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1.
Liver Int ; 2024 Jul 16.
Article in English | MEDLINE | ID: mdl-39011756

ABSTRACT

The erythropoietic protoporphyrias consist of three ultra-rare genetic disorders of the erythroid heme biosynthesis, including erythropoietic protoporphyria (EPP1), X-linked protoporphyria (XLEPP) and CLPX-protoporphyria (EPP2), which all lead to the accumulation of protoporphyrin IX (PPIX) in erythrocytes. Affected patients usually present from early childhood with episodes of severe phototoxic pain in the skin exposed to visible light. The quantification of PPIX in erythrocytes with a metal-free PPIX ≥3 times the upper limit of normal confirms the diagnosis. Protoporphyria-related complications include liver failure, gallstones, mild anaemia and vitamin D deficiency with reduced bone mineral density. The management is focused on preventing phototoxic reactions and treating the complications. Vitamin D should be supplemented, and DEXA scans in adults should be considered. In EPP1, even in cases of biochemically determined iron deficiency, supplementation of iron may stimulate PPIX production, resulting in an increase in photosensitivity and the risk of cholestatic liver disease. However, for patients with XLEPP, iron supplementation can reduce PPIX levels, phototoxicity and liver damage. Because of its rarity, there is little data on the management of EPP-related liver disease. As a first measure, any hepatotoxins should be eliminated. Depending on the severity of the liver disease, phlebotomies, exchange transfusions and ultimately liver transplantation with subsequent haematopoietic stem cell transplantation (HSCT) are therapeutic options, whereby multidisciplinary management including porphyria experts is mandatory. Afamelanotide, an alpha-melanocyte-stimulating hormone analogue, is currently the only approved specific treatment that increases pain-free sunlight exposure and quality of life.

2.
Nucleic Acids Res ; 48(9): 4658-4671, 2020 05 21.
Article in English | MEDLINE | ID: mdl-32313951

ABSTRACT

Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.


Subject(s)
Ferrochelatase/genetics , Oligonucleotides/administration & dosage , Protoporphyria, Erythropoietic/metabolism , RNA Splicing , Albumins/metabolism , Animals , Bone Marrow/metabolism , COS Cells , Chlorocebus aethiops , Disease Models, Animal , Ferrochelatase/metabolism , Humans , K562 Cells , Mice , Oligonucleotides/blood , Oligonucleotides/chemistry , Oligonucleotides/pharmacokinetics , Polymorphism, Single Nucleotide , Protoporphyria, Erythropoietic/genetics , Protoporphyria, Erythropoietic/therapy , RNA Splice Sites , Tissue Distribution
3.
Mol Genet Metab ; 128(3): 304-308, 2019 11.
Article in English | MEDLINE | ID: mdl-31076252

ABSTRACT

Deficiency in ferrochelatase (FECH), the last enzyme in the heme biosynthetic pathway, leads to an accumulation of protoporphyrin IX (PPIX) that causes a severely painful phototoxic reaction of the skin in patients with erythropoietic protoporphyria (EPP). Besides phototoxicity of the skin, EPP patients often present with symptoms of iron deficiency in form of a microcytic and hypochromic anemia with low serum iron and ferritin. In addition, elevated aminolevulinic acid synthase 2 (ALAS2) both at the mRNA and protein levels have been observed among EPP patients. ALAS is the first enzyme in the pathway and exists in two isoforms, whereby the isoform 2 (ALAS2) is expressed exclusively in erythropoiesis. The mRNA of ALAS2 contains an iron response element (IRE) at its 5'UTR. When iron is limited, iron response element binding protein 2 (IRP2) binds to the IRE of ALAS2 mRNA and suppresses its translation. In this study, we demonstrated that iron deprivation increased the amount of ALAS2 mRNA as well as the ratio of ALAS2 to FECH mRNAs in cultured erythroleukemic K562 cells. At the protein level, however, iron deprivation in the cell line caused reductions in both enzymes as shown by the Western blot analysis. A comparable increase in the ratio of ALAS2 to FECH mRNAs was also found in EPP patients indicating an imbalance in heme biosynthesis. As iron cannot be completely missing from an organism, we assume that in EPP patients, a certain amount of ALAS2 mRNA is translated despite a partial deficiency of FECH. The increase in ALAS2 enzyme contributes to the accumulation in PPIX in the patients. Targeted inhibition of ALAS2 could therefore be a treatment option for EPP.


