Ferrochelatase Gene Research Articles

X-linked Dominant Erythropoietic Protoporphyria with early-onset manifestations caused by a hemizygous ALAS2 variant.

Erythropoietic protoporphyria (EPP) is usually caused by autosomal dominant mutations FECH, blocking the conversion of protoporphyrin to haem. The resulting accumulation of protoporphyrin causes itching, burning and erythema after sun exposure, with facial scarring and, sometimes, liver damage. We report a 4-year-old boy from India with XLDPP with a previously unreported manifestation of photo-onycholysis and an unusually severe phenotype, possibly attributable to extensive sun exposure.

When the diagnosis is written in the DNA: a case of erythropoietic protoporphyria in a patient with a chromosome-18 deletion.

In this case study, we describe a 21-year-old man with erythropoietic protoporphyria who sought medical attention in April 2022 for diffuse edema and erythema of the hands. These symptoms had been present since childhood and usually occurred soon after sun exposure. The patient's medical history showed that chromosome 18's long arm had partially deleted. We performed a number of tests, including measuring total erythrocyte protoporphyrin levels and utilizing a spectrofluorometer to assess the fluorometric emission peak of plasma porphyrins, based on the patient's medical history and clinical symptoms. Furthermore, a genetic analysis identified an intronic variant on one allele, c.315-48T>C (IVS3-48T>C), which is categorized as a susceptibility polymorphism, and a complete deletion of the ferrochelatase gene on the other allele. The patient's clinical condition improved following the June 2022 afamelanotide implant procedure.

Heme Biosynthetic Gene Expression Analysis With dPCR in Erythropoietic Protoporphyria Patients.

Background: The heme biosynthesis (HB) involves eight subsequent enzymatic steps. Erythropoietic protoporphyria (EPP) is caused by loss-of-function mutations in the ferrochelatase (FECH) gene, which in the last HB step inserts ferrous iron into protoporphyrin IX (PPIX) to form heme. Aim and method: The aim of this work was to for the first time analyze the mRNA expression of all HB genes in peripheral blood samples of patients with EPP having the same genotype FECH c.[215dupT]; [315-48T > C] as compared to healthy controls by highly sensitive and specific digital PCR assays (dPCR). Results: We confirmed a decreased FECH mRNA expression in patients with EPP. Further, we found increased ALAS2 and decreased ALAS1, CPOX, PPOX and HMBS mRNA expression in patients with EPP compared to healthy controls. ALAS2 correlated with FECH mRNA expression (EPP: r = 0.63, p = 0.03 and controls: r = 0.68, p = 0.02) and blood parameters like PPIX (EPP: r = 0.58 p = 0.06). Conclusion: Our method is the first that accurately quantifies HB mRNA from blood samples with potential applications in the monitoring of treatment effects of mRNA modifying therapies in vivo, or investigation of the HB pathway and its regulation. However, our findings should be studied in separated blood cell fractions and on the enzymatic level.

The role of the genetic variant FECH rs11660001 in the occurrence of anti-tuberculosis drug-induced liver injury.

The pathogenic mechanism of anti-tuberculosis drug-induced liver injury (AT-DILI) is still largely unknown. Recent studies have indicated that rifampicin and isoniazid cotreatment causes the accumulation of endogenous protoporphyrin IX in the liver through the haem biosynthesis pathway. Alanine synthase 1 (ALAS1) and ferrochelatase (FECH) are the rate-limiting enzymes in the production of haem. The present study aimed to investigate the genetic contribution of the ALAS1 and FECH genes to the risk of AT-DILI in an Eastern Chinese Han population. A 1:4 matched case-control study was conducted, and eight SNPs in the ALAS1 and FECH genes were detected and assessed. A multivariate conditional logistic regression model was used to estimate the association between genotypes and the risk of AT-DILI by the odds ratios (ORs) with 95% confidence intervals (CIs), with liver disease history, hepatoprotectant use, smoking and drinking history as covariates. Overall, 202 AT-DILI cases and 808 controls were included in this study. The female patients carrying polymorphisms of rs11660001 in FECH had an increased risk of AT-DILI under the dominant and additive models (OR=1.831, 95% CI: 1.014-3.307, p=0.045; OR=1.673, 95% CI: 1.015-2.760, p=0.044, respectively). The peak aspartate transaminase level was significantly higher in female patients carrying the GA+AA genotype of rs11660001 than in those with the GG genotype during anti-TB treatment (p=0.032). Based on this 1:4 individual matched case-control study, SNP rs11660001 in the FECH gene may be associated with susceptibility to AT-DILI in Chinese female anti-TB treatment patients. Further studies in larger varied populations are needed to validate our findings.

