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Effects of CYP2R1 gene variants on vitamin D levels and status: A systematic review and meta-analysis
Gene 678 (2018) 361–369
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Review
Effects of CYP2R1 gene variants on vitamin D levels and status: A systematic review and meta-analysis Leizhen Duana, Zonggui Xuea, Huanwen Jia, Dongdong Zhangb, Yan Wangb, a b
T
⁎
Department of Medical Services, Zhengzhou Central Hospital affiliated to Zhengzhou University, Zhengzhou 450001, China Department of Nutrition and Food Hygiene, College of Public Health, Zhengzhou University, Zhengzhou 450001, China
A R T I C LE I N FO
A B S T R A C T
Keywords: CYP2R1 Polymorphism 25(OH)D Vitamin D deficiency Meta-analysis
Background and objective: CYP2R1 is a key gene in the vitamin D metabolic pathway. It has been suggested that CYP2R1 gene variants in European populations are associated with concentrations of 25(OH)D, a biomarker of vitamin D levels and status in peripheral blood. However, a comprehensive meta-analysis of this effect including different ethnicities has never been conducted. The objective of this meta-analysis was to evaluate the association between CYP2R1 gene variants and 25(OH)D levels and vitamin D status. Methods: PubMed, EMBASE, Web of Science, CNKI and Wanfang databases were systematically searched up to May 2018. Reporting followed PRISMA guidelines. The quality of the evidence was assessed using the STREGA system. Random or fixed effects model combined estimates and sub-group tested for ethnic differences. The I2 statistic quantified between-study variation due to heterogeneity. Results: Sixteen articles with a total of 52,417 participants met the inclusion criteria and were included in the meta-analysis. For rs10741657, GG genotype was associated with a clear descending trend of 25(OH)D levels when compared with the AA genotype [SMD = −2.32, 95% CI (−4.42, −0.20); SMD = −3.46, 95% CI (−6.60, −0.33) and SMD = −0.24, 95% CI (−0.51, −0.03) for total, Caucasian and Asian groups, respectively] with the following heterogeneities I2 = 37.9%, 69.2% and 24.5%, respectively. However, under the AG/ AA genetic model, significant changes in 25(OH)D levels [SMD and 95% CI: −1.27(−2.32, −0.23)] were only evident in the Caucasian population. The meta-analysis on vitamin D deficiency showed that the risk-allele G was associated with an increased risk of vitamin D deficiency (OR = 1.09; 95% CI = 1.03–1.15, P = 0.002). The association between rs10741657 and increased risk of vitamin D deficiency was significant (OR = 1.42; 95% CI = 1.11–1.83, P = 0.006) under the dominant model (GG + AG/AA), but not under the recessive model (GG/ AG + AA), (OR = 1.28; 95% CI = 0.89–1.84, P = 0.181). There was no evidence of publication bias. Conclusion: Published articles provide evidence supporting a major role for the rs10741657 polymorphism of the CYP2R1 gene in determining 25(OH)D levels and the presence of vitamin D deficiency.
1. Introduction
asthma, diabetes, cardiovascular disease and some forms of cancer (Mccullough and Mayo, 2009; Abudawood et al., 2018; Parr et al., 2018). Factors which influence the concentrations of 25-hydroxyvitamin D [25(OH)D], a vitamin D biomarker that can be measured in blood, include sun exposure, latitude, race, age, gender and dietary supplements. (Fohner et al., 2016). However, these environmental factors appear to only contribute to 13% of the individual heterogeneity in vitamin D levels (Burgaz et al., 2007). Accumulating evidence indicates that differences in vitamin D levels, including vitamin D
Vitamin D is a fat-soluble vitamin and steroid pro-hormone that is involved in the regulation of various biological processes, such as immunoregulation and insulin resistance and also has anti-inflammatory and anti-cancer effects (Mccullough and Mayo, 2009; Sharma et al., 2017; Tabatabaeizadeh et al., 2017; Abudawood et al., 2018). Accumulating evidence supports the notion that low vitamin D concentrations are strongly associated with various adverse outcomes, such as
Abbreviations list: CYP2R1, cytochrome P450 family 2, subfamily R, peptide 1; PRISMA, preferred reporting items for systematic reviews and meta-analyses; STREGA, strengthening report of genetic association studies; SMD, standardized mean difference; OR, odds ratio; CI, confidence intervals; 25(OH)D, 25-hydroxyvitamin D; 7-DHC, 7-dehydrocholesterol; GWAS, genome-wide association studies; IOM, Institute of Medicine; HWE, Hardy-Weinberg equilibrium; VDD, vitamin D deficiency ⁎ Corresponding author at: Department of Nutrition and Food Hygiene, College of Public Health, Zhengzhou University, Zhengzhou, 450001, Henan, China. E-mail address: [email protected] (Y. Wang). https://doi.org/10.1016/j.gene.2018.08.056 Received 5 June 2018; Received in revised form 13 August 2018; Accepted 14 August 2018 Available online 16 August 2018 0378-1119/ © 2018 Elsevier B.V. All rights reserved.
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Fig. 1. The vitamin D metabolic process in vivo and the role of the CYP2R1 gene.
2. Methods
deficiency, are under strong genetic control. Several twin and family studies have reported that 23% to 80% of the heterogeneity in vitamin D levels was attributable to genetic factors (Bahrami et al., 2017). Attention has now turned to the gene effects that could have an impact on vitamin D metabolism. The production of 1,25(OH)2D, an active form of vitamin D, relies on a series of enzymatic processes. Sun light (UVB at 270 nm–290 nm), acting on 7-dehydrocholesterol (7-DHC) in the epidermis of the skin, generates pre-vitamin D. Pre-vitamin D is an inactive precursor, requiring 25-hydroxylase (encoded by the cytochrome P450 CYP2R1 gene in the liver) to yield the 25(OH)D. Subsequently, 1α-hydroxylase in the kidney (encoded by the CYP27B1 gene) catalyzes the conversion of 25(OH)D to 1,25(OH)2D. (Fig. 1). The human CYP2R1 gene contains 5 exons and spans about 15.5 kb. It is located on chromosome 11p15.2 (gene ID: 120227), and contains 501 amino acids (Cheng et al., 2003). It encodes a microsomal vitamin D 25-hydroxylase which is considered to be the most important enzyme in the metabolism of vitamin D. In mice, the combined knockout of both Cyp2r1 and Cyp27b1, only reduced levels of 25(OH)D by 50%, did not affect circulating 1,25(OH)2D (Burgaz et al., 2007). In humans, Cheng et al. first identified a missense mutation in exon 2 of the CYP2R1 gene that could result in vitamin D deficiency (Cheng et al., 2004). Subsquently, several genome-wide association studies (GWAS) performed in European populations (Ahn et al., 2010; Wang et al., 2010; Anderson et al., 2014; O'Brien et al., 2018; Xia et al., 2018), identified > 25 SNPs of the CYP2R1 gene associated with vitamin D levels or vitamin D deficiency. Numerous replication studies were conducted in other ethnic/racial populations (Wen, 2012; Robien et al., 2013; Xu et al., 2015; Yu, 2016; Mao, 2017). However, the results of these studies did not seem to support the conclusions observed in the European populations. Here, we conducted a meta-analysis including a large scale and unrelated population with general health condition to evaluate more precisely the association between CYP2R1 gene polymorphisma and 25(OH)D levels and vitamin D status.
2.1. Data sources and search strategies This study was performed following a predefined protocol and under the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) (Table S1) (Moher et al., 2009). The searches were conducted by LZ.D and YW from Jan 2010 to May 2018 using PubMed, EMBASE, Web of Science, CNKI and WanFang databases with the following search terms: (“CYP2R1” or “25-hydroxylase” or “cytochrome P450 2R1”) and (“SNP” or “polymorphism” or “variants” or “mutation”) and (“vitamin D" or “25(OH)D” or “calcitriol” or “vitamin D deficiency”). The search was restricted to studies published in English or Chinese. To complement the electronic searches, we also perused studies included in relevant systematic reviews and reference lists of pertinent articles. 2.2. Inclusion and exclusion criteria Papers were included if they contained the following data: (1) primary outcome of interest was vitamin D deficiency or 25(OH)D levels; (2) the gene of interest was CYP2R1; (3) the definition of vitamin D deficiency was 25(OH)D levels below 20 ng/mL (50 nmol/L), as described by the Institute of Medicine (IOM) (Ross et al., 2011); (4) either (a) or (b), as follows: (a) the odds ratio (OR) estimates and their 95% confidence intervals (CI) were given or sufficient data were available to evaluate the association between the rs10741657 polymorphism and vitamin D deficiency. (b) the values for 25(OH)D levels, including mean and standard deviation (SD), as well as the sample sizes (n) for the rs10741657, rs12794714 and rs10766197 genotypes were provided. (5) If the subjects were included in a study of a disease or an interventional experiment, only the normal control group or baseline population was included. We excluded studies if: (1) data were not fully available after 362
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Fig. 2. Flow diagram showing the number of studies included and excluded.
2.5. Data synthesis and statistical analysis
contacting the authors by E-mail; (2) they were meta-analysis or systematic reviews; (3) they were duplicate studies; (4) participants were children, pregnant women, and subjects diagnosed with a chronic medical illness, (such as malignancy), or were documented to have any organ dysfunction that could affect vitamin D metabolism.
