Clinical, Biochemical, and Genetic Study of 11 Patients With Erythropoietic Protoporphyria Including One With Homozygous Disease FREE

Erythropoietic protoporphyria (EPP) (Mendelian Inheritance in Man 177000) is an inherited disorder of porphyrin metabolism caused by a partial deficiency of ferrochelatase (FECH) (European Community 4.99.1.1), which catalyzes the insertion of iron into protoporphyrin IX to form heme. Patients with EPP accumulate free protoporphyrin mainly in erythropoietic tissues, but the metabolite may also reach the skin, absorb light at certain wavelengths, and generate free radicals, resulting in phototoxic cell injury. Most patients with EPP develop solely photosensitivity commencing in childhood, but in some cases liver complications also appear. The strong hydrophobic nature of the molecule does not allow renal clearance but only bile secretion. Therefore, massive hepatic accumulation of protoporphyrin in the liver may cause cholestasis and liver damage in a minority of patients with EPP. Hepatic involvement may vary in severity, ranging from abnormalities in 20% of the cases to cirrhosis and liver failure in less than 5%.^1- 3 Blood disorders such as mild anemia with hypochromia and microcytosis may also be detected in 20% to 50% of these patients.^2,4- 5

The FECH gene is assigned to chromosome 18q21.3 (GenBank No. D00726), with more than 110 different mutations having been described in EPP families to date.⁶ Individuals who are heterozygous for these mutations remain asymptomatic, despite having FECH activity reduced by about 50%. Clinical expression of the disease in most cases results from the coinheritance of a null FECH allele plus a wild-type low-expressed allele, which further reduces FECH activity to below a critical threshold of about 35%.⁷ In a haplotype segregation analysis,⁸ it was found that the low-expression allele is the C variant of an intronic single-nucleotide polymorphism (SNP) IVS3-48T/C in intron 3 of the FECH gene. Because the IVS3-48C allele is relatively common among European populations,⁶ overt EPP is normally inherited in such a way that it resembles an autosomal dominant disease with low clinical penetrance.

Erythropoietic protoporphyria is rarely inherited as an autosomal recessive disease with true mutations in both alleles.^9- 12

In some areas of southern Europe, the prevalence of EPP and the genetic defects underlying the disease are not sufficiently elucidated. Therefore, we investigated the mutations in the FECH gene and the phenotypic expression of the disease in a series of Spanish patients with EPP. The molecular, biochemical, and clinical study included a rare case of recessive EPP.

We studied 11 unrelated patients (6 female and 5 male, with a mean age of 12.8 years [range, 4-26 years]) who attended the Department of Dermatology of the Hospital Clinic of Barcelona (Barcelona, Spain) for photosensitivity and suspicion of porphyria. The diagnosis of EPP was established initially by clinical and biochemical criteria and was later confirmed by DNA analysis of mutations in the FECH gene. A complete hematological and biochemical profile and specific liver function tests were performed in every case. In addition, 19 asymptomatic relatives from 8 families were also included in the genetic study. The study was conducted in accordance with the Declaration of Helsinki principles and was approved by the hospital ethics committee of the Hospital Clinic of Barcelona. Written consent was obtained from all patients.

Protoporphyrin concentration in erythrocytes was determined fluorometrically after solvent partition according to the method described elsewhere.^13- 15 Protoporphyrin in feces was extracted according to Lockwood et al¹⁶ and was analyzed by high-performance liquid chromatography according to Lim and Peters.¹⁷ Porphyrins in urine were analyzed by standard methods of high-performance liquid chromatography.¹⁷

FECH activity in lymphocytes was determined by incubating a homogenate with zinc and mesoporphyrin and measuring zinc-mesoporphyrin formation by fluorometry according essentially to the method of Rossi et al,¹⁸ as modified by Gouya el al.⁶FECH activity was expressed as nanomoles of zinc-mesoporphyrin formed per hour per milligram of protein at 37°C.

We analyzed exons 1 to 11 of the FECH gene and the associated splice donor and acceptor sites. The primers used for exon amplification are available from the authors. All polymerase chain reaction (PCR) analyses were carried out using the PCR Master Mix (Promega Corporation, Madison, Wisconsin) following the manufacturer's instructions. The following PCR conditions were applied: initial denaturizing step (5 minutes at 95°C), 35 cycles (1 minute at 95°C, 1 minute at 57°C, and 1 minute at 72°C), followed by final extension (10 minutes at 72°C) and maintenance at 4°C until single-strand conformational polymorphism or sequencing studies were carried out. Part of intron 3 was sequenced in all samples to test for the IVS3-48T/C polymorphism.

