Family‐based Association between Allele T of rs4646536 in CYP27B1 and vitamin D deficiency

Background The circulating concentration of 25(OH)D is widely applied to indicate the vitamin D status, as the directly metabolic genes of 25(OH)D, CYP2R1, and CYP27B1 are associated with the concentration of 25(OH)D. However, the contributions of allelic transmission disequilibrium of single nucleotide polymorphisms (SNPs) in these genes to vitamin D deficiency remain unclear. We aimed at investigating the family‐based association between SNPs of CYP2R1 and CYP27B1 and vitamin D deficiency. Method First, SNPs selected in family‐based study were screened by a pilot case‐control study. Second, allelic transmissions of the selected SNPs were investigated with family‐based study (n = 880). Finally, associations between selected SNPs and the concentration of 25(OH)D were verified in siblings (n = 120). Results The results of the pilot case‐control study indicated that both CT and TT genotypes of rs4646536 in CYP27B1 could increase the susceptibility of vitamin D deficiency when compared with CC genotype. The adjusted ORs were 2.846 (95%CI 1.312‐6.174, P = 0.008) and 2.609 (95%CI 1.197‐5.687, P = 0.016), respectively. Furthermore, the results of family‐based association test suggested that there was transmission disequilibrium for allele T of rs4646536 in vitamin D deficiency families. In addition, the concentration of 25(OH)D3 for CC genotype was higher than CT genotype between siblings (P = 0.016). Conclusions Transmission disequilibrium of allele T of rs4646536 is associated with vitamin D deficiency.


| INTRODUC TI ON
The circulating concentration of 25(OH)D is often applied to evaluate the vitamin D status. It is estimated that the heritability of 25(OH)D ranged from 23% to 80%. 1 25(OH)D is produced by hydroxylation at the C 25 position of vitamin D in the liver, which is catalyzed by 25-hydroxylase encoded by CYP2R1. 25(OH)D is further hydroxylated at the C 1 position in the kidney to form 1,25(OH) 2 D, which is catalyzed by 1α-hydroxylase encoded by CYP27B1, 2 as the key metabolic genes of 25(OH)D, CYP2R1, and CYP27B1 play direct roles in circulating concentration of 25(OH)D, 3 which is often used as indicator of vitamin D status. 4 Genetic variants in these genes have been reported to be associated with the concentration of circulating 25(OH)D. Bu et al reported that single nucleotide polymorphism (SNP) in CYP2R1 was associated with 25(OH)D concentration in Caucasian. 5 Significant associations between SNPs in CYP27B1 and 25(OH)D concentration were observed by Orton. 6 However, whether there are transmission disequilibriums for these genetic variants in vitamin D deficiency families remains unclear.
Transmission disequilibrium is used to describe the association between a genetic marker and a trait, which is tested by a familybased study. In this study, we hypothesized that transmission disequilibrium of SNPs in CYP2R1 and CYP27B1 contributed to the heritability of 25(OH)D. Thus, we aimed at investigating the familybased associations of SNPs in CYP2R1 and CYP27B1 with vitamin D deficiency. First, SNPs selected in family-based study were screened by a pilot case-control study. Second, allelic transmissions of the selected SNPs were investigated in a family-based study. Finally, associations between selected SNPs having allelic transmission disequilibrium and the concentration of 25(OH)D were verified in siblings. This work would shed more light on the heritability of 25(OH)D.

| Study subjects
Participant flowchart is shown in Figure 1

| 25(OH)D 3 measurement
A certificated third-party medical laboratory of Kingmed Center for Clinical Co., Ltd. (Guangzhou, China), was entrusted to determine the concentration of serum 25(OH)D 3 with electrochemical luminescence.

| SNP selection and genotyping
Many studies have reported that SNPs in CYP2R1 and CYP27B1 were significantly associated with the concentration of 25(OH)D, including rs12794714, rs1993116, rs10766197, and rs10741657 in CYP2R1, rs10877012, and rs4646536 in CYP27B1 (1). Thus, these reported SNPs were selected in this study. The SNPs associated with vitamin D deficiency in the pilot case-control study were further investigated in family-based study.
Single nucleotide polymorphism genotyping was completed in fluorescence quantitative PCR instrument (7500 Fast, Applied Biosystems, California, USA). All the reagents and consumables were supplied by Applied Biosystems (California, USA). All the operations were according to the manufacturer manual.

| Statistical analysis
Categorical variable was described as frequency and percentage and compared by chi-square test. Continuous variable with normal distribution was presented as means ± SD and compared with Student's t test. Continuous variable with skew distribution was presented as 1. rs4646536 is associated with vitamin D deficiency in case-control study.

