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
Malhotra et al. Sports Medicine - Open
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
Seema Malhotra 0
Kiran Preet 0
Arvind Tomar 1
Shweta Rawat 0
Sayar Singh 0
Inderjeet Singh 0
L. Robert Varte 0
Tirthankar Chatterjee 0
M. S. Pal 0
Soma Sarkar 0
0 Defence Institute of Physiology and Allied Sciences (DIPAS), Ministry of Defence. Government of India , Lucknow Road, Delhi 110054 , India
1 Defence Research and Development Establishment (DRDE). Ministry of Defence, Government of India , Jhansi Road, Gwalior 474002, Madhya Pradesh , India
Background: Gorkhas, a sub-mountainous population of the Himalayan region, are known for strength and bravery. In the present study when “Gorkha” is used without brackets, we are mentioning Gorkhas of Tibeto-Burman origin. Physical capability, strength and endurance are important components of fitness associated with genetic traits. The aim of this study was to examine the endurance potential of male Gorkha soldiers, based on endurance-related genetic markers ACE I/D, ACTN3 Arg (R)577Ter(X), CKMM A/G NcoI and eNOS Glu(G)298Asp(T). Methods: Genotypic and allelic frequencies were determined in 374 male Gorkha soldiers (Tibeto-Burman). These frequencies were compared with frequencies obtained from Gorkha (Indo-Aryan), high-altitude natives (Tibeto-Burman) and Indian lowlanders (Indo-Aryan). “Total genotype score” (TGS) was calculated from accumulated combination of polymorphisms with maximum value “100” for theoretically “optimal” polygenic score. Probability of occurrence of “optimal” endurance profile was also determined. Results: ACE II genotypic frequency was highest in Tamangs followed by Gurungs, Rais, Limbus and Magars. No statistical difference in genotypic and allelic frequency of ACTN3 Arg(R)577Ter(X) was noted within the groups. Rais showed the highest CKMM A allele frequency (0.908) compared to other Gorkha (Tibeto-Burman) groups. Limbus and Tamangs showed the highest eNOS G allele frequency (0.938 and 0.915, respectively) compared to that of other groups. Probability of male Gorkha soldiers possessing a theoretically optimal polygenic endurance profile for four candidate polymorphisms was ~3.35% (1 in 30). Four percent of the population of male Gorkha soldiers (15 in 374) exhibited an optimal TGS 100, and 16% exhibited TGS 87 for endurance compared to male Indian soldiers belonging to the lowland (Indo-Aryan) and Gorkha (Indo-Aryan) populations suggesting an overall more “favourable” polygenic profile in the male Gorkha soldier (Tibeto-Burman) population. Conclusions: This study presents evidence of higher frequency of endurance-associated genes in the Gorkhas implying thereby that such genetically endowed individuals from the population may be selected and trained for achieving excellence in endurance-related elite sports activities.
Polygenic; Endurance; Gorkha; Indian; ACE; ACTN3; CKMM; eNOS
This study was conducted on male Gorkha soldiers
of the Tibeto-Burman linguistic phyla wherein
genetic studies are limited. The study reports for the
first time the genotypic and allelic frequencies of
four endurance-associated genetic markers in the
male Gorkha soldiers.
The study shows that nearly 4% of the male Gorkha
soldiers exhibit an “optimal” total genotypic score
(100) for endurance based on four polymorphisms,
indicating the genetic potential of this population for
achieving excellence in endurance-related elite
Gorkhas (also spelled as Gurkhas1) 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 . The soldiers in the British army were
mainly recruited from the “true Gorkha martial
tribes” of Gurung, Magar, Rai, Limbu, Thakur, Chhetri
and Sunawar ; 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 .
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 .
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 (VO2max) 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 VO2max . Higher
frequency of I allele of ACE (angiotensin-converting
enzyme gene, location: 17q23.3) have been reported in
Sherpas . ACE I allele is associated with
endurancerelated events [7, 8] and exercise performance in
atmospheric hypoxia . Predominance of ACE II genotype
and I allele was demonstrated in male Gorkha soldiers .
Many other polymorphisms are associated with
endurancerelated performance . 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  and
muscle performance  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 . 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 . A allele and AA genotype were
significantly higher in endurance athletes and were associated
with high values of VO2max . Physical fitness test scores
in military recruits were also associated with A allele .
