Microcytosis in Erythropoietic Protoporphyria
Authors: Graziadei Giovanna , Duca Lorena , Granata Francesca , De Luca Giacomo , Brancaleoni Valentina , Nava Isabella , Tavazzi Dario , Fustinoni Silvia , Di Pierro Elena 
Published: February 2021

Background: Erythropoietic Protoporphyria (EPP) is a rare inherited hematological disorder resulting from reduced activity of the ferrochelatase (FECH), which catalyzes the insertion of iron into protoporphyrin IX (PPIX) to form heme, in the last step of the biosynthetic pathway. FECH reduced activity leads to significantly elevated PPIX levels mainly in erythrocytes and subsequently in skin and liver, causing severe photosensitivity with a childhood onset and acute liver failure. Unexpectedly no iron overload is reported, on the contrary iron deficiency anemia is often observed. The mechanisms involved in this last feature of the disease are not fully understood.
Aims: This study aimed to evaluate anemia and iron status in 20 EPP patients attending the Rare Disease Centre at Fondazione Ca' Granda Policlinico in Milan, Italy, from January 2009 to present.
Methods: Nine females and 11 males (age 23-57 yrs.) were diagnosed by the clinical history of photosensitivity in the presence of plasma porphyrin peak at 635nm, high levels of free PPIX in the erythrocytes and feces, and detection of genetic defects in the FECH coding gene. In all patients, iron, ferritin, transferrin (Tf), Tf saturation % (TS%), haemoglobin (Hb) and mean corpuscular volume (MCV) in peripheral venous blood were measured. Hepcidin and soluble transferrin receptor (sTfr) were also determined using commercially available immunoassays.
Results: The 44% (4/9) of female patients had mild microcytic anemia (Hb 10-11.9g/dl; MCV 70.6-77.7 fl; TS% 8-16) while the 64% (7/11) male patients presented microcytosis despite normal Hb and iron levels (MCV 72-78.9 fl; Hb 13.6-14.9g/dl; iron 66-168; TS% 17-52). In addition, males with microcytosis showed normal sTfr (0.98-2.80mcg/ml) and hepcidin (7.5-24ng/ml) levels compared to higher sTfr (1.45-5.76mcg/ml) and lower hepcidin levels (0.01-15ng/ml) in microcytic females. Indeed, in 7/9 females both ferritin (4-13 ng/ml) and TS% (6-18%) were also reduced suggesting iron deficiency without anemia. As expected, hecpidin levels directly correlated with iron (r2=0.334; p&lt;0.001), ferritin (r2=0.717; p&lt;0.0001) and TS% (r2=0.459; p=0.002) confirming that the iron regulation mechanism in these patients is not disrupted. Although hepcidin mean levels were higher than normal in all normocytic patients, no significant difference was detected between EPP patients and normal subjects except for a larger dispersion of values. Moreover, no significant correlation was found between iron, ferritin, TS% and MCV while an inverse correlation trend was detected between MCV and PPIX (r2=0.22; p=0.04). In addition, all microcytic patients showed PPIX mean levels significantly higher compared to normocytic patients (p&lt;0.0001 in males and p=0.03 in females). Finally, a positive trend of correlation between serum malondialdehyde (MDA), the most biomarkers for evaluation of membrane lipid peroxidation and PPIX amount was also observed (r2=0.833; p&lt;0.011). In addition, MDA mean levels were significantly higher in all EPP patients with respect to normal subjects (p=0.024).
Conclusion: This study shows that microcytosis is a hematological feature of EPP patients also in absence of anemia and iron deficiency. Moreover, it suggests an active role of PPIX accumulation in reducing MCV probably through a toxic effect on the red blood cell membrane. Further investigations are however required to better clarify the hepcidin-independent mechanisms involved in the iron uptake down-regulation in EPP.

Disclosures
No relevant conflicts of interest to declare.