Ultraviolet radiation exposure accelerates the ... - Wiley Online Library

Aging Cell (2007) 6, pp557–564

Doi: 10.1111/j.1474-9726.2007.00310.x

Ultraviolet radiation exposure accelerates the accumulation of the aging-dependent T414G mitochondrial DNA mutation in human skin

Blackwell Publishing Ltd

Matthew J. Birket and Mark A. Birch-Machin Dermatological Sciences, Institute of Cellular Medicine, School of Clinical and Laboratory Sciences, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK

Summary The accumulation of mitochondrial DNA (mtDNA) mutations has been proposed as an underlying cause of the aging process. Such mutations are thought to be generated principally through mechanisms involving oxidative stress. Skin is frequently exposed to a potent mutagen in the form of ultraviolet (UV) radiation and mtDNA deletion mutations have previously been shown to accumulate with photoaging. Here we report that the age-related T414G point mutation originally identified in skin fibroblasts from donors over 65 years also accumulates with age in skin tissue. Moreover, there is a significantly greater incidence of this mutation in skin from sun-exposed sites χ2 = 6.8, P < 0.01). Identification and quantification of the (χ T414G mutation in dermal skin tissue from 108 donors ranging from 8 to 97 years demonstrated both increased occurrence with photoaging as well as an increase in the proportion of molecules affected. In addition, we have discovered frequent genetic linkage between a common photoaging-associated mtDNA deletion and the T414G mutation. This linkage indicates that mtDNA mutations such as these are unlikely to be distributed equally across the mtDNA population within the skin tissue, increasing their likelihood of exerting focal effects at the cellular level. Taken together, these data significantly contribute to our understanding of the DNA damaging effects of UV exposure and how resultant mutations may ultimately contribute towards premature aging. Key words: aging; fibroblast; mtDNA; mutation; skin; UV.

Correspondence Mark A. Birch-Machin, Dermatological Sciences, Institute of Cellular Medicine, School of Clinical and Laboratory Sciences, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK. Tel.: 0191 222 5841; fax: 0191 222 7094; e-mail: [email protected] Accepted for publication 17 April 2007

Introduction Current evidence suggests the accumulation of damage to the mitochondrial genome may be a fundamental cause of aging. Consistent with this hypothesis is the discovery of an agedependent increase in mitochondrial DNA (mtDNA) deletion and point mutations across a range of tissues (Chomyn & Attardi, 2003). Initial studies examining evidence for the accumulation of mtDNA point mutations with aging in human tissues focused on known pathogenic base substitutions such as the specific mitochondrial disease causing A3243G mutation. However, in skin this mutation has been reported to affect only a very low proportion of the mtDNA population in normal individuals (up to 0.12%) and mutations at such low levels may not have severely deleterious consequences in normal aging. Furthermore, no age-dependent increase of this mutation has been observed in skin. In contrast to this, a large accumulation of age-associated mtDNA point mutations have been identified in skin fibroblasts in a region of the genome thought to be crucial for the processes of transcription and replication (Michikawa et al., 1999). This particular study highlighted a T414G base change in fibroblasts from more than 50% of donors over the age of 65, and in a generally high proportion of molecules (up to 50%). The T414G mutation lies within the promoter for the synthesis of the RNA primer of mtDNA heavy (H)-strand synthesis and for light (L)-strand transcription, pointing towards a functional relevance (Ghivizzani et al., 1994). However, the inference of mutant levels in cultured fibroblasts to the in vivo situation in the skin tissue is somewhat indirect and the relevance of this finding in these rapidly proliferating cultures has been questioned (Chinnery et al., 2001). The matter of whether point mutations in the control region of mtDNA do actually occur at high levels in skin tissue has not yet been addressed. Information regarding the accumulation of mtDNA deletion mutations in skin tissue is more comprehensive. Reports indicate an age-dependent increase in the incidence and abundance of a 4977-bp deletion (Liu et al., 1998; Lu et al., 1999), although it is clear that the accumulation of mtDNA deletions in skin is largely dependent on solar ultraviolet (UV) radiation exposure rather than chronological aging (Berneburg & Krutmann, 1998; Birch-Machin et al., 1998; Birch-Machin, 2006). The functional consequences of such mtDNA damage in skin remain undefined, but in other body sites including muscle, brain neurons and intestinal crypt cells, high levels of deletions or point mutations have been shown to correlate with respiratory defects (Cao et al., 2001; Bender et al., 2006; Greaves et al., 2006).

