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Ornithine transcarbamylase deficiency (OTCD) is the most common disorder of the urea cycle and is caused by a mutation of the OTC gene, located on chromosome X. Its prevalence is estimated at 1 in 80,000 to 56,500 births, but this X-chromosomal inheritance results in males being more affected than females. In neonates affected with this disorder, hyperammonemia after birth can lead to neurological and liver damage that can be fatal. We present a child with a prenatal diagnosis based on an older sibling with the same pathology, which led us to adopt an intensive treatment since the delivery. He was admitted in a neonatal unit and treatment with protein restriction, 10% glucose saline serum and glycerol phenylbutyrate was initiated. To date, after 3.5 years of follow up, growth and neurological development have been adequate, biochemical control has been appropriate except for a simple and mild decompensation during the course of a gastroenteritis. This case emphasises the importance of early diagnosis and treatment to avoid potential complications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor CP-G declared a past collaboration with the authors ÁM-R and EM-H., (Copyright © 2024 Martín-Rivada, Murray Hurtado and Martín-Hernández.)

Biochar obtained from sewage sludge serves as a valuable soil amendment in agriculture, enhancing soil properties by increasing the nutrient content, cation exchange capacity, water retention, and oxygen transmission. However, its utilisation is hampered by the presence of micropollutants such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and volatile organic compounds (VOCs). Previous studies indicate that the type and amount of micropollutants can be significantly adjusted by selecting the right process parameters. This literature review provides an overview of how (1) pyrolysis temperature, (2) carrier gas flow and type, (3) heating rate, and (4) residence time affect the concentration of micropollutants in biochar produced from sewage sludge. The micropollutants targeted are those listed by the European Biochar Certificate (EBC) and by the International Biochar Institution (IBI), including PAHs, PCDD/Fs, PCBs and VOCs. In addition, per- and poly-fluoroalkyl substances (PFAS) are also considered due to their presence in sewage sludge. The findings suggest that higher pyrolysis temperatures reduce micropollutant levels. Moreover, the injection of a carrier gas (N 2 or CO 2 ) during the pyrolysis and cooling processes effectively lowers PAHs and PCDD/Fs, by reducing the contact of biochar with oxygen, which is crucial in mitigating micropollutants. Nevertheless, limited available data impedes an assessment of the impact of these parameters on PFAS in biochar. In addition, further research is essential to understand the effects of carrier gas type, heating rate, and residence time in order to determine the optimal pyrolysis process parameters for generating clean biochar., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The application of sewage sludge to agricultural land is facing increasing restrictions due to concerns about various micropollutants, including polycyclic aromatic hydrocarbons (PAHs), dioxins and furans (PCDD/Fs), polychlorinated biphenyls (PCBs), per- and poly-fluoroalkyl substances (PFAS), and heavy metals (HMs). As an alternative approach to manage this residue, the use of pyrolysis, a process that transforms sludge into biochar, a carbon-rich solid material, is being explored. Despite the potential benefits of pyrolysis, there is limited data on its effectiveness in removing micropollutants and the potential presence of harmful elements in the resulting biochar. This study aims to evaluate the impact of the temperature and the use of a carrier gas (N 2 ) during a two-stage pyrolysis and cooling on micropollutant removal. Pilot-scale tests showed that a higher temperature (650 °C) and the use of a carrier gas (0.4 L/min N 2 ) during the pyrolysis and the cooling process led to a reduction of PAHs, PCDD/Fs, PCBs and PFAS below their detection limits. As such, the generated biochar aligns with the guidelines set by the International Biochar Initiative (IBI) and the European Biochar Certificate (EBC) for all micropollutants, except for zinc and copper. Additional investigation is required to determine whether the micropollutants undergo destruction or transition into other pyrolysis end-products, such as the gas or liquid phase., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Intensive livestock farming generates vast amounts of organic materials, which are an important source of nitrogen releases. These anthropogenic nitrogen releases contribute to multiple environmental problems, including eutrophication of water systems, contamination of drinking water sources, and greenhouse gas emissions. Nitrogen recovery and recycling are technically feasible, and there exists a number of processes for nitrogen recovery from livestock material in the form of different products. In this work, a multi-scale techno-economic assessment of techniques for nitrogen recovery and recycling is performed. The assessment includes a material flow analysis of each process, from material collection to final treatment, to determine nitrogen recovery efficiency, losses, and recovery cost, as well as an environmental cost-benefit analysis to compare the nitrogen recovery cost versus the economic losses derived from its uncontrolled release into the environment. The results show that transmembrane chemisorption process results in the lowest recovery cost, 3.4-10.4 USD per kilogram of nitrogen recovered in the range of studied processing scales. The recovery of nitrogen from livestock material through three technologies, i.e., transmembrane chemisorption, MAPHEX, and stripping in packed bed, reveales to be cost-effective. Since the economic losses due to the harmful effects of nitrogen into the environment are estimated at 32-35 USD per kilogram of nitrogen released, nitrogen recycling is an environmentally and economically beneficial approach to reduce nutrient pollution caused by livestock operations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Objectives: Identify the efficacy variables collected in the literature for therapies used in lysosomal storage diseases (LDS), evaluate the quality of this evidence, and know the effectiveness and safety of these treatments., Material and Methods: Retrospective observational study that included patients with LDS treated with enzyme replacement therapy (ERT) or substrate reduction therapy (SRT). Published clinical trials (CT) and LDS treatment guidelines were reviewed to select efficacy variables. Data to measure them (and adverse effects) were obtained from the medical history., Results: No CTs have been found in which efficacy is evaluated with final variables, all have been surrogated. Twenty-two patients were included: eight with Gaucher disease, six with Niemann-PickC disease, two with Hunter disease, one with Morquio-A disease, and five with Pompe disease. Eight patients have responded to ERT and one to SRT with eliglustat. ERT has not been associated with adverse effects. Miglustat has produced tolerance problems, requiring a change in a patient., Conclusions: The effectiveness was variable according to the pathology. Regarding safety, manageable adverse reactions to SRT were associated with dosage adjustments., (Copyright © 2022 Elsevier España, S.L.U. All rights reserved.)

