Biallelic Variants of MRPS36 Cause a New Form of Leigh Syndrome.

BACKGROUND: The MRPS36 gene encodes a recently identified component of the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme of the Krebs cycle catalyzing the oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA. Defective OGDHC activity causes a clinically variable metabolic disorder characterized by global developmental delay, severe neurological impairment, liver failure, and early-onset lactic acidosis. METHODS: We investigated the molecular cause underlying Leigh syndrome with bilateral striatal necrosis in two siblings through exome sequencing. Functional studies included measurement of the OGDHC enzymatic activity and MRPS36 mRNA levels in fibroblasts, assessment of protein stability in transfected cells, and structural analysis. A literature review was performed to define the etiological and phenotypic spectrum of OGDHC deficiency. RESULTS: In the two affected brothers, exome sequencing identified a homozygous nonsense variant (c.283G>T, p.Glu95*) of MRPS36. The variant did not affect transcript processing and stability, nor protein levels, but resulted in a shorter protein lacking nine residues that contribute to the structural and functional organization of the OGDHC complex. OGDHC enzymatic activity was significantly reduced. The review of previously reported cases of OGDHC deficiency supports the association of this enzymatic defect with Leigh phenotypic spectrum and early-onset movement disorder. Slightly elevated plasma levels of glutamate and glutamine were observed in our and literature patients with OGDHC defect. CONCLUSIONS: Our findings point to MRPS36 as a new disease gene implicated in Leigh syndrome. The slight elevation of plasma levels of glutamate and glutamine observed in patients with OGDHC deficiency represents a candidate metabolic signature of this neurometabolic disorder. © 2024 International Parkinson and Movement Disorder Society.

A biallelic pathogenic variant in the OGDH gene results in a neurological disorder with features of a mitochondrial disease.

2-Oxoglutarate dehydrogenase (OGDH) is a rate-limiting enzyme in the mitochondrial TCA cycle, encoded by the OGDH gene. α-Ketoglutarate dehydrogenase (OGDH) deficiency was previously reported in association with developmental delay, hypotonia, and movement disorders and metabolic decompensation, with no genetic data provided. Using whole exome sequencing, we identified two individuals carrying a homozygous missense variant c.959A>G (p.N320S) in the OGDH gene. These individuals presented with global developmental delay, elevated lactate, ataxia and seizure. Fibroblast analysis and modeling of the mutation in Drosophila were used to evaluate pathogenicity of the variant. Skin fibroblasts from subject # 2 showed a decrease in both OGDH protein and enzyme activity. Transfection of human OGDH cDNA in HEK293 cells carrying p.N320S also produced significantly lower protein levels compared to those with wild-type cDNA. Loss of Drosophila Ogdh (dOgdh) caused early developmental lethality, rescued by expressing wild-type dOgdh (dOgdhWT ) or human OGDH (OGDHWT ) cDNA. In contrast, expression to the mutant OGDH (OGDHN320S ) or dOgdh carrying homologous mutations to human OGDH p.N320S variant (dOgdhN324S ) failed to rescue lethality of dOgdh null mutants. Knockdown of dOgdh in the nervous system resulted in locomotion defects which were rescued by dOgdhWT expression but not by dOgdhN324S expression. Collectively, the results indicate that c.959A>G variant in OGDH leads to an amino acid change (p.N320S) causing a severe loss of OGDH protein function. Our study establishes in the first time a genetic link between an OGDH gene mutation and OGDH deficiency.

OGDHL silencing promotes hepatocellular carcinoma by reprogramming glutamine metabolism.

