Mutational analysis of mitochondrial tRNA genes in patients with lung cancer

Over the last century, lung cancer from the rarest of diseases became the biggest cancer killer of men worldwide and in some parts of the world also of women. It is the most common type of cancer diagnosed in the world and is the number one cancer killer in the US [1]. Survival rates in patients with lung cancer are much lower than patients with other common cancers, such as breast, colon or prostate cancer. It is estimated that by 2035, the number of lung cancer deaths will increase globally by 86.0% when compared to 2012 [2]. However, to date, the molecular mechanism underlying this disease remains poorly understood.

Mitochondria are cellular organelles with distinct features that belie their origins and unique functions. Originally derived from ancient aerobic bacteria, mitochondria are critical for meeting cellular energy demands by driving the synthesis of ATP [3]. Mitochondria also influence cellular signaling and survival pathways, including apoptosis [4]. Mitochondrial dysfunction has been implicated in a plethora of human diseases, most notably in cancer [5]. In addition, due to the lack of protective histones, introns and efficient DNA repair systems, mitochondrial DNA (mtDNA) acquires 10-fold more mutations than nuclear genomic DNA [6]. Among these mutations, mitochondrial tRNA (mt-tRNA) genes are hot-spots for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states [7,8]. Nevertheless, little is known regarding the mt-tRNA mutations in lung cancer.

In this study, we performed a systematic and extensive mutational screening for 22 mt-tRNA genes with lung cancer. We also used the pathogenicity scoring system for these mutations.

Materials and Methods

Blood Samples

Since January 2015, a total of 200 lung cancer patients (45.0% females and 55.0% males, aged 45 to 55 years) were recruited from the First Affiliated Hospital, Soochow University, Suzhou, People’s Republic of China (PRC). Moreover, 100 unrelated healthy controls, age- and gender-matched, were collected in the same area. Blood and experimental procedures were approved by the Ethics Committee of Soochow University, Suzhou, PRC. A signed informed consent was obtained from by all participants.

DNA Extraction, Polymerase Chain Reaction (PCR) Amplification and Sequence Analysis

The genomic DNA was extracted using standard phenol/ chloroform methodology, and stored at –20 °C for future use. The 22 mt-tRNA genes were amplified by PCR, the primers information are listed in Table 1. The PCR mixture included 200 mm dNTP, 10X buffer, Taq DNA polymerase and 15 mmol/L Mg²⁺ (Takara Biotechnology Co., Ltd., Dalian, China). Each amplified DNA sample was purified and analyzed using the ABI PRISM® 3700 automated DNA sequencer and the BigDye Terminator Cycle sequencing reaction kit (Applied Biosystems; Thermo Fisher Scientific, Waltham, MA, USA). The sequence data were compared with the reversed consensus Cambridge sequence to screen the mutations (GenBank Accession No. NC_012920) [9].

Table 1

Characterization of lung cancer associated mt-tRNA mutations.

tRNA Species	Sequence Alteration	Location	Conservation Index (%)	Frequency (%)
tRNA^Ala	T5655C	Acceptor arm	100.0	0.5
tRNA^Arg	T10454C	T loop	100.0	1.0
tRNALeu(CUN)	A12330G	Acceptor arm	100.0	1.5
tRNASer(UCN)	T7505C	DHU stem	100.0	0.5
tRNA^Thr	G15927A	Anticodon stem	100.0	2.0

Phylogenetic Conservation Analysis

A total of 16 vertebrates’ mtDNA sequences were used in the interspecific analysis. These included: Bos Taurus, Cebus albifrons, Gorilla gorilla, Homo sapiens, Hylobates lar, Lemur catta, Macaca mulatta, Macaca sylvanus, Mus musculus, Nycticebus coucang, Pan paniscus, Pan troglodytes, Pongo pygmaeus, Pongo abelii, Papio hamadryas and Tarsius bancanus. The conservation index (CI) was then calculated by comparing the human nucleotide variants with another 15 vertebrates. The CI was then defined as the percentage of species from the list of 15 different vertebrates that have the wild-type nucleotide at that position.

