Gene 238 (1999) 211–230

www.elsevier.com/locate/gene

Mitochondrial DNA variation in human evolution and disease

Douglas C. Wallace *, Michael D. Brown, Marie T. Lott
Center for Molecular Medicine, Emory University School of Medicine, 1462 Clifton Rd., Atlanta, GA 30322, USA

Received 21 June 1999; accepted 6 July 1999

Abstract

Analysis of mitochondrial DNA (mtDNA) variation has permitted the reconstruction of the ancient migrations of women.
This has provided evidence that our species arose in Africa about 150 000 years before present ( YBP), migrated out of Africa into
Asia about 60 000 to 70 000 YBP and into Europe about 40 000 to 50 000 YBP, and migrated from Asia and possibly Europe to
the Americas about 20 000 to 30 000 YBP. Although much of the mtDNA variation that exists in modern populations may be
selectively neutral, studies of the mildly deleterious mtDNA mutations causing Leber’s hereditary optic neuropathy (LHON ) have
demonstrated that some continent-specific mtDNA lineages are more prone to manifest the clinical symptoms of LHON than
others. Hence, all mtDNA lineages are not equal, which may provide insights into the extreme environments that were encountered
by our ancient ancestor, and which may be of great importance in understanding the pathophysiology of mitochondrial disease.
© 1999 Elsevier Science B.V. All rights reserved.

Keywords: Leber’s hereditary optic neuropathy; Mitochondrial disease; Mitochondrial DNA; mtDNA mutation

1. Introduction mitochondrial ATP synthase to generate ATP for work

or is short circuited (uncoupled ) to generate heat for
The mitochondrial DNA (mtDNA) is a small circular thermal regulation.
genome located within the mitochondria in the cyto- The mtDNA is strictly maternally inherited. This is
plasm of the cell. The mitochondrion arose as a symbiont because the cytoplasmic location of the mitochondria
of the proto-eukaryotic cell about 1.5 to 2 billion years and mtDNA dictates that the mitochondria and
before present ( YBP), and subsequently gave up most mtDNAs are transmitted from one generation to the
of its genes to the cell’s nucleus. Currently, mtDNA next through the oocyte cytoplasm. The sperm appears
codes for only 13 polypeptides, all of which are essential to make no genetic contribution to the mtDNA.
subunits for the mitochondrial energy-generating The mtDNAs also have a very high mutation rate.
enzymes of oxidative phosphorylation (OXPHOS). As a result, the human population currently harbors a
Today, each human cell contains hundreds of mito- high level of population-specific mtDNA polymor-
chondria and thousands of mtDNAs. Thus, mito- phisms. Analysis of the population-specific mtDNA
chondrial genetics is a population genetics, both at the polymorphism has permitted the reconstruction of
level of the intracellular colony of symbionts and at human pre-history. Moreover, deleterious mtDNA mut-
the level of human populations. ations arise frequently, and result in mitochondrial
The mitochondria provide much of the energetic disease. Hence, analysis of maternally inherited diseases
needs for our cells. Mitochondrial energy is generated has opened a new field of human genetics, and recently
by OXPHOS in which the hydrogen derived from the demonstrated that some mtDNA diseases show a strong
carbohydrates and fats in our diets is oxidized by the continental bias. In one case, this is now understood as
oxygen we breathe to give water. The released energy is the synergistic interaction between a pathogenic mtDNA
stored as an electrochemical gradient across the mito- mutation and a specific European mtDNA lineage.
chondrial inner membrane (Dy), which is utilized by the Thus various mtDNA lineages are qualitatively
different, and hence can be differentially acted on by
selection. Analysis of this mtDNA variation may ulti-
* Corresponding author. Tel.: +1-404-727-5624; mately tell us something about the pathophysiology of
fax: +1-404-727-3949. mtDNA disease.

0378-1119/99/$ – see front matter © 1999 Elsevier Science B.V. All rights reserved.
PII: S0 3 7 8 -1 1 1 9 ( 9 9 ) 0 0 29 5 - 4
212 D.C. Wallace et al. / Gene 238 (1999) 211–230

2. Mitochondrial DNA structure and genetics (Michaels et al., 1982; Chen et al., 1995a). The sperm
mtDNAs are contributed to the zygote at fertilization
The mtDNA is a 16 569 nucleotide pair (np), closed and will persist in inter-specific crosses (Gyllensten et al.,
circular molecule that codes for a small (12S, MTRNR1) 1991), but are selectively eliminated in intra-specific
and large (16S, MTRNR2) rRNA as well as 22 tRNAs. crosses ( Kaneda et al., 1995). This has recently been
These structural RNAs are used to translate the 13 correlated with the observation that the mitochondria
polypeptides that are subunits of the OXPHOS enzyme in the sperm mid-piece is ubiquinated (Hopkin, 1999)
complexes, including: seven of the approximately 43 and hence may be labeled for immediate destruction
polypeptides of complex I (MTND1, 2, 3, 4L, 4, 5, and within the oocyte cytoplasm.
6), one of the 11 polypeptides of complex III When a mutation arises in a cellular mtDNA, it
(MTCYTB), three polypeptides of complex IV creates a mixed intracellular population of mutant and
(MTCOI, COII, and COIII ), and two polypeptides of normal molecules known as heteroplasmy. When a cell
complex V (MTATP6 and 8) (Fig. 1). divides, it is a matter of chance as to whether the mutant
The human mtDNA is strictly maternally inherited mtDNAs will be partitioned into one daughter cell or
(Giles et al., 1980; Case and Wallace, 1981), due in large another. Thus, over time, the percentage of mutant
measure to the fact that the mammalian egg contains mtDNAs in different cell lineages can drift toward either
about 100 000 mitochondria and mtDNAs, whereas the pure mutant or normal (homoplasmy), a process known
sperm contains only in the order of 100 mtDNAs as replicative segregation ( Wallace, 1986).

Fig. 1. The human mtDNA map, showing the location of the genes and the ‘primary’ Leber’s hereditary optic neuropathy (LHON ) mutations.
The human mtDNA is a 16 569 base pair circular molecule that codes for seven (ND1, 2, 3, 4L, 4, 5, and 6) of the 43 subunits of complex I,
shown in pink; one (cytochrome b, cyt b) of the 11 subunits of complex III, shown in orange; three (COI, II, and III ) of the 13 subunits of complex
IV, shown in purple; and two (ATPase 6 and 8) of 16 subunits of complex V, shown in yellow. It also codes for the small and large rRNAs, shown
in green, and 22 tRNAs, shown in light yellow, with the adjacent letters indicating the cognate amino acids. The heavy (H )-strand origin of
replication (O ) and the H-strand and light (L)-strand promoters, P and P , are indicated in the control region.
H H L
 D.C. Wallace et al. / Gene 238 (1999) 211–230 213

