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.     

Hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome is caused by mutations in a gene encoding a mitochondrial ornithine transporter.

Neurospora crassa ARG13 and Saccharomyces cerevisiae ARG11 encode mitochondrial carrier family (MCF) proteins that transport ornithine across the mitochondrial inner membrane. We used their sequences to identify EST candidates that partially encode orthologous mammalian transporters. We thereby identified such a gene (ORNT1) that maps to 13q14 and whose expression, similar to that of other urea cycle (UC) components, was high in liver and varied with changes in dietary protein. ORNT1 expression restores ornithine metabolism in fibroblasts from patients with hyperammonaemia-hyperornithinaemia-homocitrullinuria (HHH) syndrome. In a survey of 11 HHH probands, we identified 3 ORNT1 mutant alleles that account for 21 of 22 possible mutant ORNT1 genes in our patients: F188delta, which is common in French-Canadian HHH patients and encodes an unstable protein; E180K, which encodes a stable, properly targeted protein that is inactive; and a 13q14 microdeletion. Our results show that ORNT1 encodes the mitochondrial ornithine transporter involved in UC function and is defective in HHH syndrome.     

Mutant deoxynucleotide carrier is associated with congenital microcephaly

The disorder Amish microcephaly (MCPHA) is characterized by severe congenital microcephaly, elevated levels of alpha-ketoglutarate in the urine and premature death. The disorder is inherited in an autosomal recessive pattern and has been observed only in Old Order Amish families whose ancestors lived in Lancaster County, Pennsylvania. Here we show, by using a genealogy database and automated pedigree software, that 23 nuclear families affected with MCPHA are connected to a single ancestral couple. Through a whole-genome scan, fine mapping and haplotype analysis, we localized the gene affected in MCPHA to a region of 3 cM, or 2 Mb, on chromosome 17q25. We constructed a map of contiguous genomic clones spanning this region. One of the genes in this region, SLC25A19, which encodes a nuclear mitochondrial deoxynucleotide carrier (DNC), contains a substitution that segregates with the disease in affected individuals and alters an amino acid that is highly conserved in similar proteins. Functional analysis shows that the mutant DNC protein lacks the normal transport activity, implying that failed deoxynucleotide transport across the inner mitochondrial membrane causes MCPHA. Our data indicate that mitochondrial deoxynucleotide transport may be essential for prenatal brain growth.     

Exome sequencing identifies ACAD9 mutations as a cause of complex I deficiency

An isolated defect of respiratory chain complex I activity is a frequent biochemical abnormality in mitochondrial disorders. Despite intensive investigation in recent years, in most instances, the molecular basis underpinning complex I defects remains unknown. We report whole-exome sequencing of a single individual with severe, isolated complex I deficiency. This analysis, followed by filtering with a prioritization of mitochondrial proteins, led us to identify compound heterozygous mutations in ACAD9, which encodes a poorly understood member of the mitochondrial acyl-CoA dehydrogenase protein family. We demonstrated the pathogenic role of the ACAD9 variants by the correction of the complex I defect on expression of the wildtype ACAD9 protein in fibroblasts derived from affected individuals. ACAD9 screening of 120 additional complex I-defective index cases led us to identify two additional unrelated cases and a total of five pathogenic ACAD9 alleles.     

HAX1 deficiency causes autosomal recessive severe congenital neutropenia (Kostmann disease)

Autosomal recessive severe congenital neutropenia (SCN) constitutes a primary immunodeficiency syndrome associated with increased apoptosis in myeloid cells, yet the underlying genetic defect remains unknown. Using a positional cloning approach and candidate gene evaluation, we identified a recurrent homozygous germline mutation in HAX1 in three pedigrees. After further molecular screening of individuals with SCN, we identified 19 additional affected individuals with homozygous HAX1 mutations, including three belonging to the original pedigree described by Kostmann. HAX1 encodes the mitochondrial protein HAX1, which has been assigned functions in signal transduction and cytoskeletal control. Here, we show that HAX1 is critical for maintaining the inner mitochondrial membrane potential and protecting against apoptosis in myeloid cells. Our findings suggest that HAX1 is a major regulator of myeloid homeostasis and underline the significance of genetic control of apoptosis in neutrophil development.     

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, SUCLA2, DGUOK 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. 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.     

