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Mitochondrial DNA Somatic Mutations (Point Mutations and Large Deletions) and Mitochondrial DNA Variants in Human Thyroid Pathology

A Study with Emphasis on Hürthle Cell Tumors
      In an attempt to progress in the understanding of the relationship of mitochondrial DNA (mtDNA) alterations and thyroid tumorigenesis, we studied the mtDNA in 79 benign and malignant tumors (43 Hürthle and 36 non-Hürthle cell neoplasms) and respective normal parenchyma. The mtDNA common deletion (CD) was evaluated by semiquantitative polymerase chain reaction. Somatic point mutations and sequence variants of mtDNA were searched for in 66 tumors (59 patients) and adjacent parenchyma by direct sequencing of 70% of the mitochondrial genome (including all of the 13 OXPHOS system genes). We detected 57 somatic mutations, mostly transitions, in 34 tumors and 253 sequence variants in 59 patients. Follicular and papillary carcinomas carried a significantly higher prevalence of nonsilent point mutations of complex I genes than adenomas. We also detected a significantly higher prevalence of complex I and complex IV sequence variants in the normal parenchyma adjacent to the malignant tumors. Every Hürthle cell tumor displayed a relatively high percentage (up to 16%) of mtDNA CD independently of the lesion’s histotype. The percentage of deleted mtDNA molecules was significantly higher in tumors with D-loop mutations than in mtDNA stable tumors. Sequence variants of the ATPase 6 gene, one of the complex V genes thought to play a role in mtDNA maintenance and integrity in yeast, were significantly more prevalent in patients with Hürthle cell tumors than in patients with non-Hürthle cell neoplasms. We conclude that mtDNA variants and mtDNA somatic mutations of complex I and complex IV genes seem to be involved in thyroid tumorigenesis. Germline polymorphisms of the ATPase 6 gene are associated with the occurrence of mtDNA CD, the hallmark of Hürthle cell tumors.
      Hürthle (oxyphil) cells are found in a minority of thyroid tumors, either benign (Hürthle cell adenoma) or malignant (Hürthle cell variants of follicular and papillary carcinoma), as well as in other types of thyroid tumors and several nonneoplastic thyroid disorders.
      • Müller-Höcker J
      • Jacob U
      • Seibel P
      Hashimoto thyroiditis is associated with defects of cytochrome-c oxidase in oxyphil Askanazy cells and with the common deletion (4,977) of mitochondrial DNA.
      • Máximo V
      • Sobrinho-Simões M
      Hurthle cell tumours of the thyroid. A review with emphasis on mitochondrial abnormalities with clinical relevance.
      Hürthle cells are characterized by a large, granular, eosinophilic cytoplasm, which is filled with abnormal mitochondria. Most Hürthle cell tumors are sporadic and frequently occur in association with autoimmune thyroiditis, but their occurrence in a familial setting has also been reported.
      • Katoh R
      • Harach HR
      • Williams ED
      Solitary, multiple, and familial oxyphil tumours of the thyroid gland.
      • Canzian F
      • Amati P
      • Harach HR
      • Kraimps JL
      • Lesueur F
      • Barbier J
      • Levillain P
      • Romeo G
      • Bonneau D
      A gene predisposing to familial thyroid tumors with cell oxyphilia maps to chromosome 19p13.2.
      The abundance of abnormal mitochondria makes Hürthle cell tumors a good model to study mtDNA abnormalities in human cancer.
      Mitochondrial DNA (mtDNA) is thought to be more susceptible than nuclear DNA to mutagen-induced damage for several reasons: mtDNA polymerase γ replicates the DNA with poor fidelity,
      • Kunkel TA
      • Loeb LA
      Fidelity of mammalian DNA polymerases.
      mtDNA is a naked (without histones) molecule to which chemical carcinogens can easily bind,
      • Backer JM
      • Weinstein IB
      Mitochondrial DNA is a major cellular target for a dihydrodiol-epoxide derivative of benzo[a]pyrene.
      • Allen JA
      • Coombs MM
      Covalent binding of polycyclic aromatic compounds to mitochondrial and nuclear DNA.
      and mtDNA is particularly susceptible to the high concentration of reactive oxygen species in mitochondria.
      • Oberley LW
      • Buettner GR
      Role of superoxide dismutase in cancer: a review.
      Nuclear microsatellite instability (nMSI) is related to functional loss of mismatch repair genes, including the hMSH2, hMLH1, hPMS1, and hPMS2 genes.
      • Parsons R
      • Li GM
      • Longley MJ
      • Fang WH
      • Papadopoulos N
      • Jen J
      • de la Chapelle A
      • Kinzler KW
      • Vogelstein B
      • Modrich P
      Hypermutability and mismatch repair deficiency in RER+ tumor cells.
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      • Umar A
      • Risinger JI
      • Lipford JR
      • Kane M
      • Yin S
      • Barrett JC
      • Kolodner RD
      • Kunkel TA
      Microsatellite instability, mismatch repair deficiency, and genetic defects in human cancer cell lines.
      In the mitochondrial genome, the mismatch repair 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 RA
      • Kolodner RD
      Characterization of insertion mutations in the Saccharomyces cerevisiae MSH1 and MSH2 genes: evidence for separate mitochondrial and nuclear functions.
      No MSH1 homologue has been found in mammalian cells and it remains uncertain whether a mismatch repair system plays a role in the maintenance of the mammalian mitochondrial genome.
      The term mitochondrial microsatellite instability (mtMSI) was introduced by Habano and colleagues,
      • Habano W
      • Nakamura S
      • Sugai T
      Microsatellite instability in the mitochondrial DNA of colorectal carcinomas: evidence for mismatch repair systems in mitochondrial genome.
      in a study on colorectal tumors, to describe alterations in repetitive regions of mtDNA. For the evaluation of mtMSI, Habano and colleagues
      • Habano W
      • Nakamura S
      • Sugai T
      Microsatellite instability in the mitochondrial DNA of colorectal carcinomas: evidence for mismatch repair systems in mitochondrial genome.
      quantified the alterations in two simple repeat sequences in a noncoding displacement-loop (D-loop) region in mtDNA. Additional studies have addressed the issue of mtMSI in human cancers
      • Tamura G
      • Nishizuka S
      • Maesawa C
      • Suzuki Y
      • Iwaya T
      • Sakata K
      • Endoh Y
      • Motoyama T
      Mutations in mitochondrial control region DNA in gastric tumours of Japanese patients.
      • Habano W
      • Sugai T
      • Nakamura SI
      • Uesugi N
      • Yoshida T
      • Sasou S
      Microsatellite instability and mutation of mitochondrial and nuclear DNA in gastric carcinoma.
      • Richard SM
      • Bailliet G
      • Paez GL
      • Bianchi MS
      • Peltomaki P
      • Bianchi NO
      Nuclear and mitochondrial genome instability in human breast cancer.
      • Máximo V
      • Soares P
      • Seruca R
      • Sobrinho-Simoe˜s M
      Comments on mutations in mitochondrial control region DNA in gastric tumours of Japanese patients.
      • Máximo V
      • Soares P
      • Seruca R
      • Rocha AS
      • Castro P
      • Sobrinho-Simões M
      Microsatellite instability, mitochondrial DNA large deletions, and mitochondrial DNA mutations in gastric carcinoma.
      without reaching concordant conclusions about the relationship between the instability of nuclear and mitochondrial genomes.
      • Habano W
      • Nakamura S
      • Sugai T
      Microsatellite instability in the mitochondrial DNA of colorectal carcinomas: evidence for mismatch repair systems in mitochondrial genome.
      • Tamura G
      • Nishizuka S
      • Maesawa C
      • Suzuki Y
      • Iwaya T
      • Sakata K
      • Endoh Y
      • Motoyama T
      Mutations in mitochondrial control region DNA in gastric tumours of Japanese patients.
      • Habano W
      • Sugai T
      • Nakamura SI
      • Uesugi N
      • Yoshida T
      • Sasou S
      Microsatellite instability and mutation of mitochondrial and nuclear DNA in gastric carcinoma.
      • Richard SM
      • Bailliet G
      • Paez GL
      • Bianchi MS
      • Peltomaki P
      • Bianchi NO
      Nuclear and mitochondrial genome instability in human breast cancer.
      • Máximo V
      • Soares P
      • Seruca R
      • Sobrinho-Simoe˜s M
      Comments on mutations in mitochondrial control region DNA in gastric tumours of Japanese patients.
      • Máximo V
      • Soares P
      • Seruca R
      • Rocha AS
      • Castro P
      • Sobrinho-Simões M
      Microsatellite instability, mitochondrial DNA large deletions, and mitochondrial DNA mutations in gastric carcinoma.
