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

Background FTO gene variants have been associated with obesity phenotypes in sedentary and obese populations, but rarely with skeletal muscle and elite athlete phenotypes. Methods In 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. Results In 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). Conclusion Collectively, 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.


Background
Fat mass and obesity associated (FTO) is the most investigated gene in obesity and has complex molecular mechanisms that are yet to be elucidated. Recent genome-wide association studies (GWAS) have identified several common single nucleotide polymorphisms (SNP) in the human FTO gene associated with obesity, body mass index BMI; [1], cardiovascular disease and hypertension [2,3]. These FTO SNPs, which are in strong linkage disequilibrium (r 2 > 0.80), are located in a cluster on the first intron of the gene on chromosome 16 and consequently exhibit similar obesityrelated traits [4]. Thus, within different FTO variants, those alleles that have been positively associated with obesity-related phenotypes are referred to as risk alleles, while those negatively associated with such traits are referred to as protective alleles. Homozygotes for the minor risk allele consistently demonstrate greater BMI and body mass (3-10 kg) in comparison to protective allele carriers [5,6]. This greater body mass is likely to be adipose tissue [7][8][9][10][11], although there exist some suggestions of greater lean mass (LM) in addition to fat mass [9,12] and independent of fat intake and physical activity [9]. This suggests that FTO genotype may be related to muscle properties and is supported by evidence from a large UK twin study that related FTO SNPs with body composition while controlling for lean mass and fat mass (separately and combined). The authors concluded that FTO SNP associations with body size were a composite of both lean and fat mass, not fat mass alone [13].
Environmental lifestyle factors (diet and physical activity) have also been investigated for FTO gene-environment interactions. Risk allele carriers are more likely to choose a high fat diet than protective allele carriers [11,14,15]. However, with administration of a high protein diet (25% energy intake) risk allele carriers demonstrated a greater reduction in body mass, fat mass and percentage body fat [16], due to greater appetite suppression than in protective allele carriers [17]. Additionally, physically active risk allele carriers have a 30% reduction in likelihood of becoming obese and have 36% less body fat compared to inactive risk allele carrying individuals [18]. In contrast, data from the HERITAGE Family Study showed that following 20 weeks of endurance training, protective allele carriers exhibited reductions in fat mass three times greater than risk allele homozygotes [19]. Interestingly, when comparing normal weight and obese individuals who participate in sport, no differences in FTO genotype were observed (P = 0.97), which was contrasted by those not participating P = 0.02; [20]. Considering the attenuation of FTO-associated obesity with environmental factors and the greater FTO-associated LM reported in obese populations [9,12], investigating LM and associated phenotypes in healthy, non-obese, nonresistance trained individuals and habitually trained elite athletes would be worthwhile.
To date, there have been no investigations of in vivo skeletal muscle phenotypes in trained or non-resistance trained populations for associations with FTO genotype. Eynon et al. [21] investigated FTO rs9939609 in three European cohorts of power (n = 258; 58.3% elite) and endurance athletes (n = 266; 57.1% elite) from a variety of sporting disciplines -but identified no associations. This lack of association was likely due to the considerable differences in physiological demand between the various athletic disciplines included, plus further variability in the standard of athlete. We have recently shown the ability of genetic research in a single sport with player roles that differ distinctly, namely rugby union (RU), to reveal context-specific competitive advantages provided by particular alleles [22]. Therefore, as RU includes athletes of remarkably distinct anthropometric and body composition phenotypes, elite RU provides a unique opportunity to investigate FTO in individuals at the extreme upper end of physical fitness [23].
Therefore, the main aims of the present study were to (1) investigate any association of FTO rs9939609 with body composition and muscle structural and functional parameters in a homogenous, healthy, non-obese nonresistance trained population (2) investigate whether FTO rs9939609 genotype differed between elite rugby athletes and a control population, and/or between RU player positions. Based on prior data in obese populations, it was hypothesised that the rs9939609 risk (A) allele would be associated with greater body mass, fat mass, BMI, LM, muscle volume and muscle strength in non-resistance trained individuals. Secondly, for the elite rugby cohort, it was hypothesised that the FTO A allele would be overrepresented in player positions typically requiring greater body and muscle mass while the protective (T) allele would be more common in positions requiring a lean phenotype.

