Very Long-chain acyl-CoA Dehydrogenase Deficiency Research Articles

Fasting induces prominent proteomic changes in liver in very long chain Acyl-CoA dehydrogenase deficient mice

Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is a clinically heterogeneous disorder of mitochondrial fatty acid β-oxidation usually identified through newborn screening. Genotype-phenotype correlations have been defined, but considerable clinical heterogeneity still exists. Symptoms are often induced by physiological stress such as fasting or intercurrent illness, setting it as an important example of environmental effects altering clinical course in an individual with a genetic disease. However, neither the cellular changes that predispose to this phenomenon nor the alterations it induces are well characterized. We examined the effects of fasting in a knockout mouse model to explore changes in global mitochondria protein profiles in liver and to investigate the physiologically relevant changes that lead to the clinical presentations. An isobaric tags for relative and absolute quantification (iTRAQ) labeling approach was employed to examine mitochondrial proteome changes in VLCAD deficient compared to wild type mice in the fed and fasted states. We identified numerous proteomic changes associated with the gene defect and fasting within relevant metabolic pathways. Few changes induced by fasting were shared between the VLCAD deficient and wild type mice, with more alterations found in the deficient mice on fasting. Particularly, fasting in the deficient mice could reverse the protective response in oxidative phosphorylation pathway seen in wild type animals. In addition, we found that changes in chaperone proteins including heat shock protein 60 (HSP60) and 10 (HSP10) during fasting differed between the two genotypes, highlighting the importance of these proteins in VLCAD deficiency. Finally, the effects on the liver proteome imposed by changes in fasted VLCAD deficient mice indicates that this environmental factor may be an inducer of both cellular and physiological changes.

P.385 - Genotype–phenotype correlation in VLCAD deficiency

P.385 - Genotype–phenotype correlation in VLCAD deficiency

VLCAD deficiency: Follow-up and outcome of patients diagnosed through newborn screening in Victoria.

Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is an inherited metabolic disorder of fatty acid oxidation. Treatment practices of the disorder have changed over the past 10-15years since this disorder was included in newborn screening programs and patients were diagnosed pre-symptomatically. A genotype-phenotype correlation has been suggested but the discovery of novel mutations make this knowledge limited. Herein, we describe our experience in treating patients (n=22) diagnosed through newborn screening and mutational confirmation and followed up over a median period of 104months. We report five novel mutations. In 2013 we formalised our treatment protocol, which essentially follows a European consensus paper from 2009 and our own experience. The prescribed low natural fat diet is relaxed for patients who are asymptomatic when reaching age 5years but medium-chain triglyceride oil is recommended before and after physical activity regardless of age. Metabolic stability, growth, development and cardiac function are satisfactory in all patients. There were no episodes of encephalopathy or hypoglycaemia but three patients had episodes of muscle pain with our without rhabdomyolysis. Body composition studies showed a negative association between dietary protein intake and percent body fat. Larger patient cohort and longer follow up time are required for further elucidation of genotype-phenotype correlations and for establishing the role of dietary protein in metabolic stability and long-term healthier body composition in patients with VLCAD deficiency.

Outcomes and genotype-phenotype correlations in 52 individuals with VLCAD deficiency diagnosed by NBS and enrolled in the IBEM-IS database.

Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency can present at various ages from the neonatal period to adulthood, and poses the greatest risk of complications during intercurrent illness or after prolonged fasting. Early diagnosis, treatment, and surveillance can reduce mortality; hence, the disorder is included in the newborn Recommended Uniform Screening Panel (RUSP) in the United States. The Inborn Errors of Metabolism Information System (IBEM-IS) was established in 2007 to collect longitudinal information on individuals with inborn errors of metabolism included in newborn screening (NBS) programs, including VLCAD deficiency. We retrospectively analyzed early outcomes for individuals who were diagnosed with VLCAD deficiency by NBS and describe initial presentations, diagnosis, clinical outcomes and treatment in a cohort of 52 individuals ages 1-18years. Maternal prenatal symptoms were not reported, and most newborns remained asymptomatic. Cardiomyopathy was uncommon in the cohort, diagnosed in 2/52 cases. Elevations in creatine kinase were a common finding, and usually first occurred during the toddler period (1-3years of age). Diagnostic evaluations required several testing modalities, most commonly plasma acylcarnitine profiles and molecular testing. Functional testing, including fibroblast acylcarnitine profiling and white blood cell or fibroblast enzyme assay, is a useful diagnostic adjunct if uncharacterized mutations are identified.