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 . Higher frequency of wild G allele of eNOS
Glu298Arg (G894T) polymorphism was reported in
highaltitude 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)  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.
(a) Three hundred ninety-four healthy male Gorkha
individuals serving in the Gorkha regiment of Indian
Army, mean age 29 ± 8 years, participated in the
study. The clan ties were determined based on
self-report, and ethnic backgrounds were ascertained
through ethno-linguistic questionnaire. Classification
of linguistic phyla and clusters was as per van Driem
. For ascertaining that the individuals were
unmixed, both parents had to belong to the same
group. Based on the ethno-linguistic questionnaire,
the male Gorkha soldier participants belonged to five
ethnic groups of Tibeto-Burman linguistic phyla:
Gurungs (28 participants were from Nepal and 47
were from India), Magars (25 participants were from
Nepal and 101 were from India), Rais (37 participants
were from Nepal and 37 were from India), Tamangs
(36 participants were from Nepal and 18 were from
India) and Limbus (25 participants were from Nepal
and 40 were from India) (Table 1).
(b)Repository DNA samples from (i) Gorkhas
belonging to the Indo-Aryan linguistic phylum, (ii)
Indian lowlanders belonging to the Indo-Aryan
linguistic phylum and (iii) high-altitude natives of
Ladakh (elevation ~3500 m) belonging to
Tibeto-Burman linguistic phylum were used for
generating genotypic and allelic frequencies for
comparison with male Gorkha soldiers (Tibeto-Burman).
The repository DNA samples were from healthy male
soldiers of Indian Army.
Body weight, height, and body mass index (BMI), heart
rate, systolic and diastolic blood pressures were
measured prior to blood draw. Height was measured with an
Table 1 Details of participants based on ethno-linguistic
grouping (n = 394)
Region of origin
Linguistic phylum and
Note: Of the 394 participants, 20 individuals were hypertensive and excluded
from subsequent studies. The participants were two Gurungs (one from Nepal
and one from India), three Magars (two from Nepal and one from India), nine
Rais (six from Nepal and three from India), one Tamang (from Nepal) and five
Limbus (three from Nepal and one from India)
aClassification according to van Driem 
anthropometer (GPM, Switzerland). Body weight and
body mass index was measured by Body Composition
Analyser (Tanita, Korea). Resting heart rate, resting
systolic (SBP) and diastolic (DBP) blood pressures were
measured with digital blood pressure monitor (M2
Model, Omron Health Care Company Limited, Japan).
Hypertension was defined as BP ≥140/90 mmHg. Out of
394 individuals who initially participated (Table 1), 20
individuals were found to be hypertensive and were
excluded from all further studies. The rest of the
participants (n = 374) were normotensive and not on any
medications. Of the normotensive participants, 122 volunteers
consented for maximal oxygen uptake (VO2max). VO2max
was assessed on a bicycle ergometer (Ergoline Gmbh,
Lindenstr, Germany) using published protocol .
Briefly, the initial workload was 50 W, the increment
was 25 W · min−1, and the target cadence was 60–
70 rpm. Subjects kept the pedal rotational speed
between 60–65 rpm throughout the test. After warm up
for 2 min at 50 W, increments of 25 W were made
every minute till exhaustion. The test was completed
within 10–12 min including the warm up with
maximum load of 200–225 W at 60 rpm. During the
test, oxygen consumption (VO2), carbon dioxide
production (VCO2) and heart rate (HR) were
continuously recorded using a portable breath by breath gas
analysis system (K4b2, Cosmed Srl, Italy). Criteria for
reaching VO2max were assessed as reaching a plateau
despite the increase in work rate or a respiratory
exchange ratio value >1.1 or heart rate (HR) reaching
>90% of age-predicted maximum HR. All subjects
showed at least one of the above-mentioned criteria.
Venous blood samples (2–3 ml) were obtained at rest in
EDTA anticoagulant vacutainers (Beckton Dickinson,
CA, USA) and stored at −20 °C till further processing.
