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

Abstract

Aim: Previous studies have shown a high prevalence of vitamin D deficiency among Jordanians despite adequate exposure to sunlight, suggesting the presence of other causes for this deficiency. The aim of this study was to identify the relationship between 25-hydroxyvitamin D [25-(OH) VD] status and the nonsynonymous single-nucleotide polymorphisms (SNPs) (rs7041 and rs4588) of the GC gene, which encodes the vitamin D binding protein, and one SNP (rs10741657) near the CYP2R1 gene. Methods: Blood samples from 381 subjects (74 males and 307 females, 18-60 years of age) were obtained from the “National Center for Diabetes, Endocrinology and Genetics” (Amman, Jordan). The subjects were classified as “apparently healthy” if they did not suffer from chronic diseases and as “unhealthy” if they suffered from certain chronic diseases. Subjects' genotypes for GC; rs7041 and rs4588; CYP2R1; rs10741657 were determined by the polymerase chain reaction-restriction fragment length polymorphism assay method. Results: Apparently, healthy subjects had significantly higher 25-(OH) VD levels than unhealthy patients. In apparently healthy subjects, the rs10743657 genotypes containing the variant allele A (AA, GA) were associated with higher 25-(OH) VD levels than the homozygous wild-type genotype (GG). The genotypes containing the variant allele of rs7041 (TT, TG) and rs4588 (AA, AC) were associated with lower 25-(OH) VD levels than the wild-type genotypes (GG and CC, respectively). Haplotype analysis of rs7041 and rs4588 revealed that the haplotypes GC1S and GC1S/S were associated with 25-(OH) VD sufficiency, whereas haplotypes GC1F/S, GC1F/2, GC1S/2, GC2, and GC2/2 were associated with 25-(OH) VD deficiency. In unhealthy patients, only the homozygous genotype of the variant allele of rs7041 (TT) was associated with higher 25-(OH) VD levels, which is the reverse of what had been observed in apparently healthy subjects. Conclusions: The rs70141657G/A of CYP2R1 and rs7041T/G and rs4588C/A of vitamin D binding protein genetic polymorphisms were associated with increased risk of vitamin D deficiency among apparently healthy Jordanians.

Introduction

Vitamin D is a fat-soluble sterol derivative and a prohormone that exerts a hormone-like function. It is essential for prevention of rickets and osteomalacia (Holick, 2006). Its deficiency has been recognized as a contributing factor in the development of many serious diseases such as heart diseases, viral infections, multiple sclerosis, cardiac diseases, diabetes, cancer, and many other illnesses (Holick, 2006; Cannell and Hollis, 2008). Vitamin D inadequacy is fairly common all over the world. It has been reported in about 36% of healthy young adults and 57% of general medicine inpatients in the United States (Holick, 2006). The percentages were found to be higher in Europe (Holick, 2006). In Saudi Arabia, low vitamin D levels were seen in 28-37% of studied groups (Sadat-Ali et al., 2009). Three studies were conducted in Jordan to assess the vitamin D status. All pointed to the presence of high prevalence of vitamin D deficiency despite adequate sun exposure in the country (Mishal, 2001; Batieha et al., 2011; Mallah et al., 2011). One of these studies was a nationwide study that involved 5640 subjects. Vitamin D deficiency was observed in 37.3% of females and 5.1% of males (Batieha et al., 2011). These studies addressed the possibility of the presence of other unidentified causes other than low sun exposure or low dietary intake behind vitamin D insufficiency.

According to genome-wide association studies, genetic variants at the gene-encoding vitamin D binding protein (rs2282679, rs7041, and rs1155563), and in or near genes involved in vitamin D synthesis or activation (nicotinamide adenine dinucleotide synthetase [rs3829251], acyl-coenzyme A dehydrogenase [rs6599638], 7-dehydrocholesterol reductase [DHCR7; rs1790349], and CYP2R1 [rs2060793]) (Ahn et al., 2010) were found to have association with 25-hydroxyvitamin D [25-(OH) VD] concentrations. Another genome-wide association study reported on variants at three loci that are associated with 25-hydroxyvitamin D concentrations (vitamin D-binding protein [rs2282679], DHCR7 [rs12785878], and CYP2R1 [rs10741657]) (Wang et al., 2010). In a systematic review of the literature to study the relationship between 25-hydroxyvitamin D concentrations and common single-nucleotide polymorphisms (SNPs), a relationship was found with the vitamin D binding protein (SNPs rs4588 and rs7041), the vitamin D receptor (SNP rs10735810), and a range of cytochrome P450 enzymes (one SNP of CYP27B1 rs10877012) (McGrath et al., 2010).

The association of vitamin D status with various genetic polymorphisms was not studied previously in Jordan. Thus, the purpose of this study was to investigate the effects of the nonsynonymous polymorphisms rs7041 and rs4588 on the GC gene that codes the vitamin D binding protein and one SNP rs10741657 near the CYP2R1 gene and on the 25-(OH) VD levels among Jordanians.