BACKGROUND & AIMS: Mitochondrial dysfunction and subsequent metabolic deregulation are commonly observed in cancers, including hepatocellular carcinoma (HCC). When mitochondrial function is impaired, reductive glutamine metabolism is a major cellular carbon source for de novo lipogenesis to support cancer cell growth. The underlying regulators of reductively metabolized glutamine in mitochondrial dysfunction are not completely understood in tumorigenesis. METHODS: We systematically investigated the role of oxoglutarate dehydrogenase-like (OGDHL), one of the rate-limiting components of the key mitochondrial multi-enzyme OGDH complex (OGDHC), in the regulation of lipid metabolism in hepatoma cells and mouse xenograft models. RESULTS: Lower expression of OGDHL was associated with advanced tumor stage, significantly worse survival and more frequent tumor recurrence in 3 independent cohorts totaling 681 postoperative HCC patients. Promoter hypermethylation and DNA copy deletion of OGDHL were independently correlated with reduced OGDHL expression in HCC specimens. Additionally, OGDHL overexpression significantly inhibited the growth of hepatoma cells in mouse xenografts, while knockdown of OGDHL promoted proliferation of hepatoma cells. Mechanistically, OGDHL downregulation upregulated the α-ketoglutarate (αKG):citrate ratio by reducing OGDHC activity, which subsequently drove reductive carboxylation of glutamine-derived αKG via retrograde tricarboxylic acid cycling in hepatoma cells. Notably, silencing of OGDHL activated the mTORC1 signaling pathway in an αKG-dependent manner, inducing transcription of enzymes with key roles in de novo lipogenesis. Meanwhile, metabolic reprogramming in OGDHL-negative hepatoma cells provided an abundant supply of NADPH and glutathione to support the cellular antioxidant system. The reduction of reductive glutamine metabolism through OGDHL overexpression or glutaminase inhibitors sensitized tumor cells to sorafenib, a molecular-targeted therapy for HCC. CONCLUSION: Our findings established that silencing of OGDHL contributed to HCC development and survival by regulating glutamine metabolic pathways. OGDHL is a promising prognostic biomarker and therapeutic target for HCC. LAY SUMMARY: Hepatocellular carcinoma (HCC) is one of the most prevalent tumors worldwide and is correlated with a high mortality rate. In patients with HCC, lower expression of the enzyme OGDHL is significantly associated with worse survival. Herein, we show that silencing of OGDHL induces lipogenesis and influences the chemosensitization effect of sorafenib in liver cancer cells by reprogramming glutamine metabolism. OGDHL is a promising prognostic biomarker and potential therapeutic target in OGDHL-negative liver cancer.

Reductions in the mitochondrial enzyme α-ketoglutarate dehydrogenase complex in neurodegenerative disease - beneficial or detrimental?

Reductions in metabolism and excess oxidative stress are prevalent in multiple neurodegenerative diseases. The activity of the mitochondrial enzyme α-ketoglutarate dehydrogenase complex (KGDHC) appears central to these abnormalities. KGDHC is diminished in multiple neurodegenerative diseases. KGDHC can not only be rate limiting for NADH production and for substrate level phosphorylation, but is also a source of reactive oxygen species (ROS). The goal of these studies was to determine how changes in KGDHC modify baseline ROS, the ability to buffer ROS, baseline glutathionylation, calcium modulation and cell death in response to external oxidants. In vivo, reducing KGDHC with adeno virus diminished neurogenesis and increased oxidative stress. In vitro, treatments of short duration increased ROS and glutathionylation and enhanced the ability of the cells to diminish the ROS from added oxidants. However, long-term reductions lessened the ability to diminish ROS, diminished glutathionylation and exaggerated oxidant-induced changes in calcium and cell death. Increasing KGDHC enhanced the ability of the cells to diminish externally added ROS and protected against oxidant-induced changes in calcium and cell death. The results suggest that brief periods of diminished KGDHC are protective, while prolonged reductions are harmful. Furthermore, elevated KGDHC activities are protective. Thus, mitogenic therapies that increase KGDHC may be beneficial in neurodegenerative diseases. Read the Editorial Highlight for this article on Page 689.

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.

The negative impact of α-ketoglutarate dehydrogenase complex deficiency on matrix substrate-level phosphorylation.

