Update on the Porphyrias.

The porphyrias are a group of rare diseases, each resulting from a defect in a different enzymatic step of the heme biosynthetic pathway. They can be broadly divided into two categories, hepatic and erythropoietic porphyrias, depending on the primary site of accumulation of heme intermediates. These disorders are multisystemic with variable symptoms that can be encountered by physicians in any specialty. Here, we review the porphyrias and describe their clinical presentation, diagnosis, and management. We discuss novel therapies that are approved or in development. Early diagnosis is key for the appropriate management and prevention of long-term complications in these rare disorders.

RNA interference therapy in acute hepatic porphyrias.

The acute hepatic porphyrias (AHPs) are inherited disorders of heme biosynthesis characterized by life-threatening acute neurovisceral attacks precipitated by factors that upregulate hepatic 5-aminolevulinic acid synthase 1 (ALAS1) activity. Induction of hepatic ALAS1 leads to the accumulation of porphyrin precursors, in particular 5-aminolevulinic acid (ALA), which is thought to be the neurotoxic mediator leading to acute attack symptoms such as severe abdominal pain and autonomic dysfunction. Patients may also develop debilitating chronic symptoms and long-term medical complications, including kidney disease and an increased risk of hepatocellular carcinoma. Exogenous heme is the historical treatment for attacks and exerts its therapeutic effect by inhibiting hepatic ALAS1 activity. The pathophysiology of acute attacks provided the rationale to develop an RNA interference therapeutic that suppresses hepatic ALAS1 expression. Givosiran is a subcutaneously administered N-acetylgalactosamine-conjugated small interfering RNA against ALAS1 that is taken up nearly exclusively by hepatocytes via the asialoglycoprotein receptor. Clinical trials established that the continuous suppression of hepatic ALAS1 mRNA via monthly givosiran administration effectively reduced urinary ALA and porphobilinogen levels and acute attack rates and improved quality of life. Common side effects include injection site reactions and increases in liver enzymes and creatinine. Givosiran was approved by the US Food and Drug Administration and European Medicines Agency in 2019 and 2020, respectively, for the treatment of patients with AHP. Although givosiran has the potential to decrease the risk of chronic complications, long-term data on the safety and effects of sustained ALAS1 suppression in patients with AHP are lacking.

Brazilian registry of patients with porphyria: REBRAPPO study.

BACKGROUND: Porphyrias are a rare group of disease due to inherited defects of heme synthesis with important systemic manifestations and great burden of disease for patients and families due to the exceptional course of disease with disabling chronic symptoms interposed by life-threatening acute attacks. Unfortunately, the porphyrias are usually underrecognized reflecting a lack of medical and disease awareness as well as few studies about natural history in large cohorts of patients. The main aim of this article is present consistent data about natural history and burden of disease in a large Brazilian cohort. METHODS: We conducted a national cross-sectional registry with retrospective clinical data of Brazilian patients with porphyria collected with Brazilian patients Association with Porphyria in collaboration with a tertiary care center for rare diseases. RESULTS: A cohort of 172 patients was analyzed in which 148 (86%) patients had the diagnosis of acute hepatic porphyria [AHP] that needed a mean of 62.04 medical visits and 9.6 years to achieve a definitive diagnosis. About AHP cohort, the most common first clinical manifestation were abdominal pain in 77 (52%) patients and acute muscle weakness in 23 (15.5%) with 73 (49.3%) patients presenting only one attack during disease course and 37 (25%) exhibiting 4 or more attacks in the last year. Of note, 105 patients with AHP reported chronic manifestations and the scores for quality of life are lower when compared with general healthy population. CONCLUSIONS: Brazilian patients with AHP had a higher prevalence of chronic disabling manifestations and a poor quality of life like other cohorts and a higher proportion of patients with recurrent attacks than previously reported.

New Avenues of Heme Synthesis Regulation.

