Quantitative assessment of a novel mtDNA deletion in the minor deletion arc by realtime PCR
Authors: Andrew Harbottle, Mark A. Birch-Machin
Journal: Cancer Research (2004)

Proc Amer Assoc Cancer Res, Volume 45, 2004

1538 

Mitochondria have been implicated in the carcinogenic process because of their role in apoptosis and other aspects of tumour biology; in particular, somatic mutations of mitochondrial DNA (mtDNA) have been observed in a wide variety of human tumours. In addition we have recently pinpointed mtDNA damage in human skin cancer for the first time in the skin cancer literature (Durham et al., 2003). In our current study we have established a quantitative real time PCR methodology to determine the levels of a novel mtDNA deletion in the minor arc of the mitochondrial genome, in tumour and histologically normal perilesional skin. We have compared the incidence of a novel mtDNA deletion, using three separate methodologies, namely: long extension PCR, a deletion specific PCR and a quantitative real time Taqman PCR. Long extension PCR of a 5.5kb region of the minor arc detected the deletion in 6 out of 30 samples, primarily in the tumour or the perilesional dermis rather than the epidermis. Using a non quantitative deletion specific PCR technique, the deletion was detected in 73% of our 30 samples. The pattern of expression of the deletion was dependent upon the type of tumour. For example, the deletion was found in 3 of the 5 Basal cell carcinomas (BCCs), while in Squamous cell carcinomas (SCCs) the deletion was generally observed in the perilesional dermis, rather than the tumour. In concordance with the Long PCR data, those samples with the strongest PCR product were those that contained the corresponding deletion band in the Long PCR. Using cloned wild type and deleted templates, we have established a real time Taqman PCR assay which is specific for the novel minor arc mtDNA deletion. Realtime PCR analysis of our 30 samples, showed a maximal level of deletion of around 5% compared to wild type although most samples had levels of the deletion of less than 1%. As our tumour and normal skin samples are from sun exposed sites we examined whether the novel minor arc mtDNA deletion is useful as a marker of sun exposure. A comparison of the expression of the deletion in sun exposed to sun intermittent revealed differences in the levels of the deletion between the two body sites. In exposed dermis, 100% (n=10) samples contained levels of deletion greater than 0.1% with the highest containing 2.6% deleted mtDNA. In contrast, in the sun intermittent samples only 33% (n=6) dermis contained levels of deletion greater than 0.1%, with the highest being only 0.2%. For epidermis 50% of the sun exposed samples were higher than 0.1%, while no sun intermittent samples were over 0.1%. In summary we have established a quantitative real time PCR methodology for the detection of a novel mitochondrial deletion which may serve as a highly sensitive marker of sun exposure in skin and consequently may be used as an early marker of skin cancer development. Durham, S.E., Krishnan, K.J., Betts, J. & Birch-Machin, M.A. (2003). Mitochondrial DNA damage in non-melanoma skin cancer. Br J Cancer , 88, 90-5.