Abstract
R577XandACE I/Dpolymorphisms are associated with endurance exercise ability. This case–control study explored the association ofACTN3andACEgene polymorphisms with elite pure endurance in Korean athletes, hypothesizing that individuals with bothACTN3 XXand ACE IIgenotypes would exhibit superior endurance. We recruited 934 elite athletes (713 males, 221 females) and selected 45 pure endurance athletes (36 males, 9 females) requiring “≥90% aerobic energy metabolism during sports events”, in addition to 679 healthy non-athlete Koreans (361 males, 318 females) as controls. Genomic DNA was extracted and genotyped forACTN3 R577XandACE I/Dpolymorphisms.ACE ID(p= 0.090) andACTN3 RX+XX(p= 0.029) genotype distributions were significantly different between the two groups. ComplexACTN3-ACEgenotypes also exhibited significant differences (p= 0.014), with dominant complex genotypes positively affecting endurance (p= 0.039). The presence ofRX+IIorXX+IIwas associated with a 1.763-fold higher likelihood of possessing a superior endurance capacity than that seen in healthy controls (90% CI = 1.037–3.089). Our findings propose an association of combinedACTN3 RX+XXandACE IIgenotypes with enhanced endurance performance in elite Korean athletes. While causality remains to be confirmed, our study highlights the potential ofACTN3-ACEpolymorphisms in predicting elite endurance. Keywords:ACTN3;ACE; polymorphism; genotype; alleles; endurance;
associated with a 1.763-fold higher likelihood of possessing a superior endurance capacity than that seen in healthy controls (90% CI = 1.037–3.089). Our findings propose an association of combinedACTN3 RX+XXandACE IIgenotypes with enhanced endurance performance in elite Korean athletes. While causality remains to be confirmed, our study highlights the potential ofACTN3-ACEpolymorphisms in predicting elite endurance. Keywords:ACTN3;ACE; polymorphism; genotype; alleles; endurance; elite athletes; Koreans 1. Introduction Recent research has highlighted DNA polymorphisms contributing to prowess in certain types of sports [1]. Polymorphisms with a frequency of≥1% can be classified as genetic markers of power, muscle strength, and endurance [2]. According to studies by Ahmetov et al. [1] and Semenova et al. [3], the number of known markers positively correlated with specific abilities in elite athletes has increased from 11 to 41 between 2014 and 2023. Over 70% of the athletic ability in elite athletes is considered to have a genetic basis, with the rest being derived from the interactions of genetics with exogenous factors [4–6]. Endurance, which is the ability to repeatedly perform a physical movement under minimal load over a long duration, is determined by cardiovascular factors that affect oxygen supply to skeletal muscle components and shows considerable variation among individuals[7–11] . Endurance can thus be defined as a phenotype arising from athletic ability-related geno- types. Because it is a complex phenotype, thousands of DNA polymorphisms need to be considered, and a consistent, strong level of evidence is required to prove their contri- bution [1,12–15]. To date,ACTN3 R577XandACE I/Dhave shown stronger associations Genes2024,15, 1110.
