AIMS:Nephrolithiasis is a major global health challenge, with oxidative stress and mitochondrial dysfunction emerging as key drivers of renal injury and stone formation. l-ergothioneine (l-Erg), a naturally occurring antioxidant transported by OCTN1, has shown promising effects in cystinuria models, preventing stone formation. Despite evidence supporting an indirect mechanism of action, key mechanistic aspects have yet to be fully clarified. This study aimed to evaluate whether l-Erg can prevent stone progression in cystinuria and in other types of lithiasis, such as calcium oxalate nephrolithiasis, and to further elucidate its mechanistic basis. RESULTS:Using mouse models, l-Erg significantly reduced cystine stone growth and renal inflammation, and its combination with d-penicillamine enhanced stone dissolution and mitigated drug-related toxicity. In calcium oxalate nephrolithiasis, l-Erg decreased crystal deposition, preserved renal architecture, normalized glutathione levels, and restored mitochondrial respiration. Transcriptomic analysis revealed downregulation of immune pathways and activation of cell cycle genes, suggesting attenuation of inflammation and promotion of tubular repair. INNOVATION:This study is the first to demonstrate that l-Erg exerts renoprotective effects through combined antioxidant and mitochondrial mechanisms in two major forms of nephrolithiasis and introduces a dual therapeutic approach combining an antioxidant with a cystine-solubilizing agent. CONCLUSION:By targeting oxidative stress and mitochondrial dysfunction, l-Erg represents a promising therapeutic strategy for nephrolithiasis, either alone or as an adjunct to current treatments. Antioxid. Redox Signal. 44, 878-891.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is closely linked to systemic metabolic disturbances, yet the early hepatic mechanisms contributing to disease initiation and progression remain incompletely understood. Alterations in nucleoside metabolism and signalling have been implicated in metabolic liver disease, but the role of nucleoside transport has not been defined. Here, we investigated the contribution of equilibrative nucleoside transporter 1 (ENT1), the predominant hepatic nucleoside transporter, to MASLD pathophysiology. By integrating human liver biopsies, public transcriptomic datasets, and diet-induced mouse models, including ENT1-deficient mice, we analysed ENT1 expression and its functional impact on hepatic and systemic metabolism. We show that hepatic ENT1 expression changes during MASLD, with increased expression at early disease stages in both humans and mice, followed by a decline with disease progression. Under basal conditions, ENT1 deficiency induces subtle metabolic alterations in the liver. Notably, loss of ENT1 decreases hepatic adenosine and is associated with protection against diet-induced steatosis, inflammation, and fibrosis, and with improved glucose tolerance and insulin sensitivity, potentially through systemic metabolic effects. In humans with MASLD, hepatic ENT1 expression correlates positively with insulin levels and HOMA-IR. Together, these findings identify ENT1 as a regulator of systemic nucleoside handling and metabolic homeostasis and highlight nucleoside transport as a previously underappreciated axis contributing to MASLD development and progression.

Individuals with phenylketonuria (PKU), caused by different variants of the phenylalanine hydroxylase gene, need to restrict their intake of phenylalanine. This study evaluated dietary patterns and physical activity levels in children with different PKU phenotypes compared to healthy controls. Eighty-two children were recruited (22 classic PKU [cPKU], 21 BH4-responsive PKU, 19 hyperphenylalaninemia, and 20 controls). Anthropometric data, dietary intake, biochemical markers, and physical activity were assessed. Classic PKU (cPKU) subjects exhibited higher carbohydrate and sugar intake than other PKU phenotypes and controls. Notably, 42% of carbohydrate and 17% of sugar intake was from special low-protein foods, and 20% of carbohydrate and 29% of sugar intake was from protein substitutes. Compared to controls, the cPKU group was less physically active and reported a higher frequency of sweet consumption. Ninety percent of PKU had good metabolic control and carbohydrate intake was significantly correlated with HOMA-IR; however, after adjusting for age, only a trend remained (p = 0.08). Participants in the PKU group following a low natural protein diet consumed more carbohydrate and sugars than those on a normal-protein diet. Multivariate regression analysis showed that the low natural protein diet group was significantly associated with higher levels of vitamin B12, linoleic acid, α-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid, and with lower levels of total cholesterol and HDL-C compared to the normal-protein diet group. In conclusion, children with PKU, particularly those with classical PKU following low-protein diets, showed higher carbohydrate intake and distinct micronutrient and lipid profiles compared with healthy controls.

