An evolutionary perspective on pathogenic mtDNA mutations: haplogroup associations of clinical disorders.

More than 75 human diseases have been associated with mitochondrial dysfunction, and many of these are directly caused by overtly pathogenic mutations in the mitochondrial genome (mtDNA). In addition, there have been a number of reports that posit a different, subtler role for mtDNA substitutions in the disease process. As we review here, mtDNA evolution has resulted in the distribution of sequences into continent-specific haplogroups, which are defined by a relatively small number of polymorphisms. Thus, mtDNA sequences can be assigned to European, African, or Asian/Native American haplogroups. There are numerous reports that various diseases are haplogroup-associated, and it has been suggested that some of these haplogroup-associated polymorphisms act as risk factors in these disorders. It has also been suggested that there are haplogroup-associations for aging. As we note here, however, such associations have usually been observed only in single studies and it is difficult to draw broad conclusions on the basis of the available evidence. At a minimum, we suggest that, a haplogroup-group association must be detected in multiple subpopulations or in a large, carefully controlled population survey.

A high frequency of mtDNA polymorphisms in HeLa cell sublines.

The complete mtDNA sequences from the uncloned "founder" HeLa cells and from five sublines have been determined. These sequences all carry a common "core" of 38 single basepair alterations relative to the revised Cambridge Reference Sequence (CRS). The HeLa mitochondrial genome is of African descent and it is a member of the African L3 haplogroup. The sequence of the HeLa mtDNA resolves the uncertainty surrounding the mosaic composition of the original CRS for human mtDNA. Most importantly, we detected a total of eight polymorphisms that have arisen in the mtDNA coding region of different HeLa sublines. These observations suggest that HeLa mtDNA has a high rate of sequence divergence, relative to the phylogenetically-derived divergence rate for mtDNAs in the human population, which results from a relaxation of negative selection against the fixation of deleterious mutations. Furthermore, this high frequency of polymorphisms in HeLa mtDNA may reflect a process similar to the accumulation of somatic mtDNA mutations in human cancers. Preliminary analysis of single-cell derived subclone lines revealed the occurrence of another polymorphism and provided evidence for a large number of mtDNA segregation units.

Does the mitochondrial genome play a role in the etiology of Alzheimer's disease?

We report here the analyses of complete mtDNA coding region sequences from more than 270 Alzheimer's disease (AD) patients and normal controls to determine if inherited mtDNA mutations contribute to the etiology of AD. The AD patients and normal individuals were carefully screened and drawn from two populations of European descent in an effort to avoid spurious effects due to local population anomalies. Overall, there were no significant haplogroup associations in the combined AD and normal control sequence sets. Reduced median network analysis revealed that the AD mtDNA sequences contained a higher number of substitutions in tRNA genes, and that there was an elevated frequency of replacement substitutions in the complex I genes of the control sequences. Analysis of the replacement substitutions indicated that those arising in the AD mtDNAs were no more deleterious, on average, than those in the control mtDNAs. The only evidence for the synergistic action of mutations was the presence of both a rare non-conservative replacement substitution and a tRNA mutation in 2 AD mtDNAs, from a total of 145, whereas such a combination of mutations was not observed in the control sequences. Overall, the results reported here indicate that pathogenic inherited mtDNA mutations do not constitute a major etiological factor in sporadic AD. At most, a small proportion of AD patients carry a pathogenic mtDNA mutation and a small proportion of cognitively normal aged individuals carry a mtDNA mutation that reduces the risk of AD.

The matrilineal ancestry of Ashkenazi Jewry: portrait of a recent founder event.

Both the extent and location of the maternal ancestral deme from which the Ashkenazi Jewry arose remain obscure. Here, using complete sequences of the maternally inherited mitochondrial DNA (mtDNA), we show that close to one-half of Ashkenazi Jews, estimated at 8,000,000 people, can be traced back to only 4 women carrying distinct mtDNAs that are virtually absent in other populations, with the important exception of low frequencies among non-Ashkenazi Jews. We conclude that four founding mtDNAs, likely of Near Eastern ancestry, underwent major expansion(s) in Europe within the past millennium.

Low penetrance of the 14484 LHON mutation when it arises in a non-haplogroup J mtDNA background.

