AGE (2013) 35:2477–2483

DOI 10.1007/s11357-013-9526-y

Association between polymorphisms in the TRHR gene, fat-free

mass, and muscle strength in older women
Cláudia C. Lunardi & Ricardo M. Lima &
Rinaldo W. Pereira & Tailce K. M. Leite &
Ana B. M. Siqueira & Ricardo J. Oliveira

Received: 16 August 2012 / Accepted: 12 March 2013 / Published online: 2 April 2013
# American Aging Association 2013

Abstract A previous genome-wide association study average, an extra 1 kg and 900 g of AFFM when com-
suggested that polymorphisms in the thyrotrophin- pared to C/C genotype carriers. Also, the C/C genotype
releasing hormone receptor (TRHR) gene contribute to group presented a significantly higher chance to have
fat-free mass (FFM) variation. The aim of the present reduced muscle strength. The observations presented
study was to examine the association between polymor- here provide further evidence that the rs16892496 poly-
phisms in the TRHR gene with FFM and muscle strength morphism in the TRHR gene may play a role in FFM
in older women. Volunteers (n = 241; age = 66.65 ± variation. Moreover, the results bring the novel insight
5.5 years) underwent quadriceps strength assessment that this genetic variant can present a modest contribu-
using isokinetics and fat-free mass by dual-energy X- tion to muscle strength in older women.
ray absorptiometry. TRHR polymorphisms and ancestry-
informative markers were genotyped through standard Keywords Aging . Sarcopenia . Genetic variation
procedures. No significant difference was observed for
rs7832552. Regarding the rs16892496, ANCOVA re-
vealed that appendicular fat-free mass (AFFM) and rel- Introduction
ative AFFM were significantly different between groups
(p=0.04 and p=0.05, respectively). Individuals carrying Sarcopenia is described as the loss of skeletal muscle
A/A and A/C genotypes respectively showed, on mass and strength related to aging (Forbes 1976;
Gallagher et al. 1997; Hughes et al. 2001). This process
is associated with negative consequences among the
C. C. Lunardi (*) elderly, such as loss of functional capacity and autonomy
College of Physical Education, University of Brasilia,
(Fleg and Lakatta 1988; Baumgartner et al. 1998; Lima
SMU/QRS/RCG, Rua E, Casa 504,
Brasília, Distrito Federal, Brazil et al. 2009), increased risk of falls (Whipple et al. 1987),
e-mail: [email protected] metabolic impairments (Bloesch et al. 1988), and
reduced bone mineral density (Gentil et al. 2007a, b,
R. W. Pereira : T. K. M. Leite : A. B. M. Siqueira
2009; Lima et al. 2009). Previous reports provide evi-
College of Physical Education,
Catholic University of Brasilia, dence that sarcopenia has significant health-care cost
Brasilia, Brazil implications that warrant efforts to understand and coun-
teract this age-related muscle mass and strength decline
R. M. Lima : R. J. Oliveira
(Janssen et al. 2004).
College of Physical Education, University of Brasilia,
Campus Universitário Darcy Ribeiro Asa Norte, The etiology of sarcopenia is not completely known
Brasília, DF, Brazil but seems to be influenced by a variety and interrelated
2478 AGE (2013) 35:2477–2483