Materials and Methods

Study participants

A total of 381 participants were recruited from patients attending the Clinics of the National Center for Diabetes, Endocrinology and Genetics (NCDEG) (Amman, Jordan). The participants were Jordanians with ages ranging from 18 to 60 years. Their vitamin D levels were predetermined by NCDEG laboratories, using the enzyme-linked immunosorbent assay (immunodiagnostic system [IDS] kit). The results were interpreted (according to NCDEG laboratories) as follows: vitamin D deficiency, serum 25-(OH) VD concentration <20 ng/mL; vitamin D insufficiency, serum 25-(OH) VD (20-29 ng/mL); and vitamin D sufficiency, serum 25-(OH) VD ≥30 ng/mL. Some sociodemographic characteristics of the participants are presented in Table 1. The participants were divided into two groups: “apparently healthy” if they do not suffer from chronic diseases and “unhealthy” if they suffer from chronic diseases as shown in Table 2.

Table 1.

Sociodemographic Characteristics of the Study Population

Variable	N (%)
Male	74 (19.2)
Female	311 (80.8)
Hijab	217 (82.5)
Niqab	14 (5.3)
Western dress style	32 (12.2)
Wheatish skin color	124 (39.6)
Dark skin color	12 (3.9)
White skin color	177 (56.5)
Nonsmoker	252 (83.2)
Ex-smoker	6 (2.0)
Current smoker	45 (14.8)
Unemployed	145 (45.9)
Indoor job	151 (47.8)
Outdoor job	20 (6.3)

Table 2.

Characteristics of the Study Population and Vitamin D Status

	25-(OH) vitamin D status
	Deficient n (%)	Insufficient n (%)	Sufficient n (%)	Mean 25-(OH) vitamin D level ± SD (ng/mL)	Odds ratio ^a	95% CI	Z statistic	p-Value
Total (n = 381)	212 (55.6)	60 (15.8)	109 (28.6)	24.2 ± 18.1	—	—	—	—
Males (n = 74)	43 (58.1)	11 (14.9)	20 (27)	26.0 ± 19.7	—	—	—	>0.05
Females (n = 307)	169 (55.0)	49 (16.0)	89 (29.0)	23.8 ± 17.6	—	—	—
Apparently healthy subjects (n = 200)	92 (46.0)	21 (10.5)	87 (43.5)	29.7 ± 20.8	—	—	—	—
Unhealthy patients (n = 181)
Cardiovascular diseases (n = 87)	60 (69.0)	15 (17.2)	12 (13.8)	18.1 ± 13.3	2.61	1.53-4.44	3.53	0.0004
Bone and joints disease (n = 17)	10 (58.8)	5 (29.4)	2 (11.8)	18.5 ± 8.9	1.68	0.61-4.58	1.01	0.31
Thyroid and parathyroid diseases (n = 54)	34 (63.0)	15 (27.8)	5 (9.3)	17.8 ± 8.3	2.00	1.08-3.70	2.19	0.029
Other diseases (n = 23)	16 (69.6)	4 (17.4)	3 (13.0)	18.3 ± 14.5	2.68	1.06-6.81	2.08	0.038

Odds ratio was calculated for subjects with diseases and deficient in 25-hydroxyvitamin and compared with that of apparently healthy subjects.

25-Hydroxyvitamin D, 25-(OH) vitamin D; CI, confidence intervals of the odds ratio; n, number of subjects; SD, standard deviation.

Study design

Participants were divided into three groups according to their vitamin D levels: deficient, insufficient, and sufficient. The genomic DNA of each participant was examined for SNP rs10741657 in the gene of 25-hydroxyvitamin D hydroxylation enzyme, CYP2R1, and rs7041 and rs4588 SNPs in the vitamin D binding protein gene (GC-gene).

Genomic DNA extraction

DNA was extracted from lymphocytes of peripheral blood using commercial kits for genomic DNA extraction according to the manufacturer's protocol (Wizard Genomic DNA Purification Kit; Promega).

Polymorphism analysis of vitamin D binding protein (GC-gene)

The polymerase chain reaction (PCR) of rs4588 and rs7041 SNPs was performed using forward and reverse primers: 5′-AAATAATGAGCAAATGAAAGAAGAC3′ and 5′-CAATAACAGCAAAGAAATGAGTAGA-3′ (Li et al., 2011). The PCR was performed by initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 40 min, and a final extension at 72°C for 7 min. The different loci were recognized by the following restriction endonucleases (New England Biolab): HaeIII for T/G at 37°C and StyI for C/A at 37°C. After restriction fragment length polymorphism (RFLP) analysis, the 483 bp PCR product was uncleaved in rs7041T and rs4588C alleles and then cleaved into two fragments of 297 and 186 bp and 305 and 178 bp in rs7041G and rs4588A alleles, respectively.

Polymorphism analysis of the CYP2R1 gene

Subjects were genotyped for rs10741657 G/A SNP of the CYP2R1 gene by PCR-RFLP. The region surrounding the polymorphism was amplified using the following forward and reverse primers:

5′-GGGAAGAGCAATGACATGGA-3′ and 5′-GCCCTGGAAGACTCATTTTG-3 (Hussein et al., 2012).

