Introduction
Vitamin D regulates calcium and phosphate homeostasis and reportedly has other roles in human physiology[
1,
2]. Vitamin D deficiency is associated with the occurrence of osteoporosis, autoimmune diseases, cardiovascular disease, type 1 and type 2 diabetes mellitus, and several types of cancer[
3‐
7]. Vitamin D also plays an important role in the maintenance of the musculoskeletal system. It is positively associated with muscle strength and physical performance, and is negatively associated with fall and fracture risk[
8‐
11].
Vitamin D deficiency has been reported to be common in patients with rheumatoid arthritis (RA), and more than 70% of Japanese patients with RA had vitamin D deficiency[
12,
13]. Significant associations of vitamin D deficiency were found with some independent clinical risk factors: female gender, younger age, high disability score in the Japanese version of the Health Assessment Questionnaire (J-HAQ), low serum total protein level, low serum total cholesterol level, high serum alkaline phosphate (ALP) level, and use of non-steroidal anti-inflammatory drugs (NSAIDs)[
12].
Bone mineral density (BMD) is the major predictor of osteoporotic fracture, and previous studies have reported that patients with RA have a lower BMD and are at greater risk of hip fracture than healthy individuals[
14,
15]. We have previously shown that a high J-HAQ disability score, advanced age, history of total knee replacement (TKR), and low body mass index (BMI) were clinical risk factors for the occurrence of hip fracture in Japanese patients with RA[
16].
Prior twin and family studies suggested that genetic factors also influence serum vitamin D concentration[
17,
18]. Genetic variants that affect serum 25-hydroxyvitamin D (25(OH)D) concentration, an indicator of vitamin D status, were recently identified in a meta-analysis of genome-wide association studies (GWAS) in Caucasian populations[
19,
20]. Though the presence of heterogeneity in genes related to RA has been suggested in many population-based studies[
21], these associations remain unknown in the Japanese population.
The purpose of this study was to validate the possible association between genetic variants and serum 25(OH)D concentration and to test whether the serum 25(OH)D-linked variants were associated with the occurrence of hip fracture in Japanese patients with RA.
Discussion
In this study, an association between a polymorphism of rs2282679 in the GC locus and serum 25(OH)D concentration was validated in Japanese patients with RA. Minor alleles of rs2282679 had additive effects on decreasing serum 25(OH)D concentrations. In addition, rs2282679 was significantly associated with the occurrence of hip fracture in Japanese patients with RA. This is the first report that a SNP P7 in the GC locus was associated with the risk for hip fracture.
The
GC gene encodes the group-specific component known as the vitamin D binding protein (DBP) that plays an important role in the vitamin D metabolic pathway[
25]. Most circulating vitamin D metabolites are bound to DBP to be transported to target organs. In the previous candidate gene studies and the recent GWAS, some
GC polymorphisms were associated with serum 25(OH)D concentration, and the strongest association was observed for rs2282679[
19,
20,
26]. Our results provide supportive evidence that serum 25(OH)D concentration might partly be affected by a polymorphism of rs2282679 or the other variants that are in tight linkage disequilibrium with rs2282679.
Vitamin D is an important factor in mineral metabolism, bone growth and maintenance of the skeleton[
1]. In addition, 1,25(OH)
2D, one of the vitamin D metabolites, has direct action on muscle strength and function modulated by vitamin D receptors expressed in human muscle tissue[
8]. Therefore, vitamin D deficiency can lead to low bone density and muscle weakness, resulting in falls and fractures[
8,
27,
28]. In many studies, vitamin D supplementation has been reported to reduce the risk for falls and fractures among older individuals[
29,
30].
To date, many genetic factors associated with low-trauma fracture including hip fracture have been reported in genome-wide meta-analysis studies[
31,
32]. Many variants with small effects may contribute to fracture risk, but only a few vitamin D-related genetic polymorphisms have been reported to be associated with fracture risk[
33]. We explored the genetic risk of hip fracture in variants demonstrated to be associated with lower serum vitamin D concentration and found an association between rs2282679 in
GC and the occurrence of hip fracture. Our results indicated that the risk allele carriers of the
GC gene polymorphism tend to have low vitamin D levels that lead to greater risk of hip fracture.
Though
DHCR7/NADSYN1 and
CYP2R1 polymorphisms were associated with serum 25(OH)D concentration in the recent GWAS, we could not validate such an association in this study[
19,
20]. There are a number of possible explanations for the lack of an association. One is the insufficient statistical power to validate the associations. The number of samples was smaller than in the previous reports[
20]. The SNP with highest statistical power to validate the association in this study was rs2282679 in the
GC locus with a value of 0.72, and the others had comparatively lower statistical power (for example, 0.22 with rs3829251 in the
DHCR7/NADSYN1 locus). Another reason for the lack of an association is that all studied subjects were RA patients, whereas the recent GWAS were in healthy individuals[
19,
20]. Vitamin D is related to immunological processes, and vitamin D status has been reported to be associated with the risk of developing autoimmune diseases including RA[
1,
14]. In addition, serum vitamin D concentration has been shown to be lower in patients with greater disease activity[
34]. Although the disease activity of the patients might affect the results of this study, DAS28 was not associated with serum 25(OH)D level in the studied population (data not shown). The difference in the genetic background between Caucasian and Japanese populations might also affect the results, which suggests genetic heterogeneity in
NADSYN1,
DHCR7 and
CYP2R1.
The strength of this study is that the datasets were relatively large and based on a single-institution cohort study of Japanese patients with RA. Serum 25(OH)D concentration was measured in the same season of the same year. Therefore, the differences between regions, heterogeneity and seasons had less influence on the results.
The potential limitation of this study is that the serum 25(OH)D concentration data were available from only 899 of the 1,957 patients with DNA samples, and the study on serum 25(OH)D concentration was a cross-sectional study. The smaller sample size reduced the statistical power to detect minor effects on events. Though the measurement from multiple time points would provide more valid estimates of the results, there was only one blood sample assayed for serum 25(OH)D concentration for each person. Further studies are required to confirm these associations.
Acknowledgements
We appreciate all DNA donors for making this study possible. We thank all members of the Institute of Rheumatology, Tokyo Women’s Medical University for their successful management of the IORRA cohort. We are also grateful to Ms Kaori Arai and Ms Akiko Sato for their technical assistance and to Dr Eisuke Inoue for his guidance and suggestions in statistical analyses. This work was supported by grants provided by the Japanese Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research (KI, SM), the Japanese Ministry of Health, Labour and Welfare (SM, HY) and the Japan Science and Technology Agency (HY).
Competing interests
The authors declared that they have no competing interests.
Authors’ contributions
KI designed the study. KI, TF, YT, AT, HY and SM collected DNA samples and the data on fracture. SY and KI performed genotyping. SY and KI contributed to the statistical analyses. SY and KI wrote most of the manuscript. All authors contributed to writing and correcting the manuscript and have approved the final version.