Single-strand conformation analysis was used to detect mutations. Three microliters of denatured PCR product was combined with loading buffer and loaded into GeneGel Excel 12.5 acrylamide gels (Amersham Biosciences, Uppsala, Sweden) and run following the manufacturer's instructions. The different migrations were silver stained.¹⁹ DNA samples with abnormal migrations were sequenced. The PCR products were purified using the GFX PCR DNA and gel band purification kit (Amersham Biosciences) and automatically sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California) and an ABI3100 automatic sequencer (Applied Biosystems).

Mature ferrochelatase was expressed in Escherichia coli strain JM109 (Promega Corporation) using the expression vector pKK223-3 (Pharmacia Biotech Inc, Piscataway, New Jersey). The wild-type human ferrochelatase complementary DNA (cDNA) was synthesized by PCR excluding the region encoding the mitochondrial leader sequence and including a new ATG start site of translation, and cloned into the EcoRI-HindIII sites of pKK223-3. This construct was designated pKK-FECH-wt. To generate the mutant construct pKK-FECH-A185T, a fragment of FECH cDNA, containing the A185T mutation was generated by PCR site-directed mutagenes²⁰ using the pKK-FECH-wt plasmid as templated. Then, the PCR product was digested with the restriction endonucleases MfeI and CveI (New England Biolabs, Beverly, Massachusetts) and the MfeI-CveI fragment was ligated as a cassette into the corresponding sites in pKK-FECH-wt, and transformed in E coli strain JM109. The integrity of each expression construct was checked by automated sequencing. The primers are available from the authors.

Bacterial clones, each of them containing one of the plasmids pKK223-3, pKK-FECH-wt, or pKK-FECH-A185T, were grown to log phase and were induced with 5mM isopropylthiogalactoside for 3 hours. Then, cells were harvested by centrifugation and washed twice in phosphate-buffered saline. The pellets were resuspended in 250 μL of lysis buffer (100mM Tris hydrochloride, pH 7.6; 0.1% Triton-X 100) and sonicated on ice. After centrifugation, ferrochelatase activity was measured in the supernatant using protoporphyrin IX and zinc sulfate as substrates, according to the method described by Camadro and Labbe.²¹

The main clinical and biochemical outcomes of the patients with EPP are given in the Table. All presented acute cutaneous photosensitivity, with the symptoms beginning in early childhood in most cases. Only 3 cases presented with chronic skin thickening over the knuckles. Microcytic anemia was present in 4 cases. Liver function test results were persistently normal in 10 cases, most with a long clinical follow-up (mean, 8.6 years; range 1-26 years). Only 1 case showed alterations in the liver function test results. The main characteristics of this patient are described in the “Report of a Case” subsection.

Ten different mutations in the FECH gene were found, thereby confirming the molecular heterogeneity of EPP (Table). Four novel mutations were found: 553G>A in exon 5 (A185T); IVS4 + 1delG and 347-351delC, both in exon 4; and 130_147dupl 18 in exon 1. The A185T mutation was homozygous, while the other mutations were heterozygous. Only the already described 575del5 mutation was present in 2 patients. Patients 7, 8, 9, and 10 (I186T, H386P, 843delC, and R115X, respectively) were previously studied by Gouya et al,²² but the status of the SNP of the allele trans to mutant allele was not determined in that study. All patients, except patient 1 who was homozygous for IVS3-48T/T, were heterozygous for IVS3-48C/T.

The A185T mutation was expressed in E coli.FECH specific activity was determined in 4 independent experiments. The residual activity was calculated from the value of the normal FECH construct (pKK-FECH-wt) and was 4% of normal activity.

Molecular analysis of 19 asymptomatic relatives from patients 1 through 6 and 8 through 10 were performed. This allowed the identification of 10 relatives who were carriers of heterozygous mutations. Inspection of the intron 3 SNP showed that none harbored the IVS3-48C allele. All of these relatives had normal values of protoporphyrin in blood. Analysis of 180 nonporphyric unrelated Spanish individuals showed that the IVS3-48C allele was present in 10.5% (allele frequency, 5.2%).

