Anibh M. Das

IDH2 Mutations in Patients with d-2-Hydroxyglutaric Aciduria

A mutation that changes the specificity of an enzyme in human cancer is also found in an inherited metabolic disorder. Heterozygous somatic mutations in the genes encoding isocitrate dehydrogenase-1 and -2 (IDH1 and IDH2) were recently discovered in human neoplastic disorders. These mutations disable the enzymes’ normal ability to convert isocitrate to 2-ketoglutarate (2-KG) and confer on the enzymes a new function: the ability to convert 2-KG to d-2-hydroxyglutarate (D-2-HG). We have detected heterozygous germline mutations in IDH2 that alter enzyme residue Arg140 in 15 unrelated patients with d-2-hydroxyglutaric aciduria (D-2-HGA), a rare neurometabolic disorder characterized by supraphysiological levels of D-2-HG. These findings provide additional impetus for investigating the role of D-2-HG in the pathophysiology of metabolic disease and cancer.

Enzyme Replacement Therapy and/or Hematopoietic Stem Cell Transplantation at diagnosis in patients with Mucopolysaccharidosis type I: results of a European consensus procedure

BackgroundMucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder that results in the accumulation of glycosaminoglycans causing progressive multi-organ dysfunction. Its clinical spectrum is very broad and varies from the severe Hurler phenotype (MPS I-H) which is characterized by early and progressive central nervous system (CNS) involvement to the attenuated Scheie phenotype (MPS I-S) with no CNS involvement. Indication, optimal timing, safety and efficacy of the two available treatment options for MPS I, enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT), are subject to continuing debate. A European consensus procedure was organized to reach consensus about the use of these two treatment strategies.MethodsA panel of specialists, including 8 specialists for metabolic disorders and 7 bone marrow transplant physicians, all with acknowledged expertise in MPS I, participated in a modified Delphi process to develop consensus-based statements on MPS I treatment. Fifteen MPS I case histories were used to initiate the discussion and to anchor decisions around either treatment mode. Before and at the meeting all experts gave their opinion on the cases (YES/NO transplantation) and reasons for their decisions were collected. A set of draft statements on MPS I treatment options composed by a planning committee were discussed and revised during the meeting until full consensus.ResultsFull consensus was reached on several important issues, including the following: 1) The preferred treatment for patients with MPS I-H diagnosed before age 2.5 yrs is HSCT; 2) In individual patients with an intermediate phenotype HSCT may be considered if there is a suitable donor. However, there are no data on efficacy of HSCT in patients with this phenotype; 3) All MPS I patients including those who have not been transplanted or whose graft has failed may benefit significantly from ERT; 4) ERT should be started at diagnosis and may be of value in patients awaiting HSCT.ConclusionsThis multidisciplinary consensus procedure yielded consensus on the main issues related to therapeutic choices and research for MPS I. This is an important step towards an international, collaborative approach, the only way to obtain useful evidence in rare diseases.

One Liver for Four Children: First Clinical Series of Liver Cell Transplantation for Severe Neonatal Urea Cycle Defects

Background. Urea cycle disorders (UCD) have a poor prognosis despite dietary and pharmacologic therapy, especially if the onset of the disease is within the neonatal period. They are promising target diseases for liver cell transplantation (LCT), which may be a less invasive alternative or supplementation to orthotopic liver transplantation. Methods. Cryopreserved hepatocytes were isolated under good manufacturing practice conditions. Four children with severe neonatal UCD (age 1 day–3 years) received multiple intraportal infusions of cryopreserved hepatocytes from that same donor, a 9-day old neonate. Portal vein access was achieved surgically in two children, whereas the umbilical vein was suitable for interventional catheter placement in two neonates. Cell applications were carefully monitored by means of Doppler ultrasound and portal vein pressure. Results. LCT was feasible in all children. No signs of portal vein thrombosis or extrahepatic shunting were observed. All children showed metabolic stabilization during observation periods of 4 to 13 months. One child with prenatally diagnosed ornithine transcarbamylase deficiency died after 4 months from a fatal metabolic decompensation. Conclusions. Given the poor prognosis of UCD with conservative therapy, LCT caused considerable beneficial effects. Periods of hyperammonemia and clinically relevant crises could be reduced during an observation period of up to 13 months. Though cell therapy is not a permanent therapeutic option, bridging to liver transplantation may be substantially improved.