© 2007 The Authors Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007

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558 Photoaging accumulation of the T414G mutation in human skin, M. J. Birket and M. A. Birch-Machin

Following the exciting discovery of age-associated mtDNA control region mutations in skin fibroblasts, a question remains regarding the mutational status of skin tissue itself as opposed to proliferating cultured cells. Therefore, the central aim of the current study was to obtain conclusive data on the incidence (frequency of occurrence) of the T414G mutation in dermal skin tissue, the base change previously found to be most frequent in fibroblasts, and how its incidence may be affected by exposure to UV radiation, as shown with mtDNA deletions (Berneburg et al., 1999). To address this we have identified and quantified the T414G mutation in dermal skin samples from 108 donors using denaturing high performance liquid chromatography TM (DHPLC) performed on the WAVE System, in combination with direct DNA sequencing. For a mutation to exert a phenotypic effect on a cell it is thought that the level within the cell’s mtDNA population may have to reach a certain threshold (Porteous et al., 1998). In many tissues mutations can accumulate clonally and exist at high levels within individual cells even though their frequency may be low at the tissue level (Khrapko et al., 1999; Nekhaeva et al., 2002). While nothing is known about the distribution of mtDNA mutations in skin, there is some evidence to suggest that the T414G mutation may be clonally distributed in vitro in fibroblasts (Michikawa et al., 2002). To address the distribution of mtDNA mutations in photoaged skin in vivo we have investigated the occurrence of compound mutations in the same molecules and evaluated the results against theoretical values assuming a random distribution across the skin. The mutation we have analysed in combination with the T414G change is a 3895-bp deletion where the deleted region encompasses the heavy strand promoter but neither the heavy nor light strand origins of mtDNA replication. This 3895-bp deletion is similar to the commonly reported 4977-bp deletion in that it is flanked by two 13-bp direct repeat sequences. The rationale for choosing the 3895-bp deletion is that, first, studies have shown it to be common in photoaged skin (Krishnan et al., 2004; Harbottle & Birch-Machin, 2006) and second, it has a break-point in close proximity to nucleotide 414 thereby facilitating analysis. The linkage we describe between the two genetic alterations indicates that mtDNA mutations such as these are unlikely to be distributed equally across the mtDNA population, which increases their likelihood of exerting focal effects at the cellular level.

Results The T414G mutation is observed in human skin and is associated with photoaging TM

To determine the applicability of the WAVE system for the identification and quantification of a heteroplasmic point mutation in mtDNA, plasmids pT414T and pT414G were titrated and used as templates. The optimal melting temperature for detection of a mutation at nucleotide 414 within our amplicon using the TM WAVE system was ascertained and using this we were able to detect a heteroduplex peak, indicative of the T-G mismatch,

TM

at levels of heteroplasmy down to 1%. Although the WAVE analysis allows for the detection of a mutation within this area of the 203-bp amplicon, this assay alone was not confirmatory of base specificity. Therefore, to confirm that the heteroduplex peak was the T414G base change, we directly sequenced the region in all the tested samples. Samples with a heteroduplex TM peak on the WAVE system clearly showed a mutant 414 peak on the sequence chromatogram and those showing no heteroduplex showed a clear absence of a mutant peak in the sequencing, and this methodology proved to be reliable and reproducible (Fig. 1). For added confirmation, in a number of cases we also eluted the heteroduplex peak with a fraction collector and sequenced the DNA to confirm enrichment of the T414G base change (data not shown). Results from the mutation screen were divided into two donor age groups: under 65 years and 65 or over, based on a previously suggested cut-off value (Michikawa et al., 1999). By screening 108 dermal skin samples we observed that the T414G mutation increases in its incidence (frequency of occurrence) as a function of age. In addition, we observed an increased incidence with increasing sun-exposure according to body site. In detail, evaluation of skin from donors over the age of 65 revealed the T414G mutation in 6 out of 17 sun-protected skin samples compared to 36 out of 38 sun-exposed skin samples (Fig. 2A). This photoaging association was further supported by the presence of the mutation in 10 out of 40 sun-exposed compared with 0 out of 13 sun-protected skin samples from donors under the age of 65. A comparison of the presence or absence of the mutation in all sun-protected vs. sun-exposed samples resulted in strong statistical significance (χ2 = 6.8, P < 0.01). Furthermore, our results demonstrate that the heteroplasmy (percentage of mutant out of the total mtDNA population) of the T414G mutation also increased with age in both sun-protected and sun-exposed skin, reaching a maximum of around 30% mutant mtDNA molecules across the dermis (Fig. 2B). The heteroplasmy was also generally higher in sun-exposed than sun-protected skin. Furthermore, intraindividual variation confirmed a role for solar UV exposure in the incidence of the mutation. For example, two cases were studied where sun-exposed (outer arm) and sunprotected skin (buttock) was assayed from the same individual. Both cases showed the presence of the T414G mutation only in skin from the sun-exposed site, one example of which is shown in Fig. 1C,D. In a number of samples we compared the T414G mutation level in the dermal skin tissue to the corresponding cultured fibroblast population (first passage), and found that the T414G mutation was also present in the fibroblasts and often at a higher level (Fig. 3), although the degree of difference deviated from pair to pair.

