Targeted ultra performance liquid chromatography tandem mass spectrometry procedures for the diagnosis of inborn errors of metabolism: validation through ERNDIM external quality assessment schemes.

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.

Exploring the Contribution of the Transporter AGT1/rBAT in Cystinuria Progression: Insights from Mouse Models and a Retrospective Cohort Study.

More than 20 years have passed since the identification of SLC3A1 and SLC7A9 as causative genes for cystinuria. However, cystinuria patients exhibit significant variability in the age of lithiasis onset, recurrence, and response to treatment, suggesting the presence of modulatory factors influencing cystinuria severity. In 2016, a second renal cystine transporter, AGT1, encoded by the SLC7A13 gene, was discovered. Although it was discarded as a causative gene for cystinuria, its possible effect as a modulatory gene remains unexplored. Thus, we analyzed its function in mouse models of cystinuria, screened the SLC7A13 gene in 34 patients with different lithiasic phenotypes, and functionally characterized the identified variants. Mice results showed that AGT1/rBAT may have a protective role against cystine lithiasis. In addition, among the four missense variants detected in patients, two exhibited a 25% impairment in AGT1/rBAT transport. However, no correlation between SLC7A13 genotypes and lithiasis phenotypes was observed in patients, probably because these variants were found in heterozygous states. In conclusion, our results, consistent with a previous study, suggest that AGT1/rBAT does not have a relevant effect on cystinuria patients, although an impact in patients carrying homozygous pathogenic variants cannot be discarded.

Association of Dickkopf-1 Polymorphisms With Radiological Damage and Periodontal Disease in Patients With Early Rheumatoid Arthritis: A Cross-Sectional Study.

BACKGROUND: Rheumatoid arthritis (RA) is a systemic autoimmune disease that increased bone resorption. Periodontal disease (PD) is an associated risk factor of RA. Studies suggest an association between bone markers such as the dickkopf-related protein 1 (DKK-1) and progression of radiological damage. We aimed to evaluate the marker DKK-1, its polymorphisms in patients with early rheumatoid arthritis (eRA), and its association with rheumatic, radiological, and periodontal variables. METHODS: This is a cross-sectional study. Samples were obtained from 63 patients with eRA. Radiographs of hands and feet were evaluated by Sharp-van der Heijde score (SHS) and Simple Erosion Narrowing Score (SENS). Serum DKK-1 levels and high-resolution fusion analysis was used for polymorphisms (rs1896368, rs1896367, rs1528873). Bivariate analyses were performed. RESULTS: Individuals heterozygous for rs1896367 had more frequent erosions (p = 0.026) and joint space narrowing (p = 0.005) in the feet, higher SHS (p = 0.016), and higher SENS (p ≤ 0.001). Patients homozygous for rs1896368 had less frequent joint space narrowing in hands and feet as assessed by SHS and less presence of erosions by SENS (odds ratio, 0.04; 95% confidence interval, 0.00-0.93; p < 0.05). The presence of PD was associated with the homozygous of rs1896367 (p = 0.009) and the heterozygous of rs1896368 (p = 0.033). CONCLUSIONS: Polymorphism rs1896367 seems to be associated with greater radiological compromise; rs1896368 confers protection against bone damage in Colombian eRA patients.

Mutations in TIMM50 cause severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology.

3-Methylglutaconic aciduria (3-MGA-uria) syndromes comprise a heterogeneous group of diseases associated with mitochondrial membrane defects. Whole-exome sequencing identified compound heterozygous mutations in TIMM50 (c.[341 G>A];[805 G>A]) in a boy with West syndrome, optic atrophy, neutropenia, cardiomyopathy, Leigh syndrome, and persistent 3-MGA-uria. A comprehensive analysis of the mitochondrial function was performed in fibroblasts of the patient to elucidate the molecular basis of the disease. TIMM50 protein was severely reduced in the patient fibroblasts, regardless of the normal mRNA levels, suggesting that the mutated residues might be important for TIMM50 protein stability. Severe morphological defects and ultrastructural abnormalities with aberrant mitochondrial cristae organization in muscle and fibroblasts were found. The levels of fully assembled OXPHOS complexes and supercomplexes were strongly reduced in fibroblasts from this patient. High-resolution respirometry demonstrated a significant reduction of the maximum respiratory capacity. A TIMM50-deficient HEK293T cell line that we generated using CRISPR/Cas9 mimicked the respiratory defect observed in the patient fibroblasts; notably, this defect was rescued by transfection with a plasmid encoding the TIMM50 wild-type protein. In summary, we demonstrated that TIMM50 deficiency causes a severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology, such as the maintenance of proper mitochondrial morphology, OXPHOS assembly, and mitochondrial respiratory capacity.