As the percentage of deleterious mutant mtDNAs the recognition sequence, but the polymorphic nucleo-
increases, cellular energy output declines. Since different tide may be different. For example, the HpaI np 3592
tissues and organs rely on mitochondrial energy genera- site change is caused by a C-to-T transition at np 3594.
tion to different extents, this results in tissue-specific In contrast to African mtDNAs, about 13% of Asians
symptoms for systemic genetic defects. The differential lacked an HpaI restriction site at np 12 406 (G to A at
organ sensitivities to mitochondrial defects, in decreasing np 12 406), which was present in all other mtDNAs
order, are the central nervous system including the eye, (Denaro et al., 1981). A further survey of the mtDNA
the heart and skeletal muscle, renal system, endocrine variation detected using six highly informative restric-
system and liver ( Wallace, 1987, 1995; Wallace et al., tion enzymes (HpaI, BamHI, HaeII, MspI, and AvaII )
1988a, 1994; Shoffner et al., 1990). and Southern blotting confirmed that mtDNA variation
The mtDNA has a very high sequence evolution was high and correlated strongly with the geographic
(Brown et al., 1979), in the order of 10 to 17 times origin of the individual. It also showed that all mtDNAs
faster than nuclear DNA genes of similar function were part of a single phylogenetic tree, that the greatest
(Neckelmann et al., 1987; Wallace et al., 1987). This variation was in Africa, and that the tree was about
has resulted in the accumulation of a broad spectrum 100 000 years old (Johnson et al., 1983). Extensive
of mtDNA sequence polymorphisms in human popula- studies by our group, as well as by others, ultimately
tions, but also may be a common source of mutations led to the characterization of 3065 mtDNAs from 62
causing mitochondrial disease. geographic samples using these six enzymes. This
revealed 149 haplotypes and 81 polymorphic sites. This
analysis confirmed: (1) that the mtDNA polymorphisms
3. MtDNA variation in human populations within each mtDNA were virtually in total linkage
disequilibrium, consistent with a low frequency of
The mtDNA sequence evolution rate is in the same recombination; that mtDNA variation correlated highly
time frame as the origin and radiation of human conti- with the ethnic and geographic origin of the individual;
nental populations. As a result, mtDNA polymorphisms (2) that there was a single mtDNA tree; (3) that the
have accumulated sequentially as women migrated west greatest variation and deepest root of the tree was in
of Africa and into the various continents. Generally, for Africa, consistent with an African origin of humans.
mtDNA variants to reach polymorphic frequencies, they The extent of mtDNA sequence differences between
need to be selectively neutral or near neutral to avoid continental populations was estimated from this data
being eliminated by selection and thus become prevalent by calculating the GST statistic. For the mtDNA, the
through genetic drift. In fact, the rapid shift in mtDNA GST was 0.35±0.025, implying that about 35% of the
lineages that is observed between continent may have mtDNA variation was continent-specific. By contrast,
been influenced by selection as well as by drift. the comparable nDNA value was 0.12. Hence, the
mtDNA encompasses much greater continent-specific
3.1. World mtDNA phylogeny and the origin of women sequence diversity than the nDNA (Merriwether et al.,
1991).
Because the mtDNA is strictly maternally inherited, A recent African origin of human mtDNAs was also
the mtDNA sequence has evolved by the sequential demonstrated by the investigations of Cann et al. (1987).
accumulation of base substitutions along radiating These investigators purified the individual mtDNAs
maternal lineages. Thus, as women migrated out of from cells or tissues, digested the DNA with 12 restric-
Africa into the different continents about 150 000 YBP tion endonucleases (Hpal, AvaII, FnuDII, HhaI, HpaII,
they accumulated mtDNA mutations that today are Mbol, TaqI, Rsal, HinfI, HaeIII, AluI, and DdeI ), end-
seen as high frequency, continent-specific mtDNA labeled the fragments, and resolved the fragments using
sequence polymorphisms. These polymorphisms are polyacrylamide gels and autoradiography (Brown,
associated with specific mtDNA haplotypes, and groups 1980). A survey of 147 mtDNAs, including 34 Asians,
of related haplotypes (haplogroups) ( Torroni and 21 Australian aboriginals, 26 aboriginal New Guineans,
Wallace, 1994; Wallace, 1995). 46 Caucasians, and 20 Africans (18 of whom were Black
The first clear evidence that mtDNA variation corre- Americans), also revealed that there was a single
lated with the ethnic and geographic origin of the mtDNA tree, that the deepest root occurred in Africa,
individual came from our survey of HpaI RFLPs in and that Africa harbored the greatest sequence diversity.
African, Asian, and European–American mtDNAs. This Hence, Africa is the origin of Homo sapiens. Using an
revealed that, in Africans, 96% of Pygmies, 93% of San estimated sequence evolution rate of 2–4% per million
Bushman, and 71% of Bantus harbored an HpaI restric- years (MYR), the human mtDNA tree was calculated
tion site at np 3592 not seen in Asians or Europeans. to be about 200 000 years old (Cann et al., 1987).
By convention, all polymorphic restriction sites, includ- This analysis was extended to include 62 Japanese
ing the HpaI np 3592 site, are defined by the 5∞ end of ( Horai and Matsunaga, 1986) and 119 Papua New
214 D.C. Wallace et al. / Gene 238 (1999) 211–230

Guineans (Stoneking et al., 1990). The Papua New and became established by genetic drift, resulting in
Guineans were sampled from 25 localities, and signifi- continent-specific mtDNA variation. Today, these pop-
cant differences in mtDNA variation were found ulation-specific polymorphisms constitute the back-
between the highland and coastal populations. ground on which potentially pathogenic mtDNA
Combining the Papua New Guinea data with the previ- mutations must be identified.
ous European, Asian, and African data permitted calcu-
lation of a global GST of 0.31 (Stoneking et al., 1990), 3.2. Cataloging continent-specific mtDNA variation
a value similar to that found for the six-enzyme analysis
discussed above. Although the above methods permitted elucidation
The African origin of mtDNA variation was also of the general features of human mtDNA evolution, a
supported through sequence analyses of the 1121 np more detailed analysis of mtDNA variation has been
non-coding control region of the mtDNA. This region necessary for clinical studies and for addressing addi-
has a three- to four-fold greater sequence diversity than tional anthropological questions on the age and origin
the coding region. Analysis of the control-region of Africans, Europeans, Asians, and Native Americans.
sequences from 189 individuals, 121 of whom were To increase the sensitivity of our analyses, we developed
native African, once again confirmed that the greatest a new mtDNA analysis procedure — high resolution
sequence diversity was in Africans, that the deepest root RFLP analysis — in which the mtDNAs from a variety
was between Africans, and that the coalescence time of of human samples could be amplified by using PCR in
the mtDNA tree (phylogeny) was between 166 000 and nine overlapping fragments. Each fragment was then
249 000 YBP ( Vigilant et al., 1991). The African root digested with 14 restriction endonucleases (AluI, AvaII,
of this phylogeny was subsequently challenged on the BamHI, Ddel, HaeII, HhaI, HinfI, HincII, HpaI, Hpal,
basis that multiple equally probable parsimony trees MspI, Mbol, RsaI, and TaqI ), and the fragments
could be generated from the data ( Templeton, 1992). resolved on agarose gels and detected by ethidium
However, other phylogenetic analysis procedures, such bromide staining and UV fluorescence. This procedure
as neighbor-joining trees, have reaffirmed the cohesive- surveys >20% of the mtDNA sequence, and the aggre-
ness of the deepest African associations and thus support gate of the restriction-site polymorphisms for each
the African origin of the mtDNA phylogeny (Hedges mtDNA is used to define the mtDNA haplotype
et al., 1991). (Ballinger et al., 1992; Torroni et al., 1992). The regional
Analysis of the control-region sequence of 95 individ- PCR fragments can also be sequenced, permitting exten-
uals, including 61 Japanese, confirmed that the greatest sion of the analysis to areas of interest such as the
diversity and deepest root occurred in Africa and hypervariable control region ( Torroni et al., 1993a,b).
revealed that ‘Mongoloid’ mtDNAs were subdivided The sequence differences between mtDNAs can then be
into two distinct groups (Horai and Hayasaka, 1990). compared by using various phylogenetic procedures
Analysis of 117 Caucasian mtDNAs confirmed the including parsimony, neighbor-joining, and unweighted
distinctive nature of many European mtDNAs and pair-group analyses. These phylogenetic trees reveal the
revealed that the various mtDNA lineages were widely relatedness of the mtDNAs, with the more similar
disseminated throughout Europe (Di Rienzo and mtDNAs clustering together. The extent of sequence
Wilson, 1991). diversity within or between groups of related haplotypes
Finally, comparison of the original European (haplogroup) can also be calculated ( Tateno et al., 1982;
mtDNA sequence (Anderson et al., 1981) with that Nei and Tajima, 1983; Saitou and Nei, 1987; Swofford,
from an African, a Japanese, and four African apes 1993).
(common and pygmy chimpanzees, gorilla, and orang-
utan) revealed that the European and Japanese mtDNAs 3.3. African mtDNA variation
were most similar, that the African mtDNA was more
divergent, and that the nearest ape relatives, the chim- To characterize better African mtDNA variation, we
panzees, were 10 times more divergent from humans have surveyed, using high-resolution RFLP analysis, the
than Africans are from Asians and Europeans. Using mtDNAs from 214 Africans: 101 from Senegal (60
the orangutan–African ape divergence time of 13 million Mandenkalu, 20 Wolof, 8 Pular, 13 others from eight
YBP as reference, this study gave an age for human tribes); 22 Mbuti ( Eastern) Pygmies from Zaire and 17
mtDNA radiation of 143 000±18 000 YBP and a time Biaka ( Western) Pygmies from Central African
for European and Japanese radiation of 70 000±13 000 Republic; 74 South Africans, including 43 Kung and 31
YBP (Horai et al., 1995). Khwe (Chen et al., 1995b, 1999). This survey revealed
All of these studies lead to the same conclusion. The 105 haplotypes defined by greater than 157 polymorphic
human mtDNA tree appears to have originated in Africa sites. Phylogenetic analysis revealed that 75 of the
about 150 000 YBP. As women migrated from Africa to haplotypes formed a single, coherent, African-specific
colonize new lands, additional mtDNA mutations arose haplogroup designated ‘L’ (Fig. 2), which is defined by
 D.C. Wallace et al. / Gene 238 (1999) 211–230 215

