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Short communication

Mitochondrial haplogroup T is negatively associated with the status of elite endurance athlete
nica G. Castro a, Nicola s Terrados b,c, Julia n R. Reguero d, Mo a Victoria Alvarez , Eliecer Coto a,*
a

tica Molecular-Instituto de Estudios Nefrolo gicos, Hospital Central de Asturias, Oviedo, Spain Gene b a Funcional-Univ., Oviedo, Oviedo, Spain Dept. de Biolog c n Dep., Municipal Avile s, Avile s, Spain Fundacio d a, Hospital Central de Asturias, Oviedo, Spain Cardiolog Received 14 April 2007; received in revised form 30 May 2007; accepted 20 June 2007 Available online 27 June 2007

Abstract Mitochondrial function is absolutely necessary to supply the energy required for muscles, and germ line mutations in mitochondrial genes have been related with impaired cardiac function and exercise intolerance. In addition, alleles at several polymorphic sites in mtDNA dene nine common haplogroups, and some of these haplogroups have been implicated in the risk of developing several diseases. In this study, we analysed the association between mtHaplogroups and the capacity to reach the status of elite endurance athlete. DNA was obtained from blood leukocytes of 95 Spanish elite endurance athletes and 250 healthy male population controls. We analysed eight mitochondrial polymorphisms and the frequencies were statistically compared between elite athletes and controls. Haplogroup T, specically dened by 13368A, was signicantly less frequent among elite endurance athletes (p = 0.012, Fishers exact test). Our study suggests that allele 13368A and mitochondrial haplogroup T might be a marker negatively associated with the status of elite endurance athlete. This mitochondrial variant could be related with a lower capacity to respond to endurance training, through unknown mechanisms involving a less ecient mitochondrial workload. 2007 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
Keywords: Mitochondrial DNA; Endurance capacity; Mitochondrial haplogroups; Genetic association

1. Introduction Exercise capacity depends on various factors, such as oxygen uptake and oxygen utilization at the mitochondria. The maximal oxygen uptake (VO2 max) reaches a maximum value after optimal endurance training, and sets the upper limit of endurance capacity (Lortie et al., 1984; DiPrampero, 2003). As endurance capacity improves, several physiological and metabolic adaptations occur, such as an increase in capillary and mitochondria density in muscles (Holloszy and Coyle, 1984; Hoppeler et al., 1985; Zoll

Corresponding author. Tel.: +34 985 10 79 68. E-mail address: eliecer.coto@sespa.princast.es (E. Coto).

et al., 2002). Mitochondria are the cellular organelles that perform the metabolic reactions necessary to generate energy as adenosine triphosphate (ATP). Most of the mitochondrial proteins are encoded by genes in the nucleus, and these gene products are imported from the cytoplasm, but mitochondria also contain their own DNA (mtDNA) in a single circular chromosome, and their own machinery for synthesising RNA and proteins. The mitochondrial genome consists of 37 intronless genes that encode 13 subunits of the electron-transfer chain, 2 ribosomal RNAs (rRNA), and 22 transfer RNAs (tRNA). Because mitochondria are in every cell of each tissue, mutations in mtDNA are frequently associated with multisystemic diseases (DiMauro and Schon, 2003). In some cases, the disease is directly linked to mtDNA-mutations,

1567-7249/$ - see front matter 2007 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2007.06.002

