Ferrochelatase Mutation Research Articles

Seasonal Palmar Keratoderma in Erythropoietic Protoporphyria Indicates Autosomal Recessive Inheritance

Erythropoietic protoporphyria (EPP) is an inherited disorder that results from partial deficiency of ferrochelatase (FECH). It is characterized clinically by acute photosensitivity and, in 2% of patients, liver disease. Inheritance is usually autosomal dominant with low penetrance but is recessive in about 4% of families. A cross-sectional study of 223 patients with EPP in the United Kingdom identified six individuals with palmar keratoderma. We now show that these and three additional patients, from six families, have an inherited subtype of EPP which is characterized by seasonal palmar keratoderma, relatively low erythrocyte protoporphyrin concentrations, and recessive inheritance. No patient had evidence of liver dysfunction; four patients had neurological abnormalities. Patients were hetero- or homoallelic for nine different FECH mutations; four of which were previously unreported. Prokaryotic expression predicted that FECH activities were 2.7-25% (mean 10.6%) of normal. Neither mutation type nor FECH activity provided an explanation for the unusual phenotype. Our findings show that palmar keratoderma is a clinical indicator of recessive EPP, identify a phenotype that occurs in 38% of reported families with recessive EPP that to our knowledge is previously unreported, and suggest that patients with this phenotype may carry a lower risk of liver disease than other patients with recessive EPP.

Biochemical and molecular diagnosis of erythropoietic protoporphyria in an Ashkenazi Jewish family

Erythropoietic protoporphyria (EPP) is a rare hereditary disorder due to a partial deficiency of ferrochelatase (FECH). The genotype of EPP patients features a mutation on one allele of the FECH gene and a common hypomorphic FECH IVS3-48c on the other allele (M/c). The resulting enzyme activity in patients is ∼35% of that in normal individuals. Ferrochelatase deficiency results in the accumulation of protoporphyrin in the skin, which is responsible for the clinical symptom of cutaneous photosensitivity in patients. In this study, we report the identification of a novel FECH mutation delT23 in an 11-member EPP family of Jewish origin. Two EPP siblings shared an identical genotype of delT23/IVS3-48c (M/c). They were both photosensitive and showed highly increased erythrocyte protoporphyrin. The genotype of the patients' mother, who did not present with any EPP clinical symptoms, was delT23/IVS3-48t (M/t). The patients' father, an offspring of consanguineous parents, was homozygous IVS3-48 c/c. He exhibited a mild photosensitivity, and an increase of 4-fold in erythrocyte protoporphyrin. His FECH mRNA amount was 71% of that of genotype t/t. It is the first reported case of an individual with c/c genotype who exhibits both biochemical and clinical indications of EPP. These results suggest that IVS3-48c is a functional variant of ferrochelatase. The clinical symptoms and biochemical abnormalities in the patients' father could be the result of an interaction between genetic and environmental factors. In addition, the frequency of IVS3-48c in the Ashkenazi Jewish population was estimated at 8%, which is similar to that in the European populations.

Gene Dosage Analysis Identifies Large Deletions of the FECH Gene in 10% of Families with Erythropoietic Protoporphyria

Erythropoietic protoporphyria (EPP) is an inherited cutaneous porphyria characterized by partial deficiency of ferrochelatase (FECH), accumulation of protoporphyrin IX in erythrocytes, skin, and liver, and acute photosensitivity. Genetic counseling in EPP requires identification of FECH mutations, but current sequencing-based procedures fail to detect mutations in about one in six families. We have used gene dosage analysis by quantitative PCR to identify large deletions of the FECH gene in 19 (58%) of 33 unrelated UK patients with EPP in whom mutations could not be detected by sequencing. Seven deletions were identified, six of which were previously unreported. Breakpoints were identified for six deletions (c.1-7887-IVS1+2425insTTCA; c.1-9629-IVS1+2437; IVS2-1987-IVS4+352del; c.768-IVS7+244del; IVS7+2784-IVS9+108del; IVS6+2350-TGA+95del). Five breakpoints were in intronic repeat sequences (AluSc, AluSq, AluSx, L1MC4). The remaining deletion (Del Ex3-4) is likely to be a large insertion-deletion. Combining quantitative PCR with routine sequencing increased the sensitivity of mutation detection in 189 unrelated UK patients with EPP from 83% (95% CI: 76-87%) to 93% (CI: 88-96%) (P=0.003). Our findings show that large deletions of the FECH gene are an important cause of EPP. Gene dosage analysis should be incorporated into routine procedures for mutation detection in EPP.

