Erythropoietic protoporphyria (EPP) is a rare autosomal recessive disorder caused by the moderate to severe deficiency (<20% activity) of ferrochelatase (FECH). Homozygous or compound heterozygous mutations of the FECH gene have in fact been identified in a small number of patients ^[1][44]. In another 5–10% of cases, the defect is a gain-of-function mutation in the aminolevulinate synthase 2 (ALAS2) gene, which is encoded on the X chromosome and therefore determines the X-linked protoporphyria (XLEPP).

EPP is characterized by severe photo-toxicity induced by accumulation of PPIX in the skin, with development of redness, swelling and lesions in the affected areas and a systemic inflammatory response if the person remains exposed to the noxious agent (sunlight or strong light). This greatly impacts the quality of life of patients, who have been forced to avoid sunlight since infancy. Other potential major complications of EPP/XLP are the development of symptomatic gallstones and/or acute or chronic liver disease (i.e., protoporphyric hepatopathy) due to the accumulation of protoporphyrins in the liver. As for blood-test alterations, EPP patients frequently present with a microcytic, hypochromic anemia, which is consistent with a pattern of iron-deficiency ^[2][3][45,46] or impaired hemoglobin production.

There has long been debate on the role of iron supplementation in such patients. In fact, since iron is required for FECH activity and heme completion, it would be reasonable to suppose that iron supplementation would facilitate the deficient FECH activity, reduce the amount of accumulated PPIX and therefore ameliorate the phenotype in such patients. Indeed, in some case reports, iron supplementation has been shown to reduce PPIX levels in the erythrocytes and plasma of subjects with EPP ^[4][5][47,48]. However, its association with the exacerbation and worsening of symptoms has also been reported ^[6][7][49,50], likely due to the presence of a 5′-IRE in ALAS2 mRNA, which would be bound by IRP under conditions of iron scarcity or released, with consequently increased AlLAS2 levels and activity, under conditions of iron availability ^[8][51].

Furthermore, Barman-Aksozen et al. described three EPP patients (2 females and 1 male with FECH-related EPP) with decreased PPIX levels in red blood cells when they were in an iron-deficient status, with worsening microcytic, hypochromic anemia. They also documented a significant increase in ALAS2 mRNA and protein amount. To test the hypothesis of a connection between FECH deficiency and ALAS2 over-expression, FECH was inhibited in cultured cells and a subsequent increase in ALAS2 mRNA was shown, leading to the plausible conclusion that a deficiency in FECH leads to a secondary increase in ALAS2 expression ^[8][51]. Overall, these data do not support the indiscriminate regular iron administration to all patients with EPP but rather a tailored iron supplementation for iron-deficient patients, especially when anemic, with a biochemical and clinical surveillance for potential detrimental effects related to biosynthetic-intermediate accumulation.

Additionally, it can be hypothesized that the inflammatory state following the episodic cutaneous reactions could trigger hepcidin expression, leading to reduced circulatory-iron levels.

In a small case series of nine patients with EPP and one patient with XLP, Bossi et al. demonstrated a higher prevalence of iron deficiency with or without anemia (5/10 had low Ft levels (<25 ng/mL), 8/10 had low Tf saturation; 2/10 had low Hb and hematocrit per sex and age). The only female EPP subject proved to be iron deficient (serum ferritin = 4 ng/mL; transferrin saturation = 5%). The EPP patient with higher circulating iron also had classical hereditary hemochromatosis (HFE C282Y+/+). Serum and urine hepcidin levels were lower in subjects with EPP/XLP compared to healthy volunteers, and no clear relationship with serum ferritin was observed. More importantly, it was shown that intestinal adsorption of ferrous sulfate was not altered in EPP and XLP patients ^[9][52], excluding a strong inhibition of iron release form enterocytes by hepcidin and suggesting that other still-unknown factors must be in play to account for the iron-deficient phenotype. Furthermore, these preliminary data indicate that hepcidin levels are mainly driven by anemia and iron deficiency in EPP patients, which is in line with the concept that these two pathological conditions are hierarchically prevalent over inflammatory signals in hepcidin regulation in humans ^[10][53].

