Valeria Tiranti

Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria

The gene products involved in mammalian mitochondrial DNA (mtDNA) maintenance and organization remain largely unknown. We report here a novel mitochondrial protein, Twinkle, with structural similarity to phage T7 gene 4 primase/helicase and other hexameric ring helicases. Twinkle colocalizes with mtDNA in mitochondrial nucleoids. Screening of the gene encoding Twinkle in individuals with autosomal dominant progressive external ophthalmoplegia (adPEO), associated with multiple mtDNA deletions, identified 11 different coding-region mutations co-segregating with the disorder in 12 adPEO pedigrees of various ethnic origins. The mutations cluster in a region of the protein proposed to be involved in subunit interactions. The function of Twinkle is inferred to be critical for lifetime maintenance of human mtDNA integrity.

Mutations of SURF-1 in Leigh Disease Associated with Cytochrome c Oxidase Deficiency

Leigh disease associated with cytochrome c oxidase deficiency (LD[COX-]) is one of the most common disorders of the mitochondrial respiratory chain, in infancy and childhood. No mutations in any of the genes encoding the COX-protein subunits have been identified in LD(COX-) patients. Using complementation assays based on the fusion of LD(COX-) cell lines with several rodent/human rho0 hybrids, we demonstrated that the COX phenotype was rescued by the presence of a normal human chromosome 9. Linkage analysis restricted the disease locus to the subtelomeric region of chromosome 9q, within the 7-cM interval between markers D9S1847 and D9S1826. Candidate genes within this region include SURF-1, the yeast homologue (SHY-1) of which encodes a mitochondrial protein necessary for the maintenance of COX activity and respiration. Sequence analysis of SURF-1 revealed mutations in numerous DNA samples from LD(COX-) patients, indicating that this gene is responsible for the major complementation group in this important mitochondrial disorder.

MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion

The mitochondrial (mt) DNA depletion syndromes (MDDS) are genetic disorders characterized by a severe, tissue-specific decrease of mtDNA copy number, leading to organ failure. There are two main clinical presentations: myopathic (OMIM 609560) and hepatocerebral (OMIM 251880). Known mutant genes, including TK2 (ref. 2), SUCLA2 (ref. 3), DGUOK (ref. 4) and POLG, account for only a fraction of MDDS cases. We found a new locus for hepatocerebral MDDS on chromosome 2p21-23 and prioritized the genes on this locus using a new integrative genomics strategy. One of the top-scoring candidates was the human ortholog of the mouse kidney disease gene Mpv17 (ref. 8). We found disease-segregating mutations in three families with hepatocerebral MDDS and demonstrated that, contrary to the alleged peroxisomal localization of the MPV17 gene product, MPV17 is a mitochondrial inner membrane protein, and its absence or malfunction causes oxidative phosphorylation (OXPHOS) failure and mtDNA depletion, not only in affected individuals but also in Mpv17−/− mice.

Loss of ETHE1, a mitochondrial dioxygenase, causes fatal sulfide toxicity in ethylmalonic encephalopathy

Ethylmalonic encephalopathy is an autosomal recessive, invariably fatal disorder characterized by early-onset encephalopathy, microangiopathy, chronic diarrhea, defective cytochrome c oxidase (COX) in muscle and brain, high concentrations of C4 and C5 acylcarnitines in blood and high excretion of ethylmalonic acid in urine. ETHE1, a gene encoding a β-lactamase–like, iron-coordinating metalloprotein, is mutated in ethylmalonic encephalopathy. In bacteria, ETHE1-like sequences are in the same operon of, or fused with, orthologs of TST, the gene encoding rhodanese, a sulfurtransferase. In eukaryotes, both ETHE1 and rhodanese are located within the mitochondrial matrix. We created a Ethe1−/− mouse that showed the cardinal features of ethylmalonic encephalopathy. We found that thiosulfate was excreted in massive amounts in urine of both Ethe1−/− mice and humans with ethylmalonic encephalopathy. High thiosulfate and sulfide concentrations were present in Ethe1−/− mouse tissues. Sulfide is a powerful inhibitor of COX and short-chain fatty acid oxidation, with vasoactive and vasotoxic effects that explain the microangiopathy in ethylmalonic encephalopathy patients. Sulfide is detoxified by a mitochondrial pathway that includes a sulfur dioxygenase. Sulfur dioxygenase activity was absent in Ethe1−/− mice, whereas it was markedly increased by ETHE1 overexpression in HeLa cells and Escherichia coli. Therefore, ETHE1 is a mitochondrial sulfur dioxygenase involved in catabolism of sulfide that accumulates to toxic levels in ethylmalonic encephalopathy.

Mutations of mitochondrial DNA polymerase γA are a frequent cause of autosomal dominant or recessive progressive external ophthalmoplegia

One form of familial progressive external ophthalmoplegia with multiple mitochondrial DNA deletions recently has been associated with mutations in POLG1, the gene encoding pol γA, the catalytic subunit of mitochondrial DNA polymerase. We screened the POLG1 gene in several PEO families and identified five different heterozygous missense mutations of POLG1 in 10 autosomal dominant families. Recessive mutations were found in three families. Our data show that mutations of POLG1 are the most frequent cause of familial progressive external ophthalmoplegia associated with accumulation of multiple mitochondrial DNA deletions, accounting for approximately 45% of our family cohort.

