Manuel Mendez

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.

The molecular basis of porphyria cutanea tarda in Chile: Identification and functional characterization of mutations in the uroporphyrinogen decarboxylase gene

Abstract:  The porphyrias are heterogeneous disorders arising from predominantly inherited catalytic deficiencies of specific enzymes in heme biosynthesis. Porphyria cutanea tarda (PCT) results from a decreased activity of uroporphyrinogen decarboxylase, the fifth enzyme in heme biosynthesis. The disorder represents the only porphyria that is not exclusively inherited monogenetically. In PCT, at least two different types can be distinguished: acquired/sporadic (type I) PCT, in which the enzymatic deficiency is limited to the liver and inherited/familial (type II) PCT, which is inherited as an autosomal dominant trait with a decrease of enzymatic activity in all tissues. In an effort to characterize the molecular basis of PCT in Chile, we identified eight mutations in 18 previously unclassified PCT families by polymerase chain reaction, heteroduplex analysis, and automated sequencing. To study the role of these mutations in disease causality, in vitro expression of all novel missense mutations was studied. Our results indicate that the frequency of familial PCT in Chile is approximately 50%, thus, to our knowledge, representing the highest incidence of familial PCT reported to date. The data further emphasize the molecular heterogeneity in type II PCT and demonstrate the advantages of molecular genetic techniques as a diagnostic tool and in the detection of clinically asymptomatic mutation carriers.

Acute intermittent porphyria: Biochemical and clinical analysis in the Argentinean population

Acute intermittent porphyria (AIP) is the most common type of hepatic acute porphyria. In this work, we have analyzed the biochemical data of all Argentinean AIP families studied in the Porphyrins and Porphyrias Research Centre (CIPYP). We have shown that: (i) the prevalence for this population is about 1:125,000; (ii) the disease is more frequent in women than in men (7:3); (iii) about 60% are latent carriers; (iv) 15% of patients with symptomatic AIP died during an acute attack; (v) the most important precipitating factors of acute attacks in our population were the ingestion of therapeutic drugs (25%), anesthetics in surgical interventions (25%) and infections (20%); (vi) the initial symptom in Argentinean AIP individuals is severe abdominal pain (100%), and it is often accompanied by constipation (37%), anorexia (37%) and tachycardia (30%); and (vii) the percentage of recurrence of the acute attacks is high (81%).

Molecular heterogeneity of familial porphyria cutanea tarda in Spain: characterization of 10 novel mutations in the UROD gene

Background  Porphyria cutanea tarda (PCT) results from decreased hepatic uroporphyrinogen decarboxylase (UROD) activity. In the majority of patients, the disease is sporadic (S‐PCT or type I) and the enzyme deficiency is limited to the liver. Familial PCT (F‐PCT or type II) is observed in 20–30% of patients in whom mutations on one allele of the UROD gene reduce UROD activity by approximately 50% in all tissues. Another variant of PCT (type III) is characterized by family history of the disease although it is biochemically indistinguishable from S‐PCT.

A homozygous mutation in the ferrochelatase gene underlies erythropoietic protoporphyria associated with palmar keratoderma

