Ferrochelatase Mutation Research Articles

Two Novel FECH Mutations in a Patient with Erythropoietic Protoporphyria

Introduction: Erythropoietic protoporphyria (EPP) is a rare photodermatosis mainly caused by deficiency of the enzyme ferrochelatase (FECH). We herein report a case of EPP associated with two novel mutations in FECH. Case presentation: A 15-year-old boy experienced pain and pruritus after sunlight exposure. He had occasional claret-red urine, hepatomegaly with increased alanine aminotransferase and aspartate aminotransferase levels, and an elevated free erythrocyte protoporphyrin level. He was treated with oral β-carotene and cholestyramine and avoidance of sunlight as much as possible. Discussion: Genome sequencing revealed two novel FECH mutations that had been inherited from his healthy parents. Pathogenicity analysis involving prediction using PolyPhen-2, SIFT, and Mutation Taster revealed that the two novel mutations were likely pathogenic. Although the patient’s parents were healthy, they each had one of these two mutations. This finding is consistent with previous reports stating that individuals carrying low-expression alleles can be asymptomatic. The pathogenesis of the disease caused by these two mutations requires verification by larger and more detailed studies. Conclusion: Although the precise role of these mutations in EPP is not clear, the findings in the present case expand the genotypic spectrum of the disease.

Digital PCR Assay to Assess the Alternative Splicing and Absolute Ferrochelatase Expression

Ferrochelatase (FECH), the last enzyme of heme biosynthetic pathway, is responsible for the insertion of ferrous iron into protoporphyrin IX (PPIX) to form a molecule of heme. Mutations in the FECH gene cause Erythropoietic Protoporphyria (EPP, MIM#17700), which is clinically characterized by acute and painful cutaneous photosensibility and biochemically by the accumulation of PPIX in erythrocytes and plasma. The autosomal recessive form of EPP requires the presence of FECH mutations in homozygosis or a single mutation in trans to a ‘low expressed allele’. This allele originates from an abnormal event of alternative splicing of FECH intron 3, creating an aberrant mRNA that has a non-sense mediated decay. The presence of the c.315-48T>C polymorphism in intron 3 is able to raise the physiologic amount of this aberrant splicing, causing EPP when associated to a FECH mutation.To evaluate the role of the aberrant splicing on FECH expression, we developed a Digital PCR assay which allows to evaluate in vivo the absolute quantification of the aberrant splicing and the FECH global expression. In this study 91 subjects were analyzed: 27 EPP patients (c.315-48T>C+ mutation), 13 carrying only the FECH mutation, 17 carriers of c.315-48T>C, 5 homozygous for c.315-48T>C and 29 wild type subjects as control. Digital PCR on mRNA was performed in duplex with two differently labelled probes: one specific for the normal FECH mRNA; the second one was specifically designed to detect the inserted mRNA. Data analysis was performed with ordinary one-way ANOVA with Tukey-Dunnet's post-hoc test; nominal statistical significance was set at p<0.05.We observed that the percentage of the aberrant mRNA is lower compared to what is reported in literature (Gouya et al. 2002 - Barman-Aksozen J. et al. 2013) both in wild type and in c.315-48T>C carriers, even thought the aberrant mRNA in the latter is doubled compared to the wild type (Tab.1). A significant difference was observed (Fig.1a) when comparing wild type class to other classes: vs c.315-48T>C (p<0.0001), vs homozygote for c.315-48T>C (p<0.0001) and vs c.315-48T>C+mut (p<0.0001). These data were confirmed when analyzing the number of single events of insertion. Interestingly, there was no significant difference between wild type subjects and carriers of FECH mutations.Evaluating the total FECH expression, a significant difference were observed when comparing the wild type class vs c.315-48T>C+mut class (p<0.0001) and vs homozygotes for c.315-48T>C class (p=0.0085) (Fig.1b). The same was true when we compared the class of FECH mutation carriers vs c.315-48T>C+mut class (p=0.0004) and vs the class of homozygotes for c.315-48T>C (p=0.0189) (Fig. 1c). These data suggest that homozygosis for c.315-48T>C could be considered pathological. As a matter of fact in this cohort, 4 out of 5 homozygous cases for c.315-48T>C presented with typical clinical signs of EPP.In conclusion, it is known that c.315-48T>C reduces the global FECH expression to a pathologic level when associated to a FECH mutation in trans, but alone in heterozygosity has no effect, whereas in homozygosis, the c.315-48T>C is able to reduce the FECH gene expression to a pathologic level, causing EPP phenotype. [Display omitted] DisclosuresCappellini:Sanofi-Genzyme: Honoraria, Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Celgene: Honoraria; Vifor: Honoraria.