Genomic DNA samples had A260/280 ratio of 1.8–1.9
and were adjusted to 20 ng/μl. ~100 to 150 ng of
genomic DNA was used for polymerase chain reaction
(PCR) amplification in a total volume of 25 μl. Gene
variants studied for endurance status are shown in
Table 2. Primer sequences and detection of
polymorphism in ACTN3 Arg(R)577Ter(X) (rs1815739), ACE Ins/
Del (rs4646994) and eNOS Glu298Asp (rs1799983) were
performed as per published protocol [10, 23], and
detection of polymorphism in CKMM A/G (rs1803285) was
performed as per Rivera and co-workers . PCR
reaction was run on Gene Amp PCR system 9700 (Applied
Biosystem). Samples found to have ACE DDand ID
genotypes were reconfirmed by a second, independent
PCR amplification having an insertion-specific sequence
. Genotypic profiling for ACTN3 R577X, CKMM A/G
NcoI and eNOS Glu298Asp was performed on repository
T = 0.4008
C = 0.3403
T = 0.1763
Table 2 Gene variants studied for endurance status
DNA samples of Gorkhas (Indo-Aryan), Indian lowlanders
(Indo-Aryan) and high-altitude natives of Ladakh
(TibetoBurman). The experiments were conducted in accordance
with the quality control measures at the Department of
Molecular Biology, Defence Institute of Physiology and
Allied Sciences, Delhi, which is an accredited laboratory
Allele frequencies and genotype frequencies were
calculated by allele counting and analysed by Pearson
chisquare (χ2) and Fisher’s exact test, respectively.
Deviations from the Hardy-Weinberg equilibrium (HWE)
were tested for the polymorphisms by comparing
observed and expected genotype frequencies with an exact
goodness of fit test. For comparison of genotype
frequencies within Gorkha sub groups, statistical
significance was accepted at p < 0.0025 after adjustment with
Bonferroni’s correction (alpha = 0.05/20). For inter
population comparisons, statistical significance was accepted
at p < 0.05.
Comparison of maximal oxygen uptake (VO2max)
between the groups and association of VO2max with
genotypes and TGS was performed by one-way analysis of
variance (ANOVA) using the software SPSS (Statistical
Package for Social Sciences, version 17.0 for Windows;
SPSS Inc., Chicago, IL, USA).
Probability of “optimal” polygenic profile for endurance
in the Gorkha soldiers (Tibeto-Burman)
Probability of any given individual possessing the
“optimal” genotype from one up to all four polymorphisms,
ranked on official gene symbols placed in an alphabetical
order, was calculated by using the typical frequency
distribution of the genotypes. Based on the typical
frequencies of the “optimal” genotypes in male Gorkha soldiers
(Tibeto-Burman) and Indian lowlander soldiers
(IndoAryan), a scale was generated indicating the probability of
possessing the “optimal” genetic profile which was then
applied to the population. The genetic potential for
endurance phenotype of the population was computed by using
the algorithm proposed by Williams and Folland , and
a total genotype score (TGS) was produced. TGS is a
bioinformatic approach using predictive algorithm
“genotype score” for finding the probability of individuals
carrying the preferable genotype for each polymorphism linked
to a phenotype . TGS modelling approach uses a
simple algorithm resulting from the best accumulated
combination of candidate gene polymorphisms. We chose
this approach to provide a quantitative way of combining
genotype data to predict a complex phenotype. Each
genotype within each polymorphism was scored and a
combined influence of polymorphisms was computed. A
genotype score (GS) of 2 was assigned to the “optimal”
homozygous genotype for endurance. A genotypic score
of 0 was assigned to “less favourable” homozygous
genotype for endurance while the heterozygous genotype
received a score of 1. The genotype scores of each
single genotype (GSACE + GSACTN3 + GSCKMM + GSeNOS)
were added up and a TGS was produced as follows:
where n is the number of polymorphisms studied. As
suggested by Williams and Folland , a TGS of 100
represents the “perfect” polygenic profile of endurance
(all GS are 2) and a TGS of 0 represents the “not
perfect” profile of endurance. Frequency distribution of the
“optimal” endurance genotype in the Gorkha soldiers
(Tibeto-Burman) obtained in the present study was
compared with the frequency distribution of the
optimal endurance genotype in other populations viz.,
Gorkhas (Indo-Aryan), high-altitude natives (HAN)
from Ladakh (Tibeto-Burman), Indian lowlanders
(Indo-Aryan), Indian Gujaratis from Houston
(IndoAryan), HAN Chinese Beijing (Sino-Tibetan), Japanese
from Tokyo and Caucasian (European ancestry) taken
from published literature and databases.
