RESUMEN
BACKGROUND: Synthesis and apoenzyme attachment of lipoic acid have emerged as a new complex metabolic pathway. Mutations in several genes involved in the lipoic acid de novo pathway have recently been described (i.e., LIAS, NFU1, BOLA3, IBA57), but no mutation was found so far in genes involved in the specific process of attachment of lipoic acid to apoenzymes pyruvate dehydrogenase (PDHc), α-ketoglutarate dehydrogenase (α-KGDHc) and branched chain α-keto acid dehydrogenase (BCKDHc) complexes. METHODS: Exome capture was performed in a boy who developed Leigh disease following a gastroenteritis and had combined PDH and α-KGDH deficiency with a unique amino acid profile that partly ressembled E3 subunit (dihydrolipoamide dehydrogenase / DLD) deficiency. Functional studies on patient fibroblasts were performed. Lipoic acid administration was tested on the LIPT1 ortholog lip3 deletion strain yeast and on patient fibroblasts. RESULTS: Exome sequencing identified two heterozygous mutations (c.875C > G and c.535A > G) in the LIPT1 gene that encodes a mitochondrial lipoyltransferase which is thought to catalyze the attachment of lipoic acid on PDHc, α-KGDHc, and BCKDHc. Anti-lipoic acid antibodies revealed absent expression of PDH E2, BCKDH E2 and α-KGDH E2 subunits. Accordingly, the production of 14CO2 by patient fibroblasts after incubation with 14Cglucose, 14Cbutyrate or 14C3OHbutyrate was very low compared to controls. cDNA transfection experiments on patient fibroblasts rescued PDH and α-KGDH activities and normalized the levels of pyruvate and 3OHbutyrate in cell supernatants. The yeast lip3 deletion strain showed improved growth on ethanol medium after lipoic acid supplementation and incubation of the patient fibroblasts with lipoic acid decreased lactate level in cell supernatants. CONCLUSION: We report here a putative case of impaired free or H protein-derived lipoic acid attachment due to LIPT1 mutations as a cause of PDH and α-KGDH deficiencies. Our study calls for renewed efforts to understand the mechanisms of pathology of lipoic acid-related defects and their heterogeneous biochemical expression, in order to devise efficient diagnostic procedures and possible therapies.
Asunto(s)
Aciltransferasas/genética , Enfermedad de Leigh/genética , Aminoácidos/sangre , Aminoácidos/líquido cefalorraquídeo , Aminoácidos/orina , Proteínas Portadoras/genética , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Immunoblotting , Complejo Cetoglutarato Deshidrogenasa/deficiencia , Complejo Cetoglutarato Deshidrogenasa/genética , Cetona Oxidorreductasas/deficiencia , Cetona Oxidorreductasas/genética , Enfermedad de Leigh/sangre , Enfermedad de Leigh/orina , Piruvato Deshidrogenasa (Lipoamida)/genética , Ácido Tióctico/sangre , Ácido Tióctico/líquido cefalorraquídeo , Ácido Tióctico/orinaRESUMEN
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.
Asunto(s)
Anemia Megaloblástica/genética , Diabetes Mellitus/genética , Pérdida Auditiva Sensorineural/genética , Heterocigoto , Proteínas de Transporte de Membrana/genética , Mutación Missense , Anemia Megaloblástica/diagnóstico , Anemia Megaloblástica/etnología , Austria , Preescolar , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/etnología , Marcadores Genéticos , Pérdida Auditiva Sensorineural/diagnóstico , Pérdida Auditiva Sensorineural/etnología , Humanos , Complejo Cetoglutarato Deshidrogenasa/deficiencia , Complejo Cetoglutarato Deshidrogenasa/genética , Masculino , Deficiencia de Tiamina/congénito , Población BlancaRESUMEN
The sucABCD genes of Escherichia coli encode subunits for two enzymes of the tricarboxylic acid (TCA) cycle, alpha-ketoglutarate dehydrogenase (sucAB) and succinyl coenzyme A synthetase (sucCD). To examine how these genes are expressed in response to changes in oxygen and carbon availability, a set of sucA-lacZ, sucC-lacZ, sdhCDAB-sucA-lacZ, and sdhC-lacZ fusions were constructed and analyzed in vivo. While the expression of a sucA-lacZ fusion was low under all cell growth conditions tested, the expression of the sucA gene from the upstream sdhC promoter was considerably higher and varied by up to 14-fold depending on the carbon substrate used. Expression of the sdhCDAB-sucA-lacZ fusion varied by fourfold in response to oxygen. In contrast, no expression was seen from a sucC-lacZ reporter fusion, indicating that no promoter immediately precedes the sucCD genes. Taken together, these findings demonstrate that the oxygen and carbon control of sucABCD gene expression occurs by transcriptional regulation of the upstream sdhC promoter. The weaker sucA promoter provides an additional low constitutive level of sucABCD gene expression to supplement transcription from the sdhC promoter. The negative control of sucABCD gene expression seen under anaerobic conditions, like that for the sdhCDAB genes, is provided by the arcA and fnr gene products. These findings establish that the differential expression of eight genes for three of the TCA cycle enzymes in E. coli is controlled from one regulatory element.