Meta-analysis methods were used to assess the association between CYP2R1 gene variants and vitamin D levels and status. Statistical heterogeneity between different studies was measured using the Chisquare based Q and I2 statistics. Depending on the heterogeneity, either a fixed-effect model or a random-effect model was adopted (if the heterogeneity I2 was over 50% or the P value of the Q test > 0.05, the random-effect model was used; otherwise, the fixed-effect model was applied). The pooled OR and 95% CI were calculated to determine the association between rs10741657 and vitamin D deficiency under allele, dominant and recessive genetic models. The combined standardized mean difference (SMD) and 95% CI were used to assess the relationship between 25(OH)D levels and polymorphisms in rs10741657, rs12794714 and rs10766197 of CYP2R1 gene. Separate meta-analyses were carried out for different ethnic groups (Asian and Caucasian groups). Sensitivity analyses were performed to assess the stability of the results by omitting one study at a time and repeating the meta-analysis with the rest. Publication bias was assessed through visual inspection of Begg's funnel plots and Egger's linear regression tests. Statistical evidence of an association was considered significant when P < 0.05. Data were analyzed using the Stata software, version 11 (Stata Corp, College Station, TX, USA).
2.3. Data extraction Data on the studies' characteristics and related information were collected in Excel format using a premade checklist by one reviewer (LZD), and then double-checked by another author (YW). The following information was extracted: (1) surname of the first author, year of publication, sample size and country of origin; (2) information about participants and methods, including gender, age, BMI, genotyping methods, measurement of 25(OH)D; (3) primary information on vitamin D status, including risk allele, genotypes and their frequency or numbers, OR and the corresponding 95% CI, and the P-value for the Hardy–Weinberg equilibrium (HWE) test in the control group; (4) primary information on vitamin D levels, including the mean and SD values of the 25(OH)D concentrations, and sample sizes for every genotype. 2.4. Quality assessment Two reviewers (LZD and YW) independently assessed the quality of all included studies by following a quality score system- STREGA (strengthening report of genetic association studies) (Little et al., 2009). The STREGA system includes 22 items with scores ranging from 0 to 22 (Table S2). The quality of the studies was regarded as “high” when the score was 18–22, “moderate-high” when the score was 13–17, and “low” when the score was 0–12. Discrepancies were resolved through consensus and third-party adjudication.
3. Results 3.1. Results of the search strategy A systematic search of the literature led to the identification of 311 possibly relevant articles. One hundred and fifty-nine duplicate studies 363
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Table 1 Characteristics of the studies on the association of rs10741657 variants with vitamin D deficiency. First author (year)
Wang, 2010 Cheung, 2013 Li, 2014 Lafi, 2015 Xu, 2015 Xu, 2015 Sadat-Ali, 2016
Country (ethnicity)
n
European (C-p) China (A-p) China (A-p) Jordan (A-p) China (A-p) China (A-p) Saudi-Arab (A-p)
Age
BMI
VDD(+) (n)
VDD(−) (n)
GG
GA
AA
GG
GA
AA
HWE
STREGA score
30,000
NA
NA
NA
NA
NA
NA
NA
NA
√
20
712
48.41 ± 15.34
22.76 ± 3.97
NA
NA
NA
NA
NA
NA
√
16
1199
48.3 ± 12.5
23.5 ± 3.3
196
218
51
278
347
109
√
18
200
NA
NA
52
33
7
39
53
16
√
17
300
47 (39–56.4)
27.5 (24.4–30.6)
93
162
45
75
186
39
√
17
300
44 (40–55)
24 (21.9–27.3)
111
150
39
75
168
57
√
17
284
48 ± 16.7
25.28 ± 3.91
112
114
22
17
18
0
√
19
A-p: Asian population; C-p: Caucasian population; n:number of participants; NA: not available; VDD (+): vitamin D deficiency; VDD(−): no-vitamin D deficiency; HEW: Hardy–Weinberg equilibrium; STREGA: strengthening report of genetic association studies. Table 2 Characteristics of the studies on the association of three SNPs with 25(OH)D levels. rs97 (risk allele)
STREGA score
Country (ethnicity)
n
Age
BMI
GG 25(OH)D mean (sd)
AG 25(OH)D mean (sd)
AA 25(OH)D mean (sd)
HWE
Bu, 2010
American (C-p) England (C-p) American (C-p) Singapore (C-p)
496
65.3 ± 6.7
29.2 ± 5.4
76.6 ± 23.3
74.4 ± 20.8
65.2 ± 17.0
√
6552
NA
NA
56.8 ± 0.34
60.2 ± 0.32
61.1 ± 0.3
√
(G)
5656
NA
NA
75.4 ± 0.87
78.6 ± 0.76
81.6 ± 1.26
√
(G)
504
55.7 ± 7.8
22.8 ± 3
64.9 ± 16.7
67.6 ± 16.6
70 ± 18.03
√
(G)
Hassanein, 2014
Germany (C-p)
109
NA
NA
69.2 ± 17.5 61.2 ± 5.6
66.0 ± 16.7 67.2 ± 5.7
58.6 ± 14.8 69.7 ± 4.8
√ √
(G)
Nissen, 2014
Denmark (C-p)
762
30
21.8 ± 5.5
65.2 ± 6.1 67.2 ± 18.8
63.3 ± 4.7 73.9 ± 21.0
80.5 ± 7.4 76.6 ± 21.4
√ √
(G)
Clendenen, 2015
Swiss (C-p) Jordan (A-p) China (A-p)
1435
NA
NA
74.3 ± 20.8 49.8 ± 2.4
72.9 ± 20.8 51.8 ± 2.0
66.9 ± 19.0 56.5 ± 3.3
√ √
(G)
17
200
18–60
NA
25.3 ± 18.1
31.8 ± 21.4
38.7 ± 24.9
√
(G)
17
2215
52 ± 17.3
NA
27.1 ± 22.4
28.3 ± 23.1
26.7 ± 19.9
√
(G)
17
27.1 ± 21.4 27.4 ± 21.6 41.8 ± 21.5
28.0 ± 22.7 27.9 ± 22.4 41.9 ± 22.8
27.4 ± 23.6 27.2 ± 24.1 47.9 ± 23.1
√ √ √
19.4 ± 0.7
19.7 21.9 22.1 18.8
20.9 20.2 20.1 19.2
22.1 19.2 19.1 20.5
√ √ √ √
NA
18.8 ± 6.7 18.9 ± 7.3 35.7 ± 17.6
Wang, 2010
Robien, 2013
Lafi, 2015 Yu, 2016
Jolliffe, 2016 Arabi, 2017
Mao, 2017
Størdal, 2017
American (C-p) American (C-p) China (A-p)
Norway (C-p)
222
72 ± 9.2
29.3 ± 6.8
218
71.0 ± 4.7
30.3 ± 4.6
480
1063
53.2 ± 7.3
NA
± ± ± ±
7.3 8.4 8.2 7.0
± ± ± ±
7.2 7.4 7.6 8.8
20.0 ± 9.7 19.8 ± 9.0 35.2 ± 17.5
± ± ± ±
10 6.5 6.4 8.7
18.2 ± 7.2 18.5 ± 7.6 35.4 ± 17.1
√ √ √
rs57 (risk allele)
rs14 (risk allele)
First author (year)
(G)
20 20
19 (G) 18 (G) 19 (A)
(G) (A) (A)
16 20
(G) (G) (A) (G)
16 (G) (A)
(G)
18
A-p: Asian population; C-p: Caucasian population; n: number of participants; NA: not available; BMI: body mass index; HWE: Hardy–Weinberg equilibrium; rs57: rs10741657; rs14: rs12794714; rs97: rs10766197; STREGA: strengthening report of genetic association studies.
articles and the reasons for exclusion are shown in the flow chart (Fig. 2).
