Article

Fatty acid oxidation flux predicts the clinical severity of VLCAD deficiency

April 2015
Genetics in medicine: official journal of the American College of Medical Genetics 17(12)

April 2015
17(12)

DOI:10.1038/gim.2015.22

Source
PubMed

Authors:

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.Genet Med advance online publication 02 April 2015Genetics in Medicine (2015); doi:10.1038/gim.2015.22.

Thermo‐sensitive mitochondrial trifunctional protein deficiency presenting with episodic myopathy

Article

Full-text available

Apr 2022

Mitochondrial trifunctional protein (MTP) is involved in long-chain fatty acid β-oxidation (lcFAO). Deficiency of one or more of the enzyme activities as catalyzed by MTP causes generalized MTP deficiency (MTPD), long-chain hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), or long-chain ketoacyl-CoA thiolase deficiency (LCKATD). When genetic variants result in thermo-sensitive enzymes, increased body temperature (e.g. fever) can reduce enzyme activity and be a risk factor for clinical decompensation. This is the first description of five patients with a thermo-sensitive MTP deficiency. Clinical and genetic information was obtained from clinical files. Measurement of LCHAD and LCKAT activities, lcFAO-flux studies and palmitate loading tests were performed in skin fibroblasts cultured at 37°C and 40°C. In all patients (four MTPD, one LCKATD), disease manifested during childhood (manifestation age: 2-10 years) with myopathic symptoms triggered by fever or exercise. In four patients, signs of retinopathy or neuropathy were present. Plasma long-chain acylcarnitines were normal or slightly increased. HADHB variants were identified (at age: 6-18 years) by whole exome sequencing or gene panel analyses. At 37°C, LCHAD and LCKAT activities were mildly impaired and lcFAO-fluxes were normal. Remarkably, enzyme activities and lcFAO-fluxes were markedly diminished at 40°C. Preventive (dietary) measures improved symptoms for most. In conclusion, all patients with thermo-sensitive MTP deficiency had a long diagnostic trajectory and both genetic and enzymatic testing were required for diagnosis. The frequent absence of characteristic acylcarnitine abnormalities poses a risk for a diagnostic delay. Given the positive treatment effects, upfront genetic screening may be beneficial to enhance early recognition.

Nutrition management guideline for very-long chain acyl-CoA dehydrogenase deficiency (VLCAD): An evidence- and consensus-based approach

Article

Full-text available

Oct 2020

The nutrition management guideline for very-long chain acyl-CoA dehydrogenase deficiency (VLCAD) is the fourth in a series of web-based guidelines focusing on the diet treatment for inherited metabolic disorders and follows previous publication of guidelines for maple syrup urine disease (2014), phenylketonuria (2016) and propionic acidemia (2019). The purpose of this guideline is to establish harmonization in the treatment and monitoring of individuals with VLCAD of all ages in order to improve clinical outcomes. Six research questions were identified to support guideline development on: nutrition recommendations for the healthy individual, illness management, supplementation, monitoring, physical activity and management during pregnancy. This report describes the methodology used in its development including review, critical appraisal and abstraction of peer-reviewed studies and unpublished practice literature; expert input through two Delphi surveys and a nominal group process; and external review from metabolic physicians and dietitians. It includes the summary statements of the nutrition management recommendations for each research question, followed by a standardized rating based on the strength of the evidence. Online, open access of the full published guideline allows utilization by health care providers, researchers and collaborators who advise, advocate and care for individuals with VLCAD and their families and can be accessed from the Genetic Metabolic Dietitians International (https://GMDI.org) and Southeast Regional Genetics Network (https://southeastgenetics.org/ngp) websites.

A Decade of Newborn Screening for Very-Long-Chain Acyl-CoA Dehydrogenase Deficiency (VLCADD): Benefits, Complications and the Need for Long-Term Follow-Up

Article

Full-text available

Jan 2018

Prediction of VLCAD deficiency phenotype by a metabolic fingerprint in newborn screening bloodspots

Article

Full-text available

Feb 2020
BBA-MOL BASIS DIS

Purpose: Newborns who test positive for very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) in newborn screening may have a severe phenotype with early onset of life-threatening symptoms but may also have an attenuated phenotype and never become symptomatic. The objective of this study is to investigate whether metabolomic profiles in dried bloodspots (DBS) of newborns allow early phenotypic prediction, permitting tailored treatment and follow-up. Methods: A metabolic fingerprint was generated by direct infusion high resolution mass spectrometry in DBS of VLCADD patients (n = 15) and matched controls. Multivariate analysis of the metabolomic profiles was applied to differentiate subgroups. Results: Concentration of six acylcarnitine species differed significantly between patients and controls. The concentration of C18:2- and C20:0-carnitine, 13,14-dihydroretinol and deoxycytidine monophosphate allowed separation between mild and severe patients. Two patients who could not be prognosticated on early clinical symptoms, were correctly fitted for severity in the score plot based on the untargeted metabolomics. Conclusion: Distinctive metabolomic profiles in DBS of newborns with VLCADD may allow phenotypic prognostication. The full potential of this approach as well as the underlying biochemical mechanisms need further investigation.

Prediction of disease severity in multiple acyl-CoA dehydrogenase deficiency: A retrospective and laboratory cohort study

Article

Full-text available

Jul 2019

Introduction: Multiple acyl-CoA dehydrogenase deficiency (MADD) is an ultra-rare inborn error of mitochondrial fatty acid oxidation (FAO) and amino acid metabolism. Individual phenotypes and treatment response can vary markedly. We aimed to identify markers that predict MADD phenotypes. Methods: We performed a retrospective nationwide cohort study; then developed an MADD -disease severity scoring system (MADD-DS3) based on signs and symptoms with weighed expert opinions; and finally correlated phenotypes and MADD-DS3 scores to FAO flux (oleate and myristate oxidation rates) and acylcarnitine profiles after palmitate loading in fibroblasts. Results: Eighteen patients, diagnosed between 1989 and 2014, were identified. The MADD-DS3 entails enumeration of eight domain scores, which are calculated by averaging the relevant symptom scores. Lifetime MADD-DS3 scores of patients in our cohort ranged from 0-29. FAO flux and [U-13 C]C2-, C5- and [U-13 C]C16-acylcarnitines were identified as key variables that discriminated neonatal from later onset patients (all p<0.05) and strongly correlated to MADD-DS3 scores (oleate: r=-0.86; myristate: r=-0.91; [U-13 C]C2-acylcarnitine: r=-0.96; C5-acylcarnitine: r=0.97; [U-13 C]C16-acylcarnitine: r=0.98, all p<0.01). Conclusion: Functional studies in fibroblasts were found to differentiate between neonatal and later onset MADD-patients and were correlated to MADD-DS3 scores. Our data may improve early prediction of disease severity in order to start (preventive) and follow-up treatment appropriately. This is especially relevant in view of the inclusion of MADD in population newborn screening programs. This article is protected by copyright. All rights reserved.

A Nationwide Retrospective Observational Study Of Population Newborn Screening For Medium‐Chain Acyl‐CoA Dehydrogenase (MCAD) Deficiency In The Netherlands

Article

Full-text available

Apr 2019

To evaluate the Dutch newborn screening (NBS) for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency since 2007, a nationwide retrospective, observational study was performed of clinical, laboratory and epidemiological parameters of patients with MCAD deficiency born between 2007 and 2015. Severe MCAD deficiency was defined by ACADM genotypes associated with clinical ascertainment, or variant ACADM genotypes with a residual MCAD enzyme activity <10%. Mild MCAD deficiency was defined by variant ACADM genotypes with a residual MCAD enzyme activity ≥10%. The prevalence of MCAD deficiency was 1/8300 (95% CI: 1/7300-1/9600). Sensitivity of the Dutch NBS was 99% and specificity ~100%, with a positive predictive value of 86%. Thirteen newborns with MCAD deficiency suffered from neonatal symptoms, three of them died. Of the 189 identified neonates, 24% had mild MCAD deficiency. The acylcarnitine ratio octanoylcarnitine (C8)/decanoylcarnitine (C10) was superior to C8 in discriminating between mild and severe cases and more stable in the first days of life. NBS for MCAD deficiency has a high sensitivity, specificity, and positive predictive value. In the absence of a golden standard to confirm the diagnosis, the combination of acylcarnitine (ratios), molecular and enzymatic studies allows risk stratification. To improve evaluation of NBS protocols and clinical guidelines, additional use of acylcarnitine ratios and multivariate pattern-recognition software may be reappraised in the Dutch situation. Prospective recording of NBS and follow-up data is warranted covering the entire health care chain of preventive and curative medicine.

Impact of NBS for VLCAD deficiency on genetic, enzymatic and clinical outcomes

Article

Full-text available

Feb 2019

Most infants with very‐long‐chain acyl‐CoA dehydrogenase deficiency (VLCADD) identified by newborn screening (NBS) are asymptomatic at the time of diagnosis and remain asymptomatic. If this outcome is due to prompt diagnosis and initiation of therapy, or because of identification of individuals with biochemical abnormalities who will never develop symptoms, is unclear. Therefore, a 10‐year longitudinal national cohort study of genetically confirmed VLCADD patients born before and after introduction of NBS was conducted. Main outcome measures were clinical outcome parameters, acyl‐CoA dehydrogenase very long chain gene analysis, VLCAD activity, and overall capacity of long‐chain fatty acid oxidation (LC‐FAO flux) in lymphocytes and cultured skin fibroblasts. Median VLCAD activity in lymphocytes of 54 patients, 21 diagnosed pre‐NBS and 33 by NBS was, respectively, 5.4% (95% confidence interval [CI]: 4.0‐8.3) and 12.6% (95% CI: 10.7‐17.7; P < 0.001) of the reference mean. The median LC‐FAO flux was 33.2% (95% CI: 22.8‐48.3) and 41% (95% CI: 40.8‐68; P < 0.05) of the control mean, respectively. Clinical characteristics in 23 pre‐NBS and 37 NBS patients revealed hypoglycemic events in 12 vs 2 patients, cardiomyopathy in 5 vs 4 patients and myopathy in 14 vs 3 patients. All patients with LC‐FAO flux <10% developed symptoms. Of the patients with LC‐FAO flux >10% 7 out of 12 diagnosed pre‐NBS vs none by NBS experienced hypoglycemic events. NBS has a clear beneficial effect on the prevention of hypoglycemic events in patients with some residual enzyme activity, but does not prevent hypoglycemia nor cardiac complications in patients with very low residual enzyme activity. The effect of NBS on prevalence and prevention of myopathy‐related complications remains unclear.

Proposal for an individualized dietary strategy in patients with very long-chain acyl-CoA dehydrogenase deficiency

Article

Full-text available

Feb 2019

Background Patients with very long chain acyl‐CoA dehydrogenase deficiency (VLCADD), a long chain fatty acid oxidation disorder, are traditionally treated with a long chain triglyceride (LCT) restricted and medium chain triglyceride (MCT) supplemented diet. Introduction of VLCADD in newborn screening (NBS) programs has led to the identification of asymptomatic newborns with VLCADD, who may have a more attenuated phenotype and may not need dietary adjustments. Objective To define dietary strategies for individuals with VLCADD based on the predicted phenotype. Method We evaluated long‐term dietary histories of a cohort of individuals diagnosed with VLCADD identified before the introduction of VLCADD in NBS and their beta‐oxidation (LC‐FAO) flux score (rate of oleate oxidation) in cultured skin fibroblasts in relation to the clinical outcome. Based on these results a dietary strategy is proposed. Results Sixteen individuals with VLCADD were included. One had an LC‐FAO flux score >90%, was not on a restricted diet and is asymptomatic to date. Four patients had an LC‐FAO flux score <10%, and significant VLCADD related symptoms despite the use of strict diets including LCT restriction, MCT supplementation and nocturnal gastric drip feeding. Patients with an LC‐FAO flux score between 10 and 90% (n = 11) showed a more heterogeneous phenotype. Conclusions This study shows that a strict diet cannot prevent poor clinical outcome in severely affected patients and that the LC‐FAO flux is a good predictor of clinical outcome in individuals with VLCADD identified before its introduction in NBS. Hereby, we propose an individualized dietary strategy based on the LC‐FAO flux score.

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

Article

Sep 2018

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is the most common defect of mitochondrial β-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.

Proposal for an individualized dietary strategy in patients with very long-chain acyl-CoA dehydrogenase deficiency

Article

Full-text available

Mar 2018

Background: Patients with very long chain acyl-CoA dehydrogenase deficiency (VLCADD), a long chain fatty acid oxidation disorder, are traditionally treated with a long chain triglyceride (LCT) restricted and medium chain triglyceride (MCT) supplemented diet. Introduction of VLCADD in newborn screening (NBS) programs has led to the identification of asymptomatic newborns with VLCADD, who may have a more attenuated phenotype and may not need dietary adjustments. Objective: To define dietary strategies for individuals with VLCADD based on the predicted phenotype. Method: We evaluated long-term dietary histories of a cohort of individuals diagnosed with VLCADD identified before the introduction of VLCADD in NBS and their beta-oxidation (LC-FAO) flux score (rate of oleate oxidation) in cultured skin fibroblasts in relation to the clinical outcome. Based on these results a dietary strategy is proposed. Results: Sixteen individuals with VLCADD were included. One had an LC-FAO flux score >90%, was not on a restricted diet and is asymptomatic to date. Four patients had an LC-FAO flux score <10%, and significant VLCADD related symptoms despite the use of strict diets including LCT restriction, MCT supplementation and nocturnal gastric drip feeding. Patients with an LC-FAO flux score between 10 and 90% (n = 11) showed a more heterogeneous phenotype. Conclusions: This study shows that a strict diet cannot prevent poor clinical outcome in severely affected patients and that the LC-FAO flux is a good predictor of clinical outcome in individuals with VLCADD identified before its introduction in NBS. Hereby, we propose an individualized dietary strategy based on the LC-FAO flux score.

