Defects in gene involved in heme biosynthesis: first molecular step for the development of potential salivary biomarkers correlated with porphyria.

Heme is an important molecule involved in several biological processes such as oxygen transport and its storage in myocytes as a component of the respiratory proteins haemoglobin and myoglobin. Moreover, heme is a component of various other haemoproteins such as cytochromes, catalase, peroxidase, tryptophan pyrroles, and nitric oxide synthase. Heme plays a role in the pathogenesis of diseases, in fact defects in any enzyme of the pathway may cause porphyric disorders. Porphyrias are a group of metabolic and genetic diseases characterized by a deficiency in the activity of specific enzymes in the heme pathway. The abnormal function of one of the eight enzymes involved in this pathway, has as a result eight different types of porphyria that lead to accumulation of pathway intermediates. Porphyrias are divided in acute porphyrias which included autosomal dominant porphyrias such as Acute Intermittent Porphyria (AIP), Variegated Porphyria (VP), Hereditary Coproporphyria (HCP) and a rare autosomal recessive porphyria 5-Aminolevulinc Acid Dehydratase deficiency Porphyria (ALADP); and non-acute or cutaneous porphyrias that included Congenital Erythropoietic Porphyria (CEP), Porphyria Cutanea Tarda (PCT), Erythropoietic Protoporphyria (EPP) and X-Linked Protoporphyria (XLP).In the first part of this thesis, we have conducted molecular studies on DNA extracted from saliva and blood samples obtained from Italian and Spaniard patients. The analysis conducted on the CPOX and FECH gene of an Italian patient affected by VP, revealed some polymorphisms not directly associated with the patient phenotype. Numerous nucleotides present in heterozygosity and an insertion of six nucleotides, (18505_6insTATTCT), were found in the 3’ UTR region of the CPOX gene, which is an important region for the half-life of the messenger. Analysis of the PPOX gene have shown frameshift mutations between exons 2 and 3 (c.5710_5711ins.CG, c.5714_5715ins.A, c.5720_5721ins.CT), which led on a stop signal in exon 4. Results obtained at Hospital Universitario 12 de Octubre (Madrid, Spain) confirmed two new mutations in Spaniard patients with AIP: Ivs11+3A>T in intron 11 and c.41_42ins.A in exon 3. In the second part we analyzed porphyrin intermediates in body biofluids (blood, urine and faeces) in patients with suspected porphyria. In particular, we quantified, by micro chromatography and spectrophotometry, the presence of these metabolites in patients diagnosed with porphyria. The results obtained showed that porphyrin precursor concentrations were particularly high in urine and faeces during acute porphyria attacks and also in blood in patients with cutaneous porphyrias when exposed to sunlight.In the third part we focused on the quantitative and qualitative proteomic study of proteins present in saliva of porphyric patients. Proteomics is a valid investigation tool in the search for biomarkers in specific pathologies. In patients with cutaneous porphyria skin lesions, caused by porphyrin precursors that are activated by exposure to the sun in parts of the body such as the face, affecting also the oral cavity, creating serious lesions to the oral mucosa. The accumulation of these precursors may also cause a brownish teeth discoloration. The salivary study of these patients aims to identify specific biomarkers for the diagnosis of different types of porphyria, through the use of bottom-up approach based on SDS-PAGE technique used for the separation of the proteins. After the separation of the proteins, followed the in-gel trypsin digestion and the characterization of proteins by high-resolution mass-spectrometry. We created 3 separate saliva pools for patients with porphyria, divided into: Pool 1 for IAP patients, Pool 2 for VP patients and Pool 3 for HCP patients, and 1 saliva pool from healthy subjects with the same sex-age ratio as patients and the same final concentration. Unfortunately, due to the Covid-19 emergency, the results are still being processed.