Method
Participants A total of 1089 individuals were recruited and gave written informed consent to participate in the present study. The total sample comprised elite Caucasian male rugby athletes (n = 530; height 1.85 (0.07) m, mass 101 (14) kg, age 29 (7) yr, BMI 29.4 (3.7) kg•m -2 ; mean (standard deviation (SD)) including 73% British, 16% South African, 7% Irish and 4% from other nationalities and non-athlete Caucasian control participants (male and female; n = 559; height 1.75 (0.10) m, mass 75 (13) kg, age 29 (16) yr, BMI 24.5 (3.6) kg•m -2 ) including 86% British, 12% South African, 1% Irish and 1% from other nationalities. Athletes were considered elite if they had competed regularly (> 5 matches) since 1995 in the highest professional league in the UK, Ireland or South Africa for RU or the highest professional league in the UK for rugby league (RL). Of the RU athletes, 52.7% had competed at an international level for a "High Performance Union" (Regulation 16, worldrugby.org) and 43.2% of RL had competed at international level. All data for the athlete group's international status were confirmed as of 1st June 2016. Furthermore, within the rugby cohort, a subsample (n = 77) were examined for performance-related muscle phenotypes. Within the control group were a subgroup of non-resistance trained healthy males (NT; Table 1). NT participants were aged 18-39 years, had a BMI 18.5-30 kg•m -2 , had not undertaken any structured resistance training in the preceding 12 months and had no history of neurological or musculoskeletal disorders. Additionally, only those participants undertaking less than 3 h of low-to-moderate physical activity per week, assessed via questionnaire [24], were included.

Muscle properties in NT
An isokinetic dynamometer (Cybex Norm, Cybex International Inc., NY, USA) was used to assess maximal isometric knee extension (MVC KE ) and maximal isometric knee flexion (MVC KF ) torque at knee joint angles of 70°, 80°and 90°(full knee extension = 0°). The angle of peak torque was taken as the optimal knee joint angle and used to estimate antagonist muscle co-activation during MVC, which assumed a linear relationship between biceps femoris EMG activity and knee flexion torque [25]. Together with antagonist co-activation, quadriceps femoris (QF) voluntary activation capacity, determined using the interpolated twitch technique [26], allowed for the calculation of net MVC KE torque. Subsequently, patella tendon moment arm length (d PT ) was measured using dual energy x-ray absorptiometry DXA; [27] and patella tendon force calculated as net MVC KE torque/ d PT . The contribution of the vastus lateralis (VL) muscle (MF VL ) to patella tendon force was calculated by estimating the relative physiological cross-sectional area (PCSA) of the VL as~21% of the QF [28]. VL muscle architecture (fascicle length, L f and pennation angle, θ) was measured at 50% of VL length during MVC KE at the optimal knee joint angle using ultrasound (AU5, Esaote, Italy) and VL fascicle force estimated as MF VL /cosine θ [29]. At rest, ultrasound was also used to obtain a series of transverse plane scans at 50% of VL muscle length from the medial to lateral borders, which were contour matched to determine VL anatomical cross-sectional area [ACSA ; 29]. With VL length and VL ACSA used to estimate VL volume (V VL ) as previously [30]. Subsequently, VL PCSA was calculated as V VL / L f and, VL specific force calculated by dividing VL fascicle force by VL PCSA [29]. Finally, quantification of whole body and appendicular LM was completed using DXA (Hologic Discovery: Vertec Scientific Ltd, UK) following a 12 h overnight fast. Participants lay in a supine position, avoiding any contact between the trunk and the appendicular mass during a 7 min whole body scanning procedure (effective dose, 8.4 μSv). Appendicular lean mass was estimated from the DEXA as the total muscle mass of both the upper and lower limbs, where LM is body mass excluding fat and bone mass.

