation arrest and XPC mRNA template degradation by nonsense mediated decay. Our previous studies have shown treatment with aminoglycosides promote readthrough of PTC in XP-C cells. Amlexanox, an anti-inflammatory, antiallergic and immunomodulator, was reported to increase readthrough in human cells with PTC associated diseases. We treated parallel pairs of fibroblasts and lymphoblasts derived from two unrelated XP-C patients along with 2 normal control cells with amlexanox and/or previously tested aminoglycosides. Levels of XPC mRNA increased in XPC fibroblasts exposed to both G418 and amlexanox to a different extent in different cells. We are also developing a more direct assessment of level of XPC protein in human skin by use of immunohistochemistry with XPC antibodies. Two mm skin punch biopsies are stained with IHC and assessed microscopically. The slides are then scanned and the intensity of the staining is assessed by computer algorithm. In skin from normal donors XPC staining was prominent in the nuclei of epidermal cells and sweat glands in the dermis. In contrast, only background XPC staining was observed in cells from XPC patients. These preclinical tests are a step towards precision medicine that should assist patient selection and determining which drug combination would be optimal to increase XPC function for each patient.