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Mitochondrial DNA: The Journal of DNA Mapping, Sequencing, and Analysis Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/imdn20
Age-related decrease in mtDNA content as a consequence of mtDNA 4977 bp deletion a
a
a
Leila Zabihi Diba , Seyed Mojtaba Mohaddes Ardebili , Jalal Gharesouran & Massoud b
Houshmand a
Department of Biochemistry and Clinical Laboratory, Division of Medical Genetics, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran and b
Department of Medical Genetics, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran Published online: 08 Jul 2015.
To cite this article: Leila Zabihi Diba, Seyed Mojtaba Mohaddes Ardebili, Jalal Gharesouran & Massoud Houshmand (2015): Age-related decrease in mtDNA content as a consequence of mtDNA 4977 bp deletion, Mitochondrial DNA: The Journal of DNA Mapping, Sequencing, and Analysis To link to this article: http://dx.doi.org/10.3109/19401736.2015.1063046
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–5 ! 2015 Informa UK Ltd. DOI: 10.3109/19401736.2015.1063046
RESEARCH ARTICLE
Age-related decrease in mtDNA content as a consequence of mtDNA 4977 bp deletion Leila Zabihi Diba1, Seyed Mojtaba Mohaddes Ardebili1, Jalal Gharesouran1, and Massoud Houshmand2 1
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Department of Biochemistry and Clinical Laboratory, Division of Medical Genetics, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran and 2Department of Medical Genetics, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran
Abstract
Keywords
As one of the most frequent somatic mutations accumulated during aging in human mitochondrial DNA, the 4977 bp deletion has intrigued scientific interest in recent years. Although many studies have shown a significant increase in the amount of 4977 bp deletion, the findings with respect to an age-dependent escalate of DmtDNA4977 bp in blood are still disputatious. Therefore, we investigated the presence of common deletion and mtDNA deletion level in whole blood samples of 100 old individuals (60–90 years). We detected the accumulation of common deletion in 46 old individuals. Consequently, there was statistically significant difference between the aged and young individuals in mitochondrial content (p ¼ 0.01) and deletion levels ranged from 2% to 17% of the total mtDNA (mean: 10% ± 0.02%). We conclude that common deletion has decreased the mtDNA content; however, it is not clearly detectable in the blood as one of the fast replicating tissues comparing with tissues with low mitotic activity.
Aging, common deletion, mitochondria, mtDNA deletion, DmtDNA 4977 bp
Introduction Aging is an inevitable natural phenomenon wherein there is a gradual decline in the physical and mental faculties of an individual (Poulose & Raju, 2014). Aging is generally describe as accumulation of a variety of adverse changes in different cells and tissues which result in alteration in macromolecules including DNA (Harman, 2003). According to the free radical theory of aging which was originally coined by Herman, accumulation of free radical produced by mitochondria postulated as the major reason led to cumulative and extensive damages that are responsible for aging and disease. (Finkel & Holbrook, 2000; Harman, 1956; Piotrowska & Bartnik, 2014) After indicating that mitochondria are the huge intracellular source of reactive oxygen species (ROS), it is hypothesized that deterioration in mitochondrial function result in generating more free radical and eventually more accumulation of DNA damage lead to cell failure and death (Lauri et al., 2014 Stuart et al., 2014; Wallace, 2008). Mitochondria are distinctive among the cellular organelles, as they contain their own unique genetic content, mtDNA, a doublestranded circular molecule of 16.5 kb encoding 13 nuclear Correspondence: Seyed Mojtaba Mohaddes Ardebili, Professor of Medical Genetics, Department of Biochemistry and Clinical Laboratory, Division of Medical Genetics, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. Tel: +98 9126628091. E-mail: [email protected] Dr Massoud Houshmand, Associate Professor of Medical Genetics, Department of Medical Genetics, National Institute for Genetic Engineering and Biotechnology, Tehran, Islamic Republic of Iran. Tel: +98 9123369613. E-mail: [email protected], [email protected]
History Received 21 April 2015 Accepted 14 June 2015 Published online 7 July 2015
proteins, 22 transfer RNAs (tRNAs), and two ribosomal RNAs in mammals (Bratic & Larsson, 2013; Yu et al., 2007). Mitochondria consider as a key organelle in producing cellular power that has also significant role in cell proliferation and death (Fang et al., 2010). Mitochondria are semi-autonomous elements, the biogenesis of which depends on the interconnected expression of the genome located in two cellular compartments: the nucleus and the mitochondrial matrix (Osiewacz & Bernhardt, 2013). Apart from playing a role as ATP factory of cells, mitochondria perform a multiplicity of functions like signaling, redox homeostasis, calcium storage and regulation of membrane potential, apoptosis and inflammasome activation. It clearly demonstrates the importance of age-dependent decline in mitochondrial function as a determinant factor which adversely affects the cells’ bio-energetic and survival mechanisms (Poulose & Raju, 2014). Alterations in both quality of mtDNA (mutations) (Schon et al., 1997; Shen et al., 2011; Wallace et al., 1995) and quantity of mtDNA (copy number) (Clay Montier et al., 2009) have been associated with many human diseases including neurodegenerative diseases, metabolic diseases, and variety types of cancer and also in aging process (Brandon et al., 2006; Chatterjee et al., 2006; Shen et al., 2010). There is concrete evidence that the amount of mtDNA mutations increases with age in humans; as an exemplary case, deletions in mtDNA have been identified in the aged human central nervous system, skeletal muscle, and hepatocytes (Corral-Debrinski et al., 1992; Fayet et al., 2002; Yen et al., 1991). A human somatic cell typically comprises myriad copies of mtDNA, whereas an oocyte comprises roughly 105 copies. Replication of mtDNA is independent of the cell cycle, and the replication of a particular mtDNA molecule may,
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therefore, occurs numerous times or not at all as a cell divides (Clayton, 1982). In order to identify the role of the mtDNA 4977-bp deletion in aging process and testing the possibility of indicating mtDNA 4977-bp deletion as a biomarker of aging, we studied 100 old subjects who referred to medical diagnostic Laboratory and carried out a systematic investigation in order to evaluate mtDNA 4977-bp deletion and the mtDNA content in peripheral blood samples and their association with human aging process. These findings heighten our comprehension of the role of mtDNA alterations in aging.
