1. Introduction

The recent VTD insufficiency pandemic has been influencing people of all age groups across different countries. Studies among U.S population demonstrated that more than 70% of children and around 77% of adults have insufficient VTD status with race ethnic differences ^{[1, 2]}. Several research investigations have revealed that VTD insufficiency is associated with higher risk of cardiovascular mortality ^[3], cancer incidence ^[4], and autoimmune diseases like multiple sclerosis ^[5].

Although the body can normally secure its adequate VTD requirement by directly exposing skin to sunlight and consuming VTD rich/enriched foods, the VTD obtained from such sources is inactive and needs further activation within the body before achieving full hormonal function. Metabolic activation of VTD requires two hydroxylation steps, the first of which occurs in the liver and the second in the kidney. Following the metabolic action of hepatic and renal enzymes, the active hormone, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃, calcitriol] is produced. Inadequate sunlight exposure and intake of dietary VTD, liver or kidney diseases can significantly reduce the level of active VTD and hence increase the risk of VTD insufficiency ^[6].

VTD metabolism is complex and involves several key enzymes which might alter the status of VTD. This includes 25-hydroxylase (CYP2R1), a hepatic enzyme which converts VTD₃ to 25-hydroxyvitamin D₃ [25(OH)D_3, Calcifediol] ^[7], another essential enzyme is 1-hydroxylase (CYP27B1), which is found mainly in the kidneys, and converts Calcifediol to calcitriol the bioactive form of vitamin D3, by hdroxylating 25(OH)D₃ into 1,25(OH)₂D₃. The last key enzyme is 24-hydroxylase (CYP24A1), that regulates the level of 1,25(OH)₂D₃ via hydroxylation at C24 which results in a sensational diminishing in the biological activities of VTD, prompts further oxidation to produce calcitroic acid which is passed in the urine ^[8].

Researchers identified SNPs that are associated with the modification of 25(OH)D₃ level in response to VTD supplementation; two SNPs in CYP24A1 (rs2209314 and rs2762939), one SNP rs10766197 in CYP2R1, another rs6013897 in CYP24A1 and rs7968585 in VDR are well represented in several populations ^[7]. Several studies have shown that the Gulf region has one of the highest rates of VTD insufficiency and deficiency around the globe, paradoxically despite the abundance of sunlight throughout the entire year ^[9]. According to recent study, 63.2% of UAE residents who work indoors (N=141) suffer from VTD deficiency due to the sun avoidance behaviors ^[10]. A current study conducted at a hospital in Dubai (UAE) reviewed the medical records of 2,736 patients between 2008 and 2012. All individuals examined were not taking VTD supplements upon first visit and it was found that 81.9 % suffered from VTD insufficiency. Three quarters of these patients were UAE nationals ^[11]. Another study conducted in Al Ain, (UAE), illustrated that 45.4% of adolescents (N=315) were VTD insufficient ^[12]. Additionally, a recent study at Zayed University in Abu-Dhabi (UAE) tested a random sample of 138 females and 70 males for serum 25(OH)D₃ levels. The mean concentrations for serum 25(OH)D₃ for females and males were 20.9 ± 14.9 nmol/L and 27.3 ± 15.7 nmol/L, respectively and were below the adequacy threshold ^[13].

Recently, genetic polymorphism of VTD metabolizing enzymes has gained interest because of the association of VTD metabolism with T2DM, CVDs and many other chronic illnesses ^[14]. Therefore this study aimed to examine the association of genetic polymorphism between VTD insufficiency and the metabolizing enzymes; CYP2R1, CYP27B1 and CYP24A1 among a representative sample of Emirati Youth.

2. Materials and Methods

A total of 163 unrelated healthy UAE national students (111 female and 52 male) were recruited for the study at Khalifa University of Science, Technology and Research (KUSTAR) from November 2014 to November 2015. Registered nurses from Burjeel/VPS healthcare hospital in Abu Dhabi collected samples. The ethical review committee of KUSTAR and Burjeel/VPS healthcare hospital approved the study. The subjects were informed about the study and signed an informed written consent prior to participation. Each participant filled out a validated questionnaire that provided the needed information that could play a role in the predisposition to VTD insufficiency which includes lifestyle and environmental factors.

Based on serum 25(OH)D₃ levels, the participants were categorized into either VTD sufficient or insufficient. VTD insufficiency was defined as serum 25(OH)D₃ level ≤50nmol/l while VTD sufficiency was defined as serum 25(OH)D₃ level >50nmol/l ^[15]. Individual with Vitamin D supplementation were excluded from this study. Body Mass Index (BMI) was determined and recorded for all the subjects using the Standard BMI formula, as body weight (kg) divided by height (m) squared. Individuals with a BMI score of greater than or equal to 30 were considered to be ‘obese’ and those with a BMI greater than or equal to 25 were grouped into the overweight group ^[16].

