A thermolabile aldolase A mutant causes fever-induced recurrent rhabdomyolysis without hemolytic anemia.

Aldolase A deficiency has been reported as a rare cause of hemolytic anemia occasionally associated with myopathy. We identified a deleterious homozygous mutation in the ALDOA gene in 3 siblings with episodic rhabdomyolysis without hemolytic anemia. Myoglobinuria was always triggered by febrile illnesses. We show that the underlying mechanism involves an exacerbation of aldolase A deficiency at high temperatures that affected myoblasts but not erythrocytes. The aldolase A deficiency was rescued by arginine supplementation in vitro but not by glycerol, betaine or benzylhydantoin, three other known chaperones, suggesting that arginine-mediated rescue operated by a mechanism other than protein chaperoning. Lipid droplets accumulated in patient myoblasts relative to control and this was increased by cytokines, and reduced by dexamethasone. Our results expand the clinical spectrum of aldolase A deficiency to isolated temperature-dependent rhabdomyolysis, and suggest that thermolability may be tissue specific. We also propose a treatment for this severe disease.

Functional and electrophysiological characterization of four non-truncating mutations responsible for creatine transporter (SLC6A8) deficiency syndrome.

Intellectual disability coupled with epilepsy are clinical hallmarks of the creatine (Cr) transporter deficiency syndrome resulting from mutations in the SLC6A8 gene. So far characterization of pathogenic mutations of SLC6A8 has been limited to Cr uptake. The aim of our study was to characterize the electrogenic and pharmacological properties of non truncating SLC6A8 mutations identified in patients presenting variable clinical severity. Electrophysiological and pharmacological properties of four mutants (including two novel ones) were studied in X. laevis oocyte expression system. Creatine uptake was assessed with [(14)C]-Cr in X. laevis and patients' fibroblasts. Subcellular localization was determined by immunofluorescence and western blot. All mutants were properly targeted to the plasma membrane in both systems. Mutations led to the complete loss of both electrogenic and transport activities in X. laevis and Cr uptake in patients' fibroblasts. Among the Cr analogs tested, guanidinopropionate induced an electrogenic activity with the normal SLC6A8 transporter similar to creatine whereas a phosphocreatine derivative, PCr-Mg-CPLX, resulted in partial activity. SLC6A8 mutants displayed no electrogenic activity with all Cr analogs tested in X. laevis oocytes. Although the mutations altered various domains of SLC6A8 Cr uptake and electrogenic properties were completely inhibited and could not be dissociated. Besides the metabolic functions of Cr, the loss of SLC6A8 electrogenic activity, demonstrated here for the first time, may also play a role in the altered brain functions of the patients.

Mutations in human lipoyltransferase gene LIPT1 cause a Leigh disease with secondary deficiency for pyruvate and alpha-ketoglutarate dehydrogenase.

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.