Material and methods Sample collection and DNA extraction Blood samples from 100 old individuals (60–90) who were referred to a medical diagnosis laboratory for regular check were obtained. At the time of sampling, a complete history, including
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Table 1. Distribution of gender and age among people included in study. Gender
Frequency (%)
Average age
Male Female
54 46
70.55 69.97
Table 2. Sequences of primers used in the multiplex PCR reactions. Control 4977-bp Within Deletion region B-actin
86 89 25 74 71 46
50 -CCCTTACCACGCTACTCCTA-30 50 -GGCGGGAGAAGTAGATTGAA-30 50 - CTACGGTCAATGCTCTGAAA-30 50 -GGTTGACCTGTTAGGGTGAG-30 50 - TGCTAGTAACCACGTTCTCC-30 50 - TTTGTTAGGGTTAACGAGGG-30
F 50 - AGACGCAGGATGGCATGGG-30 R 50 - GAGACCTTCAACACCCCAGCC-30
Figure 1. A schematic design used for determination of individuals with 4977 bp mtDNA deletion. This diagram is not drawn to scale.
neurodegenerative disease like Alzheimer and Parkinson was collected for all subjects. We used a structured questionnaire to identify disease-free samples and to exclude subjects who were suspected of having our targeted neurodegenerative disease. The study was ratified by the Ethics Committee of the Tabriz University of Medical Sciences. The mean age of the examinees was 70.29 years. The youngest examinee was 60-year old, while the oldest 90. The gender ratio in both groups was about 46% females to 54% males (Table 1). All individuals were drawn from population-representative collections. Blood samples were collected from peripheral blood and genomic DNA was isolated from the blood samples using purification kit according to the instructions of the manufacturer (DNA fast, QIAGEN, Hilden, Germany). The quality of extracted DNA was quantified by spectrometric analysis (NanoDropÔ 2000c, UV-Vis spectrophotometer, Thermo Fisher Scientific, Waltham, MA). Detection and measurement of mtDNA4977 bp deletion The detection of mtDNA 4977-bp deletion was performed using a multiplex polymerase chain reaction (PCR) protocol. Two pairs of primers for detection of the 4977-bp deletion were described in Table 2. The PCR condition was set as the following: pre denaturation at 94 C for 5 min; then 35 cycles at 94 C for 1 min, 57 C for 1 min, and 72 C for 35 s; and a final extension at 72 C for 10 min. PCR products were then electrophoresed on a 1.5% agarose gel at 100 V (the buffer fluid was 1 TAE buffer). By design, PCR with primer pairs (86/89) yields the 297-bp product and the presence of the 4977-bp deletion was revealed by the appearance of a 497-bp band (Figure 1), which was confirmed by sequencing analysis. To illustrate, primer oligonucleotides (25/74) were designed to anneal outside the common deletion. Indeed, wild-type mtDNA as the template would not produce any PCR products under such circumstances because of the large flanking region (45-kb). Co-amplification of mtDNA4977 bp and wildtype mtDNA showed no interference between the primers pairs hence, the PCR products could be clearly distinguishable by separating in agarose gel electrophoresis. As a positive control, DNA from a sample with confirmed 497 bp deletion was obtained for the presence of dmtDNA4977 bp (Figure 1).
Mitochondrial 4977 bp deletion and aging
DOI: 10.3109/19401736.2015.1063046
Sequencing analysis To confirm the specificity of the PCR product contain the deletion junction created by common deletion, the 503 bp PCR band fragment was purified using an agarose gel DNA fragment recovery kit, Ver.2.0 (TaKaRa, Shiga, Japan) and sequenced using an ABI PRISM 3730 sequence analyzer (gene Fanavaran, Macrogene, Seoul, Korea) (Figure 2). Sequencing was performed in both strands using the original primers. Sequence analysis was carried out using the Finch TV 1.4.0 softwareÕ (Geospiza, Inc., Seattle, WA). The sequences were compared with the human mtDNA reference sequence using the NCBI BLAST sequence analysis tool (Macrogene, Seoul, Korea).