Inborn errors of metabolism (IEM) constitute a huge group of rare diseases affecting 1 in every 1000 newborns. Next-generation sequencing has transformed the diagnosis of IEM, leading to its proposed use as a second-tier technology for confirming cases detected by clinical/biochemical studies or newborn screening. The diagnosis rate is, however, still not 100%. This paper reports the use of a personalized multi-omics (metabolomic, genomic and transcriptomic) pipeline plus functional genomics to aid in the genetic diagnosis of six unsolved cases, with a clinical and/or biochemical diagnosis of galactosemia, mucopolysaccharidosis type I (MPS I), maple syrup urine disease (MSUD), hyperphenylalaninemia (HPA), citrullinemia, or urea cycle deficiency. Eight novel variants in six genes were identified: six (four of them deep intronic) located in GALE , IDUA , PTS , ASS1 and OTC , all affecting the splicing process, and two located in the promoters of IDUA and PTS , thus affecting these genes' expression. All the new variants were subjected to functional analysis to verify their pathogenic effects. This work underscores how the combination of different omics technologies and functional analysis can solve elusive cases in clinical practice.

We report the clinical and genetic features of a Caucasian girl who presented a severe neurodevelopmental disorder with drug-resistant epilepsy, hypotonia, severe gastro-esophageal reflux and brain magnetic resonance imaging anomalies. WES uncovered a novel variant in homozygosis (g.197092814_197092824delinsC) in HECW2 gene that encodes the E3 ubiquitin-protein ligase HECW2. This protein induces ubiquitination and is implicated in the regulation of several important pathways involved in neurodevelopment and neurogenesis. Furthermore, de novo heterozygous missense variants in this gene have been associated with neurodevelopmental disorder with hypotonia, seizures, and absent language (NDHSAL). The homozygous variant of our patient disrupts the splice donor site of intron 22 and causes the elimination of exon 22 (r.3766_3917+1del) leading to an in-frame deletion of the protein (p.Leu1256_Trp1306del). Functional studies showed a twofold increase of its RNA expression, while the protein expression level was reduced by 60%, suggesting a partial loss-of-function mechanism of pathogenesis. Thus, this is the first patient with NDHSAL caused by an autosomal recessive splicing variant in HECW2., (© 2022 Wiley Periodicals LLC.)

Objectives: We present the results of our experience in the diagnosis and follow up of the positive cases for propionic, methylmalonic acidemias and cobalamin deficiencies (PA/MMA/MMAHC) since the Expanded Newborn Screening was implemented in Madrid Region., Methods: Dried blood samples were collected 48 h after birth. Amino acids and acylcarnitines were quantitated by MS/MS. Newborns with alterations were referred to the clinical centers for follow-up. Biochemical and molecular genetic studies for confirmation of a disease were performed., Results: In the period 2011-2020, 588,793 children were screened, being 953 of them were referred to clinical units for abnormal result (192 for elevated C3 levels). Among them, 88 were false positive cases, 85 maternal vitamin B12 deficiencies and 19 were confirmed to suffer an IEM (8 PA, 4 MMA, 7 MMAHC). Ten out 19 cases displayed symptoms before the NBS results (6 PA, 1 MMA, 3 MMAHC). C3, C16:1OH+C17 levels and C3/C2 and C3/Met ratios were higher in newborns with PA/MMA/MMAHC. Cases diagnosed with B12 deficiency had mean B12 levels of 187.6 ± 76.9 pg/mL and their mothers 213.7 ± 95.0; 5% of the mothers were vegetarian or had poor eating while 15% were diagnosed of pernicious anemia. Newborns and their mothers received treatment with B12 with different posology, normalizing their levels and the secondary alterations disappeared., Conclusions: Elevated C3 are a frequent cause for abnormal result in newborn screening with a high rate of false positive cases. Presymptomatic diagnosis of most of PA and some MMA/MMAHC is difficult. Vitamin B12 deficiency secondary to maternal deprivation is frequent with an heterogenous clinical and biochemical spectrum., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

Modeling and optimization are essential tasks that arise in the analysis and design of supply chains (SCs). SC models are essential for understanding emergent behavior such as transactions between participants, inherent value of products exchanged, as well as impact of externalities (e.g., policy and climate) and of constraints. Unfortunately, most users of SC models have limited expertise in mathematical optimization, and this hinders the adoption of advanced decision-making tools. In this work, we present ADAM, a web platform that enables the modeling and optimization of SCs. ADAM facilitates modeling by leveraging intuitive and compact graph-based abstractions that allow the user to express dependencies between locations, products, and participants. ADAM model objects serve as repositories of experimental, technology, and socio-economic data; moreover, the graph abstractions facilitate the organization and exchange of models and provides a natural framework for education and outreach. Here, we discuss the graph abstractions and software design principles behind ADAM, its key functional features and workflows, and application examples., Competing Interests: Declaration of Competing Interest This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue. The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript

Background and objectives: Glycerol phenylbutyrate (GPB) has demonstrated safety and efficacy in patients with urea cycle disorders (UCDs) by means of its clinical trial program, but there are limited data in clinical practice. In order to analyze the efficacy and safety of GPB in clinical practice, here we present a national Spanish experience after direct switching from another nitrogen scavenger to GPB. Methods: This observational, retrospective, multicenter study was performed in 48 UCD patients (age 11.7 ± 8.2 years) switching to GPB in 13 centers from nine Spanish regions. Clinical, biochemical, and nutritional data were collected at three different times: prior to GPB introduction, at first follow-up assessment, and after one year of GPB treatment. Number of related adverse effects and hyperammonemic crisis 12 months before and after GPB introduction were recorded. Results: GPB was administered at a 247.8 ± 102.1 mg/kg/day dose, compared to 262.6 ± 126.1 mg/kg/day of previous scavenger (46/48 Na-phenylbutyrate). At first follow-up (79 ± 59 days), a statistically significant reduction in ammonia (from 40.2 ± 17.3 to 32.6 ± 13.9 μmol/L, p < 0.001) and glutamine levels (from 791.4 ± 289.8 to 648.6 ± 247.41 μmol/L, p < 0.001) was observed. After one year of GPB treatment (411 ± 92 days), we observed an improved metabolic control (maintenance of ammonia and glutamine reduction, with improved branched chain amino acids profile), and a reduction in hyperammonemic crisis rate (from 0.3 ± 0.7 to less than 0.1 ± 0.3 crisis/patients/year, p = 0.02) and related adverse effects (RAE, from 0.5 to less than 0.1 RAEs/patients/year p < 0.001). Conclusions: This study demonstrates the safety of direct switching from other nitrogen scavengers to GPB in clinical practice, which improves efficacy, metabolic control, and RAE compared to previous treatments.