To examine the distribution of TGS in the Gorkhas
(Tibeto-Burman), we created a dataset of 100,000 virtual
Gorkha (Tibeto-Burman) individuals from genotypic
frequencies of the four polymorphisms obtained in the
present study with randomly generated genotypes
computed by online software GEMINI . Similarly, a
hypothetical data of 100,000 virtual individuals in each
population of Gorkhas (Indo-Aryan), Indian lowlanders
(Indo-Aryan) and high-altitude natives (Tibeto-Burman)
was computed with randomly generated genotypes based
on the population-specific genotype frequencies.
Distribution of TGS within these virtual populations was
examined, and mean and kurtosis statistics were
calculated using SPSS, v 17.0 for Windows. The TGS of
simulated Gorkha (Tibeto-Burman) population was then
compared to the average genotype score of the three
hypothetical population datasets.
Of the 394 individuals, 151 were from Nepal and 243
were from India. The participants belonged to Gurung,
Magar, Rai, Tamang and Limbu groups of
TibetoBurman linguistic phyla (Table 1). Twenty individuals
(3.26%) were found to be hypertensive [systolic blood
pressure (SBP), 152.65 ± 10.08 mmHg; diastolic blood
pressure (DBP), 98.25 ± 6.67 mmHg] and were excluded.
Within the remaining population, systolic blood pressure
and heart rate were observed to be statistically similar
(Table 3). Body mass index and diastolic blood pressure
(DBP) were significantly different within the population
with Rais and Limbus showing the highest DBP (Table 3).
Statistically significant difference in VO2max was noted
between the groups (Table 4). VO2max was highest in
Tamangs (55.80 ± 8.27) followed by Limbus (55.34 ± 6.26)
(Table 4, Additional file 1: Table S1).
Genotypic and allelic frequency distribution of the
Overall genotypic frequency of the studied polymorphism
in the male Gorkha soldiers is shown in Table 5.
Distribution of homozygous II genotype was highest in Tamangs
followed by Rais, Gurungs, Limbus and Magars; although,
the difference was not statistically significant within the
subgroups after Bonferroni correction (p < 0.0025).
Genotypic frequency of ACTN3 Arg(R)577Ter(X) was
statistically similar across the Gorkha subpopulation. Both
ACE Ins/Del and ACTN3 Arg(R)577Ter(X) polymorphisms
were in Hardy-Weinberg Equilibrium (HWE). Statistical
difference was noted in allele frequency distribution
of ACTN3 Arg(R)577Ter(X) between Gorkhas
(TibetoBurman), Gorkhas (Indo-Aryan) and high-altitude
natives (p < 0.05) (Table 6).
Higher frequency of A (major) allele of CKMM A/G
and G (major) allele of eNOS Glu298Argpolymorphism
was observed in the population (Table 5). Frequency of
homozygous AA genotype of CKMM A/G was higher
than homozygous mutant GG genotype in the population.
Rais showed the highest CKMM A allele frequency
compared to other Gorkha (Tibeto-Burman) groups.
Frequency of homozygous GG genotype of eNOS Glu298Arg
was also higher than homozygous mutant TT genotype.
Limbus and Tamangs showed the highest eNOS G allele
frequency compared to other groups. Homozygous
mutant TT genotype of eNOS Glu298Arg was not observed in
Tamang and Limbu groups (Table 5). Both the
polymorphisms were in HWE. Of the 81 possible combinatorial
genotypic profiles, 34 profiles were not observed. The
predominant genotype combination was ACE ID/ACTN3
RX/CKMM AA/eNOS GG which was present in
approximately 11.5% of the population (Additional file 2:
Table S2). The optimal endurance-associated homozygous
genotypic combination of ACE II/ACTN3 XX/CKMM
AA/eNOS GG was present in nearly 4% of the male
Gorkha soldier population (Additional file 2: Table S2).
Although VO2max values showed statistical difference
within the subgroups, it did not show any statistically
significant association with the genotypic profiles either
individually or in combination (Additional file 3: Table S3
and Additional file 4: Table S4).