The T414G mutation is common within a 3895-bp deleted mtDNA population One of the break-points of a reported photoaging-associated minor-arc deletion lies in relatively close proximity to the T414G


Photoaging accumulation of the T414G mutation in human skin, M. J. Birket and M. A. Birch-Machin 559

Fig. 1 Identification of the T414G mutation. A combination of WAVE™ and direct DNA sequencing was used to identify the T414G mutation as exemplified here with four human skin samples. (A) Wild-type skin from a 28-year-old (sun-exposed site), showing the absence of a heteroduplex peak on the WAVE™ chromatogram as well as in the sequencing. (B) T414G mutant skin from an 83-year-old (sun-exposed site), showing the presence of a heteroduplex peak on the WAVE™ chromatogram and characteristic double peak in the sequencing. (C) Sun-protected skin (buttock) and (D) sun-exposed (outer arm) from the same 73-year-old individual. A small heteroduplex peak on the WAVE™ trace as well as in the sequencing chromatogram in (D) but not (C) shows the presence of the T414G mutation in only the sun-exposed skin of this individual.

mutation (nucleotide position; np 536 –548) (Krishnan et al., 2004), and this enabled us to address the distribution of these two representative photoaging-associated mutations. The close proximity of the 414 nucleotide to the 3895-bp deletion break-points facilitated the use of an alternative, deletionspecific reverse primer, in order to amplify and sequence the 414 nucleotide in linkage with the deletion. Primers 1 and 2 were used for sequencing the full length/mixed population (Fig. 4A), while a short extension time ensures that Primers 1 and 3 will only generate a 482-bp polymerase chain reaction (PCR) product from molecules containing the 3895-bp deletion (Fig. 4C). Examples of the relative sequence chromatograms from the mixed and 3895-bp deleted populations of one particular sample are shown by Fig. 4B,D. In this example the 3895-bp deleted population contained higher levels of the T414G mutation than the mixed population (~100% vs. ~26%, respectively). We describe the non-3895-bp deleted population as ‘mixed’ because it will include molecules with other deletions provided that the primer binding sites are intact. Amplification of the 3895-bp deleted population was successful in 28 sun-exposed skin samples and permitted sequencing of the population in each case. Comparison of the level of T414G in the mixed population vs. the level within this specific 3895-bp deleted population revealed a significantly skewed distribution, which indicated that these two mutations are present together more commonly than predicted (P < 0.01 Wilcoxon signedranks, Fig. 4E). This prediction is based on the null hypothesis

that the point mutation will be at the same level within these different genetic populations.

Discussion The results of the present study have extended previous work evaluating the T414G point mutation by (i) identifying the mutation for the first time in skin tissue and (ii) demonstrating increased incidence in skin of sun-exposed origin. Additionally, we have discovered an interesting genetic linkage between a 3895-bp deletion and the T414G base change, giving an insight into the in vivo distribution of such mutations in skin. It was important to establish whether mtDNA control region point mutations were present in skin tissue biopsies, as their presence in cultured fibroblasts may not be reflective of the in vivo situation because levels may diverge rapidly in culture, as demonstrated for other mtDNA mutations (Koch et al., 2001; Krishnan & Birch-Machin, 2006). Therefore, in vitro analyses may not give a clear quantitative picture of age-related changes in the skin itself. Here we show that the T414G mutation, which was previously shown to be the most prevalent control region mutation in fibroblasts from older donors (Michikawa et al., 1999), can also be identified in dermal skin tissue. We also report the very striking observation that UV radiation exposure accelerates the accumulation of this mutation, thereby suggesting a direct influence for UV on the accumulation of mtDNA control region point mutations in the skin. UV radiation is known to



Fig. 3 T414G mutation level in dermal skin (closed bars) and corresponding fibroblast cultures (open bars). Fresh skin was split and some of the dermis was used directly for DNA isolation and the remainder was used for the isolation of fibroblasts. DNA was isolated from fibroblasts as soon as they became confluent within the flask.