Fig. 2. Phylogeny of African mtDNA haplotypes. This tree summarizes the results from 214 Africans, including 101 Senegalese, 22 Mbuti Pygmies,
17 Biaka Pygmies, 43 Kung and 31 Khwe, encompassing 105 haplotypes. Each haplotype is positioned relative to the others based on number of
genetic similarities or differences. The closer together two haplotypes are in the tree, the more similar is their sequence. MtDNA polymorphisms
that arose recently are present in only one or a few mtDNAs and define the terminal twigs of the tree. By contrast, polymorphisms that arose early
in human mtDNA evolution are shared by larger groups of related mtDNAs and define major branches of the tree. These groups of related mtDNA
haplotypes are called haplogroups. To identify the oldest mtDNA haplogroups, the human mtDNA haplotypes are compared with that of a more
distantly related mtDNA, the outgroup, in this case chimpanzee mtDNA. This permits the ‘rooting’ of the tree. For African mtDNAs, one of the
oldest and most significant polymorphisms is the HpaI site at np 3592. This site is African-specific with the oldest 2/3 of the tree having the HpaI
3592 site (+3592 HpaI ) and being defined as macro-haplogroup L, with haplogroup subdivisions L1 and L2. The absence of this site is defined as
haplogroup L3. Haplogroup L3 forms the bridge between African mtDNAs and European and Asian mtDNAs. Within the L1, L2, and L3 divisions
are multiple haplogroups and sub-haplogroups, some of which correlate highly with the population sampled. Thus a core block of related haplotypes
in L1a, designated a, are specific for the Kung; a second block in L1b , labeled b, are specific for the Biaka Pygmies; a third block in L2a, designated
2
c, are specific for Mbuti Pygmies; a fourth block in L2c, labeled d, are specific for the Senegalese. This implies that these core haplotypes arose
with these populations. The fact that the a block of the Kung and the b block the Biaka Pygmies are closest to the root of the tree implies that
these are among the oldest African populations. Although the deep branches of this tree are very robust, the most distal twigs are subject to greater
interpretive fluctuation. Hence, this tree is one of the thousands that could be drawn in which the major branches are retained but the precise
branching order of the individual haplotypes can vary. This is standard. This tree is reproduced from Chen et al. (1999) with permission.
216 D.C. Wallace et al. / Gene 238 (1999) 211–230

the African-specific Hpal site at np 3592 together with haplogroup L2 and are delineated by a DdeI site loss at
the DdeI site at np 10 394 (A to G at np 10 398). This np 13 065, and an RsaI site gain at np 11 776. Finally,
lineage is subdivided into two sublineages, L1 and L2. the Bantu-derived Senegalese core haplotypes also
L1 encompasses 52% of the L haplotypes and 29% of belong to haplogroup L2 and are defined by an HaeIII
all African mtDNAs and is defined by an additional site loss at np 322, a DdeI site loss at np 679, and an
HinfI site at np 10 806 ( T to C at np 10 810). L2 HaeIII site loss at np 13 957. Calculation of the sequence
encompasses 48% of the L haplotypes and 34% of the divergence of the core haplotypes for each population
African mtDNAs and is defined by an additional com- reveals that the Vasekela Kung a lineage and Biaka
bined HinfI site gain at np 16 389 and AvaII site loss at Pygmy b lineages are the oldest, whereas the Mbuti
np 16 390 (G to A at np 16 390). All HpaI np 3592 Pygmy and Senegalese lineages are much younger.
positive haplotypes are of African origin, with the only Hence, the Kung and Biaka Pygmies are more represen-
exceptions occurring in populations known from histori- tative of the proto-Africans and the Biaka and Mbuti
cal evidence to have had Africa contact. Pygmies may have had independent origins.
Several other features of haplogroup L are of interest. Calculation of the accumulated sequence diversity of
Two length mutations have been observed in L1: a nine the African-specific haplogroup L and its subhaplo-
np COII/tRNALys deletion between nps 8272 and 8289 groups L1 and L2 gave values of 0.356%, 0.328%, and
(Cann and Wilson, 1983; Wrischnik et al., 1987) found 0.171% respectively. The total African mtDNA sequence
in two African haplotypes, AFR 60 (representing 27% diversity was 0.364%. This means that haplogroup L
of Mbuti Pygmies) and AFR 61 (representing 24% of has the highest sequence diversity of any continent-
Biaka Pygmies); and a 10–12 bp insertion of cytosines specific haplogroup and that Africa encompasses the
(Cs) between the tRNATyr and COI gene (nps 5895– greatest diversity of any continent. Using our estimate
5899) in Biaka Pygmies with the ARF66 haplotype of the mtDNA sequence evolution rate of 2.2–
(Fig. 2). 2.9%/MYR ( Torroni et al., 1994a), the L haplogroup is
The remaining African mtDNAs form a hetero- between 123 000 and 162 000 years old, and the total
geneous array of four lineages, designated haplogroup African mtDNA lineage is between 126 000 and
L3, each defined by specific restriction-site gains or 166 000 years old ( Fig. 2).
losses ( Watson et al., 1996). One of these lineages is Calculation of the intragroup and intergroup
defined by loss of the DdeI site at np 10 394. This lineage sequence variations of the various African groups
represents only a few percent of the African mtDNAs, strengthen these observations. The Biaka Pygmies and
yet it appears to be the progenitor of roughly half of all the Vakekela Kung had the greatest intragroup sequence
European, Asian, and Native American mtDNAs. variation: 0.342% and 0.320% respectively, comparable
Within this lineage are mtDNAs that also lack an HinfI to that of Africa as a whole. This further supports the
site at np 12 308. This mtDNA haplotype is closely conclusion that these are among the oldest populations.
related to the European-specific haplogroup H. The Mbuti Pygmies and Senegalese have intragroup
Analysis of African mtDNA control region sequences sequence diversities in the range of 0.241 to 0.277,
reveals many of the same population subdivisions. confirming that these populations are younger. A neigh-
However, in some cases the control region sequences bor-joining tree analysis clearly separates the Biaka
subdivide haplotypes, and in others the haplotype mark- Pygmies from the Mbuti Pygmies, and places the Mbuti
ers subdivide control region sequence groups (Chen Pygmies on the same side of the tree as the Bantu-
et al., 1999). derived populations. Hence, the Mbuti and Biaka pyg-
Analyses of the population distribution of the African mies probably are distinct populations (Chen et al.,
haplotypes revealed that each of the four primary pop- 1999).
ulations studied (the Senegalese of West Africa, the Overall, the mtDNA data show that African mtDNAs
Mbuti Pygmies, the Biaka Pygmies and the Vasikela are distinct, they are the oldest with the greatest diversity
Kung) has a distinctive set of related core haplotypes and deepest root, that the Vasikela Kung and Biaka
that are specific for that population (Fig. 2). The core Pygmies are among the original populations of Africa,
haplotypes of the Vasikela Kung of South Africa, desig- and that the ‘Pygmy’ morphology arose two independent
nated a, occur in haplogroup L1 and are defined by times in Africa.
MspI site losses at np 8112 and 8150, and AvaII site
gain at np 8249, and an HaeIII site loss at np 8250. 3.4. European mtDNA variation
This cluster of Vasikela Kung haplotypes is at the
deepest root of the African phylogeny, suggesting that European mtDNA sequence variation has been
the Kung are one of the oldest populations. The Biaka defined by the analysis of 259 samples from individuals
Pygmy core haplotypes, designated b, also reside in of European ancestry living in the United States,
haplogroup L1 and are defined by an AluI site gain at Canada, Finland, Italy, and Sweden ( Torroni et al.,
np 10 319. The Mbuti Pygmies, designated c, reside in 1994b, 1996a). Restriction analysis revealed 178 poly-
 D.C. Wallace et al. / Gene 238 (1999) 211–230 217

morphic sites that define in the order of 170 haplotypes at np 15 606 and accounts for 15.2% of European
( Torroni et al., 1994b, 1996a). Phylogenetic analysis mtDNAs. Haplogroup U is defined by the presence of
showed that all European mtDNAs could be subdivided an HinfI site at np 12 308 and accounts for 14.7% of
into two groups by the presence (1/4) or absence (3/4) European mtDNAs. Haplogroup V is delineated by the
of the Ddel site at np 10 394 (Fig. 3). Thus, Europeans loss of an NlaIII site at np 4577 and is found in 4.8%
exhibit a marked increase in the proportion of −10 394 of Europeans, whereas haplogroup X is, in part, defined
DdeI mtDNAs over the 4% seen in Africans. by the loss of a DdeI site at np 1715 and is found in
In addition to the macro-subdivision of European about 6.9% of European mtDNAs. Of the haplotypes
mtDNAs by the Ddel site at np 10 394, nine distinct that retain the DdeI site, haplogroup I is defined by the
European mtDNA haplogroups have also been loss of the DdeI np 1715 site, and the gain of an AvaII
observed. Those lacking the DdeI site at np 10 394 are site at 8249 and an AluI site at np 10 028 and represents
haplogroups H, T, U, V, W, and X; those retaining the 6.7% of European mtDNAs; haplogroup J is identified
DdeI np 10 394 site are I, J, and K ( Fig. 3). by the loss of a BstNI site at np 13 708 and is found in
Of the haplogroups that lack the DdeI np 10 394 site, about 11.3% of European mtDNAs; and haplogroup K
haplogroup H also lacks an AluI site at np 7025 (C to is delineated by the loss of an HaeII site at np 9052 and
T at np 7028). This haplogroup encompasses 40.5% of the gain of an HinfI site at np 12 308 and is found in
European mtDNAs. Haplogroup T is defined by the 9.1% of Europeans (Fig. 3). The sequence diversity of
presence of a BamHI site at np 13 366 and an AluI site haplogroup H is 0.065%, giving an age of this lineage
of 22 000 to 30 000 YBP. However, the sequence diver-
gence of haplogroup U, which is shared between
Europeans and African Bantu, is 0.148%, giving an age
of 51 000 to 67 000 YBP. Hence, haplogroup U may
represent one of the founder lineages of Europe ( Torroni
et al., 1996a). The overall sequence divergence between
the two major branches of the European phylogeny is
0.113%, giving an age for the colonization of Europe of
between 39 000 and 51 000 YBP ( Torroni et al., 1994b).
Analysis of the control regions of the European
mtDNAs revealed additional continent-specific markers
(Di Rienzo and Wilson, 1991). However, one control-
region mutation in haplogroup I proved to be totally
novel, with potential implications for the evolution of
the human mtDNAs. All haplogroup I mtDNAs were
found to have a homoplasmic insertion of two to six Cs
within a cluster of Cs in the sequence ACCCCCC (Box
2), where the A is located at np 567. This germ-line
mutation increases the homology between this sequence
and the nearby control-region sequence ACGCCCCC-
TCCCCCGCT (Box 1), where the A is located at np
302. Because of this homology, every individual who
inherits the Box 2 germ-line insertion mutation becomes
prone to undergo a somatic mutation during develop-
ment. In this somatic mutation, the region between
Boxes 1 and 2 is duplicated as a 270 np direct repeat,
possibly through slipped misreplication ( Torroni et al.,
1994b). The somatic 270 bp duplication duplicates the
Fig. 3. Phylogeny of European mtDNA haplotypes. This representa- H-strand promoter (nps 545–567), the L-strand pro-
tive tree encompasses the analysis of 86 samples from individuals of moter (nps 392–445), the two intervening mitochondrial
European ancestry. The tree is bifurcated by the presence or absence
of an ancient polymorphism, the DdeI site at np 10 394. European transcription factor binding sites (nps 418–445 and nps
mtDNAs lacking the 10 394 site fall into six distinct haplogroups: H, 523–550), CSBIII (nps 346–363), part of CSBII (nps
T, U, V, W, and X; those retaining the site fall into three major 299–315), and the putative replication primer processing
haplogroups: I, J, and K. Each haplogroup is defined by distinctive site (nps 317–321) (Brockington et al., 1993; Torroni
polymorphisms (see text). These nine European-specific haplogroups et al., 1994b). This raises the possibility that the dupli-
account for over 98% of all mtDNAs found in Europe ( Torroni et al.,
1994a,b). As for all parsimony trees, there are many alternative cated molecules are transcribed twice as frequently and
European mtDNA phylogenies, but the central branches are quite may be preferentially replicated, providing selective
robust. advantage for this mutation. This may explain why the
218 D.C. Wallace et al. / Gene 238 (1999) 211–230

