Genetic Profile in Genes Associated with Cardiorespiratory Fitness in Elite Spanish Male Endurance Athletes

ground: most of the research concerning the in uence of genetics on endurance performance has been carried out by investigating target genes separately. However, endurance performance is a complex trait that can stem from the interaction of several genes. The objective of this study was to compare the frequencies of polymorphisms in target genes involving cardiorespiratory functioning in elite endurance athletes vs. non-athlete controls. Methods: genotypic frequencies were determined in 123 elite endurance athletes and in 122 non-athletes. Genotyping ofACE (rs4340),NOS3(rs2070744 and rs1799983),ADRA2a(rs1800544 and rs553668),ADRB2(rs1042713 and rs1042714), and BDKRB2 (rs5810761) was performed by polymerase chain reaction. The total genotype score (TGS: from 0 to 100 arbitrary units; a.u.) was calculated from the genotype score in each polymorphism. Results: the mean TGS in non-athletes (47.72 11.29 a.u.) was similar to elite endurance athletes (46.54 11.32 a.u.,p= 0.415). The distribution of TGS frequencies were also similar in non-athletes and elite endurance athletes (p= 0.333). There was no TGS cut-off point to discriminate being elite endurance athletes. Conclusions: the genetic pro le in

from the genotype score in each polymorphism. Results: the mean TGS in non-athletes (47.72 11.29 a.u.) was similar to elite endurance athletes (46.54 11.32 a.u.,p= 0.415). The distribution of TGS frequencies were also similar in non-athletes and elite endurance athletes (p= 0.333). There was no TGS cut-off point to discriminate being elite endurance athletes. Conclusions: the genetic pro le in the selected genes was similar in elite endurance athletes and in controls, suggesting that the combination of these genes does not determine endurance performance. Keywords:endurance; physical performance; sports performance; genotype score 1. Introduction Exercise performance is a complex trait resulting in different environmental factors, such as training, nutrition, social status, and gender. However, inherited features, such as genetics, also play a key role in the probability of becoming an elite athlete [1]. This is because genetics might impact muscle and cardiorespiratory function and adaptation to training stimuli, ultimately modifying exercise performance [2]. Recently, it has been shown that at least 120 polymorphisms in target genes are linked to the capacity of being an elite athlete by measuring genotypic frequencies in elite athletes and sedentary population. These studies have found genetic variants that are more prevalent in elite athletes in athlet- ics [3], soccer [4], triathlon [5], and other power-based sports disciplines [1]. However, only a few of these target genes have been directly associated with endurance performance [6,7]. Most of the research on the in uence of genetics on endurance performance has been carried out by investigating target genes separately. Nevertheless, the combined in uence of several genetic variants, each with a signi cant contribution, as well as the complex interaction of genetic variants, is likely the best approach to explain individual variations in endurance performance [8]. Several polymorphisms reportedly correlating to athlete performance have gained attention; however, inconsistent research design and varying sports make it dif cult to ascertain the relevance of these genes to the wider sporting population [9]. Previous inves- tigations have pointed out that the addition of the in uence of several polymorphisms by using a total genotype score (TGS), might predict the likelihood of becoming

correlating to athlete performance have gained attention; however, inconsistent research design and varying sports make it dif cult to ascertain the relevance of these genes to the wider sporting population [9]. Previous inves- tigations have pointed out that the addition of the in uence of several polymorphisms by using a total genotype score (TGS), might predict the likelihood of becoming an endurance Genes2021,12, 1230.

