Hum Genet (2006) 119: 241–254

DOI 10.1007/s00439-005-0123-8

O RI GI N AL IN V ES T IG A T IO N

Joanna L. Elson Æ Corinna Herrnstadt

Gwen Preston Æ Leon Thal Æ Christopher M. Morris
J. A. Edwardson Æ M. Flint Beal
Douglass M. Turnbull Æ Neil Howell

Does the mitochondrial genome play a role in the etiology

of Alzheimer’s disease?

Received: 4 October 2005 / Accepted: 12 December 2005 / Published online: 12 January 2006

Springer-Verlag 2006

Abstract 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
Electronic Supplementary Material Supplementary material is
available for this article at http://dx.doi.org/10.1007/s00439-005-
0123-8 and is accessible for authorized users.
J. L. Elson Æ D. M. Turnbull
Mitochondrial Research Group, School of Neurology,
Neurobiology, and Psychiatry,
The University of Newcastle upon Tyne,
Newcastle upon Tyne, United Kingdom
C. Herrnstadt Æ G. Preston
MitoKor Inc. (now MIGENIX Corp.), San Diego, CA, USA
L. Thal
Department of Neurosciences, University of California San Diego,
San Diego, CA, USA
C. M. Morris Æ J. A. Edwardson
Institute for the Health of the Elderly, Newcastle General Hospital,
Newcastle upon Tyne, United Kingdom
spurious effects due to local population anomalies.
Overall, there were no significant haplogroup associa-
tions 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 pa-
tients carry a pathogenic mtDNA mutation and a small
proportion of cognitively normal aged individuals carry
a mtDNA mutation that reduces the risk of AD.

M. F. Beal Introduction
Department of Neurology, Cornell University Medical College,
New York, NY, USA Alzheimer’s disease [AD (MIM 104300)] is the most
D. M. Turnbull
prevalent late-onset neurodegenerative disorder with an
MRC/University of Newcastle upon Tyne Development Centre estimated 3–4 million affected individuals in the US
for Clinical Brain Ageing, Newcastle upon Tyne, United Kingdom (Mayeux 2003). AD is a major public health problem,
and a special concern in view of the increasing age of
N. Howell (&) Western populations as the incidence of AD rises rapidly
MIGENIX Corp., Suite 210, 12780 High Bluff Drive,
San Diego, CA 92130, USA over the age of 60. Although there are a small number of
E-mail: NHowell@migenix.com families with autosomal dominant AD, more than 90%
Tel.: +1-858-5095616 of the cases are classified as sporadic in origin. The eti-
Fax: +1-858-7937805 ology of sporadic AD is complex and a number of risk
N. Howell modifiers— genetic, behavioral, and environmen-
Department of Radiation Oncology, tal—appear to play a role in the disease process (re-
The University of Texas Medical Branch, Galveston, TX, USA viewed in Mayeux 2003). The neuropathology of AD is
242
also complex with abnormalities in many neuronal
functions. In addition to the characteristic late-stage
amyloid plaques and neurofibrillary tangles, there is
considerable evidence for abnormal mitochondrial
function and oxidative stress in AD patients (for
example, Hirai et al. 2001 and Smith et al. 2002).
Parker et al. (1990) reported that platelet fractions
from AD patients had lower levels of cytochrome oxi-
dase activity (COX), complex IV of the mitochondrial
electron transfer chain, than did those from aged-mat-
ched controls. Several groups of investigators have
subsequently reported decreased mitochondrial COX in
multiple tissues from AD patients, including autopsied
brain samples (Kish et al. 1992, 1999; Mutisya et al.
1994; Parker et al. 1994; Chagnon et al. 1995; Maurer
et al. 2000; Bosetti et al. 2002; Cardoso et al. 2004).
Additional support for a COX defect came from the
histochemical analyses of Simonian and Hyman (1993,
1994). Cottrell et al. (2001, 2002) showed that COX-
negative neurons occurred more frequently in AD brains
than in those from normal controls although these
COX-negative neurons were not associated with amyloid
plaques, neurofibrillary tangles, or markers of apoptosis.
Similarly, Valla et al. (2001) detected a selective COX
deficiency, relative to the COX activity in motor cortex,
in the posterior cingulate cortex of AD patients. The
decrement in COX activity was correlated with disease
duration and symptom severity, but it was not corre-
lated with histopathological markers such as amyloid
plaques and neurofibrillary tangles.
The etiologic origin of the mitochondrial functional
defect in AD—and its pathogenic significance—remains
unclear (Castellani et al. 2002; Schon and Manfredi
2003; Beal 2005). Some investigators have reported that
the COX defect in patients could be cytoplasmically
transferred to normal cell lines, thereby indicating that
the defect was due to inherited mtDNA mutations
(Swerdlow et al. 1997; Ghosh et al. 1999), although Ito
et al. (1999) obtained no evidence for cybrid transmis-
sion. More recently, it has been reported that AD cybrid
lines have increased intracellular amyloid accumulation
and increased amyloid secretion (Khan et al. 2000), as
well as an increased proportion of morphologically
abnormal mitochondria (Trimmer et al. 2000). Trimmer
et al. (2004) observed that AD cybrid lines show a bio-
energetic defect that becomes more severe with passage
in culture. Despite these reports, a number of technical
issues have confounded cybrid studies (Schon et al. 1998;
Danielson et al. 2005). Furthermore, no candidate
mtDNA mutations in such AD cybrids have been
identified, a situation which is difficult to reconcile with
the reported results.
Genetic epidemiological analysis, haplogroup asso-
ciation studies, and case-control analysis of mtDNA
sequence changes have also been used to ascertain the
presence of pathogenic mtDNA mutations. Despite
these efforts, the issue remains unresolved (reviewed in
Howell et al. 2005). As part of our efforts to identify the
role of the mitochondrial genome in the etiology of AD,
we report here our population-based sequence analyses
of the mtDNA coding region. To increase the rigor of
our study, the following steps were taken: (1) a large set
of mtDNA sequences were obtained to increase signal
(>270 sequences were analyzed); (2) the AD and normal
control cohorts were subject to stringent inclusion cri-
teria; (3) results from two independent populations of
European descent were analyzed to overcome the effects
of ‘‘local’’ population sampling; and (4) efforts were
made to ensure the accuracy of the mtDNA sequences.
Subjects and methods
Subjects and DNA samples
Two independent collections of autopsied brain samples
were analyzed for this study, and these are termed the
MitoKor (MK) and Medical Research Council (MRC)
sets. The MitoKor sequence set comprises the complete
mtDNA coding regions for 75 AD patients and 64 normal
controls, whereas the MRC set contains such sequences
for 70 AD patients and 64 normal controls. The MitoKor
and MRC mtDNA sequences were obtained from indi-
viduals who lived in the US and UK, respectively, and
only mtDNAs were analyzed that belonged to one of the
major European haplogroups. It should be noted that,
since the initiation of this study, MitoKor has become
MIGENIX Corp., a subsidiary of MIGENIX Inc.
(Vancouver). To maintain clarity and continuity with
previous reports, we will continue to designate the mtD-
NAs as ‘‘MitoKor’’ sequences. The MRC and MitoKor
mtDNA coding region sequences, formatted as character
stage changes (that is, differences from the rCRS) are
available in Supplementary Table 1 attached to this
report, by an email request addressed to the corre-
sponding author, and at the University of Newcastle
website (http://www.ncl.ac.uk/nnp/staff/profile/j.l.elson).
Note that we do not include expansions/contractions
of simple repeat sequences in our analyses.
Alzheimer’s disease was confirmed in all brains from
the disease groups by standard and accepted neuro-
pathological criteria. Stringent inclusion criteria for the
normal controls were used. For the MRC samples,
controls were included only if there was an absence of
known or recorded neuropsychiatric disease including
stroke (or other major cerebrovascular disease),
dementia, depression, or psychosis. At the neuropatho-
logical level, only appropriate levels of age-related
change were allowed, including the presence of some
small-vessel disease. Finally, the MRC control brains
had to have <2 tangles/mm2 in cortical lobes (and
usually <1) and <5 senile plaques/mm2. The 64 Mito-
Kor control sequences were derived from three sample
sets: (1) 13 autopsy-confirmed brain samples of normal
histology from individuals with a mean age of 81 years;
(2) 21 blood samples from a cohort of healthy elderly
individuals (mean age = 88 years); and (3) 30 blood
samples from a cohort of individuals that were
 243

