Polygenic study of endurance-associated genetic markers ACE I/D, ACTN3 Arg(R)577Ter(X), CKMM A/G NcoI and eNOS Glu(G)298Asp(T) in male Gorkha soldiers

Gorkhas (also spelled as Gurkhas ¹) are a sub-mountainous population of the Himalayan region (Nepal) and make excellent soldiers. During the Anglo-Nepalese war of 1814–1816, the British were greatly impressed by the bravery of the Nepalese soldiers and started recruiting Nepalese to the Gurkha regiments of the British Indian Army [1]. The soldiers in the British army were mainly recruited from the “true Gorkha martial tribes” of Gurung, Magar, Rai, Limbu, Thakur, Chhetri and Sunawar [2]; the Indian Army continues to recruit from the same brigade of Gorkhas. Gurungs, Magars, Rais, Limbus, Tamangs and Sherpas are associated with Tibeto-Burmese cultural traditions and physical features conventionally labelled as Mongoloid while Thakur (Brahmin) and Chhetri castes are associated with Aryan cultural traditions and have physical features conventionally labelled as Caucasoid [3]. Gurkha Service opened an opportunity for the soldiers to settle in different parts of the British Empire including India, and their descendants are present in Assam, Sikkim, Darjeeling and Dehradun [3].

The Gorkha soldiers are best known for their physical strength, fighting tenacity, bravery and fearlessness in battle [1, 4]. Physical capability, strength and power are important components of fitness. Himalayan Sherpas are well known for their physical strength and endurance in the high-altitude terrain. Himalayan Sherpa elite climbers demonstrated high functional reserve with maximal oxygen uptake (VO_2max) of 66.7 ± 3.7 ml min⁻¹ kg⁻¹, maximal cardiac frequency of 199 ± 7 beats min⁻¹ and ventilatory anaerobic threshold of 62 ± 4% of VO_2max [5]. Higher frequency of I allele of ACE (angiotensin-converting enzyme gene, location: 17q23.3) have been reported in Sherpas [6]. ACE I allele is associated with endurance-related events [7, 8] and exercise performance in atmospheric hypoxia [9]. Predominance of ACE II genotype and I allele was demonstrated in male Gorkha soldiers [10]. Many other polymorphisms are associated with endurance-related performance [11]. Arg(R)577Ter(X) polymorphism of ACTN3 (alpha actinin 3 gene, location: 11q 13.1) (functional R allele and non-functional X allele) is associated with generation of rapid forceful contractions [12] and muscle performance [13] with frequency of XX-null genotype (loss of alpha actinin 3) being higher in endurance athletes [14, 15]. In an earlier study, XX genotype of ACTN3 Arg(R)577Ter(X) polymorphism was observed to be present in 23% of Gorkhas [10]. The A/G NcoI polymorphism of CKMM (muscle-specific creatine kinase gene, location: 19q13.32) is associated with energy-buffering in the skeletal muscle fibres along with tolerance to skeletal muscle damage [16]. A allele and AA genotype were significantly higher in endurance athletes and were associated with high values of VO_2max [17]. Physical fitness test scores in military recruits were also associated with A allele [18]. eNOS Glu(G)298Arg(T) (endothelial nitric oxide gene, location: 7q36) polymorphism is linked with endurance performance and endurance elite status [19, 20]. eNOS encodes the rate-limiting enzyme for nitric oxide (NO) products [21]. Higher frequency of wild G allele of eNOS Glu298Arg (G894T) polymorphism was reported in high-altitude natives from Ladakh suggesting advantageous consequences in high-altitude environment [22, 23].