unstable Box 2 insertion has been maintained through- T ); and, for haplogroup D, variants at nps 16 362 ( T
out the 34 000 year history of haplogroup I ( Torroni to C ) and 16 223 (C to T ) ( Torroni et al., 1993a).
et al., 1994b). Three other prominent Asia haplogroups are E, F,
and G. Haplogroups E and G have the combined DdeI
3.5. Asian mtDNA variation and AluI sites at nps 10 394 and 10 397, whereas haplo-
group F lacks these sites. Haplogroup E is further
To define further the Asian mtDNA sequence varia- defined by an HpaI site loss at np 7598, haplogroup G
tion, we have analyzed the mtDNAs from 153 Central by the presence of an HaeIII site at np 4830 and an
and Southeast Asians, including aboriginal Malays and HpaI site at np 4831; haplogroup F is delineated by the
Orang Ash, aboriginal Borneans, Han Chinese, combined HpaI/HincII site loss at np 12 406, the first
Vietnamese, Koreans, and Malaysian Indians (Ballinger Asian-specific polymorphism observed (Denaro et al.,
et al., 1992) as well as 54 Tibetans ( Torroni et al., 1981; Blanc et al., 1983). All of these haplogroups
1994c) and 758 Siberians from 11 aboriginal popula- show marked frequency variation throughout Asia.
tions, including the Chukchi and Koryaks from Haplogroup F is prominent in southern Asian popula-
northeastern-most Siberia (Torroni et al., 1993b; tions, being found in 32% of Vietnamese mtDNAs and
Starikovskaya et al., 1998; Schurr et al., 1999). A 21% of Malay mtDNAs. It is present in about 15% of
representative Asian phylogenetic tree encompassing 42 Koreans and Tibetans, but is virtually absent in Siberia.
Tibetan haplotypes, 106 Asian haplotypes, and 34 By contrast, haplogroups A, C, D, E, and G are absent
Siberian haplotypes is presented in Fig. 4. This phylog- in southern Asian populations, including Vietnamese,
eny shows that all Asian mtDNAs can be subdivided Malays, Sabah, Malay aboriginals, and New Guineans,
into two macro-haplogroups defined by the presence or but these groups are found at significant frequencies in
absence of the polymorphic site at DdeI at np 10 394, Tibetans, Koreans, and Han Chinese. This north–south
which also bifurcates the European mtDNA lineages. distinction supports the dichotomization of Asians into
Moreover, every Asian mtDNA that harbors the Ddel the Sinodont (northern) and Sunodont (southern) Asian
site at np 10 394 also has an adjacent AluI site at np populations (Turner, 1983, 1987). Furthermore, haplo-
10 397 (C to T at np 10 400). The macro-haplogroup groups A, C, and D extend into the Siberia populations
defined by the presence of the DdeI np 10 394 and the analyzed, reaching maximum frequencies of 68%, 84%
AluI np 10 397 sites has been designated macro-haplo- and 28% respectively. Haplogroup A reaches its highest
group M [or also as (+/+)]. The constant association frequencies in the Chukchi and Koryaks, the northeast-
of the DdeI np 10 394 and AluI np 10 394 in Asians, but ern-most populations of Siberia and likely progenitors
not in Africans or Europeans, implies that the AluI np of Native Americans. The haplogroup frequencies of
10 397 mutation must have arisen on an mtDNA carry- the Koryaks are 5% A, 36% C, 1% D, and 42% G, 10%
ing the DdeI np 10 394 mutation as women migrated Y, and 6% other, whereas those of the Chukchi are 68%
out of Africa and into Asia ( Fig. 4). A, 11% C, 12% D, and 9% G ( Torroni et al., 1993b;
In addition to this major bifurcation of Asian Starikovskaya et al., 1998; Schurr et al., 1999).
mtDNAs, there are a number of distinctive sublineages Haplogroup B, defined by the 9 np COII-tRNALys
of relevance to Asian and Native American prehistory. deletion, displays a markedly different distribution. It is
Haplogroups A, B, C, and D have proved to be the common throughout central and southern Asia and is
progenitors of virtually all Native American mtDNAs. prominent in coastal Asian populations, approaching
Haplogroups A and B lack both the DdeI site at np fixation (100%) in certain Pacific island populations
10 394 and the AluI site at np 10 397, whereas haplo- ( Hertzberg et al., 1989; Stoneking et al., 1990; Ballinger
groups C and D have these sites. In addition, haplogroup et al., 1992). It is virtually absent from all nine Siberian
A is defined by an HaeIII site at np 663 (A to G at np populations analyzed, yet it reappears throughout
663), haplogroup B by an independent occurrence of North, Central, and South American Native American
the 9 np deletion between the COII and tRNALys genes, populations (Schurr et al., 1990; Torroni et al., 1993a,b).
haplogroup C by the simultaneous HincII site loss at The high frequency of this haplogroup among coastal
np 13 259 and an AluI site gain at np 13 262 (A to G at Asian and Pacific island populations, and its striking
np 13 262), and haplogroup D by the loss of an AluI absence in Siberians relative to Central Asians and
site at np 5176 (C to A at np 5178). These haplogroups Native Americans, raises the possibility that haplogroup
are further delineated in most Asians and Native B mtDNAs did not come to the Americas via a trans-
Americans by specific control-region variants. For Siberian migration, but rather may have crossed from
haplogroup A, these include variants at nps 16 362 ( T Asia to the Americas by migration along the Siberian
to C ), 16 319 (G to A), 16 290 (C to T ), and 16 223 (C coast. This deduction has been questioned, however,
to T ); for haplogroup B, variants at nps 16 217 ( T to based on mathematical analysis of control-region
C ) and 16 189 (T to C ); for haplogroup C, variants at sequence diversity (Forster et al., 1996).
nps 16 327 (C to T ), 16 298 ( T to C ), and 16 223 (C to The co-occurrence of the AluI site at np 10 397 and
 D.C. Wallace et al. / Gene 238 (1999) 211–230 219

Fig. 4. Phylogeny of Asian mtDNA Haplotypes. This representative tree is based on data from 153 Central and Southeast Asians, 54 Tibetans,
and 758 aboriginal Siberians. The Asian mtDNA phylogeny is bifurcated into two macro-haplogroups. One macro-haplogroup lacks the DdeI site
at np 10 394, as well lacking an AluI site at np 10 397, and thus is designated (−/−), whereas the other macro-haplogroup has these restriction
sites and is thus designated (+/+). The later group is called macro-haplogroup ‘M’. Within these two major Asian mtDNA lineages are multiple
important haplogroups. Haplogroup F is (−/−) and is found at high frequency in Southeast Asia, but declines toward Northeast Asia. Haplogroups
A and B are −/−, whereas haplogroups C and D are (+/+). These four mtDNA lineages are at a low frequency in Southern Asia, but rise to
high frequencies in Northeast Asia, where they participated in the peopling of the Americas. This tree is reprinted from Torroni et al. (1994c) with
permission.