Fig. 2 The T414G point mutation accumulates in a photoaging-dependent manner in dermal skin. (A) Presence or absence of T414G mutation. Samples are grouped into those obtained from donors < 65 years old or those ≥ 65 years, and additionally by sun-exposure status. The values above each bar indicate the actual proportion of T414G positive samples. The association with both the ≥ 65-year-old population and the sun-exposed skin sites is statistically significant (χ2 = 18.5 and 6.8, respectively, P < 0.01). (B) T414G mutation heteroplasmy level in dermal skin samples from either sun-exposed (closed boxes), or sun-protected sites (open circles), from donors of varying ages. Samples where the precise donor age was unknown were omitted.

increase oxidative stress (Rezvani et al., 2006), and this is particularly true for the longer wavelengths that are able to penetrate through to the dermis. Our results are therefore suggestive of a direct link between oxidative stress and the presence of the T414G mutation. The proximity of the 414 nucleotide to the origin of heavy-strand replication could make this region particularly vulnerable to free radical mediated damage during mtDNA replication because of the prolonged period of time that this region will be in single-strand form. This connection to a stressful environment is also evident given the identification

of the T414G mutation in the brains of Alzheimer’s patients (Coskun et al., 2004); a pathological condition known to be associated with mitochondrial oxidative stress. Interestingly, distinct mtDNA mutations in this region of the genome have been shown to accumulate with age in skeletal muscle, demonstrating a tissue-specific pattern of occurrence (Wang et al., 2001). Our analyses have focussed on the dermal compartment of the skin because mtDNA mutations are observed more commonly in the dermis compared to the epidermis (Krishnan et al., 2004). In this respect, we did detect the T414G mutation in a small number of epidermal samples that were analysed; however, the exact frequency of occurrence within the epidermis was not determined (data not shown). mtDNA deletions accumulate in skin in a similar photoagingdependent fashion to that observed for the T414G mutation (Berneburg et al., 1997; Ray et al., 2000; Eshaghian et al., 2006). This led us to consider whether a random distribution of deletion and point mutations exist across the population of mtDNA molecules or whether there is evidence for such mutations being present together more commonly than would be predicted by their individual frequencies. If mutations were independently distributed we would have expected to see the 3895-bp deleted molecules carrying the same abundance of T414G as the mixed population. Interestingly, our results demonstrated a significantly skewed distribution indicating that these two mutations are present together more commonly than predicted. The most extreme example of this trend is where one skin sample harboured the T414G mutation at a level of 13% within the mixed population, whereas in the same skin sample all the 3895bp deleted molecules appeared to carry the T414G mutation. We propose a few possible explanations for this phenomenon. First, one mutation could predispose to the induction of the other perhaps through mechanisms involving elevated oxidative stress; however, to date there is no evidence to suggest that either mutation has this effect. Second, the 3895-bp deleted mtDNA molecules also carrying the T414G mutation may have a selective advantage over those not carrying the T414G mutation,



Fig. 4 The T414G mutation is common within a 3895-bp deleted population. (A) A schematic representation of full length mtDNA. Primers 1 and 2 can bind and generate a polymerase chain reaction (PCR) product. (B) Sequencing the 522-bp product generated from Primers 1 and 2 gives a representation of the level of T414G on the full length molecules (or deleted molecules where the annealing sites of Primers 1 and 2 are still intact). A PCR with Primers 1 and 3 will not generate a product from molecules not carrying the 3895-bp deletion when a short extension time is used in the PCR assay. (C) A schematic representation of 3895-bp deleted mtDNA. On 3895-bp deleted molecules the annealing site of Primer 3 is brought closer to Primer 1, allowing the amplification of this region. (D) Sequencing the 482-bp PCR product generated using Primers 1 and 3 gives a representation of the level of T414G on molecules carrying this specific deletion. (E) T414G level within the mixed population plotted against the level within the 3895-bp deleted population in 28 sun-exposed skin samples where the deletion could be amplified. The percentage of 3895-bp deleted molecules carrying the T414G mutation is significantly higher than the percentage of non-3895-bp deleted (mixed) molecules (P < 0.01, Wilcoxon signed-ranks). The dashed line indicates the expected correlation if the point mutation had been approximately the same level within the different mtDNA populations.