Design and synthesis of 2,6-disubstituted-8-amino imidazo[1,2a]pyridines, a promising privileged structure.

Imidazo[1,2a]pyridines have gained much interest in the field of medicinal chemistry research. In the aim of accessing new privileged structure, we decided to design and synthesize 8-aminated-imidazo[1,2a]pyridines substituted on positions 2 and 6. This scaffold, rarely found in the literature, was obtained via palladium-catalyzed coupling reactions (Suzuki reaction or N-hydroxysuccinimidyl activated ester method) and tested on adenosine receptor A2A. We demonstrated how incorporation of an exocyclic amine enhanced affinity towards this receptor while maintaining low cytotoxicity.

Free-thiamine is a potential biomarker of thiamine transporter-2 deficiency: a treatable cause of Leigh syndrome.

Thiamine transporter-2 deficiency is caused by mutations in the SLC19A3 gene. As opposed to other causes of Leigh syndrome, early administration of thiamine and biotin has a dramatic and immediate clinical effect. New biochemical markers are needed to aid in early diagnosis and timely therapeutic intervention. Thiamine derivatives were analysed by high performance liquid chromatography in 106 whole blood and 38 cerebrospinal fluid samples from paediatric controls, 16 cerebrospinal fluid samples from patients with Leigh syndrome, six of whom harboured mutations in the SLC19A3 gene, and 49 patients with other neurological disorders. Free-thiamine was remarkably reduced in the cerebrospinal fluid of five SLC19A3 patients before treatment. In contrast, free-thiamine was slightly decreased in 15.2% of patients with other neurological conditions, and above the reference range in one SLC19A3 patient on thiamine supplementation. We also observed a severe deficiency of free-thiamine and low levels of thiamine diphosphate in fibroblasts from SLC19A3 patients. Surprisingly, pyruvate dehydrogenase activity and mitochondrial substrate oxidation rates were within the control range. Thiamine derivatives normalized after the addition of thiamine to the culture medium. In conclusion, we found a profound deficiency of free-thiamine in the CSF and fibroblasts of patients with thiamine transporter-2 deficiency. Thiamine supplementation led to clinical improvement in patients early treated and restored thiamine values in fibroblasts and cerebrospinal fluid.

Mutations in the lipoyltransferase LIPT1 gene cause a fatal disease associated with a specific lipoylation defect of the 2-ketoacid dehydrogenase complexes.

Cofactor disorders of mitochondrial energy metabolism are a heterogeneous group of diseases with a wide variety of clinical symptoms, particular metabolic profiles and variable enzymatic defects. Mutations in NFU1, BOLA3, LIAS and IBA57 have been identified in patients with deficient lipoic acid-dependent enzymatic activities and defects in the assembly and activity of the mitochondrial respiratory chain complexes. Here, we report a patient with an early onset fatal lactic acidosis presenting a biochemical phenotype compatible with a combined defect of pyruvate dehydrogenase (PDHC) and 2-ketoglutarate dehydrogenase (2-KGDH) activities, which suggested a deficiency in lipoic acid metabolism. Immunostaining analysis showed that lipoylated E2-PDH and E2-KGDH were extremely reduced in this patient. However, the absence of glycine elevation, the normal activity of the glycine cleavage system and the normal lipoylation of the H protein suggested a defect of lipoic acid transfer to particular proteins rather than a general impairment of lipoic acid biosynthesis as the potential cause of the disease. By analogy with yeast metabolism, we postulated LIPT1 as the altered candidate gene causing the disease. Sequence analysis of the human LIPT1 identified two heterozygous missense mutations (c.212C>T and c.292C>G), segregating in different alleles. Functional complementation experiments in patient's fibroblasts demonstrated that these mutations are disease-causing and that LIPT1 protein is required for lipoylation and activation of 2-ketoacid dehydrogenases in humans. These findings expand the spectrum of genetic defects associated with lipoic acid metabolism and provide the first evidence of a lipoic acid transfer defect in humans.