A three-gene cluster in Trichoderma reesei reveals a potential role of dmm2 in DNA repair and cellulase production

BackgroundThe ascomycete Trichoderma reesei is one of the most efficient industrial producers of cellulase. Gene targeting by homologous recombination is a key technique for improving strains and constructing mutants. In T. reesei, tku70 (homologous to human KU70) was deleted to block non-homologous end-joining, which led to 95% of transformants exhibiting homologous recombination.ResultsTwo genes located in close proximity to tku70 were identified: the ferrochelatase gene hem8 (tre78582, homologous to Aspergillus niger hemH and Cryptococcus neoformans HEM15) and a putative DNA methylation modulator-2 gene dmm2 (tre108087, homologous to Neurospora crassa dmm-2). Genome-wide surveys of 324 sequenced fungal genomes revealed that the homologues of the three genes of interest are encoded in tandem in most Sordariomycetes. The expression of this three-gene cluster is regulated by blue light. The roles of these three genes were analyzed via deletion and complementation tests. The gene hem8 was originally described as a novel and highly distinct auxotrophic marker in T. reesei and we found that the product protein, HEM8, catalyzes the final step in heme biosynthesis from highly photoreactive porphyrins. The lethal phenotype of the hem8 deletion could be overcome by hematin supplementation. We also studied the functions of tku70 and dmm2 in DNA repair using mutagen sensitivity experiments. We found that the Δtku70 strain showed increased sensitivity to bleomycin, which induces DNA double-strand breaks, and that the Δdmm2 strain was sensitive to bleomycin, camptothecin (an inhibitor of type I topoisomerases), and hydroxyurea (a deoxynucleotide synthesis inhibitor). The double-mutant Δtku70&dmm2 showed higher sensitivity to hydroxyurea, camptothecin, and bleomycin than either of the single mutants. Knockout of dmm2 significantly increased cellulase production.ConclusionsOur data show, for the first time, that ferrochelatase encoded by hem8 catalyzes the final step in heme biosynthesis from highly photoreactive porphyrins and that dmm2 encodes a putative DNA methylation modulator-2 protein related to DNA repair and cellulase expression in T. reesei. Our data provide important insights into the roles of this three-gene cluster in T. reesei and other Sordariomycetes and show that the DNA methylation modulator DMM2 affects cellulase gene expression in T. reesei.

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.

Differential gene content and gene expression for bacterial evolution and speciation of Shewanella in terms of biosynthesis of heme and heme-requiring proteins