Attenti al sole e pensa al fegato: due casi pediatrici di protoporfiria eritropoietica

The article reports two cases of a rare paediatric disease: erythropoietic protoporphyria (EPP) characterized by burning erythema and variable liver damage. EPP is the most common porphyria in children, caused by mutations in ferrochelatase (FECH) gene, which is the last enzyme along the haem biosynthetic pathway. Consequently, haem precursor protoporphyrin IX accumulates in erythrocytes, plasma, skin and liver. The clinical features are: photosensitivity (pain, itching, burning along with oedema and/or erythema in sun-exposed areas), mild anaemia, and a variable degree of hepato-biliary disease with potential cholelithiasis and, in 1-5% of patients, end-stage liver disease. Diagnosis is made by finding elevated erythrocyte protoporphyrin and confirmed by molecular genetic testing in FECH gene. Therapy is based on sun protection, vitamin D, and beta-carotene, along with ursodeoxycholic acid and/or cholestyramine if hepatobiliary disease is present. Afamelanotide and cimetidine have shown efficacy in treating photo-sensitivity, but do not prevent liver damage, so that a tight follow-up is needed.

Erythroid-Progenitor-Targeted Gene Therapy Using Bifunctional TFR1 Ligand-Peptides in Human Erythropoietic Protoporphyria

Erythropoietic protoporphyria (EPP) is a hereditary disease characterized by a deficiency in ferrochelatase (FECH) activity. FECH activity is responsible for the accumulation of protoporphyrin IX (PPIX). Without etiopathogenic treatment, EPP manifests as severe photosensitivity. 95% of affected individuals present a hypomorphic FECH allele trans to a loss-of-function (LOF) FECH mutation, resulting in a reduction in FECH activity in erythroblasts below a critical threshold. The hypomorphic allele promotes the use of a cryptic acceptor splice site, generating an aberrant FECH mRNA, which is responsible for the reduced level of wild-type FECH mRNA and, ultimately, FECH activity. We have previously identified an antisense oligonucleotide (AON), AON-V1 (V1), that redirects splicing to the physiological acceptor site and reduces the accumulation of PPIX. Here, we developed a specific strategy that uses transferrin receptor 1 (TRF1) as a Trojan horse to deliver V1 to erythroid progenitors. We designed a bifunctional peptide (P1-9R) including a TFR1-targeting peptide coupled to a nine-arginine cell-penetrating peptide (CPP) that facilitates the release of the AON from TFR1 in endosomal vesicles. We demonstrated that the P1-9R/V1 nanocomplex promotes the efficient and prolonged redirection of splicing towards the physiological splice site and subsequent normalization of WT FECH mRNA and protein levels. Finally, the P1-9R/V1 nanocomplex increases WT FECH mRNA production and significantly decreases PPIX accumulation in primary cultures of differentiating erythroid progenitors from an overt EPP-affected individual. P1-9R is a method designed to target erythroid progenitors and represents a potentially powerful tool for the invivo delivery of therapeutic DNA in many erythroid disorders.

Congenital erythropoietic porphyria and erythropoietic protoporphyria: Identification of 7 uroporphyrinogen III synthase and 20 ferrochelatase novel mutations