factors. It is well established that heritability significantly (rs16892496 and rs7832552) and muscle strength and
contributes to the interindividual variability of muscle FFM phenotypes in a sample of Brazilian elderly wom-
mass and strength (Arden and Spector 1997; Tiainen et en. Aware of the high admixture in Brazilians and of its
al. 2009) and to the decrease of strength due to aging potential complications when performing association
(Carmelli and Reed 2000). Previous studies provide studies in such population (Lins et al. 2011), 19
evidence that genetic factors are responsible for about ancestry-informative single-nucleotide polymorphisms
60–80 % of the interindividuals variation in fat-free mass were genotyped, and the estimated genetic ancestry
(Seeman et al. 1996). However, although the heritability values were included as covariates during the analysis.
of muscle-related phenotypes is well documented in the
literature, the identification of genes and polymorphisms
associated with these phenotypes requires further stud- Methodology
ies. A reasonable number of allelic variants have been
studied, but lack of consistency on replicating the find- Subjects
ings makes the identification of genes with significant
contribution still unclear. Detection of significant con- After applying the exclusion criteria, this study includ-
tributor's genetic variants may assist in improving the life ed 241 women aged between 60 to 82 years and
quality of seniors, enabling to target preventive practices participants in a voluntary project developed at the
best suited to each individual, reducing the risk of devel- university. The following were excluded from the
oping dependence and related complications. The advent study participants: those who (a) did not have
of biotechnology and reduced costs of genotyping have Brazilian nationality, (b) were not able to walk inde-
made genome-wide association studies (GWAS) an im- pendently, (c) had unilateral or bilateral hip prosthetic,
portant tool in the search for candidate genes. (d) had a prosthetic heart valve, (e) were smokers, (f)
Regarding muscle-related phenotypes, few GWAS had endocrine or metabolic disorder known to affect
have been conducted, and few candidate genes have the muscular system, and (g) had abnormal cardiac
been identified. De Mars et al. (2008) sought to iden- conduction or infusion that would contraindicate the
tify chromosomal regions linked to muscle area and practice of physical activities. To take part in the study,
bone cross-sectional, knee flexors, and extensors iso- volunteers signed a written consent. The study was
metric torque in twins. The authors concluded that approved by the Research Ethics Committee of the
many genes still have little explanation for muscle university (CEP/UCB 014/2007).
strength determination (De Mars et al. 2008). Liu et The official Portuguese long version of the
al. (2009) studied the association of the thyrotrophin- International Physical Activity Questionnaire (IPAQ)
releasing hormone receptor (TRHR) gene and fat-free was used to determine the physical activity level of
mass (FFM) in 1,000 individuals. The authors reported each volunteer. The questionnaire was performed in
that the polymorphisms rs16892496 (GG) and face-to-face interviews as recommended. The IPAQ was
rs7832552 (TT) in the TRHR gene are associated with developed by investigators from various countries with
FFM and replicated these findings in three independent the support of the World Health Organization. It has
samples. According to the authors, subjects with these been reported as an instrument with acceptable measure-
genotypes have, on average, 2.70 and 2.55 kg less FFM, ment properties in various countries (Craig et al. 2003).
respectively, when compared to those with other geno-
types. However, it was not in the scope of the report to Assessment of body composition
examine the association with muscle strength.
Studies conducted in different populations are Body composition measurements were conducted using
needed to better understand the influence of the dual-energy X-ray absorptiometry (DXA) (DPX-L,
TRHR gene on muscle phenotypes. Accordingly, this Lunar Radiation Corporation, Madison, WI). All mea-
study sought to examine candidate genes previously surements were carried out by the same trained techni-
identified through GWAS against muscle phenotypes. cian according to the procedures previously described
Specifically, the objective was to replicate the results (Lima et al. 2007). Besides total FFM (TFFM) and fat
of previous studies, to verify whether there is an mass, computer-generated lines with subsequent manual
association between polymorphisms in the TRHR gene adjustment enabled specification of FFM for the arms,
AGE (2013) 35:2477–2483 2479