were removed using the “Find Duplicates” tool in EndNote X7. An initial examination identified 46 articles by screening the titles and abstracts. After manual examination and detailed consideration, 16 articles with a total of 52,417 participants were included in the present meta-analysis. Of these, 7 articles investigated rs10741657 and vitamin D deficiency, 10 articles investigated rs10741657 and 25(OH)D levels, 6 articles studied rs12794714 and 25(OH)D levels, and 5 articles investigated rs10766197 and 25(OH)D levels. A list of the excluded
3.2. Study characteristics Briefly, a total of 6 articles with 32,695 participants assessed the association between the rs10741657 polymorphism and vitamin D deficiency (Wang et al., 2010; Cheung et al., 2013; Li et al., 2014; Lafi 364
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Table 3 Meta-analysis of CYP2R1 gene polymorphisma and 25(OH)D levels or vitamin D deficiency. SNP/genetic model
Association of CYP2R1 polymorphisms with 25(OH)D levels N
Rs10741657 G/A (overall) Asian Caucasian GG + AG/AA Asian GG/AG + AA Asian GG/AA (overall) Asian Caucasian AG/AA (overall) Asian Caucasian Rs12794714 GG/AA (overall) Asian Caucasian AG/AA (overall) Asian Caucasian Rs10766197 AA/GG (overall) Asian Caucasian AG/GG (overall) Asian Caucasian
n
SMD (95% CI)
P
Association of CYP2R1 polymorphisms with vitamin D status
2
I (%)
P-H
10
19,194
−2.31 (−4.42, −0.20)
0.032
37.9
0.101
4 6 10
3399 15,795 19,194
−0.24 (−0.51, −0.03) −3.46 (−6.60, −0.33) −0.86 (−1.80, 0.08)
0.049 0.034 0.073
24.5 69.2 43.7
0.108 0.008 0.064
4 6 6
3399 15,795 4022
−0.06 (−0.23, 0.10) −1.27 (−2.32, −0.23) −0.03 (−0.47, 0.41)
0.456 0.017 0.884
39.5 69.7 53.6
0.175 0.027 0.047
3 3 6 3 3 5
3199 823 4022 3199 823 3897
0.21 (−0.14, 0.57) −0.43 (−1.71, 0.86) −0.17 (−0.63, 0.30) 0.20 (−0.04, 0.45) −0.73 (−2.15, 0.69) −0.13 (−0.34, 0.09)
0.236 0.518 0.481 0.108 0.313 0.242
36.5 66.4 50.8 41.5 67.7 72.3
0.201 0.009 0.049 0.043 0.014 0.006
2 3 5 2 3
2695 1202 3897 2695 1202
−0.02 (−0.13, 0.10) −0.2 (−0.57, 0.17) −0.001 (−0.08, 0.08) 0.04 (−0.05, 0.12) −0.09 (−0.22, 0.05)
0.781 0.295 0.988 0.379 0.207
0 73.5 9.5 0 0
0.774 0.023 0.352 0.456 0.471
N
n
OR (95% CI)
P
I2 (%)
P-H
7
32,995
1.09 (1.03–1.15)
0.002
15.4
0.308
6 1 4
2995 30,000 2283
1.17 (1.06–1.29) 1.06 (1.00–1.13) 1.41 (1.11–1.85)
0.002 0.048 0.006
16.0 – 50.0
0.412 – 0.092
4
2283
1.28 (0.89–1.84)
0.181
47.7
0.105
N: numbers of studies; n: numbers of participants; P-H: p value of the heterogeneity; SMD: standardized mean difference; OR: odds ratio; CI: confidence interval.
in Asian populations, where the SMD and 95% CI values were [−0.06 (−0.23, 0.1), P = 0.456], with low heterogeneity (I2 = 39.5%, P = 0.175). The pooled results are presented in Table 3 and Fig. 3B. The rs12794714 and rs10766197 polymorphisms were not found to be associated with 25(OH)D levels, either in pooled or subgroup analyses. (Table 3 and Fig. 4).
et al., 2015; Xu et al., 2015; Sadat-Ali et al., 2016). The cut-off point for vitamin D deficiency was 25 (OH) D level < 20 ng/mL (50 nmol/L) (Ross et al., 2011). The studies were conducted in China, America, Suidi Arabia, Jordan and European countries. A summary of the characteristics of the included studies is provided in Table 1. Twelve articles comprising 19,722 participants from 9 geographical areas were included in the meta-analysis to investigate the association between rs10741657, rs12794714 and rs10766197 and 25(OH)D levels (Bu et al., 2010; Wang et al., 2010; Robien et al., 2013; Hassanein et al., 2014; Nissen et al., 2014; Clendenen et al., 2015; Lafi et al., 2015; Jolliffe et al., 2016; Yu, 2016; Arabi et al., 2017; Mao, 2017; Størdal et al., 2017). All the selected loci were in accordance with HWE. Descriptive study information is shown in Table 2.
3.4. Meta-analysis of vitamin D status Under the allelic genetic model (G/A), we found that the 25(OH)D decreasing allele rs10741657 (G) was associated with an increased risk of vitamin D deficiency (OR = 1.09; 95% CI = 1.03–1.15, P = 0.002), with low heterogeneity among studies (I2 = 15.4%, P = 0.308). (Fig. 5A). Under the dominant model (GG + AG/AA), the association between rs10741657 and increased risk of vitamin D deficiency was significant (OR = 1.42; 95% CI = 1.11–1.83, P = 0.006), with low heterogeneity (I2 = 50%, P = 0.092). (Fig. 5B). However, under the recessive model (GG/AG + AA), no significant association was found (OR = 1.28; 95% CI = 0.89–1.84, P = 0.181), with low heterogeneity among studies (I2 = 47.7%, P = 0.105). (Fig. 5C).
3.3. Meta-analysis of 25(OH)D levels Meta-analysis estimates the association between the different SNPs and 25(OH)D levels are shown in Table 3. For rs10741657, GG genotype showed an obvious decreasing trend of 25(OH)D levels when compared with the AA reference genotype [SMD = −2.32, 95% CI = (−4.42, −0.20); SMD = −3.46, 95% CI = (−6.60, −0.33) and SMD = −0.24, 95% CI = (−0.51, −0.03) for total, Caucasian and Asian groups, respectively] with heterogeneities (I2 = 37.9%, P = 0.101; I2 = 69.2%, P = 0.008 and I2 = 24.5%, P = 0.108). (Table 3 and Fig. 3A). Under the AG/AA genetic model, no association was found between rs10741657 and 25(OH)D levels in total populations [SMD = −0.86, 95% CI = (−1.80, 0.08), P = 0.073], with low heterogeneity (I2 = 43.7%, P = 0.064). However, when the study population was divided into two ethnic subgroups, we found evidence of greater change in 25(OH)D levels in the Caucasian population [SMD and 95% CI = −1.27 (−2.32, −0.23), P = 0.017], with high heterogeneity (I2 = 69.7%, P = 0.027). This decreasing trend was not been detected
3.5. Sensitivity and publication bias analysis Sensitivity analysis utilizing the leave-one-out method did not show any major change in primary outcomes, an indication of the good stability of the results. Funnels plots for each meta-analysis appeared to be symmetrical. Egger's test indicated that all the P values were over 0.1, therefore, there is no publication bias in this meta-analysis. (data not shown).
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Fig. 3. Forest plot for the association between rs10741657 and 25(OH)D levels under the additive model. (A) GG/AA. (B) AG/AA.
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Fig. 4. The pooled forest plot for the association of rs12794714 and rs10766197 with 25(OH)D levels.
4. Disscusion To our knowledge, this is the first comprehensive meta-analysis evaluating the association between CYP2R1 SNPs and 25(OH)D levels and vitamin D status. The meta-analysis of 25(OH)D levels and vitamin D status in European and Asian populations identified rs10741657 variants that showed a large effect and strong association with 25(OH)D levels and vitamin D deficiency. The gene of CYP2R1 encodes 25-hydroxylase which can convert vitamin D into 25(OH)D (Cheng et al., 2003). Growing evidence suggests that the CYP2R1 gene mutation have the capacity to directly impact 25(OH)D levels and correlate with diseases related to vitamin D deficits (Ahn et al., 2010; Wang et al., 2010; Elkum et al., 2014; Hassanein et al., 2014; Bahrami et al., 2017). The rs10741657 polymorphism is located in the non-coding region 5′-UTR, which may regulate gene expression, and therefore modulate the levels of expression and activity of 25-hydroxylase (Ramos-Lopez et al., 2007). The rs12794714 and rs10766197 polymorphisms are located in the coding region of introns, which may be involved in gene regulation and selective splicing regulation (Ramos-Lopez et al., 2007). In our meta-analysis of the association between rs10741657 and 25(OH)D levels, GG carriers showed a decreasing trend of 25(OH)D levels compared with the no-risk genotype AA, the calculated SMD and 95% CI values were −2.31(−4.42, −0.20), −0.24 (−0.51, −0.03), and −3.46 (−6.60, −0.33) for total, Asian and Caucasian populations, respectively. Our results are in agreement with those reported by Zheng et al., who found an association between the rs10741657 polymorphism and 25(OH)D levels (Ye et al., 2015). However, this significant decreasing trend was only observed in the Caucasian population when comparing the AG genotype with AA genotype [SMD and 95% CI = −1.27 (−2.32, −0.23), P = 0.017]. There may be some reasonable explanations for these differences between ethnic groups. First, the sample size of the Asian population is much smaller than that of Caucasian population (3399 vs. 15,795). Second, compared with the GG/AA additive model, the AG/AA model has a lower cumulative risk 366
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Fig. 5. The pooled forest plot for the association of rs10741657 with vitamin D deficiency under three genetic model. (A) G/A. (B) GG + AG/AA. (C) GG/AG + AA. 367
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Authors contributions
effect. Third, the effects of CYP2R1 SNPs on serum 25(OH)D concentrations may vary between different ethnic groups (Thacher and Levine, 2016). Our analysis of the association between rs12794714 or rs10766197 and 25(OH)D levels, under the GG/AA (AA/GG) or AG/AA (AG/GG) genetic models did not showed any associations between SNPs and 25(OH)D levels in the total or subgroup populations. Rs12794714 and rs10766197 are located in the intron region, and since these synonymous mutations do not modify the protein sequence, 25-hydroxylase activity and function may not be affected (Manousaki et al., 2017). Therefore, this may explain the lack of meaningful results. In addition, only 6 articles on rs12794714 and 5 articles on rs10766197 fulfilled the inclusion criteria were included in the meta-analysis, and all of them studied Asian populations. This could also be an important reason why the association was not significant. Therefore, our conclusions need to be verified with larger samples and including other ethnic groups. Analysis of the association between rs10741657 and vitamin D deficiency showed that the risk allele G had an effect size 1.09-fold, 1.17-fold and 1.06-fold larger than the no-risk allele A in total, Asian and Caucasian populations, respectively. Our finding is in agreement with the results of some published studies (Wang et al., 2010; Lafi et al., 2015; Slater et al., 2015). The rs10741657 polymorphism was associated with a 1.42-fold increase in the risk of vitamin D deficiency under the dominant genetic model (GG + AG/AA) in Asian populations. However, this association was not observed under the recessive genetic model (GG/AG + AA) in Asian populations [OR and 95% CI = 1.28 (0.89–1.84), P = 0.181]. A weakening of the cumulative effect of risk alleles could be a reasonable explanation for this difference between genetic models (Thakkinstian et al., 2005). A major strength of this study is that it only included in the healthy participants, which is a very reasonable approach to eliminate the effects of disease on the levels of vitamin D and thus reduce heterogeneity between studies (Munafò and Flint, 2004; Teriaky et al., 2017). This is the first meta-analysis to evaluate the relationship between CYP2R1 gene polymorphisms and vitamin D levels and status in Asian populations. No publication bias was observed and the results were stable and reliable. However, several limitations should also be acknowledged. First, this meta-analysis did not include studies with African populations, so it's conclusions cannot be extrapolated. Second, the association between rs12794714 and rs10766197 polymorphisms and vitamin D deficiency was not analyzed because the number of studies and participants was insufficient. Third, the blood collection times and 25(OH)D measurement methods were not controlled.