BACKGROUND & AIMS:Copper is a trace element essential for enzymatic reactions, but excessive accumulation can cause toxicity. Accurate interpretation of serum copper concentrations is crucial for diagnosing conditions such as Wilson's disease, liver dysfunction, and nutritional deficiencies. Current reference intervals often ignore the effect of inflammation and are typically established using discrete age groups rather than modelling age as a continuous variable. This study aimed to establish continuous, age-adjusted reference intervals for serum copper and to develop a method for correcting inflammation-related variability. METHODS:We retrospectively analyzed serum copper concentrations in a pediatric cohort of 4,368 unique samples. Inflammatory status was assessed using erythrocyte sedimentation rate (ESR), fibrinogen, and C-reactive protein (CRP). Samples without inflammation were used to generate age-continuous reference intervals through polynomial regression. To quantify and adjust for inflammation effects, we developed a composite inflammation score using partial least squares regression on standardized values of the three acute-phase markers and applied it to correct copper concentrations in samples exhibiting inflammation. RESULTS:Serum copper showed a nonlinear relationship with age. Inflammation elevated copper concentrations by approximately 24 %. The composite inflammation score independently predicted this variability in copper concentrations, and adjustment using the score restored copper concentrations within reference limits, reducing the risk of data misinterpretation. CONCLUSION:Our findings underscore the necessity of considering both age and inflammation variables when interpreting pediatric serum copper concentrations. We provide continuous, age-adjusted reference intervals and a method to correct for inflammation-related variability, enhancing data interpretation. We propose a proof-of-concept potentially applicable to other biomarkers related with metabolic and nutritional disturbances in chronic and acute diseases.

Phenylketonuria (PKU) is a rare metabolic disorder caused by a deficiency in the enzyme phenylalanine hydroxylase, leading to the accumulation of phenylalanine (Phe). Raised Phe levels can result in neurocognitive deficits, intellectual disabilities, and behavioral or psychiatric disorders. To conduct a systematic review of human studies on metabolites identified through metabolomics in individuals with PKU, compared to healthy controls, and to provide insights into their biological significance. A total of 26 human studies analyzing metabolites in urine and blood met the inclusion criteria. In total, 544 metabolites that differed between patients with PKU and healthy controls were identified through different metabolomic techniques (LC-MS, GC-MS, NMR). Differences were primarily observed in blood samples, which accounted for 95

Certain diseases are marked by elevated ammonium levels in the blood, a condition known as hyperammonemia. Prompt detection and medical intervention are crucial to prevent potentially fatal outcomes. Therefore, ammonium levels should be monitored regularly, typically in referral hospitals where specialized and costly equipment is available. Although compact commercial devices are available for this purpose, none of them meet all the technical and analytical requirements needed for direct blood analysis, and current reported strategies have not been validated with enough samples to confirm results reliably. We present a robust and reliable automated point-of-care (POC) analyzer for the potentiometric determination of ammonium in blood. Comprising three computer-controlled modules—fluid management, detection, and data acquisition and transmission—this system combines portability, ease of use, and affordability. It can directly measure untreated blood samples, significantly reducing analysis time. Fully automated, it operates unsupervised with minimal lab personnel intervention. Analytical quality parameters include 5% RSD repeatability (n = 8), a limit of detection of 24 μM, a working range of 30–1000 µM and a sample volume of 215 µL. Successfully implemented in a hospital for 2 months, it analyzed 238 blood samples in parallel with the hospital’s reference method showing comparable results (paired t -test, Passing-Bablok regression and Bland–Altman Plot) and randomly distributed errors, with a 4% accuracy calculated as mean error. Results indicate the POC analyzer effectiveness and reliability in a clinical setting compared to currently reported or commercially available equipment, being suitable for bedside monitoring of conditions associated with hyperammonemia in healthcare centers, including emergency rooms and clinics in developing countries. Graphical Abstract