Mitochondrial morphology, bioenergetics and proteomic responses in fatty acid oxidation disorders

Article

Full-text available

Mar 2021

Mutations in nuclear genes encoding for mitochondrial proteins very long-chain acyl-CoA dehydrogenase (VLCAD) and trifunctional protein (TFP) cause rare autosomal recessive disorders. Studies in fibroblasts derived from patients with mutations in VLCAD and TFP exhibit mitochondrial defects. To gain insights on pathological changes that account for the mitochondrial deficits we performed quantitative proteomic, biochemical, and morphometric analyses in fibroblasts derived from subjects with three different VLCAD and three different TFP mutations. Proteomic data that was corroborated by antibody-based detection, indicated reduced levels of VLCAD and TFP protein in cells with VLCAD and TFP mutations respectively, which in part accounted for the diminished fatty acid oxidation capacity. Decreased mitochondrial respiratory capacity in cells with VLCAD and TFP mutations was quantified after glucose removal and cells with TFP mutations had lower levels of glycogen. Despite these energetic deficiencies, the cells with VLCAD and TFP mutations did not exhibit changes in mitochondria morphology, distribution, fusion and fission, quantified by either confocal or transmission electron microscopy and corroborated by proteomic and antibody-based protein analysis. Fibroblasts with VLCAD and to a lesser extend cells with TFP mutations had increased levels of mitochondrial respiratory chain proteins and proteins that facilitate the assembly of respiratory complexes. With the exception of reduced levels of catalase and glutathione S-transferase theta-1 in cells with TFP mutations, the levels of 45 proteins across all major intracellular antioxidant networks were similar between cells with VLCAD and TFP mutations and non-disease controls. Collectively the data indicate that despite the metabolic deficits, cells with VLCAD and TFP mutations maintain their proteomic integrity to preserve cellular and mitochondria architecture, support energy production and protect against oxidative stress.

Clinical and biochemical outcome of patients with very long-chain acyl-CoA dehydrogenase deficiency

Article

Apr 2019

Background: Very-Long-Chain Acyl-CoA Dehydrogenase (VLCAD) deficiency is a disorder of fatty acid oxidation included in the recommended uniform newborn screening (NBS) panel in the USA. It can have variable clinical severity and there is limited information on the natural history of this condition, clinical presentation according to genotype and effectiveness of newborn screening. Methods: Retrospective data (growth parameters, morbidity, biochemical and genetic testing results) were collected from patients with VLCAD deficiency, to evaluate biochemical and clinical outcomes. Descriptive statistics was used for qualitative variables, while linear regression analysis was used to correlate continuous variables. Results: VLCAD deficiency (screened by measuring elevated levels of C14:1-carnitine in blood spots) was more frequent in Utah than the national average (1:27,617 versus 1:63,481) in the first ten years of screening. Twenty-six patients had a confirmed diagnosis of VLCAD deficiency using DNA testing or functional studies. The c.848T>C (p.V283A) variant in the ACADVL gene was the most frequent in our population. Novel variants (c.623-21A>G (IVS7-21A>G); c.1052C>T (p.T351I); c.1183-7A>G (IVS11-7A>G); c.1281G>C (p.W427C); c.1923G>C (p.L641F); c.1924G>A (p.V642M)) were identified in this study, with their pathogenicity remaining unclear in most cases. C14:1-carnitine levels decreased with age and significantly correlated with CK levels as index of muscle involvement. There were no cases of HELLP syndrome nor liver disease during pregnancies in the mothers of VLCAD patients. None of our patients developed cardiac involvement after birth and all patients had normal growth parameters while on treatment. Clinical manifestations were related to concomitant infections and altered biochemical parameters. Discussion: VLCAD deficiency can be identified by neonatal screening. Most patients compliant with therapy normalized biochemical parameters and had no major clinical manifestations. Complications were completely prevented with a relatively low number of pre-emptive ER visits or hospital admissions. It remains unclear whether neonatal screening is now identifying less severely affected patient or if complications will arise as subjects become older. Observation beyond puberty is necessary to fully understand the impact of VLCAD deficiency on morbidity in patients with VLCAD deficiency.

Mitochondrial Genetic Disorders: Cell Signaling and Pharmacological Therapies

Article

Full-text available

Mar 2019

Mitochondrial fatty acid oxidation (FAO) and respiratory chain (RC) defects form a large group of inherited monogenic disorders sharing many common clinical and pathophysiological features, including disruption of mitochondrial bioenergetics, but also, for example, oxidative stress and accumulation of noxious metabolites. Interestingly, several transcription factors or co-activators exert transcriptional control on both FAO and RC genes, and can be activated by small molecules, opening to possibly common therapeutic approaches for FAO and RC deficiencies. Here, we review recent data on the potential of various drugs or small molecules targeting pivotal metabolic regulators: peroxisome proliferator activated receptors (PPARs), sirtuin 1 (SIRT1), AMP-activated protein kinase (AMPK), and protein kinase A (PKA)) or interacting with reactive oxygen species (ROS) signaling, to alleviate or to correct inborn FAO or RC deficiencies in cellular or animal models. The possible molecular mechanisms involved, in particular the contribution of mitochondrial biogenesis, are discussed. Applications of these pharmacological approaches as a function of genotype/phenotype are also addressed, which clearly orient toward personalized therapy. Finally, we propose that beyond the identification of individual candidate drugs/molecules, future pharmacological approaches should consider their combination, which could produce additive or synergistic effects that may further enhance their therapeutic potential.

A Case Study of Very Long Fatty Acid Dehydrogenase Deficiency Presented by Irritability and Seizures

Article

Jan 2024

Magdy Khames Aly

This case study presents the clinical course and management of an 18-year-old male patient diagnosed with very long fatty acid dehydrogenase deficiency (VLCADD). The patient exhibited recurrent episodes of rhabdomyolysis accompanied by acute kidney injury (AKI) and elevated liver enzymes. Presented to us by irritability and restlessness and 3 episodes of seizures. The purpose of this case study is to highlight the challenges in diagnosing and managing this rare metabolic disorder and to emphasize the importance of early recognition and intervention to prevent further complications.

A Case Study of Very Long Fatty Acid Dehydrogenase Deficiency Presented by Irritability and Seizures

Article

Full-text available

Mar 2024

This case study presents the clinical course and management of an 18-year-old male patient diagnosedwith very long fatty acid dehydrogenase deficiency (VLCADD). The patient exhibited recurrent episodes ofrhabdomyolysis accompanied by acute kidney injury (AKI) and elevated liver enzymes. Presented to us byirritability and restlessness and 3 episodes of seizures. The purpose of this case study is to highlight the challenges in diagnosing and managing this rare metabolic disorder and to emphasize the importance of early recognition and intervention to prevent further complications

Lower Metabolic Potential and Impaired Metabolic Flexibility in Human Lymph Node Stromal Cells from Patients with Rheumatoid Arthritis

Article

Full-text available

Dec 2022

Cellular metabolism is important for determining cell function and shaping immune responses. Studies have shown a crucial role for stromal cells in steering proper immune responses in the lymph node microenvironment. These lymph node stromal cells (LNSCs) tightly regulate immune tolerance. We hypothesize that malfunctioning LNSCs create a microenvironment in which normal immune responses are not properly controlled, possibly leading to the development of autoimmune diseases such as rheumatoid arthritis (RA). Therefore, we set out to determine their metabolic profile during health and systemic autoimmunity. We included autoantibody positive individuals at risk of developing RA (RA-risk individuals), RA patients and healthy volunteers. All study subjects underwent lymph node biopsy sampling. Mitochondrial function in cultured LNSCs was assessed by quantitative PCR, flow cytometry, Seahorse and oleate oxidation assays. Overall, mitochondrial respiration was lower in RA(-risk) LNSCs compared with healthy LNSCs, while metabolic potential was only lower in RA LNSCs. To maintain basal mitochondrial respiration, all LNSCs were mostly dependent on fatty acid oxidation. However, RA(-risk) LNSCs were also dependent on glutamine oxidation. Finally, we showed that RA LNSCs have impaired metabolic flexibility. Our results show that the metabolic landscape of LNSCs is not only altered during established disease, but partly already in individuals at risk of developing RA. Future studies are needed to investigate the impact of restoring metabolic capacity in LNSC-mediated immunomodulation and disease progression.

Altered lipid metabolism in synovial fibroblasts of individuals at risk of developing rheumatoid arthritis

Article

Full-text available

Jan 2023
J AUTOIMMUN

Objective Fibroblast-like synoviocytes (FLS) can augment the inflammatory process observed in synovium of patients with rheumatoid arthritis (RA). A recent transcriptomic study in synovial biopsies revealed changes in metabolic pathways before disease onset in absence of synovial tissue inflammation. This raises the question whether alterations in cellular metabolism in tissue resident FLS underlie disease pathogenesis. Materials and methods To study this, we compared the metabolic profile of FLS isolated from synovial biopsies from individuals with arthralgia who were autoantibody positive but without any evidence of arthritis (RA-risk individuals, n = 6) with FLS from patients with RA (n = 6), osteoarthritis (OA, n = 6) and seronegative controls (n = 6). After synovial digestion, FLS were cultured in vitro and cellular metabolism was assessed using quantitative PCR, flow cytometry, XFe96 Seahorse Analyzer and tritium-labelled oleate oxidation assays. Results Real-time metabolic profiling revealed that basal (p < 0.0001) and maximum mitochondrial respiration (p = 0.0024) were significantly lower in RA FLS compared with control FLS. In all donors, basal respiration was largely dependent on fatty acid oxidation while glucose was only highly used by FLS from RA patients. Moreover, we showed that RA-risk and RA FLS are less metabolically flexible. Strikingly, mitochondrial fatty acid β-oxidation was significantly impaired in RA-risk (p = 0.001) and RA FLS (p < 0.0001) compared with control FLS. Conclusion Overall, this study showed several metabolic alterations in FLS even in absence of synovial inflammation, suggesting that these alterations already start before clinical manifestation of disease and may drive disease pathogenesis.

Treatment of VLCAD-Deficient Patient Fibroblasts with Peroxisome Proliferator-Activated Receptor δ Agonist Improves Cellular Bioenergetics

Article

Full-text available

Aug 2022

Background: Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an autosomal recessive disease that prevents the body from utilizing long-chain fatty acids for energy, most needed during stress and fasting. Symptoms can appear from infancy through childhood and adolescence or early adulthood, and include hypoglycemia, recurrent rhabdomyolysis, myopathy, hepatopathy, and cardiomyopathy. REN001 is a peroxisome-proliferator-activated receptor delta (PPARδ) agonist that modulates the expression of the genes coding for fatty acid β-oxidation enzymes and proteins involved in oxidative phosphorylation. Here, we assessed the effect of REN001 on VLCAD-deficient patient fibroblasts. Methods: VLCAD-deficient patient and control fibroblasts were treated with REN001. Cells were harvested for gene expression analysis, protein content, VLCAD enzyme activity, cellular bioenergetics, and ATP production. Results: VLCAD-deficient cell lines responded differently to REN001 based on genotype. All cells had statistically significant increases in ACADVL gene expression. Small increases in VLCAD protein and enzyme activity were observed and were cell-line- and dose-dependent. Even with these small increases, cellular bioenergetics improved in all cell lines in the presence of REN001, as demonstrated by the oxygen consumption rate and ATP production. VLCAD-deficient cell lines containing missense mutations responded better to REN001 treatment than one containing a duplication mutation in ACADVL. Discussion: Treating VLCAD-deficient fibroblasts with the REN001 PPARδ agonist results in an increase in VLCAD protein and enzyme activity, and a decrease in cellular stress. These results establish REN001 as a potential therapy for VLCADD as enhanced expression may provide a therapeutic increase in total VLCAD activity, but suggest the need for mutation-specific treatment augmented by other treatment measures.

Skin fibroblast metabolomic profiling reveals that lipid dysfunction predicts the severity of Friedreich’s ataxia

Article

Full-text available

Jul 2022
J LIPID RES

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a triplet guanine-adenine-adenine (GAA) repeat expansion in intron 1 of the FXN gene, which leads to decreased levels of the frataxin protein. Frataxin is involved in the formation of iron-sulfur (Fe-S) cluster prosthetic groups for various metabolic enzymes. To provide a better understanding of the metabolic status of FRDA patients, here we used patient-derived fibroblast cells as a surrogate tissue for metabolic and lipidomic profiling by liquid-chromatography high resolution-mass spectrometry (LC-HRMS). We found elevated HMG-CoA and β-hydroxybutyrate (BHB)-CoA levels, implying dysregulated fatty acid oxidation, which was further demonstrated by elevated acyl-carnitine levels. Lipidomic profiling identified dysregulated levels of several lipid classes in FRDA fibroblast cells when compared with non-FRDA fibroblast cells. For example, levels of several ceramides were significantly increased in FRDA fibroblast cells; these results positively correlated with the GAA repeat length and negatively correlated with the frataxin protein levels. Furthermore, stable isotope tracing experiments indicated increased ceramide synthesis, especially for long chain fatty acid-ceramides, in FRDA fibroblast cells compared to ceramide synthesis in healthy control fibroblast cells. In addition, PUFA containing triglycerides and phosphatidylglycerols were enriched in FRDA fibroblast cells and negatively correlated with frataxin levels, suggesting lipid remodeling as a result of FXN deficiency. Altogether, we demonstrate patient-derived fibroblast cells exhibited dysregulated metabolic capabilities, and their lipid dysfunction predicted the severity of FRDA, making them a useful surrogate to study the metabolic status in FRDA.