Assembly of the oxidative phosphorylation system in humans: what we have learned by studying its defects.

Assembly of the oxidative phosphorylation (OXPHOS) system in the mitochondrial inner membrane is an intricate process in which many factors must interact. The OXPHOS system is composed of four respiratory chain complexes, which are responsible for electron transport and generation of the proton gradient in the mitochondrial intermembrane space, and of the ATP synthase that uses this proton gradient to produce ATP. Mitochondrial human disorders are caused by dysfunction of the OXPHOS system, and many of them are associated with altered assembly of one or more components of the OXPHOS system. The study of assembly defects in patients has been useful in unraveling and/or gaining a complete understanding of the processes by which these large multimeric complexes are formed. We review here current knowledge of the biogenesis of OXPHOS complexes based on investigation of the corresponding disorders.

Mutations of ANT1, Twinkle, and POLG1 in sporadic progressive external ophthalmoplegia (PEO)

To verify the impact of mutations in ANT1, Twinkle, and POLG1 genes in sporadic progressive external ophthalmoplegia associated with multiple mitochondrial DNA (mtDNA) deletions, DNA samples from 15 Italian and 12 British patients were screened. Mutations in ANT1 were found in one patient, in Twinkle in two patients, and in POLG1 in seven patients. Irrespective of the inheritance mode, screening of these genes should be performed in all patients with progressive external ophthalmoplegia with multiple mtDNA deletions.

Infantile Encephalopathy and Defective Mitochondrial DNA Translation in Patients with Mutations of Mitochondrial Elongation Factors EFG1 and EFTu

Mitochondrial protein translation is a complex process performed within mitochondria by an apparatus composed of mitochondrial DNA (mtDNA)-encoded RNAs and nuclear DNA-encoded proteins. Although the latter by far outnumber the former, the vast majority of mitochondrial translation defects in humans have been associated with mutations in RNA-encoding mtDNA genes, whereas mutations in protein-encoding nuclear genes have been identified in a handful of cases. Genetic investigation involving patients with defective mitochondrial translation led us to the discovery of novel mutations in the mitochondrial elongation factor G1 (EFG1) in one affected baby and, for the first time, in the mitochondrial elongation factor Tu (EFTu) in another one. Both patients were affected by severe lactic acidosis and rapidly progressive, fatal encephalopathy. The EFG1-mutant patient had early-onset Leigh syndrome, whereas the EFTu-mutant patient had severe infantile macrocystic leukodystrophy with micropolygyria. Structural modeling enabled us to make predictions about the effects of the mutations at the molecular level. Yeast and mammalian cell systems proved the pathogenic role of the mutant alleles by functional complementation in vivo. Nuclear-gene abnormalities causing mitochondrial translation defects represent a new, potentially broad field of mitochondrial medicine. Investigation of these defects is important to expand the molecular characterization of mitochondrial disorders and also may contribute to the elucidation of the complex control mechanisms, which regulate this fundamental pathway of mtDNA homeostasis.

Cloning of human and rat cDNAs encoding the mitochondrial single-stranded DNA-binding protein (SSB)

We have retro-transcribed and amplified by PCR the full-length cDNAs specifying the rat and human precursors of the single-stranded mitochondrial DNA (mtDNA)-binding protein (mtSSB). Each deduced sequence is composed of a 16-amino-acid (aa) N-terminal basic pre-sequence and a mature protein (132 aa in humans and 135 aa in the rat). The mature proteins are highly conserved among themselves and with the mtSSB from Xenopus laevis (Xl). Moreover, three regions of the protein are similar to corresponding domains of the SSB of Escherichia coli and to the E. coli F-sex factor SSB, indicating the existence of a broad class of DNA-binding proteins with structural and functional similarities both in prokaryotes and in prokaryote-derived organelles of higher organisms.

A novel mtDNA mutation in the ND5 subunit of complex I in two MELAS patients

We identified a novel heteroplasmic mutation in the mitochodrial DNA gene encoding the ND5 subunit of complex I. This mutation (13514A→G) hits the same codon affected by a previously reported mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS)‐associated mutation (13513G→A), but the amino acid replacement is different (D393G vs D393N). The 13514A→G mutation was found in two unrelated MELAS‐like patients. However, in contrast to typical MELAS, lactic acidosis was absent or mild and the muscle biopsy was morphologically normal. Strongly positive correlation between the percentage of heteroplasmy and defective activity of complex I was found in cybrids. We found an additional 13513G→A‐positive case, affected by a progressive mitochondrial encephalomyopathy. Our results clearly demonstrate that the amino acid position D393 is crucial for the function of complex I. Search for D393 mutations should be part of the routine screening for mitochondrial disorders. Ann Neurol 2001;49:106–110