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Biochemical Abnormality in Erythropoietic Protoporphyria: Cause and Consequences

Bloomer, Joseph R.; Wang, Yongming; Singhal, Anuj; Risheg, Hiba

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Journal of Pediatric Gastroenterology and Nutrition: July 2006 - Volume 43 - Issue 1 - p S36-S40
doi: 10.1097/01.mpg.0000226388.56612.fa
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The porphyrias are a group of genetic/metabolic disorders in which abnormalities of the heme biosynthetic pathway cause the excessive production and excretion of porphyrins and/or the porphyrin precursors delta-aminolevulinic acid (ALA) and porphobilinogen (PBG). Clinical reports of the porphyrias first appeared in the latter part of the 19th century. During the 20th century, the clinical and biochemical features of the individual porphyrias were described. Each was found to have a unique pattern of biochemical abnormalities that could be used to establish the diagnosis in an individual with consistent clinical features. A major breakthrough in understanding the pathogenesis of the biochemical abnormalities was made in 1970, when it was demonstrated that patients with acute intermittent porphyria have a deficiency of porphobilinogen deaminase activity, which causes increased urinary excretion of ALA and PBG (1). Following this, a defect in an enzymatic step of heme biosynthesis was defined for each of the other porphyrias (Table 1).

Gene mutations in the porphyrias

Although the clinical features of the porphyrias are protean, they can be grouped into the categories of neurovisceral dysfunction (responsible for the acute porphyric attack), photocutaneous lesions and structural liver disease. The clinical features are linked to the biochemical abnormalities. The acute porphyrias, in which neurovisceral dysfunction occurs, all have increased urinary excretion of ALA and, with one exception, PBG. Clinical and experimental studies indicate that ALA may be acting as a neurotoxin. Another possibility to explain neurovisceral dysfunction is that a heme deficiency state in liver leads to altered nerve function (2). Photocutaneous lesions in the porphyrias are caused by the phototoxic properties of porphyrins in blood circulating through dermal blood vessels (3). Structural liver damage, which occurs in patients with porphyria cutanea tarda and some patients with erythropoietic protoporphyria (EPP), also may be caused by the toxic effects of porphyrins.

Because there is a link between the biochemical abnormalities and clinical features in the porphyrias, study of the molecular pathogenesis of the biochemical abnormalities should help in understanding phenotype and may also provide the basis for molecular therapy. The cloning and sequencing of DNA that encodes the enzymes of the heme biosynthetic pathway has made it possible to identify the gene mutations that cause the enzyme defects that underlie the biochemical abnormalities (Table 1). Acute intermittent porphyria has been most intensively studied, and more than 200 different mutations have been identified in the porphobilinogen deaminase gene. The mutations identified cause abnormal splicing of mRNA, insertions or deletions in exons that cause premature termination of protein synthesis, single nucleotide changes that produce stop codons and prevent complete translation of mRNA and single nucleotide changes that cause amino acid substitutions and thus alter catalytic activity of the enzyme (4). Genetic heterogeneity has also been demonstrated in the other types of porphyria. Unfortunately, the different mutations found have not satisfactorily explained the different phenotypic manifestations of the disorder that occur in patients. In acute intermittent porphyria, 80-90% of individuals who carry the gene defect do not have clinical manifestations of the disease, and approximately two thirds have neither clinical nor biochemical abnormalities (4). Thus, the gene mutation is necessary but not sufficient to explain the phenotypic severity that occurs, and other genetic and/or acquired factors are also important.

This article will focus on the current understanding of the molecular basis of biochemical abnormalities in EPP, which was first described in 1961 (5). The major clinical feature is acute photosensitivity that causes painful swelling and erythema of skin exposed to sunlight. Some patients also develop hepatobiliary disease because of the toxic effect of protoporphyrin on the liver and the biliary tract when this compound is excreted in bile (6-9). The enzyme abnormality that underlies overproduction of protoporphyrin is a deficiency of ferrochelatase (FECH) activity (10,11), which catalyzes the insertion of ferrous iron into protoporphyrin to form heme. FECH mRNA has an open reading frame of 1269 base pairs that encode a protein of 423 amino acid residuals (12). The FECH gene contains 11 exons and maps to chromosome 18 (13).


