Hervé Puy

Contribution of a Common Single-Nucleotide Polymorphism to the Genetic Predisposition for Erythropoietic Protoporphyria

Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis that results from a partial deficiency of ferrochelatase (FECH). Recently, we have shown that the inheritance of the common hypomorphic IVS3-48C allele trans to a deleterious mutation reduces FECH activity to below a critical threshold and accounts for the photosensitivity seen in patients. Rare cases of autosomal recessive inheritance have been reported. We studied a cohort of 173 white French EPP families and a group of 360 unrelated healthy subjects from four ethnic groups. The prevalences of the recessive and dominant autosomal forms of EPP are 4% (95% confidence interval 1-8) and 95% (95% confidence interval 91-99), respectively. In 97.9% of dominant cases, an IVS3-48C allele is co-inherited with the deleterious mutation. The frequency of the IVS3-48C allele differs widely in the Japanese (43%), southeast Asian (31%), white French (11%), North African (2.7%), and black West African (<1%) populations. These differences can be related to the prevalence of EPP in these populations and could account for the absence of EPP in black subjects. The phylogenic origin of the IVS3-48C haplotypes strongly suggests that the IVS3-48C allele arose from a single recent mutational event. Estimation of the age of the IVS3-48C allele from haplotype data in white and Asian populations yields an estimated age three to four times younger in the Japanese than in the white population, and this difference may be attributable either to differing demographic histories or to positive selection for the IVS3-48C allele in the Asian population. Finally, by calculating the KA/KS ratio in humans and chimpanzees, we show that the FECH protein sequence is subject to strong negative pressure. Overall, EPP looks like a Mendelian disorder, in which the prevalence of overt disease depends mainly on the frequency of a single common single-nucleotide polymorphism resulting from a unique mutational event that occurred 60,000 years ago.

Variegate Porphyria in Western Europe: Identification of PPOX Gene Mutations in 104 Families, Extent of Allelic Heterogeneity, and Absence of Correlation between Phenotype and Type of Mutation

Variegate porphyria (VP) is a low-penetrance, autosomal dominant disorder characterized clinically by skin lesions and acute neurovisceral attacks that occur separately or together. It results from partial deficiency of protoporphyrinogen oxidase encoded by the PPOX gene. VP is relatively common in South Africa, where most patients have inherited the same mutation in the PPOX gene from a common ancestor, but few families from elsewhere have been studied. Here we describe the molecular basis and clinical features of 108 unrelated patients from France and the United Kingdom. Mutations in the PPOX gene were identified by a combination of screening (denaturing gradient gel electrophoresis, heteroduplex analysis, or denaturing high-performance liquid chromatography) and direct automated sequencing of amplified genomic DNA. A total of 60 novel and 6 previously reported mutations (25 missense, 24 frameshift, 10 splice site, and 7 nonsense) were identified in 104 (96%) of these unrelated patients, together with 3 previously unrecognized single-nucleotide polymorphisms. VP is less heterogeneous than other acute porphyrias; 5 mutations were present in 28 (26%) of the families, whereas 47 mutations were restricted to 1 family; only 2 mutations were found in both countries. The pattern of clinical presentation was identical to that reported from South Africa and was not influenced by type of mutation. Our results define the molecular genetics of VP in western Europe, demonstrate its allelic heterogeneity outside South Africa, and show that genotype is not a significant determinant of mode of presentation.

Human hereditary hepatic porphyrias

The human hereditary hepatic porphyrias are diseases due to marked deficiencies of enzymes in the heme biosynthetic pathway. Porphyrias can be classified as either hepatic or erythroid, depending on the major production site of porphyrins or their precursors. The pathogenesis of inherited hepatic porphyrias has now been defined at the molecular level. Some gene carriers are vulnerable to a range of exogenous and endogenous factors, which may trigger neuropsychiatric and/or cutaneous symptoms. Early diagnosis is of prime importance since it makes way for counselling. In this article we present an overview of recent advances on hepatic porphyrias: 5-aminolevulinic acid dehydratase deficiency porphyria, acute intermittent porphyria (AIP), porphyria cutanea tarda (PCT), hereditary coproporphyria (HC), and variegate porphyria (VP).

