Mitochondrial energy deficiency leads to hyperproliferation of skeletal muscle mitochondria and enhanced insulin sensitivity.

Diabetes is associated with impaired glucose metabolism in the presence of excess insulin. Glucose and fatty acids provide reducing equivalents to mitochondria to generate energy, and studies have reported mitochondrial dysfunction in type II diabetes patients. If mitochondrial dysfunction can cause diabetes, then we hypothesized that increased mitochondrial metabolism should render animals resistant to diabetes. This was confirmed in mice in which the heart-muscle-brain adenine nucleotide translocator isoform 1 (ANT1) was inactivated. ANT1-deficient animals are insulin-hypersensitive, glucose-tolerant, and resistant to high fat diet (HFD)-induced toxicity. In ANT1-deficient skeletal muscle, mitochondrial gene expression is induced in association with the hyperproliferation of mitochondria. The ANT1-deficient muscle mitochondria produce excess reactive oxygen species (ROS) and are partially uncoupled. Hence, the muscle respiration under nonphosphorylating conditions is increased. Muscle transcriptome analysis revealed the induction of mitochondrial biogenesis, down-regulation of diabetes-related genes, and increased expression of the genes encoding the myokines FGF21 and GDF15. However, FGF21 was not elevated in serum, and FGF21 and UCP1 mRNAs were not induced in liver or brown adipose tissue (BAT). Hence, increased oxidation of dietary-reducing equivalents by elevated muscle mitochondrial respiration appears to be the mechanism by which ANT1-deficient mice prevent diabetes, demonstrating that the rate of mitochondrial oxidation of calories is important in the etiology of metabolic disease.

Mitochondrial DNA associations with East Asian metabolic syndrome.

Mitochondrial dysfunction has repeatedly been reported associated with type 2 diabetes mellitus (T2DM) and metabolic syndrome (MS), as have mitochondrial DNA (mtDNA) tRNA and duplication mutations and mtDNA haplogroup lineages. We identified 19 Taiwanese T2DM and MS pedigrees from Taiwan, with putative matrilineal transmission, one of which harbored the pathogenic mtDNA tRNALeu(UUR) nucleotide (nt) 3243A>G mutation on the N9a3 haplogroup background. We then recruited three independent Taiwanese cohorts, two from Taipei (N = 498, mean age 52 and N = 1002, mean age 44) and one from a non-urban environment (N = 501, mean age 57). All three cohorts were assessed for an array of metabolic parameters, their mtDNA haplogroups determined, and the haplogroups correlated with T2DM/MS phenotypes. Logistic regression analysis revealed that mtDNA haplogroups D5, F4, and N9a conferred T2DM protection, while haplogroups F4 and N9a were risk factors for hypertension (HTN), and F4 was a risk factor for obesity (OB). Additionally, the 5263C>T (ND2 A165V) variant commonly associated with F4 was associated with hypertension (HTN). Cybrids were prepared with macro-haplogroup N (defined by variants m.ND3 10398A (114T) and m.ATP6 8701A (59T)) haplogroups B4 and F1 mtDNAs and from macro-haplogroup M (variants m.ND3 10398G (114A) and m.ATP6 8701G (59A)) haplogroup M9 mtDNAs. Additionally, haplogroup B4 and F1 cybrids were prepared with and without the mtDNA variant in ND1 3394T>C (Y30H) reported to be associated with T2DM. Assay of mitochondria complex I in these cybrids revealed that macro-haplogroup N cybrids had lower activity than M cybrids, that haplogroup F cybrids had lower activity than B4 cybrids, and that the ND1 3394T>C (Y30H) variant reduced complex I on both the B4 and F1 background but with very different cumulative effects. These data support the hypothesis that functional mtDNA variants may contribute to the risk of developing T2DM and MS.

Progressive increase in mtDNA 3243A>G heteroplasmy causes abrupt transcriptional reprogramming.

