The ferrochelatase gene structure and molecular defects associated with erythropoietic protoporphyria

Ferrochelatase [heme synthase, protoheme ferrolyase (EC 4.99.1.1)], the terminal enzyme of the heme biosynthetic pathway, catalyzes the incorporation of ferrous ion into protoporphyrin IX to form protoheme IX. The genes and cDNAs for ferrochelatase from mammals and microorganisms have been isolated. The gene for human ferrochelatase has been mapped to chromosome 18q 21.3 and consists of 11 exons with a size of about 45 kilodaltons. The induction of ferrochelatase expression occurs during erythroid differentiation, and can be attributed to the existence of the promoter sequences of erythroid-related genes. Analysis of the ferrochelatase gene in patients with erythropoietic protoporphyria, an inherited disease caused by ferrochelatase defects, revealed that molecular anomalies of ferrochelatase from 11 patients were found in 9 patients as autosomal dominant type, and 2 patients as recessive type. Diversity of the mutations of the ferrochelatase gene is also briefly described.     

Systematic analysis of molecular defects in the ferrochelatase gene from patients with erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP; MIM 177000) is an inherited disorder caused by partial deficiency of ferrochelatase (FECH), the last enzyme in the heme biosynthetic pathway. In EPP patients, the FECH deficiency causes accumulation of free protoporphyrin in the erythron, associated with a painful skin photosensitivity. In rare cases, the massive accumulation of protoporphyrin in hepatocytes may lead to a rapidly progressive liver failure. The mode of inheritance in EPP is complex and can be either autosomal dominant with low clinical penetrance, as it is in most cases, or autosomal recessive. To acquire an in-depth knowledge of the genetic basis of EPP, we conducted a systematic mutation analysis of the FECH gene, following a procedure that combines the exon-by-exon denaturing-gradient-gel-electrophoresis screening of the FECH genomic DNA and direct sequencing. Twenty different mutations, 15 of which are newly described here, have been characterized in 26 of 29 EPP patients of Swiss and French origin. All the EPP patients, including those with liver complications, were heterozygous for the mutations identified in the FECH gene. The deleterious effect of all missense mutations has been assessed by bacterial expression of the respective FECH cDNAs generated by site-directed mutagenesis. Mutations leading to a null allele were a common feature among three EPP pedigrees with liver complications. Our systematic molecular study has resulted in a significant enlargement of the mutation repertoire in the FECH gene and has shed new light on the hereditary behavior of EPP.     

A novel mutation in erythropoietic protoporphyria: an aberrant ferrochelatase mRNA caused by exon skipping during RNA splicing

An aberrant ferrochelatase mRNA lacking exon 10 was found in a patient with erythropoietic protoporphyria (EPP). In her genomic DNA an A → T transversion at position ?3 of the donor site of intron 10 appeared to be responsible for the exon skipping. Both the patient and her sister were heterozygous for this mutation.     

Human erythropoietic protoporphyria: two point mutations in the ferrochelatase gene.

The molecular basis of the ferrochelatase defect responsible for human Erythropoietic Protoporphyria (EPP), a usually autosomal dominant disease, was investigated in a family with an apparently homozygous patient. Two mutations of the ferrochelatase gene were identified by sequencing the proband's cDNA after in vitro amplification of the mRNA and subcloning of the amplified products. One mutation results from a G to T transition at nucleotide 163 which produces a glycine to cysteine substitution at amino-acid residue 55 (G-55-C). The other one was a G to A change at nucleotide 801, leading to a methionine to isoleucine substitution at amino-acid residue 267 (M-267-I). This EPP patient was then double heterozygous and as expected each of his parents carried one of the mutations. A second similar EPP patient was screened for these mutations with negative results, showing a genetic heterogeneity in EPP.     

Zebrafish dracula encodes ferrochelatase and its mutation provides a model for erythropoietic protoporphyria

Exposure to light precipitates the symptoms of several genetic disorders that affect both skin and internal organs. It is presumed that damage to non-cutaneous organs is initiated indirectly by light, but this is difficult to study in mammals. Zebrafish have an essentially transparent periderm for the first days of development. In a previous large-scale genetic screen we isolated a mutation, dracula (drc), which manifested as a light-dependent lysis of red blood cells [1]. We report here that protoporphyrin IX accumulates in the mutant embryos, suggesting a deficiency in the activity of ferrochelatase, the terminal enzyme in the pathway for heme biosynthesis. We find that homozygous drc(m248) mutant embryos have a G-->T transversion at a splice donor site in the ferrochelatase gene, creating a premature stop codon. The mutant phenotype, which shows light-dependent hemolysis and liver disease, is similar to that seen in humans with erythropoietic protoporphyria, a disorder of ferrochelatase.     