The authors' responsibilities were as follows: YW: conceived and designed the study; LZD, ZGX and HWJ: performed literature search and Data collection; LZD: analyzed data and wrote the manuscript; YW and DDZ: modified the language of the manuscript. All authors read and provided critical comment on the manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2018.08.056. References Abudawood, M., Tabassum, H., Ansar, S., Almosa, K., Sobki, S., Ali, M.N., Aljohi, A., 2018. Assessment of gender-related differences in vitamin D levels and cardiovascular risk factors in Saudi patients with type 2 diabetes mellitus. Saudi J. Biol. Sci. 25, 31–36. Ahn, J., Yu, K., Stolzenberg-Solomon, R., Simon, K.C., Mccullough, M.L., Gallicchio, L., Jacobs, E.J., Ascherio, A., Helzlsouer, K., Jacobs, K.B., 2010. Genome-wide association study of circulating vitamin D levels. Hum. Mol. Genet. 19, 2739–2745. Anderson, D., Holt, B.J., Pennell, C.E., Holt, P.G., Hart, P.H., Blackwell, J.M., 2014. Genome-wide association study of vitamin D levels in children: replication in the western Australian pregnancy cohort (Raine) study. Genes Immun. 15, 578–583. Arabi, A., Khoueiry-Zgheib, N., Awada, Z., Mahfouz, R., Al-Shaar, L., Hoteit, M., Rahme, M., Baddoura, R., Halabi, G., Singh, R., 2017. CYP2R1 polymorphisms are important modulators of circulating 25-hydroxyvitamin D levels in elderly females with vitamin insufficiency, but not of the response to vitamin D supplementation. Osteoporos. Int. 28, 279–290. Bahrami, A., Sadeghnia, H.R., Tabatabaeizadeh, S.A., Bahrami-Taghanaki, H., Behboodi, N., Esmaeili, H., G, A.F., Mobarhan, M.G., Avan, A., 2017. Genetic and epigenetic factors influencing vitamin D status. J. Cell. Physiol. 233, 4033–4043. Bu, F.X., Armas, L., Lappe, J., Zhou, Y., Gao, G., Wang, H.W., Recker, R., Zhao, L.J., 2010. Comprehensive association analysis of nine candidate genes with serum 25-hydroxy vitamin D levels among healthy Caucasian subjects. Hum. Genet. 128, 549–556. Burgaz, A., Åkesson, A., Öster, A., Michaëlsson, K., Wolk, A., 2007. Associations of diet, supplement use, and ultraviolet B radiation exposure with vitamin D status in Swedish women during winter. Am. J. Clin. Nutr. 86, 1399–1404. Cheng, J.B., Motola, D.L., Mangelsdorf, D.J., Russell, D.W., 2003. De-orphanization of cytochrome P450 2R1 a microsomal vitamin D 25-hydroxylase. J. Biol. Chem. 278, 38084–38093. Cheng, J.B., Levine, M.A., Bell, N.H., Mangelsdorf, D.J., Russell, D.W., 2004. Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase. Proc. Natl. Acad. Sci. U. S. A. 101, 7711–7715. Cheung, C.L., Lau, K.S., Sham, P.C., Tan, K.C., Kung, A.W., 2013. Genetic variant in vitamin D binding protein is associated with serum 25-hydroxyvitamin D and vitamin D insufficiency in southern Chinese. J. Hum. Genet. 58, 749–751. Clendenen, T.V., Ge, W., Koenig, K.L., Axelsson, T., Liu, M., Afanasyeva, Y., Andersson, A., Arslan, A.A., Chen, Y., Hallmans, G., 2015. Genetic polymorphisms in vitamin D metabolism and signaling genes and risk of breast cancer: a nested case-control study. PLoS One 10, e0140478. Elkum, N., Alkayal, F., Noronha, F., Ali, M.M., Melhem, M., Alarouj, M., Bennakhi, A., Behbehani, K., Alsmadi, O., Abubaker, J., 2014. Vitamin D insufficiency in Arabs and south Asians positively associates with polymorphisms in GC and CYP2R1 genes. PLoS One 9, e113102. Fohner, A.E., Wang, Z., Yracheta, J., O'Brien, D.M., Hopkins, S.E., Black, J., Philip, J., Wiener, H.W., Tiwari, H.K., Stapleton, P.L., 2016. Genetics, diet, and season are associated with serum 25-hydroxycholecalciferol concentration in a Yup'ik study population from southwestern Alaska. J. Nutr. 146, 318–325. Hassanein, S.I., Ma, A.E.M., Sleem, H.M., Gad, M.Z., 2014. Triangular relationship between single nucleotide polymorphisms in the CYP2R1 gene (rs10741657 and rs12794714), 25-hydroxyvitamin d levels, and coronary artery disease incidence. Biomarkers 19, 488–492. Jolliffe, D.A., Hanifa, Y., Witt, K.D., Venton, T.R., Rowe, M., Timms, P.M., Hyppönen, E., Walton, R.T., Griffiths, C.J., Martineau, A.R., 2016. Environmental and genetic determinants of vitamin D status among older adults in London, UK. J. Steroid Biochem. Mol. Biol. 164, 30–35. Lafi, Z.M., Irshaid, Y.M., El-Khateeb, M., Ajlouni, K.M., Hyassat, D., 2015. Association of rs7041 and rs4588 polymorphisms of the vitamin D binding protein and the rs10741657 polymorphism of CYP2R1 with vitamin D status among Jordanian patients. Genet. Test. Mol. Biomarkers 19, 629–636. Li, L.H., Yin, X.Y., Wu, X.H., Zhang, L., Pan, S.Y., Zheng, Z.J., Wang, J.G., 2014. Serum 25(OH)D and vitamin D status in relation to VDR, GC and CYP2R1 variants in Chinese. Endocr. J. 61, 133–141. Little, J., Higgins, J.P.T., Ioannidis, J.P.A., Moher, D., Gagnon, F., Von Elm, E., Khoury, M.J., Cohen, B., Davey-Smith, G., Grimshaw, J., 2009. STrengthening the REporting of genetic association studies (STREGA) – an extension of the STROBE statement. Eur. J. Clin. Investig. 39, 247–266. Manousaki, D., Dudding, T., Haworth, S., Hsu, Y.H., Liu, C.T., Medinagómez, C., Voortman, T., Van, D.V.N., Melhus, H., Robinsoncohen, C., 2017. Low-frequency
5. Conclusion The present meta-analysis confirmed that a statistically significant association exists between the rs10741657 polymorphism and 25(OH)D levels and vitamin D deficiency in Caucasian and Asian populations. However, these results may not be generalizable to all races, because our meta-analysis did not included African populations. Therefore, in the future we plan to conduct a well-designed, more comprehensive study with a larger sample size study to validate our conclusions. Acknowledgments We thank all the investigators in this study. Funding sources No funding supports this work. Disclosure We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. 368
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Slater, N.A., Rager, M.L., Havrda, D.E., Harralson, A.F., 2015. Genetic variation in CYP2R1 and GC genes associated with vitamin D deficiency status. J. Pharm. Pract. 30, 31–36. Størdal, K., Mårild, K., Tapia, G., Haugen, M., Cohen, A.S., Lie, B.A., Stene, L.C., 2017. Fetal and maternal genetic variants influencing neonatal vitamin D status. J. Clin. Endocrinol. Metab. 102, 4072–4079. Tabatabaeizadeh, S.A., Avan, A., Bahrami, A., Khodashenas, E., Esmaeili, H., Ferns, G.A., Abdizadeh, M.F., Ghayour-Mobarhan, M., 2017. High-dose supplementation of vitamin D affects measures of systemic inflammation: reductions in high-sensitivity Creactive protein level and neutrophil to lymphocyte ratio (NLR) distribution. J. Cell. Biochem. 118, 4317–4322. Teriaky, A., Mosli, M., Chandok, N., Aljudaibi, B., Marotta, P., Qumosani, K., 2017. Prevalence of fat-soluble vitamin (A, D, and E) and zinc deficiency in patients with cirrhosis being assessed for liver transplantation. Acta Gastroenterol. Belg. 80, 237–241. Thacher, T.D., Levine, M.A., 2016. CYP2R1 mutations causing vitamin D-deficiency rickets. J. Steroid Biochem. Mol. Biol. 173, 333–336. Thakkinstian, A., Mcelduff, P., D'Este, C., Duffy, D., Attia, J., 2005. A method for metaanalysis of molecular association studies. Stat. Med. 24, 1291–1306. Wang, T.J., Zhang, F., Richards, J.B., Kestenbaum, B., Meurs, J.B.V., Berry, D., Kiel, D., Streeten, E.A., Ohlsson, C., Koller, D.L., 2010. Common genetic determinants of vitamin D insufficiency: a genome-wide association study. Lancet 376, 180–188. Wen, X., 2012. Association of GC and CYP2R1 Gene Polymorphisms With Serum Vitamin D Levels in Postmenopausal Women of Han Nationality in Beijing. Beijing Union Medical College. Xia, J., O'Reilly, P.F., Aschard, H., Hsu, Y.H., Richards, J.B., Dupuis, J., Ingelsson, E., Karasik, D., Pilz, S., Berry, D., 2018. Genome-wide association study in 79,366 European-ancestry individuals informs the genetic architecture of 25-hydroxyvitamin D levels. Nat. Commun. 9, 260. Xu, X., Mao, J., Zhang, M., Liu, H., Li, H., Lei, H., Han, L., Gao, M., 2015. Vitamin D deficiency in Uygurs and Kazaks is associated with polymorphisms in CYP2R1 and DHCR7/NADSYN1 genes. Med. Sci. Monit. 21, 1960–1968. Ye, Z., Sharp, S.J., Burgess, S., Scott, R.A., Imamura, F., Consortium, I.A., Langenberg, C., Wareham, N.J., Forouhi, N.G., 2015. Association between circulating 25-hydroxyvitamin D and incident type 2 diabetes: a mendelian randomisation study. Lancet Diabetes Endocrinol. 3, 35–42. Yu, F., 2016. The Association of Vitamin D Pathway Genes VDR and CYP2R1 Polymorphisms with Type 2 Diabetes Mellitus. Zhengzhou University.
