CHARACTERIZATION OF THE FUNCTIONAL ROLE OF NEW VARIANTS INVOLVED IN VARIEGATE PORPHYRIA AND HIPSC DERIVED HEPATOCYTE LIKE CELLS TO MODEL HEPATIC PORPHYRIAS

Porphyrias are a group of inherited metabolic disorders of heme biosynthesis. Each porphyria derives from an alteration in the heme biosynthetic pathway resulting in a specific accumulation of heme precursors. These rare diseases are characterized by an extended heterogeneity of mutations affecting the coding region of the genes directly or indirectly involved in the pathway. In this study, we assessed three new variants identified in the regulatory regions of the PPOX gene using human hepatocarcinoma (Hep3B) and erythroleukemia (K562) cell lines. We demonstrated a lower expression of the PPOX gene through luciferase assays and RNA analysis for the c.1-883G>C promoter mutant and we suggested a post-transcriptional role for the c.1-413G>T and c.1-176G>A variants in the 5′ UTR of PPOX mRNA variant 2. Transfection experiments of mutant -413T reporter plasmid indicate that this variant inhibits translation of the downstream firefly luciferase mRNA. In fact, the reduced firefly luciferase activity did not correlate with the proportional reduction in firefly luciferase mRNA expression. Normal values of PPOX mRNA level validated with Digital PCR in the patient carrying the c.1-413G>T substitution support this evidence. Data for the c.1-176G>A variant show a possible role in the spicing regulation for it. The qualitative RNA analysis confirms that this variant is involved in the alteration of the normal splicing between exon 1 and exon 2 of PPOX due to a 4 bp deletions in exon 1. The relation between these post-transcriptional alterations and the variegate porphyria remains to be investigated. These results suggest that the regulatory regions have to be considered in the diagnostic process but more studies are required to clarify their role in the disease. To model hepatic porphyrias, we derived hepatocyte-like cells from hiPSC generated from blood of two patients affected by acute intermittent porphyria, a hepatic porphyria. We hypothesized that this can serve as an in vitro model to study different pathways linked with acute intermittent porphyria. The data suggest a general comparable profile of the heme biosynthec pathway between hiPSC-HLC and primary hepatocytes. Although some immature features, probably linked with the in vitro condition, could directly or indirectly affect the metabolism network links with hepatic porphyrias, now hiPSC-HLCs are one of the most useful model available for hepatic diseases. The patient derived hepatocyte-like cells showed a basal overexpression of ALAS1 and its regulator PPARA and PGC1A with induction of oxidative damage response genes. Moreover, the induction of ketogenetic and lipid metabolic genes highlighted a different metabolic profile in the acute intermittent porphyria patient lines. The in vitro simulation of acute attack mediated by ALA administration, one of the principal compound accumulated during the attack, showed a negative feedback regulation on ALAS1 with massive induction of HMOX1 in the control cell line. On the other hand, phenobarbital response in control line suggested the link between cholesterol synthesis and gluconeogenesis with the heme biosynthetic pathway. The drug metabolism in fact, induces the heme biosynthetic pathway through ALAS1, and this gene results constantly overexpressed in the derived patient cell lines. In conclusion, this study focus the attention on the importance of the regulatory regions in the diagnostic process of porphyrias and supply an alternative in vitro model to study the metabolic alterations linked with porphyrias. Further experiments are required to better understand the direct effect of alteration in the regulatory regions of porphyria genes and are necessary to analyse the involvement of different pathways in the onset of the disease.