The penetrance in Leber's hereditary optic neuropathy (LHON) pedigrees is determined primarily by a mutation in the mitochondrial genome (mtDNA), but secondary factors are also necessary for manifestation of the disorder. It has been proposed that mtDNA polymorphisms affect penetrance in LHON pedigrees. In particular, it has been postulated that one or more polymorphisms associated with European haplogroup J mtDNAs substantially increase the penetrance of the primary LHON mutation at nucleotide 14484. We report here a haplogroup H matrilineal pedigree (VIC14) in which the single affected member carries the 14484 LHON mutation, but who manifested a milder and atypical optic nerve disorder. In addition, during a population screen, we identified an individual who carried the 14484 mutation but who had normal vision. Finally, the 14484 mutation is under-represented among haplogroup H mtDNAs that carry a LHON mutation. These results, in conjunction with other studies that are reviewed, indicate that 14484 LHON mutations have a low penetrance when they arise in a haplogroup H mtDNA background.

African Haplogroup L mtDNA sequences show violations of clock-like evolution.

A set of 96 complete mtDNA sequences that belong to the three major African haplogroups (L1, L2, and L3) was analyzed to determine if mtDNA has evolved as a molecular clock. Likelihood ratio tests (LRTs) were carried out with each of the haplogroups and with combined haplogroup sequence sets. Evolution has not been clock-like, neither for the coding region nor for the control region, in combined sets of African haplogroup L mtDNA sequences. In tests of individual haplogroups, L2 mtDNAs showed violations of a molecular clock under all conditions and in both the control and coding regions. In contrast, haplogroup L1 and L3 sequences, both for the coding and control regions, show clock-like evolution. In clock tests of individual L2 subclades, the L2a sequences showed a marked violation of clock-like evolution within the coding region. In addition, the L2a and L2c branch lengths of both the coding and control regions were shorter relative to those of the L2b and L2d sequences, a result that indicates lower levels of sequence divergence. Reduced median network analyses of the L2a sequences indicated the occurrence of marked homoplasy at multiple sites in the control region. After exclusion of the L2a and L2c sequences, African mtDNA coding region evolution has not significantly departed from a molecular clock, despite the results of neutrality tests that indicate the mitochondrial coding region has evolved under nonneutral conditions. In contrast, control region evolution is clock-like only at the haplogroup level, and it thus appears to have evolved essentially independently from the coding region. The results of the clock tests, the network analyses, and the branch length comparisons all caution against the use of simple mtDNA clocks.

Lightning strikes twice: Leber hereditary optic neuropathy families with two pathogenic mtDNA mutations.

OBJECTIVE: To report the clinical and mitochondrial genetic analyses of two families, each of which carries both the 11778 and 14484 Leber hereditary optic neuropathy (LHON) mutations in mitochondrial DNA. METHODS: In addition to detailed clinical histories, the complete sequence of the mitochondrial DNA (mtDNA) from each family was determined. RESULTS: A small Australian LHON family (Vic20) and a family from the United States carry the 11778 and 14484 LHON mutations. In addition to the optic neuropathy, one branch of the Baltimore LHON pedigree had a high incidence of a fatal infantile encephalopathy. In both families, the 14484 LHON mutation was homoplasmic, whereas the 11778 LHON mutation was heteroplasmic. CONCLUSIONS: There are no additional mtDNA sequence changes that explain the encephalopathy in the Baltimore LHON family, and a nuclear gene involvement is an alternative explanation that is supported by the available data. The ophthalmological characteristics and penetrance in the 11778 and 14484 "two-mutation" LHON families are not markedly more severe than those of classic LHON families who carry a single mtDNA mutation.

The pedigree rate of sequence divergence in the human mitochondrial genome: there is a difference between phylogenetic and pedigree rates.

We have extended our previous analysis of the pedigree rate of control-region divergence in the human mitochondrial genome. One new germline mutation in the mitochondrial DNA (mtDNA) control region was detected among 185 transmission events (generations) from five Leber hereditary optic neuropathy (LHON) pedigrees. Pooling the LHON pedigree analyses yields a control-region divergence rate of 1.0 mutation/bp/10(6) years (Myr). When the results from eight published studies that used a similar approach were pooled with the LHON pedigree studies, totaling >2,600 transmission events, a pedigree divergence rate of 0.95 mutations/bp/Myr for the control region was obtained with a 99.5% confidence interval of 0.53-1.57. Taken together, the cumulative results support the original conclusion that the pedigree divergence rate for the control region is approximately 10-fold higher than that obtained with phylogenetic analyses. There is no evidence that any one factor explains this discrepancy, and the possible roles of mutational hotspots (rate heterogeneity), selection, and random genetic drift and the limitations of phylogenetic approaches to deal with high levels of homoplasy are discussed. In addition, we have extended our pedigree analysis of divergence in the mtDNA coding region. Finally, divergence of complete mtDNA sequences was analyzed in two tissues, white blood cells and skeletal muscle, from each of 17 individuals. In three of these individuals, there were four instances in which an mtDNA mutation was found in one tissue but not in the other. These results are discussed in terms of the occurrence of somatic mtDNA mutations.