Iron status and inflammatory biomarkers in patients with acutely decompensated heart failure: early in-hospital phase and 30-day follow-up

Iron deficiency (ID) is an important comorbidity in heart failure (HF).1 Its reported prevalence in chronic heart failure (CHF) is 30–50% and it constitutes an independent predictor of morbidity, mortality and cardiac transplantation.2,3 Current European Society of Cardiology (ESC) guidelines for the diagnosis and management of acute and chronic HF recommend assessment of iron parameters in symptomatic HF patients with reduced ejection fraction, with ensuing iron therapy in cases of ID, as defined by the criteria used in the FAIR-HF (Ferinject Assessment in patients with IRon deficiency and chronic Heart Failure) trial.1,4 Data on ID in acutely decompensated HF (ADHF) are scarce, yet in the CardioFer study, conducted in 865 patients in France, ID showed a higher prevalence in ADHF patients (60–80%).5 In this study, we performed serial blood sampling in ADHF patients and assessed the reliability of ID diagnosis during ADHF. In addition, we wanted to evaluate the associations between parameters of iron metabolism and biomarkers of inflammation, cardiovascular stress, fibrosis and renal function. Patients were derived from the Biomarcoeurs cohort (ClinicalTrials.gov: NCT 01374880), detailed previously.6 This substudy included patients with decompensated CHF and de novo HF. Patients with a concomitant infection or myocardial ischaemia were excluded. The protocol of the study was approved by the local ethics committee and patients provided written informed consent. Following admission, blood sampling was performed at day 0 (D0) and day 30 (D30). Iron status was assessed in several ways: we considered absolute and functional ID as defined by the ESC1 thus: ID was considered to be absolute if serum ferritin was <100 μg/L, and functional when serum ferritin was 100–299 μg/L and transferrin saturation (TSAT) was <20%. Furthermore, we assessed plasma levels of hepcidin and soluble transferrin receptor (sTfR) and defined ID as represented by serum hepcidin of <14.5 ng/mL (5th percentile among healthy controls; depleted iron stores) and/or sTfR of ≥1.59 mg/L (95th percentile among healthy controls; depleted intracellular iron content in metabolizing cells).7 Several biomarkers were analysed, including brain natriuretic peptide (BNP), mid-regional–proadrenomedullin (MRproADM), procalcitonin (PCT), interleukin-6 (IL-6), high-sensitivity C-reactive protein (hsCRP), growth/differentiation factor-15 (GDF15), galectin-2, fibrinogen, soluble ST2, tumour necrosis factor-α (TNF-α) and myeloperoxidase (MPO). Statistical analyses were performed using STATA version 14.2 (StataCorp LLC, College Station, TX, USA). Groups were compared with the Student’s t-test, Wilcoxon matched-pairs signed-rank sum test, Pearson’s χ2 test or McNemar test when appropriate. A P-value of <0.05 was considered to indicate statistical significance. Forty-seven ADHF patients (32 men and 15 women) in whom assessments of ferritin and TSAT had been made at both D0 and D30 were included (data are available in supplementary material online, Tables S1 and S2). Their age (mean± standard deviation) was 70.4±13.7 years. Median BNP was 1004 pg/mL [interquartile range (IQR): 652–1676 pg/mL]. At D0, the median ferritin value was 93 μg/L (IQR: 76–107 μg/L) and median TSAT was 13% (IQR: 6–20%). Twenty-seven (57%) patients fulfilled criteria for absolute ID and 12 (26%) patients did so for functional ID. Thus, 83% of ADHF patients fulfilled the criteria for ID at admission. At D30 of follow-up and guideline-based treatments, the median ferritin value was found to have increased to 159 μg/L (IQR: 134–190 μg/L; P< 0.0001), median TSAT was 17% (IQR: 12–23%; P= 0.0176), and median BNP was 261 pg/mL (IQR: 176–462 pg/mL; P< 0.0001). Frequencies of absolute and functional ID were 11% (five patients) and 57% (27 patients), respectively. The remaining 15 patients (32%) were not ID at D30 of follow-up. Iron status changed significantly between D0 and D30 (P= 0.00001). The difference between the absence and presence of ID is clinically more important than the difference between absolute and functional ID; therefore, absolute and functional ID were combined for the purpose of comparing overall prevalences of ID between D0 and D30, which showed a trend toward statistical significance (P= 0.07) (Figure 1A) and only a moderate association between ID status at D0 and D30 (φ coefficient: 0.26). Circulating levels of hepcidin and sTfR were available for 41 patients at D0; 34 (83%) patients were identified as ID. Comparisons of ID status at D0 using either ferritin/transferrin or hepcidin/sTfR criteria are presented in Figure 1B. Twenty-eight patients (68%) were similarly categorized by both definitions, 13 patients (32%) were considered to be ID according to one but not the other definition. Based on our subsample of 41 patients, there was no difference between these iron parameters in sensitivity to detect ID at admission (P= 0.78); further analysis revealed a weak association between both tests (φ coefficient: 0.04). To explain the variation in ferritin and TSAT in parallel with adequate treatment of ADHF, we correlated the iron parameters studied with several cardiovascular and inflammatory biomarkers. ΔFerritin (ferritin at D30 minus ferritin at D0) correlated with Δsoluble ST2 (Spearman’s ρ=−0.4028; P= 0.0082) and ΔIL-6 (ρ=−0.3569; P= 0.0138), but not with ΔMR-proADM, ΔBNP, ΔPCT, ΔTNF-α, ΔhsCRP, ΔGDF15, Δgalectin-3, Δfibrinogen or ΔMPO. ΔTransferrin saturation correlated only with Δgalectin-3 (ρ=−0.3858; P= 0.0074). We did not detect a correlation between Δhepcidin or ΔsTfR and the other biomarkers studied. In conclusion, biochemical evidence of ID is common at admission for ADHF. Transferrin saturation and ferritin increase significantly over a period of 30 days following ADHF admission, which changes the ID status of patients significantly between D0 and D30 following ADHF. Thus, iron status is not stationary in ADHF patients, even during short periods of follow-up. Changing levels of circulating markers of inflammation weakly correlated with changing circulating iron parameters, which supports the suggestion that systemic inflammation contributes to alterations in iron status between admission and steady state. By contrast, changing iron

