MITOCH-00919; No of Pages 7 Mitochondrion xxx (2014) xxx–xxx
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Ozgur Guney, Handan Ak, Sevcan Atay, Ali Burak Ozkaya, Hikmet Hakan Aydin ⁎
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Ege University School of Medicine, Department of Medical Biochemistry, Izmir, Turkey
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a r t i c l e
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Article history: Received 3 February 2014 received in revised form 16 April 2014 accepted 21 April 2014 Available online xxxx
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Keywords: Aging Mitochondrial DNA Sequencing Longevity Turkish
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Mitochondrial DNA polymorphisms associated with longevity in the Turkish population
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The accumulation of mutations in mitochondrial DNA is a widely recognized mechanism for aging and age related diseases. However, studies indicate that some mutations could be beneficial to longevity by slowing down the function of the electron transport chain, reducing free radical production. In this study, we re-sequenced the entire mitochondrial DNA from 50 individuals and examined aging-related variations in the Turkish population. We evaluated sequence data by comparing whole SNP frequencies, individual SNP frequencies, the effect of SNPs, SNP accumulation in certain mtDNA regions and haplotype profiles between elderly and control groups. The frequency of total mitochondrial SNPs was significantly higher in nonagenarians than controls (p = 0.0094). Furthermore, non-coding, synonymous and tRNA mutations were more prevalent in the 90+ group compared to controls (p = 0.0001, p b 0.001, p = 0.0096, respectively). A73G and C152T polymorphisms were significantly associated with longevity in the Turkish population (p = 0.0086 and p = 0.004, respectively). Additionally, C150T was specific to the 90+ group, but the difference failed to reach statistical significance (p = 0.053). We also detected a novel transversion in the ATPase6 gene (C8899A) that was negatively associated with longevity (p = 0.0016). Examining the distribution of SNPs among genes and functionally associated gene regions revealed a significant accumulation of mutations in the D-loop region and genes encoding Complex I subunits (ND1-6) (p b 0.0001, p = 0.0302, respectively). Moreover, there was an increase in the non-synonymous mutation frequency of Complex I genes in aged subjects (p b 0.0001). Haplotype H was also significantly increased in the control group (p = 0.0405). Overall, our findings support a role for mitochondrial genome variations and the functionality of oxidative phosphorylation in longevity. In this report, we sequenced the whole mtDNA of the Turkish population for the first time. © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
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Aging is a multiple factor process that is associated with a progressive decrease in physiological function, decreased ability for reproduction and reduced survival. For centuries, numerous theories have been proposed to explain the multifactorial aging process, but none are sufficient to fully explain this phenomenon (Jin, 2010). One widely recognized aging theory is the mitochondrial free radical theory, which suggests a causative link between mitochondrial dysfunction and human senescence (Beckman and Ames, 1998). According to this theory, the production of reactive oxygen species (ROS) during oxidative phosphorylation in mitochondria has the potential to damage the macromolecular components of cells, including DNA, and contribute to physiological aging. In particular, mitochondrial DNA is believed to be more susceptible to oxidative damage and mutation than nuclear DNA due to inadequate repair mechanisms, the absence of histones and the
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1. Introduction
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⁎ Corresponding author at: Ege University, School of Medicine, Department of Medical Biochemistry, Bornova, Izmir 35100, Turkey. Tel.: +90 232 390 3139; fax: +90 232 373 7034. E-mail addresses: [email protected], [email protected] (H.H. Aydin).
lack of genome recombination. Together with oxidative damage, increased mtDNA replication errors also generate mutations (Kennedy et al., 2013), resulting in the production of dysfunctional electron transport chain (ETS) proteins. Studies indicate that dysfunctional ETS proteins could increase the escape of free electrons from the ETS, resulting in more oxidative damage (Harman, 1956). In concordance with these theories, studies have revealed that somatic mutations and large deletions in mtDNA accumulate with age, especially in high oxygen consumer tissues (Arnheim and Cortopassi, 1992), such as the brain (Bandy and Davison, 1990; Corral-Debrinski et al., 1992a; Soong et al., 1992), skeletal muscle (Cao et al., 2001; Del Bo et al., 2002b) and heart (Corral-Debrinski et al., 1992b; Hattori et al., 1991). These genetic alterations eventually give rise to age-associated phenotypes and reduced lifespan, as demonstrated in mtDNA mutator mice (Trifunovic et al., 2004). Moreover, many studies in humans have revealed the effect of mtDNA variability on mitochondrial dysfunction, age related diseases and aging (for a review, see Lagouge and Larsson, 2013). Alternatively, studies on aged individuals suggest that some haplotypes and/or variations could be beneficial and associated with longevity (Beekman et al., 2013; Dato et al., 2004a; Feng et al., 2011; Kenney et al., 2013; Niemi et al., 2003; Rea et al., 2013; Takasaki, 2008, 2009). These studies also
http://dx.doi.org/10.1016/j.mito.2014.04.013 1567-7249/© 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
Please cite this article as: Guney, O., et al., Mitochondrial DNA polymorphisms associated with longevity in the Turkish population, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.013
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2.5. Statistical analysis
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2. Material and method
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2.1. Subjects
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The study protocol was approved by the Ege University Medical School Ethical Committee, and all recruited participants provided written informed consent prior to inclusion in the study. A complete physical examination and detailed clinical history were obtained from all participants. Twenty-five healthy, unrelated Turkish individuals (13 females and 12 males) were selected for the study. Inclusion criteria for the study group were as follows: age N 90 years, absence of acute or chronic disease and absence of drug intake. Twenty-five healthy, unrelated Turkish participants (10 females and 15 males) served as the control group, and they were selected according to the following inclusion criteria: 40–65 years of age, absence of acute or chronic disease and absence of drug intake.
Fisher's exact test was used to evaluate the differences in the relevant haplogroup frequencies. Unpaired T tests were used to evaluate differences between the numbers of polymorphisms in each group. Tests for statistical significance were two-sided, and p b 0.05 was considered to be statistically significant. Data are presented as the mean ± standard deviation for continuous variables, as appropriate. Statistical calculations were performed using GraphPad InStat version 3.10 (GraphPad Software, San Diego California, USA).
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2.2. Sample preparation
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Genomic DNA was isolated from the peripheral blood using QIAamp DNA Blood Mini Kits (Qiagen, Alameda, CA), according to the manufacturer's instructions. The quantity and purity of gDNA were verified spectrophotometrically (NanoDrop ND-1000 Spectrophotometer, Thermo Fisher Scientific, USA). DNA samples were stored at − 80 °C until mtDNA sequence analysis.
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2.3. mtDNA sequencing
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The GeneChip® Human Mitochondrial Resequencing Array 2.0 (Affymetrix) was used to sequence the 16.5-kb mtDNA (Maitra et al., 2004). Briefly, mtDNA was amplified by long range PCR using the LA PCR Kit Ver. 2.1 (TaKaRa Bio Inc. Shiga, Japan) to produce three overlapping fragments and equimolar amounts of amplicons were pooled using the protocol from the Affymetrix GeneChip Resequencing Reagent Kit (Affymetrix Inc., Santa Clara, CA). Prehybridization, hybridization, washing and scanning of the GeneChip were performed according to the Affymetrix CustomSeq Resequencing Array Protocol v 2.1, and the chips were run on Affymetrix GeneChip equipment (GeneChip Scanner 3000 7G System).
