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European Journal of Human Genetics (2001) 9, 797 ± 801 ã 2001 Nature Publishing Group All rights reserved 1018-4813/01 $15.00www.nature.com/ejhg The angiotensin converting enzyme I/D polymorphism Igor B Nazarov*,1,3, David R Woods2, Hugh E Montgomery2, Olga V Shneider1,3, Vasiliy I Kazakov1, Nikolai V Tomilin1 and Viktor A Rogozkin3 1Institute of Cytology of the Russian Academy of Sciences, Tikchoretski Ave. 4, Saint Petersburg, 194064, Russia; 2Department of Cardiovascular Genetics, 3rd floor Rayne Institute, University College London, 5 University Street, London WC1E 6JJ, UK; 3Saint-Petersburg Scientific Research Institute of Physical Culture, 2 Dinamo Street, Saint The deletion (D) allele of the human ACE gene is associated with higher ACE activity than the insertion (I) allele. There is controversy as to whether the ACE genotype may be associated with elite athletic status; recent studies have identified no significant associations amongst those drawn from mixed sporting disciplines.
However, such lack of association may reflect the mixed nature of such cohorts, given that an excess frequency of the I allele has been reported amongst elite endurance athletes, and an excess of the D allele amongst those engaged in more power-orientated sports. We examined this hypothesis by determining ACE I/D allele frequency amongst 217 Russian athletes (swimmers, skiers, triathletes and track-and-field participants) prospectively stratified by performance (`outstanding' or `average'), and the duration of their event (SDA (51 min), MDA (1 to 20 min), and LDA (420 min): short, middle and long distance athletes respectively). ACE genotype and allele frequencies were compared to 449 controls. ACE genotype frequency amongst the whole cohort, or the outstanding athletes alone, was no different to that amongst sedentary controls. However, there was an excess of the D allele (frequency 0.72, P=0.001) amongst the outstanding SDA group, and an excess of the I allele (frequency 0.63, P=0.032) amongst the outstanding MDA group. These findings were replicated in the outstanding swimmers, with track and field SDA similarly demonstrating an excess of the D allele (P=0.01). There was no association found between the outstanding LDA and ACE genotype (P=0.27).
These data not only confirm an excess of the D allele in elite SDA, and I allele in elite MDA, but also offer an explanation as to why any such association may be hard to detect amongst a heterogeneous cohort of mixed athletic ability and discipline. European Journal of Human Genetics (2001) 9, 797 ± 801.
Keywords: angiotensin converting enzyme; ACE; athletes; sport An excess of the I allele has been associated with some A polymorphism in intron 16 of the human angiotensin I- aspects of endurance performance, being identified in elite converting enzyme (ACE) gene has been identified in which British distance runnersand mountaineers.In addition, an the presence (insertion, I allele) rather than the absence excess of the I allele is present in Australianand Croatian (deletion, D allele) of a 287 bp Alu-sequence insertion rowers as well as Spanish elite athletes.Conversely, an excess fragment is associated with lower serumand ACE of the D allele has been reported amongst elite athletes in more power-oriented events such as short distance swim-mingand However, there has been debate as to the reproducibility of *Correspondence: Igor B Nazarov, Med. Biological Chemistry, 4107 such associations. Several studies have failed to identify any Tupper Hall, One Shields Avenue, University of California at Davis, Davis, association with elite endurance performance.Taylor et CA 95616, USA. Tel: +1 530 7525913; Fax: +1 530 7523516; alexamined hockey players, cyclists, skiers, track and field Received 2 April 2001; revised 28 June 2001; accepted 5 July 2001 athletes, swimmers, rowers, gymnasts and `others'. Similarly, ACE genotype frequencies in Russian athletes the mixed cohort examined by Karjalainenincluded `Outstanding' athletes (n=141) were at least national diverse sports such as long distance running, orienteering, representatives. This group included 81 European and cross country skiing and triathlon. The 192 athletes studied Russian champions and 19 Olympic or World champions.
by Rankinen et alalso included skiers, long and middle `Average' athletes (n=76) were regional competitors with distance runners, cyclists and biathletes (the latter would also no less than 7 years experience participating in their sport, have to to demonstrate more than a proficiency at rifle but who had never been selected for the national team.
marksmanship). Such studies thus comprise individuals This may have introduced unintended selection bias but selected from diverse sporting disciplines, with potential was fundamental to the study aim. In order to further variation in standard, with events of varying duration and avoid selection bias, three methods were used to recruit the outstanding athletes; targeting of national teams, In general, therefore, the association of ACE genotype with information provided by national coaching staff, and sporting prowess is recognised in studies of elite athletes athletes attending national training camps.