synonymous coding variation in CYP2R1 has large effects on vitamin D levels and risk of multiple sclerosis. Am. J. Hum. Genet. 101, 227–238. Mao, Y.Y., 2017. Correlation between GC、CYP2R1 Genetic Polymorphism and 25Hydroxyvitamin D in Ningxia Hui Postmenopousal Women. Ningxia Medical University. Mccullough, M.L., Mayo, B.T.L., 2009. Vitamin D gene pathway polymorphisms and risk of colorectal, breast, and prostate cancer. Annu. Rev. Nutr. 29, 111–132. Moher, D., Liberati, A., Tetzlaff, J., Altman, D.G., 2009. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. Revista Española De Nutrición Humana Y Dietética. 18, e123. Munafò, M.R., Flint, J., 2004. Meta-analysis of genetic association studies. Trends Genet. 20, 439–444. Nissen, J., Vogel, U., Ravn-Haren, G., Andersen, E.W., Nexø, B.A., Andersen, R., Mejborn, H., Madsen, K.H., Rasmussen, L.B., 2014. Real-life use of vitamin D 3 -fortified bread and milk during a winter season: the effects of CYP2R1 and GC genes on 25-hydroxyvitamin D concentrations in Danish families, the VitmaD study. Genes Nutr. 9, 413. O'Brien, K.M., Sandler, D.P., Shi, M., Harmon, Q.E., Taylor, J.A., Weinberg, C.R., 2018. Genome-wide association study of serum 25-hydroxyvitamin D in US women. Front. Genet. 9, 67. Parr, C.L., Magnus, M.C., Karlstad, O., Holvik, K., Lund-Blix, N.A., Haugen, M., Page, C.M., Nafstad, P., Ueland, P.M., London, S.J., Haberg, S.E., Nystad, W., 2018. Vitamin a and D intake in pregnancy, infant supplementation, and asthma development: the Norwegian mother and child cohort. Am. J. Clin. Nutr. 107, 789–798. Ramos-Lopez, E., Brueck, P., Jansen, T., Herwig, J., Badenhoop, K., 2007. CYP2R1 (vitamin D 25-hydroxylase) gene is associated with susceptibitity to type 1 diabetes and vitamin D levels in Germans. Diabetes Metab. Res. Rev. 23, 631–636. Robien, K., Butler, L.M., Wang, R., Beckman, K.B., Walek, D., Koh, W.P., Yuan, J.M., 2013. Genetic and environmental predictors of serum 25(OH)D concentrations among middle-aged and elderly Chinese in Singapore. Br. J. Nutr. 109, 493–502. Ross, A.C., Manson, J.A.E., Abrams, S.A., Aloia, J.F., Brannon, P.M., Clinton, S.K., Durazoarvizu, R.A., Gallagher, J.C., Gallo, R.L., Jones, G., 2011. The 2011 report on dietary reference intakes for calcium and vitamin D from the Institute of Medicine: what clinicians need to know. J. Clin. Endocrinol. Metab. 96, 53–58. Sadat-Ali, M., Al-Turki, H.A., Azam, M.Q., Al-Elq, A.H., 2016. Genetic influence on circulating vitamin D among Saudi Arabians. Saudi Med. J. 37, 996–1001. Sharma, S., Biswal, N., Bethou, A., Rajappa, M., Kumar, S., Vinayagam, V., 2017. Does vitamin D supplementation improve glycaemic control in children with type 1 diabetes mellitus? - a randomized controlled trial. J. Clin. Diagn. Res. 11, SC15.
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Review
Effects of CYP2R1 gene variants on vitamin D levels and status: A systematic review and meta-analysis Leizhen Duana, Zonggui Xuea, Huanwen Jia, Dongdong Zhangb, Yan Wangb, a b
T
⁎
Department of Medical Services, Zhengzhou Central Hospital affiliated to Zhengzhou University, Zhengzhou 450001, China Department of Nutrition and Food Hygiene, College of Public Health, Zhengzhou University, Zhengzhou 450001, China
A R T I C LE I N FO
A B S T R A C T
Keywords: CYP2R1 Polymorphism 25(OH)D Vitamin D deficiency Meta-analysis
Background and objective: CYP2R1 is a key gene in the vitamin D metabolic pathway. It has been suggested that CYP2R1 gene variants in European populations are associated with concentrations of 25(OH)D, a biomarker of vitamin D levels and status in peripheral blood. However, a comprehensive meta-analysis of this effect including different ethnicities has never been conducted. The objective of this meta-analysis was to evaluate the association between CYP2R1 gene variants and 25(OH)D levels and vitamin D status. Methods: PubMed, EMBASE, Web of Science, CNKI and Wanfang databases were systematically searched up to May 2018. Reporting followed PRISMA guidelines. The quality of the evidence was assessed using the STREGA system. Random or fixed effects model combined estimates and sub-group tested for ethnic differences. The I2 statistic quantified between-study variation due to heterogeneity. Results: Sixteen articles with a total of 52,417 participants met the inclusion criteria and were included in the meta-analysis. For rs10741657, GG genotype was associated with a clear descending trend of 25(OH)D levels when compared with the AA genotype [SMD = −2.32, 95% CI (−4.42, −0.20); SMD = −3.46, 95% CI (−6.60, −0.33) and SMD = −0.24, 95% CI (−0.51, −0.03) for total, Caucasian and Asian groups, respectively] with the following heterogeneities I2 = 37.9%, 69.2% and 24.5%, respectively. However, under the AG/ AA genetic model, significant changes in 25(OH)D levels [SMD and 95% CI: −1.27(−2.32, −0.23)] were only evident in the Caucasian population. The meta-analysis on vitamin D deficiency showed that the risk-allele G was associated with an increased risk of vitamin D deficiency (OR = 1.09; 95% CI = 1.03–1.15, P = 0.002). The association between rs10741657 and increased risk of vitamin D deficiency was significant (OR = 1.42; 95% CI = 1.11–1.83, P = 0.006) under the dominant model (GG + AG/AA), but not under the recessive model (GG/ AG + AA), (OR = 1.28; 95% CI = 0.89–1.84, P = 0.181). There was no evidence of publication bias. Conclusion: Published articles provide evidence supporting a major role for the rs10741657 polymorphism of the CYP2R1 gene in determining 25(OH)D levels and the presence of vitamin D deficiency.