legs, and trunk. Appendicular FFM (AFFM) was calcu- City, CA, USA) followed by purification with shrimp
lated as the sum of both arms and legs FFM. In addition alkaline phosphatase. An aliquot of this purified prod-
to the use of absolute TFFM and AFFM, these variables uct, prepared with a molecular size standard (GS120
were considered relative to body height squared (in Liz, Applied Biosystems) and highly deionized form-
kilogram per square meter) (Baumgartner et al. 1998). amide, was denatured at 95 °C to the subsequent capil-
Coefficients of variation observed for the DXA were 2.1 lary electrophoresis (ABI 3130xl, Applied Biosystems).
and 1.9 % for fat mass and FFM, respectively. The electropherograms were analyzed using the
GeneMapper™ 4.0 software (Applied Biosystems,
Isokinetic muscle peak torque Foster City, CA, USA). Genotyping of 19 ancestry-
informative markers was performed under the same
Quadriceps strength was measured using the Biodex conditions described above.
System 3 isokinetic dynamometer (Biodex Medical
Systems, New York, USA). Before each test, partici- Genetic ancestry evaluation
pants underwent 5 min of warm-up in a cycle ergom-
eter with low load and comfortable speed. After a To estimate genomic ancestry, 19 ancestry-informative
detailed explanation of the evaluation procedures, vol- markers were genotyped for all the samples, and indi-
unteers were carefully positioned on the seat of the vidual ancestry in each parental population was esti-
equipment. The rotation axis from the dynamometer mated using the software ADMIXMAP (http://
arm was aligned with the dominant femoral homepages.ed.ac.uk/pmckeigu/admixmap/), with
epicondyle. The force application point was posi- 2,500 iterations for the burn-in period and 10,000
tioned 2 cm above the medial malleolus. Fastened iterations to measure parameter data.
Velcro belts were used on the trunk, pelvis, and thigh
to avoid compensatory movements. The protocol Statistical analysis
consisted of three sets of four concentric muscle con-
tractions (60°/s) with 30-s rest intervals between sets Normality of data distribution was examined using the
(Bottaro et al. 2005). The recorded value for analysis Kolmogorov–Smirnov test. Data are presented using
was the highest peak torque (PT) of the three series, descriptive statistics, with mean and standard deviations
which was expressed in absolute values (in normal procedures (unless otherwise noted). To verify if the
meter) and relative to body mass (in normal meter allelic frequencies were in accordance with the Hardy–
per kilogram). Participants were asked to perform the Weinberg equilibrium, the chi-square test was used.
contractions with the greatest possible force, and ver- Analysis of variance (ANOVA) was applied to test for
bal encouragement was offered during the measure- differences between genotypes in the following vari-
ment. The calibration of the equipment was performed ables: age, weight, height, BMI, and fat percentage. To
according to manufacturer's specifications at the be- verify differences between genotypes for noncontinuous
ginning of the evaluation sessions, and the same eval- variables such as the prevalence of sarcopenia, calcium
uator carried out the procedures. supplementation, use of hormone replacement therapy,
and physical activity level, the chi-square test was
Genotyping conducted. To test the association between genotypes
and muscle-related phenotypes, analysis of covariance
Blood samples from all participants were collected in models was performed with LSD post hoc covariates
the antecubital vein, and genomic DNA of high molec- which included years of menopause, hormone replace-
ular weight was extracted from peripheral leukocytes by ment, calcium supplementation, and physical activity
the “salting out” method (Miller et al. 1988). levels. The inclusion of individual African genomic
Genotyping of the rs16892496 (A/C) and rs7832552 ancestry was also used as covariate as an attempt to
(A/C) SNPs was performed using a multiplex PCR correct for population stratification. Mantel–Haenszel
(Qiagen® Multiplex PCR kit), followed by purification common odds ratio analysis was used to investigate
with exonuclease I and shrimp alkaline phosphatase, the association between outcomes (phenotypes) and
and single-base extension method (ABI Prism® risks (genotype), adjusted for age and genomic ancestry.
SNaPshot® Multiplex kit, Applied Biosystems, Foster To reach this end, muscle phenotypes variables were
2480 AGE (2013) 35:2477–2483

divided into quartiles, where Q1 was the lower quartile, significantly higher for those with genotype C/C and,
and Q4, the highest. The significance level was p≤0.05, therefore, was used as covariate in subsequent analyses
and data were analyzed using SPSS 13.0 for Windows. along with African genomic ancestry. The results of
body composition and muscle-related phenotypes
(i.e., FFM and strength) according to the rs16892496
Results genotypes are shown in Table 2. No significant differ-
ences were observed for BMI or body fat percentage.
The descriptive data of the sample such as age, weight, ANOVA revealed that AFFM and PT were significantly
height, BMI, years since menopause, peak torque, and different between groups, with lower values of AFFM in
body composition are shown in Table 1. According to subjects carrying C/C genotype. However, when age
the approach proposed by Baumgartner et al. (1998), and genomic ancestry were entered as covariates, the
17.3 % of subjects were classified as sarcopenic. In results did not reach a statistical significance, but only a
relation to hormone replacement therapy and calcium trend (p values ranging from 0.07 to 0.15; Table 2).
supplements, 19 (7.9 %) and 56 (23.7 %) women were When genotype C/C was compared to the group com-
using both, respectively. Physical activity levels were posed by A allele carriers (i.e., A/A+A/C), significant
as follows: 5 (2.1 %) were sedentary, 69 (28.6 %) were differences were observed for AFFM and relative
insufficiently active, 163 (67.6 %) were active, and 4 AFFM with lower values for the C/C genotype (after
(1.7 %) were very active. adjustment for age and African genomic ancestry).
The genotype distribution of the TRHR gene Appendicular FFM relative to height squared and PT
(rs16892496 and rs7832552) was consistent with relative to body weight were divided into quartiles, and
Hardy–Weinberg equilibrium (p>0.05). No significant the odds ratio in relation to genotype groups are
difference was observed for rs7832552, and thus, the presented in Table 3. It was observed that the C/C
results are all related to the rs16892496. The prevalence genotype group presented a significantly higher chance
of sarcopenia was 27.6, 17.8, and 13.3 %, respectively, to be classified in the lower quartile of AFFM when
for genotypes C/C, A/C, and A/A; however, the chi- compared to both A/A genotypes. Regarding PT relative
square test did not reveal significant differences. to body weight, the C/C genotype presented a threefold
Furthermore, ANOVA revealed no significant differ- increase in the chance to be classified in the lower
ence for weight, height, or BMI; however, age was quartile when compared to the A/A genotype.