Analysis of clinical and genetic results in one case of very long chain acyl-CoA dehydrogenase deficiency

Clinical characteristics were analyzed in a child with very long chain acyl-CoA dehydrogenase deficiency(VLCADD). The gene analysis was performed in 20 exon all coding regions and 10 bp shear zone in the very long chain acyl-CoA dehydrogenase(VLCAD)gene of the case and his family members by direct sequencing of PCR-DNA from peripheral blood. The results showed that the patient presented with acute onset, clinical manifestations of repeated vomiting, poor spirit, abnormal liver function, increased myocardial enzyme kinase. At the age of one year old, this child was diagnosed with Reye's syndrome for liver injury. Genetic testing results revealed that E14 c. 1349G>A, p. R450H heterozygous mutation in VLCAD gene was found in this case, his mother, and his younger sister, and E15 c. 1532G>A, p. R511Q heterozygous mutation was found in this case and his father. The pathogenic genes of the case are from his mother and the younger sister is a carrier. (Chin J Endocrinol Metab, 2016, 32: 309-311) Key words: Very long chain acyl-CoA dehydrogenase; Very long chain acyl-CoA dehydrogenase deficiency; Muscle disease; Tandem mass spectrometry; Gene diagnosis

Altered Energetics of Exercise Explain Risk of Rhabdomyolysis in Very Long-Chain Acyl-CoA Dehydrogenase Deficiency.

Rhabdomyolysis is common in very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and other metabolic myopathies, but its pathogenic basis is poorly understood. Here, we show that prolonged bicycling exercise against a standardized moderate workload in VLCADD patients is associated with threefold bigger changes in phosphocreatine (PCr) and inorganic phosphate (Pi) concentrations in quadriceps muscle and twofold lower changes in plasma acetyl-carnitine levels than in healthy subjects. This result is consistent with the hypothesis that muscle ATP homeostasis during exercise is compromised in VLCADD. However, the measured rates of PCr and Pi recovery post-exercise showed that the mitochondrial capacity for ATP synthesis in VLCADD muscle was normal. Mathematical modeling of oxidative ATP metabolism in muscle composed of three different fiber types indicated that the observed altered energy balance during submaximal exercise in VLCADD patients may be explained by a slow-to-fast shift in quadriceps fiber-type composition corresponding to 30% of the slow-twitch fiber-type pool in healthy quadriceps muscle. This study demonstrates for the first time that quadriceps energy balance during exercise in VLCADD patients is altered but not because of failing mitochondrial function. Our findings provide new clues to understanding the risk of rhabdomyolysis following exercise in human VLCADD.

