Pediatric Exercise Science, 2015, 27, 102-112 http://dx.doi.org/10.1123/pes.2014-0050 © 2015 Human Kinetics, Inc.
Physical-Capacity-Related Genetic Polymorphisms in Children with Cystic Fibrosis Thomas Yvert, Catalina Santiago, Elena Santana-Sosa, Zoraida Verde, Felix GómezGallego, Luis Lopez-Mojares, and Margarita Pérez Universidad Europea de Madrid
Nuria Garatachea University of Zaragoza
Alejandro Lucia Universidad Europea de Madrid In patients with cystic fibrosis (CF), physical capacity (PC) has been correlated with mortality risk. In turn, PC is dependent on genetic factors. This study examines several polymorphisms associated with PC and healthrelated phenotype traits (VO2peak, FEV1, FVC, PImax and muscular strength) in a group of children with CF (n = 66, primary purpose). The same analyses were also performed in a control group of healthy children (n = 113, secondary purpose). The polymorphisms determined were classified as muscle function polymorphisms (ACE rs1799752; AGT rs699; ACTN3 rs1815739; PTK2 rs7843014 and rs7460; MSTN rs1805086; TRHR rs7832552; NOS3 rs2070744) or energy metabolism polymorphisms (PPARGC1A rs8192678; NRF1 rs6949152; NRF2 rs12594956; TFAM rs1937; PPARD rs2267668; ACSL1 rs6552828). No significant polymorphism/phenotype correlations were detected in children with CF, with marginal associations being observed between NOS3 rs2070744 and VO2peak and FEV1, as well as between PPARGC1A rs8192678 and FEV1. Overall, similar findings were observed in the control group, i.e., no major associations. The PC-related polymorphisms examined seem to have no effects on the PC or health of children with CF. Keywords: SNP, exercise, disease, pediatrics
Background Cystic fibrosis (CF) is the most common life-limiting autosomal recessive genetic disorder to affect Caucasians, with an incidence estimated at 1 in 2,500 Caucasian newborns (10). CF is a multisystemic disease affecting several vital organs, particularly the lungs, pancreas, and digestive system. Its cause has been identified as a mutation in the gene cystic fibrosis transmembrane conductance regulator (CFTR). Despite several possible treatments, there is currently no curative treatment for CF. Yvert, Santiago, Gómez-Gallego, Pérez, and Lucia are with the School of Doctorate Studies and Research, and SantanaSosa and Verde the Dept. of Morphological and Biomedical Sciences, Lopez-Mojares the Faculty of Health Sciences of Sport and Physical Activity, Universidad Europea de Madrid, Madrid, Spain. Garatachea is with the Dept. of Physiotherapy and Nursing, University of Zaragoza, Zaragoza, Spain. Address author correspondence to Thomas Yvert at yvert.thomas.paul@ gmail.com.
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As part of therapy and rehabilitation programs in the CF management, physical activity (PA) and exercise training are generally recommended to improve lung function (66). In fact, several health and physical fitness indicators have been positively correlated with lung function, quality of life, and survival in these patients. These indicators include peak oxygen uptake (VO2peak) (49), forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), maximal inspiratory pressure (PImax) (13), and muscular strength (59). However, wide interindividual differences in the aforementioned indicators have been shown depending on PA level, training status and genetic factors (46,49). In the last 10 years, over 200 genes with polymorphisms related to physical fitness have been identified (6,51,64). Among the most well-studied of these polymorphisms are the following: angiotensin converting enzyme (ACE) gene I/D (rs1799752) (24,39,53); angiotensinogen (AGT) M235T (rs699) (25); α-actinin 3 (ACTN3) R577X (rs1815739) (16,39); protein-tyrosine kinase 2 (PTK2) A > C (rs7843014) and PTK2 T > A (rs7460) (14); myostatin
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(MSTN) K153R (rs1805086) (22); thyrotropin-releasing hormone receptor (TRHR) C > T (rs7832552) (35); nitric oxide synthase 3 (NOS3) C > T (rs2070744) (21,23); peroxisome proliferator-activated receptor-γ coactivator-1 α (PPARGC1A) G482S (rs8192678) (17,37); nuclear respiratory factor 1 (NRF1) A > G (rs6949152) (29) and nuclear respiratory factor 2 (NRF2) A > C (rs12594956) (15,18); mitochondrial transcription factor-A (TFAM) S12T (rs1937) (2); peroxisome proliferator-activated receptor-δ (PPARD) A > G (rs2267668) (40) and acylCoA synthase long-chain-family-member-1 (ACSL1) G > A (rs6552828) (5). Most findings come from case:control designs (some of which have included several geographically/ethnically independent cohorts, e.g.; 19), and fewer evidence arises from genome wide scan association studies, e.g., for polymorphisms ACSL1 rs6552828 (5) and TRHR rs7832552 (35). Thus, owing to the important role of the abovementioned genes and polymorphisms in several body functions and systems, we hypothesized that some of them would be also associated with physical fitness phenotypes in children with CF. In fact, we previously showed that some of the abovementioned genetic polymorphisms can influence the physical fitness and clinical severity of patients with another genetic disorder, McArdle disease: the D-allele or DD genotype of the ACE I/D polymorphism and the variant R-allele of the MSTN K153R variation are associated with lower VO2peak (26,28) or greater clinical severity (28,55), while carriage of the variant ACTN3 577X-allele would play a favorable role in exercise capacity (38). Genetic association studies are performed to determine whether a genetic variant is associated with a trait, so the purpose of this study was to determine genotype frequencies for these polymorphisms in children with CF and to examine their association with physical fitness and health-related traits as VO2peak, FVC, FEV1, PImax and muscular strength. As a secondary study purpose, the same analyses were replicated in a group of age and sex-matched healthy controls.
Methods Subjects This gene candidate association study was conducted in 66 children with CF and 113 healthy children. Children aged 4–17 years were invited to participate if they had been diagnosed with CF by means of the classic sweat test (until identification of pathogenic CFTR mutations) and treated at the Hospital Niño Jesús or the Hospital La Paz in Madrid, Spain. All the children recruited had mild (FEV1 ≥ 70% of predicted), moderate (FEV1 = 60–69% of predicted) or moderately to severe CF (FEV1 = 50–59% of predicted) (50), and their clinical condition was stable. Exclusion criteria were: (i) had severe lung deterioration (defined as an FEV1 below 50% of the expected value), (ii) had been hospitalized within the previous 3 months, or (iii) had Burkholderia cepacia infection. The healthy
children recruited were 4–14 years of age with no chronic disease. Each child and one of his/her parents or caregivers provided written informed consent before the study onset. The study protocol adhered to the tenets of the Declaration of Helsinki, and received institutional review board approval from the Hospital Niño Jesús in Madrid (Approval number 006–09).
Procedures Each child received 3 familiarization sessions, during which they learned to walk on treadmill during 5–10 min at 2.5 kph, and learned the correct technique of the strength movements with 3 × 15 repetitions of each movement at minimum weight (2.25 kg). Afterward, the children have performed all the tests on one day, first with the graded treadmill test and then, after sufficient recovery time, with the 5RM test of each movement. In the children with CF, cardio-respiratory fitness was assessed in the exercise physiology laboratory of the Hospital Niño Jesús. Tests in the control children were performed in the exercise physiology laboratory of the Universidad Europea de Madrid (Spain) using the same equipment.
Phenotype Assessment Anthropometry. Body weight was measured using a digital balance (Seca, Madrid, Spain) to the nearest 0.1 kg (kg), with the child wearing no shoes. Height, without shoes, was measured to the nearest 0.1 cm using a stadiometer (Seca, Madrid, Spain) with the child standing with heels and head against the wall. Body mass index was calculated by dividing body weight (kg) by height in meters (m2) squared (kg.m-2). Triceps, abdominal, supra-iliac, and thigh skin-folds were measured using a caliper (Holtan Crymych, United Kingdom). Body fat percentage was estimated using the equation described by Jackson and Pollock (52). Lung-Pancreatic Status. Lung-pancreatic status was established using the classification scheme of Cleveland et al. (9) in which the CFTR class of mutation is used to define the groups S (severe disease) and M (mild disease). Within group S, the following subgroups were defined: A, patients with two class I alleles; B, those with a class I allele and class II or III allele; or C, those with a class II allele and class II or III allele. Group M was comprised of patients with at least one class IV–VI allele. Genotyping for CFTR gene mutations was performed using the kits Inno-lipa CFTR19 and Inno-lipa CFTR17+update from Innogenetics (Innogenetics NV, BTW BE 0427.550.660, RPR Gent, Technologiepark 6, B-9052 Gent, Belgium). Pulmonary Function. Pulmonary function was
determined through a spirometry protocol designed by the Spanish Society of Pneumology and Thoracic Surgery (SEPAR) (57), according to which FVC and FEV1 (L) are expressed as percentages of expected reference
104 Yvert et al.
values (27). A portable spirometer (CareFusion 232 Ltd., Chatham Maritime, Kent, UK) was used to determine PImax (mmHg) at residual volume in accordance with established standards (3) recorded as the best result of three attempts performed at intervals of at least 1 min.
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Cardiorespiratory Fitness. VO2peak (ml·kg-1·min-1) was
determined by open-circuit spirometry using pediatric facemasks during a graded treadmill test (Technogym Run Race 1400HC; Gambettola, Italy). Gas exchange was measured using a breath-by-breath system (Vmax 29c; Sensormedics; CA, USA). The test was performed after familiarization sessions; treadmill speed began at 1.5 km·h-1 (for children .0036) for any of the phenotypes we studied (VO2peak, FEV1, FVC, PImax, muscle strength), except for the following marginally significant correlations (Figure 2): between NOS3 rs2070744 T allele and top decile children according to the VO2peak and FEV1 data (p = .002 in both associations); and between PPARGC1A rs8192678 SS genotype and top decile children according to the FEV1 data (p = .003). Overall, similar findings were obtained in the healthy children group, where the only association found was for the NOS3 rs2070744 polymorphism and VO2peak, where the mean value of this variable was higher in CC (42.3 ± 6.8 ml·kg-1·min-1) compared with CT children (35.4 ± 8.8 ml·kg-1·min-1, p = .002), with no significant difference for the comparison with the TT genotype (37.9 ± 7.2 ml·kg-1·min-1, p = .079).