Subject(s)
5-Aminolevulinate Synthetase/genetics , 5-Aminolevulinate Synthetase/metabolism , Iron/metabolism , Protoporphyria, Erythropoietic/enzymology , Biosynthetic Pathways , Ferrochelatase/genetics , Humans , Iron/blood , Iron Regulatory Protein 2/metabolism , Iron-Regulatory Proteins/metabolism , K562 Cells , Protoporphyria, Erythropoietic/therapy , Protoporphyrins/metabolism
4.
Scand J Clin Lab Invest ; 79(5): 305-313, 2019 Sep.
Article in English | MEDLINE | ID: mdl-31154864

ABSTRACT

Molecular diagnosis of autosomal dominant acute hepatic porphyrias (AHPs) plays an important role in the management of these disorders. To introduce next generation sequencing (NGS) to the porphyria diagnosis, we designed a panel that contained four genes, ALAS1, HMBS, CPOX and PPOX for mutational analysis of acute intermittent porphyria (AIP), hereditary coproporphyria (HCP) and variegate porphyria (VP). To validate the AHP panel, 30 samples with known pathogenic variants as determined by Sanger sequencing, were analyzed using the Ion PGM™. Among them, nine have so far not been reported. The pathogenic variants were identified and annotated manually in IGV by three individuals who were blinded to the Sanger results. The AHP panel consists of 95 amplicons that covers 92% of the coding region of the four genes. Of the 95 amplicons, 93 had an average read-depth of >500 reads. In 29 of the 30 tested samples, pathogenic variants were correctly identified and annotated. The number of reads from the mutated alleles were approximately 50% of the total. The annotation of a 22-bp duplication with NGS differed from that of Sanger by one nucleotide. NGS showed an advantage in allelic discrimination over Sanger sequencing and was also able to detect a known somatic variant in the HMBS gene. The AHP panel will be applied in the initial diagnosis of new patients. Any sequence variations with a frequency of ≥10% will be confirmed by Sanger sequencing. The cost-effectiveness of a NGS approach for AHP in a diagnostic laboratory needs to be further assessed.


Subject(s)
DNA Mutational Analysis/methods , High-Throughput Nucleotide Sequencing/methods , Porphobilinogen Synthase/deficiency , Porphyrias, Hepatic/genetics , Alleles , Base Sequence , Cohort Studies , Humans , Mutation/genetics , Polymorphism, Single Nucleotide/genetics , Porphobilinogen Synthase/genetics
5.
Ther Umsch ; 75(4): 225-233, 2018 Nov.
Article in German | MEDLINE | ID: mdl-30468116

ABSTRACT

Porphyria - when to think about how to clarify and treat? Abstract. Porphyrias are a group of metabolic disorders that are mostly hereditary. They manifest either as abdominal colic or as skin changes at light-exposed areas. During the symptomatic phase the diagnosis of porphyria can be made by cost-effective screening tests. If the screening gives a positive result, further testing is required to determine the exact type of porphyria and to establish the best therapeutic option for the patient in a specialized porphyria center.


Subject(s)
Porphyrias , Humans , Skin
6.
J Inherit Metab Dis ; 40(3): 433-441, 2017 05.
Article in English | MEDLINE | ID: mdl-28185024

ABSTRACT

Patients with erythropoietic protoporphyria (EPP) have reduced activity of the enzyme ferrochelatase that catalyzes the insertion of iron into protoporphyrin IX (PPIX) to form heme. As the result of ferrochelatase deficiency, PPIX accumulates and causes severe photosensitivity. Among different patients, the concentration of PPIX varies considerably. In addition to photosensitivity, patients frequently exhibit low serum iron and a microcytic hypochromic anemia. The aims of this study were to (1) search for factors related to PPIX concentration in EPP, and (2) characterize anemia in EPP, i.e., whether it is the result of an absolute iron deficiency or the anemia of chronic disease (ACD). Blood samples from 67 EPP patients (51 Italian and 16 Swiss) and 21 healthy volunteers were analyzed. EPP patients had lower ferritin (p = 0.021) and hepcidin (p = 0.031) concentrations and higher zinc-protoporphyrin (p < 0.0001) and soluble-transferrin-receptor (p = 0.0007) concentrations compared with controls. This indicated that anemia in EPP resulted from an absolute iron deficiency. Among EPP patients, PPIX concentrations correlated with both growth differentiation factor (GDF) 15 (p = 0.012) and male gender (p = 0.015). Among a subgroup of patients who were iron replete, hemoglobin levels were normal, which suggested that iron but not ferrochelatase is the limiting factor in heme synthesis of individuals with EPP.