Activation of PPARα by Fatty Acid Accumulation Enhances Fatty Acid Degradation and Sulfatide Synthesis.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first reaction in the mitochondrial fatty acid β-oxidation pathway. VLCAD deficiency is associated with the accumulation of fat in multiple organs and tissues, which results in specific clinical features including cardiomyopathy, cardiomegaly, muscle weakness, and hepatic dysfunction in infants. We speculated that the abnormal fatty acid metabolism in VLCAD-deficient individuals might cause cell necrosis by fatty acid toxicity. The accumulation of fatty acids may activate peroxisome proliferator-activated receptor (PPAR), a master regulator of fatty acid metabolism and a potent nuclear receptor for free fatty acids. We examined six skin fibroblast lines, derived from VLCAD-deficient patients and identified fatty acid accumulation and PPARα activation in these cell lines. We then found that the expression levels of three enzymes involved in fatty acid degradation, including long-chain acyl-CoA synthetase (LACS), were increased in a PPARα-dependent manner. This increased expression of LACS might enhance the fatty acyl-CoA supply to fatty acid degradation and sulfatide synthesis pathways. In fact, the first and last reactions in the sulfatide synthesis pathway are regulated by PPARα. Therefore, we also measured the expression levels of enzymes involved in sulfatide metabolism and the regulation of cellular sulfatide content. The levels of these enzymes and cellular sulfatide content both increased in a PPARα-dependent manner. These results indicate that PPARα activation plays defensive and compensative roles by reducing cellular toxicity associated with fatty acids and sulfuric acid.

Elevations of C14:1 and C14:2 Plasma Acylcarnitines in Fasted Children: A Diagnostic Dilemma

To test whether follow-up testing for very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency uncovers a diagnosis in patients with elevations of C14:1 and C14:2 plasma acylcarnitines after a controlled fasting study performed for clinically suspected hypoglycemia and to compare the acylcarnitine profiles from fasted patients without VLCAD deficiency vs patients with known VLCAD deficiency to determine whether metabolite testing distinguishes these groups. We performed a retrospective chart review and identified 17 patients with elevated C14:1 and C14:2 plasma acylcarnitine levels after a controlled fast and with testing for VLCAD deficiency (ACADVL sequencing or fibroblast fatty acid oxidation studies). The follow-up testing in all patients was inconsistent with a diagnosis of VLCAD deficiency. We compared the plasma acylcarnitine profiles from these fasted patients vs patients with VLCAD deficiency. C14:1/C12:1 was significantly lower (P<.001) in fasted patients vs patients with VLCAD deficiency. Metabolomics analysis performed in 2 fasted patients and 1 patient with VLCAD deficiency demonstrated evidence for up-regulated lipolysis and β-oxidation in the fasted state. Elevations of plasma C14:1 and C14:2 acylcarnitines appear to be a physiologic result of lipolysis that occurs with fasting. Both metabolomics analysis and/or C14:1/C12:1 may distinguish C14:1 elevations from physiologic fasting-induced lipolysis vs VLCAD deficiency.

Denovo fatty acid biosynthesis and elongation in very long-chain acyl-CoA dehydrogenase-deficient mice supplemented with odd or even medium-chain fatty acids.

An even medium-chain triglyceride (MCT)-based diet is the mainstay of treatment in very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD). Previous studies with magnetic resonance spectroscopy have shown an impact of MCT on the average fatty acid chain length in abdominal fat. We therefore assume that medium-chain fatty acids (MCFAs) are elongated and accumulate in tissue as long-chain fatty acids. In this study, we explored the hepatic effects of long-term supplementation with MCT or triheptanoin, an odd-chain C7-based triglyceride, in wild-type and VLCAD-deficient (VLCAD(-/-) ) mice after 1year of supplementation as compared with a control diet. The denovo biosynthesis and elongation of fatty acids, and peroxisomal β-oxidation, were quantified by RT-PCR. This was followed by a comprehensive analysis of hepatic and cardiac fatty acid profiles by GC-MS. Long-term application of even and odd MCFAs strongly induced denovo biosynthesis and elongation of fatty acids in both wild-type and VLCAD(-/-) mice, leading to an alteration of the hepatic fatty acid profiles. We detected denovo-synthesized and elongated fatty acids, such as heptadecenoic acid (C17:1n9), eicosanoic acid (C20:1n9), erucic acid (C22:1n9), and mead acid (C20:3n9), that were otherwise completely absent in mice under control conditions. In parallel, the content of monounsaturated fatty acids was massively increased. Furthermore, we observed strong upregulation of peroxisomal β-oxidation in VLCAD(-/-) mice, especially when they were fed an MCT diet. Our data raise the question of whether long-term MCFA supplementation represents the most efficient treatment in the long term. Studies on the hepatic toxicity of triheptanoin are still ongoing.

