Influence of vitamin D binding protein polymorphism, demographics and lifestyle factors on vitamin D status of healthy Malaysian pregnant women

Vitamin D is well known for its role in regulating bone metabolism by stimulating intestinal calcium and phosphorus absorption [1]. Recent findings showed that vitamin D also plays a vital role in cellular proliferation and regulation, trophoblast invasion, and immunomodulation at the maternal-fetal interface [2, 3]. Maternal vitamin D deficiency (VDD) has been suggested as a potential mechanism in mediating the pathogenesis of adverse pregnancy and neonatal outcomes, including preeclampsia [4, 5], gestational diabetes [6], small for gestational age [7, 8] and preterm birth [9].

Globally, it was estimated that 50–80% of pregnant women are deficient of vitamin D (25-hydroxyvitamin D (25OHD) < 50 nmol/L) [10]. Findings from previous epidemiological studies have demonstrated that VDD was high in countries that are located at a high latitude [10]. Recent studies also reported a high prevalence of VDD in countries with abundant sunshine such as Spain [11], India [12], Thailand [13, 14], and Malaysia [15]. In countries located at high latitudes such as the United Kingdom [16] and the Netherlands [17], a routine vitamin D supplementation is recommended for a specific group at risk, particularly pregnant women. Despite the high prevalence of VDD among pregnant women in the region with abundant sunshine like Malaysia, routine vitamin D supplementation has not been established due to insufficient data to inform vitamin D supplementation for pregnant women.

Previously, VDD has been related to environmental and lifestyle factors including latitude, season, sun exposure, skin type, clothing, dietary vitamin D intake, Body Mass Index (BMI) and ethnicity [18]. Several genome-wide association studies (GWAS) and candidate gene studies have shown that single nucleotide polymorphisms (SNPs) were associated with vitamin D status [19‐22]. These SNPs located in or close to the genes encode the key enzymes or proteins for the metabolism, transportation and action of the mechanism of vitamin D, such as cytochrome P450-2R1 (CYP2R1), cytochrome P450-27B1 (CYP27B1). Nonetheless, the studies related the association of the SNPs and vitamin D status were largely conducted for the non-pregnant women [19‐22]. Also, previous studies focused on the factors associated with VDD during pregnancy were restricted to the study of only environmental [15, 23, 24] or genetic factors only [25‐28].

In a review [29] that included a total of 120 genetic associations studies, it was reported that SNPs in GC, particularly rs7041 and rs4588 are the most reported SNPs associated with the level of 25OHD. Furthermore, SNP rs7041 and rs4588 are missense SNPs (functional SNPs) where the variation in the nucleotide resulted in the change in amino acid and vitamin D binding protein glycosylation pattern. For rs7041, A to C allele transversion causes the change of amino acid aspartic acid (CTA) to glutamic acid (CTC), whereas, for rs4588, G to T allele transversion causes the change of amino acid Threonine (TGC) to Lysine (TTC) [30]. The combination of the two SNPs (rs4588 and rs7041) has resulted in three different GC haplotypes/isoforms: GC1f (A allele rs7041 and G allele rs4588), GC1 s (C allele rs7041 and G allele rs4588) and GC2 (A allele rs7041 and T allele rs4588). These three GC haplotype/isoforms gave rise to 6 GC diplotypes: 1f/1f, 1s/1s, 2/2, 1f/1s, 1f/2, 1s/2 with different binding affinities and post-translational glycosylation [31, 32].

Besides being the major transport protein for all the vitamin D metabolites, vitamin D binding protein is important to keep 25OHD in the circulation pool [33]. Different GC isoforms have been linked to different vitamin D binding protein concentrations in postmenopausal women [33], infants and toddlers [34]. The variation in the level and functionality of vitamin D binding protein has been postulated to directly or indirectly affect the 25OHD level in the blood circulation. During pregnancy, the concentration of vitamin D binding protein is elevated by two to three folds compared to the non-pregnant (normal) state [35]. Thus, the effect of GC SNPs on vitamin D status among pregnant women may be different from that reported in the non-pregnant population. Therefore, this study aimed to (1) assess the prevalence of VDD, and (2) determine the associations between demographics, lifestyle and GC gene polymorphism (rs7041, rs4588 and the GC diplotypes) with VDD among healthy Malaysian pregnant women.

