Heptanoate Improves Compensatory Mechanism of Glucose Homeostasis in Mitochondrial Long-Chain Fatty Acid Oxidation Defect.

Defects in mitochondrial fatty acid ß-oxidation (FAO) impair metabolic flexibility, which is an essential process for energy homeostasis. Very-long-chain acyl-CoA dehydrogenase (VLCADD; OMIM 609575) deficiency is the most common long-chain mitochondrial FAO disorder presenting with hypoglycemia as a common clinical manifestation. To prevent hypoglycemia, triheptanoin-a triglyceride composed of three heptanoates (C7) esterified with a glycerol backbone-can be used as a dietary treatment, since it is metabolized into precursors for gluconeogenesis. However, studies investigating the effect of triheptanoin on glucose homeostasis are limited. To understand the role of gluconeogenesis in the pathophysiology of long-chain mitochondrial FAO defects, we injected VLCAD-deficient (VLCAD-/-) mice with 13C3-glycerol in the presence and absence of heptanoate (C7). The incorporation of 13C3-glycerol into blood glucose was higher in VLCAD-/- mice than in WT mice, whereas the difference disappeared in the presence of C7. The result correlates with 13C enrichment of liver metabolites in VLCAD-/- mice. In contrast, the C7 bolus significantly decreased the 13C enrichment. These data suggest that the increased contribution of gluconeogenesis to the overall glucose production in VLCAD-/- mice increases the need for gluconeogenesis substrate, thereby avoiding hypoglycemia. Heptanoate is a suitable substrate to induce glucose production in mitochondrial FAO defect.

Altered markers of mitochondrial function in adults with autism spectrum disorder.

Previous research suggests potential mitochondrial dysfunction and changes in fatty acid metabolism in a subgroup of individuals with autism spectrum disorder (ASD), indicated by higher lactate, pyruvate levels, and mitochondrial disorder prevalence. This study aimed to further investigate potential mitochondrial dysfunction in ASD by assessing blood metabolite levels linked to mitochondrial metabolism. Blood levels of creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate, pyruvate, free and total carnitine, as well as acylcarnitines were obtained in 73 adults with ASD (47 males, 26 females) and compared with those of 71 neurotypical controls (NTC) (44 males, 27 females). Correlations between blood parameters and psychometric ASD symptom scores were also explored. Lower CK (pcorr = 0.045) levels were found exclusively in males with ASD compared to NTC, with no such variation in females. ALT and AST levels did not differ significantly between both groups. After correction for antipsychotic and antidepressant medication, CK remained significant. ASD participants had lower serum lactate levels (pcorr = 0.036) compared to NTC, but pyruvate and carnitine concentrations showed no significant difference. ASD subjects had significantly increased levels of certain acylcarnitines, with a decrease in tetradecadienoyl-carnitine (C14:2), and certain acylcarnitines correlated significantly with autistic symptom scores. We found reduced serum lactate levels in ASD, in contrast to previous studies suggesting elevated lactate or pyruvate. This difference may reflect the focus of our study on high-functioning adults with ASD, who are likely to have fewer secondary genetic conditions associated with mitochondrial dysfunction. Our findings of significantly altered acylcarnitine levels in ASD support the hypothesis of altered fatty acid metabolism in a subset of ASD patients.

Riboflavin 1 Transporter Deficiency: Novel SLC52A1 Variants and Expansion of the Phenotypic Spectrum.

