1

Supplementary Section 1: Phenotype testing

Tests were chosen to measure the three parameters (obesity, strength/power and endurance) postulated to be under the influence of R577X in Yang et al.8. During physical education classes, subjects’ physical characteristics (height (m), weight (kg) and skinfolds (mm)) were measured. Height and weight were used to calculate BMI using Quatelet’s index (weight/height2 (kg/m2)), skinfold thickness was measured in triplicate, using Harpenden Callipers, and the average of the three measures was used in the analysis. These were used as obesity-related measures. Subjects also completed a series of tasks designed to measure physical and physiological attributes. We selected tasks used by the Australian Sports Commission for their ‘Sport Search’18,19 to allow direct comparison of the results. Briefly, strength/power-related phenotypes included hand-grip strength (kg), basketball throw (m)20,21, vertical jump (cm)22-25, 40m sprint (s) and agility run (s)26. To estimate endurance ability, we employed the multistage shuttle run test15,16 to estimate max (weight-corrected maximum rate of oxygen consumption in mlkg-1min-1) with correction for age as described by Leger et al.17. Assessment protocols for 4 of the tests can be found below.

BASKETBALL THROW

Purpose

To measure upper body power by determining the distance the participant can project the basketball in a forward direction from a sitting position.

Equipment

Standard size and weight senior basketball

15 meter tape measure accurate to 5 centimetres

Procedure

  1. The participant sits with their buttocks, back and head resting against a wall. The legs rest on the floor horizontally in front of the body.
  2. The participant uses a two-handed chest pass to push the ball in the horizontal direction as far as possible. A one arm or shoulder pass is not allowed.
  3. Ensure that the participant keeps the head, shoulders and buttocks in contact with the wall and the ball is thrown only using the arm and shoulder muscles.
  4. Allow two trials for each participant.

Scoring

  • Record the longest distance thrown to the nearest 5 cm (measure from the base of the ball where it makes contact with the ground on the first bounce).

40 METER SPRINT

Purpose

To measure the ability to run as fast as possible from a stationary position to a point 40 m further.

Equipment

Stopwatch

10 marker cones

40 m running track that is straight, level and placed cross wind (if a grass surface is used ensure that it is dry).

Procedure

  1. Mark a 40-m running track with marker cones placed at 10-m intervals.
  2. The participant starts in a standing position, one foot in front of the other -the front foot placed exactly on the line.
  3. The tester should stand at the finish line, call ready and sweep down their arm quickly to start the participant (do not call 'go'). As the arm sweeps down the tester should simultaneously start the stopwatch which is held in the descending hand.
  4. Stop the stopwatch when the participant’s chest crosses the line.
  5. Emphasise to the participant to run as quickly as possible.
  6. Allow two trials for each participant, with a short rest between the trials.

Scoring

  • Record the time taken to run the fastest trial to the nearest 0.1 of a second

AGILITY RUN

Purpose

To measure the ability to change direction of the body quickly whilst moving (i.e., agility).

Equipment

Stopwatch

Two parallel lines (1.2 meters in length) marked on the ground 5 meters apart (measured between the two inside edges of the line).

Marker cones (4)

The floor surface should be flat, even and slip resistant. A gymnasium floor is often dusty and slippery. If this is the case it is better to conduct the test outside on a concrete or bitumen level surface.

Procedure

  1. The subject starts from behind one line with their front foot exactly on the line.
  2. On the command 'go' the participant runs forward as quickly as possible to the other line, pivots and returns to the start line.
  3. This constitutes one cycle, with five cycles required in total.
  4. The participant must touch both feet beyond the lines and between the marker cones except at the end of the 5th cycle when they should run past the finish line without slowing.
  5. Start the stopwatch on the command 'go' and stop it when the subject’s chest crosses the line.
  6. Allow two trials for each participant.
  7. If the participant slips, do not include this result but conduct another trial.

Scoring

  • Record the time taken to the nearest 0.1 of a second for the fastest trial.

The score should be written in tenths of a second

e.g. 21.6 seconds scores 216

MULTI-STAGE SHUTTLE RUN

Purpose

To assess aerobic fitness (endurance ability).

Equipment

Cadence compact disc for the shuttle run (This will be supplied by the Project Team)

Masking Tape

Compact Disc player

Marker cones (8)

20 meter marked distance on a surface that is flat, even and slip resistant.

Stop watch

Shuttle run data collection forms

Procedure

  1. Check the speed of the compact disc player using the one-minute calibration period.
    Measure the 20 meter distance and mark with tape (if available) and marker cones.
  2. Instruct the participant to run to the opposite end and place one foot behind the line by the time the next beep sounds. If the participant arrives before the whistle (s)he should turn (pivot) and wait for the signal, then run to the opposite line to reach it in time for the next signal.
  3. At the end of each minute the time interval between "beeps" is decreased and the running speed becomes progressively faster.
  4. Ensure that the participant reaches the end line each time and does not turn short. Emphasise to the participant to turn and pivot rather than run in an arc, which takes more time.
  5. Each participant continues running for as long as possible until (s)he can no longer keep up with the indicated tempo.
  6. The criterion for eliminating a participant is two lengths consecutively where (s)he is more than two steps from the end when the whistle blows.

Scoring

  • Record the last level and shuttle the participant successfully completed.

