Chain acyl-CoA Dehydrogenase Research Articles

Human inborn errors of long-chain fatty acid oxidation show impaired inflammatory responses to TLR4-ligand LPS.

Stimulation of mammalian cells with inflammatory inducers such as lipopolysaccharide (LPS) leads to alterations in activity of central cellular metabolic pathways. Interestingly, these metabolic changes seem to be important for subsequent release of pro-inflammatory cytokines. This has become particularly clear for enzymes of tricarboxylic acid (TCA) cycle such as succinate dehydrogenase (SDH). LPS leads to inhibition of SDH activity and accumulation of succinate to enhance the LPS-induced formation of IL-1β. If enzymes involved in beta-oxidation of fatty acids are important for sufficient responses to LPS is currently not clear. Using cells from various patients with inborn long-chain fatty acid oxidation disorders (lcFAOD), we report that disease-causing deleterious variants of Electron Transfer Flavoprotein Dehydrogenase (ETFDH) and of Very Long Chain Acyl-CoA Dehydrogenase (ACADVL), both cause insufficient inflammatory responses to stimulation with LPS. The insufficiencies included reduced TLR4 expression levels, impaired TLR4 signaling, and reduced or absent induction of pro-inflammatory cytokines such as IL-6. The insufficient responses to LPS were reproduced in cells from healthy controls by targeted loss-of-function of either ETFDH or ACADVL, supporting that the deleterious ETFDH and ACADVL variants cause the attenuated responses to LPS. ETFDH and ACADVL encode two distinct enzymes both involved in fatty acid beta-oxidation, and patients with these deficiencies cannot sufficiently metabolize long-chain fatty acids. We report that genes important for beta-oxidation of long-chain fatty acids are also important for inflammatory responses to an acute immunogen trigger like LPS, which may have important implications for understanding infection and other metabolic stress induced disease pathology in lcFAODs.

Significance of Variants of Uncertain Significance: The Human Cost of Genetic Uncertainty.

This piece narrates the journey of Maria (name of the mother has been altered to protect the family's privacy), a new mother confronting her newborn's unexpected diagnosis of very long chain acyl-CoA dehydrogenase (VLCAD) deficiency, despite undergoing proactive genetic carrier screening within a consanguineous marriage. It highlights the emotional and systemic challenges arising from the lack of diversity in genetic databases, which, in this case, failed to detect pathogenic variants in Maria and her husband. Maria's story sheds light on situations where a masked variant of uncertain significance (VUS) necessitates consultation with a trained genetics specialist and underscores the urgent need for a more equitable healthcare system.

Diagnostic challenges and outcome of fatty acid oxidation defects in a tertiary care center in Lebanon

BackgroundFatty acid oxidation defects are rare autosomal recessive disorders with variable clinical manifestations and outcome. Early detection by systematic neonatal screening may improve their prognosis. Long-term outcome studies of these disorders in the Middle East and North Africa region are limited. The purpose of this study is to report the diagnostic challenges and outcome of fatty acid oxidation defects in a major tertiary care center in Lebanon, a resource-constrained country in the Middle East.MethodsA retrospective review of charts of all fatty acid oxidation defects sequential patients diagnosed and followed at our center was conducted. Collected data included: parental consanguinity, age at diagnosis, clinical presentation, biochemical profile, confirmatory diagnosis, treatment and outcome. A genotype–phenotype correlation was also performed, when available.ResultsSeven types of fatty acid oxidation defects were identified in a total of 34 patients from 21 families. Most families (79%) were consanguineous (first-degree cousins). The majority were diagnosed when clinically symptomatic (78%), at various ages between 10 days and 19 years (average: 2 years). Follow-up duration spanned between 2 months and 15 years (average: 5 years). The remainder of the patients were detected while still asymptomatic by systematic neonatal screening (9%) or due to positive family history (9%). The most common defect was carnitine transporter deficiency (50%) with an exclusive cardiac presentation related to a founder variant c.981C > T, (p.Arg254*) in the SLC22A5 gene. Medium chain acyl-CoA dehydrogenase deficiency was found in 13% only, which could be explained by the absence of systematic neonatal screening. Rare gene variants were detected in very long chain and multiple acyl-CoA dehydrogenase deficiency. The worse prognosis was observed in very long chain acyl-CoA dehydrogenase deficiency. The overall survival at last follow-up reached 75% with a complete reversal of symptoms with treatment in most patients (63%), despite their late diagnosis.ConclusionsOur experience highlights the diagnostic challenges and outcome of fatty acid oxidation defects in a resource-constrained country with high consanguinity rates. Physicians’ awareness and systematic neonatal screening are key for diagnosis. Larger genotype–phenotype studies are still needed to understand the natural history of these rare diseases and possibly improve their outcome.

Structural basis for expanded substrate specificities of human long chain acyl-CoA dehydrogenase and related acyl-CoA dehydrogenases

Crystal structures of human long-chain acyl-CoA dehydrogenase (LCAD) and the catalytically inactive Glu291Gln mutant, have been determined. These structures suggest that LCAD harbors functions beyond its historically defined role in mitochondrial β-oxidation of long and medium-chain fatty acids. LCAD is a homotetramer containing one FAD per 43 kDa subunit with Glu291 as the catalytic base. The substrate binding cavity of LCAD reveals key differences which makes it specific for longer and branched chain substrates. The presence of Pro132 near the start of the E helix leads to helix unwinding that, together with adjacent smaller residues, permits binding of bulky substrates such as 3α, 7α, l2α-trihydroxy-5β-cholestan-26-oyl-CoA. This structural element is also utilized by ACAD11, a eucaryotic ACAD of unknown function, as well as bacterial ACADs known to metabolize sterol substrates. Sequence comparison suggests that ACAD10, another ACAD of unknown function, may also share this substrate specificity. These results suggest that LCAD, ACAD10, ACAD11 constitute a distinct class of eucaryotic acyl CoA dehydrogenases.

A newborn Screening Programme for Inborn errors of metabolism in Galicia: 22 years of evaluation and follow-up

BackgroundThere is a notable lack of harmonisation in newborn screening (NBS) programmes worldwide. The Galician programme for early detection of inborn errors of metabolism (IEM) was one of the first NBS programmes in Europe to incorporate mass spectrometry (July 2000). This programme currently screens for 26 IEMs in dried blood and urine samples collected 24–72 h after birth.ResultsIn its 22-year history, this programme has analysed samples from 440,723 neonates and identified 326 cases of IEM with a prevalence of 1:1351. The most prevalent IEMs were hyperphenylalaninaemia (n = 118), followed by medium chain acyl-CoA dehydrogenase deficiency (MCADD, n = 26), galactosaemia (n = 20), and cystinurias (n = 43). Sixty-one false positives and 18 conditions related to maternal pathologies were detected. Urine samples have been identified as a useful secondary sample to reduce the rate of false positives and identify new defects. There were 5 false negatives. The overall positive value was 84.23%. The fatality rate over a median of 12.1 years of follow-up was 2.76%. The intelligence quotient of patients was normal in 95.7% of cases, and school performance was largely optimal, with pedagogic special needs assistance required in < 10% of cases. Clinical onset of disease preceded diagnosis in 4% of cases. The age at which first NBS report is performed was reduced by 4 days since 2021.ConclusionsThis study highlights the benefits of collecting urine samples, reduce NBS reporting time and expanding the number of IEMs included in NBS programmes.

ACADVL−/−/ACADL−/− mice demonstrate symptoms of very long chain acyl-CoA dehydrogenase deficiency that are rescued by AAV9 gene therapy

ACADVL−/−/ACADL−/− mice demonstrate symptoms of very long chain acyl-CoA dehydrogenase deficiency that are rescued by AAV9 gene therapy

Medium Chain Acyl CoA Dehydrogenase Deficiency and Eating Disorders: An Underreported Coincidence

Medium Chain Acyl CoA Dehydrogenase Deficiency and Eating Disorders: An Underreported Coincidence

Acylcarnitines’ Level in the Dried Blood Spot Samples of Healthy Newborns in Serbia-The Pilot Study

Abstract Analysis of the acylcarnitines’ (ACs) is the mainstay for screening for fatty acid oxidation disorders (FAOD). Data about the ACs profile in the dried blood spot samples of healthy newborns in Serbia are not at disposal. Therefore, we determined the ACs levels and established the cut-offs. Between August 2018 and August 2019 a total of 1771 samples had been analysed. Cut-offs, established using a non-parametric approach, were verified in comparison with the worldwide target ranges and the data for several Caucasian populations. The majority of ACs had comparable distribution in Serbian and the worldwide population. In case of discrepancy, the individual alterations had a frequency of less than 10%. Seventeen out of 25 established cutoffs were in the worldwide target range. Reliability of the cut-offs positioning out of the target ranges is not jeopardized, since alterations are negligible or similar findings were reported for other Caucasian populations. The established and verified set of cut-offs can be used in the future screening for carnitine uptake/transport defect, medium-chain acyl-CoA dehydrogenase deficiency, very long-chain acyl-CoA dehydrogenase deficiency, long-chain L-3 hydroxyacyl-CoA dehydrogenase deficiency, trifunctional protein deficiency, carnitine palmitoyltransferase deficiency Ia and II, as well as carnitine:acylcarnitine translocase deficiency.

De l’intérêt du test de Guthrie dans le dépistage du déficit en MCAD

AbstractA child was born at term from an uncomplicated pregnancy. Biochemical and hematological tests were unremarkable. The Guthrie test was performed, which involves taking a blood sample on a filter paper to screen for serious diseases. These diseases must be treated early in the patient’s life to avoid complication. Newborn screening revealed a deficiency in MCAD (Medium Chain Acyl-CoA Dehydrogenase). This is an inherited metabolic disease that leads to the accumulation of medium-chain fatty acids due to a defect in mitochondrial beta-oxidation. During catabolic situations, this condition can cause hypocetotic hypoglycemia and symptoms that can range from malaise to coma or even death.

Heptanoic and medium branched-chain fatty acids as anaplerotic treatment for medium chain acyl-CoA dehydrogenase deficiency

Triheptanoin (triheptanoylglycerol) has shown value as anaplerotic therapy for patients with long chain fatty acid oxidation disorders but is contraindicated in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. In search for anaplerotic therapy for patients with MCAD deficiency, fibroblasts from three patients homozygous for the most common mutation, ACADMG985A/G985A, were treated with fatty acids hypothesized not to require MCAD for their metabolism, including heptanoic (C7; the active component of triheptanoin), 2,6-dimethylheptanoic (dMC7), 6-amino-2,4-dimethylheptanoic (AdMC7), or 4,8-dimethylnonanoic (dMC9) acids. Their effectiveness as anaplerotic fatty acids was assessed in live cells by monitoring changes in cellular oxygen consumption rate (OCR) and mitochondrial protein lysine succinylation, which reflects cellular succinyl-CoA levels, using immunofluorescence (IF) staining. Krebs cycle intermediates were also quantitated in these cells using targeted metabolomics. The four fatty acids induced positive changes in OCR parameters, consistent with their oxidative catalysis and utilization. Increases in cellular IF staining of succinylated lysines were observed, indicating that the fatty acids were effective sources of succinyl-CoA in the absence of media glucose, pyruvate, and lipids. The ability of MCAD deficient cells to metabolize C7 was confirmed by the ability of extracts to enzymatically utilize C7-CoA as substrate but not C8-CoA. To evaluate C7 therapeutic potential in vivo, Acadm-/- mice were treated with triheptanoin for seven days. Dose dependent increase in plasma levels of heptanoyl-, valeryl-, and propionylcarnitine indicated efficient metabolism of the medication. The pattern of the acylcarnitine profile paralleled resolution of liver pathology including reversing hepatic steatosis, increasing hepatic glycogen content, and increasing hepatocyte protein succinylation, all indicating improved energy homeostasis in the treated mice. These results provide the impetus to evaluate triheptanoin and the medium branched chain fatty acids as potential therapeutic agents for patients with MCAD deficiency.

Remodeling of lipid landscape in high fat fed very-long chain acyl-CoA dehydrogenase null mice favors pro-arrhythmic polyunsaturated fatty acids and their downstream metabolites

Very-long chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial step of mitochondrial long chain (LC) fatty acid β-oxidation (FAO). Inherited VLCAD deficiency (VLCADD) predisposes to neonatal arrhythmias whose pathophysiology is still not understood. We hypothesized that VLCADD results in global disruption of cardiac complex lipid homeostasis, which may set conditions predisposing to arrhythmia. To test this, we assessed the cardiac lipidome and related molecular markers in seven-month-old VLCAD−/− mice, which mimic to some extent the human cardiac phenotype. Mice were sacrificed in the fed or fasted state after receiving for two weeks a chow or a high-fat diet (HFD), the latter condition being known to worsen symptoms in human VLCADD. Compared to their littermate counterparts, HFD/fasted VLCAD−/− mouse hearts displayed the following lipid alterations: (1) Lower LC, but higher VLC-acylcarnitines accumulation, (2) higher levels of arachidonic acid (AA) and lower docosahexaenoic acid (DHA) contents in glycerophospholipids (GPLs), as well as (3) corresponding changes in pro-arrhythmogenic AA-derived isoprostanes and thromboxane B2 (higher), and anti-arrythmogenic DHA-derived neuroprostanes (lower). These changes were associated with remodeling in the expression of gene or protein markers of (1) GPLs remodeling: higher calcium-dependent phospholipase A2 and lysophosphatidylcholine-acyltransferase 2, (2) calcium handling perturbations, and (3) endoplasmic reticulum stress. Altogether, these results highlight global lipid dyshomeostasis beyond FAO in VLCAD−/− mouse hearts, which may set conditions predisposing the hearts to calcium mishandling and endoplasmic reticulum stress and thereby may contribute to the pathogenesis of arrhythmias in VLCADD in mice as well as in humans.

Analysis of clinical characteristics and ACADM gene variants in four children with Medium chain acyl-CoA dehydrogenase deficiency

To explore the clinical and genetic characteristics of four patients with medium-chain acyl-CoA dehydrogenase deficiency (MCADD). Four children who had presented at the Children's Hospital Affiliated to Zhengzhou University between August 2019 and August 2021 were selected as the study subjects. Clinical data of the children were collected. The children were subjected to whole exome sequencing (WES). All of the four children were diagnosed with MCADD. Blood amino acid and ester acyl carnitine spectrum test showed that the concentration of octanoyl carnitine (C8) was significantly increased. The main clinical manifestations included poor mental response (3 cases), intermittent diarrhea with abdominal pain (1 case), vomiting (1 case), increased transaminase (3 cases), and metabolic acidosis (2 cases). Five variants were identified by genetic testing, among which c.341A>G (p.Y114C) was unreported previously. Three were missense variants, one was frameshift variant and one was splicing variant. The clinical heterogeneity of MCADD is obvious, and the severity of the disease may vary. WES can assist with the diagnosis. Delineation of the clinical symptoms and genetic characteristics of the disease can facilitate early diagnosis and treatment of the disease.

Functional and structural impact of 10 ACADM missense mutations on human medium chain acyl-Coa dehydrogenase

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency (MCADD) is associated with ACADM gene mutations, leading to an impaired function and/or structure of MCAD. Importantly, after import into the mitochondria, MCAD must incorporate a molecule of flavin adenine dinucleotide (FAD) per subunit and assemble into tetramers. However, the effect of MCAD amino acid substitutions on FAD incorporation has not been investigated. Herein, the commonest MCAD variant (p.K304E) and 11 additional rare variants (p.Y48C, p.R55G, p.A88P, p.Y133C, p.A140T, p.D143V, p.G224R, p.L238F, p.V264I, p.Y372N, and p.G377V) were functionally and structurally characterized. Half of the studied variants presented a FAD content <65 % compared to the wild-type. Most of them were recovered as tetramers, except the p.Y372N (mainly as dimers). No correlation was found between the levels of tetramers and FAD content. However, a correlation between FAD content and the cofactor's affinity, proteolytic stability, thermostability, and thermal inactivation was established. We showed that the studied amino acid changes in MCAD may alter the substrate chain-length dependence and the interaction with electron-transferring-flavoprotein (ETF) necessary for a proper functioning electron transfer thus adding additional layers of complexity to the pathological effect of ACADM missense mutations. Although the majority of the variant MCADs presented an impaired capacity to retain FAD during their synthesis, some of them were structurally rescued by cofactor supplementation, suggesting that in the mitochondrial environment the levels and activity of those variants may be dependent of FAD's availability thus contributing for the heterogeneity of the MCADD phenotype found in patients presenting the same genotype.

Acylglycine Analysis by Ultra‐Performance Liquid Chromatography‐Tandem Mass Spectrometry (UPLC‐MS/MS)

Quantitative analysis of urine acylglycines has shown to be a highly sensitive and specific method with proven clinical utility for the diagnosis of several inherited metabolic disorders including: medium chain acyl-CoA dehydrogenase deficiency, multiple acyl-CoA dehydrogenase deficiency, short chain acyl-CoA dehydrogenase deficiency, 3-methylcrotonyl-CoA carboxylase deficiency, 2-methylbutyryl-CoA dehydrogenase deficiency, isovaleric acidemia, propionic academia, and isobutyryl-CoA dehydrogenase deficiency. Here, a method that is currently performed using ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) is described. © 2023 Wiley Periodicals LLC. Basic Protocol: Urinary acylglycine analysis by UPLC-MS/MS Support Protocol 1: Quality control preparation Support Protocol 2: Internal standard (ISTD) preparation Support Protocol 3: Standard (STD)/calibrator preparation.

Klf4-Sirt3/Pparα-Lcad pathway contributes to high phosphate-induced lipid degradation

BackgroundPhosphorus commonly reduces lipid deposition in the vertebrates. However, the underlying mechanisms involved in the process remain unclear.MethodsYellow catfish were given three experimental diets with dietary phosphate levels of 3.22, 6.47 and 7.99 g Pi kg− 1, respectively, for 8 weeks. The contents of triglyceride, non-esterified free fatty acids, adenosine triphosphate, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide, enzymatic activities, mRNA and protein expression were determined in the intestinal tissues. Hematoxylin and eosin, Oil Red O staining, and transmission electron microscope were performed for intestinal tissues. Primary intestinal epithelial cells were isolated from yellow catfish intestine. Western blot analysis, Immunoprecipitation assays, Immunofluorescence staining, and RNA extraction and quantitative real-time PCR were decided. Luciferase reporter assays and electrophoretic mobility shift assay were used to evaluate the function of Sirt3, PPARα and Lcad promoters.ResultsHigh dietary phosphate intake activated intestinal phosphate absorption and excretion, and reduced lipid deposition through increasing lipolysis in the intestine. Moreover, phosphate incubation increased the mRNA and protein expression of krüppel like factor 4 (klf4), silent mating-type information regulation 2 homolog 3 (sirt3), peroxisome proliferator activated receptor alpha (pparα) and long chain acyl-CoA dehydrogenase (lcad) in the intestinal epithelial cells (IECs), and klf4 knockdown attenuated the phosphate-induced increase of protein levels of Sirt3, Pparα and Lcad. Further investigation found that Klf4 overexpression increased the activity of sirt3 and pparα promoters, which in turn reduced the acetylation and protein level of Lcad.ConclusionDietary Pi excess induced lipid degradation by the activation of the Klf4-Sirt3/Pparα-Lcad pathway in the intestine and primary IECs.Graphical 2J2H-vAY8k8xL15UHAsvLYVideo

Newborn screening and genetic variation of medium chain acyl-CoA dehydrogenase deficiency in the Chinese population.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is an autosomal recessive disorder of thefatty acid oxidative metabolism. This study aimed toinvestigate the epidemiological characteristics, the spectrum of variation, clinical phenotype, and prognosis ofMCADD in Chinese newborns. We retrospectively analysed newborn screening (NBS) data in the Zibo area from January 2016 to March 2022 and summarized 42 cases recently reported in Chinese neonates. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and next-generation sequencing (NGS) were used to detect the concentrations ofcarnitine in the blood spots and for diagnosis. A total of 183,082 newborns were detected, andsix patients were diagnosed with MCADD (1/3,0514). The primary octanoylcarnitine (C8) and the octanoylcarnitine/decanoylcarnitine ratio (C8/C10) were elevated in allpatients. Gene analysis revealed four known and four novel variants of the ACADM gene. Five patients were asymptomatic and developed normally under dietary guidance. One child died of vaccination-induced MCADD, presenting with hypoglycemia and elevated acylcarnitines. The incidence of MCADD in Chinese newborns varies geographically from 1/222,903 to 1/30,514, andthe most common pathogenic variant isc.449_452 delCTGA (p. T150Rfs∗4) in ACADM gene with a frequency of 27.7%. HPLC-MS/MS and genetic analysis are beneficial for early prevention and good prognosis of MCADD.

Medium-chain acyl-CoA dehydrogenase deficiency: prevalence of ACADM pathogenic variants c.985A&gt;G and c.199T&gt;C in a healthy population in Rio Grande do Sul, Brazil

Abstract Objectives: To investigate the prevalence of ACADM pathogenic variants, c.985A&gt;G and c.199T&gt;C, for medium chain acyl CoA dehydrogenase deficiency (MCADD) in a healthy population in the southern region of Brazil. Methods: This was an observational cross-sectional study with a convenience sampling strategy. The participants were recruited from the blood bank of the Hospital de Clínicas of Porto Alegre, Brazil. A total of 1000 healthy individuals from the state of Rio Grande do Sul were included. Genotyping for the c.199T&gt;C and c.985A&gt;G variants was performed using real-time polymerase chain reaction (PCR) and the PCR-restriction fragment length polymorphism (RFLP) technique, respectively. Individuals considered heterozygous for c.985A&gt;G were subjected to additional acylcarnitine profile analysis using tandem mass spectrometry. Carrier frequency was obtained by calculating the ratio of heterozygous individuals to the total number of individuals analyzed and reported with a 95% confidence interval. Allele and genotype frequencies were calculated based on the Hardy–Weinberg equilibrium. Results: The c.985A&gt;G variant was detected as heterozygotes in three individuals (frequency of the heterozygous genotype = 1:333, allele frequency = 0.0015, minimum frequency of MCADD = 1:444,444) whose acylcarnitine profiles were within normal limits. The c.199T&gt;C variant was not identified. Conclusions: Considering the small sample size and associated allelic heterogeneity with MCADD, these findings are believed to denote the rarity or underdiagnosis of MCADD in southern Brazil. This study provides evidence for the need for further investigation to ascertain the contribution of these diseases to child morbidity and mortality in the country.

CircRNA and miRNA expression profiles during remote ischemic postconditioning attenuate brain ischemia/reperfusion injury

Remote ischemic postconditioning (RIPostC) is a protective procedure for brain damage caused by ischemia/reperfusion (IR), yet the mechanism of this treatment remains to be elucidated. Circular RNAs (circRNAs) are endogenous non-coding RNAs that have recently been recognized to play vital roles in ischemic brain injury. The aim of this study was to explore the role of circRNAs in the protective mechanism of RIPostC and to analyze the circRNA–microRNA (miRNA) regulation network in RIPostC. Nine rats were assigned randomly into three groups (three rats per group): sham, IR, and RIPostC. Their brain tissues were extracted for next-generation RNA sequencing and bioinformatics analysis was performed for two comparisons: sham vs. IR and IR vs. RIPostC. The expression patterns of selected circRNAs and miRNAs were validated by quantitative real-time PCR (qPCR). We detected 82 upregulated and 51 downregulated circRNAs and 137 upregulated and 127 downregulated miRNAs in the IR group compared with the sham group, and 41 upregulated and 100 downregulated circRNAs and 45 upregulated and 64 downregulated miRNAs in the RIPostC group compared with the IR group. The proposed competitive endogenous RNA (ceRNA) network, which included 24 circRNAs, 20 miRNAs, and 145 mRNAs, indicated that the dysregulated circRNAs played important roles in brain IR injury. On the basis of the expression patterns of selected circRNAs, miRNAs, and mRNAs obtained by qPCR, we proposed a circRNA_0002286–miR-124–3p–VLCAD pathway. In PC12 cell, the expression level of miR-124–3p was significantly upregulated when the expression of circRNA_0002286 was repressed and the expression level of VLCAD (very-long chain acyl-CoA dehydrogenase) was significantly downregulated, which suggested that circRNA_0002286 may act as a miRNA sponge for miR-124–3p to regulate the expression of VLCAD. We found that upregulation of circRNA_0002286 attenuated IR injury and was associated with downregulation of miR-124–3p and upregulation of VLCAD. This is the first time that circRNAs have been shown to be closely related to brain IR injury and RIPostC and suggests that targeting the circRNA_0002286–miR-124–3p–VLCAD pathway might attenuate brain IR injury.

EP001: Newborn screening experience for very long chain Acyl-CoA Dehydrogenase (VLCAD) deficiency in Kuwait

Among the various inborn errors of fatty acid oxidation disorders, very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is the most common disorder in Kuwaiti population, which has been noticed especially following the launch of the expanded newborn screening program in Kuwait in October 2014. VLCAD deficiency is a rare autosomal recessive disorder with a worldwide incidence of 1:30,000 to 1:100,000 births. It is caused by deficiency of VLCAD coenzyme, encoded by ACADVL gene which converts very-long-chain fatty acids into energy. In October 2014, the Kuwait Ministry of Health has started a publicly funded expanded newborn screening program meeting the highest international standards to screen for a wide range of metabolic and endocrine disorders including a total of 22 disorders via testing dried blood spots and thus replacing the old, limited newborn screening for congenital hypothyroidism and phenylketonuria that was introduced in 2005. A retrospective analysis of the data registry for the newborn screening over the 6-year period between January 2015 and December 2020 in Kuwait has been conducted after obtaining consent from the newborn screening program. This data included newborns delivered in hospitals all over Kuwait. Data on metabolite concentrations in dried blood spots at the time of screening obtained from all newborns were reviewed and only dried blood spots detecting elevated blood C14:1 (cutoff 0.29 mmol/L) and C14:1/C2 ratio (cutoff 0.03) via tandem mass spectrometry (MS/MS) were included in this study. The positive initial screening is followed by a confirmatory plasma or dried blood acylcarnitine analysis (cutoff ≥ 1 mmol/L) with or without a follow-up genetic analysis of ACADVL gene, either targeted variant testing using PCR amplification followed by Sanger sequencing, or through next generation sequencing technology. Total of 36 cases (19 male/17 female) out of 304,086 screened newborns have been identified and confirmed to have VLCAD deficiency with an incidence of ∼1:8300. The diagnosis was based on the detection of elevated blood C14:1 and C14:1/C2 ratio in the initial dried blood spots in the newborn screening, followed by a confirmatory plasma or dried blood acylcarnitines profile for VLCAD deficiency with or without a follow-up genetic analysis, except for three babies who had positive initial screen but have died before obtaining confirmatory acylcarnitine or genetic analyses. Out of the 36 individuals, there were 3 cases from Kingdom of Saudi Arabia, 2 cases from India and the rest were Kuwaiti. Molecular testing of 24 of them has revealed a founder truncating pathogenic variant in exon 2 of the ACADVL gene, * c.65C>A; p.(Ser22Ter); the reference transcript is NM_001178008.2(hg19/GRCh3). We have identified three genetically confirmed cases with VLCAD deficiency following a positive initial screen but negative confirmatory acylcarnitine analysis. Furthermore, we identified four heterozygous individuals for VLCAD deficiency via molecular testing, after a positive initial newborn screening and normal confirmatory acylcarnitine profile. This is the first study to review the Kuwaiti newborn screening experience of VLCAD deficiency since its launch in October 2014. We recommend including molecular genetic testing for ACADVL gene as part of the newborn screening for VLCAD deficiency particularly for the cases with negative confirmatory acylcarnitine profile. Our study provides evidence that the expanded newborn screening in Kuwait has led to the early detection of VLCAD deficiency cases and thus the initiation of the adequate management plan for these individuals aiming to prevent death and disability.

Decreased Expression of ACADSB Predicts Poor Prognosis in Clear Cell Renal Cell Carcinoma.

BackgroundPrevious reports have shown that short/branched chain acyl-CoA dehydrogenase (ACADSB) plays an important role in glioma, but its role in clear cell renal carcinoma (ccRCC) has not been reported.MethodsThe TIMER and UALCAN databases were used for pan-cancer analysis. RNA sequencing and microarray data of patients with ccRCC were downloaded from the Cancer Genome Atlas and Gene Expression Omnibus database. The differential expression of ACADSB in ccRCC and normal kidney tissues was tested. Correlations between ACADSB expression and clinicopathological parameters were assessed using the Wilcoxon test. The influences of ACADSB expression and clinicopathological parameters on overall survival were assessed using Cox proportional hazards models. Gene set enrichment analysis (GSEA) was performed to explore the associated gene sets enriched in different ACADSB expression phenotypes. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on genes with similar expression patterns to ACADSB. Correlations between ACADSB and ferroptosis-related genes were assessed using Spearman’s correlation analysis.ResultsPan-cancer analysis revealed that ACADSB is down-regulated in multiple cancers, and decreased expression of ACADSB correlates with poor prognosis in certain types of cancer. Differential expression analyses revealed that ACADSB was down-regulated in ccRCC, indicating that ACADSB expression could be a single significant parameter to discriminate between normal and tumor tissues. Clinical association analysis indicated that decreased ACADSB expression was associated with high tumor stage and grade. The Cox regression model indicated that low ACADSB expression was an independent risk factor for the overall survival of patients with ccRCC. GSEA showed that 10 gene sets, including fatty acid (FA) metabolism, were differentially enriched in the ACADSB high expression phenotype. GO and KEGG pathway enrichment analysis revealed that ACADSB-related genes were significantly enriched in categories related to FA metabolism, branched-chain amino acid (BCAA) metabolism, and iron regulation. Spearman’s correlation analysis suggested that the expression of ACADSB was positively correlated with the expression of ferroptosis driver genes.ConclusionsACADSB showed good diagnostic and prognostic abilities for ccRCC. The downregulation of ACADSB might promote tumorigenesis and tumor progression by inhibiting FA catabolism, BCAA catabolism, and ferroptosis in ccRCC.