The erythropoietic porphyrias are inborn errors of heme biosynthesis with prominent cutaneous manifestations. They include autosomal recessive Congenital Erythropoietic Porphyria (CEP) due to loss-of-function (LOF) mutations in the Uroporphyrinogen III Synthase (UROS) gene, Erythropoietic Protoporphyria (EPP) due to LOF mutations in the ferrochelatase (FECH) gene, and X-Linked Protoporphyria (XLP) due to gain-of-function mutations in the terminal exon of the Aminolevulinic Acid Synthase 2 (ALAS2) gene. During the 11-year period from 01/01/2007 through 12/31/2017, the Mount Sinai Porphyrias Diagnostic Laboratory provided molecular diagnostic testing for one or more of these disorders in 628 individuals, including 413 unrelated individuals. Of these 628, 120 patients were tested for CEP, 483 for EPP, and 331 for XLP, for a total of 934 tests. For CEP, 24 of 78 (31%) unrelated individuals tested had UROS mutations, including seven novel mutations. For EPP, 239 of 362 (66%) unrelated individuals tested had pathogenic FECH mutations, including twenty novel mutations. The IVS3-48 T > C low-expression allele was present in 231 (97%) of 239 mutation-positive EPP probands with a pathogenic FECH mutation. In the remaining 3%, three patients with two different FECH mutations in trans were identified. For XLP, 24 of 250 (10%) unrelated individuals tested had ALAS2 exon 11 mutations. No novel ALAS2 mutations were identified. Among family members referred for testing, 33 of 42 (79%) CEP, 62 of 121 (51%) EPP, and 31 of 81 (38%) XLP family members had the respective family mutation. Mutation-positive CEP, EPP, and XLP patients who had been biochemically tested had marked elevations of the disease-appropriate porphyrin intermediates. These results expand the molecular heterogeneity of the erythropoietic porphyrias by adding a total of 27 novel mutations. The results document the usefulness of molecular testing to confirm the positive biochemical findings in these patients and to identify heterozygous family members.

Modeling the ferrochelatase c.315-48C modifier mutation for erythropoietic protoporphyria (EPP) in mice.

ABSTRACTErythropoietic protoporphyria (EPP) is caused by deficiency of ferrochelatase (FECH), which incorporates iron into protoporphyrin IX (PPIX) to form heme. Excitation of accumulated PPIX by light generates oxygen radicals that evoke excessive pain and, after longer light exposure, cause ulcerations in exposed skin areas of individuals with EPP. Moreover, ∼5% of the patients develop a liver dysfunction as a result of PPIX accumulation. Most patients (∼97%) have a severe FECH mutation (Mut) in trans to an intronic polymorphism (c.315-48C), which reduces ferrochelatase synthesis by stimulating the use of an aberrant 3′ splice site 63 nt upstream of the normal site for exon 4. In contrast, with the predominant c.315-48T allele, the correct splice site is mostly used, and individuals with a T/Mut genotype do not develop EPP symptoms. Thus, the C allele is a potential target for therapeutic approaches that modify this splicing decision. To provide a model for pre-clinical studies of such approaches, we engineered a mouse containing a partly humanized Fech gene with the c.315-48C polymorphism. F1 hybrids obtained by crossing these mice with another inbred line carrying a severe Fech mutation (named m1Pas) show a very strong EPP phenotype that includes elevated PPIX in the blood, enlargement of liver and spleen, anemia, as well as strong pain reactions and skin lesions after a short period of light exposure. In addition to the expected use of the aberrant splice site, the mice also show a strong skipping of the partly humanized exon 3. This will limit the use of this model for certain applications and illustrates that engineering of a hybrid gene may have unforeseeable consequences on its splicing.

Mitochondrial Protein Kinase A Regulates Heme Biosynthesis

During erythropoiesis, heme production becomes dramatically increased to support the production of oxygen-carrying hemoglobin. Heme biosynthesis requires an eight-step enzymatic cascade culminating with the insertion of ferrous iron into protoporphyrin IX by the mitochondrial enzyme ferrochelatase (FECH). This last step is the rate-limiting step in red blood cells (RBCs) and represents a critical regulatory juncture in RBC biology and human blood diseases. Most notably, in humans, FECH mutations are strongly associated with a disorder called erythropoietic protoporphyria (EPP) that typically presents with mild anemia and porphyria resulting from inadequate heme production and upstream accumulation of porphyrin intermediates, respectively. Disease severity can be highly heterogeneous, suggesting that additional modulators of FECH likely exist. However, despite its implications in EPP and other human blood disorders, very little is known regarding such regulatory mechanisms.Here, using complementary biochemical and genetics approaches, we identify FECH as a direct physiologic target for protein kinase A (PKA) phosphorylation during erythroid maturation. Quantitative proteomics revealed that PKA becomes enriched in mitochondria of differentiating murine erythroid cells. Phosphorylation of a highly conserved Thr116 in the catalytic domain of human FECH by PKA increases its activity resulting in elevated hemoglobinization of erythroblasts. We examined the importance of this phosphorylation in vivo by taking advantage of CRISPR/Cas9-mediated genome editing to knock-in the analogous Thr115Ala substitution into the endogenous Fech gene in murine RBCs. This approach allowed us to examine FECH function in a more physiologic context. Murine erythroid cells harboring only Thr115Ala Fech show reduced ferrous iron incorporation into protoporphyrin IX and, consequently, compromised hemoglobinization. In primary murine erythroid cells, we demonstrate that a distinct PKA gene expression signature is induced early in erythropoiesis. This suggests that the PKA pathway is engaged by physiologic signaling mechanisms during RBC development.Together, our results support a model where in maturing RBCs, PKA becomes enriched in the mitochondria where it phosphorylates FECH. Phosphorylation of FECH is required for full activity to support elevated heme biosynthesis and hemoglobin production. Our data also uncover PKA as a novel mechanism that links early erythroid signaling pathways to regulate heme biosynthesis and suggests that porphyrin accumulation can result not only from defective porphyrin transport but also aberrant cell signaling. [Display omitted] DisclosuresBauer:Biogen: Research Funding; Editas Medicine: Consultancy.