Hyperammonaemia is a metabolic derangement that may cause severe neurological damage and even death due to cerebral oedema, further complicating the prognosis of its triggering disease. In small children it is a rare condition usually associated to inborn errors of the metabolism. As age rises, and especially in adults, it may be precipitated by heterogeneous causes such as liver disease, drugs, urinary infections, shock, or dehydration. In older patients, it is often overlooked, or its danger minimized. This protocol was drafted to provide an outline of the clinical measures required to normalise ammonia levels in patients of all ages, aiming to assist clinicians with no previous experience in its treatment. It is an updated protocol developed by a panel of experts after a review of recent publications. We point out the importance of frequent monitoring to assess the response to treatment, the nutritional measures that ensure not only protein restriction but adequate caloric intake and the need to avoid delays in the use of specific pharmacological therapies and, especially, extrarenal clearance measures. In this regard, we propose initiating haemodialysis when ammonia levels are >200−350 µmol/L in children up to 18 months of age and >150−200 µmol/L after that age.

The determination of acylcarnitines (AC) in dried blood spots (DBS) by tandem mass spectrometry in newborn screening (NBS) programs has enabled medium-chain acyl-coA dehydrogenase deficiency (MCADD) to be identified in presymptomatic newborns. Nevertheless, different confirmatory tests must be performed to confirm the diagnosis. In this work, we have collected and analyzed the NBS results and confirmatory test results (plasma AC, molecular findings, and lymphocyte MCAD activity) of forty individuals, correlating them with clinical outcomes and treatment, with the aim of obtaining useful diagnostic information that could be applied in the follow-up of the patients. Our results led us to classify patients into two groups. The first group (14 cases) had high increased octanoylcarnitine (C8) levels, biallelic pathogenic variants, and severe impaired enzyme activity (<10% of the intra-assay control (IAC)); all of these cases received nutritional therapy and required carnitine supplementation during follow-up, representing the most severe form of the disease. The second group (16 patients) was a heterogeneous group presenting moderate increases in C8, biallelic likely pathogenic/pathogenic variants, and intermediate activity (<41% IAC). All of them are currently asymptomatic and could be considered as having a milder form of the disease. Finally, eight cases presented a normal−mild increase in plasma C8, with only one pathogenic variant detected, and high−intermediate residual activity (15−100%). Based on our results, we confirm that combined evaluation of acylcarnitine profiles, genetic findings, and residual enzyme activities proves useful in predicting the risk of future metabolic decompensation, in making decisions regarding future treatment or follow-up, and also in confirming the clinical effects of unknown clinical variants.

Livestock operations have been highly intensified over the last decades, resulting in the advent of large concentrated animal feeding operations (CAFOs). Intensification decreases production costs but also leads to substantial environmental impacts. Specifically, nutrient runoff from livestock waste results in eutrophication, harmful algal blooms, and hypoxia. The implementation of nutrient recovery systems in CAFOs can abate nutrient releases and negative ecosystem responses, although they might negatively affect the economic performance of CAFOs. We design and analyze potential incentive policies for the deployment of phosphorus recovery technologies at CAFOs considering the geospatial vulnerability to nutrient pollution. The case study demonstration consists of 2217 CAFOs in the U.S. Great Lakes area. The results reveal that phosphorus recovery is more economically viable in the largest CAFOs due to economies of scale, although they also represent the largest eutrophication threats. For small and medium-scale CAFOs, phosphorus credits progressively improve the profitability of nutrient management systems. The integration of biogas production does not improve the economic performance of phosphorus recovery systems at most of CAFOs, as they lack enough size to be cost-effective. Phosphorus recovery proves to be economically beneficial by comparing the net costs of nutrient management systems with the negative economic impact derived from phosphorus releases. The incentives necessary for avoiding up to 20.7×10 3 ton/year phosphorus releases and achieve economic neutrality in the Great Lakes area are estimated at $223 million/year. Additionally, the fair distribution of limited incentives is studied using a Nash allocation scheme, determining the break-even point for allocating monetary resources., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

We present the results of our experience in the diagnosis of inborn errors of metabolism (IEM) since the Expanded Newborn Screening was implemented in our Region. Dried blood samples were collected 48 h after birth. Amino acids and acylcarnitines were quantitated by mass spectrometry (MS)/MS. Newborns with alterations were referred to the clinical centers for follow-up. Biochemical and molecular genetic studies for confirmation of a disease were performed. In the period 2011 to 2019, 592 822 children were screened: 902 of them were referred for abnormal results. An IEM was confirmed in 222 (1/2670): aminoacidopathies: 89 hyperphenylalaninemia (HPA) (51 benign HPA, 32 phenylketonuria, 4 DNAJC12 defect, and 2 primapterinuria), 6 hypermethioninemia, 3 tyrosinemia type 1 (TYR-1), 1 TYR-3, 4 maple syrup urine disease (MSUD), 2 branched-chain amino acid transferase 2 deficiency, 2 homocystinuria, 1 cystinuria, 2 ornithine transcarbamylase (OTC) deficiency, 2 citrullinemia type I (CTLN1); FAO defects: 43 medium-chain acyl-CoA dehydrogenase deficiency (MCADD), 13 very long-chain acyl-CoA dehydrogenase deficiency, 2 long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), 1 multiple acyl-coA dehydrogenation deficiency, 11 systemic primary carnitine deficiency, 2 carnitine palmitoyltransferase type 2 (CPT-II) deficiency, 1 CPT-I deficiency; organic acidurias: 12 glutaric aciduria type 1 (GA-1), 4 methylmalonic acidemia (MMA), 7 MMA including combined cases with homocystinuria (MMAHC), 6 propionic acidemia (PA), 7 3-methylcrotonyl-CoA carboxylase, 1 3-hydroxy-3-methylglutaryl-CoA lyase deficiency lyase deficiency. Only 19 infants (8.5%) were symptomatic at newborn screening result (1 LCHADD, 5 PA, 1 CPT-II deficiency, 1 MMA, 3 MMAHC, 2 MSUD, 2 OTC deficiency, 1 CTLN1, 1 MCADD, 2 TYR-1). No false negative cases were identified. Genetic diagnosis was conclusive in all biochemically confirmed cases, except for two infants with HPA, identifying pathogenic variants in 32 different genes. The conditions with the highest incidence were HPA (1/6661) and MCAD deficiencies (1/13 787)., Competing Interests: The authors declare no potential conflict of interest., (© 2021 The Authors. JIMD Reports published by John Wiley & Sons Ltd on behalf of SSIEM.)