      Alterations of mtDNA have been demonstrated in various types of human cancer and include large deletions, missense mutations, frameshift mutations, and small deletions/insertions.
      • Müller-Höcker J
      • Jacob U
      • Seibel P
      Hashimoto thyroiditis is associated with defects of cytochrome-c oxidase in oxyphil Askanazy cells and with the common deletion (4,977) of mitochondrial DNA.
      • Habano W
      • Nakamura S
      • Sugai T
      Microsatellite instability in the mitochondrial DNA of colorectal carcinomas: evidence for mismatch repair systems in mitochondrial genome.
      • Tamura G
      • Nishizuka S
      • Maesawa C
      • Suzuki Y
      • Iwaya T
      • Sakata K
      • Endoh Y
      • Motoyama T
      Mutations in mitochondrial control region DNA in gastric tumours of Japanese patients.
      • Habano W
      • Sugai T
      • Nakamura SI
      • Uesugi N
      • Yoshida T
      • Sasou S
      Microsatellite instability and mutation of mitochondrial and nuclear DNA in gastric carcinoma.
      • Richard SM
      • Bailliet G
      • Paez GL
      • Bianchi MS
      • Peltomaki P
      • Bianchi NO
      Nuclear and mitochondrial genome instability in human breast cancer.
      • Máximo V
      • Soares P
      • Seruca R
      • Sobrinho-Simoe˜s M
      Comments on mutations in mitochondrial control region DNA in gastric tumours of Japanese patients.
      • Máximo V
      • Soares P
      • Seruca R
      • Rocha AS
      • Castro P
      • Sobrinho-Simões M
      Microsatellite instability, mitochondrial DNA large deletions, and mitochondrial DNA mutations in gastric carcinoma.
      • Máximo V
      • Soares P
      • Rocha AS
      • Sobrinho-Simões M
      The common deletion of mitochondrial DNA is found in goitres and thyroid tumors with and without oxyphil cell change.
      • Burgart LJ
      • Zheng J
      • Shu Q
      • Strickler JG
      • Shibata D
      Somatic mitochondrial mutation in gastric cancer.
      • Habano W
      • Sugai T
      • Yoshida T
      • Nakamura S
      Mitochondrial gene mutation, but not large-scale deletion, is a feature of colorectal carcinomas with mitochondrial microsatellite instability.
      • Polyak K
      • Li Y
      • Zhu H
      • Lengauer C
      • Willson JK
      • Markowitz SD
      • Trush MA
      • Kinzler KW
      • Vogelstein B
      Somatic mutations of the mitochondrial genome in human colorectal tumours.
      • Fliss MS
      • Usadel H
      • Caballero OL
      • Wu L
      • Buta MR
      • Eleff SM
      • Jen J
      • Sidransky D
      Facile detection of mitochondrial DNA mutations in tumors and bodily fluids.
      • Yeh JJ
      • Lunetta KL
      • van Orsouw NJ
      • Moore FD
      • Mutter GL
      • Vijg J
      • Dahia PL
      • Eng C
      Somatic mitochondrial DNA (mtDNA) mutations in papillary thyroid carcinomas and differential mtDNA sequence variants in cases with thyroid tumours.
      • Máximo V
      • Sobrinho-Simões M
      Mitochondrial DNA ‘common’ deletion in Hurthle cell lesions of the thyroid.
      • Lewis PD
      • Baxter P
      • Paul Griffiths A
      • Parry JM
      • Skibinski DO
      Detection of damage to the mitochondrial genome in the oncocytic cells of Warthin's tumour.
      • Tallini G
      • Ladanyi M
      • Rosai J
      • Jhanwar SC
      Analysis of nuclear and mitochondrial DNA alterations in thyroid and renal oncocytic tumors.
      mtDNA is a hot spot for mutations in cancer as it is preferentially damaged by many carcinogens.
      • Backer JM
      • Weinstein IB
      Mitochondrial DNA is a major cellular target for a dihydrodiol-epoxide derivative of benzo[a]pyrene.
      • Allen JA
      • Coombs MM
      Covalent binding of polycyclic aromatic compounds to mitochondrial and nuclear DNA.
      The role of mtDNA somatic mutations in this setting is not yet understood.
      • Habano W
      • Sugai T
      • Nakamura SI
      • Uesugi N
      • Yoshida T
      • Sasou S
      Microsatellite instability and mutation of mitochondrial and nuclear DNA in gastric carcinoma.
      • Polyak K
      • Li Y
      • Zhu H
      • Lengauer C
      • Willson JK
      • Markowitz SD
      • Trush MA
      • Kinzler KW
      • Vogelstein B
      Somatic mutations of the mitochondrial genome in human colorectal tumours.
      • Fliss MS
      • Usadel H
      • Caballero OL
      • Wu L
      • Buta MR
      • Eleff SM
      • Jen J
      • Sidransky D
      Facile detection of mitochondrial DNA mutations in tumors and bodily fluids.
      • Yeh JJ
      • Lunetta KL
      • van Orsouw NJ
      • Moore FD
      • Mutter GL
      • Vijg J
      • Dahia PL
      • Eng C
      Somatic mitochondrial DNA (mtDNA) mutations in papillary thyroid carcinomas and differential mtDNA sequence variants in cases with thyroid tumours.
      We have previously detected the mitochondrial common deletion (mtDNA CD) in a small series of thyroid tumors composed of Hürthle (oxyphil) cells, as well as in some nonneoplastic thyroid lesions with incipient Hürthle cell changes.
      • Máximo V
      • Soares P
      • Rocha AS
      • Sobrinho-Simões M
      The common deletion of mitochondrial DNA is found in goitres and thyroid tumors with and without oxyphil cell change.
      • Máximo V
      • Sobrinho-Simões M
      Mitochondrial DNA ‘common’ deletion in Hurthle cell lesions of the thyroid.
      The mtDNA CD has also been detected in Hashimoto’s thyroiditis displaying oxyphilic cells.
      • Müller-Höcker J
      • Jacob U
      • Seibel P
      Hashimoto thyroiditis is associated with defects of cytochrome-c oxidase in oxyphil Askanazy cells and with the common deletion (4,977) of mitochondrial DNA.
      Very few studies analyzing mtDNA mutations in thyroid have been published to date.
      • Müller-Höcker J
      • Jacob U
      • Seibel P
      Hashimoto thyroiditis is associated with defects of cytochrome-c oxidase in oxyphil Askanazy cells and with the common deletion (4,977) of mitochondrial DNA.
      • Máximo V
      • Soares P
      • Rocha AS
      • Sobrinho-Simões M
      The common deletion of mitochondrial DNA is found in goitres and thyroid tumors with and without oxyphil cell change.
      • Yeh JJ
      • Lunetta KL
      • van Orsouw NJ
      • Moore FD
      • Mutter GL
      • Vijg J
      • Dahia PL
      • Eng C
      Somatic mitochondrial DNA (mtDNA) mutations in papillary thyroid carcinomas and differential mtDNA sequence variants in cases with thyroid tumours.
      • Máximo V
      • Sobrinho-Simões M
      Mitochondrial DNA ‘common’ deletion in Hurthle cell lesions of the thyroid.
      • Tallini G
      • Ladanyi M
      • Rosai J
      • Jhanwar SC
      Analysis of nuclear and mitochondrial DNA alterations in thyroid and renal oncocytic tumors.
      • Ebner D
      • Rodel G
      • Pavenstaedt I
      • Haferkamp O
      Functional and molecular analysis of mitochondria in thyroid oncocytoma.
      Such studies were limited by the small size of the samples and the small percentage of mtDNA analyzed per case.
      • Müller-Höcker J
      • Jacob U
      • Seibel P
      Hashimoto thyroiditis is associated with defects of cytochrome-c oxidase in oxyphil Askanazy cells and with the common deletion (4,977) of mitochondrial DNA.
      • Máximo V
      • Soares P
      • Rocha AS
      • Sobrinho-Simões M
      The common deletion of mitochondrial DNA is found in goitres and thyroid tumors with and without oxyphil cell change.
      • Yeh JJ
      • Lunetta KL
      • van Orsouw NJ
      • Moore FD
      • Mutter GL
      • Vijg J
      • Dahia PL
      • Eng C
      Somatic mitochondrial DNA (mtDNA) mutations in papillary thyroid carcinomas and differential mtDNA sequence variants in cases with thyroid tumours.
      • Máximo V
      • Sobrinho-Simões M
      Mitochondrial DNA ‘common’ deletion in Hurthle cell lesions of the thyroid.
      • Tallini G
      • Ladanyi M
      • Rosai J
      • Jhanwar SC
      Analysis of nuclear and mitochondrial DNA alterations in thyroid and renal oncocytic tumors.
      • Ebner D
      • Rodel G
      • Pavenstaedt I
      • Haferkamp O
      Functional and molecular analysis of mitochondria in thyroid oncocytoma.
      In an attempt to progress in the understanding of the putative relationship between mtDNA alterations in thyroid tumors in general, and Hürthle cell tumors in particular, we searched for mtDNA alterations in a large series of thyroid tumors, including both benign and malignant lesions, paying a special attention to the different histotypes of Hürthle cell neoplasms. In each case we have also analyzed the mtDNA of normal adjacent parenchyma in an attempt to find sequence variants of mtDNA putatively associated with the occurrence of Hürthle cell tumors.

