Shane M. Heffernan

Genomics in rugby union: A review and future prospects

Abstract This article introduces some aspects of sports genomics in a rugby union context, considers the rugby-specific genetic data in the published literature and outlines the next research steps required if the potential applications of genetic technology in rugby union, also identified here, are to become possible. A substantial proportion of the inter-individual variation for many traits related to rugby performance, including strength, short-term muscle power, VO2 max, injury susceptibility and the likelihood of being an elite athlete is inherited and can be investigated using molecular genetic techniques. In sports genomics, significant efforts have been made in recent years to develop large DNA biobanks of elite athletes for detailed exploration of the heritable bases of those traits. However, little effort has been devoted to the study of rugby athletes, and most of the little research that has focused on rugby was conducted with small cohorts of non-elite players. With steadily growing knowledge of the molecular mechanisms underpinning complex performance traits and the aetiology of injury, investigating sports genomics in the context of rugby is now a viable proposition and a worthwhile endeavour. The RugbyGene project we describe briefly in this article is a multi-institutional research collaboration in rugby union that will perform molecular genetic analyses of varying complexity. Genetic tests could become useful tools for rugby practitioners in the future and provide complementary and additional information to that provided by the non-genetic tests currently used.

Association of ACTN3 R577X but not ACE I/D gene variants with elite rugby union player status and playing position

We aimed to quantify the ACE I/D and ACTN3 R577X (rs1815739) genetic variants in elite rugby athletes (rugby union and league) and compare genotype frequencies to controls and between playing positions. The rugby athlete cohort consisted of 507 Caucasian men, including 431 rugby union athletes that for some analyses were divided into backs and forwards and into specific positional groups: front five, back row, half backs, centers, and back three. Controls were 710 Caucasian men and women. Real-time PCR of genomic DNA was used to determine genotypes using TaqMan probes and groups were compared using χ2 and odds ratio (OR) statistics. Correction of P values for multiple comparisons was according to Benjamini-Hochberg. There was no difference in ACE I/D genotype between groups. ACTN3 XX genotype tended to be underrepresented in rugby union backs (15.7%) compared with forwards (24.8%, P = 0.06). Interestingly, the 69 back three players (wings and full backs) in rugby union included only six XX genotype individuals (8.7%), with the R allele more common in the back three (68.8%) than controls (58.0%; χ2 = 6.672, P = 0.04; OR = 1.60) and forwards (47.5%; χ2 = 11.768, P = 0.01; OR = 2.00). Association of ACTN3 R577X with playing position in elite rugby union athletes suggests inherited fatigue resistance is more prevalent in forwards, while inherited sprint ability is more prevalent in backs, especially wings and full backs. These results also demonstrate the advantage of focusing genetic studies on a large cohort within a single sport, especially when intrasport positional differences exist, instead of combining several sports with varied demands and athlete characteristics.

TTN genotype is associated with fascicle length and marathon running performance.

Titin provides a molecular blueprint for muscle sarcomere assembly, and sarcomere length can vary according to titin isoform expression. If variations in sarcomere length influence muscle fascicle length, this may provide an advantage for running performance. Thus, the aim of this study was to investigate whether the titin (TTN) rs10497520 polymorphism was associated with muscle fascicle length in recreationally active men (RA; n=137) and marathon personal best time in male marathon runners (MR; n=141). Fascicle length of the vastus lateralis was assessed in vivo using B‐mode ultrasonography at 50% of muscle length in RA. All participants provided either a whole blood, saliva or buccal cell sample, from which DNA was isolated and genotyped using real‐time polymerase chain reaction. Vastus lateralis fascicle length was 10.4% longer in CC homozygotes, those carrying two copies of the C‐allele, than CT heterozygotes (P=.003) in RA. In the absence of any TT homozygotes, reflective of the low T‐allele frequency within Caucasian populations, it is unclear whether fascicle length for this group would have been smaller still. No differences in genotype frequency between the RA and MR groups were observed (P=.500), although within the MR group, the T‐allele carriers demonstrated marathon personal best times 2 minutes 25 seconds faster than CC homozygotes (P=.020). These results suggest that the T‐allele at rs10497520 in the TTN gene is associated with shorter skeletal muscle fascicle length and conveys an advantage for marathon running performance in habitually trained men.