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Quantitative analysis When comparing the relative amount of dmtDNA4977 bp, only individuals who carried the DmtDNA4977 bp mutation were considered in the analysis. Real-time quantitative PCR was performed using the ROTOR GENE 6000 REAL-TIME PCR MACHINE (Corbett Life Science, Mortlake, Australia). Each PCR was performed using the SYBR Premix Ex Taq reagent (Takara Bio, Kyoto, Japan) according to the protocol of the manufacturer. To determine the percent of the common deletion, a specific primer pair (71/46) which is located within the common deletion region and the specific product of these primers was amplified just in wild-type mtDNA without deletion (Table 2). To specify the implication of common deletion in the amount of mtDNA, the whole mtDNA PCR product amplified using the primer pair (86/89) and normalized by simultaneous measurement of nuclear DNA encoded B-actin genes the amplification condition was 1 ml of total DNA extracted in a 15 ml reaction. Predenaturtion at 95 C for 90 s, 50 cycles of three-step PCR followed 95 C for 15 s, 60 C for 20 s, and 72 C for 15 s. A linear regression model evaluated the quantification of wild-type and deleted mtDNA. Ct values of all samples were within the linear range. Two independent measurements of each PCR product were performed in all samples, and it was confirmed that there was significant correlation in the ratio of the common deletion/wildtype mtDNA between these two measurements. The ratio was calculated from the mean values of two measurements.
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Kolmogorov–Smirnov test. The p value50.05 was considered significant. The association between mtDNA alteration and age characteristics of our samples was evaluated using one-way ANOVA analysis.
Result Multiplex PCR (Figure 3) identified the presence of the 4977-bp deletion in the 46 sample of all 100 individuals (46%) from aged people (Figure 4). To determine if the 4977-bp deletion was implicate in the alteration of mtDNA content in aging, the mtDNA copy numbers in 46 cases with desire deletion were analyzed. The amount of mitochondrial copy number in these individuals has been evaluated by measuring the proportion of mtDNA/B-actin. The average DCT value for mtDNA/B-actin for aged individuals was 4.7052 ± 1.8691 (mean ± SD). The average DCT value for mtDNA/B-actin for young individuals as the control group was 6.6170 ± 2.8602 (mean ± SD). As a result, the amount of mitochondrial content was decreased nearly three-fold compared with the control group. Furthermore, the differences between aged people and young people was statistically significant (p value ¼ 0.01). However, there was no significant association between mtDNA alterations and age of subjects. In addition, the percent of deletion was assessed by evaluating the proportion of common deletion region/mtDNA. The results showed that
Statistical analysis Statistical analysis was performed using SPSS for Windows (version 22, SPSS Inc., Chicago, IL). All numerical variables were checked for normal distribution using the one-sample
Figure 3. The number of tested subjects with and without 4977 bp mtDNA deletion.
Figure 2. Schematic diagram of mtDNA 4977 bp deletion. Positive PCR products of mtDNA 4977 bp deletion were confirmed by DNA sequencing. The arrows are at the breakpoint position.
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Figure 4. Electrophoregram of multiplex PCR analysis (agarosis gel 1.5% photographed under UV light).
average percent of deletion among tested individuals were 10% (min 2%, max 17%).
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Discussion In this paper, a study has been conducted based on the mitochondrial theory of aging indicating that cumulative changes in human mitochondrial DNA are associated with aging. Indeed, It has been hypothesized that oxidative stress is responsible for the generation of mitochondrial DNA mutations in human cells (Beckman & Ames, 1998) and since these oxidative stresses increase with age, large scale deletions of mitochondrial DNA such as the common deletion are thought to play a pivotal role in aging process (Berneburg et al., 1999). Most frequently reported mutation is the 4977-bp deletion which is positioned between nucleotide 8470 and 13 447 bp, nominated as the most common and ample large-scale deletion of mtDNA in various human tissues, accounting for 30–50% of all deletions, and has been widely used as a scale of somatic mtDNA injury. The common deletion region is responsible for encoding seven of 13 polypeptides that are crucial subunits for the oxidative phosphorylation pathway (Zhong et al., 2011). Therefore, to determine the common deletion, a multiplex PCR method was performed, and as a result, the presence of 4977 bp deletion in 46% of aged people was observed. In order to quantify the amount of mtDNA of individuals who represent 4977 bp deletion, a real-time PCR was performed. This method has been used according to N. von wrumb et al.’s study. In which, they have indicated that the real time is a suitable method to determine the ratio of 4977 bp deleted mtDNA (von Wurmb-Schwark et al., 2002). These findings illustrate that the incidence of the 4977 bp dmtDNA in aged human normal blood is not clearly detectable during ageing process since it has not detected in all individuals. One of the possible reasons for these results can be explained by referring to Lee et