The PCR was performed by initial denaturation at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 40 s. A final extension step was carried out at 72°C for 7 min. After RFLP analysis using enzyme MnII at 37°C, the 288-bp PCR product was cleaved into two fragments of 256 and 32 bp in A allele and cleaved into three fragments of 151, 105, and 32 bp when allele G was present.

Statistical analyses

Statistical analyses were performed using Statistical Package for Social Sciences (SPSS Inc.) version 17.0 software for Windows. Continuous variables were analyzed using analysis of variance. Odds ratios (ORs) were calculated using the Web OR calculator at www.medcalc.org/calc/odds_ratio.php. p-Value of < 0.05 was considered significant. The differences between allele frequencies in different populations were evaluated by the Z test using the online z test calculator at www.socscistatistics.com/tests/ztest/Default2.aspx. The “Haploview” program was used to compare the haplotypes (Barrett et al., 2005).

Results

Of the 381 subjects included in this study, 74 (19.4%) were male and 307 (80.6%) were female. The ages ranged from 18 to 60 years. Some other sociodemographic characteristics of the study group are shown in Table 1. No significant difference was found between males and females in 25-(OH) VD mean concentration (p > 0.5). 25-(OH) VD deficiency was seen in 55.6% of the studied subjects, while insufficiency was seen in 15.8% of them. The frequency of 25-(OH) VD-deficient subjects was apparently higher in those with diseases, such as cardiovascular diseases and thyroid and parathyroid diseases, than in apparently healthy subjects. Subjects with diseases also had lower mean 25-(OH) VD levels than apparently healthy individuals (Table 2). The minor allele frequency for rs70143657A was 0.35. The minor allele frequency of rs7041T was 0.46, while that of rs4588A was 0.19 (Table 3).

Table 3.

Allele and Gene Frequencies of rs10743657, rs7041, and rs4588 Polymorphisms Among Jordanian Subjects

rs10743657		rs7041		rs4588		Haplotypes of rs7041 and rs4588
Alleles (n = 762)
Allele	Frequency	Allele	Frequency	Allele	Frequency	Alleles	Frequency
G	0.653	G	0.543	C	0.810	T C	0.268
						G C	0.545
A	0.347	T	0.457	A	0.190	T A	0.187
						G A	0.000
Genotypes (N = 381)
Genotype	Frequency	Genotype	Frequency	Genotype	Frequency	Haplotype	Frequency
GG	0.418	GG	0.290	CC	0.662	TT CC	0.094
						TG CC	0.283
GA	0.470	TG	0.500	CA	0.294	TT CA	0.065
						GG CC	0.294
AA	0.112	TT	0.210	AA	0.044	TG CA	0.221
						TT AA	0.044

N, number of study subjects; n, number of alleles.

The association of the minor alleles of the three polymorphisms (rs7041T, rs4588A, and rs10741657A) with deficient 25-(OH) VD levels for apparently healthy and unhealthy subjects is shown in Tables 4 and 5, respectively. For apparently healthy people, the two minor alleles (rs7041T and rs4588A) were found to be associated with low vitamin D levels. The minor allele of the third SNP (rs10741657A) was found to be associated with a high vitamin D level (Table 4). In contrast, for unhealthy people, the minor allele (rs7041T) was found to be significantly associated with a high vitamin D status. Actually, this result is completely the opposite of the results of the healthy people. The other two SNPs (rs10741657A and rs4588A) were not found to be associated with vitamin D status (Table 5). The mean ± standard deviation (SD) of vitamin D levels in the various genotypes of the three SNPs in both apparently healthy and unhealthy subjects is presented in Table 6. There were significant differences in vitamin D levels among the various genotypes of rs10741657 in apparently healthy but not in the unhealthy individuals, with higher levels in the genotypes having the variant allele. There were also significant differences in vitamin D levels among the various genotypes of rs7041 in both apparently healthy and unhealthy subjects, but genotypes containing the variant allele showed lower levels of vitamin D in apparently healthy individuals and higher levels in unhealthy patients. Concerning the third SNP (rs4588), there were differences in vitamin D levels among the genotypes in apparently healthy subjects but not in unhealthy subjects, with lower levels in genotypes containing the minor allele.

Table 4.