Identification of a ferrochelatase mutation in a Chinese family with erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) is a rare autosomal dominant disorder of heme biosynthesis characterized by a partial decrease in ferrochelatase (FECH) activity leading to excessive accumulation of protoporphyrin. While a majority of EPP patients only exhibit photosensitivity, a small percentage of patients also develop liver complications and need liver transplantation. In this study, we have sequenced the ferrochelatase gene of a Chinese EPP patient who suffered from EPP-related liver complications. A nonsense mutation in exon 4, 343C>T, introducing a premature stop codon at position arginine 115, was identified in the proband as well as her symptomatic mother and brother, but was absent in her father. All the family members with overt photosensitivity also carried the low-expressed allele IVS3-48c, whose prevalence in the Chinese Han population was determined to be 41.35% and which was also functional in producing an aberrant 63 bp insertion. We describe the first FECH mutation identified in the Chinese Han population and report a high frequency of the hypomorphic IVS3-48c allele in China.

Abnormal Expression of Human Mitoferrin (SLC25A37) Is Associated with a Variant of Erythropoietic Protoporphyria.

Mitoferrin (mfrn, slc25a37) is the high-affinity transporter of iron into mitochondria of developing erythroblasts and is essential for normal erythropoiesis. Recently, we described the cloning and characterization of mfrn in the zebrafish frascati (frs) mutant, in which homozygous null mutation leads to profound hypochromic anemia and embryonic lethality (Shaw GC, et al. 2006 Nature 440:96–100). Here we characterize the human MFRN orthologue and implicate a specific disease process purportedly caused by defects in MFRN expression. We studied human MFRN expression by a) analysis of human-tissue northern blots, b) fluorescent immunohistochemistry and confocal microscopy for subcellular localization, c) genetic complementation in MRS3/4 deficient yeast strains, and d) microinjection and rescue efficiency of frs mutant zebrafish. Human MFRN, mapping to chromosome 8p21, shows 82% and 64% similarity at the amino acid level to orthologous mouse and zebrafish Mfrn proteins respectively, and 60% similarity to a paralogous MFRN2 (SLC25A28). MFRN mRNA is highly expressed in fetal and adult hematopoietic tissues, including fetal liver and bone marrow, similar to zebrafish and mice. The MFRN protein localizes to the mitochondria like other SLC25 solute carriers. Ectopic expression of human MFRN capably restores hemoglobinized cells in anemic frs embryos, programs increased 55Fe-labeled heme synthesis in K562 cells, and rescues MRS3/4 mutant yeast for growth on iron-deficient media, demonstrating conservation of MFRN function. We evaluated 6 unrelated probands with a variant of erythropoietic protoporphyria (EPP) who harbor no mutations in ferrochelatase, the genetic cause of classic EPP. Five of the 6 index cases had severe hepatic disease necessitating liver transplantation. Our analysis of all 6 patients revealed the presence of an aberrantly spliced MFRN transcript, which was absent in normal individuals and patients with classic EPP. This aberrant spliceoform results in insertion of a 477 bp fragment from intron 2 and leads to an early nonsense codon after amino acid 156, prematurely truncating the MFRN protein. In contrast to normal MFRN cDNA, the variant MFRN cDNA from EPP patients fails to complement MRS3/4 mutant yeasts in low-iron media, demonstrating its loss-of-function activity. Our data demonstrate conserved function of human MFRN in erythroid mitochondrial iron metabolism and an association between a loss-of-function MFRN transcript and a variant of erythropoietic protoporphyria.