Nutrient pollution of waterbodies is a major worldwide water quality problem. Excessive use and discharge of nutrients can lead to eutrophication and algal blooms in fresh and marine waters, resulting in environmental problems associated with hypoxia, public health issues related to the release of toxins and freshwater scarcity. A promising option to address this problem is the recovery of nutrient releases prior to being discharged into the environment. Driven by the sustainable materials management concept, the COW2NUTRIENT (Cattle Organic Waste to NUTRIent and ENergy Technologies) framework is developed for the techno-economic evaluation and selection of nutrient recovery systems at livestock facilities. Environmental vulnerability to nutrient pollution determined through a geographic information system (GIS)-based model and techno-economic information of different state-of-the-art nutrient management technologies are combined in a multi-criteria decision analysis (MCDA) model, resulting in the selection and economic analysis of the most suitable process for each studied livestock facility. This framework has been employed for studying the implementation of sustainable phosphorus management systems at 2,217 livestock facilities in the Great Lakes area, resulting in capital expenses of 2.5 billion USD if only phosphorus recovery technologies are installed, and up to 5.2 billion USD if nutrient management is combined with biogas and power production. However, considering potential economic incentives for the recovery of phosphorus, net revenues up to 230 million USD per year can be achieved. Therefore, the framework presented reveals the potential of implementing nutrient management systems at regional scale for the abatement of phosphorus releases from livestock facilities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Background: Mucopolysaccharidosis (MPS) IVA or Morquio A syndrome is a progressive and disabling disease characterized by a deficiency of the enzyme N-acetylgalactosamine-6-sulphate sulphatase. Its clinical presentation is very heterogeneous and poorly understood in adults. The aim of this study was to describe the clinical manifestations of MPS IVA in adult patients in Spain and to assess their health-related quality of life (HRQoL)., Results: Thirty-three patients from nine reference centres participated in the study. The median age was 32 (interquartile range [IQR]: 20.5-40.5) years. The phenotype was classical in 54.5% of patients, intermediate in 33.3% of patients, and non-classical in 12.1% of patients. The most common clinical manifestation was bone dysplasia, with a median height of 118 (IQR: 106-136) cm. Other frequent clinical manifestations were hearing loss (75.7%), ligamentous laxity (72.7%), odontoid dysplasia (69.7%), limb deformities that required orthopaedic aids (mainly hip dysplasia and genu valgus) (63.6%), and corneal clouding (60.6%). In addition, 36.0% of patients had obstructive sleep apnoea/hypopnoea syndrome and 33.3% needed non-invasive ventilation. Cervical surgery and varisation osteotomy were the most common surgical interventions (36.4% each). Almost 80% of patients had mobility problems and 36.4% used a wheelchair at all times. Furthermore, 87.9% needed help with self-care, 33.3% were fully dependent, and 78.8% had some degree of pain. HRQoL according to the health assessment questionnaire was 1.43 (IQR: 1.03-2.00) in patients with the non-classical phenotype, but 2.5 (IQR: 1.68-3.00) in those with the classical phenotype. Seven patients were initiated on enzyme replacement therapy (ERT), but two of them were lost to follow-up. Lung function improved in four patients and slightly worsened in one patient. The distance achieved in the six-minute walk test increased in the four patients who could perform it. HRQoL was better in patients treated with elosulfase alfa, with a median (IQR) of 1.75 (1.25-2.34) versus 2.25 (1.62-3.00) in patients not treated with ERT., Conclusions: The study provides real-world data on patients with MPS IVA. Limited mobility, difficulties with self-care, dependence, and pain were common, together with poor HRQoL. The severity and heterogeneity of clinical manifestations require the combined efforts of multidisciplinary teams., (© 2021. The Author(s).)

Atypical microcytic anemias are rare diseases of iron/heme metabolism that can be diagnostically challenging. We report the case of a 2-year-old twin boy with neurodevelopmental delay and persistent microcytosis in whom atypical microcytic anemias was initially suspected. He had low blood iron and transferrin saturation with normal/high ferritin despite iron therapy. Hemoglobinopathies were excluded by conventional/DNA studies. Hepcidin was high but iron-refractory-iron-deficiency anemia was ruled out by a genetic panel. Bone marrow aspiration revealed foamy cells and iron depletion. A genetic study confirmed the diagnosis of Niemann-Pick disease type C which was finally considered the origin of microcytosis through anemia of chronic disease., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

