Mitochondrial D-Loop instability in thyroid tumours is not a marker of malignancy

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Mitochondrial D-Loop instability in thyroid tumours

is not a marker of malignancy

Valdemar Maximoa, Jorge Limaa, Paula Soaresa,b, Tiago Botelhoa,

Lıgia Gomesa, Manuel Sobrinho-Simoesa,b,c,*

aInstitute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, PortugalbDepartment of Pathology, Medical Faculty of the University of Porto, Porto, Portugal

cDepartment of Pathology, Hospital Sao Joao, Porto, Portugal

Received 4 February 2005; received in revised form 20 May 2005; accepted 1 June 2005

Abstract

Despite the numerous studies describing a high frequency of mitochondrial DNA (mtDNA) somatic mutations in many types

of human primary tumors the mechanisms that generate such mutations and the role of mtDNA mutations in tumor development

remain unclear. We present the results obtained in the study of mtDNA displacement-loop (D-Loop) region in a series of 66

thyroid tumors, and respective adjacent parenchyma, including benign (adenomas, nZ30) and malignant tumors (follicular

carcinomas, nZ17 and papillary carcinomas, nZ19). Three repetitive regions were analyzed [two mononucleotide repetitive

(D310 and D568) and one dinucleotide repetitive (D514)]. Thirty-two (48.5%) of the 66 tumors [15/30 (50.0%) adenomas, 8/17

(47.1%) follicular carcinomas and 9/19 (47.4%) papillary carcinomas] harbored somatic insertions in D-Loop repetitive

regions. Twenty (30.3%) of the 66 tumors [12/30 (40%) adenomas, 3/17 (17.6%) follicular carcinomas and 5/19 (26.3%)

papillary carcinomas] harbored somatic insertions at the D310 mononucleotide repeat. Three (4.6%) of the 66 tumors [1/30

(3.3%) adenomas and 2/17 (11.8%) follicular carcinomas] harbored somatic insertions at the D568 mononucleotide repeat.

Fifteen (22.7%) of the 66 tumors [3/30 (10.0%) adenomas, 5/17 (29.4%) follicular carcinomas and 7/19 (36.8%) papillary

carcinomas] harbored somatic insertions at the D514 dinucleotide repeat. Five (7.6%) of the 66 tumors [1/30 (3.3%) adenomas,

1/17 (5.9%) follicular carcinomas and 2/19 (10.5%) papillary carcinomas] harbored somatic insertions in more than one region,

and in one of them (a carcinoma) alterations were detected in the three regions. We conclude that mutations in the mtDNA

D-Loop region are frequent in benign and malignant thyroid tumors and cannot be considered a marker of malignancy. Our

study shows, furthermore, two repetitive regions (D310 and D514) that appear to be susceptible to mutation in thyroid tumors.

q 2005 Published by Elsevier B.V. on behalf of Mitochondria Research Society.

Keywords: Thyroid tumors; mtDNA; D-Loop instability; Hurthle cell tumors; D310

1567-7249/$ - see front matter q 2005 Published by Elsevier B.V. on be

doi:10.1016/j.mito.2005.06.003

* Corresponding author. Address: IPATIMUP, Rua Dr Roberto

Frias s/n, 4200-465 Porto, Portugal. Tel.: C351 22 55 70 700; fax:

C351 22 55 70 799.

E-mail address: [email protected] (M. Sobrinho-Simoes).

1. Introduction

The human mtDNA is a 16-kb circular, double

stranded DNA that encodes 13 polypeptides of the

mitochondrial respiratory chain (MRC), 22 transfer

Mitochondrion xx (2005) 1–8

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half of Mitochondria Research Society.

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RNAs and 2 ribosomal RNAs required for protein

synthesis (Anderson et al., 1981; Andrews et al.,

1999). The mutation rate of mtDNA is about 10!20

times higher than that of the nuclear DNA (nDNA)

(Wallace et al., 1998; Brown and Wallace 1994). This

high mutation rate may be due to one or more of the

following factors: a high concentration of oxygen

radicals at the mitochondrial inner membrane, the

lack of efficient mtDNA repair mechanisms, and/or

the absence of DNA-coating proteins such as the

histones (Anderson et al., 1981; Wallace et al., 1998).