Patient 1 was a 15-year-old girl with microcytic anemia since she was 4 months old and photosensitive dermatitis since she was 4 years old. When she was 10 years old, alterations in liver function had been detected, with the persistence of microcytic anemia. She presented an itchy erythema and edema on the hands and face after sun exposure. On examination, pitted and linear scars on the nose, cheeks, and forehead and mild skin thickening over the knuckles were observed. There was no family history of photosensitivity and no apparent familial consanguinity.

Results from porphyrin analyses included the following: erythrocyte protoporphyrin level, 32 μmol/L (reference value, < 1.5 μmol/L; > 90% free protoporphyrin); and fecal protoporphyrin level, 1066 nmol/g of dried feces (reference value, < 100 nmol/g), which allowed for the diagnosis of EPP. The excretion of coproporphyrins in urine was normal. Results from biochemical analysis included the following: aspartate transaminase level, 52 U/L (reference value, < 40 U/L), alanine aminotransferase level, 109 U/L (reference value, < 40 U/L), and γ-glutamyltransferase level, 57 U/L (reference value, < 40 U/L) (to convert to microkatals per liter, multiply by 0.0167); hemoglobin level, 11.5 g/dL (reference range, 12.0-17.0 g/dL) (to convert to grams per liter, multiply by 10); median corpuscular volume, 73 fL (reference range, 80-100 fL); median corpuscular hemoglobin concentration, 22.7 pg (26.7-33.3 pg); serum iron level, 59 μg/dL (reference range, 50-150 μg/dL) (to convert to micromoles per liter, multiply by 0.179); ferritin level, 28 ng/mL (reference range, 15-200 ng/mL) (to convert to picomoles per liter, multiply by 2.247); and soluble transferrin receptor level, −0.089 mg/dL (reference range, 0.083-0.176 mg/dL) (to convert to micromoles per liter, multiply by 0.0123). The appearance of the liver on echography was normal. A hepatic biopsy revealed normal liver architecture and the presence of brown material without birefringence at polarized light. Hemoglobin electrophoresis demonstrated normal percentages of hemoglobin A₂ and F, and findings from DNA analysis excluded the thalassemia trait. Bone marrow examination showed normal percentages of the hematopoietic cell lineages with dysplasia signs in the erythroid cells. The bone marrow karyotype was 46,XX[20].

Findings from a molecular study of the DNA showed that this patient was apparently homozygous for a novel A185T missense mutation and homozygous for the T variant of the IVS3-48C/T polymorphism. The residual activity of the mutant FECH in E coli was 4% of normal activity. The FECH activity in peripheral lymphocytes was 0.4 nmol of zinc-mesoporphyrin per hour per milligram of protein at 37°C (reference value, mean ± SD, 4.5 ± 1). Two years later, the erythrocyte protoporphyrin level had increased to 77 μmol/L (fecal protoporphyrin level, 1221 nmol/g of dried feces).

Familial investigation showed that both parents and a sister were heterozygous for the A185T mutation, and none harbored the low-expression IVS3-48C allele, thus confirming recessive EPP. All were asymptomatic and had an erythrocyte protoporphyrin concentration of less than 1.5 μmol/L.

We present the clinical, biochemical, and molecular characteristics of 11 cases of EPP and 19 asymptomatic relatives from 10 Spanish families. Among the series of patients with EPP, 1 had homozygous disease. Recessive inheritance of EPP, with patients presenting mutations in both alleles is rare. Only 13 cases (12 families) have been reported to date,^6,9- 12 most being compound heterozygotes. To our knowledge, the patient who was homozygous for an A185T mutation is the second case of true homozygous EPP, with the same mutation in both alleles confirmed by molecular analysis.¹² The A185T mutation is located within the β₃-strand of the enzyme and may produce a significant structural alteration owing to the replacement of a hydrophobic amino acid by a polar amino acid. Although the alanine residue at this position is not invariant among different species, it is conserved in mammals, yeast, and some prokaryotic species.^23- 24 Expression of the A185T allele revealed a low but still detectable residual activity of 4% in a crude bacterial extract. These data indicate that the mutation produces important structural and/or functional consequences in the FECH enzyme but is not completely deleterious.