Recurrent ACADVL molecular findings in individuals with a positive newborn screen for very long chain acyl-coA dehydrogenase (VLCAD) deficiency in the United States.

Very long chain acyl-coA dehydrogenase deficiency (VLCADD) is an autosomal recessive inborn error of fatty acid oxidation detected by newborn screening (NBS). Follow-up molecular analyses are often required to clarify VLCADD-suggestive NBS results, but to date the outcome of these studies are not well described for the general screen-positive population. In the following study, we report the molecular findings for 693 unrelated patients that sequentially received Sanger sequence analysis of ACADVL as a result of a positive NBS for VLCADD. Highlighting the variable molecular underpinnings of this disorder, we identified 94 different pathogenic ACADVL variants (40 novel), as well as 134 variants of unknown clinical significance (VUSs). Evidence for the pathogenicity of a subset of recurrent VUSs was provided using multiple in silico analyses. Surprisingly, the most frequent finding in our cohort was carrier status, 57% all individuals had a single pathogenic variant or VUS. This result was further supported by follow-up array and/or acylcarnitine analysis that failed to provide evidence of a second pathogenic allele. Notably, exon-targeted array analysis of 131 individuals screen positive for VLCADD failed to identify copy number changes in ACADVL thus suggesting this test has a low yield in the setting of NBS follow-up. While no genotype was common, the c.848T>C (p.V283A) pathogenic variant was clearly the most frequent; at least one copy was found in ~10% of all individuals with a positive NBS. Clinical and biochemical data for seven unrelated patients homozygous for the p.V283A allele suggests that it results in a mild phenotype that responds well to standard treatment, but hypoglycemia can occur. Collectively, our data illustrate the molecular heterogeneity of VLCADD and provide novel insight into the outcomes of NBS for this disorder.

Retraction.

RetractionRetractionPublished Online:15 Apr 2015https://doi.org/10.1152/ajpheart.zh4-1470-retr.2015Retracted articleMoreSectionsPDF (50 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Volume 292, May 2007 Volume 61, May 2007Werdich AA, Baundenbacher F, Dzhura I, Jeyakumar LH, Kannankeril PJ, Fleischer S, LeGrone A, Milatovic D, Aschner M, Strauss AW, Anderson ME, Exil VJ. Polymorphic ventricular tachycardia and abnormal Ca2+ handling in very-long-chain acyl-CoA dehydrogenase null mice. Am J Physiol Heart Circ Physiol 292: H2202–H2211, 2007; doi: https://doi.org/10.1152/ajpheart.00382.2006.We regret that the data shown in Fig. 6 of this article were falsified and/or fabricated. The author who contributed these data recently acknowledged committing scientific misconduct (http://ori.hhs.gov/content/case-summary-dzhura-igor). Therefore, this article is being retracted by the American Physiological Society in agreement with the authors. However, the authors believe that the remaining data shown in Figs.1–5 and 7 are consistent and sufficient to establish the cellular electrophysiological phenotype of VLCAD deficiency in mice and that the overall conclusions from this article are valid. We apologize to the scientific community for any inconvenience or challenges resulting from the publication and retraction of this article.Please note that Dr. Igor Dzhura could not be reached to sign the notice.This article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformationRelated ArticlesPolymorphic ventricular tachycardia and abnormal Ca2+ handling in very-long-chain acyl-CoA dehydrogenase null mice 01 May 2007American Journal of Physiology-Heart and Circulatory Physiology More from this issue > Volume 308Issue 8April 2015Pages H963-H963 Copyright & PermissionsCopyright © 2015 the American Physiological Societyhttps://doi.org/10.1152/ajpheart.zh4-1470-retr.2015PubMed25878245History Published online 15 April 2015 Published in print 15 April 2015 Metrics