Genes2021,12, 1230 2 of 11 or power sports elite athlete [10,11]. Thus, the use of investigations that include several genes might increase the probability of explaining the in uence of genetics on the different traits associated with exercise performance [12]. Among the candidate genes associated with endurance performance, an insertion (I)/deletion (D) polymorphism in the gene that codi es the angiotensin I-converting en- zyme (ACE) (rs4340) has been widely studied. Speci cally, the insertion (I) rather than deletion (D) is associated with lower circulating angiotensin I-converting enzyme activ- ity and with higher endurance performance [13], although is not always the case [14]. Two polymorphisms have been associated with important phenotypes for endurance per- formance in the gene that codi es nitric oxide synthase 3 (NOS3). The T allele in the NOS3c.-786T/C polymorphism (rs2070744) has been related to both power and endurance exercise performance [15] due to enhanced ef ciency in the functioning of the athletes' cardiorespiratory systems during exercise [16]. However, the T allele in c.894G/T poly- morphism (rs1799983) is a genetic factor for hypertension [17]. Genotypic variations in the gene that codi es 2a-adrenoceptor (ADRA2A) c.-1291C/G (rs553668) and c.1780A/G (rs553668) have been associated with elite endurance athlete status [18], but the informa- tion about this gene is scarce and contradictory despite the key role of 2a-adrenoceptor in regulating neurotransmitter release from sympathetic nerves and regulating vascular adaptations to endurance training [19]. In the gene that codi es -2-adrenergic receptors (ADRB2), two polymorphisms (46A/G and 79C/G) promote positive aerobic phenotypes upregulating lipolysis during exercise. However, there is no demonstrable evidence of the predictive ability of this genotype for identifying potential elite athletes [20]. Lastly, the polymorphism 9/+9 in the gene of the bradykinin receptor B2 (BDKRB2) has been associated with endurance performance. The absence ( 9) of a 9 bp repeat sequence in exon 1 of theBDKRB2has been associated with the ef ciency of muscular contraction during running [21]. In addition, the 9/ 9 genotype was prevalent in elite triathletes compared with a control group [20]. The aim of our research was to compare the frequencies of the polymorphic variations

endurance performance. The absence ( 9) of a 9 bp repeat sequence in exon 1 of theBDKRB2has been associated with the ef ciency of muscular contraction during running [21]. In addition, the 9/ 9 genotype was prevalent in elite triathletes compared with a control group [20]. The aim of our research was to compare the frequencies of the polymorphic variations of these target genes involving cardiorespiratory functioning in elite endurance athletes vs. non-athlete controls. By adding the in uence of each gene through a total genotype score, we will try to predict the likelihood of becoming an endurance elite athlete by providing a genotype score cut-off point. 2. Materials and Methods 2.1. Design A prospective transversal study with case (elite endurance athletes) and controls. 2.2. Subjects We studied 123 elite endurance athletes and 122 non-athlete's subjects, all of them males. Non-athlete subjects and elite endurance athletes' three previous generations were of Caucasian descent. Elite endurance runners had a certi ed high level according to their times (<2 h 10 m in marathon or <1 h 3 min in 1 2 marathon, or <29 min in 10 k or<14 min in 5 k for runners) or because they had competed in one-day endurance competition or/and Grand Tours (Tour de France, Giro d'Italia and Vuelta a España for cyclists). All subjects involved in the study signed the informed consent. The study protocol was approved by the Committee of Institutional Ethics of University of Valladolid (protocol code UVa-21/2019) and complied with the Declaration of Helsinki for Human Research of 1974 (last modi ed in 2003). Participants' rights and con dentiality were protected during the whole experiment, and the genetic information was used only for the purposes included in this investigation. 2.3. Genetic Analyses We analysed the genetic variants at the Institute of Biology and Molecular Genetics (IBMG), University of Valladolid, Spain. Genomic DNA was obtained from ethylenedi-

Genes2021,12, 1230 3 of 11 aminetetraacetic acid (EDTA) anticoagulated blood samples according to standard phenol- chloroform procedures, followed by precipitation with ethanol. All DNA samples were then stored in the same conditions at 20 C until subsequent processes were performed. The samples were genotyped using Mastercycler ep gradient S Eppendorf ® Thermocycler (Eppendorf, Hamburg, Germany). 2.3.1. ACE Genotyping The I/D variant (rs4340) of theACEgene was studied by direct genotyping, using for- ward 5 0 -CTGGAGACCACTCCCATCCTTTCT-3 0 and reverse 5 0 -GATGTGGCCATCACATT CGGTCAGA-3 0 primers. The PCR mixture was denatured at 94 C for 10 min, amplifying in 35 cycles of 30 s at 94 C, 30 s at 58 C, and 1 min at 72 C, nal extension of 7 min at 72 C. When homozygous individuals were observed for the D (D/D) allele, we per- formed a second round of ampli cation to avoid mistyping produced by the D allele that prevents the existence of non-ampli ed allele I. In this second round was used forward 5 0 - TGGGACAGCGCCCGCCACTAC-3 0 and reverse 5 0 -TCGCCAGCCCTCCCATGCCCATAA- 3 0 primers. The PCR mixture was denatured at 94 C for 10 min, amplifying in 35 cycles of 30 s at 94 C, 30 s at 67 C, and 1 min at 72 C, nal extension of 10 min at 72 C. PCR products were separated through a 2% agarose gel. 2.3.2. NOS3 Genotyping TheNOS3c.-786T/C polymorphism (rs2070744) was genotyped using forward 5 0 - GAGGTCTCGAAATCACGAGG-3 0 and reverse 5 0 -ATACAAGAACTCCTGGATCC-3 0 primers. The PCR mixture was denatured at 95 C for 5 min, amplifying in 40 cycles of 30 s at 95 C , 30 s at 60 C, and 45 s at 72 C, nal extension of 7 min at 72 C, follow by a restriction using MspI enzyme (Thermo Fisher Scienti c, Waltham, MA, USA), separated through a 2% agarose gel. For theNOS3c.894G/T polymorphism (rs1799983), a forward 5 0 -AAGGCAGGAGACAAGTGGATG-3 0 and reverse 5 0 -CAGTCAATCCCTTTGGTGCT- 3 0 primers were used. The PCR mixture was denatured at 95 C for 5 min, amplifying in 30 cycles of 1 min