Subject(s)
Growth Differentiation Factor 15/metabolism , Iron/metabolism , Protoporphyria, Erythropoietic/metabolism , Anemia, Hypochromic/metabolism , Case-Control Studies , Erythrocytes/metabolism , Female , Ferritins/metabolism , Ferrochelatase/metabolism , Hemoglobins/metabolism , Hepcidins/metabolism , Humans , Male , Photosensitivity Disorders/metabolism , Protoporphyrins/metabolism , Severity of Illness Index
7.
Hum Mol Genet ; 22(7): 1280-8, 2013 Apr 01.
Article in English | MEDLINE | ID: mdl-23263862

ABSTRACT

Frameshift mutations in the last coding exon of the 5-aminolevulinate synthase (ALAS) 2 gene were described to activate the enzyme causing increased levels of zinc- and metal-free protoporphyrin in patients with X-linked dominant protoporphyria (XLDPP). Only two such so-called gain-of-function mutations have been reported since the description of XLDPP in 2008. In this study of four newly identified XLDPP families, we identified two novel ALAS2 gene mutations, a nonsense p.Q548X and a frameshift c.1651-1677del26bp, along with a known mutation (delAGTG) found in two unrelated families. Of relevance, a de novo somatic and germinal mosaicism was present in a delAGTG family. Such a phenomenon may explain the high proportion of this mutation in XLDPP worldwide. Enhancements of over 3- and 14-fold in the catalytic rate and specificity constant of purified recombinant XLDPP variants in relation to those of wild-type ALAS2 confirmed the gain of function ascribed to these enzymes. The fact that both p.Q548X and c.1651-1677del26bp are located in close proximity and upstream from the two previously described mutations led us to propose the presence of a large gain-of-function domain within the C-terminus of ALAS2. To test this hypothesis, we generated four additional nonsense mutants (p.A539X, p.G544X, p.G576X and p.V583X) surrounding the human XLDPP mutations and defined an ALAS2 gain-of-function domain with a minimal size of 33 amino acids. The identification of this gain-of-function domain provides important information on the enzymatic activity of ALAS2, which was proposed to be constitutively inhibited, either directly or indirectly, through its own C-terminus.


Subject(s)
5-Aminolevulinate Synthetase/genetics , Genetic Diseases, X-Linked/genetics , Protoporphyria, Erythropoietic/genetics , 5-Aminolevulinate Synthetase/chemistry , 5-Aminolevulinate Synthetase/deficiency , 5-Aminolevulinate Synthetase/metabolism , Amino Acid Sequence , Base Sequence , Child, Preschool , Codon, Nonsense , DNA Mutational Analysis , Exons , Female , Frameshift Mutation , Genetic Association Studies , Genetic Diseases, X-Linked/enzymology , Humans , Infant , Kinetics , Molecular Sequence Data , Mosaicism , Mutagenesis, Site-Directed , Pedigree , Protein Structure, Tertiary , Protoporphyria, Erythropoietic/enzymology , Sequence Analysis, DNA , Young Adult
8.
J Hepatol ; 62(3): 734-8, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25445397

ABSTRACT

Variegate porphyria (VP) and acute intermittent porphyria (AIP), the two most common types of acute porphyrias (AHPs), result from a partial deficiency of protoporphyrinogen oxidase (PPOX) and hydroxymethylbilane synthase (HMBS), respectively. A rare but serious complication in the AHPs is hepatocellular carcinoma (HCC). However, the underlying pathomechanisms are yet unknown. We performed DNA sequence analysis in cancerous and non-cancerous liver tissue of a VP and an AIP patient, both with HCC. In samples of both cancerous and non-cancerous liver tissues from the patients, we identified the underlying PPOX and HMBS germline mutations, c.1082dupC and p.G111R, respectively. Additionally, we detected a second somatic mutation, only in the cancer tissue i.e., p.L416X in the PPOX gene of the VP patient and p.L220X in the HMBS gene of the AIP patient, both located in trans to the respective germline mutations. Both somatic mutations were not detected in 10 non-porphyria-associated HCCs. Our data demonstrate that in the hepatic cancer tissue of AHP patients, somatic second-hit mutations result in nearly complete inactivation of the enzymes catalyzing major steps in the heme biosynthetic pathway. Both PPOX and HMBS, which might act as tumor suppressors, play a crucial role in the development of HCC in these individuals.