Controls consisted of 449 healthy unrelated volunteers (111 students of St Petersburg University, aged 18 ± 20, and 338 blood donors, age 25 ± 45). The athletes and control groups were all Caucasian Russians, with an equivalent ratio from European and Siberian descent (3 : 1 in both groups).
We have tested this hypothesis in the study of a mixed Further characteristics are presented in cohort of Russian athletes. ACE gene I/D allele frequencies forthe cohort overall were first compared to those in a control sample. Subsequently, allele frequency was compared across DNA was extracted from white blood cells or mouthwash event duration in individual sporting disciplines, for those samples as previously described.ACE genotype was determined using a three-primer method,yielding ampli-fication products of 65 bp (I allele) and 84 bp (D allele). Thesewere separated by electrophoresis on a 7.5% polyacrylamide gel and visualised using ethidium bromide. Genotyping was The University of St Petersburg Ethics Committee approved performed by experienced staff blind to subject data.
the study and written informed consent was obtained fromeach participant.
Statistical analysisAllele frequencies were determined by gene counting.
Genotype distribution and allele frequencies between groups Two hundred and seventeen male and female Russian of athletes and controls were then compared by w2 test. P athletes of regional or national competitive standard were values of 50.05 were considered statistically significant. By a recruited from the following sports: swimming (n=66), track- priori hypothesis, primary independent analyses explored and-field athletics (n=81), cross-country skiing (n=52), and differences in allele frequency between mixed athletes and controls, and by event duration in truly elite athletes Our a priori intention was to clarify whether previously competing in sports in which an association had been documented associations between the ACE polymorphism and elite athletes found in the study of a single sportingdiscipline could be duplicated in Russian athletes. Sec-ondly, we wanted to determine whether negative studies Table 1 ACE genotype distribution of the athletes and that have found no association were due to the combining controls with sex (frequencies) and age (average+SE) of athletes from different sports with different elements ofpower/endurance (anaerobic/aerobic) activity. The athletes were therefore prospectively stratified into groups accord-ing to event duration, covering a spectrum from the more `power'-oriented to the more endurance-oriented. SDA (short distance athletes) consisted of `sprinters', performing their given task in under 1 min (predominantly anaerobic energy production). MDA (middle distance athletes) were those competing over 1 to 20 min (mixed anaerobic and aerobic energy production), and LDA (long distance athletes) were those performing over more than 20 min (aerobic). The athletes were further classified by their standard of competition based on previous performances.
ACE genotype frequencies in Russian athletes confined to the elite swimmers and were not replicated The subjects' age, male/female ratio and years in their chosen sport did not differ by ACE genotype The track and field athletes demonstrated an excess of the ACE genotype distributions amongst subjects and controls D allele in outstanding SDA (P=0.01), with an excess of the were in Hardy-Weinberg equilibrium, being similar to that DD genotype (P=0.018), which was not present in the average Figure 1 I and D allele frequencies in 141 outstanding Russian athletes, by duration of event, and sedentary controls. As In considering individual sporting disciplines the hypothesised, the overall cohort of `outstanding' athletes failed mixed cohort of average and elite swimmers again demon- to demonstrate any association with the ACE genotype (P=0.78, strated no association with the ACE genotype (P=0.67).
and 0.55 by w2 test compared to controls, for genotype and allele frequency respectively). However, ACE genotype was However, again examining allele frequencies by duration of associated with event duration: an excess of D alleles (P=0.001) event, a significant excess of the D allele was evident in the being noted in the SDA (short-distance athletes) group, and an SDA (P=0.042), with an excess of the I allele in the MDA elevated frequency of the I allele in the MDA (middle-distance (P=0.042). As previously reported these findings were Table 2 ACE genotype distribution and frequencies of ACE gene D allele in athletes stratified by sporting discipline, standard and duration of event. Comparison with controls was by w2 test *P50.05, **P50.02. SDA, short distance athletes; MDA, middle distance althletes; LDA, long distance athletes.
ACE genotype frequencies in Russian athletes athletes. These were responsible for the significant excess of former elite, but not the average, MDA. This emphasises the the DD genotype (P=0.043), and D allele (P=0.018), in track need to examine outstanding athletes with stratification by the nature and duration of the event.