Mitochondrial aspartyl-tRNA synthetase deficiency causes leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation

Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) has recently been defined based on a highly characteristic constellation of abnormalities observed by magnetic resonance imaging and spectroscopy. LBSL is an autosomal recessive disease, most often manifesting in early childhood. Affected individuals develop slowly progressive cerebellar ataxia, spasticity and dorsal column dysfunction, sometimes with a mild cognitive deficit or decline. We performed linkage mapping with microsatellite markers in LBSL families and found a candidate region on chromosome 1, which we narrowed by means of shared haplotypes. Sequencing of genes in this candidate region uncovered mutations in DARS2, which encodes mitochondrial aspartyl-tRNA synthetase, in affected individuals from all 30 families. Enzyme activities of mutant proteins were decreased. We were surprised to find that activities of mitochondrial complexes from fibroblasts and lymphoblasts derived from affected individuals were normal, as determined by different assays.     

ABCB6 is dispensable for erythropoiesis and specifies the new blood group system Langereis

The human ATP-binding cassette (ABC) transporter ABCB6 has been described as a mitochondrial porphyrin transporter essential for heme biosynthesis, but it is also suspected to contribute to anticancer drug resistance, as do other ABC transporters located at the plasma membrane. We identified ABCB6 as the genetic basis of the Lan blood group antigen expressed on red blood cells but also at the plasma membrane of hepatocellular carcinoma (HCC) cells, and we established that ABCB6 encodes a new blood group system (Langereis, Lan). Targeted sequencing of ABCB6 in 12 unrelated individuals of the Lan(-) blood type identified 10 different ABCB6 null mutations. This is the first report of deficient alleles of this human ABC transporter gene. Of note, Lan(-) (ABCB6(-/-)) individuals do not suffer any clinical consequences, although their deficiency in ABCB6 may place them at risk when determining drug dosage.     

Fatal infantile cardioencephalomyopathy with COX deficiency and mutations in SCO2, a COX assembly gene.

Mammalian cytochrome c oxidase (COX) catalyses the transfer of reducing equivalents from cytochrome c to molecular oxygen and pumps protons across the inner mitochondrial membrane. Mitochondrial DNA (mtDNA) encodes three COX subunits (I-III) and nuclear DNA (nDNA) encodes ten. In addition, ancillary proteins are required for the correct assembly and function of COX (refs 2, 3, 4, 5, 6). Although pathogenic mutations in mtDNA-encoded COX subunits have been described, no mutations in the nDNA-encoded subunits have been uncovered in any mendelian-inherited COX deficiency disorder. In yeast, two related COX assembly genes, SCO1 and SCO2 (for synthesis of cytochrome c oxidase), enable subunits I and II to be incorporated into the holoprotein. Here we have identified mutations in the human homologue, SCO2, in three unrelated infants with a newly recognized fatal cardioencephalomyopathy and COX deficiency. Immunohistochemical studies implied that the enzymatic deficiency, which was most severe in cardiac and skeletal muscle, was due to the loss of mtDNA-encoded COX subunits. The clinical phenotype caused by mutations in human SCO2 differs from that caused by mutations in SURF1, the only other known COX assembly gene associated with a human disease, Leigh syndrome.     

Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy

Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy occurring in 1 in 50,000 individuals that features progressive loss in visual acuity leading, in many cases, to legal blindness. Phenotypic variations and loss of retinal ganglion cells, as found in Leber hereditary optic neuropathy (LHON), have suggested possible mitochondrial impairment. The OPA1 gene has been localized to 3q28-q29 (refs 13-19). We describe here a nuclear gene, OPA1, that maps within the candidate region and encodes a dynamin-related protein localized to mitochondria. We found four different OPA1 mutations, including frameshift and missense mutations, to segregate with the disease, demonstrating a role for mitochondria in retinal ganglion cell pathophysiology.     

Mitochondrial point mutations do not limit the natural lifespan of mice

Whether mitochondrial mutations cause mammalian aging, or are merely correlated with it, is an area of intense debate. Here, we use a new, highly sensitive assay to redefine the relationship between mitochondrial mutations and age. We measured the in vivo rate of change of the mitochondrial genome at a single-base pair level in mice, and we demonstrate that the mutation frequency in mouse mitochondria is more than ten times lower than previously reported. Although we observed an 11-fold increase in mitochondrial point mutations with age, we report that a mitochondrial mutator mouse was able to sustain a 500-fold higher mutation burden than normal mice, without any obvious features of rapidly accelerated aging. Thus, our results strongly indicate that mitochondrial mutations do not limit the lifespan of wild-type mice.     