the DdeI site at np 10 394 in macro-haplogroup M Ethiopians. Either the haplogroup M mtDNA entered
mtDNAs throughout Asia indicates that the AluI site East Africa through relatively recent migrations from
gain occurred at the beginning of Asian habitation. This Asia, or they originated in East Africa. If the latter is
hypothesis has been supported by the discovery of true, this would place the origin of M at close to the
macro-haplogroup M mtDNAs in East African time of the migration of proto-Asians out of Africa and
220 D.C. Wallace et al. / Gene 238 (1999) 211–230

into Asia (Passarino et al., 1996, 1998). Consequently, 10 394 and AluI np 10 397 site gains found in their Asian
the sequence diversity that has accumulated in the DdeI progenitors (Schurr et al., 1990; Torroni et al., 1992,
np 10 394+AluI np 10 397 lineage should be indicative 1994d ) ( Fig. 5). Each of the four primary Native
of the age of the Asian population. The overall sequence American haplogroups traces back to a single nodal
diversity in this lineage is 0.161%. This gives an age for mtDNA haplotype that is shared by Asia and the
the Asian population of 56 000–73 000 YBP. Americas and that initiated the mtDNA radiation in the
Americas. However, none of the derived haplotypes is
3.6. Native American mtDNA variation shared by Asians and Americans, as demonstrated by
analyzing haplogroup C and D mtDNAs from Siberians
To learn more about the origin of Native Americans, and Native Americans ( Torroni et al., 1993b). Hence,
we analyzed 743 Native American mtDNAs. Multiple it appears that, primarily, these four mtDNA haplotypes
hypotheses have been put forward to explain the origin crossed from Siberia into the Americas. All four of these
and radiation of Native Americans. One hypotheses is haplogroups are distributed throughout the Paleo-
based on the classification of Native American languages Indians of North, Central, and South America, though
by Greenberg et al. (1986). These authors divided all individual tribes may have lost one or more of the
Native American languages into three major groups: haplogroups through genetic drift. The broad distribu-
Amerind, which encompasses the great diversity of tion of all four mtDNA haplogroups suggests that they
languages spoken by the Paleo-Indian peoples occupying either came together or were subsequently thoroughly
most of North America and all of Central and South mixed.
America; Na-Déné, which is spoken by the Athapaskans The calculation of the mtDNA sequence diversity
of the northwestern United States, Canada, and Alaska, that has accumulated within each haplogroup revealed
as well as by the Navajo and Apache, who migrated that haplogroups A, C, and D had relatively similar
south through the great plains around 1000 AD; the values: A=0.075%, C=0.096%, and D=0.053%, with
Eskaleut languages, which are spoken by the Eskimos a mean value of 0.075%. By contrast, haplogroup B had
and Aleuts of the Arctic region. Greenberg et al. (1986) a much lower value, 0.034%, suggesting that haplogroup
hypothesized that each of these language groups corres- B arrived in the Americas much more recently than A,
ponded to a different migration, arising in a different C, and D (Schurr et al., 1999). This difference is
geographic homeland. Using glottochronology-dating consistent with the absence of haplogroup B in Siberia,
based on the divergence rate of languages, they estimated even though haplogroups A, C, and D are prevalent.
that these migrations occurred at about 11 000 YBP, These two results imply that the Paleo-Indians of the
9000 YBP, and 5000 YBP respectively. Amerind linguistic group may have been derived from
In our first studies on Native American mtDNA two migrations. The first migration moved up from
variation, we focused on the Pima and the Papago, central Asia through Siberia, during which it became
Paleo-Indians of the southwestern United States. Using progressively enriched for the founder haplotypes of
Southern blot analysis and our initial six informative haplogroups A, C, and D. Ultimately, only these haplo-
restriction endonucleases, we discovered that about 40% types crossed the Bering land bridge to found the Paleo-
of these Native American mtDNAs lacked the HincII Indians. The second migration came much later, bearing
site at np 13 259 ( Wallace et al., 1985), whereas only the founder haplotype of haplogroup B. This migration
1.8% of central Asian mtDNAs lacked this site (Blanc bypassed Siberia, possibly moving along the Siberian
et al., 1983). This led to the hypothesis that Native and Alaskan coasts, and entered the Americas, where it
American mtDNAs were derived from a limited number interspersed with the already present haplogroups A, C,
of founding mtDNA haplotypes that crossed the Bering and D (Schurr et al., 1990; Torroni et al., 1992, 1993a)
land bridge in distinct migrations ( Wallace et al., 1985; ( Fig. 5). An independent analysis of the number of
Schurr et al., 1990; Wallace and Torroni, 1992). This Native American migration based on mtDNA control
hypothesis has subsequently been confirmed by our region sequence data concluded that all Paleo-Indians
more extensive analysis encompassing 563 Paleo-Indians (Amerinds) were derived from a single migration. That
from 24 tribes, 130 Na-Déné representing five tribes, is, B came with A, C, and D ( Forster et al., 1996).
and 50 Eskimos ( Torroni et al., 1992, 1993a, 1994a,d ). To investigate further the Paleo-Indian tribalization
Our analysis of mtDNA variation in Paleo-Indians process, we examined the mtDNAs from an isolated
revealed a dramatic result. Virtually all of the mtDNAs groups of Aztec descendants in central Mexico: the
fell into one of the four Asian haplogroups: A (HaeIII Mixtec of Alta and Baja, the Zapotecs, and the adjacent
site at np 663), B (9 np deletion between Mixe. These tribes were compared with the Pima of
COII/tRNALys), C (HincII site loss at np 13 259 and Arizona, the Maya of Yucatan, the Chibchan-speakers
AluI site gain at np 13 262), and D (AluI site loss at np of Panama, the Bella Coola and the Nuu-Chah-Nulth
5176), with haplogroups C and D also belonging to of North America, and the Yanomama and Wapishana of
macro-haplogroup M and thus harboring the Ddel np South America. In aggregate, the Aztec-derived Mixtec
 D.C. Wallace et al. / Gene 238 (1999) 211–230 221

Fig. 5. Phylogeny of Native American mtDNAs. This representative tree was derived from the haplotype data obtained from approximately 563
Paleo-Indians, 130 Na-Déné, and 50 Eskimos, and reveals a dramatic point: all Native American mtDNAs were derived from only five founder
mtDNAs. These founders originated from haplogroups A, B, C, and D, found in Asia, and from haplogroup X, found only in Europe. The
Amerind-speaking Paleo-Indians of North, Central, and South America encompass haplogroups A, B, C, and D. The Paleo-Indians of North
Central, North America also encompass haplogroup X. The Na-Déné of the Pacific Northwest and Southwest (Apache and Navajo) are primarily
A with 1/3 having the Na-Déné-specific marker, an RsaI site loss at np 16 329. The Eskimos of the sub-Arctic are primarily haplogroups A and
D. This phylogeny is derived from data reported by Wallace and coworkers ( Wallace et al., 1985; Schurr et al., 1990, 1999; Torroni et al., 1992,
1993a, 1994a,d; Torroni and Wallace, 1995; Brown et al., 1998; Starikovskaya et al., 1998).

and Zapotecs harbored three haplogroups: 66% A, 18% The prevalence of haplogroup A, B, C, and D
B, and 16% C. The linguistically related Pima of Arizona mtDNAs in Native American populations has now been
harbored the same haplogroups, as did the Mixe, sug- confirmed by multiple investigators ( Ward et al., 1991,
gesting a common ancestry. The Maya were more similar 1993; Horai et al., 1993; Santos and Barrantes, 1994).
to the North American Paleo-Indians, whereas the These same four haplogroups have also been found in
Chibchans and South American tribes differed from the the Native American skeletons excavated from a pre-
more northern tribes and from each other. These results Columbian burial site in Central North America (Stone
suggest that the Maya and Aztecs may have been derived and Stoneking, 1993). Although some Native American
from different populations ( Torroni et al., 1994d ). mtDNAs have been found not to exhibit one of the four
222 D.C. Wallace et al. / Gene 238 (1999) 211–230

