ACE I/D, ACTN3 R577X, PPARD T294C and PPARGC1A Gly482Ser polymorphisms and physical fitness in Taiwanese late adolescent girls

Abstract

Physical performance of youth is influenced by various factors, including body composition, biological maturity status, level of habitual physical activity, and muscular strength. Muscular strength has been largely attributed to genetic effects. To exclude possible confounding effects from various acquired factors, this study examined the relationships between polymorphisms of the angiotensin-converting enzyme (ACE), α-actinin-3 (ACTN3), peroxisome proliferator-activated receptor delta (PPARD), and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PPARGC1A) genes and performance as measured by six fitness tests (handgrip strength of dominant hand, 30- and 60-s sit-ups, standing long jump, 60-m dash, and 800-m run) in 170 sedentary adolescent girls with the adjustment of anthropometric characteristics. We found that subjects with the ACE DD genotype were significantly heavier than those with I allele, while those with the ACTN3 RR genotype had higher fat-free mass percentage (FFM%) than those with the XX genotype. In addition, those with the PPARD TT genotype were significantly taller, heavier, and had a greater FFM than those with the CC genotype. Subjects with the ACE DD, ACTN3 RR and PPARD TC genotype had better performance in handgrip strength, 30- and 60-s sit-up tests, and standing long jump, respectively, when individual gene was analyzed independently after adjusting anthropometric characteristics. In the gene combination analysis, subjects with ACE DD, ACTN3 RR and PPARD TT genotype had significantly greater performance in handgrip strength. Overall, the results indicate that the genes studied have a modest influence on individual performance as assessed by specific fitness and strength tests in female late adolescents.

Introduction

It has been reported that the physical performance of youth is influenced by a variety of factors, including age, sex, body size, and composition, biological maturity status, level of habitual physical activity and muscular strength [1, 2]. Among them, muscular strength can be attributed to genetic effects varying from 0.27 to 0.58 based on family studies and between 0.14 and 0.83 based on twin studies [3]. Recently, the development of technology for rapid DNA sequencing and genotyping has allowed the identification of some individual genetic variations that contribute to physical performance. Bray et al. [4] has comprehensively reviewed genes and markers that show evidence of association with performance or fitness phenotypes in sedentary or active people, in responses to acute exercise, and in terms of training-induced adaptation. Among the 214 listed autosomal genes, angiotensin-converting enzyme (ACE) and α-actinin-3 (ACTN3) are the genes with the highest number of positive findings. Most studies have linked the ACE I allele to endurance performance [5] and the D allele to muscle strength and power-oriented performance [6–8]. The ACTN3 R allele is prevalent among sprint athletes and the X allele is less common among sprinters, particularly in the homozygous form (XX) [9–13].

In addition to ACE and ACTN3, there is emerging evidence that the peroxisome proliferator-activated receptor delta (PPARD) [14, 15] and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PPARGC1A) [16] gene may play an important role in physical performance. Recently, Eynon et al. [17] found that the PPARD T294C polymorphism together with peroxisome proliferator-activated receptor γ coactivator-1α (PPARGC1A) play an important role in endurance-type performance.

There are few data available on the combined influence of polymorphisms of ACE, ACTN3 and other genes on physical capability phenotypes, especially in non-athletic populations [18–21]. Furthermore, physical performance is also determined by a range of acquired factors such as age, body composition, and physical training. To exclude possible confounding effects from these factors, the present study was carried out to examine the relationships between the ACE, ACTN3, PPARD, and PPARGC1A genotypes and performance in fitness tests by sedentary female adolescents (16–18 years).

Methods

Subjects

This study was conducted according to the Harriss and Atkinson Statement [22] and approved by the Institutional Review Board of Chang Gung Memorial Hospital. To exclude the possible influences of menstrual and disease status on physical performances, we first surveyed the disease history as well as the menstrual cycle length and the first day of the latest menstrual bleeding for all the 11th grade (16–18 years old) female students. Only those students with a regular menstrual cycle length of 28–30 days and the first day of their latest menstrual bleeding were within just 3 days were asked to participate the present study. Finally, a total of 170 sedentary female students without cardiovascular, metabolic, or musculoskeletal diseases were included for the present analysis. All parents gave written consent and each girl also provided individual written assent. Body mass index (BMI) was calculated as weight (kg) divided by square of the height (m²). The percentage of body fat was estimated by bioelectrical impedance analysis using an OMRON (HBF-355) hand-to-foot body composition monitor (Omron Healthcare, Kyoto, Japan) [23] and was used to calculate the fat-free mass FFM (kg) and the fat-free mass percentage (FFM%). The fat-free mass index (FFMI) was calculated as the FFM (kg) divided by the square of the height (m²).