Riboflavin transporter 1 (RFVT1) deficiency is an ultrarare metabolic disorder due to autosomal dominant pathogenic variants in SLC52A1. The RFVT1 protein is mainly expressed in the placenta and intestine. To our knowledge, only five cases of RFVT1 deficiency from three families have been reported so far. While newborns and infants with SLC52A1 variants mainly showed a multiple acyl-CoA dehydrogenase deficiency-like presentation, individuals identified in adulthood were usually clinically asymptomatic. We report two patients with novel heterozygous SLC52A1 variants. Patient 1 presented at the age of 62 with mild hyperammonemia following gastroenteritis. An acylcarnitine analysis in dried blood spots was abnormal with a multiple acyl-CoA dehydrogenase deficiency-like pattern, and genetic analysis confirmed a heterozygous SLC52A1 variant, c.68C > A, p. Ser23Tyr. Patient 2 presented with recurrent seizures and hypsarrhythmia at the age of 7 months. Metabolic investigations yielded unremarkable results. However, whole exome sequencing revealed a heterozygous start loss variant, c.3G > A, p. Met1Ile in SLC52A1. These two cases expand the clinical spectrum of riboflavin transporter 1 deficiency and demonstrate that symptomatic presentation in adulthood is possible.

Mitochondrial reprogramming in peripheral blood mononuclear cells of patients with glycogen storage disease type Ia.

BACKGROUND: Glycogen storage disease type Ia (GSDIa) is an inborn metabolic disorder caused by the deficiency of glucose-6-phospatase-α (G6Pase-α) leading to mitochondrial dysfunction. It remains unclear whether mitochondrial dysfunction is present in patients' peripheral blood mononuclear cells (PBMC) and whether dietary treatment can play a role. The aim of this study was to investigate mitochondrial function in PBMC of GSDIa patients. METHODS: Ten GSDIa patients and 10 age-, sex- and fasting-time matched controls were enrolled. Expression of genes involved in mitochondrial function and activity of key fatty acid oxidation (FAO) and Krebs cycle proteins were assessed in PBMC. Targeted metabolomics and assessment of metabolic control markers were also performed. RESULTS: Adult GSDIa patients showed increased CPT1A, SDHB, TFAM, mTOR expression (p < 0.05) and increased VLCAD, CPT2 and citrate synthase activity in PBMC (p < 0.05). VLCAD activity directly correlated with WC (p < 0.01), BMI (p < 0.05), serum malonycarnitine levels (p < 0.05). CPT2 activity directly correlated with BMI (p < 0.05). CONCLUSION: Mitochondrial reprogramming is detectable in PBMC of GSDIa patients. This feature may develop as an adaptation to the liver enzyme defect and may be triggered by dietary (over)treatment in the frame of G6Pase-α deficiency. PBMC can represent an adequate mean to assess (diet-induced) metabolic disturbances in GSDIa.

An Altered Sphingolipid Profile as a Risk Factor for Progressive Neurodegeneration in Long-Chain 3-Hydroxyacyl-CoA Deficiency (LCHADD).

Long-chain 3-hydroxyacyl-CoA deficiency (LCHADD) and mitochondrial trifunctional protein (MTPD) belong to a group of inherited metabolic diseases affecting the degradation of long-chain chain fatty acids. During metabolic decompensation the incomplete degradation of fatty acids results in life-threatening episodes, coma and death. Despite fast identification at neonatal screening, LCHADD/MTPD present with progressive neurodegenerative symptoms originally attributed to the accumulation of toxic hydroxyl acylcarnitines and energy deficiency. Recently, it has been shown that LCHADD human fibroblasts display a disease-specific alteration of complex lipids. Accumulating fatty acids, due to defective ß-oxidation, contribute to a remodeling of several lipid classes including mitochondrial cardiolipins and sphingolipids. In the last years the face of LCHADD/MTPD has changed. The reported dysregulation of complex lipids other than the simple acylcarnitines represents a novel aspect of disease development. Indeed, aberrant lipid profiles have already been associated with other neurodegenerative diseases such as Parkinson's Disease, Alzheimer's Disease, amyotrophic lateral sclerosis and retinopathy. Today, the physiopathology that underlies the development of the progressive neuropathic symptoms in LCHADD/MTPD is not fully understood. Here, we hypothesize an alternative disease-causing mechanism that contemplates the interaction of several factors that acting in concert contribute to the heterogeneous clinical phenotype.

