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 mttRNAPhe 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: tRNAAsp G7521A, tRNAArg T10411C and T10463C, tRNALeu(CUN) A12308G, tRNAIle G4292C and C4312T, tRNAAla 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.
We systematically searched for unrestricted language articlesincluded in PubMed, Embase, Google Scholar, Cochrane Library, China National Knowledge Infrastructure (CNKI), Chinese VIP, and Chinese Wan-fang databases from inception to December 2015. The literature search in PubMed was carried out using the following keywords “mitochondrial tRNA mutations, thyroid carcinoma,” or “mt-tRNA mutations, thyroid cancer,” or “mt-tRNA variants, thyroid cancer.” We excluded studies if the crucial data were not reported in original papers, or if they had a very high probability of inaccurate reporting.
Data were independently extracted by two authors (F. Lv and G. Qian), and checked by another author (W. You). Any disagreement was resolved by discussions until consensus was reached.
With the purpose of understanding the possible role of mt-tRNA variants in thyroid cancer, we performed a phylogentic approach to see the conservation index (CI) of each mt-tRNA variant. In brief, the mtDNA sequences of 15 vertebrates were used in the inter-specific analyses; these species included
To see whether these variants affected the ⊿G of mt-tRNAs, we used the RNA Fold Web server program to measure the ⊿G of each mt-tRNA with and without these variants (
We then performed the polymerase chain reaction (PCR)-Sanger sequence to detect the occurrence of the “well-known” A12308G variant in patients with thyroid cancer. Briefly, a total of 300 blood samples of thyroid cancer (150 male and 150 females; average age 52 years) and 200 healthy subjects (100 males and 100 females; average age 49 years) were recruited from the Henan Provincial People’s Hospital, Zhengzhou, People’s Republic of China (PRC). Informed consent and clinical evaluation were obtained from all these subjects. The study protocol was approved by the Ethics Committee of Henan Provincial People’s Hospital.
We first extracted the genomic DNA from each sample, then we used the PCR to amplify the gene using the following primer sequences: forward (5’-TGC TAG TAA CCA CGT TCT CC-3’); reverse (5’-TTT GTT AGG GTT AAC GAG GG-3’). The PCR product was subsequently examined for specificity using 1.5% agarose gel electrophoresis. Double-stranded automated sequencing was performed using an ABI PRISM™ 3700 sequencing machine (Applied Biosystems Inc., Foster City, CA, USA). The sequence was then compared with the human mitochondrial reference sequence (NC_012920) [6].
As a result, three studies were identified using the keywords mentioned in the previous section. After a full-text review, we noticed that one of them concerned the somatic mtD-NA mutations in PTC [12]. Another article that met our inclusion criteria was about the association of mtDNA transversion mutations with familiar MTC [13], whereas another article talked mainly about the mtDNA mutations causing defective OXPHOS in thyroid carcinoma [14].
We further screened the mt-tRNA variants that had been reported with thyroid cancer. Consequently, a total of seven mt-tRNA variants were described: tRNAAsp G7521A, tRNAArg T10411C and T10463C, tRNALeu(CUN) A12308G, tRNAIle G4292C and C4312T, and tRNAAla T5655C. The molecular characterization of these mt-tRNA variants are listed in Table 1 and Figure 1.
Molecular characterization of mt-tRNA variants for thyroid cancer.
tRNA | tRNAAsp | tRNAArg | tRNALeu(CUN) | tRNAIle | tRNAAla | ||
---|---|---|---|---|---|---|---|
Nt changes | G7521A | T10411C | T10463C | A12308G | G4292C | C4312T | T5655C |
Number of nts in tRNA | 4 | 7 | 67 | 44 | 34 | 54 | 1 |
Position | acceptor arm | acceptor arm | acceptor arm | variable loop | anticodon arm | T arm | acceptor arm |
Homoplasmy/heteroplasmy | homoplasmy | homoplasmy | homoplasmy | homoplasmy | homoplasmy | homoplasmy | homoplasmy |
CI (%) | 13.5 | 21.1 | 94.2 | 93.3 | 10.0 | 19.0 | 71.2 |
G (wild type)(kcal/mol) | ‒10.59 | ‒10.58 | ‒10.86 | ‒16.01 | ‒8.98 | ‒8.98 | ‒18.47 |
G (mutant)(kcal/mol) | ‒11.11 | ‒10.13 | ‒10.13 | ‒15.88 | ‒9.06 | ‒9.12 | ‒18.81 |
CI: conservation index.
Assessing pathogenicity of a nt substitution in a mttRNA gene involved evaluation of the evolutionary conservation of the base involved. For this purpose, we analyzed the CIs of these mt-tRNA variants. Briefly, we chose 15 animals for inter-species analysis, these species included
We used the RNA Fold Web Server (
By comparing the human mitochondrial genome sequence, we found that there were five thyroid cancer patients and three control subjects carrying the homoplasmic A12308G variant, suggesting that it may be very polymorphic in the human population.
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
In order to examine structure-function relationships, we used the RNA Fold Web Server (
Previous studies showed a positive association between the known tRNALeu(CUN) A12308G variant and clinical phenotypes. However, Deschauer
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
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.