primary mtDNA restriction site markers ( Torroni et al., Analysis of northern Na-Déné, including Dogrib,
1992, 1993a; Bailliet et al., 1994), most of these mtDNAs Tlingit, and Haida, indicates that these people harbor
can be shown to result from either recent genetic admix- mtDNAs from only one of the founding mtDNA haplo-
ture with European or African immigrants or the second- groups, that of haplogroup A ( Torroni et al., 1992,
ary gain or loss of informative restriction sites ( Torroni 1993a). This is substantiated by the southern Na-Déné,
and Wallace, 1995). The major exception to this is found including the Apache and Navajo, who are >60%
in the Native Americans of Central North America. haplogroup A. The remaining Apache and Navajo
These populations have been discovered to harbor a mtDNAs are from haplogroups B, C, and D and
fifth ancient founding mtDNA lineage, designated probably represent recent admixture with adjacent
haplogroup X ( Fig. 5). Analysis of the mtDNA of 42 Paleo-Indian tribes. The distinctive nature of the
Ojibwa of the Great Lakes region revealed 11 (26%) Na-Déné mtDNAs is confirmed by the fact that about
that were not from haplogroups A, B, C, or D. Similarly, a third of all Na-Déné haplogroup A mtDNAs carry a
of the Nuu-Chah-Nulth and the Bella Coola of the novel variant, an RsaI site loss at np 16 329 (A to G at
Pacific northwest, 13.3% and 4% respectively were non- 16 331). Interestingly, this variant is found in all
A–D. Extensive analysis of these mtDNAs revealed that Na-Déné except the Haida. This may suggest that the
they had some features in common with the rare RsaI site loss occurred in the original Beringian popula-
European haplogroup X. The shared markers included tions that gave rise to the Na-Déné in the Americas.
restriction site polymorphisms for an AvaII site at np Analyses of the sequence diversity of the Na-Déné
14 465, the absence of DdeI sites at np 1715 and at np haplogroup A mtDNAs gave a value of 0.021%. This is
10 394, and the presence of the HaeIII site at np 16 517. substantially lower than the diversity of the Paleo-Indian
Control-region sequence analysis further revealed that haplogroups A+C+D and also different from the
both the Native American and European haplogroup X Paleo-Indian haplogroup B. Hence, the Na-Déné do
mtDNAs shared additional nucleotide variants, located appear to have arrived as a single independent migration,
at 16 189C, 16 223T, 16 278T, 73G, 153G, 195G, 225A, which occurred more recently than the Paleo-Indians
and 263B. However, comparison of the European and migration ( Torroni et al., 1992, 1993a). This conclusion
Native American control-region sequences also revealed has been confirmed by analysis of control-region
clear differences, indicating that the last common ances- sequence data (Forster et al., 1996).
tor of the European and Native American haplogroup Analysis of the mtDNA variation in Eskimos has
X mtDNAs lived long ago. Moreover, whereas the been more difficult, because of the limited availability
above-mentioned survey of the pre-Columbian burial of samples and their recent divergence from ancestral
site revealed mostly mtDNAs of haplogroups A, B, C, populations. However, a survey of 129 Siberian Yupik
or D, two skeletons were found to be different and to Eskimos, who represent Eskimo peoples inhabiting both
have the same control region sequence as a subset of sides of the Bering Strait, revealed only haplogroups A
the Ojibwa haplogroup X mtDNAs (Brown et al., 1998). and D. Hence, the Eskimos may also be distinct from
Hence, the Central North American haplogroup X the Na-Déné and Paleo-Indians, as predicted from lin-
mtDNAs are not the result of recent European admix- guistic associations ( Torroni et al., 1993b; Starikovskaya
ture with Native Americans, but arrived in an ancient, et al., 1998).
pre-Columbian, migration (Brown et al., 1998). A Times of the Native American migrations, as in the
survey of the distribution of haplogroup X throughout cases of the African, European, and Asian populations,
Asia, including 411 Siberians ( Torroni et al., 1993b) were calculated using the haplogroup-specific mtDNA
and 207 Asians (Ballinger et al., 1992; Torroni et al., sequence diversity and the mtDNA sequence evolution
1994c), failed to reveal a single haplogroup X mtDNA. rate of 2.2–2.9%/MYR. This rate was calculated using
This implies that the Native American haplogroup X the Chibcha-speaking peoples of Central America, who
may not be of Asian ancestry. This raises the possibility are estimated from anthropological and nuclear genetic
that haplogroup X came to the Americas in a separate data to have originated about 8000–10 000 YBP.
migration originating in Europe. Analysis of the haplo- Analyzing the mtDNA variation of 110 Chibchans
group X restriction and control region sequence data representing five tribes revealed that all but one of the
has permitted us to estimate the divergence time of the Chibchan mtDNAs were from haplogroups A or B, the
American and European haplogroup X mtDNAs, which exception being a single haplogroup D. Moreover, 62%
proved to be in the range of 15 000 to 30 000 YBP. of the Chibchan haplogroup A mtDNAs showed a
Thus, haplogroup X must have come to the Americas distinctive private polymorphism, the loss of an MspI
with the original Native American migrations and repre- site at np 104, the result of a small deletion in the 3∞
sents a novel founder lineage of possibly European end of the D-loop hypervariable region II (Santos and
origin (Brown et al., 1998). Barrantes, 1994; Torroni et al., 1994a). Averaging the
Analysis of mtDNA variation of the Na-Déné has sequence diversity of the haplogroup A and B mtDNAs
confirmed that they are distinct from the Paleo-Indians. and dividing by the putative age of the population gave
 D.C. Wallace et al. / Gene 238 (1999) 211–230 223

the sequence evolution rate of 2.2–2.9%/MYR (Torroni sion that gave rise to the Na-Déné and Eskimo popula-
et al., 1994a). tions (Starikovskaya et al., 1998).
Using this Native American sequence evolution rate, Analysis of the Koryaks and Itel’men of the
we calculated that the first Paleo-Indian migration carry- Kamchatka peninsula revealed a very different haplo-
ing haplogroups A, C, and D (sequence diversity of group distribution. The Koryaks proved to be 5.2% A;
0.075%) arrived 26 000–34 000 YBP and that the second 36.1% C, 1.3% D, 41.9% G, 9.7% Y, and 5.8% Z. The
Paleo-Indian migration bringing haplogroup B (0.034%) Itel’men were 6.4% A, 14.9% C, 68.1% G, 4.3% Y, and
arrived 12 000–15 000 YBP. The Na-Déné migration 6.4% Z. Thus, even though the Chukotka and
with a sequence diversity of 0.021% was estimated to Kamchatka populations are proximate to each other
have arrived 7200–9000 YBP, a value strikingly similar and the Chukchi and Koryaks have related languages,
to the 9500 YBP value estimated by glottochronology the mtDNAs of these are strikingly distinct. Indeed, in
( Torroni et al., 1994a) ( Fig. 5). a neighbor-joining tree analysis, the Eskimos and
Comparable estimates from control-region sequence Chukchi were located at one end of the tree with the
data have suggested an initial major expansion out of Amerinds and Na-Déné, whereas the Koryaks and
northeastern Siberia into the Americas to generate the Itel’men were at the other end of the tree with the Ainu,
Paleo-Indians (Amerind ) occurring about 20 000 to Japanese, Koreans, and other Siberians (Schurr et al.,
25 000 YBP, followed by a second rapid expansion out 1999).
of Beringia about 11 300 YBP giving rise to the Na-Déné The predominance of haplogroup C in the Paleo-
and Eskimos (Forster et al., 1996). Indians (Amerind ) is most similar to the haplogroup
distribution seen in the central Siberian, where the
3.7. Siberian antecedents to Native American migrations Evenks and Udeguys are 84.3% C and 17.8% C respec-
tively. This raises the possibility that the progenitors of
With the Siberian origin of Native Americans estab- the Paleo-Indians may currently reside in central Siberia
lished, it was of interest to determine which Siberian (Starikovskaya et al., 1998; Schurr et al., 1999).
populations are the most likely progenitors of the vari-
ous Native American migrations. To address this ques-
tion, we have performed an extensive analysis of the 4. Leber’s hereditary optic neuropathy (LHON ) and the
interaction between mtDNA disease mutations and
mtDNAs of the Siberian Eskimos and Chukchi of the
background mtDNA variation
Chukotka peninsula, and of the Koryaks and Itel’men
of the Kamchatka peninsula. The Chukotka peninsula
Whereas some germline mtDNA mutations are neut-
is the region of northeastern Siberia closest to Alaska,
ral and become established in the human populations
and the Kamchatka peninsula extends south from the
by genetic drift, many are deleterious and result in
Chukotcha peninsula forming the eastern border of the
genetic disease. Deleterious mtDNA mutations vary
Sea of Okhotsk. Analysis of 79 Siberian Eskimos
widely in their severity, but the mildest mutations result
revealed an mtDNA haplogroup distribution of 77.2%
in an acute onset blindness syndrome known as LHON.
A, 2.5% C, and 20.3% D. Similarly, the haplotype
Extensive analysis of the mtDNAs of LHON patients
distribution of the Chuckchi was 68.2% A, 10.6% C, has revealed that certain mtDNA haplotypes are prone
12.1% D, and 9.1% G. Control-region sequence analysis to expression of LHON more than others. This indicates
of the Chukotka haplogroup A mtDNAs, and compari- that different population-specific mtDNA may be func-
son with those of Native American Na-Déné and tionally different and hence might have been influenced
Northwestern Amerinds, revealed a 16 111 C to T trans- by selection.
ition that appears to delineate an ‘American’ enclave of LHON classically manifests as acute-onset, bilateral,
northeastern Siberian mtDNAs. Furthermore, deriva- central vision loss associated with the degeneration of
tives of this sublineage were found to include some of the retinal ganglion cell layer and optic nerve. Typically,
the progenitors of the coastal Amerinds, some bearing the onset and progression of blindness is relatively rapid,
an additional 16 129A variant; the Haida and Bella with both eyes developing vision loss within a year of
Coola bear an additional 16 355T variant, and some each other and with blindness usually the only clinical
Eskimos bear an additional 16 265G variant. The sign. However, in some LHON cases other more sever
16 111T lineage of haplogroup A is further subdivided neurological abnormalities are also observed, the most
by the additional 16 192T variant. In addition to being common of which is early-onset dystonia associated
found in Chuckotka, this 16 111T+16 192T lineage is with basal ganglia degeneration.
the progenitor of the Na-Déné lineage, which bears the LHON is strictly maternally inherited, though the
variants 16 233G and 16 331G, the latter site generating penetrance is highly variable and there is an unexplained
the characteristic Na-Déné RsaI site loss at np 16 329 predilection for males to be preferentially affected. A
(16 331 A to G). Hence Chukotka seems to harbor typical pedigree for the most common mtDNA muta-
some remnants of the progenitors of the Beringia expan- tion, MTND41LHON11778A, is shown in Fig. 6.
224 D.C. Wallace et al. / Gene 238 (1999) 211–230