Probability of optimal polygenic profile for endurance
performance in the Gorkha (Tibeto-Burman) population
Typical genotypic frequencies were used for calculating the
probability of possessing optimal genetic profile for
endurance in the male Gorkha soldiers (Tibeto-Burman) as
compared to lowlander Indian population (Table 7). The typical
genotypic frequencies of the four polymorphisms ranged
Body weight (Kg)
(n = 374)
(n = 73)
(n = 123)
(n = 60)
(n = 53)
Values are mean ± SD, significant value <0.05. The significant p values are italicized
The values are from participants who were normotensive
BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, HR heart rate, TB Tibeto-Burman
(n = 65)
Level of significance (p)
from 25% for II genotype of ACE to 65% of GG genotype of
eNOS in lowlanders and 43% for II genotype of ACE to 80%
of GG genotype of eNOS in male Gorkha soldiers (Table 7).
Probability of any given lowlander soldier possessing the
“favourable” genotype for endurance at one locus (II
genotype of ACE) was ~25% which was reduced to ~6.5% when
the second polymorphism (XX genotype of ACTN3) was
added. The probability of any lowlander possessing the
optimal polygenic profile for endurance at all four loci further
reduced to 1.88% with an approximate odds ratio of 1:78
(i.e., one in 78 individuals) (Table 7). In the male Gorkha
soldiers (Tibeto-Burman), probability of possessing the
favourable genotype for endurance for one polymorphism
(II genotype of ACE) was ~43% which was reduced to
~6.5% when the second polymorphism (XX genotype of
ACTN3) was added. The probability of any male Gorkha
soldier possessing the optimal polygenic profile for
endurance at all four loci was ~3.35% with an approximate odds
ratio of 1:30 (i.e., one in 30 individuals) (Table 7). The
typical endurance-associated optimal genotypic frequency
in the male Gorkha soldiers (Tibeto-Burman) ranged from
15% for XX genotype of ACTN3 to 80% of GG genotype of
eNOS (Table 8). Frequency distribution of the optimal
endurance genotype in the male Gorkha soldiers and other
Asian and Caucasian populations is shown in Table 8.
The mean ± SD of total genotype score (TGS) was
69.05 ± 15.08 and kurtosis statistics 0.106 ± 0.252, and
distribution was shifted towards the right in the male
Gorkha soldiers (Fig. 1). About 4% of the male Gorkha
soldiers (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
(Fig. 1, Additional file 5: Table S5). The lowest limit of
genetic potential for endurance in male Gorkha soldiers
was TGS ~25 with only 4 individuals having this score.
On scrutiny, it was seen that two of these four
individuals possessed ACE DD/ACTN3 RR genotypic
combination, another possessed ACE ID/ACTN3 RR and the
fourth one possessed ACE DD/ACTN3RX (Additional
file 6: Table S6), genotypic combinations which are
suggested to be favourably associated with muscle strength/
power phenotypes . Comparison of TGS (maximum
100 and minimum 25) with physiological characteristics
(SBP, DBP and HR) and VO2max did not show any
statistical difference (Additional file 7: Table S7).
Frequency distribution of TGS calculated from the
virtual samples is depicted in Fig. 2. In the simulated
population of Gorkhas (Tibeto-Burman), mean ± SD
TGS was 69.01 ± 15.40 and kurtosis statistics (−) 0.094 ±
0.015 (SE); 4% (4046 individuals out of 100,000) showed
TGS 100 while 17.6% (17,591 individuals out of 100,000)
had TGS 87 (Fig. 2) corroborating the observation
obtained from the representative 374 participants of the
present study (4% with TGS 100 and 16% with TGS of
87 (Fig. 1, Additional file 5: Table S5). In the simulated
population of high-altitude natives, predicted mean ± SD
TGS was 70.06 ± 15.66 and kurtosis statistics (−) 0.168 ±
0.015 (SE) with 5.2% (5174 individuals out of 100,000)
having optimal TGS 100 and another 19.5% (19,487
individuals out of 100000) having TGS 87 (Fig. 2). In
simulated Gorkha (Indo-Aryan) population, mean ± SD TGS
was 67.44 ± 16.10 and kurtosis statistics (−) 0.176 ± 0.015
(SE); TGS 100 was observed in 3.8% (3761 out of 100,000
individuals), and TGS 87 was observed in 16.2% (16,199
individuals out of 100,000) (Fig. 2). In the simulated
population of Indian lowlanders, the predicted mean ± SD TGS
was 62.46 ± 16.66 and kurtosis statistics (−) 0.217 ± 0.015
(SE); TGS 100 was observed in 2.1% (2061 out of 100,000
individuals) and TGS 87 was in 10.5% (10530 individuals
out of 100000) (Fig. 2). Number of Gorkha soldiers with
Genotypica frequency Alleleb frequency
Table 6 Comparison of genotypic and allelic frequencies of studied polymorphisms in male soldiers belonging to Gorkha (TB),
Gorkha (IA), HAN (TB) and Indian lowlander (IA)
Indian Gorkha (TB) vs Gorkha (TB)
lowlander (IA) Gorkha (IA) vs HAN (TB)