The hypothesis of ACD due to EPP was instead supported by an EPP mouse model in which the affected animals had an identical total-body-iron content to their control littermates but showed abnormal iron distribution in their bodies ^[11][54]. In FECH-deficient mice, most of the iron was stored in the enlarged spleen, whereas in the liver, kidney, and heart, iron content was decreased. Hepcidin expression in the liver of FECH-deficient animals was not suppressed, although these animals exhibited severe anemia, suggesting that erythropoiesis was not restricted by an absolute iron deficiency but likely limited by iron compartmentalization (i.e., functional iron deficiency), and/or the presence of strong up-regulators of hepcidin in this experimental setting. In addition, those animals showed a markedly increased transferrin expression (a typical feature of iron-deficient anemia, whereas inflammation down-regulates transferrin expression) and concomitantly decreased transferrin saturation (which is associated with both iron-deficient and inflammatory anemia), with overall normal serum iron.

In a French cohort of 55 EPP patients, Delaby et al. noted a mild hypochromic microcytic anemia and thrombocytopenia, but normal serum iron and soluble transferrin receptor (sTfR). A positive correlation between erythrocyte PPIX and Tf levels was reported, suggesting a positive effect of PPIX on the synthesis on Tf, which could facilitate the mobilization of tissue-iron stores to meet the erythropoiesis requirement and therefore a possible role of the PPIX–transferrin pathway in the regulation of iron distribution between organs ^[2][45]. This hypothesis might also explain the observation seen in the above murine model.

In another study, 178 patients with dominant EPP were evaluated; erythropoiesis was impaired in all patients, with 48% of women and 33% of men satisfying the criteria for mild microcytic, hypochromic anemia. Iron stores in the form of serum ferritin were decreased compared to the controls, with normal levels of serum iron and sTfR1, suggesting that erythropoiesis was not limited by iron supply ^[12][55].

A recent investigation of 67 EPP patients (51 Italian: 35 male and 16 female; 16 Swiss: 5 male and 11 female) compared to 21 healthy volunteers excluded the presence of anemia of chronic disease (ACD) in such patients, who had significantly decreased levels of hepcidin compared to the healthy volunteers, supporting the hypothesis of an absolute iron deficiency, possibly secondary to an inefficient iron adsorption at the intestinal level (through a hepcidin-independent mechanism) or to an undetected iron loss ^[13][56].

Overall, these data suggest that other, hepcidin-independent mechanisms could be at least partially responsible for the iron deficiency in such diseases. Furthermore, absolute and functional iron deficiency could be alternatively or concomitantly present in EPP patients, and the response to iron supplementation could be influenced by their relative weight and by the patient’s genetics (including variants in FECH, ALAS2 and other genes involved in iron homeostasis and heme metabolism).

Congenital erythropoietic porphyria (CEP) is an autosomal recessive disease caused by mutations in the uroporphyrinogen III synthase (UROS) gene, with the consequent nonenzymatic conversion of hydroxymethylbilane to isomer I porphyrin metabolites, which accumulate in red blood cells and their late precursors, resulting in ineffective erythropoiesis, hemolysis and splenomegaly and, when disseminated into the tissues and skin, severe photosensitivity.

More than 45 mutations of UROS have been founds with other factors that can influence disease phenotype; particularly, combined mutations of ALAS2 have been associated with more severe CEP symptoms ^[14][57].

Beyond sunlight avoidance, the suppression of bone-marrow activity using different strategies was attempted in the past, such as multiple transfusions and the use of hydroxyurea; bone marrow transplants have resulted in the successful reduction of symptoms in a series of patients ^[15][16][17][58,59,60]; iron chelation has been used to ameliorate the accompanying iron overload that often develops in CEP patients (both for transfusion regimen and secondary to hemolytic anemia).

Additionally, considering the iron-dependent post-transcriptional regulation of ALAS2, the hypothesis that iron chelation with deferiprone could decrease ALAS2 expression via IRE/IRP was tested both in vitro and in a murine model of CEP, proving its efficiency at reducing porphyrin production; porphyrin accumulation progressively decreased in red blood cells and urine, and skin photosensitivity ameliorated in CEP mice treated with deferiprone (1 or 3 mg/mL in drinking water) for 26 weeks. Hemolysis and iron overload improved upon iron chelation as well, with a full correction of anemia in the CEP mice treated at the highest dose of deferiprone ^[17][60] indicating that a main effect of iron chelation is ALAS2 down-regulation. Another explanation might be related to the removal of the inhibitory effects on erythropoiesis ^[18][61].

Porphyria cutanea tarda (PCT) is the most common type of porphyria worldwide, with an incidence between 20,000–70,000, and it encompasses a group of disorders caused by an insufficient/altered UROD enzymatic activity. Such deficiency leads to an accumulation of porphyrins (URO and 7-carboxyl porphyrins) in the liver.