A genotype-phenotype correlation between null-allele mutations in the ferrochelatase gene and liver complication in patients with erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP), an inborn error of heme metabolism, causes in the majority of the patients only a symptom of photosensitivity. However, around 2% of the EPP sufferers develop liver complication in the form of liver cirrhosis and progressive liver failure. Mutations in the human ferrochelatase (FECH) gene causing EPP are highly heterogeneous and mostly family-specific. Actually, 62 FECH mutations have been published, 48 of them are "null allele" mutations inducing the formation of a truncated protein. The remaining 14 are missense mutations. In contrast to the null allele mutations, the latter lead to substitution of a single amino acid residue in the protein molecule and generate an enzyme that, although functionally impaired, is in its full length. In order to study the association between "null allele" mutation and liver complication, we combined our data with those in the literature. A total of 112 EPP patients were counted among 93 EPP families with a known FECH mutation. All 18 EPP patients who had severe liver complication carried a "null allele" mutation. In contrast, none of the 20 patients who carried a missense mutation had developed liver complication till the time of study (Fisher's exact test, p<0.05). High protoporphyrin blood concentration are considered to be a sign of an increased risk of liver disease. No correlation of protoporphyrin blood level with the type of mutation, was found, if patients with overt liver disease were excluded from the sample. Furthermore, no significant association of the liver complication with the location of the mutation within the FECH gene was found (Fisher exact test p = 0.46). These available data indicate a significant genotype-phenotype correlation between "null allele" mutation and protoporphyrin related liver disease in EPP. Although the risk for a EPP patient with a missense mutation to develop liver disease cannot be totally eliminated based on these data, it is comparably low.     

Regulation of ferrochelatase gene expression by hypoxia

Ferrochelatase (FECH), the last enzyme of the heme biosynthetic pathway, catalyzes the insertion of iron into protoporphyrin to form heme. This pathway provides heme for hemoglobin and other essential hemoproteins. The regulatory role of oxygen in the pathway has not been clearly established. In this study, we examined whether FECH gene expression is upregulated during hypoxia by a mechanism which involves the hypoxia-inducible factor 1 (HIF-1). Two HIF-1 binding motifs were identified within the -150 bp FECH minimal promoter sequence. Exposure of HEL, K562, and Hep-G2 cells to hypoxia for 18 hours resulted in a significant increase in FECH mRNA expression (p < 0.05). Hypoxia also transactivated the minimal promoter for the FECH gene in the cells. Transient co-expression of wild-type HIF-1alpha or a dominant negative HIF-1alpha with the FECH minimal promoter luciferase construct stimulated or blocked FECH promoter activity, respectively. Expression of the von Hippel-Lindau (VHL) tumor suppressor factor blocked the expression of both FECH mRNA and HIF-1alpha protein during normoxic culture of renal carcinoma cell line (RCC4). The results suggest that the FECH gene is a target for HIF-1 during hypoxia.     

Ferrochelatase gene mutations in erythropoietic protoporphyria: focus on liver disease.

A deficiency of ferrochelatase (FECH) activity underlies the excess accumulation of protoporphyrin that occurs in erythropoietic protoporphyria (EPP). In some patients, protoporphyrin accumulation causes liver damage that necessitates liver transplantation. The purpose of this study was to determine if specific mutations in the FECH gene are present in patients who develop liver disease. FECH cDNA and all 11 exons and their flanking intron regions in the FECH gene were amplified and sequenced by specific polymerase chain reactions. Gene mutations were determined in 34 individuals from 24 families: 14 had liver disease, 10 necessitating liver transplantation. All individuals were heterozygous for mutations that altered the coding region of FECH mRNA. The mutations in patients with liver disease were heterogenous, but usually caused a major structural alterations in the FECH protein, most commonly as a result of exon skipping in FECH mRNA. However, the mutations could not account for the severe phenotype by themselves, since the same mutations were found in asymptomatic family members of patients with liver disease and in patients from families in which liver disease was not present. Other genetic factors, and possibly acquired factors, also must be critical to the development of this severe phenotype in EPP.     