causing the compound mutant molecules to become the more common deleted species. For this mechanism to explain such data this would require a very strong selective advantage and this seems unlikely. An alternative explanation is that the two mutations are independent in their induction and all species have a similar rate of accumulation but the mutations are only generated in a subset of cells within the skin. Therefore, by sequencing the mixed population we may be analysing the majority of the cells across the dermis, but when we limit this to the 3895-bp deleted population we may be restricting this to a subset of cells that for some reason have been more prone to this deletion. The direct influence of UV exposure on the incidence of both the 3895-bp deletion (previous studies) and the T414G mutation (this study) might suggest these species may occur more frequently in the cells within the upper dermis

where penetration will be greater. This latter explanation has the advantage that it assumes no direct phenotypic influence from either mutation. Given this scenario UV-associated mtDNA mutations may be generated within a subset of cells rather than being dispersed across the whole cell population in the skin. Our present study has shown that multiple mutations can exist within single cells and this may have implications regarding the threshold of accumulation required before a cell is affected phenotypically. It is possible that the effects of a multiplicity of mtDNA mutations may combine to influence the overall bioenergetic capacity of the cell. Consistent with this is a report showing a correlation between aggregate mutational load in the mtDNA control region and cytochrome c oxidase deficiency in individual muscle fibres from aged donors (Del Bo et al., 2003).



The generally higher level of the T414G mutation in the cultured fibroblasts compared to the corresponding dermal tissue might suggest that the fibroblast is the primary cell type harbouring this mutation within the dermis. It is worth noting that these are very early passage cell cultures and so any selective mechanism of this mutation may not be fully operating (Michikawa et al., 2002). Future studies will be required to investigate the behaviour of these fibroblasts in order to determine whether the T414G mutation is of functional importance in these cultured cells. In summary, these data demonstrate an age-dependent accumulation of the T414G mutation in skin tissue, accumulation that is strongly influenced by exposure to UV radiation, parallel to that previously observed with mtDNA deletions. This suggests a common stress-related mechanism is involved in the induction and/or accumulation of both mtDNA deletions and control region point mutations in skin. Importantly, the genetic linkage we report between the T414G mutation and a 3895-bp deletion could be indicative of a spatially restricted generation of mtDNA damage in photoaged skin. This would increase the likelihood of a cell acquiring multiple mtDNA alterations, the accumulation of which may ultimately lead to the onset of mitochondrial dysfunction and a consequential aging phenotype.

Experimental procedures Skin and fibroblast samples Sun-exposed skin samples were derived from usually (i.e. scalp, face, neck, ears and forearms) or occasionally (i.e. shoulders, back and chest) exposed skin areas. The majority of these were obtained with informed consent from non-melanoma skin cancer (NMSC) patients attending the skin cancer excision clinic at the Royal Victoria Infirmary, Newcastle, UK (n = 66). In addition, normal skin was donated as a gift from Unilever, Colworth, UK (n = 10); or obtained from previous post-mortem samples (n = 2). Sunprotected skin samples were derived from rarely exposed sites including solely: foreskin, buttock, heel or inner upper arm. These comprised previously obtained post-mortem skin (n = 16); samples offered as a gift from Unilever (n = 10); circumcisions from the Royal Victoria Infirmary (n = 2); and from NMSC patients attending the skin cancer excision clinic at the Royal Victoria Infirmary (n = 2). The male to female donor ratio in this study was relatively even (56.5% female). None of the patients used had a mitochondrial disease. Epidermis and dermis were separated using 0.25% dispase at 4 °C overnight (Durham et al., 2003) and DNA was extracted using a Qiagen DNeasy (Crawley, UK) tissue extraction kit. Fibroblasts were isolated by explanting dermal skin and the outgrowing dermal fibroblasts were harvested once they 2 approached confluence within the first 75 cm flask.

Creation of control plasmids pT414T and pT414G The region surrounding the mtDNA np 414 was amplified from a heteroplasmic fibroblast sample using primers (np 16 –35)

5′-AACCTAT TAACCACTCACGG-3′ and (np 611–591) 5′CAGTGTAT TGCT T TGAGGAGG-3′. The resulting PCR product was cloned into a TOPO TA cloning vector (Invitrogen, Carlsbad, CA, USA). Positive colonies were picked and analysed by PCR amplification and T414T and T414G plasmid containing colonies were selected. Plasmid preparations obtained using a DNA Midi-prep (Qiagen) were standardized using multiple pico green dsDNA binding assays measured with a fluorimeter, using λ / HindIII DNA as a standard. The plasmids were then titrated to create the desired heteroplasmic levels for calibration.

