Preventing hyperhomocysteinemia using vitamin B6 supplementation in Givosiran-treated acute intermittent porphyria: Highlights from a case report and brief literature review.

Acute hepatic porphyrias are inherited metabolic disorders of heme biosynthesis characterized by the accumulation of toxic intermediate metabolites responsible for disabling acute neurovisceral attacks. Givosiran is a newly approved siRNA-based treatment of acute hepatic porphyria targeting the first and rate-limiting δ-aminolevulinic acid synthase 1 (ALAS1) enzyme of heme biosynthetic pathway. We described a 72-year old patient who presented with severe inaugural neurological form of acute intermittent porphyria evolving for several years which made her eligible for givosiran administration. On initiation of treatment, the patient developed a major hyperhomocysteinemia (>400 µmol/L) which necessitated to discontinue the siRNA-based therapy. A thorough metabolic analysis in the patient suggests that hyperhomocysteinemia could be attributed to a functional deficiency of cystathionine ß-synthase (CBS) enzyme induced by givosiran. Long-term treatment with vitamin B6, a cofactor of CBS, allowed to normalize homocysteinemia while givosiran treatment was maintained. We review the recently published cases of hyperhomocysteinemia in acute hepatic porphyria and its exacerbation under givosiran therapy. We also discuss the benefits of vitamin B6 supplementation in the light of hypothetic pathophysiological mechanisms responsible for hyperhomocysteinemia in these patients. Our results confirmed the importance of monitoring homocysteine metabolism and vitamin status in patients with acute intermittent porphyria in order to improve management by appropriate vitamin supplementation during givosiran treatment.

Postauthorization safety study of betaine anhydrous.

Patient registries for rare diseases enable systematic data collection and can also be used to facilitate postauthorization safety studies (PASS) for orphan drugs. This study evaluates the PASS for betaine anhydrous (Cystadane), conducted as public private partnership (PPP) between the European network and registry for homocystinurias and methylation defects and the marketing authorization holder (MAH). Data were prospectively collected, 2013-2016, in a noninterventional, international, multicenter, registry study. Putative adverse and severe adverse events were reported to the MAH's pharmacovigilance. In total, 130 individuals with vitamin B6 nonresponsive (N = 54) and partially responsive (N = 7) cystathionine beta-synthase (CBS) deficiency, as well as 5,10-methylenetetrahydrofolate reductase (MTHFR; N = 21) deficiency and cobalamin C (N = 48) disease were included. Median (range) duration of treatment with betaine anhydrous was 6.8 (0-9.8) years. The prescribed betaine dose exceeded the recommended maximum (6 g/day) in 49% of individuals older than 10 years because of continued dose adaptation to weight; however, with disease-specific differences (minimum: 31% in B6 nonresponsive CBS deficiency, maximum: 67% in MTHFR deficiency). Despite dose escalation no new or potential risk was identified. Combined disease-specific treatment decreased mean ± SD total plasma homocysteine concentrations from 203 ± 116 to 81 ± 51 µmol/L (p < 0.0001), except in MTHFR deficiency. Recommendations for betaine anhydrous dosage were revised for individuals ≥ 10 years. PPPs between MAH and international scientific consortia can be considered a reliable model for implementing a PASS, reutilizing well-established structures and avoiding data duplication and fragmentation.

Amino acids and vitamins status during continuous renal replacement therapy: An ancillary prospective observational study of a randomised control trial.

BACKGROUND: Continuous renal replacement therapy (CRRT) is associated with micronutrients loss. Current recommendations are to administer 1-1.5g/kg/day of proteins during CRRT. We aim to evaluate the net effect of CRRT on amino acids (AA), vitamins A and C (Vit A, Vit C) levels. METHODS: This is a prospective observational study embedded within a randomised controlled trial comparing two CRRT doses in patients with septic shock. CRRT was provided in continuous veno-venous haemofiltration mode at a dose of either 35ml/kg/h or 70ml/kg/h. All patients received parenteral nutrition with standard trace elements and vitamins (protein intake 1g/kg/d). We measured serum levels of glutamine, valine and alanine as well as Vit A and Vit C upon randomisation, study day four and eight. In addition, we measured a larger panel of AA in a subset of 11 patients. RESULTS: We included 30 patients (17 allocated to 70ml/kg/h and 13 to 35ml/kg/h CRRT). Before CRRT initiation, mean plasma levels of glutamine and valine, Vit A and Vit C were low. CRRT was not associated with any significant change in AA levels except for a decrease in cystein. It was associated with an increase in Vit A and a decrease in Vit C levels. CRRT dose had no impact on those nutrients blood levels. CONCLUSIONS: Irrespective of dose, CRRT was associated with a decrease in cysteine and Vit C and an increase in Vit A with no significant change in other AA. Further studies should focus on lean mass wasting during CRRT.

