Mutations in COQ4, an essential component of coenzyme Q biosynthesis, cause lethal neonatal mitochondrial encephalomyopathy.

BACKGROUND: The identification of the molecular basis of mitochondrial disorders continues to be challenging and expensive. The increasing usage of next-generation sequencing is facilitating the discovery of the genetic aetiology of heterogeneous phenotypes associated with these conditions. Coenzyme Q(10) (CoQ(10)) is an essential cofactor for mitochondrial respiratory chain complexes and other biochemical pathways. Mutations in genes involved in CoQ(10) biosynthesis cause primary CoQ(10) deficiency syndromes that can be treated with oral supplementation of ubiquinone. METHODS: We used whole exome sequencing to evaluate six probands from four unrelated families with clinical findings suggestive of a mitochondrial disorder. Clinical data were obtained by chart review, parental interviews, direct patient assessment and biochemical and pathological evaluation. RESULTS: We identified five recessive missense mutations in COQ4 segregating with disease in all four families. One mutation was found in a homozygous state in two unrelated Ashkenazi Jewish probands. All patients were female, and presented on the first day of life, and died in the neonatal period or early infancy. Clinical findings included hypotonia (6/6), encephalopathy with EEG abnormalities (4/4), neonatal seizures (3/6), cerebellar atrophy (4/5), cardiomyopathy (5/6) and lactic acidosis (4/6). Autopsy findings in two patients revealed neuron loss and reactive astrocytosis or cerebellar and brainstem hypoplasia and microdysgenesis. CONCLUSIONS: Mutations in COQ4 cause an autosomal recessive lethal neonatal mitochondrial encephalomyopathy associated with a founder mutation in the Ashkenazi Jewish population. The early mortality in our cohort suggests that COQ4 is an essential component of the multisubunit complex required for CoQ(10) biosynthesis.

Engineering the gut microbiota to treat hyperammonemia.

Increasing evidence indicates that the gut microbiota can be altered to ameliorate or prevent disease states, and engineering the gut microbiota to therapeutically modulate host metabolism is an emerging goal of microbiome research. In the intestine, bacterial urease converts host-derived urea to ammonia and carbon dioxide, contributing to hyperammonemia-associated neurotoxicity and encephalopathy in patients with liver disease. Here, we engineered murine gut microbiota to reduce urease activity. Animals were depleted of their preexisting gut microbiota and then inoculated with altered Schaedler flora (ASF), a defined consortium of 8 bacteria with minimal urease gene content. This protocol resulted in establishment of a persistent new community that promoted a long-term reduction in fecal urease activity and ammonia production. Moreover, in a murine model of hepatic injury, ASF transplantation was associated with decreased morbidity and mortality. These results provide proof of concept that inoculation of a prepared host with a defined gut microbiota can lead to durable metabolic changes with therapeutic utility.

Down-regulation of hepatic urea synthesis by oxypurines: xanthine and uric acid inhibit N-acetylglutamate synthase.

We previously reported that isobutylmethylxanthine (IBMX), a derivative of oxypurine, inhibits citrulline synthesis by an as yet unknown mechanism. Here, we demonstrate that IBMX and other oxypurines containing a 2,6-dione group interfere with the binding of glutamate to the active site of N-acetylglutamate synthetase (NAGS), thereby decreasing synthesis of N-acetylglutamate, the obligatory activator of carbamoyl phosphate synthase-1 (CPS1). The result is reduction of citrulline and urea synthesis. Experiments were performed with (15)N-labeled substrates, purified hepatic CPS1, and recombinant mouse NAGS as well as isolated mitochondria. We also used isolated hepatocytes to examine the action of various oxypurines on ureagenesis and to assess the ameliorating affect of N-carbamylglutamate and/or l-arginine on NAGS inhibition. Among various oxypurines tested, only IBMX, xanthine, or uric acid significantly increased the apparent K(m) for glutamate and decreased velocity of NAGS, with little effect on CPS1. The inhibition of NAGS is time- and dose-dependent and leads to decreased formation of the CPS1-N-acetylglutamate complex and consequent inhibition of citrulline and urea synthesis. However, such inhibition was reversed by supplementation with N-carbamylglutamate. The data demonstrate that xanthine and uric acid, both physiologically occurring oxypurines, inhibit the hepatic synthesis of N-acetylglutamate. An important and novel concept emerging from this study is that xanthine and/or uric acid may have a role in the regulation of ureagenesis and, thus, nitrogen homeostasis in normal and disease states.

Ifosfamide-induced nephrotoxicity: mechanism and prevention.

The efficacy of ifosfamide (IFO), an antineoplastic drug, is severely limited by a high incidence of nephrotoxicity of unknown etiology. We hypothesized that inhibition of complex I (C-I) by chloroacetaldehyde (CAA), a metabolite of IFO, is the chief cause of nephrotoxicity, and that agmatine (AGM), which we found to augment mitochondrial oxidative phosphorylation and beta-oxidation, would prevent nephrotoxicity. Our model system was isolated mitochondria obtained from the kidney cortex of rats treated with IFO or IFO + AGM. Oxidative phosphorylation was determined with electron donors specific to complexes I, II, III, or IV (C-I, C-II, C-III, or C-IV, respectively). A parallel study was done with (13)C-labeled pyruvate to assess metabolic dysfunction. Ifosfamide treatment significantly inhibited oxidative phosphorylation with only C-I substrates. Inhibition of C-I was associated with a significant elevation of [NADH], depletion of [NAD], and decreased flux through pyruvate dehydrogenase and the TCA cycle. However, administration of AGM with IFO increased [cyclic AMP (cAMP)] and prevented IFO-induced inhibition of C-I. In vitro studies with various metabolites of IFO showed that only CAA inhibited C-I, even with supplementation with 2-mercaptoethane sulfonic acid. Following IFO treatment daily for 5 days with 50 mg/kg, the level of CAA in the renal cortex was approximately 15 micromol/L. Taken together, these observations support the hypothesis that CAA is accumulated in renal cortex and is responsible for nephrotoxicity. AGM may be protective by increasing tissue [cAMP], which phosphorylates NADH:oxidoreductase. The current findings may have an important implication for the prevention of IFO-induced nephrotoxicity and/or mitochondrial diseases secondary to defective C-I.