A decline in α-ketoglutarate dehydrogenase complex (KGDHC) activity has been associated with neurodegeneration. Provision of succinyl-CoA by KGDHC is essential for generation of matrix ATP (or GTP) by substrate-level phosphorylation catalyzed by succinyl-CoA ligase. Here, we demonstrate ATP consumption in respiration-impaired isolated and in situ neuronal somal mitochondria from transgenic mice with a deficiency of either dihydrolipoyl succinyltransferase (DLST) or dihydrolipoyl dehydrogenase (DLD) that exhibit a 20-48% decrease in KGDHC activity. Import of ATP into the mitochondrial matrix of transgenic mice was attributed to a shift in the reversal potential of the adenine nucleotide translocase toward more negative values due to diminished matrix substrate-level phosphorylation, which causes the translocase to reverse prematurely. Immunoreactivity of all three subunits of succinyl-CoA ligase and maximal enzymatic activity were unaffected in transgenic mice as compared to wild-type littermates. Therefore, decreased matrix substrate-level phosphorylation was due to diminished provision of succinyl-CoA. These results were corroborated further by the finding that mitochondria from wild-type mice respiring on substrates supporting substrate-level phosphorylation exhibited ~30% higher ADP-ATP exchange rates compared to those obtained from DLST(+/-) or DLD(+/-) littermates. We propose that KGDHC-associated pathologies are a consequence of the inability of respiration-impaired mitochondria to rely on "in-house" mitochondrial ATP reserves.

Thiamine-responsive megaloblastic anemia syndrome with Ebstein anomaly: a case report.

UNLABELLED: Thiamine-responsive megaloblastic anemia (TRMA) or Roger syndrome is a rare autosomal recessive disorder characterized by the occurrence of multiple clinical manifestations including megaloblastic anemia, diabetes mellitus, and sensorineural deafness. A few patients have been also described with congenital cardiac malformations. The patients usually respond to treatment with pharmacological doses of thiamine. Mutations in the SLC19A2 gene, located at chromosome 1q24.2, are responsible for this syndrome. Here, we present two new Iranian TRMA patients who were homozygous for c.697C > T mutation in the SLC19A2 gene. On follow-up, one of the patients showed Ebstein anomaly. CONCLUSION: The present study confirms the variability of the clinical manifestations caused by the same mutation within patients with TRMA syndrome. Therefore, follow-up of the affected children should be considered.

Rare case with megaloblastic anaemia.

A nine years old boy presented with history of pallor and anaemia since early infancy along with neural hearing loss responding to empirical multivitamin and folic acid therapy started on basis of blood complete picture showing anaemia and megaloblastic anaemia. On investigation he was diagnosed with Thiamine Responsive Megaloblastic Anaemia, a very rare condition in our settings.

Thiamine responsive megaloblastic anemia: a novel SLC19A2 compound heterozygous mutation in two siblings.

Thiamine responsive megaloblastic anemia (TRMA) is an autosomal recessive disease caused by loss of function mutations in the SLC19A2 gene. TRMA is characterized by anemia, deafness, and diabetes. In some cases, optic atrophy or more rarely retinitis pigmentosa is noted. We now report two sisters, the eldest of which presented to a different hospital during childhood with sensorineural deafness, which was treated with a hearing prosthesis, insulin requiring diabetes, retinitis pigmentosa, optic atrophy, and macrocytic anemia. These features initially suggested a clinical diagnosis of Wolfram syndrome (WS). Therapy with thiamine was initiated which resulted in the resolution of the anemia. The younger sister, who was affected with sensorineural deafness, was referred to our hospital for non-autoimmune diabetes. She was found to have macrocytosis and ocular abnormalities. Because a diagnosis of TRMA was suspected, therapy with insulin and thiamine was started. Sequencing analysis of the SLC19A2 gene identified a compound heterozygous mutation p.Y81X/p.L457X (c.242insA/c.1370delT) in both sisters. Non-autoimmune diabetes associated with deafness and macrocytosis, without anemia, suggests a diagnosis of TRMA. Patients clinically diagnosed with WS with anemia and/or macrocytosis should be reevaluated for TRMA.