Subject(s)
Carcinoma, Hepatocellular/etiology , Carcinoma, Hepatocellular/genetics , Flavoproteins/genetics , Hydroxymethylbilane Synthase/genetics , Liver Neoplasms/etiology , Liver Neoplasms/genetics , Mitochondrial Proteins/deficiency , Mitochondrial Proteins/genetics , Mutation , Porphyria, Acute Intermittent/complications , Porphyria, Acute Intermittent/genetics , Porphyria, Variegate/complications , Porphyria, Variegate/genetics , Protoporphyrinogen Oxidase/genetics , Aged , Aged, 80 and over , Carcinoma, Hepatocellular/enzymology , Female , Germ-Line Mutation , Humans , Liver Neoplasms/enzymology , Porphyria, Acute Intermittent/enzymology , Porphyria, Variegate/enzymology , Tumor Suppressor Proteins/deficiency , Tumor Suppressor Proteins/genetics
9.
Blood Cells Mol Dis ; 54(1): 71-7, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25179834

ABSTRACT

The activity of the erythroid-specific isoenzyme of 5-aminolevulinic acid synthase (ALAS2), the first and rate-limiting enzyme in heme biosynthesis, is down-regulated during iron-deficiency. Ferrochelatase (FECH), the last enzyme of this pathway, inserts iron into protoporphyrin IX (PPIX) to form heme. Patients with erythropoietic protoporphyria (EPP), an inherited deficiency in FECH, often show signs of iron deficiency in addition to phototoxicity which is caused by PPIX accumulation. However, iron supplementation often leads to exacerbation of phototoxicity. We report three EPP patients who had reduced erythrocytic PPIX concentrations when they were iron-deficient and their microcytic and hypochromic anemia deteriorated. Additionally, we found a significant increase in the amount of ALAS2 mRNA and protein among EPP patients. To verify the connection between FECH deficiency and ALAS2 over-expression, we inhibited FECH in cultured cells and found a subsequent increase in ALAS2 mRNA. We conclude that the primary deficiency in ferrochelatase leads to a secondary increase in ALAS2 expression. The combined action of these two enzymes within the heme biosynthetic pathway contributes to the accumulation of PPIX. Furthermore, we hypothesize that EPP patients may benefit from a mild iron deficiency since it would limit PPIX production by restricting ALAS2 over-expression.


Subject(s)
5-Aminolevulinate Synthetase/biosynthesis , Erythrocytes/enzymology , Gene Expression Regulation, Enzymologic , Iron/metabolism , Protoporphyria, Erythropoietic/enzymology , Protoporphyrins/metabolism , Adolescent , Adult , Erythrocytes/pathology , Female , Humans , Male , Middle Aged , Protoporphyria, Erythropoietic/pathology , RNA, Messenger/biosynthesis
10.
Skin Pharmacol Physiol ; 28(2): 103-13, 2015.
Article in English | MEDLINE | ID: mdl-25402764

ABSTRACT

UNLABELLED: Afamelanotide is an α-melanocyte-stimulating hormone (α-MSH) agonist with proven efficacy in photodermatoses such as erythropoietic protoporphyria (EPP). This peptide drug, repeatedly administered over prolonged time, may induce anti-drug antibodies (ADA). Here, we describe a new ELISA method developed to monitor the occurrence of ADA against afamelanotide as well as against α-MSH. Covalent binding instead of absorption of antigen onto the microtitre wells prevented antigen leakage and enabled extensive washings followed by lower background. The cut-off between antibody-negative and -positive sera was determined. Inhibition of the antigen-antibody reaction by excess soluble antigen tested for specificity. The sensitivity of the ELISA was 608 and 1,390 ng/ml of specific ADA against afamelanotide and α-MSH, respectively. This ELISA method enabled us to investigate the occurrence of ADA during long-term administration of afamelanotide. No immunoreactivity was found in 23 of the 26 EPP patients exposed to the drug for up to 6 years. Pre-existing immunoreactivity against afamelanotide as well as α-MSH was found in 3 patients, whose titres did not change during afamelanotide administration. CONCLUSION: The new ELISA is suitable to determine ADA against afamelanotide and α-MSH. Afamelanotide did not elicit ADA during long-term administration in patients with EPP.


Subject(s)
Antibodies/blood , Enzyme-Linked Immunosorbent Assay , Porphyria, Erythropoietic/drug therapy , Porphyria, Erythropoietic/immunology , alpha-MSH/analogs & derivatives , Compassionate Use Trials , Drug Implants , Humans , Porphyria, Erythropoietic/blood , Predictive Value of Tests , Time Factors , Treatment Outcome , alpha-MSH/administration & dosage , alpha-MSH/immunology , alpha-MSH/therapeutic use
11.
Blood Cells Mol Dis ; 51(3): 151-61, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23787363