Gorkha (TB) Gorkha (IA) Gorkha (IA)
vs Indian vs HAN vs Indian
lowlander (IA) (TB) lowlander (IA) lowlander (IA)
Level of significance (p)*
rs1799752 ACE I/D
TB Tibeto-Burman, IA Indo-Aryan, HAN high-altitude native from Ladakh
*Significance for genotypic and allelic frequency is assumed when p < 0.05. The significant p values are italicized
aData from 
Table 7 Probability of possessing “optimal” genetic profile by number of polymorphism in the male Gorkha (TB) and Indian
lowlander (IA) soldiers
Typical frequency (%)
of “optimal” genotype
Typical frequency (%)
of “optimal” genotype
Probability of possessing
Probability of possessing
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TGS 100 was significantly higher than the Indian lowlander
soldiers with TGS 100 (p < 0.0001) (2 × 2 contingency table;
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
Fig. 2 Frequency distribution of total genotype scores (TGS) derived from dataset of 100,000 randomly generated individuals of Indian lowlanders
(IA), high-altitude native (TB), Gorkhas (IA) and Gorkhas (TB)
(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 . 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,  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 . 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
ACTN3 gene is associated with performance and
genotype across multiple cohorts of elite power athletes and
also supported by gene knockout mouse model . 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)  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
populationspecific difference between Caucasian and Asian
(TibetoBurman/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
Creatine kinase is an important enzyme in energy
metabolism which catalyzes phosphorylation of creatine
to phosphocreatine, an energy storage molecule and
source of ATP . 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
endurancerelated sports activity . 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 . The frequency
of the CKMM A allele varies from 85% in the Chinese
population  to 68% in white Americans  with
Caucasoids having 65–71% . 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  and Quechuas of Andean
altpino . Higher exhaled NO has also been observed
in Tibetan and Bolivian Aymara population [48, 49].
Maximum oxygen uptake capacity (VO2max), 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 . 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 VO2max 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 VO2max is reflected in better
performance in running, hill climbing and endurance
work [53, 54]. VO2max 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
(TibetoBurman) at the physiological level. We did not,
however, observe statistically significant association
between VO2max, 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 VO2max (Additional file 3: Table S3 and
Additional file 4: Table S4). It is probable that the small
cohort of individuals for VO2max may be limiting the
interpretation of the result or it may also be probable that
VO2max is indeed not associated with the studied
polymorphisms. Rankinen and co-workers  analysed elite
endurance athletes with VO2max 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 . It
appears logical to suggest that the study of influence of
genotypes on the VO2max 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 . 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.  studied association of
324,611 SNPs with the response of VO2max 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
VO2max trainability . 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 .
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 . 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 . 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.vassar
stats.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 . 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.
It may be argued that the soldiers used in the present
study may not be representative of the entire Gorkha
population as recruitment in Indian Army may be
selective. For examining this issue, we compared the genotypic
frequencies obtained from participants of Indian Army
with those reported from general Indian population
(Table 9). The genotypic frequency distribution in both
the army and general population was not statistically
different (except for ACTN3), substantiating the fact that
the genotypic frequency distribution obtained from
soldier population of the Indian Army is generally
reflective of the frequency in general Indian population. As an
extension of this observation, we state that the data
obtained in the Gorkhas (Tibeto-Burman) in the present
study reflects the general Gorkha population frequency.
