Mutation in PNPT1, which encodes a polyribonucleotide nucleotidyltransferase, impairs RNA import into mitochondria and causes respiratory-chain deficiency.

Multiple-respiratory-chain deficiency represents an important cause of mitochondrial disorders. Hitherto, however, mutations in genes involved in mtDNA maintenance and translation machinery only account for a fraction of cases. Exome sequencing in two siblings, born to consanguineous parents, with severe encephalomyopathy, choreoathetotic movements, and combined respiratory-chain defects allowed us to identify a homozygous PNPT1 missense mutation (c.1160A>G) that encodes the mitochondrial polynucleotide phosphorylase (PNPase). Blue-native polyacrylamide gel electrophoresis showed that no PNPase complex could be detected in subject fibroblasts, confirming that the substitution encoded by c.1160A>G disrupts the trimerization of the protein. PNPase is predominantly localized in the mitochondrial intermembrane space and is implicated in RNA targeting to human mitochondria. Mammalian mitochondria import several small noncoding nuclear RNAs (5S rRNA, MRP RNA, some tRNAs, and miRNAs). By RNA hybridization experiments, we observed a significant decrease in 5S rRNA and MRP-related RNA import into mitochondria in fibroblasts of affected subject 1. Moreover, we found a reproducible decrease in the rate of mitochondrial translation in her fibroblasts. Finally, overexpression of the wild-type PNPT1 cDNA in fibroblasts of subject 1 induced an increase in 5S rRNA import in mitochondria and rescued the mitochondrial-translation deficiency. In conclusion, we report here abnormal RNA import into mitochondria as a cause of respiratory-chain deficiency.

Toward genotype phenotype correlations in GFM1 mutations.

Multiple respiratory chain deficiencies represent a common cause of mitochondrial diseases. We report two novel GFM1 mutations in two unrelated patients with encephalopathy and liver failure respectively. The first patient had intrauterine growth retardation, seizures, encephalopathy and developmental delay. Brain MRI showed hypoplasia of the vermis and severe pontine atrophy of the brainstem that were similar to those reported in patients with mitochondrial translation deficiencies. The second patient had liver failure with hypoglycemia. Respiratory chain analysis showed a complex IV deficiency in muscle of both patients. A 10K SNP genotyping detected several regions of homozygosity in the two patients. In vitro translation deficiency prompted us to study genes involved in mitochondrial translation. Therefore, we sequenced the GFM1 gene, encoding the mitochondrial translation factor EFG1, included in a shared homozygous region and identified two different homozygous mutations (R671C and L398P). Modeling studies of EFG1 protein suggested that the R671C mutation disrupts an inter-subunit interface and could locally destabilize the mutant protein. The second mutation (L398P) disrupted the H-bond network in a rich-beta-sheet domain, and may have a dramatic effect on local structure. GFM1 mutations have been seldom reported and are associated with different clinical presentation. By modeling the structure of the protein and the position of the various mutations we suggest that the clinical phenotypes of the patients could be related to the localization of the mutations.