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and follows a maternal transmission pattern (mitochondrial DNA is inherited from the mother). Mutations in mtDNA could reduce the capacity to produce ATP, and this impairment in energy supply could aect cardiac muscle contraction and neuronal function, among others. In addition to rare mutations, commonly associated with diseases, mtDNA is highly polymorphic. These single nucleotide polymorphisms (SNPs) have accumulated sequentially along radiating female lineages, giving rise to a wide variety of mitochondrial haplogroups (Torroni et al., 1996). The characterisation of these mtDNA-haplogroups is an important tool to investigate the origin and relationships of human populations. In addition, this mtDNA-variation could contribute to the expression of mitochondrial-related diseases, and carriers of a particular allele or haplogroup could be at a higher or lower risk for these diseases (Van der Walt et al., 2003; Nishigaki et al., 2007). It is well known that trainability and exercise capacity diers from one person to another, and this individual difference is determined by environmental/life-style and genetic (inherited) factors (Lesage et al., 1985; Bouchard and Lortie, 1986; Hamel et al., 1986). Maximal aerobic power (VO2 max) and other determinants of endurance performance are largely inherited, and the response of VO2 max to endurance training diers between individuals (Bouchard et al., 1998). In recent years, the polymorphisms at several genes have been related with individual dierences in the optimization of endurance training, and these polymorphisms could be related with the capacity to reach the status of elite endurance athlete (Montgomery et al., 1998; Alvarez et al., 2000; Rankinen et al., 2004). Taking into account the role of mitochondrias oxidative activity on endurance capacity, the mtDNA-variation could be related with individual dierences in trainability and physical capacity (Dionne et al., 1991; Murakami et al., 2001). In this work, we genotyped endurance elite athletes and healthy controls for several mitochondrial polymorphisms. Our aim was to determine the role of these mtDNA-polymorphisms in the capacity to reach the status of endurance elite athlete. 2. Materials and methods

(n = 25), and long-distance rowers (n = 20). They were n Deportiva Municipalrecruited through the Fundacio s, a Sports-Medicine Centre in the region of Asturias Avile (Northern Spain). Table 1 summarizes the main physical parameters in these individuals, including the maximum VO2 and heart beats. A total of 250 Spanish healthy male, aged 2042 years, were used as population controls. They were blood bank donors and University students, did not have a history of cardiovascular diseases, and were not involved in competitive physical exercise. Both, athletes and controls were Caucasian. All the individuals participating in the study gave their informed consent, and the study was approved by the Ethical Committee of Hospital Central Asturias. 2.2. mtDNA polymorphisms genotyping DNA was obtained from 10 ml of blood. Eight mitochondrial DNA fragments were polymerase chain reaction (PCR) amplied, and the genotype corresponding to each polymorphism was determined through digestion of the PCR-fragment with a restriction enzyme which target sequence was aected by the nucleotide change (RFLP). PCR-primers and the restriction enzymes used to analyse these polymorphisms had been previously reported (Castro et al., 2006). Each PCR contained approximately 100 ng of genomic DNA, 10 pmol of each primer-pair, and 2 mM of each dNTP, in a nal volume of 20 ll. PCR consisted on a initial denaturation at 95 C for 5 min, followed by 35 cycles of 95 C for 30 s, the annealing temperature for 1 min, and 72 C for 1 min, followed by a nal extension at 72 C for 5 min. Ten microlitres of each PCR product was digested with the appropriated restriction enzyme (New England Biolabs), digestions were electrophoresed on 3% agarose gels, and fragments were visualised after ethidium bromide staining. To determine the accuracy of this genotyping method, DNAs representative of each RFLP-genotype were amplied, the PCR-fragments were puried, and both strands were sequenced in an automated system (ABI310), using Big-Dye terminator v3.1 cycle sequencing kit chemistry (Applied Biosystems). 2.3. Haplogroups classication

2.1. Elite athletes and controls The study included a total of 95 male elite endurance athletes. They were Spanish, aged 20- to 40-years-old, and professional cyclists (n = 50), long-distance runners
Table 1 Mean values for the 95 elite endurance athletes included in the study Age (range) years Total (n = 95) Cyclists (n = 50) Runners (n = 25) Rowers (n = 20) 32 31 29 27 (2040) (2139) (2038) (2035) VO2 max (range) ml/min 5100 5400 4995 5105 (38535877) (44725809) (38535335) (45455877) VO2 max/kg (range) ml/kg/min 72 79 74 71 (5684) (6684) (5678) (5975) Heart beats max (range) beats/min 192 190 189 193 (181203) (185202) (181195) (189203)

Haplogroups are dened by nucleotides at specic known polymorphic sites in the mtDNA (Torroni et al., 1996). These polymorphisms have been previously described, at positions 1719 G/A, 4580 G/A, 7028 C/T,