A Novel Mutation in the FECH Gene in a Czech Family with Erythropoietic Protoporphyria and a Population Study of IVS3-48C Variant Contributing to the Disease

Erythropoietic protoporphyria (EPP), a chronic erythropoietic porphyria, is characterized by excess accumulation of protoporphyrin, particularly in erythroid cells. EPP inheritance is complex, almost always associated with two molecular defects. In most EPP patients, clinical expression requires coinheritance of a private ferrochelatase (FECH) mutation trans- to a hypomorphic FECH*IVS3-48C allele. This leads to a decrease of FECH activity below the critical threshold. This is characterized by cutaneous photosensitivity in early childhood such as itching, burning, swelling and redness in sun-exposed areas. Hepatic failure occurs in some patients (about 1–10 % of EPP patients), which may necessitate liver transplantation. We investigated a Czech family with two patients with manifested EPP in four generations. We found a novel mutation, c.84G &gt;A, in the FECH gene in four individuals including proband and his mother (G84A transition in exon 2; p.W28*). Both clinically manifested probands inherited the hypomorphic IVS3-48C allele as well, while two clinically latent individuals with FECH mutation did not. To address the question whether the relatively low incidence of EPP in the Czech Republic might be due to lower frequency of the IVS3-48C allele, we screened for the frequency of the low expression allele in a control Czech (West Slaves) Caucasian population. Such study has not been performed in any Slavic population. Among 312 control individuals, there were no IVS3-48C/C (c.68-23C-T) homozygotes; 35 IVS3-48C/T heterozygous individuals were detected. The frequency of IVS3-48C allele was thus found to be 5.5 % in the Czech population, comparable to most West Caucasian populations.