Variation in the intracellular percentage of normal and mutant mitochondrial DNAs (mtDNA) (heteroplasmy) can be associated with phenotypic heterogeneity in mtDNA diseases. Individuals that inherit the common disease-causing mtDNA tRNA(Leu(UUR)) 3243A>G mutation and harbor ∼10-30% 3243G mutant mtDNAs manifest diabetes and occasionally autism; individuals with ∼50-90% mutant mtDNAs manifest encephalomyopathies; and individuals with ∼90-100% mutant mtDNAs face perinatal lethality. To determine the basis of these abrupt phenotypic changes, we generated somatic cell cybrids harboring increasing levels of the 3243G mutant and analyzed the associated cellular phenotypes and nuclear DNA (nDNA) and mtDNA transcriptional profiles by RNA sequencing. Small increases in mutant mtDNAs caused relatively modest defects in oxidative capacity but resulted in sharp transitions in cellular phenotype and gene expression. Cybrids harboring 20-30% 3243G mtDNAs had reduced mtDNA mRNA levels, rounded mitochondria, and small cell size. Cybrids with 50-90% 3243G mtDNAs manifest induction of glycolytic genes, mitochondrial elongation, increased mtDNA mRNA levels, and alterations in expression of signal transduction, epigenomic regulatory, and neurodegenerative disease-associated genes. Finally, cybrids with 100% 3243G experienced reduced mtDNA transcripts, rounded mitochondria, and concomitant changes in nuclear gene expression. Thus, striking phase changes occurred in nDNA and mtDNA gene expression in response to the modest changes of the mtDNA 3243G mutant levels. Hence, a major factor in the phenotypic variation in heteroplasmic mtDNA mutations is the limited number of states that the nucleus can acquire in response to progressive changes in mitochondrial retrograde signaling.

Mitochondrial DNA variant associated with Leber hereditary optic neuropathy and high-altitude Tibetans.

The distinction between mild pathogenic mtDNA mutations and population polymorphisms can be ambiguous because both are homoplasmic, alter conserved functions, and correlate with disease. One possible explanation for this ambiguity is that the same variant may have different consequences in different contexts. The NADH dehydrogenase subunit 1 (ND1) nucleotide 3394 T > C (Y30H) variant is such a case. This variant has been associated with Leber hereditary optic neuropathy and it reduces complex I activity and cellular respiration between 7% and 28% on the Asian B4c and F1 haplogroup backgrounds. However, complex I activity between B4c and F1 mtDNAs, which harbor the common 3394T allele, can also differ by 30%. In Asia, the 3394C variant is most commonly associated with the M9 haplogroup, which is rare at low elevations but increases in frequency with elevation to an average of 25% of the Tibetan mtDNAs (odds ratio = 23.7). In high-altitude Tibetan and Indian populations, the 3394C variant occurs on five different macrohaplogroup M haplogroup backgrounds and is enriched on the M9 background in Tibet and the C4a4 background on the Indian Deccan Plateau (odds ratio = 21.9). When present on the M9 background, the 3394C variant is associated with a complex I activity that is equal to or higher than that of the 3394T variant on the B4c and F1 backgrounds. Hence, the 3394C variant can either be deleterious or beneficial depending on its haplogroup and environmental context. Thus, this mtDNA variant fulfills the criteria for a common variant that predisposes to a "complex" disease.

Association Between Mitochondrial DNA Haplogroup Variation and Autism Spectrum Disorders.

