Association of rs699 (M235T) Polymorphism in the AGT Gene With Power but Not Endurance Athlete Status

Abstract

Zarębska, A, Sawczyn, S, Kaczmarczyk, M, Ficek, K, Maciejewska-Karłowska, A, Sawczuk, M, Leońska-Duniec, A, Eider, J, Grenda, A, and Cięszczyk, P. Association of rs699 (M235T) polymorphism in the AGT gene with power but not endurance athlete status. J Strength Cond Res 27(10): 2898–2903, 2013—Thus far, genetic studies of the renin-angiotensin system (RAS) with respect to athletic performance or athlete status have mainly focused on the angiotensin-converting enzyme gene and its insertion/deletion polymorphism. The aim of this study was to investigate the functional rs699 (M235T) polymorphism in angiotensinogen (AGT), the second most important gene of the RAS, for association with athletic status and level of performance. The study included 123 endurance athletes and 100 power-oriented athletes, who were classified as elite or sub-elite according to competitive achievements at the international level, and 354 unrelated sedentary control subjects. The M235T genotype and allele distributions differed significantly between power and endurance athletes (p < 0.0001 and p < 0.0002, genotypes and alleles, respectively) and between power athletes and control subjects (p < 0.0001 and p < 0.0002, genotypes and alleles, respectively). The frequency of the CC genotype in the power athlete group was 2.2 times higher and 3.1 times higher than in the control and endurance groups, respectively. No difference was found in M235T allele distribution between elite and sub-elite athletes, either in power- or endurance-oriented athletes. We conclude that the CC genotype of the M235T polymorphism is overrepresented in Polish power athletes, suggesting that the AGT M235T variant is associated with power athletes' status.

Introduction

Montgomery et al. (²³) reported a genetic factor that strongly influenced human physical performance, a common insertion/deletion (I/D) polymorphism in the gene encoding angiotensin-converting enzyme (ACE). Since then, research on exercise, fitness, and performance genomics has continued constantly, and at the end of 2007, the human gene map for physical performance and health-related fitness phenotypes included 239 gene entries and quantitative trait loci (⁶). Recently, studies have culminated in attempts to identify a compound genotype or “optimum” polygenic profile that could predict an individual's potential in either endurance- or power-oriented sports (^7,31,36).

The effects of the renin-angiotensin system (RAS), which has been classically regarded as one of the most important humoral factors in the regulation of blood pressure, body salt, and fluid balance in both healthy and ill states (⁴⁰), are not restricted to cardiovascular and renal systems only. The RAS modulates free radical production and the cellular synthesis of several molecules such as cytokines, chemokines, and transcription factors (⁸). Furthermore, angiotensin II (ANGII), the primary mediator of the RAS, is a pleiotropic peptide which by acting on ANGII type 1 receptors (AGT1R) and ANGII type 2 receptors (AGT2R) in various tissues, is involved in inflammation, cell growth, proliferation, immune response regulation, and central neuromodulation (¹¹).

Thus far, genetic studies of the RAS with respect to athletic performance or athlete status have mainly focused on the ACE gene and its I/D polymorphism. The ACE I/D polymorphic variant has been associated with endurance performance or endurance athlete status (^9,14,23–25), power performance or power athlete status (^24,25,39), or the response to exercise or training (^15,22,23,37). Nevertheless, it must be noted that several studies have demonstrated no association between the ACE I/D polymorphism and athlete status (³³), and in some instances provided conflicting findings concerning which alleles are associated with power/strength and endurance sports or phenotypes (^3,16), which in sum demonstrate the complex nature of these traits.

Angiotensinogen (AGT), a globular glycoprotein, is encoded by the AGT gene and located on chromosome 1q42 (¹⁰). As the AGT concentration seems to impose a rate limitation on the generation of both plasma angiotensin I (ANG I) and ANG II, the in vivo AGT concentration may be of great importance for the activation of the RAS (¹⁰). Among the many single-nucleotide polymorphisms (SNPs) described in the AGT gene: −532C>T, −217A>G, −20A>C, +31T>C, −6G>A, M235T—the latter two, being in strong linkage disequilibrium with one another and with the remaining ones, have been particularly thoroughly studied, especially in conjunction with human hypertension (¹⁹). The M235T (rs699, 4072T>C) is a missense polymorphism, in which a T to C transition at position 4,072 in exon 2 results in a change of the amino acid methionine (M) to threonine (T) at residue 235 of mature AGT. It has been shown that the M235T polymorphism has a moderate effect on the plasma AGT concentration, with a 10–30% increase (both in men and women) among those carrying the C allele (²⁰).