High functional reserve, physical strength and endurance in the hostile high altitudes coupled with higher frequency distribution of some of the endurance-related performance enhancing genetic markers in the mountain population indicates possibility of the mountain population being genetically endowed for endurance- related activities. This natural endowment would set the stage for investigation of prospects of the mountain people for distinctive performance in endurance-related elite sports activities. In the present study, we chose to investigate in male Gorkha soldiers, (i) the genotypic and allelic frequency distribution of four genetic variants associated with endurance performance: ACE I/D (rs4646994), ACTN3 Arg(R)577Ter(X) (rs1815739 C/T), CKMM A/G NcoI (rs8111989 T/C) and eNOS Glu298Asp (rs1799983 G/T); (ii) determine the probability for the occurrence of an “optimal” polygenic endurance profile using the four polymorphisms in the population and assess whether individuals were likely to exist who harboured “preferable” genotypes for endurance; and (iii) generate a “total genotype score” (TGS) [11] for finding a likely distribution of genetic endurance potential of the male Gorkha soldiers and compare with virtual data of other populations of male Indian soldiers.

The Gorkha population is largely an understudied population and genetic information on the population is scanty. This study reports for the first time the genotypic and allelic frequency distribution of endurance-related four polymorphic markers in the male Gorkha soldiers of Tibeto-Burman linguistic cluster and the polygenic endurance potential of the population. Ethnic heterogeneity with respect to ACE I/D polymorphism was noted with a trend of higher frequency of ACE II genotype and I allele in Tamangs. It may be mentioned here that the Gorkhas included in this study were main ethnics (tribes): Gurungs, Magars, Rais, Tamangs and Limbus. They look similar but are very different ethnics and could be having their own distinct genetic makeup. Thus, the frequency differences within the subgroups (although not significant after Bonferroni correction, p < 0.00250), could be attributed to their evolutionary adaptation. The observed genotypic frequency of ACE I/D in Tamangs was in agreement with frequency distribution reported from inhabitants of Kotyang, majority of whom were Tamangs [30]. Tamangs are indigenous inhabitants of the western Himalayan regions and one of the major Tibeto-Burman speaking communities who trace their ancestry to Tibet and further back to Mongolia (Tamang people. World Public Library-eBooks). Higher I allele frequency in Tamangs, similar to that found in Sherpas, suggests enhanced physical activity in the group. Predominance of homozygous II genotype and I allele of ACE in Gorkhas in the present study is in agreement with that observed in Gorkhas reported earlier, [10] majority of whom belonged to Indo-Aryan ethnicity. Predominance of ACE I allele in the Gorkhas suggests endurance and muscle efficiency [31, 32], the key determinants of performance and also associated with enhanced performance at high altitudes [31]. ACE I allele influences human physical performance and trainability [33, 34] and is also related to cardiorespiratory efficiency [35, 36]; although, contrary reports also exist showing no relationship between ACE I/D polymorphism and cardiorespiratory fitness [37].

ACTN3 gene is associated with performance and genotype across multiple cohorts of elite power athletes and also supported by gene knockout mouse model [38]. Ter (X) allele affects endurance ability of elite athletes while Arg (R) allele affects sprinting [13, 39]. It would be interesting to further investigate the polygenic potential of ACTN3 RR genotype (associated with sprinting) along with power-related muscle performance genes in this population. ACTN3 Ter (X) allele and the TerTer (XX) genotypes are significantly associated with certain groups of elite endurance athletes [14, 40]. The frequency distribution of Ter (X) allele in the male Gorkha soldiers is observed to be 0.394 with genotype frequency being 0.155 XX and 0.366 RR. Interestingly, the XX genotype frequency in the male Gorkha soldiers was observed to be lower than Gorkha and Indian lowlander soldiers belonging to Indo-Aryan linguistic phylum as well as Caucasians (Table 8). Similar to the frequency observed in male Gorkha soldiers (Tibeto-Burman), the XX genotype frequency has been reported to be ~0.170 in other Asian populations viz., HAN Chinese (CHB, Sino-Tibetan linguistic phylum) [41] and Japanese (HapMap Phase 3) while in Caucasians, the XX frequency is 0.22 (HapMap Phase 3). At the moment, we do not have an explanation for less XX genotype frequency in the Gorkha (Tibeto-Burman) population compared to Caucasian population; this appears to be a population-specific difference between Caucasian and Asian (Tibeto-Burman/Sino-Tibetan) population. The derived 577X allele has been shown to increase in frequency with distance from Africa, reaching the highest frequencies on the American continent [42].