Importance: Autism spectrum disorders (ASD) are characterized by impairments in social interaction, communication, and repetitive or restrictive behavior. Although multiple physiologic and biochemical studies have reported defects in mitochondrial oxidative phosphorylation in patients with ASD, the role of mitochondrial DNA (mtDNA) variation has remained relatively unexplored. Objective: To assess what impact mitochondrial lineages encompassing ancient mtDNA functional polymorphisms, termed haplogroups, have on ASD risk. Design, Setting, and Participants: In this cohort study, individuals with autism and their families were studied using the Autism Genetic Resource Exchange cohort genome-wide association studies data previously generated at the Children's Hospital of Philadelphia. From October 2010 to January 2017, we analyzed the data and used the mtDNA single-nucleotide polymorphisms interrogated by the Illumina HumanHap 550 chip to determine the mtDNA haplogroups of the individuals. Taking into account the familial structure of the Autism Genetic Resource Exchange data, we then determined whether the mtDNA haplogroups correlate with ASD risk. Main Outcomes and Measures: Odds ratios of mitochondrial haplogroup as predictors of ASD risk. Results: Of 1624 patients with autism included in this study, 1299 were boys (80%) and 325 were girls (20%). Families in the Autism Genetic Resource Exchange collection (933 families, encompassing 4041 individuals: 1624 patients with ASD and 2417 healthy parents and siblings) had been previously recruited in the United States with no restrictions on age, sex, race/ethnicity, or socioeconomic status. Relative to the most common European haplogroup HHV, European haplogroups I, J, K, O-X, T, and U were associated with increased risk of ASD, as were Asian and Native American haplogroups A and M, with odds ratios ranging from 1.55 (95% CI, 1.16-2.06) to 2.18 (95% CI, 1.59-3) (adjusted P < .04). Hence, mtDNA haplogroup variation is an important risk factor for ASD. Conclusions and Relevance: Because haplogroups I, J, K, O-X, T, and U encompass 55% of the European population, mtDNA lineages must make a significant contribution to overall ASD risk.

mtDNA Variation and Analysis Using Mitomap and Mitomaster.

The Mitomap database of human mitochondrial DNA (mtDNA) information has been an important compilation of mtDNA variation for researchers, clinicians and genetic counselors for the past twenty-five years. The Mitomap protocol shows how users may look up human mitochondrial gene loci, search for public mitochondrial sequences, and browse or search for reported general population nucleotide variants as well as those reported in clinical disease. Within Mitomap is the powerful sequence analysis tool for human mitochondrial DNA, Mitomaster. The Mitomaster protocol gives step-by-step instructions showing how to submit sequences to identify nucleotide variants relative to the rCRS, to determine the haplogroup, and to view species conservation. User-supplied sequences, GenBank identifiers and single nucleotide variants may be analyzed.

Association between mitochondrial DNA variations and Alzheimer's disease in the ADNI cohort.

Despite the central role of amyloid deposition in the development of Alzheimer's disease (AD), the pathogenesis of AD still remains elusive at the molecular level. Increasing evidence suggests that compromised mitochondrial function contributes to the aging process and thus may increase the risk of AD. Dysfunctional mitochondria contribute to reactive oxygen species (ROS) which can lead to extensive macromolecule oxidative damage and the progression of amyloid pathology. Oxidative stress and amyloid toxicity leave neurons chemically vulnerable. Because the brain relies on aerobic metabolism, it is apparent that mitochondria are critical for the cerebral function. Mitochondrial DNA sequence changes could shift cell dynamics and facilitate neuronal vulnerability. Therefore we postulated that mitochondrial DNA sequence polymorphisms may increase the risk of AD. We evaluated the role of mitochondrial haplogroups derived from 138 mitochondrial polymorphisms in 358 Caucasian Alzheimer's Disease Neuroimaging Initiative (ADNI) subjects. Our results indicate that the mitochondrial haplogroup UK may confer genetic susceptibility to AD independently of the apolipoprotein E4 (APOE4) allele.

A mitochondrial etiology of neurodegenerative diseases: evidence from Parkinson's disease.

Evidence continues to accrue implicating mitochondrial dysfunction in the etiology of a number of neurodegenerative diseases. For example, Parkinson's disease (PD) can be induced by mitochondrial toxins, and nuclear DNA (nDNA) loci linked to PD have been associated with mitochondrial dysfunction. Although conclusions about the role of mitochondrial DNA (mtDNA) variants in PD vary, we argue here that this is attributable to the novel genetics of the mtDNA and the fact that clinically relevant mtDNA variation encompasses ancient adaptive polymorphisms, recent deleterious mutations, and somatic mutations. An mtDNA association with PD is supported by an analysis of the Russian Tatar population which revealed that polymorphisms associated with haplogroup H mtDNAs increased PD risk (odds ratio [OR]= 2.58, P= 0.0001), whereas those associated with haplogroup UK cluster mtDNAs were protective (OR = 0.38, P= 0.003). Moreover, mtDNA sequencing revealed that PD patients with either haplogroup H or UK cluster mtDNAs can harbor additional recent variants that might further modulate PD risk. Therefore, the complexity of PD genetics may reflect the complex mitochondrial genetics.

