Relationship of vitamin D receptor gene polymorphism with sarcopenia and muscle traits based on propensity score matching

Abstract Background Vitamin D receptor (VDR) gene polymorphism is reported to be associated with muscle mass and muscle strength. Loss of skeletal muscle mass and decreased muscle strength are the main characteristics of sarcopenia. In this study, the relationship of VDR gene polymorphism with muscle traits (muscle mass, muscle strength, and physical performance) and sarcopenia were studied in Xinjiang, China. Methods Totally, 205 sarcopenia patients were enrolled. Propensity score method was used to match control group. FokI and BsmI polymorphisms were genotyped using improved multiplex ligation detection reaction (iMLDR). Results Fok1, but not Bsm1, polymorphism was significantly associated with sarcopenia. Patients with Fok1 GG genotype were more likely to have sarcopenia. Both Bsm1 and Fok1 polymorphism were related to muscle traits. Patients with Bsm1 CT genotype had lower gait speed (GS) but higher skeletal mass index. Patients with Fok1 GG genotype had lower GS, and female subjects with the Fok1 GG genotype had lower handgrip strength (HS). GS was decreased in Bsm1 CT genotype than CC carriers. HS and GS were decreased in Fok1 GG genotype than AA carriers. Conclusion Fok1, but not Bsm1, polymorphism is associated with sarcopenia. Both Bsm1 and Fok1 polymorphism affect both HS and GS.

different demographic characteristics (such as age and sex). 6 Sarcopenia is also related to nutrition status (eg, vitamin D level), 6,12,13 physical activity, [13][14][15] and sleep quality. 4,16,17 On the other hand, a number of studies have shown that skeletal muscle strength and mass are highly heritable, indicating a strong genetic contribution. 18,19 However, only a few genes have been confirmed to be related to skeletal muscle strength and mass. There are few reports on the association of genetic polymorphisms with sarcopenia.
There is a certain relationship among skeletal muscle mass, strength, and vitamin D levels, as well as vitamin D receptor (VDR) gene polymorphisms. Studies have shown that low vitamin D is associated with low muscle strength, muscle mass, 20 and muscle function. 21 Geusens et al 22 found that grip strength was associated with VDR genotype in older women (age ≥ 70 years). The Fok1 and Bsm1 loci of the VDR gene are associated with muscle mass and muscle strength. Subjects with the FF/AA genotype of Fok1 were more likely to have higher skeletal muscle strength and quality than FF/ GG carriers. [23][24][25] Meanwhile, the carriers with BB/CC genotype of Bsm1 were more likely to have higher skeletal muscle strength than those with BB/TT genotype. 25,26 All these studies have highlighted the role of VDR gene polymorphisms in muscle mass and muscle strength.
Randomized controlled trial (RCT) is considered as the optimal study method. 27 However, RCT is subject to ethical, economic, and other conditions. Propensity score matching (PSM) aims to reduce or eliminate differences between baseline features. 28 PSM method can effectively balance the confounding factors between the diseased group and the control group. Compared to RCT, PSM is widely used in non-RCT.
Here, in this study, we studied the relationship of VDR gene polymorphism with muscle traits (muscle mass, muscle strength, and physical performance) and sarcopenia. The subjects were Urumqi residents from Tianshan district of Xinjiang, China. The control group was matched by PSM method (in 1:2 matching).

| Ethics
All subjects provided written informed consent. The study was conducted in accordance with the Declaration of Helsinki, and the study protocol was approved by the Ethics Committee of Xinjiang Medical University.

| Subjects
This study was carried out between March 2017 and July 2018. A total of 1886 subjects were enrolled in this study. They were subjects who underwent physical examination in Tianshan district of Urumqi, China.
The inclusion criteria were as follows: (a) Urumqi residents, (b) aged ≥50 years. The exclusion criteria were as follows: (a) subjects with limited mobility, (b) subjects with disorder of communication or mental illness, (c) subjects with hyperthyroidism or hypothyroidism, (d) subjects with long-term use of steroids, (e) subjects taking weight-loss drugs and glucocorticoids that might affect body composition in the past three weeks, (f) subjects with metal stents or pacemaker, and (g) physically disabled or amputee.
Asian Working Group for Sarcopenia (AWGS) 29 defined sarcopenia as reduction in skeletal muscle mass, skeletal muscle strength, and/or physical performance. According to AWGS, 205 subjects were diagnosed as sarcopenia in our study and 410 subjects were matched by PSM method (in 1:2 matching) as the control group.
The matched covariate variables included age, sex, ethnicity, educational level, monthly income, marital status, smoking, and alcohol consumption.