Targeting the mitochondrial trifunctional protein restrains tumor growth in oxidative lung carcinomas.

Metabolic reprogramming is a common hallmark of cancer, but a large variability in tumor bioenergetics exists between patients. Using high-resolution respirometry on fresh biopsies of human lung adenocarcinoma, we identified 2 subgroups reflected in the histologically normal, paired, cancer-adjacent tissue: high (OX+) mitochondrial respiration and low (OX-) mitochondrial respiration. The OX+ tumors poorly incorporated [18F]fluorodeoxy-glucose and showed increased expression of the mitochondrial trifunctional fatty acid oxidation enzyme (MTP; HADHA) compared with the paired adjacent tissue. Genetic inhibition of MTP altered OX+ tumor growth in vivo. Trimetazidine, an approved drug inhibitor of MTP used in cardiology, also reduced tumor growth and induced disruption of the physical interaction between the MTP and respiratory chain complex I, leading to a cellular redox and energy crisis. MTP expression in tumors was assessed using histology scoring methods and varied in negative correlation with [18F]fluorodeoxy-glucose incorporation. These findings provide proof-of-concept data for preclinical, precision, bioenergetic medicine in oxidative lung carcinomas.

Downregulation of Glutamine Synthetase, not glutaminolysis, is responsible for glutamine addiction in Notch1-driven acute lymphoblastic leukemia.

The cellular receptor Notch1 is a central regulator of T-cell development, and as a consequence, Notch1 pathway appears upregulated in > 65% of the cases of T-cell acute lymphoblastic leukemia (T-ALL). However, strategies targeting Notch1 signaling render only modest results in the clinic due to treatment resistance and severe side effects. While many investigations reported the different aspects of tumor cell growth and leukemia progression controlled by Notch1, less is known regarding the modifications of cellular metabolism induced by Notch1 upregulation in T-ALL. Previously, glutaminolysis inhibition has been proposed to synergize with anti-Notch therapies in T-ALL models. In this work, we report that Notch1 upregulation in T-ALL induced a change in the metabolism of the important amino acid glutamine, preventing glutamine synthesis through the downregulation of glutamine synthetase (GS). Downregulation of GS was responsible for glutamine addiction in Notch1-driven T-ALL both in vitro and in vivo. Our results also confirmed an increase in glutaminolysis mediated by Notch1. Increased glutaminolysis resulted in the activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway, a central controller of cell growth. However, glutaminolysis did not play any role in Notch1-induced glutamine addiction. Finally, the combined treatment targeting mTORC1 and limiting glutamine availability had a synergistic effect to induce apoptosis and to prevent Notch1-driven leukemia progression. Our results placed glutamine limitation and mTORC1 inhibition as a potential therapy against Notch1-driven leukemia.

Clinical and molecular characterization of adult patients with late-onset MTHFR deficiency.

5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency usually presents as a severe neonatal disease. This study aimed to characterize natural history, biological and molecular data, and response to treatment of patients with late-onset MTHFR deficiency. The patients were identified through the European Network and Registry for Homocystinuria and Methylation Defects and the Adult group of the French Society for Inherited Metabolic Diseases; data were retrospectively colleted. To identify juvenile to adult-onset forms of the disease, we included patients with a diagnosis established after the age of 10 years. We included 14 patients (median age at diagnosis: 32 years; range: 11-54). At onset (median age: 20 years; range 9-38), they presented with walking difficulties (n = 8), cognitive decline (n = 3) and/or seizures (n = 3), sometimes associated with mild mental retardation (n = 6). During the disease course, symptoms were almost exclusively neurological with cognitive dysfunction (93%), gait disorders (86%), epilepsy (71%), psychiatric symptoms (57%), polyneuropathy (43%), and visual deficit (43%). Mean diagnostic delay was 14 years. Vascular events were observed in 28% and obesity in 36% of the patients. One patient remained asymptomatic at the age of 55 years. Upon treatment, median total homocysteine decreased (from 183 µmol/L, range 69-266, to 90 µmol/L, range 20-142) and symptoms improved (n = 9) or stabilized (n = 4). Missense pathogenic variants in the C-terminal regulatory domain of the protein were over-represented compared to early-onset cases. Residual MTHFR enzymatic activity in skin fibroblasts (n = 4) was rather high (17%-58%). This series of patients with late-onset MTHFR deficiency underlines the still unmet need of a prompt diagnosis of this treatable disease.