Very long-chain acyl-CoA dehydrogenase deficiency in a Swedish cohort: Clinical symptoms, newborn screening, enzyme activity, and genetics.

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is a recessive disorder of fatty acid beta-oxidation with variable phenotype. Patients may present during the neonatal period with lethal multi-organ failure or during adulthood with a myopathic phenotype. VLCADD is included in the Swedish newborn screening (NBS) program since 2010. The study describes the phenotype and biochemical findings in relation to the genotype, enzyme activity, and screening data in a Swedish cohort of pediatric patients with VLCADD. A total of 22 patients (20 diagnosed via NBS between 2010 and 2019, two diagnosed pre NBS) were included. Parameters analyzed were enzyme activity (palmitoyl CoA oxidation rate); ACADVL genotype; NBS results including Collaborative Laboratory Integrated Reports (CLIR) score; biochemical findings; treatment; clinical outcome. A clinical severity score (CSS) was compiled using treatment interventions and clinical symptoms. A possible correlation between CSS and VLCAD residual enzyme activity and NBS CLIR score was analyzed. The most common ACADVL variant (c.848T>C) was identified in 24/44 alleles. Five novel variants were detected. Clinical manifestations varied from asymptomatic to severe. There was a correlation between CSS, residual enzyme activity, and CLIR scores. Most patients diagnosed via NBS had less severe disease compared to those clinically diagnosed. In conclusion, the identified correlation between the NBS CLIR score, residual enzyme activity, and clinical outcome suggests that information available neonatally may aid in treatment decisions.

Lipidomic and Proteomic Alterations Induced by Even and Odd Medium-Chain Fatty Acids on Fibroblasts of Long-Chain Fatty Acid Oxidation Disorders.

Medium-chain fatty acids (mc-FAs) are currently applied in the treatment of long-chain fatty acid oxidation disorders (lc-FAOD) characterized by impaired ß-oxidation. Here, we performed lipidomic and proteomic analysis in fibroblasts from patients with very long-chain acyl-CoA dehydrogenase (VLCADD) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHADD) deficiencies after incubation with heptanoate (C7) and octanoate (C8). Defects of ß-oxidation induced striking proteomic alterations, whereas the effect of treatment with mc-FAs was minor. However, mc-FAs induced a remodeling of complex lipids. Especially C7 appeared to act protectively by restoring sphingolipid biosynthesis flux and improving the observed dysregulation of protein homeostasis in LCHADD under control conditions.

Different Lipid Signature in Fibroblasts of Long-Chain Fatty Acid Oxidation Disorders.

Long-chain fatty acid oxidation disorders (lc-FAOD) are a group of diseases affecting the degradation of long-chain fatty acids. In order to investigate the disease specific alterations of the cellular lipidome, we performed undirected lipidomics in fibroblasts from patients with carnitine palmitoyltransferase II, very long-chain acyl-CoA dehydrogenase, and long-chain 3-hydroxyacyl-CoA dehydrogenase. We demonstrate a deep remodeling of mitochondrial cardiolipins. The aberrant phosphatidylcholine/phosphatidylethanolamine ratio and the increased content of plasmalogens and of lysophospholipids support the theory of an inflammatory phenotype in lc-FAOD. Moreover, we describe increased ratios of sphingomyelin/ceramide and sphingomyelin/hexosylceramide in LCHAD deficiency which may contribute to the neuropathic phenotype of LCHADD/mitochondrial trifunctional protein deficiency.

Altered Metabolic Flexibility in Inherited Metabolic Diseases of Mitochondrial Fatty Acid Metabolism.