al.’s study. They indicated that although the 4977 bp deletion has been observed nearly in all of the tissues of aged humans, in the some of the old individual’s tissues they could not find 4977 bp deletion (Lee et al., 1994). According to earlier studies, the 4977 bp deletion is just one of the many possible deletions (4834 bp, 3617 bp, 10 422 bp, 7663 bp, 13 162 bp, etc.) in mtDNA which are frequently found in ageing process (Zheng et al., 2012; Zhong et al., 2011) Furthermore, it is interesting to note that the frequency of 4977 bp deletion is higher in some specific tissues; therefore, evaluating this deletion in whole blood may have been influenced the results. One of the evidences for this is Cortopassi et al.’s investigation. Their results showed that the pattern of accumulation of the mtDNA4977 bp deletion in different human tissues is different. Their study revealed that adult heart muscle, brainstem, brain cortex, psoas muscle, and diaphragm muscle have higher levels of the deletion than liver, kidney, skin, spleen, and lung (Cortopassi et al., 1992). Following this further, it has been shown
that the heteroplasmic loads of common deletion were higher in buccal mucosa (37%) and hair (32%) than in blood (17%) (Mkaouar-Rebai et al., 2010). Furthermore, higher degree of mtDNA deletions has been demonstrated in muscle samples, previously. Bua et al. (2006) demonstrated deletion-containing mtDNA genomes increases up to480% and He et al. showed high level of deletion up to 60% (Bua et al., 2006). These results may vindicate that why our findings showed the lower incidence of common deletion in blood samples of old subjects. Indeed, blood as one of fast replicatory tissues compared with low mitotic activity and high oxygen demand tissues like skeletal muscles turn over fairly more rapidly (Lee et al., 1994). Consequently, it has been shown that dmtDNA 4977 bp can be detected in very low amounts in fast replicating tissues such as blood and, in accordance to our study, they concluded that it is uncertain whether an age-dependent distribution of dmtDNA 4977 bp occurs in blood (von Wurmb-Schwark et al., 2010). In other words, the exclusive tracing of the deletions in the mtDNA extracted from the blood does not omit their presence in other tissues, such as muscle (which is not available in our case). Moreover, in a study, when the subjects were divided into two age classes (60–72 years versus 73–95 years), the deletion had significantly higher levels in the older subjects (Bogliolo et al., 1999) and since the average age of our subjects was 70 years old, if we had chosen elder individuals, the result could be different One of the potential possibilities for our findings could relate to other biological factors in the lifestyle of tested individuals. According to N. von Wrumb et al.’s examinations, the variation in 4977 bp concentration among individuals who are in the same or identical age may also result from mtDNA 4977 bp deletion that had been exposed to other biological factors independent of age, like some genotoxic stresses including cigarettes and UVA radiation (von Wurmb-Schwark et al., 2002). Consequently, the mtDNA4977 bp levels could display a snapshot of the mutational incidents that occurred in a relatively short period of time before taking the blood samples (Iwai et al., 2003). Besides, another important factor that should consider is the Involvement of genetic factors in mtDNA4977 bp levels. It has been suggested that variability in specified genes (in both the mitochondrial and the nuclear genome) helps to modulate somatic mtDNA damages through an efficient repair system or a more capable ROS-scavenging process (von Wurmb-Schwark et al., 2010).
Conclusion In the present study, we showed existence of 46% percent of 4977 bp deletion in the blood of aged people and investigated the implication of this deletion on mitochondrial DNA content compared to young individuals. There was statistically significant difference between mtDNA content in aged individuals carrying common deletion and young individuals. From what has been discussed above, we conclude that mtDNA 4977-bp deletion have
DOI: 10.3109/19401736.2015.1063046
decreased the mtDNA content in a age-dependent manner; however, it is not clearly detectable in the blood samples as one of the fast replicating tissues that compensate the mtDNA damage faster than tissues with low mitotic activity and high oxygen demand (skeletal muscle and heart).
Acknowledgements We appreciate all patients for blood donation for scientific research as well as Jorjani clinical diagnostic laboratory for their cooperation and support. The study was approved by the Tabriz University of Medical Sciences Human Research Ethics Committee.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. The research was supported by Tabriz University of Medical Sciences.