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2.4. mtDNA SNP and haplotype analysis
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Sequence data were analyzed using GeneChips Sequence Analysis Software 4.0 (GSEQ 4.1) and GeneChip Operating Software (GCOS v.1) (Affymetrix Inc., Santa Clara, CA). Mitochondrial DNA sequences were aligned and compared with the complete Cambridge Reference Sequence (rCRS; GenBank accession no. NC_012920.1) (Bandelt et al., 2013). Sequence variants were classified and evaluated using MITOMAP (Ruiz-Pesini et al., 2007) and MtDNAprofiler (Yang et al., 2013). The identified SNPs were also verified manually by searching PhyloTree, the Human Phylogenetic tree (mtDNA tree Build 15, rCRS oriented version) (Van Oven and Kayser, 2009). The mitochondrial DNA haplogroup was defined using Haplofind (Vianello et al., 2013) and the MITOMAP database. To examine sequence variation patterns, multiple-alignment of sequence data was performed using the Clustal Omega program, and an un-rooted neighbor-joining tree (Saitou and Nei, 1987) was constructed on the Kimura 2-parameter distance matrix (500 bootstrap replicates) (Kimura, 1980) using MEGA6 software (Tamura et al., 2013). A rooted neighbor-joining tree with African
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119 120 121 122 123 124 125 126 127 128 129 130 131 132 133
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3. Results
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3.2. Sequence analysis
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The mean age of the subjects was 93.76 ± 2.84 years in the 90 + group and 49.66 ± 6.99 years in the control group. The distribution of subject birthplaces is shown in S. Fig. 1. The following numbers of samples were collected from each region: 6 from the north, 14 from the west, 1 from the north-east, 6 from the central, 13 from the northwest, 2 from the south and 6 from the east Anatolia.
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All mtDNA sequence data were compared to reference sequence 156 (rCRS). Sequencing of 16.544 mtDNA bases yielded a total of 326 differ- 157 ent single nucleotide variations at 959 positions in the nonagenarians 158 and control groups. Most of the detected polymorphisms were transi- 159 tions (95% for elderly group and 94.5% for controls), while transversions 160 were less common. No insertions or deletions were detected (Table S1). 161 Investigation on SNP distribution revealed considerable variability 162 between groups. As shown in the Venn diagram, 147 SNPs were defined 163 as 90+ specific, and 91 SNPs were only observed in the control group 164 (Fig. 1). Concomitantly, the number of polymorphisms was significantly 165 higher in the elderly group (p = 0.0094). Notably, in subjects over 166 95 years of age (n = 8), which were included in 90+ group, the num- 167 ber of SNPs was lower compared to other subjects in the corresponding 168 group (p = 0.8142), but this value was not significantly higher than the 169 control group (p = 0.3292) (Table 1). 170 The frequency of non-coding, synonymous and tRNA mutations was 171 increased in the 90 + group compared to controls (p = 0.0001, p 172 b 0.001, p = 0.0096, respectively), whereas the opposite profile was 173 observed for non-synonymous mutations (p N 0.05). No significant dif- 174 ferences were found in rRNA mutations between groups (Fig. 2). Analy- 175 sis of the detected SNPs revealed that the noncoding 73G and 152C 176 polymorphisms in the HV2 (Hypervariable segment 2) of the D-loop 177
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(Uganda) sequence bearing haplotype L0a was constructed based on percentage identity distances (the number of identical bases per 100 base pairs) between samples using Jalview (www.jalview.org) and FigTree Software v1.4.0.
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reveal that longevity is profoundly affected by environmental factors and ethnicity. Although mitochondrial sequence data from the European and Asian populations are well defined, the whole mitochondrial sequence of the Anatolian population has been published. In this study, we re-sequenced whole mitochondrial DNA and examined aging-related variations in the Turkish population for the first time.
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Fig. 1. Venn diagram comparing SNPs associated with the 90+ and control groups.
Please cite this article as: Guney, O., et al., Mitochondrial DNA polymorphisms associated with longevity in the Turkish population, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.013
O. Guney et al. / Mitochondrion xxx (2014) xxx–xxx
Female
20.87 ± 11.97 22.92 ± 9.8 16.2 ± 7.57
19.33 ± 14.81 24.76 ± 10.39 15.8 ± 7.41
21.8 ± 9.76 20.91 ± 9.29 16.8 ± 8.16
188 189 190 191 192 193 194 195 196 197 198 Q4 199 200 201 202 203 204 205 206 207 208 209 210 211 212
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186 187
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were significantly associated with longevity in the Turkish population (p = 0.0086 and p = 0.004, respectively). A non-synonymous C8899A polymorphism in the ATPase 6 gene was also observed at a higher frequency in the control group compared to the 90+ group (p = 0.0016). Thus, we identified frequent mtDNA polymorphisms in the Turkish population; 263G, 750G, 1438G, 4769G, 8860G and 15326G polymorphisms were detected in over 95% of the samples (Table 2). Furthermore, to determine the possibility that mutations accumulate in certain mtDNA locations, we classified the detected SNPs by genes, gene groups or regions in each sample and compared the frequency of SNPs between the 90+ and control groups. The SNP frequency in the control, HV1 and HV2 regions was significantly higher (p b 0.0001, separately) in the 90+ group compared to controls (Fig. 3). Next, we evaluated the distribution of SNPs in genes and the partitioned D-loop to subfragments belonging to these regions. Mutations in the ND1 (p = 0.0489) and ND3 (p = 0.0111), ND4 (p = 0.0164) genes, as well as one subfragment of the D-loop (150T, 152T and 88G), were significantly more prevalent in the 90+ group (Fig. 4). To better understand the relationship between mutation accumulation and aging, we investigated the functionally of associated gene groups in nonagenarians and controls. D-loop, ATPase 6–8 (Complex V), ND1-6 (Complex I), COI–III (Cytochrome c oxidase) and Cytb (Complex III) regions of the mtDNA were evaluated for SNP frequency in the subjects. The average number of SNPs located in these regions was higher in the 90 + group, except for the region encoding ATPase6–8 genes (Fig. 5). In particular, significant SNP accumulation was detected in the ND1-6 and D-loop regions in the elderly group compared to controls (p = 0.0302 and p b 0.0001, respectively). To determine whether non-synonymous polymorphisms belonging to these regions contribute to aging, the elderly and control groups were analyzed based on missense mutation frequency (Fig. 6). The frequency of missense mutations in the ND1-6 region was significantly higher in the 90 + group compared to controls (p b 0.0001). No significant difference was detected in the Cytb, COI–III and ATPase6/8 regions (p = 0.2635, p = 0.0606 and p = 0.2635, respectively).