72 C, follow by a restriction using MspI enzyme (Thermo Fisher Scienti c, Waltham, MA, USA), separated through a 2% agarose gel. For theNOS3c.894G/T polymorphism (rs1799983), a forward 5 0 -AAGGCAGGAGACAAGTGGATG-3 0 and reverse 5 0 -CAGTCAATCCCTTTGGTGCT- 3 0 primers were used. The PCR mixture was denatured at 95 C for 5 min, amplifying in 30 cycles of 1 min at 94 C, 1 min at 56 C, and 1:30 min at 72 C, nal extension of 5 min at 72 C , followed by a restriction by MboI enzyme (Thermo Fisher Scienti c, USA), separated through a 2% agarose gel. 2.3.3. ADRA2A Genotyping TheADRA2Ac.-1291C/G polymorphism (rs553668) was genotyped with forward 5 0 -TCACACCGGAGGTTACTTCCCTCG-3 0 and reverse 5 0 -TCCGACGACAGCGCGAGT-3 0 primers. The PCR mixture was denatured at 94 C for 3 min, amplifying in 40 cycles of 30 s at 95 C, 45 s at 60 C, and 45 s at 72 C, nal extension of 10 min at 72 C. After restriction by DraI enzyme (Thermo Fisher Scienti c, Waltham, MA, USA), restric- tion products were separated through a 2% agarose gel. For theADRA2A1780A/G (rs553668) polymorphism the forward 5 0 -CAGAGCAGCACTGGACTAC-3 0 and reverse 5 0 -TGGAAGGCATCTCTCCCAAG-3 0 primers were used. The PCR mixture was denatured at 95 C for 5 min, amplifying in 40 cycles of 40 s at 95 C, 40 s at 60 C, and 40 s at 72 C, nal extension of 7 min at 72 C, followed by restriction by DraI enzyme (Thermo Fisher Scienti c, Waltham, MA, USA), separated through a 2% agarose gel. 2.3.4. ADRB2 Genotyping In theADRB2gene, we studied two polymorphisms; c.46A/G Arg16Gly (ADRB2R16G; rs1042713) and c.79C/G Gln27Glu (ADRB2Q27E; rs1042714). For both polymorphisms, the forward 5 0 -GCCTTCTTGCTGGCACCCCAT-3 0 and reverse 5 0 -CAGACGCTCGAACTTGG CCATG-3 0 primers were used. The PCR mixture was rst denatured at 94 C for 2 min, amplifying in 40 cycles of 40 s at 94 C, 40 s at 64 C, and 50 s at 72 C, nal extension of 7 minat 72 C. Was used restriction by NcoI (Thermo Fisher Scienti c, Waltham, MA,

reverse 5 0 -CAGACGCTCGAACTTGG CCATG-3 0 primers were used. The PCR mixture was rst denatured at 94 C for 2 min, amplifying in 40 cycles of 40 s at 94 C, 40 s at 64 C, and 50 s at 72 C, nal extension of 7 minat 72 C. Was used restriction by NcoI (Thermo Fisher Scienti c, Waltham, MA, USA) for ADRB2R16Gand by BseXI (BbvI) enzymes forADRB2Q27E(Thermo Fisher Scienti c, Waltham, MA, USA), separated through a 2% agarose gel.

