Profiling of Mitochondrial DNA Heteroplasmy in a Prospective Oral Squamous Cell Carcinoma Study

Abstract

While a shift in energy metabolism is essential to cancers, the knowledge about the involvement of the mitochondrial genome in tumorigenesis and progression in oral squamous cell carcinoma (OSCC) is still very limited. In this study, we evaluated 37 OSCC tumors and the corresponding benign mucosa tissue pairs by deep sequencing of the complete mitochondrial DNA (mtDNA). After extensive quality control, we identified 287 variants, 137 in tumor and 150 in benign samples exceeding the 1% threshold. Variant heteroplasmy levels were significantly increased in cancer compared to benign tissues (p = 0.0002). Furthermore, pairwise high heteroplasmy frequency difference variants (∆HF% > 20) with potential functional impact were increased in the cancer tissues (p = 0.024). Fourteen mutations were identified in the protein-coding region, out of which thirteen were detected in cancer and only one in benign tissue. After eight years of follow-up, the risk of mortality was higher for patients who harbored at least one ∆HF% > 20 variant in mtDNA protein-coding regions relative to those with no mutations (HR = 4.6, (95%CI = 1.3–17); p = 0.019 in primary tumor carriers). Haplogroup affiliation showed an impact on survival time, which however needs confirmation in a larger study. In conclusion, we observed a significantly higher accumulation of somatic mutations in the cancer tissues associated with a worse prognosis.

Analyzing Low-Level mtDNA Heteroplasmy—Pitfalls and Challenges from Bench to Benchmarking

“…It is worth noting that the arguable enzyme was the cheapest in the observed group. In our previous work [ 12 ] this variant was described as heteroplasmic, but in the light of this observation it would probably be more appropriate to classify it as an artefact. This heteroplasmic site at position 3210 manifested itself in one benign tumor pair with heteroplasmy levels of 1.8% and 1.7%, respectively (Patient KT013), and one additional cancer sample (Patient KT012 with 6.2% heteroplasmy level) but not in the corresponding benign control.…”

“…Peripheral blood was collected for earlier sequencing projects [ 1 , 49 ] approved by the Medical University of Innsbruck (study codes UN3564 and AN 4837 respectively) and sequenced as part of Fendt et al [ 12 ] (Ref. No.…”

“…To validate the MPS analysis we performed gold standard Sanger-type sequencing of the entire mtDNA of the two mixture component samples, as presented in Kloss-Brandstätter et al [ 1 ]. In brief, whole mtDNA was amplified as two overlapping 8.5 kb fragments as described in Fendt et al [ 12 , 52 ]. Libraries for the HiSeq 2500 of the unmixed DNA reference samples were prepared as described in Kloss-Brandstätter et al [ 1 ].…”

“…Libraries for the Illumina MiSeq of the DNA and PCR mixtures were constructed as presented in Fendt et al [ 12 ] by preparing 500–800 bp inserts of each 200 ng of pooled fragment A and B. In a first step, the pooled samples were enzymatically fragmented in a final volume of 20 µL containing 2 µL of 2 × dsDNA Fragmentase Reaction Buffer v2 and 2 µL of 200 mM MgCl 2 and 2 µL NEBNext dsDNA Fragmentase (New England Biolabs, Ipswich, MA, USA) by incubation for approximately 6 min at 37 °C.…”

“…In forensic genetics, the discernibility of two sequence profiles is essentially elevated if heteroplasmy can be taken into account [ 6 , 7 ]. The detection of low-level variants is also of great importance in medical genetics for diagnosis and prognosis of a mitochondrial disease [ 8 ] but also in cancer research [ 9 , 10 , 11 , 12 ] or aging-related research [ 13 ]. A study of Ye et al [ 14 ] reporting extensive pathogenicity of mitochondrial heteroplasmy in healthy human individuals was the starting point of a discussion about published sequence quality derived from MPS platforms [ 15 , 16 ].…”