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95+ group (n = 8) 90+ group (n = 25) Control group (n = 25)
Control (n = 25)
90 + Group (n = 25)
Total (n = 50)
p
t2:5
73G 150T 152C 263G 489C 709A 750G 1438G 1811G 1888A 2706G 4216C 4769G 4917G 7028T 8697A 8701G 8701G 8860G 8899A 10398G 11251G 11467G 11719A 12308G 12372A 12705T 14766T 15326G 16223T 16304C
5 0 0 25 1 3 22 22 3 3 11 3 25 3 12 2 1 1 24 9 1 2 4 9 2 3 2 10 23 2 2
15 5 8 25 5 3 23 24 3 2 18 5 25 3 18 3 3 3 25 0 6 5 6 15 6 6 5 15 25 4 5
20 5 8 50 6 6 45 44 6 5 29 8 50 6 30 5 4 4 49 9 7 7 10 24 8 9 7 25 48 6 7
0.0086 0.0502 0.004 1.000 0.1895 1.000 1.000 0.6092 1.000 1.000 0.0845 0.7019 1.000 1.000 0.1482 1.000 0.6092 0.6092 1.000 0.0016 0.0983 0.4174 0.7252 0.1564 0.2467 0.4635 0.4174 0.2578 0.4898 0.6671 0.4174
t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27 t2:28 t2:29 t2:30 t2:31 t2:32 t2:33 t2:34 t2:35 t2:36
3.3. Haplogroup analysis
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To determine the genetic distance between samples, we constructed a Neighbor-joining tree with Kimura's two parameter distance from the entire mtDNA and samples were distributed into haplotypes (Fig. 7). The frequencies of mtDNA haplogroups in the 90 + and controls are summarized in Table 3. The haplogroup analysis suggests that H, U and T are common haplogroups in the Turkish population (40%, 18% and 10%, respectively). Haplogroup H was significantly lower in the 90+ group (p = 0.0405). Similar results were obtained with the rooted neighbor-joining tree, which was constructed using percentage identity distances (S. Fig. 2). The following three distinct, major groups were determined according to the analysis of the rooted NJ tree: U, JT and H haplogroups. A fourth major group was also identified, which included
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Number of polymorphisms (±SD)
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Table 2 The most frequent single nucleotide polymorphisms in the Turkish population. p value indicates the significance of the difference between the control and 90+ groups (p b 0.05, Fisher's exact test).
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Table 1 Number of polymorphisms in the 90 +, 95 + and control groups. Data represent the mean ± SD.
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t1:1 t1:2 t1:3
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Fig. 2. Number of polymorphisms in the 90+ and control groups. SNPs were classified through their effects. Statistical analyses were performed using the average number of polymorphisms per subject. *Indicates statistical significance (p b 0.05).
Please cite this article as: Guney, O., et al., Mitochondrial DNA polymorphisms associated with longevity in the Turkish population, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.013
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To date, several studies have elucidated the Turkish mtDNA sequence data of hypervariable segments 1 and 2 (Calafell et al., 1996; Comas et al., 1996; Mergen et al., 2004; Richards et al., 1996). In this study, for the first time, we determined the entire mtDNA sequence from 50 Turkish individuals and evaluated age-related variations. We evaluated sequence data by comparing i) whole SNP frequencies, ii) individual SNP frequencies, iii) effects of SNPs, iv) SNP accumulation in certain mtDNA regions and v) haplotype profiles between groups. Consistent with the geographic position of Turkey, 92% of our study population harbored common haplogroups detected in Europe (H, U, T, J, V, N, X, R, K and HV), as well as Asian haplogroups (C and D) (Herrnstadt et al., 2002; Kong et al., 2003). Among these, H, U and T derivative haplogroups constituted 68% of the total identified haplogroups. Analysis of haplotype profiles between groups revealed that the H cluster was significantly less frequent in the 90+ group. Additionally, U derivative clusters were twice as frequent in nonagenarians, but this association did not reach statistical significance. Niemi et al. obtained similar results in Finnish nonagenarians (Niemi et al., 2003), but no difference was
248 249 250
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4. Discussion
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detected in a study consisting of samples from 11 European countries (Raule et al., 2013). Previous studies demonstrated that haplogroup J is associated with longevity in Italy (Dato et al., 2004b; De Benedictis et al., 1999), Northern Ireland (Ross et al., 2001) and Finland (Niemi et al., 2003). In our study, haplogroup J1 was only found in nonagenarians, but this result did not reach statistical significance. The Asian haplogroup D, which was previously associated with longevity in the Japanese population (Alexe et al., 2007; Bilal et al., 2008), was equally prevalent in both groups. Additionally, the C haplotype was found only in nonagenarians, and this haplotype has not been associated with longevity in previous studies. Consistent with other reports, analysis of the entire SNP data revealed that total SNP frequency was significantly higher in the aged group (Corral-Debrinski et al., 1992a; Meissner et al., 2008). In addition, striking SNP variation was observed between groups, emphasizing the difference in the genetic signatures between nonagenarians and controls. We also detected a significant increase in the frequency of noncoding, synonymous and tRNA mutations in the 90+ group, while no significance was detected in the prevalence of rRNA and nonsynonymous mutations compared to the control group. Mitochondrial tRNA mutations contribute to the development of diseases, including cancer, stroke, altered brain PH, hearing loss, diabetes and hypertension (for a review see Yarham et al., 2010). Thus, the accumulation of mutations in mt-tRNA genes in nonagenarians could promote age-associated phenotypes. Similar to our results, numerous studies reported that the frequency of mutations in the D-loop region increases with age (Del Bo et al., 2002a; Ren et al., 2008; Zhang et al., 1998). Here, we detected
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haplotypes N, R, C, D and X. As shown in S. Fig. 2, the number of aged subjects was significantly lower in “group 2”, which included samples from haplogroup H, where as the other three groups were defined as “group 1” (p = 0.0475). Moreover, the C, V, and J haplogroups were observed only in the 90 + group. No association was detected between gender and haplogroup status.
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Fig. 3. SNP frequency in the control and HV1-3 regions of the mtDNA in the 90+ and control groups. *Indicates a significant difference between groups (p b 0.05).
Fig. 4. Number of polymorphisms in the 90+ and control groups. SNPs were classified according to the genes they belong to. Statistical analyses were performed using the average number of polymorphisms in current gene regions per subject. *Indicates statistical significance (p b 0.05).