Discussion This study was designed to address the potential influence of PC-related genes on PC phenotypes in children with CF. Our results suggest that the genetic polymorphisms
Table 1 Main Demographic Data (Mean ± SD) Children with CF (Girls = 31 (47%), Boys = 35) n
Mean ± SD
Age (years)
66
9.0 ± 4.0
Height (cm)
66
Body weight (kg)
66
(kg·m-2)
66
BMI
Healthy Children (Girls = 61 (54%), Boys= 52) n
Mean ± SD
Age (years)
113
9.0 ± 2.5
134.1 ± 20.0
Height (cm)
113
139.4 ± 15.9
32.5 ± 13.7
Body weight (kg)
113
36.6 ± 12.2
17.2 ± 2.8
(kg·m-2)
113
18.3 ± 2.9
111
77.8 ± 7.0
BMI
Obese: 4.5%
Obese: 13.3%
Underweight: 9.1%
Underweight: 0.9%
Lean mass (%)
59
80.3 ± 6.5
Lean mass (%)
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Note. BMI = body mass index, CF = cystic fibrosis.
Table 2 Cardiorespiratory and Muscular Fitness and Pulmonary Function (Mean ± SD) in the Two Study Groups Children with CF
Healthy Children n
Mean ± SD
n
Mean ± SD
VO2peak (ml·kg-1·min-1)
66
36.0 ± 7.3
112
38.0 ± 7.4
HRpeak (beats·min-1)
66
187 ± 6
112
193 ± 8
FEV1 (%)
62
85.3 ± 28.1
24
108.9 ± 17.3
FVC (%)
62
86.1 ± 32.0
22
109.9 ± 18.0
PImax (mmHg)
62
63.9 ± 28.2
103
69.4 ± 25.6
Seated lateral row/kg (kg)
66
0.9 ± 0.2
111
0.9 ± 0.2
Seated bench press/kg (kg)
66
0.5 ± 0.3
111
0.9 ± 0.2
Seated leg press/kg (kg)
66
1.6 ± 0.5
113
3.2 ± 0.8
Overall strength/kg (kg)
66
3.5 ± 0.8
111
4.6 ± 0.9
Cardiorespiratory fitness
Pulmonary function
Muscular fitness
Note. Strength values are expressed in relation to the total body weight of each child. CF = cystic fibrosis, FVC = forced vital capacity, FEV1 = forced expiratory volume in 1 s; HRpeak = peak heart rate, VO2peak = peak oxygen uptake, PImax = maximum inspiratory pressure.
Table 3 Available Data of CFTR Mutation Class (Percentages) and Type According to Parental Contribution and Lung-Pancreatic Status in the Children with CF (All Diagnosed with the Classic Sweat Test) CFTR Mutation
Paternal % (n = 49)
Maternal % (n = 30)
Lung-pancreatic status
Percentage (n = 31)
Class 1
8.2 (G542×)
13.3 (R1162×; G542×)
Severe A
0.0
Class 2
87.8 (ΔF508; N1303K)
63.3 (ΔF508; N1303K)
Severe B
16.1
Class 3
0.0
3.3 (S549R)
Severe C
58.1
Class 4
4.1 (G85E; L206W)
13.3 (R334W; G85E)
Mild
25.8
Class 5
0.0
6.7 (3839+10Kb C > T)
Class 6
0.0
0.0
105
106
Figure 1 — Genotype frequencies (%) for the polymorphisms examined in children with cystic fibrosis (CF) (A) (n = 66) and in healthy children (B) (n = 113).
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Figure 2 — Genotype/allele frequencies for those polymorphisms showing a significant association for a given outcome variable (e.g., VO2peak) in the CF children, that is, showing a significant difference in genotype/allele frequencies when comparing the children in top or lowest decile vs. the rest of children, respectively. The following marginally significant associations were detected: between the NOS3 rs2070744 T allele and the top decile according to VO2peak (A) or FEV1 (B) (n = 65, * and † p = .002); and between the PGC-1α rs8192678 SS genotype (C) and the top decile according to FEV1 (‡ p = .003).
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108 Yvert et al.
involved in enhancing PC do not have a major effect on such phenotypes in these patients. Our data are however limited by the small sample size of our cohorts, which reduces the statistical power and generalizability of our findings. Further research with larger samples (ideally from multicenter cohorts of different ethnic/geographic origins) is needed in the field of genetics and PC related traits (20). Future studies might also corroborate the results of the current study in CF children showing more severe disease manifestations. Indeed, the CF children assessed here had a stable clinical condition and no severe lung deterioration, as reflected by their mean VO2peak values (36.0 ± 7.3 mL·kg-1·min-1), which were comparable to those of the healthy children (38.0± 7.4 mL·kg-1·min-1). We detected no significant relationship between polymorphisms of the genes associated with strength, muscle mass or muscle fiber contractile function (ECA, AGT, ACTN3, PTK2, MSTN, NOS3 and TRHR) and PC and health indicators in the CF children. Similar findings were obtained in the healthy children. However, marginal correlations were observed for polymorphisms in the genes NOS3 and PPARGC1A. A significant correlation was detected between the T allele of the NOS3 C > T (rs2070744) polymorphism and the children with CF belonging to the 10% upper decile for the data of VO2peak and FEV1. This could mean that this polymorphism in the gene (NOS3) encoding endothelial nitric oxide (NO) synthase (eNOS) is positively associated with health status and survival in children with CF. As far as we are aware, no such link has been reported in healthy children or those with CF. The eNOS is responsible for the generation of NO in blood vessels, where this molecule plays an important vasodilatory and regulatory role. It is also involved in muscle glucose uptake during exercise and in modulating blood flow and oxygen consumption in working muscles (43). In contrast with other linked polymorphisms (-922A/G and -1468T/A) that are not associated with changes in gene transcription, the T→C mutation of the NOS3 rs2070744 polymorphism results in significantly reduced gene promoter activity and reduced endothelial NO synthesis (47). In turn, the T-allele would result in higher NO synthesis There is strong rationale in postulating that higher endothelial NO production could lead to improved muscle hyperemia and oxygen supply to working fibers, and thus to higher VO2peak values. Several studies in isolated hindlimb and diaphragm muscle in dogs and rats, have reported decreased muscle VO2 during high-intensity evoked contractions with eNOS inhibition (4,30,31,33,56,62,63), although these findings have not been corroborated in human research using positron emission tomography (30). Higher plasma levels of nitrite (the main oxidation product of NO in plasma, that sensitively reflects changes in eNOS activity and serves as an endocrine NO donor), are positively associated with performance in high-intensive endurance exercise (12) or stress testing until exhaustion in nonathletes (54), as well as with exercise capacity in highly trained athletes (60). The NO can also be produced from dietary nitrate, and nitrate supplementation has been shown to improve arterial and muscle oxygenation during hypoxic exercise (42).
No correlation was observed between polymorphisms of the ACE and AGT genes, involved in angiotensin II production (a powerful vasoconstrictor and muscular growth factor), and indicators of PC and health in the two groups of children. A relationship has been reported between the ACE I/D polymorphism and the load-capacity ratio of the inspiratory muscles in children (11) and disease severity in children with CF (41). Despite the lack of correlation observed here between these polymorphisms and PC, there have been numerous reports of such links although these have been inconsistent. Ahmetov et al. related the D allele of the ACE I/D polymorphism to long jump ability in 219 children (1). In contrast, Moran et al. described an association between the I allele of this polymorphism and grip strength and high jump capacity in 484 teenage girls (45). The literature lacks information on the AGT M235T polymorphism and PC in children. In our study, allele frequencies for this polymorphism in the children with CF were inconsistent with HWE and were, therefore, excluded from the study. No significant correlations for the ACTN3 R577X polymorphism were observed in the two groups of children. α-actinin3 contributes to the contractile superstructure of type II muscle fibers, mainly by helping to anchor actin microfilaments in Z discs (65). Few studies have analyzed the effect of this polymorphism on the PC of children. Ahmetov et al. noted a positive association between the R allele of the ACTN3 R577X polymorphism (only in combination with other PPARA or ACE polymorphisms) and the grip strength and long jump capacity of 219 children (1). Chiu et al. positively related the RR genotype of the ACTN3 R577X polymorphism (also only in combination with other ACE and PPARD polymorphisms) and grip strength capacity in 170 adolescent girls (7). However, as in the current study, no association has been reported between this polymorphism alone and the PC of healthy children or children with CF. We detected no relationship between MSTN and TRHR gene polymorphisms and factors associated with health and PC in our two study groups. Myostatin is a protein secreted by muscle cells that inhibits their own growth, acting as a limiting factor in muscle development (44). The role of myostatin was discovered through the use of transgenic knockout mice for this gene (44). These mice showed up to double the muscle mass and lower fat tissue than control wild-type mice. The lack of an association with the MSTN K153R polymorphism noted in our cohort may be due to the low presence of the mutant R allele in Caucasians, with estimates running at 2.0% (48). The TRH hormone (thyrotropin-releasing hormone) exerts its effect by binding to its receptor TRHR activating pathways whose ultimate goal is the liberation of thyroxine, an important hormone for skeletal muscle development (34). Liu et al. (35) reported a link between lean body mass and the TRHR C > T (rs7832552) polymorphism. However, no study has addressed the relationship between MSTN K153R or TRHR C > T (rs7832552) polymorphisms and PC in children. In effect, our findings indicate no such effects of these polymorphisms.
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Genes and Cystic Fibrosis 109
In eukaryotic cells, mitochondria play a crucial role in energy production. Mitochondria are responsible for most of the useful energy derived from the degradation of carbohydrates and fatty acids, which are converted to adenosine-triphosphate (ATP) through oxidative phosphorylation, thus playing an important role in exercise. New mitochondria in a cell are formed via a process known as mitochondrial biogenesis, which is activated by several different signals in response to extracellular stimuli such as muscular exercise, exposure to cold or oxidative stress. Mitochondrial biogenesis requires the expression of a large number of genes among which we find PPARGC1A, PPARD, NRF1, NRF2, and TFAM (58). The ACSL1 gene plays a role in fatty acid metabolism (36). We observed a marginally significant relationship between having the SS genotype of the PPARGC1A G482S polymorphism and being a child with CF belonging to the top decile according to FEV1 data. To the best of our knowledge, no investigation has explored the effect of this polymorphism on the PC of children. This discrepancy determines a need for further work designed to elucidate the mechanisms involved in a possible positive effect of this allele on such an important factor as respiratory capacity in children with CF. No correlation was observed here between PPARD, ACSL1, NRF1, NRF2, or TFAM gene polymorphisms and factors associated with PC and health in the two groups examined (the NRF1 rs6949152 polymorphism in children with CF did not meet HWE and was excluded from the study). No prior study has attempted to link these polymorphisms with child PC, thus we have no data with which to compare our results. Phenotypic traits of physical capacity are determined by a wide range of variables, both genetic and environmental, such as exercise, nutrition, rest, psychology, advances in technology and equipment, among others. According to our data, we may state that the polymorphisms analyzed, despite being candidates for significantly influencing human physical performance, have no effect on physical capacity and performance in children with CF, with the exception of a few marginal cases for which further research is needed to draw any firm conclusions. Acknowledgments This study was supported by a Grant from Fondo de Investigaciones Sanitarias (FIS, grant # PS09/00194).