Pathogenicity Scoring System for These Mitochondrial tRNA Mutations

McFarland et al. [10] provided a program for assigning pathogenicity to mt-tRNA mutations. Their weighting scoring system was revised in 2011 [11]. According to this standard, we classified a mutation with a score of ≤6 as a “neutral polymorphism,” while a mutation with a score of 7-10 was a “possible pathogenic,” and a mutation with a score of >13 points was classed as “defnitely pathogenic.”

Results

Mutational Analysis of the Mitochondrial tRNA Genes

Mutational screening of the 22 mt-tRNA genes led us to identify five potential pathogenic mutations: tRNA^Ala T5655C, tRNA^Arg T10454C, tRNA^Leu(CUN) A12330G, tRNA^Ser(UCN) T7505C and tRNA^Thr G15927A. Of these, the T5655C mutation was detected in one out of 200 patients (0.5%), the T10454C mutation was detected in two patients (1.0%), the A12330G mutation was detected in three patients (1.5%), the T7505C mutation was presented in one patient (0.5%) and the G15927A mutation was presented in four patients (2.0%). However, none of these mutations were found in the healthy controls. The characterization of each mt-tRNA mutation is listed in Table 1 and Figure 1.

Phylogenetic Conservation Analysis

To see whether these mutations contributed to the genetic susceptibility of lung cancer, we performed the phylogenetic conservation analysis for each mt-tRNA mutations. We found that all these mutations exhibited high levels of CI (100.0%), indicating that these mutations may play important roles in lung cancer.

Cloverleaf structure of mt-tRNA^AIa, mt-tRNA^Arg, mt-tRNA^Leu(CUN) mt-tRNA^Ser(UCN) and mt-tRNA^Thr. The arrows indicate the position of the T5655C, T10454C, A12330G, T7505C and Gl 5927A mutations.

The Pathogenicity Scoring System for These Mitochondrial tRNA Mutations

According to the revised pathogenicity scoring system, we found that the total score of the T5655C, T10454C, A12330G, T7505C and G15927A mutations were 11, 6, 14, 11 and 15 points, respectively, it seemed that except for the T10454C mutation, other mutations should be classified as “defnitely pathogenic” (Table 2).

Table 2

The pathogenicity scoring system for the mt-tRNA mutations for lung cancer.

Scoring Criteria	T5655C	Score Score out of 20.	T10454C	Score Score out of 20.	A12330G	Score Score out of 20.	T7505C	Score Score out of 20.	G15927A	Score Score out of 20.	Classification
More than one independent report	yes	2	yes	2	yes	2	yes	2	yes	2	-
Evolutionary conservation of the bp	no changes	2	no changes	2	no changes	2	no changes	2	no changes	2	–
Variant heteroplasmy	no	0	no	0	no	0	no	2	no	2	≤6 points: neutral polymorphisms
Segregation of the mutation with disease	yes	2	yes	2	yes	2	yes	2	yes	2	7-10 points: possibly pathogenic
Histochemical evidence of mt disease	no evidence	0	no	0	yes	2	no	0	no evidence	0	≥ 11 points: definitely pathogenic
Biochemical defect in complexes I, III or IV	no	0	no	0	yes	2	no	0	yes	2	–
Evidence of mutation segregation with biochemical defect from single-fiber studies	no	0	no	0	yes	2	no	0	yes	2	–
Mutant tRNA steady-state level of evidence of pathogenicity in transmt cybrid studies	strong evidence	5	no	0	no	0	strong evidence	5	strong evidence	5	–
Maximum score	definitely pathogenic	Ρ	neutral polymorphism	6	definitely pathogenic	14	definitely pathogenic	11	definitely pathogenic	15

bp: base pair; mt: mitochondrial.

Discussion

In this study, we screened mt-tRNA mutations in patients with lung cancer. To the best of our knowledge, this is the first report dealing with the association between mt-tRNA mutations and lung cancer. Lung cancer is a common type of cancer diagnosed all over the world. Screening options for this disease are very limited and only 15.0% of cases are diagnosed at an early stage [12]. Thus, there is an urgent need for developing a biomarker for early detection for lung cancer.

Mitochondrial tRNA genes are hot-spots for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation, disrupting this primary function affects protein synthesis and expression, folding and function of oxidative phosphorylation enzymes [13]. However, we noticed that a certain amount of tRNA mutations have been wrongly classified as “pathogenic,” as in a recent article concerning the association between mt-tRNA mutations and thyroid carcinoma [14].