Clinical and functional characterization of a human ORNT1 mutation (T32R) in the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome.

We studied two related families (HHH013 and HHH015) with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, a disorder of the urea cycle and ornithine degradation pathway, who have the same novel ornithine transporter (ORNT1) genotype (T32R) but a variable phenotype. Both HHH015 patients are doing well in school and are clinically stable; conversely, the three affected HHH013 siblings had academic difficulties and one suffered recurrent episodes of hyperammonemia and ultimately died. Overexpression studies revealed that the product of the ORNT1-T32R allele has residual function. Ornithine transport studies in HHH015 fibroblasts, however, showed basal activity similar to fibroblasts carrying nonfunctional ORNT1 alleles. We also examined two potential modifying factors, the ORNT2 gene and the mitochondrial DNA lineage (haplogroup). Haplogroups, associated with specific diseases, are hypothesized to influence mitochondrial function. Results demonstrated that both HHH015 patients are heterozygous for an ORNT2 gain of function polymorphism and belong to haplogroup A whereas the HHH013 siblings carry the wild-type ORNT2 and are haplogroup H. These observations suggest that the ORNT1 genotype cannot predict the phenotype of HHH patients. The reason for the phenotypic variability is unknown, but factors such as redundant transporters and mitochondrial lineage may contribute to the neuropathophysiology of HHH patients.

Mitochondrial DNA diversity in indigenous populations of the southern extent of Siberia, and the origins of Native American haplogroups.

In search of the ancestors of Native American mitochondrial DNA (mtDNA) haplogroups, we analyzed the mtDNA of 531 individuals from nine indigenous populations in Siberia. All mtDNAs were subjected to high-resolution RFLP analysis, sequencing of the control-region hypervariable segment I (HVS-I), and surveyed for additional polymorphic markers in the coding region. Furthermore, the mtDNAs selected according to haplogroup/subhaplogroup status were completely sequenced. Phylogenetic analyses of the resulting data, combined with those from previously published Siberian arctic and sub-arctic populations, revealed that remnants of the ancient Siberian gene pool are still evident in Siberian populations, suggesting that the founding haplotypes of the Native American A-D branches originated in different parts of Siberia. Thus, lineage A complete sequences revealed in the Mansi of the Lower Ob and the Ket of the Lower Yenisei belong to A1, suggesting that A1 mtDNAs occasionally found in the remnants of hunting-gathering populations of northwestern and northern Siberia belonged to a common gene pool of the Siberian progenitors of Paleoindians. Moreover, lineage B1, which is the most closely related to the American B2, occurred in the Tubalar and Tuvan inhabiting the territory between the upper reaches of the Ob River in the west, to the Upper Yenisei region in the east. Finally, the sequence variants of haplogroups C and D, which are most similar to Native American C1 and D1, were detected in the Ulchi of the Lower Amur. Overall, our data suggest that the immediate ancestors of the Siberian/Beringian migrants who gave rise to ancient (pre-Clovis) Paleoindians have a common origin with aboriginal people of the area now designated the Altai-Sayan Upland, as well as the Lower Amur/Sea of Okhotsk region.

Traces of early Eurasians in the Mansi of northwest Siberia revealed by mitochondrial DNA analysis.