ABSTRACT

Erythropoietic protoporphyria (EPP) results from partial deficiency of ferrochelatase (FECH). Genetically, EPP patients differ from asymptomatic mutation carriers at the unmutated FECH allele, the expression of which is modulated by single nucleotide polymorphism IVS3-48C/T. The IVS3-48C genotype, which is present among patients, leads to correct splicing of 60% of the pre-mRNA and to alternative splicing of 40%, the latter mRNA-product being destroyed by nonsense-mediated decay. An IVS3-48T genotype generates 80% correct and 20% aberrant products. Our study demonstrated that under iron deficient conditions, the aberrant splice product was increased to 56% and 50% of total FECH mRNA in erythroleukemic K562 and lymphoblastoid cell lines, respectively, both being homozygous for IVS3-48T. Concomitantly, FECH protein was decreased. Iron deficiency had less effect on the FECH splice ratio in an IVS3-48C/C lymphoblastoid cell line. Effects similar to iron deficiency were generated by siRNA knockdown of either splicing factor U2AF(65) or Fe(II)- and 2-oxoglutarate-dependent dioxygenase Jumonji domain-containing protein 6 (Jmjd6), which interacts with U2AF(65) by lysyl-hydroxylation. Based on these results, we propose that the availability of iron, a co-factor of Jmjd6, modulates U2AF(65)-lysyl-hydroxylation. This in turn, influences the relative amounts of correct and aberrant FECH mRNA splice products and thus, regulates the FECH enzyme activity.


Subject(s)
Alternative Splicing , Ferrochelatase/genetics , Iron/metabolism , Jumonji Domain-Containing Histone Demethylases/metabolism , Ketoglutaric Acids/metabolism , Nuclear Proteins/metabolism , Ribonucleoproteins/metabolism , Base Sequence , Case-Control Studies , Cell Line , Cobalt/pharmacology , Deferoxamine/metabolism , Deferoxamine/pharmacology , Ferrochelatase/metabolism , Gene Expression Regulation/drug effects , Gene Order , Gene Silencing , Genotype , Humans , Introns , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , K562 Cells , Molecular Sequence Data , Mutation , Polymorphism, Single Nucleotide , Protoporphyria, Erythropoietic/genetics , Protoporphyria, Erythropoietic/metabolism , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Splicing Factor U2AF
12.
Life (Basel) ; 13(4)2023 Apr 21.
Article in English | MEDLINE | ID: mdl-37109595

ABSTRACT

In animal models, melanocyte-stimulating hormones (MSHs) protect the liver from various injuries. Erythropoietic protoporphyria (EPP), a metabolic disorder, leads to the accumulation of protoporphyrin (PPIX). In addition to the most prominent symptom of incapacitating phototoxic skin reactions, 20% of EPP patients exhibit disturbed liver functioning and 4% experience terminal liver failure caused by the hepatobiliary elimination of excess PPIX. Skin symptoms are mitigated through the application of the controlled-release implant afamelanotide, an α-MSH analog, every sixty days. Recently, we showed that liver function tests (LFTs) improved during afamelanotide treatment when compared to before treatment. The present study investigated whether this effect is dose-dependent, as the evidence of dose dependency would support a beneficial influence of afamelanotide. METHODS: In this retrospective observational study, we included 2933 liver-function tests, 1186 PPIX concentrations and 1659 afamelanotide implant applications in 70 EPP patients. We investigated whether the number of days since the preceding afamelanotide dose or the number of doses during the preceding 365 days had an effect on LFTs and PPIX levels. In addition, we assessed the effect of global radiation. RESULTS: Inter-patient differences exerted the most prominent effect on PPIX and LFTs. In addition, PPIX increased significantly with an increase in the number of days since the last afamelanotide implant (p < 0.0001). ALAT and bilirubin decreased significantly with an increasing number of afamelanotide doses in the preceding 365 days (p = 0.012, p = 0.0299, respectively). Global radiation only influenced PPIX (p = 0.0113). CONCLUSIONS: These findings suggest that afamelanotide ameliorates both PPIX concentrations and LFTs in EPP in a dose-dependent manner.

13.
Life (Basel) ; 13(9)2023 Sep 10.
Article in English | MEDLINE | ID: mdl-37763293

ABSTRACT

Acute porphyrias are a group of monogenetic inborn errors of heme biosynthesis, characterized by acute and potentially life-threatening neurovisceral attacks upon exposure to certain triggering factors. Biochemical analyses can determine the type of acute porphyria, and subsequent genetic analysis allows for the identification of pathogenic variants in the specific gene, which provides information for family counselling. In 2017, a male Swiss patient was diagnosed with an acute porphyria while suffering from an acute attack. The pattern of porphyrin metabolite excretion in urine, faeces, and plasma was typical for an acute intermittent porphyria (AIP), which is caused by inherited autosomal dominant mutations in the gene for hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway. However, the measurement of HMBS enzymatic activity in the erythrocytes was within the normal range and Sanger sequencing of the HMBS gene failed to detect any pathogenic variants. To explore the molecular basis of the apparent AIP in this patient, we performed third-generation long-read single-molecule sequencing (nanopore sequencing) on a PCR product spanning the entire HMBS gene, including the intronic sequences. We identified a known pathogenic variant, c.77G>A, p.(Arg26His), in exon 3 at an allelic frequency of ~22% in the patient's blood. The absence of the pathogenic variant in the DNA of the parents and the results of additional confirmatory studies supported the presence of a de novo mosaic mutation. To our knowledge, such a mutation has not been previously described in any acute porphyria. Therefore, de novo mosaic mutations should be considered as potential causes of acute porphyrias when no pathogenic genetic variant can be identified through routine molecular diagnostics.

