Variants in mitochondrial tRNA gene may not be associated with thyroid carcinoma

With an incidence of 2.0%, thyroid carcinoma is the most common form of endocrine system malignancy [1,2]. Thyroid carcinomas are histologically classified as papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), anaplastic thyroid carcinoma (ATC) and medullary thyroid carcinoma (MTC), accounting for approximately 80.0, 15.0, 2.0 and 4.0% of all thyroid malignancies, respectively [3]. Decreased survival in patients with oncocytic carcinomas may be due to reduced competence in iodine uptake by the tumor cells, resulting in poor response to radioiodine treatment. However, to date, the molecular mechanism underlying this disease remain largely unknown.

Since Warburg proposed that cancer originated from a non neoplastic cell that adopted anaerobic metabolism as a means of survival after injury to its respiratory system [4], changes in the number, shape, and function of mitochondria have been reported in various cancers [5]. The mitochondrial genome is a closed double-stranded circular molecule consisting of 16,569 bp coding for 37 genes, including 13 polypeptides, 22 tRNAs and two rRNAs necessary for function of the respiratory chain [6]. Due to the lack of histone protection and a poor repair system, mtDNA is thought to be more susceptible than nuclear DNA to mutagen-induced damage [7]. Of these, mt-tRNA is the hot-spot for mutations in cancers as it is preferentially damaged by many carcinogens [8]. However, some of these mutations are single nucleotide polymorphisms (SNPs) and may not cause mitochondrial dysfunction, such as the mttRNA^Phe C628T variant in deafness expression [9]. Distinguishing the SNPs and mutations is important, because failure to do so will inevitably lead to poor diagnosis and genetic advice.

In this study, we reassess seven reported mttRNA variants: tRNA^Asp G7521A, tRNA^Arg T10411C and T10463C, tRNA^Leu(CUN) A12308G, tRNA^Ile G4292C and C4312T, tRNA^Ala T5655C, in clinical manifestation of thyroid cancer. First, we carried out database searches for the allele frequencies of these variants, and then the genotype to phenotype association of these variants. Moreover, we performed the phylogenetic conservation analysis of these variants. We further utilized the bioinformatic tool to predict the ⊿G of mt-tRNAs with and without these variants. To determine the frequency of A12308G variant, we screened this variant in 300 patients with thyroid cancer and 200 controls.

Mutations of mtDNA, as well as nuclear genome, are associated with various human diseases and may play important roles in age-related disorders, including cancer and aging [16]. Alterations in OXPHOS in tumor cells were originally believed to play a causative role in malignant growth and tumorigenesis [17]. On the other hand, mutations in the mt-tRNA genes have impact on the secondary and tertiary tRNA structure, and may consequently cause transcriptional and translational defects and mitochondrial respiratory chain dysfunction. More than half of mitochondrial mutations have been located in mt-tRNA genes which are hot-spots for mitochondrial pathogenesis [18]. However, we noticed that a certain amount of mt-tRNA mutations were wrongly classified as a “pathogenic” mutation, such as the C628T variant in deafness expression [9].

In this study, we evaluated seven reported mttRNA variants with thyroid cancer by employing phylogenetic conservation analysis (Table 1 and Figure 1). Of these variants, four (G7521A, T10411C, T10463C and T5655C) were located at the acceptor arm, one variant (A12308G) localized at the variable loop, one variant (G4292C) was in the anticodon loop, while the C4312T variant was at the T arm. By systematic review and literature searching, we found that only the A12308G and T5655C variants had been reported to be associated with mitochondrial diseases, while other variants were rare polymorphisms and had not been reported before. Of these, the common variant, A12308G, had been described with a wide range of clinical disorders such as stroke [19], and this variant may increase the risk of developing pigmentary retinal degeneration, short stature, dysphasia-dysarthria and cardiac conduction defects [20]. In addition, the T5655C variant had been reported to increase the penetrance and expressivity of non syndromic deafness associated with the tRNA^Ser(UCN)T7511C mutation [21]. However, as the T5655C variant caused a slight change of ⊿G between the wild-type and the mutant tRNA^Ala, it indicated that it may have been served as a polymorphism.

In order to examine structure-function relationships, we used the RNA Fold Web Server (http:// rna.tbi.univie. ac.at/cgi-bin/RNAfold.cgi) program to predict the optimal secondary structures for the mt-tRNAs through free energy minimization [22,23], in which the size of the minimum free energy change indicated the “driving force” behind the reaction, and the more negative the ⊿G is, the more likely was the process. We found that a wide spectrum of differences in the ⊿G of the mt-tRNAs. The largest negative differences in ⊿G (wild-type)‒⊿G(mutant)<‒3 were found in all these variants, suggesting that the polymorphic variant was energetically less favorable than the wild-type form.

Previous studies showed a positive association between the known tRNA^Leu(CUN) A12308G variant and clinical phenotypes. However, Deschauer et al. [24] considered that this mutation may not increase the risk of stroke in patients with the tRNA^Leu(UUR) A3243G mutation. To address this problem, we performed a mutational analysis for the A12308G in thyroid cancer individuals and healthy controls. To our surprise, there were five patients with this variant, as well as three control subjects. This finding was consistent with a recent study by Mohammed et al. [25], and our data suggested that the A12308G variant was most probably a neutral polymorphism as it was a common event in the human genetic population.

Analysis of the CIs of these variants showed that three of them exhibited high levels of conservation, including the A12308G, T10463C and T5655C (CI >70.0%). However, we noticed that not all the pathogenic mutations were conserved between different species, such as the Leber’s Hereditary Optic Neuropathy (LHON) associated ND4 G11778Amutation [26]. Most importantly, using the cybrid cells carrying these variants, by examining the steady-state level, aminoacylation ability were the “gold standards” to detect the polymorphisms and mutations [27]. Therefore, based on this study, we found that mt-tRNA variants may not be involved in the pathogenesis of thyroid carcinoma.

This study was partly supported by Zhejiang Provincial Natural Science Foundation (LQ13H280002, LY12H15001 and LY12H03001), Henan Basic and Frontier Technology Research Projects (142300410388 and 132300410048), Wenling Foundation of Science and Technology (2011WLCB0109, 2014C311051 and 2015C312055), Project of Medical Technology of Zhejiang Province (2013KYA130, 2015KYB234, 2015KYA 154, 2016KYA166 and 2016KYB275), Public Project of Science and Technology of Wenzhou City (Y20140739 and Y20150094), Ningbo Natural Science Foundation (2015A610234), and Xiangshan Science and Technology Project (2015C6005). The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.