There was no relationship with ACE genotype amongst the The lack of an association between ACE genotype and elite skiers and triathletes may help explain the failure to find anexcess of the I allele in other cohortswhich haveincluded a substantial proportion of such athletes. I allele- associated endurance may be an important, but not prime, These data confirm our hypothesis that the study of a determinant of success in cross-country skiing and the mixed cohort of athletes from various sporting disciplines, triathlon. A cohort such as that previously which or the inclusion of non-elite athletes, may result in failure includes swimmers and track and field athletes, without to demonstrate an association between elite athletes and deference to event duration, is even less likely to demonstrate the ACE genotype. However, in considering only out- an ACE genotype effect, especially in light of the excess of D standing athletes by duration of event (and hence relative alleles in short distance swimmers and track and field athletes contribution of anaerobic/power component compared to aerobic/endurance), we confirm an excess of the D allele Although the D allele has been associated with greater in the SDA with an excess of the I allele in the MDA.
training-related changes in left ventricular and VO2 Such findings are consistent with those previously max risethe mechanism underlying the association of the D allele with power oriented, anaerobic sports is most likely Statistical adjustment for multiple comparison was not mediated through differences in skeletal muscle strength required in this study, as separate primary analysis for a single gain. A greater training-related increase in quadriceps muscle gene were performed according to a priori criteria, which strength has been associated with the D Such effects aimed to confirm previously-identified associations.
may, in turn, depend upon increased ACE-mediated activa- It is interesting that we failed to demonstrate an excess of tion of the growth factor angiotensin II,and increased the I allele in the outstanding long-distance athletes, and hence did not find an increasing linear trend of I allele with sely, the I allele may influence endurance performance increasing aerobic component as has previously been through improvements in substrate deliveryand the This may relate to differences in categorising efficiency of skeletal muscle,with subsequent conservation athletes; Myerson et alexamined allele frequency by distance; 200 m, 400 ± 3000 m, and symbol 55000 m, The II genotype is strongly associated with lower ACE whereas in this study we categorised athletes by duration of activity,perhaps caused by an Alu associated transcription event (under 1 min, 1 to 20, and over 20 min). This was done silencer,or by an unidentified polymorphic variant of to enable reasonable comparison between outstanding the ACE gene promoter in linkage disequilibrium with the I athletes of different disciplines and in order to account for alleleIndeed, the gene for growth hormone lies in close the different times taken to complete the same distance. An proximity. Although linkage to the ACE I/D polymorphic site elite cross country skier will complete the 5000 m in around seems unlikely,a physiological interaction seems possible 12 min, an elite 5000 m runner in around 13 (current world as elevated angiotensin II stimulates growth hormone release record 12 m in 39 s). However, a swimmer will have only in humansvia the angiotensin II type 1 receptor completed less than 1500 m by similar time points (the In conclusion, these data support the suggestion that current swimming world 1500 m record being 14 min 41 s).
subsequent studies might best focus upon only truly elite Hence, a 5000 m runner or skier would be categorised as a athletes, that only those of a single sporting discipline should MDA by our criteria, but a long distance competitor by the be considered, and that cohort stratification should be criteria of Myerson et al.Indeed, only the symbol 55000 m undertaken in some fashion according to anaerobic/aerobic runners reported by Myerson et al showed an excess of I or power/endurance component, as has been previously alleles compared to controls,which equates to our similar descriThe ACE I/D polymorphism should not be finding in outstanding MDA (I allele frequency 0.62 and 0.63, considered a `gene for human performance', but a marker of respectively). Secondly, there was no excess of I alleles in modulation such that one would expect association only in symbol 55000 m runners when compared to middle the truly elite athlete according to the nature of the specific distance athletes in the cohort of Myerson et alThe trend of rising I allele frequency, from short to longer distance,partly reflects an excess of the D allele in anaerobic events,which we have confirmed.
Important additional findings include the excess of D We thank Valentina Saburova for technical assistance. This work was alleles amongst only the outstanding SDA swimmers, and supported by grants from the Russian Fund for Basic Research 00-04- track and field athletes, with an excess of the I allele in the ACE genotype frequencies in Russian athletes 15 Rankinen T, Perusse L, Gagnon J et al. Angiotensin-converting 1 Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier enzyme ID polymorphism and fitness phenotype in the F. An insertion/deletion polymorphism in the angiotensin-1- HERITAGE Family Study. J Appl Physiol 2000; 88: 1029 ± 1035.
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