Combined malonic and methylmalonic aciduria (CMAMMA) is an inborn error of metabolism caused by a deficiency in mitochondrial malonyl-CoA synthetase, the enzyme responsible for activating malonic acid (MA) to malonyl-CoA, a precursor of lipoic acid. The clinical phenotype is highly heterogeneous, ranging from asymptomatic cases to severe neurological impairment. We describe a patient affected by CMAMMA. The patient presented in the neonatal period with hyperinsulinemic hypoglycemia. Urinary organic acid analysis revealed elevated levels of both malonic and methylmalonic acids. The diagnosis of CMAMMA was confirmed through molecular testing of the ACSF3 gene. Levels of both lipoylated pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (αKGDH) were decreased. Given the role of lipoic acid in regulating insulin secretion, the involvement of impaired mitochondrial lipoic acid biosynthesis in the clinical presentation of hyperinsulinemic hypoglycemia cannot be excluded. We describe a case of CMAMMA associated with hyperinsulinemic hypoglycemia. While a definitive association between CMAMMA and hyperinsulinism cannot be established based on a single case, the observed reduction in lipoic acid levels may suggest a mechanistic connection between the two disorders. We suggest considering urinary organic acid testing in patients with hyperinsulinism, especially when the cause is unknown.

BOLA3 is one of the proteins involved in the assembly and transport of [4Fe-4S] clusters, which are incorporated into mitochondrial respiratory chain complexes I and II, aconitase, and lipoic acid synthetase. Pathogenic variants in the BOLA3 gene cause a rare condition known as multiple mitochondrial dysfunctions syndrome 2 with hyperglycinemia, characterized by life-threatening lactic acidosis, nonketotic hyperglycinemia, and hypertrophic cardiomyopathy. The aim of this study was to elucidate the biochemical characteristics of patients with BOLA3 variants and to clarify the role of BOLA3 protein in humans. The characteristics, clinical course, and biochemical data of eight Japanese patients with BOLA3 pathogenic variants were collected. In addition, metabolomic analyses were performed using capillary electrophoresis time-of-flight mass spectrometry, blue native polyacrylamide gel electrophoresis (BN-PAGE)/Western blot analysis of mitochondrial respiratory chain complexes, and in-gel enzyme staining of mitochondrial respiratory chain complexes of fibroblasts from all eight patients. Metabolomic data were compared between the eight patients with BOLA3 variants and three control samples using Welch's t-test. In the metabolomic analysis, levels of lactic acid, pyruvic acid, alanine, tricarboxylic acid (TCA) cycle intermediates (such as α-ketoglutaric acid and succinic acid), branched-chain amino acids, and metabolites of lysine and tryptophan were significantly elevated in the BOLA3 group. Data collected during the patients' lives showed increased lactic acid and glycine levels. In BN-PAGE/Western blot analysis and in-gel enzyme staining, bands for complexes I and II were barely detectable in all eight cases. These results indicate that BOLA3 variants decrease the activity of lipoic acid-dependent proteins (pyruvate dehydrogenase complex, α-ketoglutarate dehydrogenase, 2-oxoadipate dehydrogenase, branched-chain ketoacid dehydrogenase, and the glycine cleavage system), as well as mitochondrial respiratory chain complexes I and II, but do not affect aconitase. Thus, it is believed that BOLA3 is involved in transporting [4Fe-4S] clusters to respiratory chain complexes I and II and lipoic acid synthetase, but does not interfere with aconitase. These findings suggest that while lipoic acid supplementation or vitamin cocktails may provide benefits, the impaired [4Fe-4S] cluster pathway itself should be targeted for treatment to improve the extensive metabolic abnormalities caused by BOLA3 deficiency.

