A. Fontanellas

Successful therapeutic effect in a mouse model of erythropoietic protoporphyria by partial genetic correction and fluorescence-based selection of hematopoietic cells

Erythropoietic protoporphyria is characterized clinically by skin photosensitivity and biochemically by a ferrochelatase deficiency resulting in an excessive accumulation of photoreactive protoporphyrin in erythrocytes, plasma and other organs. The availability of the Fechm1Pas/Fechm1Pas murine model allowed us to test a gene therapy protocol to correct the porphyric phenotype. Gene therapy was performed by ex vivo transfer of human ferrochelatase cDNA with a retroviral vector to deficient hematopoietic cells, followed by re-injection of the transduced cells with or without selection in the porphyric mouse. Genetically corrected cells were separated by FACS from deficient ones by the absence of fluorescence when illuminated under ultraviolet light. Five months after transplantation, the number of fluorescent erythrocytes decreased from 61% (EPP mice) to 19% for EPP mice engrafted with low fluorescent selected BM cells. Absence of skin photosensitivity was observed in mice with less than 20% of fluorescent RBC. A partial phenotypic correction was found for animals with 20 to 40% of fluorescent RBC. In conclusion, a partial correction of bone marrow cells is sufficient to reverse the porphyric phenotype and restore normal hematopoiesis. This selection system represents a rapid and efficient procedure and an excellent alternative to the use of potentially harmful gene markers in retroviral vectors.

Clinical utility of fluorometric scanning of plasma porphyrins for the diagnosis and typing of porphyrias

The fluorometric emission scanning (using excitation at 405 nm) of plasma samples, simply diluted five‐fold in phosphate‐buffered saline, allows the differentiation of three conditions according to their porphyrin content. The emission maximum at 626–628 nm is a specific finding in variegate porphyria, while in erythropoietic protoporphyria a characteristic peak is found at 636 nm. A fluorescence emission maximum at 618–622 nm corresponds to a third group that includes normal subjects, non‐porphyria patients and patients suffering from acute intermittent porphyria, hereditary coproporphyria, congenital erythropoietic porphyria (Günther disease) and porphyria cutanea tarda.

Human leukocyte antigen haplotypes and HFE mutations in Spanish hereditary hemochromatosis and sporadic porphyria cutanea tarda

Background and Aims:  It has been postulated that the HFE C282Y mutation (linked to human leukocyte antigen [HLA]‐A3‐B7 haplotype) is not only responsible for hereditary hemochromatosis; HLA class I alleles would also contribute to the disease pathogenesis. In addition, H63D mutation linked to HLA‐A29‐B44 would also be pathogenetic, particularly in the Mediterranean Basin and throughout the world. However, sporadic porphyria cutanea tarda (s‐PCT) has also been linked to these HFE mutations. In the present work, we have studied HFE mutations and HLA genes to test these hypotheses.

Correlation between levels of free and protein-bound plasma porphyrin and urinary porphyrins in porphyria cutanea tarda

Porphyria cutanea tarda (PCT) is a disorder of porphyrin metabolism that leads to massive overproduction and excretion of uroporphyrin. Most plasma porphyrins are bound to albumin and hemopexin. The aim of this work was to analyze the relationship between the concentrations of serum albumin and hemopexin, the levels of total, free and protein-bound plasma porphyrins and the urinary coproporphyrin and uroporphyrin excretion, in PCT patients at different stages of the disease. Urinary porphyrins showed a stronger correlation with total plasma porphyrin levels (r = 0.863) than with the free fraction of plasma porphyrins (r = 0.608). Patients considered in an active stage of PCT, have a higher mean level of total plasma porphyrins (8.80 micrograms/dl +/- 8.75) and a lower mean percentage of free plasma porphyrins (12.79% +/- 11.21) than those patients on remission (0.71 +/- 0.5 microgram/dl and 44.3 +/- 35.3%, respectively). 30% of patients showed hypohemopexinaemia, presumably due to hepatic damage. Despite the high affinity of this protein for porphyrins, no significant correlation was found between plasma porphyrin levels and hemopexin or albumin. It is concluded that (i) the kidney is not merely a passive filter for free plasma porphyrins and (ii) that the formation of hemopexin-porphyrin complex occurs when plasma porphyrins concentrations are increased (i.e. in those patients in an active stage of PCT).

Salivary porphyrin concentration in porphyria cutanea tarda

To assess the clinical utility of saliva samples, serum, urine and saliva porphyrin concentration were fluorometrically measured in 31 patients with porphyria cutanea tarda. In comparison with normal values, porphyrin mean levels were 20-fold increased in serum and urine but only 3-fold in saliva. Saliva porphyrin concentration exhibited significant but weak correlations with porphyrin levels in serum (r = 0.69) and urine (r = 0.67). However, saliva porphyrins were not more closely correlated with protein-unbound serum porphyrins (r = 0.57). This finding suggests that saliva porphyrin concentration does not simply correspond to the filtered free fraction of serum porphyrins. Nevertheless, measurement of saliva porphyrins could represent a valuable non-invasive alternative to serum porphyrins in the monitoring of patients with porphyria cutanea tarda.

Hepatic porphyrin concentration and uroporphyrinogen decarboxylase activity in hepatitis C virus infection: HPC and UROD in HCV infection

Previous studies have shown a high prevalence of hepatitis C virus (HCV) infection in patients with porphyria cutanea tarda (PCT). The aim of this study was to assess hepatic porphyrin concentrations (HPC) and hepatic uroporphyrinogen decarboxylase (UROD) activity in HCV‐infected patients free of PCT. Thirty‐two HCV‐infected patients (20 M, 12 F, mean age 51 years) and seven control patients (4 M, 3 F, mean age 59 years) free of liver disease, were studied. Knodell’s score was determined on liver biopsy by two independent anatomopathologists. Measurement of HPC and hepatic UROD activity levels were carried out on liver biopsy. Relative to controls, HCV‐infected patients had high HPC levels (mean ± SD: 47 ± 20 vs. 17 ± 6 pmol/mg protein, P < 0.001) and low hepatic UROD activity levels (514 ± 95 vs. 619 ± 125 pmol Copro/h/mg protein, P < 0.05). HPC was not correlated with hepatic UROD activity and the increase was due to coproporphyrin accumulation. No correlation was observed between HPC or hepatic UROD activity values and HCV‐RNA concentrations, Knodell’s score, hepatic fibrosis, periportal necrosis, periportal inflammation or hepatic iron content in HCV‐infected patients. Hepatocellular necrosis was significantly correlated with HPC value (P < 0.005). Hence, in HCV‐infected patients, HPC is significantly increased and hepatic UROD activity is very slightly decreased as compared to controls. HPC values and UROD activity are not correlated with HCV‐RNA concentrations, hepatic iron content and hepatic fibrosis. The small increase in HPC values in hepatitis C infection is linked with hepatic injury and not with a direct effect on hepatic UROD enzyme.