Tracer-based lipidomics enables the discovery of disease-specific candidate biomarkers in mitochondrial ß-oxidation disorders.

Carnitine derivatives of disease-specific acyl-CoAs are the diagnostic hallmark for long-chain fatty acid ß-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.

Long-term monitoring of fatty acid oxidation defects: results from a MetabERN survey.

BACKGROUND: Implementation of long-chain fatty acid oxidation defects (LCFAOD) in newborn screening (NBS) programs allows for pre-symptomatic diagnosis and treatment. The long-term natural history of NBS LCFAOD patients is largely unknown and may differ from clinically diagnosed pre-NBS patients. This complicates long-term monitoring of LCFAOD and may cause high monitoring variability. To gain insight in current clinical practice, we performed a web-based questionnaire among all metabolic members of the European Reference Network for Hereditary Metabolic Disorders (MetabERN). RESULTS: Thirty-seven colleagues representing at least 35 European metabolic centres shared their experience and results were discussed at the European Metabolic Group (EMG) meeting 2022. The centres concurred in many aspects of long-term monitoring of LCFAOD including the frequency of clinical visits, determination of laboratory parameters, cardiac monitoring and retinopathy screening. Main discrepancies comprised hepatic imaging, glucose monitoring and electrophysiological investigations. CONCLUSIONS: Discrepancies may reflect differences in local availability of monitoring tools, the inclusion of LCFAOD in NBS programs as well as differences in local genotypes and phenotypes. Because monitoring strategies are largely based on the natural disease course of clinically identified patients, there might be over-monitoring of some NBS patients. Nevertheless, we advocate long-term monitoring because resulting information is essential to further characterize the natural disease course, develop evidence-based guidelines and provide a basis for evaluation of future therapies.

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

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.

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

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 also the other deficiencies of the mitochondrial trifunctional protein (MTP), a multi-enzyme complex involved in long-chain fatty acid ß-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). To gain insight in the outcomes of MTP-deficient patients diagnosed after the introduction of NBS for LCHADD in the Netherlands, a retrospective evaluation of genetic, biochemical, and clinical characteristics of MTP-deficient patients, identified since 2007, was carried out. 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 1 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. In 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 the 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.

Thermo-sensitive mitochondrial trifunctional protein deficiency presenting with episodic myopathy.

Mitochondrial trifunctional protein (MTP) is involved in long-chain fatty acid ß-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.

Glucose transporter type 1 deficiency syndrome and the ketogenic diet.

Glucose transporter type 1 deficiency syndrome (GLUT1DS) is characterised by deficient glucose transport over the blood-brain barrier and reduced glucose availability in the brain. This causes epilepsy, movement disorders, and cognitive impairment. Treatment with ketogenic diet provides ketones as alternative energy source. However, not all GLUT1DS patients are on dietary treatment (worldwide registry: 77/181 [43%] of patients). The current 25-year experience allows evaluation of effects and tolerability of dietary treatment for GLUT1DS. To this end, literature was searched up to January 2019 for individual case reports and series reporting (side) effects of dietary treatment for GLUT1DS. Upon aggregation of data for analysis, we identified 270 GLUT1DS patients with dietary treatment with a mean follow-up of 53 months. Epilepsy improved for 83% of 230 patients and remained unchanged for 17%, movement disorders improved for 82% of 127 patients and remained unchanged for 17%, and cognition improved for 59% of 58 patients and remained stable for 40%. Effects on epilepsy were seen within days/weeks and were most pronounced in patients with early treatment initiation. Effects on movement disorders were noticed within months and were strongest in patients with higher cerebrospinal fluid-to-blood glucose ratio. Although side effects were minimal, 18% of 270 patients reported poor compliance. In individual patients, symptoms deteriorated upon low ketosis, poor compliance, or treatment discontinuation. Based on the good tolerability and strong favourable effect of dietary treatment on GLUT1DS symptoms, we advocate dietary treatment in all GLUT1DS patients and prompt diagnosis or screening to allow early treatment.

Genetic defect of the sodium-dependent multivitamin transporter: A treatable disease, mimicking biotinidase deficiency.

The sodium-dependent multivitamin transporter that facilitates the uptake of the water-soluble vitamins biotin, pantothenic acid, and the vitamin-like substance lipoate is coded by the SLC5A6 gene. Variants in this gene cause a relatively novel treatable metabolic disorder. Here we describe the second case. A 17-month-old girl presented with hypoglycemia (2.0 mmol/L) and severe metabolic acidosis (pH 6.87), leading to resuscitation. Her history revealed feeding problems from birth and poor weight gain. Metabolic investigation showed elevated plasma C3-carnitine and C5-OH-carnitine. Urine analysis showed persistently elevated excretion of 3-OH-isovaleric acid. Biochemically, the combination of elevated C5-OH-carnitine and increased excretion of 3-OH-isovaleric acid seemed compatible with biotinidase deficiency. Supplementation with biotin was started. Biotinidase activity in plasma showed only marginally decreased activity, which was considered insufficient explanation for her clinical symptoms. Subsequent trio-based whole exome sequencing revealed compound heterozygosity for variants in the SLC5A6 gene. Upon increasing the dosage of biotin supplementation and introduction of pantothenic acid supplementation, a striking clinical improvement was seen.