Disruptive mitochondrial DNA mutations in complex I
subunits are markers of oncocytic phenotype
in thyroid tumors
Giuseppe Gasparre*, Anna Maria Porcelli†, Elena Bonora*‡, Lucia Fiammetta Pennisi*, Matteo Toller§, Luisa Iommarini¶,
Anna Ghelli†, Massimo Moretti§, Christine M. Betts?, Giuseppe Nicola Martinelli**, Alberto Rinaldi Ceroni††,
Francesco Curcio§, Valerio Carelli¶, Michela Rugolo†, Giovanni Tallini‡‡, and Giovanni Romeo*
*Unita ` di Genetica Medica, Policlinico Universitario S. Orsola-Malpighi,†Dipartimento di Biologia Evoluzionistica Sperimentale,¶Dipartimento di Scienze
Neurologiche,?Dipartimento di Patologia Sperimentale, **Dipartimento di Anatomia Patologica, Policlinico S. Orsola-Malpighi,††Dipartimento di Scienze
Chirurgiche ed Anestesiologiche, and‡‡Dipartimento di Anatomia Patologica, Ospedale Bellaria, University of Bologna, 40126 Bologna, Italy; and
§Dipartimento di Patologia e Medicina Sperimentale e Clinica and Centro Interdipartimentale di Medicina Rigenerativa, University of Udine,
33100 Udine, Italy
Communicated by Victor A. McKusick, Johns Hopkins University School of Medicine, Baltimore, MD, April 4, 2007 (received for review November 28, 2006)
Oncocytic tumors are a distinctive class of proliferative lesions
composed of cells with a striking degree of mitochondrial hyper-
plasia that are particularly frequent in the thyroid gland. To
understand whether specific mitochondrial DNA (mtDNA) muta-
tions are associated with the accumulation of mitochondria, we
sequenced the entire mtDNA in 50 oncocytic lesions (45 thyroid
tumors) and 52 control cases (21 nononcocytic thyroid tumors, 15
breast carcinomas, and 16 gliomas) by using recently developed
technology that allows specific and reliable amplification of the
whole mtDNA with quick mutation scanning. Thirteen oncocytic
lesions (26%) presented disruptive mutations (nonsense or frame-
shift), whereas only two samples (3.8%) presented such mutations
in the nononcocytic control group. In one case with multiple
thyroid nodules analyzed separately, a disruptive mutation was
found in the only nodule with oncocytic features. In one of the five
mitochondrion-rich breast tumors, a disruptive mutation was iden-
tified. All disruptive mutations were found in complex I subunit
genes, and the association between these mutations and the
oncocytic phenotype was statistically significant (P ? 0.001). To
study the pathogenicity of these mitochondrial mutations, primary
cultures from oncocytic tumors and corresponding normal tissues
were established. Electron microscopy and biochemical and mo-
lecular analyses showed that primary cultures derived from tumors
bearing disruptive mutations failed to maintain the mutations and
complex I subunits are markers of thyroid oncocytic tumors.
oncocytic tumors ? heteroplasmy ? homoplasmy ? damaging mutation ?
sequences have been reported in databases such as Mitomap (2)
and HmtDB (3). Several groups have focused on the regulatory
region of mtDNA, the displacement loop (D-loop) (4), whereas
others investigated the D-loop in conjunction with various
coding regions (5) or only a single coding region (6). Different
approaches were undertaken to analyze the frequency of single
polymorphisms in association with a specific tumor prevalence
(7). The question regarding the pathogenic role for mtDNA
mutations in cancer has already been debated (8, 9). However,
the difficult task of attributing a causal role to mitochondrial
In fact, most variants found in tumors are also present as
polymorphic variants in the control population, whereas for
those inducing amino acid changes, it is difficult to prove a
functional role relevant for tumor pathology (8).
utations in mitochondrial DNA (mtDNA) have been
widely described in many types of tumors (1), and variant
Recently, we have attempted to clarify this point by clearly
demonstrating the association between mtDNA mutations and
defective oxidative phosphorylation in a cell line model of
thyroid oncocytic tumor (10). The term ‘‘oncocytic’’ is used to
designate lesions composed of cells with aberrant accumulation
of mitochondria, resulting in a distinctive granular eosinophilic
appearance on conventional histology. Tumors composed of
oncocytic cells occur at various sites and are particularly com-
mon among thyroid neoplasms of follicular cell derivation (11).