Treatment of VLCAD deficient patient fibroblasts with peroxisome-proliferator activated receptor δ agonist improves cellular bioenergetics

Preprint

Full-text available

Apr 2022

Very long chain acyl-CoA dehydrogenase deficiency (VLCADD) is an autosomal recessive disease that prevents the body from utilizing long chain fatty acids for energy, most needed during stress and fasting. Symptoms can appear from infancy through childhood and adolescence or early adulthood, and include hypoglycemia, recurrent rhabdomyolysis, myopathy, hepatopathy, and cardiomyopathy. REN001 is a peroxisome proliferator activated receptor delta (PPARδ) agonist that modulates gene expression of fatty acid β-oxidation enzymes and oxidative phosphorylation proteins. VLCADD fibroblasts responded differently to REN001 based on genotype. All cells had statistically significant increases in ACADVL gene expression. Small increases in VLCAD protein and enzyme activity were observed and were cell line and dose dependent. Cellular bioenergetics improved in all REN001 treated fibroblasts as demonstrated by oxygen consumption rate and ATP production. VLCADD fibroblasts containing missense mutations responded better to REN001 treatment than one containing a duplication mutation in ACADVL . REN001 treated VLCADD fibroblasts results in an increase in VLCAD protein, enzyme activity, and a decrease in cellular stress. These results establish REN001 as a potential therapy for VLCADD as enhanced expression may provide therapeutic increase in total VLCAD activity but suggests the need to mutation specific treatment augmented by other treatment measures.

Genetic, biochemical and clinical spectrum of patients with mitochondrial trifunctional protein deficiency identified after introduction of newborn screening in the Netherlands

Article

Full-text available

Apr 2022

Introduction: Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is included in many newborn screening (NBS) programs. Acylcarnitine-based NBS for LCHADD not only identifies LCHADD, but all different deficiencies of the mitochondrial trifunctional protein (MTP), a multi-enzyme complex involved in long-chain fatty acid β-oxidation. Besides LCHAD, MTP harbors two additional enzyme activities: long-chain enoyl-CoA hydratase (LCEH), and long-chain ketoacyl-CoA thiolase (LCKAT). Deficiency of one or more MTP activities causes generalized MTP deficiency (MTPD), LCHADD, LCEH deficiency (not yet reported), or LCKAT deficiency (LCKATD). Aim: To gain insight in the outcomes of MTP-deficient patients diagnosed after introduction of NBS for LCHADD in the Netherlands. Methods: Retrospective evaluation of genetic, biochemical and clinical characteristics of MTP-deficient patients, identified since 2007. Results: Thirteen patients were identified: seven with LCHADD, five with MTPD and one with LCKATD. All LCHADD patients (one missed by NBS, clinical diagnosis) and one MTPD patient (clinical diagnosis) were alive. Four MTPD patients and one LCKATD patient developed cardiomyopathy and died within one month and 13 months of life, respectively. Surviving patients did not develop symptomatic hypoglycemia, but experienced reversible cardiomyopathy and rhabdomyolysis. Five LCHADD patients developed subclinical neuropathy and/or retinopathy. Conclusion: Patient outcomes were highly variable, stressing the need for accurate classification of and discrimination between the MTP deficiencies to improve insight in the yield of NBS for LCHADD. NBS allowed prevention of symptomatic hypoglycemia, but current treatment options failed to treat cardiomyopathy and prevent long-term complications. Moreover, milder patients, who might benefit from NBS, were missed due to normal acylcarnitine profiles.

Severity estimation of very-long-chain acyl-CoA dehydrogenase deficiency via 13C-fatty acid loading test

Article

Feb 2022

Background: The clinical severity of very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is difficult to predict using conventional diagnostic methods. Methods: Peripheral blood mononuclear cells obtained from 14 VLCAD deficiency patients and 23 healthy adults were loaded with carbon-13-universally labeled (U-13C-) fatty acids. Differences in acylcarnitine ratios between the patients and healthy groups and correlations between acylcarnitine ratios and a newly established clinical severity score (CSS) in the patient group were statistically examined. Results: There was a significant decrease in the 13C-C2/13C-C18 and 13C-C12/13C-C14 ratios in the U-13C-stearic acid loading test and in the 13C-C2/13C-C18:1 and 13C-C12:1/13C-C14:1 ratios in the U-13C-oleic acid loading test in the patient group. The values of each ratio were significantly correlated with the CSS, suggesting that they could predict disease severity. Additionally, patients with a higher 13C-C16/13C-C18 ratio than the 13C-C14/13C-C18 ratio in the U-13C-stearic acid loading test had a significantly higher CSS and were presumed to have more severe disease. Conclusions: Our data indicated that this method could be used to predict the clinical severity of VLCAD deficiency, and identify patients at a risk of severe disease. Impact: We established a novel method to predict the severity of VLCAD deficiency by performing a loading test with carbon-13-labeled fatty acids on peripheral blood mononuclear cells. The U-13C-oleic acid loading test was useful for comparing the patient group with the control group in terms of disease severity. The U-13C-stearic acid loading test was useful for identifying the more severely affected patients. These methods are relatively less invasive and enable rapid evaluation of the clinical severity.

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

Article

Full-text available

Jan 2022

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.

Biochemical Markers for the Diagnosis of Mitochondrial Fatty Acid Oxidation Diseases

Article

Full-text available

Oct 2021

Mitochondrial fatty acid β-oxidation (FAO) contributes a large proportion to the body’s energy needs in fasting and in situations of metabolic stress. Most tissues use energy from fatty acids, particularly the heart, skeletal muscle and the liver. In the brain, ketone bodies formed from FAO in the liver are used as the main source of energy. The mitochondrial fatty acid oxidation disorders (FAODs), which include the carnitine system defects, constitute a group of diseases with several types and subtypes and with variable clinical spectrum and prognosis, from paucisymptomatic cases to more severe affectations, with a 5% rate of sudden death in childhood, and with fasting hypoketotic hypoglycemia frequently occurring. The implementation of newborn screening programs has resulted in new challenges in diagnosis, with the detection of new phenotypes as well as carriers and false positive cases. In this article, a review of the biochemical markers used for the diagnosis of FAODs is presented. The analysis of acylcarnitines by MS/MS contributes to improving the biochemical diagnosis, both in affected patients and in newborn screening, but acylglycines, organic acids, and other metabolites are also reported. Moreover, this review recommends caution, and outlines the differences in the interpretation of the biomarkers depending on age, clinical situation and types of samples or techniques.

The potential and limitations of intrahepatic cholangiocyte organoids to study inborn errors of metabolism

Article

Full-text available

Oct 2021

Inborn errors of metabolism (IEMs) comprise a diverse group of individually rare monogenic disorders that affect metabolic pathways. Mutations lead to enzymatic deficiency or dysfunction, which results in intermediate metabolite accumulation or deficit leading to disease phenotypes. Currently, treatment options for many IEMs are insufficient. Rarity of individual IEMs hampers therapy development and phenotypic and genetic heterogeneity suggest beneficial effects of personalized approaches. Recently, cultures of patient-own liver-derived intrahepatic cholangiocyte organoids (ICOs) have been established. Since most metabolic genes are expressed in the liver, patient-derived ICOs represent exciting possibilities for in vitro modelling and personalized drug testing for IEMs. However, the exact application range of ICOs remains unclear. To address this, we examined which metabolic pathways can be studied with ICOs and what the potential and limitations of patient-derived ICOs are to model metabolic functions. We present functional assays in patient ICOs with defects in branched-chain amino acid metabolism (methylmalonic acidemia), copper metabolism (Wilson disease) and transporter defects (cystic fibrosis). We discuss the broad range of functional assays that can be applied to ICOs, but also address the limitations of these patient-specific cell models. In doing so, we aim to guide the selection of the appropriate cell model for studies of a specific disease or metabolic process. This article is protected by copyright. All rights reserved.

Very Long-Chain Acyl-CoA Dehydrogenase Deficiency: High Incidence of Detected Patients With Expanded Newborn Screening Program

Article

Full-text available

Apr 2021

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is a rare autosomal recessive disorder of fatty acid metabolism with a variable presentation. The aim of this study was to describe five patients with VLCADD diagnosed through the pilot study and expanded newborn screening (NBS) program that started in 2018 in Slovenia. Four patients were diagnosed through the expanded NBS program with tandem mass spectrometry; one patient was previously diagnosed in a pilot study preceding the NBS implementation. Confirmatory testing consisted of acylcarnitines analysis in dried blood spots, organic acids profiling in urine, genetic analysis of ACADVL gene, and enzyme activity determination in lymphocytes or fibroblasts. Four newborns with specific elevation of acylcarnitines diagnostic for VLCADD and disease-specific acylcarnitines ratios (C14:1, C14, C14:2, C14:1/C2, C14:1/C16) were confirmed with genetic testing: all were compound heterozygotes, two of them had one previously unreported ACDVL gene variant each (NM_000018.3) c.1538C > G; (NP_000009) p.(Ala513Gly) and c.661A > G; p.(Ser221Gly), respectively. In addition, one patient diagnosed in the pilot study also had a specific elevation of acylcarnitines. Subsequent ACDVL genetic analysis confirmed compound heterozygosity. In agreement with the diagnosis, enzyme activity was reduced in five patients tested. In seven other newborns with positive screening results, only single allele variants were found in the ACDVL gene, so the diagnosis was not confirmed. Among these, two variants were novel, c.416T > C and c.1046C > A, respectively (p.Leu139Pro and p.Ala349Glu). In the first 2 years of the expanded NBS program in Slovenia altogether 30,000 newborns were screened. We diagnosed four cases of VLCADD. The estimated VLCADD incidence was 1:7,500 which was much higher than that of the medium-chain acyl-CoA dehydrogenase deficiency (MCADD) cases in the same period. Our study also provided one of the first descriptions of ACADVL variants in Central-Southeastern Europe and reported on 4 novel variants.

Mitochondrial Fatty Acid Oxidation Disorders: Laboratory Diagnosis, Pathogenesis, and the Complicated Route to Treatment

Article

Full-text available

Sep 2020

Mitochondrial fatty acid (FA) oxidation deficiencies represent a genetically heterogeneous group of diseases in humans caused by defects in mitochondrial FA beta-oxidation (mFAO). A general characteristic of all mFAO disorders is hypoketotic hypoglycemia resulting from the enhanced reliance on glucose oxidation and the inability to synthesize ketone bodies from FAs. Patients with a defect in the oxidation of long-chain FAs are at risk to develop cardiac and skeletal muscle abnormalities including cardiomyopathy and arrhythmias, which may progress into early death, as well as rhabdomyolysis and exercise intolerance. The diagnosis of mFAO-deficient patients has greatly been helped by revolutionary developments in the field of tandem mass spectrometry (MS) for the analysis of acylcarnitines in blood and/or urine of candidate patients. Indeed, acylcarnitines have turned out to be excellent biomarkers; not only do they provide information whether a certain patient is affected by a mFAO deficiency, but the acylcarnitine profile itself usually immediately points to which enzyme is likely deficient. Another important aspect of acylcarnitine analysis by tandem MS is that this technique allows high-throughput analysis, which explains why screening for mFAO deficiencies has now been introduced in many newborn screening programs worldwide. In this review, we will describe the current state of knowledge about mFAO deficiencies, with particular emphasis on recent developments in the area of pathophysiology and treatment.

β-Hydroxybutyrate Increases Exercise Capacity Associated with Changes in Mitochondrial Function in Skeletal Muscle

Article

Full-text available

Jun 2020

β-hydroxybutyrate is the main ketone body generated by the liver under starvation. Under these conditions, it can sustain ATP levels by its oxidation in mitochondria. As mitochondria can modify its shape and function under different nutritional challenges, we study the chronic effects of β-hydroxybutyrate supplementation on mitochondrial morphology and function, and its relation to exercise capacity. Male C57BL/6 mice were supplemented with β-hydroxybutyrate mineral salt (3.2%) or control (CT, NaCl/KCl) for six weeks and submitted to a weekly exercise performance test. We found an increase in distance, maximal speed, and time to exhaustion at two weeks of supplementation. Fatty acid metabolism and OXPHOS subunit proteins declined at two weeks in soleus but not in tibialis anterior muscles. Oxygen consumption rate on permeabilized fibers indicated a decrease in the presence of pyruvate in the short-term treatment. Both the tibialis anterior and soleus showed decreased levels of Mitofusin 2, while electron microscopy assessment revealed a significant reduction in mitochondrial cristae shape in the tibialis anterior, while a reduction in the mitochondrial number was observed only in soleus. These results suggest that short, but not long-term, β‑hydroxybutyrate supplementation increases exercise capacity, associated with modifications in mitochondrial morphology and function in mouse skeletal muscle.

Electrophysiological Abnormalities in VLCAD Deficient hiPSC-Cardiomyocytes Can Be Improved by Lowering Accumulation of Fatty Acid Oxidation Intermediates

Article

Full-text available

Apr 2020
INT J MOL SCI

Patients with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) can present with life-threatening cardiac arrhythmias. The pathophysiological mechanism is unknown. We reprogrammed fibroblasts from one mildly and one severely affected VLCADD patient, into human induced pluripotent stem cells (hiPSCs) and differentiated these into cardiomyocytes (VLCADD-CMs). VLCADD-CMs displayed shorter action potentials (APs), more delayed afterdepolarizations (DADs) and higher systolic and diastolic intracellular Ca2+ concentration ([Ca2+]i) than control CMs. The mitochondrial booster resveratrol mitigated the biochemical, electrophysiological and [Ca2+]i changes in the mild but not in the severe VLCADD-CMs. Accumulation of potentially toxic intermediates of fatty acid oxidation was blocked by substrate reduction with etomoxir. Incubation with etomoxir led to marked prolongation of AP duration and reduced DADs and [Ca2+]i in both VLCADD-CMs. These results provide compelling evidence that reduced accumulation of fatty acid oxidation intermediates, either by enhanced fatty acid oxidation flux through increased mitochondria biogenesis (resveratrol) or by inhibition of fatty acid transport into the mitochondria (etomoxir), rescues pro-arrhythmia defects in VLCADD-CMs and open doors for new treatments.