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Harman D. (1956). Aging: A theory based on free radical and radiation chemistry. J Gerontol 11:298–300. Harman D. (2003). The free radical theory of aging. Antioxid Redox Signal 5:557–61. Iwai K, Miyao M, Wadano Y, Iwamura Y. (2003). Dynamic changes of deleted mitochondrial DNA in human leucocytes after endurance exercise. Eur J Appl Physiol 88:515–19. Lauri A, Pompilio G, Capogrossi MC. (2014). The mitochondrial genome in aging and senescence. Ageing Res Rev 18c:1–15. Lee HC, Pang CY, Hsu HS, Wei YH. (1994). Differential accumulations of 4,977 bp deletion in mitochondrial DNA of various tissues in human ageing. Biochim Biophys Acta 1226:37–43. Mkaouar-Rebai E, Chamkha I, Kammoun T, Chabchoub I, Aloulou H, Fendri N, Hachicha M, Fakhfakh F. (2010). A case of Kearns–Sayre Syndrome with two novel deletions (9.768 and 7.253 kb) of the mtDNA associated with the common deletion in blood leukocytes, buccal mucosa and hair follicles. Mitochondrion 10:449–55. Osiewacz HD, Bernhardt D. (2013). Mitochondrial quality control: Impact on aging and life span – A mini-review. Gerontology 59: 413–20. Piotrowska A, Bartnik E. (2014). [The role of reactive oxygen species and mitochondria in aging]. Postepy Biochem 60:240–7. Poulose N, Raju R. (2014). Aging and injury: Alterations in cellular energetics and organ function. Aging Dis 5:101–8. Schon EA, Bonilla E, Dimauro S. (1997). Mitochondrial DNA mutations and pathogenesis. J Bioenerg Biomembr 29:131–49. Shen L, Fang H, Chen T, He J, Zhang M, Wei X, Xin Y, et al. (2010). Evaluating mitochondrial DNA in cancer occurrence and development. Ann N Y Acad Sci 1201:26–33. Shen L, Wei J, Chen T, He J, Qu J, He X, Jiang L, et al. (2011). Evaluating mitochondrial DNA in patients with breast cancer and benign breast disease. J Cancer Res Clin Oncol 137:669–75. Stuart JA, Maddalena LA, Merilovich M, Robb EL. (2014). A midlife crisis for the mitochondrial free radical theory of aging. Longev Healthspan 3:4. Von Wurmb-Schwark N, Higuchi R, Fenech AP, Elfstroem C, Meissner C, Oehmichen M, Cortopassi GA. (2002). Quantification of human mitochondrial DNA in a real time Pcr. Forensic Sci Int 126:34–9. Von Wurmb-Schwark N, Schwark T, Caliebe A, Drenske C, Nikolaus S, Schreiber S, Nebel A. (2010). Low level of the mtDNA(4977) deletion in blood of exceptionally old individuals. Mech Ageing Dev 131: 179–84. Wallace DC. (2008). Mitochondria as Chi. Genetics 179:727–35. Wallace DC, Shoffner JM, Trounce I, Brown MD, Ballinger SW, CorralDebrinski M, Horton T, et al. (1995). Mitochondrial DNA mutations in human degenerative diseases and aging. Biochim Biophys Acta 1271: 141–51. Yen TC, Su JH, King KL, Wei YH. (1991). Ageing-associated 5 kb deletion in human liver mitochondrial DNA. Biochem Biophys Res Commun 178:124–31. Yu M, Zhou Y, Shi Y, Ning L, Yang Y, Wei X, Zhang N, et al. (2007). Reduced mitochondrial DNA copy number is correlated with tumor progression and prognosis in Chinese breast cancer patients. IUBMB Life 59:450–7. Zheng Y, Luo X, Zhu J, Zhang X, Zhu Y, Cheng H, Xia Z, et al. (2012). Mitochondrial DNA 4977 bp deletion is a common phenomenon in hair and increases with age. Bosn J Basic Med Sci 12:187–92. Zhong Y, Hu YJ, Yang Y, Peng W, Sun Y, Chen B, Huang X, Kong WJ. (2011). Contribution of common deletion to total deletion burden in mitochondrial DNA from inner ear of D-galactose-induced aging rats. Mutat Res 712:11–19.
Mitochondrial DNA: The Journal of DNA Mapping, Sequencing, and Analysis Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/imdn20
Age-related decrease in mtDNA content as a consequence of mtDNA 4977 bp deletion a
a
a
Leila Zabihi Diba , Seyed Mojtaba Mohaddes Ardebili , Jalal Gharesouran & Massoud b
Houshmand a
Department of Biochemistry and Clinical Laboratory, Division of Medical Genetics, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran and b
Department of Medical Genetics, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran Published online: 08 Jul 2015.
To cite this article: Leila Zabihi Diba, Seyed Mojtaba Mohaddes Ardebili, Jalal Gharesouran & Massoud Houshmand (2015): Age-related decrease in mtDNA content as a consequence of mtDNA 4977 bp deletion, Mitochondrial DNA: The Journal of DNA Mapping, Sequencing, and Analysis To link to this article: http://dx.doi.org/10.3109/19401736.2015.1063046
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
http://informahealthcare.com/mdn ISSN: 1940-1736 (print), 1940-1744 (electronic) Mitochondrial DNA, Early Online: 1–5 ! 2015 Informa UK Ltd. DOI: 10.3109/19401736.2015.1063046
RESEARCH ARTICLE
Age-related decrease in mtDNA content as a consequence of mtDNA 4977 bp deletion Leila Zabihi Diba1, Seyed Mojtaba Mohaddes Ardebili1, Jalal Gharesouran1, and Massoud Houshmand2 1
Downloaded by [UQ Library] at 06:15 15 July 2015
Department of Biochemistry and Clinical Laboratory, Division of Medical Genetics, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran and 2Department of Medical Genetics, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran
Abstract
Keywords
As one of the most frequent somatic mutations accumulated during aging in human mitochondrial DNA, the 4977 bp deletion has intrigued scientific interest in recent years. Although many studies have shown a significant increase in the amount of 4977 bp deletion, the findings with respect to an age-dependent escalate of DmtDNA4977 bp in blood are still disputatious. Therefore, we investigated the presence of common deletion and mtDNA deletion level in whole blood samples of 100 old individuals (60–90 years). We detected the accumulation of common deletion in 46 old individuals. Consequently, there was statistically significant difference between the aged and young individuals in mitochondrial content (p ¼ 0.01) and deletion levels ranged from 2% to 17% of the total mtDNA (mean: 10% ± 0.02%). We conclude that common deletion has decreased the mtDNA content; however, it is not clearly detectable in the blood as one of the fast replicating tissues comparing with tissues with low mitotic activity.