It has been suggested that recessive EPP forms may have a higher risk of liver failure than the more usual dominant forms.¹⁰ According to published reports, severe liver disease has been observed in 4 of 13 patients with recessive EPP detected to date.^6,10,12 This is a high proportion, taking into account that liver failure appears in only in less than 5% of patients with EPP dominant forms.^6,25

The determinants of liver injury in EPP are not well understood because genetic factors may modulate the accumulation of protoporphyrin in the liver.²⁶ It is likely, however, that the amount of residual FECH activity in cells^12,25 may play a key role in determining the course of the disease. Most of the missense mutations found in heterozygous EPP cases show a drastic reduction or even undetectable activity when expressed in prokaryotes.^27- 29 In contrast, in a report of recessive EPP, all the mutations retained a FECH residual activity ranging from 12% to 50% when expressed in E coli.¹² This is in accordance with the general view that homozygous cases of porphyria must retain a substantial residual enzyme activity to be compatible with life. The A185T mutation, however, showed only 4% of the normal activity in E coli, thus suggesting a severe impairment in functionality. Consequently, even if this homozygous patient is free of cholestasis signs and the liver markers remain stable to date, the progression of the disease should be considered of risk and advocates close clinical follow-up.

Except for the recessive case, all the other patients in our study showed mutations in heterozygosity within the FECH gene. Some of the new mutations can also cause severe impairment in enzyme activity. The c.351delC mutation is a single C deletion from a stretch of 5 C that causes a frameshift and introduces a premature termination codon of translation in exon 4. As a consequence, we can predict the rapid degradation of the mutant transcript due to nonsense-mediated messenger RNA decay.^30- 31 The c.130_147dupl 18 mutation does not result in a frameshift, but its location within the mitochondrial leader sequence could result in a defective targeting to mitochondria or a deficient processing of the polypeptide. The novel splicing defect IVS4 + 1delG affects the consensus donor splice site of intron 4.³² Unfortunately, RNA from this patient was not available for analysis. Nonetheless, this mutation probably results in exon 4 skipping, as was observed in a previously reported mutation (IVS4 + 1G > C) in this donor site.²⁷

All heterozygous cases presented the low-expression IVS3-48C allele, thereby confirming the common view that clinically overt EPP is associated with inheritance of this allele in trans to a severe FECH mutation.^6,12,33- 34 The study of the relatives yielded additional confirmation of the importance of this polymorphism in EPP, since all asymptomatic carriers had the IVS3-48T/T genotype. Familial investigation of patient 1 showed that several relatives harbored the A185T mutation, but none had the IVS3-48C allele. Therefore, the phenotype of a hypothetical carrier of the A185T mutation with the low-expression allele in trans remains unknown.

In conclusion, among our series of Spanish patients with EPP, we have found 10 cases with coinheritance of 1 FECH gene mutation with IVS3-48C and 1 rare case with a severe homozygous mutation with very low residual activity. This patient presented a cutaneous photosensitivity similar to the heterozygous cases but had a higher protoporphyrin accumulation, pronounced microcytic anemia, and early signs of liver involvement. These findings emphasize the need for further research on the genotypic-phenotypic relationships in porphyria.

Correspondence: Jordi To-Figueras, PhD, Department of Biochemistry and Molecular Genetics, Villarroel 170, Hospital Clinic Barcelona, 08036 Barcelona, Spain (JTO@clinic.ub.es).

Financial Disclosure: None reported.

Accepted for Publication: January 26, 2007.

Author Contributions: Drs To-Figueras and Carmen Herrero had full access to all the data and take full responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Herrero, To-Figueras, Méndez, Enríquez-Salamanca, and Lecha. Acquisition of data: Herrero, To-Figueras, Badenas, Méndez, and Serrano. Analysis and interpretation of data: Herrero, To-Figueras, and Méndez. Drafting of the manuscript: Herrero, To-Figueras, Méndez, and Serrano. Critical revision of the manuscript for important intellectual content: Badenas, Enríquez-Salamanca, and Lecha. Obtained funding: Herrero and To-Figueras. Administrative, technical, and material support: Badenas. Study supervision: Méndez, Enríquez-Salamanca, and Lecha.

Funding/Support: This study was supported in part by grants from “Fondo de Investigación Sanitaria” (03/0489) and “Fundación Mutua Madrileña.” Both are nonprofit organizations that supported the University-based research as a contribution to health progress.

Role of the Sponsors: The sponsors did not participate in the conduction of the study; collection, management, analysis, interpretation of the data; or preparation, review, or approval of the manuscript.

Additional Contributions: We appreciate the participation of the family members in this study. M. Bruguera, MD, provided help in the control of hepatic disease, J. M, Mascaró, MD, collaborated in the recruitment of patients, and M. Sala provided technical assistance.