Fatty acid oxidation flux predicts the clinical severity of VLCAD deficiency.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is an inherited disorder of mitochondrial long-chain fatty acid β-oxidation (LC-FAO) and is included in many newborn screening (NBS) programs worldwide. Patients may present with hypoketotic hypoglycemia, cardiomyopathy, and/or myopathy, but clinical severity varies widely and the clinical outcome is unpredictable. We investigated predictive markers that may determine clinical severity. We developed a clinical severity score (CSS), which was determined for 13 Dutch patients with VLCADD, all of whom were diagnosed before the introduction of VLCADD in NBS to prevent bias from early diagnosis. In cultured skin fibroblasts from these patients, we measured LC-FAO flux (the rate of oleate oxidation), VLCAD activity, and acylcarnitine profiles following palmitate loading. The strongest correlation (r = 0.93; P < 0.0001) was observed between LC-FAO flux and the CSS. VLCAD activity measurement and the C14/C16-to-acylcarnitine ratio correlated much less. A median LC-FAO flux of 6% of control values (range 5.6-6.8%) was associated with cardiomyopathy (P < 0.01), and 32.4% (range 5.6-50.5%) was associated with myopathy (P < 0.05). Our results demonstrate a very strong correlation between LC-FAO flux in fibroblasts and the clinical severity of VLCADD. LC-FAO flux measurements may thus predict whether patients are likely to develop symptoms.

Clinical features and ACADVL gene mutation spectrum analysis of 11 Chinese patients with very long chain acyl-CoA dehydrogenase deficiency

To investigate the clinical and laboratory features of very long chain acyl-CoA dehydrogenase deficiency ( VLCADD ) and the correlations between its genotype and phenotype. Eleven patients diagnosed as VLCADD of Shanghai Jiaotong University School of Medicine seen from September 2006 to May 2014 were included. There were 9 boys and 2 girls, whose age was 2 d-17 years. Analysis was performed on clinical features, routine laboratory examination, and tandem mass spectrometry (MS-MS) , gas chromatography mass spectrometry (GC-MS) and genetic analysis were conducted. All cases had elevated levels of blood tetradecanoylcarnitine (C14:1) recognized as the characteristic biomarker for VLCADD. The eleven patients were classified into three groups: six cases in neonatal onset group, three in infancy onset group form patients and two in late onset group. Neonatal onset patients were characterized by hypoactivity, hypoglycemia shortly after birth. Infancy onset patients presented hepatomegaly and hypoglycemia in infancy. The two adolescent patients showed initial manifestations of exercise intolerance or rhabdomyolysis. Six of the eleven patients died at the age of 2-8 months, including four neonatal onset and two infant onset patients, with one or two null mutations. The other two neonatal onset patients were diagnosed since early birth through neonatal screening and their clinical manifestation are almost normal after treatments. Among 11 patients, seventeen different mutations in the ACADVL gene were identified, with a total mutation detection rate of 95.45% (21/22 alleles), including eleven reported mutations ( p. S22X, p. G43D, p. R511Q, p. W427X, p. A213T, p. C215R, p. G222R, p. R450H, p. R456H, c. 296-297delCA, c. 1605 + 1G > T) and six novel mutations (p. S72F, p. Q100X, p. M437T, p. D466Y, c. 1315delG insAC, IVS7 + 4 A > G). The p. R450H was the most frequent mutation identified in three alleles (13.63%, 3/22 alleles), followed by p. S22X and p. D466Y mutations which were detected in two alleles (9.09%, 2/22 alleles). The ACADVL gene mutations were heterozygous in our patients. The mortality of neonatal onset form and infant onset form is much higher than the late onset form patients, suggesting a certain correlation between the genotype and phenotype was found. The earlier diagnosis and treatment of VLCADD are of vital importance for the improvement of the prognosis of the patients.