Please cite this article as: Guney, O., et al., Mitochondrial DNA polymorphisms associated with longevity in the Turkish population, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.013
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293 294 295 296 297 298 299 300 301 302 303 304 305 306
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associated with respiratory capacity, mtDNA level, mitochondrial gene expression level, or growth rate, suggesting a complicated mechanism that includes an interaction between 150T and multiple variations belonging to the haplogroups investigated, J2 and U (Chen et al., 2012). We detected the 150T transition in five aged individuals, three of whom harbored U derivative haplogroups, while the others harbored D5a and HV1 haplotypes, suggesting a potential interaction between 150T and other haplogroups. An association between a common polymorphism in Caucasians (A73G) and longevity, which was also observed in our population, was detected in the Bama Country population (Yang et al., 2012). The results of previous studies are rather confusing. Reports indicate a significantly lower incidence of 73G in breast cancer patients (Czarnecka et al., 2010), higher prevalence in pancreatic cancer patients (Navaglia et al., 2006) and higher frequency in choroidal neovascularization patients compared to controls (Mueller et al., 2012). These results suggest that an interaction among mitochondrial SNPs and communication between the mitochondrial genome and nuclear DNA could be the main effectors in the emergence of a specific phenotype instead of a single point mutation. Our results also revealed that a C N A transversion at nucleotide position 8899 was significantly over represented in the control group. This novel polymorphism is located in the highly conserved position of the ATPase6 gene, leading to a leucine to methionine substitution. Further studies are required to determine the importance of 8899A. The 146C, 150T, 152C, 195C, and 199C polymorphisms were defined as the most common polymorphisms in the HVS II region in the Turkish population by Mergen et al., but the age range of the samples was not noted (Mergen et al., 2004). In our study, the frequency of 263G,
E
291 292
T
290
C
288 289
E
286 287
R
284 285
R
282 283
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280 281
a significant accumulation of mutations in a specific D-loop region, HVS2/OH/ATT/D-loop/7S DNA, which includes the following SNPs: 150T, 152T, and 188G. However, analysis of the distribution of these SNPs revealed that the aggregated profile of SNPs is largely due to the high frequency of 150T and 152T polymorphisms in nonagenarians. Additionally, only one aged individual was found to carry these two SNPs together, suggesting that 150T and 152T were not transmitted together. We detected another SNP in the D-loop region, A73G, which was significantly higher in the 90+ group compared to controls. Interestingly, all individuals bearing the 150T polymorphism and 62.5% of the individuals carrying 152T also carried 73G, suggesting that 150T and 152T tend to co-occur with 73G in aged individuals. The frequency of the C150T polymorphism, which was previously identified as a longevity region by similar studies, was specific for the 90+ group, but this result failed to reach statistical significance (p = 0.053). It has been suggested that other polymorphism in this region could have a similar effect, such as 152T and 189A (Niemi et al., 2005), and the accumulation of these mitochondrial SNPs has been shown in the fibroblasts (Michikawa et al., 1999) and muscle (Wang et al., 2001), respectively, of aged individuals. The C150T polymorphism is located in the secondary origins of the H-strand replication of mtDNA, and the presence of this mutation is thought to impart a replicative advantage to mtDNA (Zhang et al., 2003). However, the mechanism underlying the contribution of 150T to long-term survival remains unclear. One explanation is that a reduction in OXPHOS efficiency caused by an interaction of SNPs with 150T results in decreased ROS production, which decreases apoptosis and increases longevity (Coskun et al., 2003). In accordance with this hypothesis, another study revealed that the presence of 150T transition alone is not
U
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Fig. 5. The distribution of the SNPs in functionally connected gene regions of the mtDNA from the 90+ and control groups. Frequency indicates the average number of mutations per subject. *Indicates a significant difference between groups (p b 0.05).
Fig. 6. Non-synonymous SNP frequency in functionally associated gene regions of mtDNA from the 90+ and control groups. Frequency indicates the average number of non-synonymous polymorphism per subject. *Indicates statistical significance (p b 0.05).
Please cite this article as: Guney, O., et al., Mitochondrial DNA polymorphisms associated with longevity in the Turkish population, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.013
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A263G, A2076G, A8860G, G11719A, C14766T, and A15326G) in the Bama Country (Guangxi, China) (Yang et al., 2012). These data emphasize that the association between longevity and mtDNA variations is highly dependent on population and geography. Investigation of the distribution of SNPs between functionally related mitochondrial genes revealed that SNPs in OXPHOS Complex I genes (ND1-6) were significantly higher in the 90+ group compared to controls. In particular, significant SNP accumulation was detected in the ND1, ND3 and ND4 regions in the elderly group compared to controls, suggesting that SNPs in these regions might be beneficial. To determine whether an alteration in protein structure could contribute to this hypothetical beneficial effect, we examined the non-synonymous mutation frequency in these OXPHOS genes. The non-synonymous SNP frequency was significantly increased in the genes of Complex I, suggesting that an alteration of the functionality of that protein (NADH dehydrogenase) might confer a selective advantage for longevity. Similar results were obtained in Finns, but higher mutation frequency in Complex I genes was detected in controls compared to the 90+ group in Danish and in southern European populations (Raule et al., 2013). Although no statistical significance was detected, the frequency of non-synonymous SNPs in Complex V (ATPase6/8) and COI–III (Cytochrome c oxidase subunits I–III) genes was reduced, suggesting that SNPs in these regions might decrease resistance to age related diseases. Interestingly, the decreased frequency of non-synonymous SNPs and the increased frequency of total SNPs in COI–III genes in the 90 + group highlighted the importance of these proteins and the detrimental potential of altered Cytochrome c oxidase functionality. However, further studies are required to support these findings. Our data suggest that the accumulation of mutations in Complex I could be beneficial, and it is likely highly dependent on genetic background and the occurrence of other SNPs in mitochondrial DNA. We observed an association between 150T, 152T and 73G SNPs in the D-loop region and longevity in the Turkish population. Furthermore, haplogroup H was negatively associated with longevity. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mito.2014.04.013.
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Acknowledgments
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R
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Fig. 7. An unrooted neighbor-joining (NJ) tree of the haplotypes of Turkish individuals. Individuals belonging to the 90+ group are represented by solid circles, and controls are represented by open circles. *Indicates a subject over the age of 95. The optimal tree with the sum of branch length of 0.02506385 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary distances were computed using the Kimura 2-parameter method, and the units are the number of base substitutions per site. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted using MEGA6.
t3:1 t3:2
Table 3 Frequency (%) of mtDNA major haplogroups in the elderly and control groups.