References 1. Ahmetov II, Gavrilov DN, Astratenkova IV, et al. The association of ACE, ACTN3 and PPARA gene variants with strength phenotypes in middle school-age children. J Physiol Sci. 2013; 63(1):79–85. PubMed doi:10.1007/ s12576-012-0233-8 2. Akhmetov II, Popov DV, Missina SS, Vinogradova OL, Rogozkin VA. Association of the mitochondrial transcription factor (TFAM) gene polymorphism with
physical performance of athletes. Fiziol Cheloveka. 2010; 36(2):121–125. PubMed 3. American Thoracic Society/European Respiratory Society. ATS/ERS Statement on respiratory muscle testing. Am J Respir Crit Care Med. 2002; 166(4):518–624. PubMed doi:10.1164/rccm.166.4.518 4. Baker DJ, Krause DJ, Howlett RA, Hepple RT. Nitric oxide synthase inhibition reduces O2 cost of force development and spares high-energy phosphates following contractions in pump-perfused rat hindlimb muscles. Exp Physiol. 2006; 91(3):581–589. PubMed doi:10.1113/expphysiol.2005.032698 5. Bouchard C, Sarzynski MA, Rice TK, et al. Genomic predictors of the maximal O2 uptake response to standardized exercise training programs. J Appl Physiol. (1985) 2011;110(5):1160-70. 6. Bray MS, Hagberg JM, Perusse L, et al. The human gene map for performance and health-related fitness phenotypes: the 2006-2007 update. Med Sci Sports Exerc. 2009; 41(1):35–73. PubMed doi:10.1249/ MSS.0b013e3181844179 7. Chiu LL, Chen TW, Hsieh SS, Hsieh LL. ACE I/D, ACTN3 R577X, PPARD T294C and PPARGC1A Gly482Ser polymorphisms and physical fitness in Taiwanese late adolescent girls. J Physiol Sci. 2012; 62(2):115–121. PubMed doi:10.1007/s12576-011-0189-0 8. Chomczynski P, Sacchi N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc. 2006; 1(2):581–585. PubMed doi:10.1038/nprot.2006.83 9. Cleveland RH, Zurakowski D, Slattery D, Colin AA. Cystic fibrosis genotype and assessing rates of decline in pulmonary status. Radiology. 2009; 253(3):813–821. PubMed doi:10.1148/radiol.2533090418 10. Cystic Fibrosis Foundation. Cystic Fibrosis Foundation Patient Registry 2007 Annual data report. Bethesda, Maryland: Cystic Fibrosis Foundation; 2008. 11. Dimitriou G, Papakonstantinou D, Stavrou EF, et al. Angiotensin-converting enzyme gene polymorphism and respiratory muscle function in infants. Pediatr Pulmonol. 2010; 45(12):1233–1239. PubMed doi:10.1002/ ppul.21316 12. Dreissigacker U, Wendt M, Wittke T, et al. Positive correlation between plasma nitrite and performance during high-intensive exercise but not oxidative stress in healthy men. Nitric Oxide. 2010; 23(2):128–135. PubMed doi:10.1016/j.niox.2010.05.003 13. Enright S, Chatham K, Ionescu AA, Unnithan VB, Shale DJ. Inspiratory muscle training improves lung function and exercise capacity in adults with cystic fibrosis. Chest. 2004; 126(2):405–411. PubMed doi:10.1378/chest.126.2.405 14. Erskine RM, Williams AG, Jones DA, Stewart CE, Do Degens H. PTK2 gene polymorphisms contribute to the interindividual variability in muscle strength and the response to resistance training? A preliminary report. J Appl Physiol. 2012; 112(8):1329–1334. PubMed doi:10.1152/japplphysiol.01137.2011 15. Eynon N, Alves AJ, Sagiv M, Yamin C, Sagiv M, Meckel Y. Interaction between SNPs in the NRF2 gene and
Downloaded by Teachers College Library on 09/16/16, Volume 27, Article Number 1
110 Yvert et al.
elite endurance performance. Physiol Genomics. 2010; 41(1):78–81. PubMed doi:10.1152/physiolgenomics.00199.2009 16. Eynon N, Hanson ED, Lucia A, et al. Genes for elite power and sprint performance: ACTN3 leads the way. Sports Med. 2013; 43(9):803–817. PubMed doi:10.1007/ s40279-013-0059-4 17. Eynon N, Meckel Y, Sagiv M, et al. Do PPARGC1A and PPARalpha polymorphisms influence sprint or endurance phenotypes? Scand J Med Sci Sports. 2010; 20(1):e145– e150. PubMed doi:10.1111/j.1600-0838.2009.00930.x 18. Eynon N, Ruiz JR, Bishop DJ, et al. The rs12594956 polymorphism in the NRF-2 gene is associated with top-level Spanish athlete’s performance status. J Sci Med Sport. 2013; 16(2):135–139. PubMed doi:10.1016/j. jsams.2012.05.004 19. Eynon N, Ruiz JR, Femia P, et al. The ACTN3 R577X polymorphism across three groups of elite male European athletes. PLoS ONE. 2012; 7(8):e43132. PubMed doi:10.1371/journal.pone.0043132 20. Eynon N, Ruiz JR, Oliveira J, Duarte JA, Birk R, Lucia A. Genes and elite athletes: a roadmap for future research. J Physiol. 2011; 589(Pt 13):3063–3070. PubMed doi:10.1113/jphysiol.2011.207035 21. Eynon N, Ruiz JR, Yvert T, et al. The C allele in NOS3 -786 T/C polymorphism is associated with elite soccer player’s status. Int J Sports Med. 2012; 33(7):521–524. PubMed doi:10.1055/s-0032-1306337 22. Ferrell RE, Conte V, Lawrence EC, Roth SM, Hagberg JM, Hurley BF. Frequent sequence variation in the human myostatin (GDF8) gene as a marker for analysis of musclerelated phenotypes. Genomics. 1999; 62(2):203–207. PubMed doi:10.1006/geno.1999.5984 23. Gomez-Gallego F, Ruiz JR, Buxens A, et al. The -786 T/C polymorphism of the NOS3 gene is associated with elite performance in power sports. Eur J Appl Physiol. 2009; 107(5):565–569. PubMed doi:10.1007/s00421-0091166-7 24. Gomez-Gallego F, Santiago C, Gonzalez-Freire M, et al. Endurance performance: genes or gene combinations? Int J Sports Med. 2009; 30(1):66–72. PubMed doi:10.1055/s-2008-1038677 25. Gomez-Gallego F, Santiago C, Gonzalez-Freire M, et al. The C allele of the AGT Met235Thr polymorphism is associated with power sports performance. Appl Physiol Nutr Metab. 2009; 34(6):1108–1111. PubMed doi:10.1139/ H09-108 26. Gomez-Gallego F, Santiago C, Moran M, et al. The I allele of the ACE gene is associated with improved exercise capacity in women with McArdle disease. Br J Sports Med. 2008; 42(2):134–140. PubMed doi:10.1136/ bjsm.2007.038992 27. Gonzalez Barcala FJ, Cadarso Suarez C, et al. Lung function reference values in children and adolescents aged 6 to 18 years in Galicia. Arch Bronconeumol. 2008; 44(6):295– 302. PubMed doi:10.1016/S0300-2896(08)70436-8 28. Gonzalez-Freire M, Santiago C, Gomez-Gallego F, et al. Does the K153R variant of the myostatin gene influence the clinical presentation of women with McArdle disease?