The present study, however, is not without limitation
as only four endurance-associated polymorphisms
were studied. These four polymorphisms were chosen
because of definitive information about their
association with endurance, exercise performance, muscle
performance, generation of rapid forceful contraction,
high values of VO2max and endurance elite status and
also for reason that frequency data for these
polymorphisms were available in the Indian population for
comparison and computation of TGS. Further studies
Table 9 Comparison of genotypic and allelic frequency distribution between participants of the Indian Army and general
Indian Army General population
rs41317140 REN C/T
are required in larger cohorts and with additional
endurance-related polymorphisms to evaluate the role
of genetic variations in determining sports
performance. It is also mentioned that in absence of objective
data, we have used the extension of the observation
seen in general Indian population and Indian army to
state that the data obtained in the male Gorkha
soldiers in the present study reflects the general Gorkha
The findings of the present study imply that young
talented adolescents from the high hill population,
having polygenic endurance profiles linked with higher TGS
could be selected and given appropriate training in
endurance-related sports activities with prospects of
competing in Olympics and other sports events held in
high-altitude environment. Earlier studies have shown
that whole body physiological variables related to O2
consumption are influenced by the level of training of
the individual [68–71]. It has been shown that long-term
endurance training in a coordinated fashion impacts
muscle epigenetics and affects thousands of methylation
sites and genes associated with improvement in muscle
function and health . Appropriate training combined
with favourable genetic profile will definitely be
advantageous for achievement of elite athletic status by this
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
(TibetoBurman) exhibit an optimal total genotypic score (100)
for endurance, indicating genetic potential of this
population for achieving excellence in endurance-related
elite sports activities.
1The Gorkhas derive their name from Guru Gorakhanath
of eighth century. The shrine dedicated to Guru
Gorakhnath was part of Gorkha Durbar built on a
rocky crag in Nepal in 1639 from where the Gorkhas
began their soldiering and where the ancient army
called “Gurujiko Paltan” was raised. The
400-yearold shrine dedicated to Guru Gorkhnath was
devastated in the massive earthquake that struck Nepal on
25 April 2015.
Additional file 1: Table S1. One-way ANOVA (PostHocTukey test) of
VO2max in five subpopulation of male Gorkha soldiers (TB). (DOCX 22 kb)
Additional file 2: Table S2. Frequency of combined genotype
distribution in male Gorkha soldiers. (DOC 106 kb)
Additional file 3: Table S3. Descriptive statistics and comparative
analysis of maximal oxygen uptake (VO2 max in ml kg−1 min−1) between
genotypes and groups. (DOC 36 kb)
Additional file 4: Table S4. Association analysis of combinatorial
genotype profiles with maximal oxygen uptake (VO2max in ml kg−1 min−1).
(DOC 36 kb)
Additional file 5: Table S5. Frequency distribution of total genotype
score (TGS) from male Gorkha soldiers (n = 374). (a) Tabular information
of TGS. (b) Graphical representation of TGS. (DOC 85 kb)
Additional file 6: Table S6. Total genotype score and percentage of
frequency for all the possible combinations of four polymorphisms in
male Gorkha soldiers. (DOC 89 kb)
Additional file 7: Table S7. Comparison of physiological characteristics
and VO2max in male Gorkha soldiers grouped according to total genotype
score (TGS). (DOCX 32 kb)
Authors wish to thank the Army HQ SD branch, the Commanding Officers of
the Units and the volunteers who participated in the study. Authors also
thank Neha Thakur for the typing of the manuscript.
This work was supported by the Defence Research and Development
Organization Grant No. S&T-09/DIP-251 (C6.0. SR) at the Defence Institute of
Physiology and Allied Sciences.
SS conceived the study and coordinated and wrote the manuscript. SR,
SaS, IS, LRV, TC and MSP collected the samples and participated in the
physiological measurements. SM, KP and AT performed the genetic
experiments and statistical analysis. All authors read and approved the
The authors, Seema Malhotra, Kiran Preet, Arvind Tomar, Shweta Rawat, Sayar
Singh, Inderjeet Singh, L. Robert Varte, Tirthankar Chatterjee, M.S Pal and
Soma Sarkar declare that they have no conflict of interest.
Ethics approval and consent to participate
This study was approved by the Institutional Ethics Committee of Defence
Institute of Physiology and Allied Sciences (DIPAS), Delhi. All experiments
were conducted as per Ethical Guidelines for Biomedical Research on
Human Participants of Indian Council of Medical Research (2006), and the
procedures and protocols followed were in accordance with the Declaration
of Helsinki for Human Research. The participants were part of an ongoing
research study at the Institute, and provided written informed consent after
explanation of the study.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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