The mitochondrial DNA (mtDNA) of 98 Mansi, an ancient group (formerly known as "Vogul") of Uralic-speaking fishers and hunters on the eastern slope of the northern Ural Mountains, were analyzed for sequence variants by restriction fragment--length polymorphism analysis, control-region sequencing, and sequencing of additional informative sites in the coding region. Although 63.3% of the mtDNA detected in the Mansi falls into western Eurasian lineages (e.g., haplogroups UK, TJ, and HV), the remaining 36.7% encompass a subset of eastern Eurasian lineages (e.g., haplogroups A, C, D, F, G, and M). Among the western Eurasian lineages, subhaplogroup U4 was found at a remarkable frequency of 16.3%, along with lineages U5, U7, and J2. This suggests that the aboriginal populations residing immediately to the east of the Ural Mountains may encompass remnants of the early Upper Paleolithic expansion from the Middle East/southeastern Europe. The added presence of eastern Eurasian mtDNA lineages in the Mansi introduces the possibilities that proto-Eurasians encompassed a range of macrohaplogroup M and N lineages that subsequently became geographically distributed and that the Paleolithic expansion may have reached this part of Siberia before it split into western and eastern human groups.

Analysis of mitochondrial DNA diversity in the aleuts of the commander islands and its implications for the genetic history of beringia.

The Aleuts are aboriginal inhabitants of the Aleutian archipelago, including Bering and Copper (Medny) Islands of the Commanders, and seem to be the survivors of the inhabitants of the southern belt of the Bering Land Bridge that connected Chukotka/Kamchatka and Alaska during the end of the Ice Age. Thirty mtDNA samples collected in the Commanders, as well as seven mtDNA samples from Sireniki Eskimos in Chukotka who belong to the Beringian-specific subhaplogroup D2, were studied through complete sequencing. This analysis has provided evidence that all 37 of these mtDNAs are closely related, since they share the founding haplotype for subhaplogroup D2. We also demonstrated that, unlike the Eskimos and Na-Dene, the Aleuts of the Commanders were founded by a single lineage of haplogroup D2, which had acquired the novel transversion mutation 8910A. The phylogeny of haplogroup D complete sequences showed that (1) the D2 root sequence type originated among the latest inhabitants of Beringia and (2) the Aleut 8910A sublineage of D2 is a part of larger radiation of rooted D2, which gave rise to D2a (Na-Dene), D2b (Aleut), and D2c (Eskimo) sublineages. The geographic specificity and remarkable intrinsic diversity of D2 lineages support the refugial hypothesis, which assumes that the founding population of Eskimo-Aleut originated in Beringan/southwestern Alaskan refugia during the early postglacial period, rather than having reached the shores of Alaska as the result of recent wave of migration from interior Siberia.

The role of mtDNA background in disease expression: a new primary LHON mutation associated with Western Eurasian haplogroup J.

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted form of blindness caused by mitochondrial DNA (mtDNA) mutations. Approximately 90% of LHON cases are caused by 3460A, 11778A, or 14484C mtDNA mutations. These are designated "primary" mutations because they impart a high risk for LHON expression. Although the 11778A and 14484C mutations unequivocally predispose carriers to LHON, they are preferentially associated with mtDNA haplogroup J, one of nine Western Eurasian mtDNA lineages, suggesting a synergistic and deleterious interaction between these LHON mutations and haplogroup J polymorphism(s). We report here the characterization of a new primary LHON mutation in the mtDNA ND4L gene at nucleotide pair 10663. The homoplasmic 10663C mutation has been found in three independent LHON patients who lack a known primary mutation and all of which belong to haplogroup J. This mutation has not been found in a large number of haplotype-matched or non-haplogroup-J control mtDNAs. Phylogenetic analysis with primarily complete mtDNA sequence data demonstrates that the 10663C mutation has arisen at least three independent times in haplogroup J, indicating that it is not a rare lineage-specific polymorphism. Analysis of complex I function in patient lymphoblasts and transmitochondrial cybrids has revealed a partial complex I defect similar in magnitude to the 14484C mutation. Thus, the 10663C mutation appears to be a new primary LHON mutation that is pathogenic when co-occurring with haplogroup J. These results strongly support a role for haplogroup J in the expression of certain LHON mutations.