During erythropoiesis, there is an enormous demand for the synthesis of the essential cofactor of hemoglobin, heme. Heme is synthesized de novo via an eight enzyme-catalyzed pathway within each developing erythroid cell. A large body of data exists to explain the transcriptional regulation of the heme biosynthesis enzymes, but until recently much less was known about alternate forms of regulation that would allow the massive production of heme without depleting cellular metabolites. Herein, we review new studies focused on the regulation of heme synthesis via carbon flux for porphyrin synthesis to post-translations modifications (PTMs) that regulate individual enzymes. These PTMs include cofactor regulation, phosphorylation, succinylation, and glutathionylation. Additionally discussed is the role of the immunometabolite itaconate and its connection to heme synthesis and the anemia of chronic disease. These recent studies provide new avenues to regulate heme synthesis for the treatment of diseases including anemias and porphyrias.

Risk of primary liver cancer in acute hepatic porphyria patients: A matched cohort study of 1244 individuals.

BACKGROUND: The acute hepatic porphyrias (AHP) are associated with a risk of primary liver cancer (PLC), but risk estimates are unclear, and what AHP characteristics that predict PLC risk are unknown. In this register-based, matched cohort study, we assessed the PLC risk in relation to biochemical and clinical porphyria severity, genotype, age, and sex. METHODS: All patients in the Swedish porphyria register with acute intermittent porphyria (AIP), variegate porphyria (VP), or hereditary coproporphyria (HCP) during 1987-2015 were included. This AHP cohort was compared with age-, sex-, and county-matched reference individuals from the general population. National register-based hospital admissions for AHP were used to indicate the clinical severity. For AIP, the most common AHP type, patients were stratified by genotype and urinary porphobilinogen (U-PBG). Incident PLC data were collected from national health registers. RESULTS: We identified 1244 individuals with AHP (1063 [85%] AIP). During a median follow-up of 19.5 years, we identified 108 incident PLC cases, including 83 AHP patients (6.7%) and 25 of 12,333 reference individuals (0.2%). The adjusted hazard ratio for AHP-PLC was 38.0 (95% confidence interval: 24.3-59.3). Previously elevated U-PBG and hospitalizations for porphyria, but not AIP genotype or sex, were associated with increased PLC risk. Patients aged >50 years with previously elevated U-PBG (n = 157) had an annual PLC incidence of 1.8%. CONCLUSION: This study confirmed a high PLC risk and identified a strong association with clinical and biochemical AIP activity. Regular PLC surveillance is motivated in patients older than 50 years with a history of active AIP.

ABCB6 polymorphisms are not overly represented in patients with porphyria.

The Mendelian inheritance pattern of acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria is autosomal dominant, but the clinical phenotype is heterogeneous. Within the general population, penetrance is low, but among first-degree relatives of a symptomatic proband, penetrance is higher. These observations suggest that genetic factors, in addition to mutation of the specific enzyme of the biosynthetic pathway of heme, contribute to the clinical phenotype. Recent studies by others suggested that the genotype of the transporter protein ABCB6 contribute to the porphyria phenotype. Identifying the molecule(s) that are transported by ABCB6 has been problematic and has led to uncertainty with respect to how or if variants/mutants contribute to phenotypic heterogeneity. Knockout mouse models of Abcb6 have not provided a direction for investigation as homozygous knockout animals do not have a discrete phenotype. To address the proposed link between ABC6 genotype and porphyria phenotype, a large cohort of patients with acute hepatic porphyria and erythropoietic protoporphyria was analyzed. Our studies showed that ABCB6 genotype did not correlate with disease severity. Therefore, genotyping of ABCB6 in patients with acute hepatic porphyria and erythropoietic protoporphyria is not warranted.

Two Zn(II)-based Coordination Polymers: Treatment Activity on Chronic Periodontitis by Inhibiting the Relative Expression of the Porphyria gingivalis Survival Gene.