Energy Metabolism Rewiring Precedes UVB-Induced Primary Skin Tumor Formation.

Although growing evidence indicates that bioenergetic metabolism plays an important role in the progression of tumorigenesis, little information is available on the contribution of reprogramming of energy metabolism in cancer initiation. By applying a quantitative proteomic approach and targeted metabolomics, we find that specific metabolic modifications precede primary skin tumor formation. Using a multistage model of ultraviolet B (UVB) radiation-induced skin cancer, we show that glycolysis, tricarboxylic acid (TCA) cycle, and fatty acid ß-oxidation are decreased at a very early stage of photocarcinogenesis, while the distal part of the electron transport chain (ETC) is upregulated. Reductive glutamine metabolism and the activity of dihydroorotate dehydrogenase (DHODH) are both necessary for maintaining high ETC. Mice with decreased DHODH activity or impaired ETC failed to develop pre-malignant and malignant lesions. DHODH activity represents a major link between DNA repair efficiency and bioenergetic patterning during skin carcinogenesis.

Coagulopathies frequency in aseptic osteonecrosis patients.

Many risk factors of aseptic osteonecrosis (AO) are well-known, even if 40% of events are idiopathic. Intravascular thrombosis is one of the physiopathological mechanisms of AO. The aim of this study is to determine the influence of coagulopathies on AO set-up. We performed a prospective case-control study, with 39 cases and 39 controls matched on age and sex. Cases are defined according to radiological criteria, and controls as non-affected by renal or hepatic insuffiency, and without inflammatory syndrome. Well-known AO risk factors were studied. Assessment of thrombosis was based on anti-phospholipid, anti beta2 Glycoprotein I, antiprothrombin, anti-cardiolipin, antithrombin, protein S and C, factor V Leiden, prothrombin gene and MTHFR mutations. 71% of cases presented a classical AO risk factor, vs. 38% of the controls. A significant association was also found between smoking and risk of AO. No significant difference in coagulopathy frequency was shown between cases and controls (56.4% vs. 48.7% respectively, p>0.05). Only abusive consumption of alcohol and tobacco is associated with risk of AO. Our study did not demonstrate any implication of coagulopathies in AO susceptibility. Further studies are needed to investigate more precisely these features.

Detection of an intragenic deletion expands the spectrum of CTSC mutations in Papillon-Lefèvre syndrome.

The Papillon-Lefèvre syndrome (PLS) is an autosomal recessive disorder. The gene responsible for the disease, cathepsin C (CTSC), is localized in 11q14.1-q14.21. We performed mutational and functional analyses of CTSC in two patients affected by this condition. Three previously unreported CTSC mutations were identified. The first patient had a compound heterozygous status with a p.G386R missense mutation and an intragenic deletion spanning exons 3-7. Second patient carried a homozygous splice site mutation, p.A253SfsX30. CTSC activity was undetectable in both patients, thus demonstrating the pathological effect of these mutations. We describe early evidence of an original intragenic deletion reported in PLS. Since this mutational mechanism could not be detected by direct sequencing, intragenic deletion has to be specifically investigated using gene dosage analysis techniques such as quantitative multiplex fluorescent polymerase chain reaction. We consider that this technique should be performed in patients with apparently homozygous CTSC mutations when one parent does not carry the expected mutation or is not available for analysis.

[Fabry disease: proposed guidelines from a French expert group for its diagnosis, treatment and follow-up]. / Maladie de Fabry: propositions d'un groupe d'experts français concernant le diagnostic, le traitement et le suivi des patients.

