Low level of the mtDNA 4977 deletion in blood of exceptionally old individuals Nicole von Wurmb-Schwark a , Thorsten Schwark a , Amke Caliebe b , Carolin Drenske a , Susanna Nikolaus c,d , Stefan Schreiber c,e , Almut Nebel e, * a Institute of Legal Medicine, Christian-Albrechts-University and the University Hospital Schleswig-Holstein, Kiel, Germany b Institute of Medical Informatics and Statistics, Christian-Albrechts-University and the University Hospital Schleswig-Holstein, Kiel, Germany c Clinic of Internal Medicine I, Christian-Albrechts-University and the University Hospital Schleswig-Holstein, Kiel, Germany d Popgen Biobank, Christian-Albrechts-University and the University Hospital Schleswig-Holstein, Kiel, Germany e Institute of Clinical Molecular Biology, Christian-Albrechts-University and the University Hospital Schleswig-Holstein, Schittenhelmstrasse 12, 24105 Kiel, Germany 1. Introduction Somatic mutations in the mitochondrial DNA (mtDNA) have been shown to accumulate with age in a variety of tissues in various species, including humans (Cortopassi and Arnheim, 1990; Wei, 1992; Melov et al., 1995; Ozawa, 1995; Wang et al., 1997). These mutations seem to be primarily caused by reactive oxygen species (ROS) that arise as a by-product of oxidative phosphorylation in mitochondria (Harman, 1956; reviewed in Galtier et al., 2009; Wei et al., 2009). Mutated and wildtype mtDNA molecules often coexist in the heteroplasmic state within the same cell, tissue or organ. Many different types of somatic mtDNA mutations have been observed, with the 4977-bp deletion (dmtDNA 4977 ) being the most common in humans (Cortopassi and Arnheim, 1990; Lee et al., 1994). The dmtDNA 4977 deletion occurs frequently in tissues of high oxygen demand and low mitotic activity, e.g. brain (Corral-Debrinski et al., 1992; Soong et al., 1992; Storm et al., 2002; Fuke et al., 2008), heart (Ozawa, 1995), skeletal muscle (Cortopassi et al., 1992; Meissner et al., 1997, 1999; Liu et al., 1998; Zhang et al., 1998; von Wurmb-Schwark et al., 2002) or skin (Yang et al., 1994; Berneburg et al., 1997). In many of these tissues, dmtDNA 4977 shows an age-related increase and can reach surprisingly high proportions though they still lie far below the assumed critical threshold for physiological effects (Prigione and Cortopassi, 2007). The dmtDNA 4977 mutation can also be detected – albeit in much lower amounts – in fast replicating cells such as blood leukocytes (Gattermann et al., 1995; von Wurmb et al., 1998). Here, the findings with respect to an age-dependent increase of dmtDNA 4977 are still controversial. Some researchers described an accumulation as a function of age (Li et al., 2006), others found no association (Berneburg et al., 1997) or obtained ‘‘inconclusive’’ results (von Wurmb et al., 1998). The various reports are difficult to reconcile, and the studies differed considerably in terms of the sensitivity of the methodologies applied and the number and age of the examined participants. Very few elderly or exceptionally old individuals (beyond 90 years) have so far been investigated for dmtDNA 4977 in blood (Table 1). Many of the studies included only a small number of individuals and had therefore very low power to detect an age dependency. What further adds to the controversy is that transient Mechanisms of Ageing and Development 131 (2010) 179–184 ARTICLE INFO Article history: Received 30 June 2009 Received in revised form 8 January 2010 Accepted 24 January 2010 Available online 1 February 2010 Keywords: Mitochondrial DNA Deletion dmtDNA 4977 Blood Centenarians Human longevity Ageing ABSTRACT The common 4977-bp deletion in mitochondrial DNA (dmtDNA 4977 ) occurs frequently in tissues of high oxygen demand and low mitotic activity, e.g. brain, heart and skeletal muscle, where it appears to show an age-related accumulation. Although dmtDNA 4977 can also be detected in very low amounts in fast replicating tissues such as blood, it is still unclear whether an age-dependent distribution of dmtDNA 4977 occurs in blood. In view of these uncertainties, we investigated the presence of the mutation and changes in the dmtDNA 4977 level in whole blood samples from 473 individuals who belong to two different age groups, i.e. elderly (aged 61–75 years) and long-lived individuals (LLI, aged 95–109 years). We applied a highly sensitive and reliable duplex-PCR method that allowed relative quantification of dmtDNA 4977 . For validation, we additionally performed absolute quantification on a subset of samples using real time- PCR. Our results showed that the proportion of dmtDNA 4977 carriers was very similar in both groups, but that the individual mutational load was on average much lower in the nonagenarians and centenarians than in the elderly. The finding was independent of smoking habits, gender or variation in APOE and FOXO3A but could be caused by other environmental and/or genetic factors. ß 2010 Elsevier Ireland Ltd. All rights reserved. * Corresponding author. Tel.: +49 431 597 1373; fax: +49 431 597 1842. E-mail address: a.nebel@mucosa.de (A. Nebel). Contents lists available at ScienceDirect Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev 0047-6374/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2010.01.005