Pathogenic variants inGCSHencoding the moonlighting H-protein cause combined nonketotic hyperglycinemia and lipoate deficiency

Abstract

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 modelling, expression analysis in GCSH knock-down 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.

A hub for regulation of mitochondrial metabolism: Fatty acid and lipoic acid biosynthesis

“…33 In humans, both functions of the H protein (Gcv3) are critical for human health; mutations in the GCSH gene cause both lipoylation defects and hyperglycinemia. 77 As has been discussed in this review, fatty acids with chains longer than eight carbons are crucial in several aspects of mitochondrial function. One function in yeast mitochondria is to promote 5 0 processing of pre-tRNAs.…”

“…Mutations in human FAS II-LAS pathway genes are rarely found in newborns because they affect very early steps in development, most often leading to premature death of the fetus. 15,77,85 There may be some potential for alleviating symptoms of more minorly affected individuals with mitochondrially directed transgene products. Supplementation with free fatty acids or lipoic acids cannot rescue these children because there are no known mitochondrial enzymes that can utilize the free molecules.…”

“…Mutations in human genes LIPT1, LIPT2, and LIAS are reviewed in Reference[15]. Mutations in human gene GCSH are reported in Reference[77]. 0 phosphopantetheine is depicted as an asterisk *) and the synthesis of acetyl CoA followed by malonyl CoA precede the transfer of the malonyl group to *Acp1.…”

Deep postnatal phenotyping of a new mouse model of nonketotic hyperglycinemia

“…Glycine levels were determined by HPLC in mouse plasma and brain tissue (cortex, cerebellum, and hippocampus) as previously described. 4,5 In brief, plasma samples were combined with 3:1 volumes of methanol containing 133 µM L-homocysteic acid as an internal standard, and placed on ice for 10 minutes to precipitate the proteins. Brain tissues were homogenized in 2 volumes of phosphate-buffered saline using 0.5 mm zirconium oxide beads in a Storm Pro Bullet Blender (Next Advance, Troy, NY) at speed 4 for 5 minutes, followed by deproteinization as above.…”

“…In 80% of patients with NKH, deficient GCS activity is caused by pathogenic loss of function variants in the GLDC gene encoding the P-protein, while the remaining 20% of patients have defects in the AMT gene encoding the T-protein, and in very rare patients the defect resides in the GCSH gene encoding the H-protein. 1,4 Most patients have the severe form of NKH and present with neonatal epileptic encephalopathy, absent psychomotor development, spasticity, and therapy-resistant epilepsy. 2,5,6 An attenuated form of NKH is present in 15% of patients characterized by developmental progress and treatable or no epilepsy.…”