1. Introduction
asthma, diabetes, cardiovascular disease and some forms of cancer (Mccullough and Mayo, 2009; Abudawood et al., 2018; Parr et al., 2018). Factors which influence the concentrations of 25-hydroxyvitamin D [25(OH)D], a vitamin D biomarker that can be measured in blood, include sun exposure, latitude, race, age, gender and dietary supplements. (Fohner et al., 2016). However, these environmental factors appear to only contribute to 13% of the individual heterogeneity in vitamin D levels (Burgaz et al., 2007). Accumulating evidence indicates that differences in vitamin D levels, including vitamin D
Vitamin D is a fat-soluble vitamin and steroid pro-hormone that is involved in the regulation of various biological processes, such as immunoregulation and insulin resistance and also has anti-inflammatory and anti-cancer effects (Mccullough and Mayo, 2009; Sharma et al., 2017; Tabatabaeizadeh et al., 2017; Abudawood et al., 2018). Accumulating evidence supports the notion that low vitamin D concentrations are strongly associated with various adverse outcomes, such as
Abbreviations list: CYP2R1, cytochrome P450 family 2, subfamily R, peptide 1; PRISMA, preferred reporting items for systematic reviews and meta-analyses; STREGA, strengthening report of genetic association studies; SMD, standardized mean difference; OR, odds ratio; CI, confidence intervals; 25(OH)D, 25-hydroxyvitamin D; 7-DHC, 7-dehydrocholesterol; GWAS, genome-wide association studies; IOM, Institute of Medicine; HWE, Hardy-Weinberg equilibrium; VDD, vitamin D deficiency ⁎ Corresponding author at: Department of Nutrition and Food Hygiene, College of Public Health, Zhengzhou University, Zhengzhou, 450001, Henan, China. E-mail address: [email protected] (Y. Wang). https://doi.org/10.1016/j.gene.2018.08.056 Received 5 June 2018; Received in revised form 13 August 2018; Accepted 14 August 2018 Available online 16 August 2018 0378-1119/ © 2018 Elsevier B.V. All rights reserved.
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Fig. 1. The vitamin D metabolic process in vivo and the role of the CYP2R1 gene.
2. Methods
deficiency, are under strong genetic control. Several twin and family studies have reported that 23% to 80% of the heterogeneity in vitamin D levels was attributable to genetic factors (Bahrami et al., 2017). Attention has now turned to the gene effects that could have an impact on vitamin D metabolism. The production of 1,25(OH)2D, an active form of vitamin D, relies on a series of enzymatic processes. Sun light (UVB at 270 nm–290 nm), acting on 7-dehydrocholesterol (7-DHC) in the epidermis of the skin, generates pre-vitamin D. Pre-vitamin D is an inactive precursor, requiring 25-hydroxylase (encoded by the cytochrome P450 CYP2R1 gene in the liver) to yield the 25(OH)D. Subsequently, 1α-hydroxylase in the kidney (encoded by the CYP27B1 gene) catalyzes the conversion of 25(OH)D to 1,25(OH)2D. (Fig. 1). The human CYP2R1 gene contains 5 exons and spans about 15.5 kb. It is located on chromosome 11p15.2 (gene ID: 120227), and contains 501 amino acids (Cheng et al., 2003). It encodes a microsomal vitamin D 25-hydroxylase which is considered to be the most important enzyme in the metabolism of vitamin D. In mice, the combined knockout of both Cyp2r1 and Cyp27b1, only reduced levels of 25(OH)D by 50%, did not affect circulating 1,25(OH)2D (Burgaz et al., 2007). In humans, Cheng et al. first identified a missense mutation in exon 2 of the CYP2R1 gene that could result in vitamin D deficiency (Cheng et al., 2004). Subsquently, several genome-wide association studies (GWAS) performed in European populations (Ahn et al., 2010; Wang et al., 2010; Anderson et al., 2014; O'Brien et al., 2018; Xia et al., 2018), identified > 25 SNPs of the CYP2R1 gene associated with vitamin D levels or vitamin D deficiency. Numerous replication studies were conducted in other ethnic/racial populations (Wen, 2012; Robien et al., 2013; Xu et al., 2015; Yu, 2016; Mao, 2017). However, the results of these studies did not seem to support the conclusions observed in the European populations. Here, we conducted a meta-analysis including a large scale and unrelated population with general health condition to evaluate more precisely the association between CYP2R1 gene polymorphisma and 25(OH)D levels and vitamin D status.
2.1. Data sources and search strategies This study was performed following a predefined protocol and under the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) (Table S1) (Moher et al., 2009). The searches were conducted by LZ.D and YW from Jan 2010 to May 2018 using PubMed, EMBASE, Web of Science, CNKI and WanFang databases with the following search terms: (“CYP2R1” or “25-hydroxylase” or “cytochrome P450 2R1”) and (“SNP” or “polymorphism” or “variants” or “mutation”) and (“vitamin D" or “25(OH)D” or “calcitriol” or “vitamin D deficiency”). The search was restricted to studies published in English or Chinese. To complement the electronic searches, we also perused studies included in relevant systematic reviews and reference lists of pertinent articles. 2.2. Inclusion and exclusion criteria Papers were included if they contained the following data: (1) primary outcome of interest was vitamin D deficiency or 25(OH)D levels; (2) the gene of interest was CYP2R1; (3) the definition of vitamin D deficiency was 25(OH)D levels below 20 ng/mL (50 nmol/L), as described by the Institute of Medicine (IOM) (Ross et al., 2011); (4) either (a) or (b), as follows: (a) the odds ratio (OR) estimates and their 95% confidence intervals (CI) were given or sufficient data were available to evaluate the association between the rs10741657 polymorphism and vitamin D deficiency. (b) the values for 25(OH)D levels, including mean and standard deviation (SD), as well as the sample sizes (n) for the rs10741657, rs12794714 and rs10766197 genotypes were provided. (5) If the subjects were included in a study of a disease or an interventional experiment, only the normal control group or baseline population was included. We excluded studies if: (1) data were not fully available after 362
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Fig. 2. Flow diagram showing the number of studies included and excluded.
2.5. Data synthesis and statistical analysis
contacting the authors by E-mail; (2) they were meta-analysis or systematic reviews; (3) they were duplicate studies; (4) participants were children, pregnant women, and subjects diagnosed with a chronic medical illness, (such as malignancy), or were documented to have any organ dysfunction that could affect vitamin D metabolism.
Meta-analysis methods were used to assess the association between CYP2R1 gene variants and vitamin D levels and status. Statistical heterogeneity between different studies was measured using the Chisquare based Q and I2 statistics. Depending on the heterogeneity, either a fixed-effect model or a random-effect model was adopted (if the heterogeneity I2 was over 50% or the P value of the Q test > 0.05, the random-effect model was used; otherwise, the fixed-effect model was applied). The pooled OR and 95% CI were calculated to determine the association between rs10741657 and vitamin D deficiency under allele, dominant and recessive genetic models. The combined standardized mean difference (SMD) and 95% CI were used to assess the relationship between 25(OH)D levels and polymorphisms in rs10741657, rs12794714 and rs10766197 of CYP2R1 gene. Separate meta-analyses were carried out for different ethnic groups (Asian and Caucasian groups). Sensitivity analyses were performed to assess the stability of the results by omitting one study at a time and repeating the meta-analysis with the rest. Publication bias was assessed through visual inspection of Begg's funnel plots and Egger's linear regression tests. Statistical evidence of an association was considered significant when P < 0.05. Data were analyzed using the Stata software, version 11 (Stata Corp, College Station, TX, USA).
2.3. Data extraction Data on the studies' characteristics and related information were collected in Excel format using a premade checklist by one reviewer (LZD), and then double-checked by another author (YW). The following information was extracted: (1) surname of the first author, year of publication, sample size and country of origin; (2) information about participants and methods, including gender, age, BMI, genotyping methods, measurement of 25(OH)D; (3) primary information on vitamin D status, including risk allele, genotypes and their frequency or numbers, OR and the corresponding 95% CI, and the P-value for the Hardy–Weinberg equilibrium (HWE) test in the control group; (4) primary information on vitamin D levels, including the mean and SD values of the 25(OH)D concentrations, and sample sizes for every genotype. 2.4. Quality assessment Two reviewers (LZD and YW) independently assessed the quality of all included studies by following a quality score system- STREGA (strengthening report of genetic association studies) (Little et al., 2009). The STREGA system includes 22 items with scores ranging from 0 to 22 (Table S2). The quality of the studies was regarded as “high” when the score was 18–22, “moderate-high” when the score was 13–17, and “low” when the score was 0–12. Discrepancies were resolved through consensus and third-party adjudication.