In general, metabolic flexibility refers to an organism's capacity to adapt to metabolic changes due to differing energy demands. The aim of this work is to summarize and discuss recent findings regarding variables that modulate energy regulation in two different pathways of mitochondrial fatty metabolism: ß-oxidation and fatty acid biosynthesis. We focus specifically on two diseases: very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and malonyl-CoA synthetase deficiency (acyl-CoA synthetase family member 3 (ACSF3)) deficiency, which are both characterized by alterations in metabolic flexibility. On the one hand, in a mouse model of VLCAD-deficient (VLCAD-/-) mice, the white skeletal muscle undergoes metabolic and morphologic transdifferentiation towards glycolytic muscle fiber types via the up-regulation of mitochondrial fatty acid biosynthesis (mtFAS). On the other hand, in ACSF3-deficient patients, fibroblasts show impaired mitochondrial respiration, reduced lipoylation, and reduced glycolytic flux, which are compensated for by an increased ß-oxidation rate and the use of anaplerotic amino acids to address the energy needs. Here, we discuss a possible co-regulation by mtFAS and ß-oxidation in the maintenance of energy homeostasis.

The spectrum of peripheral neuropathy in disorders of the mitochondrial trifunctional protein.

Peripheral neuropathy is a known irreversible long-term complication of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and mitochondrial trifunctional protein deficiency (MTPD), two inherited disorders of mitochondrial long-chain fatty acid oxidation. The underlying pathophysiology of neuropathy is still not fully understood. We report electrophysiological studies and neurological findings in a series of 8 LCHAD-deficient and 11 MTP-deficient patients. The median age at time of the study was 8.0 years (0.5-25 years). The overall prevalence of neuropathy was 58% with neuropathic symptoms being slightly more common in MTPD compared to LCHADD (70% vs 50%, respectively). Onset of neuropathy was significantly earlier in MTPD patients compared to LCHADD patients (median age at onset 4.7 vs 15.3 years, respectively, P = .047). In four patients, isolated peripheral neuropathy was the first and only presenting symptom, and in all four the diagnosis was missed by newborn screening. About half of the patients (45.5%) had a sensorimotor neuropathy, while 27.3% showed a pure motor form and another 27.3% an isolated sensory form. Despite early diagnosis by newborn screening and early initiation of therapy, peripheral neuropathy cannot be prevented in all patients with LCHADD/MTPD and has severe impact on the life of affected patients. Electrophysiology classifies LCHADD/MTPD neuropathy as axonal with secondary demyelination. A novel observation is that in patients with acute, fulminant onset of neuropathy, symptoms can be partly reversible. Further studies are needed to elucidate the underlying pathophysiology of axonal damage and possible therapeutic targets.

Genotype and residual enzyme activity in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: Are predictions possible?

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common defect of mitochondrial ß-oxidation. Confirmation diagnostics after newborn screening (NBS) can be performed either by enzyme testing and/or by sequencing of the ACADM gene. Here, we report the results from enzyme testing in lymphocytes with gene variants from molecular analysis of the ACADM gene and with the initial acylcarnitine concentrations in the NBS sample. From April 2013 to August 2019, in 388 individuals with characteristic acylcarnitine profiles suggestive of MCADD the octanoyl-CoA-oxidation was measured in lymphocytes. In those individuals with residual activities <50%, molecular genetic analysis of the ACADM gene was performed. In 50% of the samples (195/388), MCADD with a residual activity ranging from 0% to 30% was confirmed. Forty-five percent of the samples (172/388) showed a residual activity >35% excluding MCADD. In the remaining 21 individuals, MCAD residual activity ranged from 30% to 35%. The latter group comprised both heterozygous carriers and individuals carrying two gene variants on different alleles. Twenty new variants could be identified and functionally classified based on their effect on enzyme function. C6 and C8 acylcarnitine species in NBS correlated with MCAD activity and disease severity. MCADD was only confirmed in half of the cases referred suggesting a higher false positive rate than expected. Measurement of the enzyme function in lymphocytes allowed fast confirmation diagnostics and clear determination of the pathogenicity of new gene variants. There is a clear correlation between genotype and enzyme function underlining the reproducibility of the functional measurement in vitro.