FLVCR1a But Not FLVCR1b Is Required For Effective Erythropoiesis In Adult Mice

There are two characterized isoforms of FLVCR. The long form, FLVCR1a, is a broadly expressed cell surface heme exporter (Cell 118: 757, 2004) which exports heme from the cytoplasm to an extracellular carrier protein (JBC 285: 28874, 2010). The short isoform, FLVCR1b, is transcribed off an internal promoter and localizes to the mitochondria where it exports heme out of mitochondria into the cytoplasm (JCI 112: 4569, 2012). Adult mice with an induced deletion of both isoforms of FLVCR develop severe macrocytic anemia (Science 319: 825, 2008), indicating that FLVCR is essential for erythropoiesis. Mice constitutively lacking both isoforms of FLVCR die midgestation with craniofacial and digit abnormalities and lack definitive erythropoiesis while mice lacking only FLVCR1a die midgestation with craniofacial and digit abnormalities yet appear to have intact erythropoiesis (Science 319: 825, 2008, JCI 112: 4569, 2012). These results suggest that FLVCR1b, and not FLVCR1a, is required for erythropoiesis.To definitively test whether FLVCR1a or FLVCR1b is sufficient for red cell development, we transplanted mice with marrow lacking both isoforms of FLVCR that had been transduced with either FLVCR1a or FLVCR1b along with a GFP marker. Both cohorts of mice had comparable marking frequency in granulocytes (31.1±16.4%, N=15, FLVCR1a vs 32.7±16.9%, N=8, FLVCR1b) at 4 weeks post-transplant. By 7 wks post-transplant, 71.3±19.7% of the RBC in mice that received FLVCR1a were derived from transduced cells, while only 1.0±0.8% of the RBC in mice that received FLVCR1b were derived from transduced cells. Mice that received FLVCR1a are healthy and have normal CBC parameters (WBC 8.34±4.7 k/ml, RBC 7.95±1.2 M/ml, HGB 12.5±1.7 g/dl, MCV 47.9±2.0 fl , PLT 879±387 k/ml) which persist 9 months later, while mice that received FLVCR1b are severely anemic (WBC 1.9±1.3 k/ml, RBC 1.5±0.6 M/ml, HGB 2.0±0.8 g/dl, MCV 36.5±0.8 fl , PLT 1442±1232 k/ml) and die by 8 weeks post-transplant. This demonstrates that only the FLVCR1a isoform is capable of reconstituting erythropoiesis in adult mice lacking both isoforms in hematopoietic cells. One possible way to reconcile these data with the reported role of FLVCR1b, would be if FLVCR1b were needed during fetal, but not adult, erythropoiesis.As mentioned above, adult mice with an induced deletion of FLVCR develop severe macrocytic anemia (HBG 6.5±2.2 g/dl, MCV 67.9±7.3 fL; vs controls 14.6±0.7, 44.7±3.6), in contrast, over expression of FLVCR1a results in mild hypochromic microcytic anemia (HBG 13.2±1.4 g/dl, MCV 41±5.2 fL; vs controls 15.4±0.5, 49.3±1.3). Because hypochromasia and microcytosis only result from heme or hemoglobin deficiency, FLVCR1a must export heme from differentiating erythroblasts in vivo. To confirm this, we sorted developing erythroblasts from FLVCR-deleted and control mice and measured heme content at each stage. Terminally differentiating erythroid precursors (populations I-IV, PNAS 106: 17413, 2009) from FLVCR-deficient mice have significantly more heme than those from control mice (I&II 359.6±99.9 pg/cell, III 702.2±302.2 pg/cell, IV 657.8±292.9 pg/cell, versus controls 131.4±65.2, 153.9±27.1, and 269.1±102.7 respectively, all p<0.05) and have significantly more apoptosis. To definitively demonstrate that heme toxicity causes proerythroblast apoptosis and macrocytic anemia, we are using existing mouse model systems to modulate impact heme synthesis or degradation and test whether they alter the effect of FLVCR deletion on erythropoiesis. The presence of the ferrochelatase mutation, Fechm1Pas/m1Pas, does not rescue FLVCR deficiency, most likely because the accumulation of toxic precursor products which are also substrates of FLVCR (JBC 285: 28874, 2010). We are currently evaluating whether over-expression of HO-1 or restricted expression of the transferrin receptor can mitigate the effect of FLVCR deletion. Disclosures:No relevant conflicts of interest to declare.

Loss-of-Function Ferrochelatase and Gain-of-Function Erythroid-Specific 5-Aminolevulinate Synthase Mutations Causing Erythropoietic Protoporphyria and X-Linked Protoporphyria in North American Patients Reveal Novel Mutations and a High Prevalence of X-Linked Protoporphyria

Erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are inborn errors of heme biosynthesis with the same phenotype but resulting from autosomal recessive loss-of-function mutations in the ferrochelatase (FECH) gene and gain-of-function mutations in the X-linked erythroid-specific 5-aminolevulinate synthase (ALAS2) gene, respectively. The EPP phenotype is characterized by acute, painful, cutaneous photosensitivity and elevated erythrocyte protoporphyrin levels. We report the FECH and ALAS2 mutations in 155 unrelated North American patients with the EPP phenotype. FECH sequencing and dosage analyses identified 140 patients with EPP: 134 with one loss-of-function allele and the common IVS3-48T>C low expression allele, three with two loss-of-function mutations and three with one loss-of-function mutation and two low expression alleles. There were 48 previously reported and 23 novel FECH mutations. The remaining 15 probands had ALAS2 gain-of-function mutations causing XLP: 13 with the previously reported deletion, c.1706_1709delAGTG, and two with novel mutations, c.1734delG and c.1642C>T(p.Q548X). Notably, XLP represented ~10% of EPP phenotype patients in North America, two to five times more than in Western Europe. XLP males had twofold higher erythrocyte protoporphyrin levels than EPP patients, predisposing to more severe photosensitivity and liver disease. Identification of XLP patients permits accurate diagnosis and counseling of at-risk relatives and asymptomatic heterozygotes.