3. Results 3.1. Results of the search strategy A systematic search of the literature led to the identification of 311 possibly relevant articles. One hundred and fifty-nine duplicate studies 363
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Table 1 Characteristics of the studies on the association of rs10741657 variants with vitamin D deficiency. First author (year)
Wang, 2010 Cheung, 2013 Li, 2014 Lafi, 2015 Xu, 2015 Xu, 2015 Sadat-Ali, 2016
Country (ethnicity)
n
European (C-p) China (A-p) China (A-p) Jordan (A-p) China (A-p) China (A-p) Saudi-Arab (A-p)
Age
BMI
VDD(+) (n)
VDD(−) (n)
GG
GA
AA
GG
GA
AA
HWE
STREGA score
30,000
NA
NA
NA
NA
NA
NA
NA
NA
√
20
712
48.41 ± 15.34
22.76 ± 3.97
NA
NA
NA
NA
NA
NA
√
16
1199
48.3 ± 12.5
23.5 ± 3.3
196
218
51
278
347
109
√
18
200
NA
NA
52
33
7
39
53
16
√
17
300
47 (39–56.4)
27.5 (24.4–30.6)
93
162
45
75
186
39
√
17
300
44 (40–55)
24 (21.9–27.3)
111
150
39
75
168
57
√
17
284
48 ± 16.7
25.28 ± 3.91
112
114
22
17
18
0
√
19
A-p: Asian population; C-p: Caucasian population; n:number of participants; NA: not available; VDD (+): vitamin D deficiency; VDD(−): no-vitamin D deficiency; HEW: Hardy–Weinberg equilibrium; STREGA: strengthening report of genetic association studies. Table 2 Characteristics of the studies on the association of three SNPs with 25(OH)D levels. rs97 (risk allele)
STREGA score
Country (ethnicity)
n
Age
BMI
GG 25(OH)D mean (sd)
AG 25(OH)D mean (sd)
AA 25(OH)D mean (sd)
HWE
Bu, 2010
American (C-p) England (C-p) American (C-p) Singapore (C-p)
496
65.3 ± 6.7
29.2 ± 5.4
76.6 ± 23.3
74.4 ± 20.8
65.2 ± 17.0
√
6552
NA
NA
56.8 ± 0.34
60.2 ± 0.32
61.1 ± 0.3
√
(G)
5656
NA
NA
75.4 ± 0.87
78.6 ± 0.76
81.6 ± 1.26
√
(G)
504
55.7 ± 7.8
22.8 ± 3
64.9 ± 16.7
67.6 ± 16.6
70 ± 18.03
√
(G)
Hassanein, 2014
Germany (C-p)
109
NA
NA
69.2 ± 17.5 61.2 ± 5.6
66.0 ± 16.7 67.2 ± 5.7
58.6 ± 14.8 69.7 ± 4.8
√ √
(G)
Nissen, 2014
Denmark (C-p)
762
30
21.8 ± 5.5
65.2 ± 6.1 67.2 ± 18.8
63.3 ± 4.7 73.9 ± 21.0
80.5 ± 7.4 76.6 ± 21.4
√ √
(G)
Clendenen, 2015
Swiss (C-p) Jordan (A-p) China (A-p)
1435
NA
NA
74.3 ± 20.8 49.8 ± 2.4
72.9 ± 20.8 51.8 ± 2.0
66.9 ± 19.0 56.5 ± 3.3
√ √
(G)
17
200
18–60
NA
25.3 ± 18.1
31.8 ± 21.4
38.7 ± 24.9
√
(G)
17
2215
52 ± 17.3
NA
27.1 ± 22.4
28.3 ± 23.1
26.7 ± 19.9
√
(G)
17
27.1 ± 21.4 27.4 ± 21.6 41.8 ± 21.5
28.0 ± 22.7 27.9 ± 22.4 41.9 ± 22.8
27.4 ± 23.6 27.2 ± 24.1 47.9 ± 23.1
√ √ √
19.4 ± 0.7
19.7 21.9 22.1 18.8
20.9 20.2 20.1 19.2
22.1 19.2 19.1 20.5
√ √ √ √
NA
18.8 ± 6.7 18.9 ± 7.3 35.7 ± 17.6
Wang, 2010
Robien, 2013
Lafi, 2015 Yu, 2016
Jolliffe, 2016 Arabi, 2017
Mao, 2017
Størdal, 2017
American (C-p) American (C-p) China (A-p)
Norway (C-p)
222
72 ± 9.2
29.3 ± 6.8
218
71.0 ± 4.7
30.3 ± 4.6
480
1063
53.2 ± 7.3
NA
± ± ± ±
7.3 8.4 8.2 7.0
± ± ± ±
7.2 7.4 7.6 8.8
20.0 ± 9.7 19.8 ± 9.0 35.2 ± 17.5
± ± ± ±
10 6.5 6.4 8.7
18.2 ± 7.2 18.5 ± 7.6 35.4 ± 17.1
√ √ √
rs57 (risk allele)
rs14 (risk allele)
First author (year)
(G)
20 20
19 (G) 18 (G) 19 (A)
(G) (A) (A)
16 20
(G) (G) (A) (G)
16 (G) (A)
(G)
18
A-p: Asian population; C-p: Caucasian population; n: number of participants; NA: not available; BMI: body mass index; HWE: Hardy–Weinberg equilibrium; rs57: rs10741657; rs14: rs12794714; rs97: rs10766197; STREGA: strengthening report of genetic association studies.
articles and the reasons for exclusion are shown in the flow chart (Fig. 2).
were removed using the “Find Duplicates” tool in EndNote X7. An initial examination identified 46 articles by screening the titles and abstracts. After manual examination and detailed consideration, 16 articles with a total of 52,417 participants were included in the present meta-analysis. Of these, 7 articles investigated rs10741657 and vitamin D deficiency, 10 articles investigated rs10741657 and 25(OH)D levels, 6 articles studied rs12794714 and 25(OH)D levels, and 5 articles investigated rs10766197 and 25(OH)D levels. A list of the excluded
3.2. Study characteristics Briefly, a total of 6 articles with 32,695 participants assessed the association between the rs10741657 polymorphism and vitamin D deficiency (Wang et al., 2010; Cheung et al., 2013; Li et al., 2014; Lafi 364
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Table 3 Meta-analysis of CYP2R1 gene polymorphisma and 25(OH)D levels or vitamin D deficiency. SNP/genetic model
Association of CYP2R1 polymorphisms with 25(OH)D levels N
Rs10741657 G/A (overall) Asian Caucasian GG + AG/AA Asian GG/AG + AA Asian GG/AA (overall) Asian Caucasian AG/AA (overall) Asian Caucasian Rs12794714 GG/AA (overall) Asian Caucasian AG/AA (overall) Asian Caucasian Rs10766197 AA/GG (overall) Asian Caucasian AG/GG (overall) Asian Caucasian
n
SMD (95% CI)
P
Association of CYP2R1 polymorphisms with vitamin D status
2
I (%)
P-H
10
19,194
−2.31 (−4.42, −0.20)
0.032
37.9
0.101
4 6 10
3399 15,795 19,194
−0.24 (−0.51, −0.03) −3.46 (−6.60, −0.33) −0.86 (−1.80, 0.08)
0.049 0.034 0.073
24.5 69.2 43.7
0.108 0.008 0.064
4 6 6
3399 15,795 4022
−0.06 (−0.23, 0.10) −1.27 (−2.32, −0.23) −0.03 (−0.47, 0.41)
0.456 0.017 0.884
39.5 69.7 53.6
0.175 0.027 0.047
3 3 6 3 3 5
3199 823 4022 3199 823 3897
0.21 (−0.14, 0.57) −0.43 (−1.71, 0.86) −0.17 (−0.63, 0.30) 0.20 (−0.04, 0.45) −0.73 (−2.15, 0.69) −0.13 (−0.34, 0.09)
0.236 0.518 0.481 0.108 0.313 0.242
36.5 66.4 50.8 41.5 67.7 72.3
0.201 0.009 0.049 0.043 0.014 0.006
2 3 5 2 3
2695 1202 3897 2695 1202
−0.02 (−0.13, 0.10) −0.2 (−0.57, 0.17) −0.001 (−0.08, 0.08) 0.04 (−0.05, 0.12) −0.09 (−0.22, 0.05)
0.781 0.295 0.988 0.379 0.207
0 73.5 9.5 0 0
0.774 0.023 0.352 0.456 0.471
N
n
OR (95% CI)
P
I2 (%)
P-H
7
32,995
1.09 (1.03–1.15)
0.002
15.4
0.308
6 1 4
2995 30,000 2283
1.17 (1.06–1.29) 1.06 (1.00–1.13) 1.41 (1.11–1.85)
0.002 0.048 0.006
16.0 – 50.0
0.412 – 0.092
4
2283
1.28 (0.89–1.84)
0.181
47.7
0.105
N: numbers of studies; n: numbers of participants; P-H: p value of the heterogeneity; SMD: standardized mean difference; OR: odds ratio; CI: confidence interval.
in Asian populations, where the SMD and 95% CI values were [−0.06 (−0.23, 0.1), P = 0.456], with low heterogeneity (I2 = 39.5%, P = 0.175). The pooled results are presented in Table 3 and Fig. 3B. The rs12794714 and rs10766197 polymorphisms were not found to be associated with 25(OH)D levels, either in pooled or subgroup analyses. (Table 3 and Fig. 4).
et al., 2015; Xu et al., 2015; Sadat-Ali et al., 2016). The cut-off point for vitamin D deficiency was 25 (OH) D level < 20 ng/mL (50 nmol/L) (Ross et al., 2011). The studies were conducted in China, America, Suidi Arabia, Jordan and European countries. A summary of the characteristics of the included studies is provided in Table 1. Twelve articles comprising 19,722 participants from 9 geographical areas were included in the meta-analysis to investigate the association between rs10741657, rs12794714 and rs10766197 and 25(OH)D levels (Bu et al., 2010; Wang et al., 2010; Robien et al., 2013; Hassanein et al., 2014; Nissen et al., 2014; Clendenen et al., 2015; Lafi et al., 2015; Jolliffe et al., 2016; Yu, 2016; Arabi et al., 2017; Mao, 2017; Størdal et al., 2017). All the selected loci were in accordance with HWE. Descriptive study information is shown in Table 2.