Due to this high mutation rate, mtDNA is likely a

hotspot for mutations in cancer as it is preferentially

modified by many carcinogens (Backer et al., 1980).

Alterations of mtDNA have been demonstrated in

various types of primary human cancers and include

large deletions, missense mutations, frameshift

mutations and small insertions/deletions in repetitive

regions (Sanchez-Cespedes et al., 2001; Maximo et

al., 2001, 2002; Habano et al., 1998, 2000; Richard et

al., 2000; Burgart et al., 1995; Polyak et al., 1998;

Fliss et al., 2000; Yeh et al., 2000; Lewis et al., 2000).

Despite the high number of studies on mtDNA

mutations in human primary neoplasms, the mechan-

isms that generate such mutations and the putative

role played by mtDNA mutations in tumor develop-

ment remain unclear.

In a previous study, Sanchez-Cespedes et al.

(2001) reported the identification of a specific and

highly polymorphic homopolymeric C stretch (D310),

located within the displacement-Loop (D-Loop) of the

mtDNA, as a mutational hotspot in human benign

and malignant primary tumors. Moreover, these

alterations were also present in 2 out of 14

preneoplastic lesions from head and neck tumors

(Sanchez-Cespedes et al., 2001). Most of the somatic

alterations found were deletion/insertions generating

D310 variants identical to constitutive polymorph-

isms described previously (Sanchez-Cespedes et al.,

2001; MITOMAP: A Human Mitochondrial Genome

Database, 2001).

In an attempt to clarify the putative pathogenic

meaning of somatic mitochondrial D-Loop instability

in thyroid tumors, we have studied three repetitive

regions of the D-Loop region in a series of 66 benign

and malignant thyroid tumors and adjacent thyroid

parenchyma by PCR/SSCP and automated

sequencing.

2. Material and methods

2.1. Material

Sixty-six thyroid tumors from 59 patients were

studied. In 7 patients there were two distinct lesions

that were separately studied. The 66 lesions were

classified according to Hedinger et al. (1988) and

Rosai et al. (1992) as follicular adenoma (nZ10),

Hurthle cell follicular adenoma (nZ20), follicular

carcinoma (nZ4), Hurthle cell follicular carcinoma

Polyak et al. (1998), papillary carcinoma (nZ12) and

Hurthle cell papillary carcinoma (nZ7). Samples

from 27 lesions were obtained at the time of surgery,

together with the corresponding normal adjacent

tissues; these samples were carefully dissected by

expert pathologists and snap frozen. In the remaining

33 cases, microdissected paraffin-embedded material

was used for the screening of mtDNA D-Loop

mutations, due to the absence of representative

tumor tissue in the frozen samples.

2.2. DNA extraction

DNA was extracted from microdissected frozen

and/or paraffin-embedded pathologic and normal

thyroid tissue pairs using the NucleoSpinw Tissue

Kit (Macherey-Nagel, Duren, Germany).

2.3. Screening of mtDNA D-Loop mutations

The screening of mtDNA D-Loop mutations was

performed by PCR/SSCP and automated sequencing.

Two independent fragments of 157 bp (between nt

267 and 463) and 185 bp (between nt 453 and 637),

using the following primers: the forward primer used

for the 157 bp fragment amplification was 5 0-

TCCACACAGACATCATAACA-3 0 and the reverse

was 5 0-AAAGTGCATACCGCCAAAAG-3 0; the for-

ward primer used for the 185 bp fragment amplifi-

cation was 5 0-TTTCCCCTCCCACTCCCATACT-3 0

and the reverse was 5 0-GTGATGTGAGCCCGTC-

TAAACA-3 0. PCR amplifications were performed in

a 25 ml volume containing 200 mM of each dNTP,

12.5 pmol of each of the forward and reverse primers,

50 mM KCl, 10 mM Tris–HCl (pH 9.0), 1.5 mM

MgCl2 and 1 U of Taq DNA polymerase (Amersham

Pharmacia Biotech). Cycling conditions were a single

Fig. 1. Electropherograms showing mtDNA D-Loop D310

mutations in case 18, (A) normal tissue; (B) tumor tissue; and

mtDNA D-Loop D514 mutations in case 53, (C) normal tissue and

(D) tumor tissue).