Biochemical Abnormality in Erythropoietic Protoporphyria

Erythropoietic protoporphyria (EPP) is a genetic disorder in which deficient ferrochelatase (FECH) activity causes the excessive production and excretion of protoporphyrin. This in turn causes the major clinical manifestation of EPP, photosensitivity and, in some patients, hepatobiliary disease that may be severe. The objective of this study was to define genotypic determinants of phenotype in EPP. FECH activity was measured in 30 tissue samples from 26 patients with symptomatic EPP to determine the degree of deficient activity. FECH DNA analysis was also done in 26 families with EPP to identify mutations and examine for the presence of a polymorphism (IVS3-48c) that causes low gene expression. The level of residual FECH activity that was measured in tissue samples of patients with symptomatic EPP was <or=30% of the mean normal level in all patients except one. Lowest levels (4-20% normal) were in patients with advanced EPP liver disease. Heterozygous FECH mutations were found in 45 individuals from 26 families with EPP. In 94% of the 32 symptomatic individuals, 15 of whom had liver disease, the polymorphism was present in the nonmutant allele. In 13 asymptomatic patients, the polymorphism was absent. Patients with symptomatic EPP (photosensitivity with/without hepatobiliary disease) usually have a mutation in 1 FECH allele that alters enzyme structure/function, together with a polymorphism in the nonmutant allele that causes low gene expression. This leads to a significant reduction in FECH activity that causes symptomatic disease to develop because of the excess protoporphyrin produced.

Ferrochelatase Mutation Screening by Denaturing High Performance Liquid Chomatography in Idiopathic Acquired Sideroblastic Anemia (Refractory Anemia with Ringed Sideroblasts) and Erythrocytic Protoporphyria.

BACKGROUND: The molecular etiology of idiopathic acquired sideroblastic anemia (IASA), now considered a form of myelodysplastic syndrome, is currently unknown. Romslo et al (Blood 1982) reported a patient with IASA who had moderately elevated free erythrocytic protoporphyrin (FEP); this observation is now frequently made in IASA, helping distinguish IASA (high FEP) from inherited sideroblastosis (low FEP). In addition, rare patients with erythropoietic protoporphyria (EPP)—defined by cutaneous photosensitivity, dramatically elevated FEP levels, and germline point mutations in the ferrochelatase (FECH) gene at 18q21.3—have ringed sideroblasts in their marrow. We hypothesized that patients with IASA might have acquired somatic FECH mutations.METHODS AND RESULTS: We designed a denaturing high performance liquid chromatography (DHPLC) assay to explore this possibility and to avoid problems related to mutation screening in the setting of mixed clonality. To validate the DHPLC assay, the coding region of the FECH gene from 2 molecularly undiagnosed EPP patients without liver disease was analyzed. In one patient, both a heterozygous 69delG mutation (GenBank Accession NM_000140) and heterozygosity for the FECH expression-modulating IVS3-48C/T polymorphism were detected. In the other patient, no FECH coding mutations or polymorphisms were detected, either by DHPLC or conventional dye sequencing. FEP measurements were then obtained on 2 IASA patients and were elevated (65 and 115 mcg/dL; normal 1–10 mcg/dL) with normal urine and fecal porphyrins. Genomic DNA obtained from these 2 patients as well as archival DNA from 30 other patients with IASA was amplified and tested for FECH mutations. No coding region mutations were detected. Synonymous polymorphisms in exon 7 (rs536765) and 9 (rs536560) were found in 6/32 (19%; normal heterozygosity 0.398) and 14/32 (44%; normal heterozygosity 0.402) samples, respectively. The IVS3-48C/T polymorphism (rs2272783) was found in 3/32 (9%) (prevalence in the general population is 11%, Gouya Nat Genet 2002). In addition, 3 intronic polymorphisms not in the refSNP database were detected: IVS8+34 C/T (2/32), IVS8-61delG (5/32), and IVS9-59delA (2/32).CONCLUSION: IASA is not associated with coding mutations in FECH. Elevation of FEP in ASA must instead be due to the lack of mitochondrial iron in the proper form for incorporation into the porphyrin ring; attention should instead focus on factors responsible for maintaining the appropriate redox state and compartmentalization of iron. DHPLC can detect FECH mutations and polymorphisms, but because of the high frequency of the latter and the consequent need to sequence multiple exons, it is not a practical screening tool for studying undiagnosed EPP patients.