Muscle power in RU athletes
Using a portable force platform (Type 92866AA, Kistler, Germany) peak power output (PPO) was determined during a bilateral countermovement jump (CMJ) according to methods described previously [31]. Body mass and the vertical component of the ground reaction force during the CMJ (sampled at 1000 Hz) were used to determine instantaneous velocity and displacement of the participant's centre of gravity. Instantaneous power output was determined using the following equation: Power (W) = vertical GRF (N) x vertical velocity of centre of gravity (m · s -1 ), with the highest value produced deemed the PPO.

Sample collection and genotyping
Description of all molecular procedures have previously been described in detail [22]. Briefly, blood (~70% of all

RU positional groups
As established previously [22], to compare genotype and allele frequencies within the RU group, athletes were allocated to subgroups; forwards (props, hookers, locks, flankers, number eights) and backs (scrum halves, fly halves, centres, wings, full backs). Also, due to diverse physiological demands within RU [33,34], athletes were further divided into established positional groups according to their movement patterns [33]; front five (props, hookers, locks), back row (flankers, number eights), half backs (scrum halves, fly halves), back three (wings and full backs) and centres. For example, in one study the front five travelled~136 m at > 5 m · s -1 compared to~566 m for the back three [35]. Comparisons between positions were not performed for the RL cohort due to low statistical power when it was subdivided.

Data analysis
SPSS for Windows version 22 (SPSS Inc., Chicago, IL) software was used to conduct statistical analyses. Oneway analysis of variance (ANOVA) was used to compare height, mass, BMI, age and PPO between sample populations and genotype groups. When genotype groups were compared using a recessive model, an independent samples t-test was used. Furthermore, genotype effects on muscle phenotypes of interest were assessed for linear trend. Pearson's χ 2 tests compared genotype and allelic frequencies between athlete and control groups and between RU positional subgroups. There were 30 comparisons for genotype frequency between groups and 28 tests of genotype differences in phenotype in NT that were subjected to Benjamini-Hochberg corrections to control false discovery rate and corrected probability values are reported [36]. Where appropriate, odds ratio (OR) and eta squared (η P 2 ) were calculated to estimate effect size. Alpha was set at 0.05.

Results
Genotype calling was successful in all samples. There was 100% agreement among reference samples genotyped in the three genotyping centres, i.e. Glasgow, Northampton and MMU laboratories. Genotype frequencies were in Hardy-Weinberg equilibrium for the entire control group (P = 0.871), NT (P = 0.988) and athlete groups (RL, P = 0.183; RU, P = 0.076).
In terms of player position, backs had a greater frequency of T allele carriers than forwards (P = 0.03, Table 2, Fig. 1) and showed greater odds of being T allele carriers than AA genotype (OR = 1.84, Table 3). When combined, the back three and centres group contained less AA homozygotes and more T allele carriers (P = 0.03, P = 0.02, respectively; Fig. 1a and Table 2) than controls. Additionally, controls had more than twice the odds of being AA than the back three and centres group, with greater odds of T allele carriers in the back three and centres than controls (Table 3). Compared to forwards and all other RU athletes, TT genotype (P = 0.03; P = 0.03, respectively) and T allele carriers (P = 0.02; P = 0.02, respectively) were more common in the back three and centres group (Fig. 1a and Table 2). Likewise, forwards and all other RU athletes had greater than three times the odds of being AA genotype than the back three and centres group, with greater odds of T allele carriers in the back three and centres group than forwards and all other RU athletes (Table 3). Furthermore, the back three and centres group showed a greater T allele frequency than both forwards and all other RU athletes (Fig. 1b) and almost one and a half times greater odds of possessing the T allele (Table 3).