Allele, genotype and haplotype frequencies of 5 SNPs in 3 enzymes involved in VTD metabolism, CYP27B1 (rs10877012; rs4646536), CYP24A1 (rs2762939; rs6013897) and CYP2R1 (rs10741657) were compared between participants with VTD levels >50nmol/l (sufficient) and those with levels ≤50nmol/l (insufficient).

Four ml of blood was collected from each subject using anti-coagulant tubes (BD Vacutainer ®, Franklin Lakes, New Jersey, USA). Samples were aliquoted equally into two tubes, one for measuring serum 25(OH)D₃ as an indicator for VTD level in the body by DiaSorin (LIAISON ^®, Saluggia, Italy) ^[17] and another for DNA extraction ^[18]. DNA from all samples was extracted within 48 hours.

Genomic DNA was extracted from blood samples using QIAamp DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA was quantified using NanoDrop 2000c UV-Vis spectrophotometer (Thermo scientific, USA). DNA samples were diluted to 10 ng/µL and frozen for subsequent PCR reactions. Five SNPs in VTD metabolism genes were selected CYP24A1 (rs2762939, rs6013897), CYP27B1 (rs4646536, rs10877012) and CYP2R1 (rs10741657) for genotyping. This selection was based on data from population-based studies that revealed the association of specific SNPs with VTD status [19-24]^[19]. Genotyping was performed using TaqMan^® real-Time PCR assays (Applied Biosystems, Foster City, CA) ^[18]. PCR amplification reactions were carried out in 96 well plates (Applied Biosystems) by using the ViiA™ 7 Real-Time PCR System (Applied Biosystems). Each reaction was performed according to the manufacturer’s instructions with a final reaction volume of 10 μl that contained 10 ng of genomic DNA, 5 μl of TaqMan GTXpress Master Mix (Applied Biosystems) and 0.5 μl of primers and probes (20X). Primer and TaqMan^® minor groove binding group (MGB) probe (FAM^™ and VIC^® dye-labeled) sets used for TaqMan real-Time PCR. Amplification reactions began with incubation at 95°C for 20s and was followed by 40 cycles of 95°C for 3s (denaturing) and 60°C for 20s (annealing/extension). Result assessment of the SNPs’ amplifications and genotyping was carried out using the ViiA™ 7 Software (Applied Biosystems). Samples were run in duplicates, with positive, negative controls and blanks. SNPs call rate was 99.65%.

For determining the sample size, we utilized the Power Calculator for Genetic Studies developed by Skol and his team (https://www.sph.umich.edu/csg/abecasis/CaTS/index.html). Using VTD prevalence rate of 20% in the adult population of United Arab Emirates as reported by Muhairi et al., 2013 ^[12]. The major allele frequencies of ≥ 0.50 were predicted, and a multiplicative disease model was assumed. From the power calculator: 100-250 controls were needed for this study to be able to reject the null hypothesis with an OR of ≥ 1.5 reaching at least 85% power.

The data was analyzed using the statistical program Stata version 13 (StataCorp. College Station, Texas, USA). The results for continuous variables were expressed as mean ± SE and as percentages for categorical variables. The Hardy-Weinberg equilibrium at individual loci was assessed by χ2 test using de Finetti program (https://ihg.gsf.de/cgi-bin/hw/hwa1.pl). The categorical variables in Table 1 were tested by χ2 test and the continuous variables by Fisher’s exact test. A χ2 test was used to compare the genotype and allele frequencies for each SNP between insufficient and sufficient groups. Allele frequency was calculated as the number of occurrences of the test allele in the population divided by the total number of alleles. All p values were two sided, and differences were considered statistically significant for p< 0.05. Odds ratio (OR) were set at 95% confidence interval (CI). The SHEsis online haplotype analysis software (https://analysis.bio-x.cn/myAnalysis.php) was used to test the haplotypes.

3. Results

The study population consisted of a total of 163 participants (111 female, 52 male) as a representative sample of the young adult Emiratis within Khalifa University. The mean ± SE age of the VTD insufficient group (n=136) was 19.72 ± 0.19 years while the mean age of the sufficient group (n=27) was 20.15 ± 0.55 years. The characteristics of the healthy unrelated Emirati subjects were summarized in Table 1. Mean ± SE serum 25(OH)D₃ concentration was 71.92 ± 4.43 nmol/l in sufficient group and significantly lower in insufficient group (26.23 ± 0.97; p=0.00001). Compared to the sufficient group, the insufficient subjects were less physically active (p=0.023).