Malaysia is a tropical country where sunlight is available throughout the year. However, the prevalence of VDD among pregnant women is high. An estimate of 82.0% of maternal plasma 25OHD < 50 nmol/L was consistent with previous studies conducted in the local urban area (Kuala Lumpur), where 90 and 72% of VDD (25OHD < 50 nmol/L) were found among pregnant women in the first trimester [15] and at delivery [24], respectively. The VDD prevalence reported in the present study (82.2%) was higher compared to the previous study (61.0%) conducted among pregnant women in a private hospital in Kuala Lumpur [44]. The variation could be attributed to differences in the proportion of ethnicity and socio-economic background among study participants in present and previous studies. The prevalence of maternal VDD reported in the present study was comparable to that reported in countries located at high latitude, which included Ireland (80.0%) [45] , Germany (77.0%) [46] , China (75.0%) [47] and Japan (73.0%) [48]. Nevertheless, the VDD prevalence found in the present study was higher compared to that reported in Thailand (20–40%) [13, 14] and India (66.0%) [12] among pregnant women at delivery and third trimester.

In previous studies, the factors that had been reported to be significantly associated with maternal vitamin D status differ accordingly [49] owing to vitamin D status is country-, ethnic- or subgroup-specific. Besides geographical factor (latitude and UV availability), each country has its own dietary recommendation, vitamin D food fortification strategies and supplementation policy. Likewise, each subgroup or ethnic groups has a different eating habit, clothing, skin colour, physical activity, attitude towards sun exposure and genetic make-up. A combination and interaction of these factors may lead to a risk of VDD in the population. Therefore, there is a need to explore various factors that can increase the risk of VDD for a different country, ethnics, and sub-groups. In this study, the demographic characteristics, lifestyle factors and GC polymorphism that might associated with VDD were comprehensively assessed.

Despite abundance of sunlight throughout the year, no association between time spent outdoors and maternal VDD was noted in this study, likewise the skin type variable. These null results are consistent with that reported in previous local studies, which examined factors associated with VDD in early pregnancy [15]. No association was reported between Fitzpatrick classification, melanin indices, sun protection score and sunlight exposure with maternal VDD [15]. The null results could be due to a high proportion of Malay ethnic group in the study with majority of them (82.5%) were veiled. In support of this, veiled was identified as a risk of VDD in our study where veiled women were about 4 times at higher risk than unveiled women as UVB rays do not transmit through clothing. This finding is in an agreement with a study conducted in Saudi Arabia [50] where the researchers reported that veiled clothing was significantly associated with increased risk of VDD.

The positive association between vitamin D intake from food and supplements and maternal VDD is in agreement with most of the previous findings [40, 48, 51-56]except a study [46]. It was found that vitamin D intake from foods and supplements contributed significantly to 25OHD level in population where exposure to sun is minimum or dermal synthesis of 25OHD is limited. For instances, recent studies from two high latitude countries, Sweden [57] and Switzerland [40, 55]reported that consumption of supplements but not time spent outdoors was associated with decreased risk of VDD. Similarly, in a large Chinese study, Yun and colleagues [47] demonstrated that vitamin D supplement consumption was positively associated with vitamin D status during winter when the exposure to sunlight is limited but not significantly associated during autumn. In the current study, it was found that exposure to sun did not contribute to the risk of VDD, but dietary vitamin D intake did which is consistent with the findings by Yun and colleagues [47].