Two mixed-ligand complexes on the basis of L ligand [L = 3,6-bis(imidazol-1-yl)pyridazine] have been prepared under the solvothermal reaction conditions via the Zn(II) salts reacting with the ligands of L in the existence of two positional isomerous carboxylic acid ligands and their chemical formula respectively are [Zn5(L)(1,2-BDC)4(µ3-OH)2] n (1, 1,2-H2BDC = 1,2-benzenedicarboxylic acid ) and {[Zn4(L)2(1,3-HBDC) (1,3-BDC)(µ3-OH)4]·ClO4·3H2O} n (2, 1,3-H2BDC = 1,3-benzenedicarboxylic acid). The inhibitory influence of the two compounds against the inflammatory response in periodontium was evaluated by measuring the inflammatory cytokines releasing with ELISA detection kit. The results of ELISA assay indicated that compound 1 showed much stronger inhibitory influences than compound 2 against the inflammatory cytokines releasing. In addition to this, the suppression activity of the compounds against the survival gene of Porphyria gingivalis was detected via the real time Reverse Transcription-Polymerase Chain Reaction, and the results suggested that compound 1 could evidently suppresses the survival gene expression of Porphyria gingivalis, which is much better than the biological activity of compound 2. Above all, compound 1 was more outstanding than compound 2 on chronic periodontitis treatment by inhibiting the Porphyria gingivalis survival.

Mutation-Specific Guide RNA for Compound Heterozygous Porphyria On-target Scarless Correction by CRISPR/Cas9 in Stem Cells.

CRISPR/Cas9 is a promising technology for gene correction. However, the edition is often biallelic, and uncontrolled small insertions and deletions (indels) concomitant to precise correction are created. Mutation-specific guide RNAs were recently tested to correct dominant inherited diseases, sparing the wild-type allele. We tested an original approach to correct compound heterozygous recessive mutations. We compared editing efficiency and genotoxicity by biallelic guide RNA versus mutant allele-specific guide RNA in iPSCs derived from a congenital erythropoietic porphyria patient carrying compound heterozygous mutations resulting in UROS gene invalidation. We obtained UROS function rescue and metabolic correction with both guides with the potential of use for porphyria clinical intervention. However, unlike the biallelic one, the mutant allele-specific guide was free of on-target collateral damage. We recommend this design to avoid genotoxicity and to obtain on-target scarless gene correction for recessive disease with frequent cases of compound heterozygous mutations.

Nutrients and Porphyria: An Intriguing Crosstalk.

Porphyria refers to a group of fascinating diseases from a metabolic and nutritional standpoint as it provides an example of how metabolic manipulation can be used for therapeutic purposes. It is characterized by defects in heme synthesis, particularly in the erythrocytes and liver. Specific enzymes involved in heme biosynthesis directly depend on adequate levels of vitamins and minerals in the tissues. Moreover, micronutrients that are required for producing succinyl CoA and other intermediates in the Krebs (TCA) cycle are indirectly necessary for heme metabolism. This review summarizes articles that describe the nutritional status, supplements intake, and dietary practices of patients affected by porphyria, paying special attention to the therapeutic use of nutrients that may help or hinder this group of diseases.

Glycogen branching enzyme controls cellular iron homeostasis via Iron Regulatory Protein 1 and mitoNEET.

Iron Regulatory Protein 1 (IRP1) is a bifunctional cytosolic iron sensor. When iron levels are normal, IRP1 harbours an iron-sulphur cluster (holo-IRP1), an enzyme with aconitase activity. When iron levels fall, IRP1 loses the cluster (apo-IRP1) and binds to iron-responsive elements (IREs) in messenger RNAs (mRNAs) encoding proteins involved in cellular iron uptake, distribution, and storage. Here we show that mutations in the Drosophila 1,4-Alpha-Glucan Branching Enzyme (AGBE) gene cause porphyria. AGBE was hitherto only linked to glycogen metabolism and a fatal human disorder known as glycogen storage disease type IV. AGBE binds specifically to holo-IRP1 and to mitoNEET, a protein capable of repairing IRP1 iron-sulphur clusters. This interaction ensures nuclear translocation of holo-IRP1 and downregulation of iron-dependent processes, demonstrating that holo-IRP1 functions not just as an aconitase, but throttles target gene expression in anticipation of declining iron requirements.

Heme biosynthesis and the porphyrias.