Accumulation of long-chain fatty acids in the tumor microenvironment drives dysfunction in intrapancreatic CD8+ T cells.

CD8+ T cells are master effectors of antitumor immunity, and their presence at tumor sites correlates with favorable outcomes. However, metabolic constraints imposed by the tumor microenvironment (TME) can dampen their ability to control tumor progression. We describe lipid accumulation in the TME areas of pancreatic ductal adenocarcinoma (PDA) populated by CD8+ T cells infiltrating both murine and human tumors. In this lipid-rich but otherwise nutrient-poor TME, access to using lipid metabolism becomes particularly valuable for sustaining cell functions. Here, we found that intrapancreatic CD8+ T cells progressively accumulate specific long-chain fatty acids (LCFAs), which, rather than provide a fuel source, impair their mitochondrial function and trigger major transcriptional reprogramming of pathways involved in lipid metabolism, with the subsequent reduction of fatty acid catabolism. In particular, intrapancreatic CD8+ T cells specifically exhibit down-regulation of the very-long-chain acyl-CoA dehydrogenase (VLCAD) enzyme, which exacerbates accumulation of LCFAs and very-long-chain fatty acids (VLCFAs) that mediate lipotoxicity. Metabolic reprogramming of tumor-specific T cells through enforced expression of ACADVL enabled enhanced intratumoral T cell survival and persistence in an engineered mouse model of PDA, overcoming one of the major hurdles to immunotherapy for PDA.

Primary carnitine deficiency - diagnosis after heart transplantation: better late than never!

BACKGROUND: Primary carnitine deficiency due to mutations in the SLC22A5 gene is a rare but well-treatable metabolic disorder that puts patients at risk for metabolic decompensations, skeletal and cardiac myopathy and sudden cardiac death. RESULTS: We report on a 7-year-old boy diagnosed with primary carnitine deficiency 2 years after successful heart transplantation thanks his younger sister's having been identified via expanded newborn screening during a pilot study evaluating an extension of the German newborn screening panel. CONCLUSION: As L-carnitine supplementation can prevent and mostly reverse clinical symptoms of primary carnitine deficiency, all patients with cardiomyopathy should be investigated for primary carnitine deficiency even if newborn screening results were unremarkable.

Sex-specific perturbation of complex lipids in response to medium-chain fatty acids in very long-chain acyl-CoA dehydrogenase deficiency.

Very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD) is the most common defect of long-chain fatty acid ß-oxidation. The recommended treatment includes the application of medium-chain triacylglycerols (MCTs). However, long-term treatment of VLCAD-/- mice resulted in the development of a sex-specific metabolic syndrome due to the selective activation of the ERK/mTORc1 signalling in females and ERK/peroxisome proliferator-activated receptor gamma pathway in males. In order to investigate a subsequent sex-specific effect of MCT on the lipid composition of the cellular membranes, we performed lipidomic analysis, SILAC-based quantitative proteomics and gene expression in fibroblasts from WT and VLCAD-/- mice of both sexes. Treatment with octanoate (C8) affected the composition of complex lipids resulting in a sex-specific signature of the molecular profile. The content of ceramides and sphingomyelins in particular differed significantly under control conditions and increased markedly in cells from mutant female mice but remained unchanged in cells from mutant males. Moreover, we observed a specific upregulation of biosynthesis of plasmalogens only in male mice, whereas in females C8 led to the accumulation of higher concentration of phosphatidylcholines and lysophosphatidylcholines. Our data on membrane lipids in VLCAD after supplementation with C8 provide evidence of a sex-specific lipid perturbation. We hypothesize a likely C8-induced pro-inflammatory response contributing to the development of a severe metabolic syndrome in female VLCAD-/- mice on long-term MCT supplementation.

Brain metabolism and neurological symptoms in combined malonic and methylmalonic aciduria.