Table 1 Haplogroup distribution of AD and control mtDNAs

Haplogroup MK-CON MK-AD MRC-CON MRC-AD M+M-CON M+M-AD Total-CON Total-AD

HVa 46 (72%) 42 (56%) 22 (34%) 36 (51%) 68 (53%) 78 (54%) 120 (50%) 130 (50%)
U 4 (6%) 8 (11%) 11 (17%) 15 (21%) 15 (12%) 23 (16%) 36 (15%) 42 (16%)
K 6 (9%) 7 (9%) 9 (14%) 8 (11%) 15 (12%) 15 (10%) 27 (11%) 28 (11%)
T 3 (5%) 6 (8%) 5 (8%) 3 (4%) 8 (6%) 9 (6%) 21 (9%) 22 (8%)
J 3 (5%) 7 (9%) 13 (20%) 3 (4%) 16 (13%) 10 (7%) 28 (12%) 24 (10%)
W 0 (0%) 1 (1%) 1 (2%) 3 (4%) 1 (1%) 4 (3%) 1 (0%) 4 (2%)
I 1 (2%) 2 (3%) 2 (3%) 0 (0%) 3 (2%) 2 (1%) 4 (2%) 5 (2%)
X 1 (2%) 1 (1%) 1 (2%) 1 (1%) 2 (2%) 2 (1%) 1 (0%) 1 (0%)
Otherb 0 (0%) 1 (1%) 0 (0%) 1 (1%) 0 (0%) 2 (1%) 5 (2%) 4 (2%)
Total 64 (100%) 75 (100%) 64 (100%) 70 (100%) 128 (100%) 145 (100%) 243 (100%) 260 (100%)

M+M refers to the combined MitoKor and MRC sequence sets, whereas ‘‘Total’’ refers to the combined data of the MitoKor sequences
and the haplogroup analyses of Chinnery et al. (2000), which involved analysis of the entire MRC set of AD and control brain samples.
The number of mtDNAs for each haplogroup are shown whereas the percentages of each sequence set in that haplogroup are shown in
parentheses
MK-CON the number of MitoKor control mtDNA sequences that belong to the different haplogroups; MK-AD MitoKor AD mtDNA
sequences; MRC-CON MRC control mtDNA sequences; and MRC-AD MRC AD mtDNA sequences
a
For this table, H and V mtDNAs were identified by the presence of a polymorphism at nt 14766, and they were distinguished by the
presence of a polymorphism at nt 2706 (H), or its absence (V), relative to an L3e outgroup sequence. Pre-HV mtDNAs are also included
(Figs. 1 and 2)
b
The approach of Chinnery et al. (2000) did not specifically identify haplogroups X and V. In our analyses here, the numbers of
haplogroup V (as well as V* and preHV) mtDNAs are included in total for the HV superhaplogroup, while we count the numbers of
haplogroup X mtDNAs separately, rather than as ‘‘Other’’. As a result, the numbers in the ‘‘Total’’ columns have some inaccuracies
because of the different assignment schemes, but the anomalies are minor and they do not affect the overall conclusions. The two ‘‘Other’’
AD mtDNAs (mk68 and mrc70) were found to belong to Asian haplogroup A and were omitted from subsequent analysis

>80 years of age and cognitively normal upon evalua- would be included in our analyses, but such occurrences
tion over a period of at least 6 years (and extending up are too rare to invalidate our overall conclusions (for
to 16 years). example, Howell et al. 2003b).

DNA sequencing Statistical tests of association

Coding region mtDNA sequences were obtained and
analyzed as described previously (Herrnstadt et al.
2002). Our sequencing approach was designed to pro-
vide up to a fourfold redundancy to minimize errors,
and other quality control measures were also incorpo-
rated (Herrnstadt et al. 2003). Reduced median network
analyses of the coding region sequences were carried out
as described previously (Bandelt et al. 1995; Herrnstadt
et al. 2002). Sequence changes refer to those of the L-
strand of the Revised CRS (Andrews et al. 1999).
The analyses here are limited to the mitochondrial
coding region (nucleotide positions 577–16023). At the
time of this analysis, almost all—and perhaps all—ac-
cepted mitochondrial pathogenic single base pair
mutations have been mapped to the coding region (see
Keers et al. 2004 and references therein). Furthermore,
this study was designed to test the role of inherited
mtDNA mutations in AD and only substitutions that
were operationally homoplasmic were analyzed. That is,
our sequencing approach detected a nucleotide change
only if its allele proportion was >50%. We made no
efforts to detect low-level heteroplasmic substitutions
because these could represent somatic mutations (see
Discussion), and because the pathogenicity of low-level
heteroplasmic remains in dispute. Somatic mutations
that have segregated to an allele proportion >50%
Unless otherwise noted, the statistical significances of
differences between AD and control groups were deter-
mined with the chi-square (v2) test. This approach was
chosen because it is a non-parametric test for bivariate
tabular analyses (in this case, AD and control groups)
that makes fewer assumptions about the data than
equivalent parametric tests.
Results
Testing the role of mtDNA mutations in AD
On the basis of previous analyses of diseases known to
have, or suspected of having, a mitochondrial genetic
component, there are three patterns of results that could
be obtained if inherited mtDNA mutations play a sig-
nificant role in the etiology of AD:
1. It has been observed in several instances that a dis-
order or condition is significantly associated with one
mtDNA haplogroup (Herrnstadt and Howell 2004), a
result that suggests one or more haplogroup-associ-
ated polymorphisms modify risk.
2. A specific substitution occurs at a significant fre-
quency in either the patient cohort (pathogenic
244