Introduction: Methylmalonic acid (MMA) is the key metabolite for genetic methylmalonic acidurias (MMAs) with or without homocystinuria, and also, the second-line test recommended in acquired vitamin B12 (Cbl) deficiency. Besides, MMA quantification is a tool for the treatment monitoring, thereby, a sensitive methodology enable to detect low and precise concentrations is required. We propose and validate a simple, sensitive and highthroughput method to obtain accurate quantification of plasma MMA using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Methods: We performed the analysis in a 96-well plate format, using aqueous extraction, deproteinization by ultrafiltration, and UPLC-MS/MS analysis. We validated the method on plasma samples from 218 healthy controls, 58 patients (6 MUT, 11 MMACHC, 2 MMAB, 1 TCN2, and 38 acquired Cbl deficiencies), and 8 external quality controls. Results: The analytical validation demonstrated good performance with excellent linearity, good precision (coefficient of variation < 14 %), accuracy (>92 %), and recovery (>78 %). Plasma MMA was elevated in MMAs, being higher in genetic cases (97.5th percentile = 3190 mu mol/L) than in acquired cases (97.5th percentile = 33 mu mol/L). Plasma MMA levels correlated well with urine and DBS MMA levels. We observed a decrease and normalization of plasma MMA concentrations post-Cbl-treatment. Conclusions: This novel UPLC-MS/MS method is a simple, sensitive and high-throughput assay to analyse plasma MMA. We have implemented this assay in our routine laboratory practice as a complementary tool for the biochemical diagnosis and treatment response monitoring of both genetic and acquired MMAs.

This study addresses challenges in cerebrospinal fluid (CSF) amino acid profiling in pediatric neurological disorders by establishing age-continuous reference intervals. It examines amino acid variations in epilepsy of different etiologies and evaluates the effects of antiseizure medications (ASMs), resolving inconsistencies in previous research. We retrospectively analyzed 410 CSF samples from pediatric patients (201 with epilepsy). Reference intervals were established using linear regression, adjusting for age and sex as confounders. Statistical analysis of age-normalized data included tests to assess the relationship between clinical features and altered or normal amino acid levels, multiple regression models evaluating the effects of ASMs on CSF amino acid levels, and hierarchical clustering for pattern identification. Age significantly influenced CSF amino acid levels, with higher concentrations observed in neonates, except for aspartic acid and arginine. Sex was not a significant predictor. Over 90 % of patients with epilepsy had normal levels for most amino acids, except for glutamine, which was more frequently elevated in epilepsy. Valproate and GABAA receptor agonists were linked to elevated glutamine, while vigabatrin therapy was associated with increased levels of ornithine, leucine, and isoleucine. Hierarchical clustering identified a subcluster with elevated essential amino acid levels, predominantly comprising patients with generalized seizures or status epilepticus. This study establishes new CSF amino acid reference intervals for pediatric patients and finds no significant associations between individual amino acid levels and epilepsy-related clinical features. However, a subgroup with high essential amino acid levels, primarily associated with generalized epilepsy, suggests potential links to generalized epileptogenic discharges and blood-brain barrier disruption.

BACKGROUND:Cerebrospinal fluid (CSF) homovanillic (HVA), and 5-hydroxyindoleacetic acids (5-HIAA) are biomarkers of neurological diseases affecting the dopaminergic and serotoninergic pathways. Establishing reference intervals for these metabolites faces the challenges of a lack of healthy controls and a negative correlation with age, making stratified intervals unrealistic. We propose a pipeline to determine continuous reference intervals for HVA and 5-HIAA using an indirect method. We also studied the confounding effects of different variables and explored the impact of antiepileptic and neuroleptic treatments on HVA and 5-HIAA values. METHODS:The study used least squares regression to fit age-concentration curves from a cohort of pediatric patients (n = 1533), where the residuals represent metabolite values excluding age effect. Presuming that HVA and 5-HIAA primary deficiencies characterize a distinct subpopulation, we fitted a two-component finite mixture model in age-normalized data and set reference intervals at the central 95% of the nondeficient population. RESULTS:Patients with primary genetic deficiencies of HVA and/or 5-HIAA consistently fall outside the proposed continuous reference intervals. Using the new continuous reference intervals reduces the number of secondary deficiencies detected compared with using stratified values. No correlations were observed between CSF HVA and 5-HIAA values across the studied drug categories (antiseizure and neuroleptic medications). In addition, biopterin values positively influenced both metabolite concentrations. CONCLUSION:The proposed continuous reference intervals caused a substantial reduction in the number of secondary deficiencies detected, most of which demonstrated no conclusive correlations between the diseases and altered HVA and 5-HIAA values.

