Balkan Journal of Medical Genetics

ANALYSIS OF MITOCHONDRIAL TRANSFER RNA MUTATIONS IN BREAST CANCER
Ding H.J.1, Zhao Y.P.2, Jiang Z.C.3, Zhou D.T.4, Zhu R.1* Han-Jie Ding and Ya-Ping Zhao contribute equally for this work
*Corresponding Author: Ph.D. Rui Zhu, School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Binwen Road No. 548, Hangzhou, P.R. China. Phone/Fax: 0086-0571-86633133, E-mail: zhuruizjtcm@yeah.net
page: 15

MATERIALS AND METHODS

Samples and Clinical Assessments. This study enrolled 80 breast cancer samples, as well as 80 controls. The breast cancer tissues were obtained after surgical resection and stored under -80℃ conditions for further experiments. Furthermore, the normal adjacent tissues were assessed by a pathologist. All of these tissues were obtained from Second Affiliated Hospital of Zhejiang University School of Medicine, and this work was approved by the Ethics Committees of Second Affiliated Hospital of Zhejiang University School of Medicine and Zhejiang Chinese Medical University. Every subject provided his/ her written informed consent. Notably, participants who received chemotherapy or radiotherapy before the surgical treatment would not be enrolled, as well as patients who had mitochondrial disorders such as neurological disease, cardiovascular diseases, and hearing loss were excluded. Analysis of Mt-tRNA Mutations. The genomic DNA of fresh frozen tissues was isolated as described previously [18]. The DNA quality and quantity were assessed using a BioSpec Nano spectrophotometer. For the amplification of 22 mt-tRNA genes, a total of 14 primers were used for PCR reaction, as suggested previously [19]. The PCR products were further purified and sequenced. Finally, the mt-tRNA mutations were detected by comparison to the revised Cambridge sequence (GenBank accession number: NC_012920.1) [20]. Analysis of Conservation Index (CI). To determine the CI, 17 species’ mtDNA sequences were used. The CI≥75% was believed to have functional significance. Analysis of MtDNA Haplogroup. The mtDNA haplogroups of seven patients with breast cancer who carried pathogenic mt-tRNA mutations were classified using the PhyloTree database (http://www.phylotree.org) [21]. Analysis of mtDNA Copy Number. Total genomic DNA of seven breast cancer patients with putative pathogenic mt-tRNA mutations and matched controls were isolated, and subsequently the mtDNA copy number was measured by relative quantitative real-time PCR method [22]. Briefly, the DNA was extracted from tissue samples using the NucleoSpin® Tissue kit (Macherey-Nagel, Hoerdt, France), according to the manufacturer’s recommendations. The mtDNA specific primers for the tRNALeu(UUR) gene (forward primer: 5’-CACCCAAGAACAGGGTTTGT-3’ and reverse primer: 5’-TGGCCATGGGTATGTTGTTAA-3’) and nuclear DNA primers for 18s rRNA gene (forward primer: 5’-TAGAGGGACAAGTGGCGTTC-3’ and reverse primer: 5’-CGCTGAGCCAGTCAGTGT-3’) were used for quantitative PCR to determine the mtDNA copy number, according to the method described previously [23]. Analysis of ATP Production. The cancer tissues and normal controls from seven patients with mt-tRNA pathogenic/likely pathogenic mutations were used for ATP measurement. The ATP concentrations in tissues were analyzed using ATP assay kit (Molecular Probes, Carlsbad, CA, USA), according to the manufacturer’s protocols instructions [24]. Statistical Analyses. All statistical analyses were performed using the unpaired, two-tailed Student’s t test contained in the GraphPad Prism 5 program (GraphPad Software). p<0.05 was considered as statistically significant. Classifications of the Pathogenic Mt-tRNA Mutations. To assess the potential pathogenicity, Yarham and colleagues designed a weighted scoring system for mt-tRNA mutations [25]: if the score of a tRNA mutation <6 points, it was a “neutral polymorphism”; while a score of 7-10 points was classified as “possible pathogenic”, and a score of >11 points was classified as “definitely pathogenic”.

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