The Galactose Index measured in fibroblasts of GALT deficient patients distinguishes variant patients detected by newborn screening from patients with classical phenotypes

Article

Full-text available

Jan 2020

Background: The high variability in clinical outcome of patients with Classical Galactosemia (CG) is poorly understood and underlines the importance of prognostic biomarkers, which are currently lacking. The aim of this study was to investigate if residual galactose metabolism capacity is associated with clinical and biochemical outcomes in CG patients with varying geno- and phenotypes. Methods: Galactose Metabolite Profiling (GMP) was used to determine residual galactose metabolism in fibroblasts of CG patients. The association between the galactose index (GI) defined as the ratio of the measured metabolites [U13C]Gal-1-P/ [13C6]UDP-galactose, and both intellectual and neurological outcome and galactose-1-phosphate (Gal-1-P) levels was investigated. Results: GMP was performed in fibroblasts of 28 patients and 3 control subjects. The GI of the classical phenotype patients (n = 22) was significantly higher than the GI of four variant patients detected by newborn screening (NBS) (p = .002), two homozygous p.Ser135Leu patients (p = .022) and three controls (p = .006). In the classical phenotype patients, 13/18 (72%) had a poor intellectual outcome (IQ < 85) and 6/12 (50%) had a movement disorder. All the NBS detected variant patients (n = 4) had a normal intellectual outcome (IQ ≥ 85) and none of them has a movement disorder. In the classical phenotype patients, there was no significant difference in GI between patients with a poor and normal clinical outcome. The NBS detected variant patients had significantly lower GI levels and thus higher residual galactose metabolism than patients with classical phenotypes. There was a clear correlation between Gal-1-P levels in erythrocytes and the GI (p = .001). Conclusions: The GI was able to distinguish CG patients with varying geno- and phenotypes and correlated with Gal-1-P. The data of the NBS detected variant patients demonstrated that a higher residual galactose metabolism may result in a more favourable clinical outcome. Further research is needed to enable individual prognostication and treatment in all CG patients.

Complex patterns of inheritance, including synergistic heterozygosity, in inborn errors of metabolism: Implications for precision medicine driven diagnosis and treatment

Article

Jul 2019

Inborn errors of metabolism have traditionally been viewed as the quintessential single gene disorders; defects in one gene leads to loss of activity of one enzyme causing a metabolic imbalance and clinical disease. However, reality has never been quite that simple, and the classic "one gene-one enzyme" paradigm has been upended in many ways. Multiple gene defects can lead to the same biochemical phenotype, often with different clinical symptoms. Additionally, different mutations in the same gene can cause variable phenotypes, often most dramatic when a disease can be identified by pre-symptomatic screening. Moreover, response to therapy is not homogeneous across diseases and specific mutations. Perhaps the biggest deviation from traditional monogenic inheritance is in the setting of synergistic heterozygosity, a multigenic inheritance pattern in which mutations in multiple genes in a metabolic pathway lead to sufficient disruption of flux through the pathway, mimicking a monogenic disorder caused by homozygous defects in one gene in that pathway. In addition, widespread adoption of whole exome and whole genome sequencing in medical genetics has led to the realization that individual patients with apparently hybrid phenotypes can have mutations in more than one gene, leading to a mixed genetic disorder. Each of these situations point to a need for as much precision as possible in diagnosing metabolic disease, and it is likely to become increasingly critical to drive therapy. This article examines examples in traditional monogenic disorders that illustrates these points and define inborn errors of metabolism as complex genetic traits on the leading edge of precision medicine.

Translational Metabolism: A multidisciplinary approach towards precision diagnosis of inborn errors of metabolism in the omics era

Article

Feb 2019

The laboratory diagnosis of inborn errors of metabolism has been revolutionized in recent years, thanks to the amazing developments in the field of DNA sequencing including whole exome and whole genome sequencing (WES and WGS). Interpretation of the results coming from WES and/or WGS analysis is definitely not trivial especially since the biological relevance of many of the variants identified by WES and/or WGS, have not been tested experimentally and prediction programs like POLYPHEN‐2 and SIFT are far from perfect. Correct interpretation of WES and/or WGS results can only be achieved by performing functional studies at multiple levels (different metabolomics platforms, enzymology, in vitro and in vivo flux analysis), often requires studies in model organisms like zebra fish, Caenorhabditis elegans, Saccharomyces cerevisiae, mutant mice and others, and also requires the input of many different disciplines to make this Translational Metabolism approach effective.

Profiling of intracellular metabolites produced from galactose and its potential for galactosemia research

Article

Full-text available

Dec 2018
ORPHANET J RARE DIS

Background: Clinical outcome of patients with a classical presentation of galactosemia (classical patients) varies substantially, even between patients with the same genotype. With current biomarkers, it is not possible to predict clinical outcome early in life. The aim of this study was to develop a method to provide more insight into galactose metabolism, which allows quantitative assessment of residual galactose metabolism in galactosemia patients. We therefore developed a method for galactose metabolite profiling (GMP) in fibroblasts using [U-13C]-labeled galactose. Methods: GMP analysis was performed in fibroblasts of three classical patients, three variant patients and three healthy controls. The following metabolites were analyzed: [U13C]-galactose, [U13C]-galactose-1-phosphate (Gal-1-P) and [13C6]- uridine diphosphate(UDP)-galactose. The ratio of [U13C]-Gal-1-P/ [13C6]-UDP-galactose was defined as the galactose index (GI). Results: All patient cell lines could be distinguished from the control cell lines and there was a clear difference between variant and classical patients. Variant patients had lower levels of [U13C]-galactose and [U13C]-Gal-1-P than classical patients (though substantially higher than healthy controls) and higher levels of [13C6]-UDP-galactose than classical patients (though substantially lower than healthy controls) resulting in a different GI in all groups. Conclusions: GMP in fibroblasts is a sensitive method to determine residual galactose metabolism capacity, which can discriminate between patients with a classical presentation of galactosemia, patients with a variant presentation and healthy controls. GMP may be a useful method for early prognostication after further validation in a larger cohort of patients representing the full phenotypic spectrum of galactosemia.

Target Identification of a Small Molecule Rescuing Monastrol-Induced Spindle Defects : Can Lipid Metabolism Govern Cytoskeletal Architecture?

Thesis

Jan 2016

Naowras Al-Obaidi

Disorders of mitochondrial long-chain fatty acid oxidation and the carnitine shuttle

Article

Full-text available

Mar 2018
REV ENDOCR METAB DIS

Mitochondrial fatty acid oxidation is an essential pathway for energy production, especially during prolonged fasting and sub-maximal exercise. Long-chain fatty acids are the most abundant fatty acids in the human diet and in body stores, and more than 15 enzymes are involved in long-chain fatty acid oxidation. Pathogenic mutations in genes encoding these enzymes result in a long-chain fatty acid oxidation disorder in which the energy homeostasis is compromised and long-chain acylcarnitines accumulate. Symptoms arise or exacerbate during catabolic situations, such as fasting, illness and (endurance) exercise. The clinical spectrum is very heterogeneous, ranging from hypoketotic hypoglycemia, liver dysfunction, rhabdomyolysis, cardiomyopathy and early demise. With the introduction of several of the long-chain fatty acid oxidation disorders (lcFAOD) in newborn screening panels, also asymptomatic individuals with a lcFAOD are identified. However, despite early diagnosis and dietary therapy, a significant number of patients still develop symptoms emphasizing the need for individualized treatment strategies. This review aims to function as a comprehensive reference for clinical and laboratory findings for clinicians who are confronted with pediatric and adult patients with a possible diagnosis of a lcFAOD.

Metabolite accumulation in VLCAD deficiency markedly disrupts mitochondrial bioenergetics and Ca 2+ homeostasis in the heart

Article

Feb 2018

Prediction of phenotypic severity in mucopolysaccharidosis type IIIA: Phenotypic severity prediction in MPS IIIA (42 characters)

Article

Full-text available

Oct 2017

Objective: Mucopolysaccharidosis IIIA or Sanfilippo disease type A is a progressive neurodegenerative disorder presenting in early childhood, caused by an inherited deficiency of the lysosomal hydrolase sulfamidase. New missense mutations, for which genotype-phenotype correlations are currently unknown, are frequently reported, hampering early prediction of phenotypic severity and efficacy assessment of new disease-modifying treatments. We aimed to design a method to determine phenotypic severity early in the disease course. Methods: Fifty-three patients were included of whom skin fibroblasts and data on disease course and mutation analysis were available. Patients were phenotypically characterized on clinical data as 'rapidly progressing' or 'slowly progressing'. Sulfamidase activity was measured in fibroblasts cultured at 37°C and at 30°C. Results: Sulfamidase activity in fibroblasts from patients homozygous or compound heterozygous for a combination of known severe mutations remained below the limit of quantification under both culture conditions. In contrast, sulfamidase activity in fibroblasts from patients homozygous or compound heterozygous for a known mild mutation increased above the limit of quantification when cultured at 30°C. With division on the basis of the patients phenotype, fibroblasts from 'slowly progressing' patients could be separated from 'rapid progressing' patients by increase in sulfamidase activity when cultured at 30°C (p<0.001, sensitivity 96%, specificity 93%). Interpretation: Phenotypic severity strongly correlates with the potential to increase sulfamidase activity in fibroblasts cultured at 30°C, allowing reliable distinction between patients with 'rapidly progressing' or 'slowly progressing' phenotypes. This method may provide an essential tool for assessment of treatment effects and for healthcare and life planning decisions. This article is protected by copyright. All rights reserved.

Clinical, Biochemical, and Molecular Features in 37 Saudi Patients with Very Long Chain Acyl CoA Dehydrogenase Deficiency

Chapter

Full-text available

Oct 2017

Background: Very long chain acyl CoA dehydrogenase (VLCAD) deficiency (OMIM#201475) is an autosomal recessive disorder of fatty acid beta oxidation caused by defect in the ACADVL. The aim of this study was to analyze the clinical, biochemical, and molecular features of VLCAD deficiency in Saudi Arabia, including the treatment and outcome. Methods: We carried out a retrospective chart review analysis of 37 VLCAD deficiency patients from two tertiary centers in Saudi Arabia, over a 14-year period (2002-2016). Twenty-three patients were managed at King Abdul-Aziz Medical City and fourteen patients at King Fahad Medical City. Results: Severe early onset VLCAD deficiency is the most frequent phenotype in our patients, caused by four different mutations in ACADVL; 31 patients (83.7%) had a homozygous nonsense mutation in exon 2 of ACADVL c.65C>A;p. Ser22X. Twenty-three patients died before the age of 2 years, despite early detection by newborn screening and implementation of treatment, including supplementation with medium chain triglycerides. Conclusion: This study reports the clinical, biochemical, molecular findings, treatment, and outcome of patients with VLCAD deficiency over the last 14 years. We identified the most common variant and one new variant in ACADVL. Despite early diagnosis and treatment, the outcome of VLCAD deficiency in this Saudi Arabian population remains poor. Preventive measures, such as prenatal diagnosis, could be implemented.

Fibroblast Fatty-Acid Oxidation Flux Assays Stratify Risk in Newborns with Presumptive-Positive Results on Screening for Very-Long Chain Acyl-CoA Dehydrogenase Deficiency

Article

Full-text available

Feb 2017

Very-long chain acyl-CoA dehydrogenase deficiency (VLCADD) is a clinically heterogeneous disorder with three major phenotypes: severe neonatal/infantile, milder childhood and late onset myopathic. VLCADD is genetically heterogeneous with numerous pathogenic mutations and variants of uncertain significance. VLCADD is included in many newborn screening programs but these suffer from high false positive rates, primarily due to positive screens in heterozygotes. Separating these and newborns with two low-risk “mild” variants from clinically at risk patients can be problematic, as clinical and biochemical markers are often unreliable, particularly in stable neonates. We have measured fibroblast fatty acid oxidation flux using [9,10-H ³]myristic acid and [9,10-H ³]oleic acid from 69 clinically presenting VLCADD patients including myopathic and infantile phenotypes and 13 positive newborn screened patients. We also measured fibroblast VLCADD enzyme activity by UV-HPLC detection of product in a sub-set of patients and compared these results to oleate FAO-flux. Fibroblast enzyme assay by UV-HPLC detection failed to clearly discriminate between some clinically presenting VLCADD patient cell lines and cell lines from some simple heterozygotes. FAO-flux clearly discriminated between clinically presenting VLCADD patients and the false positive screened patients. FAO-flux at 37 °C provides information as to the likely clinical phenotype but FAO-flux at 41 °C is the best discriminator for identifying clinically at risk patients. View Full-Text

Very Long-Chain Acyl-Coenzyme A Dehydrogenase Deficiency and Perioperative Management in Adult Patients

Chapter

Full-text available

Aug 2016

Surgery and anesthesia pose a threat to patients with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), because prolonged fasting, stress, and pain are known risk factors for the induction of metabolic derangement. The optimal perioperative management in these patients is unknown and the use of volatile agents and agents dissolved in fatty acids has been related to postoperative metabolic complications. However, the occurrence of metabolic derangement is multifactorial and depends, amongst others, on the severity of the mutation and residual enzyme activity. Current guidelines suggest avoiding both volatile anesthetics as well as propofol, which seriously limits the options for providing safe anesthesia. Therefore, we reviewed the available literature on the perioperative management of patients with VLCADD. We concluded that the use of some medications, such as volatile anesthetics, in patients with VLCADD might be wrongfully avoided and could in fact prevent metabolic derangement by the adequate suppression of pain and stress during surgery. We will illustrate this with a case report of an adult VLCADD patient undergoing minor surgery. Besides the use of remifentanil, anesthesia was uneventfully maintained with the use of sevoflurane, a volatile agent, and continuous glucose infusion. The patient was monitored with a continuous glucose meter and creatinine kinase measurements.