OBJECTIVES:Early diagnosis of inborn errors of metabolism (IEM) is crucial to ensure early detection of conditions which are treatable. This study reports on targeted metabolomic procedures for the diagnosis of IEM of amino acids, acylcarnitines, creatine/guanidinoacetate, purines/pyrimidines and oligosaccharides, and describes its validation through external quality assessment schemes (EQA). METHODS:Analysis was performed on a Waters ACQUITY UPLC H-class system coupled to a Waters Xevo triple-quadrupole (TQD) mass spectrometer, operating in both positive and negative electrospray ionization mode. Chromatographic separation was performed on a CORTECS C18 column (2.1 × 150, 1.6 µm). Data were collected by multiple reaction monitoring. RESULTS:The internal and EQA results were generally adequate, with a few exceptions. We calculated the relative measurement error (RME) and only a few metabolites displayed a RME higher than 30 % (asparagine and some acylcarnitine species). For oligosaccharides, semi-quantitative analysis of an educational panel clearly identified the 8 different diseases included. CONCLUSIONS:Overall, we have validated our analytical system through an external quality control assessment. This validation will contribute to harmonization between laboratories, thus improving identification and management of patients with IEM.

GRIN-related disorders are rare developmental encephalopathies with variable manifestations and limited therapeutic options. Here, we present the first non-randomized, open-label, single-arm trial (NCT04646447) designed to evaluate the tolerability and efficacy of L-serine in children with GRIN genetic variants leading to loss-of-function. In this phase 2A trial, patients aged 2-18 years with GRIN loss-of-function pathogenic variants received L-serine for 52 weeks. Primary end points included safety and efficacy by measuring changes in the Vineland Adaptive Behavior Scales, Bayley Scales, age-appropriate Wechsler Scales, Gross Motor Function-88, Sleep Disturbance Scale for Children, Pediatric Quality of Life Inventory, Child Behavior Checklist and the Caregiver-Teacher Report Form following 12 months of treatment. Secondary outcomes included seizure frequency and intensity reduction and EEG improvement. Assessments were performed 3 months and 1 day before starting treatment and 1, 3, 6 and 12 months after beginning the supplement. Twenty-four participants were enrolled (13 males/11 females, mean age 9.8 years, SD 4.8), 23 of whom completed the study. Patients had GRIN2B, GRIN1 and GRIN2A variants (12, 6 and 5 cases, respectively). Their clinical phenotypes showed 91% had intellectual disability (61% severe), 83% had behavioural problems, 78% had movement disorders and 58% had epilepsy. Based on the Vineland Adaptive Behavior Composite standard scores, nine children were classified as mildly impaired (cut-off score > 55), whereas 14 were assigned to the clinically severe group. An improvement was detected in the Daily Living Skills domain (P = 0035) from the Vineland Scales within the mild group. Expressive (P = 0.005), Personal (P = 0.003), Community (P = 0.009), Interpersonal (P = 0.005) and Fine Motor (P = 0.031) subdomains improved for the whole cohort, although improvement was mostly found in the mild group. The Growth Scale Values in the Cognitive subdomain of the Bayley-III Scale showed a significant improvement in the severe group (P = 0.016), with a mean increase of 21.6 points. L-serine treatment was associated with significant improvement in the median Gross Motor Function-88 total score (P = 0.002) and the mean Pediatric Quality of Life total score (P = 0.00068), regardless of severity. L-serine normalized the EEG pattern in five children and the frequency of seizures in one clinically affected child. One patient discontinued treatment due to irritability and insomnia. The trial provides evidence that L-serine is a safe treatment for children with GRIN loss-of-function variants, having the potential to improve adaptive behaviour, motor function and quality of life, with a better response to the treatment in mild phenotypes.

