Modelling porokeratosis to identify therapeutic targets

Porokeratosis, a genodermatosis caused by variants in mevalonate pathway enzymes, poses challenges in treating severe phenotypes despite various symptomatic and cholesterol-statin therapies due to an incompletely understood pathogenic mechanism. We conducted RNA sequencing on a linear porokeratosis patient with hyperkeratosis, severe inflammation lesions. Dysregulated genes involved like the mevalonate pathway, cholesterol metabolism, and keratinocyte differentiation. The inflammatory profile resembled psoriasis, skewing the T-helper 17 cell pathway. Immunohistochemical staining confirmed high interleukin-17A expression in lesions from the patient and eight other porokeratosis cases. An anti-interleukin-17A treatment with Secukinumab, showed significant therapeutic efficacy, offering a promising option for porokeratosis with severe inflammation and hyperkeratosis in porokeratosis. We generated and characterized two stable PMVK-deficient keratinocytes cell lines to establish vitro models: one lacked activity of PMVK, derived from the patient lesions, while the other exhibited strongly reduced amounts of PMVK achieved through lentivirus shRNA knockdown in normal keratinocytes. The PMVK-deficient severely impacts keratinocytes viability, resulting in elongation over increasing passages, inhibited cell proliferation, and an increase in mRNA expression of the CARF, interleukin-6 and interleukin-8. The abnormal cholesterol metabolism was evident not only in gene expression studies but also visualized through filipin staining, demonstrating aberrant cholesterol/precursor accumulation within the cornoid lamellae area of the patient's lesions. In PMVK-deficient keratinocytes, cholesterol levels exhibited uneven distribution between cells, with higher content compared to normal cells. Additionally, gene expression studies prominently displayed the dysregulation of keratinocyte differentiation. In both PMVK-deficient keratinocytes and calcium-induced differentiated PMVK-deficient keratinocytes, the mRNA expression of differentiation markers, including involucrin and keratin 10, was lower compared to normal controls. We confirmed that the PMVK R138* nonsense mutation leads to mRNA degradation, contrasting the R110Q missense mutation, potentially leading to proteins, accumulation, and additional stress. Our research highlighted the gene expression profile of porokeratosis and the critical role of Interleukin-17A in inflammation, demonstrating its effectiveness in anti-Interleukin-17A treatment. We established two stable in vitro models, mimicking in vivo abnormal physiological processes, and revealed the correlation between mutation types and severity, laying a solid foundation for precision therapies.