15.
Blood ; 115(5): 1062-9, 2010 Feb 04.
Article in English | MEDLINE | ID: mdl-19965637

ABSTRACT

Splicing mutations account for approximately 10% of lesions causing genetic diseases, but few branchpoint sequence (BPS) lesions have been reported. In 3 families with autosomal recessive congenital erythropoietic porphyria (CEP) resulting from uroporphyrinogen III synthase (URO-synthase) deficiency, sequencing the promoter, all 10 exons and the intron/exon boundaries did not detect a mutation. Northern analyses of lymphoblast mRNAs from 2 patients and reverse-transcribed polymerase chain reaction (RT-PCR) of lymphoblast mRNAs from all 3 patients revealed multiple longer transcripts involving intron 9 and low levels of wild-type message. Sequencing intron 9 RT-PCR products and genomic DNA in each case revealed homozygosity for a novel BPS mutation (c.661-31T-->G) and alternatively spliced transcripts containing 81, 246, 358, and 523 nucleotides from intron 9. RT-PCR revealed aberrant transcripts in both wild-type and CEP lymphoblasts, whereas BPS mutation reduced the wild-type transcript and enzyme activity in CEP lymphoblasts to approximately 10% and 15% of normal, respectively. Although the +81-nucleotide alternative transcript was in-frame, it only contributed approximately 0.2% of the lymphoblast URO-synthase activity. Thus, the BPS mutation markedly reduced the wild-type transcript and enzyme activity, thereby causing the disease. This is the first BPS mutation in the last intron, presumably accounting for the observed 100% intron retention without exon skipping.


Subject(s)
Alternative Splicing/genetics , Mutation , Porphyria, Erythropoietic/genetics , Uroporphyrinogen III Synthetase/genetics , Adolescent , Adult , Blotting, Northern , DNA Mutational Analysis , Exons/genetics , Family Health , Female , Humans , Introns/genetics , Lymphocytes/metabolism , Male , Middle Aged , Porphyria, Erythropoietic/enzymology , Porphyria, Erythropoietic/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Young Adult
16.
Front Genet ; 13: 993453, 2022.
Article in English | MEDLINE | ID: mdl-36386813

ABSTRACT

Background and aims: Porphyrias constitute a group of rare genetic diseases due to various, mostly autosomal dominant mutations, causing enzymatic deficiency in heme biosynthesis. As a result, neurotoxic porphyrin precursors and light-sensitive porphyrins accumulate, while dysfunction in their targets determines the disease symptoms. Variegate porphyria (VP), one of the acute hepatic porphyrias, is caused by a protoporphyrinogen oxidase (PPOX) mutation. During acute attacks, among other factors, triggered by drugs, stressors, or fasting, an increase in urinary and fecal porphobilinogen (PBG), aminolevulinic acid (ALA), and porphyrins occurs, damaging the autonomous, peripheral, or central nervous system. The disease remains often latent or displays minimal symptoms usually overlooked, exposing undiagnosed patients to potentially serious complications in the presence of the aforementioned triggers. Case report: This 46-year-old woman presented, some days after a bariatric surgery, with severe flaccid tetraparesis and neuropathic pain, initially misdiagnosed as a functional neurological disorder. The severe axonal sensorimotor polyneuropathy led to further investigations, disclosing high urinary porphobilinogen, ALA, and porphyrin levels due to a new PPOX mutation. Retrospectively, it appeared that the patient had had typical VP symptoms (abdominal pain, fragile skin, and dark urine episodes) for years prior to the surgery. Treated with carbohydrate load, neurorehabilitation, and analgesics, she slowly recovered to full mobility, with partial autonomy in her daily life activities, although fatigue and severe pain persisted, preventing her from returning to work. Conclusion: This case documents gastric bypass surgery as a trigger of severe VP invalidating neurological symptoms and illustrates how the delayed diagnosis and post-interventional complications could have been prevented by screening for porphyria cardinal symptoms prior to the intervention. Likewise, this cost-effective screening should be performed before any treatment influencing the diet, which would dramatically improve the porphyria diagnosis rate and outcome.