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9055 G/A, 12308 A/G, 13368 G/A, 13708 G/A, and 16391 G/A. We genotyped athletes and controls for these SNPs, and determined the haplogroup of each individual (Castro et al., 2006). 2.4. Statistical analysis Allele and haplogroup frequencies between elite athletes and controls were compared through a Fisher exact test. For the haplogroups analysis, we compared each haplogroup with all other haplogroups pooled into one group, and with haplogroup H (the most common haplogroup). A p < 0.05 was considered as the level of statistical signicance. 3. Results and discussion In this study, we analysed eight mitochondrial polymorphisms in 95 elite endurance athletes and 250 controls. Allele 13368A was at a higher frequency in controls compared to athletes (8% vs. 1%, p = 0.012, Fishers exact test). These data indicated that 13368A could be a mitochondrial DNA marker related with a lower adaptation to endurance training, thus being a negative factor to become an elite endurance athlete. The eight polymorphic sites analysed dene nine common mitochondrial haplogroups. Haplogroup T was signicantly less frequent in athletes compared to controls (1% vs. 8%; p = 0.012; Table 2). Because haplogroup T is the only containing 13368A, this was in agreement with a higher frequency of this allele in controls compared to athletes. The association between some mitochondrial polymorphisms and the status of elite endurance athlete has been previously examined, with conicting results (Rivera et al., 1998; Niemi and Majamaa, 2005; Scott et al., 2005). Niemi and Majamaa genotyped 52 endurance Finnish athletes as well as sprint athletes and controls, and found a signicantly reduced frequency of haplogroups J and K among the endurance athletes. In particular, haplogroup K was found in 4.5% of controls and in none of the 52 endurance athletes. They hypothesised that these mtDNA haplogroups could be associated with lower ATP production, thus being negative genetic markers for
Table 2 Frequencies of the dierent mtHaplogroups in elite endurance athletes and controls Haplogroups H Athletes n = 95 Controls n = 275 43 43% 105 42% I 1 1% 2 1% J 15 15% 38 15% K 12 12% 22 9% T* 1 1% 21 8% U 13 13% 30 12% V 5 5% 13 5% W 4 4% 7 3% X 2 2% 4 2% Other 5 5% 8 3%

physical performance. In contrast with the results of these authors, the frequencies of haplogroups J and K in our study were similar between athletes and controls. Interestingly, T and J dene one of the four haplogroup clusters (HV, KU, IWX and JT), and the dierence between the two studies could reect the dierent genetic background between the Spanish and Finnish populations (Ross et al., 2003; Ruiz-Pesini et al., 2004). This is supported in part by the fact that haplogroup T was found in 3.6% of Finnish, but in 8% of our controls. No previous studies describing the biochemical properties of mtHaplogroups have been reported. The G13368A that dene haplogroup T is a silent change at the ND5 gene and would not be directly responsible for a less ecient trainability and physical capacity. However, sperm cells from Spanish males with haplogroup T should have a slower motility compared to those with haplogroup H (Ruiz-Pesini et al., 2000). This and other ndings suggest dierent functional properties among the dierent mtDNA lineages (Dionne et al., 1991; Ross et al., 2003). In particular, haplogroup T should be intrinsically prone to development of defects in the mitochondrial oxidative phosphorylation system, thus aecting the performance of mitochondria ATP production. Interesting, haplogroup T was signicantly more frequent among Spanish patients with Left Ventricular Hypertrophy (Castro et al., 2006). This suggests that the lower frequency of this haplotype among the elite athletes could be related with a negative eect on cardiac adaptation to endurance training (Hoppeler and Weibel, 1998). Finally, because G13368A is a silent ND5-polymorphism, sequencing of the mitochondrial genome in elite athletes with haplogroup T should be necessary to dene if a particular nucleotide change is directly responsible for this eect. In conclusion, we found a signicantly negative association between mitochondrial haplogroup T, dened by allele 13368A, and the condition of elite endurance athlete. This genetic marker could be involved in a lower adaptability to endurance training, thus determining a less chance to reach the condition of elite endurance athlete. Acknowledgements This work has been supported by Premio Nacional de n en Medicina del Deporte-Univ. OviedoInvestigacio 2005. Authors wish to thank all the individuals participating in this research. References
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*p = 0.012, elite endurance athletes vs. controls; T vs. all the other haplogroups pooled. *p = 0.02; T vs. H.

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