BackgroundMost species of Shewanella harbor two ferrochelatase paralogues for the biosynthesis of c-type cytochromes, which are crucial for their respiratory versatility. In our previous study of the Shewanella loihica PV-4 strain, we found that the disruption of hemH1 but not hemH2 resulted in a significant accumulation of extracellular protoporphyrin IX (PPIX), but it is different in Shewanella oneidensis MR-1. Hence, the function and transcriptional regulation of two ferrochelatase genes, hemH1 and hemH2, are investigated in S. oneidensis MR-1.ResultIn the present study, deletion of either hemH1 or hemH2 in S. oneidensis MR-1 did not lead to overproduction of extracellular protoporphyrin IX (PPIX) as previously described in the hemH1 mutants of S. loihica PV-4. Moreover, supplement of exogenous hemins made it possible to generate the hemH1 and hemH2 double mutant in MR-1, but not in PV-4. Under aerobic condition, exogenous hemins were required for the growth of MR-1ΔhemH1ΔhemH2, which also overproduced extracellular PPIX. These results suggest that heme is essential for aerobic growth of Shewanella species and MR-1 could also uptake hemin for biosynthesis of essential cytochrome(s) and respiration. Besides, the exogenous hemin mediated CymA cytochrome maturation and the cellular KatB catalase activity. Both hemH paralogues were transcribed in wild-type MR-1, and the hemH2 transcription was remarkably up-regulated in MR-1ΔhemH1 mutant to compensate for the loss of hemH1. The periplasmic glutathione peroxidase gene pgpD, located in the same operon with hemH2, and a large gene cluster coding for iron, heme (hemin) uptake systems are absent in the PV-4 genome.ConclusionOur results indicate that the genetic divergence in gene content and gene expression between these Shewanella species, accounting for the phenotypic difference described here, might be due to their speciation and adaptation to the specific habitats (iron-rich deep-sea vent versus iron-poor freshwater) in which they evolved and the generated mutants could potentially be utilized for commercial production of PPIX.

A Novel FECH Mutation Causes Erythropoietic Protoporphyria with Severe Liver Dysfunction

Background: Erythropoietic protoporphyria (EPP) is a rare autosomal dominant disease that is caused by partial ferrochelatase (FECH) deficiency because of a genetic mutation in the FECH gene. Objectives: The aim of this study was to investigate the genetic mechanism of EPP in a Chinese family. Methods: Clinical physical examination, laboratory testing, and medical history tracking were performed for phenotyping. The FECH gene was sequenced for genotyping. Results: The proband presented EPP with severe liver dysfunction. A novel nonsense mutation of c.910 C>T in the FECH gene, resulting in p.Arg304Ter, was identified in the proband as well as her symptomatic mother, aunt, and asymptomatic male cousin. The proband and her living family members, except the asymptomatic male cousin, carried the alleles IVS 3-48C and IVS 1-23T on the FECH gene. The mutation of c.910 C>T in the FECH gene presented its heterozygous autosomal dominant inherent clinical manifestations, only in the presence of the heterozygous alleles IVS 3-48C and IVS 1-23T. The frequencies of the alleles IVS 3-48C and IVS 1-23T were 30.8% and 27.9% in a northeast Chinese Han population, respectively. Conclusions: A new nonsense mutation of c.910 C>T was identified in the FECH gene, which expressed EPP with severe liver dysfunction when it was co-inherited with the heterozygous alleles IVS 3-48C and IVS 1-23T, in a Chinese family.

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.

Digital PCR (dPCR) analysis reveals that the homozygous c.315–48T>C variant in the FECH gene might cause erythropoietic protoporphyria (EPP)

Alterations in the ferrochelatase gene (FECH) are the basis of the phenotypic expressions in erythropoietic protoporphyria. The phenotype is due to the presence of a mutation in the FECH gene associated in trans to the c.315–48 T > C variant in the intron 3. The latter is able to increase the physiological quota of alternative splicing events in the intron 3. Other two variants in the FECH gene (c.1–252A > G and c.68–23C > T) have been found to be associated to the intron 3 variant in some populations and together, they constitute a haplotype (ACT/GTC), but eventually, their role in the alternative splicing event has never been elucidated.The absolute number of the aberrantly spliced FECH mRNA molecules and the absolute expression of the FECH gene were evaluated by digital PCR technique in a comprehensive cohort. The number of splicing events that rose in the presence of the c.315–48 T > C variant, both in the heterozygous and homozygous condition was reported for the first time. Also, the percentage of the inserted FECH mRNA increased, even doubled in the T/C cases, compared to T/T cases. The constant presence of variants in the promoter and intron 2 did not influence or modulate the aberrant splicing. The results of FECH gene expression suggested that the homozygosity for the c.315–48 T > C variant could be considered pathological.Thus, this study identified the homozygotes for the c.315–48 T > C variant as pathological. By extension, when the samples were categorised according to the haplotypes, the GTC haplotype in homozygosis was pathological.