Combined malonic and methylmalonic aciduria (CMAMMA) is an inborn error of metabolism which has been proposed being a benign condition. However, older patients may present with neurological manifestations such as seizures, memory problems, psychiatric problems and/ or cognitive decline. In fibroblasts from CMAMMA patients we have recently demonstrated a dysregulation of energy metabolism with increased dependency on ß-oxidation for energy production. Because of the inability of the brain to rely efficiently on this pathway to retrieve the required energy to a great extent, we hypothesize an alternative disease-causing mechanism that does not only include the accumulation of the metabolites malonic and methylmalonic acids. Here, we suggest a novel hypothesis on the possible pathophysiological mechanism responsible for the development of neurological symptoms in the long-run.

Therapeutic potential of triheptanoin in metabolic and neurodegenerative diseases.

In the past 15 years the potential of triheptanoin for the treatment of several human diseases in the area of clinical nutrition has grown considerably. Use of this triglyceride of the odd-chain fatty acid heptanoate has been proposed and applied for the treatment of several conditions in which the energy supply from citric acid cycle intermediates or fatty acid degradation are impaired. Neurological diseases due to disturbed glucose metabolism or metabolic diseases associated with impaired ß-oxidation of long chain fatty acid may especially take advantage of alternative substrate sources offered by the secondary metabolites of triheptanoin. Epilepsy due to deficiency of the GLUT1 transporter, as well as diseases associated with dysregulation of neuronal signalling, have been treated with triheptanoin supplementation, and very recently the advantages of this oil in long-chain fatty acid oxidation disorders have been reported. The present review summarises the published literature on the metabolism of triheptanoin including clinical reports related to the use of triheptanoin.

The fate of medium-chain fatty acids in very long-chain acyl­CoA dehydrogenase deficiency (VLCADD): A matter of sex?

Medium-chain-triglycerides (MCT) are widely applied in the treatment of long-chain fatty acid oxidation disorders (lcFAOD). Long-term treatment with MCT led to a sexually dimorphic response in the mouse model of very-long-chain-acyl-CoA-dehydrogenase-deficiency (VLCAD-/-) with the subsequent development of a metabolic syndrome in female mice. In order to evaluate the molecular mechanisms responsible for this sex specific response we performed a comprehensive metabolic phenotyping, SILAC-based quantitative proteomics and characterized the involved signaling pathways by western blot analysis and gene expression. WT and VLCAD-/- mice showed strong sex-dependent differences in basal metabolism and expression of proteins involved in the distinct metabolic pathways, even more prominent after treatment with octanoate. The investigation of molecular mechanisms responsible for the sexual dimorphisms delineated the selective activation of the ERK/mTORc1 signaling pathway leading to an increased biosynthesis and elongation of fatty acids in VLCAD-/- females. In contrast, octanoate induced the activation of ERK/PPARγ pathway and the subsequent upregulation of peroxisomal ߭oxidation in males. We here provide first evidence that sex has to be considered as important variable in disease phenotype. These findings may have implications on treatment strategies in the different sexes.

The emerging role of the mitochondrial fatty-acid synthase (mtFASII) in the regulation of energy metabolism.

Malonyl-CoA synthetase (ACSF3) catalyzes the first step of the mitochondrial fatty acid biosynthesis (mtFASII). Mutations in ACSF3 cause CMAMMA a rare inborn error of metabolism. The clinical phenotype is very heterogeneous, with some patients presenting with neurologic manifestations. In some children, presenting symptoms such as coma, ketoacidosis and hypoglycemia are suggestive of an intermediary metabolic disorder. The overall pathophysiological mechanisms are not understood. In order to study the role of mtFASII in the regulation of energy metabolism we performed a comprehensive metabolic phenotyping with Seahorse technology proteomics in fibroblasts from healthy controls and ACSF3 patients. SILAC-based proteomics and lipidomic analysis were performed to investigate the effects of hypofunctional mtFASII on proteome and lipid homeostasis of complex lipids. Our data clearly confirmed an impaired mitochondrial flexibility characterized by reduced mitochondrial respiration and glycolytic flux due to a lower lipoylation degree. These findings were accompanied by the adaptational upregulation of ß-oxidation and by the reduction of anaplerotic amino acids as compensatory mechanism to address the required energy need. Finally, lipidomic analysis demonstrated that the content of the bioactive lipids sphingomyelins and cardiolipins was strongly increased. Our data clearly demonstrate the role of mtFASII in metabolic regulation. Moreover, we show that mtFASII acts as mediator in the lipid-mediated signaling processes in the regulation of energy homeostasis and metabolic flexibility.