4.1. MtDNA mutations and LHON detected in a large number of control mtDNAs (Brown
and Wallace, 1994a; Brown et al., 1995; Howell et al.,
A total of 23 mtDNA missense mutations have been 1995; Howell, 1997). In patients of European descent,
associated with LHON patients ( Table 1). However, the MTND41LHON11778A mutation accounts for
detailed genetic analysis has revealed that only a few about 50% of cases, whereas the MTND11LHON3460A
‘primary’ mutations contribute in a major way to the and MTND61LHON14484C mutations encompass
development of blindness. The remaining ‘secondary’ roughly 15% each ( Table 1). In Asia, the MTND41
mutations may contribute to LHON by increasing the LHON11778A mutation accounts for 95% of patients
probability of expressing the phenotype or may simply (Mashima et al., 1993). Such continental differences
be linked to other clinically important variants in the suggest that genetic backgrounds may affect the expres-
same mtDNA haplotype. sion of the different LHON mutations.
The four ‘primary’ LHON mutations, listed in order The MTND61LDYT14459A mutation, while rela-
of decreasing severity, are MTND61LDYT14459A (Jun tively rare, is the most severe of the primary LHON
et al., 1994; Shoffner et al., 1995), MTND41LHON mutations (Jun et al., 1994; Shoffner et al., 1995). This
11778A ( Wallace et al., 1988b), MTND11LHON3460A mutation can be manifested in two very different pheno-
(Howell et al., 1991; Huoponen et al., 1991, 1993), and types, LHON and/or generalized dystonia. The
MTND61LHON14484C (Johns et al., 1992a; Mackey MTND61LDYT14459A mutation converts the highly
and Howell, 1992) ( Table 1). The individual primary conserved alanine at codon 72 in the ND6 polypeptide
mutations represent strong risk factors for expression to a valine, has been found to be heteroplasmic in at
of maternally inherited LHON, they have been observed least some family members in every pedigree studied
in a number of unrelated LHON families, they rarely ( Table 1) (Jun et al., 1994; Shoffner et al., 1995). The
co-occur with each other, and they have not been optic atrophy associated with his mutation is similar to

Fig. 6. Maternally inherited pedigree of LHON due to the MTND41LHON11778A mutation. Affected individuals (filled symbols) experience acute
onset optic atrophy and central vision loss, generally as young adults. Even though the mutation is essentially homoplasmic, penetrance among
maternal relatives is highly variable. Moreover, males are about three to four times more likely to lose their vision than females ( Wallace et al.,
1988b; Newman et al., 1991).
 D.C. Wallace et al. / Gene 238 (1999) 211–230 225

Table 1
LHON disease mutations

No.a Mutation Classb Other Amino acidsc Approx. Controls Heteroplasmy Penetranced (%) Recoverye
neurol. European (%) (%)
disease Cons Change patients (%) Relatives Males

1 MTND61LDYT14459A Primary +/− A72V M Rare 0 + 61 58 Low

2 MTND41LHON11778A Primary +/− R340H H 50 0 +/− 33–60 82 4
3 MTND11LHON3460A Primary +/− A52T M 15 0 +/− 14–75 40–80 22
4 MTND61LHON14484C Primary − M64V L 15 0 +/− 27–80 68 37-50
5 MTND21LHON5244A Primary − G2595 H Rare 0 + NA NA UN
6 MTND51LHON13730A Primary − G465E M Rare 0 + NA NA Yes
7 MTCO31LHON9804A Primary? − A200T H 1.5 0 − UN UN UN
8 MTND31LHON10663C Primary? − V65A L Rare 0 − 56 60 UN
9 MTATP61LHON9101C Primary? − I192T L Rare 0 − NA NA UN
10 MTND41LDYT11696G Primary? +/− V312I L Rare 0 − UN UN UN
11 MTND61LHON14482G Primary? − M64I L Rare 0 − UN 89 UN
12 MTND61LHON14498T Primary? − Y59C M Rare 0 + 31 50 UN
13 MTND61LHON14568T Primary? − G36S L Rare 0 − NA NA UN
14 MTND61LDYT14596A Primary? +/− I26M M Rare 0 + UN UN UN
15 MTCYB1LHON15257A Intermediate − D171N H 9 0.4 − NA NA NA
16 MTND51LHON13708A Secondary − A458T M 30 6 − NA NA NA
17 MTND11LHON3394C Secondary − Y30H H Rare 0.9 − NA NA NA
18 MTND11LHON4160C Secondary ++ L285P H Rare 0 − 76 54 0
19 MTND11LHON4216C Secondary − Y304H L ~40 13 − NA NA NA
20 MTND21LHON4917G Secondary − D150N H 3 3 − NA NA NA
21 MTCO11LHON7444A Secondary − TerK NA 5 1 − NA NA UN
22 MTCO31LHON9438A Secondary − G78S H 2.5 4.6 − NA UN NA
23 MTCYB1LHON15812A Secondary − V357M M 4 0.1 − NA NA NA

a The first 10 LHON-associated mtDNA mutations are listed in order of estimated severity (see text).
b A question mark in classification indicates transient assignment pending more data.
c H=high amino acid conservation; M=moderate; L=low; NA=not applicable; Ter=termination codon.
d NA=not applicable; UN=unknown; penetrance estimate for the 10 663 mutation is from a single LHON family that does not harbor a common
primary LHON mutation by complete mtDNA sequence analysis.
e Low=anecdotal low degree of vision recovery; UN=unknown; NA=not applicable.

that seen for other LHON mutations. By contrast, the et al., 1991; Brown and Wallace, 1994b). Furthermore,
dystonia caused by this mutation has a mean age of only about 4% of affected individuals experience visual
onset of 4 years, with a range of 1.5 to 9 years. In these recovery. Hence, the MTND41LHON11778A mutation
dystonia patients, the motor system was primarily has a slightly lower penetrance than the MTND61
involved, resulting in gait disturbance and rigidity of LDYT14459A mutation, and a much greater bias
the lower extremities, which advances with age to include towards males being affected.
the upper extremities. Patients also developed pseudo- Of the affected individuals, the mean age of onset is
bulbar syndrome (swallowing and speech problems), 27.6 years, with a range of onset from 8 to 60 years.
impaired intelligence, short stature and myopathic fea- About 58% of patients show additional ophthalmologi-
tures. These symptoms are often associated with bilateral cal features, including peripapillary telangiectasias,
striatal necrosis (Novotny et al., 1986), the loss of cells microangiopathy, disk pseudo-edema, and vascular tor-
in the striatum, putamen and caudate. The penetrance tuosity. A total of 55% of patients have a simultaneous
of the MTND61LDYT14459A mutation among mater- onset of vision loss in both eyes, and once vision loss
nal relatives is about 61%, with 58% of affected individ- begins it can progress rapidly or slowly, with a mean
uals being male. There is no record of affected length of progression for 3.7 months and a range of 0
individuals recovering ( Table 1). to 24 months. In about 98% of cases, the final visual
The MTND41LHON11778A mutation is the most acuity is 20/200 or worse, and only 2% are better than
common cause of LHON, and the second most severe 20/200 (Newman et al., 1991). Occasional individuals
mutation. It converts the highly conserved ND4 codon also show additional neurological symptoms, reminis-
340 from an arginine to a histidine ( Wallace et al., cent of those of the MTND61LDYT14459A mutation
1988b). Among MTND41LHON11778A families, including ataxia, spastic paraparesis, extrapyramidal
about 33–60% of maternal relatives are affected, with signs, dystonic rigidity associated with bilateral basal
82% of the affected individuals being male and 18% ganglia, and cerebellar and pontine atrophy, etc.
female. About 14% of cases are heteroplasmic (Newman (Larsson et al., 1991; Funakawa et al., 1995; Vergani
226 D.C. Wallace et al. / Gene 238 (1999) 211–230