mutation) or in the control cohort (protective muta- is that there might be a mitochondrial genetic contri-
tion). This is the most straightforward scenario for a bution to cognition in the very elderly, a concern that
mitochondrial genetic etiology. However, this situa- was heightened by the recent report of Roubertoux et al.
tion becomes increasingly complex if there are large (2003). With this possibility in mind, our subsequent
numbers of rare mtDNA mutations in the popula- analyses were designed to avoid bias from differences in
tion, any one of which increases the risk of develop- haplogroup distributions.
ing AD. This is a very difficult hypothesis to test, in
large part because the high rate of mtDNA evolution
makes it difficult to identify rare pathogenic muta- Reduced median network analysis
tions.
3. The third possibility is that, in an AD patient, the Reduced median networks were constructed with con-
cumulative effects of multiple phenotypically subtle trol and AD coding region sequence sets to obtain a
mtDNAs mutations are risk factors. To test this eti- finer-grained analysis of the non-synonymous substitu-
ology entails determining whether there is an in- tions in these mtDNAs (Figs. 1, 2). Silent polymor-
creased ‘‘mtDNA mutation load’’ in AD patients, phisms are omitted from these networks, because of our
which we do here in multiple ways. In this scenario, focus on potentially pathogenic mutations. The net-
the increased mutation load might occur throughout works are similar with the exception that the AD net-
the mitochondrial genome, only in a single gene, or work (Fig. 2) has a greater number of pre-HV and pre-V
within a functionally related set of genes (for exam- sequences than does the control network (Fig. 1). The
ple, the seven genes that encode complex I subunits). control set contains one HV sequence (mrc104) and two
V*/V sequences (mk132 and mk91, respectively). In
contrast, the AD sequence set contains two pre-HV
sequences (mrc11 and mrc12) and nine HV/pre-V/V
mtDNA haplogroup distributions
sequences (mrc13-mrc20 and mk66). Ten of these 11 AD
sequences were present in the MRC sequence set,
The haplogroup distributions of MitoKor and MRC
but only one in the MitoKor set. If the relatively high
control and AD mtDNAs are shown in Table 1. In the
frequency of pre-HV and HV sequences was due to an
MitoKor sequences, there is no statistically significant
association with AD, then such sequences should have
association between haplogroup and disease status, al-
been observed among both the MRC and MitoKor AD
though there was a relatively high frequency of haplo-
sequences. On the other hand, if these sequences repre-
group H mtDNAs in the control group. Among the
sented some specific feature of the population of the
MRC sequences, there was a significant excess (P0.04)
northeast of England from which the MRC samples
of haplogroup J mtDNAs in the control set of sequences
were drawn (and, thus, were not disease-related), then
relative to the mtDNAs from AD patients. However,
such sequences should have been observed in both the
this is a spurious result, because we analyzed only a
MRC AD and control sequences. There is thus no
portion of the entire MRC sample collection. In a pre-
consistent explanation for these results, but the differ-
vious study, there were no significant haplogroup asso-
ence in distribution between AD sequence sets was not
ciations when the entire set of MRC samples was
statistically significant (using a chi-squared test) and it
assayed (Chinnery et al. 2000). Furthermore, when the
might simply represent the vagaries of sampling.
MitoKor and MRC sequence sets are pooled, none of
the haplogroups show any association with AD. Finally,
we compared the haplogroup distributions from the
Analyses of mtDNA mutation load
combined MitoKor sequences and Chinnery et al. (2000)
results. No associations were observed, either for single
The networks of mtDNA coding region sequences from
haplogroups or for superhaplogroups.
AD and control sets, with the exception of the pre-HV/
In our previous study (Herrnstadt et al. 2002), ha-
HV sequences (see above), do not show any AD-specific
plogroup H accounted for 52% of the European mtD-
branches or phylogenetic clusters. That is, there is no
NAs carried by individuals in this sample of the US
evidence for an ‘‘old’’—in the phylogenetic
population. However, almost 70% of the mtDNAs in
sense—pathogenic mtDNA mutation, or set of muta-
the MitoKor cohort of normal controls belonged to
tions, that accounts for a subset of AD cases. To assess
haplogroup H (Table 1). While there was no haplogroup
whether multiple slightly deleterious mutations act as
H association in the combined datasets, a potential
AD risk factors, we then used the networks to determine
complication had arisen. The mean age (and standard
if the mutation load was increased in AD sequences.
deviation) of the MRC control group was 77.2 years
Both synonymous and non-synonymous substitutions
(9.6 years), whereas the mean age of the MitoKor con-
were grouped according to respiratory chain function:
trol group was 83.4 years (5.0 years). A two-tailed t-test
(a) the seven mtDNA genes that encode complex I su-
(without an assumption of equal standard deviations)
bunits (ND); (b) the mitochondrial cytochrome b gene,
indicates that the MitoKor control group is significantly
which is the principal catalytic subunit of complex III
older than the MRC controls. The upshot of this finding
(CYB); (c) the three cytochrome oxidase, or complex IV,
 245

mk76
mk77
8705 I
2868 x 10034 G
8557
mrc71 8393
13966 10389 13780 8616 mrc72
13708
15924 T
15927 T 15758
mrc115 mrc114 1719
W mrc73
5824 C
4908 709 3505
9300 5319 mrc74
8348 K 1243 5046
12346 1406 5460
mrc75
mrc134
mrc116 7245 mrc76
2757 12705
mrc117 15924 T
3212 1850 mrc85
mk78
4129
mrc128 1700 mrc129
mrc127 14793 8557 4674
709 1721
4732 15218 J2 13879
mrc118 9531 13637 2158
10031 G 7476 S 5460 mrc119
mk130 15257
U5
3197
9477
J1 13934
J 3010
1811 14798
12308 L 10398
U9 2294 7772
13708 mrc77 15947 T mrc84
3010 mrc130 11204
10506 4216
mrc111 15452 3394
mrc113
mrc78 13681
15693 U4 mrc79
9055 12311 L
mrc112 12135 709 10463 K 12083 mk79
11778 K 14798 U8 13145
mk131
3721 15119
mk115 T 1888 15928 T
4917 8734
mrc81
K1 mrc105 12937 mrc83
mrc82
14002
1189 13528 mk116
mrc133 10398 K2 13565
mrc82 709 961 745
T2
2217 mk117
mk139 4561 3204 12030 mk80
14053 13135 mk128 930 mrc131
5913 15650 mk111 5277 mrc120
mrc126
mrc106 mk110 6489
mk114 4295 I mrc110 mk129
3394 1735 2098
10750
9752
mrc86
mk113 mk112
mrc109 7468 S
mrc108 10680
14110 15928 T 13651