Fabry disease is a rare and under-recognized disease associated with an altered X-linked gene controlling hydrolase alpha-galactosidase A activity. This mutation impairs the glycosphingolipid metabolism. A multisystemic disease with a highly variable clinical presentation, its principal symptom is acroparesthesia. Manifestations of Fabry disease occur mostly in hemizygous males but also in heterozygous females. Before enzyme replacement therapy was available, life expectancy was about 50 years in men and 70 years in women. Early diagnosis is essential to prevent irreversible organ damage. Diagnosis is based on an assay of alpha-galactosidase A activity in male patients and on genetic analysis in female patients. Prognosis is related principally to three complications: involvement of the central nervous system, kidneys, and heart. Management of Fabry patients should in all cases combine symptomatic therapy and regular clinical, laboratory and morphological follow-up by specialists in genetic metabolic diseases. Enzyme replacement therapy should be considered in all adult male patients and should probably begin early. In adult heterozygous female patients and in children, this treatment should be considered only for patients with severe pain, organ damage, or central nervous system, kidney, or heart involvement. After a proband is identified, a genealogical tree should be used to identify other affected members of the family.

A bicistronic SIN-lentiviral vector containing G156A MGMT allows selection and metabolic correction of hematopoietic protoporphyric cell lines.

BACKGROUND: Erythropoietic protoporphyria (EPP) is an inherited disease characterised by a ferrochelatase (FECH) deficiency, the latest enzyme of the heme biosynthetic pathway, leading to the accumulation of toxic protoporphyrin in the liver, bone marrow and spleen. We have previously shown that a successful gene therapy of a murine model of the disease was possible with lentiviral vectors even in the absence of preselection of corrected cells, but lethal irradiation of the recipient was necessary to obtain an efficient bone marrow engraftment. To overcome a preconditioning regimen, a selective growth advantage has to be conferred to the corrected cells. METHODS: We have developed a novel bicistronic lentiviral vector that contains the human alkylating drug resistance mutant O(6)-methylguanine DNA methyltransferase (MGMT G156A) and FECH cDNAs. We tested their capacity to protect hematopoietic cell lines efficiently from alkylating drug toxicity and correct enzymatic deficiency. RESULTS: EPP lymphoblastoid (LB) cell lines, K562 and cord-blood-derived CD34(+) cells were transduced at a low multiplicity of infection (MOI) with the bicistronic constructs. Resistance to O(6)-benzylguanine (BG)/N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) was clearly shown in transduced cells, leading to the survival and expansion of provirus-containing cells. Corrected EPP LB cells were selectively amplified, leading to complete restoration of enzymatic activity and the absence of protoporphyrin accumulation. CONCLUSIONS: This study demonstrates that a lentiviral vector including therapeutic and G156A MGMT genes followed by BG/BCNU exposure can lead to a full metabolic correction of deficient cells. This vector might form the basis of new EPP mouse gene therapy protocols without a preconditioning regimen followed by in vivo selection of corrected hematopoietic stem cells.

Highly efficient lentiviral gene transfer in CD34+ and CD34+/38-/lin- cells from mobilized peripheral blood after cytokine prestimulation.

Because mobilized peripheral blood (mPB) represents an attractive source of cells for gene therapy, we investigated lentiviral gene transfer in CD34(+) cells and the stem/progenitor-cell-enriched CD34(+)/38(-)/lin(-) cell subset isolated from mPB. In this study, we used an optimized third-generation self-inactivating lentiviral vector containing both the central polypurine tract and the woodchuck hepatitis posttranscriptional regulatory element sequences and encoding enhanced green fluorescent protein (EGFP) under the control of the elongation factor lalpha promoter. This lentivector was first used to compare multiplicity of infection (MOI)-dependent gene transfer efficiency in cord blood (CB) versus mPB CD34(+)-derived cells, colony-forming cells (CFCs), and long-term culture-initiating cells (LTC-ICs). Results showed a difference in the percentage of transduced cells particularly significant at low MOIs. A plateau was reached where 15% and 25% of CB and mPB cells, respectively, remained refractory to lentiviral trans-duction. Effects of a cytokine prestimulation period (18 hours) with interleukin-3, stem cell factor, Flt-3 ligand, and thrombopoietin were then analyzed in total cells, CFCs, and LTC-ICs derived from mPB CD34(+) cells. Transduction levels in those conditions demonstrated a two- and fourfold increase in CFCs and LTC-ICs, respectively, compared with unstimulated (<3 hours) control cells. Moreover, using the same transduction protocol, we were able to efficiently transduce CD34(+)/38(-)/lin(-) cells isolated from mPB, with up to >85% of colonies derived from LTC-ICs expressing EGFP and gene transfer levels remaining stable for 10 weeks in liquid culture. We therefore demonstrate a highly efficient gene transfer in this therapeutically relevant target cell population.