Implementation of a fast method for the measurement of carnitine palmitoyltransferase 2 activity in lymphocytes by tandem mass spectrometry as confirmation for newborn screening.

Carnitine palmitoyltransferase II (CPT2) is a rare autosomal recessive inherited disorder affecting mitochondrial ß-oxidation. Confirmation diagnostics are mostly based on molecular sequencing of the CPT2 gene, especially to distinguish CPT2 and carnitine:aclycarnitine translocase deficiencies, which present with identical acylcarnitine profiles on newborn screening (NBS). In the past, different enzyme tests in muscle biopsies have been developed in order to study the functional effect in one of the main target organs. In this study, we implemented a method for measurement of CPT2 enzyme activity in human lymphocytes with detection of the reaction products via liquid chromatography mass spectrometry to enable the simultaneous evaluation of the functional impairment and the clear diagnosis of the disease. CPT2 activity was measured in samples collected from CPT2 patients (n = 11), heterozygous carriers (n = 6), and healthy individuals (n = 52). Seven patients out of 11 were homozygous for the common mutation c.338T>C and showed a residual activity with median values of 19.2 ± 3.7% of healthy controls. Heterozygous carriers showed a residual activity in the range of 42% to 75%. Four individuals carrying the heterozygous mutation c.338T>C showed a 2-fold higher residual activity as compared to homozygous individuals. Our optimized method for the measurement of CPT2 activity is able to clearly discriminate between patients and healthy individuals and offers the possibility to determine CPT2 activity in human lymphocytes avoiding the need of an invasive muscle biopsy. This method can be successfully used for confirmation diagnosis in case of positive NBS and would markedly reduce the time to define diagnosis.

The diagnostic challenge in very-long chain acyl-CoA dehydrogenase deficiency (VLCADD).

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is the most common defect of mitochondrial ß-oxidation of long-chain fatty acids. However, the unambiguous diagnosis of true VLCADD patients may be challenging, and a high rate of false positive individuals identified by newborn screening undergo confirmation diagnostics. In this study, we show the outcome of enzyme testing in lymphocytes as a confirmatory tool in newborns identified by screening, and the correlation with molecular sequencing of the ACADVL gene. From April 2013 to March 2017, in 403 individuals with characteristic acylcarnitine profiles indicative of VLCADD, palmitoyl-CoA oxidation was measured followed by molecular genetic analysis in most of the patients with residual activity (RA) <50%. In almost 50% of the samples (209/403) the RA was >50%, one-third of the individuals (125/403) displayed a RA of 30-50% and 69/403 individuals showed a residual activity of 0-30%. Sequencing of the ACADVL gene revealed that all individuals with activities below 24% were true VLCADD patients, individuals with residual activities between 24 and 27% carried either one or two mutations. Twenty new mutations could be identified and functionally classified based on their effect on enzyme function. Finally, we observed an up-regulation of MCAD-activity in many patients. However, this did not correlate with the degree of VLCAD RA. Although the likely clinical phenotype cannot be fully foreseen by genetic and functional tests as it depends on many factors, our data demonstrate the strength of this functional enzyme test in lymphocytes as a quick and reliable method for confirmation diagnostics of VLCADD.