Aging, common deletion, mitochondria, mtDNA deletion, DmtDNA 4977 bp
Introduction Aging is an inevitable natural phenomenon wherein there is a gradual decline in the physical and mental faculties of an individual (Poulose & Raju, 2014). Aging is generally describe as accumulation of a variety of adverse changes in different cells and tissues which result in alteration in macromolecules including DNA (Harman, 2003). According to the free radical theory of aging which was originally coined by Herman, accumulation of free radical produced by mitochondria postulated as the major reason led to cumulative and extensive damages that are responsible for aging and disease. (Finkel & Holbrook, 2000; Harman, 1956; Piotrowska & Bartnik, 2014) After indicating that mitochondria are the huge intracellular source of reactive oxygen species (ROS), it is hypothesized that deterioration in mitochondrial function result in generating more free radical and eventually more accumulation of DNA damage lead to cell failure and death (Lauri et al., 2014 Stuart et al., 2014; Wallace, 2008). Mitochondria are distinctive among the cellular organelles, as they contain their own unique genetic content, mtDNA, a doublestranded circular molecule of 16.5 kb encoding 13 nuclear Correspondence: Seyed Mojtaba Mohaddes Ardebili, Professor of Medical Genetics, Department of Biochemistry and Clinical Laboratory, Division of Medical Genetics, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. Tel: +98 9126628091. E-mail: [email protected] Dr Massoud Houshmand, Associate Professor of Medical Genetics, Department of Medical Genetics, National Institute for Genetic Engineering and Biotechnology, Tehran, Islamic Republic of Iran. Tel: +98 9123369613. E-mail: [email protected], [email protected]
History Received 21 April 2015 Accepted 14 June 2015 Published online 7 July 2015
proteins, 22 transfer RNAs (tRNAs), and two ribosomal RNAs in mammals (Bratic & Larsson, 2013; Yu et al., 2007). Mitochondria consider as a key organelle in producing cellular power that has also significant role in cell proliferation and death (Fang et al., 2010). Mitochondria are semi-autonomous elements, the biogenesis of which depends on the interconnected expression of the genome located in two cellular compartments: the nucleus and the mitochondrial matrix (Osiewacz & Bernhardt, 2013). Apart from playing a role as ATP factory of cells, mitochondria perform a multiplicity of functions like signaling, redox homeostasis, calcium storage and regulation of membrane potential, apoptosis and inflammasome activation. It clearly demonstrates the importance of age-dependent decline in mitochondrial function as a determinant factor which adversely affects the cells’ bio-energetic and survival mechanisms (Poulose & Raju, 2014). Alterations in both quality of mtDNA (mutations) (Schon et al., 1997; Shen et al., 2011; Wallace et al., 1995) and quantity of mtDNA (copy number) (Clay Montier et al., 2009) have been associated with many human diseases including neurodegenerative diseases, metabolic diseases, and variety types of cancer and also in aging process (Brandon et al., 2006; Chatterjee et al., 2006; Shen et al., 2010). There is concrete evidence that the amount of mtDNA mutations increases with age in humans; as an exemplary case, deletions in mtDNA have been identified in the aged human central nervous system, skeletal muscle, and hepatocytes (Corral-Debrinski et al., 1992; Fayet et al., 2002; Yen et al., 1991). A human somatic cell typically comprises myriad copies of mtDNA, whereas an oocyte comprises roughly 105 copies. Replication of mtDNA is independent of the cell cycle, and the replication of a particular mtDNA molecule may,
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therefore, occurs numerous times or not at all as a cell divides (Clayton, 1982). In order to identify the role of the mtDNA 4977-bp deletion in aging process and testing the possibility of indicating mtDNA 4977-bp deletion as a biomarker of aging, we studied 100 old subjects who referred to medical diagnostic Laboratory and carried out a systematic investigation in order to evaluate mtDNA 4977-bp deletion and the mtDNA content in peripheral blood samples and their association with human aging process. These findings heighten our comprehension of the role of mtDNA alterations in aging.
Material and methods Sample collection and DNA extraction Blood samples from 100 old individuals (60–90) who were referred to a medical diagnosis laboratory for regular check were obtained. At the time of sampling, a complete history, including
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Table 1. Distribution of gender and age among people included in study. Gender
Frequency (%)
Average age
Male Female
54 46
70.55 69.97
Table 2. Sequences of primers used in the multiplex PCR reactions. Control 4977-bp Within Deletion region B-actin
86 89 25 74 71 46
50 -CCCTTACCACGCTACTCCTA-30 50 -GGCGGGAGAAGTAGATTGAA-30 50 - CTACGGTCAATGCTCTGAAA-30 50 -GGTTGACCTGTTAGGGTGAG-30 50 - TGCTAGTAACCACGTTCTCC-30 50 - TTTGTTAGGGTTAACGAGGG-30
F 50 - AGACGCAGGATGGCATGGG-30 R 50 - GAGACCTTCAACACCCCAGCC-30
Figure 1. A schematic design used for determination of individuals with 4977 bp mtDNA deletion. This diagram is not drawn to scale.
neurodegenerative disease like Alzheimer and Parkinson was collected for all subjects. We used a structured questionnaire to identify disease-free samples and to exclude subjects who were suspected of having our targeted neurodegenerative disease. The study was ratified by the Ethics Committee of the Tabriz University of Medical Sciences. The mean age of the examinees was 70.29 years. The youngest examinee was 60-year old, while the oldest 90. The gender ratio in both groups was about 46% females to 54% males (Table 1). All individuals were drawn from population-representative collections. Blood samples were collected from peripheral blood and genomic DNA was isolated from the blood samples using purification kit according to the instructions of the manufacturer (DNA fast, QIAGEN, Hilden, Germany). The quality of extracted DNA was quantified by spectrometric analysis (NanoDropÔ 2000c, UV-Vis spectrophotometer, Thermo Fisher Scientific, Waltham, MA). Detection and measurement of mtDNA4977 bp deletion The detection of mtDNA 4977-bp deletion was performed using a multiplex polymerase chain reaction (PCR) protocol. Two pairs of primers for detection of the 4977-bp deletion were described in Table 2. The PCR condition was set as the following: pre denaturation at 94 C for 5 min; then 35 cycles at 94 C for 1 min, 57 C for 1 min, and 72 C for 35 s; and a final extension at 72 C for 10 min. PCR products were then electrophoresed on a 1.5% agarose gel at 100 V (the buffer fluid was 1 TAE buffer). By design, PCR with primer pairs (86/89) yields the 297-bp product and the presence of the 4977-bp deletion was revealed by the appearance of a 497-bp band (Figure 1), which was confirmed by sequencing analysis. To illustrate, primer oligonucleotides (25/74) were designed to anneal outside the common deletion. Indeed, wild-type mtDNA as the template would not produce any PCR products under such circumstances because of the large flanking region (45-kb). Co-amplification of mtDNA4977 bp and wildtype mtDNA showed no interference between the primers pairs hence, the PCR products could be clearly distinguishable by separating in agarose gel electrophoresis. As a positive control, DNA from a sample with confirmed 497 bp deletion was obtained for the presence of dmtDNA4977 bp (Figure 1).
