Implications of using the ND1 gene as a control region for real-time PCR analysis of mitochondrial DNA deletions in human skin.

Previous work by our group and others has demonstrated that mitochondrial DNA (mtDNA) deletions may be useful as a biomarker of cumulative ultraviolet radiation exposure in human skin (Birch-Machin, 2000). Of particular importance is the incidence of various mtDNA deletions, including the 4977 bp ‘‘common’’ deletion, which has been strongly associated with increased sun exposure in human skin (BirchMachin et al, 1998; Ray et al, 2000) and UV irradiation in cultured skin cells (Berneburg et al, 1999). Until recently, a rapid reliable technique has not been available for quantifying low levels (i.e., o1%) of these mitochondrial deletions (Krishnan et al, 2003). For this reason, researchers have begun to look towards real-time PCR assays to provide reliable quantification of low levels of deletions. To date, the only published real-time PCR assay for a single deletion is that for the ‘‘common’’ deletion (Koch et al, 2001). The incidence of the common deletion, however, is likely to be only the tip of the deletion iceberg (Ray et al, 2000), and so the spectrum of the many other mtDNA deletions has been investigated in sun-exposed skin and skin cancer (Ray et al, 2000; Durham et al, 2003). These latter studies have been limited however due to the qualitative nature of the methodology. Recently, a real-time Taqman PCR assay has been described, which attempts to solve this problem by claiming to quantify the level of the mtDNA deletion spectrum in muscle cells (He et al, 2002). The assay uses two probe regions in the mitochondrial genome, firstly the control probe region in the ND1 gene, and secondly the test probe region in the ND4 gene (Fig 1). The ND1 gene lies in the part of the genome that has been historically called the minor deletion arc, because it is thought to have very few deletions (Mitomap, 2001). This has prompted its use as the control region in the assay. In contrast, the ND4 probe resides in the major deletion arc, an area that has been frequently reported to contain many deletions (Mitomap, 2001). In order to estimate the level of total major arc deletions within a sample, the assay calculates the ratio of the copy number of the ND1 probe to the ND4 probe. Using a qualitative long extension PCR technique, we have previously described a spectrum of large-scale mtDNA deletions in non-melanoma skin cancer (NMSC) samples and their histologically normal perilesional skin (Durham et al, 2003). This study addresses the question of whether the real-time PCR deletion assay described by He et al (2002) is able to provide a robust and reliable means of quantifying the deletion spectrum that has been identified in these NMSC and perilesional skin samples.