Neuromuscul Disord. 2009; 19(3):220–222. PubMed doi:10.1016/j.nmd.2009.01.001 29. He Z, Hu Y, Feng L, et al. NRF-1 genotypes and endurance exercise capacity in young Chinese men. Br J Sports Med. 2008; 42(5):361–366. PubMed doi:10.1136/ bjsm.2007.042945 30. Heinonen I, Saltin B, Kemppainen J, et al. Skeletal muscle blood ñow and oxygen uptake at rest and during exercise in humans: a pet study with nitric oxide and cyclooxygenase inhibition. Am J Physiol Heart Circ Physiol. 2011; 300:H1510–H1517. PubMed 31. King-VanVlack CE, Mewburn JD, Chapler CK, MacDonald PH. Endothelial modulation of skeletal muscle blood flow and [UNKNOWN ENTITY &Vdot;]o2 during low- and high-intensity contractions. J Appl Physiol. 2002; 92(2):461–468. PubMed 32. Kraemer W, Fry A. Strength testing development and evaluation of methodology. Physiological Assessment of Human Fitness. Champaign (IL): Human Kinetics ed.; 1995. 33. Krause DJ, Hagen JL, Kindig CA, Hepple RT. Nitric oxide synthase inhibition reduces the O2 cost of force development in rat hindlimb muscles pump perfused at matched convective O2 delivery. Exp Physiol. 2005; 90(6):889–900. PubMed doi:10.1113/expphysiol.2005.031567 34. Larsson L, Li X, Teresi A, Salviati G. Effects of thyroid hormone on fast- and slow-twitch skeletal muscles in young and old rats. J Physiol. 1994; 481(Pt 1):149–161. PubMed doi:10.1113/jphysiol.1994.sp020426 35. Liu XG, Tan LJ, Lei SF, et al. Genome-wide association and replication studies identified TRHR as an important gene for lean body mass. Am J Hum Genet. 2009; 84(3):418–423. PubMed doi:10.1016/j.ajhg.2009.02.004 36. Lobo S, Wiczer BM, Bernlohr DA. Functional analysis of long-chain acyl-CoA synthetase 1 in 3T3-L1 adipocytes. J Biol Chem. 2009; 284(27):18347–18356. PubMed doi:10.1074/jbc.M109.017244 37. Lucia A, Gomez-Gallego F, Barroso I, et al. PPARGC1A genotype (Gly482Ser) predicts exceptional endurance capacity in European men. J Appl Physiol. 2005; 99(1):344– 348. PubMed doi:10.1152/japplphysiol.00037.2005 38. Lucia A, Gomez-Gallego F, Santiago C, et al. The 577X allele of the ACTN3 gene is associated with improved exercise capacity in women with McArdle’s disease. Neuromuscul Disord. 2007; 17(8):603–610. PubMed doi:10.1016/j.nmd.2007.04.006 39. Ma F, Yang Y, Li X, et al. The association of sport performance with ACE and ACTN3 genetic polymorphisms: a systematic review and meta-analysis. PLoS ONE. 2013; 8(1):e54685. PubMed doi:10.1371/journal.pone.0054685 40. Maciejewska-Karlowska A, Hanson ED, Sawczuk M, Cieszczyk P, Eynon N. Genomic haplotype within the Peroxisome Proliferator-Activated Receptor Delta (PPARD) gene is associated with elite athletic status. Scand J Med Sci Sports. 2014; 24(3):e148–e155. PubMed doi:10.1111/ sms.12126 41. Marson FA, Bertuzzo CS, Hortencio TD, Ribeiro JD, Bonadia LC, Ribeiro AF. The ACE gene D/I polymorphism as a modulator of severity of cystic fibrosis. BMC Pulm Med. 2012; 12:41. PubMed doi:10.1186/1471-2466-12-41
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42. Masschelein E, Van Thienen R, Wang X, et al. Dietary nitrate improves muscle but not cerebral oxygenation status during exercise in hypoxia. J Appl Physiol. 2012; 113:736– 745. PubMed doi:10.1152/japplphysiol.01253.2011 43. McConell GK, Rattigan S, Lee-Young RS, Wadley GD, Merry TL. Skeletal muscle nitric oxide signaling and exercise: a focus on glucose metabolism. Am J Physiol Endocrinol Metab. 2012; 303(3):E301–E307. PubMed doi:10.1152/ajpendo.00667.2011 44. McPherron AC, Lawler AM, Lee SJ. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature. 1997; 387(6628):83–90. PubMed doi:10.1038/387083a0 45. Moran CN, Vassilopoulos C, Tsiokanos A, et al. The associations of ACE polymorphisms with physical, physiological and skill parameters in adolescents. Eur J Hum Genet. 2006; 14(3):332–339. PubMed doi:10.1038/ sj.ejhg.5201550 46. Moskowitz SM, Chmiel JF, Sternen DL, et al. Clinical practice and genetic counseling for cystic fibrosis and CFTR-related disorders. Genet Med. 2008; 10(12):851– 868. PubMed doi:10.1097/GIM.0b013e31818e55a2 47. Nakayama M, Yasue H, Yoshimura M, et al. T-786→C mutation in the 5′-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation. 1999; 99(22):2864–2870. PubMed doi:10.1161/01.CIR.99.22.2864 48. NCBI. 2014; Available at: http://www.ncbi.nlm.nih.gov/ projects/SNP/snp_ref.cgi?rs=1805086. 49. Nixon PA, Orenstein DM, Kelsey SF, Doershuk CF. The prognostic value of exercise testing in patients with cystic fibrosis. N Engl J Med. 1992; 327(25):1785–1788. PubMed doi:10.1056/NEJM199212173272504 50. Pellegrino R, Viegi G, Brusasco V, et al. Interpretative strategies for lung function tests. Eur Respir J. 2005; 26(5):948–968. PubMed doi:10.1183/09031936.05.00 035205 51. Pitsiladis Y, Wang G, Wolfarth B, et al. Genomics of elite sporting performance: what little we know and necessary advances. Br J Sports Med. 2013; 47(9):550–555. PubMed doi:10.1136/bjsports-2013-092400 52. Pollock ML, Jackson AS. Research progress in validation of clinical methods of assessing body composition. Med Sci Sports Exerc. 1984; 16(6):606–615. PubMed doi:10.1249/00005768-198412000-00016 53. Puthucheary Z, Skipworth JR, Rawal J, Loosemore M, Van Someren K, Montgomery HE. The ACE gene and human performance: 12 years on. Sports Med. 2011; 41(6):433– 448. PubMed doi:10.2165/11588720-000000000-00000 54. Rassaf T, Lauer T, Heiss C, et al. Nitric oxide synthasederived plasma nitrite predicts exercise capacity. Br J Sports Med. 2007; 41(10):669–673. PubMed doi:10.1136/ bjsm.2007.035758 55. Rubio JC, Gomez-Gallego F, Santiago C, et al. Genotype modulators of clinical severity in McArdle disease. Neurosci Lett. 2007; 422(3):217–222. PubMed doi:10.1016/j. neulet.2007.06.025 56. Sahach VF, Bohuslavs’kyi A, Dmytriieva AV, Nadtochii SM. Role of nitric oxide and mitochondrial permeability
pore in changes of oxygen consumption in the working skeletal muscle. Fiziol Zh. 2004; 50(2):19–26. PubMed 57. Sanchís Aldás J, Casán Clará P, Castillo Gómez J, González Mangado N, Palenciano Ballesteros L, Roca Torrent J. Normativa para la espirometría forzada. Recomendaciones SEPAR N° 1. Sociedad Española de Neumología y Cirugía Torácica; 1985. 58. Scarpulla RC. Nuclear control of respiratory chain expression by nuclear respiratory factors and PGC-1-related coactivator. Ann N Y Acad Sci. 2008; 1147:321–334. PubMed doi:10.1196/annals.1427.006 59. Strauss GD, Osher A, Wang CI, et al. Variable weight training in cystic fibrosis. Chest. 1987; 92(2):273–276. PubMed doi:10.1378/chest.92.2.273 60. Totzeck M, Hendgen-Cotta UB, Rammos C, et al. Higher endogenous nitrite levels are associated with superior exercise capacity in highly trained athletes. Nitric Oxide. 2012; 27(2):75–81. PubMed doi:10.1016/j.niox.2012.05.003 61. Troosters T, Langer D, Vrijsen B, et al. Skeletal muscle weakness, exercise tolerance and physical activity in adults with cystic fibrosis. Eur Respir J. 2009; 33(1):99–106. PubMed doi:10.1183/09031936.00091607 62. Ward ME, Hussain SN. Effect of inhibition of nitric oxide release on the diaphragmatic oxygen delivery-consumption relationship. J Crit Care. 1994; 9(2):90–99. PubMed doi:10.1016/0883-9441(94)90019-1 63. West TG, Curtin NA, Ferenczi MA, et al. Actomyosin energy turnover declines while force remains constant during isometric muscle contraction. J Physiol. 2004; 555(Pt 1):27–43. PubMed doi:10.1113/jphysiol.2003.040089 64. Wolfarth B, Rankinen T, Hagberg JM, et al. Advances in exercise, fitness, and performance genomics in 2013. Med Sci Sports Exerc. 2014; 46(5):851–859. PubMed doi:10.1249/MSS.0000000000000300 65. Yang N, Garton F, North K. Alpha-Actinin-3 and performance. Med Sport Sci. 2009; 54:88–101. PubMed doi:10.1159/000235698 66. Yankaskas JR, Marshall BC, Sufian B, Simon RH, Rodman D. Cystic fibrosis adult care: consensus conference report. Chest. 2004; 125(1, Suppl.):1S–39S. PubMed doi:10.1378/ chest.125.1_suppl.1S
Supplement (Genotyping Methods) 1) ACE polymorphism. Polymerase chain reaction (PCR) followed by electrophoresis by the agarose gel method. The primers used were: 5¢-CTGGAGAGCCACTCCCATCCTTTCT-3¢ and 5¢-GACGTGGCCATCACATTCGTCAGAT-3¢, and PCR conditions were as follows: initial denaturing at 96 °C 5 min; 35 cycles at 94 °C 30 s, 58 °C 30 s, 72 °C 1 min and a final extension at 72 °C 10 min. 2) AGT polymorphism. The restriction fragment length polymorphism method (RFLP) was used with the SfaNI 10U/μl (New England Biolabs) restriction enzyme. The PCR primers used were: 5¢-TGGATGCGCACAAGGTCCTGTC-3¢ and 5¢-AGGGTGCTGTCCACACTGGCTCGC-3¢, and
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112 Yvert et al.
PCR conditions were as follows: initial denaturing at 95 °C 5 min; 35 cycles at 95 °C 30 s, 61 °C 30 s, 72 °C 1 min and a final extension at 72 °C 5 min. The resulting PCR products were detected in an ABI PRISM analyzer (Applied Biosystems, Foster City, CA). 3) MSTN polymorphism. The PCR primers used were: 5¢-GAAAACCCAAATGTTGCTTC-3¢ and 5¢-TGTCTAGCTTATGAGCTTAGGG-3¢ and PCR conditions were as follows: initial denaturing at 95 °C 10 min; 35 cycles at 95 °C 1 min, 52 °C 45 s, 72 °C 1 min and a final extension at 72 °C 5 min. The resulting PCR products were genotyped by single base extension (SBE). The primer used was 5¢-TTTAATACAATACAATAAAGTAGTAA-3¢. Conditions for SBE PCR were: 96 °C 10 s; 25 cycles at 50 °C 5 s and 60 °C 30 s. The resulting PCR products were detected in an ABI PRISM analyzer. 4) Polymorphisms in the genes PPARGC1A, NRF1, NRF2, TFAM, ACSL1, PPARD, ACTN3, TRHR, PTK2 and NOS3 were detected using predesigned Applied Biosystems TaqMan SNP Genotyping Assays for the polymorphisms:
PGC-1α G482S (rs8192678) (ID: C_1643192_20); NRF1 A > G (rs6949152) (ID: C_29144830_10); NRF2 A > C (rs12594956) (ID: C_32072163_20); TFAM S12T (rs1937) (ID: C_8975662_10); ACSL1 G > A (rs6552828) (ID: C_30469648_10); PPARD A > G (rs2267668) (ID: C_15872729_10); ACTN3 R577X (rs1815739) (ID: C_590093_1_); TRHR C > T (rs7832552) (ID: C_26370395_20); PTK2 A > C (rs7843014) (ID: C_11605645_10); PTK2 T > A (rs7460) (ID: C_243385_10); NOS3 C > T (rs2070744) (ID: C_15903863_10). PCR amplification was performed using a StepOnePlus Real-Time PCR System (Applied Biosystems) under the conditions: denaturation at 95 °C for 10 min, followed by 50 cycles of denaturation at 92 °C for 15 s, annealing/ extension at 60 °C for 1 min, and a final extension stage of 30 s at 60 °C.
