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Maintaining protein lipoylation is vital for cell metabolism. The H-protein encoded by GCSH has a dual role in protein lipoylation required for bioenergetic enzymes including pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase, and in the one-carbon metabolism through its involvement in glycine cleavage enzyme system, intersecting two vital roles for cell survival. Here, we report six patients with biallelic pathogenic variants in GCSH and a broad clinical spectrum ranging from neonatal fatal glycine encephalopathy to an attenuated phenotype of developmental delay, behavioral problems, limited epilepsy and variable movement problems. The mutational spectrum includes one insertion c.293-2_293-1insT, one deletion c.122_(228 + 1_229-1) del, one duplication of exons 4 and 5, one nonsense variant p.Gln76*and four missense p.His57Arg, p.Pro115Leu and p.Thr148Pro and the previously described p.Met1?. Via functional studies in patient's fibroblasts, molecular modeling, expression analysis in GCSH knockdown COS7 cells and yeast, and in vitro protein studies, we demonstrate for the first time that most variants identified in our cohort produced a hypomorphic effect on both mitochondrial activities, protein lipoylation and glycine metabolism, causing combined deficiency, whereas some missense variants affect primarily one function only. The clinical features of the patients reflect the impact of the GCSH changes on any of the two functions analyzed. Our analysis illustrates the complex interplay of functional and clinical impact when pathogenic variants affect a multifunctional protein involved in two metabolic pathways and emphasizes the value of the functional assays to select the treatment and investigate new personalized options., (© The Author(s) 2022. Published by Oxford University Press.)

The uptake of radioactive biotin has been studied in glial cell cultures from dissociated cerebral hemispheres of newborn rats. We describe saturable kinetics for the biotin uptake at biotin concentrations of less than 60 nM and linear at greater than 60 nM. The uptake appeared temperature sensitive, Na+ independent, nonsensitive to valinomycin, and not affected by metabolic inhibitors such as sodium fluoride or azide. Lipoic acid and biocytin were effective in inhibiting the biotin uptake. These findings are consistent with biotin uptake by the primary culture of astrocytes as a process of facilitated diffusion. Moreover, biotin uptake in astrocytes grown in biotin-restricted conditions was significantly higher compared with the control. This increase appeared mediated through a pronounced increase (10-fold) in the Vmax of the biotin uptake without any change in the apparent Km.

The biochemical characteristics of a patient with most of the clinical symptoms of nonketotic-hyperglycinemia have been studied. Despite the extremely low plasma/cerebrospinal fluid glycine ratio of the patient, typical of the nonketotic syndrome, at autopsy we found no enhancement of glycine levels in brain and glycine cleavage enzyme activities in the brain and liver that were similar to those of a control newborn. In order to ascertain the basic defect responsible for glycine accumulation in the cerebrospinal fluid and for the neurological damage, we examined the possible existence of altered glycine transport systems in nervous cells. Our findings indicate that (1) low and high affinity, Na+-dependent transport systems for glycine can be detected in both rat and control human postmortem tissue and (2) the low affinity glycine transport system is absent in the brain of the patient, whereas an alteration of transport systems occurs in the patient's spinal cord, probably involving the high-affinity component. These alterations could account for the clinical features of the patient. Since the feasibility of determining glycine transport parameters in postmortem tissue has been established, we think it would be of interest to investigate such systems in cases where the etiology of hyperglycinemia is not clear.