Patient Population

The patient population consisted of individuals from North American families in which at least one family member had symptomatic EPP. The diagnosis was made on the basis of characteristic photosensitivity and elevated red blood cell protoporphyrin level (>100 μg/dL in this laboratory). The age range was 10-91 years. Fifteen individuals had liver disease, in 11 of whom this necessitated liver transplantation.

Measurement of FECH Activity

Ferrochelatase activity was measured in liver tissue from 12 patients, cultured skin fibroblasts from 7 patients and Epstein-Barr virus (EBV)-transformed lymphoblasts from 11 patients. Four patients had measurement done in both liver tissue and lymphoblasts.

Three different assays were used to measure FECH activity. These were a radiochemical assay which measures the formation of radiolabeled heme (11). a pyridine hemochromogen assay which measures the formation of deuteroheme (14) and a fluorometric assay which measures the formation of zinc deuteroporphyrin (14) To combine the results of these different studies, the level of FECH activity was expressed as a percent of the mean normal value established for the method used. Some of this data has been previously published (14,15).

FECH DNA Analysis

Total RNA was isolated from liver tissue, peripheral white blood cells and EBV-transformed lymphoblasts by Trio reagent (Life Technologies, Rockville, MD). Genomic DNA was isolated using the Pure-Gene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). Seven specific primer pairs were used in polymerase chain reactions (PCR) to amplify and sequence FECH cDNA using methods previously described (16,17). Amplification and sequencing of the 11 exons of the FECH gene and their flanking intron regions was done on genomic DNA by PCR using oligonucleotides that were specific for the particular exon of interest. Sequencing of the purified PCR products was done using an ABI 373 DNA sequencer (Applied Biosystems, Foster City, CA).

Specific primers were also used to amplify a 212 base pair segment of intron 3 that contains the -48t/c base (16,17). The amplified product was purified using QIA Quick Gel Extraction kit (Quiagen, Valencia, CA) and sequenced on an ABI 373A DNA sequencer (Applied Biosystems, Inc, Foster City, CA). The amplified product was also digested with BbVI Restriction Enzyme (New England Biotabs, Beverly, MA) according to the manufacturer's instructions. Individuals who were heterozygous for IVS3-48c (a transition from t > c in intron 3 of the FECH gene) produced 2 additional bands with fragments sizes of 126 and 84 bp.

The results of the FECH DNA analysis have been reported elsewhere in different form (16,17).


The level of FECH activity in liver tissue and/or cells of patients with symptomatic EPP was ≤ 30% of the mean normal level in 29 of the 30 measurements made (Fig. 1). This provided a clue to the possibility that both FECH alleles might be altered in patients with symptomatic EPP. The lowest levels of FECH activity were found in patients with advanced protoporphyric liver disease (Fig. 1), ranging from 4-20% of the mean normal level in liver tissue and 5-15% of the mean normal level in EBV-transformed lymphoblasts. Thus, patients with the most severe phenotype of EPP seem to have the greatest deficiency of FECH activity, which in turn would lead to greater overproduction and excretion of protoporphyrin. FECH DNA analysis helped to clarify the reason for the diminished FECH activity found in tissues of patients with symptomatic EPP (Table 2). There were 45 individuals found to be heterozygous for FECH gene mutations which cause structural defects in the FECH protein. Thirteen were asymptomatic and had either normal or minimally increased red cell protoporphyrin levels. The remaining 32 individuals were symptomatic and included 15 patients with liver disease, 11 of whom have had liver transplantation. None of the individuals with symptomatic EPP, including those with liver disease, was homozygous or compound heterozygous for a FECH mutation. However, the striking difference between asymptomatic and symptomatic individuals was that none of the asymptomatic individuals carried the polymorphism IVS3-48c, whereas 30 of the 32 symptomatic individuals carried the c polymorphism (P < 0.009). Haplotype analysis in 4 families showed that symptomatic members had the IVS3-48c polymorphism in the nonmutant FECH allele, and the levels of normal FECH mRNA were correspondingly lower in their cultured lymphoblasts. Studies by Gouya et al have shown that the c polymorphism at position -48 in intron 3 increases formation of aberrantly spliced FECH mRNA, causing the insertion of 63 base pairs of intron 3 between exons 3 and 4 (18). This transcript contains a stop codon and is rapidly degraded by a surveillance mechanism known as nonsense mediated mRNA decay (18).