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predenaturation step at 94 8C for 5 min followed by 35

cycles of denaturation at 94 8C for 30 s, annealing at

58 8C for 20 s and elongation at 72 8C for 30 s, and a

final incubation at 72 8C for 5 min. Electrophoresis of

the denatured PCR products was carried out in

MDEe gel solution (FMCw Bioproducts, Rockland,

Maine USA) and runned at 160 V, 8 8C. MDE gels

were stained using silver nitrate staining as described

(Budowle et al., 1991), and blotted onto 3MM

Whatman paper (Maidstone, United Kingdom) and

dried. All cases presenting band shifts were submitted

to a second analysis (new PCR amplifications and

SSCP analysis, using the initial DNA samples), and

only reproducible bands were considered. Only cases

with mtDNA alterations in tumoral and not in adjacent

normal tissue were considered to harbor somatic

mutations. Abnormal bands, as well as the corre-

sponding normal bands, detected by SSCP were

recovered from MDE gels and submitted to PCR

reamplification with the original set of primers.

Reamplification products were purified and sequenced

using the ABI Prism BigDye Terminator Cycle

Sequencing Kit (Perkin–Elmer, Foster City, CA) and

an ABI Prism 377 DNA Sequencer (Perkin–Elmer).

Sequencing was performed in both strands using the

original primers. Sequences were compared against a

comprehensive mitochondrial databank (MITOMAP:

A Human Mitochondrial Genome Database, 2001).

All mtDNA altered samples were subjected to an

additional complete analysis: new PCR followed by

SSCP and sequencing.

2.4. Statistical analysis

The statistical analysis of the results was per-

formed using the Chi-square test with the Yates

correction, Fisher’s exact test and Student’s t-test. A P

value !0.05 was considered statistically significant.

3. Results

3.1. MtDNA somatic mutations in D-Loop region

The results are summarized in Table 1. MtDNA

somatic mutations in D-Loop region were detected in

32 of the 66 tumors (48.5%). The mutations included

insertions/deletion in repetitive regions (D310, D514

and D568) (Fig. 1) as well as mtDNA D-Loop point

mutations [all of them were transitions (data not

shown)]. Three out of the 32 cases with mtDNA

D-Loop alterations had two lesions: in case 10 (10A,

papillary carcinoma and 10B, Hurthle cell adenoma)

the same alteration in the D310 repetitive region was

present in both lesions (Table 1), but the Hurthle cell

adenoma showed an extra D-Loop point mutation

(data not shown); case 16 (16A and 16B, two Hurthle

cell adenomas) displayed the same alteration in the

D310 repetitive region in both lesions (Table 1), but

16A showed an extra D-Loop point mutation (data not

shown); in case 12 (12A, Hurthle cell adenoma and

12B, papillary carcinoma) only the Hurthle cell

adenoma harbored a D-Loop mutation (Table 1).

The frequency mtDNA D-Loop insertions/

deletions in at least one of the three repetitive regions

(D310, D514 and D568) is similar (PO0.999) in

benign (50.0%, 15 of 30) and in malignant tumors

(47.2%, 17 of 36). The frequency of Hurthle cell

tumors with mtDNA D-Loop insertions/deletions in at

least one of the three repetitive regions—D310, D514

and D568—is higher (57.5%, 23 of 40) than that of

non-Hurthle cell tumors (34.6%, 9 of 26), although the

difference does reach the threshold of statistical

significance (PZ0.083). The presence of mtDNA

D-Loop mutations was not significantly associated

with the age and/or gender of the patients (data not

shown), nor with the presence or absence of

lymphocytic thyroiditis in normal adjacent thyroid

(data not shown).

