Disruptive mitochondrial DNA mutations in complex I subunits are markers of oncocytic phenotype in thyroid tumors

Abstract

Oncocytic tumors are a distinctive class of proliferative lesions composed of cells with a striking degree of mitochondrial hyperplasia that are particularly frequent in the thyroid gland. To understand whether specific mitochondrial DNA (mtDNA) mutations are associated with the accumulation of mitochondria, we sequenced the entire mtDNA in 50 oncocytic lesions (45 thyroid tumors of epithelial cell derivation and 5 mitochondrion-rich breast tumors) and 52 control cases (21 nononcocytic thyroid tumors, 15 breast carcinomas, and 16 gliomas) by using recently developed technology that allows specific and reliable amplification of the whole mtDNA with quick mutation scanning. Thirteen oncocytic lesions (26%) presented disruptive mutations (nonsense or frameshift), whereas only two samples (3.8%) presented such mutations in the nononcocytic control group. In one case with multiple thyroid nodules analyzed separately, a disruptive mutation was found in the only nodule with oncocytic features. In one of the five mitochondrion-rich breast tumors, a disruptive mutation was identified. All disruptive mutations were found in complex I subunit genes, and the association between these mutations and the oncocytic phenotype was statistically significant (P=0.001). To study the pathogenicity of these mitochondrial mutations, primary cultures from oncocytic tumors and corresponding normal tissues were established. Electron microscopy and biochemical and molecular analyses showed that primary cultures derived from tumors bearing disruptive mutations failed to maintain the mutations and the oncocytic phenotype. We conclude that disruptive mutations in complex I subunits are markers of thyroid oncocytic tumors.

Fig. 1.

Analysis of separate hyperplastic nodules with and without oncocytic phenotype from the same thyroid (case HTC1, Table 1). (Upper) Histologic appearance of the hyperplastic oncocytic nodule (A) and of one hyperplastic nodule without oncocytic change (B). Immunohistochemistry with antibodies specific for human mitochondria confirms the increased mitochondrial mass in the oncocytic nodule (C) compared with the nononcocytic one (D). (Lower) The heteroplasmic nonsense mutation of the ND5 (G13414A) gene in the oncocytic and nononcocytic nodule. (Magnification: ×400.)

Fig. 2.

Disruptive mtDNA mutation in a mitochondrion-rich breast carcinoma (case BRCA13, Table 1). (A) Electropherograms showing the heteroplasmic deletion in the ND1 gene in tumor (Upper) and perilesional normal (Lower) tissue. (B) Histologic appearance of the breast carcinoma showing neoplastic cells with abundant eosinophilic cytoplasm and oncocytic features. (C and D) Immunohistochemistry with antibodies specific for human mitochondria confirms the increased mitochondrial mass in the tumor (D) compared with the nonneoplastic perilesional breast parenchyma (C). (Magnification: ×400.)

Fig. 3.

Characterization of normal and tumor primary cell cultures. (A–C) Ultrastructure of thyroid tumor biopsy shows mitochondrial hyperplasia (A Upper) lacking in normal and tumor primary cultures (B Upper and C Upper). Electropherograms showing the mutated base (bold and underlined) in the tumor biopsy and the wild-type base at the same position in both normal and tumor primary culture (A Lower, B Lower, and C Lower). (D–F) Citrate synthase activity (D), total ATP levels in glucose (E), and activity during incubation in galactose-containing medium (F) are shown. Data points F are means ± SD of at least five different experiments.