Physical-Capacity-Related Genetic Polymorphisms in Children with Cystic Fibrosis Thomas Yvert, Catalina Santiago, Elena Santana-Sosa, Zoraida Verde, Felix GómezGallego, Luis Lopez-Mojares, and Margarita Pérez Universidad Europea de Madrid
Nuria Garatachea University of Zaragoza
Alejandro Lucia Universidad Europea de Madrid In patients with cystic fibrosis (CF), physical capacity (PC) has been correlated with mortality risk. In turn, PC is dependent on genetic factors. This study examines several polymorphisms associated with PC and healthrelated phenotype traits (VO2peak, FEV1, FVC, PImax and muscular strength) in a group of children with CF (n = 66, primary purpose). The same analyses were also performed in a control group of healthy children (n = 113, secondary purpose). The polymorphisms determined were classified as muscle function polymorphisms (ACE rs1799752; AGT rs699; ACTN3 rs1815739; PTK2 rs7843014 and rs7460; MSTN rs1805086; TRHR rs7832552; NOS3 rs2070744) or energy metabolism polymorphisms (PPARGC1A rs8192678; NRF1 rs6949152; NRF2 rs12594956; TFAM rs1937; PPARD rs2267668; ACSL1 rs6552828). No significant polymorphism/phenotype correlations were detected in children with CF, with marginal associations being observed between NOS3 rs2070744 and VO2peak and FEV1, as well as between PPARGC1A rs8192678 and FEV1. Overall, similar findings were observed in the control group, i.e., no major associations. The PC-related polymorphisms examined seem to have no effects on the PC or health of children with CF. Keywords: SNP, exercise, disease, pediatrics
Background Cystic fibrosis (CF) is the most common life-limiting autosomal recessive genetic disorder to affect Caucasians, with an incidence estimated at 1 in 2,500 Caucasian newborns (10). CF is a multisystemic disease affecting several vital organs, particularly the lungs, pancreas, and digestive system. Its cause has been identified as a mutation in the gene cystic fibrosis transmembrane conductance regulator (CFTR). Despite several possible treatments, there is currently no curative treatment for CF. Yvert, Santiago, Gómez-Gallego, Pérez, and Lucia are with the School of Doctorate Studies and Research, and SantanaSosa and Verde the Dept. of Morphological and Biomedical Sciences, Lopez-Mojares the Faculty of Health Sciences of Sport and Physical Activity, Universidad Europea de Madrid, Madrid, Spain. Garatachea is with the Dept. of Physiotherapy and Nursing, University of Zaragoza, Zaragoza, Spain. Address author correspondence to Thomas Yvert at yvert.thomas.paul@ gmail.com.
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As part of therapy and rehabilitation programs in the CF management, physical activity (PA) and exercise training are generally recommended to improve lung function (66). In fact, several health and physical fitness indicators have been positively correlated with lung function, quality of life, and survival in these patients. These indicators include peak oxygen uptake (VO2peak) (49), forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), maximal inspiratory pressure (PImax) (13), and muscular strength (59). However, wide interindividual differences in the aforementioned indicators have been shown depending on PA level, training status and genetic factors (46,49). In the last 10 years, over 200 genes with polymorphisms related to physical fitness have been identified (6,51,64). Among the most well-studied of these polymorphisms are the following: angiotensin converting enzyme (ACE) gene I/D (rs1799752) (24,39,53); angiotensinogen (AGT) M235T (rs699) (25); α-actinin 3 (ACTN3) R577X (rs1815739) (16,39); protein-tyrosine kinase 2 (PTK2) A > C (rs7843014) and PTK2 T > A (rs7460) (14); myostatin
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Genes and Cystic Fibrosis 103
(MSTN) K153R (rs1805086) (22); thyrotropin-releasing hormone receptor (TRHR) C > T (rs7832552) (35); nitric oxide synthase 3 (NOS3) C > T (rs2070744) (21,23); peroxisome proliferator-activated receptor-γ coactivator-1 α (PPARGC1A) G482S (rs8192678) (17,37); nuclear respiratory factor 1 (NRF1) A > G (rs6949152) (29) and nuclear respiratory factor 2 (NRF2) A > C (rs12594956) (15,18); mitochondrial transcription factor-A (TFAM) S12T (rs1937) (2); peroxisome proliferator-activated receptor-δ (PPARD) A > G (rs2267668) (40) and acylCoA synthase long-chain-family-member-1 (ACSL1) G > A (rs6552828) (5). Most findings come from case:control designs (some of which have included several geographically/ethnically independent cohorts, e.g.; 19), and fewer evidence arises from genome wide scan association studies, e.g., for polymorphisms ACSL1 rs6552828 (5) and TRHR rs7832552 (35). Thus, owing to the important role of the abovementioned genes and polymorphisms in several body functions and systems, we hypothesized that some of them would be also associated with physical fitness phenotypes in children with CF. In fact, we previously showed that some of the abovementioned genetic polymorphisms can influence the physical fitness and clinical severity of patients with another genetic disorder, McArdle disease: the D-allele or DD genotype of the ACE I/D polymorphism and the variant R-allele of the MSTN K153R variation are associated with lower VO2peak (26,28) or greater clinical severity (28,55), while carriage of the variant ACTN3 577X-allele would play a favorable role in exercise capacity (38). Genetic association studies are performed to determine whether a genetic variant is associated with a trait, so the purpose of this study was to determine genotype frequencies for these polymorphisms in children with CF and to examine their association with physical fitness and health-related traits as VO2peak, FVC, FEV1, PImax and muscular strength. As a secondary study purpose, the same analyses were replicated in a group of age and sex-matched healthy controls.
Methods Subjects This gene candidate association study was conducted in 66 children with CF and 113 healthy children. Children aged 4–17 years were invited to participate if they had been diagnosed with CF by means of the classic sweat test (until identification of pathogenic CFTR mutations) and treated at the Hospital Niño Jesús or the Hospital La Paz in Madrid, Spain. All the children recruited had mild (FEV1 ≥ 70% of predicted), moderate (FEV1 = 60–69% of predicted) or moderately to severe CF (FEV1 = 50–59% of predicted) (50), and their clinical condition was stable. Exclusion criteria were: (i) had severe lung deterioration (defined as an FEV1 below 50% of the expected value), (ii) had been hospitalized within the previous 3 months, or (iii) had Burkholderia cepacia infection. The healthy
children recruited were 4–14 years of age with no chronic disease. Each child and one of his/her parents or caregivers provided written informed consent before the study onset. The study protocol adhered to the tenets of the Declaration of Helsinki, and received institutional review board approval from the Hospital Niño Jesús in Madrid (Approval number 006–09).
Procedures Each child received 3 familiarization sessions, during which they learned to walk on treadmill during 5–10 min at 2.5 kph, and learned the correct technique of the strength movements with 3 × 15 repetitions of each movement at minimum weight (2.25 kg). Afterward, the children have performed all the tests on one day, first with the graded treadmill test and then, after sufficient recovery time, with the 5RM test of each movement. In the children with CF, cardio-respiratory fitness was assessed in the exercise physiology laboratory of the Hospital Niño Jesús. Tests in the control children were performed in the exercise physiology laboratory of the Universidad Europea de Madrid (Spain) using the same equipment.
Phenotype Assessment Anthropometry. Body weight was measured using a digital balance (Seca, Madrid, Spain) to the nearest 0.1 kg (kg), with the child wearing no shoes. Height, without shoes, was measured to the nearest 0.1 cm using a stadiometer (Seca, Madrid, Spain) with the child standing with heels and head against the wall. Body mass index was calculated by dividing body weight (kg) by height in meters (m2) squared (kg.m-2). Triceps, abdominal, supra-iliac, and thigh skin-folds were measured using a caliper (Holtan Crymych, United Kingdom). Body fat percentage was estimated using the equation described by Jackson and Pollock (52). Lung-Pancreatic Status. Lung-pancreatic status was established using the classification scheme of Cleveland et al. (9) in which the CFTR class of mutation is used to define the groups S (severe disease) and M (mild disease). Within group S, the following subgroups were defined: A, patients with two class I alleles; B, those with a class I allele and class II or III allele; or C, those with a class II allele and class II or III allele. Group M was comprised of patients with at least one class IV–VI allele. Genotyping for CFTR gene mutations was performed using the kits Inno-lipa CFTR19 and Inno-lipa CFTR17+update from Innogenetics (Innogenetics NV, BTW BE 0427.550.660, RPR Gent, Technologiepark 6, B-9052 Gent, Belgium). Pulmonary Function. Pulmonary function was
determined through a spirometry protocol designed by the Spanish Society of Pneumology and Thoracic Surgery (SEPAR) (57), according to which FVC and FEV1 (L) are expressed as percentages of expected reference
104 Yvert et al.
values (27). A portable spirometer (CareFusion 232 Ltd., Chatham Maritime, Kent, UK) was used to determine PImax (mmHg) at residual volume in accordance with established standards (3) recorded as the best result of three attempts performed at intervals of at least 1 min.
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Cardiorespiratory Fitness. VO2peak (ml·kg-1·min-1) was
determined by open-circuit spirometry using pediatric facemasks during a graded treadmill test (Technogym Run Race 1400HC; Gambettola, Italy). Gas exchange was measured using a breath-by-breath system (Vmax 29c; Sensormedics; CA, USA). The test was performed after familiarization sessions; treadmill speed began at 1.5 km·h-1 (for children .0036) for any of the phenotypes we studied (VO2peak, FEV1, FVC, PImax, muscle strength), except for the following marginally significant correlations (Figure 2): between NOS3 rs2070744 T allele and top decile children according to the VO2peak and FEV1 data (p = .002 in both associations); and between PPARGC1A rs8192678 SS genotype and top decile children according to the FEV1 data (p = .003). Overall, similar findings were obtained in the healthy children group, where the only association found was for the NOS3 rs2070744 polymorphism and VO2peak, where the mean value of this variable was higher in CC (42.3 ± 6.8 ml·kg-1·min-1) compared with CT children (35.4 ± 8.8 ml·kg-1·min-1, p = .002), with no significant difference for the comparison with the TT genotype (37.9 ± 7.2 ml·kg-1·min-1, p = .079).