      Materials and Methods

      Materials

      Seventy-nine thyroid tumors from 68 patients were studied. In 11 patients there were two distinct lesions that were separately studied. The 79 lesions were classified according to Hedinger and colleagues
      • Hedinger CE
      • Williams ED
      • Sobin LH
      Histological typing of thyroid tumors.
      and Rosai and colleagues
      • Rosai J
      • Carcangiu ML
      • DeLellis RA
      Tumors of the thyroid gland.
      as follicular adenoma (n = 15), follicular Hürthle cell adenoma (n = 20), follicular carcinoma (n = 5), follicular Hürthle cell carcinoma (n = 13), papillary carcinoma (n = 16), and papillary Hürthle cell carcinoma (n = 10). Samples from 32 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 47 cases, microdissected paraffin-embedded material was used for the screening of mtDNA mutations because of the absence of representative tumor tissue in the frozen samples.

      DNA Extraction

      DNA was extracted from microdissected frozen and/or paraffin-embedded pathological and normal thyroid tissue pairs using the NucleoSpin Tissue Kit (Macherey-Nagel, Düren, Germany).

      Screening of mtDNA CD by Polymerase Chain Reaction (PCR)

      The detection of mtDNA CD was performed using two sets of primers: Mitout-F and Mitout-R (outside the deletion region) and Mitin-F and Mitin-R (within the deletion region).
      • Máximo V
      • Soares P
      • Seruca R
      • Sobrinho-Simoe˜s M
      Comments on mutations in mitochondrial control region DNA in gastric tumours of Japanese patients.
      • Máximo V
      • Soares P
      • Rocha AS
      • Sobrinho-Simões M
      The common deletion of mitochondrial DNA is found in goitres and thyroid tumors with and without oxyphil cell change.
      In the wild-type mtDNA only the Mitin primer set gives a PCR product with 142 bp. In cases with the mtDNA CD, Mitin primers amplify a 142-bp target sequence and Mitout primers an aberrant PCR product with 214 bp.
      • Máximo V
      • Soares P
      • Seruca R
      • Sobrinho-Simoe˜s M
      Comments on mutations in mitochondrial control region DNA in gastric tumours of Japanese patients.
      PCR amplifications were performed in a 25-μl volume containing 200 μmol/L of each dNTP, 12.5 pmol of each of the forward and reverse primers, 50 mmol/L KCl, 10 mmol/L Tris-HCl, (pH 9.0), 1.5 mmol/L MgCl2, and 1 U of Taq DNA polymerase (Amersham Biosciences, Lda, Buckinghamshire, England). Cycling conditions were a single predenaturation step at 94°C for 5 minutes followed by 35 cycles of denaturation at 94°C for 20 seconds, annealing at 60°C for 20 seconds, and elongation at 72°C for 20 seconds, and a final incubation at 72°C for 2 minutes. PCR products were inspected by electrophoresis on 2% agarose gels.

      Semiquantitative PCR

      For the quantitation of the percentage of mtDNA molecules deleted in each sample, PCR co-amplification of two fragments of mtDNA (one within and the other outside the deletion region) were performed. PCR co-amplifications were performed in a 25-μl volume containing 200 μmol/L of each dNTP, 12.5 pmol of each of the forward and reverse of both sets of primers, 50 mmol/L KCl, 10 mmol/L Tris-HCl, (pH 9.0), 1.5 mmol/L MgCl2, and 1 U of Taq DNA polymerase (Amersham Biosciences, Lda). Cycling conditions were a single predenaturation step at 94°C for 5 minutes followed by 18 cycles of denaturation at 94°C for 20 seconds, annealing at 62°C for 20 seconds, and elongation at 72°C for 20 seconds, and a final incubation at 72°C for 2 minutes. PCR products were inspected by electrophoresis on 2% agarose gels. The optimal number of cycles of amplification to allow quantitation of the two PCR products was determined using three samples of normal thyroid. Two hundred ng of each DNA sample were subjected to a number of amplification cycles ranging from 10 to 25 cycles. PCR products were separated on a 2% agarose gel and stained with ethidium bromide. The intensity of the fluorescence was automatically measured and integrated with the genescan software Image Master (Amersham Biosciences, Lda). A close to exponential increase in the amount of PCR product was obtained between 15 and 23 cycles for both fragment products. In every semiquantitative PCR experiment 18 cycles were used. The determination of the optimal annealing temperature of the two sets of primers was performed using the same three samples of DNA of normal thyroid samples used in the determination of the optimal number of cycles for amplification. PCR triplicates of 200 ng of DNA of each sample were used for co-amplification of both fragments. Cycling conditions were a single predenaturation step at 94°C for 5 minutes followed by 18 cycles of denaturation at 94°C for 20 seconds, annealing varying between 54°C and 65°C for 20 seconds, and elongation at 72°C for 20 seconds, and a final incubation at 72°C for 2 minutes. PCR products were inspected by electrophoresis on 2% agarose gels. At 62°C the amount of PCR products of both fragments was similar. In all quantitation analyses, 18 cycles of PCR amplification and an annealing temperature of 62°C were used.