HV mrc87 mrc88
mrc107 mk91
mrc91

9391
pre v mrc90
mk108 mrc104
14199 3105 9210
mrc101 3472 13105 T
15904 mk83 5298
mrc92 15928 T
14766 10365
mk107 4454 M 14180
2851 mk132
7853 15758 mk85
9055 mrc121 8764
mrc102 mrc103 10026 G
870 mk92 4384 Q
mrc89 6237
mk106 mk109 2857 13708 9300 5460 8839 mk84
721 4561 9073
2706 mk93 15789
mk137 7444
6367
3027 2626 mk81 7922
H5 8417
5319 mk120 mrc123
mk105 8563 4336 Q
mk94 3010 mk119 mk118 7270
15773 mk82
mk138 mrc124 mk135
14871 mk134 mk126 8308 K mrc122 mk121
mrc100 mrc80 13105 mk90
10750
1007 14405 5788 C
mk123 mk122 mk136
mrc99 mk125 mk124 mrc93
1438 mk89 2098 15930 T
mk104 H2 3796
8602 11204
10845
mk88 15153
11253 3992 mk87

mk103 4024 3866 12811 mrc95

4216 951 mk86
10044 G mrc94
mk127 14582 750 mk95 H1
2243 1462
mrc98 mrc96
5785 C mk100 mk99
mrc125 mrc132 mk133
mk98

980 2284 3992 3388

6339 2708 4418 M
14970 13708 8950 8860 mk96
H4 H3 15326

mrc97
mk102 mk101 mk97

Fig. 1 Reduced median network of coding region sequences from color-coded according to the genes affected: ND (green); cyto-
the normal controls. Substitutions that affect tRNA gene sequences chrome oxidase (dark blue); ATPase (red); cytochrome b (cyan);
are distinguished with a one-letter superscript to identify the amino tRNA (dark pink italic font); and rRNA (orange italic font).
acid normally acylated. The sequence changes in the networks are Network-specific mutations are underlined
246

x I
8393 10398 15758 3398 mk2
mrc1 mk3 13966
15927T 13780 15924T 10034G

1719 W 10005 G mrc6 8508

14178 669
709 5046 2702
mk52 mk53
U6 1243 5460
3505 4363Q mk8 mk1
mk54 mk4 mrc5
mrc54 6498 10410 R
mrc53 mrc55 mk65 mrc57
14769 12705 8659 15947T
mrc56 2833 8887 mrc4 mk5
3397
3745 10398 Mk6
mk51 1700 9667 14684E mk35 13934 mk9
896 15924T 960del
mrc52 2218 1809
15218 3159ins 5554W 3394
723 789
4732 7581D 7805
961ins 4084 14798 mrc2
8896 15380 8430
J1
3221 5836Y 5773c
mrc51 9300 1721 14793
12634 mk7 mrc3
13889 13637
13630 3010 1007
5418
mrc50 5076 mk64 mk11 10463 R
U5 10398
3197 J 1850
13708 mk10
mk69 15734 9477 7476S
U9 J2 15257
mrc7
10506
mrc66 1811 12308 L 709 T
13934 4216 T2 6480 mk14
15452 1888
7521D mk71
mrc49 4917 930
14258 5686N mrc9
9055 15693 U4 10463 R 10750 mrc10
12557 mk12
K 14798 15928T
mrc65 11204
mrc62 15937T
K2 mrc8
2217 mrc61
mrc48 7853
13135
mrc47 15803
mk62
709 K1 mk63 13528 2355 5277
6261
mrc64 1189 mrc11
4561 15674 6489
10398 5913 2442 mk13
1832 mrc12
12173H
mk49 6367 mk67 6480 mrc46 mk15
mk16
13565 15326
4295I
14002 15924T mrc43 mk46
mrc13 mrc14
mrc63 13943 mrc15
mrc31 mk61 14766 HV 4123 15930T 606F mrc16
5846Y 15924 T
5460
mk48 593F mrc20 16298*
2067 mk66 15904T mrc17 V
mrc45 2483
2905
mk47 3397 4312I mrc18
mrc44 mrc19 11337
mk18
mrc37 mrc25 mrc21 mrc26
2706 H mk19
mrc38
H2 mrc41 15152
mk43 870
H3 7521D
mrc40 mrc23 4763
mrc24 10007 G 15326
12236 S 593F
14798 12217 s
4812
8557
15024 mk72
mrc67 2352
H1
14687 E 15236
5516 W
mk45
mrc22 8764 5460 9804
593F 750 3796 9861 mk20
mrc39
2259 1508
mrc42 951 mk17 6022
mk42 1438 mk58 6480
10448 R 7830
5773C mrc29
mrc69 mk33 mrc30
13889 3992 mk55 mrc58
H4 mk34 mk70
4024 mk59 mk57 8839
3010 mk73 mk74 mk21
709 14582 mrc59 mk36 15789
mk40 10044G mk50 mrc60 mk60 mk75
mk41 6261
10192 14978
mrc36
5634A mk56 mrc28 15153 8602 mk22
827 5979 15924T
709 961
mk39 11253 mrc27
13711 8448 mk23
4336Q
1393 13759 mk24
9007 1719 8255 H5 mk28 mk25
mk26
8752 mk27
9202 mrc68
mrc35 mrc32
5460 3421
827 mk44
mrc33 mk37 mk29
5785C mk32 2851
mk38 9948 9055

mrc34 mk30
mk31

Fig. 2 Reduced median network of coding region sequences from the AD patients. The substitutions are marked as in Fig. 1
 247

genes (COX); and (d) the two genes that encode subunits
of the ATPase, or complex V (ATP). In addition, we
include mutations in the two rRNA and in the 22 tRNA
genes with the non-synonymous substitutions, because a
number of pathogenic mutations in these genes have
been identified (this aggregate is referred to here as
‘‘non-silent’’).
When all substitutions within each network are con-
sidered (Table 2), there is no significant difference in the
distributions (P=0.18 using a 2· 6 v2 test). While there
are some differences within any one particular class (see
further analysis below), the cumulative effect is non-sig-
nificant. The similarity between sequence sets is also seen
when one calculates the average number of substitu-
tions/mtDNA. The average values for both synonymous
and non-silent changes are very similar (less than 10%
difference) in the AD and control sequence sets.
The next step in the network analyses was to separate
substitutions into ‘‘interior’’ and ‘‘tip’’. Interior substi-
tutions are more likely to show the effects of selection
because they are older—on average—than tip substitu-
tions (Elson et al. 2004). While there is no obvious reason
why mtDNA mutations that cause a late-onset disorder
such as AD should be subject to selection, there is no
need to introduce a bias at this stage of the analysis. The
distributions of interior substitutions were similar
between the AD and control mtDNA sequences, and we
observed only a slight increase in the average number
of synonymous substitutions in the AD sequences
(Table 2).
When we analyzed tip substitutions, we observed
that, for both synonymous and non-silent changes, the
overall numbers of substitutions/mtDNA were the same
for the AD and control sequences (Table 2). However,
the distributions of non-silent substitutions among the
sets of functionally related genes were significantly
different (P0.03 using a 2· 6 v2 test). In the first place,
the AD sequences contain a larger number of tRNA
substitutions at the tips of the network. When consid-
ering all substitutions within the tRNA genes, there was
an ‘‘excess’’ of 18 such changes in the AD sequences, all
of which occur at tip positions (Table 2). Furthermore,
we note that six of the tRNA substitutions in the AD
sequences occurred in the HV and pre-HV sequences
(Fig. 2). In other words, some of the ‘‘excess’’ in tRNA
mutations, but not all of it, is due to the over-repre-
sentation of these haplogroups among the AD sequences
obtained from the MRC brain samples (see the further
analyses below).
The statistically significant difference between AD
and control sequence sets is also caused by an excess
of non-synonymous tip substitutions in the ND genes
of controls. Thus, there are 21 more tip substitutions
in the ND genes of control sequences, whereas there
are an additional five interior branch substitutions in
the AD sequences. The latter result is in accord with