Cholestane-3ß,5α,6ß-triol: high levels in Niemann-Pick type C, cerebrotendinous xanthomatosis, and lysosomal acid lipase deficiency.

Niemann-Pick type C (NPC) is a progressive neurodegenerative disease characterized by lysosomal/endosomal accumulation of unesterified cholesterol and glycolipids. Recent studies have shown that plasma cholestane-3ß,5α,6ß-triol (CT) and 7-ketocholesterol (7-KC) could be potential biomarkers for the diagnosis of NPC patients. We aimed to know the sensitivity and specificity of these biomarkers for the diagnosis of NPC compared with other diseases that can potentially lead to oxysterol alterations. We studied 107 controls and 122 patients including 16 with NPC, 3 with lysosomal acid lipase (LAL) deficiency, 8 with other lysosomal diseases, 5 with galactosemia, 11 with cerebrotendinous xanthomatosis (CTX), 3 with Smith-Lemli-Opitz, 14 with peroxisomal biogenesis disorders, 19 with unspecific hepatic diseases, 13 with familial hypercholesterolemia, and 30 with neurological involvement and no evidence of an inherited metabolic disease. CT and 7-KC were analyzed by HPLC-ESI-MS/MS as mono-dimethylglycine derivatives. Levels of 7-KC were high in most of the studied diseases, whereas those of CT were only high in NPC, LAL, and CTX patients. Consequently, although CT is a sensitive biomarker of NPC disease, including those cases with doubtful filipin staining, it is not specific. 7-KC is a very unspecific biomarker.

Treatment effect of coenzyme Q(10) and an antioxidant cocktail in fibroblasts of patients with Sanfilippo disease.

Coenzyme Q10 (CoQ10) plays a key role in the exchange of electrons in lysosomal membrane, which contributes to protons' translocation into the lumen and to the acidification of intra-lysosomal medium, which is essential for the proteolytic function of hydrolases responsible -when deficient- of a wide range of inherited lysosomal diseases such as Sanfilippo syndromes. Our aim was to evaluate whether treatment with CoQ10 or with an antioxidant cocktail (α-tocopherol, N-acetylcysteine and α-lipoic acid) were able to ameliorate the biochemical phenotype in cultured fibroblasts of Sanfilippo patients. Basal CoQ10 was analyzed in fibroblasts and Sanfilippo A patients showed decreased basal levels. However, no dysfunction in the CoQ10 biosynthesis pathways was found, revealing for the first time a secondary CoQ10 deficiency in Sanfilippo A fibroblasts. Cultured fibroblasts from five patients affected by Sanfilippo A and B diseases were treated with CoQ10 and an antioxidant cocktail. Upon CoQ10 treatment, none of the Sanfilippo A fibroblasts increased their residual enzymatic activity, but the two Sanfilippo B cell lines showed a statistically significant increase of their residual activity. The antioxidant treatment had no effect on the residual activity in all tested cell lines. Moreover, one Sanfilippo A and two Sanfilippo B fibroblasts showed a statistically significant reduction of glycosaminoglycans accumulation both, after 50 µmol/L CoQ10 and antioxidant treatment. Fibroblasts responsive to treatment enhanced their exocytosis levels. Our results are encouraging as some cellular alterations observed in Sanfilippo syndrome can be partially restored by CoQ10 or other antioxidant treatment in some patients.

Characterization of CoQ10 biosynthesis in fibroblasts of patients with primary and secondary CoQ10 deficiency.