Protein lipoylation, a vital lysine post-translational modification, plays a crucial role in the function of key mitochondrial tricarboxylic acid cycle enzymatic complexes. In eukaryotes, lipoyl post-translational modification synthesis occurs exclusively through de novo pathways, relying on lipoyl synthesis/transfer enzymes, dependent upon mitochondrial fatty acid and Fe-S cluster biosynthesis. Dysregulation in any of these pathways leads to diminished cellular lipoylation. Efficient restoration of lipoylation in lipoylation deficiency cell states using either chemical or genetic approaches has been challenging because of pathway complexity and multiple upstream regulators. To address this challenge, we explored the possibility that a bacterial lipoate protein ligase A (lplA) enzyme, which can salvage free lipoic acid bypassing the dependency on de novo synthesis, could be engineered to be functional in human cells. Overexpression of the engineered lplA in lipoylation null cells restored lipoylation levels, cellular respiration, and growth in low glucose conditions. Engineered lplA restored lipoylation in all tested lipoylation null cell models, mimicking defects in mitochondrial fatty acid synthesis (MECR KO), Fe-S cluster biosynthesis (BOLA3 KO), and specific lipoylation-regulating enzymes (FDX1 [ferredoxin 1], LIAS [lipoyl synthase], and LIPT1 [lipoyl (octanoyl) transferase 1] KOs). Furthermore, we describe a patient with a homozygous c.212C>T variant LIPT1 with a previously uncharacterized syndromic congenital sideroblastic anemia. K562 erythroleukemia cells engineered to harbor this missense LIPT1 allele recapitulate the lipoylation-deficient phenotype and exhibit impaired proliferation in low glucose that is completely restored by engineered lplA. This synthetic approach offers a potential therapeutic strategy for treating lipoylation disorders.

Glutamate-cysteine ligase catalytic subunit (GCLC), previously known as gamma-glutamyl-cysteine synthetase, is an essential rate-limiting step in glutathione synthesis. Glutathione modulates multitudes of critical cellular processes and scavenges free radicals. Its deficiency is reported to cause hemolysis of variable severity and is a rare cause of neurological abnormalities such as spinocerebellar ataxia. We report a 55-year-old female patient with progressive late-onset ataxia, lower limb spasticity, and chronic hemolytic anemia found to have a GCLC pathogenic variant and low glutathione level. Magnetic resonance imaging of the head and cervical spine showed global cerebellar atrophy with widened folia and decreased diameter of the upper cervical spine. Blood workup revealed hemolytic anemia with genetic testing confirmed a homozygous variant, c.514 T>A in exon 4 of the GCLC gene, resulting in Ser172Thr (TCC>ACC). Management encompassed a multidisciplinary approach with a trial of high-dose alpha-lipoic acid, glutathione supplement, and physical therapy. GCLC deficiency manifesting with hemolysis has been reported in 12 cases worldwide from 6 independent families, with only 4 cases having additional neurological manifestations. To date, no specific GCLC gene mutation has been attributed to the reported neurological constellation of symptoms. To the best of our knowledge, this is the first case report of late-onset spinocerebellar degeneration as a manifestation of c.514T>A (p. S172T) GCLC pathological variant genetic mutation.

The tripeptide thiol antioxidant glutathione (GSH) has multiple physiological functions. Female mice lacking the modifier subunit of glutamate cysteine ligase (GCLM), the rate-limiting enzyme in GSH synthesis, have decreased GSH concentrations, ovarian oxidative stress, preimplantation embryonic mortality, and accelerated age-related decline in ovarian follicles. We hypothesized that supplementation with thiol antioxidants, N-acetyl cysteine (NAC), or α-lipoic acid (ALA) will rescue this phenotype. Gclm-/- and Gclm+/+ females received 0 or 80 mM NAC in drinking water from postnatal day (PND) 21-30; follicle growth was induced with equine chorionic gonadotropin (eCG) on PND 27, followed by an ovulatory dose of human CG and mating with a wild type male on PND 29 and zygote harvest 20 h after hCG. N-acetyl cysteine supplementation failed to rescue the low rate of second pronucleus formation in zygotes from Gclm-/- versus Gclm+/+ females. In the second study, Gclm-/- and Gclm+/+ females received diet containing 0, 150, or 600 mg/kg ALA beginning at weaning and were mated with wild type males from 8 to 20 weeks of age. α-Lipoic acid failed to rescue the decreased offspring production of Gclm-/- females. However, 150 mg/kg diet ALA partially rescued the accelerated decline in primordial follicles, as well as the increased recruitment of follicles into the growing pool and the increased percentages of follicles with γH2AX positive oocytes or granulosa cells of Gclm-/- females. We conclude that ovarian oxidative stress is the cause of accelerated primordial follicle decline, while GSH deficiency per se may be responsible for preimplantation embryonic mortality in Gclm-/- females.