Association Between Vitamin D Levels in Apparently Healthy Subjects (n = 200) with Genotypes of rs10743657, rs7041, and rs4588

		25(OH) vitamin D status
Genotypes	Mean ± SD of 25(OH) vitamin D level	Deficient n (%)	Insufficient n (%)	Sufficient n (%)	Odds ratio ^a	95% CI	Z statistic	p-Value
rs10743657
AA (n = 23)	38.7 ± 24.9	7 (7.6)	1 (4.8)	15 (17.2)	0.33	0.12-0.87	2.23	0.026
AG (n = 86)	31.8 ± 21.4	33 (35.9)	11 (52.4)	42 (48.3)	0.47	0.26-0.85	2.48	0.013
GG (n = 91)	25.3 ± 18.1	52 (56.5)	9 (42.9)	30 (34.5)	—	—	—	—
Total		92 (100.0)	21 (100.0)	87 (100.0)
AA+AG (n = 109)	33.3 ± 22.3	40 (43.5)	12 (57.2)	57 (65.5)	0.44	0.25-0.77	2.87	0.004
GG+AG (n = 177)	28.5 ± 20.0	85 (92.4)	20 (95.3)	72 (82.8)	0.69	0.42-1.15	1.41	0.158
rs7041
TT (n = 48)	24.6 ± 17.7	26 (28.3)	6 (28.6)	16 (18.4)	3.31	1.46-7.50	2.87	0.004
GT (n = 95)	28.5 ± 21.8	51 (55.4)	8 (38.1)	36 (41.4)	3.25	1.59-6.63	3.23	0.001
GG (n = 57)	36.1 ± 20.4	15 (16.3)	7 (33.3)	35 (40.2)	—	—	—	—
Total		92 (100.0)	21 (100.0)	87 (100.0)
TT+GT (n = 143)	27.2 ± 20.5	77 (83.7)	14 (66.7)	52 (59.8)	3.27	1.66-6.42	3.44	0.0006
GG+GT (n = 152)	31.3 ± 21.5	66 (71.7)	15 (71.4)	71 (81.6)	2.15	1.10-4.20	2.23	0.026
rs4588
AA (n = 8)	12.4 ± 6.9	7 (7.6)	1 (4.8)	0 (0.0)	11.58	1.39-96.78	2.26	0.024
AC (n = 54)	24.6 ± 20.0	33 (35.9)	4 (19.0)	17 (19.5)	2.60	1.36-4.96	2.90	0.004
CC (n = 138)	32.7 ± 20.9	52 (56.5)	16 (76.2)	70 (80.5)	—	—	—	—
Total		92 (100.0)	21 (100.0)	87 (100.0)
AA+AC (n = 62)	18.5 ± 15.6	40 (43.5)	5 (23.8)	17 (19.5)	3.01	1.61-5.61	3.46	0.0005
CC+AC (n = 192)	28.7 ± 20.5	85 (92.4)	20 (95.2)	87 (100.0)	7.95	4.48-14.12	7.08	<0.0001

Odds ratios for vitamin D deficiency each compared with the homozygous wild-type genotype.

Table 5.

Association Between Vitamin D Levels in Unhealthy Subjects (n = 181) with Genotypes of rs10743657, rs7041, and rs4588

		25(OH) vitamin D status
Genotypes	Mean ± SD of 25(OH) vitamin D level	Deficient n (%)	Insufficient n (%)	Sufficient n (%)	Odds ratio ^a	95% CI	Z statistic	p-Value
rs10743657
AA (n = 20)	14.3 ± 6.9	15 (12.5)	5 (12.8)	0 (0.0)	1.71	0.55-5.31	0.94	0.350
AG (n = 95)	17.6 ± 10.5	63 (52.5)	19 (48.7)	13 (59.1)	1.13	0.58-2.17	0.35	0.730
GG (n = 66)	20.1 ± 4.1	42 (35.0)	15 (38.5)	9 (40.9)	—	—	—	—
Total		120 (100.0)	39 (100.0)	22 (100.0)
AA+AG (n = 115)	17.0 ± 10.0	78 (65.0)	24 (61.5)	13 (59.1)	1.21	0.64-2.28	0.574	0.566
GG+AG (n = 161)	18.6 ± 12.1	105 (87.5)	34 (87.2)	22 (100.0)	1.07	0.59-1.95	0.226	0.821
rs7041
TT (n = 31)	24.1 ± 18.9	15 (12.5)	6 (15.4)	10 (45.5)	0.27	0.10-0.70	2.71	0.007
GT (n = 96)	17.8 ± 10.4	63 (52.5)	22 (56.4)	11 (50.0)	0.55	0.25-1.18	1.55	0.122
GG (n = 54)	15.1 ± 6.5	42 (35.0)	11 (28.2)	1 (4.5)	—	—	—	—
Total		120 (100.0)	39 (100.0)	22 (100.0)
TT+GT (n = 127)	19.6 ± 13.2	78 (65.0)	28 (71.8)	21 (95.5)	0.46	0.22-1.95	2.10	0.036
GG+GT (n = 150)	16.9 ± 9.2	105 (87.5)	33 (84.6)	12 (54.5)	0.67	0.32-1.38	1.09	0.277
rs4588
AA (n = 9)	24.2 ± 19.8	5 (4.2)	1 (2.6)	3 (13.6)	0.55	0.14-2.19	0.84	0.399
AC (n = 58)	19.3 ± 13.3	36 (30.0)	14 (35.9)	8 (36.4)	0.73	0.37-1.41	0.95	0.342
CC (n = 114)	17.0 ± 9.8	79 (65.8)	24 (61.5)	11 (50.0)	—	—	—	—
Total		120 (100.0)	39 (100.0)	22 (100.0)
AA+AC (n = 67)	20 ± 14.3	41 (34.2)	15 (38.5)	11 (50.0)	0.70	0.38-1.32	1.11	0.266
CC+AC (n = 172)	17.8 ± 11.2	115 (95.8)	38 (97.4)	19 (86.4)	0.89	0.54-1.49	0.43	0.666

Odds ratios for vitamin D deficiency each compared with the homozygous wild-type genotype.