Table 1. General characteristics of healthy unrelated Emirati subjects enrolled in the study

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Table 2 presents the correlation between five different SNPs in CYP2R1, CYP24A1 and CYP27B1 genes and serum 25(OH)D₃ levels. None of the genotype distributions for the SNPs showed deviations from Hardy-Weinberg Equilibrium proportions. Our results indicated that some genotypes of the VTD metabolizing enzymes were significantly more frequent among VTD insufficient subjects, such as the GT+TT genotype of CYP27B1 (rs10877012) (OR: 6.13, 95% CI [1.76-21.33], p=0.001) and the AG+GG genotype of CYP27B11 (rs4646536) (OR: 2.57, 95% CI [1.02-6.49], p=0.040), while on the other hand the GG genotype of CYP24A1 (rs2762939) (OR: 0.10, 95% CI [0.006-1.81], p=0.036) and the TT genotype of CYP24A1 (rs6013897) (OR: 0.10, 95% CI [0.006-1.76], p=0.033) were significantly more frequent among VTD sufficient subjects. No significant association was found between the genetic variant in CYP2R1 and the status of VTD among the studied Emirati subpopulation.

Table 2. Genotype and allele frequencies of the CYP24A1, CYP27B1 and CYP2R1 SNPs in the context of serum 25(OH)D₃ levels

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Table 3. Haplotype frequency in CYP24A1 and CYP27B1 in the context of serum 25(OH)D₃ levels

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Table 3 depicts haplotype frequencies of the 4 SNPs in CYP24A1 and CYP27B1 and demonstrates that almost all haplotypes from SNPs rs2762393, rs6013897 in CYP24A1 and rs10877012, rs4646536 in CYP27B1 were predominant among VTD insufficient young adult Emirati participants. The haplotype with the four alleles GTGA (rs2762393, rs6013897, rs10877012, rs4646536) exhibited the highest frequency of 69.9% among the sufficient group (OR: 0.23, 95% CI 0.11-0.46], p=1.22x10-⁵), while the GTTG haplotype was more frequent in the insufficient group (p=0.004).

In addition, comparing the BMI values among sufficient and insufficient participant mean BMI was 23.77 ± 0.47 in the insufficient group and 23.29 ± 0.92 in the sufficient group.

4. Discussion

Recent studies revealed that several polymorphisms of VTD metabolism genes have been linked to T2DM, CVDs, cancer and other chronic illnesses ^[25]. However, conflicting results have been reported regarding the association of the genetic variants of VTD metabolism genes in different ethnic groups.

There are three known cytochrome P450s (CYPs) and possibly other VTD-related CYPs linked to the metabolism of VTD. The major enzymes in VTD metabolism are the hepatic 25-hydroxylase (CYP2R1), renal 25-hydroxyvitamin D-1a-hydroxylase (CYP27B1), and 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) ^[26].

Studies of genetic variants that specifically affect 25(OH)D₃ concentrations can provide another route to interpret the underlying cause of VTD insufficiency. Therefore in the present prospective study, we examined the association of genetic polymorphism of the metabolizing enzymes; CYP2R1, CYP27B1 and CYP24A1 with VTD status among a representative sample of young adult Emiratis. Specifically, we have investigated five SNPs; rs2762939, rs6013897 in CYP24A1, rs4646536, rs10877012 in CYP27B1and rs10741657 in CYP2R1.

CYP2R1 is a liver enzyme that catalyzes the hydroxylation of VTD₃ to its active form, 25(OH)D₃. Cheng et al., 2003 identified a polymorphism in CYP2R1 which was found to be associated with VTD metabolism ^[27]. The rs10741657 variant located in the promoter region of the CYP2R1 gene affects the enzymatic activity of CYP2R1 and cause a relative lack of 25(OH)D₃ ^[28]_. However, our results showed no association between rs10741657 variant and the lower VTD levels in young adult Emirati subpopulation. There was no significant difference between the allele and genotype frequencies of rs10741657 in (CYP2R1) in both sufficient and insufficient subgroups.

CYP24A1 is the cytochrome P450 component of the 25-hydroxyvitamin D-24-hydroxylase enzyme that catalyzes the conversion of 25(OH)D₃ and 1,25(OH)₂D₃ into the less active 24-hydroxylated products, which lead to inadequate VTD status due to degradation of active VTD ^[29]. This had been linked to obesity one of the significant parameters of Type 2 Diabetes ^[30]. A common variant in CYP24A1, is associated with obesity and its related phenotypes in Chinese Han population ^[31]. The rs2762939 variant located in intron 5 and the rs6013897 located in the intergenic region downstream of CYP24A1, were associated with high 25(OH)D₃ levels. In our, the mutant alleles G (rs2762939) and T (rs6013897) of CYP24A1 were significantly more frequent in VTD sufficient group than the insufficient. Based on such results, we can conclude that two variants of CYP24A1 were associated with reduced risk of VTD insufficiency among young adult Emiratis. This result was in concordance with another study conducted in western New York which reported an association between rs2762939 (CYP24A1) and reduced risk of VTD insufficiency among colorectal cancer patients ^[21].