Table 1

Summary of mtDNA D-Loop alterations in 66 thyroid tumors

Case Age Diagnosis mtDNA D-Loop alterations

D310 D514 D568

1 57 PC – CA7/CA4 –

2 51 HCFC C6/C6,7 – –

3 32 HCFC C6/C8,9 CA5/CA6 C6/C8

4 39 HCFC – – C6/C8–10

5 33 HCFC – CA5/CA7 –

6 76 HCFC – CA5/CA6 –

7 62 HCPC C7/C8 CA5/CA6 –

9 49 HCA C6/C8 – –

10A 63 PC C7/C8 – –

10B 63 HCA C7/C8 – –

12A 45 HCA C6/C8 – –

14 34 HCA C7/C9,10 – –

15 59 HCA C7/C8 – –

16A 67 HCA C7/C8 – –

16B 67 HCA C7/C8,9 – –

17 57 HCA C7/C8 – –

18 38 HCA C7/C8,9 – C6/C7

19 54 HCFC – CA5/CA6 –

21 8 HCPC – CA4/CA4–6 –

22 46 HCPC C7/C8 CA5/CA6 –

27 48 HCFC – CA5/CA6 –

28 42 A C7/C9,10 – –

32 32 HCPC C7/C9,10 – –

39 53 PC – CA5/CA8 –

40 77 HCA – CA5/CA6,7 –

41 40 FC C7/C8–10 – –

46 67 A – CA7/CA6 –

51 54 PC C7/C8–12 CA5/CA6,7 –

53 37 A – CA4/CA5 –

54 56 HCA C7/C8 – –

57 34 HCA C7/C8,9 – –

60 56 PC – CA5/CA6 –

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3.2. D310

Twenty (30.3%) of the 66 tumors [12/30 (40%)

adenomas, 5/19 (26.3%) papillary carcinomas and

3/17 (17.6%) follicular carcinomas] harbored somatic

insertions at the polymorphic homopolymeric C

stretch (D310). The mutations were insertions that

varied from 1 to 5 bp (Tables 1 and 2). The prevalence

of D310 alterations was higher, though not statisti-

cally significant (PZ0.178), in adenomas (40%, 12/

30) than in carcinomas (22.2%, 8/36; follicular

carcinoma K17.6%, 3 of 17: papillary carcinoma

K26.3%, 5 of 19). There were 11 tumors with 2 bp or

more insertions (7 adenomas, 2 follicular carcinomas

and 2 papillary carcinomas) (Tables 1 and 2). Five out

the 20 (25.0%) D310 alterations were outside the

usual polymorphic length range (between 7C and 9C)

and included unusual variants (10C, 11C and 12C).

Four of the 59 (6.1%) patients presented constitu-

tionally a deletion of 1 bp (6C).

3.3. D514

Somatic mutations in the dinucleotide (CA) repeat

region D514 were detected in 15 cases (22.7%). The

prevalence of mutations was significantly lower

(PZ0.038) in adenomas (10.0%, 3/30) than in

carcinomas (33.3%, 12/36; follicular carcinoma

K29.4%; 5 of 17: papillary carcinoma K36.8%; 7

of 19). The type of alterations (number of CA repeats)

Table 2

Somatic insertions (1-bp or higher) in the D310 repeat in 66 thyroid tumors

Histotype No. of alterations (total) 1 bp O1 bp

Adenoma (nZ30) 12 (40.0%) 5 (16.7%) 7 (23.3%)

Carcinoma (nZ36) 8 (22.2%) 4 (11.1%) 4 (11.1%)

Follicular carcinoma (nZ17) 3 (17.6%) 1 (5.8%) 2 (11.8%)

Papillary carcinoma (nZ19) 5 (26.3%) 3 (15.8%) 2 (10.5%)

PZ0.178 (Adenomas vs. carcinomas)

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in the three groups of tumors is summarized in

Tables 1 and 3. The alterations we have found vary

from deletion of 3 CA to addition of 3 CA. Two of the

59 patients presented constitutionally an insertion of

4 bp (2CA) and another two presented constitutionally

a deletion of 2 bp (1CA).

3.4. D568

There were alterations in the mononucleotide (C)

repeat region D568 in 3 out of the 66 (4.5%): one

adenoma and two follicular carcinomas. The adenoma

showed an insertion of 2 bp (2C), one follicular

carcinoma showed a mixture of mtDNA molecules

with a range varying from 8C to 10C in the D568

stretch—addition of 2 bp to 4 bp (C) and the other

follicular carcinoma presented an insertion of 1 bp

(1C) (Table 1). All the 59 patients presented

constitutionally 6C in the D568 region.