Table 6.

Mean ± SD of 25(OH) Vitamin D Level in the Various Genotypes of the Three SNPs (rs10743657, rs7041, and rs4588)

	Mean ± SD of 25(OH) vitamin D level
Genotypes (N = 381)	Apparently healthy (n = 200)	Unhealthy (n = 181)	p-Value^a	p-Value^b	p-Value^c
rs10743657 SNP
AA (n = 43)	38.7 ± 24.9	14.3 ± 6.9	0.008	0.130	0.001
AG (n = 181)	31.8 ± 21.4	17.6 ± 10.5			0.001
GG (n = 157)	25.3 ± 18.1	20.1 ± 14.1			0.052
AA+AG (n = 224)	33.4 ± 22.3	17 ± 10	0.027	0.130	0.001
GG+AG (n = 338)	28.5 ± 20.0	18.6 ± 12.1			0.001
rs7041 SNP
TT (n = 79)	24.6 ± 17.7	24.1 ± 18.9	0.013	0.002	0.900
GT (n = 191)	28.5 ± 21.8	17.8 ± 10.4			0.001
GG (n = 111)	36.1 ± 20.4	15.1 ± 6.5			0.001
TT+GT (n = 270)	27.2 ± 20.5	19.4 ± 13.2	0.008	0.020	0.001
GG+GT (n = 302)	31.3 ± 21.5	16.9 ± 9.2			0.001
rs4588 SNP
AA (n = 17)	12.4 ± 6.9	24.2 ± 19.8	0.003	0.130	0.130
AC (n = 112)	24.6 ± 20.0	19.3 ± 13.3			0.100
CC (n = 252)	32.7 ± 20.9	17.0 ± 9.8			0.001
AA+AC (n = 129)	23.0 ± 19.2	20 ± 14.3	0.002	0.120	0.300
CC+AC (n = 364)	30.4 ± 20.9	17.8 ± 11.2			0.001

Analyses of variance test:

p-Value for mean 25(OH) vitamin D level of healthy individuals and different genotypes.

p-Value for mean 25(OH) vitamin D level of unhealthy individuals and different genotypes.

p-Value for mean 25(OH) vitamin D level between healthy and unhealthy individuals for different genotypes.

SNPs, single-nucleotide polymorphisms.

There were also significant differences in vitamin D levels among most genotypes when apparently healthy individuals were compared with unhealthy patients.

The association between 25(OH) vitamin D status and the haplotypes of rs7041 and rs4588 is presented in Table 7. Only haplotype (GC1F/F) and haplotype 1 (GC1F) were not significantly different from haplotype (GC1S/S) and haplotype 1 (GC1F), respectively, concerning 25(OH) vitamin D status. The other haplotypes listed showed association with 25(OH) vitamin D status. Haplotype (GC1S/2), haplotype (GC2/2), and haplotype 3 (GC2) were strongly associated with 25(OH) vitamin D deficiency. In contrast, haplotype (GC1S/S) and haplotype 2 (GC1S) were associated with 25(OH) vitamin D sufficiency.

Table 7.

Association Between 25(OH) Vitamin D Status and the Haplotypes of rs7041 and rs4588 in Apparently Healthy Subjects

		25(OH) vitamin D status
Haplotype name	Combination of genotypes of rs7041, rs4588 (N)	Deficient N (%)	Insufficient N (%)	Sufficient N (%)	Odds ratio	95% CI (p-value)	Chi-square (χ²)	Power 95%
Haplotype (GC1F/F)	TT:CC (25)	11 (44.0)	4 (16.0)	10 (40.0)	2.3	0.84-6 (0.106)	2.64	0.37
Haplotype (GC1F/S)	TG:CC (56)	26 (46.5)	5 (8.9)	25 (44.6)	2.5	1.1-5.5 (0.024)	5.19	0.62
Haplotype (GC1F/2)	TT:CA (15)	8 (53.3)	1 (6.7)	6 (40.0)	3.3	1-10.6 (0.047)	4.11	0.51
Haplotype (GC1S/S)	GG:CC (58)	15 (25.9)	7 (12.1)	36 (62)	—	—
Haplotype (GC1S/2)	TG:CA (38)	25 (65.8)	3 (7.9)	10 (26.3)	5.5	2.3-13.4 (0.0002)	14.9	0.96
Haplotype (GC2/2)	TT:AA (8)	7 (87.5)	1 (12.5)	0 (0)	20.1	2.3-177 (0.007)	11.8	0.77
Total	200	92	21	87
Haplotype 1 (GC1F)	T-C (121)	56 (46.3)	14 (11.6)	51 (42.1)	1.37	0.87-2.15 (0.17)	1.88	0.28
Haplotype 2 (GC1S)	G-C (210)	81 (38.5)	22 (10.5)	107 (51)	—	—
Haplotype 3 (GC2)	T-A (69)	47 (68.1)	6 (8.7)	16 (23.2)	3.4	1.9-6.1 (<0.0001)	18.19	0.99
Total	400	184	42	174

Haplotypes analyzed using haploview program.