The most frequent PCT is the acquired/sporadic type, which accounts for 75–80% of cases, in which the deficiency of UROD is limited to hepatocytes; affected patients usually present with other known risk or triggering factors such as other genetic factors (i.e., HFE mutations), viral infections (i.e., HCV-hepatitis) and exposure to certain chemical substances (i.e., alcohol, smoking, estrogens) ^[19][62].

A second type of PCT, which accounts for 20–30% of cases, is caused by mutations of the uroporphyrinogen III decarboxylase (UROD) gene in all tissues; the gene defect is transmitted in an autosomal-dominant manner with incomplete penetrance ^[20][63]. There is also a third type, which is very rare and is characterized by an apparently genetic predisposition that leads to decreased UROD activity in hepatocytes ^[19][62].

The pathogenesis of PCT is complex, but hereditary or acquired factors that lead to increased oxidative stress and hepatic-iron loading are critical in producing the clinical expression of the first and second forms of the disease ^[20][63].

From the clinical point of view, cutaneous lesions on the sunlight-exposed areas, particularly the hands and face, are the only consistent clinical feature of PCT. The most common lesions are superficial erosions from increased mechanical fragility of the skin, subepidermal bullae, hypertrichosis, and pigmentation. The age at presentation is usually lower for the familial type, while the sporadic type presents more frequently at middle age, with variations in gender likely reflecting differences in exposure to provoking factors, particularly alcohol or estrogenic therapy ^[21][64].

Liver histopathology includes the red fluorescence of unfixed hepatic tissue (as in various types of porphyria), necrosis, inflammation, and varying degrees of siderosis or steatosis.

At least 80% of patients with PCT show some degree of hepatic siderosis, especially of periportal hepatocytes ^[22][65], and total-body-iron stores are increased in 60–65% of patients ^[23][66].

Therefore, iron seems to play a role in the development of type I PCT; an imbalance in iron homeostasis may provide an oxidative environment in hepatocytes, contributing to the generation of a UROD inhibitor, likely uroporphomethene, which causes the expression of uroporphyria in mice and PCT in humans ^[24][67]. Despite the importance of liver siderosis in PCT, iron by itself is insufficient to cause uroporphyrin overproduction in the absence of other predisposing factors, emphasizing the multifactorial pathogenesis of PCT ^[20][63].

Therefore, conditions linked to increased iron accumulation, especially in the liver, have been studied and seen as possible co-risk factors for PCT development.

The prevalence of HFE mutations in PCT subjects has been evaluated with contrasting results in different geographical regions, although the net prevalence of both p.Cys282TyrY and p.His63Asp variants seem to be higher in affected patients compared to the general population ^[25][26][68,69]. Animal models with a disrupted hemochromatosis gene (Hfe −/−) and a disruption of one of the Urod alleles developed uroporphyria, which is the equivalent of human PCT in mice, whereas Urod −/+ mice were not affected ^[27][28][70,71], reinforcing the concept of an iron-driven second hit.

Hepatic hepcidin may also be reduced in PCT patients without HFE mutations, suggesting that other susceptibility factors may lower the expression of this hormone and cause hepatic siderosis in PCT ^[29][72]. Additionally, other genes important for iron regulation could be involved, as indagated in a population of 74 PCT South African patients, who underwent sequencing analysis of the promoter region of four genes that are involved in iron metabolism (ceruloplasmin (CP), cytochrome b reductase 1 (CYBRD1), hepcidin antimicrobial peptide (HAMP) and ferroportin or solute carrier family 40 member A1 (SLC40A1)). Some polymorphic loci were exhibited by PCT patients, which could contribute to the disease development by affecting iron metabolism. ^[30][73] In accordance, researchers recently reported an enrichment of non-HFE gene variants in a population of NAFLD patients with hepatic-iron deposition; particularly, CP variants were associated with hyperferritinemia, hepatic-iron staining and fibrosis worsening ^[31][74], indicating that genes other than HFE may affect the expression and course of chronic liver disease.

Chronic hepatitis C (CHC) is another known risk factor for PCT, and it is still not clear whether this association is secondary to iron overload due to the oxidative stress that often accompanies CHC ^[32][75] or to the hepatitis C infection (HCV) infection per se, or if both factors are relevant. As matter of fact, HCV-induced reactive oxygen species have been shown to down-regulate hepcidin transcription through inhibition of C/EBPalpha DNA binding activity in murine models, leading to increased duodenal iron transport and macrophage iron release, causing hepatic-iron accumulation ^[33][76]. In agreement, HCV infection appeared to down-regulate hepcidin expression in patients who developed PCT ^[29][72].