C
N
Haplogroup
90+
Control
(n = 50)
Total
Group
Group
%
(n = 25)
(n = 25)
%
%
14 12 6 4 4 2 2 4 2 2 2 2
26 6 4 2 – 2 2 – – 2 2 –
U
t3:3
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339 340
750G, 1438G, 4769G, 8860G and 15326G polymorphisms was greater than 95% in the general population. 15326G, 7028T, 11719A and 14766T were also present at a relatively high proportion (50–60%). Interestingly, most of these frequent mitochondrial mutations in the Turkish population coincide with variants associated with longevity (A73G,
337 Q6 338
t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18
H U T N C D R J X K HV V
40 18 10 6 4 4 4 4 2 4 4 2
343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376
This project was supported by the Ege University Research Funds 378 (Project No. 2010 TIP 013). 379 References
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Please cite this article as: Guney, O., et al., Mitochondrial DNA polymorphisms associated with longevity in the Turkish population, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.013
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Mitochondrion journal homepage: www.elsevier.com/locate/mito
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Ozgur Guney, Handan Ak, Sevcan Atay, Ali Burak Ozkaya, Hikmet Hakan Aydin ⁎
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Ege University School of Medicine, Department of Medical Biochemistry, Izmir, Turkey
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Article history: Received 3 February 2014 received in revised form 16 April 2014 accepted 21 April 2014 Available online xxxx
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Keywords: Aging Mitochondrial DNA Sequencing Longevity Turkish
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Mitochondrial DNA polymorphisms associated with longevity in the Turkish population
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The accumulation of mutations in mitochondrial DNA is a widely recognized mechanism for aging and age related diseases. However, studies indicate that some mutations could be beneficial to longevity by slowing down the function of the electron transport chain, reducing free radical production. In this study, we re-sequenced the entire mitochondrial DNA from 50 individuals and examined aging-related variations in the Turkish population. We evaluated sequence data by comparing whole SNP frequencies, individual SNP frequencies, the effect of SNPs, SNP accumulation in certain mtDNA regions and haplotype profiles between elderly and control groups. The frequency of total mitochondrial SNPs was significantly higher in nonagenarians than controls (p = 0.0094). Furthermore, non-coding, synonymous and tRNA mutations were more prevalent in the 90+ group compared to controls (p = 0.0001, p b 0.001, p = 0.0096, respectively). A73G and C152T polymorphisms were significantly associated with longevity in the Turkish population (p = 0.0086 and p = 0.004, respectively). Additionally, C150T was specific to the 90+ group, but the difference failed to reach statistical significance (p = 0.053). We also detected a novel transversion in the ATPase6 gene (C8899A) that was negatively associated with longevity (p = 0.0016). Examining the distribution of SNPs among genes and functionally associated gene regions revealed a significant accumulation of mutations in the D-loop region and genes encoding Complex I subunits (ND1-6) (p b 0.0001, p = 0.0302, respectively). Moreover, there was an increase in the non-synonymous mutation frequency of Complex I genes in aged subjects (p b 0.0001). Haplotype H was also significantly increased in the control group (p = 0.0405). Overall, our findings support a role for mitochondrial genome variations and the functionality of oxidative phosphorylation in longevity. In this report, we sequenced the whole mtDNA of the Turkish population for the first time. © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
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Aging is a multiple factor process that is associated with a progressive decrease in physiological function, decreased ability for reproduction and reduced survival. For centuries, numerous theories have been proposed to explain the multifactorial aging process, but none are sufficient to fully explain this phenomenon (Jin, 2010). One widely recognized aging theory is the mitochondrial free radical theory, which suggests a causative link between mitochondrial dysfunction and human senescence (Beckman and Ames, 1998). According to this theory, the production of reactive oxygen species (ROS) during oxidative phosphorylation in mitochondria has the potential to damage the macromolecular components of cells, including DNA, and contribute to physiological aging. In particular, mitochondrial DNA is believed to be more susceptible to oxidative damage and mutation than nuclear DNA due to inadequate repair mechanisms, the absence of histones and the
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⁎ Corresponding author at: Ege University, School of Medicine, Department of Medical Biochemistry, Bornova, Izmir 35100, Turkey. Tel.: +90 232 390 3139; fax: +90 232 373 7034. E-mail addresses: [email protected], [email protected] (H.H. Aydin).
lack of genome recombination. Together with oxidative damage, increased mtDNA replication errors also generate mutations (Kennedy et al., 2013), resulting in the production of dysfunctional electron transport chain (ETS) proteins. Studies indicate that dysfunctional ETS proteins could increase the escape of free electrons from the ETS, resulting in more oxidative damage (Harman, 1956). In concordance with these theories, studies have revealed that somatic mutations and large deletions in mtDNA accumulate with age, especially in high oxygen consumer tissues (Arnheim and Cortopassi, 1992), such as the brain (Bandy and Davison, 1990; Corral-Debrinski et al., 1992a; Soong et al., 1992), skeletal muscle (Cao et al., 2001; Del Bo et al., 2002b) and heart (Corral-Debrinski et al., 1992b; Hattori et al., 1991). These genetic alterations eventually give rise to age-associated phenotypes and reduced lifespan, as demonstrated in mtDNA mutator mice (Trifunovic et al., 2004). Moreover, many studies in humans have revealed the effect of mtDNA variability on mitochondrial dysfunction, age related diseases and aging (for a review, see Lagouge and Larsson, 2013). Alternatively, studies on aged individuals suggest that some haplotypes and/or variations could be beneficial and associated with longevity (Beekman et al., 2013; Dato et al., 2004a; Feng et al., 2011; Kenney et al., 2013; Niemi et al., 2003; Rea et al., 2013; Takasaki, 2008, 2009). These studies also
http://dx.doi.org/10.1016/j.mito.2014.04.013 1567-7249/© 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
Please cite this article as: Guney, O., et al., Mitochondrial DNA polymorphisms associated with longevity in the Turkish population, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.013
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The study protocol was approved by the Ege University Medical School Ethical Committee, and all recruited participants provided written informed consent prior to inclusion in the study. A complete physical examination and detailed clinical history were obtained from all participants. Twenty-five healthy, unrelated Turkish individuals (13 females and 12 males) were selected for the study. Inclusion criteria for the study group were as follows: age N 90 years, absence of acute or chronic disease and absence of drug intake. Twenty-five healthy, unrelated Turkish participants (10 females and 15 males) served as the control group, and they were selected according to the following inclusion criteria: 40–65 years of age, absence of acute or chronic disease and absence of drug intake.
Fisher's exact test was used to evaluate the differences in the relevant haplogroup frequencies. Unpaired T tests were used to evaluate differences between the numbers of polymorphisms in each group. Tests for statistical significance were two-sided, and p b 0.05 was considered to be statistically significant. Data are presented as the mean ± standard deviation for continuous variables, as appropriate. Statistical calculations were performed using GraphPad InStat version 3.10 (GraphPad Software, San Diego California, USA).
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Genomic DNA was isolated from the peripheral blood using QIAamp DNA Blood Mini Kits (Qiagen, Alameda, CA), according to the manufacturer's instructions. The quantity and purity of gDNA were verified spectrophotometrically (NanoDrop ND-1000 Spectrophotometer, Thermo Fisher Scientific, USA). DNA samples were stored at − 80 °C until mtDNA sequence analysis.
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The GeneChip® Human Mitochondrial Resequencing Array 2.0 (Affymetrix) was used to sequence the 16.5-kb mtDNA (Maitra et al., 2004). Briefly, mtDNA was amplified by long range PCR using the LA PCR Kit Ver. 2.1 (TaKaRa Bio Inc. Shiga, Japan) to produce three overlapping fragments and equimolar amounts of amplicons were pooled using the protocol from the Affymetrix GeneChip Resequencing Reagent Kit (Affymetrix Inc., Santa Clara, CA). Prehybridization, hybridization, washing and scanning of the GeneChip were performed according to the Affymetrix CustomSeq Resequencing Array Protocol v 2.1, and the chips were run on Affymetrix GeneChip equipment (GeneChip Scanner 3000 7G System).