Primary coenzyme Q10 (CoQ10) deficiencies are associated with mutations in genes encoding enzymes important for its biosynthesis and patients are responsive to CoQ10 supplementation. Early treatment allows better prognosis of the disease and therefore, early diagnosis is desirable. The complex phenotype and genotype and the frequent secondary CoQ10 deficiencies make it difficult to achieve a definitive diagnosis by direct quantification of CoQ10. We developed a non-radioactive methodology for the quantification of CoQ10 biosynthesis in fibroblasts that allows the identification of primary deficiencies. Fibroblasts were incubated 72 h with 28 µmol/L (2)H3-mevalonate or 1.65 mmol/L (13)C6-p-hydroxybenzoate. The newly synthesized (2)H3- and (13)C6- labelled CoQ10 were analysed by high performance liquid chromatography-tandem mass spectrometry. The mean and the reference range for (13)C6-CoQ10 and (2)H3-CoQ10 biosynthesis were 0.97 (0.83-1.1) and 0.13 (0.09-0.17) nmol/Unit of citrate synthase, respectively. We validated the methodology through the study of one patient with COQ2 mutations and six patients with CoQ10 deficiency secondary to other inborn errors of metabolism. Afterwards we investigated 16 patients' fibroblasts and nine showed decreased CoQ10 biosynthesis. Therefore, the next step is to study the COQ genes in order to reach a definitive diagnosis in these nine patients. In the patients with normal rates the deficiency is probably secondary. In conclusion, we have developed a non-invasive non-radioactive method suitable for the detection of defects in CoQ10 biosynthesis, which offers a good tool for the stratification of patients with these treatable mitochondrial diseases.

Structure and mechanisms of transport of human Asc1/CD98hc amino acid transporter.

Recent cryoEM studies elucidated details of the structural basis for the substrate selectivity and translocation of heteromeric amino acid transporters. However, Asc1/CD98hc is the only neutral heteromeric amino acid transporter that can function through facilitated diffusion, and the only one that efficiently transports glycine and D-serine, and thus has a regulatory role in the central nervous system. Here we use cryoEM, ligand-binding simulations, mutagenesis, transport assays, and molecular dynamics to define human Asc1/CD98hc determinants for substrate specificity and gain insights into the mechanisms that govern substrate translocation by exchange and facilitated diffusion. The cryoEM structure of Asc1/CD98hc is determined at 3.4-3.8 Å resolution, revealing an inward-facing semi-occluded conformation. We find that Ser 246 and Tyr 333 are essential for Asc1/CD98hc substrate selectivity and for the exchange and facilitated diffusion modes of transport. Taken together, these results reveal the structural bases for ligand binding and transport features specific to human Asc1.

Role of creatine as biomarker of mitochondrial diseases.

Recent investigations have suggested creatine (Cr) as an additional biomarker of mitochondrial diseases. With the aim of corroborating previous findings, we have studied plasma Cr in a cohort of 33 patients with different mitochondrial diseases. Cr was clearly increased in 9 out of 33 patients. Therefore, positive patients represent only 28% of the total number, suggesting that Cr is not a sensitive biomarker of mitochondrial diseases although it does present an acceptable specificity (83%). High plasma Cr, together with other biomarkers, might be useful to reinforce the diagnosis of mitochondrial diseases.

Fibroblast phenylalanine concentration as a surrogate biomarker of cellular number.

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.

Defining the pathogenicity of creatine deficiency syndrome.

This work examined nine patients with creatine deficiency syndrome (CDS): six with a creatine transport (CRTR) defect and three with a GAMT defect. Eleven nucleotide variations were detected: six in SLC6A8 and five in GAMT. These changes were analyzed at the mRNA level and specific alleles (most of which bore premature stop codons) were selected as nulls because they provoked nonsense-mediated decay activation. The impact of these CDS mutations on metabolic stress (ROS production, p38MAPK activation, aberrant proliferation and apoptosis) was analyzed in patient fibroblast cultures. Oxidative stress contributed toward the severe form of CDS, with increases seen in the intracellular ROS content and the percentage of apoptotic cells. An altered cell cycle was also seen in a number of CRTR and GAMT fibroblast cell lines (mostly those carrying null alleles). p38MAPK activation only correlated with oxidative stress in the CRTR cells. Based on intracellular creatine levels, the contribution of energy depletion toward metabolic stress was demonstrable only in selected CRTR cells. Together, these findings suggest that the apoptotic response to genotoxic damage in the present CDS cells may have been triggered by different cell signaling pathways. They also suggest that reducing oxidative stress could be helpful in treating CDS. Hum Mutat 32:1-10, 2011. © 2011 Wiley-Liss, Inc.