Thiamine-responsive megaloblastic anemia (TRMA) in an Austrian boy with compound heterozygous SLC19A2 mutations.

Thiamine-responsive megaloblastic anemia (TRMA) is a rare disorder typically characterized by megaloblastic anemia, non-type I diabetes and sensorineural deafness. It is caused by various mutations in the SLC19A2 gene that impair the encoded thiamine transporter. So far, only 70 affected individuals mainly from consanguineous families of Middle and Far Eastern origin with a wide spectrum of signs and symptoms, variable onset of disease, and primarily homozygote mutations in SLC19A2 have been reported. We present the first genuine central European descendent with combined heterozygote mutations in SLC19A2, an Austrian boy suffering from pancytopenia and non-type I diabetes. Both manifestations resolved completely under continuous oral thiamine supplementation. Our observation underlines that despite its rarity, TRMA must be considered as an important differential diagnosis in native central European patients with suggestive signs and symptoms. An early molecular genetic verification of the diagnosis provides a sound basis for a successful and simple treatment that helps to prevent severe sequelae.

Thiamine-responsive megaloblastic anemia syndrome: a novel mutation.

The thiamine-responsive megaloblastic anemia syndrome (TRMA) is an autosomal recessive disorder characterized by diabetes mellitus, megaloblastic anemia and sensorineural hearing loss due to mutations in SLC 19A2 that encodes a thiamine transporter protein. The disease can manifest at any time between infancy and adolescence, and not all cardinal findings are present initially. The anemia typically improves significantly with pharmacological doses of thiamine. Variable improvement in diabetes is also noted. However, the hearing loss is apparently irreversible, although a delay in the onset of deafness may be possible. We present a 2-year old girl with non-autoimmune diabetes mellitus and anemia in whom we found a novelc.95T>A (leu32X) mutation in the SLC19A2 gene in this study.Our patient with this new mutation did not suffer from hearing loss.

Thiamine-responsive megaloblastic anemia syndrome with atrial standstill: a case report.

Thiamine-responsive megaloblastic anemia (TRMA) syndrome is an uncommon autosomal recessive disorder. The disease is caused by mutations in the gene, SLC19A2, encoding a high-affinity thiamine transporter, which disturbs the active thiamine uptake into cells. Major features include megaloblastic anemia, diabetes mellitus, and sensorineural deafness. Cardiac malformations with conduction defects and/or dysrhythmias, have also been described in some patients. To our knowledge, only 13 TRMA patients with cardiac defects have been reported. Here, we describe the first case of TRMA syndrome with atrial standstill, probably caused by a 2 base-pair deletion in exon 4 (1147delGT) of the gene SLC19A2.

[Ultrastructure of coronary microvessels in conditions of heart reperfusion following prolonged ischemia while applying various methods of artificial hypothermia].

It has been found out that in children with Roger's disease corrected in the conditions of two fundamentally different procedures of anesthetic management, myocardial reperfusion after cardiac arrest under artificial hypothermic circulation is accompanied by obstruction of more than 30% of coronary bed microvessels with hydropic endothelial cells or their cystiform fragments. The content of necrotic cells increases, while the "working" cells demonstrate a decrease in myocropinocytotic transport characteristics. Circulatory arrest under perfusionless hypothermia and immersion reperfusion do not result in a dramatic change of general morphology of microvessels as compared to the control group, while a heterogenic response of the structures responsible for transendothelial transfer of macromolecules provides the basis for recovery of the endothelium structure and function, as a patient's temperature reaches a standard value.

Mice deficient in dihydrolipoyl succinyl transferase show increased vulnerability to mitochondrial toxins.