ESSENTIAL FATTY ACID DEFICIENCY IN VERY LONG-CHAIN ACYL-COA DEHYDROGENASE DEFICIENT PATIENTS

Introduction: Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), a long-chain fatty acid (LCFA) beta-oxidation dis order, may be treated with LCFA restriction. As Essential Fatty Acids (EFAs) are LCFAs, patients may be at risk for EFA deficiency. Objectives: Investigate whether LCFA restrictions lead to EFA deficiency in VLCADD and which markers are indicative of EFA deficiency. Methods: Thirty-nine LCFA profiles of 16 VLCADD patients were determined in erythrocytes and compared to 48 healthy controls. The predictive value of EFA deficiency markers was calculated from data of a historic cohort (n=4523, 0-39yrs). Results: Linoleic acid (LA), dihomo-γ-linolenic acid (DHLA) and eicosapentaenoic acid (EPA) were significantly decreased in VLCADD patients. Patients on docosahexaenoic acid (DHA) and arachidonic acid (AA) supplementation exhibited even lower LA. Mead acid, a presumed marker for EFA-deficiency, was not increased in patients. In the historic cohort, sensitivity of MA was low for LA deficiency (24% for levels <2.5 percentile) and for DHA+AA deficiency (12% for levels <2.5 percentile). Discussion: VLCADD patients on LCFA restriction are prone to develop LA deficiency. Furthermore, MA is a specific, but not a sensitive marker for LA or EFA deficiency, neither in VLCADD patients, nor in healthy controls, nor in a large patient cohort.

The Newborn Screening Paradox: Sensitivity vs. Overdiagnosis in VLCAD Deficiency.

To improve the efficacy of newborn screening (NBS) for very long chain acyl-CoA dehydrogenase deficiency (VLCADD). Data on all dried blood spots collected by the Dutch NBS from October 2007 to 2010 (742.728) were included. Based solely on the C14:1 levels (cutoff ≥0.8μmol/L), six newborns with VLCADD had been identified through NBS during this period. The ratio of C14:1 over C2 was calculated. DNA of all blood spots with a C14:1/C2 ratio of ≥0.020 was isolated and sequenced. Children homozygous or compound heterozygous for mutations in the ACADVL gene were traced back and invited for detailed clinical, biochemical, and genetic evaluation. Retrospective analysis based on the C14:1/C2 ratio with a cutoff of ≥0.020 identified an additional five children with known ACADVL mutations and low enzymatic activity. All were still asymptomatic at the time of diagnosis (age 2-5 years). Increasing the cutoff to ≥0.023 resulted in a sensitivity of 93% and a positive predictive value of 37%. The sensitivity of the previously used screening approach (C14:1 ≥0.8) was 50%. This study shows that the ratio C14:1/C2 is a more sensitive marker than C14:1 for identifying VLCADD patients in NBS. However, as these patients were all asymptomatic at the time of diagnosis, this suggests that a more sensitive screening approach may also identify individuals who may never develop clinical disease. Long-term follow-up studies are needed to establish the risk of these VLCADD-deficient individuals for developing clinical signs and symptoms.

Application of an Image Cytometry Protocol for Cellular and Mitochondrial Phenotyping on Fibroblasts from Patients with Inherited Disorders.

Cellular phenotyping of human dermal fibroblasts (HDFs) from patients with inherited diseases provides invaluable information for diagnosis, disease aetiology, prognosis and assessing of treatment options. Here we present a cell phenotyping protocol using image cytometry that combines measurements of crucial cellular and mitochondrial parameters: (1) cell number and viability, (2) thiol redox status (TRS), (3) mitochondrial membrane potential (MMP) and (4) mitochondrial superoxide levels (MSLs). With our protocol, cell viability, TRS and MMP can be measured in one small cell sample and MSL on a parallel one. We analysed HDFs from healthy individuals after treatment with various concentrations of hydrogen peroxide (H2O2) for different intervals, to mimic the physiological effects of oxidative stress. Our results show that cell number, viability, TRS and MMP decreased, while MSL increased both in a time- and concentration-dependent manner. To assess the use of our protocol for analysis of HDFs from patients with inherited diseases, we analysed HDFs from two patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD), one with a severe clinical phenotype and one with a mild one. HDFs from both patients displayed increased MSL without H2O2 treatment. Treatment with H2O2 revealed significant differences in MMP and MSL between HDFs from the mild and the severe patient. Our results establish the capacity of our protocol for fast analysis of cellular and mitochondrial parameters by image cytometry in HDFs from patients with inherited metabolic diseases.

