Detecting Rare Variants and Heteroplasmy of Mitochondrial DNA from High-Throughput Sequencing in Patients with Coronary Artery Disease

BACKGROUND: Although mutations and dysfunction of mitochondrial DNA (mtDNA) are related to a variety of diseases, few studies have focused on the relationship between mtDNA and coronary artery disease (CAD), especially the relationship between rare variants and CAD. MATERIAL/METHODS: Two-stage high-throughput sequencing was performed to detect mtDNA variants or heteroplasmy and the relationship between them and CAD phenotypes. In the discovery stage, mtDNA was analyzed by high-throughput sequencing of long-range PCR products generated from the peripheral blood of 85 CAD patients and 80 demographically matched controls. In the validation stage, high-throughput sequencing for mtDNA target regions captured by GenCap Kit was performed on 100 CAD samples and 100 controls. Finally, tRNA fine mapping was performed between our study and the reported Chinese CAD study. RESULTS: Among the tRNA genes, we confirmed a highly conserved rare variant, A5592G, previously reported in the Chinese CAD study, and 2 novel rare mutations that reached Bonferroni’s correction significance in the combined analysis were found (P=7.39×10(−4) for T5628C in tRNA(Ala) and P=1.01×10(−5) for T681C in 12S rRNA) in the CAD study. Both of them were predicted to be pathological, with T5628C disrupting an extremely conservative base-pairing at the AC stem of tRNA(Ala). Furthermore, we confirmed the controversial issue that the number of non-synonymous heteroplasmic sites per sample was significantly higher in CAD patients. CONCLUSIONS: In conclusion, our study confirmed the contribution of rare variants in CAD and showed that CAD patients had more non-synonymous heterogeneity mutations, which may be helpful in identifying the genetic and molecular basis of CAD.