Denaturing high performance liquid chromatography WAVETM analysis A 203-bp amplicon was amplified around np 414 using primers: (np 362–368) 5′-CAA AGA ACC CTA ACA CCA GCC-3′ and (np 564–544) 5′-CT T TGG GGT T TG GTT GGT TCG-3′. PCRs were performed in 50-µL volumes using 200 µM dNTPs, 15 pM primers, 1× PCR buffer containing 1.5 mM MgSO4, 2.5 units Optimase polymerase (Transgenomic, Cramlington, UK) and ~10 ng total genomic DNA or 1 ng plasmid DNA as template. Reaction conditions were 94 °C for 5 min and 30 cycles of 94 °C for 30 s, 62.5 °C for 45 s, and 72 °C for 45 s, followed by a final 7-min extension at 72 °C. Heteroduplexing was performed by heating the PCR products at 95 °C for 5 min, then cooling at a rate of 1.5 °C per min until a temperature of 25 °C was reached. The reactions were then analysed immediately or stored at 4 °C until analysis. TM The PCRs were screened with the WAVE System (Transgenomic) at a temperature of 57 °C using a gradient of Buffer B from 51.9 to 59.9% in 6 min (2% slope) and a sample injection volume of 10 µL. A standard curve was generated for each run using the titrated plasmids as templates. Mutant heteroplasmy was calculated from the area of the heteroduplex peak (reflecting the T-G mismatch) relative to the homoduplex peak (reflecting T-T or G-G pairing). Samples were only regarded as mutation positive if the level was equal to or greater than 2%. To confirm the identity of the mutant peak, some of the samples were subject to fraction collection and sequencing using the reverse primer.

Direct automated sequencing A 522-bp amplicon was amplified around np 414 using M13 tagged primers: (np 315–332) 5′-CGCT TCTGGCCACAGCAC-3′ (with M13 uni tag) and (np 803–786) 5′-GGTGTGGCTAGGCTAAGC-3′ (with M13 rev tag). PCRs were performed in 50-µL reactions using 200 µM dNTPs, 0.4 µM primers, 1× PCR buffer containing 1.5 mM MgCl2, 2.6 units Expand Hi-Fidelity DNA polymerase (Roche, Burgess Hill, UK) and ~10 ng total genomic DNA as template. Reaction conditions were 94 °C for 2 min and 30 cycles of 94 °C for 15 s, 58 °C for 30 s and 72 °C for 1 min, followed by a final 7-min extension at 72 °C. The PCR products were separated on a 1% agarose TAE gel, excised under a UV lamp and purified using a QIAquick Gel Extraction Kit (Qiagen). The purified DNA was quantified and 100 ng was air dried and sequenced by MWG Biotech, Ebersberg, Germany.



3895-bp deletion amplification and control region sequencing The control region of 3895-bp deleted mtDNA was amplified using primers spanning the deletion break-point. A short extension time was used to ensure that only deleted molecules are amplified. Primers were as follows: (np 315–332) 5′-CGCT TCTGGCCACAGCAC-3′ (with M13 uni tag) and (np 4657– 4676) 5′-GAT TATGGATGCGGT TGCT T-3′. PCRs were performed in 50 µL reactions using 200 µM dNTPs, 0.6 µM primers, 1× PCR buffer containing 1.8 mM MgCl2, 2.5 units FastStart Hi-Fidelity DNA polymerase (Roche) and ~10 ng total genomic DNA as template. Reaction conditions were 95 °C for 2 min and 30 cycles of 95 °C for 30 s, 57 °C for 30 s, and 72 °C for 45 s, followed by a final 5-min extension at 72 °C. The PCR products were separated on a 1% agarose TAE gel, the 482-bp band was excised under a UV lamp and purified using a QIAquick Gel Extraction Kit (Qiagen). The purified DNA was quantified and 100 ng was air dried and directly sequenced by MWG Biotech.

