Management of erythropoietic protoporphyria with cholestatic liver disease: A case report.

Erythropoietic protoporphyria (EPP) is a rare metabolic disease of the heme biosynthetic pathway where an enzymatic dysfunction results in protoporphyrin IX (PPIX) accumulation in erythroid cells. The porphyrins are photo-reactive and are responsible for severe photosensitivity in patients, thus drastically decreasing their quality of life. The liver eliminates PPIX and as such, the main and rare complication of EPP is progressive cholestatic liver disease, which can lead to liver failure. The management of this complication is challenging, as it often requires a combination of approaches to promote PPIX elimination and suppress the patient's erythropoiesis. Here we described a 3-year follow-up of an EPP patient, with three episodes of liver involvement, aggravated by the coexistence of a factor VII deficiency. It covers all the different types of intervention available for the management of liver disease, right through to successful allogeneic hematopoietic stem cell transplantation.

Givosiran in acute intermittent porphyria: A personalized medicine approach.

BACKGROUND: In patients with acute intermittent porphyria (AIP), induction of delta aminolevulinic acid synthase 1 (ALAS1) leads to haem precursor accumulation that may cause recurring acute attacks. In a recent phase III trial, givosiran significantly reduced the attack rate in severe AIP patients. Frequent adverse events were injection-site reaction, fatigue, nausea, chronic kidney disease and increased alanine aminotransferase. OBJECTIVES: To describe the efficacy and safety of givosiran based on a personalized medical approach. METHODS: We conducted a retrospective patient file study in 25 severe AIP patients treated with givosiran in France. We collected data on clinical and biochemical efficacy along with reports of adverse events. RESULTS: Givosiran drastically reduced the attack rate in our cohort, as 96% were attack-free at the time of the study. The sustained efficacy of givosiran in most patients allowed us to personalize dosing frequency. In 42%, givosiran was only given when haem precursor levels were increasing. Our data suggest that givosiran is most effective when given early in the disease course. We confirmed a high prevalence of adverse events. One patient discontinued treatment due to acute pancreatitis. All patients had hyperhomocysteinemia, and all patients with initial homocysteine levels available showed an increase under treatment. In this context, one patient was diagnosed with pulmonary embolism. CONCLUSION: The sustained effect of givosiran allowed a decrease in dosing frequency without compromising treatment efficacy. The high prevalence of adverse events emphasizes the importance of restricting the treatment to severe AIP and administering the minimum effective dose for each patient.

Iron, Heme Synthesis and Erythropoietic Porphyrias: A Complex Interplay.

Erythropoietic porphyrias are caused by enzymatic dysfunctions in the heme biosynthetic pathway, resulting in porphyrins accumulation in red blood cells. The porphyrins deposition in tissues, including the skin, leads to photosensitivity that is present in all erythropoietic porphyrias. In the bone marrow, heme synthesis is mainly controlled by intracellular labile iron by post-transcriptional regulation: translation of ALAS2 mRNA, the first and rate-limiting enzyme of the pathway, is inhibited when iron availability is low. Moreover, it has been shown that the expression of ferrochelatase (FECH, an iron-sulfur cluster enzyme that inserts iron into protoporphyrin IX to form heme), is regulated by intracellular iron level. Accordingly, there is accumulating evidence that iron status can mitigate disease expression in patients with erythropoietic porphyrias. This article will review the available clinical data on how iron status can modify the symptoms of erythropoietic porphyrias. We will then review the modulation of heme biosynthesis pathway by iron availability in the erythron and its role in erythropoietic porphyrias physiopathology. Finally, we will summarize what is known of FECH interactions with other proteins involved in iron metabolism in the mitochondria.

Renal Function Decline With Small Interfering RNA Silencing Aminolevulinic Acid Synthase 1 (ALAS1).

INTRODUCTION: Givosiran is an RNA interference therapeutic designed to block the synthesis of the aminolevulinic acid (ALA) synthase 1 (ALAS1) enzyme in patients with acute intermittent porphyria (AIP). Givosiran may have adverse effects on the kidney. METHODS: We performed a descriptive case series of renal function parameters of all the patients who received givosiran in France. Twenty patients receiving givosiran between March 2018 and July 2020 in France were analyzed: 7 patients in the ENVISION trial and 13 patients treated in collaboration with the Centre de Référence Maladies Rares Prophyries. RESULTS: A transient decrease in renal function was observed in all but 2 patients (90%) within the 3 months following givosiran initiation. None of the patients developed acute kidney injury or disease. Patients of the ENVISION cohort were followed for at least 30 months: 2 patients did not experience estimated glomerular filtration rate (eGFR) loss, 3 patients experienced a modest decline in renal function (-3.4 ml/min per 1.73 m2 per year in average), and 2 patients had a clearly abnormal eGFR loss (-5.8 ml/min per 1.73 m2 per year in average). None of the patients had biochemical signs of active tubular or glomerular injury. One patient's kidney was biopsied without finding any signs of an active kidney disease and with normal ALAS1 tubular expression. CONCLUSIONS: Givosiran is associated with a transient moderate increase in serum creatinine (sCr) without sign of kidney injury. A long-term deleterious impact of ALAS1 inhibition on renal function is not excluded. Because AIP promotes chronic kidney disease, it is difficult to separate the long-term effects of givosiran from the natural progression of the renal disease.

TSPO2 translocates 5-aminolevulinic acid into human erythroleukemia cells.