Lysine restricted diet for pyridoxine-dependent epilepsy: First evidence and future trials

OBJECTIVE To evaluate the efficacy and safety of dietary lysine restriction as an adjunct to pyridoxine therapy on biochemical parameters, seizure control, and developmental/cognitive outcomes in children with pyridoxine-dependent epilepsy (PDE) caused by antiquitin (ATQ) deficiency. METHODS In this observational study, seven children with confirmed ATQ deficiency were started on dietary lysine restriction with regular nutritional monitoring. Biochemical outcomes were evaluated using pipecolic acid and α-aminoadipic semialdehyde (AASA) levels in body fluids; developmental/cognitive outcomes were evaluated using age-appropriate tests and parental observations. RESULTS Lysine restriction was well tolerated with good compliance; no adverse events were reported. Reduction in biomarker levels (measurement of the last value before and first value after initiation of dietary lysine restriction) ranged from 20 to 67% for plasma pipecolic acid, 13 to 72% for urinary AASA, 45% for plasma AASA and 42% for plasma P6C. For the 1 patient in whom data were available and who showed clinical deterioration upon interruption of diet, cerebrospinal fluid levels decreased by 87.2% for pipecolic acid and 81.7% for AASA. Improvement in age-appropriate skills was observed in 4 out of 5 patients showing pre-diet delays, and seizure control was maintained or improved in 6 out 7 children. CONCLUSIONS This observational study provides Level 4 evidence that lysine restriction is well tolerated with significant decrease of potentially neurotoxic biomarkers in different body compartments, and with the potential to improve developmental outcomes in children with PDE caused by ATQ deficiency. To generate a strong level of evidence before this potentially burdensome dietary therapy becomes the mainstay treatment, we have established: an international PDE consortium to conduct future studies with an all-inclusive integrated study design; a website containing up-to-date information on PDE; a methodological toolbox; and an online registry to facilitate the participation of interested physicians, scientists, and families in PDE research.

Developmental changes in the L-arginine/nitric oxide pathway from infancy to adulthood: plasma asymmetric dimethylarginine levels decrease with age.

BACKGROUND The L-arginine/nitric oxide (NO) pathway has multiple physiological functions including vasodilation, inhibition of platelet aggregation and neurotransmission. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of all known NO synthase isoforms, has adverse effects on renal and cardiovascular function in adults. It is unknown whether ADMA might also exert similar effects in younger individuals including infants. Also, reference data for important members of the L-arginine/NO family, notably ADMA and the NO metabolites, nitrite and nitrate, in infancy are lacking. METHODS In the present study, we investigated the status of the L-arginine/NO pathway in 34 healthy volunteers aged 2 days to 24 years by measuring the concentration of ADMA, nitrite, nitrate and L-arginine in plasma and urine using gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry methods. RESULTS We found that ADMA levels in plasma decreased with age (Pearson correlation coefficient r=-0.619, p<0.001). In contrast, urinary excretion of nitrate (r=0.471, p=0.036) and nitrite increased with age (r=0.484, p=0.037). CONCLUSIONS Our study suggests that in infants ADMA biosynthesis accompanied by an inhibition of NO synthesis is higher than in adults and diminishes considerably with age.

Impact of high glucose and transforming growth factor–β on bioenergetic profiles in podocytes

Diabetic nephropathy is the most common cause of chronic renal failure in industrialized countries. Depletion of podocytes plays an important role in the progression of diabetic glomerulopathy. Various factors in the diabetic milieu lead to serious podocyte stress driving the cells toward cell cycle arrest (p27(Kip1)), hypertrophy, detachment, and apoptosis. Mitochondria are responsible for oxidative phosphorylation and energy supply in podocytes. Recent studies indicated that mitochondrial dysfunction is a key factor in diabetic nephropathy. In the present study, we investigated metabolic profiles of podocytes under diabetic conditions. We examined oxygen consumption rates (OCRs) and oxidative phosphorylation complex activities in murine podocytes. Cells were exposed to high glucose for 48 hours, cultured for 10 passages under high-glucose conditions (30 mmol/L), or incubated with transforming growth factor-β (5 ng/mL) for 24 hours. After prolonged exposure to high glucose, podocytes showed a significantly increased OCR at baseline and also a higher OCR after addition of oligomycin, indicating significant changes in mitochondrial energy metabolism. Higher OCRs after inhibition of respiration by rotenone also indicated changes in nonmitochondrial respiration. Podocytes stimulated with a proapoptotic concentration of transforming growth factor-β displayed similar bioenergetic profiles, even with decreased citrate synthase activity. In all tested conditions, we found a higher cellular nicotinamide adenine dinucleotide content and changes in activities of respiratory chain complexes. In summary, we provide for the first time evidence that key factors of the diabetic milieu induce changes in glucose metabolism and mitochondrial function in podocytes.