We report the clinical, biochemical and genetic findings from a Spanish boy of Caucasian origin who presented with fever-dependent RALF (recurrent acute liver failure) and osteogenesis imperfecta (OI). Whole-exome sequencing (WES) uncovered two compound heterozygous variants in NBAS (c.[1265 T > C];[1549C > T]:p.[(Leu422Pro)];[(Arg517Cys)]), and a heterozygous variant in P4HB (c.[194A > G];[194=]:p.[(Lys65Arg)];[(Lys65=)]) that was transmitted from the clinically unaffected mother who was mosaic carrier of the variant. Variants in NBAS protein have been associated with ILFS2 (infantile liver failure syndrome-2), SOPH syndrome (short stature, optic nerve atrophy, and Pelger-Huët anomaly syndrome), and multisystem diseases. Several patients showed clinical manifestations affecting the skeletal system, such as osteoporosis, pathologic fractures and OI. Experiments in the patient's fibroblasts demonstrated that mutated NBAS protein is overexpressed and thermally unstable, and reduces the expression of MGP, a regulator of bone homeostasis. Variant in PDI (protein encoded by P4HB) has been associated with CLCRP1 (Cole-Carpenter syndrome-1), a type of severe OI. An increase of COL1A2 protein retention was observed in the patient's fibroblasts. In order to study if the variant in P4HB was involved in the alteration in collagen trafficking, overexpression experiments of PDI were carried out. These experiments showed that overexpression of mutated PDI protein produces an increase in COL1A2 retention. In conclusion, these results corroborate that the variants in NBAS are responsible for the liver phenotype, and demonstrate that the variant in P4HB is involved in the bone phenotype, probably in synergy with NBAS variants., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: Tandem mass spectrometry (MS/MS) is being used for newborn screening since this laboratory testing technology increases the number of metabolic disorders that can be detected from dried blood-spot specimens. In the Community of Madrid, it was implemented in March 2011 and it includes 13 aminoacidopathies, fatty acid oxidation disorders and organic acidemias. The aim of this study was to describe our experience and evaluate the screening positive cases in a period of 9 years (2011-2019)., Methods: During the period of the study, a total of 592.822 neonates were screened with this expanded program by MS/MS in the Community of Madrid. Amino acids, acylcarnitines, and succinylacetone were quantified in all samples that met the quality criteria. Means, medians, percentiles and standard deviation of the analytes and ratios of interest were calculated., Results: 901 patients (0,15 %) with a positive screening test were referred to clinical evaluation. 230 patients were diagnosed of 30 different inborn errors of metabolism (prevalence 1:2577), 11 of which were not included as a target in the Community of Madrid newborn screening program. The global positive predictive value was 25,6 %. During this period of time, two false negative cases were detected. The most prevalent disorders were phenylketonuria/hyperphenylalaninemia and medium chain acyl-CoA dehydrogenase deficiency (1:6444 and 1:13174 respectively). 93 % of the patients were detected in the presymptomatic stage., Conclusions: During the last 9 years a large number of cases of IEM have been detected with an acceptable global positive predictive value. These results confirm the utility of inborn errors of metabolism newborn screening as a public health program., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Nutrient pollution is one of the major worldwide water quality problems, resulting in environmental and public health issues. Agricultural activities are the main source of nutrient release emissions, and the livestock industry has been proven to be directly related to the presence of high concentrations of phosphorus in the soil, which potentially can reach waterbodies by runoff. To mitigate the phosphorus pollution of aquatic systems, the implementation of nutrient recovery processes allows the capture of phosphorus, preventing its release into the environment. Particularly, the use of struvite precipitation produces a phosphorus-based mineral that is easy to transport, enabling redistribution of phosphorus to deficient locations. However, livestock leachate presents some characteristics that hinder struvite precipitation, preventing extrapolation of the results obtained from wastewater studies to cattle waste. Consideration of these elements is essential to determine the optimal operating conditions for struvite formation, and for predicting the amount of struvite recovered. In this work, a detailed thermodynamic model for precipitates formation from cattle waste is used to develop surrogate models to predict the formation of struvite and calcium precipitates from cattle waste. The variability in the organic waste composition, and how it affects the production of struvite, is captured through a probability framework based on the Monte Carlo method embedded in the model. Consistent with the developed surrogate models, the potential of struvite production to reduce the phosphorus releases from the cattle industry to watersheds in the United States has been assessed. Also, the more vulnerable locations to nutrient pollution were determined using the techno-ecological synergy sustainability metric (TES) by evaluating the spatial distribution and balance of phosphorus from agricultural activities. Although only struvite formation from cattle operations is considered, reductions between 22% and 36% of the total phosphorus releases from the agricultural sector, including manure releases and fertilizer application, can be achieved., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Urea cycle disorders are congenital metabolism errors that affect ammonia elimination. Clinical signs and prognosis are strongly influenced by peak ammonia levels. Numerous triggers associated with metabolic decompensation have been described with many of them, including fasting or stress, being related to the perioperative period., Aims: We aimed to assess perioperative complications in pediatric patients with urea cycle disorders requiring general anesthesia in our center., Methods: We reviewed the clinical history of all the pediatric patients with a confirmed urea cycle disorders diagnosis requiring surgery or a diagnostic procedure with anesthesia between January 2002 and June 2018., Results: We included 33 operations (major surgery, minor surgery, and diagnostic procedures) carried out on 10 patients via different anesthetic techniques. We observed the following complications: intraoperative hyperglycemia in one case, postoperative vomiting in eight cases, and slightly increased postoperative ammonia levels (54, 59, and 69 µmol/L) with normal preoperative levels in three cases without associated metabolic decompensation. There were two cases of perioperative hyperammonemia (72 and 69 µmol/L) secondary to preoperative metabolic decompensation (137 and 92 µmol/L) with the levels progressively dropping and normalizing in the first 24-48 hours, respectively., Conclusions: Procedures under anesthesia on pediatric patients with urea cycle diseases should be performed by experienced multidisciplinary teams at specialized centers. Perioperative management focused on avoiding catabolism (especially during fasting) and monitoring signs associated with metabolic decompensation to allow for its early treatment should be included in routine anesthetic techniques for children with urea cycle disorders., (© 2020 John Wiley & Sons Ltd.)

We report the case of a Caucasian Spanish origin female who showed severe psychomotor developmental delay, hypotonia, strabismus, epilepsy, short stature, and poor verbal language development. Brain magnetic resonance imaging scans showed thickened corpus callosum, cortical malformations, and dilated and abnormal configuration of the lateral ventricles without hydrocephalus. Whole-exome sequence uncovered a de novo variant in the microtubule associated serine/threonine kinase 1 gene (MAST1; NM&#95;014975.3:c.1565G>A:p.(Gly522Glu)) that encodes for the MAST1. Only 12 patients have been identified worldwide with 10 different variants in this gene: six patients with mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations; two patients with microcephaly and cerebellar hypoplasia; two patients with autism, one patient with diplegia, and one patient with microcephaly and dysmorphism. Our patient shows a new phenotypic subtype defined by mega-corpus-callosum syndrome with cortical malformations without cerebellar hypoplasia. In conclusion, our data expand the phenotypic spectrum associated to MAST1 gene variants., (© 2020 Wiley Periodicals, Inc.)