FIG. 1
FIG. 1:
FECH activity in liver tissue and cells of patients with symptomatic EPP is significantly decreased compared with that in normal tissue. Measurements in 30 samples from 26 patients with symptomatic EPP showed that levels were ≤30% of the mean normal level in all patients except one. The lowest levels of FECH activity were found in patients with advanced EPP liver disease, shown by the closed circles.
FIG. 2
FIG. 2:
FECH DNA analysis is shown for a family in which the 18-year old proband developed severe EPP liver disease that required liver transplantation. The patient inherited a splicing mutation (1135A→T that caused skipping of exon 10) from her mother, as did her asymptomatic brother. The proband also inherited a polymorphism in the nonmutant allele from her father that causes low expression of the allele (IVS3-48c). Her asymptomatic brother and asymptomatic mother both had the normal polymorphism (IVS3-48t) in the nonmutant allele.
FECH DNA analysis in EPP

Thus, most symptomatic patients with EPP have a mutation in 1 FECH allele which causes a structural alteration in the protein, together with a polymorphism in the nonmutant FECH allele which lowers its expression. This combination significantly decreases the amount of normal FECH protein made, causing a reduction of FECH activity to less than 30% of normal.


DNA analysis in patients with EPP has identified several different mutations (genetic heterogeneity) (14-20), as has been the situation in the other types of porphyria. Although the mutations found in patients with liver disease, which is the most severe phenotype in EPP, are most frequently those that abolish enzyme activity (null mutation) through a major alteration of the protein structure (Table 2), the same mutations are seen in patients with less severe phenotype and in individuals who are asymptomatic. Thus, heterozygous mutations in the FECH gene by themselves do not satisfactorily explain the variance of phenotype in EPP. However, patients with symptomatic EPP usually also have a polymorphism in the nonmutant allele that causes low expression of the allele. The combination of mutation and polymorphism produces a significant reduction of FECH activity, which brings out symptomatic disease.

This finding is of considerable significance when genetic counseling is provided to a patient with symptomatic EPP and his/her family members. If the symptomatic patient has both the mutation and the polymorphism in FECH DNA, it does not necessarily mean that his/her children will have severe EPP unless the spouse has the polymorphism, which occurs in approximately 10% of the normal population (17,18). Conversely, asymptomatic family members who carry only the mutation can have children with severe EPP if the spouse carries the polymorphism. Thus, the patient and other family members should be advised that FECH DNA analysis in them and their spouses should be done to provide more precise genetic counseling.

However, the combination of the FECH mutation and polymorphism still does not satisfactorily explain why some symptomatic individuals develop severe liver disease, whereas others only have photosensitivity. Thus, there are likely other genetic, epigenetic and acquired factors that affect disease severity. The proximal 5-untranslated region of the FECH gene contains a CpG island that houses the minimal promoter and 2 Sp1 transcription factor-binding sites (21,22), and methylation within this region could be another reason for variable disease expression among individuals with symptomatic EPP (23). There could also be a difference in transacting factors that modulate FECH gene expression and in genes with products that alter FECH enzyme activity. These additional factors are yet to be identified and will be important to a more complete understanding of the severity of disease expression in EPP.

Anything which affects protoporphyrin uptake and excretion by the liver could also alter the severity of phenotype in EPP. Acquired factors that may impair protoporphyrin excretion include alcoholism (24), drugs (6) and other liver disease such as viral hepatitis (25). Endogenous factors include hepatobiliary transport proteins, particularly those involved in transport and canalicular secretion of protoporphyrin (26-31). However, the fact that there is a high recurrence of EPP liver disease in the graft after liver transplantation suggests that genetic defects in hepatic protoporphyrin excretion are unlikely to be of major significance (32).


This study was supported by Research Grant DK026466 from the National Institutes of Health. The authors thank Sheri McFall for her excellent assistance in preparation of the manuscript.


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Erythropoietic protoporphyria; Heme biosynthesis; Ferrochelatase activity; Ferrochelatase DNA analysis

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