1

1

Supplementary Section 2: New RFLP assay for R577X genotype determination

New primers were designed for R577X genotyping to enable more convenient visualisation. Primer design was based on GenBank Accession no. NT_033903. Genotypes were determined using polymerase chain reaction (PCR) amplification followed by restriction fragment length polymorphism (RFLP) analysis. A pair of primers, forward (ACTN3CM0945F; 5’-CTGGGCTGGAAGACAGGAG-3’) and reverse (ACTN3CM1234R; 5’-AGGGTGATGTAGGGATTGGTG-3’), was used to amplify a region containing the R577X polymorphism. PCR reactions were performed in a final volume of 25 l comprised of ReddyMixTM PCR mastermix containing 1.5mM MgCl2 (ABgene, Epsom, Surrey, UK), 400nM each primer (Sigma-Genosys, Haverhill, Cambridgeshire, UK), water and genomic DNA (20-100ng). Thermocycling consisted of 2.5 minutes at 94 oC followed by 35 cycles of 94 oC for 45 seconds, 59 oC for 30 seconds and 72 oC for 30 seconds and was completed with 10 minutes at 72 oC. PCR product amplified in these reactions was subjected to restriction enzyme digestion using DdeI. One unit of DdeI (New England Biolabs UK Ltd., Hitchin, Hertfordshire, UK) was used to digest 5 l of PCR product at 37 oC, made up to a final volume of 10 l with distilled water. The resulting bands were separated on a 2% agarose gel (Supplementary Figure 1). Representative products giving all three banding patterns were sequenced to confirm the predicted genotypes.

Supplementary Figure 1. Genotyping of ACTN3 rs2228325 (R577X) polymorphism.

The figure shows an ethidium bromide stained 2% agarose gel of DdeI digested fragments of the ACTN3 -PCR amplified product. Lane 1: 100 bp DNA ladder (Promega, Southampton, UK). Lane 2: Uncut PCR product (control). Lane 3: No DNA PCR (control). Lane 4: Bands produced by an individual homozygous for the C-allele (577R; 290 bp). Lane 5: Bands produced by a heterozygous individual. Lane 6: Bands produced by an individual homozygous for the T-allele (577X; 192 bp and 98 bp bands). Lane 7: 100 bp DNA ladder (Promega, Southampton, UK).

1

1

Supplementary Section 3: Phenotypic correlations

Supplementary Table 1. Summary of correlations between the assessed phenotypes used for calculating the Sidak correction.

For each pair of phenotypes, the correlation coefficient (r) is shown, with the coefficient of determination (r2) shown in parentheses, both to 3 significant figures. At the bottom of the table, the average values for r and r2 are shown.

r
(r2) / Gender / BMI / Triceps Skinfolds / Subscapular Skinfolds / Handgrip strength / Basketball Throw / Vertical Jump / 40 m Sprint / Agility Run
BMI / 0.030
(0.001)
Triceps Skinfolds / 0.024
(0.001) / 0.730
(0.533)
Subscapular Skinfolds / 0.018
(0.000) / 0.807
(0.651) / 0.775
(0.601)
Handgrip strength / 0.013
(0.000) / 0.360
(0.130) / 0.069
(0.005) / 0.185
(0.034)
Basketball Throw / 0.022
(0.000) / 0.150
(0.023) / 0.019
(0.000) / 0.054
(0.003) / 0.328
(0.108)
Vertical Jump / -0.002
(0.000) / -0.183
(0.033) / -0.305
(0.093) / -0.277
(0.077) / 0.271
(0.073) / 0.179
(0.032)
40 m Sprint / -0.017
(0.000) / 0.298
(0.089) / 0.394
(0.155) / 0.380
(0.144) / -0.287
(0.082) / -0.195
(0.038) / -0.525
(0.276)
Agility Run / -0.017
(0.000) / 0.205
(0.042) / 0.261
(0.068) / 0.274
(0.075) / -0.157
(0.025) / -0.106
(0.011) / -0.395
(0.156) / 0.507
(0.257)
max / -0.003
(0.000) / -0.357
(0.127) / -0.383
(0.147) / -0.425
(0.181) / 0.079
(0.006) / 0.101
(0.010) / 0.278
(0.077) / -0.437
(0.191) / -0.314
(0.099)
Average / 0.054
(0.003)

1

1

Supplementary References

18Tomkinson GR, Olds TS, Gulbin J: Secular trends in physical performance of Australian children. J. Sports Med. Phys. Fit. 2003; 43: 90-98.

19Australian Sports Commission. Sport Search - the search is over. Canberra, Australia, Australian Sports Commission, 1993.

20Viitasalo JT: Evaluation of Explosive Strength for Young and Adult Athletes. Res. Q. Exerc. Sport 1988; 59: 9-13.

21Gillespie J, Keenum S: A Validity and Reliability-Analysis of the Seated Shot Put as a Test of Power. J. Hum. Movement Stud. 1987; 13: 97-105.

22Draper JA, Minikin B, Telford R. Test Methods Manual. Canberra, National Sports Research Centre, Australian Sports Commission, 1991.

23Simons J, Beunen GP, Renson R, Claessens ALM, Van Reusel B, Lefevre JAV: Growth and fitness of Flemish girls: The Leuven growth study. Champaign, Illinois, 1990.

24Bloomfield J, Blanksby BA, Elliot BC, Ackland TR, Morton AR. University of Western Australia Growth and Development Study, University of Western Australia: Department of Human Movement Studies, 1983.

25Ostyn M, Simons J, Beunen G, Renson R, Van Gerven D: Somatic and Motor Development of Belgian Secondary Schoolboys: Norms and Standards. Leuven, Leuven University Press, 1980.

26Adam C, Klissouras V, Ravassolo M: Eurofit. Handbook for the Eurofit test of physical fitness. Rome, Council of Europe. Committee for the Development of Sport, 1988.

1