DNA of Erythroid Origin Is Present in Human Plasma and Informs the Types of Anemia.

There is much interest in the tissue of origin of circulating DNA in plasma. Data generated using DNA methylation markers have suggested that hematopoietic cells of white cell lineages are important contributors to the circulating DNA pool. However, it is not known whether cells of the erythroid lineage would also release DNA into the plasma. Using high-resolution methylation profiles of erythroblasts and other tissue types, 3 genomic loci were found to be hypomethylated in erythroblasts but hypermethylated in other cell types. We developed digital PCR assays for measuring erythroid DNA using the differentially methylated region for each locus. Based on the methylation marker in the ferrochelatase gene, erythroid DNA represented a median of 30.1% of the plasma DNA of healthy subjects. In subjects with anemia of different etiologies, quantitative analysis of circulating erythroid DNA could reflect the erythropoietic activity in the bone marrow. For patients with reduced erythropoietic activity, as exemplified by aplastic anemia, the percentage of circulating erythroid DNA was decreased. For patients with increased but ineffective erythropoiesis, as exemplified by β-thalassemia major, the percentage was increased. In addition, the plasma concentration of erythroid DNA was found to correlate with treatment response in aplastic anemia and iron deficiency anemia. Plasma DNA analysis using digital PCR assays targeting the other 2 differentially methylated regions showed similar findings. Erythroid DNA is a hitherto unrecognized major component of the circulating DNA pool and is a noninvasive biomarker for differential diagnosis and monitoring of anemia.

Erythropoietic protoporphyria a clinical and molecular study from Lebanon: Ferrochelatase a potential tumor suppressor gene in colon cancer.

Erythropoietic protoporphyria (EPP) is a rare cutaneous and systemic disease caused by mutations in the ferrochelatase gene (FECH). The molecular underpinnings of EPP in Middle Eastern populations and relative to other ethnic groups secondary to increased consanguinity are unknown. To understand the molecular pathogenesis of Middle Eastern EPP, we surveyed clinicopathological and molecular features in 6 large consanguineous families from Lebanon and Syria presenting with cutaneous and systemic features consistent with EPP. We observed 30% increased liver disease and 20% elevated end-stage liver complications in our EPP cohort compared to EPP patients previously reported elsewhere. In addition, Middle Eastern EPP patients in our cohort exhibited uniquely an increased incidence of colon cancer. Sequence analysis revealed 2 novel non-synonymous FECH mutations in the studied families designated p.M294T and p.I230M. In addition, FECH activity was significantly decreased (6%) in fibroblasts obtained from sun-exposed sites in a patient with p.M294T mutation, whereas in sharp contrast, protected sites from the same patient exhibited 54% activity for the gene. We also found that sun-exposed fibroblasts, relative to sun-protected and control fibroblasts, exhibited suppressed growth and atypical morphology in vitro, and that these effects were alleviated when the cells were co-cultured with sun-protected fibroblasts. Our findings on the increased incidence of colon cancer in EPP patients prompted us to survey FECH expression patterns in cancer. Using publicly available microarray datasets we found that FECH mRNA was largely significantly decreased in colon adenocarcinomas relative to normal colon tissues. Our findings suggest that families with autosomal recessive EPP should be screened more extensively for systemic involvement including liver diseases and colon cancer, and point to a previously unknown yet plausible tumor suppressor role for FECH in colon malignancy.

A novel genetic technique in Plasmodium berghei allows liver stage analysis of genes required for mosquito stage development and demonstrates that de novo heme synthesis is essential for liver stage development in the malaria parasite.