Although, the M235T polymorphism has been reported to modify the influence of training or exercise on various physical performance and health-related fitness phenotypes, such as cardiorespiratory endurance (²), blood pressure, and heart morphology (^2,28–30), to date, only one study yielded a positive result for the M235T polymorphism in relation to athlete status (¹⁷). In a study of Spanish Caucasians, Gomez-Gallego et al. (¹⁷) demonstrated significantly different M235T genotype distributions between elite power– and elite endurance–oriented athletes and nonathlete controls, suggesting an association of this genetic marker with power athlete status. Specifically, the CC genotype was overrepresented in power-oriented athletes. However, the allele distribution did not differ significantly between all groups studied and between Olympic level and national level power athletes (¹⁷). The authors concluded that the C allele, associated with higher AGT and subsequent ANG II level—a skeletal muscle growth factor, might be beneficial in power (sprint) sport performance (¹⁷). Later, Buxens et al. (⁷) conducted a study within the same population (Spanish Caucasians) designed to examine genetic differences between power- and endurance-oriented athletes, and they found only marginally significant M235T genotype and allele frequency differences between the 2 groups of elite athletes (⁷).

As the results obtained so far are uncertain and inconsistent, we designed a study to verify the previous findings of the association of M235T polymorphism in the AGT gene with athlete status and to test whether this variant is linked to athletic performance. Several studies have shown the association of the I/D ACE polymorphism, a known genetic contributor to ACE enzyme activity (³⁴) with power or endurance performance (^24,25). The relationship between I/D ACE polymorphism and ANGII level is less clear. Abraham et al. (¹) found that not only plasma ACE activity but also plasma ANGII concentrations are highest in DD homozygotes. Thus, it is likely, that M235T variant having a moderate effect on AGT level—a rate limiting in the synthesis of ANGII (¹⁰)—may be associated with athletic performance. Therefore, the aim of this study was first, to investigate the AGT M235T polymorphism for association with athletic status in the Polish population, and second, to search for possible associations between the M235T variants and athletic performance.

Methods

Experimental Approach to the Problem

To accomplish the objectives, we designed a case-control study in which all participants were Polish Caucasians. Two hundred twenty-three athletes and 344 sedentary volunteers were recruited for this study. To accomplish the first objective, we compared the genotype and allele frequency between athlete groups and controls, sedentary subjects, and the second objective was fulfilled through a comparison of allele distribution in athletes stratified by competitive level. Various methods were used to obtain the samples, including targeting national teams and providing information to national coaching staffs and athletes attending training camps. All athletes and controls were Caucasian to reduce the possibility of racial gene skew and to overcome any potential problems because of population stratification.

Subjects

The study was done using a group of 223 Polish athletes of the highest nationally competitive standard (males, n = 156, and females, n = 67). The group of athletes was composed of 2 subgroups:

1. Endurance athletes (END) (n = 123), characterized by predominantly aerobic energy production (duration of exertion over 5 minutes, intensity of exertion moderate to high). This group included triathletes (n = 4), race walkers (n = 6), road cyclists (n = 14), 15- to 50-km cross-country skiers (n = 6), marathon runners (n = 12), rowers (n = 53), 3- to 10-km runners (n = 17), and 800- to 1,500-m swimmers (n = 11);
2. Power athletes (PWR) (n = 100) with predominantly anaerobic energy production (duration of exertion <1 minute, intensity of exertion submaximal to maximal): 100- to 400-m runners (n = 29), powerlifters (n = 22), weightlifters (n = 20), throwers (n = 14), and jumpers (n = 15).

There were 133 athletes classified as “elite,” 39 of whom were “top-elite” (gold medalists in the World and European Championships, World Cups, or Olympic Games), plus 94 athletes who were silver or bronze medalists in the World and European Championships, World Cups, or Olympic Games. The other athletes (n = 90) were classified as “sub-elite” (participants in international competitions). There were 69 elite and 54 sub-elite athletes in the endurance class and 64 elite and 36 sub-elite in the power class. The age of subjects of this study is: athletes 27 ± 7 years old and controls 20 ± 2 years old.

Control samples (C) were prepared from 354 unrelated sedentary volunteers (students of the University of Szczecin, aged 19–23 years; 81 females and 273 males).

The Pomeranian Medical University Ethics Committee approved all the procedures. All participants gave informed consent to genotyping with the understanding that it was anonymous and obtained results would have confidential status.