      Screening of mtDNA Somatic Mutations and mtDNA Variants by Direct Sequencing

      By PCR/direct sequencing we analyzed 66 thyroid tumors and the respective adjacent normal thyroid tissue, surgically excised from 59 patients. In the remaining 13 tumors the study could not be performed for technical reasons. In seven cases (patients 52 to 58) blood samples were also analyzed. Using fragments varying from 0.6 to 1.4 kb we have screened 70% of the mitochondrial genome: all mtDNA coding genes, 46% of tRNA genes (tRNAPhe, Gly, Lys, Asp, Leu1, Ser1, His, Leu2, Ile, Ser2, Glu, Arg) and 52% of D-loop region. Details of PCR primers and sequencing primers are available on request from the authors. All PCR amplifications were performed in a 25-μl volume containing 200 μmol/L of each dNTP, 12.5 pmol of each of the forward and reverse primers, 50 mmol/L KCl, 10 mmol/L Tris-HCl, (pH 9.0), 1.5 mmol/L MgCl2, and 1 U of Taq DNA polymerase (Amersham Biosciences Lda). Cycling conditions were a single predenaturation step at 94°C for 5 minutes followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 20 seconds, and elongation at 72°C for 1 minute, and a final incubation at 72°C for 5 minutes. PCR products were separated by electrophoresis on 2% agarose gels and purified using the NucleoSpin Extract Kit (Macherey-Nagel, Düren, Germany). Sequencing analysis was then performed on purified products using the ABI Prism dGTP BigDye Terminator Ready Reaction Kit (Perkin-Elmer, Foster City, CA) and an ABI Prism 377 DNA sequencer (Perkin-Elmer). Both strands were screened using the original primers. Sequences were compared against a comprehensive mitochondrial databank (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000). All mtDNA-altered samples were subjected to an additional complete analysis.

      Statistical Analysis

      The statistical analysis of the results was performed using the chi-square test with the Yates correction, Fisher’s exact test, and Student’s t-test. A nonparametric test (Mann-Whitney) was also used whenever appropriate. A P value <0.05 was considered statistically significant.

      Results

      MtDNA CD Screening

      The overall results are summarized in Table 1. The mtDNA CD was found in all Hürthle cell tumors (100%, n = 43), in 5 of 15 (33.3%) adenomas, and in 3 of 16 (18.8%) papillary carcinomas without Hürthle cell features. The occurrence of mtDNA CD was significantly associated (P < 0.001) with Hürthle cell tumors. The mtDNA CD was also detected at very low levels in the normal adjacent thyroid tissue of some cases (Table 1). The histological study of the specimens in which mtDNA CD was observed, including the positive peritumoral tissues, revealed either typical Hürthle cells or follicular cells with relatively abundant, granular, oxyphilic cytoplasm of a kind we have previously designated as incipient Hürthle cell transformation.
      • Máximo V
      • Sobrinho-Simões M
      Hurthle cell tumours of the thyroid. A review with emphasis on mitochondrial abnormalities with clinical relevance.
      • Máximo V
      • Soares P
      • Rocha AS
      • Sobrinho-Simões M
      The common deletion of mitochondrial DNA is found in goitres and thyroid tumors with and without oxyphil cell change.
      Table 1Screening and Evaluation of the mtDNA CD in Seventy-Nine Tumors and in the Corresponding Adjacent Parenchyma
      In two cases there was no adjacent parenchyma available for study.
      DiagnosisNo. of casesPercentage of cases showing the mtDNA CD, %Percentage of deleted mtDNA (mean ± SD), %
      AP1500
      A1533.30.5 ± 0.7
      AP2025.00.3 ± 1.0
      HCA201004.3 ± 1.6
      AP500
      FC500
      AP1233.30.3 ± 0.4
      HCFC131007.4 ± 2.2
      AP1612.50.04 ± 0.2
      PC1618.80.2 ± 0.3
      AP933.30.1 ± 0.3
      HCPC101004.3 ± 1.6
      AP, Adjacent parenchyma; A, adenoma; HCA, Hürthle cell adenoma; FC, follicular carcinoma; HCFC, Hürthle cell follicular carcinoma; PC, papillary carcinoma; HCPC, Hürthle cell papillary carcinoma.
      * In two cases there was no adjacent parenchyma available for study.

      Percentage of mtDNA CD

      The average percentage and the SD of mtDNA CD in the lesions of each group are summarized in Table 1. The amount of mtDNA CD was significantly higher (P < 0.0001) in Hürthle cell tumors than in non-Hürthle cell tumors independently of the benignity or malignancy of the lesions. The percentage of mtDNA CD was significantly higher (P < 0.0001 and P = 0.0012, respectively) in Hürthle cell follicular carcinomas (7.4 ± 2.2%) than in Hürthle cell adenomas (4.3 ± 1.6%) and Hürthle cell papillary carcinomas (4.3 ± 1.6%) (Table 1).

      MtDNA Somatic Point Mutations

      D-Loop Region

      The results of the screening of D-loop somatic mutations are summarized in Table 2. Most of the mutations found in the D-loop region were located in repetitive regions. Following Habano and colleagues
      • Habano W
      • Sugai T
      • Yoshida T
      • Nakamura S
      Mitochondrial gene mutation, but not large-scale deletion, is a feature of colorectal carcinomas with mitochondrial microsatellite instability.
      we classified the tumors displaying somatic mutations in repetitive regions (variations in the number of repetitive units) as lesions with mtDNA D-loop instability. MtDNA D-loop instability was detected in 32 of 66 (48.5%) tumors (Table 2). By tumor type, mtDNA D-loop instability was found in 3 of 10 (30.0%) adenomas, 12 of 20 (60.0%) Hürthle cell adenomas, 1 of 4 (25.0%) follicular carcinomas, 7 of 13 (53.9%) Hürthle cell follicular carcinomas, 5 of 12 (41.7%) papillary carcinomas, and in 4 of 7 (57.1%) Hürthle cell papillary carcinomas. The percentage of tumors with D-loop instability is similar (P = 0.822) in benign (50.0%, 15 of 30) and in malignant tumors (47.2%, 17 of 36). The percentage of Hürthle cell tumors with mtDNA D-loop instability (57.5%, 23 of 40) is higher, although not significantly (P = 0.069), than the percentage of non-Hürthle cell tumors with mtDNA D-loop instability (34.6%, 9 of 26).
      Table 2Summary of mtDNA D-Loop Alterations in Sixty-Six Tumors
      CaseAgeDiagnosisNucleotide positionNucleotide change
      157PC514CA7 → CA4
      251HCFC303C6 → C6,7
      497C → T
      499G → A
      332HCFC303C6 → C8,9
      325C → T
      514CA5 → CA6
      568C6 → C8
      439HCFC568C6 → C8–10
      533HCFC460T → C
      514CA5 → CA7
      676HCFC514CA5 → CA6
      762HCPC303C7 → C8
      514CA5 → CA6
      949HCA303C6 → C8
      10A63PC303C7 → C8
      10B63HCA195T → C
      303C7 → C8
      12A45HCA303C6 → C8
      481C → T
      1434HCA303C7 → C9,10
      1559HCA303C7 → C8
      16A67HCA303C7 → C8
      456C → T
      16B67HCA303C7 → C8,9
      1757HCA462C → T
      303C7 → C8
      1838HCA207G → A
      303C7 → C8,9
      568C6 → C7
      1954HCFC115T → C
      514CA5 → CA6
      218HCPC185G → A
      514CA4 → CA4–6
      2246HCPC303C7 → C8
      514CA5 → CA6
      2748HCFC514CA5 → CA6
      2842A303C7 → C9,10
      3232HCPC303C7 → C9,10
      3953PC514CA5 → CA8
      549C → T
      4077HCA514CA5 → CA6,7
      4140FC150C → T
      303C7 → C8–10
      4667A514CA7 → CA6
      5154PC303C7 → C8–12
      325C → T
      514CA5 → CA6,7
      5337A98C del
      514CA4 → CA5
      5456HCA303C7 → C8
      5734HCA73A → G
      107G del
      303C7 → C8,9
      6056PC150C → T
      514CA5 → CA6