A comparison between the variant allele frequencies of the three SNPs of this study and some other populations is presented in Table 8. The minor allele frequency of rs7041T in Jordanians was similar to that in Canadian Caucasians, but significantly different from that in Hispanics, and it tended to be different from that in South Brazilians. The minor allele frequency of rs4588A in Jordanians was significantly different from that in Hispanics, Canadian Caucasians, South Brazilians, and Danish Caucasians. The minor allele frequency of rs70143657A in Jordanians was different from that in Egyptians, Austrian Caucasians, and Danish Caucasians, but similar to that in Lebanese.

Table 8.

Variant Allele Frequencies of the Three SNPs (rs7041T, rs4588A, and rs70141657A) Among Some Ethnic Groups

	Sample size	Allele frequency	Z statistic	p-Value^a	Reference
rs7041T SNP
Jordanian	381	0.46	—	—	This study
Hispanics	504	0.59	−5.47	<0.0001	Engelman et al. (2008)
Canadian European	111	0.41	1.30	0.194	Gozdzik et al. (2011)
South Brazil	198	0.52	−1.97	0.049	Santos et al. (2013)
rs4588A SNP
Jordanian	381	0.19	—	—	This study
Hispanics	504	0.25	−2.98	0.003	Engelman et al. (2008)
Canadian European	111	0.28	−2.86	0.004	Gozdzik et al. (2011)
South Brazil	198	0.26	−2.75	0.006	Santos et al. (2013)
Caucasian/Denmark	758	0.28	−4.30	<0.0001	Nissen et al. (2014)
rs70141657A SNP
Jordanians	381	0.35	—	—	This study
Egyptians	120	0.43	−2.21	0.027	Hussein et al. (2012)
Lebanese	172	0.29	1.95	0.051	Zgheib et al. (2013)
Caucasian (Austria)	298	0.42	−2.60	0.009	Trummer et al. (2012)
Caucasian/Denmark	758	0.41	2.50	0.012	Nissen et al. (2014)

Significance level, 0.05.

The minor allele frequencies of the three SNPs were compared between “apparently healthy” subjects and patients with cardiovascular diseases or thyroid diseases. The rs7041T minor allele frequency was significantly less (0.37) in patients with thyroid diseases than in healthy subjects (0.48), whereas the rs4588A minor allele frequency was significantly more in patients with cardiovascular diseases than in apparently healthy subjects. There were no differences in the minor allele frequencies of rs70143657A between apparently healthy subjects and patients with cardiovascular or thyroid diseases (Table 9).

Table 9.

Minor Allele Frequencies of the Three SNPs (rs7041T, rs4588A, and rs70141657A) Among Healthy Subjects and Patients with Cardiovascular and Thyroid Diseases

	Total number of alleles	Allele frequency	Z statistic	p-Value^a
rs7041T SNP
Apparently healthy subjects	400	0.48	—	—
Patients with cardiovascular diseases	174	0.50	−0.50	0.617
Patients with thyroid diseases	108	0.37	1.98	0.048
rs4588A SNP
Apparently healthy subjects	400	0.18	—	—
Patients with cardiovascular diseases	174	0.21	−2.90	0.004
Patients with thyroid diseases	108	0.16	0.43	0.667
rs70141657A SNP
Apparently healthy subjects	400	0.33	—	—
Patients with cardiovascular diseases	174	0.37	−1.01	0.313
Patients with thyroid diseases	108	0.33	−0.065	0.944

Significance level, 0.05.

Discussion

The aim of this study was to investigate the association of three polymorphisms in the GC gene (rs7041 and rs4588) and CYP2R1 gene (rs10741657) on 25-(OH) VD serum concentration among Jordanians. We noticed that the frequency of vitamin D deficiency is high among Jordanians. The frequency of vitamin D deficiency was 55.6%, and the mean ± SD value of 25-(OH) VD was 24.2 ng/mL ± 18.1 for the study sample. However, the frequency of vitamin D deficiency of healthy subjects was 46%, and the mean ± SD value of 25-(OH) VD was 29.7 ± 20.8. These percentages were less than those of Mishal (2001) and Mallah et al. (2011) among samples of Jordanian subjects. In contrast, these percentages were higher than those of the larger nationwide study (Batieha et al., 2011). These differences might be attributed to differences of sample selection, since the purpose of our study was to identify any association between the three polymorphisms and vitamin D status rather than to study the prevalence of vitamin D deficiency.