Hepatic complications of erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) is caused by mutations in ferrochelatase (<i>FECH</i>), the last step in the haem biosynthesis pathway. EPP is inherited in a pseudodominant fashion, most commonly due to presence of a low-expression splicing variant IVS3-48C <i>in trans</i> to a disease causing mutation. Reduction in fer-rochelatase activity leads to accumulation of protoporphyrin IX (PPIX) and photosensitivity. Hepatotoxicity is a rare complication of EPP, seen in 1–5% of patients.¹ Here we present the first reported case of fatal liver failure secondary to EPP in New South Wales, Australia. The patient was found to carry a known mutation (p.Phe260Leu), but IVS3–48C was not detected. The search for additional modifier genes at the genomic level found several potential candidates. These modifier genes may provide an insight on genotype-phenotype correlations in porphyrias.

Genetic study in a Singaporean patient with erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) is a rare autosomal dominant disorder of haem biosynthesis resulting from a partial decrease in ferrochelatase (FECH) activity which leads to the excessive accumulation of protoporphyrin in blood, erythrocytes and tissues. Cutaneous manifestations of photosensitivity usually appear in early infancy upon the first sun exposures. This normally requires the co-inheritance of a common hypomorphic FECH allele and a deleterious FECH mutation. Here, we report the first Singaporean Chinese patient with EPP characterized at the molecular level.

Coding region polymorphisms in the indoleamine 2,3-dioxygenase(INDO) gene and recurrent spontaneous abortion

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.

Oxidative stress, Nrf2 and keratin up-regulation associate with Mallory-Denk body formation in mouse erythropoietic protoporphyria

Mallory-Denk bodies (MDBs) are hepatocyte inclusions commonly seen in steatohepatitis. They are induced in mice by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) for 12 weeks, which also causes porphyrin accumulation. Erythropoietic protoporphyria (EPP) is caused by mutations in ferrochelatase (fch), and a fraction of EPP patients develop liver disease that is phenocopied in Fech(m1Pas) mutant (fch/fch) mice, which have an inactivating fch mutation. fch/fch mice develop spontaneous MDBs, but the molecular factors involved in their formation and whether they relate to DDC-induced MDBs are unknown. We tested the hypothesis that fch mutation creates a molecular milieu that mimics experimental drug-induced MDBs. In 13- and 20-week-old fch/fch mice, serum alkaline phosphatase, alanine aminotransferase, and bile acids were increased. The 13-week-old fch/fch mice did not develop histologically evident MDBs but manifested biochemical alterations required for MDB formation, including increased transglutaminase-2 and keratin overexpression, with a greater keratin 8 (K8)-to-keratin 18 (K18) ratio, which are critical for drug-induced MDB formation. In 20-week-old fch/fch mice, spontaneous MDBs were readily detected histologically and biochemically. Short-term (3-week) DDC feeding markedly induced MDB formation in 20-week-old fch/fch mice. Under basal conditions, old fch/fch mice had significant alterations in mitochondrial oxidative-stress markers, including increased protein oxidation, decreased proteasomal activity, reduced adenosine triphosphate content, and Nrf2 (redox sensitive transcription factor) up-regulation. Nrf2 knockdown in HepG2 cells down-regulated K8, but not K18. Fch/fch mice develop age-associated spontaneous MDBs, with a marked propensity for rapid MDB formation upon exposure to DDC, and therefore provide a genetic model for MDB formation. Inclusion formation in the fch/fch mice involves oxidative stress which, together with Nrf2-mediated increase in K8, promotes MDB formation.

Protoporphyrin Retention in Hepatocytes and Kupffer Cells Prevents Sclerosing Cholangitis in Erythropoietic Protoporphyria Mouse Model