Human ferrochelatase: a novel mutation in patients with erythropoietic protoporphyria and an isoform caused by alternative splicing

Erythropoietic protoporphyria (EPP), attributable to deficiency of ferrochelatase activity (FECH), is characterised mainly by cutaneous photosensitivity. To define the molecular defect in two EPP-affected siblings and their parents in a Swiss family, ferrochelatase cDNA was amplified by the polymerase chain reaction (PCR) and subjected to sequence analysis. A 5-bp deletion (T580G584) was identified on one allele of the ferrochelatase gene in both patients and their mother. Screening of the mutation among family members by RsaI digestion of PCR-amplified genomic DNA revealed autosomal dominant inheritance associated with abnormal protoporphyrin concentration and enzyme activity. We also isolated ferrochelatase cDNAs containing a 18-bp insertion (part of the intron 2 sequence) between exons 2 and 3; this corresponded to six extra amino acids (YESNIR) inserted between Arg-65 and Lys-66 of the known ferrochelatase. This isoform was identified initially in mRNAs derived from both alleles of the ferrochelatase gene in one patient. Its existence was confirmed in six additional EPP patients, in five out of seven controls, and in four different cell lines (fibroblast, muscle, hepatoma and myelogenous leukaemia). This isoform, roughly 20% of the total ferrochelatase mRNA, was generated through splicing at a second donor site in intron 2 and its presence was not linked to EPP.     

Modulation of the phenotype in dominant erythropoietic protoporphyria by a low expression of the normal ferrochelatase allele.

Erythropoietic protoporphyria (EPP) is a monogenic inherited disorder of the heme biosynthetic pathway due to ferrochelatase (FC) deficiency. EPP is generally considered to be transmitted as an autosomal dominant disease with incomplete penetrance, although autosomal recessive inheritance has been documented at the enzymatic and molecular level in some families. In the dominant form of EPP, statistical analysis of FC activities documented a significantly lower mean value in patients than in asymptomatic carriers, suggesting a more complex mode of inheritance. To account for these findings, we tested a multiallelic inheritance model in one EPP family in which the enzymatic data were compatible with this hypothesis. In this EPP family, the specific FC gene mutation was an exon 10 skipping (delta Ex10), resulting from a G deletion within the exon 10 consensus splice donor site. The segregation of all FC alleles within the family was followed using the delta Ex10 mutation and a new intragenic dimorphism (1520 C/T). mRNAs transcribed from each FC allele were then subjected to relative quantification by a primer extension assay and to absolute quantification by a ribonuclease protection assay. The data support the hypothesis that in this family the EPP phenotype results from the coinheritance of a low output normal FC allele and a mutant delta Ex10 allele.     

Regulation of haem biosynthesis in normoblastic erythropoiesis: role of 5-aminolaevulinic acid synthase and ferrochelatase

The development of haem biosynthetic enzyme activity during normoblastic human erythropoiesis was examined in seven patients. The first and last enzymes of the haem biosynthetic pathway, ALA synthase and ferrochelatase, were assayed by radiochemical/high performance liquid chromatographic (HPLC) methods. An assay for ferrochelatase activity in human bone marrow was developed. Enzyme substrates were protoporphyrin IX and ⁵⁹Fe²⁺ ions. ⁵⁹Fe-labelled haem was isolated by organic solvent extraction/sorbent extraction followed by reversed-phase HPLC. Optimal activity occurred at pH 7.3 in the presence of ascorbic acid, in darkness and under anaerobic conditions. Haem production was proportional to cell number and was linear with time to 30 min. The assay was sensitive to the picomolar range of haem production. ALA synthase and ferrochelatase activity was assayed in four highly purified age-matched erythroid cell populations. ALA synthase activity was maximal in the most immature erythoid cells and diminished as the cells matured with an overall five fold loss of activity from proerythroblast to late erythroblast development. Ferrochelatase activity was, however, more stable with less than a two fold change in activity observed during the same period of erythroid differentiation. Maximal activity occurred in erythroid fractions enriched with intermediate erythroblasts. These results support sequential rather than simultaneous appearance of these enzymes during normoblastic erythropoiesis. Quantitative analysis of relative enzyme activity however indicates that at all times during erythroid differentiation ferrochelatase activity is present in excess to that theoretically required relative to ALA synthase activity since ALA and haem are not produced in stoichiometric amounts. The lability of ALA synthase versus the stability and gross relative excess of ferrochelatase activity indicates a far greater role for ALA synthase in the regulation of erythroid haem biosynthesis than for ferrochelatase.     