This study suggested a strong association between the vitamin D status of the healthy subjects of the study sample and the nonsynonymous polymorphisms rs7041 and rs4588. T allele of rs7041 and A allele of rs4588 were found to be associated with low 25-(OH) VD serum concentrations (p < 0.05). In addition, the two SNPs (rs7041 and rs4588) were found to be in linkage disequilibrium (ID´’I = 1, r² = 0.28), which meant that there was a nonrandom association between alleles of the two SNPs. Haplotype analysis of the two SNPs in apparently healthy individuals revealed that haplotypes GC1F/S, GC1F/2, GC1S/2, GC2/2, and GC2 were associated significantly with low 25-(OH) VD status. However, in unhealthy patients, only the homozygous variant allele (TT) of rs7041 was found to be associated with low 25-(OH) VD status (p = 0.007). This discrepancy might be due to the disease factors that might by themselves affect 25-(OH) VD status. SNPs rs7041 and rs4588 were found to be associated with 25-(OH) VD in diabetic subjects with insulin resistance from African American and Hispanic ancestry (Engelman et al., 2008). In elderly patients with osteoporosis (Caucasians from the Rotterdam study), haplotype 1 (GC1S) was associated with increased 25-(OH) VD serum levels, while haplotype 2 (GC2) was associated with decreased 25-(OH) VD serum levels (Fang et al., 2009). In premenopausal women mostly of French descent, both rs7041 and rs4588 SNPs were strongly associated with lower 25-(OH) VD concentrations (Sinotte et al., 2009). In apparently healthy girls (10-18 years old), the TT genotype of rs7041 and the AA genotype of rs4588, as well as the diplotype GC 1F2/2-2, were significantly associated with lower 25-(OH) VD levels (Santos et al., 2013). The rs4588 but not the rs7041 SNP was related to lower 25(OH) VD levels in Polish Graves disease patients (Kurylowicz et al., 2006). SNP rs7041 T allele was associated with low 25(OH) VD levels in patients with chronic obstructive pulmonary disease (COPD) in Belgium, which was independent of the COPD severity (Janssens et al., 2010). In a study on healthy young Canadian subjects of different ancestry, rs4588 and rs7041 SNPs were genotyped by a method that ascertains the GC diplotype of each subject. It was demonstrated that the number of GC-2 alleles was associated with low 25(OH) VD levels in the Eastern Asian sample in fall and winter. It also showed such an association in the European sample, but not in the South Asian sample (Gozdzik et al., 2011). In contrast, both rs4588 and rs7041 were not found to be associated with lower levels of 25(OH) VD in northeastern Han Chinese children (Zhang et al., 2012).

The rs10741657 variant allele A was associated with higher 25(OH) VD levels in apparently healthy individuals, but not in unhealthy patients. Unhealthy patients had lower 25(OH) VD than apparently healthy subjects in general. The reason behind this discrepancy is not known but might be due to the presence of other disease factors that affect 25(OH) VD concentrations other than the genetic factors. The rs10741657 polymorphism was associated with 25(OH) VD status (Wang et al., 2010; Zhang et al., 2012). The GG or GA genotypes of the rs10741657 polymorphism had lower levels of 25(OH) VD than the AA genotypes in patients with type 1 diabetes mellitus (Ramos-Lopez et al., 2007). The homozygous variant allele of rs10741657 was associated with higher 25(OH) VD levels in South Asians, but not in Arabs and South East Asian subjects (Elkum et al., 2014). No association was seen between A allele and high 25(OH) VD levels in Austrian Caucasians (Trummer et al., 2012).

The minor allele frequency of rs7041T (0.37) was significantly lower in patients with thyroid disease than in apparently healthy subjects (0.48), whereas the minor allele frequency of rs4588A (0.21) was significantly higher in cardiovascular disease patients than in apparently healthy subjects (0.18). The minor allele frequency of rs10741657A in patients with cardiovascular disease or thyroid disease was not different from that in apparently healthy subjects.

On the ethnicity side, the minor allele frequency of rs7041T of 0.46 in this study is similar to that in Canadian Europeans (0.41), but significantly different from that in Hispanics (0.59) and to a lesser extent significantly different from that in south Brazilians (0.52). The minor allele frequency of rs4588A in this study of 0.19 is significantly different from that in Canadian Europeans (0.28), Hispanics (0.25), and south Brazilians (0.26). Concerning the variant allele rs70141657A, the frequency in this study was 0.35, which is found to be different from that in Caucasians from Austria (0.42) and in Egyptians (0.43), but not different from that in Lebanese (0.29).

In conclusion, this study demonstrates that the polymorphisms rs70141657G/A of CYP2R1 gene and rs7041T/G and rs4588C/A of the vitamin D binding protein gene were associated with increased risk of vitamin D deficiency among apparently healthy Jordanians.

Footnotes

Acknowledgment

This research was supported by grant 6/2011-2012, Deanship of Research, the University of Jordan.

Author Disclosure Statement

No competing financial interests exist.

References

Ahn

, Yu

, Stolzenberg-Solomon

, et al. (2010) Genome-wide association study of circulating vitamin D levels. Hum Mol Genet, 19:2739-2745.