An association between reduced 25(OH)D₃ levels and rs10877012 in CYP27B1 promoter polymorphism leading to reduced 1,25(OH)₂D₃ levels among the British population ^{[32, 33]} has been reported in literature. Our results support this finding, as rs10877012 was associated with increased risk of VTD insufficiency. Specifically the GT+TT genotype of rs10877012 and the AG+GG genotype of rs4646536 were significantly more frequent in VTD insufficient subjects. The data strongly indicates that genetic variants relevant to VTD metabolism could play an important role in defining VTD status among the young adult Emiratis. CYP27B1 also known as 1α-hydroxylase is the enzyme responsible for the hydroxylation of 25(OH)D₃ to form 1,25(OH)₂D₃. This enzyme carries out the final reaction to convert VTD to its active form, 1,25(OH)₂D₃ in the kidneys ^[13]. The two variant that we have studied here, rs10877012 in the 5’region and rs4646536 in intron 6, reduce the activity of CYP27B1 leading to decreased the level of 1,25(OH)₂D₃ ^[33]. Thus, even if subjects who carry this genotype receive a sufficient amount of sun exposure, they would still suffer from VTD insufficiency because of inefficient conversion of VTD into the active form. The inconvenience of measuring serum 1,25(OH)₂D₃ levels adds more emphasis on the need to screen for such risk genotypes and to identify individuals properly so as to tailor supplementation therapy.

Furthermore, our results indicated that the haplotype of four SNPs rs2762939, rs6013897 in CYP24A1 and rs10877012, rs4646536 in CYP27B1 could be associated with lower VTD levels. Specifically, the haplotype carrying G allele from rs2762393 and T from rs6013897 in CYP24A1 and T allele from rs10877012 and G allele from rs4646536 in CYP27B1 are more frequent in insufficient subjects. This could have important implications for predicting the VTD status and further direct prognosis for a number of clinically significant diseases such as cancer and T2D in subjects with these haplotypes.

Therefore, in light of these associations, it is feasible to conclude that the optimal concentration of 25(OH)D₃ in the body is correlated to CYP24A1 and CYP27B1 genotype. Future work will include measuring 1,25(OH)₂D₃ in patients along with 25(OH)D₃ to strengthen our results.

In conclusion, this unique study illustrated the association between genetic variants at the VTD metabolism sites and VTD insufficiency among young adult Emiratis. We have determined the frequency of CYP2R1, CYP24A1 and CYP27B1 polymorphism in young Emirati subpopulation. In addition, the highest haplotype frequency of CYP24A1 and CYP27B1 was presented by the GTGA haplotype and this indicates that individuals with a haplotype of GTGA are highly to be VTD sufficient. Therefore, this study shed light on a serious public health issue that should definitely get much more attention in the Gulf region and correlated the genotype of VTD metabolism genes with the level of serum 25(OH)D₃.

Therefore, this study shed light on a serious public health issue that should definitely get much more attention in the Gulf region and correlated the genotype of VTD metabolism genes with the level of serum 25(OH)D₃. Further research investigations need to be conducted to identify any link between specific VTD metabolism gene haplotypes and chronic diseases.

List of Abbreviations

1,25(OH)₂D₃: 1,25-dihydroxyvitamin D₃

25(OH)D₃: 25-hydroxyvitamin D₃

95% CI: 95% confidence interval

BMI: Body Mass Index

CVDs: Cardiovascular diseases

CYPs: cytochrome P450s

KUSTAR: Khalifa University of Science, Technology and Research

OR: Odds ratio

SNPs: Single nucleotide polymorphisms

T2DM: Type 2 Diabetes Mellitus

UAE: United Arab Emirates

VTD: Vitamin D

Statement of Competing Interests

All the authors declare no conflict of interest.

Acknowledgments

We gratefully acknowledge the contribution of participating individuals members whose cooperation made this study possible. This study was supported by research incentive funds from Zayed University granted to F. Al Anouti.

Author Contribution

Drs. Alsafar, and Al Anouti have designed the study, prepared the manuscript and performed all the data analyses with assistance from co-authors. Specifically, Ms Sarah El Hajj Chehadeh has performed all laboratory work in Molecular Cell Biology laboratory at Khalifa University Biotechnology center. Drs El Ghazali provided endless support by contributing to the design and discussion of this study.

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