Study of inborn errors of metabolism in urine from patients with unexplained mental retardation.

Mental retardation (MR) is a common disorder frequently of unknown origin. Because there are few studies regarding MR and inborn errors of metabolism (IEM), we aimed to identify patients with IEM from a cohort of 944 patients with unexplained MR. Biochemical examinations such as determination of creatine (Cr) metabolites, acylcarnitines, purine, and pyrimidines in urine were applied. We found seven patients with IEM [three with cerebral Cr deficiency syndromes (CCDS)], one with adenylosuccinate lyase (ADSL) deficiency, and three, born before the neonatal metabolic screening program in Catalonia, with phenylketonuria (PKU). All told, they represent 0.8% of the whole cohort. All of them had additional symptoms such as epilepsy, movement disorders, autism, and other psychiatric disturbances. In conclusion, in patients with MR, it is essential to perform a thorough appraisal of the associated signs and symptoms, and in most disorders, it is necessary to apply specific analyses. In some cases, it is important to achieve an early diagnosis and therapy, which may reduce the morbimortality, and to offer genetic counselling.

[Cerebral creatine deficiency: first Spanish patients harbouring mutations in GAMT gene]. / Deficiencia cerebral de creatina: primeros pacientes españoles con mutaciones en el gen GAMT.

BACKGROUND AND OBJECTIVE: Brain creatine (Cr) deficiencies are a group of inborn errors of metabolism that are characterized by an absence or severe reduction of brain Cr. Clinically, these patients can display psychomotor/mental retardation and language disorders, commonly associated with epilepsy or movement disorders. Three metabolic defects are known: two affect synthesis - guanidinoacetate metiltransferase (GAMT) and glycine amidinotransferase (AGAT) deficiencies- and one affect the Cr transporter (CRTR). We present the first three Spanish patients with GAMT deficiency, and we compare their clinical phenotype and treatment response with other published cases. PATIENTS AND METHOD: The three patients presented mental retardation, epilepsy and autistic behaviour. Patient 1 also had severe chorea. Diagnosis was done by biochemical and genetic procedures (guanidinoacetate quantification, determination of GAMT activity and mutation analysis in the GAMT gene). RESULTS: An increase of guanidinoacetate was detected in urine and plasma. Brain magnetic resonance spectroscopy revealed low Cr levels. Enzymatic studies revealed a decreased GAMT activity in fibroblasts. Molecular analysis detected pathogenic mutations in the GAMT gene. After the deficiency was confirmed, the patients started treatment with Cr. In addition, patient 2 and 3 received an arginine-restricted diet and ornithine supplements. All them showed a partial improvement. CONCLUSIONS: Patients with GAMT deficiency have an unspecific but relatively constant clinical presentation. Brain Cr deficiency should be considered in patients with mental retardation of unknown aetiology, specially in those with movement disorders or epilepsy. Early diagnosis is important in cases with known treatment such as GAMT deficiency.

Creatine transporter deficiency: prevalence among patients with mental retardation and pitfalls in metabolite screening.

OBJECTIVES: To report the prevalence of creatine transporter deficiency in males with mental retardation and to study whether a protein-rich food intake might be a potential diagnostic pitfall. DESIGN AND METHODS: We determined creatine/creatinine ratio in urine samples from 1600 unrelated male patients with mental retardation and/or autism. Urine creatine was analyzed by HPLC-MS/MS. RESULTS: Thirty-three of 1600 cases showed increased urine creatine/creatinine ratio. Four out of these thirty-three cases were definitively diagnosed with creatine transporter deficiency, while the other 29 were false positive results. Significantly higher values were observed for urine Cr/Crn ratio in healthy volunteers after a meal based on beef or oily fish as compared to eggs, pasta or salad (Wilcoxon test: p<0.005). CONCLUSIONS: False positive results may be observed in biochemical screening for creatine transporter deficiency, and they may be due to intake of meals rich in creatine prior to urine samples analysis.