According to the Taiwan physical fitness test manual [24], six fitness tests were carried out without any prior training. They were (1) handgrip strength of the dominant hand, (2) 30- and 60-s sit-ups, (3) a standing long jump, (4) a 60-m dash, and (5) a 800-m endurance run. All the tests were executed in the morning and all the subjects completed the same test on the same day. In addition, 5 mL of saliva was collected from each participant and centrifuged at 800g for 10 min at room temperature to obtain oral mucosa cells for genotyping.

Genotyping

Genomic DNA was purified from oral mucosa cells by digestion with proteinase K and then extracted using a conventional phenol/chloroform procedure. Genotyping of the ACE I/D (rs1799752) was performed using polymerase chain reaction (PCR) as previously described [25]. The genotypes ACTN3 R577X (rs1815739) and PPARD T294C (rs2016520) were determined by PCR restriction fragment length polymorphism (RFLP) as described by Mills et al. [26] and Ahmetov et al. [14], respectively. The conditions for PCR were as shown in Table 1. Since it has been reported that amplification of the ACE I allele is sometimes suppressed in ID heterozygotes and mistyped as the DD genotype [27], all the samples classified as DD genotype were checked with a second PCR reaction using an I-specific primer pair: 5′-TGGGACCACAGCGCCCGCCACTAC-3′ (forward) and 5′-TCGCCAGCCCTCCCATGCCCATAA-3′ (reverse) [28]. The ACTN3 and PPARD genotypes were determined by enzymatic digestion of their amplicons with Dde I and Bsc4I, respectively. The PPARGC1A Gly482Ser (rs8192678) genotype was determined as described previously [29] using TaqMan-based allelic discrimination assay on a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

Table 1 Forward/reverse primers and PCR conditions for ACE, ACTN3, and PPARD genotyping

Total genotype score (TGS) determination

The combined influence of the four studied polymorphisms was determined in a similar manner to the previous study by Ruiz et al. [20]. A genotype score (GS) for the ‘optimal’ or preferable endurance genotype in each polymorphism was assigned as 2, whereas a GS of 0 was assigned to the least optimal genotype (Table 2). The sum of the GS from the genes studied (i.e. GS _ACE  + GS _ACTN3  + GS _PPARD ) was designated as the TGS _{ACE+ACTN3+PPARD} .

Table 2 Genotype score (GS) and frequency distribution of ACE, ACTN3, PPARD, and PPARGC1A studied in Taiwanese female late adolescents

Statistical analysis

All statistical analyses were performed using SPSS v.13.0 (SPSS, Chicago, IL, USA). The distribution of genotypes studied was tested for the fulfilment of Hardy–Weinberg equilibrium status by using a Chi-square test with one degree of freedom. The differences in anthropometric characteristics among the different genotypes were compared by one-way analysis of variance (ANOVA) or the Kruskal–Wallis test depending on the normality of the variables. The differences in physical performance among the different genotypes were compared by analysis of covariance (ANCOVA) adjusted for height, body weight, BMI, FFM%, FFM, and FFMI. The level of significance was set at 0.05. All reported p values are 2-sided.

Results

The distribution of genotype for these four genes studied was not deviated from Hardy–Weinberg equilibrium (all p values >0.05). The association between anthropometric characteristics and ACE, ACTN3, PPARD, and PPARGC1A polymorphisms were shown in Table 3. Subjects with the ACE DD genotype (61.6 kg) were significantly heavier than those with the ID (54.9 kg) and the II (55.4 kg) genotype. Subjects with the ACTN3 RR genotype (71.0%) had higher FFM% values than those with the XX genotype (68.6%). In addition, those with the PPARD TT genotype were significantly taller (161.0 cm), heavier (57.1 kg), and had a greater FFM (39.3 kg) than those with the CC genotype (157.1 cm, 51.8 kg, and 36.2 kg). On the other hand, BMI and FFMI themselves were not significantly associated with the ACE, ACTN3, PPARD, and PPARGC1A polymorphisms.

Table 3 Association between anthropometric characteristics and ACE, ACTN3, PPARD, and PPARGC1A polymorphisms

After adjustment of anthropometric characteristics (height, weight, BMI, FFM%, FFM and FFMI), subjects with the ACE DD genotype had greater handgrip strength (28.3 kg) than those with the ID (25.0 kg) and the II (25.6 kg) genotype (Table 4). Individuals with the ACTN3 RR genotype performed better in the 30- and 60-s sit-up tests (18.7 and 34.1 counts) than those with the RX genotype (17.0 and 30.3 counts). Subjects with the PPARD TC genotype (150.1 cm) performed significantly better in the standing long jump test than those with the CC genotype (136.9 cm). Subjects with the PPARGC1A Gly/Gly genotype (34.4 counts) performed significantly better in the 60-s sit-up test than those with the Gly/Ser genotype (30.5 counts). However, there were no associations between the genotypes and either 60-m dash or 800-m endurance run test.