Procedures

The buccal cells donated by the participants were collected in Resuspension Solution (GenElute Mammalian Genomic DNA Miniprep Kit; Sigma, Steinheim, Germany) with the use of sterile foam-tipped applicators (Puritan, Guilford, Maine, USA). DNA was extracted from the buccal cells using a GenElute Mammalian Genomic DNA Miniprep Kit (Sigma, Steinheim, Germany) according to the manufacturer's protocol. Genotyping of the M235T polymorphism (rs699, also designated as 4072T>C or c.803T>C [cDNA label], and p.M268T [protein label], depending on the position at which the numbering was started) (³⁵) was done using an allelic discrimination assay on a Rotor-Gene real-time polymerase chain reaction instrument (Corbett, Sydney, Australia) with TaqMan probes. To distinguish between AGT M235 and T235 alleles, TaqMan Pre-Designed SNP Genotyping Assays were used (assay ID: C_1985481_20; Applied Biosystems, Carlsbad, California, USA), including primers and fluorescent-labeled (FAM and VIC) MGB probes for the detection of both alleles.

Statistical Analyses

Any differences in genotype and allele frequency were analyzed using χ² tests (or Fisher exact tests). Odds ratios with 95% confidence intervals (95% CI) were calculated. Statistical power to detect the relative differences of 10, 20, and 30% in CC genotype frequency between groups were, 53, 96, and 100%; 59, 98, and 100%; 44, 91, and 100%; for PWR vs. C, END vs. C, PWR vs. END, respectively. All calculations were performed using STATISTICA (StatSoft, Inc., 2011; data analysis software system, version 10; www.statsoft.com), except Hardy-Weinberg equilibrium, which was tested with the programming language and environment R (http://www.r-project.org). Bonferroni adjusted p values (p_adj) were obtained by multiplying the observed p values from the significance tests by the number of tests (k), and any k × p which exceeded 1 were ignored (⁴). The p values <0.05 were considered statistically significant.

Results

Genotype distributions met Hardy-Weinberg proportions in the control group (χ² = 2.43, p = 0.124) and in the endurance group (χ² = 1.07, p = 0.343). There was, however, a significant deviation from Hardy-Weinberg equilibrium in the power group (χ² = 13.87, p = 0.0004).

The genotype distribution in the power athletes differed significantly from those in controls (χ² = 21.29, df = 2, p = 0.00002, p_adj = 0.00006) and the endurance group (χ² = 22.34, df = 2, p = 0.00001, p_adj = 0.00003). No difference in genotype distribution was found between the endurance and control groups (Table1).

Table 1: Genotype distribution and allele frequencies of the M235T polymorphism in Polish athletes and control group.

The frequency of the CC genotype in the power athlete group was 2.2 times higher and 3.1 times higher than in the control and endurance groups, respectively. When compared with control subjects, the chance of being a power athlete was 3.02 (95% CI, 1.85–5.04, p < 0.00001, p_adj < 0.00003) times higher among CC homozygotes than among carriers of the T allele (TT + TC). When compared with endurance athletes, the chance of being a power athlete was 4.46 (95% CI, 2.20–9.12, p < 0.00001, p_adj < 0.00003) times higher among CC homozygotes than among carriers of the T allele (TT + TC) The frequency of the C allele was the highest in the power group (55.5%), and it was significantly higher compared with controls (40.1%, p = 0.0001, p_adj = 0.0003) and endurance athletes (39.0%, p = 0.0005, p_adj = 0.0015). No difference in the C allele distribution was found between the endurance and nonathlete groups (Table 1). No difference was found in M235T allele distribution between elite and sub-elite athletes, either in power-oriented (p = 0.758) or endurance-oriented (p = 0.275) athletes (Figure 1).

Figure 1: The M235T allele frequencies in elite level and sub-elite level athletes, both endurance and power oriented. χ² = 1.19, df = 1, p = 0.275 in elite vs. sub-elite endurance athletes. χ² = 0.10, df = 1, p = 0.758 in elite vs. sub-elite power athletes.

Discussion

The main finding of our study is a highly significant overrepresentation of both the CC genotype and the C (T235) allele of the M235T polymorphism in the AGT gene among power-oriented athletes compared with endurance and control participants, suggesting that the C allele of the M235T variant may be associated with a predisposition to power-oriented events. Although an excess of CC homozygotes has already been demonstrated among top national and Olympic level Spanish male power athletes by Gomez-Gallego et al. (¹⁷), this finding has not been supported in the literature. The only other study to investigate the M235T variant in relation to athlete status was done by Buxens et al. (⁷). It was conducted on the same population of Spanish Caucasians and was primarily designed to determine the polygenic predisposition toward endurance or power sports. In the study by Buxens et al. (⁷), the M235T genotype frequency difference between elite endurance and elite power athletes (32% vs. 16%, respectively) was only marginally significant. Although the authors failed to demonstrate a significant difference in the genotype distribution between the 2 groups of athletes originating from the same population, it is noteworthy that the power athletes' group size was smaller in comparison with the initial study by Gomez-Gallego et al. (¹⁷).