17.
BMC Clin Pathol ; 11: 6, 2011 May 10.
Article in English | MEDLINE | ID: mdl-21569233

ABSTRACT

BACKGROUND: The effect of centrifugation time of heparinized blood samples on clinical chemistry and immunology results has rarely been studied. WHO guideline proposed a 15 min centrifugation time without citing any scientific publications. The centrifugation time has a considerable impact on the turn-around-time. METHODS: We investigated 74 parameters in samples from 44 patients on a Roche Cobas 6000 system, to see whether there was a statistical significant difference in the test results among specimens centrifuged at 2180 g for 15 min, at 2180 g for 10 min or at 1870 g for 7 min, respectively. Two tubes with different plasma separators (both Greiner Bio-One) were used for each centrifugation condition. Statistical comparisons were made by Deming fit. RESULTS: Tubes with different separators showed identical results in all parameters. Likewise, excellent correlations were found among tubes to which different centrifugation conditions were applied. Fifty percent of the slopes lay between 0.99 and 1.01. Only 3.6 percent of the statistical tests results fell outside the significance level of p < 0.05, which was less than the expected 5%. This suggests that the outliers are the result of random variation and the large number of statistical tests performed. Further, we found that our data are sufficient not to miss a biased test (beta error) with a probability of 0.10 to 0.05 in most parameters. CONCLUSION: A centrifugation time of either 7 or 10 min provided identical test results compared to the time of 15 min as proposed by WHO under the conditions used in our study.

18.
Ther Adv Rare Dis ; 2: 26330040211065453, 2021.
Article in English | MEDLINE | ID: mdl-37181106

ABSTRACT

Erythropoietic protoporphyria (EPP) is an ultra-rare inherited disorder with overproduction of protoporphyrin in maturating erythroblasts. This excess protoporphyrin leads to incapacitating phototoxic burns in sunlight exposed skin. Its biliary elimination causes cholestatic liver injury in 20% and terminal liver failure in 4% of EPP patients. Thereby, the risk of liver injury increases with increasing erythrocyte protoporphyrin concentrations. Afamelanotide, an α-melanocyte-stimulating hormone (MSH) analog inducing skin pigmentation, was shown to improve sunlight tolerance in EPP. Beyond this well-known effect on pigmentation, the MSHs have liver-protective effects and improve survival of maturating erythroblasts, effects described in animal or in vitro models to date only. We investigated whether afamelanotide treatment in EPP has effects on erythropoiesis, protoporphyrin concentrations, and liver injury by analyzing retrospectively our long-term safety data. Methods: From the 47 Swiss EPP-patients treated at our center since 2006, we included those 38 patients in the current analysis who received at least one afamelanotide dose between 2016 and 2018 and underwent regular laboratory testing before and during the treatment. We compared the means of pretreatment measurements with those during the treatment. Results: Protoporphyrin concentrations dropped from 21.39 ± 11.12 (mean ± SD) before afamelanotide to 16.83 ± 8.24 µmol/L (p < .0001) during treatment. Aspartate aminotransferase decreased from 26.67 ± 13.16 to 22.9 ± 7.76 IU/L (p = .0146). For both entities, patients with higher values showed a more progressive decrease, indicating a risk reduction of EPP-related liver disease. The pre-existing hypochromia and broad mean red-cell distribution width were further augmented under afamelanotide. This was more likely due to an influence of afamelanotide on maturating erythroblasts than due to an exacerbated iron deficiency, as mean zinc-protoporphyrin decreased significantly and ferritin remained unchanged. No serious afamelanotide-related adverse events were observed for a total of 240 treatment years. Conclusion: Our findings point to a protective effect of afamelanotide on erythroblast maturation and protoporphyrin-induced liver injury. Plain Language summary: Afamelanotide, a skin tanning hormone, may protect patients with erythropoietic protoporphyria not only from skin burns, but also from liver injury associated with the disease. Patients with erythropoietic protoporphyria (EPP), an inherited metabolic disease, suffer from light-induced skin burns and liver injury elicited by the accumulated light sensitizer protoporphyrin. The excess protoporphyrin is produced in red cell precursors in the bone marrow, and it is eliminated from the body via the liver and bile. A high protoporphyrin excretion burden damages the liver cells, the risk for this increases with higher protoporphyrin concentrations. About 20% of EPP patients show some sign of liver injury and 4% develop life-threatening liver dysfunction.Afamelanotide, closely related to natural α-melanocyte stimulating hormone (MSH), induces skin tanning. This effect protects EPP patients from light-induced skin burns as shown in previous studies. We have treated Swiss EPP patients with afamelanotide since 2006, and we regularly perform safety tests of this treatment.Recent in vitro and animal studies demonstrated α-MSH effects other than skin tanning, including an improved synthesis of red blood cell precursors in the bone-marrow and protection of the liver from experimentally induced damage. Until now, it is unknown whether afamelanotide has similar effects in the human organism.To study this question, we analyzed retrospectively the safety laboratory data of 38 Swiss patients, who received at least one dose of afamelanotide from 2016 to 2019. We found that both, the average protoporphyrin concentrations and aspartate aminotransferase, a test for liver function, improved during afamelanotide treatment as compared to before.We concluded that afamelanotide applied to EPP patients to protect them from light-induced skin burns also may reduce their risk of liver injury.

