Assessing vitamin D related genetic variants, status, and influence factors in pregnant women in Eastern and Central China

Abstract Vitamin D deficiency has recently become a global public health problem. However, it is still unclear if gene polymorphisms in the vitamin D pathway influence vitamin D levels among pregnant women in Eastern and Central China. The objective of this study was to assess factors influencing vitamin D levels in pregnant women. A total of 326 participants in Shandong and Henan provinces in China were enrolled from August 2017 to April 2019. Serum 25(OH)D levels and single nucleotide polymorphisms (SNPs) in the vitamin D pathway were measured using the blood samples collected in the first trimester, second trimester, and third trimester. Data on demographics, lifestyle, and health behavior were collected using a questionnaire. Statistical analyses were performed using the R software. The prevalence of 25(OH)D deficiency was significantly more severe in pregnant women. The average 25(OH)D value of all enrolled pregnant women was 14.57 ± 7.21 ng/ml (deficiency). Only 15 (4.60%) participants had a 25(OH)D concentration ≥30 ng/ml (sufficient). The prevalence of four ranks of vitamin D levels from severe 25(OH)D deficiency to 25(OH)D sufficiency (<10, 10–20, 20–30, and ≥30 ng/ml) was 29.14%, 52.45%, 13.80%, and 4.60%, respectively. Variants of GC (rs1155563) and CYP24A1 (rs6013897) were significantly associated with both 25(OH)D concentrations and vitamin D deficiency among pregnant women, respectively. Our findings suggest that pregnant women in Eastern and Central China are at high risk of vitamin D deficiency. Genetic mutants in the vitamin D pathway (GC and CYP24A1) were significantly associated with 25(OH)D levels in pregnant women in Eastern and Central China.


| INTRODUC TI ON
Vitamin D plays an important role in regulating calcium and phosphorus metabolism and maintaining normal bone mineral homeostasis in the human body (Holick, 2007;White, 2008). The risk of osteomalacia and fracture is proportional to vitamin D deficiency (Bischoff-Ferrari et al., 2009). Recent research has found that vitamin D deficiency is also related to immune dysfunction, tumors, cardiovascular diseases (Adams & Hewison, 2010), and blood glucose metabolic disorders (Leung, 2016). Vitamin D deficiency can cause systemic diseases and warrants attention as a global health problem. Nowadays, compared with men, women have fewer outdoor activities and often use sunscreen; thus, pregnant women's demand for vitamin D has increased significantly (Daly et al., 2012;Zhen, Liu, Guan, Zhao, & Tang, 2015), and vitamin D deficiency is more common in pregnant women. Vitamin D deficiency or insufficiency may be related to pregnancy complications and adverse pregnancy outcomes, including preeclampsia, gestational diabetes mellitus, and preterm birth (Bodnar et al., 2007(Bodnar et al., , 2013Brannon, 2012). As has been well researched, 25-hydroxyvitamin D (25(OH)D) is currently regarded as the only representative biomarker of serum vitamin D and is supplied by cutaneous synthesis and dietary intake (Zerwekh, 2008).  (Carter, 2011). Thus, the genes involved in vitamin D synthesis and metabolism pathways would influence the circulating level of vitamin D. Two genome-wide association studies have found strong associations between polymorphisms of GC and CYP2R1 genes with vitamin D levels (Ahn et al., 2010;Wang, Zhang, et al., 2010). Variants near the genes involved in cholesterol synthesis, hydroxylation, and vitamin D transport affect the vitamin D status. Genetic variations at these loci could identify individuals with a high risk of vitamin D insufficiency (Wang, Zhang, et al., 2010). Barry et al. (2014) revealed that the increase in serum (25(OH) D) attributable to vitamin D 3 supplementation may vary according to common genetic differences in vitamin D 25-hydroxylase (CYP2R1), 24-hydroxylase (CYP24A1), and the vitamin D receptor (VDR) genes. Other previous studies have also reported significant associations of variants in VDR, CYP27B1, CYP24A1, DHCR7/ NADSYN1, CYP27A1, and CYP3A4 (Barry et al., 2014;Gupta, Patrick, & Bell, 2007;Lu, Sheng, et al., 2012), and LRP2 with vitamin D concentrations (Nykjaer et al., 1999).
Recent studies have found that pregnant women in South China are at high risk of vitamin D insufficiency (Wang, Yang, et al., 2010;Xiang et al., 2013;Xiao et al., 2015). However, there are very few studies on the association between genetic polymorphisms and vitamin D concentrations among pregnant women in China. Thus, we conducted a study among pregnant women in Eastern and Central China to evaluate the relationships between SNPs of vitamin D pathway genes and serum vitamin D levels in the first trimester, second trimester, and third trimester. Some studies have evaluated vitamin D status in healthy Chinese populations. For example, Hjelmesaeth, Roislien, Hofso, and Bollerslev's (2010) revealed the relationship between vitamin D and metabolic syndrome in middle-aged and elderly Chinese individuals, while Foo et al. (2009) evaluated vitamin D levels in girls aged 12-14 years in Beijing. Another study described the vitamin D status in Chinese subjects by collecting data from five cities, namely Dalian, Beijing, Hangzhou, Guangzhou, and Urumqi (Yu et al., 2015). However, the status of vitamin D is mediated by different factors, including gender, age, region, ethnicity, clothing habits, and supplement intake. China covers a vast territory and regions of different latitudes receiving different amounts of sunlight. Therefore, to evaluate the general vitamin D levels of the Chinese population, it is important to collect data from Eastern and Central China, such as Shandong and Henan Provinces, which are higher in elevation than Southern China, with thin air and less rainfall. In particular, Tengzhou City is located in the North China Plain in the north latitude of 34.5°, where the average annual number of hours of sunshine is 1,791, and Dengzhou City is located in the Nanyang Basin in the north latitude of 32.5°, where the average annual number of hours of sunshine reaches 1,935. Although another study has examined vitamin D levels by collecting data from several community centers (Lu, Zhang, et al., 2012), they used the immunoassay method to analyze vitamin D levels. Until now, immunoassays have been the most commonly used method to test 25(OH)D levels in China.
Considering these limitations of immunoassays, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the gold standard for testing 25(OH)D levels (Mineva et al., 2015;Tai, Bedner, & Phinney, 2010). The LC-MS/MS assay achieves higher sensitivity, specificity, and reproducibility compared to immunoassays and can determine 25(OH)D 2 and 25(OH)D 3 separately (Maunsell, Wright, & Rainbow, 2005). Vitamin D insufficiency is usually defined by 25(OH)D levels of ≤30 ng/ml, while a concentration of ≤20 ng/ml could represent vitamin D deficiency. Because of the various immunoassay methods, it is difficult to compare inter-study data. There is currently no consensus on a cutoff value for confirming vitamin D deficiency (Holick et al., 2011;Ross et al., 2011;Yates, Schlicker, & Suitor, 1998).
Therefore, one of the goals of our study was to examine the prevalence of vitamin D insufficiency in pregnant women in Eastern and Central China and to evaluate all samples using the gold-standard vitamin D LC-MS/MS analysis method in a central laboratory.
Additionally, we conducted a study to determine whether environmental factors would modify the link between 25(OH)D levels and SNPs during pregnancy to guide pregnant women in order to elevate their 25(OH)D levels.