Muscle power
While PPO tended to be greater in back row players with lowest power in the halfbacks (5792 vs 5000 W; P = 0.09), PPO relative to body mass did differ according to playing position ( Table 4). The centre and back 3 players (59 W•kg -1 ) were 9.8% more powerful than back row (54 W•kg -1 ; P = 0.025) and 20.2% more powerful than front 5 players (47 W•kg -1 ; P = 6 x 10 -7 ; Table 4).

Discussion
We have shown that individuals possessing the FTO rs9939609 T allele and TT genotype had greater LM, while no differences in leg muscle size or strength were observed (Table 1), thus rejecting our first hypothesis that the risk (A) allele would be associated with greater LM and muscle volume in a healthy non-resistance trained population. That greater LM in T allele and TT genotype individuals was observed despite A allele carriers having greater body mass and BMI as reported previously [5,6]. In agreement with our second hypothesis, we report a greater T allele and TT genotype frequency in elite rugby athlete playing positions more reliant on a lean phenotype for success [37], while the A allele is more common in those positions where total body mass is more important [38]; Fig. 1, Tables 2 and 3. The ability to rapidly produce high levels of power relative to body mass using the leg musculature was greater in those playing positions more reliant on a lean phenotype (Table 4) and is in agreement with previously published data of elite RU players [39]. One possible biological mechanism underlying the present results may be the action of the iroquois homeobox 3 (IRX3) protein through its FTO genomic loci interaction. Eight athletes competed in both elite RL and RU and were included in both groups that were analysed separately. *Different from controls (P < 0.04). ‡ Different from forwards (P = 0.03) Until recently, little was known about the molecular basis for FTO SNP associations with any reported phenotype measure, because there was no association between FTO SNPs and expression of the FTO protein [40,41]. However, FTO has recently been found to influence IRX3 protein expression, through evolutionarily conserved long-range chromatin looping. Individuals possessing the protective FTO genotype/allele (TT/T) display lower IRX3 expression than AA homozygotes [42]. Furthermore, in contrast to IRX3 knockout (KO) mice, wild type mice exhibited similar FTO SNP risk (A) allele-associated phenotypes, such as greater BMI, body mass and body fat percentage [42]. Interestingly, IRX3 KO mice expended more energy, particularly at night, due to a greater browning of white fatty tissue [42] and recent findings show a link between brown fat and muscle developmental precursor Myf5 [43] which may provide a mechanism for the observation of greater LM in FTO T allele carriers in our NT cohort. Moreover,  using a transgenic mouse model (Rosa26 Enr-Irx3 ) that disrupts IRX3 function whilst maintaining the genomic interaction between IRX3 and FTO (mimicking more accurately the human in vivo state than the aforementioned KO model), the authors showed retention of the KO model phenotype traits [42]. These FTO-IRX3 protein interactions suggest an explanation for the greater LM seen with the T allele carriers of the present study and possibly the association of the T allele with muscle power relative to body mass and its relationship with playing position in RU athletes (Tables 1, 2, and 4; Fig. 1). The precise mechanisms of action of IRX3 in mammalian physiology are not fully understood, however the primary role of IRX3 in embryonic development and future actions in motor neuron restriction is relevant to this discussion. During neuronal development, IRX3 expression plays a key role in N-tubulin development and initiation of neuronal programming. High levels of IRX3 protein promote early tissue development resulting in a lack of cell differentiation [44]. Thus, it is possible that because the FTO T allele is associated with lower IRX3 expression, greater early motor neuron differentiation might subsequently lead to greater LMas we observed ( Table 1). As such, for predeterminant neuronal cells to differentiate into a progenitor motor neuron domain and subsequently motor neurons, it appears IRX3 must be repressed by the microRNA mir-17-3p in order for OLIG2 to regulate the development of ventral spinal motor neurons [45]. Thus, as the expression of OLIG2 increases, the yield of motor neurons increases in tandem [46]. Considering FTO T allele carriers have a lower embryonic expression of IRX3, T allele carriers may have a predisposition for greater LM through enhanced life-long motor neuron availability via OLIG2 expression and therefore, may be at an advantage for certain forms of athletic ability and associated performance phenotypes (Tables 2, 3 and 4; Fig. 1). This rationale and the present results may represent a small portion of the 85% heritability of adult muscle neuronal function [47].
Recent associations between FTO variants and IGF-1, specifically that serum IGF-1 levels were greater in T allele carriers [48], may provide a second mechanism to explain the observed genotype differences in LM (Table 1). It is well known that IGF-1 is upregulated as a consequence of mechanical load/exercise and plays an important role in the cellular development of muscle hypertrophy [49].
Hence, T allele carriers, who in the NT group had significantly greater LM, may experience upregulation of IGF-1 compared to AA genotype counterparts. Furthermore, serum IGF-1 levels have been positively associated with quadriceps torque [50] and explosive muscle power [51] in older men. These data provide a further potential basis for our observation that RU athletes who require greater muscle power relative to body mass (Table 4) show a greater frequency of the T allele than other playing positions (Table 2 and 3; Fig. 1).
We observed a lower frequency of the AA genotype in back three and centre playing positions (OR = 2.53; Table 3), although there was no difference between the entire rugby cohort and controls. That latter observation is similar to that we reported previously regarding another genetic variant (ACTN3 rs1815739) where there was no difference between the entire rugby cohort and controls despite differences in genotype frequency according to playing position [22]. This again demonstrates the importance of defining athletes very carefully when conducting such comparisons. Global positioning system (GPS) data provide evidence for the relevance of our finding regarding FTO genotype in elite athletes. Jones et al. [35] showed that -at an elite competitive level -the back three and centre players express the greatest 'instantaneous and accumulative demands for exercise' (exertion index; EI) than all other athletes and spent more time at sprinting intensities. Thus, there is congruence between our dual observations of firstly greater LM in NT associated with the T allele, and secondly a greater frequency of the T allele in certain elite rugby athletes who rely on greater power outputs relative to body mass to be successful in those specific playing positions Table 3; [39].
These data suggest the relevance of the FTO rs9939609 T allele to muscle-related phenotypes and subsequently, athletic success. When considering the possible molecular mechanism from FTO via IRX3 to OLIG2 resulting in greater lifelong motor neuron availability, this may have implications for muscle size-related disorders such as sarcopenia and cachexia.