| Body composition
The appendicular skeletal muscle mass (ASM) was measured using bioelectrical impedance analysis on a body composition analyzer (InBody 720, Korea). During measurement, the participants stood on the device in bare feet with legs slightly apart while holding the electrodes in both hands with arms away from the trunk. The skeletal mass index (SMI) was defined as ASM/height. 2 We used the AWGS to determine the cutoff value of SMI, which is 7.0 and 5.7 kg/m 2 for male subjects and female subjects, respectively.

| Muscle strength (Hand grip strength)
Muscle strength was assessed by HS. HS was tested three times for the dominant and non-dominant hand using Jamar Hand Dynamometer (Sammons Preston Inc) with a break of 30 seconds between each test. Grip width was adjusted individually. The average value of the three tests was used for analysis. According to AWGS, the cutoff value of HS is 26 kg and 18 kg for male subjects and female subjects, respectively.

| Physical performance (gait speed)
Physical performance was examined by 10-meter GS. 29 In order to allow participants to accelerate/decelerate outside the data collection area, 5-meter distance was provided at the beginning and end of the walkway, respectively. We marked 4 positions (0, 5, 15, and 20 m), and walking time was collected from 5 to 15 m. Participants were instructed to "walk at your usual pace." Then, GS was calculated as 10(m)/time(s). According to AWGS, the cutoff value of GS is 0.8m/s for both male subjects and female subjects.
Sarcopenia was defined as presentation of both low muscle mass and function (strength or performance) following the AWGS criteria.

| Genotyping
Blood samples were collected during physical examination. Genomic DNA was extracted using the Whole Blood Extraction kit (Tiangen Biotech). The Fok1 and Bsm1 genotyping were performed using improved multiplex ligation detection reaction (iMLDR, Genesky, Shanghai, China). Briefly, the genomic DNA was amplified using the PCR instrument (Mycycler, Bio-Rad). The procedure was 25 cycles of 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 30 seconds.
PCR product (10 μL) was then digested with enzymes. The digested fragments were subjected to electrophoresis on 2% agarose gels and visualized with UV light. A double-blind control was set up during the genetic polymorphism detection, and 1% of the samples were randomly selected for repeated detection.

| Statistical analyses
R statistical software was used to match case and control group (in 1:2 matching). Caliper matching method was used, and the caliper value was 0.2. All other data were analyzed by SPSS21.0 (IBM). Shapiro-Wilk method was used to test normality of quantitative data. If normality was satisfied, the data were described as mean ± standard deviation (SD). Qualitative data were described as percentage. Chi-square (χ 2 ) test was used to analyze categorical variables (ie, genotype and Hardy-Weinberg equilibrium).
Univariate logistic regression was used to determine the effect of different genotypes on the risk of sarcopenia. The odds ratios (OR) and 95% confidence intervals (CI) were calculated. The comparisons

| Comparisons of covariate before and after PSM
As shown in Table 1, there was a significant difference in age, ethnicity, educational level, marital status, and alcohol consumption between sarcopenia and control groups (P < .05) before PSM.
However, there was no significant difference in them between the two groups after PSM. The distribution of PS consistently demonstrated an improvement in the covariate balance after PSM (Figures 1 and 2).

| Hardy-Weinberg equilibrium tests and the distribution of the Bsm1 and Fok1 genotypes
The distribution of the Bsm1 and Fok1 genotypes met with Hardy-Weinberg equilibrium (P > .05). As shown in Table 2

| Comparison of HS, GS, and SMI across the Bsm1 and Fok1 genotypes
Comparisons of HS, GS, and SMI among the three Bsm1 genotypes by sex were showed in The bold values present these comparisons were statistically significant.

| Multivariate analyses of relationship of the Bsm1 and Fok1 genotypes with HS, GS, and SMI
Multivariate linear regression analyses of the association between Bsm1 genotypes and HS, GS, and SMI were shown in Abbreviations: GS, gait speed; HS, handgrip strength; SMI, skeletal mass index.
The bold values present these comparisons were statistically significant. The bold values present these comparisons were statistically significant.
Multivariate linear regression analyses of the association between Fok1 genotypes and HS, GS, and SMI were shown in Table 6.
In Model 1, subjects with Fok1 GG genotype were more likely to report less GS compared to those with AA genotype (β = −0.06,  changes in aging population in muscles and bones. 36 The association between VDR gene polymorphism and osteoporosis has been confirmed. 37

| CON CLUS IONS
In conclusion, subjects with the Fok1 GG genotype are more likely to have sarcopenia. Even after adjusting potentially confounding factors, there is lower GS in Bsm1 CT compared to CC carriers and lower HS and GS in Fok1 GG compared to AA carriers. Our findings indicate that VDR genotypes can affect muscle traits and sarcopenia.

AUTH O R CO NTR I B UTI O N S
HX involved in conceptualization, writing-review and editing, supervision, project administration, and funding acquisition; XY, LY, and ML involved in methodology, software, and formal analysis; XY involved in data curation and writing-original draft preparation.