Table 2 Network, tip, and interior substitutions in control and AD mtDNAs

Gene class Subs./mtDNA

tRNA rRNA COX ATP ND CYB

I. Network substitutions
CON–SYNa – – 61 13 115 23 1.66
CON–NS 27 46 18 16 73 18 1.51
AD–SYN – – 72 20 144 28 1.82
AD–NS 46 47 21 16 57 23 1.41
II. Interior substitutions
CON–SYN – – 9 6 26 5 0.35
CON–NS 9 14 1 1 23 8 0.38
AD–SYN – – 19 3 34 8 0.44
AD–NS 9b 14 2 1 28 7 0.38
III. Tip substitutions
CON–SYN – – 52 7 90 18 1.30
CON–NS 19 32 17 15 50 10 1.13
AD–SYN – – 53 17 110 20 1.38
AD–NS 37c 33 19 15 29 16 1.03
a
SYN and NS refer to synonymous and non-silent/non-synonymous substitutions, respectively. Note that all substitutions in the tRNA
and rRNA genes are considered to be non-silent for this analysis and, as a result, there are no data for synonymous changes in these genes
b
One of the branches of haplogroup U5 carries an interior tRNA mutation at nucleotide 15924 (Fig. 1). However, the same branch in the
network of AD mtDNA sequences (Fig. 2) has the 15924 site change in a tip position. For the purposes of the present analysis, we classify
this substitution as an interior change in both networks. The same situation (that is, a tip location in one network but an interior location
in the other) occurs at other shared sites for the two networks: 1700 (haplogroup U5), 3394 (haplogroup J), 10044 (haplogroup H4), and
13528 (haplogroup U4). For all of these sequence changes, they have been classified here as interior. The sequence change at site 8764 (on a
branch of haplogroup H1) is an unusual situation. In both networks, the change occurs in a tip position. However, because of the
additional presence of a site change at 10026 in the mk83 sequence (control), site 8764 would be located at an interior position in a
combined AD + CONTROL network. Therefore, we have classified the 8764 substitution as ‘‘interior’’ for the present analysis, although
classifying it as ‘‘tip’’ would not change the overall results
c
Haplogroup T sequences characteristically carry a forward mutation at nucleotide 15928 of the tRNAthr gene. However, AD sequence
mrc87 has undergone a reverse mutation at this site in a tip position, thus explaining why a branch of the haplogroup T network displays
the same mutation twice (Fig. 2)
248
the greater number of AD versus control sequences
(145 and 128, respectively). That is, on a ‘‘per
mtDNA’’ basis, the excess of tip ND mutations in
mtDNAs from controls is even more pronounced than
shown by the total counts used in Table 2. We are
unable to detect, upon inspection of the networks, any
apparent mutation ‘‘hot spots’’, either with regard to
the particular ND gene or to the various haplogroups
and subclades.
The finding that there were excess tRNA mutations in
the AD sequences, and excess ND mutations in the
control sequences, prompted us to follow up these
analyses with a specific determination of whether there
was a subset of sequences (AD or control) with ‘‘clus-
tered’’ mutations. Therefore, we determined the number
of non-silent tip mutations in each mtDNA. The results
(Table 3) show that the AD and control mtDNAs show
very similar distributions of mutations, and there is thus
no evidence for hypermutated mitochondrial genomes in
either of the two sequence sets.
Analyses of mutation load adjusted for Haplogroup
distribution
The analyses in Table 2 did not factor in differences in
haplogroup frequencies among the AD and control
coding region sequence sets. The major European ha-
plogroups have significantly different extents of se-
quence divergence, a phenomenon most likely indicating
differing evolutionary ages (for example, Richards et al.
2000). As a result, even slight differences in haplogroup
distribution might affect the apparent mutation loads
between the two sequence sets (especially as regards the
differences in mutations in the tRNA and ND genes).
One way to ‘‘filter out’’ the effects of haplogroup
distribution is to analyze mutation loads in individual
haplogroups or in superhaplogroups, the latter com-
prising two or more closely related haplogroups. Be-
cause the frequency of single haplogroups, with the
exception of H, is relatively low, we analyzed the HV,
TJ, and UK superhaplogroups to maintain sufficient
group sizes for valid v2-square statistical tests. An
additional advantage of this approach is that it should
indicate if the relative excess of haplogroup H mtDNAs
in the MitoKor sequences from controls is having a
significant effect on the results. The results are summa-
rized in Table 4. There were no significant differences in
Table 3 Distribution of non-silent tip substitutions/mtDNA
Subs./genome AD Control
0 52 (36%) 43 (34%)
1 51 (35%) 50 (39%)
2 27 (19%) 20 (16%)
3 8 (5%) 9 (7%)
4 6 (4%) 4 (3%)
5 1 (1%) 1 (1%)
6 0 (0%) 1 (1%)
the distributions of synonymous and non-synonymous
substitutions in any of the three superhaplogroups.
These analyses considered all substitutions in the se-
quences. In addition, we also carried out tests of sub-
stitutions that were unique in each network, and these
distributions are also not significantly different.
Use of mtDNA networks to identify candidate
pathogenic mutations
It is difficult to identify rare mtDNA mutations that are
pathogenic, because of the high rate of mtDNA muta-
tion. As one identification approach, we reasoned that
rare coding region substitutions which arise indepen-
dently two or more times and which are disease associ-
ated are plausible candidates for pathogenic mutations.
Our mtDNA networks (Figs. 1 and 2) were used to
identify such substitutions, which we term ‘‘homopla-
sic private’’ mutations, in both the control and AD
sequence sets.
The first step was to determine the number of ho-
moplasic synonymous substitutions, and we observed six
such polymorphisms in each sequence set. The nucleo-
tide positions of these substitutions are as follows:
1. AD: 3591, 4991, 6293, 8485, 10978, 11152
2. Control: 3447, 3531, 8020, 10915, 12966, 14305
For example, the substitution at nucleotide 4991 arose
independently in two AD patients, one of whom carried a
haplogroup H mtDNA and another who carried a
haplogroup K mtDNA. As expected for silent poly-
morphisms (that is, that are clinically benign), the fre-
quencies of homoplasic silent substitutions were
essentially identical between the AD and control se-
quence sets. These results further underscore the high
rate of homoplasy in the human mitochondrial genome,
even in the coding region (see also Herrnstadt et al. 2002;
Moilanen and Majamaa 2003).
The results for homoplasic non-synonymous muta-
tions are shown in Table 5. Whereas there were three
such substitutions among the normal controls, there
were six examples in the AD mtDNAs (the difference is
not statistically significant), one of which (at nt 6480)
arose on three occasions. These six mutations in the AD
mtDNA sequences are spread throughout the mito-
chondrial genome and there is no functional clustering
beyond the fact that two of them map to the CO1 sub-
unit of cytochrome oxidase.
Assessing pathogenicity of mutations in tRNA
and ND Genes
In an effort to improve the identification of tRNA
mutations that are associated with mitochondrial dis-
orders, we have developed a weighted scoring system
(see McFarland et al. 2004a for details) that involves
multiple criteria including evolutionary conservation,