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Sequence data were analyzed using GeneChips Sequence Analysis Software 4.0 (GSEQ 4.1) and GeneChip Operating Software (GCOS v.1) (Affymetrix Inc., Santa Clara, CA). Mitochondrial DNA sequences were aligned and compared with the complete Cambridge Reference Sequence (rCRS; GenBank accession no. NC_012920.1) (Bandelt et al., 2013). Sequence variants were classified and evaluated using MITOMAP (Ruiz-Pesini et al., 2007) and MtDNAprofiler (Yang et al., 2013). The identified SNPs were also verified manually by searching PhyloTree, the Human Phylogenetic tree (mtDNA tree Build 15, rCRS oriented version) (Van Oven and Kayser, 2009). The mitochondrial DNA haplogroup was defined using Haplofind (Vianello et al., 2013) and the MITOMAP database. To examine sequence variation patterns, multiple-alignment of sequence data was performed using the Clustal Omega program, and an un-rooted neighbor-joining tree (Saitou and Nei, 1987) was constructed on the Kimura 2-parameter distance matrix (500 bootstrap replicates) (Kimura, 1980) using MEGA6 software (Tamura et al., 2013). A rooted neighbor-joining tree with African
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The mean age of the subjects was 93.76 ± 2.84 years in the 90 + group and 49.66 ± 6.99 years in the control group. The distribution of subject birthplaces is shown in S. Fig. 1. The following numbers of samples were collected from each region: 6 from the north, 14 from the west, 1 from the north-east, 6 from the central, 13 from the northwest, 2 from the south and 6 from the east Anatolia.
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All mtDNA sequence data were compared to reference sequence 156 (rCRS). Sequencing of 16.544 mtDNA bases yielded a total of 326 differ- 157 ent single nucleotide variations at 959 positions in the nonagenarians 158 and control groups. Most of the detected polymorphisms were transi- 159 tions (95% for elderly group and 94.5% for controls), while transversions 160 were less common. No insertions or deletions were detected (Table S1). 161 Investigation on SNP distribution revealed considerable variability 162 between groups. As shown in the Venn diagram, 147 SNPs were defined 163 as 90+ specific, and 91 SNPs were only observed in the control group 164 (Fig. 1). Concomitantly, the number of polymorphisms was significantly 165 higher in the elderly group (p = 0.0094). Notably, in subjects over 166 95 years of age (n = 8), which were included in 90+ group, the num- 167 ber of SNPs was lower compared to other subjects in the corresponding 168 group (p = 0.8142), but this value was not significantly higher than the 169 control group (p = 0.3292) (Table 1). 170 The frequency of non-coding, synonymous and tRNA mutations was 171 increased in the 90 + group compared to controls (p = 0.0001, p 172 b 0.001, p = 0.0096, respectively), whereas the opposite profile was 173 observed for non-synonymous mutations (p N 0.05). No significant dif- 174 ferences were found in rRNA mutations between groups (Fig. 2). Analy- 175 sis of the detected SNPs revealed that the noncoding 73G and 152C 176 polymorphisms in the HV2 (Hypervariable segment 2) of the D-loop 177
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(Uganda) sequence bearing haplotype L0a was constructed based on percentage identity distances (the number of identical bases per 100 base pairs) between samples using Jalview (www.jalview.org) and FigTree Software v1.4.0.
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reveal that longevity is profoundly affected by environmental factors and ethnicity. Although mitochondrial sequence data from the European and Asian populations are well defined, the whole mitochondrial sequence of the Anatolian population has been published. In this study, we re-sequenced whole mitochondrial DNA and examined aging-related variations in the Turkish population for the first time.
U
77 78
O. Guney et al. / Mitochondrion xxx (2014) xxx–xxx
Fig. 1. Venn diagram comparing SNPs associated with the 90+ and control groups.
Please cite this article as: Guney, O., et al., Mitochondrial DNA polymorphisms associated with longevity in the Turkish population, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.013
O. Guney et al. / Mitochondrion xxx (2014) xxx–xxx
Female
20.87 ± 11.97 22.92 ± 9.8 16.2 ± 7.57
19.33 ± 14.81 24.76 ± 10.39 15.8 ± 7.41
21.8 ± 9.76 20.91 ± 9.29 16.8 ± 8.16
188 189 190 191 192 193 194 195 196 197 198 Q4 199 200 201 202 203 204 205 206 207 208 209 210 211 212
T
186 187
C
184 185
E
183
R
181 182
were significantly associated with longevity in the Turkish population (p = 0.0086 and p = 0.004, respectively). A non-synonymous C8899A polymorphism in the ATPase 6 gene was also observed at a higher frequency in the control group compared to the 90+ group (p = 0.0016). Thus, we identified frequent mtDNA polymorphisms in the Turkish population; 263G, 750G, 1438G, 4769G, 8860G and 15326G polymorphisms were detected in over 95% of the samples (Table 2). Furthermore, to determine the possibility that mutations accumulate in certain mtDNA locations, we classified the detected SNPs by genes, gene groups or regions in each sample and compared the frequency of SNPs between the 90+ and control groups. The SNP frequency in the control, HV1 and HV2 regions was significantly higher (p b 0.0001, separately) in the 90+ group compared to controls (Fig. 3). Next, we evaluated the distribution of SNPs in genes and the partitioned D-loop to subfragments belonging to these regions. Mutations in the ND1 (p = 0.0489) and ND3 (p = 0.0111), ND4 (p = 0.0164) genes, as well as one subfragment of the D-loop (150T, 152T and 88G), were significantly more prevalent in the 90+ group (Fig. 4). To better understand the relationship between mutation accumulation and aging, we investigated the functionally of associated gene groups in nonagenarians and controls. D-loop, ATPase 6–8 (Complex V), ND1-6 (Complex I), COI–III (Cytochrome c oxidase) and Cytb (Complex III) regions of the mtDNA were evaluated for SNP frequency in the subjects. The average number of SNPs located in these regions was higher in the 90 + group, except for the region encoding ATPase6–8 genes (Fig. 5). In particular, significant SNP accumulation was detected in the ND1-6 and D-loop regions in the elderly group compared to controls (p = 0.0302 and p b 0.0001, respectively). To determine whether non-synonymous polymorphisms belonging to these regions contribute to aging, the elderly and control groups were analyzed based on missense mutation frequency (Fig. 6). The frequency of missense mutations in the ND1-6 region was significantly higher in the 90 + group compared to controls (p b 0.0001). No significant difference was detected in the Cytb, COI–III and ATPase6/8 regions (p = 0.2635, p = 0.0606 and p = 0.2635, respectively).
