Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase

Abstract

The synthesis of bioactive vitamin D requires hydroxylation at the 1 alpha and 25 positions by cytochrome P450 enzymes in the kidney and liver, respectively. The mitochondrial enzyme CYP27B1 catalyzes 1 alpha-hydroxylation in the kidney but the identity of the hepatic 25-hydroxylase has remained unclear for >30 years. We previously identified the microsomal CYP2R1 protein as a potential candidate for the liver vitamin D 25-hydroxylase based on the enzyme's biochemical properties, conservation, and expression pattern. Here, we report a molecular analysis of a patient with low circulating levels of 25-hydroxyvitamin D and classic symptoms of vitamin D deficiency. This individual was found to be homozygous for a transition mutation in exon 2 of the CYP2R1 gene on chromosome 11p15.2. The inherited mutation caused the substitution of a proline for an evolutionarily conserved leucine at amino acid 99 in the CYP2R1 protein and eliminated vitamin D 25-hydroxylase enzyme activity. These data identify CYP2R1 as a biologically relevant vitamin D 25-hydroxylase and reveal the molecular basis of a human genetic disease, selective 25-hydroxyvitamin D deficiency.

Fig. 1.

Structure of the human CYP2R1 gene. The predicted exon–intron structure of the human CYP2R1 gene is shown together with six pairs of oligonucleotides used to amplify and sequence fragments of the DNA. Exon 3 was amplified with primer pair 5, 8, and was sequenced with primers 5, 6, 7, and 8. The gene is drawn to scale with the exception of intron 1, which is estimated to be 9.1 kb in length. The autosomal location of the gene was deduced from the Human Genome database, which can be accessed at www.ncbi.nlm.nih.gov/mapview/maps.cgi?taxid=9606&chr=11.

Fig. 2.

DNA sequence analysis of mutation in CYP2R1 vitamin D 25-hydroxylase. The partial DNA sequence of exon 2 from the CYP2R1 gene is shown from a normal individual (Upper) and from an individual with selective 25-hydroxyvitamin D deficiency (Lower). The codon specifying amino acid 99 is CTT in the normal gene and CCT (arrow) in the affected individual. The T → C transition mutation in the second nucleotide causes a change from leucine to proline at residue 99. All other nucleotides in the CYP2R1 gene of the proband were identical to those in normal individuals.

Fig. 3.

Biochemical assay of CYP2R1 vitamin D 25-hydroxylase enzyme activity. HEK 293 cells were transfected with the indicated expression plasmids for a period of 18–20 h. Thereafter, the medium was made 4.6 × 10^-7 M in [4-¹⁴C]vitamin D₃ and the incubation continued for an additional 96 h. Lipids were extracted from cells and medium into chloroform:methanol (2:1, vol/vol), and vitamin D metabolites and standards were separated by TLC on 150-Å silica gel plates (Whatman, catalog no. 4855–821) in a solvent system containing cyclohexane:ethyl acetate (3:2, vol/vol). After development, radioactivity was detected by PhosphorImager analysis, and the positions to which authentic vitamin D₃ and 25-hydroxyvitamin D₃ migrated on the plate were determined by staining with iodine.

Fig. 4.

L99P mutation in CYP2R1 causes loss of vitamin D₃ activation. Expression vectors encoding no protein, the normal CYP2R1 enzyme (CYP2R1), or a version containing a proline substituted for leucine at amino acid 99 (CYP2R1L99P), were transfected in triplicate into HEK 293 cells together with the indicated VDR-reporter gene systems. The concentrations of vitamin D₃ added to the medium ranged from 0.25 to 1.0 μM. After 16–20 h, cells were lysed and assayed for enzyme activities. Relative LUC activity was calculated by dividing units of LUC enzyme activity measured on a Dynex MLX luminometer by units of β-galactosidase enzyme activity measured on a Dynex Opsys MR spectrophotometer. Means ± SE of measurement were calculated and plotted in histogram form. The data are representative of two separate experiments carried out on different days.

Fig. 5.

Response of normal and L99P CYP2R1 enzymes to increasing concentrations of vitamin D₃ (A) and vitamin D₂ (B). The indicated expression plasmids were introduced into HEK 293 cells together with DNAs constituting the VDR/VDRE-LUC reporter gene system. After an expression period (8 h), different amounts of the two forms of vitamin D were added to the medium and the incubation was continued for an additional 16 h. Thereafter, cells were lysed and assayed for LUC and β-galactosidase enzyme activities. Points on the graphs represent means of triplicate values established at each concentration of secosteroid. These experiments were repeated at least two times each.