There are few causes of treatable neurodevelopmental diseases described to date. Branched-chain ketoacid dehydrogenase kinase (BCKDK) deficiency causes branched-chain amino acid (BCAA) depletion and is linked to a neurodevelopmental disorder characterized by autism, intellectual disability and microcephaly. We report the largest cohort of patients studied, broadening the phenotypic and genotypic spectrum. Moreover, this is the first study to present newborn screening findings and mid-term clinical outcome. In this cross-sectional study, patients with a diagnosis of BCKDK deficiency were recruited via investigators' practices through a MetabERN initiative. Clinical, biochemical and genetic data were collected. Dried blood spot (DBS) newborn screening (NBS) amino acid profiles were retrieved from collaborating centres and compared to a healthy newborn reference population. Twenty-one patients with BCKDK mutations were included from 13 families. Patients were diagnosed between 8 months and 16 years (mean: 5.8 years, 43% female). At diagnosis, BCAA levels (leucine, valine and isoleucine) were below reference values in plasma and in CSF. All patients had global neurodevelopmental delay; 18/21 had gross motor function (GMF) impairment with GMF III or worse in 5/18, 16/16 intellectual disability, 17/17 language impairment, 12/17 autism spectrum disorder, 9/21 epilepsy, 12/15 clumsiness, 3/21 had sensorineural hearing loss and 4/20 feeding difficulties. No microcephaly was observed at birth, but 17/20 developed microcephaly during follow-up. Regression was reported in six patients. Movement disorder was observed in 3/21 patients: hyperkinetic movements (1), truncal ataxia (1) and dystonia (2). After treatment with a high-protein diet (>= 2 g/kg/day) and BCAA supplementation (100-250 mg/kg/day), plasma BCAA increased significantly (P < 0.001), motor functions and head circumference stabilized/improved in 13/13 and in 11/15 patients, respectively. Among cases with follow-up data, none of the three patients starting treatment before 2 years of age developed autism at follow-up. The patient with the earliest age of treatment initiation (8 months) showed normal development at 3 years of age. NBS in DBS identified BCAA levels significantly lower than those of the normal population. This work highlights the potential benefits of dietetic treatment, in particular early introduction of BCAA. Therefore, it is of utmost importance to increase awareness about this treatable disease and consider it as a candidate for early detection by NBS programmes. Tangeraas et al. describe the largest series of BCKDK deficiency patients to date, including responses to dietetic treatment. Early introduction of BCAA ameliorates the BCKDK deficiency phenotype. This treatable neurodevelopmental disease should be considered for inclusion in newborn screening programmes.

In inborn errors of intermediate protein metabolism (IEM), the effect of special low-protein foods (SLPFs) on dietary intake has been scarcely studied. The aim of this study was to compare the nutritional profile of SLPFs with usual foods and to assess whether their intake determines the dietary pattern and affects the plasma biochemical profile in children with IEMs with different protein restrictions. A database with the nutritional composition of 250 SLPFs was created. A total of 59 children with IEMs were included in this cross-sectional observational study. The greatest significant differences in macronutrient composition were observed between dairy, meat, fish, and egg SLPFs and regular foods. After stratifying subjects by SLPFs, the participants with the highest intake (>32%) had a higher total energy intake and lower intake of natural protein than those in the lowest tertile (<24%) (p < 0.05). However, when stratifying subjects by dairy SLPF intake, children in the highest tertile (>5%) showed a higher intake of sugars, total and saturated fats, and higher plasma levels of total and low-density lipoprotein cholesterol than those in the first tertile (<1%) (p < 0.05). The variability in the nutritional composition of SLPFs highlights the need for up-to-date databases which would greatly assist in optimizing individualized recommendations for children with IEMs and protein restrictions.

Metabolomics studies in human dermal fibroblasts can elucidate the biological mechanisms associated with some diseases, but several methodological issues that increase variability have been identified. We aimed to quantify the amino acid levels in cultured fibroblasts and to apply different sample-based normalization approaches. Forty-four skin biopsies from control subjects were collected. Amino acids were measured in fibroblasts supernatants by UPLC-MS/MS. Statistical supervised and unsupervised studies were used. Spearman's test showed that phenylalanine displayed the second highest correlation with the remaining amino acids (mean r = 0.8), whereas the total protein concentration from the cell pellet showed a mean of r = 0.67. The lowest percentage of variation was obtained when amino acids were normalized by phenylalanine values, with a mean of 42% vs 57% when normalized by total protein values. When amino acid levels were normalized by phenylalanine, Principal Component Analysis and clustering analyses identified different fibroblasts groups. In conclusion, phenylalanine may be a suitable biomarker to estimate cellular content in cultured fibroblasts.