BACKGROUND: 5-Aminolevulinic acid (ALA) is the first precursor of heme biosynthesis pathway. The exogenous addition of ALA to cells leads to protoporphyrin IX (PPIX) accumulation that has been exploited in photodynamic diagnostic and photodynamic therapy. Several types of ALA transporters have been described depending on the cell type, but there was no clear entry pathway for erythroid cells. The 18 kDa translocator protein (TSPO) has been proposed to be involved in the transport of porphyrins and heme analogs. RESULTS: ALA-induced PPIX accumulation in erythroleukemia cells (UT-7 and K562) was impaired by PK 11195, a competitive inhibitor of both transmembrane proteins TSPO (1 and 2). PK 11195 did not modify the activity of the enzymes of heme biosynthesis, suggesting that ALA entry at the plasma membrane was the limiting factor. In contrast, porphobilinogen (PBG)-induced PPIX accumulation was not affected by PK 11195, suggesting that plasma membrane TSPO2 is a selective transporter of ALA. Overexpression of TSPO2 at the plasma membrane of erythroleukemia cells increased ALA-induced PPIX accumulation, confirming the role of TSPO2 in the import of ALA into the cells. CONCLUSIONS: ALA-induced PPIX accumulation in erythroid cells involves TSPO2 as a selective translocator through the plasma membrane. SIGNIFICANCE: This is the first characterisation of molecular mechanisms involving a new actor in ALA transport in ALA-induced PPIX accumulation in erythroleukemia cells, which could be inhibited by specific drug ligands.

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.

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

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

From a dominant to an oligogenic model of inheritance with environmental modifiers in acute intermittent porphyria.

Acute intermittent porphyria (AIP) is a disease affecting the heme biosynthesis pathway caused by mutations of the hydroxymethylbilane synthase (HMBS) gene. AIP is thought to display autosomal dominant inheritance with incomplete penetrance. We evaluated the prevalence, penetrance and heritability of AIP, in families with the disease from the French reference center for porphyria (CFP) (602 overt patients; 1968 relatives) and the general population, using Exome Variant Server (EVS; 12 990 alleles) data. The pathogenicity of the 42 missense variants identified was assessed in silico, and in vitro, by measuring residual HMBS activity of the recombinant protein. The minimal estimated prevalence of AIP in the general population was 1/1299. Thus, 50 000 subjects would be expected to carry the AIP genetic trait in France. Penetrance was estimated at 22.9% in families with AIP, but at only 0.5-1% in the general population. Intrafamily correlation studies showed correlations to be strong overall and modulated by kinship and the area in which the person was living, demonstrating strong influences of genetic and environmental modifiers on inheritance. Null alleles were associated with a more severe phenotype and a higher penetrance than for other mutant alleles. In conclusion, the striking difference in the penetrance of HMBS mutations between the general population and the French AIP families suggests that AIP inheritance does not follow the classical autosomal dominant model, instead of being modulated by strong environmental and genetic factors independent from HMBS. An oligogenic inheritance model with environmental modifiers might better explain AIP penetrance and heritability.

Antisense oligonucleotide-based therapy in human erythropoietic protoporphyria.

In 90% of people with erythropoietic protoporphyria (EPP), the disease results from the inheritance of a common hypomorphic FECH allele, encoding ferrochelatase, in trans to a private deleterious FECH mutation. The activity of the resulting FECH enzyme falls below the critical threshold of 35%, leading to the accumulation of free protoporphyrin IX (PPIX) in bone marrow erythroblasts and in red cells. The mechanism of low expression involves a biallelic polymorphism (c.315-48T>C) localized in intron 3. The 315-48C allele increases usage of the 3' cryptic splice site between exons 3 and 4, resulting in the transcription of an unstable mRNA with a premature stop codon, reducing the abundance of wild-type FECH mRNA, and finally reducing FECH activity. Through a candidate-sequence approach and an antisense-oligonucleotide-tiling method, we identified a sequence that, when targeted by an antisense oligonucleotide (ASO-V1), prevented usage of the cryptic splice site. In lymphoblastoid cell lines derived from symptomatic EPP subjects, transfection of ASO-V1 reduced the usage of the cryptic splice site and efficiently redirected the splicing of intron 3 toward the physiological acceptor site, thereby increasing the amount of functional FECH mRNA. Moreover, the administration of ASO-V1 into developing human erythroblasts from an overtly EPP subject markedly increased the production of WT FECH mRNA and reduced the accumulation of PPIX to a level similar to that measured in asymptomatic EPP subjects. Thus, EPP is a paradigmatic Mendelian disease in which the in vivo correction of a common single splicing defect would improve the condition of most affected individuals.

Survivin gene polymorphism association with papillary thyroid carcinoma.

Survivin expression is correlated with suppression of apoptosis in human solid tumors. A polymorphism at position -31 (G/C) (rs 9904341) has been associated with cancer risk in several studies. We evaluated the correlation of this polymorphism with the risk of papillary thyroid carcinoma (PTC) in an Irananian population. The cases consisted of patients with PTC (n=123) and normal controls, composed of non-related healthy people (n=131). The frequency of GC or CC genotype in patients with PTC was significantly higher than in the controls [GC+CC vs GG, p=0.02 OR; 1.7, 95%CI (1.05-3.04)]. There was a significant difference between patients with more aggressive clinical manifestations, including lymphatic involvement compared to the controls [GC+CC vs GG, p=0.0006, OR; 3.7, 95%CI (1.6-9.2)]. The presence of C allele was significantly associated with the presence of more profound manifestations, including lymph node involvement, vascular involvement and multifocality.