Table 4 Distribution of tip substitutions among superhaplo-
groups
Sequences Non-silent Synonymous Pa
I AD–HV 80 96 0.26
Control–HV 73 67
II AD–UK 48 76 1.00
Control–UK 37 60
III AD–TJ 18 36 0.25
Control–TJ 25 31
a
To determine if the distribution of numbers of substitutions
differed between AD and control sequence sets, Fisher’s exact
test was used
results from functional studies, and pathogenicity crite-
ria proposed by earlier studies (DiMauro and Schon
2001). The score ranges from 0 to 20 and is assigned to
the following groups: (a) neutral variants (a score of 6
points or less), (b) possibly pathogenic (7–10 points); (c)
probably pathogenic (11–13 points), and (d) definitely
pathogenic (14–20 points). Unfortunately, for most of
the tRNA mutations that occur in the control and AD
sequences analyzed here (Table 2), there is insufficient
information and only a small number could be analyzed
with this approach:
1. Five tRNA mutations were observed in both AD and
control sequences: 4295/tRNAile, 4336/tRNAglu,
10044/tRNAgly, 12308/tRNAleu, and 15924/tRNAthr.
The pathogenicity scores are 8, 6, 6, 2, and 0 points,
respectively. Although the nt 4295 mutation falls into
the ‘‘possibly pathogenic’’ class, the available evi-
dence indicates that these are benign polymorphisms.
Some studies have posited an association of the 4336
substitution with AD but we believe that the cumu-
lative evidence argues against such an association
(Howell et al. 2005).
2. Mutations 8348/tRNAlys and 12311/tRNAleu oc-
curred only in the sequences from controls. The
pathogenicity scores were 4 and 6 points, respectively.
Neither of these mutations can have a pathogenic role
in AD.
3. The 606/tRNAphe mutation occurred only in the AD
sequence set. The tRNA mutation at nt 606 was ini-
tially thought to be the primary pathogenic factor in
a case of acute rhabdomyolysis (Chinnery et al.
1997). Follow-up studies, however, have indicated
that this mutation is not pathogenic (McFarland et al.
2004b).
If one subtracts away the tRNA mutations that
occur in both the control and AD mtDNAs, there are a
net total of 27 tRNA mutational events specific to the
AD group; 19 patients carried one such mutation and 4
patients carried two mutations each. Six of these 27
mutations were also identified in our set of European
mtDNAs (Herrnstadt et al. 2002) and/or in the set of
Finnish mtDNAs (Vilmi et al. 2005), but—at this
stage—we cannot exclude their pathogenicity (for
249
Table 5 Homoplasic non-silent mutations in AD and control
mtDNAs
ADa Controls
827 A:G/12S rRNA 2098 G:A/16S rRNA (H1, K2)
(H1, H6)
3397 A:G / ND1/M31V 5319 A:G/ND2/T284A (H5, U5)
(Pre-V, U5)
6261 G:A / CO1/A120T 13105 A:G/ND5/I257V (H, V)
(H6, T2)
6480 G:A / CO1/V193I
(H1, T2, K1)
7521 G:A/tRNAasp
(H1, U9)
13889 G:A/ND5/C518Y
(H4, U)
a
The results are presented as nucleotide position, nucleotide change
on the L-strand/gene/amino acid at that position in the gene with
the first letter denoting the predicted amino acid in rCRS and the
second letter denoting the predicted amino acid that results from
the substitution. In parentheses, we show the haplogroups in which
the homoplasic substitutions have arisen. For example, the ho-
moplasic substitution at nt 827 arose independently in a haplo-
group H1 mtDNA from an AD patient and in a haplogroup H6
mtDNA. All of these homoplasic substitutions show two inde-
pendent origins, with the exception of the substitution at nt 6480,
which arose on three occasions within this set of AD mtDNAs
example, those individuals might develop AD at some
point in the future). Three of these 28 mutations arose
independently on two (5773/tRNAcys and 7521/
tRNAglu) or three (593/tRNAphe) occasions. These
tRNA mutations are distributed among 15 of the 22
mitochondrial tRNA genes, but there are insufficient
data to determine if there are AD mutational hot spots
in the tRNA genes that differ from those in the general
population (Herrnstadt et al. 2002; Vilmi et al. 2005).
We also assessed the potential pathogenicity of
replacement substitutions in mtDNAs from AD patients
and from controls by scoring the predicted functional
effects of the amino acid substitutions with three dif-
ferent systems that were specifically constructed to assess
the functional consequences of amino acid replacements
in transmembrane proteins. We included only network-
specific non-synonymous mutations in this analysis. On
average, the substitutions in the AD mtDNAs were no
more deleterious than those in the control sequences. Of
particular note, the substitutions that arose multiple
times in the mtDNAs of AD patients (Table 5) do not
appear deleterious, and they are thus probably clinically
benign. One of the control sequences carried a mutation
at nt 7444 that causes a LYS for the chain termination
codon. We have not estimated the functional conse-
quences of this mutation, but it has been reported to be
mildly deleterious (Brown et al. 1992).
There is one noteworthy result to emerge from
Table 6. There were two AD mtDNAs, but no control
mtDNAs, that carried both a replacement substitution
with a high penalty score and a rare mutation in a tRNA
or rRNA gene:
250

Table 6 Mutation index analysis of network-specific replacement Table 6 (Contd.)