The activity of a key mitochondrial tricarboxylic acid cycle enzyme, alpha-ketoglutarate dehydrogenase complex (KGDHC), declines in many neurodegenerative diseases. KGDHC consists of three subunits. The dihydrolipoyl succinyl transferase (DLST) component is unique to KGDHC. DLST(+/-) mice showed reduced mRNA and protein levels and decreased brain mitochondrial KGDHC activity. Neurotoxic effects of mitochondrial toxins were exacerbated in DLST(+/-) mice. MPTP produced a significantly greater reduction of striatal dopamine and tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta of DLST(+/-) mice. DLST deficiency enhanced the severity of lipid peroxidation in the substantia nigra after MPTP treatment. Striatal lesions induced by either malonate or 3-nitropropionic acid (3-NP) were significantly larger in DLST(+/-) mice than in wildtype controls. DLST deficiency enhanced the 3-NP inhibition of mitochondria enzymes, and 3-NP induced protein and DNA oxidations. These observations support the hypothesis that reductions in KGDHC may impair the adaptability of the brain and contribute to the pathogenesis of neurodegenerative diseases.

A novel mutation in the dihydrolipoamide dehydrogenase E3 subunit gene (DLD) resulting in an atypical form of alpha-ketoglutarate dehydrogenase deficiency.

The alpha-ketoglutarate dehydrogenase complex (KGDC) catalyses the decarboxylation of alpha-ketoglutarate into succinyl-coenzyme A in the Krebs cycle. This enzymatic complex is made up of three subunits (E1, encoded by PDHA1; E2, encoded by DLST; and E3, encoded by DLD). The E3 subunit is common to two other enzymatic complexes, namely pyruvate dehydrogenase complex (PDC) and branched-chain ketoacid dehydrogenase complex (BCKDC). KGDC deficiency is a rare autosomal recessive disorder, most often presenting with severe encephalopathy and hyperlactatemia with neonatal onset. We found a KGDC deficiency in cultured skin fibroblasts from three siblings born to consanguinous parents. E3 subunit activity was shown to be deficient (20% of control values), despite the absence of usual clinical clues to E3 deficiency, i.e. accumulation of pyruvate and branched-chain amino acids in plasma and branched-chain alpha-ketoacids in urine. RT-PCR of E3 mRNA from the three patients, followed by sequencing, revealed an homozygous c.1444A>G substitution located in E3 exon 13, predictive of a p.R482G (or R447G in the processed gene product) substitution in a highly conserved domain of the protein. Only eleven E3 mutations have been reported so far. The only other case of E3 deficiency without clinical or biochemical evidences of PDC and BCKDC deficiencies has been ascribed to a c.1436A>T (p.D479V; or D444V in the processed gene product) mutation, very close to the mutation reported herein. Since c.1444A>G (p.R482G; or R447G in the processed gene product) and c.1436A>T (p.D479V; or D444V in the processed gene product) lie within the interface domain of E3 with E2 (KGDC and BCKDC) or the E3-binding protein (PDC), our data suggest that interaction of E3 with these other subunits differs in some extent among KGDC, PDC, and BCKDC.

Inborn errors of the Krebs cycle: a group of unusual mitochondrial diseases in human.

Krebs cycle disorders constitute a group of rare human diseases which present an amazing complexity considering our current knowledge on the Krebs cycle function and biogenesis. Acting as a turntable of cell metabolism, it is ubiquitously distributed in the organism and its enzyme components encoded by supposedly typical house-keeping genes. However, the investigation of patients presenting specific defects of Krebs cycle enzymes, resulting from deleterious mutations of the considered genes, leads to reconsider this simple envision by revealing organ-specific impairments, mostly affecting neuromuscular system. This often leaves aside organs the metabolism of which strongly depends on mitochondrial energy metabolism as well, such as heart, kidney or liver. Additionally, in some patients, a complex pattern of tissue-specific enzyme defect was also observed. The lack of functional additional copies of Krebs cycle genes suggests that the complex expression pattern should be ascribed to tissue-specific regulations of transcriptional and/or translational activities, together with a variable cell adaptability to Krebs cycle functional defects.