Genes2024,15, 1110 2 of 14 with endurance exercise ability than other candidate genes and thus have been extensively researched [16–18]. TheACTN3gene, which codes forα-actinin-3, a structural protein that forms part of the cytoskeleton at the Z-line, providing spatial and structural stability, is located on chromosome 11 (11q13.2) [19,20]. TheR577Xnull polymorphism exhibits a C to T change in the 1747th base on exon 16, resulting in an early stop codon in place of arginine (Arg; R) at position 577 (R577X) [21–24]. Individuals homozygous for theXallele show a complete absence ofα-actinin-3, which is expressed only in type-II (fast twitch) muscle fibers, those responsible for rapid force production [25–29]. Deficiency or low expression of this protein in type-II fibers does not cause muscle disease [30]. Rather, as a compensation, structural proteinα-actinin-2, which can be found in all types of fatigue-tolerable muscle fibers (such as skeletal, cardiac, and smooth muscle fibers), is upregulated. Therefore,α-actinin-2 is compensated forα-actinin-3 on theXallele, which functions as fatigue-tolerable continuous contractile muscle fiber [28,29]. TheACTN3 R577Xpolymorphism, which can determineα-actinin-3 protein expres- sion, has direct functional effects that arise from the variation in structure and fiber pheno- type of skeletal muscles [26,31]. The deficit or low expression of theα-actinin-3 protein was found to improve endurance in Australian elite athletes [31]. Moreover, a study of profes- sional soccer players in Brazil found that athletes with theXXgenotype, which results in completeα-actinin-3 protein deficiency, exhibited greater aerobic ability than athletes with theRRgenotype possessing the full amount ofα-actinin-3 protein [32]. Further, a number of studies have indicated thatACTN3deficiency and low expression are significant predictors of endurance, affecting muscle fiber composition and metabolic efficiency [28,31,32]. TheACEgene, another endurance-associated candidate gene, codes for the angiotensin I-converting enzyme that catalyzes angiotensin I (C62H89N17O14) degradation to itsinacti- vated state and converts it to the physiologically active peptide angiotensin II (C50H71N13O12) within the renin-angiotensin system [33,34]. ACE activity is closely regulated by genotype- dependent expression as well as by endogenous inhibitory and secretory mechanisms [35]. Approximately 50% of the interindividual variance in ACE levels is determined by the ACEgene [36]. It is located on chromosome 17 (17q23.3) and is present
and converts it to the physiologically active peptide angiotensin II (C50H71N13O12) within the renin-angiotensin system [33,34]. ACE activity is closely regulated by genotype- dependent expression as well as by endogenous inhibitory and secretory mechanisms [35]. Approximately 50% of the interindividual variance in ACE levels is determined by the ACEgene [36]. It is located on chromosome 17 (17q23.3) and is present as one of two alleles—theI(insertion) allele andD(deletion) allele—depending on the insertion of a 287bp Alu repetitive sequence in intron 16 [37]. In many studies, theIIgenotype andIallele have been significantly associated with elite endurance athletes [38–41]. Compared with theDallele, theIallele shows approxi- mately one-third as much ACE activity [42,43]. According to Zhang et al., individuals with theIIgenotype, who carry theACE Iallele and thus have lower serum and tissue ACE ac- tivity, exhibited a higher proportion of type-I fibers and a lower proportion of type-II fibers in the left vastus lateralis compared to those with theDDgenotype [44]. Another study found that individuals carrying theACE Iallele (II+ID) who underwent endurance training had over three-fold the volume density of subsarcolemmal and intramyocellular lipids compared to those with theDDgenotype [45]. Consequently, individuals with lower ACE activity have relatively lower blood levels of angiotensin II [43,45], resulting in vasodilation and reduced blood pressure [46–50]. This was found to be advantageous for endurance by improving substrate delivery and skeletal muscle efficiency, thus preserving energy stores [14,41,51,52]. Conversely, research in African and Caucasian individuals revealed that, at 50% or 80% maximal oxygen consumption, the blood lactate concentration tended to be lower in those with theDDgenotype than in those with theIIgenotype, suggesting that theDDgenotype could be more efficient for endurance [21,53–56]. As of June 2023, approximately 110–120 studies have been conducted on endurance and theACTN3 R577XorACE I/Dpolymorphism, but the results have been inconsis- tent[17,57–62] . These discrepancies regarding gene polymorphisms and endurance appear to be due to differences in participant recruitment and group categorization between studies, in addition to differences between ethnicities affecting the recorded genetic charac- teristics[42,58,63–65] . As has been described in numerous review articles, ethnic diversity