Discussion This study was designed to address the potential influence of PC-related genes on PC phenotypes in children with CF. Our results suggest that the genetic polymorphisms
Table 1 Main Demographic Data (Mean ± SD) Children with CF (Girls = 31 (47%), Boys = 35) n
Mean ± SD
Age (years)
66
9.0 ± 4.0
Height (cm)
66
Body weight (kg)
66
(kg·m-2)
66
BMI
Healthy Children (Girls = 61 (54%), Boys= 52) n
Mean ± SD
Age (years)
113
9.0 ± 2.5
134.1 ± 20.0
Height (cm)
113
139.4 ± 15.9
32.5 ± 13.7
Body weight (kg)
113
36.6 ± 12.2
17.2 ± 2.8
(kg·m-2)
113
18.3 ± 2.9
111
77.8 ± 7.0
BMI
Obese: 4.5%
Obese: 13.3%
Underweight: 9.1%
Underweight: 0.9%
Lean mass (%)
59
80.3 ± 6.5
Lean mass (%)
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Note. BMI = body mass index, CF = cystic fibrosis.
Table 2 Cardiorespiratory and Muscular Fitness and Pulmonary Function (Mean ± SD) in the Two Study Groups Children with CF
Healthy Children n
Mean ± SD
n
Mean ± SD
VO2peak (ml·kg-1·min-1)
66
36.0 ± 7.3
112
38.0 ± 7.4
HRpeak (beats·min-1)
66
187 ± 6
112
193 ± 8
FEV1 (%)
62
85.3 ± 28.1
24
108.9 ± 17.3
FVC (%)
62
86.1 ± 32.0
22
109.9 ± 18.0
PImax (mmHg)
62
63.9 ± 28.2
103
69.4 ± 25.6
Seated lateral row/kg (kg)
66
0.9 ± 0.2
111
0.9 ± 0.2
Seated bench press/kg (kg)
66
0.5 ± 0.3
111
0.9 ± 0.2
Seated leg press/kg (kg)
66
1.6 ± 0.5
113
3.2 ± 0.8
Overall strength/kg (kg)
66
3.5 ± 0.8
111
4.6 ± 0.9
Cardiorespiratory fitness
Pulmonary function
Muscular fitness
Note. Strength values are expressed in relation to the total body weight of each child. CF = cystic fibrosis, FVC = forced vital capacity, FEV1 = forced expiratory volume in 1 s; HRpeak = peak heart rate, VO2peak = peak oxygen uptake, PImax = maximum inspiratory pressure.
Table 3 Available Data of CFTR Mutation Class (Percentages) and Type According to Parental Contribution and Lung-Pancreatic Status in the Children with CF (All Diagnosed with the Classic Sweat Test) CFTR Mutation
Paternal % (n = 49)
Maternal % (n = 30)
Lung-pancreatic status
Percentage (n = 31)
Class 1
8.2 (G542×)
13.3 (R1162×; G542×)
Severe A
0.0
Class 2
87.8 (ΔF508; N1303K)
63.3 (ΔF508; N1303K)
Severe B
16.1
Class 3
0.0
3.3 (S549R)
Severe C
58.1
Class 4
4.1 (G85E; L206W)
13.3 (R334W; G85E)
Mild
25.8
Class 5
0.0
6.7 (3839+10Kb C > T)
Class 6
0.0
0.0
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Figure 1 — Genotype frequencies (%) for the polymorphisms examined in children with cystic fibrosis (CF) (A) (n = 66) and in healthy children (B) (n = 113).
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Figure 2 — Genotype/allele frequencies for those polymorphisms showing a significant association for a given outcome variable (e.g., VO2peak) in the CF children, that is, showing a significant difference in genotype/allele frequencies when comparing the children in top or lowest decile vs. the rest of children, respectively. The following marginally significant associations were detected: between the NOS3 rs2070744 T allele and the top decile according to VO2peak (A) or FEV1 (B) (n = 65, * and † p = .002); and between the PGC-1α rs8192678 SS genotype (C) and the top decile according to FEV1 (‡ p = .003).
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involved in enhancing PC do not have a major effect on such phenotypes in these patients. Our data are however limited by the small sample size of our cohorts, which reduces the statistical power and generalizability of our findings. Further research with larger samples (ideally from multicenter cohorts of different ethnic/geographic origins) is needed in the field of genetics and PC related traits (20). Future studies might also corroborate the results of the current study in CF children showing more severe disease manifestations. Indeed, the CF children assessed here had a stable clinical condition and no severe lung deterioration, as reflected by their mean VO2peak values (36.0 ± 7.3 mL·kg-1·min-1), which were comparable to those of the healthy children (38.0± 7.4 mL·kg-1·min-1). We detected no significant relationship between polymorphisms of the genes associated with strength, muscle mass or muscle fiber contractile function (ECA, AGT, ACTN3, PTK2, MSTN, NOS3 and TRHR) and PC and health indicators in the CF children. Similar findings were obtained in the healthy children. However, marginal correlations were observed for polymorphisms in the genes NOS3 and PPARGC1A. A significant correlation was detected between the T allele of the NOS3 C > T (rs2070744) polymorphism and the children with CF belonging to the 10% upper decile for the data of VO2peak and FEV1. This could mean that this polymorphism in the gene (NOS3) encoding endothelial nitric oxide (NO) synthase (eNOS) is positively associated with health status and survival in children with CF. As far as we are aware, no such link has been reported in healthy children or those with CF. The eNOS is responsible for the generation of NO in blood vessels, where this molecule plays an important vasodilatory and regulatory role. It is also involved in muscle glucose uptake during exercise and in modulating blood flow and oxygen consumption in working muscles (43). In contrast with other linked polymorphisms (-922A/G and -1468T/A) that are not associated with changes in gene transcription, the T→C mutation of the NOS3 rs2070744 polymorphism results in significantly reduced gene promoter activity and reduced endothelial NO synthesis (47). In turn, the T-allele would result in higher NO synthesis There is strong rationale in postulating that higher endothelial NO production could lead to improved muscle hyperemia and oxygen supply to working fibers, and thus to higher VO2peak values. Several studies in isolated hindlimb and diaphragm muscle in dogs and rats, have reported decreased muscle VO2 during high-intensity evoked contractions with eNOS inhibition (4,30,31,33,56,62,63), although these findings have not been corroborated in human research using positron emission tomography (30). Higher plasma levels of nitrite (the main oxidation product of NO in plasma, that sensitively reflects changes in eNOS activity and serves as an endocrine NO donor), are positively associated with performance in high-intensive endurance exercise (12) or stress testing until exhaustion in nonathletes (54), as well as with exercise capacity in highly trained athletes (60). The NO can also be produced from dietary nitrate, and nitrate supplementation has been shown to improve arterial and muscle oxygenation during hypoxic exercise (42).
No correlation was observed between polymorphisms of the ACE and AGT genes, involved in angiotensin II production (a powerful vasoconstrictor and muscular growth factor), and indicators of PC and health in the two groups of children. A relationship has been reported between the ACE I/D polymorphism and the load-capacity ratio of the inspiratory muscles in children (11) and disease severity in children with CF (41). Despite the lack of correlation observed here between these polymorphisms and PC, there have been numerous reports of such links although these have been inconsistent. Ahmetov et al. related the D allele of the ACE I/D polymorphism to long jump ability in 219 children (1). In contrast, Moran et al. described an association between the I allele of this polymorphism and grip strength and high jump capacity in 484 teenage girls (45). The literature lacks information on the AGT M235T polymorphism and PC in children. In our study, allele frequencies for this polymorphism in the children with CF were inconsistent with HWE and were, therefore, excluded from the study. No significant correlations for the ACTN3 R577X polymorphism were observed in the two groups of children. α-actinin3 contributes to the contractile superstructure of type II muscle fibers, mainly by helping to anchor actin microfilaments in Z discs (65). Few studies have analyzed the effect of this polymorphism on the PC of children. Ahmetov et al. noted a positive association between the R allele of the ACTN3 R577X polymorphism (only in combination with other PPARA or ACE polymorphisms) and the grip strength and long jump capacity of 219 children (1). Chiu et al. positively related the RR genotype of the ACTN3 R577X polymorphism (also only in combination with other ACE and PPARD polymorphisms) and grip strength capacity in 170 adolescent girls (7). However, as in the current study, no association has been reported between this polymorphism alone and the PC of healthy children or children with CF. We detected no relationship between MSTN and TRHR gene polymorphisms and factors associated with health and PC in our two study groups. Myostatin is a protein secreted by muscle cells that inhibits their own growth, acting as a limiting factor in muscle development (44). The role of myostatin was discovered through the use of transgenic knockout mice for this gene (44). These mice showed up to double the muscle mass and lower fat tissue than control wild-type mice. The lack of an association with the MSTN K153R polymorphism noted in our cohort may be due to the low presence of the mutant R allele in Caucasians, with estimates running at 2.0% (48). The TRH hormone (thyrotropin-releasing hormone) exerts its effect by binding to its receptor TRHR activating pathways whose ultimate goal is the liberation of thyroxine, an important hormone for skeletal muscle development (34). Liu et al. (35) reported a link between lean body mass and the TRHR C > T (rs7832552) polymorphism. However, no study has addressed the relationship between MSTN K153R or TRHR C > T (rs7832552) polymorphisms and PC in children. In effect, our findings indicate no such effects of these polymorphisms.
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In eukaryotic cells, mitochondria play a crucial role in energy production. Mitochondria are responsible for most of the useful energy derived from the degradation of carbohydrates and fatty acids, which are converted to adenosine-triphosphate (ATP) through oxidative phosphorylation, thus playing an important role in exercise. New mitochondria in a cell are formed via a process known as mitochondrial biogenesis, which is activated by several different signals in response to extracellular stimuli such as muscular exercise, exposure to cold or oxidative stress. Mitochondrial biogenesis requires the expression of a large number of genes among which we find PPARGC1A, PPARD, NRF1, NRF2, and TFAM (58). The ACSL1 gene plays a role in fatty acid metabolism (36). We observed a marginally significant relationship between having the SS genotype of the PPARGC1A G482S polymorphism and being a child with CF belonging to the top decile according to FEV1 data. To the best of our knowledge, no investigation has explored the effect of this polymorphism on the PC of children. This discrepancy determines a need for further work designed to elucidate the mechanisms involved in a possible positive effect of this allele on such an important factor as respiratory capacity in children with CF. No correlation was observed here between PPARD, ACSL1, NRF1, NRF2, or TFAM gene polymorphisms and factors associated with PC and health in the two groups examined (the NRF1 rs6949152 polymorphism in children with CF did not meet HWE and was excluded from the study). No prior study has attempted to link these polymorphisms with child PC, thus we have no data with which to compare our results. Phenotypic traits of physical capacity are determined by a wide range of variables, both genetic and environmental, such as exercise, nutrition, rest, psychology, advances in technology and equipment, among others. According to our data, we may state that the polymorphisms analyzed, despite being candidates for significantly influencing human physical performance, have no effect on physical capacity and performance in children with CF, with the exception of a few marginal cases for which further research is needed to draw any firm conclusions. Acknowledgments This study was supported by a Grant from Fondo de Investigaciones Sanitarias (FIS, grant # PS09/00194).