Table 1 Characteristics of participants. Values are expressed as

mean ± standard deviation Discussion
Variables
Based on previous GWAS showing an association
N 241 between the rs16892496 and rs7832552 and FFM in
Age (year) 66.6±5.5 different populations with a wide age range, the pres-
Body mass (kg) 65.8±11.9 ent study was designed to examine its association in
Height (m) 1.53±0.1 older women not only with FFM but also with muscle
Years of menopause 18.0±7.5
strength. Muscle mass and strength decline with ad-
BMI (kg/m2) 28.0±4.5
vancing age (Goodpaster et al. 2008), and this process
is associated with negative clinical outcomes in the
Percentage of fat (%) 39.5±5.9
elderly (Baumgartner et al. 1998; Lima et al. 2009).
TFFM (kg) 37.9±4.9
Therefore, it is of particular interest to examine the
Relative TFFM (kg/m2) 16.1±1.7
association of gene polymorphisms and muscular phe-
AFFM (kg) 14.6±2.3
notypes in the elderly. The main findings of the pres-
Relative AFFM (kg/m2) 6.2±0.8
ent study provide further evidence of an association
PT (Nm) 95.1±22.8
between the THRH rs16892496 polymorphism and
Relative PT (Nm/kg) 145.9±31.2
FFM. Moreover, the results bring the insight that this
BMI body mass index, AFFM appendicular fat-free mass, TFFM genetic variant can present a contribution to muscle
total fat-free mass, PT peak torque strength in older women. Of note, these observations
AGE (2013) 35:2477–2483 2481

Table 2 Body composition and muscle phenotypes according to the genotypes of rs16892496. Data are presented as age- and African
genomic ancestry-adjusted mean ± standard error

Variables C/C A/C A/A p values C/C A/C+A/A p values

N (%) 29 (12) 129 (53.5) 83 (34.5) 29 (12) 212 (88.0)

Age (year)a 69.2±6.23* 66.4±5.25 66.1±5.53 0.03 69.2±6.2* 66.3±5.35 0.01
2
BMI (kg/m ) 27.2±0.85 28.1±0.40 28.1±0.50 0.63 27.2±0.85 28.1±0.31 0.33
Percentage of fat (%) 38.4±1.11 39.6±0.52 39.5±0.65 0.62 38.4±1.11 39.5±0.41 0.33
AFFM (kg) 13.7±0.43 14.6±0.20 14.7±0.25 0.13 13.7±0.43* 14.7±0.16 0.04
Relative AFFM (kg/m2) 5.9±0.15 6.2±0.07 6.2±0.09 0.14 5.9±0.15* 6.2±0.06 0.05
TFFM (kg) 36.9±0.91 37.9±0.43 38.4±0.53 0.40 36.9±0.91 38.1±0.33 0.24
2
Relative TFFM (kg/m ) 15.9±0.32 16.1±0.15 16.3±0.19 0.59 15.9±0.32 16.2±0.12 0.42
PT (Nm) 90.0±4.02 93.4±1.89 99.2±2.35 0.07 90.0±4.02 95.7±1.48 0.20
Relative PT (Nm/kg) 143.6±5.64 143.0±2.65 151.1±3.30 0.15 143.6±5.64 146.2±2.08 0.67

