O10 Reduced expression of <i>PPOX</i> , the variegate porphyria gene, causes intrinsic pathological changes to keratinocytes

Abstract Introduction and aims Variegate porphyria, caused by monoallelic mutations in protoporphyrinogen oxidase (PPOX), causes acute visceral attacks and skin photosensitivity, presenting in blistering and skin fragility. A more severe phenotype is seen with rare biallelic mutations. Fragility is due to accumulation of intermediate porphyrins from the liver, such as 5′-aminolaevulinic acid (ALA), close to the skin surface that are oxidized by sunlight and cause oxidative stress. We hypothesize that PPOX deficiency causes intrinsic pathological changes to keratinocytes that contribute to this skin phenotype. Method Short hairpin RNA was used to create a model of monoallelic (KD1) and of biallelic hypofunctional variants (KD2). A porphyria-like environment was then induced using ALA and/or an iron chelator. Porphyrin accumulation was measured using intrinsic fluorescent measurement and differentiation was assessed in monolayer and by using three-dimensional (3D) organotypics. Results Both KD lines displayed reduced PPOX expression (KD1, 50%; KD2, 25%) and significantly reduced proliferation with KD2 displaying reduced migration. Induction of a porphyria-like environment caused accumulation of intermediate porphyrins in both KDs, with significantly more in KD2, and intracellular oxidative stress, evidenced by reduced expression of electron transport complexes and altered glutathione/glutathione disulfide ratios. KD clones displayed reduced differentiation, proportional to KD, and this was replicated by 3D organotypic cultures, while KD2 models also had reduced stratification. Treatment of 3D organoids with ALA further reduced differentiation and stratification in KD models. Discussion In conclusion, we show that keratinocytes have an active haem synthetic pathway and contribute to porphyria pathology causing significant changes in keratinocyte proliferation, differentiation, migration and reducing epidermal thickness, which could facilitate the oxidation of porphyrins under the skin. The more severe effects seen with &amp;gt; 75% KD reflects the phenotype seen in individuals with biallelic mutations in PPOX. These 3D organotypics could therefore serve as models to trial therapeutic interventions to improve patient skincare.