We report the case of a Caucasian Spanish boy, who showed profound neonatal hypotonia, feeding difficulties, apnea, severe developmental delay, epilepsy, bilateral convergent strabismus, poor verbal language development and a large brainstem. Whole-exome sequence uncovered a novel de novo mutation in the purine-rich element binding protein A gene ( PURA ; NM&#95;005859.4:c.72del:p.(-Gly25AlafsTer53)) that encodes the transcriptional activator protein Pur-alpha (PURA). Mutations in this gene have been identified in patients with PURA syndrome, a rare disorder characterized by an early hypotonia, developmental delay, severe intellectual disability with or without epilepsy, and disability in expressive language development. Although, up to 75 cases have been identified worldwide, to the best of our knowledge, this is the first patient described with a brainstem larger than normal. In conclusion, our data expand both geneticand phenotypic spectrum associated with PURA gene mutations.

Purpose: Salts of phenylacetic acid (PAA) and phenylbutyric acid (PBA) have been used for nitrogen elimination as a treatment for hyperammonaemia caused by urea cycle disorders (UCD). A new analytical method for PBA measurement in urine which helps to evaluate the drug adherence has been implemented., Methods: Urine specimens from UCD patients receiving PBA were analysed by tandem mass spectrometry to measure urine phenylacetylglutamine (PAGln). Some clinical and biochemical data for each patient were collected., Results: Our study included 87 samples from 40 UCD patients. The PAGln levels did not correlate with height, weight or age. However, the PAGln values showed correlation with PBA dose (r = 0.383, P = 0.015). Plasma glutamine and ammonia levels presented a positive correlation (r = 0.537, P < 0.001). The stability for PAGln in urine was determined at different storage temperatures., Conclusions: We have developed a simple method for the determination of PAGln in urine, which acts as useful biomarker of effective drug delivery. PAGln in urine is stable at room temperature at least for 15 days, and for several months when frozen at -20 °C. This procedure is useful for the optimization and monitorization of the drug dose allowing the use of spot urine samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Congenital lactic acidosis (CLA) is a rare condition in most instances due to a range of inborn errors of metabolism that result in defective mitochondrial function. Even though the implementation of next generation sequencing has been rapid, the diagnosis rate for this highly heterogeneous allelic condition remains low. The present work reports our group's experience of using a clinical/biochemical analysis system in conjunction with genetic findings that facilitates the taking of timely clinical decisions with minimum need for invasive procedures. The system's workflow combines different metabolomics datasets and phenotypic information with the results of clinical exome sequencing and/or RNA analysis. The system's use detected genetic variants in 64% of a cohort of 39 CLA-patients; these variants, 14 of which were novel, were found in 19 different nuclear and two mitochondrial genes. For patients with variants of unknown significance, the genetic analysis was combined with functional genetic and/or bioenergetics analyses in an attempt to detect pathogenicity. Our results warranted subsequent testing of antisense therapy to rescue the abnormal splicing in cultures of fibroblasts from a patient with a defective GFM1 gene. The discussed system facilitates the diagnosis of CLA by avoiding the need to use invasive techniques and increase our knowledge of the causes of this condition.

Treatment with nitisinone (NTBC) has brought about a drastic improvement in the treatment and prognosis of hereditary tyrosinemia type I (HT1). We conducted a retrospective observational multicentric study in Spanish HT1 patients treated with NTBC to assess clinical and biochemical long-term evolution.We evaluated 52 patients, 7 adults and 45 children, treated with NTBC considering: age at diagnosis, diagnosis by clinical symptoms, or by newborn screening (NBS); phenotype (acute/subacute/chronic), mutational analysis; symptoms at diagnosis and clinical course; biochemical markers; doses of NTBC; treatment adherence; anthropometric evolution; and neurocognitive outcome.The average follow-up period was 6.1 ± 4.9 and 10.6 ± 5.4 years in patients with early and late diagnosis respectively. All patients received NTBC from diagnosis with an average dose of 0.82 mg/kg/d. All NBS-patients (n = 8) were asymptomatic at diagnosis except 1 case with acute liver failure, and all remain free of liver and renal disease in follow-up. Liver and renal affectation was markedly more frequent at diagnosis in patients with late diagnosis (P < .001 and .03, respectively), with ulterior positive hepatic and renal course in 86.4% and 93.2% of no-NBS patients, although 1 patient with good metabolic control developed hepatocarcinoma.Despite a satisfactory global nutritional evolution, 46.1% of patients showed overweight/obesity. Interestingly lower body mass index was observed in patients with good dietary adherence (20.40 ± 4.43 vs 24.30 ± 6.10; P = .08) and those with good pharmacological adherence (21.19 ± 4.68 vs 28.58 ± 213.79).intellectual quotient was ≥85 in all NBS- and 68.75% of late diagnosis cases evaluated, 15% of which need pedagogical support, and 6.8% (3/44) showed school failure.Among the 12 variants identified in fumarylacetoacetate hydrolase gene, 1 of them novel (H63D), the most prevalent in Spanish population is c.554-1 G>T.After NTBC treatment a reduction in tyrosine and alpha-fetoprotein levels was observed in all the study groups, significant for alpha-fetoprotein in no NBS-group (P = .03), especially in subacute/chronic forms (P = .018).This series confirms that NTBC treatment had clearly improved the prognosis and quality of life of HT1 patients, but it also shows frequent cognitive dysfunctions and learning difficulties in medium-term follow-up, and, in a novel way, a high percentage of overweight/obesity.

We report the clinical, biochemical and genetic findings from a Spanish girl of Caucasian origin who presented with macrocephaly, dysmorphic facial features, developmental delay, hypotonia, combined oxidative phosphorylation (OxPhos) deficiency, epilepsy and anti-phospholipid antibodies (aPL). Whole-exome sequencing (WES) uncovered a heterozygous variant in the MTOR gene (NM&#95;004958.3: c.7235A>T: p.(Asp2412Val)) that encodes for the Serine/threonine-protein kinase mTOR. The substrates phosphorylation experiments demonstrated that this variant exerts its effect by gain-of-function (GOF) and autosomal dominant mechanism. GOF variants in this protein have been associated with Smith-Kingsmore syndrome (SKS), a rare autosomal dominant disorder characterized by intellectual disability, macrocephaly, seizure, developmental delay and dysmorphic facial features. Furthermore, the mTOR pathway has been demonstrated previously to be involved in many types of endothelium injuries including the antiphospholipid syndrome (APS), a systemic autoimmune disease characterized by the production of aPL with recurrent vascular thrombosis. Therefore, our patient is the first one with an mTOR variant and diagnosed with SKS and APS. In conclusion, our data expand both the genetic and phenotypic spectrum associated with MTOR gene variants.