      Coding Genes/tRNAs

      The results of the screening of mtDNA somatic mutations in coding genes, including tRNAs genes, are summarized in Tables 3 4, and 5. We detected 57 mtDNA somatic mutations in 34 of the 66 (51.5%) tumors (Table 3). These somatic mutations include three deletions [one of 15 bp in the CytB gene (patient 3) and two in the COII gene, one of 573 bp (patient 3) and another of 569 bp (patient 9)]; three tRNA somatic point mutations, one in the tRNASer2 (patient 14), another in the tRNAAla (patient 30), and another in tRNAIle (patient 41); and 51 point mutations in the OXPHOS system genes (Table 3). From these 51 point mutations, 25 (49.0%) were silent mutations and 26 (51.0%) were missense mutations (Table 3); 44 of the 54 (81.5%) somatic point mutations (3 in tRNAs and 51 in OXPHOS system genes) were transitions. The technique we have used (automated direct sequencing) does not allow to decide with certainty if the mtDNA somatic mutations are homoplasmic or not, because it is not as sensitive as the manual sequencing. It is likely, however, that most somatic mutations are homoplasmic or near homoplasmic, because we have detected only a single signal in all but five cases in which two signals could be individualized. The same is also true for almost all mtDNA variants (see below). The results of the automated sequencing were confirmed in some samples using manual sequencing. The comparison of the prevalence of mutations in coding genes of the OXPHOS system in benign and malignant tumors did not yield any significant difference, but for the higher prevalence (P = 0.015) of missense point mutations in genes of the complex I in carcinomas than in adenomas (Table 4); the aforementioned missense mutations were detected in six of the seven genes of complex I (ND1, ND3, ND4, ND4L, ND5, and ND6) without any apparent concentration in a single gene (Table 3). The comparison of the prevalence of mutations in coding genes of the OXPHOS system in non-Hürthle cell and Hürthle cell tumors did not yield any significant differences (Table 5). There was however a trend (P = 0.096) toward a higher prevalence of missense point mutations in complex V genes in Hürthle cell tumors (Table 5); all of the missense mutations detected in complex V (n = 4) involved ATPase 6 gene (Table 3).
      Table 3Summary of mtDNA Somatic Mutations in Sixty-Six Tumors
      CaseDiagnosticAgeNucleotide positionGene/regionProtein
      1PC57C15280T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      CytB/CpIIISilent
      3HCFC32(14927–14941) del
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      CytB/CpIII5aa (TAFSS)
      (7631–8203) del
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      COII/CpIV191aa (16–206)
      4HCFC39A6650C
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      COI/CpIVSilent
      G9477TCOIII/CpIVV91F
      5HCFC33T14498A
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND6/CpIY59F
      6HCFC76G8697AATPase6/CpVSilent
      A8701GATPase6/CpVT59A
      C10272T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND3/CpIL272F
      7HCPC62G9746A
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      COIII/CpIVSilent
      8HCA60G9477ACOIII/CpIVV91I
      9HCA49A8701GATPase6/CpVT59A
      (7627–8195) del
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      COII/CpIVStop Codon
      10APC63C4940T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND2/CpISilent
      T7785C
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      COII/CpIVI67T
      10BHCA63T9137C
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ATPase6/CpVI204T
      11HCA67C12918T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND5/CpISilent
      C6473T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      COI/CpIVSilent
      12BPC45C9030T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ATPase6/CpVSilent
      G9575CCOIII/CpIVSilent
      14HCA34G12236AMTTS2
      15HCA59C7873T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      COII/CpIVSilent
      G8697AATPase6/CpVSilent
      A8706G
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ATPase6/CpVSilent
      16AHCA67C7873T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      COII/CpIVSilent
      17HCA57A15182G
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      CytB/CpIIII146V
      A4985GND2/CpISilent
      18HCA38A8716G
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ATPase6/CpVK64E
      20HCPC73C11332TND4/CpISilent
      G7775A
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      COII/CpIVV64I
      22HCPC46G9655ACOIII/CpIVS150N
      23HCFC71C10269T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND3/CpIL271F
      G14560T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND6/CpISilent
      25HCPC8A4613G
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND2/CpISilent
      G9477ACOIII/CpIVV91I
      27HCFC48C3992T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND1/CpIT229M
      C13943T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND5/CpIT536M
      28A42C10793T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND4//CpIL212L1 (Silent)
      30A54C5633TMTTA
      C7819A
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      COII/CpIVSilent
      31A62C9691T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      COIII/CpIVA162V
      C7103T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      COI/CpIVSilent
      35HCFC69G10197C
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND3/CpIA47P
      40HCA77G11016A
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND4/CpIS286N
      A10639GND4L/CpIN57S2
      41FC40C4312TMTTI
      C10691G
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND4L/CpISilent
      44FC35G3910A
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND1/CpIE202K
      46A67T15312G
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      CytB/CpIIII189S2
      51PC54C3594T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND1/CpISilent
      G10320A
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND3/CpIV88I
      C11840T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND4/CpIL2361L1 (Silent)
      56A46C9296TCOIII/CpIVSilent
      C10181TND3/CpISilent
      59PC40G3526A
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND1/CpIA74T
      C13943T
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND5/CpIT536M
      62FC54A12967C
      Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      ND5/CpIT211P
      * Unpublished sequence variants, (MITOMAP: A Human Mitochondrial Genome Database. Center for Molecular Medicine, Emory University, Atlanta, GA. http://www.gen.emory.edu/mitomap.html, 2000).
      Table 4Summary of the mtDNA Somatic Point Mutations in Coding Genes of the OXPHOS System in Benign and Malignant Tumors
      DiagnosisCpICpIIICpIVCpV
      Total point mutations
       Benign5/30 (16.7%)P = 0.0772/30 (6.7%)P = 0.4507/30 (23.3%)P = 0.7004/30 (13.3%)P = 0.274
       Malignant13/36 (36.1%)1/36 (2.8%)7/36 (19.4%)2/36 (5.6%)
      Missense point mutations
       Benign1/30 (3.3%)P = 0.0152/30 (6.7%)P = 0.1162/30 (6.7%)P = 0.3433/30 (10.0%)P = 0.221
       Malignant9/36 (25.0%)0/36 (0.0%)5/36 (13.9%)1/36 (2.8%)
      Cp, Complex of the OXPHOS system.
      Table 5Summary of the mtDNA Somatic Point Mutations in Coding Genes of the OXPHOS System in Non-Hürthle and Hürthle Cell Tumors
      DiagnosisCpICpIIICpIVCpV
      Total point mutations
       Non-Hürthle8/26 (30.8%)P = 0.6072/26 (7.7%)P = 0.3225/26 (19.2%)P = 0.7511/26 (3.8%)P = 0.232
       Hürthle10/40 (25.0%)1/40 (2.5%)9/40 (22.5%)5/40 (12.5%)
      Missense point mutations
       Non-Hürthle4/26 (15.4%)P = 0.9661/26 (3.8%)P = 0.7552/26 (7.7%)P = 0.5350/26 (0.0%)P = 0.096
       Hürthle6/40 (15.0%)1/40 (2.5%)5/40 (12.5%)4/40 (10.0%)
      Cp, Complex of the OXPHOS system.