Conclusions
The presented data show a novel dimension of FTO genetic variation in human physiology, by investigating in vivo muscle phenotypes in a healthy non-resistance trained population and relating those data to the extreme upper *Different from all other players (P = 8 x 10 -7 ), including front 5 (P = 3 x 10 -6 ) and back row (P = 0.005) end of human physical performancei.e. elite athletes. We show that the FTO rs9939609 protective T allele may be responsible for part of the inherited component of the interindividual variability in LM. Furthermore, elite athletes who rely greatly on LM relative to total body mass for athletic success (RU back three and centre players, in this study) also seem more likely to carry a protective T allele, have higher peak muscle power output relative to body mass and are likely to selected for appropriate playing positions as a result of these and other phenotypes. It is possible that this association between the FTO rs9939609 SNP, via LM, and athletic success, could be a result of the interaction with IRX3 in embryonic development of motor neuron patterning and the IGF-1 muscle development pathway. The strengths of the presented paper are the two-layered study design (NT and elite athlete cohorts) and the combination of muscle functional phenotypes with case-control data. While the finding that FTO genotype differs among elite rugby playing positions is a new insight, FTO is only one of many variants (most others unknown) that contribute to this phenotype and as such should not be used for talent identification at this time. Replication is necessary for each cohort using comparable methods, and future experimental focus should be on the proposed biological pathways of these FTO associations with muscle phenotypes.