A continuous anaerobic fluorimetric assay for ferrochelatase by monitoring porphyrin disappearance

A continuous spectrofluorimetric assay for determining ferrochelatase activity has been developed using the physiological substrates ferrous iron and protoporphyrin IX under strictly anaerobic conditions. In contrast to heme, the product of the ferrochelatase-catalyzed reaction, protoporphyrin IX is fluorescent, and therefore the progress of the reaction can be monitored by following the decrease in protoporphyrin fluorescence intensity (with excitation and emission wavelengths at 505 and 635 nm, respectively). This continuous fluorimetric assay detects activities as low as 0.01 nmol porphyrin consumed min(-1), representing an increase in sensitivity of up to two orders of magnitude over the currently used, discontinuous assays. The determination of the steady-state kinetic parameters of ferrochelatase yielded K(m)(PPIX)=1.4+/-0.2 microM, K(m)(Fe(2+))=1.9+/-0.3 microM, and k(cat)=4.0+/-0.3 min(-1). In addition to its applicability for acquisition of kinetic data to characterize ferrochelatase and recombinant variants, this new method should permit detection of low concentrations of ferrochelatase in biological samples.     

Mucopolysaccharidosis type VII: Characterization of mutations and molecular heterogeneity

We identified two different exonic point mutations causing beta-glucuronidase (beta G1) deficiency in two Japanese patients with mucopolysaccharidosis type VII (MPSVII). Enzyme assay of lysates of the lymphocytes and cultured fibroblasts showed little residual activity. The beta G1-specific mRNA levels were normal, as determined by northern blot analysis. Mutated cDNA clones, including the entire coding sequence, were isolated using the polymerase chain reaction (PCR) products derived from beta G1-deficient fibroblasts. Sequence analysis of the full-length mutated cDNAs showed C----T transitions, which resulted in a single Ala619----Val change (case 1, a 24-year-old male) and a Arg382----Cys change (case 2, a 7-year-old female). The former change was revealed by a loss of the cleavage site for the Fnu4HI in the mutated cDNA. On the basis of the loss of Fnu4HI restriction site, the patient (case 1) was a homozygote with this mutation. The mutational change in patient 2 was confirmed by direct sequencing and by demonstrating heterozygosity for the mutation in her parents. The Ala619----Val and Arg382----Cys mutations each disrupt a different domain which is highly conserved among human, rat, and Escherichia coli beta G1s. Each of these two amino acid changes reduced the beta G1 activity of the corresponding mutant beta G1 expressed following transfection of COS cells with expression vectors harboring the mutated cDNAs.     

Contribution of a common single-nucleotide polymorphism to the genetic predisposition for erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis that results from a partial deficiency of ferrochelatase (FECH). Recently, we have shown that the inheritance of the common hypomorphic IVS3-48C allele trans to a deleterious mutation reduces FECH activity to below a critical threshold and accounts for the photosensitivity seen in patients. Rare cases of autosomal recessive inheritance have been reported. We studied a cohort of 173 white French EPP families and a group of 360 unrelated healthy subjects from four ethnic groups. The prevalences of the recessive and dominant autosomal forms of EPP are 4% (95% confidence interval 1-8) and 95% (95% confidence interval 91-99), respectively. In 97.9% of dominant cases, an IVS3-48C allele is co-inherited with the deleterious mutation. The frequency of the IVS3-48C allele differs widely in the Japanese (43%), southeast Asian (31%), white French (11%), North African (2.7%), and black West African (<1%) populations. These differences can be related to the prevalence of EPP in these populations and could account for the absence of EPP in black subjects. The phylogenic origin of the IVS3-48C haplotypes strongly suggests that the IVS3-48C allele arose from a single recent mutational event. Estimation of the age of the IVS3-48C allele from haplotype data in white and Asian populations yields an estimated age three to four times younger in the Japanese than in the white population, and this difference may be attributable either to differing demographic histories or to positive selection for the IVS3-48C allele in the Asian population. Finally, by calculating the KA/KS ratio in humans and chimpanzees, we show that the FECH protein sequence is subject to strong negative pressure. Overall, EPP looks like a Mendelian disorder, in which the prevalence of overt disease depends mainly on the frequency of a single common single-nucleotide polymorphism resulting from a unique mutational event that occurred 60,000 years ago.     