      Mutations in D-Loop Region Versus Prevalence of mtDNA CD and Mutations in Coding Genes

      The percentage of tumors with mtDNA CD was higher, although not significantly (P = 0.148), in the group of tumors with D-loop instability (78.1%, 25 of 32) than in the group without D-loop instability (61.8%, 21 of 34). The amount (mean ± SD) of mtDNA CD was significantly higher (P = 0.045) in tumors with D-loop instability (4.1 ± 3.2%) than in tumors without D-loop instability (2.6 ± 2.8%). The percentage of tumors with mtDNA somatic mutations was significantly higher (P = 0.026) in tumors with D-loop instability (65.6%, 21 of 32) than in tumors without D-loop instability (38.2%, 13 of 34).

      MtDNA Variants

      The results of the study of mtDNA variants in the adjacent parenchyma of the tumors are summarized in Table 6. In the seven patients from whom blood samples were available the mtDNA variants detected in the normal adjacent parenchyma were also present in blood samples (data not shown); the mtDNA sequence variants detected in normal adjacent parenchyma were thus considered as germinal mtDNA variants.
      Table 6Number Per Case (Mean ± SD) of the mtDNA Variants in the Normal Adjacent Parenchyma of Patients with Benign and Malignant Thyroid Tumors and in the Normal Adjacent Parenchyma of Patients with Non-Hürthle and Hürthle Cell Thyroid Tumors, by OXPHOS System Complex
      DiagnosisNumber of casesTotal variants mean ± SDComplex I variants mean ± SDComplex III variants mean ± SDComplex IV variants mean ± SDComplex V variants mean ± SD
      Benign303.2 ± 1.61.4 ± 0.90.4 ± 0.50.6 ± 0.80.8 ± 0.6
      Malignant364.4 ± 1.4P = 0.0022.2 ± 0.9P = 0.0010.4 ± 0.5P = 0.7501.1 ± 0.6P = 0.0050.7 ± 0.6P = 0.628
      Non-Hürthle263.6 ± 1.41.9 ± 1.00.4 ± 0.50.9 ± 0.70.4 ± 0.5
      Hürthle404.0 ± 1.7P = 0.3781.8 ± 1.0P = 0.6010.4 ± 0.5P = 0.5570.9 ± 0.8P = 0.8300.9 ± 0.6P < 0.001
      We detected 253 sequence variants mtDNA in the normal parenchyma of the 59 patients; 112 of such mtDNA variants have not been previously published (data not shown). The distribution of the mtDNA variants by complex of the OXPHOS system is the following: complex I, 119; complex III, 26; complex IV, 60; and complex V, 48.
      The comparison of the prevalence of mtDNA variants in patients with benign and malignant tumors shows a significant higher prevalence (P = 0.001 and P = 0.005, respectively) of complex I and complex IV variants in patients with malignant tumors than in patients with benign tumors (Table 6). Complex V variants were significantly more frequent (P < 0.001) in patients with Hürthle cell tumors than in patients with tumors without Hürthle cell features (Table 6).
      Almost all of the variants detected in complex V genes of patients with Hürthle cell tumors involved the ATPase 6 gene (91.9%, 34 of 37) and most of them were nonsilent (79.4%, 27 of 34). The same does not hold true regarding the variants detected in patients with non-Hürthle cell tumors [8 of 11 (72.7%) involved ATPase6; 2 (25.0%) were nonsilent and 6 (75.0%) were silent].