Tracer‐based lipidomics enables the discovery of disease‐specific candidate biomarkers in mitochondrial β‐oxidation disorders

Article

Feb 2024

Carnitine derivatives of disease‐specific acyl‐CoAs are the diagnostic hallmark for long‐chain fatty acid β‐oxidation disorders (lcFAOD), including carnitine shuttle deficiencies, very‐long‐chain acyl‐CoA dehydrogenase deficiency (VLCADD), long‐chain 3‐hydroxyacyl‐CoA dehydrogenase deficiency (LCHADD) and mitochondrial trifunctional protein deficiency (MPTD). The exact consequence of accumulating lcFAO‐intermediates and their influence on cellular lipid homeostasis is, however, still unknown. To investigate the fate and cellular effects of the accumulating lcFAO‐intermediates and to explore the presence of disease‐specific markers, we used tracer‐based lipidomics with deuterium‐labeled oleic acid (D9‐C18:1) in lcFAOD patient‐derived fibroblasts. In line with previous studies, we observed a trend towards neutral lipid accumulation in lcFAOD. In addition, we detected a direct connection between the chain length and patterns of (un)saturation of accumulating acylcarnitines and the various enzyme deficiencies. Our results also identified two disease‐specific candidate biomarkers. Lysophosphatidylcholine(14:1) (LPC(14:1)) was specifically increased in severe VLCADD compared to mild VLCADD and control samples. This was confirmed in plasma samples showing an inverse correlation with enzyme activity, which was better than the classic diagnostic marker C14:1‐carnitine. The second candidate biomarker was an unknown lipid class, which we identified as S‐(3‐hydroxyacyl)cysteamines. We hypothesized that these were degradation products of the CoA moiety of accumulating 3‐hydroxyacyl‐CoAs. S‐(3‐hydroxyacyl)cysteamines were significantly increased in LCHADD compared to controls and other lcFAOD, including MTPD. Our findings suggest extensive alternative lipid metabolism in lcFAOD and confirm that lcFAOD accumulate neutral lipid species. In addition, we present two disease‐specific candidate biomarkers for VLCADD and LCHADD, that may have significant relevance for disease diagnosis, prognosis, and monitoring.

Tracer-based lipidomics identifies novel disease-specific biomarkers in mitochondrial β-oxidation disorders

Preprint

Aug 2023

Carnitine derivatives of disease-specific acyl-CoAs are the diagnostic hallmark for long-chain fatty acid oxidation disorders (lcFAOD), including carnitine shuttle deficiencies, very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD), long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and mitochondrial trifunctional protein deficiency (MPTD). The exact consequence of accumulating lcFAO-intermediates and possible influence on cellular lipid homeostasis are, however, still unknown. To investigate the fate and cellular effects of the accumulating lcFAO-intermediates and to explore new disease markers, we used tracer-based lipidomics with deuterium-labeled oleic acid (D9-C18:1) in lcFAOD patient-derived fibroblasts. In line with previous studies, we observed a trend towards neutral lipid accumulation in lcFAOD. In addition, we detected a direct connection between the chain length and patterns of (un)saturation of accumulating acylcarnitines and the various enzyme deficiencies. Our results also identified two new candidate disease markers. Lysophosphatidylcholine(14:1) (LPC(14:1)) was specifically increased in severe VLCADD compared to mild VLCADD and control samples. This was confirmed in plasma samples showing an inverse correlation with enzyme activity, which was better than the classic diagnostic marker C14:1-carnitine. The second biomarker is an unknown lipid class, which we identified as S-(3-hydroxyacyl)cysteamines. These are hypothesized to be degradation products of the CoA moiety of accumulating 3-hydroxyacyl-CoAs. S-(3-hydroxyacyl)cysteamines were significantly increased in LCHADD compared to controls and other lcFAOD, including MTPD. Our findings suggest extensive alternative lipid metabolism in lcFAOD and confirm that lcFAOD accumulate neutral lipid species. In addition, we present two new disease markers for VLCADD and LCHADD, that may have significant relevance for disease diagnosis, prognosis, and monitoring.

Integrative multi-omic analysis of radiation-induced skin injury reveals the alteration of fatty acid metabolism in early response of ionizing radiation

Article

Jan 2023
J DERMATOL SCI

Background: Radiation-induced skin injury is a serious concern during radiotherapy and accidental exposure to radiation. Objective: This study aims to investigate the molecular events in early response to ionizing radiation of skin tissues and underlying mechanism. Methods: Mice and rats were irradiated with an electron beam. Skin tissues were used for liquid chromatography-mass spectrometry (LC-MS)-based metabolomics, mRNA-Seq and single-cell RNA sequencing (scRNA-Seq). Human keratinocytes (HaCaT) and skin fibroblasts (WS1) were used for functional studies. Results: The integrated analysis of metabolomics and transcriptomics showed that 6 key fatty acid-associated metabolites, 9 key fatty acid-associated genes and multiple fatty acid-associated pathways were most obviously enriched and increased in the irradiated skins. Among them, acyl-CoA dehydrogenase very long chain (ACADVL) was investigated in greater detail due to its most obvious expression difference and significance in fatty acid metabolism. ScRNA-Seq of rat skin from irradiated individuals revealed that ACADVL was expressed in all subpopulations of skin tissues, with variations at different timepoints after radiation. Immunohistochemistry confirmed an increased ACADVL expression in the epidermis from human sample and various animal models, including monkeys, rats and mice. The knockdown of ACADVL increased the radiosensitivity of human keratinocytes and human skin fibroblasts. Silencing of ACADVL facilitated the expression of apoptosis and pyroptosis-related proteins following ionizing radiation. Conclusion: This study illustrated that cutaneous fatty acid metabolism was altered in the early response of ionizing radiation, and fatty acid metabolism-associated ACADVL is involved in radiation-induced cell death.

Odd- and even-numbered medium-chained fatty acids protect against glutathione depletion in very long-chain acyl-CoA dehydrogenase deficiency

Article

Nov 2022
BBA-MOL CELL BIOL L

Recent trials have reported the ability of triheptanoin to improve clinical outcomes for the severe symptoms associated with long-chain fatty acid oxidation disorders, including very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. However, the milder myopathic symptoms are still challenging to treat satisfactorily. Myopathic pathogenesis is multifactorial, but oxidative stress is an important component. We have previously shown that metabolic stress increases the oxidative burden in VLCAD-deficient cell lines and can deplete the antioxidant glutathione (GSH). We investigated whether medium-chain fatty acids provide protection against GSH depletion during metabolic stress in VLCAD-deficient fibroblasts. To investigate the effect of differences in anaplerotic capacity, we included both even-(octanoate) and odd-numbered (heptanoate) medium-chain fatty acids. Overall, we show that modulation of the concentration of medium-chain fatty acids in culture media affects levels of GSH retained during metabolic stress in VLCAD-deficient cell lines but not in controls. Lowered glutamine concentration in the culture media during metabolic stress led to GSH depletion and decreased viability in VLCAD deficient cells, which could be rescued by both heptanoate and octanoate in a dose-dependent manner. Unlike GSH levels, the levels of total thiols increased after metabolic stress exposure, the size of this increase was not affected by differences in cell culture medium concentrations of glutamine, heptanoate or octanoate. Addition of a PPAR agonist further exacerbated stress-related GSH-depletion and viability loss, requiring higher concentrations of fatty acids to restore GSH levels and cell viability. Both odd- and even-numbered medium-chain fatty acids efficiently protect VLCADdeficient cells against metabolic stress-induced antioxidant depletion.

Neonatal Long-Chain 3-Ketoacyl-CoA Thiolase deficiency: Clinical-biochemical phenotype, sodium-D,L-3-hydroxybutyrate treatment experience and cardiac evaluation using speckle echocardiography

Article

Jun 2022

Isolated long-chain 3-keto-acyl CoA thiolase (LCKAT) deficiency is a rare long-chain fatty acid oxidation disorder caused by mutations in HADHB. LCKAT is part of a multi-enzyme complex called the mitochondrial trifunctional protein (MTP) which catalyzes the last three steps in the long-chain fatty acid oxidation. Until now, only three cases of isolated LCKAT deficiency have been described. All patients developed a severe cardiomyopathy and died before the age of 7 weeks. Here, we describe a newborn with isolated LCKAT deficiency, presenting with neonatal-onset cardiomyopathy, rhabdomyolysis, hypoglycemia and lactic acidosis. Bi-allelic 185G > A (p.Arg62His) and c1292T > C (p.Phe431Ser) mutations were found in HADHB. Enzymatic analysis in both lymphocytes and cultured fibroblasts revealed LCKAT deficiency with a normal long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD, also part of MTP) enzyme activity. Clinically, the patient showed recurrent cardiomyopathy, which was monitored by speckle tracking echocardiography. Subsequent treatment with special low-fat formula, low in long chain triglycerides (LCT) and supplemented with medium chain triglycerides (MCT) and ketone body therapy in (sodium-D,L-3-hydroxybutyrate) was well tolerated and resulted in improved carnitine profiles and cardiac function. Resveratrol, a natural polyphenol that has been shown to increase fatty acid oxidation, was also considered as a potential treatment option but showed no in vitro benefits in the patient's fibroblasts. Even though our patient deceased at the age of 13 months, early diagnosis and prompt initiation of dietary management with addition of sodium-D,L-3-hydroxybutyrate may have contributed to improved cardiac function and a much longer survival when compared to the previously reported cases of isolated LCKAT-deficiency.

Consensus on diagnosis and treatment of very long-chain acyl-CoA dehydrogenase deficiency

Article

Mar 2022

Wenhui Lin

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a metabolic disease of long chain fatty acid oxidation. The clinical manifestations are heterogeneous, mainly with heart, liver, skeletal muscle and brain damage, and the onset of which can be from newborn to adult. Cardiomyopathy type is more serious with high mortality. The liver failure type and myopathy type would be potentially lethal, but generally the prognosis is relatively good. Recurrent hypoglycemia, energy metabolism disorder, liver dysfunction, cardiomyopathy and serious arrhythmia are the main causes of death. Most patients can be identified through neonatal screening, and the prognosis is usually good in patients with early diagnosis and treatment. The purpose of this consensus is to standardize the diagnosis, treatment and management of VLCAD deficiency, so as to improve the prognosis of patients and reduce death and disability.

Bezafibrate activation of PPAR drives disturbances in mitochondrial redox bioenergetics and decreases the viability of cells from patients with VLCAD deficiency

Article

Feb 2021
BBA-MOL BASIS DIS

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is the most common inborn long-chain fatty acid oxidation (FAO) disorder. VLCAD deficiency is characterized by distinct phenotypes. The severe phenotypes are potentially life-threatening and affect the heart or liver, with a comparatively milder phenotype characterized by myopathic symptoms. There is an unmet clinical need for effective treatment options for the myopathic phenotype. The molecular mechanisms driving the gradual decrease in mitochondrial function and associated alterations of muscle fibers are unclear. The peroxisome proliferator-activated receptor (PPAR) pan-agonist bezafibrate is a potent modulator of FAO and multiple other mitochondrial functions and has been proposed as a potential medication for myopathic cases of long-chain FAO disorders. In vitro experiments have demonstrated the ability of bezafibrate to increase VLCAD expression and activity. However, the outcome of small-scale clinical trials has been controversial. We found VLCAD deficient patient fibroblasts to have an increased oxidative stress burden and deranged mitochondrial bioenergetic capacity, compared to controls. Applying heat stress under fasting conditions to bezafibrate pretreated patient cells, caused a marked further increase of mitochondrial superoxide levels. Patient cells failed to maintain levels of the essential thiol peptide antioxidant glutathione and experienced a decrease in cellular viability. Our findings indicate that chronic PPAR activation is a plausible initiator of long-term pathogenesis in VLCAD deficiency. Our findings further implicate disruption of redox homeostasis as a key pathogenic mechanism in VLCAD deficiency and support the notion that a deranged thiol metabolism might be an important pathogenic factor in VLCAD deficiency.

Hedgehog Signaling in Intestinal Development and Homeostasis

Article

Oct 2020
ANNU REV PHYSIOL

The hedgehog (Hh) signaling pathway plays several diverse regulatory and patterning roles during organogenesis of the intestine and in the regulation of adult intestinal homeostasis. In the embryo, fetus, and adult, intestinal Hh signaling is paracrine: Hh ligands are expressed in the endodermally derived epithelium, while signal transduction is confined to the mesenchymal compartment, where at least a dozen distinct cell types are capable of responding to Hh signals. Epithelial Hh ligands not only regulate a variety of mesenchymal cell behaviors, but they also direct these mesenchymal cells to secrete additional soluble factors (e.g., Wnts, Bmps, inflammatory mediators) that feed back to regulate the epithelial cells themselves. Evolutionary conservation of the core Hh signaling pathway, as well as conservation of epithelial/mesenchymal cross talk in the intestine, has meant that work in many diverse model systems has contributed to our current understanding of the role of this pathway in intestinal organogenesis, which is reviewed here. Expected final online publication date for the Annual Review of Physiology, Volume 83 is February 10, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Implementation of a Fast Method for the Measurement of Carnitine Palmitoyltransferase 2 (CPT2) Activity in Lymphocytes by Tandem Mass Spectrometry as Confirmation for Newborn Screening

Article

Apr 2019

Carnitine palmitoyltransferase II (CPT2) is a rare autosomal recessive inherited disorder affecting mitochondrial β‐oxidation. Confirmation diagnostics are mostly based on molecular sequencing of the CPT2 gene, especially to distinguish CPT2 and carnitine aclycarnitine translocase (CACT) deficiencies, which present with identical acylcarnitine profiles on newborn screening. 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 LC‐MS/MS 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 42‐75%. Four individuals carrying the heterozygous mutation c.338T>C showed a two‐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. This article is protected by copyright. All rights reserved.