Mitochondrial 4977 bp deletion and aging
DOI: 10.3109/19401736.2015.1063046
Sequencing analysis To confirm the specificity of the PCR product contain the deletion junction created by common deletion, the 503 bp PCR band fragment was purified using an agarose gel DNA fragment recovery kit, Ver.2.0 (TaKaRa, Shiga, Japan) and sequenced using an ABI PRISM 3730 sequence analyzer (gene Fanavaran, Macrogene, Seoul, Korea) (Figure 2). Sequencing was performed in both strands using the original primers. Sequence analysis was carried out using the Finch TV 1.4.0 softwareÕ (Geospiza, Inc., Seattle, WA). The sequences were compared with the human mtDNA reference sequence using the NCBI BLAST sequence analysis tool (Macrogene, Seoul, Korea).
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Quantitative analysis When comparing the relative amount of dmtDNA4977 bp, only individuals who carried the DmtDNA4977 bp mutation were considered in the analysis. Real-time quantitative PCR was performed using the ROTOR GENE 6000 REAL-TIME PCR MACHINE (Corbett Life Science, Mortlake, Australia). Each PCR was performed using the SYBR Premix Ex Taq reagent (Takara Bio, Kyoto, Japan) according to the protocol of the manufacturer. To determine the percent of the common deletion, a specific primer pair (71/46) which is located within the common deletion region and the specific product of these primers was amplified just in wild-type mtDNA without deletion (Table 2). To specify the implication of common deletion in the amount of mtDNA, the whole mtDNA PCR product amplified using the primer pair (86/89) and normalized by simultaneous measurement of nuclear DNA encoded B-actin genes the amplification condition was 1 ml of total DNA extracted in a 15 ml reaction. Predenaturtion at 95 C for 90 s, 50 cycles of three-step PCR followed 95 C for 15 s, 60 C for 20 s, and 72 C for 15 s. A linear regression model evaluated the quantification of wild-type and deleted mtDNA. Ct values of all samples were within the linear range. Two independent measurements of each PCR product were performed in all samples, and it was confirmed that there was significant correlation in the ratio of the common deletion/wildtype mtDNA between these two measurements. The ratio was calculated from the mean values of two measurements.
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Kolmogorov–Smirnov test. The p value50.05 was considered significant. The association between mtDNA alteration and age characteristics of our samples was evaluated using one-way ANOVA analysis.
Result Multiplex PCR (Figure 3) identified the presence of the 4977-bp deletion in the 46 sample of all 100 individuals (46%) from aged people (Figure 4). To determine if the 4977-bp deletion was implicate in the alteration of mtDNA content in aging, the mtDNA copy numbers in 46 cases with desire deletion were analyzed. The amount of mitochondrial copy number in these individuals has been evaluated by measuring the proportion of mtDNA/B-actin. The average DCT value for mtDNA/B-actin for aged individuals was 4.7052 ± 1.8691 (mean ± SD). The average DCT value for mtDNA/B-actin for young individuals as the control group was 6.6170 ± 2.8602 (mean ± SD). As a result, the amount of mitochondrial content was decreased nearly three-fold compared with the control group. Furthermore, the differences between aged people and young people was statistically significant (p value ¼ 0.01). However, there was no significant association between mtDNA alterations and age of subjects. In addition, the percent of deletion was assessed by evaluating the proportion of common deletion region/mtDNA. The results showed that
Statistical analysis Statistical analysis was performed using SPSS for Windows (version 22, SPSS Inc., Chicago, IL). All numerical variables were checked for normal distribution using the one-sample
Figure 3. The number of tested subjects with and without 4977 bp mtDNA deletion.
Figure 2. Schematic diagram of mtDNA 4977 bp deletion. Positive PCR products of mtDNA 4977 bp deletion were confirmed by DNA sequencing. The arrows are at the breakpoint position.
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Figure 4. Electrophoregram of multiplex PCR analysis (agarosis gel 1.5% photographed under UV light).
average percent of deletion among tested individuals were 10% (min 2%, max 17%).