Oncocytic thyroid tumors have long been suspected to be more
aggressive than their nononcocytic counterparts (11), and the
prognostic indicator for follicular thyroid carcinomas (12).
The exact relationship between mitochondrial accumulation
in oncocytic cells and tumor development remains unknown.
Several authors have performed gene expression and biochem-
ical studies to investigate molecular aspects of this specific
histological phenotype (13, 14). In particular, deficient complex
I activity has been described in renal oncocytoma (15, 16), and
a correlation between mitochondrial hyperplasia and tumori-
genesis has been suggested (17). Most mtDNA changes reported
in thyroid oncocytic tumors have been identified after partial
sequencing of the mitochondrial genome, again without proven
pathogenicity (18, 19). To the best of our knowledge, a system-
atic approach with complete sequencing of the whole mtDNA
from tumor samples and strict mitochondrial genotype–
phenotype correlation has not been carried out.
In the present study, we used a recently developed approach
to sequence the whole mitochondrial genome from different
types of tumors and control tissues. In silico analysis was
performed on all amino acid changes with available software to
predict their pathogenic potential (20). This process allowed a
comprehensive investigation of all mtDNA variants in oncocytic
lesions and control cases. Our data indicate that mtDNA dis-
ruptive complex I mutations are markers for the oncocytic
Author contributions: E.B., V.C., M.R., G.T., and G.R. designed research; G.G., A.M.P., L.F.P.,
G.T. analyzed data; and G.G. wrote the paper.
The authors declare no conflict of interest.
Abbreviation: PSIC, position-specific independent count.
Data deposition: The sequences reported in this paper have been deposited in the HmtDB
database (accession nos. listed in SI Table 3).
‡To whom correspondence should be addressed at: Dipartimento Medicina Interna, Car-
dioangiologia ed Epatologia, U.O. Genetica Medica, Padiglione 11, Policlinico S. Orsola-
Malpighi, via Massarenti, 9, 40138 Bologna, Italy. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0703056104 PNAS ?
May 22, 2007 ?
vol. 104 ?
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mtDNA Sequencing. The entire mitochondrial genome was se-
quenced in 25 thyroid and 5 breast oncocytic lesions and 52
controls. More than 98% of mtDNA sequencing also was
obtained from 20 additional formalin-fixed thyroid oncocytic
samples. Sequencing results [deposited in the HmtDB database;
see supporting information (SI) Table 3] are summarized in
Tables 1 and 2 and SI Table 4. All mtDNA changes resulting in
impaired protein synthesis (nonsense and frameshift alterations)
were classified as ‘‘disruptive’’ mutations. Upon in silico predic-
tion (see Materials and Methods), ‘‘probably damaging’’ or
‘‘possibly damaging’’ mutations were classified under the cate-
gory of ‘‘potentially damaging’’ mutations.
Disruptive mutations were concentrated in a few mitochon-
drial genes coding for subunits of complex I of the respiratory
chain (ND1, ND2, ND4, and ND5). Two samples (Table 1)
harbored the same mutation (3571insC) in the ND1 gene that we
recently reported in the XTC.UC1 oncocytic cell line, in which
we demonstrated complete absence of the protein and defective
activity of complex I (10).
Because of the physiological polyploidy of the mitochondrial
genome, different variants of mtDNA can coexist in a single
mitochondrion or in a single cell, which gives rise to the
phenomena of homo- and heteroplasmy. Homoplasmy implies
that all copies of the mitochondrial genome in cells and tissues
harbor an identical sequence. Heteroplasmy denotes the coex-
istence of mtDNA copies carrying differences in their sequence.
Thus, nonsynonymous mtDNA changes may lead to different
protein variants, and the phenotypic effect of a mutation may
become evident only if a certain threshold of heteroplasmy is
reached. The heteroplasmic status of all mtDNA variants de-
tected is reported in Tables 1 and 2. Heteroplasmy of all
disruptive mutations was evaluated. As reported in Table 1, most
or with a high degree of heteroplasmy (?70%) and hence above
the likely threshold for a damaging effect. For all disruptive
mutations, the corresponding adjacent normal tissue was also
analyzed, and in all cases mutations were somatic.