Management and diagnosis of mitochondrial fatty acid oxidation disorders: focus on very-long-chain acyl-CoA dehydrogenase deficiency

Article

Nov 2018
J HUM GENET

Mitochondrial fatty acid oxidation disorders (FAODs) are caused by defects in β-oxidation enzymes, including very long-chain acyl-CoA dehydrogenase (VLCAD), trifunctional protein (TFP), carnitine palmitoyltransferase-2 (CPT2), carnitine-acylcarnitine translocase (CACT) and others. During prolonged fasting, infection, or exercise, patients with FAODs present with hypoglycemia, rhabdomyolysis, cardiomyopathy, liver dysfunction, and occasionally sudden death. This article describes the diagnosis, newborn screening, and treatment of long-chain FAODs with a focus on VLCAD deficiency. VLCAD deficiency is generally classified into three phenotypes based on onset time, but the classification should be comprehensively determined based on genotype, residual enzyme activity, and clinical course, due to a lack of apparent genotype–phenotype correlation. With the expansion of newborn screening for FAODs, several issues have arisen, such as missed detection, overdiagnosis (including detection of benign/asymptomatic type), and poor prognosis of the neonatal-onset form. Meanwhile, dietary management and restriction of exercise have been unnecessary for patients with the benign/asymptomatic type of VLCAD deficiency with a high fatty acid oxidation flux score. Although L-carnitine therapy for VLCAD/TFP deficiency has been controversial, supplementation with L-carnitine may be accepted for CPT2/CACT and multiple acyl-CoA dehydrogenase deficiencies. Recently, a double-blind, randomized controlled trial of triheptanoin (seven-carbon fatty acid triglyceride) versus trioctanoin (regular medium-chain triglyceride) was conducted and demonstrated improvement of cardiac functions on triheptanoin. Additionally, although the clinical efficacy of bezafibrate remains controversial, a recent open-label clinical trial showed efficacy of this drug in improving quality of life. These drugs may be promising for the treatment of FAODs, though further studies are required.

ACAD9, a complex I assembly factor with a moonlighting function in fatty acid oxidation deficiencies

Article

Full-text available

Oct 2013
HUM MOL GENET

Oxidative phosphorylation and fatty acid oxidation are two major metabolic pathways in mitochondria. Acyl-CoA dehydrogenase 9 (ACAD9), an enzyme assumed to play a role in fatty acid oxidation, was recently identified as a factor involved in complex I biogenesis. Here we further investigated the role of ACAD9's enzymatic activity in fatty acid oxidation and complex I biogenesis. We provide evidence indicating that ACAD9 displays enzyme activity in vivo. Knockdown experiments in very-long-chain acyl-CoA dehydrogenase (VLCAD)-deficient fibroblasts revealed that ACAD9 is responsible for the production of C14:1-carnitine from oleate and C12-carnitine from palmitate. These results explain the origin of these obscure acylcarnitines that are used to diagnose VLCAD deficiency in humans. Knockdown of ACAD9 in control fibroblasts did not reveal changes in the acylcarnitine profiles upon fatty acid loading. Next, we investigated whether catalytic activity of ACAD9 was necessary for complex I biogenesis. Catalytically inactive ACAD9 gave partial-to-complete rescue of complex I biogenesis in ACAD9-deficient cells and was incorporated in high-molecular-weight assembly intermediates. Our results underscore the importance of the ACAD9 protein in complex I assembly and suggest that the enzymatic activity is a rudiment of the duplication event.

Arginine improves peroxisome functioning in cells from patients with a mild peroxisome biogenesis disorder

Article

Full-text available

Sep 2013
ORPHANET J RARE DIS

Zellweger spectrum disorders (ZSDs) are multisystem genetic disorders caused by a lack of functional peroxisomes, due to mutations in one of the PEX genes, encoding proteins involved in peroxisome biogenesis. The phenotypic spectrum of ZSDs ranges from an early lethal form to much milder presentations. In cultured skin fibroblasts from mildly affected patients, peroxisome biogenesis can be partially impaired which results in a mosaic catalase immunofluorescence pattern. This peroxisomal mosaicism has been described for specific missense mutations in various PEX genes. In cell lines displaying peroxisomal mosaicism, peroxisome biogenesis can be improved when these are cultured at 30[degree sign]C. This suggests that these missense mutations affect the folding and/or stability of the encoded protein. We have studied if the function of mutant PEX1, PEX6 and PEX12 can be improved by promoting protein folding using the chemical chaperone arginine. Fibroblasts from three PEX1 patients, one PEX6 and one PEX12 patient were cultured in the presence of different concentrations of arginine. To determine the effect on peroxisome biogenesis we studied the following parameters: number of peroxisome-positive cells, levels of PEX1 protein and processed thiolase, and the capacity to beta-oxidize very long chain fatty acids and pristanic acid. Peroxisome biogenesis and function in fibroblasts with mild missense mutations in PEX1, 6 and 12 can be improved by arginine. Arginine may be an interesting compound to promote peroxisome function in patients with a mild peroxisome biogenesis disorder.

Newborn screening programmes in Europe; arguments and efforts regarding harmonization. Part 1 - From blood spot to screening result

Article

Full-text available

May 2012
J INHERIT METAB DIS

In many European countries neonatal screening has been introduced over the last 50 years as an important public health programme. Depending on health care structure, available funds, local politics, input from professional groups, parent groups, and the general public this introduction has led to different approaches in the way the screening programmes have been set up, financed and governed. To get some insight about the current situation, in 2009 the European Union, via its EAHC agency, put out a call for a tender that was acquired by our project group. An online survey was compiled in which the whole screening programme was covered by a questionnaire. This survey covered the EU member states, (potential) candidate member states and EFTA countries, in total 40 countries. Results showed little consensus concerning 1. information of parents including informed consent; 2. which conditions are screened for, ranging from 1 to around 30 conditions; 3. sampling time post partum; 4. screening methodology including cut-offs values even between screening laboratories within countries.; 5. storage of residual specimens, varying from 3 months to 1000 years. In addition, confirmatory diagnostics and follow-up also show large discrepancies (Burgard et al. http://www.iss.it/cnmr/prog/cont.php?id=1621&lang=1&tipo=64 2011). In addition to the current practices report an expert opinion document has been produced with recommendations to the EU Commission for future improvements, e.g. in parallel to the way the USA has harmonized its practices based on recommendations by the American College of Medical Genetics (Watson et al., Pediatrics 117: S296-S307, 2006).

VLCAD enzyme activity determinations in newborns identified by screening: A valuable tool for risk assessment

Article

Full-text available

Sep 2011
J INHERIT METAB DIS

Tandem mass spectrometry-based newborn screening correctly identifies individuals with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). However, a great number of healthy individuals present with identical acylcarnitine profiles during catabolism in the first three days of life. We routinely perform an enzyme activity assay as confirmation analysis in newborns identified by screening. Whereas VLCAD residual activities of less than 10% are clearly diagnostic and indicate patients at risk of clinical disease, the clinical relevance of higher residual activities is unclear. In this study we assess the molecular basis in 34 individuals with residual activities of 10-50%. We identify two pathogenic mutations in patients that result in residual activities as high as 22%, while individuals with residual activities of 25-50% either present with a heterozygous or no mutation in the VLCAD gene. In addition, confirmed heterozygous parents present with residual activities as low as 32%. In conclusion, we identify individuals with 2 pathogenic mutations and those with only one heterozygous mutation in the residual activity range of 20-30%. Whereas heterozygosity is generally regarded as clinically irrelevant, identification of 2 VLCAD mutations leads to precautions in the management of the children. Based on our data we anticipate that individuals with a residual enzyme activity >20% present with a biochemical phenotype but likely remain asymptomatic throughout life. Studies in greater patient numbers are needed to correlate residual activities >10% with the genotype and the outcome.

Exposure to resveratrol triggers pharmacological correction of fatty acid utilization in human fatty acid oxidation-deficient fibroblasts

Article

Full-text available

Mar 2011
HUM MOL GENET

Carnitine palmitoyl transferase 2 (CPT2) and very-long-chain Acyl-CoA dehydrogenase (VLCAD) deficiencies are among the most common inborn mitochondrial fatty acid β-oxidation (FAO) disorders. Despite advances in their clinical and molecular characterizations, few therapeutic approaches exist for these diseases. Resveratrol (RSV) is a natural polyphenol extensively studied for its potential health benefits. Indeed, it is presently thought that RSV could delay the onset of some cancers, and have protective effects against common aging disorders such as type II diabetes, cardiovascular or neurodegenerative diseases. Here, we show that exposure to RSV induces a dose- and time-dependant increase in FAO flux in human fibroblasts, and can restore normal FAO capacities in a panel of patients' fibroblasts with the mild forms (harboring various genotypes) of CPT2 or VLCAD deficiency. The correction of FAO flux correlated with a marked increase in mutant CPT2 or VLCAD protein level, in cells treated by RSV. Inhibition of sirtuin 1 (SIRT1) by Sirtinol and the use of peroxisome proliferator-activated receptor gamma co-activator-1-alpha (PGC-1α) small interfering RNAs demonstrate that the RSV-induced stimulation of FAO requires the presence of PGC-1α and SIRT1. These results show, for the first time, that RSV markedly induces mitochondrial FAO capacities in human fibroblasts, and provides the initial proof-of-concept that RSV might be efficient for correction of inherited FAO disorders.

The enzymology of mitochondrial fatty acid beta-oxidation and its application to follow-up analysis of positive neonatal screening results

Article

Full-text available

Oct 2010
J INHERIT METAB DIS

Oxidation of fatty acids in mitochondria is a key physiological process in higher eukaryotes including humans. The importance of the mitochondrial beta-oxidation system in humans is exemplified by the existence of a group of genetic diseases in man caused by an impairment in the mitochondrial oxidation of fatty acids. Identification of patients with a defect in mitochondrial beta-oxidation has long remained notoriously difficult, but the introduction of tandem-mass spectrometry in laboratories for genetic metabolic diseases has revolutionalized the field by allowing the rapid and sensitive analysis of acylcarnitines. Equally important is that much progress has been made with respect to the development of specific enzyme assays to identify the enzyme defect in patients subsequently followed by genetic analysis. In this review, we will describe the current state of knowledge in the field of fatty acid oxidation enzymology and its application to the follow-up analysis of positive neonatal screening results.

Disorders of mitochondrial fatty acyl-CoA beta-oxidation

Article

Full-text available

Jul 1999

In recent years tremendous progress has been made with respect to the enzymology of the mitochondrial fatty acid β-oxidation machinery and defects therein. Firstly, a number of new mitochondrial β-oxidation enzymes have been identified, including very-long-chain acyl-CoA dehydrogenase (VLCAD) and mitochondrial trifunctional protein (MTP). Secondly, the introduction of tandem MS for the analysis of plasma acylcarnitines has greatly facilitated the identification of patients with a defect in fatty acid oxidation (FAO). These two developments explain why the number of defined FAO disorders has increased dramatically, making FAO disorders the most rapidly growing group of inborn errors of metabolism. In this review we describe the current state of knowledge of the enzymes involved in the mitochondrial oxidation of straight-chain, branched-chain and (poly)unsaturated fatty acyl-CoAs as well as disorders of fatty acid oxidation. The laboratory diagnosis of these disorders is described, with particular emphasis on the methods used to identify the underlying enzyme defect and the molecular mutations. In addition, a simple flowchart is presented as a guide to the identification of mitochondrial FAO-disorders. Finally, treatment strategies are discussed briefly.

Improved detection of long‐chain fatty acid oxidation defects in intact cells using [9,10‐3H]oleic acid

Article

Jul 1997
J INHERIT METAB DIS

LIVER DISEASE IN MITOCHONDRIAL FATTY ACID OXIDATION DEFECTS: A FRENCH RETROSPECTIVE STUDY FROM 158 PATIENTS

Article

Aug 2010

Infants suspected to have very-long chain acyl-CoA dehydrogenase deficiency from newborn screening

Article

Jan 2014
MOL GENET METAB

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is a fatty acid oxidation disorder with widely varying presentations that has presented a significant challenge to newborn screening (NBS). The Western States Regional Genetics Services Collaborative developed a workgroup to study infants with NBS positive for VLCADD. We performed retrospective analysis of newborns with elevated C14:1-acylcarnitine on NBS in California, Oregon, Washington, and Hawai'i including available confirmatory testing and clinical information. Overall, from 2,802,504 children screened, there were 242 cases screen-positive for VLCADD. There were 34 symptomatic true positive cases, 18 asymptomatic true positives, 112 false positives, 55 heterozygotes, 11 lost to follow-up, and 12 other disorders. One in 11,581 newborns had an abnormal NBS for suspected VLCADD. Comparison of analytes and analyte ratios from the NBS demonstrated statistically significant differences between true positive and false positive groups for C14:1, C14, C14:1/C2, and C14:1/C16. The positive predictive value for all true positive cases was 94%, 54%, and 23% when C14:1 was ≥2.0μM, ≥1.0μM, and ≥0.7μM, respectively. Sequential post-analytical analysis could reduce the referral rate in 25.8% of cases. This study is the largest reported follow-up of infants with NBS screen-positive results for suspected VLCADD and demonstrates the necessity of developing comprehensive and consistent long-term follow-up NBS systems. Application of clinical information revealed differences between symptomatic and asymptomatic children with VLCADD. Comparison of NBS analytes and analyte ratios may be valuable in developing more effective diagnostic algorithms.

Newborn Screening: Gaps in the Evidence

Article

Oct 2013
SCIENCE

Bridget Wilcken

Despite improvement and increased testing, standards and practice in screening babies for rare disorders remain muddled.