      Discussion

      The occurrence of mtDNA CD had been previously reported in Hürthle cell lesions of the thyroid
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      Detection of damage to the mitochondrial genome in the oncocytic cells of Warthin's tumour.
      The present study confirms the presence and abundance of mtDNA CD in Hürthle cell tumors regardless of their benign or malignant nature and their histotype. The mtDNA CD was also found, usually in extremely low levels, in the normal adjacent thyroid tissue of some tumors, thus supporting the concept that mtDNA CD precedes oxyphilic changes.
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      • Jacob U
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      Hashimoto thyroiditis is associated with defects of cytochrome-c oxidase in oxyphil Askanazy cells and with the common deletion (4,977) of mitochondrial DNA.
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      The common deletion of mitochondrial DNA is found in goitres and thyroid tumors with and without oxyphil cell change.
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      Detection of damage to the mitochondrial genome in the oncocytic cells of Warthin's tumour.
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      Kearns-Sayre syndrome: oncocytic transformation of choroid plexus epithelium.
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      Oncocytic transformation of choroid plexus epithelium.
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      Involvement of choroid plexus in mitochondrial encephalomyopathy (MELAS).
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      Coordinate expression of nuclear and mitochondrial genes involved in energy production in carcinoma and oncocytoma.
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      The unusual structures of the hot-regions flanking large-scale deletions in human mitochondrial DNA.
      Such deletions would result in most instances from the action of reactive oxygen species over the mtDNA.
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      Mitochondrial DNA is a major cellular target for a dihydrodiol-epoxide derivative of benzo[a]pyrene.
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      This assumption is supported by the results of the present study: 81.5% of the mtDNA somatic point mutations were transitions. The presence of mtDNA deletions in normal thyroid tissue may be the result of an age-related accumulation effect of endogenous oxidative damage, aggression of lymphocytes in autoimmune thyroid diseases, or, as observed in some mitochondrial degenerative diseases, the result of alterations in a nuclear gene, or genes, affecting the mitochondrial biogenesis.
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      The oxidative pressure that results from tumor development can also produce reactive oxygen species and mtDNA damage in some lesions.
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      Coordinate expression of nuclear and mitochondrial genes involved in energy production in carcinoma and oncocytoma.
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      Recently, Savagner and colleagues
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      showed that ATP production is deficient in a series of Hürthle cell tumors, mainly because of a coupling defect in mitochondria; this defect might be related to a twofold increase in UCP2 (uncoupling protein 2) expression.
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      suggested that mitochondrial proliferation could therefore be an adaptative response to the UCP2 overexpression or, alternatively, the UCP2 overexpression might be a response to the proliferation of mitochondria compensating for the decreased mitochondrial ATP synthesis because of OXPHOS abnormalities. The results we have obtained fit better with the latter hypothesis.
      The trend to an association between D-loop instability and occurrence and amount of mtDNA CD may be a consequence of the mtDNA CD rather than its cause. Taking into consideration that the mtDNA CD leads to a higher replicative rate as a sort of compensatory effect,
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      Kearns-Sayre syndrome: oncocytic transformation of choroid plexus epithelium.
      • Wallace DC
      Mitochondrial genetics: a paradigm for aging and degenerative diseases?.
      it is possible that the instability in mtDNA may result from the high rate of mtDNA replication. Alternatively, it has been suggested that D-loop instability indicates the existence of a deficient repair mechanism of mtDNA alterations;
      • Reenan RA
      • Kolodner RD
      Characterization of insertion mutations in the Saccharomyces cerevisiae MSH1 and MSH2 genes: evidence for separate mitochondrial and nuclear functions.
      • Habano W
      • Nakamura S
      • Sugai T
      Microsatellite instability in the mitochondrial DNA of colorectal carcinomas: evidence for mismatch repair systems in mitochondrial genome.
      • Habano W
      • Sugai T
      • Nakamura SI
      • Uesugi N
      • Yoshida T
      • Sasou S
      Microsatellite instability and mutation of mitochondrial and nuclear DNA in gastric carcinoma.
      such deficiency might explain the concurrent existence of mtDNA point mutations and mtDNA large deletions in tumors with D-loop instability.
      • Reenan RA
      • Kolodner RD
      Characterization of insertion mutations in the Saccharomyces cerevisiae MSH1 and MSH2 genes: evidence for separate mitochondrial and nuclear functions.
      • Habano W
      • Nakamura S
      • Sugai T
      Microsatellite instability in the mitochondrial DNA of colorectal carcinomas: evidence for mismatch repair systems in mitochondrial genome.
      • Habano W
      • Sugai T
      • Nakamura SI
      • Uesugi N
      • Yoshida T
      • Sasou S
      Microsatellite instability and mutation of mitochondrial and nuclear DNA in gastric carcinoma.
      Most of the mtDNA point mutations are the result of mtDNA aggression by environmental factors, namely by reactive oxygen species,
      • Backer JM
      • Weinstein IB
      Mitochondrial DNA is a major cellular target for a dihydrodiol-epoxide derivative of benzo[a]pyrene.
      • Allen JA
      • Coombs MM
      Covalent binding of polycyclic aromatic compounds to mitochondrial and nuclear DNA.
      • Polyak K
      • Li Y
      • Zhu H
      • Lengauer C
      • Willson JK
      • Markowitz SD
      • Trush MA
      • Kinzler KW
      • Vogelstein B
      Somatic mutations of the mitochondrial genome in human colorectal tumours.
      • Adelman R
      • Saul RL
      • Ames BN
      Oxidative damage to DNA: relation to species metabolic rate and life span.
      • Ames BN
      • Shigenaga MK
      • Hagen TM
      Oxidants, antioxidants, and the degenerative diseases of aging.
      whereas insertions/deletions could occur via a slipped replication mechanism.
      • Shoffner JM
      • Lott MT
      • Voljavec AS
      • Soueidan SA
      • Costigan DA
      • Wallace DC
      Spontaneous Kearns-Sayre/chronic external ophthalmoplegia plus syndrome associated with a mitochondrial DNA deletion: a slip-replication model and metabolic therapy.
      No mechanism related with nuclear mismatch repair genes has been found to be active in human mtDNA repair to date. In contrast with the results of Habano and colleagues
      • Habano W
      • Sugai T
      • Nakamura SI
      • Uesugi N
      • Yoshida T
      • Sasou S
      Microsatellite instability and mutation of mitochondrial and nuclear DNA in gastric carcinoma.
      our data do not support the assumption that the instability in mitochondrial D-loop region is related with nMSI, because the level of mtDNA instability detected in the present study (48.5%) is much higher than the nMSI found in any type of thyroid tumor.
      • Vermiglio F
      • Schlumberger M
      • Lazar V
      • Lefrere I
      • Bressac B
      Absence of microsatellite instability in thyroid carcinomas.
      • Soares P
      • dos Santos NR
      • Seruca R
      • Lothe RA
      • Sobrinho-Simoes M
      Benign and malignant thyroid lesions show instability at microsatellite loci.
      • Nikiforov YE
      • Nikiforova M
      • Fagin JA
      Prevalence of minisatellite and microsatellite instability in radiation-induced post-Chernobyl pediatric thyroid carcinomas.
      • Lazzereschi D
      • Palmirotta R
      • Ranieri A
      • Ottini L
      • Veri MC
      • Cama A
      • Cetta F
      • Nardi F
      • Colletta G
      • Mariani-Costantini R
      Microsatellite instability in thyroid tumours and tumour-like lesions.
      Furthermore, the histotypes with higher levels of mtDNA instability are those displaying Hürthle cell features in which nMSI was not detected.
      • Lazzereschi D
      • Palmirotta R
      • Ranieri A
      • Ottini L
      • Veri MC
      • Cama A
      • Cetta F
      • Nardi F
      • Colletta G
      • Mariani-Costantini R
      Microsatellite instability in thyroid tumours and tumour-like lesions.
      Nuclear microsatellite instability was also not detected in any of the 12 cases in the present series that were analyzed previously (data not shown).
      Our study documents a large number of somatic mtDNA mutations in all tumor histotypes. This high frequency is probably because of the high susceptibility of mtDNA to damage by mutagens.
      • Kunkel TA
      • Loeb LA
      Fidelity of mammalian DNA polymerases.
      • Backer JM
      • Weinstein IB
      Mitochondrial DNA is a major cellular target for a dihydrodiol-epoxide derivative of benzo[a]pyrene.
      • Allen JA
      • Coombs MM
      Covalent binding of polycyclic aromatic compounds to mitochondrial and nuclear DNA.
      • Oberley LW
      • Buettner GR
      Role of superoxide dismutase in cancer: a review.
      Homoplasmic mtDNA mutations may reflect a replicative advantage for mutated mtDNA copies,
      • Habano W
      • Sugai T
      • Nakamura SI
      • Uesugi N
      • Yoshida T
      • Sasou S
      Microsatellite instability and mutation of mitochondrial and nuclear DNA in gastric carcinoma.
      • Polyak K
      • Li Y
      • Zhu H
      • Lengauer C