Fat mass and obesity associated (FTO) gene influences skeletal muscle phenotypes in non-resistance trained males and elite rugby playing position

BackgroundFTO gene variants have been associated with obesity phenotypes in sedentary and obese populations, but rarely with skeletal muscle and elite athlete phenotypes.MethodsIn 1089 participants, comprising 530 elite rugby athletes and 559 non-athletes, DNA was collected and genotyped for the FTO rs9939609 variant using real-time PCR. In a subgroup of non-resistance trained individuals (NT; n = 120), we also assessed structural and functional skeletal muscle phenotypes using dual energy x-ray absorptiometry, ultrasound and isokinetic dynamometry. In a subgroup of rugby athletes (n = 77), we assessed muscle power during a countermovement jump.ResultsIn NT, TT genotype and T allele carriers had greater total body (4.8% and 4.1%) and total appendicular lean mass (LM; 3.0% and 2.1%) compared to AA genotype, with greater arm LM (0.8%) in T allele carriers and leg LM (2.1%) for TT, compared to AA genotype. Furthermore, the T allele was more common (94%) in selected elite rugby union athletes (back three and centre players) who are most reliant on LM rather than total body mass for success, compared to other rugby athletes (82%; P = 0.01, OR = 3.34) and controls (84%; P = 0.03, OR = 2.88). Accordingly, these athletes had greater peak power relative to body mass than other rugby athletes (14%; P = 2 x 10-6).ConclusionCollectively, these results suggest that the T allele is associated with increased LM and elite athletic success. This has implications for athletic populations, as well as conditions characterised by low LM such as sarcopenia and cachexia.

P-43 Titin genotype is associated with skeletal muscle fascicle length in recreationally active men and running performance in habitually trained marathon runners

Objectives The titin gene (TTN) encodes the largest described protein to date and, due to its size, provides a molecular blueprint for the organisation and assembly of the muscle sarcomere. Differences in sarcomere length, due to the expression of different titin isoforms, have been observed previously and may influence muscle fascicle length, which could provide an advantage for running performance. Thus, the aim of this study was to investigate if the TTN rs10497520 polymorphism was associated with muscle fascicle length in recreationally active men and marathon personal best time in elite male marathon runners, and to investigate any differences in genotype frequency between RA and MR. Methods The sample comprised 278 healthy, unrelated Caucasian men who all gave written consent to take part. Participants were categorised as either recreationally active [RA; n = 137; age = 20.7 (2.7) yr; height = 1.79 (0.06) m; mass = 75.3 (10.1) kg] or marathon runners [MR; n = 141; age = 34.9 (7.8) yr; height = 1.79 (0.07) m; mass = 66.5 (6.7) kg]. MR comprised Olympic, international and national level athletes, who had all achieved marathon personal best times under 2 hr 36 mins. Resting fascicle length of the vastus lateralis muscle was assessed in vivo using B-mode ultrasonography at 50% of muscle length in RA only. All participants provided either a whole blood, saliva or buccal cell sample, from which DNA was isolated and genotyped using real-time polymerase chain reaction. Independent samples t-tests were used to determine any genotype-dependent differences in fascicle length in RA and marathon personal best time in MR. Pearson’s chi-square tests were conducted to compare genotype frequencies between RA and MR. Results Vastus lateralis fascicle length was 10.4% longer in CC homozygotes than CT heterozygotes (P = 0.003) in RA. In the absence of any TT homozygotes, reflective of the low T-allele frequency within Caucasian populations, it is unclear if fascicle length for this group would have been smaller still. No differences in genotype frequency between the RA and MR groups were observed (P = 0.500), however, within the MR group the T-allele carriers demonstrated marathon personal best times 2 min 25 s faster than CC homozygotes (P = 0.020). Conclusions These results suggest that the T-allele at rs10497520 in the TTN gene is associated with shorter skeletal muscle fascicle length and conveys an advantage for marathon running performance in habitually trained men.