The Use of [9,10- 3 H]Myristate, [9,10- 3 H]Palmitate and [9,10- 3 H]Oleate for the Detection and Diagnosis of Medium and Long-Chain Fatty Acid Oxidation Disorders in Intact Cultured Fibroblasts

Article

Jan 2002

Molecular and Cellular Pathology of Very-Long-Chain Acyl-CoA Dehydrogenase Deficiency

Article

Feb 2013
MOL GENET METAB

Bezafibrate can be a new treatment option for mitochondrial fatty acid oxidation disorders: Evaluation by in vitro probe acylcarnitine assay

Article

Jul 2012
MOL GENET METAB

The number of patients with mitochondrial fatty acid oxidation (FAO) disorders is recently becoming larger with the spread of newborn mass screening. Despite the advances in metabolic and molecular characterization of FAO disorders, the therapeutic studies are still limited. It was reported recently that bezafibrate (BEZ), an agonist of peroxisome proliferating activator receptor (PPAR), can restore FAO activity in cells from carnitine palmitoyltransferase-2 (CPT2) and very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiencies as well as clinical symptoms in the adult patients. In this study, the therapeutic effect of BEZ was determined by in vitro probe acylcarnitine (IVP) assay using cultured fibroblasts and tandem mass spectrometry on various FAO disorders. The clinical trial of BEZ treatment for a boy with the intermediate form of glutaric acidemia type 2 (GA2) was also performed. The effect of BEZ was proven in cells from various FAO disorders including GA2, deficiencies of VLCAD, medium-chain acyl-CoA dehydrogenase, CPT2, carnitine acylcarnitine translocase and trifunctional protein, by the IVP assay. The aberrantly elevated long- or medium-chain acylcarnitines that are characteristic for each FAO disorder were clearly corrected by the presence of BEZ (0.4mmol/L) in culture medium. Moreover, daily administration of BEZ in a 2-year-old boy with GA2 dramatically improved his motor and cognitive skills, accompanied by sustained reduction of C4, C8, C10 and C12 acylcarnitines in blood, and normalized the urinary organic acid profile. No major adverse effects have been observed. These results indicate that BEZ could be a new treatment option for FAO disorders.

Quantitative acylcarnitine profiling in fibroblasts using [U-13C] palmitic acid: An improved tool for the diagnosis of fatty acid oxidation defects

Article

Apr 1999
CLIN CHIM ACTA

A method was developed for the investigation of mitochondrial fatty acid β-oxidation in cultured fibroblasts. Monolayer cultures were incubated without foetal calf serum with commercially available [U-13C] palmitic acid and l-carnitine for 96 h. The acylcarnitines produced by the cells were extracted from the cell suspension and analysed either by quantitative stable isotope dilution gas chromatography chemical ionization mass spectrometry, or by fast atom bombardment mass spectrometry.Characteristic acylcarnitine profiles were obtained for all the different enzyme deficiencies investigated, with the exception of carnitine palmitoyltransferase II deficiency and carnitine/acylcarnitine carrier deficiency which showed similar patterns. Comparison between this method and the 3H-myristate and 3H-palmitate tritium release assays revealed that the method described here is superior, allowing unequivocal identification of patients.

Update on mitochondrial fatty acid oxidation disorders

Article

Oct 2010
J INHERIT METAB DIS

Mitochondrial fatty acid oxidation disorders have been included in newborn screening programs worldwide since the implementation of tandem mass spectrometry-based screening. Disease-specific acylcarnitine profiles pinpoint at the respective enzyme defect; however, the diagnosis has invariably to be confirmed by enzyme assay and/or molecular analysis. Metabolic profiles can be normal in the anabolic state and, consequently, newborn screening may miss the diagnosis when performed outside the catabolic state on days 2 and 3 of life. Mitochondrial fatty acid oxidation disorders comprise four groups: (1) disorders of the entry of long-chain fatty acids into mitochondria, (2) intramitochondrial β-oxidation defects of long-chain fatty acids affecting membrane-bound enzymes, (3) β-oxidation defects of short- and medium-chain fatty acids affecting enzymes of the mitochondrial matrix, and (4) disorders of impaired electron transfer to the respiratory chain from mitochondrial β-oxidation. The main pathogenic mechanisms of fatty acid oxidation defects include toxic effects by accumulating acylcarnitine and acyl-CoA species and energy deficiency due to impaired fatty acid oxidation and ketone body formation. The respective disorders present with heterogeneous phenotypes. Before newborn screening (NBS) was introduced, the most common clinical presentations were hypoketotic hypoglycemia and sudden death, usually precipitated by an infection or fasting in the neonatal period or early childhood. In addition, severe cardiomyopathy and arrhythmias were leading clinical signs in the first months of life. Older children or adults may present with exercise- or illness-induced rhabdomyolysis. Patients can remain asymptomatic throughout life if they have mild defects and are not exposed to metabolic stress. Correlation of genotype and/or residual enzyme activity with disease phenotype has been reported for some defects, whereas in others additional disease modifiers are suspected. With newborn screening, disease incidence has significantly increased and the proportion of milder phenotypes has grown. Newborn screening greatly reduces the morbidity and mortality, though it does not eliminate early neonatal death in severe phenotypes. With respect to the heterogeneous clinical presentation, treatment needs to be tailored to the severity of the respective phenotype and the specific disorder.

Carnitine palmitoyltransferase 2: New insights on the substrate specificity and implications for acylcarnitine profiling

Article

Sep 2010
Biochim Biophys Acta

Over the last years acylcarnitines have emerged as important biomarkers for the diagnosis of mitochondrial fatty acid beta-oxidation (mFAO) and branched-chain amino acid oxidation disorders assuming they reflect the potentially toxic acyl-CoA species, accumulating intramitochondrially upstream of the enzyme block. However, the origin of these intermediates still remains poorly understood. A possibility exists that carnitine palmitoyltransferase 2 (CPT2), member of the carnitine shuttle, is involved in the intramitochondrial synthesis of acylcarnitines from accumulated acyl-CoA metabolites. To address this issue, the substrate specificity profile of CPT2 was herein investigated. Saccharomyces cerevisiae homogenates expressing human CPT2 were incubated with saturated and unsaturated C2-C26 acyl-CoAs and branched-chain amino acid oxidation intermediates. The produced acylcarnitines were quantified by ESI-MS/MS. We show that CPT2 is active with medium (C8-C12) and long-chain (C14-C18) acyl-CoA esters, whereas virtually no activity was found with short- and very long-chain acyl-CoAs or with branched-chain amino acid oxidation intermediates. Trans-2-enoyl-CoA intermediates were also found to be poor substrates for CPT2. Inhibition studies performed revealed that trans-2-C16:1-CoA may act as a competitive inhibitor of CPT2 (K(i) of 18.8 microM). The results obtained clearly demonstrate that CPT2 is able to reverse its physiological mechanism for medium and long-chain acyl-CoAs contributing to the abnormal acylcarnitines profiles characteristic of most mFAO disorders. The finding that trans-2-enoyl-CoAs are poorly handled by CPT2 may explain the absence of trans-2-enoyl-carnitines in the profiles of mitochondrial trifunctional protein deficient patients, the only defect where they accumulate, and the discrepancy between the clinical features of this and other long-chain mFAO disorders such as very long-chain acyl-CoA dehydrogenase deficiency.

Newborn screening for disorders of fatty-acid oxidation: Experience and recommendations from an expert meeting

Article

Apr 2010
J INHERIT METAB DIS

Experience with new-born screening (NBS) for disorders of fatty-acid oxidation (FAOD) is now becoming available from an increasing number of programs worldwide. The spectrum of FAOD differs widely between ethnic groups. Incidence calculations from reports from Australia, Germany, and the USA of a total of 5,256,999 newborns give a combined incidence of all FAOD of approximately 1:9,300. However, it appears to be much lower in Asians. Consequently, a significant prevalence and evidence for a clear benefit of NBS is proven for medium-chain acyl-CoA dehydrogenase deficiency (MCAD) only in countries with a high percentage of Caucasians, with very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD) and long-chain 3-hydroxy acyl-CoA dehydrogenase deficiency (LCHAD) being additional candidates. The long-term benefit for many disorders has still to be evaluated and will require international collaboration, especially for the rarest disorders. Short-chain acyl-CoA dehydrogenase deficiency (SCAD) [as well as Systemic carnitine transporter deficiency (CTD) and dienoyl-CoA reductase deficiency (DE-RED)] are conditions of uncertain clinical significance, but most FAOD have a spectrum of clinical presentations (healthy-death). Confirmatory diagnostic procedures should be agreed upon to ensure international comparability of results and evidence-based modifications. The case of short-chain acyl-CoA dehydrogenase deficiency (SCAD) deficiency shows that even inclusion of conditions without a clearly known natural course may prove useful with respect to gain of knowledge and consecutive exclusion of a biochemical abnormality without clinical significance, although this line of argument implies the existence of structured follow-up programs and bears ethical controversies. As a final conclusion, the accumulated evidence suggests all FAOD should to be included into tandem mass spectrometry (MS/MS)-based NBS programs provided sufficient laboratory performance is guaranteed.

Mitochondrial long chain fatty acid β-oxidation in man and mouse

Article

Jun 2009
Biochim Biophys Acta

Several mouse models for mitochondrial fatty acid beta-oxidation (FAO) defects have been developed. So far, these models have contributed little to our current understanding of the pathophysiology. The objective of this study was to explore differences between murine and human FAO. Using a combination of analytical, biochemical and molecular methods, we compared fibroblasts of long chain acyl-CoA dehydrogenase knockout (LCAD(-/-)), very long chain acyl-CoA dehydrogenase knockout (VLCAD(-/-)) and wild type mice with fibroblasts of VLCAD-deficient patients and human controls. We show that in mice, LCAD and VLCAD have overlapping and distinct roles in FAO. The absence of VLCAD is apparently fully compensated, whereas LCAD deficiency is not. LCAD plays an essential role in the oxidation of unsaturated fatty acids such as oleic acid, but seems redundant in the oxidation of saturated fatty acids. In strong contrast, LCAD is neither detectable at the mRNA level nor at the protein level in men, making VLCAD indispensable in FAO. Our findings open new avenues to employ the existing mouse models to study the pathophysiology of human FAO defects.

A Delphi clinical practice protocol for the management of very long chain acyl-CoA dehydrogenase deficiency

Article

Feb 2009
MOL GENET METAB

Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is a disorder of oxidation of long chain fat, and can present as cardiomyopathy or fasting intolerance in the first months to years of life, or as myopathy in later childhood to adulthood. Expanded newborn screening has identified a relatively high incidence of this disorder (1:31,500), but there is a dearth of evidence-based outcomes data to guide the development of clinical practice protocols. This consensus protocol is intended to assist clinicians in the diagnosis and management of screen-positive newborns for VLCAD deficiency until evidence-based guidelines are available. The Oxford Centre for Evidence-based Medicine system was used to grade the literature review and create recommendations graded from A (evidence level of randomized clinical trials) to D (expert opinion). Delphi was used as the consensus tool. A panel of 14 experts (including clinicians, diagnostic laboratory directors and researchers) completed three rounds of survey questions and had a face-to-face meeting. Panelists reviewed the initial evaluation of the screen-positive infant, diagnostic testing and management of diagnosed patients. Grade C and D consensus recommendations were made in each of these three areas. The panel did not reach consensus on all issues, particularly in the dietary management of asymptomatic infants diagnosed by newborn screening.

Human trifunctional protein deficiency: A new disorder of mitochondrial fatty acid ??-oridation

Article

Dec 1992

In this paper we report the identification of a new disorder of mitochondrial fatty acid beta-oxidation in a patient which presented with clear manifestations of a mitochondrial beta-oxidation disorder. Subsequent studies in fibroblasts revealed an impairment in palmitate beta-oxidation and in addition, a combined deficiency of long-chain enoyl-CoA hydratase, long-chain 3-hydroxyacyl-CoA-dehydrogenase and long-chain 3-oxoacyl-CoA thiolase. The recent identification of a multifunctional, membrane-bound beta-oxidation enzyme protein catalyzing all these three enzyme activities (Carpenter et al. (1992) Biochem. Biophys. Res. Commun. 183, 443-448; Uchida et al. (1992) J. Biol. Chem. 267, 1034-1041) suggested an underlying basis for this peculiar combination of three enzyme deficiencies. We show by means of size-exclusion chromatography that there is, indeed, a deficiency of the multifunctional beta-oxidation enzyme protein in this patient.

A comparison of [9,10-3H]palmitic and [9,10-3H]myristic acids for the detection of defects of fatty acid oxidation in intact cultured fibroblasts

Article

Feb 1990

The production of tritiated water from [9,10-3H]myristic acid can be used as a screening assay for the detection of medium-chain acyl-CoA dehydrogenase deficiency, multiple acyl-CoA dehydrogenation defects (glutaric aciduria type 2 and ethylmalonic-adipic aciduria types), and some types of hydroxydicarboxylic aciduria. Comparison with the release of tritiated water from [9,10-3H]palmitic acid may give an indication of the chain-length specificity of the metabolic defect. In a case of ethylmalonic-adipic aciduria such a prediction has been confirmed by examination of accumulated intermediates in the affected fibroblasts.

Molecular analysis of carnitine palmitoyltransferase II deficiency with hepatocardiomuscular expression

Article

Jun 1996

Carnitine palmitoyltransferase (CPT) II deficiency, an inherited disorder of mitochondrial long-chain fatty-acid (LCFA) oxidation, results in two distinct clinical phenotypes, namely, an adult (muscular) form and an infantile (hepatocardiomuscular) form. The rationale of this phenotypic heterogeneity is poorly understood. The adult form of the disease is commonly ascribed to the Ser-113-Leu substitution in CPT II. Only few data are available regarding the molecular basis of the infantile form of the disease. We report herein a homozygous A-2399-C transversion predicting a Tyr-628-Ser substitution in a CPT II-deficient infant. In vitro expression of mutant cDNA in COS-1 cells demonstrated the responsibility of this mutation for the disease. Metabolic consequences of the SER-113-Leu and Tyr-628-Ser substitutions were studied in fibroblasts. The Tyr-628-Ser substitution (infantile form) resulted in a 10% CPT II residual activity, markedly impairing LCFA oxidation, whereas the Ser-113-Leu substitution (adult form) resulted in a 20% CPT II residual activity, with out consequence on LCFA oxidation. These data show that CPT II activity has to be reduced below a critical threshold in order for LCFA oxidation in fibroblasts to be impaired. The hypothesis that this critical threshold differs among tissues could provide a basis to explain phenotypic heterogeneity of CPT II deficiency.