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Discussion In this paper, a study has been conducted based on the mitochondrial theory of aging indicating that cumulative changes in human mitochondrial DNA are associated with aging. Indeed, It has been hypothesized that oxidative stress is responsible for the generation of mitochondrial DNA mutations in human cells (Beckman & Ames, 1998) and since these oxidative stresses increase with age, large scale deletions of mitochondrial DNA such as the common deletion are thought to play a pivotal role in aging process (Berneburg et al., 1999). Most frequently reported mutation is the 4977-bp deletion which is positioned between nucleotide 8470 and 13 447 bp, nominated as the most common and ample large-scale deletion of mtDNA in various human tissues, accounting for 30–50% of all deletions, and has been widely used as a scale of somatic mtDNA injury. The common deletion region is responsible for encoding seven of 13 polypeptides that are crucial subunits for the oxidative phosphorylation pathway (Zhong et al., 2011). Therefore, to determine the common deletion, a multiplex PCR method was performed, and as a result, the presence of 4977 bp deletion in 46% of aged people was observed. In order to quantify the amount of mtDNA of individuals who represent 4977 bp deletion, a real-time PCR was performed. This method has been used according to N. von wrumb et al.’s study. In which, they have indicated that the real time is a suitable method to determine the ratio of 4977 bp deleted mtDNA (von Wurmb-Schwark et al., 2002). These findings illustrate that the incidence of the 4977 bp dmtDNA in aged human normal blood is not clearly detectable during ageing process since it has not detected in all individuals. One of the possible reasons for these results can be explained by referring to Lee et al.’s study. They indicated that although the 4977 bp deletion has been observed nearly in all of the tissues of aged humans, in the some of the old individual’s tissues they could not find 4977 bp deletion (Lee et al., 1994). According to earlier studies, the 4977 bp deletion is just one of the many possible deletions (4834 bp, 3617 bp, 10 422 bp, 7663 bp, 13 162 bp, etc.) in mtDNA which are frequently found in ageing process (Zheng et al., 2012; Zhong et al., 2011) Furthermore, it is interesting to note that the frequency of 4977 bp deletion is higher in some specific tissues; therefore, evaluating this deletion in whole blood may have been influenced the results. One of the evidences for this is Cortopassi et al.’s investigation. Their results showed that the pattern of accumulation of the mtDNA4977 bp deletion in different human tissues is different. Their study revealed that adult heart muscle, brainstem, brain cortex, psoas muscle, and diaphragm muscle have higher levels of the deletion than liver, kidney, skin, spleen, and lung (Cortopassi et al., 1992). Following this further, it has been shown
that the heteroplasmic loads of common deletion were higher in buccal mucosa (37%) and hair (32%) than in blood (17%) (Mkaouar-Rebai et al., 2010). Furthermore, higher degree of mtDNA deletions has been demonstrated in muscle samples, previously. Bua et al. (2006) demonstrated deletion-containing mtDNA genomes increases up to480% and He et al. showed high level of deletion up to 60% (Bua et al., 2006). These results may vindicate that why our findings showed the lower incidence of common deletion in blood samples of old subjects. Indeed, blood as one of fast replicatory tissues compared with low mitotic activity and high oxygen demand tissues like skeletal muscles turn over fairly more rapidly (Lee et al., 1994). Consequently, it has been shown that dmtDNA 4977 bp can be detected in very low amounts in fast replicating tissues such as blood and, in accordance to our study, they concluded that it is uncertain whether an age-dependent distribution of dmtDNA 4977 bp occurs in blood (von Wurmb-Schwark et al., 2010). In other words, the exclusive tracing of the deletions in the mtDNA extracted from the blood does not omit their presence in other tissues, such as muscle (which is not available in our case). Moreover, in a study, when the subjects were divided into two age classes (60–72 years versus 73–95 years), the deletion had significantly higher levels in the older subjects (Bogliolo et al., 1999) and since the average age of our subjects was 70 years old, if we had chosen elder individuals, the result could be different One of the potential possibilities for our findings could relate to other biological factors in the lifestyle of tested individuals. According to N. von Wrumb et al.’s examinations, the variation in 4977 bp concentration among individuals who are in the same or identical age may also result from mtDNA 4977 bp deletion that had been exposed to other biological factors independent of age, like some genotoxic stresses including cigarettes and UVA radiation (von Wurmb-Schwark et al., 2002). Consequently, the mtDNA4977 bp levels could display a snapshot of the mutational incidents that occurred in a relatively short period of time before taking the blood samples (Iwai et al., 2003). Besides, another important factor that should consider is the Involvement of genetic factors in mtDNA4977 bp levels. It has been suggested that variability in specified genes (in both the mitochondrial and the nuclear genome) helps to modulate somatic mtDNA damages through an efficient repair system or a more capable ROS-scavenging process (von Wurmb-Schwark et al., 2010).
Conclusion In the present study, we showed existence of 46% percent of 4977 bp deletion in the blood of aged people and investigated the implication of this deletion on mitochondrial DNA content compared to young individuals. There was statistically significant difference between mtDNA content in aged individuals carrying common deletion and young individuals. From what has been discussed above, we conclude that mtDNA 4977-bp deletion have
DOI: 10.3109/19401736.2015.1063046
decreased the mtDNA content in a age-dependent manner; however, it is not clearly detectable in the blood samples as one of the fast replicating tissues that compensate the mtDNA damage faster than tissues with low mitotic activity and high oxygen demand (skeletal muscle and heart).
Acknowledgements We appreciate all patients for blood donation for scientific research as well as Jorjani clinical diagnostic laboratory for their cooperation and support. The study was approved by the Tabriz University of Medical Sciences Human Research Ethics Committee.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. The research was supported by Tabriz University of Medical Sciences.
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