Estudio In vitro de la deficiencia de Acil-CoA deshidrogenasa de cadena muy larga con afectación muscular

Introducción: Los estudios in vitro, que buscan intermediarios de la degradación mitocondrial de ácidos grasos, facilitan el diagnóstico de alteraciones hereditarias o adquiridas en esa ruta metabólica. Objetivo: Analizar la producción de metabolitos en fibroblastos de pacientes con deficiencia de acil-CoA deshidrogenasa de cadena muy larga (VLCAD) con afectación muscular, incubados con ácido oleico deuterado. Materiales y Métodos: Se incubaron fibroblastos de tres pacientes con deficiencia de acil-CoA deshidrogenasa de cadena muy larga (VLCAD) con cardiomiopatía y de 10 controles en presencia de ácido oleico deuterado. Resultados: Se encontró un perfil característico luego de la incubación de fibroblastos con esta deficiencia. Conclusión: Este sustrato podría ser usado para realizar diagnóstico in vitro de la deficiencia de VLCAD con afectación muscular.

Neurodevelopmental profiles of children with very long chain acyl-CoA dehydrogenase deficiency diagnosed by newborn screening

BackgroundVery long chain acyl-CoA dehydrogenase (VLCAD) deficiency is a disorder of fatty acid oxidation with an estimated incidence of between 1:31,500 and 1:125,000. There is limited information regarding neurodevelopmental outcomes, probably because the disorder is perceived as affecting the skeletal and heart muscles, and many children are deemed asymptomatic. The aim of this study was to utilise a comprehensive neuropsychological assessment battery that assessed IQ, language, attention, memory, executive functioning, motor skills, behaviour, and social skills in children 4 to 10years old diagnosed with VLCAD deficiency through newborn screening. MethodSeven children completed neuropsychological assessment and one child was only involved in part of the study (2 female, 6 male). Parents completed questionnaires regarding executive functioning, behaviour and social skills. ResultsIQ scores ranged from average to the superior range. No deficits were found in fine or gross motor skills. One patient had a mild language deficit, and two patients had previously required speech therapy. Verbal memory, attention and executive functioning skills were generally average or above. Visual memory scores were mostly above average. Parents' questionnaires identified one child as having social skills deficits, and two as having behavioural problems such as hyperactivity. One child rated high on an autism spectrum subscale; another was formally diagnosed with autism spectrum disorder—both children were symptomatic at birth. ConclusionsVLCAD deficiency does not have a significant impact on cognitive or motor skills. Some children may be vulnerable to speech, social and behavioural issues.

Critical illness in energy metabolism genetic disorder: rhabdomyolysis, acute kidney injury, respiratory arrest.

In very long-chain acylCoA dehydrogenase deficiency (VLCAD), the activity of this enzyme is either reduced or absent with the inability to use long-chain fatty acids as energy substrates. A 25-year old male with VLCAD was admitted to the Emergency Department of Policlinico Teaching Hospital (Modena, Italy)for generalized weakness and oliguria, after a period of physical and mental stress and inadequate compliance to a long-chain fatty acid free diet. Laboratory tests were compatible with acute kidney injury. Seventy-two hours after admission, the subject had an episode of chest pain with elevated markers of myocardial necrosis. The rapid deterioration of muscular strength and the subsequent worsening respiratory failure necessitated ventilator support within the local Medical Intensive Care Unit. There, the patient showed a prompt normalization of respiratory parameters and a steady improvement of renal function. An inadequate compliance to lifestyle and dietary restriction in VLCAD may trigger severe and potentially lethal crisis. The in-hospital management of these patients calls for early intensive care admission as their conditions may deteriorate without warning.