Porphyrias, is a general term for a group of metabolic diseases that are genetic in nature. In each specific porphyria the activity of specific enzymes in the heme biosynthetic pathway is defective and leads to accumulation of pathway intermediates. Phenotypically, each disease leads to either neurologic and/or photocutaneous symptoms based on the metabolic intermediate that accumulates. In each porphyria the distinct patterns of these substances in plasma, erythrocytes, urine and feces are the basis for diagnostically defining the metabolic defect underlying the clinical observations. Porphyrias may also be classified as either erythropoietic or hepatic, depending on the principal site of accumulation of pathway intermediates. The erythropoietic porphyrias are congenital erythropoietic porphyria (CEP), and erythropoietic protoporphyria (EPP). The acute hepatic porphyrias include ALA dehydratase deficiency porphyria, acute intermittent porphyria (AIP), hereditary coproporphyria (HCP) and variegate porphyria (VP). Porphyria cutanea tarda (PCT) is the only porphyria that has both genetic and/or environmental factors that lead to reduced activity of uroporphyrinogen decarboxylase in the liver. Each of the 8 enzymes in the heme biosynthetic pathway have been associated with a specific porphyria (Table 1). Mutations affecting the erythroid form of ALA synthase (ALAS2) are most commonly associated with X-linked sideroblastic anemia, however, gain-of-function mutations of ALAS2 have also been associated with a variant form of EPP. This overview does not describe the full clinical spectrum of the porphyrias, but is meant to be an overview of the biochemical steps that are required to make heme in both erythroid and non-erythroid cells.

Porphyrin-Induced Protein Oxidation and Aggregation as a Mechanism of Porphyria-Associated Cell Injury.

Genetic porphyrias comprise eight diseases caused by defects in the heme biosynthetic pathway that lead to accumulation of heme precursors. Consequences of porphyria include photosensitivity, liver damage and increased risk of hepatocellular carcinoma, and neurovisceral involvement, including seizures. Fluorescent porphyrins that include protoporphyrin-IX, uroporphyrin and coproporphyrin, are photo-reactive; they absorb light energy and are excited to high-energy singlet and triplet states. Decay of the porphyrin excited to ground state releases energy and generates singlet oxygen. Porphyrin-induced oxidative stress is thought to be the major mechanism of porphyrin-mediated tissue damage. Although this explains the acute photosensitivity in most porphyrias, light-induced porphyrin-mediated oxidative stress does not account for the effect of porphyrins on internal organs. Recent findings demonstrate the unique role of fluorescent porphyrins in causing subcellular compartment-selective protein aggregation. Porphyrin-mediated protein aggregation associates with nuclear deformation, cytoplasmic vacuole formation and endoplasmic reticulum dilation. Porphyrin-triggered proteotoxicity is compounded by inhibition of the proteasome due to aggregation of some of its subunits. The ensuing disruption in proteostasis also manifests in cell cycle arrest coupled with aggregation of cell proliferation-related proteins, including PCNA, cdk4 and cyclin B1. Porphyrins bind to native proteins and, in presence of light and oxygen, oxidize several amino acids, particularly methionine. Noncovalent interaction of oxidized proteins with porphyrins leads to formation of protein aggregates. In internal organs, particularly the liver, light-independent porphyrin-mediated protein aggregation occurs after secondary triggers of oxidative stress. Thus, porphyrin-induced protein aggregation provides a novel mechanism for external and internal tissue damage in porphyrias that involve fluorescent porphyrin accumulation.

[Syndromes with skin fragility]. / Syndrome mit Hautfragilität.

Syndromic disorders with skin fragility belong to different groups of genodermatoses: epidermolysis bullosa (EB), Ehlers-Danlos syndrome and porphyria. The genetic defects mainly concern structural proteins which assure the mechanical stability of the skin and other tissues. Depending on the expression pattern of the affected protein in the skin, cutaneous fragility may manifest as superficial erosions, blisters, wounds, wound healing defects or scars. Extracutaneous manifestations are manifold and involve the heart, skeletal muscles, intestine, kidneys, blood vessels or the skeleton. Syndromic types of EB include in addition to skin blistering: (i) cardiomyopathy in case of desmoplakin, plakoglobin, or kelch-like protein mutations; (ii) muscular dystrophy in case of plektin mutations; (iii) pyloric atresia in case of integrin α6ß4 or plectin mutations; (iv) nephrotic syndrome in case of CD151 or integrin α3 mutations. Lysyl hydroxylase 3 mutations affect posttranslational modifications of collagens and lead to a dystrophic epidermolysis bullosa-like multisystemic disorder. Ehlers-Danlos syndromes are due to defects of dermal collagens or their processing and affect the skin, joints and blood vessels. Finally porphyrias are complex metabolic disorders with photosensitivity and sometimes skin fragility, liver or neurologic problems. Their pathogenesis relies on the accumulation of precursors in the tissues. Although these syndromes are rare in clinical practice, knowledge of the syndromic constellation contributes to early diagnosis and detection of complications.