19.
Cell Chem Biol ; 28(8): 1221-1234.e6, 2021 08 19.
Article in English | MEDLINE | ID: mdl-33756123

ABSTRACT

Erythropoietic protoporphyria (EPP) is a rare disease in which patients experience severe light sensitivity. It is caused by a deficiency of ferrochelatase (FECH), the last enzyme in heme biosynthesis (HBS). The lack of FECH causes accumulation of its photoreactive substrate protoporphyrin IX (PPIX) in patients' erythrocytes. Here, we explored an approach for the treatment of EPP by decreasing PPIX synthesis using small-molecule inhibitors directed to factors in the HBS pathway. We generated a FECH-knockout clone from K562 erythroleukemia cells, which accumulates PPIX and undergoes oxidative stress upon light exposure. We used these matched cell lines to screen a set of publicly available inhibitors of factors in the HBS pathway. Inhibitors of the glycine transporters GlyT1 and GlyT2 lowered levels of PPIX and markers of oxidative stress selectively in K56211B4 cells, and in primary erythroid cultures from an EPP patient. Our findings open the door to investigation of glycine transport inhibitors for HBS disorders.


Subject(s)
Glycine Plasma Membrane Transport Proteins/antagonists & inhibitors , Protoporphyria, Erythropoietic/drug therapy , Protoporphyrins/pharmacology , Cells, Cultured , Erythrocytes/drug effects , Erythrocytes/metabolism , Glycine Plasma Membrane Transport Proteins/metabolism , Humans , K562 Cells , Molecular Structure , Protoporphyria, Erythropoietic/metabolism
20.
J Inherit Metab Dis ; 33 Suppl 3: S465-9, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21069463

ABSTRACT

Erythropoietic protoporphyria (EPP) is a hereditary disorder caused by deficiency of ferrochelatase, the last enzyme in the heme biosynthetic pathway. The majority of EPP patients present with a clinical symptom of painful phototoxicity. Liver damage, the most serious complication of EPP, occurs in <5% of the patients. This report describes a case of an EPP patient who complained of worsening cutaneous symptoms, nervousness, and insomnia. Laboratory tests showed highly increased protoporphyrin concentration in erythrocytes and elevated serum transaminases that are indicative of EPP-related liver damage. The subsequent finding of decreased serum thyroid-stimulating hormone (TSH) and increased free triiodothyronine (FT3) and free thyroxine (FT4) concentrations, as well antibodies against both thyroid peroxidase (TPO) and TSH receptors, led to the diagnosis of Graves' disease. The patient received 500 MBq of radioiodine (I(131)). Three months after the radioactive iodine therapy, the thyroid volume was reduced to 30% of pretherapeutic volume. Although the patient was slightly hypothyroidic, his liver enzymes returned to normal, his erythrocytic protoporphyrin concentration dropped fivefold, and his skin symptoms improved dramatically. The coexistence of Graves' disease and EPP is a statistically rare event as, besides our patient, there was one additional case reported in the literature. Although the exact mechanism whereby Graves' disease interacts with EPP is yet to be explored, we recommend testing thyroid function in EPP patients with liver complication to exclude hyperthyroidism as a potential cause.


Subject(s)
Graves Disease/complications , Liver Diseases/etiology , Porphyria, Erythropoietic/complications , Adult , Autoantigens/immunology , Biomarkers/blood , Disease Progression , Erythrocytes/metabolism , Graves Disease/blood , Graves Disease/diagnosis , Graves Disease/immunology , Graves Disease/radiotherapy , Humans , Immunoglobulins, Thyroid-Stimulating/blood , Iodide Peroxidase/immunology , Iodine Radioisotopes/therapeutic use , Iron-Binding Proteins/immunology , Liver Diseases/blood , Liver Diseases/diagnosis , Male , Porphyria, Erythropoietic/blood , Porphyria, Erythropoietic/diagnosis , Protoporphyrins/blood , Thyroid Hormones/blood , Time Factors , Transaminases/blood , Treatment Outcome
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