The combination of drug resistance, lack of an effective vaccine, and ongoing conflict and poverty means that malaria remains a major global health crisis. Understanding metabolic pathways at all parasite life stages is important in prioritising and targeting novel anti-parasitic compounds. The unusual heme synthesis pathway of the rodent malaria parasite, Plasmodium berghei, requires eight enzymes distributed across the mitochondrion, apicoplast and cytoplasm. Deletion of the ferrochelatase (FC) gene, the final enzyme in the pathway, confirms that heme synthesis is not essential in the red blood cell stages of the life cycle but is required to complete oocyst development in mosquitoes. The lethality of FC deletions in the mosquito stage makes it difficult to study the impact of these mutations in the subsequent liver stage. To overcome this, we combined locus-specific fluorophore expression with a genetic complementation approach to generate viable, heterozygous oocysts able to produce a mix of FC expressing and FC deficient sporozoites. These sporozoites show normal motility and can invade liver cells, where FC deficient parasites can be distinguished by fluorescence and phenotyped. Parasites lacking FC exhibit a severe growth defect within liver cells, with development failure detectable in the early to mid stages of liver development in vitro. FC deficient parasites could not complete liver stage development in vitro nor infect naïve mice, confirming liver stage arrest. These results validate the heme pathway as a potential target for prophylactic drugs targeting liver stage parasites. In addition, we demonstrate that our simple genetic approach can extend the phenotyping window beyond the insect stages, opening considerable scope for straightforward reverse genetic analysis of genes that are dispensable in blood stages but essential for completing mosquito development.

Synthesis and cellular activity of stereochemically-pure 2'-O-(2-methoxyethyl)-phosphorothioate oligonucleotides.

Stereochemically-pure 2'-O-(2-methoxyethyl)-phosphorothioate (PS-MOE) oligonucleotides were synthesized from new chiral oxazaphospholidine-containing nucleosides. Thermal stability studies showed that the incorporation of Rp-PS linkages increased RNA-binding affinity. In cells, a full Rp-PS-MOE splice-switching oligonucleotide targeting part of the ferrochelatase gene was more potent than its Sp-PS counterpart, but of similar potency to the stereorandom PS-parent sequence.

Differential Regulation of the Two Ferrochelatase Paralogues in Shewanella loihica PV-4 in Response to Environmental Stresses.

Determining the function and regulation of paralogues is important in understanding microbial functional genomics and environmental adaptation. Heme homeostasis is crucial for the survival of environmental microorganisms. Most Shewanella species encode two paralogues of ferrochelatase, the terminal enzyme in the heme biosynthesis pathway. The function and transcriptional regulation of two ferrochelatase genes, hemH1 and hemH2, were investigated in Shewanella loihica PV-4. The disruption of hemH1 but not hemH2 resulted in a significant accumulation of extracellular protoporphyrin IX (PPIX), the precursor to heme, and decreased intracellular heme levels. hemH1 was constitutively expressed, and the expression of hemH2 increased when hemH1 was disrupted. The transcription of hemH1 was regulated by the housekeeping sigma factor RpoD and potentially regulated by OxyR, while hemH2 appeared to be regulated by the oxidative stress-associated sigma factor RpoE2. When an oxidative stress condition was mimicked by adding H2O2 to the medium or exposing the culture to light, PPIX accumulation was suppressed in the ΔhemH1 mutant. Consistently, transcriptome analysis indicated enhanced iron uptake and suppressed heme synthesis in the ΔhemH1 mutant. These data indicate that the two paralogues are functional in the heme synthesis pathway but regulated by environmental conditions, providing insights into the understanding of bacterial response to environmental stresses and a great potential to commercially produce porphyrin compounds. Shewanella is capable of utilizing a variety of electron acceptors for anaerobic respiration because of the existence of multiple c-type cytochromes in which heme is an essential component. The cytochrome-mediated electron transfer across cellular membranes could potentially be used for biotechnological purposes, such as electricity generation in microbial fuel cells and dye decolorization. However, the mechanism underlying the regulation of biosynthesis of heme and cytochromes is poorly understood. Our study has demonstrated that two ferrochelatase genes involved in heme biosynthesis are differentially regulated in response to environmental stresses, including light and reactive oxygen species. This is an excellent example showing how bacteria have evolved to maintain cellular heme homeostasis. More interestingly, the high yields of extracellular protoporphyrin IX by the Shewanella loihica PV-4 mutants could be utilized for commercial production of this valuable chemical via bacterial fermentation.