International Porphyria Molecular Diagnostic Collaborative: an evidence-based database of verified pathogenic and benign variants for the porphyrias.

With the advent of precision and genomic medicine, a critical issue is whether a disease gene variant is pathogenic or benign. Such is the case for the three autosomal dominant acute hepatic porphyrias (AHPs), including acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria, each resulting from the half-normal enzymatic activities of hydroxymethylbilane synthase, coproporphyrinogen oxidase, and protoporphyrinogen oxidase, respectively. To date, there is no public database that documents the likely pathogenicity of variants causing the porphyrias, and more specifically, the AHPs with biochemically and clinically verified information. Therefore, an international collaborative with the European Porphyria Network and the National Institutes of Health/National Center for Advancing Translational Sciences/National Institute of Diabetes and Digestive and Kidney Diseases (NIH/NCATS/NIDDK)-sponsored Porphyrias Consortium of porphyria diagnostic experts is establishing an online database that will collate biochemical and clinical evidence verifying the pathogenicity of the published and newly identified variants in the AHP-causing genes. The overall goal of the International Porphyria Molecular Diagnostic Collaborative is to determine the pathogenic and benign variants for all eight porphyrias. Here we describe the overall objectives and the initial efforts to validate pathogenic and benign variants in the respective heme biosynthetic genes causing the AHPs.

Murine models of the human porphyrias: Contributions toward understanding disease pathogenesis and the development of new therapies.

Mouse models of the human porphyrias have proven useful for investigations of disease pathogenesis and to facilitate the development of new therapeutic approaches. To date, mouse models have been generated for all major porphyrias, with the exception of X-linked protoporphyria (XLP) and the ultra rare 5-aminolevulinic acid dehydratase deficient porphyria (ADP). Mouse models have been generated for the three autosomal dominant acute hepatic porphyrias, acute intermittent porphyria (AIP), hereditary coproporphyria (HCP), and variegate porphyria (VP). The AIP mice, in particular, provide a useful investigative model as they have been shown to have acute biochemical attacks when induced with the prototypic porphyrinogenic drug, phenobarbital. In addition to providing important insights into the disease pathogenesis of the neurological impairment in AIP, these mice have been valuable for preclinical evaluation of liver-targeted gene therapy and RNAi-mediated approaches. Mice with severe HMBS deficiency, which clinically and biochemically mimic the early-onset homozygous dominant AIP (HD-AIP) patients, have been generated and were used to elucidate the striking phenotypic differences between AIP and HD-AIP. Mice modeling the hepatocutaneous porphyria, porphyria cutanea tarda (PCT), made possible the identification of the iron-dependent inhibitory mechanism of uroporphyrinogen decarboxylase (UROD) that leads to symptomatic PCT. Mouse models for the two autosomal recessive erythropoietic porphyrias, congenital erythropoietic porphyria (CEP) and erythropoeitic protoporphyria (EPP), recapitulate many of the clinical and biochemical features of the severe human diseases and have been particularly useful for evaluation of bone marrow transplantation and hematopoietic stem cell (HSC)-based gene therapy approaches. The EPP mice have also provided valuable insights into the underlying pathogenesis of EPP-induced liver damage and anemia.

Regulation and tissue-specific expression of δ-aminolevulinic acid synthases in non-syndromic sideroblastic anemias and porphyrias.