Chronic, progressive hepatobiliary disease is the most severe complication of erythropoietic protoporphyria (EPP) and can require liver transplantation, although the mechanisms that lead to liver failure are unknown. We characterized protoporphyrin-IX (PPIX)-linked hepatobiliary disease in BALB/c and C57BL/6 (Fechm1Pas) mice with mutations in ferrochelatase as models for EPP. Fechm1Pas and wild-type (control) mice were studied at 12-14 weeks of age. PPIX was quantified; its distribution in the liver, serum levels of lipoprotein-X, liver histology, contents of bile salt and cholesterol phospholipids, and expression of genes were compared in mice of the BALB/c and C57BL/6 backgrounds. The in vitro binding affinity of PPIX for bile components was determined. Compared with mice of the C57BL/6 background, BALB/c Fechm1Pas mice had a more severe pattern of cholestasis, fibrosis with portoportal bridging, bile acid regurgitation, sclerosing cholangitis, and hepatolithiasis. In C57BL/6 Fechm1Pas mice, PPIX was sequestrated mainly in the cytosol of hepatocytes and Kupffer cells, whereas, in BALB/c Fechm1Pas mice, PPIX was localized within enlarged bile canaliculi. Livers of C57BL/6 Fechm1Pas mice were protected through a combination of lower efflux of PPIX and reduced synthesis and export of bile acid. PPIX binds to bile components and disrupts the physiologic equilibrium of phospholipids, bile acids, and cholesterol in bile. This process might be involved in pathogenesis of sclerosing cholangitis from EPP; a better understanding might improve diagnosis and development of reagents to treat or prevent liver failure in patients with EPP.

Erythropoietic protoporphyria: a family study and report of a novel mutation in the FECH gene

Erythropoietic protoporphyria (EPP) is a rare inherited disorder of heme biosynthesis mostly caused by a deficient activity of the enzyme ferrochelatase (FECH), and consequent accumulation of protoporphyrin (PP) in various tissues. Clinical manifestations include a childhood onset, cutaneous photosensitivity and, sometimes, hepatobiliary disease. We report a 16-year-old male with EPP characterized by acute episodes of painful photosensitivity since early infancy, permanent changes in the photoexposed skin, microcytic anemia, thrombocytopenia, and mild hepatic dysfunction. His 18-year-old sister presented less acute symptoms with no chronic changes. Lesional biopsy disclosed perivascular deposition of PAS positive hyaline material. Rimington-Cripps test was positive and PP erythrocyte levels were >9,000 μg/L (N<1,600), but normal in their parents and younger brother. Genetic studies in both patients and their mother revealed heterozygosity for a novel mutation (c.1052delA) in FECH gene of both children, and heterozygosity for the hypomorphic allele IVS3-48T>C in all of them. This confirms the "pseudodominant" inheritance pattern usually observed, explained by the combined presence of a disabling FECH mutation and a common intronic polymorphism affecting the counterpart allele (IVS3-48T>C). Phenotypic heterogeneity for this genotype explains the divergent clinical presentation. This is the first description of a Portuguese family with EPP characterized at the molecular level.

PROTOPORFIRIA ERITROPOIÉTICA – Caracterização de uma Família Portadora de uma Nova Mutação no Gene FECH

A protoporfiria eritropoiética (PPE), é uma genodermatose rara causada, na maioria dos doentes, pela redução da actividade da ferroquelatase (FECH), a última enzima da biossíntese do heme, e consequente acumulação de protoporfirina (PP) em vários tecidos. Os autores descrevem um adolescente de 16 anos (caso índice) que sofria de PPE desde a infância, caracterizada por episódios agudos de fotossensibilidade dolorosa nas áreas foto-expostas, alterações crónicas, anemia microcítica, trombocitopenia e disfunção hepática ligeira. A irmã de 18 anos apresentava sintomas agudos mais ligeiros e não evidenciava alterações crónicas. Os progenitores e o irmão mais novo eram assintomáticos. A biópsia lesional efectuada no caso índice mostrou deposição de material hialino, PAS-positivo, em localização perivascular. O teste de Rimington-Cripps foi positivo em ambos os doentes sintomáticos e os níveis de PPs eritrocitárias encontravam-se elevados, mas normais nos pais e no irmão mais novo. O estudo genético, efectuado em ambos os doentes e na mãe (amostra do pai não disponível por paradeiro desconhecido), revelou heterozigotia para uma nova mutação no gene FECH (c.1052delA) nos filhos e heterozigotia para o alelo hipomórfico IVS3-48T&gt;C em todos os elementos testados. Confirmou-se, assim, o padrão de hereditariedade “pseudodominante” observado na maioria das famílias, explicado pela presença simultânea de uma mutação no gene FECH e de um polimorfismo frequente, afectando o alelo oposto (IVS3-48T&gt;C). Neste caso, o alelo mutante foi provavelmente herdado do pai. Do nosso conhecimento, esta é a primeira descrição de uma família portuguesa com PPE caracterizada do ponto de vista molecular, tendo-se descrito uma nova mutação, não referida na literatura.