Sequence analysis of the mitochondrial genomes from Dutch pedigrees with Leber hereditary optic neuropathy.

The complete mitochondrial DNA (mtDNA) sequences for 63 Dutch pedigrees with Leber hereditary optic neuropathy (LHON) were determined, 56 of which carried one of the classic LHON mutations at nucleotide (nt) 3460, 11778, or 14484. Analysis of these sequences indicated that there were several instances in which the mtDNAs were either identical or related by descent. The most striking example was a haplogroup J mtDNA that carried the 14484 LHON mutation. Four different but related mitochondrial genotypes were identified in seven of the Dutch pedigrees with LHON, including six of those described by van Senus. The control region of the founder sequence for these Dutch pedigrees with LHON matches the control-region sequence that Macmillan and colleagues identified in the founder mtDNA of French Canadian pedigrees with LHON. In addition, we obtained a perfect match between the Dutch 14484 founder sequence and the complete mtDNA sequences of two Canadian pedigrees with LHON. Those results indicate that these Dutch and French Canadian 14484 pedigrees with LHON share a common ancestor, that the single origin of the 14484 mutation in this megalineage occurred before the year 1600, and that there is a 14484/haplogroup J founder effect. We estimate that this lineage--including the 14484 LHON mutation--arose 900-1,800 years ago. Overall, the phylogenetic analyses of these mtDNA sequences conservatively indicate that a LHON mutation has arisen at least 42 times in the Dutch population. Finally, analysis of the mtDNA sequences from those pedigrees that did not carry classic LHON mutations suggested candidate pathogenic mutations at nts 9804, 13051, and 14325.

Reduced-median-network analysis of complete mitochondrial DNA coding-region sequences for the major African, Asian, and European haplogroups.

The evolution of the human mitochondrial genome is characterized by the emergence of ethnically distinct lineages or haplogroups. Nine European, seven Asian (including Native American), and three African mitochondrial DNA (mtDNA) haplogroups have been identified previously on the basis of the presence or absence of a relatively small number of restriction-enzyme recognition sites or on the basis of nucleotide sequences of the D-loop region. We have used reduced-median-network approaches to analyze 560 complete European, Asian, and African mtDNA coding-region sequences from unrelated individuals to develop a more complete understanding of sequence diversity both within and between haplogroups. A total of 497 haplogroup-associated polymorphisms were identified, 323 (65%) of which were associated with one haplogroup and 174 (35%) of which were associated with two or more haplogroups. Approximately one-half of these polymorphisms are reported for the first time here. Our results confirm and substantially extend the phylogenetic relationships among mitochondrial genomes described elsewhere from the major human ethnic groups. Another important result is that there were numerous instances both of parallel mutations at the same site and of reversion (i.e., homoplasy). It is likely that homoplasy in the coding region will confound evolutionary analysis of small sequence sets. By a linkage-disequilibrium approach, additional evidence for the absence of human mtDNA recombination is presented here.

Origin and diffusion of mtDNA haplogroup X.

A maximum parsimony tree of 21 complete mitochondrial DNA (mtDNA) sequences belonging to haplogroup X and the survey of the haplogroup-associated polymorphisms in 13,589 mtDNAs from Eurasia and Africa revealed that haplogroup X is subdivided into two major branches, here defined as "X1" and "X2." The first is restricted to the populations of North and East Africa and the Near East, whereas X2 encompasses all X mtDNAs from Europe, western and Central Asia, Siberia, and the great majority of the Near East, as well as some North African samples. Subhaplogroup X1 diversity indicates an early coalescence time, whereas X2 has apparently undergone a more recent population expansion in Eurasia, most likely around or after the last glacial maximum. It is notable that X2 includes the two complete Native American X sequences that constitute the distinctive X2a clade, a clade that lacks close relatives in the entire Old World, including Siberia. The position of X2a in the phylogenetic tree suggests an early split from the other X2 clades, likely at the very beginning of their expansion and spread from the Near East.