Transmission disequilibrium for rs4646536 was found in
vitamin D deficiency families.

3.
Sibling with CT genotype of rs4646536 has lower levels of 25(OH)D 3 than CC genotype.
F I G U R E 1 Flowchart of participant selection. A total of 272 subjects aged from 18 to 79 years were randomly selected from the Henan Rural Cohort Study for case-control study. 257 pedigrees containing 880 subjects in Wuzhi County, Henan Province, China, were included in family-based study median (interquartile range) and compared with Wilcoxon rank sum test.
In order to investigate the associations of SNPs in CYP2R1 and CYP27B1 with vitamin D deficiency, as well as their transmission disequilibrium in vitamin D deficiency family, we analyzed the data through following strategy. On one hand, associations between SNPs and vitamin D deficiency were validated in a case-control study by logistic regression model. On the other hand, FBAT software (V2.0.4Q, https://www.hsph.harvard.edu/fbat/fbat.htm) was applied to investigate the transmission disequilibrium of SNP in vitamin D deficiency families. 7 The concentrations of 25(OH)D 3 between siblings were compared with Wilcoxon rank sum test.
All the statistical analysis except FBAT was completed with SPSS 21.0 (IBM SPSS, New York, USA). Two-tailed P value less than 0.05 was considered as statistical significance.

| SNPs associated with vitamin D deficiency in the pilot case-control study
The demographic and biochemical characteristics of case-control study are shown in Table 1. The results of logistic regression model indicated that both CT and TT genotypes of rs4646536 could increase the susceptibility of vitamin D deficiency when compared with CC genotype ( Table 2). The adjusted ORs were 2.846 (95%CI 1.312-6.174, P = 0.008) and 2.609 (95%CI 1.197-5.687, P = 0.016), respectively. No association was found for other SNPs (P > 0.05).

| Family-based association between rs4646536 and vitamin D deficiency
The results of family-based association test are shown in Table 3. In the additive model, 74 informative families were included. Allele T was observed to be associated with vitamin D deficiency (Z = 2.248, P = 0.025). In recessive model, 68 informative families were included for allele T, which was associated with vitamin D deficiency (Z = 2.512, P = 0.012).

| Association between rs4646536 and 25(OH) D 3 between siblings
Fourteen pairs of siblings with genotypes of CC and CT, 6 pairs of siblings with genotypes of CC and TT, and 40 pairs of siblings with genotypes of CT and TT from the 257 pedigrees were included. The concentration of 25(OH)D 3 for CC genotype was higher than CT genotype between siblings (P = 0.016, Figure 2).

| D ISCUSS I ON
We conducted three studies including case-control study, family-based study, and siblings study to investigate the associations between SNPs in CYP2R1 and CYP27B1 and vitamin D deficiency. Association between rs4646536 and vitamin D deficiency was found in case-control study. Both genotypes TT (OR 2.609, 95%CI 1.197-5.687, P = 0.016) and CT (OR 2.846, 95%CI 1.312-6.174, P = 0.008) could increase a comparable risk of vitamin D deficiency, which could be explained by a dominant model of allele T that genotypes CT would have the same phenotype as genotype TT. Furthermore, family-based associations between rs4646536 in CYP27B1 and vitamin D deficiency were also found. There was transmission disequilibrium for allele T of rs4646536 in vitamin D deficiency families. In addition, the association between rs4646536 and 25(OH)D 3 concentration was further verified between siblings. Therefore, these data revealed that transmission disequilibrium of risk allele T of rs4646536 contributed to vitamin D deficiency.
It was reported that the heritability of 25(OH)D ranged from 23% to 80%. 1 The heritability of 25(OH)D was estimated to be 28.8% in the Framingham Offspring Study. 8

| CON CLUS IONS
Allele T of rs4646536 is associated with vitamin D deficiency.
Transmission disequilibrium of rs4646536 in vitamin D deficiency families contributes to the heritability of 25(OH)D.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.