We report on ten individuals with a fatal infantile encephalopathy and/or pulmonary hypertension, leading to death before the age of 15 months. Hyperglycinemia and lactic acidosis were common findings. Glycine cleavage system and pyruvate dehydrogenase complex (PDHC) activities were low. Homozygosity mapping revealed a perfectly overlapping homozygous region of 1.24 Mb corresponding to chromosome 2 and led to the identification of a homozygous missense mutation (c.622G > T) in NFU1, which encodes a conserved protein suggested to participate in Fe-S cluster biogenesis. Nine individuals were homozygous for this mutation, whereas one was compound heterozygous for this and a splice-site (c.545 + 5G > A) mutation. The biochemical phenotype suggested an impaired activity of the Fe-S enzyme lipoic acid synthase (LAS). Direct measurement of protein-bound lipoic acid in individual tissues indeed showed marked decreases. Upon depletion of NFU1 by RNA interference in human cell culture, LAS and, in turn, PDHC activities were largely diminished. In addition, the amount of succinate dehydrogenase, but no other Fe-S proteins, was decreased. In contrast, depletion of the general Fe-S scaffold protein ISCU severely affected assembly of all tested Fe-S proteins, suggesting that NFU1 performs a specific function in mitochondrial Fe-S cluster maturation. Similar biochemical effects were observed in Saccharomyces cerevisiae upon deletion of NFU1, resulting in lower lipoylation and SDH activity. Importantly, yeast Nfu1 protein carrying the individuals' missense mutation was functionally impaired. We conclude that NFU1 functions as a late-acting maturation factor for a subset of mitochondrial Fe-S proteins.

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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.

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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.

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