Barrett

, Fry

, Maller

, Daly

(2005) Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics, 21:263-265.

Batieha

, Khader

, Jaddou

, et al. (2011) Vitamin D status in Jordan: dress style and gender discrepancies. Ann Nutr Metab, 58:10-18.

Cannell

, Hollis

(2008) Use of vitamin D in clinical practice. Altern Med Rev, 13:6-20.

Elkum

, Alkayal

, Noronha

, et al. (2014) Vitamin D insufficiency in Arabs and South Asians positively associates with polymorphism in GC and CYP2R1 genes. PLoS One, 9:e113102.

Engelman

, Fingerlin

, Langefeld

, et al. (2008) Genetic and environmental determinants of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D levels in Hispanic and African Americans. J Clin Endocrinol Metab, 93:3381-3388.

Fang

, van Meurs

JBJ

, Arp

, et al. (2009) Vitamin D binding protein genotype and osteoporosis. Calcif Tissue Int, 85:85-93.

Gozdzik

, Zhu

, Wong

BY-L

, et al. (2011) Association of vitamin D binding protein (VDBP) polymorphisms and serum 25(OH)D concentrations in a sample of young Canadian adults of different ancestry. J Steroid Biochem Mol Biol, 127:405-412.

Holick

(2006) High prevalence of vitamin D inadequacy and implications for health. Mayo Clin Proc, 81:353-373.

10.

Hussein

, Mohamed

, Alghobashy

(2012) Synergism of CYP2R1 and CYP27B1 polymorphisms and susceptibility to type 1diabetes in Egyptian children. Cell Immunol, 279:42-45.

11.

Janssens

, Bouillon

, Claes

, et al. (2010) Vitamin D deficiency is highly prevalent in COPD and correlates with variants in the vitamin D-binding gene. Thorax, 65:215-220.

12.

Kurylowicz

, Ramos-Lopez

, Bednarczuk

, et al. (2006) Vitamin D-binding protein (DBP) gene polymorphism is associated with Graves' disease and the vitamin D status in a Polish population study. Exp Clin Endocrinol Diabetes, 114:329-335.

13.

, Jiang

, Willis-Owen

, et al. (2011) Vitamin D binding protein variants associate with asthma susceptibility in the Chinese Han population. BMC Med Genet, 12:103.

14.

Mallah

, Hamad

, Elmanaseer

, et al. (2011) Plasma concentrations of 25-hydroxyvitamin D among Jordanians: effect of biological and habitual factors on vitamin D status. BMC Clin Pathol, 11:8-13.

15.

McGrath

, Saha

, Burne

THJ

, et al. (2010) A systematic review of the association between common single nucleotide polymorphisms and 25-hydroxyvitamin D concentrations. J Steroid Biochem Mol Biol, 121:471-477.

16.

Mishal

(2001) Effects of different dress styles on vitamin D levels in healthy young Jordanian women. Osteoporos Int, 12:931-935.

17.

Nissen

, Rasmussen

, Ravn-Haren

, et al. (2014) Common variants in CYP2R1 and GC genes predict vitamin D concentrations in healthy Danish children and adults. PLoS One, 9:e89907.

18.

Ramos-Lopez

, Brück

, Jansen

, et al. (2007) CYP2R1 (vitamin D 25-hydroxylase) gene is associated with susceptibility to type 1 diabetes and vitamin D levels in Germans. Diabetes Metab Res Rev, 23:631-636.

19.

Sadat-Ali

, AlElq

, Al-Turki

, et al. (2009) Vitamin D levels in healthy men in eastern Saudi Arabia. Ann Saudi Med, 29:378-382.

20.

Santos

, Mascarenhas

, Boguszewski

, et al. (2013) Variations in the vitamin D-binding protein (DBP) gene are related to lower 25-hydroxyvitamin D levels in healthy girls: a cross-sectional study. Horm Res Paediatr, 79:162-168.

21.

Sinotte

, Diorio

, Bérubé

, et al. (2009) Genetic polymorphisms of the vitamin D binding protein and plasma concentrations of 25-hydroxyvitamin D in premenopausal women. Am J Clin Nutr, 89:634-640.

22.

Trummer

, Schwetz

, Walter-Finell

, et al. (2012) Allelic determinants of vitamin D insufficiency, bone mineral density, and bone fractures. J Clin Endocrinol Metab, 97:E1234-E1240.

23.

Wang

, Zhang

, Richards

(2010) Common genetic determinants of vitamin D insufficiency: a genome-wide association study. Lancet, 376:180-188.

24.

Zgheib

, Arabi

, Mahfouz

, et al. (2013) Cyp2r1 genetic polymorphisms are associated with lower 25-hydroxy vitamin D levels in Lebanese subjects. Clin Ther, 35:e1.

25.

Zhang

, Wang

, Liu

, et al. (2012) The GC, CYP2R1 and DHCR7 genes are associated with vitamin D levels in northeastern, Han Chinese children. Swiss Med Wkly, 142:w13636 (online).