Recently, new genes and molecular mechanisms have been identified in patients with porphyrias and sideroblastic anemias (SA). They all modulate either directly or indirectly the δ-aminolevulinic acid synthase (ALAS) activity. ALAS, is encoded by two genes: the erythroid-specific (ALAS2), and the ubiquitously expressed (ALAS1). In the liver, ALAS1 controls the rate-limiting step in the production of heme and hemoproteins that are rapidly turned over in response to metabolic needs. Several heme regulatory targets have been identified as regulators of ALAS1 activity: 1) transcriptional repression via a heme-responsive element, 2) post-transcriptional destabilization of ALAS1 mRNA, 3) post-translational inhibition via a heme regulatory motif, 4) direct inhibition of the activity of the enzyme and 5) breakdown of ALAS1 protein via heme-mediated induction of the protease Lon peptidase 1. In erythroid cells, ALAS2 is a gatekeeper of production of very large amounts of heme necessary for hemoglobin synthesis. The rate of ALAS2 synthesis is transiently increased during the period of active heme synthesis. Its gene expression is determined by trans-activation of nuclear factor GATA1, CACC box and NF-E2-binding sites in the promoter areas. ALAS2 mRNA translation is also regulated by the iron-responsive element (IRE)/iron regulatory proteins (IRP) binding system. In patients, ALAS enzyme activity is affected in most of the mutations causing non-syndromic SA and in several porphyrias. Decreased ALAS2 activity results either directly from loss-of-function ALAS2 mutations as seen in X-linked sideroblastic anemia (XLSA) or from defect in the availability of one of its two mitochondrial substrates: glycine in SLC25A38 mutations and succinyl CoA in GLRX5 mutations. Moreover, ALAS2 gain of function mutations is responsible for X-linked protoporphyria and increased ALAS1 activity lead to acute attacks of hepatic porphyrias. A missense dominant mutation in the Walker A motif of the ATPase binding site in the gene coding for the mitochondrial protein unfoldase CLPX also contributes to increasing ALAS and subsequently protoporphyrinemia. Altogether, these recent data on human ALAS have informed our understanding of porphyrias and sideroblastic anemias pathogeneses and may contribute to new therapeutic strategies.

[The cutaneous porphyrias]. / Porphyries cutanées.

The porphyrias are a group of metabolic disorders resulting from an innate abnormality in haem biosynthesis, and the clinical settings of which vary according to the genetic enzyme abnormality in question. These are genetic disorders with autosomal dominant or recessive inheritance of varying penetrance, and whose clinical expression differs according to the preferential location of haem precursors. Different classifications have been proposed according to genetic inheritance, the enzyme anomaly at issue, and clinical expression. The clinical classification distinguishes between acute porphyria (acute intermittent porphyria, porphyria variegata, hereditary coproporphyria), bullous cutaneous porphyrias (porphyria cutanea tarda, porphyria variegata and hereditary coproporphyria), painful photosensitive acute cutaneous porphyrias (erythropoietic protoporphyria and X-linked dominant protoporphyria), and rare recessive porphyrias (congenital erythropoietic porphyria, Doss porphyria, hepatoerythropoietic porphyria and harderoporphyria). Treatment depends on the clinical expression of the disorder.

Recent advances on porphyria genetics: Inheritance, penetrance & molecular heterogeneity, including new modifying/causative genes.

The inborn errors of heme biosynthesis, the Porphyrias, include eight major disorders resulting from loss-of-function (LOF) or gain-of-function (GOF) mutations in eight of the nine heme biosynthetic genes. The major sites of heme biosynthesis are the liver and erythron, and the underlying pathophysiology of each of these disorders depends on the unique biochemistry, cell biology, and genetic mechanisms in these tissues. The porphyrias are classified into three major categories: 1) the acute hepatic porphyrias (AHPs), including Acute Intermittent Porphyria (AIP), Hereditary Coproporphyria (HCP), Variegate Porphyria (VP), and 5-Aminolevlulinic Acid Dehydratase Deficient Porphyria (ADP); 2) a hepatic cutaneous porphyria, Porphyria Cutanea Tarda (PCT); and 3) the cutaneous erythropoietic porphyrias, Congenital Erythropoietic Porphyria (CEP), Erythropoietic Protoporphyria (EPP), and X-Linked Protoporphyria (XLP). Their modes of inheritance include autosomal dominant with markedly decreased penetrance (AIP, VP, and HCP), autosomal recessive (ADP, CEP, and EPP), or X-linked (XLP), as well as an acquired sporadic form (PCT). There are severe homozygous dominant forms of the three AHPs. For each porphyria, its phenotype, inheritance pattern, unique genetic principles, and molecular genetic heterogeneity are presented. To date, >1000 mutations in the heme biosynthetic genes causing their respective porphyrias have been reported, including low expression alleles and genotype/phenotype correlations that predict severity for certain porphyrias. The tissue-specific regulation of heme biosynthesis and the unique genetic mechanisms for each porphyria are highlighted.