Quantification of T414G mutation on 3895-bp deleted background Using a titration series of pT414T and pT414G plasmids as template, the region around the 414 nucleotide was amplified using primers (np 315–332) 5′-CGCT TCTGGCCACAGCAC-3′ (with M13 uni tag) and 5′-CAGGAAACAGCTATGAC-3′ (sequence within the vector). Expand Hi-Fidelity DNA polymerase was used for amplification using the same reaction mix and PCR programme as described above (see Direct automated sequencing). The reaction products were purified and directly sequenced. The height of the mutant nucleotide peak relative to the height of the previous guanine nucleotide reflects the level of heteroplasmy and was used to generate a calibration curve. Detection of the point mutation and the peak ratio measurement was reproducible.

Statistical analyses 2

The χ -test was used for analysis of the data regarding the presence or absence of the T414G mutation using GraphPad QuickCalcs online software. The test was used to compare the data divided either by donor age (under 65 vs. over 65) or sun-exposure status (sun protected vs. sun exposed). The nonparametric Wilcoxon signed-ranks test was used to compare the heteroplasmy of the T414G mutation within a mixed vs. a 3895-bp deleted population performed using SPSS11.0 (SPSS, UK LTD, Waking, UK).

Acknowledgments This work was made possible by the generous support of the Biotechnology and Biological Sciences Research Council (BBSRC) (BBSRC CASE award with Unilever). The authors are grateful to Drs C. Lawrence, J. Langtree and the patients of the Royal Victoria Infirmary Dermatology Out-Patients Clinic for their help in this study. We also thank Penny Lovat for critical review of the manuscript.

References Bender A, Krishnan KJ, Morris CM, Taylor GA, Reeve AK, Perry RH, Jaros E, Hersheson JS, Betts J, Klopstock T, Taylor RW, Turnbull DM (2006) High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease. Nat. Genet. 38, 515–517. Berneburg M, Gattermann N, Stege H, Grewe M, Vogelsang K, Ruzicka T, Krutmann J (1997) Chronically ultraviolet-exposed human skin shows a higher mutation frequency of mitochondrial DNA as compared to unexposed skin and the hematopoietic system. Photochem. Photobiol. 66, 271–275. Berneburg M, Grether-Beck S, Kurten V, Ruzicka T, Briviba K, Sies H, Krutmann J (1999) Singlet oxygen mediates the UVA-induced generation of the photoaging-associated mitochondrial common deletion. J. Biol. Chem. 274, 15345–15349. Berneburg M, Krutmann J (1998) Mitochondrial DNA deletions in human skin reflect photo- rather than chronologic aging. J. Invest. Dermatol. 111, 709–710. Birch-Machin MA (2006) The role of mitochondria in ageing and carcinogenesis. Clin. Exp. Dermatol. 31, 548–552. Birch-Machin MA, Tindall M, Turner R, Haldane F, Rees JL (1998) Mitochondrial DNA deletions in human skin reflect photo- rather than chronologic aging. J. Invest. Dermatol. 110, 149–152. Cao Z, Wanagat J, McKiernan SH, Aiken JM (2001) Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection. Nucleic Acids Res. 29, 4502–4508. Chinnery PF, Taylor GA, Howell N, Brown DT, Parsons TJ, Turnbull DM (2001) Point mutations of the mtDNA control region in normal and neurodegenerative human brains. Am. J. Hum. Genet. 68, 529–532. Chomyn A, Attardi G (2003) mtDNA mutations in aging and apoptosis. Biochem. Biophys. Res. Commun. 304, 519–529. Coskun PE, Beal MF, Wallace DC (2004) Alzheimer’s brains harbor somatic mtDNA control-region mutations that suppress mitochondrial transcription and replication. Proc. Natl Acad. Sci. USA 101, 10726– 10731. Del Bo R, Crimi M, Sciacco M, Malferrari G, Bordoni A, Napoli L, Prelle A, Biunno I, Moggio M, Bresolin N, Scarlato G, Pietro Comi G (2003) High mutational burden in the mtDNA control region from aged muscles: a single-fiber study. Neurobiol. Aging 24, 829–838. Durham SE, Krishnan KJ, Betts J, Birch-Machin MA (2003) Mitochondrial DNA damage in non-melanoma skin cancer. Br. J. Cancer 88, 90–95. Eshaghian A, Vleugels RA, Canter JA, McDonald MA, Stasko T, Sligh JE (2006) Mitochondrial DNA deletions serve as biomarkers of aging in the skin, but are typically absent in nonmelanoma skin cancers. J. Invest. Dermatol. 126, 336–344. Ghivizzani SC, Madsen CS, Nelen MR, Ammini CV, Hauswirth WW (1994) In organello footprint analysis of human mitochondrial DNA: human mitochondrial transcription factor A interactions at the origin of replication. Mol. Cell. Biol. 14, 7717–7730. Greaves LC, Preston SL, Tadrous PJ, Taylor RW, Barron MJ, Oukrif D, Leedham SJ, Deheragoda M, Sasieni P, Novelli MR, Jankowski JA, Turnbull DM, Wright NA, McDonald SA (2006) Mitochondrial DNA mutations are established in human colonic stem cells, and mutated clones expand by crypt fission. Proc. Natl Acad. Sci. USA 103, 714–719. Harbottle A, Birch-Machin MA (2006) Real-time PCR analysis of a 3895 bp mitochondrial DNA deletion in nonmelanoma skin cancer and its use as a quantitative marker for sunlight exposure in human skin. Br. J. Cancer 94, 1887–1893. Khrapko K, Bodyak N, Thilly WG, van Orsouw NJ, Zhang X, Coller HA, Perls TT, Upton M, Vijg J, Wei JY (1999) Cell-by-cell scanning of whole mitochondrial genomes in aged human heart reveals a significant