Objective: Thymidine kinase 2, encoded by the nuclear gene TK2, is required for mitochondrial DNA maintenance. Autosomal recessive TK2 mutations cause depletion and multiple deletions of mtDNA that manifest predominantly as a myopathy usually beginning in childhood and progressing relentlessly. We investigated the safety and efficacy of deoxynucleoside monophosphate and deoxynucleoside therapies., Methods: We administered deoxynucleoside monophosphates and deoxynucleoside to 16 TK2-deficient patients under a compassionate use program., Results: In 5 patients with early onset and severe disease, survival and motor functions were better than historically untreated patients. In 11 childhood and adult onset patients, clinical measures stabilized or improved. Three of 8 patients who were nonambulatory at baseline gained the ability to walk on therapy; 4 of 5 patients who required enteric nutrition were able to discontinue feeding tube use; and 1 of 9 patients who required mechanical ventilation became able to breathe independently. In motor functional scales, improvements were observed in the 6-minute walk test performance in 7 of 8 subjects, Egen Klassifikation in 2 of 3, and North Star Ambulatory Assessment in all 5 tested. Baseline elevated serum growth differentiation factor 15 levels decreased with treatment in all 7 patients tested. A side effect observed in 8 of the 16 patients was dose-dependent diarrhea, which did not require withdrawal of treatment. Among 12 other TK2 patients treated with deoxynucleoside, 2 adults developed elevated liver enzymes that normalized following discontinuation of therapy., Interpretation: This open-label study indicates favorable side effect profiles and clinical efficacy of deoxynucleoside monophosphate and deoxynucleoside therapies for TK2 deficiency. ANN NEUROL 2019;86:293-303., (© 2019 American Neurological Association.)

Introduction: Population ageing requires that health and social systems focus their attention on identifying frailty in the elderly. In the Canary Islands, there are no studies to determine the prevalence of frailty among its population. The objective of this study is to determine the prevalence and profile of frailty in the island of La Palma, Canary Islands, Spain., Material and Method: A cross-sectional study was conducted to estimate the prevalence and the profile of frailty. The sample were residents over 70 years old, valued by the Fried criteria, and taking into account other related factors. The prevalence is offered with a confidence interval of 95% and is compared with that of other Spanish populations. To determine the profile, a simple comparison of variables was made, followed by using them in logistic regression models. All the tests were bilateral at a P≤0.05 level., Results: The prevalence of frailty in people over 70 years was estimated at 20% (17-23%). This prevalence shows differences with those of other Spanish populations. The factors that showed a relationship with frailty were, being female, widowed, living alone, low physical activity, cognitive impairment, depression, polymedication, and adverse clinical history. Multivariate analysis identifies factors associated with the frailty variables related to marital status, co-existence, polypharmacy, depressive states, and lack of physical exercise., Conclusions: The elderly population of La Palma have greater frailty compared to that described in other regions of Spain, with their profile being that of a widowed person, with depression, polymedicated, living alone, and not exercising., (Copyright © 2018 SEGG. Publicado por Elsevier España, S.L.U. All rights reserved.)

The congenital disorders of glycosylation (CDG) are defects in glycoprotein and glycolipid glycan synthesis and attachment. They affect multiple organ/systems, but non-specific symptoms render the diagnosis of the different CDG very challenging. Phosphomannomutase 2 (PMM2)-CDG is the most common CDG, but advances in genetic analysis have shown others to occur more commonly than previously thought. The present work reports the clinical and mutational spectrum of 25 non-PMM2 CDG patients. The most common clinical symptoms were hypotonia (80%), motor or psychomotor disability (80%) and craniofacial dysmorphism (76%). Based on their serum transferrin isoform profile, 18 were classified as CDG-I and 7 as CDG-II. Pathogenic variations were found in 16 genes (ALG1, ALG6, ATP6V0A2, B4GALT1, CCDC115, COG7, DOLK, DPAGT1, DPM1, GFPT1, MPI, PGM1, RFT1, SLC35A2, SRD5A3, and SSR4). Overall, 27 variants were identified, 12 of which are novel. The results highlight the importance of combining genetic and biochemical analyses for the early diagnosis of this heterogeneous group of disorders., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The present work describes the value of genetic analysis as a confirmatory measure following the detection of suspected inborn errors of metabolism in the Spanish newborn mass spectrometry screening program. One hundred and forty-one consecutive DNA samples were analyzed by next-generation sequencing using a customized exome sequencing panel. When required, the Illumina extended clinical exome panel was used, as was Sanger sequencing or transcriptional profiling. Biochemical tests were used to confirm the results of the genetic analysis. Using the customized panel, the metabolic disease suspected in 83 newborns (59%) was confirmed. In three further cases, two monoallelic variants were detected for two genes involved in the same biochemical pathway. In the remainder, either a single variant or no variant was identified. Given the persistent absence of biochemical alterations, carrier status was assigned in 39 cases. False positives were recorded for 11. In five cases in which the biochemical pattern was persistently altered, further genetic analysis allowed the detection of two variants affecting the function of BCAT2, ACSF3, and DNAJC12, as well as a second, deep intronic variant in ETFDH or PTS. The present results suggest that genetic analysis using extended next-generation sequencing panels can be used as a confirmatory test for suspected inborn errors of metabolism detected in newborn screening programs. Biochemical tests can be very helpful when a diagnosis is unclear. In summary, simultaneous genomic and metabolomic analyses can increase the number of inborn errors of metabolism that can be confirmed following suggestive newborn screening results.