Improved detection of long-chain fatty acid oxidation defects in intact cells using [9,10-3H]oleic acid

Article

Aug 1997

[9,10- 3 H]Myristate and [9,10- 3 H]palmitate are currently used widely in 3 H 2 O release assays for detecting medium- and long-chain fatty acid oxidation defects, both in intact fibroblasts (Manning et al 1990; Olpin et al 1992) and in lymphocytes (Brivet et al 1995). Over the past nine years we have used both substrates to screen routinely for fatty acid oxidation defects in over 900 patients and have identified 82 individuals with specific fatty acid oxidation disorders. Nearly all of these assays have been performed on fibroblasts. Medium-chain acyl-CoA dehydrogenase deficiency (McKusick 201450) is characterized by a much decreased oxidation of myristate, giving a high palmitate/myristate (P/M) ratio. The long-chain defects, very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency (McKusick 143450), generally give a low P/M ratio, though discrimination is relatively poor. This is particularly true for 'mild' VLCAD, where both absolute activities with [9,10- 3 H]myristate and [9,10- 3 H]palmitate and the P/M ratio may show overlap with the control range. Over the last few years we have increasingly encountered patients who present both clinically and biochemically as likely to have a fatty acid oxidation disorder and with mild to moderate reductions (into the VLCAD and LCHAD range) in fibroblast oxidation of [9,10- 3 H]myristate and [9,10- 3 H]palmitate but who do not meet the criteria for any of the recognized fatty acid oxidation defects. We now describe the use of [9,10- 3 H]oleic acid, a commercially available substrate, for improved discrimination in the detection of long-chain fatty acid oxidation defects.

Mitochondrial very-long-chain acyl-coenzyme A dehydrogenase deficiency: clinical characteristics and diagnostic considerations in 30 patients

Article

Feb 1998
CLIN CHIM ACTA

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is an enzyme catalyzing the dehydrogenation of long-chain fatty acids in the first step of mitochondrial fatty acid oxidation. Using an ETF (electron transfer flavoprotein, the physiological electron acceptor of VLCAD) reduction assay, we identified VLCAD deficiency in cultured skin fibroblasts or liver tissue from 30 patients in 27 families. They clinically presented two phenotypes: a 'severe' presentation characterized by an early onset of symptoms, with hypertrophic cardiomyopathy and a high incidence of death, and a 'mild' form with hypoketotic hypoglycaemia, resembling MCAD (medium-chain acyl-CoA dehydrogenase) deficiency. Cells isolated from patients who develop cardiomyopathy characteristically accumulate longer-chain length acylcarnitines (hexadecanoylcarnitine and tetradecanoylcarnitine) when incubated with palmitate. However, cells from patients with the hypoglycaemic presentation produced relatively shorter-chain-length intermediates (mainly dodecanoylcarnitine). Inhibition of carnitine palmitoyl transferase I, in vitro, eliminated these intermediates with cells from both phenotypes indicating their intramitochondrial origin. Although the explanation for these distinct biochemical findings is not obvious, the correlation with the two phenotypes provides an opportunity for accurate prognosis and early implementation of appropriate treatment. Prenatal diagnosis of this life-threatening disorder was successfully performed in seven pregnancies in six of those families by assay of trophoblasts or amniocytes. In an at risk family, diagnosis of an affected fetus by measurement of VLCAD activity in noncultured chorionic villi allowed termination of the pregnancy before 13 weeks of gestation.

Clear Correlation of Genotype with Disease Phenotype in Very–Long-Chain Acyl-CoA Dehydrogenase Deficiency

Article

Mar 1999

Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial rate-limiting step in mitochondrial fatty acid beta-oxidation. VLCAD deficiency is clinically heterogenous, with three major phenotypes: a severe childhood form, with early onset, high mortality, and high incidence of cardiomyopathy; a milder childhood form, with later onset, usually with hypoketotic hypoglycemia as the main presenting feature, low mortality, and rare cardiomyopathy; and an adult form, with isolated skeletal muscle involvement, rhabdomyolysis, and myoglobinuria, usually triggered by exercise or fasting. To examine whether these different phenotypes are due to differences in the VLCAD genotype, we investigated 58 different mutations in 55 unrelated patients representing all known clinical phenotypes and correlated the mutation type with the clinical phenotype. Our results show a clear relationship between the nature of the mutation and the severity of disease. Patients with the severe childhood phenotype have mutations that result in no residual enzyme activity, whereas patients with the milder childhood and adult phenotypes have mutations that may result in residual enzyme activity. This clear genotype-phenotype relationship is in sharp contrast to what has been observed in medium-chain acyl-CoA dehydrogenase deficiency, in which no correlation between genotype and phenotype can be established.

Molecular Heterogeneity in Very-Long-Chain Acyl-CoA Dehydrogenase Deficiency Causing Pediatric Cardiomyopathy and Sudden Death

Article

Apr 1999

Genetic defects are being increasingly recognized in the etiology of primary cardiomyopathy (CM). Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first step in the beta-oxidation spiral of fatty acid metabolism, the crucial pathway for cardiac energy production. We studied 37 patients with CM, nonketotic hypoglycemia and hepatic dysfunction, skeletal myopathy, or sudden death in infancy with hepatic steatosis, features suggestive of fatty acid oxidation disorders. Single-stranded conformational variance was used to screen genomic DNA. DNA sequencing and mutational analysis revealed 21 different mutations on the VLCAD gene in 18 patients. Of the mutations, 80% were associated with CM. Severe CM in infancy was recognized in most patients (67%) at presentation. Hepatic dysfunction was common (33%). RNA blot analysis and VLCAD enzyme assays showed a severe reduction in VLCAD mRNA in patients with frame-shift or splice-site mutations and absent or severe reduction in enzyme activity in all. Infantile CM is the most common clinical phenotype of VLCAD deficiency. Mutations in the human VLCAD gene are heterogeneous. Although mortality at presentation is high, both the metabolic disorder and cardiomyopathy are reversible.

Arrhythmias and Conduction Defects as Presenting Symptoms of Fatty Acid Oxidation Disorders in Children

Article

Dec 1999
CIRCULATION

The clinical manifestations of inherited disorders of fatty acid oxidation vary according to the enzymatic defect. They may present as isolated cardiomyopathy, sudden death, progressive skeletal myopathy, or hepatic failure. Arrhythmia is an unusual presenting symptom of fatty acid oxidation deficiencies. Over a period of 25 years, 107 patients were diagnosed with an inherited fatty acid oxidation disorder. Arrhythmia was the predominant presenting symptom in 24 cases. These 24 cases included 15 ventricular tachycardias, 4 atrial tachycardias, 4 sinus node dysfunctions with episodes of atrial tachycardia, 6 atrioventricular blocks, and 4 left bundle-branch blocks in newborn infants. Conduction disorders and atrial tachycardias were observed in patients with defects of long-chain fatty acid transport across the inner mitochondrial membrane (carnitine palmitoyl transferase type II deficiency and carnitine acylcarnitine translocase deficiency) and in patients with trifunctional protein deficiency. Ventricular tachycardias were observed in patients with any type of fatty acid oxidation deficiency. Arrhythmias were absent in patients with primary carnitine carrier, carnitine palmitoyl transferase I, and medium chain acyl coenzyme A dehydrogenase deficiencies. The accumulation of arrhythmogenic intermediary metabolites of fatty acids, such as long-chain acylcarnitines, may be responsible for arrhythmias. Inborn errors of fatty acid oxidation should be considered in unexplained sudden death or near-miss in infants and in infants with conduction defects or ventricular tachycardia. Diagnosis can be easily ascertained by an acylcarnitine profile from blood spots on filter paper.

The use of [9,10-3H]myristate, [9,10-3H]palmitate and [9,10-3H]oleate for the detection and diagnosis of medium and long-chain fatty acid oxidation disorders in intact cultured fibroblasts

Article

Feb 1999
ADV EXP MED BIOL

Mutation analysis in mitochondrial fatty acid oxidation defects: Exemplified by acyl-CoA dehydrogenase deficiencies, with special focus on genotype–phenotype relationship

Article

Sep 2001
HUM MUTAT

Mutation analysis of metabolic disorders, such as the fatty acid oxidation defects, offers an additional, and often superior, tool for specific diagnosis compared to traditional enzymatic assays. With the advancement of the structural part of the Human Genome Project and the creation of mutation databases, procedures for convenient and reliable genetic analyses are being developed. The most straightforward application of mutation analysis is to specific diagnoses in suspected patients, particularly in the context of family studies and for prenatal/preimplantation analysis. In addition, from these practical uses emerges the possibility to study genotype-phenotype relationships and investigate the molecular pathogenesis resulting from specific mutations or groups of mutations. In the present review we summarize current knowledge regarding genotype-phenotype relationships in three disorders of mitochondrial fatty acid oxidation: very-long chain acyl-CoA dehydrogenase (VLCAD, also ACADVL), medium-chain acyl-CoA dehydrogenase (MCAD, also ACADM), and short-chain acyl-CoA dehydrogenase (SCAD, also ACADS) deficiencies. On the basis of this knowledge we discuss current understanding of the structural implications of mutation type, as well as the modulating effect of the mitochondrial protein quality control systems, composed of molecular chaperones and intracellular proteases. We propose that the unraveling of the genetic and cellular determinants of the modulating effects of protein quality control systems may help to assess the balance between genetic and environmental factors in the clinical expression of a given mutation. The realization that the effect of the monogene, such as disease-causing mutations in the VLCAD, MCAD, and SCAD genes, may be modified by variations in other genes presages the need for profile analyses of additional genetic variations. The rapid development of mutation detection systems, such as the chip technologies, makes such profile analyses feasible. However, it remains to be seen to what extent mutation analysis will be used for diagnosis of fatty acid oxidation defects and other metabolic disorders.

Fatty Acid Oxidation Disorders

Article

Feb 2002

Genetic disorders of mitochondrial fatty acid beta-oxidation have been recognized within the last 20 years as important causes of morbidity and mortality, highlighting the physiological significance of fatty acids as an energy source. Although the mammalian mitochondrial fatty acid-oxidizing system was recognized at the beginning of the last century, our understanding of its exact nature remains incomplete, and new components are being identified frequently. Originally described as a four-step enzymatic process located exclusively in the mitochondrial matrix, we now recognize that long-chain-specific enzymes are bound to the inner mitochondrial membrane, and some enzymes are expressed in a tissue-specific manner. Much of our new knowledge of fatty acid metabolism has come from the study of patients who were diagnosed with single-gene autosomal recessive defects, a situation that seems to be further evolving with the emergence of phenotypes determined by combinations of multiple genetic and environmental factors. This review addresses the normal process of mitochondrial fatty acid beta-oxidation and discusses the clinical, metabolic, and molecular aspects of more than 20 known inherited diseases of this pathway that have been described to date.

MS/MS-based newborn and family screening detects asymptomatic patients with very-long-chain acyl-COA dehydrogenase deficiency

Article

Oct 2003
J Pediatr

To determine whether asymptomatic persons with biochemical evidence of very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency identified through expanded newborn screening with tandem mass spectometry have confirmed disease. We characterized 8 asymptomatic VLCAD-deficient individuals by enzyme and/or mutational analysis and compared them with clinically diagnosed, symptomatic patients with regard to mutations, enzyme activity, phenotype, and age of disease onset. VLCAD molecular analyses in 6 unrelated patients revealed the previously reported V243A mutation, associated with hepatic or myopathic phenotypes, on 7/12 alleles. All other mutations were also missense mutations. Residual VLCAD activities of 6% to 11% of normal were consistent with milder phenotypes. In these identified individuals treated prospectively with dietary modification as preventive measures, clinical symptoms did not develop during follow-up. MS/MS-based newborn screening correctly identifies VLCAD-deficient individuals. Based on mutational and enzymatic findings, these infants probably are at risk of future disease. Because life-threatening metabolic derangement can occur even in otherwise mild phenotypes, we advocate universal newborn screening programs for VLCAD deficiency to detect affected patients and prevent development of metabolic crises. Longer-term follow-up is essential to define outcomes, the definite risk of future disease, and appropriate treatment recommendations.

Characterization of L-aminocarnitine, an inhibitor of fatty acid oxidation

Article

May 2008
MOL GENET METAB

The pathogenesis of hypoketotic hypoglycemia and cardiomyopathy in patients with fatty acid oxidation (FAO) disorders is still poorly understood. In vitro studies are hampered by the lack of natural mutants to asses the effect of FAO inhibition. In addition, only a few inhibitors of FAO are known. Furthermore, most inhibitors of FAO are activating ligands of peroxisome proliferator-activated receptors (PPARs). We show that l-aminocarnitine (L-AC), a carnitine analog, inhibits FAO efficiently, but does not activate PPAR. L-AC inhibits carnitine palmitoyltransferase (CPT) with different sensitivities towards CPT1 and CPT2, as well as carnitine acylcarnitine translocase (CACT). We further characterized L-AC using fibroblasts cell lines from controls and patients with different FAO defects. In these cell lines acylcarnitine profiles were determined in culture medium after loading with [U-(13)C]palmitic acid. In control fibroblasts, L-AC inhibits FAO leading to a reduction of C2-acylcarnitine and elevation of C16-acylcarnitine. In very long-chain acyl-CoA dehydrogenase (VLCAD)-deficient fibroblasts, L-AC decreased the elevated C14-acylcarnitine and increased C16-acylcarnitine. In CACT and CPT2-deficient cell lines, L-AC did not change the already elevated C16-acylcarnitine level, showing that CPT1 is not inhibited. Oxidation of pristanic acid was only partly inhibited at high L-AC concentrations, indicating minimal CACT inhibition. Therefore, we conclude that in intact cells L-AC inhibits CPT2. Combined with our observation that l-AC does not activate PPAR, we suggest that L-AC is useful to simulate a FAO defect in cells from different origin.

Molecular analysis of carnitine palmitoyltransferase II deficiency with hepatocardiomuscular expression

Jan 1996
971

J P Bonnefont
F Taroni
P Cavadini
JP Bonnefont

Fatty acid oxidation flux predicts the clinical severity of VLCAD deficiency

Abstract

No full-text available

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