results have been inconsis- tent[17,57–62] . These discrepancies regarding gene polymorphisms and endurance appear to be due to differences in participant recruitment and group categorization between studies, in addition to differences between ethnicities affecting the recorded genetic charac- teristics[42,58,63–65] . As has been described in numerous review articles, ethnic diversity
Genes2024,15, 1110 3 of 14 within study cohorts can result in considerable genetic background variation, which trans- lates into differences in gene expression and function across ethnic groups [3,21]. It is therefore inappropriate to indiscriminately extrapolate the significance of genetic character- istics across different ethnic groups. Recently, an increasing number of studies have not only focused on single gene polymorphisms for the above-described two genes but have also combined multiple additional genes to corroborate the genetic basis of endurance [42,66]. However, there is a paucity of correlation studies that assess the relationships between these genes and endurance, particularly among Koreans. Such studies would highlight the phenotypes when combined polymorphisms ofACTN3andACEare present. In the present study, we investigated the association of complex genotypes ofACTN3 R577XandACE I/Dpolymorphisms with elite pure endurance in Korean athletes. We hypothesized that individuals of Korean descent who possessed both theACTN3 XXand theACE IIgenotypes would exhibit superior levels of elite endurance. We used a strict criterion of “≥90% aerobic energy metabolism during sporting events”, which refers to the contribution of aerobically produced ATP utilized during sports [67]. The combination of genotypes with polymorphisms in multiple genes was defined as a complex genotype (CG) and classified as either endurance dominant (EDCG), neutral (ENCG), or recessive (ERCG), based on a previous study [68]. Using these classifications, we investigated the relationships ofACTN3 R577XandACE I/Dpolymorphisms with endurance in elite Korean athletes. 2. Materials and Methods 2.1. Participants In this case–control study, we recruited 713 male and 221 female elite athletes, with at least 5 years of experience representing their nation and placements within the top three of a national or international competition. Of the 934 athletes, 36 male and 9 female athletes in a discipline requiring “≥90% aerobic energy metabolism during sporting events” (marathon, 10,000 m run, 5000 m run, and 10~20 km race walk), as suggested by Pow- ers [67], were included in the elite pure endurance athletes (EPEA) group, while the other 889 participants were excluded. The control group consisted of 679 non-athlete Korean apparently healthy individuals (361 male and 318 female participants) who participated in medical checkups. All study
events” (marathon, 10,000 m run, 5000 m run, and 10~20 km race walk), as suggested by Pow- ers [67], were included in the elite pure endurance athletes (EPEA) group, while the other 889 participants were excluded. The control group consisted of 679 non-athlete Korean apparently healthy individuals (361 male and 318 female participants) who participated in medical checkups. All study participants were healthy and without genetic disease. The general characteristics of the two groups are shown in Table. This study was approved by the institutional review board of Soonchunhyang University (number: 202211-BC-124-01) according to the Declaration of Helsinki, and written consent was obtained from all subjects before participation. Table 1.Demographics of the Korean EPEA and control groups (n= 724). EPEA Controls (n= 45) ( n= 679) Sex Male 36 (80.0%) 361 (53.2%) Female 9 (20.0%) 318 (46.8%) Age 20.6 ±4.4 32.6 ±4.8 Sport events Marathon 15 (33.3%) 10,000 m run 10 (22.2%) 5000 m run 18 (40.0%) 10~20-kmW 2 (4.4%) Values are shown as numbers (%) and as means±standard deviations. Note: EPEA, elite pure endurance athletes group; kmW, race walk.
Genes2024,15, 1110 4 of 14 2.2. Blood Sampling and Genomic DNA Extraction The participants provided 3-cc blood samples after 12 h of fasting, and the samples were stored as whole blood in anticoagulant-coated ethylenediaminetetraacetic acid (EDTA) tubes in a refrigerator at 4 ◦ C. Genomic DNA was isolated using the Puregene ® Blood Kit (Cat. 158023; QIAGEN, Hilden, North Rhine-Westphalia, Germany). 2.3. Genotyping To analyze theACTN3 R577Xpolymorphism (rs1815739) in extracted gDNA, we used the previously validated MGB TaqMan ® SNP Genotyping Assay (Cat. 4351379, C_590093_1_; Applied Biosystems, Waltham, MA, USA), and to analyze theACE I/D polymorphism (rs1799752), we used three primers (Cat. 4304970; Applied Biosystems) and two probes (Cat. 4316034; Applied Biosystems). The base sequences used in this analysis are shown in Table69]. Table 2.Sequences used forACE I/Dpolymorphism analysis. Material Designation Sequence Primers ACE111 a CCC-ATC-CTT-TCT-CCC-ATT-TCT-C ACE112 b AGC-TGG-AAT-AAA-ATT-GGC-GAA-AC ACE113 a CCT-CCC-AAA-GTG-CTG-GGA-TTA Probes Iallele-specific c AGG-CGT-GAT-ACA-GTC-A Dallele-specific d TGC-TGC-CTA-TAC-AGT-CA Note: a , forward 5 ′ -3 ′ ; b , reverse 5 ′ -3 ′ ; c , forward VIC 5 ′ -3 ′ MGB; d , forward FAM 5 ′ -3 ′ MGB. The materials for gene amplification ofACTN3(MGB TaqMan ® SNP Genotyping Assay (20×), 10–20 ng of gDNA) andACE(10 pmol of each primer, 150 nMIallele specific probe, 75 nMDallele specific probe, and 10–50 ng of gDNA) were added to the TaqMan ® Genotyping Master Mix (2X) (Cat. 4371353; Applied Biosystems) and nuclease-free water to make a reaction mix with a final volume of 10µL. Target gene amplification was performed using the CFX Connect Real-Time PCR Detection System (Cat. 1855201; Bio-Rad, Hercules, CA, USA). The cycling profile consisted of two steps, with 10 min of enzyme activation at 95 ◦ C, followed by 40 cycles of denaturation for 15 s at 95 ◦ C and annealing and extension for 1 min at 60 ◦ C. The allelic discrimination plot for identifying genotypes was plotted automatically using CFX Maestro Software 2.3 for Windows PC (Cat. 12013758; Bio-Rad). 2.4. Statistical Analysis The frequency of each genotype and allele for theACTN3 R577XandACE I/Dpoly- morphisms was calculated. The Hardy–Weinberg equilibrium (HWE) in each
s at 95 ◦ C and annealing and extension for 1 min at 60 ◦ C. The allelic discrimination plot for identifying genotypes was plotted automatically using CFX Maestro Software 2.3 for Windows PC (Cat. 12013758; Bio-Rad). 2.4. Statistical Analysis The frequency of each genotype and allele for theACTN3 R577XandACE I/Dpoly- morphisms was calculated. The Hardy–Weinberg equilibrium (HWE) in each group was estimated using a chi-squared test (p< 0.05). We compared the frequencies of genotypes (2×3) and alleles (2×2) for theACTN3 R577XandACE I/Dpolymorphisms between the EPEA and control groups. ForACTN3 R577Xpolymorphism genotypes, we used the chi-squared test to compare their frequency (2×2) with anXallele, which are expected to be beneficial for endurance since they show phenotypical characteristics of type-I (slow twitch) muscle fibers owing to compensation byα-actinin-2 [31,70,71]. Additionally, to compare the differences in frequencies of complexACTN3-ACEpoly- morphisms between the groups, we categorized genotype combinations as EDCGs, ENCGs, or ERCGs (2×3), and used the chi-squared test. If chi-squared tests indicated significant differences between the groups for a given genotype, we performed multiple comparisons (2×2) and adjusted the resultingp-value using Bonferroni correction. The trend for genotype combinations that are beneficial for endurance (2×6) was analyzed using linear-by-linear association analysis. We also performed binary logistic regression analysis to obtain odds ratios (ORs) with a 90% confidence interval (CI) for the relative frequency of genotypes that are beneficial for pure elite endurance in the control group. The significance level for all comparisons wasp< 0.10 [72–74]. Statistical analyses were performed using SPSS (version 26.0; IBM Corp., Armonk, NY, USA).
Genes2024,15, 1110 5 of 14 For the post hoc power analysis, a generic F test was performed using the G*power 3.1.9.7 software program, and the parametersα= 0.1, numerator df = 45-1, and denominator df = 679-1 were entered in the section “post hoc: compute power test”. The results of the power analysis wereβ= 0.0981 and (1-β) = 0.9019. 3. Results 3.1. Distribution of ACTN3 R577X and ACE I/D Polymorphisms among the Non-Athlete Korean Population and EPEA In our study, the frequencies ofACTN3 R577XandACE I/Dpolymorphism genotypes and alleles in the EPEA and control groups were all shown via HWE to be representative of the genotype distribution in each group (p> 0.05). As shown in Figure, the frequencies of theACE Iallele andDallele in the EPEA group were 53.3% and 46.7%, respectively, while those in the control group were 57.5% and 42.5%, respectively. For the distribution of ACE I/D, the respective frequencies of theII,ID, andDDgenotypes were 35.6%, 35.6%, and 28.9% in the EPEA group versus 32.0%, 51.1%, and 16.9% in the control group, respectively. A significant difference in the distribution of the threeACE I/D(X 2 = 5.615, df = 2,p= 0.060) andID(X 2 = 4.081, df = 1,p= 0.090) genotypes was noted between the groups.ACTN3 R577X Rallele andXallele frequencies in the EPEA group were 47.8% and 52.2%, respec- tively, whileACE Iallele andDallele frequencies in the control group were 55.2% and 44.8%, respectively. Regarding the distribution ofACTN3 R577Xgenotypes, the respective frequencies ofRR,RX, andXXgenotypes were 15.6%, 64.4%, and 20.0% in the EPEA group versus 29.5%, 51.5%, and 19.0% in the control group, respectively. Despite no significant differences in the distribution of the threeACTN3 R577Xgenotypes between the groups (X 2 = 4.215, df = 2,p= 0.122), statistically significant differences were observed in the distributions ofRX+XXalleles, which are associated with skeletal muscle characteristics beneficial for endurance, as well as in the distribution of the genotype (RR) without theX allele (X 2 = 3.994, df = 1,p= 0.029).Genes 2024, 15, x FOR PEER REVIEW 6 of 15 Figure 1. Distribution of ACTN3 R577X and ACE I/D genotypes in elite Korean endurance athletes. Values are shown as percentages (%).