3.5. D310 vs. D514 vs. D568

The frequency of alterations/instability was higher

in D310 (30.3%, 20/66) and D514 (22.7%, 15/66) than

in D568 (4.6%, 3/66) repetitive region. Alteration-

s/instability in at least one of the 3 repetitive regions

(D310, D514 and D568) were found in 32/66 (48.5%)

tumors [15/30 (50.0%) adenomas; 9/19 (47.4%)

papillary carcinomas and 8/17 (47.1%) follicular

carcinomas]. Five of the 20 cases with instability in

Table 3

Somatic insertions/deletions (1-CA repeat or higher) in the dinucleotide D

Histotype No. of alterations (total) 1

Adenoma (nZ30) 3 (10.0%) 2

Carcinoma (nZ36) 12 (33.3%) 7

Follicular carcinoma (nZ17) 5 (29.4%) 4

Papillary carcinoma (nZ19) 7 (36.8%) 3

PZ0.038 (Adenomas vs. carcinomas)

D310 region harbored also instability in one (four

cases) or both (one case) of the other repetitive

regions (D514 and D568): four cases presented

alterations in D310 and D514 regions and two cases

presented alterations in D310 and D568 regions; one

follicular carcinoma had alterations in the 3 repetitive

regions (Table 1).

4. Discussion

We detected mtDNA D-Loop somatic mutations in

almost fifty percent of the tumors, both benign and

malignant, thus supporting the assumption that these

alterations are early and common events in thyroid

carcinogenesis.

The mutations detected in our series were either

point mutation (transitions) which are the type of

mutations that may be expected as a consequence of

ROS damage (Backer et al., 1980; Beckman and

Ames, 1997; Cadet et al., 1997) or variations of

repetitive elements in monorepetitive (C) and

direpetitive (CA) D-Loop regions. Most of the

alterations we detected in D310, D514 and D568

regions were in the polymorphic length range

described previously (MITOMAP: A Human Mito-

chondrial Genome Database, 2001; Hauswirth and

Clayton, 1985; Cavelier et al., 2000; Wallace and

Mitochondrial, 1994), thus suggesting that most

mtDNA D-Loop variations detected by us in thyroid

514 repeat region in 66 thyroid tumors

CA repeat O1CA repeat

(6.7%) 1 (3.3%)

(19.4%) 5 (13.9%)

(23.5%) 1 (5.9%)

(15.8%) 4 (21.1%)

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tumors are unlikely to lead to functional impairment

of the mitochondria. Such alterations result probably

from the high ROS production in tumor cells and/or

from the high replication rate of the neoplastic cells.

Five of the 20 D310 alterations were outside the

usual polymorphic length range (between 7C and 9C)

and included unusual variants (10C, 11C and 12C).

Some of the observed D310 variants harbor large

C-tract insertions that are likely to interfere with the

initiation of mtDNA replication (Xu and Clayton,

1995; Lee and Clayton, 1998; Hauswirth and Clayton,

1985). These findings suggest that, at variance with

the aforementioned common D-Loop variations, these

unusual alterations may lead to the functional

impairment of the mitochondria. It remains to be

proved that these alterations are originated during

tumor development and may play a role in tumorigen-

esis (promoting the growth advantage of tumor cells).

Interestingly, the frequency of somatic mutations

in D310 region is higher, though not significantly

(PZ0.178), in adenomas than in carcinomas. This

finding fits with Sanchez-Cespedes et al. (2001)

description of D310 alterations in pre-neoplastic

lesions of head and neck tumors and indicates that

these alterations occur early in human carcinogenesis

(Sanchez-Cespedes et al., 2001). More recently,

Miyazono et al. (2002) verified that mtDNA D-Loop

mutations occur early and frequently in adenocarci-

nomas arising from Barrett’s esophagus.

Our results confirm that D310 is a major target

for mutations in human primary tumors

(Sanchez-Cespedes et al., 2001). The rate of D310

alterations (30.3%) lies in between the values

described by Sanchez-Cespedes et al. (2001) (0% in

ovarian and prostate tumors and 62.5% in gastric

tumors). The assumption that D310 was a hotspot was

indirectly confirmed by a very low rate of mutations/

instability in the other polymorphic homopolymeric C

stretch (D568).