Epistasis in iron metabolism: complex interactions between Cp, Mon1a, and Slc40a1 loci and tissue iron in mice

Disorders of iron metabolism are among the most common acquired and constitutive diseases. Hemochromatosis has a solid genetic basis and in Northern European populations it is usually associated with homozygosity for the C282Y mutation in the HFE protein. However, the penetrance of this mutation is incomplete and the clinical presentation is highly variable. The rare and common variants identified so far as genetic modifiers of HFE-related hemochromatosis are unable to account for the phenotypic heterogeneity of this disorder. There are wide variations in the basal iron status of common inbred mouse strains, and this diversity may reflect the genetic background of the phenotypic diversity under pathological conditions. We therefore examined the genetic basis of iron homeostasis using quantitative trait loci mapping applied to the HcB-15 recombinant congenic strains for tissue and serum iron indices. Two highly significant QTL containing either the N374S Mon1a mutation or the Ferroportin locus were found to be major determinants in spleen and liver iron loading. Interestingly, when considering possible epistatic interactions, the effects of Mon1a on macrophage iron export are conditioned by the genotype at the Slc40a1 locus. Only mice that are C57BL/10ScSnA homozygous at both loci display a lower spleen iron burden. Furthermore, the liver-iron lowering effect of the N374S Mon1a mutation is observed only in mice that display a nonsense mutation in the Ceruloplasmin (Cp) gene. This study highlights the existence of genetic interactions between Cp, Mon1a, and the Slc40a1 locus in iron metabolism, suggesting that epistasis may be a crucial determinant of the variable biological and clinical presentations in iron disorders.