Cross-sectional observational study of 208 patients with non-classical urea cycle disorders

Urea cycle disorders (UCDs) are inherited disorders of ammonia detoxification often regarded as mainly of relevance to pediatricians. Based on an increasing number of case studies it has become obvious that a significant number of UCD patients are affected by their disease in a non-classical way: presenting outside the newborn period, following a mild course, presenting with unusual clinical features, or asymptomatic patients with only biochemical signs of a UCD. These patients are surviving into adolescence and adulthood, rendering this group of diseases clinically relevant to adult physicians as well as pediatricians. In preparation for an international workshop we collected data on all patients with non-classical UCDs treated by the participants in 20 European metabolic centres. Information was collected on a cohort of 208 patients 50% of which were ≥ 16 years old. The largest subgroup (121 patients) had X-linked ornithine transcarbamylase deficiency (OTCD) of whom 83 were female and 29% of these were asymptomatic. In index patients, there was a mean delay from first symptoms to diagnosis of 1.6 years. Cognitive impairment was present in 36% of all patients including female OTCD patients (in 31%) and those 41 patients identified presymptomatically following positive newborn screening (in 12%). In conclusion, UCD patients with non-classical clinical presentations require the interest and care of adult physicians and have a high risk of neurological complications. To improve the outcome of UCDs, a greater awareness by health professionals of the importance of hyperammonemia and UCDs, and ultimately avoidance of the still long delay to correctly diagnose the patients, is crucial.

Rapid steroid hormone quantification for congenital adrenal hyperplasia (CAH) in dried blood spots using UPLC liquid chromatography-tandem mass spectrometry.

Newborn screening for congenital adrenal hyperplasia (CAH) is usually done by quantifying 17α-hydroxyprogesterone using immunoassay. However, this test produces high rates of false positive results caused by cross reacting steroids. Therefore we have developed a selective and specific method with a short run time (1.25 min) for quantification of 17α-hydroxyprogesterone, 21-deoxycortisol, 11-deoxycortisol, 11-deoxycorticosterone and cortisol from dried blood spots. The extraction procedure is very simple and steroid separation is ensured on a BEH C18 column and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Analysis was done in positive ionization mode (ESI+) and recorded in multiple reaction monitoring mode (MRM). The method gave linear results for all steroids over a range of 5-200 (cortisol: 12.5-500)nmol/L with coefficients of regression >0.992. Absolute recovery was >64.1%. Across the analytical range the inter-assay coefficient of variation (CV) was <3%. Newborn blood samples of patients with confirmed 21-CAH and 11-CAH could clearly be distinguished from samples of unaffected newborns falsely positive on immunoassay. The method is not influenced by cross reactions as found on immunoassay. Analysis of dried blood spots shows that this method is sensitive and fast enough to allow rapid analysis and can therefore improve the newborn screening program.

Inborn Errors of Energy Metabolism Associated with Myopathies

Inherited neuromuscular disorders affect approximately one in 3,500 children. Structural muscular defects are most common; however functional impairment of skeletal and cardiac muscle in both children and adults may be caused by inborn errors of energy metabolism as well. Patients suffering from metabolic myopathies due to compromised energy metabolism may present with exercise intolerance, muscle pain, reversible or progressive muscle weakness, and myoglobinuria. In this review, the physiology of energy metabolism in muscle is described, followed by the presentation of distinct disorders affecting skeletal and cardiac muscle: glycogen storage diseases types III, V, VII, fatty acid oxidation defects, and respiratory chain defects (i.e., mitochondriopathies). The diagnostic work-up and therapeutic options in these disorders are discussed.

Mitochondrial DNA depletion and respiratory chain activity in primary human subcutaneous adipocytes treated with nucleoside analogue reverse transcriptase inhibitors.

ABSTRACT Mitochondrial dysfunction as a consequence of mitochondrial DNA (mtDNA) depletion due to therapy with nucleoside analogue reverse transcriptase inhibitors (NRTI) has been proposed as a pathogenic mechanism leading to lipoatrophy in HIV-infected patients. The aim of our study was to investigate the impact of NRTI treatment on mtDNA abundance and the activities of respiratory chain complexes in primary human subcutaneous preadipocytes (phsPA). We studied adipocyte phenotypes, viability, and differentiation (CCAAT/enhancer-binding protein α [C/EBPα] and peroxisome proliferator-activated receptor γ [PPARγ] expression) and adiponectin production, mtDNA content, mitochondrial membrane potential, mitochondrial mass, and respiratory chain enzyme and citrate synthase activities in both proliferating and differentiating phsPA. Cells were exposed to zidovudine (6 μM), stavudine (d4T; 3 μM), and zalcitabine (ddC; 0.1 μM) for 8 weeks. NRTI-induced mtDNA depletion occurred in proliferating and differentiating phsPA after exposure to therapeutic drug concentrations of d4T and ddC. At these concentrations, ddC and d4T led to an almost 50% decrease in the number of mtDNA copies per cell without major impact on adipocyte differentiation. Despite mtDNA depletion by NRTI, the activities of the respiratory chain complexes, the mitochondrial membrane potential, and the mitochondrial mass were found to be unaffected. Severe NRTI-mediated mtDNA depletion in phsPA is not inevitably associated with impaired respiratory chain activity or altered mitochondrial membrane potential.