3.4. Meta-analysis of vitamin D status Under the allelic genetic model (G/A), we found that the 25(OH)D decreasing allele rs10741657 (G) was associated with an increased risk of vitamin D deficiency (OR = 1.09; 95% CI = 1.03–1.15, P = 0.002), with low heterogeneity among studies (I2 = 15.4%, P = 0.308). (Fig. 5A). Under the dominant model (GG + AG/AA), the association between rs10741657 and increased risk of vitamin D deficiency was significant (OR = 1.42; 95% CI = 1.11–1.83, P = 0.006), with low heterogeneity (I2 = 50%, P = 0.092). (Fig. 5B). However, under the recessive model (GG/AG + AA), no significant association was found (OR = 1.28; 95% CI = 0.89–1.84, P = 0.181), with low heterogeneity among studies (I2 = 47.7%, P = 0.105). (Fig. 5C).
3.3. Meta-analysis of 25(OH)D levels Meta-analysis estimates the association between the different SNPs and 25(OH)D levels are shown in Table 3. For rs10741657, GG genotype showed an obvious decreasing trend of 25(OH)D levels when compared with the AA reference genotype [SMD = −2.32, 95% CI = (−4.42, −0.20); SMD = −3.46, 95% CI = (−6.60, −0.33) and SMD = −0.24, 95% CI = (−0.51, −0.03) for total, Caucasian and Asian groups, respectively] with heterogeneities (I2 = 37.9%, P = 0.101; I2 = 69.2%, P = 0.008 and I2 = 24.5%, P = 0.108). (Table 3 and Fig. 3A). Under the AG/AA genetic model, no association was found between rs10741657 and 25(OH)D levels in total populations [SMD = −0.86, 95% CI = (−1.80, 0.08), P = 0.073], with low heterogeneity (I2 = 43.7%, P = 0.064). However, when the study population was divided into two ethnic subgroups, we found evidence of greater change in 25(OH)D levels in the Caucasian population [SMD and 95% CI = −1.27 (−2.32, −0.23), P = 0.017], with high heterogeneity (I2 = 69.7%, P = 0.027). This decreasing trend was not been detected
3.5. Sensitivity and publication bias analysis Sensitivity analysis utilizing the leave-one-out method did not show any major change in primary outcomes, an indication of the good stability of the results. Funnels plots for each meta-analysis appeared to be symmetrical. Egger's test indicated that all the P values were over 0.1, therefore, there is no publication bias in this meta-analysis. (data not shown).
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Fig. 3. Forest plot for the association between rs10741657 and 25(OH)D levels under the additive model. (A) GG/AA. (B) AG/AA.
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Fig. 4. The pooled forest plot for the association of rs12794714 and rs10766197 with 25(OH)D levels.
4. Disscusion To our knowledge, this is the first comprehensive meta-analysis evaluating the association between CYP2R1 SNPs and 25(OH)D levels and vitamin D status. The meta-analysis of 25(OH)D levels and vitamin D status in European and Asian populations identified rs10741657 variants that showed a large effect and strong association with 25(OH)D levels and vitamin D deficiency. The gene of CYP2R1 encodes 25-hydroxylase which can convert vitamin D into 25(OH)D (Cheng et al., 2003). Growing evidence suggests that the CYP2R1 gene mutation have the capacity to directly impact 25(OH)D levels and correlate with diseases related to vitamin D deficits (Ahn et al., 2010; Wang et al., 2010; Elkum et al., 2014; Hassanein et al., 2014; Bahrami et al., 2017). The rs10741657 polymorphism is located in the non-coding region 5′-UTR, which may regulate gene expression, and therefore modulate the levels of expression and activity of 25-hydroxylase (Ramos-Lopez et al., 2007). The rs12794714 and rs10766197 polymorphisms are located in the coding region of introns, which may be involved in gene regulation and selective splicing regulation (Ramos-Lopez et al., 2007). In our meta-analysis of the association between rs10741657 and 25(OH)D levels, GG carriers showed a decreasing trend of 25(OH)D levels compared with the no-risk genotype AA, the calculated SMD and 95% CI values were −2.31(−4.42, −0.20), −0.24 (−0.51, −0.03), and −3.46 (−6.60, −0.33) for total, Asian and Caucasian populations, respectively. Our results are in agreement with those reported by Zheng et al., who found an association between the rs10741657 polymorphism and 25(OH)D levels (Ye et al., 2015). However, this significant decreasing trend was only observed in the Caucasian population when comparing the AG genotype with AA genotype [SMD and 95% CI = −1.27 (−2.32, −0.23), P = 0.017]. There may be some reasonable explanations for these differences between ethnic groups. First, the sample size of the Asian population is much smaller than that of Caucasian population (3399 vs. 15,795). Second, compared with the GG/AA additive model, the AG/AA model has a lower cumulative risk 366
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Fig. 5. The pooled forest plot for the association of rs10741657 with vitamin D deficiency under three genetic model. (A) G/A. (B) GG + AG/AA. (C) GG/AG + AA. 367
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Authors contributions
effect. Third, the effects of CYP2R1 SNPs on serum 25(OH)D concentrations may vary between different ethnic groups (Thacher and Levine, 2016). Our analysis of the association between rs12794714 or rs10766197 and 25(OH)D levels, under the GG/AA (AA/GG) or AG/AA (AG/GG) genetic models did not showed any associations between SNPs and 25(OH)D levels in the total or subgroup populations. Rs12794714 and rs10766197 are located in the intron region, and since these synonymous mutations do not modify the protein sequence, 25-hydroxylase activity and function may not be affected (Manousaki et al., 2017). Therefore, this may explain the lack of meaningful results. In addition, only 6 articles on rs12794714 and 5 articles on rs10766197 fulfilled the inclusion criteria were included in the meta-analysis, and all of them studied Asian populations. This could also be an important reason why the association was not significant. Therefore, our conclusions need to be verified with larger samples and including other ethnic groups. Analysis of the association between rs10741657 and vitamin D deficiency showed that the risk allele G had an effect size 1.09-fold, 1.17-fold and 1.06-fold larger than the no-risk allele A in total, Asian and Caucasian populations, respectively. Our finding is in agreement with the results of some published studies (Wang et al., 2010; Lafi et al., 2015; Slater et al., 2015). The rs10741657 polymorphism was associated with a 1.42-fold increase in the risk of vitamin D deficiency under the dominant genetic model (GG + AG/AA) in Asian populations. However, this association was not observed under the recessive genetic model (GG/AG + AA) in Asian populations [OR and 95% CI = 1.28 (0.89–1.84), P = 0.181]. A weakening of the cumulative effect of risk alleles could be a reasonable explanation for this difference between genetic models (Thakkinstian et al., 2005). A major strength of this study is that it only included in the healthy participants, which is a very reasonable approach to eliminate the effects of disease on the levels of vitamin D and thus reduce heterogeneity between studies (Munafò and Flint, 2004; Teriaky et al., 2017). This is the first meta-analysis to evaluate the relationship between CYP2R1 gene polymorphisms and vitamin D levels and status in Asian populations. No publication bias was observed and the results were stable and reliable. However, several limitations should also be acknowledged. First, this meta-analysis did not include studies with African populations, so it's conclusions cannot be extrapolated. Second, the association between rs12794714 and rs10766197 polymorphisms and vitamin D deficiency was not analyzed because the number of studies and participants was insufficient. Third, the blood collection times and 25(OH)D measurement methods were not controlled.
The authors' responsibilities were as follows: YW: conceived and designed the study; LZD, ZGX and HWJ: performed literature search and Data collection; LZD: analyzed data and wrote the manuscript; YW and DDZ: modified the language of the manuscript. All authors read and provided critical comment on the manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2018.08.056. References Abudawood, M., Tabassum, H., Ansar, S., Almosa, K., Sobki, S., Ali, M.N., Aljohi, A., 2018. Assessment of gender-related differences in vitamin D levels and cardiovascular risk factors in Saudi patients with type 2 diabetes mellitus. Saudi J. Biol. Sci. 25, 31–36. Ahn, J., Yu, K., Stolzenberg-Solomon, R., Simon, K.C., Mccullough, M.L., Gallicchio, L., Jacobs, E.J., Ascherio, A., Helzlsouer, K., Jacobs, K.B., 2010. Genome-wide association study of circulating vitamin D levels. Hum. Mol. Genet. 19, 2739–2745. 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5. Conclusion The present meta-analysis confirmed that a statistically significant association exists between the rs10741657 polymorphism and 25(OH)D levels and vitamin D deficiency in Caucasian and Asian populations. However, these results may not be generalizable to all races, because our meta-analysis did not included African populations. Therefore, in the future we plan to conduct a well-designed, more comprehensive study with a larger sample size study to validate our conclusions. Acknowledgments We thank all the investigators in this study. Funding sources No funding supports this work. Disclosure We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. 368
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