substitutions
Sitea Nucleotide AA Gounntb PHAT Persson
Sitea Nucleotide AA Gounntb PHAT Persson change change
change change
6339 A:G T-A 0.6 0 1
I. AD sequences 7245 A:G T-A 0.6 0 1
3397 (2) A:G M-V 1.6 1 1 7270 T:C V-A 0.1 1 1
3398 T:C M-T 0.6 0 0 7444 G:A Term-K
3421 A:G V-M 1.6 1 1 7772 A:G T-A 0.6 0 1
3745 G:A A-T 0.6 0 1 7922 T:C Y-H 2.2 3 3
4084 G:A V-I 3.1 3 2 8417 C:A L-I 2.8 2 1
4123 A:G I-V 3.1 3 2 8563 A:G T-A 0.6 0 1
4763 C:A I-M 2.5 3 2 8616 G:T L-F 2 1 0
4812 G:C V-L 1.8 1 1 8705 T:C M-T 0.6 0 0
5076 C:T L-F 2 1 0 8950 G:A V-I 3.1 3 2
5418 T:G F-V 0.1 1 2 9073 A:G T-A 0.6 0 1
5979 G:A A-T 0.6 0 1 9210 A:G T-A 0.6 0 1
6022 A:G E-G 0.8 3 2 9391 C:T T-M 0.6 0 0
6261 (2) G:A A-T 0.6 0 1 9531 A:G T-A 0.6 0 1
6480 (3) G:A V-I 3.1 3 2 9752 C:A F-L 2 1 0
6489 C:G L-V 1.8 1 1 10365 G:A A-T 0.6 0 1
7805 G:A V-I 3.1 3 2 10680 G:A A-T 0.6 0 1
7830 G:A R-H 0.6 -4 2 10845 C:T T-I 0.6 1 1
8255 G:A V-M 1.6 1 1 11778c G:A R-H 0.6 -4 2
8430 T:C L-P 2.3 5 5 12030 A:G N-S 0.9 1 2
8448 T:C M-T 0.6 0 0 12083 T:G S-A 1.1 2 2
8508 A:G N-S 0.9 1 2 12135 C:A S-Y 1.9 2 3
8752 A:G I-V 3.1 3 2 12346 C:T H-Y 2.2 3 3
8896 G:A A-T 0.6 0 1 12811 T:C Y-H 2.2 3 3
9007 A:G T-A 0.6 0 1 13105 (2) A:G I-V 3.1 3 2
9202 A:G T-A 0.6 0 1 13145 G:A S-N 0.9 1 2
9667 A:G N-S 0.9 1 2 13651 A:G T-A 0.6 0 1
9804 G:A A-T 0.6 0 1 13681 A:G T-A 0.6 0 1
9861 T:C F-L 2.0 1 0 13879 (2) T:C S-P 0.4 -3 2
9948 G:A V-I 3.1 3 2 14053 A:G T-A 0.6 0 1
10192 C:T S-F 2.8 2 2 14110 T:C F-L 2 1 0
11337 A:G N-S 0.9 1 2 14180 T:C Y-C 0.5 1 2
12557 C:T T-I 0.6 1 1 14199 T:C T-A 0.6 0 1
13711 G:A A-T 0.6 0 1 14405 AG V-A 0.1 1 1
13759 G:A A-T 0.6 0 1 14871 T:C I-T 0.6 1 1
13889 (2) G:A C-Y 0.5 1 2 14970 A:G Y-C 0.5 1 2
13943 CT T-M 0.6 0 0 15650 G:A A-T 0.6 0 1
14178 T:C I-V 3.1 3 2 15773 G:A V-M 1.6 1 1
14258 G:A P-L 2.3 5 5 Average 0.9 0.4 0.7
14769 A:G N-S 0.9 1 2
a
15024 G:A C-Y 0.5 1 2 Sites of substitution are designated by their nucleotide position.
15152 G:A G-S 0.4 1 1 Independent mutations arose at some sites and the number in
15236 G:A I-V 3.1 3 2 parentheses indicates the number of times this occurred for the site
15380 A:G T-A 0.6 0 1 in question. The nucleotide change is that occurring on the L-
15674 T:C S-P 0.4 -3 2 strand of the revised CRS and the predicted amino acid changes are
15734 G:A A-T 0.6 0 1 designated with the standard one-letter system
b
15758 A:G I-V 3.1 3 2 The references for these mutation scoring systems are as follows:
15803 G:A V-M 1.6 1 1 Gounnt (Gonnet et al. 1992); PHAT (Ng et al. 2000); Persson
Average 1.0 0.4 0.6 (Persson and Argos 1994)
c
This site change, although detected in control sequences, is a
primary pathogenic mutation that causes Leber’s hereditary optic
Site Nucleotide AA Gounnt PHAT Persson neuropathy (LHON). Its omission from the analyses does not
change change change the overall results
II. Control sequences
3388 C:A L-M 2.8 2 2
3472 T:C F-L 2 1 0
3721 A:G T-A 0.6 0 1 1. mk7: replacement at nt 8430 that causes the LEU
3866 T:C I-T 0.6 1 1 residue at position 22 of the AT8 gene to be replaced
4129 A:G T-A 0.6 0 1
4561 T:C V-A 0.1 1 1
by a PRO and a substitution at nt 789 of the 12S
4674 A:G I-V 3.1 3 2 rRNA gene
4908 C:T P-S 0.4 -3 2 2. mrc49: replacement at nt 14258 that causes the PRO
5298 A:G I-V 3.1 3 2 residue at position 139 of the ND6 gene to be re-
5319 (2) A:G T-A 0.6 0 1 placed by a LEU and a substitution at nt 7521 of the
6237 C:A L-M 2.8 2 2
tRNAasp gene

