MUTATIONAL ANALYSIS OF MITOCHONDRIAL tRNA GENES IN PATIENTS WITH LUNG CANCER
He ZF, Zheng LC, Xie DY, Yu SS, Zhao J
*Corresponding Author: Dr. Jun Zhao, First Affiliated Hospital, Soochow University, Shizi Road, 215006, Suzhou, People’s Republic of China. Tel./Fax: +86-0512-65223637. E-mail: zhaojsz001@163.com
page: 45

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]. 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 “definitely pathogenic.”



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