Genes2021,12, 1230 4 of 11 2.3.5. BDKRB2 Genotyping The I/D +9 pb/ 9 pb variant (rs5810761) variant of theBDKRB2was genotyped with forward 5 0 -GCCCTTGAAAGATGAGCTG-3 0 and reverse 5 0 -AACTCCCCACGACCACAG- 3 0 primers. The PCR mixture and thermal-time pro le were rst denatured at 94 C for 5 min, amplifying in 40 cycles of 1 min at 94 C, 1 min at 53 C, and 1 min at 72 C, nal extension of 5 min at 72 C, separated through a 2% agarose gel. 2.4. TGS Determination The probability that an individual would possess the “optimal” genotype for each of the eight polymorphisms was calculated. We made a scale with the estimated probability of having a “perfect” genetic pro le, considering the number of polymorphisms included in this pro le [11]. We analysed the combined in uence of the eight polymorphisms studied, following the procedure of Williams and Folland [22]. A genotype score (GS) of two was assigned to the “optimal” or preferable endurance genotype, while a GS of 0 was assigned to the less optimal genotype [11] (Table). The GSs of all genotypes were added and the score was transformed to 0–100 arbitrary units (a.u.), namely TGS), as follows: TGS = (GSACE+ GSNOS3-786+ GSNOS3E298D+ GSADRA2A-1291+ GSADRA2A1780 + GSADRB2R16G+ GSADRB2Q27E+ GSBDKRB2) (100/16) (1) Table 1.Studied polymorphisms in elite endurance athletes and non-athletes. Symbol Gene Polymorphism dbSNP Genotype Score Elite Endurance Athletes Non- Athletes pValue ACE Angiotensin I-converting enzyme Alu 287bp (I/D) rs4340 2 = II 4.88% 10.66% <0.0011 = ID 28.46% 53.28% 0 = DD 66.67% 36.07% NOS3 Nitric Oxide Synthase 3c.-786T/C rs2070744 2 = TT 50.41% 31.97% 0.011 = TC 29.27% 44.26% 0 = CC 20.33% 23.77% NOS3 E298D Nitric Oxide Synthase 3c.894G/T rs1799983 2 = GG 43.09% 38.84% 0.1111 = GT 53.65% 51.24% 0 = TT 3.25% 9.91% ADRA2A -2a-adrenoceptor c.-1291C/Grs1800544 2 = CC 5.61% 2.46% 0.0161 = GC 25.23% 42.62% 0 = GG 69.16% 54.92% ADRA2A -2a-adrenoceptor c.1780A/G rs553668 2 = GG 76.42% 71.07% 0.2681 = GA 21.14% 22.31% 0 = AA 2.44% 6.61% ADRB2 R16G -2-adrenergic receptorc.46A/G rs1042713 2 = AA 8.94% 8.20% 0.9431 =

38.84% 0.1111 = GT 53.65% 51.24% 0 = TT 3.25% 9.91% ADRA2A -2a-adrenoceptor c.-1291C/Grs1800544 2 = CC 5.61% 2.46% 0.0161 = GC 25.23% 42.62% 0 = GG 69.16% 54.92% ADRA2A -2a-adrenoceptor c.1780A/G rs553668 2 = GG 76.42% 71.07% 0.2681 = GA 21.14% 22.31% 0 = AA 2.44% 6.61% ADRB2 R16G -2-adrenergic receptorc.46A/G rs1042713 2 = AA 8.94% 8.20% 0.9431 = GA 55.28% 54.10% 0 = GG 35.77% 37.70% ADRB2 Q27E -2-adrenergic receptorc.79C/G rs1042714 2 = CC 25.20% 17.21% 0.0031 = CG 60.98% 50.82% 0 = GG 13.82% 31.97% BDKRB2 Bradykinin Receptor B2 +9 pb/ 9 pb rs5810761 2 = 9/ 9 21.14% 24.59% 0.0841 = 9/+9 43.90% 53.28% 0 = +9/+9 34.96% 22.13% A TGS of 100 represents a “perfect” pro le and a TGS of 0 would be the “worst” pro le for endurance sports [22]. The TGSs' distribution between elite endurance athletes and non-athletes was assessed.