BACKGROUND:The cortico-cerebellar-thalamic-cortical circuit has been implicated in the emergence of psychotic symptoms in schizophrenia (SZ). The kynurenine pathway (KP) has been linked to alterations in glutamatergic and monoaminergic neurotransmission and to SZ symptomatology through the production of the metabolites quinolinic acid (QA) and kynurenic acid (KYNA).METHODS:This work describes alterations in KP in the post-mortem prefrontal cortex (PFC) and cerebellum (CB) of 15 chronic SZ patients and 14 control subjects in PFC and 13 control subjects in CB using immunoblot for protein levels and ELISA for interleukins and QA and KYNA determinations. Monoamine metabolites were analysed by high-performance liquid chromatography and SZ symptomatology was assessed by Positive and Negative Syndrome Scale (PANSS). The association of KP with inflammatory mediators, monoamine metabolism and SZ symptomatology was explored.RESULTS:In the PFC, the presence of the anti-inflammatory cytokine IL-10 together with IDO2 and KATII enzymes decreased in SZ, while TDO and KMO enzyme expression increased. A network interaction analysis showed that in the PFC IL-10 was coupled to the QA branch of the kynurenine pathway (TDO-KMO-QA), whereas IL-10 associated with KMO in CB. KYNA in the CB inversely correlated with negative and general PANSS psychopathology. Although there were no changes in monoamine metabolite content in the PFC in SZ, a network interaction analysis showed associations between dopamine and methoxyhydroxyphenylglycol degradation metabolite. Direct correlations were found between general PANSS psychopathology and the serotonin degradation metabolite, 5-hydroxyindoleacetic acid. Interestingly, KYNA in the CB inversely correlated with 5-hydroxyindoleacetic acid in the PFC.CONCLUSIONS:Thus, this work found alterations in KP in two brain areas belonging to the cortico-cerebellar-thalamic-cortical circuit associated with SZ symptomatology, with a possible impact across areas in 5-HT degradation.

Lysinuric Protein Intolerance (LPI) is an inborn error of metabolism resulting from SLC7A7 deficiency that causes diminished plasma concentration of cationic amino acids. The clinical picture is highly heterogeneous among patients, who commonly present intolerance to protein intake and more severe complications such as hematological abnormalities and kidney failure. Although current treatments aim to address the metabolic defects of LPI, they have been unsatisfactory when treating the most severe symptoms. Here we show that the absence of Slc7a7 in mice causes iron overload as a result of erythropoiesis failure. Regarding iron metabolism, we demonstrate that reduced plasma erythropoietin triggers a strong iron overload, as erythropoietin administration restores normal iron levels and mitigate hematological alterations. Interestingly, we found that human LPI is associated with hyperferritinemia but not iron overload, a trait that might be influenced by the citrulline treatment. Furthermore, we show that erythropoietin is a key factor in the hematological abnormalities in LPI. Our study reveals a mechanism leading to LPI-induced hematological complications and identifies erythropoietin supplementation as a promising therapeutic strategy for human LPI. Significance Statement The systemic metabolic environment derived from Slc7a7 -ablation in epithelial cells from kidney and intestine causes erythropoiesis failure prompting therefore iron overload. Here, we identify erythropoietin as the main driver of erythropoiesis failure as exogenous erythropoietin administration restores normal erythroblast population. In addition, we have also analyzed human data and found that patients with LPI have abnormal ferritin levels. Finally, as human LPI, citrulline treatment in mice restores normal iron homeostasis, highlighting the relevance of the systemic environment in LPI. Erythropoietin supplementation emerges as a promising therapeutic strategy for human LPI without the inflammatory effect associated with citrulline supplementation.

Introduction: Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by a deficiency of acid beta-glucosidase encoded by the GBA gene. In patients with GD, childhood onset parkinsonian features have been rarely described. Methods: Twin siblings with GD are described, including clinical follow-up and treatment response. Bone marrow, enzyme activity studies and genotyping were performed. Results: By age 9 months, symptoms at onset were thrombocytopenia and splenomegaly. By age 2, hypokinesia, bradykinesia and oculomotor apraxia were observed. By age 5 a complete rigid hypokinetic syndrome was stablished in both patients, including bradykinesia, tremor and rigidity. Treatment with imiglucerase, miglustat, ambroxol and levodopa were performed. Levodopa showed a good response with improvement in motor and non-motor skills. Foamy cells were found in the bone marrow study. Glucocerebrosidase activity was 28% and 26%. Sanger sequencing analysis identified a missense mutation and a complex allele (NP_000148: p. [(Asp448His)]; [(Leu422Profs*4)]) in compound heterozygosity in GBA gene. Conclusions: Two siblings with neuronopathic GD with an intermediate form between type 2 and 3, with a systemic and neurological phenotype are described. The complex neurological picture included a hypokineticrigid and tremor syndrome that improved with levodopa treatment. These conditions together have not been previously described in pediatric GD. We suggest that in children with parkinsonian features, lysosomal storage disorders must be considered, and a levodopa trial must be performed. Moreover, this report give support to the finding that GBA and parkinsonian features share biological pathways and highlight the importance of lysosomal mechanisms in parkinsonism pathogenesis, what might have therapeutic implications.