References 1. Ahmetov II, Gavrilov DN, Astratenkova IV, et al. The association of ACE, ACTN3 and PPARA gene variants with strength phenotypes in middle school-age children. J Physiol Sci. 2013; 63(1):79–85. PubMed doi:10.1007/ s12576-012-0233-8 2. Akhmetov II, Popov DV, Missina SS, Vinogradova OL, Rogozkin VA. Association of the mitochondrial transcription factor (TFAM) gene polymorphism with
physical performance of athletes. Fiziol Cheloveka. 2010; 36(2):121–125. PubMed 3. American Thoracic Society/European Respiratory Society. ATS/ERS Statement on respiratory muscle testing. Am J Respir Crit Care Med. 2002; 166(4):518–624. PubMed doi:10.1164/rccm.166.4.518 4. Baker DJ, Krause DJ, Howlett RA, Hepple RT. Nitric oxide synthase inhibition reduces O2 cost of force development and spares high-energy phosphates following contractions in pump-perfused rat hindlimb muscles. Exp Physiol. 2006; 91(3):581–589. PubMed doi:10.1113/expphysiol.2005.032698 5. Bouchard C, Sarzynski MA, Rice TK, et al. Genomic predictors of the maximal O2 uptake response to standardized exercise training programs. J Appl Physiol. (1985) 2011;110(5):1160-70. 6. Bray MS, Hagberg JM, Perusse L, et al. The human gene map for performance and health-related fitness phenotypes: the 2006-2007 update. Med Sci Sports Exerc. 2009; 41(1):35–73. PubMed doi:10.1249/ MSS.0b013e3181844179 7. Chiu LL, Chen TW, Hsieh SS, Hsieh LL. ACE I/D, ACTN3 R577X, PPARD T294C and PPARGC1A Gly482Ser polymorphisms and physical fitness in Taiwanese late adolescent girls. J Physiol Sci. 2012; 62(2):115–121. PubMed doi:10.1007/s12576-011-0189-0 8. Chomczynski P, Sacchi N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc. 2006; 1(2):581–585. PubMed doi:10.1038/nprot.2006.83 9. Cleveland RH, Zurakowski D, Slattery D, Colin AA. Cystic fibrosis genotype and assessing rates of decline in pulmonary status. Radiology. 2009; 253(3):813–821. PubMed doi:10.1148/radiol.2533090418 10. Cystic Fibrosis Foundation. Cystic Fibrosis Foundation Patient Registry 2007 Annual data report. Bethesda, Maryland: Cystic Fibrosis Foundation; 2008. 11. Dimitriou G, Papakonstantinou D, Stavrou EF, et al. Angiotensin-converting enzyme gene polymorphism and respiratory muscle function in infants. Pediatr Pulmonol. 2010; 45(12):1233–1239. PubMed doi:10.1002/ ppul.21316 12. Dreissigacker U, Wendt M, Wittke T, et al. Positive correlation between plasma nitrite and performance during high-intensive exercise but not oxidative stress in healthy men. Nitric Oxide. 2010; 23(2):128–135. PubMed doi:10.1016/j.niox.2010.05.003 13. Enright S, Chatham K, Ionescu AA, Unnithan VB, Shale DJ. Inspiratory muscle training improves lung function and exercise capacity in adults with cystic fibrosis. Chest. 2004; 126(2):405–411. PubMed doi:10.1378/chest.126.2.405 14. Erskine RM, Williams AG, Jones DA, Stewart CE, Do Degens H. PTK2 gene polymorphisms contribute to the interindividual variability in muscle strength and the response to resistance training? A preliminary report. J Appl Physiol. 2012; 112(8):1329–1334. PubMed doi:10.1152/japplphysiol.01137.2011 15. Eynon N, Alves AJ, Sagiv M, Yamin C, Sagiv M, Meckel Y. Interaction between SNPs in the NRF2 gene and
Downloaded by Teachers College Library on 09/16/16, Volume 27, Article Number 1
110 Yvert et al.
elite endurance performance. Physiol Genomics. 2010; 41(1):78–81. PubMed doi:10.1152/physiolgenomics.00199.2009 16. Eynon N, Hanson ED, Lucia A, et al. Genes for elite power and sprint performance: ACTN3 leads the way. Sports Med. 2013; 43(9):803–817. PubMed doi:10.1007/ s40279-013-0059-4 17. Eynon N, Meckel Y, Sagiv M, et al. Do PPARGC1A and PPARalpha polymorphisms influence sprint or endurance phenotypes? Scand J Med Sci Sports. 2010; 20(1):e145– e150. PubMed doi:10.1111/j.1600-0838.2009.00930.x 18. Eynon N, Ruiz JR, Bishop DJ, et al. The rs12594956 polymorphism in the NRF-2 gene is associated with top-level Spanish athlete’s performance status. J Sci Med Sport. 2013; 16(2):135–139. PubMed doi:10.1016/j. jsams.2012.05.004 19. Eynon N, Ruiz JR, Femia P, et al. The ACTN3 R577X polymorphism across three groups of elite male European athletes. PLoS ONE. 2012; 7(8):e43132. PubMed doi:10.1371/journal.pone.0043132 20. Eynon N, Ruiz JR, Oliveira J, Duarte JA, Birk R, Lucia A. Genes and elite athletes: a roadmap for future research. J Physiol. 2011; 589(Pt 13):3063–3070. PubMed doi:10.1113/jphysiol.2011.207035 21. Eynon N, Ruiz JR, Yvert T, et al. The C allele in NOS3 -786 T/C polymorphism is associated with elite soccer player’s status. Int J Sports Med. 2012; 33(7):521–524. PubMed doi:10.1055/s-0032-1306337 22. Ferrell RE, Conte V, Lawrence EC, Roth SM, Hagberg JM, Hurley BF. Frequent sequence variation in the human myostatin (GDF8) gene as a marker for analysis of musclerelated phenotypes. Genomics. 1999; 62(2):203–207. PubMed doi:10.1006/geno.1999.5984 23. Gomez-Gallego F, Ruiz JR, Buxens A, et al. The -786 T/C polymorphism of the NOS3 gene is associated with elite performance in power sports. Eur J Appl Physiol. 2009; 107(5):565–569. PubMed doi:10.1007/s00421-0091166-7 24. Gomez-Gallego F, Santiago C, Gonzalez-Freire M, et al. Endurance performance: genes or gene combinations? Int J Sports Med. 2009; 30(1):66–72. PubMed doi:10.1055/s-2008-1038677 25. Gomez-Gallego F, Santiago C, Gonzalez-Freire M, et al. The C allele of the AGT Met235Thr polymorphism is associated with power sports performance. Appl Physiol Nutr Metab. 2009; 34(6):1108–1111. PubMed doi:10.1139/ H09-108 26. Gomez-Gallego F, Santiago C, Moran M, et al. The I allele of the ACE gene is associated with improved exercise capacity in women with McArdle disease. Br J Sports Med. 2008; 42(2):134–140. PubMed doi:10.1136/ bjsm.2007.038992 27. Gonzalez Barcala FJ, Cadarso Suarez C, et al. Lung function reference values in children and adolescents aged 6 to 18 years in Galicia. Arch Bronconeumol. 2008; 44(6):295– 302. PubMed doi:10.1016/S0300-2896(08)70436-8 28. Gonzalez-Freire M, Santiago C, Gomez-Gallego F, et al. Does the K153R variant of the myostatin gene influence the clinical presentation of women with McArdle disease?