Genes2021,12, 1230 5 of 11 2.5. Statistical Analyses The statistical analysis was carried out using Statistical Package for the Social Sciences (SPSS), v.21.0 for Windows (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp). The Hardy–Weinberg equilibrium (HWE) was tested for each polymorphism using 2 tests. The probability of having an “optimal” endurance genotype for one to eight polymorphisms between endurance elite athletes and non-athletes was calculated using the 2 test with xed 0.05. The genotypic frequencies of the polymorphisms in target-selected genes were compared between elite endurance athletes and non-athletes, using a 2 test with xed 0.05. The ability of TGS to correctly distinguish potential elite endurance athletes from non-athletes (0 = non-athlete, 1 = elite endurance athlete) was assessed using receiver operating characteristic (ROC) curves, being calculated the area under the ROC curve (AUC) with con dence intervals of 95% (95%CI). A binary logistic regression model was used to study the relationship between TGS and the athletic status. 3. Results The individual genotype score for each of the SNPs used in this investigation are pre- sented in Figure. Elite endurance athletes had a higher genotype score than non-athletes in the NOS3 c.-786T/C polymorphism (p= 0.010) and in the ADRB2Q27E(p= 0.003). On the contrary, non-athletes had a higher genotype score than elite athletes in ACE (p< 0.001). There were no other between-group differences for ADRA2A variants, NOS3E298D, ADRB2R16Gnor for BDKRB2. However, the “optimal” genotype score for ADRA2A was higher in elite endurance athletes than non-athletes (Table; p= 0.016). Genotype frequen- cies for all polymorphisms were in HWE in both groups.Genes 2021, 12, x FOR PEER REVIEW 5 of 11 higher in elite endurance athletes than non-athletes (Table 1; p = 0.016). Genotype frequen- cies for all polymorphisms were in HWE in both groups. Figure 1. Individual genotype scores in elite endurance athletes and controls. Table 1. Studied polymorphisms in elite endurance athletes and non-athletes. Symbol Gene Polymorphism dbSNP Genotype Score Elite Endurance Athletes Non-Athletes p Value ACE Angiotensin I-con- verting enzyme Alu 287bp (I/D) rs4340 2 = II 4.88% 10.66% <0.001 1 =

cies for all polymorphisms were in HWE in both groups. Figure 1. Individual genotype scores in elite endurance athletes and controls. Table 1. Studied polymorphisms in elite endurance athletes and non-athletes. Symbol Gene Polymorphism dbSNP Genotype Score Elite Endurance Athletes Non-Athletes p Value ACE Angiotensin I-con- verting enzyme Alu 287bp (I/D) rs4340 2 = II 4.88% 10.66% <0.001 1 = ID 28.46% 53.28% 0 = DD 66.67% 36.07% NOS3 Nitric Oxide Syn- thase 3 c.-786T/C rs2070744 2 = TT 50.41% 31.97% 0.01 1 = TC 29.27% 44.26% 0 = CC 20.33% 23.77% NOS3E298D Nitric Oxide Syn- thase 3 c.894G/T rs1799983 2 = GG 43.09% 38.84% 0.111 1 = GT 53.65% 51.24% 0 = TT 3.25% 9.91% ADRA2A α-2a-adrenoceptor c.-1291C/G rs1800544 2 = CC 5.61% 2.46% 0.016 1 = GC 25.23% 42.62% 0 = GG 69.16% 54.92% ADRA2A α-2a-adrenoceptor c.1780A/G rs553668 2 = GG 76.42% 71.07% 0.268 1 = GA 21.14% 22.31% 0 = AA 2.44% 6.61% ADRB2R16G β-2-adrenergic re- ceptor c.46A/G rs1042713 2 = AA 8.94% 8.20% 0.943 1 = GA 55.28% 54.10% 0 = GG 35.77% 37.70% ADRB2Q27E β-2-adrenergic re- ceptor c.79C/G rs1042714 2 = CC 25.20% 17.21% 0.003 1 = CG 60.98% 50.82% 0 = GG 13.82% 31.97% BDKRB2 Bradykinin Recep- tor B2 +9 pb/−9 pb rs5810761 2 = −9/−9 21.14% 24.59% 0.084 1 = −9/+9 43.90% 53.28% 0 = +9/+9 34.96% 22.13% Figure 1.Individual genotype scores in elite endurance athletes and controls, *pValue < 0.01. When adding up the individual genotype scores, the mean value of the TGS in non- athletes (47.72 11.29 a.u., statistical kurtosis: 0.01 0.43 a.u.) was similar to elite endurance athletes (46.54 11.32 a.u., statistical kurtosis: 0.24 0.43 a.u.;p= 0.415). The distributions of TGS frequencies were also similar in non-athletes and elite endurance athletes (Figure; p= 0.775).

This research investigates genetic factors influencing endurance performance.