are associated with skeletal muscle characteristics beneficial for endurance, as well as in the distribution of the genotype (RR) without theX allele (X 2 = 3.994, df = 1,p= 0.029).Genes 2024, 15, x FOR PEER REVIEW 6 of 15 Figure 1. Distribution of ACTN3 R577X and ACE I/D genotypes in elite Korean endurance athletes. Values are shown as percentages (%). The number of cases is indicated above the bars. *, the mean ID genotype frequency in the EPEA group was significantly lower than that in the control group ( p < 0.10 by Bonferroni correction). †, the mean RX+XX genotype frequency in the EPEA group was significantly higher than that in the control group (p < 0.10 via chi-squared test). 3.2. Complex ACTN3-ACE Polymorphisms and Pure Elite Endurance in Koreans We divided complex ACTN3-ACE polymorphisms into EDCGs, ENCGs, and ERCGs, in order of the positive effects predicted for pure elite endurance, to construct groups of complex genotypes (Table 3). We observed significant differences in the distribution of the three complex genotype groups between the EPEA and control groups (Χ 2 = 8.460, df = 2, p = 0.014). We observed significant differences in distribution between the groups when using either the dominant complex genotypes or the recessive complex genotypes as a reference (EDCGs, Χ 2 = 5.835, df = 1, p = 0.039; ERCGs, Χ 2 = 5.641, df = 1, p = 0.021). Table 3. Complex genotype combinations of the ACTN3-ACE genes. Complex Genotypes EDCGs ENCGs ERCGs RX+XX/II+DD RX+XX/ID RR/II+DD RR/ID EPEA 23 * (51.1%) 21 (46.7%) 1 † (2.2%) Controls 227 (33.4%) 358 (52.7%) 94 (13.8%) Values are shown as numbers (%). Note: EPEA, elite pure endurance athletes group; EDCGs, endur- ance-dominant complex genotypes; ENCGs, endurance-neutral complex genotypes; ERCGs, endur- ance-recessive complex genotypes; *, the mean EDCG frequency in the EPEA group was signifi- cantly higher than that in the control group (p < 0.10 via Bonferroni correction); †, the mean ERCG frequency in the EPEA group was significantly lower than that in the control group (p < 0.10 via Bonferroni correction). Complex genotype distribution (2 × 3
ERCGs, endur- ance-recessive complex genotypes; *, the mean EDCG frequency in the EPEA group was signifi- cantly higher than that in the control group (p < 0.10 via Bonferroni correction); †, the mean ERCG frequency in the EPEA group was significantly lower than that in the control group (p < 0.10 via Bonferroni correction). Complex genotype distribution (2 × 3 contingency table), Χ 2 = 8.460, p < 0.10. EDCGs vs. remainder group (2 × 2 contingency table), Χ 2 = 5.835 [df = 1], p < 0.10 via Bonferroni correction. ENCGs vs. remainder group (2 × 2 contingency table), Χ 2 = 0.621 [df = 1], p > 0.10 via Figure 1.Distribution ofACTN3 R577XandACE I/Dgenotypes in elite Korean endurance athletes. Values are shown as percentages (%). The number of cases is indicated above the bars. *, the mean IDgenotype frequency in the EPEA group was significantly lower than that in the control group (p< 0.10by Bonferroni correction).†, the meanRX+XXgenotype frequency in the EPEA group was significantly higher than that in the control group (p< 0.10 via chi-squared test).
Description
This study explores the association of ACTN3 and ACE gene polymorphisms with elite endurance in Korean athletes.