Influence of exogenous 5-aminolevulinic acid on chlorophyll synthesis and related gene expression in oilseed rape de-etiolated cotyledons under water-deficit stress

5-aminolevulinic acid (ALA) is an essential precursor for the biosynthesis of tetrapyrrols such as heme and chlorophyll (Chl). Previous studies have focused mainly on promotive effects of exogenous ALA on plant growth, while regulatory mechanisms affecting Chl biosynthesis have been only partially discussed. In the present study, the ameliorative role of exogenous ALA was investigated on Chl and endogenous ALA biosynthesis in six-day-old etiolated oilseed rape (Brassica napus L.) cotyledons during the de-etiolation stage. We showed that exogenously applied ALA of a low dosage enhanced Chl and ALA accumulation in cotyledons, while 600 µM ALA treatment inhibited the accumulation of Chl and ALA severely. However, the gene expression levels of glutamyl-tRNA reductase (HEMA) and glutamate-1-semialdehyde aminotransferase (GSA) were not affected under either low or high ALA concentrations. Furthermore, water deficit induced by polyethylene glycol 6000 (PEG) suppressed the Chl and ALA accumulation in cotyledons, while the inhibition was partially alleviated in the cotyledons pretreated with ALA. The decrease in Chl biosynthesis induced by PEG stress was assumed to be related to downregulation of HEMA and Mg-chelatase ChlH (ChlH), and upregulation of ferrochelatase (FC) genes. Moreover, exogenously applied ALA did not show any effect on the expression of Chl synthesis-related genes under the PEG treatment. These results showed a difference in suppressing ALA synthesis due to the high concentration of ALA and PEG. Exogenously applied ALA did not affect the expression of HEMA and GSA, thus exogenous ALA regulated Chl synthesis not via the regulation of transcriptional level in ALA biosynthesis. However, the inhibition in Chl and endogenous ALA accumulation by the PEG treatment may be attributed to downregulation of HEMA and ChlH, and upregulation of FC.

Red cells from ferrochelatase-deficient erythropoietic protoporphyria patients are resistant to growth of malarial parasites

AbstractMany red cell polymorphisms are a result of selective pressure by the malarial parasite. Here, we add another red cell disease to the panoply of erythrocytic changes that give rise to resistance to malaria. Erythrocytes from individuals with erythropoietic protoporphyria (EPP) have low levels of the final enzyme in the heme biosynthetic pathway, ferrochelatase. Cells from these patients are resistant to the growth of Plasmodium falciparum malarial parasites. This phenomenon is due to the absence of ferrochelatase and not an accumulation of substrate, as demonstrated by the normal growth of P falciparum parasites in the EPP phenocopy, X-linked dominant protoporphyria, which has elevated substrate, and normal ferrochelatase levels. This observation was replicated in a mouse strain with a hypomorphic mutation in the murine ferrochelatase gene. The parasite enzyme is not essential for parasite growth as Plasmodium berghei parasites carrying a complete deletion of the ferrochelatase gene grow normally in erythrocytes, which confirms previous studies. That ferrochelatase is essential to parasite growth was confirmed by showing that inhibition of ferrochelatase using the specific competitive inhibitor, N-methylprotoporphyrin, produced a potent growth inhibition effect against cultures of P falciparum. This raises the possibility of targeting human ferrochelatase in a host-directed antimalarial strategy.