Erythropoietic protoporphyria (EPP) (OMIM 177000) is one of the nonacute, cutaneous porphyrias. The disease results from an inherited partial deficiency of ferrochelatase (FECH; EC 4.99.1.1), the eighth and ultimate enzyme in haem biosynthesis, that is located in the mitochondria and catalyses the insertion of iron into protoporphyrin IX to form haem. Although EPP is a rare disease overall, it is the most common type of porphyria manifesting in childhood with an estimated prevalence of one in 130 000. Clinically overt disease usually commences in infancy or early childhood and is characterized by moderate to severe cutaneous photosensitivity affecting body sites exposed to ultraviolet (UV) radiation. Following sun exposure, pain, burning, itching, erythema and swelling can develop within minutes to hours. Petechiae, purpura and vesicles may be seen, but are uncommon. Chronic UV exposure might lead to lichenification, subtle scarring and hyperpigmentation, particularly over the knuckles and on the nose. These clinical signs in association with the age at onset are quite characteristic for EPP and the tentative diagnosis is confirmed by biochemical and enzymatic analyses. In 2–5% of the patients, the disease can be complicated by severe liver failure with progressive hepatic dysfunction. EPP is inherited as an autosomal dominant trait with incomplete penetrance. In affected individuals, heterozygous mutations in the FECH gene on chromosome 18q21.3 lead to a decrease in activity of the encoded protein, FECH, by ~50%, which is not sufficient for the development of clinically overt disease. Rather, coinheritance of an intronic FECH polymorphism, IVS3-48C, in trans to a specific FECH mutation, is necessary to decrease the residual FECH activity to levels below 30% and cause photosensitivity. Indeed, coinheritance of a deleterious FECH mutation and the hypomorphic IVS3-48C allele has been found in up to 98% of patients with overt disease. In exceptional cases of EPP, autosomal recessive inheritance of FECH gene mutations associated with markedly decreased FECH activity of < 10% of normal has been observed. The prevalence of the recessive form of EPP is estimated to be ~4%. Beside cutaneous photosensitivity, the only other skin symptom that has been previously reported in a single patient with EPP is porokeratosis. Here, we describe the first incidence of homozygous inheritance of a novel missense mutation in the FECH gene underlying the uncommon phenotype of EPP associated with palmar keratoderma.

Acute intermittent porphyria: Characterization of two novel mutations in the porphobilinogen deaminase gene, one amino acid deletion (453‐455delAGC) and one splicing aceptor site mutation (IVS8‐1G>T)

A partial deficiency of Porphobilinogen deaminase (PBG‐D) is responsible for acute intermittent porphyria (AIP). AIP is inherited in an autosomal dominant fashion, and the prevalence in the Argentinean population is about 1:125,000. Here, two new mutations and three previously reported were found in the PBG‐D gene in 12 Argentinean AIP patients corresponding to 5 different families. To screen for AIP mutations in symptomatic patients, genomic DNA isolated was amplified in 2 Multiplex PCR reactions, then all coding exons and flanking intronic regions were sequenced. The new mutations are 453‐455delAGC in exon 9 which results in the loss of an alanine residue at position 152, and one new point mutation in the splicing aceptor site in the last position of intron 8 (IVS8‐1G>T) which leds to a 15 bp deletion because a cryptic site (first AG upstream) is used. Both mutations produce amino acid deletion without frameshift effect. To further characterize the 453‐455delAGC mutation, the pKK‐PBGD construct for the mutant allele was expressed in E. coli, the enzymatic activity of the recombinant protein was 1.3% of the mean level expressed by the normal allele. Finally, three missense mutations, previously reported, were identified in three unrelated families. Hum Mutat 14:355, 1999.

Amiodarone is a pharmacologically safe drug for porphyrias

Amiodarone (AD) is an effective antidysrythmic drug, however, there can be serious side effects, such as hepatic and neurological alterations, as well as skin photosensitization, as seen in porphyrias. Clinical signs in porphyrias might be triggered by the so-called porphyrinogenic drugs. Without sound basis, Amiodarone has been classified as an unsafe drug for porphyric patients. The aim of this work has been to study the effect of AD, both in vivo and in vitro, on heme metabolism. In the in vivo assays, the activities of 5-aminolevulinate synthetase (ALA-S), ALA dehydratase (ALA-D), porphobilinogenase (PBGase) and PBG-deaminase (PBG-D) in blood, liver, and kidney; hepatic and fecal porphyrins, urinary ALA, PBG and porphyrins in male mice strain CF1 treated with AD (100 mg i.p. daily) for 1 week and 1 month, were measured. No significanat differences were found for any of these parameters in the AD treated animals as compared to controls. In the in vitro experiments human blood, and mice blood, liver, and kidney, were used to measure the activities of ALA-S, ALA-D, PBGase, PBG-D and uroporphyrinogen decarboxylase, in the presence of varying concentrations of AD (0.0172-4.304 mM). AD did not modify any of the enzyme activities. All of the above biochemical parameters were studied in 17 cardiac patients under AD treatment for 3 to 20 years. Neither the activities of the heme enzymes, nor the levels of precursors and porphyrins in urine and plasma were altered. These findings clearly demonstrate that AD is a pharmacologically safe drug and can be used for the treatment of associated pathologies in porphyrias.