fraction of myocytes with clonally expanded deletions. Nucleic Acids Res. 27, 2434–2441. Koch H, Wittern KP, Bergemann J (2001) In human keratinocytes the common deletion reflects donor variabilities rather than chronologic aging and can be induced by ultraviolet A irradiation. J. Invest. Dermatol. 117, 892–897. Krishnan KJ, Birch-Machin MA (2006) The incidence of both tandem duplications and the common deletion in mtDNA from three distinct categories of sun-exposed human skin and in prolonged culture of fibroblasts. J. Invest. Dermatol. 126, 408–415. Krishnan KJ, Harbottle A, Birch-Machin MA (2004) The use of a 3895 bp mitochondrial DNA deletion as a marker for sunlight exposure in human skin. J. Invest. Dermatol. 123, 1020–1024. Liu VW, Zhang C, Pang CY, Lee HC, Lu CY, Wei YH, Nagley P (1998) Independent occurrence of somatic mutations in mitochondrial DNA of human skin from subjects of various ages. Hum. Mutat. 11, 191–196. Lu CY, Lee HC, Fahn HJ, Wei YH (1999) Oxidative damage elicited by imbalance of free radical scavenging enzymes is associated with largescale mtDNA deletions in aging human skin. Mutat. Res. 423, 11–21. Michikawa Y, Laderman K, Richter K, Attardi G (2002) Role of nuclear background and in vivo environment in variable segregation behavior of the aging-dependent T414G mutation at critical control site for human fibroblast mtDNA replication. Somat. Cell Mol. Genet. 25, 333–342.

Michikawa Y, Mazzucchelli F, Bresolin N, Scarlato G, Attardi G (1999) Aging-dependent large accumulation of point mutations in the human mtDNA control region for replication. Science 286, 774–779. Nekhaeva E, Bodyak ND, Kraytsberg Y, McGrath SB, Van Orsouw NJ, Pluzhnikov A, Wei JY, Vijg J, Khrapko K (2002) Clonally expanded mtDNA point mutations are abundant in individual cells of human tissues. Proc. Natl Acad. Sci. USA 99, 5521–5526. Porteous WK, James AM, Sheard PW, Porteous CM, Packer MA, Hyslop SJ, Melton JV, Pang CY, Wei YH, Murphy MP (1998) Bioenergetic consequences of accumulating the common 4977-bp mitochondrial DNA deletion. Eur. J. Biochem. 257, 192–201. Ray AJ, Turner R, Nikaido O, Rees JL, Birch-Machin MA (2000) The spectrum of mitochondrial DNA deletions is a ubiquitous marker of ultraviolet radiation exposure in human skin. J. Invest. Dermatol. 115, 674–679. Rezvani HR, Mazurier F, Cario-Andre M, Pain C, Ged C, Taieb A, de Verneuil H (2006) Protective effects of catalase overexpression on UVB-induced apoptosis in normal human keratinocytes. J. Biol. Chem. 281, 17999–18007. Wang Y, Michikawa Y, Mallidis C, Bai Y, Woodhouse L, Yarasheski KE, Miller CA, Askanas V, Engel WK, Bhasin S, Attardi G (2001) Musclespecific mutations accumulate with aging in critical human mtDNA control sites for replication. Proc. Natl Acad. Sci. USA 98, 4022– 4027.