Table 4 Association between the fitness test results and ACE, ACTN3, PPARD, and PPARGC1A polymorphisms

The combined gene influence on physical performance was further explored. As shown in Table S1 (supplementary) and Table 5, TGS _{ACE+ACTN3+PPARD+PPARGC1A} was only marginally associated with handgrip strength, while TGS _{ACE+ACTN3+PPARD} was associated with handgrip strength and 60-s sit-up. Additionally, it is worth noting that the maximum handgrip strength (41.4 kg) found among all the subjects was an individual who did not have the “optimal” gene profile for the least endurance group, namely a TGS of 0.

Table 5 Association between total genotype score (TGS _{ACE+ACTN3+PPARD} ) and performance in the fitness tests studied

Discussion

In this study, we found that performance in the handgrip strength, 30-/60-s sit-up, standing long jump, and 60-s sit-up test results were significantly associated with ACE, ACTN3, PPARD, and PPARGC1A polymorphisms, respectively, in sedentary female late adolescents after adjusting for anthropometric characteristics (Table 4). The ACE D allele is associated with higher ACE activity and thus an increased angiotensin II level [30]. Therefore, this allele would theoretically favor performance in power-oriented exercise tasks. Previous studies have reported a positive association of the ACE D allele and baseline grip strength in healthy untrained subjects [8], patients with chronic obstructive pulmonary disease [31], advanced cancer patients [32], and elite strength-trained athletes [33]. In contrast, some studies have failed to support such findings [28, 34]. Surprisingly, the Moran et al. [35] study of teenage Greeks reported that the homozygous I-allele individuals exhibited higher performance scores. In the present study, an association between ACE polymorphism and standing long jump was not observed. Similar phenomenon was also noted by Rodriguez-Romo et al. [36] in young non-athletic adults.

It has been reported that ACTN3 XX genotype precluded top-level athletic performance in ‘‘pure’’ power and sprint sports (sprinting, jumping, weightlifting, and throwing events), especially among women [13]. In the present study, we found that those with the ACTN3 RR genotype in combination with the ACE DD genotype performed significantly better in terms of handgrip strength than those with other genotype combinations (Tables S2 and S3). However, no effect on standing long jump was observed. Previous studies have also demonstrated that the ACTN3 R577X polymorphism does not seem to influence explosive leg muscle power (jumping, sprinting) alone or in combination with the ACE I/D polymorphism in a young non-athletic population, irrespective of gender [36]. Clarkson et al. [37] and Walsh et al. [38] reported that women with the ACTN3 XX genotype have lower strength than those with the RX genotype. Chiu et al. [39] reported that pre-adolescents with the ACTN3 RR genotype exhibited the best performance across all phases (before, during, and after training) of 25-m swimming performance. However, in older women (64 years), knee extensor concentric peak power was found to be higher in X allele homozygote individuals compared with RR genotype individuals [40]. These discrepancies may reflect that there is an interaction between age and genotype [41].

There is compelling evidence indicating that a functional T294C polymorphism of PPARD influences human physical performance [15]. In the present study, we found that the subjects with the TC genotype demonstrated a better performance in the lower extremity explosive power standing long jump test than those with CC genotype. This finding supports the hypothesis that the PPARD C allele is associated with a predisposition to endurance performance [17].

The combined effect of the ACE, ACTN3, PPARD, and PPARGC1A polymorphisms on performance across the six fitness tests was further evaluated using the TGS index as described previously [20]. In the present study, since there was no subject with homozygous ACE DD, ACTN3 RR, PPARD TT, and PPARGC1A Ser/Ser genotype, the greatest mean handgrip strength was observed in subjects with TGS _{ACE+ACTN3+PPARD+PPARGC1A}  = 1 as expected (Table S1). Furthermore, subjects that were homozygous ACE DD, ACTN3 RR, and PPARD TT had the greatest handgrip strength, which suggests that these “strength/power” alleles do indeed confer a performance advantage (Table 5). However, it is also interesting to note that the subject with the best handgrip strength performance did not belong to the “optimal” (TGS = 0) power genotype group. These findings indicate that the relationships between the genetic traits and physical performance are quite complex and not yet completely understood [19, 21, 42].

Handgrip strength has been linked to premature mortality, disability, and other health-related complications in middle-aged and older people [43–45]. In the present study, we demonstrated an association between three genetic polymorphisms (ACE I/D, ACTN3 R577X, and PPARD T294C) and handgrip strength in sedentary female adolescents (16–18 years). These results may provide evidence that helps the development of recommendations such as early specific nutritional and/or functional interventions (e.g., resistance training activity) for those with a high TGS index. In the future, polygenic physical fitness profiling of a larger general population linked to specific nutritional/functional intervention studies will be required to provide valid information on the true role of genetic factors on physical fitness and health.

In conclusion, the results indicate that the studied genes have a moderate influence on performance as measured by specific fitness tests and the effect of the ACE and ACTN3 polymorphisms on the strength type of fitness is greater than the effect of PPARD and PPARGC1A polymorphism among Taiwanese female late adolescents.