The presence of preferential combinations of mitochondrial
polymorphisms (haplogroups) in our oncocytic samples versus
nononcocytic tumor types was also investigated. Haplogroup
definition did not reveal any statistically significant divergence
from the previously reported haplogroup frequencies in Euro-
pean population (21).
Correlation of mtDNA Alterations with Clinico-Pathologic Features.
Immunohistochemistry and electron microscopy confirmed cy-
toplasmic accumulation of mitochondria in all oncocytic thyroid
samples analyzed and an increased mitochondrial mass in five
breast carcinomas. In the latter cases, the percentage of mito-
chondrion-rich cells was ?80% in the tumor tissue, whereas for
the thyroid oncocytic samples, the percentage was invariably
?75%. No evidence of oncocytic differentiation was detected in
the glioma group.
Table 1. mtDNA mutations in oncocytic samples
SampleDiagnosisBase change Amino acid changeGene Het., % % SequencedPSICDatabase
Het., heteroplasmy (% of mutated); OCA, oncocytic thyroid carcinoma; OCAp, oncocytic thyroid carcinoma with papillary festures; OFA, onocytic follicular
thyroid adenoma; OHTN, oncocytic hyperplastic thyroid module; BRduct, invasive ductal carcirnoma of the breast. Bold indicates disruptive mutations.
www.pnas.org?cgi?doi?10.1073?pnas.0703056104Gasparre et al.
Twenty-six of the 45 oncocytic thyroid samples analyzed
(57.8%) harbored 30 mtDNA mutations, 25 of which were in
complex I genes. In 12/45 samples (26.7%), the mutations were
disruptive and all occurred in complex I. In three samples, the
disruptive mutations coexisted with potentially damaging mis-
sense mutations (Table 1). In 14/45 samples (31.1%), the mu-
tations were potentially damaging missense changes, and in one
case, two such mutations coexisted in the same lesion (Table 1).
Fifteen of the 18 (83.3%) potentially damaging missense muta-
tions identified in thyroid oncocytic samples (12 of which
occurred in complex I) had a position-specific independent
count (PSIC) ? 2 [i.e., mutations were probably damaging (20)].
Eight of the 21 nononcocytic thyroid samples (38.1%) pre-
sented mtDNA mutations, 5 of which were in complex I. Only in
2/21 samples (9.5%) were the mutations, both occurring in
complex I, disruptive, whereas in 6/21 samples (28.5%), the
mutations were potentially damaging missense changes. Five of
the six (83.3%) potentially damaging missense mutations iden-
tified in nononcocytic thyroid samples had a PSIC ? 2 [i.e.,
mutations were possibly damaging (20)].
thyroid gland were analyzed. Only one nodule presented onco-
cytic change, and only in this nodule was a disruptive hetero-
plasmic nonsense mutation in ND5 found (Table 1 and Fig. 1).
Heteroplasmy evaluation showed that 29% of ND5 copies were
mutated in the nodule.
Among the 15 breast carcinoma samples examined, there were
phenotype from the same thyroid (case HTC1, Table 1). (Upper) Histologic
appearance of the hyperplastic oncocytic nodule (A) and of one hyperplastic
nodule without oncocytic change (B). Immunohistochemistry with antibodies
and nononcocytic nodule. (Magnification: ?400.)
mic deletion in the ND1 gene in tumor (Upper) and perilesional normal
(Lower) tissue. (B) Histologic appearance of the breast carcinoma showing
(C and D) Immunohistochemistry with antibodies specific for human mito-
chondria confirms the increased mitochondrial mass in the tumor (D) com-
pared with the nonneoplastic perilesional breast parenchyma (C). (Magnifi-
Disruptive mtDNA mutation in a mitochondrion-rich breast carci-
Table 2. mtDNA mutations in nononcocytic samples
SampleDiagnosis Base changeAmino acid changeGene Het., %PSICDatabase
Het., heteroplasmy (% of mutated); AST, astrocytoma; BRduct, invasive ductal carcinoma of the breast; BRlob, invasive lobular
carcinoma of the breast; FA, follicular thyroid adenoma; FTC, follicular thyroid carcinoma; HTN, hyperplastic thyroid module; PTC,
papillary thyroid carcinoma. Bold indicates disruptive mutations.