R
179 180
N C O
178
95+ group (n = 8) 90+ group (n = 25) Control group (n = 25)
Control (n = 25)
90 + Group (n = 25)
Total (n = 50)
p
t2:5
73G 150T 152C 263G 489C 709A 750G 1438G 1811G 1888A 2706G 4216C 4769G 4917G 7028T 8697A 8701G 8701G 8860G 8899A 10398G 11251G 11467G 11719A 12308G 12372A 12705T 14766T 15326G 16223T 16304C
5 0 0 25 1 3 22 22 3 3 11 3 25 3 12 2 1 1 24 9 1 2 4 9 2 3 2 10 23 2 2
15 5 8 25 5 3 23 24 3 2 18 5 25 3 18 3 3 3 25 0 6 5 6 15 6 6 5 15 25 4 5
20 5 8 50 6 6 45 44 6 5 29 8 50 6 30 5 4 4 49 9 7 7 10 24 8 9 7 25 48 6 7
0.0086 0.0502 0.004 1.000 0.1895 1.000 1.000 0.6092 1.000 1.000 0.0845 0.7019 1.000 1.000 0.1482 1.000 0.6092 0.6092 1.000 0.0016 0.0983 0.4174 0.7252 0.1564 0.2467 0.4635 0.4174 0.2578 0.4898 0.6671 0.4174
t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27 t2:28 t2:29 t2:30 t2:31 t2:32 t2:33 t2:34 t2:35 t2:36
3.3. Haplogroup analysis
213
To determine the genetic distance between samples, we constructed a Neighbor-joining tree with Kimura's two parameter distance from the entire mtDNA and samples were distributed into haplotypes (Fig. 7). The frequencies of mtDNA haplogroups in the 90 + and controls are summarized in Table 3. The haplogroup analysis suggests that H, U and T are common haplogroups in the Turkish population (40%, 18% and 10%, respectively). Haplogroup H was significantly lower in the 90+ group (p = 0.0405). Similar results were obtained with the rooted neighbor-joining tree, which was constructed using percentage identity distances (S. Fig. 2). The following three distinct, major groups were determined according to the analysis of the rooted NJ tree: U, JT and H haplogroups. A fourth major group was also identified, which included
214
U
t1:6 t1:7 t1:8
t2:1 t2:2 t2:3 t2:4
SNP
F
Male
O
Total
R O
Number of polymorphisms (±SD)
t1:5
P
t1:4
Table 2 The most frequent single nucleotide polymorphisms in the Turkish population. p value indicates the significance of the difference between the control and 90+ groups (p b 0.05, Fisher's exact test).
D
Table 1 Number of polymorphisms in the 90 +, 95 + and control groups. Data represent the mean ± SD.
E
t1:1 t1:2 t1:3
3
Fig. 2. Number of polymorphisms in the 90+ and control groups. SNPs were classified through their effects. Statistical analyses were performed using the average number of polymorphisms per subject. *Indicates statistical significance (p b 0.05).
Please cite this article as: Guney, O., et al., Mitochondrial DNA polymorphisms associated with longevity in the Turkish population, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.013
215 216 217 218 219 220 221 222 223 224 225
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F
4
To date, several studies have elucidated the Turkish mtDNA sequence data of hypervariable segments 1 and 2 (Calafell et al., 1996; Comas et al., 1996; Mergen et al., 2004; Richards et al., 1996). In this study, for the first time, we determined the entire mtDNA sequence from 50 Turkish individuals and evaluated age-related variations. We evaluated sequence data by comparing i) whole SNP frequencies, ii) individual SNP frequencies, iii) effects of SNPs, iv) SNP accumulation in certain mtDNA regions and v) haplotype profiles between groups. Consistent with the geographic position of Turkey, 92% of our study population harbored common haplogroups detected in Europe (H, U, T, J, V, N, X, R, K and HV), as well as Asian haplogroups (C and D) (Herrnstadt et al., 2002; Kong et al., 2003). Among these, H, U and T derivative haplogroups constituted 68% of the total identified haplogroups. Analysis of haplotype profiles between groups revealed that the H cluster was significantly less frequent in the 90+ group. Additionally, U derivative clusters were twice as frequent in nonagenarians, but this association did not reach statistical significance. Niemi et al. obtained similar results in Finnish nonagenarians (Niemi et al., 2003), but no difference was
248 249 250
C
E
246 247
R
244 245
R
242 243
O
240 241
C
238 239
N
236 237
U
234 235
R O
233
P
4. Discussion
D
232
228 229
detected in a study consisting of samples from 11 European countries (Raule et al., 2013). Previous studies demonstrated that haplogroup J is associated with longevity in Italy (Dato et al., 2004b; De Benedictis et al., 1999), Northern Ireland (Ross et al., 2001) and Finland (Niemi et al., 2003). In our study, haplogroup J1 was only found in nonagenarians, but this result did not reach statistical significance. The Asian haplogroup D, which was previously associated with longevity in the Japanese population (Alexe et al., 2007; Bilal et al., 2008), was equally prevalent in both groups. Additionally, the C haplotype was found only in nonagenarians, and this haplotype has not been associated with longevity in previous studies. Consistent with other reports, analysis of the entire SNP data revealed that total SNP frequency was significantly higher in the aged group (Corral-Debrinski et al., 1992a; Meissner et al., 2008). In addition, striking SNP variation was observed between groups, emphasizing the difference in the genetic signatures between nonagenarians and controls. We also detected a significant increase in the frequency of noncoding, synonymous and tRNA mutations in the 90+ group, while no significance was detected in the prevalence of rRNA and nonsynonymous mutations compared to the control group. Mitochondrial tRNA mutations contribute to the development of diseases, including cancer, stroke, altered brain PH, hearing loss, diabetes and hypertension (for a review see Yarham et al., 2010). Thus, the accumulation of mutations in mt-tRNA genes in nonagenarians could promote age-associated phenotypes. Similar to our results, numerous studies reported that the frequency of mutations in the D-loop region increases with age (Del Bo et al., 2002a; Ren et al., 2008; Zhang et al., 1998). Here, we detected
T
230 231
haplotypes N, R, C, D and X. As shown in S. Fig. 2, the number of aged subjects was significantly lower in “group 2”, which included samples from haplogroup H, where as the other three groups were defined as “group 1” (p = 0.0475). Moreover, the C, V, and J haplogroups were observed only in the 90 + group. No association was detected between gender and haplogroup status.
227
E
226
O
Fig. 3. SNP frequency in the control and HV1-3 regions of the mtDNA in the 90+ and control groups. *Indicates a significant difference between groups (p b 0.05).
Fig. 4. Number of polymorphisms in the 90+ and control groups. SNPs were classified according to the genes they belong to. Statistical analyses were performed using the average number of polymorphisms in current gene regions per subject. *Indicates statistical significance (p b 0.05).