Neuromuscul Disord. 2009; 19(3):220–222. PubMed doi:10.1016/j.nmd.2009.01.001 29. He Z, Hu Y, Feng L, et al. NRF-1 genotypes and endurance exercise capacity in young Chinese men. Br J Sports Med. 2008; 42(5):361–366. PubMed doi:10.1136/ bjsm.2007.042945 30. Heinonen I, Saltin B, Kemppainen J, et al. Skeletal muscle blood ñow and oxygen uptake at rest and during exercise in humans: a pet study with nitric oxide and cyclooxygenase inhibition. Am J Physiol Heart Circ Physiol. 2011; 300:H1510–H1517. PubMed 31. King-VanVlack CE, Mewburn JD, Chapler CK, MacDonald PH. Endothelial modulation of skeletal muscle blood flow and [UNKNOWN ENTITY &Vdot;]o2 during low- and high-intensity contractions. J Appl Physiol. 2002; 92(2):461–468. PubMed 32. Kraemer W, Fry A. Strength testing development and evaluation of methodology. Physiological Assessment of Human Fitness. Champaign (IL): Human Kinetics ed.; 1995. 33. Krause DJ, Hagen JL, Kindig CA, Hepple RT. Nitric oxide synthase inhibition reduces the O2 cost of force development in rat hindlimb muscles pump perfused at matched convective O2 delivery. Exp Physiol. 2005; 90(6):889–900. PubMed doi:10.1113/expphysiol.2005.031567 34. Larsson L, Li X, Teresi A, Salviati G. Effects of thyroid hormone on fast- and slow-twitch skeletal muscles in young and old rats. J Physiol. 1994; 481(Pt 1):149–161. PubMed doi:10.1113/jphysiol.1994.sp020426 35. Liu XG, Tan LJ, Lei SF, et al. Genome-wide association and replication studies identified TRHR as an important gene for lean body mass. Am J Hum Genet. 2009; 84(3):418–423. PubMed doi:10.1016/j.ajhg.2009.02.004 36. Lobo S, Wiczer BM, Bernlohr DA. Functional analysis of long-chain acyl-CoA synthetase 1 in 3T3-L1 adipocytes. J Biol Chem. 2009; 284(27):18347–18356. PubMed doi:10.1074/jbc.M109.017244 37. Lucia A, Gomez-Gallego F, Barroso I, et al. PPARGC1A genotype (Gly482Ser) predicts exceptional endurance capacity in European men. J Appl Physiol. 2005; 99(1):344– 348. PubMed doi:10.1152/japplphysiol.00037.2005 38. Lucia A, Gomez-Gallego F, Santiago C, et al. The 577X allele of the ACTN3 gene is associated with improved exercise capacity in women with McArdle’s disease. Neuromuscul Disord. 2007; 17(8):603–610. PubMed doi:10.1016/j.nmd.2007.04.006 39. Ma F, Yang Y, Li X, et al. The association of sport performance with ACE and ACTN3 genetic polymorphisms: a systematic review and meta-analysis. PLoS ONE. 2013; 8(1):e54685. PubMed doi:10.1371/journal.pone.0054685 40. Maciejewska-Karlowska A, Hanson ED, Sawczuk M, Cieszczyk P, Eynon N. Genomic haplotype within the Peroxisome Proliferator-Activated Receptor Delta (PPARD) gene is associated with elite athletic status. Scand J Med Sci Sports. 2014; 24(3):e148–e155. PubMed doi:10.1111/ sms.12126 41. Marson FA, Bertuzzo CS, Hortencio TD, Ribeiro JD, Bonadia LC, Ribeiro AF. The ACE gene D/I polymorphism as a modulator of severity of cystic fibrosis. BMC Pulm Med. 2012; 12:41. PubMed doi:10.1186/1471-2466-12-41
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42. Masschelein E, Van Thienen R, Wang X, et al. Dietary nitrate improves muscle but not cerebral oxygenation status during exercise in hypoxia. J Appl Physiol. 2012; 113:736– 745. PubMed doi:10.1152/japplphysiol.01253.2011 43. McConell GK, Rattigan S, Lee-Young RS, Wadley GD, Merry TL. Skeletal muscle nitric oxide signaling and exercise: a focus on glucose metabolism. Am J Physiol Endocrinol Metab. 2012; 303(3):E301–E307. PubMed doi:10.1152/ajpendo.00667.2011 44. McPherron AC, Lawler AM, Lee SJ. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature. 1997; 387(6628):83–90. PubMed doi:10.1038/387083a0 45. Moran CN, Vassilopoulos C, Tsiokanos A, et al. The associations of ACE polymorphisms with physical, physiological and skill parameters in adolescents. Eur J Hum Genet. 2006; 14(3):332–339. PubMed doi:10.1038/ sj.ejhg.5201550 46. Moskowitz SM, Chmiel JF, Sternen DL, et al. Clinical practice and genetic counseling for cystic fibrosis and CFTR-related disorders. Genet Med. 2008; 10(12):851– 868. PubMed doi:10.1097/GIM.0b013e31818e55a2 47. Nakayama M, Yasue H, Yoshimura M, et al. T-786→C mutation in the 5′-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation. 1999; 99(22):2864–2870. PubMed doi:10.1161/01.CIR.99.22.2864 48. NCBI. 2014; Available at: http://www.ncbi.nlm.nih.gov/ projects/SNP/snp_ref.cgi?rs=1805086. 49. Nixon PA, Orenstein DM, Kelsey SF, Doershuk CF. The prognostic value of exercise testing in patients with cystic fibrosis. N Engl J Med. 1992; 327(25):1785–1788. PubMed doi:10.1056/NEJM199212173272504 50. Pellegrino R, Viegi G, Brusasco V, et al. Interpretative strategies for lung function tests. Eur Respir J. 2005; 26(5):948–968. PubMed doi:10.1183/09031936.05.00 035205 51. Pitsiladis Y, Wang G, Wolfarth B, et al. Genomics of elite sporting performance: what little we know and necessary advances. Br J Sports Med. 2013; 47(9):550–555. PubMed doi:10.1136/bjsports-2013-092400 52. Pollock ML, Jackson AS. Research progress in validation of clinical methods of assessing body composition. Med Sci Sports Exerc. 1984; 16(6):606–615. PubMed doi:10.1249/00005768-198412000-00016 53. Puthucheary Z, Skipworth JR, Rawal J, Loosemore M, Van Someren K, Montgomery HE. The ACE gene and human performance: 12 years on. Sports Med. 2011; 41(6):433– 448. PubMed doi:10.2165/11588720-000000000-00000 54. Rassaf T, Lauer T, Heiss C, et al. Nitric oxide synthasederived plasma nitrite predicts exercise capacity. Br J Sports Med. 2007; 41(10):669–673. PubMed doi:10.1136/ bjsm.2007.035758 55. Rubio JC, Gomez-Gallego F, Santiago C, et al. Genotype modulators of clinical severity in McArdle disease. Neurosci Lett. 2007; 422(3):217–222. PubMed doi:10.1016/j. neulet.2007.06.025 56. Sahach VF, Bohuslavs’kyi A, Dmytriieva AV, Nadtochii SM. Role of nitric oxide and mitochondrial permeability
pore in changes of oxygen consumption in the working skeletal muscle. Fiziol Zh. 2004; 50(2):19–26. PubMed 57. Sanchís Aldás J, Casán Clará P, Castillo Gómez J, González Mangado N, Palenciano Ballesteros L, Roca Torrent J. Normativa para la espirometría forzada. Recomendaciones SEPAR N° 1. Sociedad Española de Neumología y Cirugía Torácica; 1985. 58. Scarpulla RC. Nuclear control of respiratory chain expression by nuclear respiratory factors and PGC-1-related coactivator. Ann N Y Acad Sci. 2008; 1147:321–334. PubMed doi:10.1196/annals.1427.006 59. Strauss GD, Osher A, Wang CI, et al. Variable weight training in cystic fibrosis. Chest. 1987; 92(2):273–276. PubMed doi:10.1378/chest.92.2.273 60. Totzeck M, Hendgen-Cotta UB, Rammos C, et al. Higher endogenous nitrite levels are associated with superior exercise capacity in highly trained athletes. Nitric Oxide. 2012; 27(2):75–81. PubMed doi:10.1016/j.niox.2012.05.003 61. Troosters T, Langer D, Vrijsen B, et al. Skeletal muscle weakness, exercise tolerance and physical activity in adults with cystic fibrosis. Eur Respir J. 2009; 33(1):99–106. PubMed doi:10.1183/09031936.00091607 62. Ward ME, Hussain SN. Effect of inhibition of nitric oxide release on the diaphragmatic oxygen delivery-consumption relationship. J Crit Care. 1994; 9(2):90–99. PubMed doi:10.1016/0883-9441(94)90019-1 63. West TG, Curtin NA, Ferenczi MA, et al. Actomyosin energy turnover declines while force remains constant during isometric muscle contraction. J Physiol. 2004; 555(Pt 1):27–43. PubMed doi:10.1113/jphysiol.2003.040089 64. Wolfarth B, Rankinen T, Hagberg JM, et al. Advances in exercise, fitness, and performance genomics in 2013. Med Sci Sports Exerc. 2014; 46(5):851–859. PubMed doi:10.1249/MSS.0000000000000300 65. Yang N, Garton F, North K. Alpha-Actinin-3 and performance. Med Sport Sci. 2009; 54:88–101. PubMed doi:10.1159/000235698 66. Yankaskas JR, Marshall BC, Sufian B, Simon RH, Rodman D. Cystic fibrosis adult care: consensus conference report. Chest. 2004; 125(1, Suppl.):1S–39S. PubMed doi:10.1378/ chest.125.1_suppl.1S
Supplement (Genotyping Methods) 1) ACE polymorphism. Polymerase chain reaction (PCR) followed by electrophoresis by the agarose gel method. The primers used were: 5¢-CTGGAGAGCCACTCCCATCCTTTCT-3¢ and 5¢-GACGTGGCCATCACATTCGTCAGAT-3¢, and PCR conditions were as follows: initial denaturing at 96 °C 5 min; 35 cycles at 94 °C 30 s, 58 °C 30 s, 72 °C 1 min and a final extension at 72 °C 10 min. 2) AGT polymorphism. The restriction fragment length polymorphism method (RFLP) was used with the SfaNI 10U/μl (New England Biolabs) restriction enzyme. The PCR primers used were: 5¢-TGGATGCGCACAAGGTCCTGTC-3¢ and 5¢-AGGGTGCTGTCCACACTGGCTCGC-3¢, and
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PCR conditions were as follows: initial denaturing at 95 °C 5 min; 35 cycles at 95 °C 30 s, 61 °C 30 s, 72 °C 1 min and a final extension at 72 °C 5 min. The resulting PCR products were detected in an ABI PRISM analyzer (Applied Biosystems, Foster City, CA). 3) MSTN polymorphism. The PCR primers used were: 5¢-GAAAACCCAAATGTTGCTTC-3¢ and 5¢-TGTCTAGCTTATGAGCTTAGGG-3¢ and PCR conditions were as follows: initial denaturing at 95 °C 10 min; 35 cycles at 95 °C 1 min, 52 °C 45 s, 72 °C 1 min and a final extension at 72 °C 5 min. The resulting PCR products were genotyped by single base extension (SBE). The primer used was 5¢-TTTAATACAATACAATAAAGTAGTAA-3¢. Conditions for SBE PCR were: 96 °C 10 s; 25 cycles at 50 °C 5 s and 60 °C 30 s. The resulting PCR products were detected in an ABI PRISM analyzer. 4) Polymorphisms in the genes PPARGC1A, NRF1, NRF2, TFAM, ACSL1, PPARD, ACTN3, TRHR, PTK2 and NOS3 were detected using predesigned Applied Biosystems TaqMan SNP Genotyping Assays for the polymorphisms:
PGC-1α G482S (rs8192678) (ID: C_1643192_20); NRF1 A > G (rs6949152) (ID: C_29144830_10); NRF2 A > C (rs12594956) (ID: C_32072163_20); TFAM S12T (rs1937) (ID: C_8975662_10); ACSL1 G > A (rs6552828) (ID: C_30469648_10); PPARD A > G (rs2267668) (ID: C_15872729_10); ACTN3 R577X (rs1815739) (ID: C_590093_1_); TRHR C > T (rs7832552) (ID: C_26370395_20); PTK2 A > C (rs7843014) (ID: C_11605645_10); PTK2 T > A (rs7460) (ID: C_243385_10); NOS3 C > T (rs2070744) (ID: C_15903863_10). PCR amplification was performed using a StepOnePlus Real-Time PCR System (Applied Biosystems) under the conditions: denaturation at 95 °C for 10 min, followed by 50 cycles of denaturation at 92 °C for 15 s, annealing/ extension at 60 °C for 1 min, and a final extension stage of 30 s at 60 °C.