Gasparre et al. PNAS ?
May 22, 2007 ?
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4 potentially damaging missense variants (26.7%) and no dis-
ruptive mutations (Table 2). Among the five mitochondrion-rich
breast tumors, one harbored a heteroplasmic disruptive mtDNA
mutation (Table 1 and Fig. 2), whereas two cases had potentially
damaging missense mutations. Two potentially damaging mis-
sense variants (12.5%) and no disruptive mutations were de-
tected in the glioma group. Overall, the oncocytic phenotype was
associated with mtDNA mutations. The association was signif-
icant in both cases: when considering only disruptive mutations
0.0013). Patient age, sex, and size of the lesion were not
associated with mtDNA mutations (SI Table 5).
Primary Cell Cultures. To address further the correlation between
mtDNA mutations and biochemical phenotype of oncocytic
tumors, primary cultures were established from thyroid samples
according to availability of material. We focused on the two
cultures derived from oncocytic tumors and bearing a disruptive
mutation. Although no unequivocal tumor markers are available
to ascertain the transformed phenotype of cultured cells, higher
production of IL-6 has been shown to occur in thyroid carcinoma
(22). Accordingly, IL-6 secretion was increased significantly in
all tumor-derived primary cultures compared with correspond-
ing normal cultures from the same patient (SI Fig. 4 and SI
Materials and Methods).
Interestingly, no mutations were detected in any of the three
primary tumor cultures originating from tumors presenting a
disruptive mutation (data not shown). Accordingly, electron
microscopy showed that both fresh and paraffin-retrieved
(data not shown) biopsies from oncocytic tumors were com-
posed of large cells rich in closely packed, swollen mitochon-
dria (Fig. 3A). In contrast, cultured cells from oncocytic lesions
retained only a few large mitochondria with frequently ob-
served secondary lysosomal structures (Fig. 3B) and showed a
filamentous mitochondrial network similar to that of their
normal cultured cell counterpart (Fig. 3C and SI Fig. 4).
Furthermore, no difference was detected in the citrate syn-
thase activity, a widely accepted biochemical indicator for
mitochondrial mass (Fig. 3D). The ATP content of normal and
tumor thyroid primary cultures was similar in glucose medium
(Fig. 3E) and also during incubation in galactose-containing
medium [i.e., under conditions leading to a dramatic reduction
of glycolytic rate and forced use of oxidative phosphorylation
for ATP production (Fig. 3F)]. Given that cells with defects in
oxidative phosphorylation are unable to maintain their ATP
content in galactose-containing medium (23), these results
lacking in normal and tumor primary cultures (B Upper and C Upper). Electropherograms showing the mutated base (bold and underlined) in the tumor biopsy
and the wild-type base at the same position in both normal and tumor primary culture (A Lower, B Lower, and C Lower). (D–F) Citrate synthase activity (D), total
ATP levels in glucose (E), and activity during incubation in galactose-containing medium (F) are shown. Data points F are means ? SD of at least five different
Characterization of normal and tumor primary cell cultures. (A–C) Ultrastructure of thyroid tumor biopsy shows mitochondrial hyperplasia (A Upper)
www.pnas.org?cgi?doi?10.1073?pnas.0703056104Gasparre et al.
indicate that oxidative phosphorylation was not impaired in
In this study, we have demonstrated that the oncocytic pheno-
type is associated with disruptive mutations in complex I sub-
The role of mitochondria in the process of tumorigenesis has
been debated widely, and the literature has flourished with
studies investigating the association between mtDNA variants
and tumors (1). However, careful survey of all mitochondrial
variants reported in association with cancer makes it difficult to
accept that silent and even missense mutations may be only
causally related or predisposing to tumorigenesis. Nevertheless,
controversial technical aspects have warranted skepticism (24).
Given the very complex nature of tumorigenesis, it is difficult to
attribute a causal role to single mitochondrial mutations, and a
more-than-one-hit hypothesis is more plausible. In this context,
mitochondrial mutations may play their part as one of the strikes
leading to tumor development.