Please cite this article as: Guney, O., et al., Mitochondrial DNA polymorphisms associated with longevity in the Turkish population, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.013
251 252 253 254 255 256 257 258 259 260 261 262 263 264 Q5 265 266 267 268 269 270 271 272 273 274 275 276 277
5
O
F
O. Guney et al. / Mitochondrion xxx (2014) xxx–xxx
293 294 295 296 297 298 299 300 301 302 303 304 305 306
D
P
associated with respiratory capacity, mtDNA level, mitochondrial gene expression level, or growth rate, suggesting a complicated mechanism that includes an interaction between 150T and multiple variations belonging to the haplogroups investigated, J2 and U (Chen et al., 2012). We detected the 150T transition in five aged individuals, three of whom harbored U derivative haplogroups, while the others harbored D5a and HV1 haplotypes, suggesting a potential interaction between 150T and other haplogroups. An association between a common polymorphism in Caucasians (A73G) and longevity, which was also observed in our population, was detected in the Bama Country population (Yang et al., 2012). The results of previous studies are rather confusing. Reports indicate a significantly lower incidence of 73G in breast cancer patients (Czarnecka et al., 2010), higher prevalence in pancreatic cancer patients (Navaglia et al., 2006) and higher frequency in choroidal neovascularization patients compared to controls (Mueller et al., 2012). These results suggest that an interaction among mitochondrial SNPs and communication between the mitochondrial genome and nuclear DNA could be the main effectors in the emergence of a specific phenotype instead of a single point mutation. Our results also revealed that a C N A transversion at nucleotide position 8899 was significantly over represented in the control group. This novel polymorphism is located in the highly conserved position of the ATPase6 gene, leading to a leucine to methionine substitution. Further studies are required to determine the importance of 8899A. The 146C, 150T, 152C, 195C, and 199C polymorphisms were defined as the most common polymorphisms in the HVS II region in the Turkish population by Mergen et al., but the age range of the samples was not noted (Mergen et al., 2004). In our study, the frequency of 263G,
E
291 292
T
290
C
288 289
E
286 287
R
284 285
R
282 283
N C O
280 281
a significant accumulation of mutations in a specific D-loop region, HVS2/OH/ATT/D-loop/7S DNA, which includes the following SNPs: 150T, 152T, and 188G. However, analysis of the distribution of these SNPs revealed that the aggregated profile of SNPs is largely due to the high frequency of 150T and 152T polymorphisms in nonagenarians. Additionally, only one aged individual was found to carry these two SNPs together, suggesting that 150T and 152T were not transmitted together. We detected another SNP in the D-loop region, A73G, which was significantly higher in the 90+ group compared to controls. Interestingly, all individuals bearing the 150T polymorphism and 62.5% of the individuals carrying 152T also carried 73G, suggesting that 150T and 152T tend to co-occur with 73G in aged individuals. The frequency of the C150T polymorphism, which was previously identified as a longevity region by similar studies, was specific for the 90+ group, but this result failed to reach statistical significance (p = 0.053). It has been suggested that other polymorphism in this region could have a similar effect, such as 152T and 189A (Niemi et al., 2005), and the accumulation of these mitochondrial SNPs has been shown in the fibroblasts (Michikawa et al., 1999) and muscle (Wang et al., 2001), respectively, of aged individuals. The C150T polymorphism is located in the secondary origins of the H-strand replication of mtDNA, and the presence of this mutation is thought to impart a replicative advantage to mtDNA (Zhang et al., 2003). However, the mechanism underlying the contribution of 150T to long-term survival remains unclear. One explanation is that a reduction in OXPHOS efficiency caused by an interaction of SNPs with 150T results in decreased ROS production, which decreases apoptosis and increases longevity (Coskun et al., 2003). In accordance with this hypothesis, another study revealed that the presence of 150T transition alone is not
U
278 279
R O
Fig. 5. The distribution of the SNPs in functionally connected gene regions of the mtDNA from the 90+ and control groups. Frequency indicates the average number of mutations per subject. *Indicates a significant difference between groups (p b 0.05).
Fig. 6. Non-synonymous SNP frequency in functionally associated gene regions of mtDNA from the 90+ and control groups. Frequency indicates the average number of non-synonymous polymorphism per subject. *Indicates statistical significance (p b 0.05).
Please cite this article as: Guney, O., et al., Mitochondrial DNA polymorphisms associated with longevity in the Turkish population, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.013
307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335
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A263G, A2076G, A8860G, G11719A, C14766T, and A15326G) in the Bama Country (Guangxi, China) (Yang et al., 2012). These data emphasize that the association between longevity and mtDNA variations is highly dependent on population and geography. Investigation of the distribution of SNPs between functionally related mitochondrial genes revealed that SNPs in OXPHOS Complex I genes (ND1-6) were significantly higher in the 90+ group compared to controls. In particular, significant SNP accumulation was detected in the ND1, ND3 and ND4 regions in the elderly group compared to controls, suggesting that SNPs in these regions might be beneficial. To determine whether an alteration in protein structure could contribute to this hypothetical beneficial effect, we examined the non-synonymous mutation frequency in these OXPHOS genes. The non-synonymous SNP frequency was significantly increased in the genes of Complex I, suggesting that an alteration of the functionality of that protein (NADH dehydrogenase) might confer a selective advantage for longevity. Similar results were obtained in Finns, but higher mutation frequency in Complex I genes was detected in controls compared to the 90+ group in Danish and in southern European populations (Raule et al., 2013). Although no statistical significance was detected, the frequency of non-synonymous SNPs in Complex V (ATPase6/8) and COI–III (Cytochrome c oxidase subunits I–III) genes was reduced, suggesting that SNPs in these regions might decrease resistance to age related diseases. Interestingly, the decreased frequency of non-synonymous SNPs and the increased frequency of total SNPs in COI–III genes in the 90 + group highlighted the importance of these proteins and the detrimental potential of altered Cytochrome c oxidase functionality. However, further studies are required to support these findings. Our data suggest that the accumulation of mutations in Complex I could be beneficial, and it is likely highly dependent on genetic background and the occurrence of other SNPs in mitochondrial DNA. We observed an association between 150T, 152T and 73G SNPs in the D-loop region and longevity in the Turkish population. Furthermore, haplogroup H was negatively associated with longevity. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mito.2014.04.013.
341 342
Acknowledgments
377
T
E
D
P
R O
O
F
6
336
R
R
E
C
Fig. 7. An unrooted neighbor-joining (NJ) tree of the haplotypes of Turkish individuals. Individuals belonging to the 90+ group are represented by solid circles, and controls are represented by open circles. *Indicates a subject over the age of 95. The optimal tree with the sum of branch length of 0.02506385 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary distances were computed using the Kimura 2-parameter method, and the units are the number of base substitutions per site. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted using MEGA6.
t3:1 t3:2
Table 3 Frequency (%) of mtDNA major haplogroups in the elderly and control groups.
C
N
Haplogroup
90+
Control
(n = 50)
Total
Group
Group
%
(n = 25)
(n = 25)
%
%
14 12 6 4 4 2 2 4 2 2 2 2
26 6 4 2 – 2 2 – – 2 2 –
U
t3:3
O
339 340
750G, 1438G, 4769G, 8860G and 15326G polymorphisms was greater than 95% in the general population. 15326G, 7028T, 11719A and 14766T were also present at a relatively high proportion (50–60%). Interestingly, most of these frequent mitochondrial mutations in the Turkish population coincide with variants associated with longevity (A73G,
337 Q6 338
t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18
H U T N C D R J X K HV V
40 18 10 6 4 4 4 4 2 4 4 2
343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376
This project was supported by the Ege University Research Funds 378 (Project No. 2010 TIP 013). 379 References
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Please cite this article as: Guney, O., et al., Mitochondrial DNA polymorphisms associated with longevity in the Turkish population, Mitochondrion (2014), http://dx.doi.org/10.1016/j.mito.2014.04.013
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