In this study, we investigated the possible relationship between mt-tRNA mutations and lung cancer by employing PCR-Sanger sequencing. As a result, five mt-tRNA mutations were identified, including tRNA^Ala T5655C, tRNA^Arg T10454C, tRNA^Leu(CUN)A12330G, tRNA^Ser(UCN) T7505C and tRNA^Thr G15927A. Of these, the T5655C mutation was located at the acceptor arm of tRNA^Ala, disrupted the highly conserved base-pairing (1A-72U). An in vitro processing analysis showed that the T5655C mutation reduced the efficiency of tRNA^Ala precursor 5’ end cleavage catalyzed by RNase P [15]. Moreover, a significant decreased of tRNA^Ala steady-state level was also observed in cybrid cells containing this mutation. While the homoplasmic T10454C mutation was localized at the Τ loop of tRNA^Arg (conventional position 55), this mutation was implicated to be associated with longevity and non syndromic hearing loss [16,17], However, aprevious study showed that the T10454C mutation may not modulate the phenotypic manifestation of deafness-associated mitochondrial 12S rRNAA1555G mutation [18], thus, we proposed that the T10454C mutation was a neutral polymorphism (Table 2). In addition, the A12330G mutation occurred at the acceptor arm of tRNA^Leu(CUN) gene, disrupting the highly conserved base-pairing (6T-67A), and may result in failure in tRNA metabolism. Aprevious study showed that this mutation, combined with the well known ND5 T12338C mutation, may account for the high penetrance and expressivity of essential hypertension in a Han Chinese family [19]. Moreover, the T7505C mutation was first described in a Han Chinese family with maternally transmitted non syndromic hearing impairment [20], structurally, the T7505C mutation was localized at the second base-pairing (10A-20U) on the D-stem of tRNA^Ser(UCN). The phylogenetic analysis of this mutation and mtDNAs from the other 10 vertebrates revealed that the nucleotide A at the conventional position 10 in tRNA^Ser(UCN) was extremely evolutionarily conserved [21], ~65.0% reductions in the level of tRNA^Ser(UCN) were observed in the lymphoblastoid cell lines carrying this mutation [20]. Furthermore, the homoplasmic Gl5927A in the anticodon stem of tRNA^Thr was also described in a Han Chinese family with maternally inherited hearing loss [22]; later, this mutation was reported to be associated with coronary heart disease [23]. Northern blotanalysis of the cell lines with the G15927Amutationrevealed~80.0% decrease in the steady-state level of tRNA^Thr, in addition, ~39.0% reduction in aminoacylated efficiency of tRNA^Thr was observed in mutant cells derived from the family members carrying the G15927A mutation. An increasing production of reactive oxygen species was observed in the mutant cells carrying the G15927A mutation. Therefore, we proposed that the G15927A mutation was a pathogenic mutation associated with lung cancer.

Based on these findings, we proposed that the molecular mechanism underlying these mt-tRNA mutations in the carcinogenesis of lung cancer may be as follows: first, these mutations disrupted the mt-tRNA secondary structure and subsequently resulted in failure in tRNA metabolism such as a CCA addition, posttranscriptional modification and aminoacylation [24]. Whatever the consequence may be, the expected net effect would be a decrease in mitochondrial protein synthesis. Defects in mitochondrial translation consequently leads to a respiratory phenotype and a decline in adenosine triphosphate (ATP) production below the threshold level required for normal cell function, thus, contributing to the tumorigenesis of lung cancer. In summary, this is the first report concerning the association between mt-tRNA mutations and lung cancer. The main limitation of this study was the sample size. Further studies including more samples are needed to verify the conclusions.

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Mutational analysis of mitochondrial tRNA genes in patients with lung cancer

Article Category: Original Article

Published Online: Dec 31, 2016

Page range: 45 - 50

DOI: https://doi.org/10.1515/bjmg-2016-0035

KeywordsLung cancer, Mitochondrial tRNA (mt-tRNA), Mutations, Pathogenic

© 2016 He ZF, Zheng LC, Xie DY, Yu SS, Zhao J

This article is distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Keywords
Lung cancer, Mitochondrial tRNA (mt-tRNA), Mutations, Pathogenic