Previous studies that investigated the association of GC rs7041 with 25OHD showed that C allele was associated with decreased risk of VDD among pregnant [25-27, 58] and non-pregnant population [19‐22]. In contrast to the previous studies, the C allele was found to increase the risk of VDD among pregnant women in the current study. The discrepancy could be due to the factors included in this study were different. For instance, the previous studies examined the factors associated with maternal VDD have been restricted to the only environmental [15, 23, 24] or genetic variables alone [25‐28]. Additionally, it is possible that changes in the metabolism of vitamin D notably the elevation of circulating vitamin D binding protein concentration may change the association of GC SNP with vitamin D status. This could be explained by the drastic change in the metabolism of vitamin D during pregnancy that causes variation in the factors associated with VDD during pregnancy compared to the non-pregnant state.

This study has several limitations as the data was collected in a public hospital where the findings could not be generalised to the pregnant women in private hospital. However, this study assessed a wide range of possible factors associated with maternal VDD: vitamin D intake from diet and supplements, estimates of sun exposure, skin type, clothing and SNPs. Owing to the limited resources, the sample size of this study was small which limited the number of SNPs that could be studied in the present study. However, two SNPs (rs4588 and rs7041) that were mostly reported to be associated with 25OHD in the previous studies were selected and investigated in this study. The sample size used in this study had a sufficient power to detect the prevalence and the associations of the demographic characteristics, lifestyle and GC SNPs (or diplotypes) with maternal VDD. To date, there is lack of consensus on the cut-off values used to define VDD. It is important to note that the results of factors associated with VDD in the present study might different if a different cut-off was used. However, the cut-off used in the present study was based on careful review by National Academy of Medicine (formerly known as Institute of Medicine) that suggests 25OHD level < 30 nmol/L is at increased risk of rickets, impaired fractional calcium absorption and decreased bone mineral content.

The high prevalence of maternal VDD reported in this study indicates the need for urgent development and implementation of strategies to improve maternal vitamin D status. In Malaysia, where sunlight is available throughout the year, advocating increasing sunlight exposure will be a cost-effective measure. Nonetheless, as a large majority of Malaysian women are veiled, advocating sun exposure may not increase the 25OHD level in majority of them. Veiled is a non-modifiable risk factor as almost all of the Malay women are veiled as part of religious requirement. Given that the food source of vitamin D is limited, it is a potential strategy to increase 25OHD level in pregnant women by vitamin D supplementation where a significant positive association of vitamin status with vitamin D intake from the supplement was found in this study. However, less than half (40.1%) of the study participants consumed supplements containing vitamin D where prenatal multivitamin was the most prevalent supplement (66.7%) consumed. Most of the pregnant women obtained their prenatal multivitamins (containing 10 μg of vitamin D per capsule) from public health clinics during their antenatal visit while few of them bought the supplement themselves. It should be noted that in the Malaysian public health clinic, the provision of prenatal multivitamin is not universal. However, the provision is dependent on the availability and the requirement of a pregnant women. Universal provision of a prenatal multivitamin may be effective in improving the vitamin D status of pregnant women. Investigating the effectiveness of supplement consumption by pregnant women in randomised controlled trials could be the next step to develop national recommendations or policies for supplementation. Additionally, the significant association of GC SNPs (and GC diplotypes) with maternal VDD showed in the present and previous studies suggests that supplementation should be more personalised based on genotype and risk factors assessment, particularly in pregnant women.

Almost half of the pregnant women participated in this study were VDD (25OHD < 30 nmol/L). Sun exposure was found not contribute to maternal vitamin D status. The contribution of GC rs4588 and rs7041 (or GC diplotypes) to maternal VDD was minimal but was significant. The findings showed that the risk of VDD was 4 times higher in veiled than unveiled women whereas vitamin D intake from foods and supplements were significantly associated with maternal VDD. Veiled clothing is a non-modifiable risk factors as majority of participants were veiled for religion requirement. Therefore, the current research suggests that food fortification and supplementation could be the potential strategy to improve vitamin D status among pregnant women.