| Study population
A total of 326 pregnant women who resided in Tengzhou and Dengzhou cities for at least 1 year were enrolled from several community centers between 2017 and 2019. Clinical information including age, weeks of gestation, body mass index (BMI), sun exposure, dietary and drinking habits, physical activities, and medical records during pregnancy was completed by all subjects.

| Single nucleotide polymorphisms
According to previous studies, relative SNPs have been selected in genes involved in vitamin D synthesis and metabolic pathways.
In addition, these SNPs are located in the functional domain and play a vital role in vitamin metabolism from the NCBI database.

| Characteristics of the study population
The study included 326 pregnant women based on biochemical tests, physical examinations, and detailed questionnaires. The levels of biochemical indicators and clinical characteristics are shown in

| Levels of 25(OH)D in pregnant women
Although nearly half of the participants had a habit of vitamin D intake (Table 1), we found that the average level of 25(OH)D among the participants was 14.57 ± 7.21 ng/ml (deficiency). Vitamin D severe deficiency and deficiency (<20 ng/ml) were found in 81.59% of pregnant women. Among them, 29.14% were seriously deficient.
Very few pregnant women had sufficient levels of 25(OH)D (4.6%).

| Correlation between 25(OH)D and other characteristics
To further determine the correlation between 25(OH)D levels and other related clinical characteristics, the influence factors including daily vitamin intake, age, body mass index (BMI), and physical activity were analyzed. As shown in Table 2, these data suggest that there was no significant correlation between 25(OH)D levels and age. The level of 25(OH)D was significantly associated with gestational week, BMI, physical activity, and vitamin D intake. Early pregnancy, lower BMI index, less physical activity, and vitamin D non-intake were significantly associated with lower 25(OH)D levels.

| Association between 25(OH)D levels and SNPs in the vitamin D pathway
To

| Effect of SNPs on 25(OH)D levels is influenced by vitamin D supplementation
We further classified Table 3 into two groups according to SNP genotype and type of the vitamin D supplement (Table 4). As shown in

| D ISCUSS I ON
Low levels of vitamin D in pregnant women are associated with an increased risk of poor maternal and neonatal outcomes, including lower birth weight, gestational diabetes, and high blood pressure in the offspring (Chun, Shin, Kim, Joung, & Chung, 2017;Meems et al., 2016;Murthi et al., 2017). Although some studies have reported vitamin D deficiency in pregnant women in China (Foo et al., 2009;Hjelmesaeth et al., 2010;Lu, Zhang, et al., 2012), their results were limited due to the use of immunoassays.
Hence, the participants in our study were enrolled from Tengzhou and Dengzhou city, which do not encompass all people in Eastern and Central China. Therefore, selection bias was a problem in our study. Another limitation of the study was that the method we used

ACK N OWLED G M ENT
Not applicable.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interests.