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.

Erythropoietic Protoporphyric Red Blood Cells Are Resistant to the Growth of Malarial Parasites

Many red cell polymorphisms are a result of selective pressure by the malarial parasite. Here we add another red cell disease to the panoply of erythrocytic changes that give rise to resistance to malaria. Erythrocytes from individuals with erythropoietic protoporphyria (EPP) have low levels of the final enzyme in the heme biosynthetic pathway, ferrochelatase. Cells from these patients are resistant to the growth of the human malarial parasite, Plasmodium falciparum.We first compared the growth and replication rates of P. falciparum cultured in red cells from EPP patients (n=4) and normal erythrocytes. There was a two to three-fold reduction in parasite growth in the patient cells. Next we sought to exclude the possible negative effects on the parasite due to elevated porphyrins and reduced cell hemoglobin. To do this we employed the EPP phenocopy, X-linked dominant protoporphyria (XLDPP), which has normal ferrochelatase activity. Cells from three individuals with XLDPP supported completely normal rates of parasite growth, proving that the EPP resistance phenomenon was due to the absence of ferrochelatase.We also tested the requirement of host ferrochelatase during malarial infection by using mice with a hypomorphic mutation in the murine ferrochelatase gene and the rodent malarial species, P. chabaudi. Mice homozygous for the mutation, Fechm1Pas, have 5% residual ferrochelatase activity compared to wild-type littermates 1. Following infection, we observed an almost two fold reduction in peak parasitemia levels and two to three times greater rates of survival in the homozygous mice. Host ferrochelatase is therefore also necessary to sustain a normal malarial infection in mice.To determine the requirement of parasite-expressed ferrochelase, we produced a P. berghei parasite line carrying a complete deletion of the ferrochelatase gene. This strain grew normally in wild-type mouse erythrocytes, indicating that parasite ferrochelatase is dispensable during the erythrocytic stage of infection.A complete absence of ferrochelatase in humans (and mice) is not compatible with life, and therefore testing parasite growth in complete knockout cells was not possible. Instead, we used a specific competitive inhibitor of the enzyme, N-methylprotoporphyrin (NMPP) to eliminate all ferrochelatase activity from the parasite and red cell. Treatment of P. falciparum cultures with NMPP resulted in potent cytocidal and growth inhibition effects against both antimalarial drug-sensitive and drug-resistant parasite lines. The activity of NMPP could be competitively removed by titration of the ferrochelatase substrate, protoporphyrin IX, proving that the effects of NMPP were due to specific enzyme inhibition and not off-target effects. Therefore ferrochelatase activity is also essential for the Plasmodium parasite.We conclude that the refractoriness of ferrochelatse-deficient red cells to Plasmodium is due to the parasite’s reliance on the host enzyme. Host ferrochelatase is probably utilized by the parasite for the biosynthesis of heme. In support of this hypothesis, others have observed that red cell ferrochelatase is imported by intraerythrocytic Plasmodium and enzymatic is retained 2,3. Finally, based on this collective data, we propose human ferrochelatase is a valid and novel “host-directed” target for an antimalarial therapy. 1.Lyoumi S, Abitbol M, Andrieu V, et al. Increased plasma transferrin, altered body iron distribution, and microcytic hypochromic anemia in ferrochelatase-deficient mice. Blood. 2007;109(2):811-818.2.Bonday ZQ, Dhanasekaran S, Rangarajan PN, Padmanaban G. Import of host delta-aminolevulinate dehydratase into the malarial parasite: identification of a new drug target. Nat Med. 2000;6(8):898-903.3.Varadharajan S, Sagar BK, Rangarajan PN, Padmanaban G. Localization of ferrochelatase in Plasmodium falciparum. Biochem J. 2004;384(Pt 2):429-436. DisclosuresNo relevant conflicts of interest to declare.