A similar consideration can be made for alcohol, which is a common susceptibility factor for PCT development ^[34][77]. Alcohol is a hepatotoxin that generates toxic metabolites able to induce liver damage via different mechanisms such as oxidative stress, endotoxin production, impaired immunity, hypoxia, and endoplasmic-reticulum malfunction ^[35][78]. Interestingly, alcohol has been associated with hepcidin down-regulation via oxidative stress both in hepatoma cell lines and rodents ^[36][79], indicating another way for alcohol to trigger or enhance the clinical expression of PCT. Alcohol may also contribute to iron-metabolism derangement via other mechanisms, including acute and chronic liver injury, the increased ability of desialylated transferrin to deliver iron to the liver, ineffective erythropoiesis and chronic hemolysis.

Overall, the data indicate that genetic and acquired factors capable of inducing iron overload in the liver, including through down-regulation of hepcidin expression, contribute to the overt clinical expression of PCT. Consistent with this, iron depletion (with phlebotomy as the first-line treatment) or the treatment of underlying disease as well (antiviral therapy in case of HCV infection, alcohol withdrawn), have been associated with the gradual improvement and clinical remission of disease expression in patients diagnosed with PCT ^[20][63].

The acute hepatic porphyrias (AHPs) include four of the rarest types of inherited porphyrias: acute intermittent porphyria (AIP), variegate porphyria, hereditary coproporphyria, and porphyria due to severe deficiency of ALA dehydratase (ADP).

Of these, AIP is by far the most prevalent. It is due to mutations of the HMBS gene (transmitted in an autosomal-dominant manner with low penetrance), which causes a deficient activity of porphobilinogen deaminase (PBG-D; the third enzyme in the heme biosynthesis pathway), with consequent accumulation of the precursors of ALA and PBG in the liver ^[37][80].

AIP is clinically characterized by some of the most serious manifestations of porphyrias, like recurring neurovisceral acute porphyric attacks (APAs), which can be triggered by certain drugs, starvation, inflammation, stress and hormones, and can be lethal if untreated.

As HMBS is less abundant than the other enzymes of the heme synthetic pathway, it becomes rate-limiting when ALAS1 is markedly increased. As a result, the porphyrin precursors are overproduced, and they markedly accumulate. The liver is the primary target of damage and, accordingly, affected patients tend to have altered-liver-function tests and a higher incidence of hepatocellular carcinoma ^[38][81].

Although recently givosiran, an siRNA directed specifically at hepatic ALA synthase-1 has been approved for the prevention of recurrent acute attacks in severely affected patients with AHPs, up to now, the standard therapy for APAs has been the intravenous administration of heme-arginate, in order to create negative feedback for ALAS1 activity. Patients who experienced recurring APAs tended to receive repeated infusions of hemin as a prophylactic treatment, with the consequent increased risk of iron overload ^[39][82]. In these patients, iron overload due to heme administration can contribute to hepatic fibrosis and the development of hepatocellular carcinoma ^[39][82], as seen in other chronic liver diseases of other origins with intercurrent iron accumulation ^[40][83].

Other than heminic treatment, mechanisms activated in response to starvation and linking APA and iron metabolism could be involved. It has been demonstrated that a transcriptional coactivator, peroxisome-proliferator-activated receptor-gamma coactivator-1 (PGC-1alpha), is a key player in the induction of porphyria by fasting. In fact, in a fasted state, the hepatic expression of PGC-1alpha is induced, which then acts as a coactivator for the transcription factors nuclear respiratory factor 1 (NRF-1) and forkhead box protein O1 (FOXO1), resulting in increased ALAS-1 expression and an increased risk of an APA.

On the other hand, it has been previously shown that during starvation PGC-1alpha also binds to the hepcidin gene promoter, as well as c-AMP responsive element binding protein 3 like 3 (Creb3l3), leading to increased hepcidin expression, ferroportin degradation and consequent hypoferremia and iron retention in the liver ^[41][84].

Thus, starvation could both trigger APAs through ALAs-1 induction and favor iron-status derangement and liver-iron damage via a common molecular co-activator. Indeed, it is not surprising that molecular crossroads exist between energy homeostasis, heme-protein synthesis and iron metabolism, and that the fine regulation of shared key players is crucial.

The liver-iron-overload issue in AIP patients is not trivial or outdated as many patients have already undergone long-term hemin treatment, and probably not all patients with AIP will be suitable or tolerant of long-life siRNA therapeutic strategies in the future.