The high prevalence (22.7%) of somatic mutations

in the dinucleotide D514 region identifies this stretch

as another mutational hotspot. The significantly

higher rate of D514 alterations in carcinomas than

in adenomas supports the assumption that such

alterations may be related to tumor progression, but

the study of a larger series of benign and malignant

tumors of the thyroid and other organs is necessary

before drawing any definitive conclusion. MtDNA

alterations, in other regions besides D-Loop, seem to

play also a role in tumor development. In previous

studies, our group (Maximo et al., 2002) and others

(Yeh et al., 2000), verified that mtDNA mutations/

variants, namely mtDNA complex I genes somatic

missense mutations, as well as mtDNA complex I

genes variants, are more common in human thyroid

malignant tumors, suggesting that mtDNA mutations

may play a role in tumor progression (Maximo et al.,

2002; Yeh et al., 2000). Recently, Petros et al. (2005)

and Shidara et al. (2005) demonstrated that mtDNA

mutations confer a positive contribution to the

promotion of cancer via the prevention from apoptosis

(Petros et al., 2005; Shidara et al., 2005). Taken

together, the aforementioned data suggest that

mtDNA mutations may play a role in tumor

progression. Our group has verified, furthermore,

that mutations in nuclear genes involved in the MRC

can also play a role in the causation and/or

development of neoplasms, namely Hurthle cell

tumors of the thyroid (Maximo et al., 2005).

The coexistence of deletions/insertions at two or

even three repetitive regions of the D-Loop in the

same tumors, reinforces the concept of mitochondrial

microsatellite instability (mtMSI) as advanced by

(Habano et al., 1998; Habano et al., 2000). The term

mtMSI was introduced by (Habano et al., 1998, 2000),

in a study on colorectal tumors, to describe alterations

(small insertions/deletions) in repetitive regions of

mtDNA D-Loop in analogy with the nuclear

microsatellite instability (nMSI). Hypermutable status

(instability) in the nuclear genome has been observed

in certain types of cancers, in which microsatellite

sequences are preferentially affected due to slippage

during the replication process (Aaltonen et al.,1993;

Ionov et al., 1993; Thibodeau et al., 1993). These

replication error phenotypes are related to functional

loss of mismatch repair (MMR) genes, including

hMSH2, hMLH1, hPMS1 and hPMS2 genes (Parsons

et al. 1993; Boyer et al., 1995). In the mitochondrial

genome, the MMR system has been found only in

yeast strains, in which MSH1 and MSH2 are

separately involved in mitochondrial and nuclear

DNA repair systems, respectively (Reenan and

Kolodner, 1992). No MSH1 homologue has been

found in mammalian cells and, therefore, it remains

uncertain whether or not an MMR system plays a role

in the maintenance of mammalian mitochondrial

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genome. (Habano et al., 1998, 2000) suggested that

this mtDNA instability was related with nuclear

microsatellite instability (nMSI), but we (Maximo

et al., 2001) and others (Richard et al., 2000) found

that these of alterations were not related to each other,

at least in thyroid (Maximo et al., 2001) and breast

(Richard et al., 2000) tumors. It seems likely that

oxidative damage plays a role in the acquisition of

concurrent alterations in several repetitive regions of

the D-Loop (Backer et al., 1980; Beckman and Ames,

1997; Cadet et al., 1997), but further studies are

necessary to clarify this point.

Regardless of the mechanism(s) that lead(s) to

mtDNA instability it is surprising the different

frequencies of instability in the different repetitive

regions studied, namely those that are similar, such as

D310 and D568 mononucleotide repetitive regions.

The different frequencies of alterations in the three

distinct mtDNA D-Loop regions may reflect the

importance of each region in mitochondrial biogen-

esis and/or tumor transformation, or differences on the

intrinsic susceptibility for mutations.

The different rates of D310 detected by Sanchez-

Cespedes et al. (2001) and Miyazono et al. (2002) in

different tumor types suggest the existence of

alternative mechanisms for the generation of some

D310 alterations, such as the rate of acquired

mutations during tumor development, the number of

mitochondria per cell, or the intrinsic differences in

the number of cell divisions prior to tumor formation

in different tissues. The results of the present study

and the findings of Sanchez-Cespedes et al. (2001)

support the assumption that mtDNA somatic

mutations may play different roles in different tumor

types. It remains to be proved, however, the

pathogenic role of mtDNA D-Loop instability in

human cancer, namely in thyroid tumors.

Acknowledgements

This study was partially supported by a Ph.D.

Grant (SFRH/BD/8425/2002 - JL) and by a Post-Doc

Grant (SFRH/BPD/14594/2003 - VM) from the

Portuguese Science and Technology Foundation

(FCT) and by further funding from the same source

(Project - POCT/41055/NSE/2001).

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