      • Willson JK
      • Markowitz SD
      • Trush MA
      • Kinzler KW
      • Vogelstein B
      Somatic mutations of the mitochondrial genome in human colorectal tumours.
      • Fliss MS
      • Usadel H
      • Caballero OL
      • Wu L
      • Buta MR
      • Eleff SM
      • Jen J
      • Sidransky D
      Facile detection of mitochondrial DNA mutations in tumors and bodily fluids.
      a growth advantage for a cell containing certain mtDNA mutations,
      • Polyak K
      • Li Y
      • Zhu H
      • Lengauer C
      • Willson JK
      • Markowitz SD
      • Trush MA
      • Kinzler KW
      • Vogelstein B
      Somatic mutations of the mitochondrial genome in human colorectal tumours.
      • Fliss MS
      • Usadel H
      • Caballero OL
      • Wu L
      • Buta MR
      • Eleff SM
      • Jen J
      • Sidransky D
      Facile detection of mitochondrial DNA mutations in tumors and bodily fluids.
      and/or tumorigenic properties of mtDNA mutations.
      • Polyak K
      • Li Y
      • Zhu H
      • Lengauer C
      • Willson JK
      • Markowitz SD
      • Trush MA
      • Kinzler KW
      • Vogelstein B
      Somatic mutations of the mitochondrial genome in human colorectal tumours.
      • Yeh JJ
      • Lunetta KL
      • van Orsouw NJ
      • Moore FD
      • Mutter GL
      • Vijg J
      • Dahia PL
      • Eng C
      Somatic mitochondrial DNA (mtDNA) mutations in papillary thyroid carcinomas and differential mtDNA sequence variants in cases with thyroid tumours.
      Coller and colleagues
      • Coller HA
      • Khrapko K
      • Bodyak ND
      • Nekhaeva E
      • Herrero-Jimenez P
      • Thilly WG
      High frequency of homoplasmic mitochondrial DNA mutations in human tumors can be explained without selection.
      and Jones and colleagues
      • Jones JB
      • Song JJ
      • Hempen PM
      • Parmigiani G
      • Hruban RH
      • Kern SE
      Detection of mitochondrial DNA mutations in pancreatic cancer offers a “mass”-ive advantage over detection of nuclear DNA mutations.
      have recently shown, by a mathematical approach, that these mutations could also arise by chance without any physiological advantage or tumorigenic requirement; this possibility is difficult to reconciliate with the occurrence of selection of mtDNA-specific variants in several disease models.
      • Polyak K
      • Li Y
      • Zhu H
      • Lengauer C
      • Willson JK
      • Markowitz SD
      • Trush MA
      • Kinzler KW
      • Vogelstein B
      Somatic mutations of the mitochondrial genome in human colorectal tumours.
      • Dunbar DR
      • Moonie PA
      • Jacobs HT
      • Holt IJ
      Different cellular backgrounds confer a marked advantage to either mutant or wild-type mitochondrial genomes.
      • Hao H
      • Morrison LE
      • Moraes CT
      Suppression of a mitochondrial tRNA gene mutation phenotype associated with changes in the nuclear background.
      • Holt IJ
      • Harding AE
      • Morgan-Hughes JA
      Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies.
      • Jenuth JP
      • Peterson AC
      • Shoubridge EA
      Tissue-specific selection for different mtDNA genotypes in heteroplasmic mice.
      • Yoneda M
      • Chomyn A
      • Martinuzzi A
      • Hurko O
      • Attardi G
      Marked replicative advantage of human mtDNA carrying a point mutation that causes the MELAS encephalomyopathy.
      The aforementioned hypotheses do not apply equally to silent and nonsilent mutations. The former, which represent almost half of the mtDNA mutations detected in our series (49%, 25 of 51), may have arisen by chance.
      • Coller HA
      • Khrapko K
      • Bodyak ND
      • Nekhaeva E
      • Herrero-Jimenez P
      • Thilly WG
      High frequency of homoplasmic mitochondrial DNA mutations in human tumors can be explained without selection.
      • Jones JB
      • Song JJ
      • Hempen PM
      • Parmigiani G
      • Hruban RH
      • Kern SE
      Detection of mitochondrial DNA mutations in pancreatic cancer offers a “mass”-ive advantage over detection of nuclear DNA mutations.
      However, the same does not hold true for nonsilent mutations. The high frequency of mtDNA somatic mutations in the malignant tumors of our series, namely in complex I genes, suggests a role for these mutations in thyroid cancer. It has been advanced the presence of functionally relevant mtDNA mutations in some types of human tumors.
      • Polyak K
      • Li Y
      • Zhu H
      • Lengauer C
      • Willson JK
      • Markowitz SD
      • Trush MA
      • Kinzler KW
      • Vogelstein B
      Somatic mutations of the mitochondrial genome in human colorectal tumours.
      • Yeh JJ
      • Lunetta KL
      • van Orsouw NJ
      • Moore FD
      • Mutter GL
      • Vijg J
      • Dahia PL
      • Eng C
      Somatic mitochondrial DNA (mtDNA) mutations in papillary thyroid carcinomas and differential mtDNA sequence variants in cases with thyroid tumours.
      • Sanchez-Cespedes M
      • Parrella P
      • Nomoto S
      • Cohen D
      • Xiao Y
      • Esteller M
      • Jeronimo C
      • Jordan RC
      • Nicol T
      • Koch WM
      • Schoenberg M
      • Mazzarelli P
      • Fazio VM
      • Sidransky D
      Identification of a mononucleotide repeat as a major target for mitochondrial DNA alterations in human tumors.
      Cell fusion experiment showed that some mtDNA mutations, namely complex I mutations, are dominant, able to replace recipient mtDNA, and achieve homoplasmy in tissue culture quite rapidly.
      • Polyak K
      • Li Y
      • Zhu H
      • Lengauer C
      • Willson JK
      • Markowitz SD
      • Trush MA
      • Kinzler KW
      • Vogelstein B
      Somatic mutations of the mitochondrial genome in human colorectal tumours.
      These findings fit with the observation that most of the mtDNA somatic mutations described in human cancers are located in complex I genes.
      • Habano W
      • Sugai T
      • Nakamura SI
      • Uesugi N
      • Yoshida T
      • Sasou S
      Microsatellite instability and mutation of mitochondrial and nuclear DNA in gastric carcinoma.
      • Fliss MS
      • Usadel H
      • Caballero OL
      • Wu L
      • Buta MR
      • Eleff SM
      • Jen J
      • Sidransky D
      Facile detection of mitochondrial DNA mutations in tumors and bodily fluids.
      Yeh and colleagues
      • Yeh JJ
      • Lunetta KL
      • van Orsouw NJ
      • Moore FD
      • Mutter GL
      • Vijg J
      • Dahia PL
      • Eng C
      Somatic mitochondrial DNA (mtDNA) mutations in papillary thyroid carcinomas and differential mtDNA sequence variants in cases with thyroid tumours.
      reported an association of complex I variants and homoplasmic mtDNA mutations and the occurrence of thyroid papillary carcinomas. Follicular and papillary carcinomas of our series carried a significantly higher prevalence of nonsilent mtDNA point mutations of complex I genes than adenomas. We also detected a significantly higher prevalence of complex I and complex IV sequence variants in the normal parenchyma adjacent to the malignant tumors. Our data thus support the hypothesis advanced by Yeh and colleagues
      • Yeh JJ
      • Lunetta KL
      • van Orsouw NJ
      • Moore FD
      • Mutter GL
      • Vijg J
      • Dahia PL
      • Eng C
      Somatic mitochondrial DNA (mtDNA) mutations in papillary thyroid carcinomas and differential mtDNA sequence variants in cases with thyroid tumours.
      that the accumulation of some mtDNA variants and mtDNA somatic mutations seem to be involved in thyroid tumorigenesis.
      Our results on the detection of mtDNA sequence variants suggest the existence of an association between germline polymorphisms of ATPase 6 and the occurrence of Hürthle cell tumors. In man, complex V (ATP synthase) of the mitochondrion comprises 10 to 16 subunits encoded by nuclear DNA and two subunits (ATPase 6 and ATPase 8) encoded by mtDNA.
      • Wallace DC
      Mitochondrial genetics: a paradigm for aging and degenerative diseases?.
      ATP synthase complex, at least in yeast, is involved in mtDNA genome maintenance and ATPase 6 seems to be one of the most important elements in this phenomenon.
      • Contamine V
      • Picard M
      Maintenance and integrity of the mitochondrial genome: a plethora of nuclear genes in the budding yeast.
      • Nagley P
      • Farrell LB
      • Gearing DP
      • Nero D
      • Meltzer S
      • Devenish RJ
      Assembly of functional proton-translocating ATPase complex in yeast mitochondria with cytoplasmically synthesized subunit 8, a polypeptide normally encoded within the organelle.
      • Foury F
      • Tzagoloff A
      Localization on mitochondrial DNA of mutations leading to a loss of rutamycin-sensitive adenosine triphosphatase.
      • Guidot DM
      • McCord JM
      • Wright RM
      • Repine JE
      Absence of electron transport (Rho0 state) restores growth of a manganese-superoxide dismutase-deficient Saccharomyces cerevisiae in hyperoxia. Evidence for electron transport as a major source of superoxide generation in vivo.
      In yeast, mutations that abrogate the synthesis of any of the ATPase subunits, even those that are unclearly encoded, cause the same phenotype, ie, high production of petites variants [variants having mtDNA deleted molecules (rho) or no mtDNA at all (rho0)].
      • Contamine V
      • Picard M
      Maintenance and integrity of the mitochondrial genome: a plethora of nuclear genes in the budding yeast.
      • Foury F
      • Tzagoloff A
      Localization on mitochondrial DNA of mutations leading to a loss of rutamycin-sensitive adenosine triphosphatase.
      The role of ATP synthase genes in mtDNA maintenance may contribute to explain our findings. It is tempting to suggest that, like in yeast, ATPase 6 may have a role in mtDNA maintenance in humans. The polymorphisms of ATPase 6 could lead to a less efficient mtDNA replication and to mtDNA abnormalities that could contribute to the occurrence of mtDNA CD and Hürthle cell tumors. A larger series is needed to confirm this hypothesis and to find whether or not mutations in ATPase 6 gene can be used as a tool to follow Hürthle cell transformation.

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