Background: ATP8A2 mutations have recently been described in several patients with severe, early-onset hypotonia and cognitive impairment. The aim of our study was to characterize the clinical phenotype of patients with ATP8A2 mutations., Methods: An observational study was conducted at multiple diagnostic centres. Clinical data is presented from 9 unreported and 2 previously reported patients with ATP8A2 mutations. We compare their features with 3 additional patients that have been previously reported in the medical literature., Results: Eleven patients with biallelic ATP8A2 mutations were identified, with a mean age of 9.4 years (range 2.5-28 years). All patients with ATP8A2 mutations (100%) demonstrated developmental delay, severe hypotonia and movement disorders, specifically chorea or choreoathetosis (100%), dystonia (27%) and facial dyskinesia (18%). Optic atrophy was observed in 78% of patients for whom funduscopic examination was performed. Symptom onset in all (100%) was noted before 6 months of age, with 70% having symptoms noted at birth. Feeding difficulties were common (91%) although most patients were able to tolerate pureed or thickened feeds, and 3 patients required gastrostomy tube insertion. MRI of the brain was normal in 50% of the patients. A smaller proportion was noted to have mild cortical atrophy (30%), delayed myelination (20%) and/or hypoplastic optic nerves (20%). Functional studies were performed on differentiated induced pluripotent cells from one child, which confirmed a decrease in ATP8A2 expression compared to control cells., Conclusions: ATP8A2 gene mutations have emerged as the cause of a novel neurological phenotype characterized by global developmental delays, severe hypotonia and hyperkinetic movement disorders, the latter being an important distinguishing feature. Optic atrophy is common and may only become apparent in the first few years of life, necessitating repeat ophthalmologic evaluation in older children. Early recognition of the cardinal features of this condition will facilitate diagnosis of this complex neurologic disorder.

Introduction: Pyridoxine-dependent epilepsy (PDE) is a rare disorder of the lysine metabolism, characterized by a pharmacoresistant epileptic encephalopathy that usually begins in the neonatal period. However, its phenotypic spectrum is wide and not limited to seizures. We report a new case of PDE who developed hydrocephalus, along with an exhaustive review of the literature., Case Report: Our patient presented with seizures at 13 h of life. Antiepileptic drugs, vitamins and cofactors were required to achieve seizure control. Laboratory tests were congruent with PDE. She remained seizure-free until age five months, when seizures reappeared in the context of increasing head size and irritability. A cranial ultrasound showed hydrocephalus, for which she underwent ventriculoperitoneal shunting., Discussion: Seven other patients with same features have been previously reported. Seizure onset occurred within the first 7 days in all patients. Most of the children developed hydrocephalus at 6-7 months of age. In 4 out of 7 a genetic mutation was identified, despite the accurate etiology of hydrocephalus was unknown in most of them. The case we report behaved similarly to the others previously described. We postulate that the pathogenesis of this complication could be related to the high expression of antiquitin in choroid plexus epithelium, where the cerebrospinal fluid is produced., Conclusions: patients with PDE should be closely monitored, since they may present severe complications. We highlight the development of hydrocephalus, an uncommon but potentially life-threatening problem reported in 8 patients up to present time., (Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

We report the clinical and biochemical findings from a patient who presented with Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS), an autosomal-dominant disorder characterized by optic atrophy, developmental delay and intellectual disability. In addition, the patient also displays hypotonia, stroke-like episodes, and complex IV deficiency of the mitochondrial respiratory chain. Whole-exome sequencing (WES) uncovered a novel heterozygous mutation in the NR2F1 gene (NM&#95;005654:c.286A>G:p.Lys96Glu) that encodes for the COUP transcription factor 1 protein (COUP-TF1). Loss-of-function mutations in this protein have been associated with BBSOAS, and a luciferase reporter assay showed that this variant, in the zinc-finger DNA-binding domain (DBD) of COUP-TF1 protein, impairs its transcriptional activity. The additional features of this patient are more related with mitochondrial diseases that with BBSOAS, indicating a mitochondrial involvement. Finally, our data expand both the genetic and phenotypic spectrum associated with NR2F1 gene mutations.

Human proteins MTO1 and GTPBP3 are thought to jointly catalyze the modification of the wobble uridine in mitochondrial tRNAs. Defects in each protein cause infantile hypertrophic cardiomyopathy with lactic acidosis. However, the underlying mechanisms are mostly unknown. Using fibroblasts from an MTO1 patient and MTO1 silenced cells, we found that the MTO1 deficiency is associated with a metabolic reprogramming mediated by inactivation of AMPK, down regulation of the uncoupling protein 2 (UCP2) and transcription factor PPARγ, and activation of the hypoxia inducible factor 1 (HIF-1). As a result, glycolysis and oxidative phosphorylation are uncoupled, while fatty acid metabolism is altered, leading to accumulation of lipid droplets in MTO1 fibroblasts. Unexpectedly, this response is different from that triggered by the GTPBP3 defect, as GTPBP3-depleted cells exhibit AMPK activation, increased levels of UCP2 and PPARγ, and inactivation of HIF-1. In addition, fatty acid oxidation and respiration are stimulated in these cells. Therefore, the HIF-PPARγ-UCP2-AMPK axis is operating differently in MTO1- and GTPBP3-defective cells, which strongly suggests that one of these proteins has an additional role, besides mitochondrial-tRNA modification. This work provides new and useful information on the molecular basis of the MTO1 and GTPBP3 defects and on putative targets for therapeutic intervention.

Identification of very long-chain acyl-CoA dehydrogenase deficiency is possible in the expanded newborn screening (NBS) due to the increase in tetradecenoylcarnitine (C14:1) and in the C14:1/C2, C14:1/C16, C14:1/C12:1 ratios detected in dried blood spots. Nevertheless, different confirmatory tests must be performed to confirm the final diagnosis. We have revised the NBS results and the results of the confirmatory tests (plasma acylcarnitine profiles, molecular findings, and lymphocytes VLCAD activity) for 36 cases detected in three Spanish NBS centers during 4 years, correlating these with the clinical outcome and treatment. Our aim was to distinguish unambiguously true cases from disease carriers in order to obtain useful diagnostic information for clinicians that can be applied in the follow-up of neonates identified by NBS.Increases in C14:1 and of the different ratios, the presence of two pathogenic mutations, and deficient enzyme activity in lymphocytes (<12% of the intra-assay control) identified 12 true-positive cases. These cases were given nutritional therapy and all of them are asymptomatic, except one. Seventeen individuals were considered disease carriers based on the mild increase in plasma C14:1, in conjunction with the presence of only one mutation and/or intermediate residual activity (18-57%). In addition, seven cases were classified as false positives, with normal biochemical parameters and no mutations in the exonic region of ACADVL. All these carriers and the false positive cases remained asymptomatic. The combined evaluation of the acylcarnitine profiles, genetic results, and residual enzyme activities have proven useful to definitively classify individuals with suspected VLCAD deficiency into true-positive cases and carriers, and to decide which cases need treatment.