Computational protocol: Mitochondrial DNA depletion, mitochondrial mutations and high TFAM expression in hepatocellular carcinoma

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Protocol publication

[…] Total DNA was isolated from 86 pairs of tumors (T) and matched non-tumor (N) liver tissues using a Tissue gDNA kit (Biomiga) according to the manufacturer's protocol. The entire mitochondrial genome of the 86 pairs was PCR amplified in 24 overlapping fragments using sets of oligonucleotide primers as described previously []. Each fragment was purified and subsequently analyzed by Sanger sequencing. Sequencing results were compared with the updated Cambridge Reference Sequence (GenBank accession number: NC_012920) [], along with the Human Mitochondrial Genome Database (mtDB) and the Mitomap database [http://www.mitomap.org/MITOMAP]. Twenty-two somatic nonsynonymous variants in the protein-coding region, with a frequency <0.5% in the Mitomap database, were further evaluated by bioinformatic programs including PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/), MutationAssessor (http://mutationassessor.org/), Provean (http://provean.jcvi.org/index.php), PANTHER (http://fathmm.biocompute.org.uk/), MToolBox (https://github.com/mitoNGS/MToolBox), and TransFIC (http://bg.upf.edu/transfic/home). Variants that were predicted as deleterious by more than half of the 6 programs listed above were considered as putative pathogenic mutations associated with HCC (). Records of the 22 somatic nonsynonymous variants were sought in the COSMIC database (http://cancer.sanger.ac.uk/cosmic). The entire mtDNA sequences of the 86 patients were assigned to the Asian mitochondrial haplogroups by using the nomenclature of mitochondrial haplogroups []. [...] All statistical analyses and graphical data were generated using GraphPad Prism software (v5.04, www.graphpad.com) or Microsoft-Excel, with data presented as Mean ± SD. The P value of mitochondrial haplotype was calculated using IBM SPSS Statistics 20. Statistical significance was established at P < 0.05 (*) and P < 0.01 (**). […]

Pipeline specifications

Software tools PolyPhen, Mutationassessor, PROVEAN, FATHMM, MToolBox, transFIC, SPSS
Databases MITOMAP mtDB
Applications Miscellaneous, WGS analysis
Diseases Carcinoma, Hepatocellular, Liver Neoplasms, Neoplasms