Creatine kinase is an important enzyme in energy metabolism which catalyzes phosphorylation of creatine to phosphocreatine, an energy storage molecule and source of ATP [43]. Muscle-specific creatine kinase gene (CKMM) correlates with athletic performance and CKMM AA genotype is considered as one of the genetic markers associated with predisposition to endurance-related sports activity [17]. The CKMM A allele probably influences gene expression and results in a decrease in muscle isoform of creatine kinase activity in myocytes leading to enhanced activation of oxidative phosphorylation and endurance development [17]. The frequency of the CKMM A allele varies from 85% in the Chinese population [44] to 68% in white Americans [16] with Caucasoids having 65–71% [45]. In the male Gorkha soldiers, frequency of CKMM A allele was 80.34% and AA genotype 65.50% which was comparable to that observed in high-altitude natives from India. Interestingly, frequency of AA genotype was significantly higher in Gorkhas of Tibeto-Burman ethnicity as compared to Gorkhas of Indo-Aryan ethnicity. Higher frequency distribution of Glu(G)allele of eNOS compared to that of Asp(T) allele in the Gorkhas, similar to the frequencies observed in high-altitude natives, suggests adaptive advantage of Glu(G) allele through increased production of nitric oxide (NO). Higher frequency of Glu(G) allele has been reported in Sherpas [46] and Quechuas of Andean altpino [47]. Higher exhaled NO has also been observed in Tibetan and Bolivian Aymara population [48, 49].

Maximum oxygen uptake capacity (VO_2max), the maximal amount of oxygen per unit of time that can be delivered to the peripheral organs including the skeletal muscle (where it is used to sustain muscular contraction at peak exercise), is a bench mark measure of physical performance/work capacity [50]. It provides an index of functional reserve of the organ systems involved and limitation that can be encountered at peak exercise [51, 52]. In the present study, the overall VO_2max in the population was found to be 51.34 ± 7.22 ml kg ⁻¹ min ⁻¹ with Tamangs showing the highest maximal oxygen uptake compared to other subgroups (p < 0.05) (Table 4). Higher VO_2max is reflected in better performance in running, hill climbing and endurance work [53, 54]. VO_2max in the male Gorkha soldiers was higher as compared to the values reported from Indian general population [55–57] suggesting overall better endurance potential of Gorkhas (Tibeto-Burman) at the physiological level. We did not, however, observe statistically significant association between VO_2max, and the studied polymorphisms in the Gorkhas either individually with four genetic variants or when combined genotype profile of each individual was analysed in the 122 volunteers who participated in the assessment of VO_2max (Additional file 3: Table S3 and Additional file 4: Table S4). It is probable that the small cohort of individuals for VO_2max may be limiting the interpretation of the result or it may also be probable that VO_2max is indeed not associated with the studied polymorphisms. Rankinen and co-workers [58] analysed elite endurance athletes with VO_2max values over 83 ml kg⁻¹ and found no trend for excess I allele or low number of DD homozygotes of ACE. A genomic scan for maximal oxygen uptake in Caucasian families of the HERITAGE Family study reported many potential candidate gene loci on many chromosomal regions, but no linkage was observed on chromosome 17 on the ACE locus [59]. It appears logical to suggest that the study of influence of genotypes on the VO_2max should be conducted in an increased cohort of healthy individuals for genetic association to be more evident. In addition to maximal rate of oxygen uptake, at least two other endurance phenotypes (economy of movement and lactate/ventilator threshold) also contribute to endurance performance phenotype (time taken to travel a given distance) as seen in elite competition [11]. A fourth phenotype, namely oxygen uptake kinetics, may also be discretely related. These phenotypes are yet to be associated with specific genetic polymorphisms in a healthy adult population. Unbiased genome-wide approaches have been used in search for genomic region, transcripts and DNA variants linked or associated with endurance performance related trait [60–63]. Bouchard et al. [60] studied association of 324,611 SNPs with the response of VO_2max to endurance training in 473 Whites from HERITAGE. None of the SNPs reached genome-wide significance even though there were several SNPs moderately associated with VO_2max trainability [60]. A meta-analysis of genome-wide association study of two cohorts of elite endurance athletes and controls revealed only one statistically significant marker (rs558129 at GALNTL6 locus, p = 0.0002), and no panel of genomic variation common to the elite candidate athletic group was identified [64].