In the oncocytic samples, we found a larger prevalence of
nonsense and frameshift mutations caused by insertions or
deletions in coding regions of mtDNA, in most cases (8 of 12)
occurring early in sequence, so that dramatic disruption of the
protein was easily predictable. Only two such mutations were
found in the control group. Statistical analysis showed a clear
correlation between the presence of such disruptive mutations
and the oncocytic phenotype. All of the disruptive mutations
were concentrated in complex I subunits, whereas analysis of the
corresponding samples from the normal adjacent tissue showed
the somatic origin of the mutations in all cases. This finding
further supports the hypothesis that dysfunction of complex I
may play a role in tumor development, as previously proposed
for thyroid (10) and renal oncocytoma (15, 16).
A higher prevalence of missense mutations was also found in
Additional functional studies will be needed to substantiate their
One case (HCT1), from which three different nodules were
analyzed, presented a heteroplasmic nonsense mutation in the
ND5 gene in only one of the nodules. Double-blinded his-
topathological examination confirmed that only the nodule with
the mutation presented oncocytic changes, suggesting that this
specific mutational event may be responsible for the oncocytic
Interestingly, the only case of breast carcinoma harboring a
disruptive mutation was a mitochondrion-rich tumor, and po-
tentially damaging mutations were present in two additional
mitochondrion-rich breast carcinomas. Oncocytic carcinomas
of the breast are rare tumors, although the prevalence of
mitochondrion-rich breast carcinomas with oncocytic features,
such as the cases presented in this study, might be underesti-
mated (25). mtDNA mutations have been described in breast
carcinomas (1, 5) without being correlated to a mitochondrion-
rich phenotype. Our findings in breast carcinoma strengthen the
link between pathogenic mtDNA changes and mitochondrial
A link between mtDNA disruptive mutations and the onco-
cytic phenotype also was provided by the thyroid primary culture
experiments. None of the primary tumor cultures showed evi-
dence of the disruptive mutations found in the original biopsies.
Accordingly, the oncocytic phenotype was lost during culture,
and no differences between tumor and the corresponding nor-
mal tissue cultures were found. These data indicate that, under
the culture conditions used in this study, cells bearing the
mutations are selected against.
The glycolytic shift in cancer cells observed initially by War-
burg (26), a phenomenon currently exploited for diagnostic
purposes by positron emission tomography, led to the idea that
mitochondrial damage, forcing cells to rely on glycolysis for ATP
production, may confer a selective advantage in the hypoxic
environment surrounding the tumor (27). Nonetheless, it has
been suggested that severe mutations impairing oxidative phos-
phorylation may be lost once the tumor cells return to a
high-oxygen environment as during cell culture (8). Hence, the
in vivo microenvironment may have a fundamental influence on
conditions that allow the mutation to arise, be propagated, and
eventually shift to homoplasmy. It is worth noting that different
mechanisms may be involved for the maintenance of the onco-
cytic phenotype. For example, the XTC.UC1 cell line is the only
existing cellular model of thyroid oncocytoma (28). XTC.UC1 is
the accumulation of genetic damage may have contributed to the
positive selection of oncocytic cells.
Mitochondrial dysfunction might also arise from mutations in
nuclear genes encoding for mitochondrial proteins (29, 30). A
mutation screening of nuclear-coded oxidative phosphorylation
subunits was not performed in this study. Mutations in the
nuclear-encoded complex I subunits may account for the per-
centage of oncocytic cases in which mitochondrially encoded
complex I subunits were not mutated; hence, the percentage of
complex I mutations in oncocytic tumors could be underesti-
mated. In fact, it has been shown that some types of hereditary
tumors are characterized by mitochondrial defects (31). The
presence of germ-line changes in mitochondria-related genes
and their potential involvement in oncocytic tumor development
further suggests a complex interplay between nuclear and mito-
chondrially encoded genes in promoting the oncocytic phenotype.
In conclusion, this study shows a statistically significant prev-
alence of disruptive mutations in genes coding for complex I of
the electron transport chain in thyroid oncocytic tumors. It is
likely that these mutations may arise as a secondary hit in tumor
development and that the oncocytic phenotype, characterized by
mitochondrial hyperplasia, may be strictly correlated with these
mutations. We therefore propose this type of mutation as a
molecular marker of oncocytic phenotype in thyroid tumors.
