JIMD Rep

Primary carnitine deficiency (PCD) is an inherited disease of fatty acid beta-oxidation with autosomal recessive inheritance. The disease manifests as metabolic decompensation with hypoketotic hypoglycaemia associated with cardiomyopathy, hepatomegaly, rhabdomyolysis, and seizures. Various outcomes are described from asymptomatic adults to dramatic sudden infant death syndrome cases. We present a severe case of PCD decompensation in an 18-week-old female. She presented with hypotonia, moaning, diarrhea, and vomiting at the pediatric emergency. Initially suspected as intracranial hypertension, the clinical condition evolved rapidly and caused a reversible cardiac arrest with profound hypoglycemia. Despite carnitine supplementation, she succumbed from cardiac arrhythmia and multivisceral failure 4 days after admission. The genetic analyses showed a PCD with biallelic pathogenic variants of gene. The case report is notable for the severity of the cardiac damage possibly favored by maternal carnitine deficiency during pregnancy. The analysis of previously published PCD cases highlights (i) the importance of having large access to emergency biochemical tests for early therapeutic care although the disease has unpredictable severity and (ii) the fact that the clinical outcome remains unpredictable if carnitine treatment is initiated late

Les cytochromes P-450 IIIA et IIC chez l'homme: analyse in vivo du groupe IIIA, analyse moleculaire du groupe IIC

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 78464 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Phlebotomy as an efficient long-term treatment of congenital erythropoietic porphyria

International audienceCongenital erythropoietic porphyria (CEP) is a rare autosomal recessive disease caused by impaired activity of uroporphyrinogen III synthase, the fourth enzyme of the heme biosynthetic pathway. Massive accumulation of porphyrins in red blood cells is responsible for hemolysis and porphyrin deposition in the skin, inducing severe bullous lesions and progressive photomutilation. Treatment options are scarce, relying mainly on supportive measures and, for severe cases, on bone marrow transplantation. In CEP, gain-of-function mutations in ALAS2 can represent an aggravating factor, and iron restriction can improve disease symptoms. Herein, we present the first case of a CEP patient significantly improved by iron deficiency induced by iterative phlebotomies for almost two years. We observed discontinuation of hemolysis and a marked decrease in plasma and urine porphyrins. The patient reported a major improvement in photosensitivity. No adverse effects were observed. The characterization of 3 CEP siblings in a consanguineous family with contrasting phenotypes modulated by iron availability highlights the importance of iron metabolism in the disease. Erythroid cultures were performed, demonstrating the role of iron in the rate of porphyrin production. Thus, we propose phlebotomy as an efficient, accessible, inexpensive and well-tolerated treatment for CEP

The SLC40A1 R178Q mutation is a recurrent cause of hemochromatosis and is associated with a novel pathogenic mechanism

Hemochromatosis type 4 is one of the most common causes of primary iron overload, after HFE-related hemochromatosis. It is an autosomal dominant disorder, primarily due to missense mutations in SLC40A1 This gene encodes ferroportin 1 (FPN1), which is the sole iron export protein reported in mammals. Not all heterozygous missense mutations in SLC40A1 are disease-causing. Due to phenocopies and an increased demand for genetic testing, rare SLC40A1 variations are fortuitously observed in patients with a secondary cause of hyperferritinemia. Structure/function analysis is the most effective way of establishing causality when clinical and segregation data are lacking. It can also provide important insights into the mechanism of iron egress and FPN1 regulation by hepcidin. The present study aimed to determine the pathogenicity of the previously reported p.Arg178Gln variant. We present the biological, clinical, histological and radiological findings of 22 patients from six independent families of French, Belgian or Iraqi decent. Despite phenotypic variability, all patients with p.Arg178Gln had elevated serum ferritin concentrations and normal to low transferrin saturation levels. In vitro experiments demonstrated that the p.Arg178Gln mutant reduces the ability of FPN1 to export iron without causing protein mislocalization. Based on a comparative model of the 3D structure of human FPN1 in an outward facing conformation, we argue that p.Arg178 is part of an interaction network modulating the conformational changes required for iron transport. We conclude that p.Arg178Gln represents a new category of loss-of-function mutations and that the study of "gating residues" is necessary in order to fully understand the action mechanism of FPN1.status: publishe

The SLC40A1 R178Q mutation is a recurrent cause of hemochromatosis and is associated with a novel pathogenic mechanism

International audienc

Comprehensive functional annotation of 18 missense mutations found in suspected hemochromatosis type 4 patients

International audienceHemochromatosis type 4 is a rare form of primary iron overload transmitted as an autosomal dominant trait caused by mutations in the gene encoding the iron transport protein ferroportin 1 (SLC40A1). SLC40A1 mutations fall into two functional categories (loss- versus gain-of-function) underlying two distinct clinical entities (hemochromatosis type4Aversus type 4B). However, the vast majority ofSLC40A1 mutations are rare missense variations, with only a few showing strong evidence of causality. The present study reports the results of an integrated approach collecting genetic and phenotypic data from 44 suspected hemochromatosis type 4 patients, with comprehensive structural and functional annotations. Causality was demonstrated for 10 missense variants, showing a clear dichotomy between the two hemochromatosis type 4 subtypes. Two subgroups of loss-of-function mutations were distinguished: one impairing cell-surface expression and one altering only iron egress. Additionally, a new gain-of-function mutation was identified, and the degradation of ferroportin on hepcidin binding was shown to probably depend on the integrity of a large extracellular loop outside of the hepcidin-binding domain. Eight further missense variations, on the other hand, were shown to have no discernible effects at either protein or RNA level; these were found in apparently isolated patients and were associated with a less severe phenotype. The present findings illustrate the importance ofcombining in silico and biochemical approaches to fully distinguish pathogenic SLC40A1 mutations from benign variants. This has profound implications for patient management

CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells

International audienceCRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers

CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations

International audienceCRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nucleaseinduced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on-and off-target indels and chromosomal truncations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies