Very Long-chain acyl-CoA Dehydrogenase Deficiency Research Articles

Ceramide – the unmasked driver of heart failure in very long-chain acyl-CoA dehydrogenase deficiency (VLCADD)

Ceramide – the unmasked driver of heart failure in very long-chain acyl-CoA dehydrogenase deficiency (VLCADD)

Management and Outcomes of Very Long-Chain Acyl-CoA Dehydrogenase Deficiency (VLCAD Deficiency): A Retrospective Chart Review.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a rare genetic condition affecting the mitochondrial beta-oxidation of long-chain fatty acids. This study reports on the clinical outcomes of patients diagnosed by newborn screening with VLCAD deficiency comparing metabolic parameters, enzyme activities, molecular results, and clinical management. It is a single-center retrospective chart review of VLCAD deficiency patients who met the inclusion criteria between January 2002 and February 2020. The study included 12 patients, 7 of whom had an enzyme activity of more than 10%, and 5 patients had an enzyme activity of less than 10%. The Pearson correlation between enzyme activity and the C14:1 level at newborn screening showed a p-value of 0.0003, and the correlation between enzyme activity and the C14:1 level at diagnosis had a p-value of 0.0295. There was no clear correlation between the number of documented admissions and the enzyme activity level. Patients who had a high C14:1 value at diagnosis were started on a diet with a lower percentage of energy from long-chain triglycerides. The C14:1 result at diagnosis is the value that has been guiding our initial clinical management in asymptomatic diagnosed newborns. However, the newborn screening C14:1 value is the most sensitive predictor of low enzyme activity and may help guide dietary management.

Using the C14:1/Medium-Chain Acylcarnitine Ratio Instead of C14:1 to Reduce False-Positive Results for Very-Long-Chain Acyl-CoA Dehydrogenase Deficiency in Newborn Screening in Japan.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is a long-chain fatty acid oxidation disorder that manifests as either a severe phenotype associated with cardiomyopathy, a hypoglycemic phenotype, or a myopathic phenotype. As the hypoglycemic phenotype can cause sudden infant death, VLCAD deficiency is included in newborn screening (NBS) panels in many countries. The tetradecenoylcarnitine (C14:1) level in dried blood specimens is commonly used as a primary marker for VLCAD deficiency in NBS panels. Its ratio to acetylcarnitine (C2) and various other acylcarnitines is used as secondary markers. In Japan, tandem mass spectrometry-based NBS, initially launched as a pilot study in 1997, was introduced to the nationwide NBS program in 2013. In the present study, we evaluated levels of acylcarnitine with various chain lengths (C18 to C2), free carnitine, and their ratios in 175 infants who tested positive for VLCAD deficiency with C14:1 and C14:1/C2 ratios. Our analyses indicated that the ratios of C14:1 to medium-chain acylcarnitines (C10, C8, and C6) were the most effective markers in reducing false-positive rates. Their use with appropriate cutoffs is expected to improve NBS performance for VLCAD deficiency.

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

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.

Very-Long-Chain Acyl-CoA Dehydrogenase Deficiency: Family Impact and Perspectives.

Very-Long-Chain Acyl-CoA Dehydrogenase Deficiency (VLCADD) is a fatty acid oxidation disorder characterized by the decreased ability of the enzyme very-long-chain acyl-CoA dehydrogenase to break down fatty acids with 14 to 20-long carbon chains. The resulting clinical manifestations are variable in severity and include hypoketotic hypoglycemia, rhabdomyolysis, and cardiomyopathy. Treatment can consist of limiting the dietary intake of long-chain fatty acids, the prevention of fasting, and the supplementation of medium-chain fats. This study, conducted in the context of a 5-year long-term follow-up on VLCADD, evaluates how the diagnosis of this fatty acid disorder impacts the family, specifically as it relates to the medical diet and barriers to care. Caregivers (n = 10) of individuals with VLCADD responded to a survey about how VLCADD potentially impacts their family. The review included the clinical outcomes of the patients (n = 11), covering instances of rhabdomyolysis, cardiomyopathy, and hospitalizations related to VLCADD. Families affected by VLCADD experience barriers to care, including difficulties with finances, ability to work, and access to nutrition.

Biochemical and molecular characteristics among infants with abnormal newborn screen for very-long-chain acyl-CoA dehydrogenase deficiency: A single center experience

ObjectiveTo define the biochemical and molecular characteristics and diagnostic outcomes of a large US cohort of VLCAD deficiency positive cases as detected by newborn screening (NBS) with MS:MS. This relatively common disorder of fatty acid oxidation is screened for in every state in America and often results in extensive testing of multiple samples to arrive at a diagnostic conclusion. Materials and methodsWe compared NBS dried blood spot (DBS) acylcarnitine profile (ACP) C14, C14:1, C14:2, C14:1/C12:1 ratio and plasma C14, C14:1, C14:2, C14:1/C12:1, C14:1/C16 and C14:1/C2 ratios among true positive and false positive cases. Results of VLCAD enzyme analysis, molecular testing and fibroblast fatty acid oxidation probe assay were analyzed. ResultsThe presence of compound heterozygous or homozygous pathogenic variants, along with elevations of C14, C14:1 and C14:1/C12:1 ratio, identified 19 VLCAD deficiency cases. All were asymptomatic at most recent follow-up visits. The C14:1/C12:1 ratio in NBS-DBS ACP and plasma acylcarnitine profiles at follow-up (follow-up plasma ACP), is the most useful marker to differentiate between true and false positive cases. Among all cases with molecular analysis data available, approximately 56.7% had a single pathogenic mutation. Lymphocyte enzyme analysis (n = 61) was uninformative in 23% of cases studied. ConclusionVLCAD deficiency NBS by MS:MS is highly effective at identifying asymptomatic affected infants. Our cohort showed that elevation of C14:1/C12:1, in both NBS DBS and plasma ACP, was informative in discriminating affected from unaffected individuals and contributes to improve the accuracy of confirmatory testing of infants with presumptive positive for VLCAD 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.

Specifications of the ACMG/AMP guidelines for ACADVL variant interpretation

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is a relatively common inborn error of metabolism, but due to difficulty in accurately predicting affected status through newborn screening, molecular confirmation of the causative variants by sequencing of the ACADVL gene is necessary. Although the ACMG/AMP guidelines have helped standardize variant classification, ACADVL variant classification remains disparate due to a phenotype that can be nonspecific, the possibility of variants that produce late-onset disease, and relatively high carrier frequency, amongst other challenges. Therefore, an ACADVL-specific variant curation expert panel (VCEP) was created to facilitate the specification of the ACMG/AMP guidelines for VLCADD. We expect these guidelines to help streamline, increase concordance, and expedite the classification of ACADVL variants.

Low Fasting Concentrations of Glucagon in Patients with Very Long-Chain Acyl-CoA Dehydrogenase Deficiency.

(1) Background: Deficiencies of mitochondrial fatty acid oxidation (FAO) define a subgroup of inborn errors of metabolism, with medium-chain acyl-CoA dehydrogenase deficiency (MCAD) and very long-chain acyl-CoA dehydrogenase deficiency (VLCAD) being two of the most common. Hypoketotic hypoglycemia is a feared clinical complication and the treatment focuses on avoiding hypoglycemia. In contrast, carnitine uptake deficiency (CUD) is treated as a mild disease without significant effects on FAO. Impaired FAO has experimentally been shown to impair glucagon secretion. Glucagon is an important glucose-mobilizing hormone. If and how glucagon is affected in patients with VLCAD or MCAD remains unknown. (2) Methods: A cross-sectional study was performed with plasma hormone concentrations quantified after four hours of fasting. Patients with VLCAD (n = 10), MCAD (n = 7) and CUD (n = 6) were included. (3) Results: The groups were similar in age, sex, weight, and height. The glucagon and insulin levels were significantly lower in the VLCAD group compared to the CUD group (p < 0.05, respectively). The patients with CUD had glucagon concentrations similar to the normative data. No significant differences were seen in GLP-1, glicentin, glucose, amino acids, or NEFAs. (4) Conclusions: Low fasting concentrations of glucagon are present in patients with VLCAD and cannot be explained by altered stimuli in plasma.

Validation of a targeted metabolomics panel for improved second-tier newborn screening.

Improved second-tier assays are needed to reduce the number of false positives in newborn screening (NBS) for inherited metabolic disorders including those on the Recommended Uniform Screening Panel (RUSP). We developed an expanded metabolite panel for second-tier testing of dried blood spot (DBS) samples from screen-positive cases reported by the California NBS program, consisting of true- and false-positives from four disorders: glutaric acidemia type I (GA1), methylmalonic acidemia (MMA), ornithine transcarbamylase deficiency (OTCD), and very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). This panel was assembled from known disease markers and new features discovered by untargeted metabolomics and applied to second-tier analysis of single DBS punches using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a 3-min run. Additionally, we trained a Random Forest (RF) machine learning classifier to improve separation of true- and false positive cases. Targeted metabolomic analysis of 121 analytes from DBS extracts in combination with RF classification at a sensitivity of 100% reduced false positives for GA1 by 83%, MMA by 84%, OTCD by 100%, and VLCADD by 51%. This performance was driven by a combination of known disease markers (3-hydroxyglutaric acid, methylmalonic acid, citrulline, and C14:1), other amino acids and acylcarnitines, and novel metabolites identified to be isobaric to several long-chain acylcarnitine and hydroxy-acylcarnitine species. These findings establish the effectiveness of this second-tier test to improve screening for these four conditions and demonstrate the utility of supervised machine learning in reducing false-positives for conditions lacking clearly discriminating markers, with future studies aimed at optimizing and expanding the panel to additional disease targets.

Synthetic mRNA rescues very long-chain acyl-CoA dehydrogenase deficiency in patient fibroblasts and a murine model.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an inborn error of long chain fatty acid β-oxidation (FAO) with limited treatment options. Patients present with heterogeneous clinical phenotypes affecting predominantly heart, liver, and skeletal muscle. While VLCAD deficiency is a systemic disease, restoration of liver FAO has the potential to improve symptoms more broadly due to increased total body ATP production and reduced accumulation of potentially toxic metabolites. We explored the use of synthetic human VLCAD (hVLCAD) mRNA and lipid nanoparticle encapsulated hVLCAD mRNA (LNP-VLCAD) to generate functional VLCAD enzyme in patient fibroblasts derived from VLCAD deficient patients, mouse embryonic fibroblasts, hepatocytes isolated from VLCAD knockout (Acadvl-/-) mice, and Acadvl-/- mice to reverse the metabolic effects of the deficiency. Transfection of all cell types with hVLCAD mRNA resulted in high level expression of protein that localized to mitochondria with increased enzyme activity. Intravenous administration of LNP-VLCAD to Acadvl-/- mice produced a significant amount of VLCAD protein in liver, which declined over a week. Treated Acadvl-/- mice showed reduced hepatic steatosis, were more resistant to cold stress, and accumulated less toxic metabolites in blood than untreated animals. Results from this study support the potential for hVLCAD mRNA for treatment of VLCAD deficiency.

Outcomes of mitochondrial long chain fatty acid oxidation and carnitine defects from a single center metabolic genetics clinic

BackgroundMitochondrial long-chain fatty acid oxidation and carnitine metabolism defects are a group of inherited metabolic diseases. We performed a retrospective cohort study to report on the phenotypic and genotypic spectrum of mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects as well as their treatment outcomes.MethodsAll patients with mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects were included. We divided patients into two groups to compare outcomes of those treated symptomatically (SymX) and asymptomatically (AsymX). We reviewed patient charts for clinical features, biochemical investigations, molecular genetic investigations, cardiac assessments, neuroimaging, treatments, and outcomes.ResultsThere were 38 patients including VLCAD (n = 5), LCHAD (n = 4), CACT (n = 3), MAD (n = 1), CPT-I (n = 13), CPT-II (n = 3) deficiencies and CTD (n = 9). Fourteen patients were diagnosed symptomatically (SymX), and 24 patients were diagnosed asymptomatically (AsymX). Twenty-eight variants in seven genes were identified in 36 patients (pathogenic/likely pathogenic n = 25; variant of unknown significance n = 3). Four of those variants were novel. All patients with LCHAD deficiency had the common variant (p.Glu474Gln) in HADHA and their phenotype was similar to the patients reported in the literature for this genotype. Only one patient with VLCAD deficiency had the common p.Val283Ala in ACADVL. The different genotypes in the SymX and AsymX groups for VLCAD deficiency presented with similar phenotypes. Eight patients were treated with carnitine supplementation [CTD (n = 6), CPT-II (n = 1), and MAD (n = 1) deficiencies]. Thirteen patients were treated with a long-chain fat restricted diet and MCT supplementation. A statistically significant association was found between rhabdomyolysis, and hypoglycemia in the SymX group compared to the AsymX group. A higher number of hospital admissions, longer duration of hospital admissions and higher CK levels were observed in the SymX group, even though the symptomatic group was only 37% of the study cohort.ConclusionSeven different mitochondrial long-chain fatty acid oxidation and carnitine metabolism defects were present in our study cohort. In our clinic, the prevalence of mitochondrial long-chain fatty acid oxidation and carnitine defects was 4.75%.

Structural basis for defective membrane targeting of mutant enzyme in human VLCAD deficiency

Very long-chain acyl-CoA dehydrogenase (VLCAD) is an inner mitochondrial membrane enzyme that catalyzes the first and rate-limiting step of long-chain fatty acid oxidation. Point mutations in human VLCAD can produce an inborn error of metabolism called VLCAD deficiency that can lead to severe pathophysiologic consequences, including cardiomyopathy, hypoglycemia, and rhabdomyolysis. Discrete mutations in a structurally-uncharacterized C-terminal domain region of VLCAD cause enzymatic deficiency by an incompletely defined mechanism. Here, we conducted a structure-function study, incorporating X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, computational modeling, and biochemical analyses, to characterize a specific membrane interaction defect of full-length, human VLCAD bearing the clinically-observed mutations, A450P or L462P. By disrupting a predicted α-helical hairpin, these mutations either partially or completely impair direct interaction with the membrane itself. Thus, our data support a structural basis for VLCAD deficiency in patients with discrete mutations in an α-helical membrane-binding motif, resulting in pathologic enzyme mislocalization.

The frequencies of very long-chain acyl-CoA dehydrogenase deficiency genetic variants in Japan have changed since the implementation of expanded newborn screening.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency has been a target of expanded newborn screening (ENBS) using tandem mass spectrometry in Japan. Since the implementation of ENBS, a number of novel ACADVL variants responsible for VLCAD deficiency have been identified. In this study, genotypic differences in Japanese patients with VLCAD deficiency were investigated before and after ENBS. The ACADVL variants in 61 subjects identified through ENBS (ENBS group) and in 40 patients who subsequently developed clinical symptoms without undergoing ENBS (pre-ENBS group) were compared. Subjects in the ENBS group underwent genetic testing and/or VLCAD enzyme activity measurements. Patients in the pre-ENBS group were stratified into three clinical phenotypes and underwent genetic testing. This study revealed that the variants p.K264E, p.K382Q and c.996dupT were found in both groups, but their frequencies were lower in the ENBS group (5.2%, 3.1% and 4.2%, respectively) than in the pre-ENBS group (16.5%, 12.7% and 10.1%, respectively). In addition, p.C607S, p.T409M, p.M478I, p.G289R, p.C237R, p.T260M, and p.R229* were exclusively identified in the ENBS group. Among these variants, p.C607S exhibited the highest frequency (18.8%). The patients who were heterozygous for p.C607S demonstrated 7-42% of control enzyme activity. p.C607S is suspected to be unique to Japanese individuals. According to a comparison of enzyme activity, patients with the p.C607S variant may exhibit higher enzyme activity than those with the p.A416T, p.A180T, p.R450H, and p.K264E variants, which are responsible for the myopathic form of the disease. The VLCAD deficiency genotypes have changed since the initiation of ENBS in Japan.