BMI body mass index, AFFM appendicular fat-free mass, TFFM total fat-free mass, PT peak torque
*p<0.05, significantly different compared with other genotypes
a
Values are presented as mean and standard deviation

remained significantly after adjustment for genomic TRHR gene were associated with FFM (by dual-energy
African ancestry. X-ray absorptiometry) in a study of 9,350,000 SNPs
A variety of previous reports had shown association examined in nearly 1,000 unrelated US whites.
between a reasonable number of candidate gene poly- Moreover, these authors replicated the significant asso-
morphisms and muscle-related phenotypes, but with lit- ciations in three independent samples. Taken together
tle agreement on significant contributors (Rankinen et al. with the present results, these observations suggest that
2010; Lima et al. 2011; Tan et al. 2012). GWAS has the TRHR gene might be an important candidate for
become an important tool for better understanding the interindividual differences in muscular phenotypes.
association between phenotypes and genotypes. Exercise This replication study in a sample of Brazilian
scientists need to prioritize high-quality research designs, elderly suggests a modest association between
and replication studies with large sample sizes are ur- rs16892496 and FFM. Individuals carrying the C/C
gently needed (Rankinen et al. 2010). Liu et al. (2009) genotype presented a higher chance for low values of
published the first GWAS focusing on skeletal muscular AFFM when compared to homozygous A/A.
traits, specifically FFM. Although muscle strength was Individuals A/A and A/C, on average, respectively
not assessed in their investigation, the authors identified showed an extra 1 kg and 900 g of AFFM compared
that rs16892496 and rs7832552 polymorphisms in the to C/C genotype carriers. Furthermore, we observed a
trend for the difference between genotypes for
Table 3 Odds ratio/association adjusted for age and ancestry isokinetic peak torque. It was observed that the C
between phenotype and genotype allele carriers were more likely to be classified in the
lower quartile of muscle strength. However, the dif-
Phenotype Genotype OR (95 % CI)
ferences observed in the present study were modest
Low relative AFFM (<5.7 kg) A/A 1,00 when considering the high heritability of muscle-
A/C+C/C 2.24 (1.12–4.49) related phenotypes (Liu et al. 2009; De Mars et al.
C/C 3.95 (1.41–11.11) 2008). Probably, many genes with small contributions,
Low relative PT A/A 1.00 rather than few genes with strong influence, are
(<126.7 Nm/kg) A/C 2.68 (1.18–6.10) expected to determine the interindividual differences
A/C+C/C 2.71 (1.63–5.8)
of such traits (Thompson et al. 2004; De Mars et al.
C/C 3.09 (1.11–8.58)
2008; Pescatello et al. 2006). Thus, the present study
provides evidence that the studied polymorphism
AFFM appendicular fat-free mass, PT peak torque (rs16892496) in the TRHR gene is one genetic variant
2482 AGE (2013) 35:2477–2483

that contributes to FFM and muscle strength in cross- strength in older women. These results remained
sectional analyses, though its clinical relevance remains unchanged with the inclusion of genomic ancestry as a
to be investigated. measure to correct for population stratification.
In fact, heritability of a variety of complex pheno- Determination of genetic variants associated with mus-
types is well documented in the literature (Thompson cular phenotypes in the elderly may be useful in identi-
et al. 2004; Tiainen et al. 2009; Tan et al. 2012); fying individuals who are more susceptible to lose
however, the contributory genes remain unclear. muscle mass and strength with advancing age. For in-
Understanding the relationship between genotypes stance, however, it seems that the adoption of preventive
and muscular phenotypes in the elderly might contrib- habits such as regular physical activity practice is the
ute to assist in an early intervention and allow preven- intervention of choice for all individuals, independent of
tive practices best suited to each individual, thus their genetic background.
ultimately improving life quality in this population.
Identification of significant genetic variants underly- Acknowledgments We thank the participants of the study.
Funding was provided by a grant 193.000.555/2009 from the
ing sarcopenia will provide valuable insights into im-
Research Support Foundation of the Federal District (FAP-DF)
portant potential targets for risk stratification, as well and by a grant from the National Council for Scientific and
as pharmacogenetic interventions aimed at increasing Technological Development (CNPq).
muscle mass and strength. Few genes have been iden-
tified to date, but lack of replication does not enable
unequivocal conclusions (Tan et al. 2012).
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