et al., 1995). In LHON pedigrees with the The MTND61LHON14484C mutation is additionally
MTND41LHON11778A mutation, about 1–2% of noteworthy in that fully 37–50% of patients report visual
female patients also manifest a multiple-sclerosis-like improvement ( Table 1) (Johns et al., 1993; Riordan-Eva
demyelination disease (Newman et al., 1991; Harding et al., 1995). Thus, the MTND61LHON14484C muta-
et al., 1992; Flanigan and Johns, 1993; Hanefeld et al., tion has the lowest pathogenicity of the primary LHON
1994; Kellar-Wood et al., 1994; Olsen et al., 1995). mutations.
Interestingly, in the Japanese there is no such association Analysis of the background mtDNA haplotypes in
between the MTND41LHON11778A mutation and LHON families harboring the various primary mut-
multiple sclerosis (Nishimura et al., 1995), again sug- ations has shown that most families are new mutations.
gesting that genetic background may be important in However, striking background haplotype associations
disease expression. have been found in patients with the milder LHON
The MTND11LHON3460A mutation is the next mutations, suggesting an interaction between the recent
most severe. It changes a moderately conserved alanine mild pathogenic mutation and the ancient background
at codon 52 in the ND1 gene to a threonine and has genotype.
been observed to be heteroplasmic in a number of The MTND61LDYT14459A mutation was first iden-
families ( Howell et al., 1991; Huoponen et al., 1991, tified in a large Hispanic family, which proved to harbor
1993; Brown and Wallace, 1994b). Generally, the clinical a Native American mtDNA from haplogroup D (Jun
manifestations of this mutation are confined to LHON, et al., 1994, fig. 20). A second case was found in an
having features quite similar to MTND41 African–American family harboring an African haplo-
LHON11778A. Only occasionally is the MTND11 group L mtDNA, and involved a mother and daughter
LHON3460A mutation associated with other neurolog- with LHON in which the daughter also had unilateral
ical signs (Table 1), though patients harboring this striatal degeneration. The third case involved a
mutation can manifest multiple sclerosis ( Kellar-Wood European child with generalized dystonia, who was
et al., 1994; Nikoskelainen et al., 1995). The number of found to harbor European haplogroup I mtDNA (Jun
affected maternal relatives has varied widely in different et al., 1994; Shoffner et al., 1995). Since mtDNA haplo-
studies, but can approach 75% (Johns et al., 1992b; types of these three families encompass most of the
Harding et al., 1995), with between 40% and 80% of all world’s mtDNA sequence diversity, the MTND61
affected individuals being male. In contrast to the LDYT14459A mutations must have arisen recently and
MTND41LHON11778A mutation, however, approxi- independently in each family (Jun et al., 1994; Shoffner
mately 22% of affected individuals with the et al., 1995). Whether the phenotype variation is in any
MDND11LHON3460A have been reported to experi- way related to variation in haplotype could not be
ence visual recovery (Johns et al., 1993). determined.
The MTND61LHON14484C mutation is the mildest A similar haplotype analysis has been performed on
of the common primary mutations. This mutation 47 LHON patients of European descent harboring the
changes the weakly conserved methionine at codon 64 three common ‘primary’ mutations: MTND41
in the ND6 protein to a valine. The pathogenicity of LHON11778A, MTND11LHON3460A, and MTND61
the MTND61LHON14484C mutation is nicely con- LHON14484C (Fig. 7) (Brown et al., 1995). This
firmed by one of the rare LHON mutations: revealed that patients harboring the MTND41
MTND61LHON14482G. This mutation alters the same LHON11778A and MTND11LHON3460A mutations
codon as the MTND61LHON14484C mutation, but had a variety of different mtDNA background haplo-
converts the methione to an isoleucine, yet both mut- types dispersed throughout the European mtDNA
ations are associated with LHON (Howell, 1998). The haplogroups. Thus, most, if not all, LHON families
MTND61LHON14482C mutation has only very rarely with these mutations are due to independent mutational
been reported to be heteroplasmic ( Table 1) (Johns events. By contrast, most patients harboring the
et al., 1992a; Mackey and Howell, 1992; Biousse et al., MTND61LHON14484C mutation clustered together on
1997), suggesting that the great majority of mtDNA a single mtDNA lineage, haplogroup J (Fig. 7).
patients must be mutant to cause a sufficient biochemical However, the MTND61LHON14484C patients were
defect to result in optic atrophy. Clinically, this mutation still dispersed among the haplogroup J controls, indicat-
is almost exclusively confined to an LHON phenotype. ing that most, if not all, MTND61LHON14484C pedi-
The penetrance of LHON in MTND61LHON14484C grees are new mutations (Brown et al., 1995). Still, the
mutation maternal relatives is about 27–80%, and the association between the MTND61LHON14484C muta-
male to female ratio of affected individuals is similarly tion and haplogroup J is striking. Haplogroup J is
high compared with that for the MTND41 present in only about 9% of the general European
LHON11778A and MTND11LHON3460A mutations population, but it was found in 37% of LHON patients
(Johns et al., 1993; Harding et al., 1995; Riordan-Eva harboring the MTND41LHON11778A mutation and
et al., 1995). 80% of LHON patients harboring the MTND61
 D.C. Wallace et al. / Gene 238 (1999) 211–230 227

Fig. 7. Phylogenetic tree of Caucasian mtDNAs from 47 LHON patients (L1–L47, bold ) and 175 Caucasian controls obtained from the United
States and Canada. Haplotypes were determined by RFLP analysis using the entire mtDNA. For the patients, all mtDNA haplotypes are shown
and the presence of a common primary mutation is indicated (see key). Additional mutations of interest are noted in parentheses. Multiple
occurrences of the three common primary LHON mutations are indicated by their presence in independent mtDNA lineages, and, when occurring
within the same large mtDNA lineage, by the presence of intermediate haplotypes lacking LHON mutations. The numbers in large, bold characters
on branches indicate mutations that define specific groups of mtDNAs, but they are not necessarily the mutational steps used to create the
phylogeny. The horizontal branch lengths are proportional to the number of mutational events that separate haplotypes. From Brown et al. (1995)
with permission.

LHON14484C mutation (Brown et al., 1997). Indeed, Why might haplogroup J augment the expression of
only rarely have European patients with the the MTND61LHON14489C mutation? Haplogroup
MTND61LHON14484C mutation been found to have J mtDNAs might harbor a number of important amino
a non-haplogroup J mtDNA (Biousse et al., 1997), and acid substitution polymorphisms that augment the
in only one case has the MTND61LHON14484C muta- expression of the MTND61LHON14484C mutation.
tion been reported to be associated with the African The two variants that define the main haplogroup
haplogroup L mtDNA (Torroni et al., 1996b). J lineage are MTND51LHON13708A and MTND11
228 D.C. Wallace et al. / Gene 238 (1999) 211–230

LHON4216C (Johns et al., 1992a, 1993; Obermaier- ulation? One possibility is that it was selected during
Kusser et al., 1994; Oostra et al., 1994; Brown et al., the ice ages.
1995; Howell et al., 1995; Riordan-Eva et al., 1995). It has been proposed that a major selective pressure
The MTND51LHON13708A mutation converts a mod- that acted on early European populations was the
erately conserved alanine at codon 458 in the ND5 repeated periods of severe cold, associated with the
protein to a threonine (Johns and Neufeld, 1991; Brown advance and retreat of the glaciers. In this context,
et al., 1992a), whereas the MTND11LHON4216C muta- mitochondrial OXPHOS has two major functions: pro-
tion converts a weakly conserved tyrosine at codon 304 viding energy for the cell in the form of ATP and
in the ND1 protein to histidine (Johns and Neufeld, generating heat of thermal regulation. However, the
1991; Brown et al., 1992a). The sequencing of multiple generation of heat is accomplished at the expense of
haplogroup J mtDNAs has failed to reveal any other ATP-generating capacity, since both processes compete
obvious sequence variants that could account for this for the potential energy stored in the electrochemical
mtDNA lineage’s predilection toward increased expres- gradient (Dy) maintained across the mitochondrial inner
sion of LHON (Brown et al., 1992b). membrane. Therefore, during the ice ages in Europe
One major sub-branch of haplogroup J is delineated there may have been strong selective advantage for
by the MTCYB1LHON15257A variant. This variant mtDNA mutations that partially uncoupled OXPHOS,
converts the highly conserved aspartate at codon 171 in biasing the use of calories toward increased heat pro-
the cytochrome b gene to an asparagine (Brown et al., duction and better thermal regulation, at the expense of
1992a), and is even more frequently associated with efficient ATP production. Although such mutations
LHON. Hence the MTCYB1LHON15257A mutation would be relatively neutral in conditions of caloric
might also contribute to the mitochondrial defect excess, and hence be maintained in the population at
(Fig. 7, Table 1). A second sub-branch of haplogroup polymorphic frequencies, they would have an increased
J is defined by the MTND11LHON3394C variant. This tendency for disease expression when combined with a
changes the highly conserved tyrosine at codon 30 in very mild pathogenic mutation such as MTND61
the ND1 protein to histidine. The MTND11 LHON14484C. Hence, the presence of this mtDNA
LHON3394C variant is prevalent in French Canadians, would increase the expressivity of subsequent pathogenic
but is also unlikely to contribute to the disease (Fig. 7, mtDNA mutations.
Table 1). If this analysis is correct, then the population distribu-
All haplogroup J mtDNAs harbor the MTND11 tions of the various population-specific mtDNA lineages
LHON4216C variant along with the MTND51LHON might not simply reflect genetic drift. Further, selection
13708A variant. However, MTND11LHON4216C can at various times in human history might have acted to
also be associated with MTND21LHON4917G. The increase the prevalence of certain lineages over others.
MTND21LHON4917G variant changes a highly con-
served aspartate at codon 150 in the ND2 protein to an
asparagine (Brown et al., 1995). The sharing of the
MTND11LHON4216C between these two lineages
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