Human physical capability is influenced by many environmental and genetic factors, and it is generally accepted that physical performance phenotypes are highly polygenic [65, 66]. Potential for elite human physical performance is limited by the similarity of polygenic profiles with 99% of people differing by no more than seven genotypes from the typical profile [11]. The predicted mean TGS for favourable endurance profile in endurance elite athletes was significantly higher (70.2 ± 15.6) compared to general Spanish population (60.70 ± 12.21) and about 3000 individuals out of a total population of ~41 million people were predicted to have theoretically optimal polygenic profile (TGS = 100) for seven candidate genes [27]. In the simulated population of Gorkhas in the present study, mean TGS obtained was 69.01 ± 15.40 for favourable endurance. It was interesting to note that 4% of the population (15 individuals out of 374) exhibited an optimal TGS (100) for endurance with another 16% of the population (60 individuals out of 374) showing a TGS of ~87. Compared to Gorkhas, only 2 and 10% of Indian lowlanders had TGS 100 and TGS 87, respectively. Analysis of TGS 100 and TGS 87.5 between Gorkha (Tibeto-Burman) and Indian lowlanders, using 3 × 2 contingency table (www.vassarstats.net/newcs.html), demonstrated significant difference between the cohorts (p < 0.0001) suggesting an overall more favourable polygenic endurance profile in Gorkha population. With 4% of the male Gorkha soldiers (who are otherwise drawn from the general population) having the optimal endurance profile, this would mean that nearly 1.7 million, out of an estimated 43 million (31 million of Nepalese from Nepal and about 12 million Nepalese domiciled in India, Nepal Census 2011) would have the optimal endurance profile. Even if new candidate polymorphisms are scored, probability of presence of individuals with optimal genotypes for endurance still remains large in this population. The Gorkhas are thus genetically endowed with the endurance phenotypes; they have the genetic advantage with higher co-occurrence of ACE II/ACTN3 XX/CKMM AA/eNOS GG in the population. Genetic endowment coupled with high-intensity training which considerably increases maximal oxygen uptake, of young adolescent Gorkhas will definitely bring performance improvement in many sporting events. It may not be farfetched to postulate that such individuals can rewrite the world records. It is accepted that in addition to genetic potential and physical environment, a performance record is a function of economic and social opportunity. Individuals from small geographical area hold many world athletic records. Although genetic explanations is lacking, there are considerable regional and ethnic variations in typical frequencies of many genotypes e.g., frequency of ACE II is higher in some regions of Oceania than most of Europe while athletes of north and east African descent excel in endurance events and those of west African descent excel in sprint events [67]. Interestingly, 5% of high-altitude natives (Tibeto-Burman) also had TGS 100 and 19% had TGS 87 similar to the TGS frequency observed in Gorkhas (Tibeto-Burman). The observation presents a scope for us to state that Tibeto-Burman mountain population per se has higher TGS score linked to endurance genotype than the lowlander Indian population.

The present study provides a novel perspective on endurance genetics based on polygenic profiling in the Gorkha population. The study reports for the first time the genotypic and allelic frequencies of four endurance-associated genetic markers in the male Gorkha soldiers (Tibeto-Burman). The study also highlights that nearly 4% of the Gorkha soldiers (Tibeto-Burman) exhibit an optimal total genotypic score (100) for endurance, indicating genetic potential of this population for achieving excellence in endurance-related elite sports activities.