Lysosomal Acid Lipase Deficiency in 23 Spanish Patients: High Frequency of the Novel c.966+2T>G Mutation in Wolman Disease.

Lysosomal acid lipase (LAL) is a lysosomal key enzyme involved in the intracellular hydrolysis of cholesteryl esters and triglycerides. Patients with very low residual LAL activity present with the infantile severe form Wolman disease (WD), while patients with some residual activity develop the less severe disorder known as Cholesteryl ester storage disorder (CESD). We present the clinical, biochemical, and molecular findings of 23 Spanish patients (22 families) with LAL deficiency. We identified eight different mutations, four of them not previously reported. The novel c.966+2T>G mutation accounted for 75% of the Wolman disease alleles, and the frequent CESD associated c.894G>A mutation accounted for 55% of the CESD alleles in our cohort. Haplotype analysis showed that both mutations co-segregated with a unique haplotype suggesting a common ancestor. Our study contributes to the LAL deficiency acknowledgement with novel mutations and with high frequencies of some unknown mutations for WD.

Methods for the diagnosis of creatine deficiency syndromes: a comparative study.

The increasing number of patients with creatine deficiency syndromes (CDS) stresses the need to develop screening procedures for the identification these inherited disorders. Guanidinoacetate (GAA) and creatine (Cr) are reliable biochemical markers of CDS and several analytical methods to measure both metabolites have been developed. High-pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is quick and sensitive but, unlike HPLC and gas chromatography-mass spectrometry (GC/MS), it is unavailable in most laboratories. Thus, we decided to evaluate comparatively HPLC-MS/MS, GC/MS and HPLC methods, as well as to establish reference values in a healthy paediatric population. According to our results, these three methods may be suitable for analysing GAA in urine. Furthermore, Passing-Bablock plots showed good agreement among all three. However, when comparing the Cr/Crn ratio, our results revealed that while HPLC-MS/MS data were in agreement with those of GC/MS, a constant and proportional error was observed when compared with those of HPLC. Consequently, the Cr/Crn ratio obtained by the last method should be evaluated with caution. Our reference values for GAA and Cr/Crn ratio in urine negatively correlate with age. Concerning GAA and Cr measurements in plasma, it is interesting to note that in contrast to what was occurring in urine, GAA concentration increased significantly with age, while we did not find any significant difference for Cr values within the same age group.

Treatment of genetic defects of thiamine transport and metabolism.

INTRODUCTION: Thiamine is a key cofactor for energy metabolism in brain tissue. There are four major genetic defects (SLC19A2, SLC19A3, SLC25A19 and TPK1) involved in the metabolism and transport of thiamine through cellular and mitochondrial membranes. Neurological involvement predominates in three of them (SLC19A3, SCL25A19 and TPK1), whereas patients with SLC19A2 mutations mainly present extra-neurological features (e.g. diabetes mellitus, megaloblastic anaemia and sensori-neural hearing loss). These genetic defects may be amenable to therapeutic intervention with vitamins supplementation and hence, constitutes a main area of research. AREAS COVERED: We conducted a literature review of all reported cases with these genetic defects, and focused our paper on treatment efficacy and safety, adverse effects, dosing and treatment monitoring. Expert commentary: Doses of thiamine vary according to the genetic defect: for SLC19A2, the usual dose is 25-200 mg/day (1-4 mg/kg per day), for SLC19A3, 10-40 mg/kg per day, and for TPK1, 30 mg/kg per day. Thiamine supplementation in SLC19A3-mutated patients restores CSF and intracellular thiamine levels, resulting in successful clinical benefits. In conclusion, evidence collected so far suggests that the administration of thiamine improves outcome in SLC19A-2, SLC19A3- and TPK1-mutated patients, so most efforts should be aimed at early diagnosis of these disorders.