Discussion
As reviewed briefly in the Introduction, it has been
proposed that mutations in the mitochondrial genome
might play a substantial—and perhaps the major—role
in the etiology of AD. To address this important issue,
we carried out a case-control mutational analysis and we
obtained the following results:
1. We did not find any evidence for an etiological role of
haplogroup-associated polymorphisms (contra Cha-
gnon et al. 1999; van der Walt et al. 2004), for a general
increase in mutational burden of non-synonymous
substitutions, or for a subset of mtDNAs from AD
patients that carry an increased number of mutations.
2. The mtDNAs from AD patients had a higher fre-
quency of tRNA mutations than did those of con-
trols, some of which are due to the over-
representation of pre-V and HV sequences in the
MRC set of AD samples. The pathogenicity of these
mutations is not known, but 17% of the AD pa-
tients carried an ‘‘extra’’ mutation in one of the
mitochondrial tRNA genes. Lacking detailed bio-
chemical analysis, it is extremely difficult to determine
if rare tRNA mutations are pathogenic. A substantial
proportion of rare tRNA mutations in the population
appear to be slightly deleterious (for example, Vilmi
et al. 2005; Kondrashov 2005), but how this opera-
tional phenomenon from molecular evolution might
translate to a pathogenic role in a disorder as com-
plex as AD remains unclear.
3. Analysis with mutation matrices indicated that the
replacement substitutions in the AD mtDNAs were,
on average, no more deleterious than those in the
control sequences. However, the ND genes from
control patients carried more replacement substitu-
tions than did those of AD patients. The significance
of this result in terms of decreasing the risk of AD is
not known at this point, but we can approximate that
one of six individuals who has ‘‘avoided’’ AD carries
an ‘‘extra’’ substitution in one of the seven mito-
chondrial genes that encode complex I subunits.
4. The AD sequence set contained an increased number
of mutations that had arisen independently two, or
more, times than did the control sequence set. How-
ever, this difference is not statistically significant and
there is no evidence that these mutations in the AD
patients are functionally deleterious.
5. We identified two AD mtDNAs that carried the
combination of a deleterious mutation in a gene: a
respiratory chain subunit and a rare mutation in a
tRNA or rRNA gene. If these combinations are
pathogenic, then we can estimate that no more than
 2% of AD cases in these populations might be
associated with this type of disease process.
6. Haplogroup H mtDNAs were over-represented
among the MitoKor control sequences, a result that
suggests a protective role in aging.
251
Taking all of these results into consideration, we do
not find evidence that pathogenic mtDNA mutations
play a major or dominant role in the development of
AD. At the same time, it remains possible that mtDNA
mutations have a pathogenic role in a small subset of
patients and that a small subset of the population that
does not develop AD carries a mtDNA mutation that
serves a protective role.
We recognize that the present studies have limitations
and that further analysis will be required to more
definitively reveal the contribution of mtDNA muta-
tions, if any, to the pathogenesis of AD. (1) While our
analyses involved more than 270 mtDNAs from care-
fully evaluated AD and control populations, this num-
ber will need to be increased by an order of magnitude to
follow-up our findings. (2) We did not analyze the
mtDNA control region and subsequent investigations
might provide convincing evidence for AD pathogenic
mutations in this region of the mitochondrial genome.
Coskun et al. (2004) have proposed that heteroplasmic
control region mutations contribute to the AD disease
process, although the available evidence does not sup-
port such a role (Howell et al. 2005). (3) It would have
been difficult to discern a disease process in which
mtDNAs from AD patients each carry a private path-
ogenic mutation. However, there was no evidence that
the non-synonymous substitutions that arose in AD
mtDNAs were clearly more deleterious than those in the
control mtDNAs, but larger datasets and more sensitive
approaches will be needed (see especially Thomas and
Kejariwal 2004). (4) Our methods would not have de-
tected heteroplasmic pathogenic mutations if the muta-
tion load was less than 50%. Parker and Parks (2005)
have recently proposed that low-level heteroplasmic
mutations in the mitochondrial ND5 gene cause Par-
kinson’s disease. (5) We did not test the role of somatic
mtDNA mutations in AD, but we note that two recent
studies have both concluded that the level of somatic
mutations is no higher in AD patients than in aged-
matched normal controls (Chinnery et al. 2001; Lin et al.
2002). (6) Finally, our analyses—as a result of the
stringent inclusion criteria—would likely ‘‘miss’’ patho-
genic mutations that are risk factors for more than one
disorder, only one of which is AD. In this regard, we
reported recently that a mutation at nucleotide 9804 is
associated with a variety of disorders that include optic
neuropathy as well as AD (Howell et al. 2003a).
The recent analyses of sensorineural hearing impair-
ment (SNHI) by Lehtonen et al. (2003) are germane to
our studies. They selected a subgroup of SNHI patients
that had at least one maternal relative that was also
affected with SNHI but who did not carry the known
pathogenic mtDNA mutations at nucleotides 3243
(MELAS) and 1555. The mtDNA sequences from these
patients were then compared to those of the general
Finnish population. They identified nine candidate
pathogenic mtDNA mutations and they observed that
there was an increased mtDNA mutation load in the
252
SNHI patients. Further investigations of the role of the
mitochondrial genome in AD would benefit from a
comparison of mtDNA sequences from patients with an
affected maternal relative to AD patients with no known
family history or an affected paternal relative.
The mtDNAs from the MitoKor cohort of normal
controls appeared to be associated more often with
haplogroup H than those of the MRC cohort. In con-
trast, previous studies of populations of European
descent have tended to show that haplogroup H is
under-represented in healthy, very elderly cohorts, and
that haplogroup J is over-represented (for example,
Rose et al. 2001; Niemi et al. 2003). The size of the
cohorts analyzed thus far is too small to draw mean-
ingful conclusions about an aging effect.
We believe that our studies were designed to reveal a
substantial role for inherited mtDNA mutations in AD,
if one existed, but we are aware that our study is unlikely
to be the ‘‘final answer’’. In fact, as we have pointed out
(Howell et al. 2005), 15 years of analysis have failed to
provide conclusive proof for, or against, a role of
mtDNA mutations in the AD disease process. Consid-
ering the unjustified publication bias against ‘‘negative’’
results (Ioannidis 2005 and references therein), a case
can be made that the balance of evidence therefore
argues against a major role for mtDNA mutations in
AD, and our results add further weight to that position.
The distinction needs to be made that failure to find a
major etiological role of inherited mtDNA mutations in
AD is not in conflict with the evidence for a defect in
mitochondrial energy production in AD patients. A key
feature of both AD and Down’s syndrome is the in-
creased levels of various cleavage forms of amyloid
precursor protein, and it has been concluded that amy-
loid protein causes a defect in mitochondrial energy
production, apparently by insertion into the mitochon-
drial membranes (Busciglio et al. 2002; Casley et al.
2002; Anandatheerthavarada et al. 2003. Recently,
Crouch et al. (2005) have shown that dimers of b-amy-
loid1–42 inhibit cytochrome oxidase. However, the
available results argue against a simple primary defect in
amyloid protein processing with a secondary effect on
mitochondrial energy production. Thus, Busciglio et al.
(2002) observed that, in astrocytes from normal indi-
viduals, inhibition of mitochondrial energy production
altered amyloid protein processing. From their results,
one possibility is that AD pathogenesis involves a feed-
forward pathway that involves both alterations in
amyloid protein processing, or some other pathway in-
volved in synaptic function, and a mitochondrial dys-
function (for example, Zhu et al. 2004). Further
investigations along these lines will be important for
understanding the pathogenesis of AD and the associ-
ated defect in mitochondrial energy production.
Acknowledgements We thank Dr. H. Payami (University of Oregon
Health Science University) for providing some of the samples used
for the present study. Dr. Eoin Fahy (MitoKor Inc.) assisted with
data storage and analysis. This research was supported by a
Wellcome Collaboration Grant (to D. M. T., N. H.), an MRC
BioInformatics Fellowship (J. L. E.), and grant AGO P50-5131
from the National Institutes of Health (L. T.).
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