Materials and Methods
Tissue Samples, Clinico-Pathologic Features, and Immunohistochem-
istry. Samples were obtained from the pathology units of Bolo-
gna University Medical School at Bellaria and S. Orsola-
Malpighi Hospitals. From 11 thyroid oncocytic samples and 48
controls, excess lesional and/or perilesional tissue was obtained
fresh and stored frozen at ?80°C before analysis. Samples were
diagnosed according to established criteria (12). Twenty-two
were hyperplastic thyroid nodules (16 of them oncocytic), 10
were follicular thyroid adenomas (7 of them oncocytic), 22 were
oncocytic thyroid carcinomas, 12 were thyroid carcinomas with-
out oncocytic features (11 papillary, 1 follicular), 20 were breast
carcinomas (5 of which had oncocytic features), and 16 were
on tissue sections by using routine immunohistochemical meth-
ods (25). At the time of diagnosis, the average patient age was
53 for patients with oncocytic lesions compared with 58 for
controls. Average lesional size was 2.6 cm for the oncocytic
lesions versus 3 cm for thyroid controls. All tumors considered
for the study were sporadic. Clinical information was obtained
by chart review. Handling of samples and clinical data proceeded
in accordance with internal review-board-approved protocols.
DNA Extraction and mtDNA Sequencing. DNA was extracted with
the Qiagen kit (Qiagen, Valencia, CA) according to the manu-
facturer’s protocols. mtDNA was sequenced with the recently
developed MitoAll resequencing kit (Applera, Foster City, CA)
and analyzed as described in ref. 10. Haplogroup and subhap-
logroup affiliations of all samples investigated were assigned as
Gasparre et al. PNAS ?
May 22, 2007 ?
vol. 104 ?
no. 21 ?
described in ref. 21, and whenever possible, heteroplasmy was
confirmed by cloning as described in ref. 10.
Prediction Analysis of Amino Acid Substitutions. PolyPhen (www.
tux.embl-heidelberg.de/ramensky/polyphen.cgi) was used to pre-
dict the possible impact of amino acid substitutions on the
protein. The program is based on sequence comparison with
allelic variants and represent the logarithmic ratio of the likeli-
hood of a given amino acid occurring at a particular site relative
to the likelihood of this amino acid occurring at any site
(background frequency). PSIC score differences ?2 indicate a
damaging effect, scores between 1.5 and 2 suggest that the
variant is possibly damaging, and scores ?1.5 indicate that the
variant is benign (20).
Electron Microscopy. For electron microscopy, small fresh-tissue
biopsies or cell pellets obtained from primary cultures of both
lesional and perilesional thyroid tissue were processed according
to previously published protocols (32).
Primary Cultures. Of 66 thyroid samples collected, 29 primary
cultures were established from both the tumor and the normal
tissue. Twelve of these were from oncocytic samples, and two of
these originated from biopsies in which a disruptive mutation
was present (HCT33 and HCT38, Table 1). One culture was
derived from a nononcocytic tumor (TC8, Table 2) bearing a
H1/10P Culture Growth Medium. H1/10P culture growth medium, a
basic culture proliferation medium with composition similar to
that previously used for human thyroid cells, was used (33, 34)
with and without 50 ?g/ml uridine to allow growth of cells with
impaired mitochondrial function.
Reverse Transcription. Reverse transcription was performed for
the following markers to confirm selection of thyroid cells:
thyroid transcription factor 1, thyroid-stimulating hormone re-
ceptor, and thyroid peroxidase (data not shown).
ATP Assay and Citrate Synthase Activity. Cells from primary culture
(3 ? 105) were seeded into six-well plates and incubated in
H1/10P medium or in glucose-free H1/10P medium supple-
mented with 5 mM galactose, 5 mM Na-pyruvate, and 10% FBS.
ATP was determined with the luciferin/luciferase assay (10).
Citrate synthase activity was measured as described in ref. 10.
Statistical Analysis. Statistical analysis was performed with the
Fisher’s exact test. A P value ?0.05 was considered to be
collection and L’Oreal Italia ‘‘Per le Donne e la Scienza’’ for fellowship
support (to A.M.P.). This work was supported by Associazione Italiana
Ricerca sul Cancro (AIRC) Grant 1145 (to G.T.) and partially supported
by grants from Fondo Italiano Ricerca di Base, Rome (FIRB), and
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