Molecular Studies of Liver Disease in Erythropoietic Protoporphyria

The goal of this study was to define molecular determinants of liver disease in erythropoietic protoporphyria (EPP). EPP is a genetic disorder in which deficient ferrochelatase activity causes excessive production of protoporphyrin, which is excreted in bile. Some patients develop liver disease that necessitates transplantation. Ferrochelatase gene analysis was done in 25 families with EPP to identify mutations and a polymorphism (IVS3-48c) that causes low gene expression. Expression of multiple hepatic genes was also examined by DNA microarray analysis in patients who had liver transplantation to identify genes with altered regulation. Heterozygous ferrochelatase mutations were found in 43 individuals. In 94% of 31 symptomatic patients, 15 of whom had liver disease, the polymorphism was also present in the nonmutant allele. Explanted liver of patients who had transplantation showed significant change in expression of several genes involved in wound healing, organic anion transport, and oxidative stress. Patients with EPP who develop liver disease usually have a mutation in one ferrochelatase allele that alters enzyme function, together with a polymorphism in the nonmutant allele that causes low gene expression. This results in significant increase in the hepatobiliary excretion of protoporphyrin, which can damage the liver through both cholestatic injury and oxidative stress.

Genotypic determinants of phenotype in North American patients with erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) is characterized by excess accumulation of protoporphyrin, which is due to deficient activity of the enzyme ferrochelatase (FECH). This results in photosensitivity and in some patients liver disease which may necessitate liver transplantation. The aim of this study was to delineate the abnormalities in the FECH gene which cause phenotypic expression in EPP. We identified 43 individuals from 25 North American families with EPP who were heterozygous for various FECH mutations, but the mutations did not adequately explain the variable phenotype. We also examined the presence of an intron polymorphism (IVS3-48c) in the FECH gene which was shown to cause the formation of aberrantly spliced FECH mRNA [1]. FECH DNA analysis demonstrated that 94% of 31 symptomatic individuals with FECH mutations were heterozygous for IVS3-48c, whereas 12 asymptomatic individuals with FECH mutations were homozygous for IVS3-48t. Haplotype analysis in four families showed that symptomatic members had the IVS3-48c polymorphism in the non-mutant FECH allele. Sequencing of the proximal FECH gene promoter showed no additional changes which might affect gene expression. The levels of normal FECH mRNA, measured by relative quantitative RT-PCR, and FECH enzyme activity were correspondingly lower in the cultured lymphoblasts of family members with the IVS3-48c polymorphism. These results indicate that symptomatic disease in most North American patients with EPP is explained by the inheritance of a mutation in one FECH allele which causes a structural alteration in the protein, together with a low expressing non-mutant FECH allele which is caused by the IVS3-48c polymorphism.

A new ferrochelatase mutation combined with low expression alleles in a Japanese patient with erythropoietic protoporphyria

A new ferrochelatase mutation combined with low expression alleles in a Japanese patient with erythropoietic protoporphyria

A new ferrochelatase mutation combined with low expression alleles in a Japanese patient with erythropoietic protoporphyria

We investigated the molecular defect of the ferrochelatase gene in a Japanese patient with erythropoietic protoporphyria (EPP), and identified a novel 16 base pair (574-589) deletion within exon 5. This deletion resulted in a frame-shift mutation and created a premature stop codon at amino acid position 198. The same molecular defect was also identified in his mother and a brother who had symptomatic EPP, but not in his father who was asymptomatic. The subjects with EPP were homozygous for the low expression haplotype, while his father was heterozygous for this haplotype. These results indicate that the combination of a 16 base pair deletion and low expression of the wild-type allelic variant is responsible for EPP in this pedigree.