La protoporphyrie érythropoïétique : une maladie, deux gènes et trois mécanismes moléculaires

Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis that results from an accumulation of protoporphyrin IX in erythroid cells, plasma, skin and liver. EPP leads to acute photosensitivity and, in about 2% of patients, liver disease. EPP is a complex syndrome in which two genes are independently involved: FECH and ALAS2. More than 96% of unrelated EPP patients have ferrochelatase (FECH) deficiency (MIM 177000). Four percent of them present with autosomal recessive inheritance with two mutated FECH alleles. In dominant cases (95%) the inheritance of a common hypomorphic IVS3-48C FECH allele trans to a deleterious FECH mutation reduces FECH activity below a critical threshold. The frequency of the IVS3-48C allele differs widely from the Japanese (45%), to Black West Africans (< 1%) populations. These differences in the frequency of this single common SNP account for the prevalence of overt EPP in different countries and for the absence of EPP in Black Africans. The phylogenic origin of the IVS3-48C haplotypes strongly suggests that the IVS3-48C allele arose from a single recent mutational event that occurred 60 Kyears ago. Acquired somatic mutation of FECH secondary to myeloid disease may also exceptionally cause EPP (< 1%). Finally, about 4% of unrelated EPP patients have X-linked dominant protoporphyria (XLDPP) (MIM 300752) caused by gain-of-function mutations in the ALAS2 gene leading to an increased erythroid heme biosynthesis and subsequently an accumulation of protoporphyrin without any FECH deficiency.

A novel splicing mutation and haplotype analysis of the FECH gene in a Chinese family with erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) is a rare autosomal dominant disorder of haeme biosynthesis resulting from a partial decrease in ferrochelatase (FECH) activity leading to excessive accumulation of protoporphyrin. Clinical manifestation normally requires coinheritance of a common hypomorphic FECH allele and a deleterious FECH mutation. The aim of this study was to characterize the inheritance of a Chinese family with EPP at molecular level, by identification and analysis of FECH sequence variation, including the IVS3-48C polymorphisms. Polymerase chain reaction amplification was employed to identify the FECH sequence variation, including the IVS3-48C polymorphism, in a Chinese EPP family and a matched control cohort. A splicing mutation in IVS3 + 1G-->A was identified in the proband as well as his symptomatic sister, cousin, his grandfather and his asymptomatic mother, but was absent in his father and grandmother. All the family members with overt photosensitivity carried the low-expressed allele IVS3-48C, which were absent in the asymptomatic EPP patients of this family. We described a novel splicing FECH mutation in a Chinese EPP family and analysed the hypomorphic IVS3-48C allele, which were believed to be responsible for generating the phenotypic symptoms in this family.

Molecular epidemiology of erythropoietic protoporphyria in the U.K.

Erythropoietic protoporphyria (EPP) is a cutaneous porphyria caused by mutations in the ferrochelatase (FECH) or, less frequently, the delta-aminolaevulinate synthase 2 (ALAS2) gene. Predictive genetic counselling requires accurate molecular diagnosis and knowledge of patterns of inheritance. To investigate the molecular epidemiology of EPP in the U.K. DNA samples from 191 unrelated patients resident in the U.K. were analysed for mutations in the FECH and ALAS2 genes and for the FECH IVS3-48 dimorphism. Mutations were identified in 179 (94%) patients. Most (169; 94%) had a FECH mutation on one allele and were classified as having pseudodominant EPP (psdEPP); seven (4%) patients had FECH mutations on both alleles (autosomal recessive EPP) and three (2%) patients had ALAS2 mutations (X-linked dominant protoporphyria). The FECH IVS3-48C allele was strongly associated with psdEPP and with the absence of mutations at the FECH or ALAS2 loci. Fifty-six FECH mutations were identified, 19 being previously unreported. Missense mutations were predominant in autosomal recessive EPP (82%) but not in psdEPP (32%). One mutation (c.314 + 2T>G) was present in 41 (24%) of EPP families, most of whom appeared to be descended from a common ancestor resident in the north of England. These data define the prevalence and molecular epidemiology of each type of EPP in the U.K.