Sportmedizinische Beratung nach akuter Rhabdomyolyse bei erblicher Myopathie – eine herausfordernde Leistungsdiagnostik

Increased serum creatinine kinase after physical activity is well known and there is currently no cut-off value. We present the systematic diagnosis after rhabdomyolysis in a rare cause. A 36-year-old soldier developed acute kidney failure due to rhabdomyolysis after intense physical exertion. Sporty habitus, CK elevation and highly normal kidney values. Massive CK increase after step loading on the bicycle ergometer. We stopped further physical stress and initiated diagnostics. The genetic diagnosis found a hereditary myopathy of the lipid metabolism (VLCAD deficiency). We used individualized performance analysis to determine the safe load limit. In the ordinary athletic testing of these performance limits, no excessive CK activation could be determined. Recurrent rhabdomyolyses characterize the late onset of VLCAD, which can occur after intense physical activity, periods of fasting or dehydration. Patients can continue physical exercise safely after determining the individual tolerance.

Characterization of exonic variants of uncertain significance in very long-chain acyl-CoA dehydrogenase identified through newborn screening.

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is an autosomal recessive disease resulting from mutations in the ACADVL gene and is among the disorders tested for in newborn screening (NBS). Confirmatory sequencing following suspected VLCADD NBS results often identifies variants of uncertain significance (VUS) in the ACADVL gene, leading to uncertainty of diagnosis and providing effective treatment regimen. Currently, ACADVL has >300 VUSs in the ClinVar database that requiring characterization to determine potential pathogenicity. In this study, CRISPR/Cas9 genome editing was used to knock out ACADVL in HEK293T cells, and targeted deletion was confirmed by droplet digital polymerase chain reaction (PCR). No VLCAD protein was detected and an 84% decrease in enzyme activity using the electron transfer flavoprotein fluorescence reduction assay and C21-CoA as substrate was observed compared to control. Plasmids containing control or variant ACADVL coding sequence were transfected into the ACADVL null HEK293T. While transfection of control ACADVL restored VLCAD protein and enzyme activity, cells expressing the VLCAD Val283Ala mutant had 18% VLCAD enzyme activity and reduced protein compared to control. VLCAD Ile420Leu, Gly179Arg, and Gln406Pro produced protein comparable to control but 25%, 4%, and 5% VLCAD enzyme activity, respectively. Leu540Pro and Asp570_Ala572dup had reduced VLCAD protein and 10% and 3% VLCAD enzyme activity, respectively. VLCADD fibroblasts containing the same variations had decreased VLCAD protein and activity comparable to the transfection experiments. Generating ACADVL null HEK293T cell line allowed functional studies to determine pathogenicity of ACADVL exonic variants. This approach can be applied to multiple genes for other disorders identified through NBS.

Tandem mass spectrometry and genetic variant analysis of four neonates with very long chain acyl-coenzyme A dehydrogenase deficiency

To analyze the clinical features and genetic variants in four neonates with very long chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency. Neonates with a tetradecenoylcarnitine (C14:1) concentration at above 0.4 μmol/L in newborn screening were recalled for re-testing. Four neonates were diagnosed with VLCAD deficiency by MS-MS and genetic testing, and their clinical features and genotypes were analyzed. All cases had elevated blood C14:1, and the values of first recalls were all lower than the initial test. In 2 cases, the C14:1 had dropped to the normal range. 1 case has remained at above 1 μmol/L after the reduction, and the remainder one case was slightly decreased. In total eight variants of the ADACVL genes were detected among the four neonates, which included 5 missense variants and 3 novel variants (p.Met344Val, p.Ala416Val, c.1077+6T>A). No neonate showed salient clinical manifestations. Above findings have enriched the spectrum of ADACVL gene mutations and provided a valuable reference for the screening and diagnosis of VLCAD deficiency.

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.

OP003: Characterization of variants of uncertain significance in very long-chain acyl-CoA dehyrogenase identified through newborn screening

Newborn screening identifies inborn errors of metabolism, which are often confirmed by DNA sequencing. However, sequencing often identifies variants of uncertain significance (VUS), leading to difficulty in diagnosis and treatment plans. Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is an autosomal recessive disease resulting from biallelic variants in ACADVL. Patients with VLCADD cannot utilize energy from long fatty acid chains and present with a variety of symptoms, including hypoglycemia and recurrent rhabdomyolysis.

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

The clinical severity of very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is difficult to predict using conventional diagnostic 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. 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. 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. 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.