Zinc chelatase in human lymphocytes: detection of the enzymatic defect in erythropoietic protoporphyria

We describe a fluorometric assay for heme synthetase, the enzyme that is genetically deficient in erythropoietic protoporphyria. The method, which can readily detect activity in 1 microliter of packed human lymphocytes, is based on the formation of zinc protoheme from protoporphyrin IX. That zinc chelatase and ferrochelatase activities reside in the same enzyme was shown by the competitive action of ferrous ions and the inhibitory effects of N-methyl protoporphyrin (a specific inhibitor of heme synthetase) on zinc chelatase. The Km for zinc was 11 micrograms and that for protoporphyrin IX was 6 microM. The Ki fro ferrous ions was 14 microM. Zinc chelatase was reduced to 15.3% of the mean control activity in lymphocytes obtained from patients with protoporphyria, thus confirming the defect of heme biosynthesis in this disorder. The assay should prove to be useful for determining heme synthetase in tissues with low specific activity and to investigate further the enzymatic defect in protoporphyria.     

Induction of porphobilinogen oxygenase and porphobilinogen deaminase in rat blood under conditions of erythropoietic stress

Porphobilinogen is the substrate of two enzymes: porphobilinogen deaminase and porphobilinogen-oxygenase. The first one transforms it into the metabolic precursors of heme and the second diverts it from this metabolic pathway by oxidizing porphobilinogen to 5-oxopyrrolinones. Rat blood is devoid of porphobilinogen-oxygenase under normal conditions while it carries porphobilinogen-deaminase activity. When the rats were submitted to hypoxia (p_O2 = 0.42 atm) for 18 days, the activity of porphobilinogen-oxygenase appeared at the tenth day of hypoxia and reached the maximum at the 14–16th day. It decreased to a half after 2 days (half-life of the enzyme) and disappeared after 4 days of return to normal oxygen pressure. Porphobilinogen-deaminase activity increased after the first day of hypoxia, reached a maximum at the 14–16th day and did not decrease to normal values until the 15th day after return to normal oxygen pressure. The activities of both prophobilinogen-oxygenase and porphobilinogen-deaminase were induced by administration of erythropoietin. When rats were made anaemic with phenylhydrazine, porphobilinogen-oxygenase activity also appeared in the blood cells. Although the reticulocyte concentration was higher when compared to that obtained under hypoxia, the activities of the oxygenase obtained under both conditions were comparable. Porphobilinogen-deaminase activity was always closely related to the reticulocyte content. The appearance of porphobilinogen-oxygenase under the described erythropoietic conditions was due to a de novo induction of the enzyme, as shown by its inhibition with actinomycin D and cycloheximide. Porphobilinogen-oxygenase as well as porphobilinogen-deaminase were present in the rat bone marrow under normal conditions. Their activities increased in phenylhydrazine treated rats. The properties and kinetics of porphobilinogen-oxygenase from the rat blood and bone marrow were determined and found to differ in several aspects.     

Effect of glucose on the induction of δ-aminolevulinic acid synthase and ferrochelatase in isolated rat hepatocytes by allylisopropylacetamide

The present work shows that allylisopropylacetamide exerts an inducing effect on δ-aminolevulinic acid synthase and ferrochelatase activities in isolated rat hepatocytes of normal adult rats. Dibutyryl cyclic AMP enhances the inducing effect produced in both enzymes. Glucose inhibits the induction of δ-aminolevulinic acid synthase and ferrochelatase in this in vitro system. A similar effect was observed with fructose and 2-deoxyglucose. No glucose effect was observed with galactose, mannose, glycerol, pyruvate and lactate. The glucose effect can be reversed with increasing concentrations of dibutyryl cyclic AMP. The simple in vitro method used in the present work promises to be a very useful tool for studies of regulatory mechanisms of porphyrin and heme biosynthesis in hepatocytes under normal and pathological conditions (hepatic porphyrias).     

Studies of porphyrin synthesis in fibroblasts of patients with congenital erythropoietic porphyria and one patient with homozygous coproporphyria

Partial deficiencies in enzymes activity of the heme biosynthesis pathway have been demonstrated in cultured skin fibroblasts and other tissues from patients suffering from congenital erythropoietic porphyria and hereditary coproporphyria. Using a new fluorimetric method, we have assessed quantitatively porphyrin biosynthesis from added δ-aminolevulinic acid in cultured fibroblasts of two congenital erythropoietic porphyria patients and one homozygous case of hereditary coproporphyria. The results were compared with those of the patients' parents and those of normal controls. All the porphyrins synthesized remained within the cells of normal subjects and of patients with congenital erythropoietic porphyria; these porphyrins were mostly (95%) protoporphyrin. The fibroblasts of the patient with homozygous hereditary coproporphyria synthesized the same amount of porphyrins, but only 25% were found within the cells, whereas 75% were found in the medium. The porphyrins found within the cells were coproporphyrin (25%) and protoporphyrin (75%); in the medium, only coproporphyrin was identified.