Molecular analysis of 19 Spanish patients with mixed porphyrias.

Porphyrias are rare diseases caused by alterations in the heme biosynthetic pathway. Depending on the afected enzyme, porphyrin precursors or porphyrins are overproduced, causing acute neurovisceral attacks or dermal photosensitivity, respectively. Hereditary Coproporphyria (HCP) and Variegate Porphyria (VP) are mixed porphyrias since they can present acute and/or cutaneous symptoms. These diseases are caused by a deficiency of coproporphyrinogen oxidase (CPOX) in HCP, and protoporphyrinogen oxidase (PPOX) in VP. Herein, we studied nineteen unrelated Spanish patients with mixed porphyrias. The diagnosis of either, HCP or VP was made on the basis of clinical symptoms, biochemical findings and the identification of the mutation responsible in the CPOX or PPOX genes. Two patients presented both acute and cutaneous symptoms. In most patients, the biochemical data allowed the diagnosis. Among eleven patients with HCP, ten CPOX mutations were identified, including six novel ones: two frameshift (c.32delG and c.1102delC), two nonsense (p.Cys239Ter and p.Tyr365Ter), one missense (p.Trp275Arg) and one amino acid deletion (p.Gly336del). Moreover, seven previously described PPOX mutations were identified in eight patients with VP. The impacts of CPOX mutations p.Trp275Arg and p.Gly336del, were evaluated using prediction softwares and their functional consequences were studied in a prokaryotic expression system. Both alterations were predicted as deleterious by in silico analysis. Aditionally, when these alleles were expressed in E. coli, only p.Trp275Arg retained some residual activity. These results emphasize the usefulness of integrated the biochemical tests and molecular studies in the diagnosis. Furthermore, they extend knowledge on the molecular heterogeneity of mixed porphyrias in Spain.

A Variant of Peptide Transporter 2 Predicts the Severity of Porphyria-Associated Kidney Disease.

CKD occurs in most patients with acute intermittent porphyria (AIP). During AIP, δ-aminolevulinic acid (ALA) accumulates and promotes tubular cell death and tubulointerstitial damage. The human peptide transporter 2 (PEPT2) expressed by proximal tubular cells mediates the reabsorption of ALA, and variants of PEPT2 have different affinities for ALA. We tested the hypothesis that PEPT2 genotypes affect the severity and prognosis of porphyria-associated kidney disease. We analyzed data from 122 individuals with AIP who were followed from 2003 to 2013 and genotyped for PEPT2 At last follow-up, carriers of the PEPT2*1*1 genotype (higher affinity variant) exhibited worse renal function than carriers of the lower affinity variants PEPT2*1/*2 and PEPT2*2/*2 (mean±SD eGFR: 54.4±19.1, 66.6±23.8, and 78.1±19.9 ml/min per 1.73 m2, respectively). Change in eGFR (mean±SD) over the 10-year period was -11.0±3.3, -2.4±1.9, and 3.4±2.6 ml/min per 1.73 m2 for PEPT2*1/*1, PEPT2*1*2, and PEPT*2*2*2 carriers, respectively. At the end of follow-up, 68% of PEPT2*1*1 carriers had an eGFR<60 ml/min per 1.73 m2, compared with 37% of PEPT2*1*2 carriers and 15% of PEPT2*2*2 carriers. Multiple regression models including all confounders indicated that the PEPT2*1*1 genotype independently associated with an eGFR<60 ml/min per 1.73 m2 (odds ratio, 6.85; 95% confidence interval, 1.34 to 46.20) and an annual decrease in eGFR of >1 ml/min per 1.73 m2 (odds ratio, 3.64; 95% confidence interval, 1.37 to 9.91). Thus, a gene variant is predictive of the severity of a chronic complication of AIP. The therapeutic value of PEPT2 inhibitors in preventing porphyria-associated kidney disease warrants investigation.