Involvement of a novel Tnf receptor homologue in hair follicle induction.

Although inductive interactions are known to be essential for specification of cell fate in many vertebrate tissues, the signals and receptors responsible for transmitting this information remain largely unidentified. Mice with mutations in the downless (dl) gene have defects in hair follicle induction, lack sweat glands and have malformed teeth. These structures originate as ectodermal placodes, which invaginate into the underlying mesenchyme and differentiate to form specific organs. Positional cloning of the dl gene began with identification of the transgenic family OVE1. One branch of the family, dl(OVE1B), carries an approximately 600-kb deletion at the dl locus caused by transgene integration. The mutated locus has been physically mapped in this family, and a 200-kb mouse YAC clone, YAC D9, has been identified and shown to rescue the dl phenotype in the spontaneous dl(Jackson) (dl(J), recessive) and Dl(sleek) (Dl(slk), dominant negative) mutants. Here we report the positional cloning of the dl gene, which encodes a novel member of the tumour necrosis factor (Tnf) receptor (Tnfr) family. The mutant phenotype and dl expression pattern suggests that this gene encodes a receptor that specifies hair follicle fate. Its ligand is likely to be the product of the tabby (Ta) gene, as Ta mutants have a phenotype identical to that of dl mutants and Ta encodes a Tnf-like protein.     

Distinct mitochondrial retrograde signals control the G1-S cell cycle checkpoint

During electron transport, the mitochondrion generates ATP and reactive oxygen species (ROS), a group of partially reduced and highly reactive metabolites of oxygen. In this in vivo genetic analysis in Drosophila melanogaster, we establish that disruption of complex I of the mitochondrial electron transport chain specifically retards the cell cycle during the G1-S transition. The mechanism involves a specific signaling cascade initiated by ROS and transduced by ASK-1, JNK, FOXO and the Drosophila p27 homolog, Dacapo. On the basis of our data combined with previous analyses of the system, we conclude that mitochondrial dysfunction activates at least two retrograde signals to specifically enforce a G1-S cell cycle checkpoint. One such signal involves an increase in AMP production and downregulation of cyclin E protein; another independent pathway involves increased ROS and upregulation of Dacapo. Thus, our results indicate that the mitochondrion can use AMP and ROS at sublethal concentrations as independent signaling molecules to modulate cell cycle progression.     

Generation of mice with mitochondrial dysfunction by introducing mouse mtDNA carrying a deletion into zygotes

Mice carrying mitochondrial DNA (mtDNA) with pathogenic mutations would provide a system in which to study how mutant mtDNAs are transmitted and distributed in tissues, resulting in expression of mitochondrial diseases. However, no effective procedures are available for the generation of these mice. Isolation of mouse cells without mtDNA (rho0) enabled us to trap mutant mtDNA that had accumulated in somatic tissues into rho0 cells repopulated with mtDNA (cybrids). We isolated respiration-deficient cybrids with mtDNA carrying a deletion and introduced this mtDNA into fertilized eggs. The mutant mtDNA was transmitted maternally, and its accumulation induced mitochondrial dysfunction in various tissues. Moreover, most of these mice died because of renal failure, suggesting the involvement of mtDNA mutations in the pathogeneses of new diseases.     

Systematic screen for human disease genes in yeast

High similarity between yeast and human mitochondria allows functional genomic study of Saccharomyces cerevisiae to be used to identify human genes involved in disease. So far, 102 heritable disorders have been attributed to defects in a quarter of the known nuclear-encoded mitochondrial proteins in humans. Many mitochondrial diseases remain unexplained, however, in part because only 40-60% of the presumed 700-1,000 proteins involved in mitochondrial function and biogenesis have been identified. Here we apply a systematic functional screen using the pre-existing whole-genome pool of yeast deletion mutants to identify mitochondrial proteins. Three million measurements of strain fitness identified 466 genes whose deletions impaired mitochondrial respiration, of which 265 were new. Our approach gave higher selection than other systematic approaches, including fivefold greater selection than gene expression analysis. To apply these advantages to human disorders involving mitochondria, human orthologs were identified and linked to heritable diseases using genomic map positions.