Novel biomarkers for diagnosing and understanding pathophysiology of metabolic diseases

BackgroundTandem mass spectrometric analysis of simultaneously measuredblood spot acylcarnitines and amino acids is a widely accepted andapplied method for the early detection of a number of disorders offatty acid, organic acid and amino acid metabolism. Adaptation of thisapproach has led to vastly improved clinical outcomes for a number ofdisorders including medium chain acyl-CoA dehydrogenase (MCAD)deficiency [1], very long-chain acyl-CoA dehydrogenase (VLCAD)deficiency, glutaric acidemia type 1 and maple syrup urine disease.Outcome data for other conditions are awaited but, intuitively, mostinvestigators believe that these conditions should also result in betteroutcomes if diagnosed presymptomatically [2]. The sensitivity andspecificity of the current process are very high for disorders such asMCAD deficiency but there are numerous examples where sensitivityis not sufficiently high such that positive cases may be missed. Thereis a significant literature documenting missed cases of subsequentlyfatal VLCAD deficiency that was not diagnosed in the newborn perioddespite screening for the disorder [3]. We are lookingfor analternativescreening method to reduce this false negative rate.MethodAcylcarnitinemeasurementinbody fluidsrepresentsbiomarkeraccu-mulation that is several steps away from the primary accumulating me-tabolites. For fatty acid oxidation and many organic acid defects theprimary accumulating metabolite is an intra-mitochondrial coenzyme Aspecies. This species is then converted to an acylcarnitine which exitsthe mitochondria, may be further metabolized elsewhere in the cytosol(peroxisomes, microsomes) and eventually reaches the circulationthrough unknown mechanisms. For VLCAD deficiency for instance, theprimaryaccumulatingmetabolitesshouldbeC16:0,C18:1andC18:2spe-cies but the diagnostic measurands ar e unsaturated C14 species suggest-ing extensive modification of the primary species. Presently, mostpublished acyl-CoA assays require a chromatographic separation stepwhich is generally regarded as too time consuming for screening pur-poses. To address this issue we have developed a flow injection tandemmass spectrometric protocol which can analyze all species of acyl-CoA'sin a Multiple Reaction Mode (MRM) [4]. Acyl-CoA's are intracellular bio-markers and would not be expected to accumulate outside of a cellulardomain so the method was developed initially for tissue analysis for val-idation purposes.ResultsWe analyzed normal mouse tissues and those from both the short-chain acyl-CoA dehydrogenase (SCAD) mouse and the short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) knockout mouse. A normalprofile from the wild-type mouse liver demonstrated the expectedspecies including C16, C18:1 and C18:1 in addition to acetyl-CoA andfree CoA. There was little intermediate species accumulation. TheSCAD knockout mouse liver demonstrated the expected accumulationof butyryl-CoA whilst the SCHAD knockout mouse demonstrated accu-mulation of medium-chain species consistent with the known chain-length specificity of the enzyme which includes medium chain species.Future directionsWearenowworkingonincreasingthesensitivityoftheassayinorderto be able to analyze acyl-CoA's in the cellular component of a wholeblood sample, a requisite for newborn screening. Initial work withwhitebloodcellssuggeststhatthisisfeasibleandmaybecomeapplicableas mass spectrometers continue to evolve in terms of sensitivity.WehavebeenabletoutilizetheassaytostudyfattyacidoxidationinthegastrointestinaltractandhaverecentlydeterminedthattheGItractcan generate energy from long-chain fatty acids derived fromthedietandshortchainfattyacidsthataregeneratedbythemicrobiomeinapathwaythatappearstobeindependentofmalonyl-CoAregulation.References