Thus, the present study is, to our knowledge, the first independent positive replication of the initial finding reported by Gomez-Gallego et al. (¹⁷) and fulfills the suggested criteria for establishing positive replication (²⁶) by having a sufficient sample size, an independent data set, a similar population (Polish Caucasian), the same genetic model, and a very similar phenotype under analysis. Moreover, in contrast to the initial study, not only was the CC genotype significantly overrepresented among the power athletes when compared with control subjects as well as endurance athletes (as in the Gomez-Gallego study (¹⁷)) but also the allele frequencies differed between groups, with a significant predominance of the C allele in the power group when compared with both controls and the endurance group. Furthermore, these associations held up after adjustments for multiple testing.

The M235T allele distribution varies widely according to the subject's ethnic origin: the T235 allele is by far the most frequent in Africans (∼0.90) and in African-Americans (∼0.80). It is also high in the Japanese population (0.65–0.75) (¹⁰). The T235 (C4027) allele distribution of the control participants in our study was lower (0.40) but was similar to that reported among Spanish Caucasians (0.41), as were the sports specialties of both the power athletes (throwers, sprinters, and jumpers) and endurance athletes (marathon runners, 3- to 10-km runners, and road cyclists), thus mirroring the aforementioned studies (^7,17). Of particular interest in our study was the lack of Hardy-Weinberg equilibrium among the power athletes. Although the departure from Hardy-Weinberg equilibrium might have been because of a genotyping error, this phenomenon could also be accounted for by common disease or complex traits such as athletic ability (³⁸). The latter possibility is more likely because a departure form Hardy-Weinberg equilibrium was found in another study of M235T with respect to athletic status (¹⁷), as well as in the other genes related to human athletic status or performance, e.g., ACTN3 (¹²) and IL6 (¹³).

So far, no article has reported significantly different M235T allele or genotype distribution in athletes stratified by their level of performance; thus, the association of this marker with athletic performance remains unknown. Gomez-Gallego et al. (¹⁷) reported the C allele frequency in Olympic level and National level power athletes; yet, no formal statistical tests were performed. Although the M235T polymorphism seems to be associated with power athlete status, we found no evidence of such association with power or endurance performance (elite status). Indeed, the M235T allele distribution did not differ between elite and sub-elite power athletes nor did it differ between elite and sub-elite endurance athletes. Cautious interpretation, however, is required, mainly because of the small sample size of the subgroups and subsequently reduced statistical power/increased probability of a type II error. Additional studies are warranted to clarify whether this polymorphism affects athletic performance.

It seems likely that the effects of the M235T variant result from modulation of ANGII levels. First, AT1R-mediated ANG II is crucial for muscle performance because of multiple mechanisms (e.g., direct hypertrophic effect on skeletal muscle, redirection of blood flow from type I fibers to fast powerful type II fibers), thereby enhancing power and strength capacity (²¹). Secondly, plasma AGT levels have been shown to significantly relate to M235T allele status, and subjects, homozygous for the M235 allele (T4072), displayed the lowest circulating AGT levels, whereas subjects, homozygous for the T235 allele (C4072), had the highest AGT levels (³²). Taken together, because the AGT concentration has been shown to be a rate-limiting factor for ANGII generation (¹⁰), the overrepresentation of the CC genotype in power athletes might be because of its advantageous effect on building muscle mass or other ANGII-dependent skeletal muscle strength phenotypes. It must be emphasized, however, that the functional consequence of M235T variants is still controversial, and in one study (¹⁸), this polymorphism was excluded as a functional variant by biochemical analysis of recombinant M235 and T235 molecules. It is possible that the potential functionality of the M235T polymorphism may be because of linkage disequilibrium with other functional AGT variants, especially −6G>A (^5,27). As in previous studies (^7,17), we also found no association between M235T and endurance status, despite the fact that this variant was reported to be associated with some endurance phenotypes, such as maximal aerobic capacity (²).

In conclusion, our study is the first independent positive replication of the previous finding and supports the hypothesis that the C allele of the M235T polymorphism in the AGT gene may confer a favorable effect in power-oriented sports, mediated at least in part through increased ANGII production in skeletal muscles. However, further studies are warranted to determine whether this polymorphism affects an athlete's level of performance.

Practical Applications

The area of genomics relating to exercise, fitness, and performance is rapidly evolving. Identifying genetic characteristics related to athletic excellence or individual predisposition to types of sports with different demands (power or endurance oriented) or even sport specialty may be decisive in recognizing athletic talent and probably will allow for greater specificity in steering of sports training programs. The present study has shown that the M235T variant in the AGT may be one of the genetic markers to investigate when an assessment of predisposition to power sports is being made.

Acknowledgments

The research was conducted in the laboratory of Department of Genetics, University of Szczecin, Szczecin, Poland. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.