P-41 ACTN3 R577x genotype is not associated with elite european caucasian marathon performance

Objectives A common nonsense polymorphism (R577X) in the ACTN3 (α-actinin-3 protein) has been associated with elite athlete status previously. Specifically, the X allele has been positively associated with elite endurance status, however, this remains inconclusive due to contradictory reports within the literature. Thus, the current study aimed to compare ACTN3 R577X genotype and allele frequency distributions in ‘elite’ and ‘sub-elite’ marathon runners with those of a non-athletic, control population and to determine whether marathon personal best time was associated with ACTN3 R577X genotype. Method Four hundred and eighty four elite and sub-elite European Caucasian marathon runners and 554 ethnically matched controls provided a DNA sample from which the ACTN3 R577X polymorphism was genotyped using real-time PCR. Personal best (PB) times were used to determine elite (men < 2 h 30 min, n = 111; women < 3 h 00 min, n = 105) or sub-elite (men 2 h 30 min – 2 h 45 min, n = 189; women 3 h 00 min – 3 h 15 min, n = 79) status. Genotype and allele frequencies were compared between athletes and controls using Chi-square analyses. One-way ANOVAs were implemented to identify any genotype-dependent differences in PB times for men and women, which were subject to correction for multiple comparisons. Results The X allele was ∼3% more frequent in the marathon runners than in non-athlete controls (see Table 1 and Figure 1), although this small difference did not approach statistical significance. There were no significant differences in genotype (χ2 = 3.40; P = 0.182) or allele (χ2 = 2.31; P = 0.128) frequency distributions between athletes (RR = 29.1%, RX = 50.6% XX = 20.2%; R = 54.4%, X = 45.6%) and controls. There were also no differences between elite and sub-elite genotype (P = 0.968, χ2 = 0.66) and allele frequencies (P = 0.916, χ2 = 0.11). Similarly, no differences in genotype or allele frequencies were found between either elite (P = 0.439, χ2 = 1.65; P = 0.265, χ2 = 1.24) or sub-elite (P = 0.254, χ2 = 2.74; P = 0.183, χ2 = 1.77) runners and the control group. Neither were PB times genotype-dependent for either men (P = 0.864) or women (P = 0.966). Abstract P-41 Table 1 ACTN3 R577X polymorphism count, genotype and allele frequencies in elite, sub-elite and control groups. Genotype count (%) Allele count (%) RR RX XX R X Elite (n = 216) 64 (29.6) 108 (50.0) 44 (20.4) 236 (54.6) 196 (45.4) Sub-elite (n = 268) 77 (28.7) 137 (51.1) 54 (20.1) 291 (54.3) 245 (45.7) Controls (n = 554) 191 (34.5) 258 (46.6) 105 (19.0) 640 (57.8) 468 (42.2) Abstract P-41 Figure 1 Genotype and allele frequencies of the ACTN3 R577X polymorphism in elite endurance athletes (n = 484) and controls (n = 554) Conclusion No differences in genotype and allele frequencies were observed between athletes and controls, elite vs sub-elite, nor elite and sub-elite comparisons with the control group. Additionally, there was no genotype-dependent influence on PB time, which further emphasises that the ACTN3 R577X polymorphism does not influence elite endurance athlete status or determine marathon performance in European Caucasian runners. This is congruent with some previous findings and suggests other genetic variants or environmental factors may play a more prominent role in achieving elite endurance athlete status.

Protocol for Multiplex Amplicon Sequencing Using Barcoded Primers

Gene differences predisposing to atherosclerosis disease are important to identify and allow us to understand cellular processes involved in the progression and development of atherosclerotic disease. This protocol describes the experimental procedure for multiplex amplicon sequencing using barcoded primers. The procedures included in this chapter focus on amplicon library preparation (including polymerase chain reaction (PCR) conditions), DNA library quantitation options, next generation pyrosequencing with the 454 GS Junior system and data analysis. The process from DNA to sequencing data can be completed within 5 days.