CYP2R1 Mutations Impair Generation of 25-hydroxyvitamin D and Cause an Atypical Form of Vitamin D Deficiency

Abstract

Context: Production of the active vitamin D hormone 1,25-dihydroxyvitamin D requires hepatic 25-hydroxylation of vitamin D. The CYP2R1 gene encodes the principal vitamin D 25-hydroxylase in humans.

Objective: This study aimed to determine the prevalence of CYP2R1 mutations in Nigerian children with familial rickets and vitamin D deficiency and assess the functional effect on 25-hydroxylase activity.

Design and participants: We sequenced the CYP2R1 gene in subjects with sporadic rickets and affected subjects from families in which more than one member had rickets.

Main outcome measures: Function of mutant CYP2R1 genes as assessed in vivo by serum 25-hydroxyvitamin D values after administration of vitamin D and in vitro by analysis of mutant forms of the CYP2R1.

Results: CYP2R1 sequences were normal in 27 children with sporadic rickets, but missense mutations were identified in affected members of 2 of 12 families, a previously identified L99P, and a novel K242N. In silico analyses predicted that both substitutions would have deleterious effects on the variant proteins, and in vitro studies showed that K242N and L99P had markedly reduced or complete loss of 25-hydroxylase activity, respectively. Heterozygous subjects were less affected than homozygous subjects, and oral administration of vitamin D led to significantly lower increases in serum 25-hydroxyvitamin D in heterozygous than in control subjects, whereas homozygous subjects showed negligible increases.

Conclusion: These studies confirm that CYP2R1 is the principal 25-hydroxylase in humans and demonstrate that CYP2R1 alleles have dosage-dependent effects on vitamin D homeostasis. CYP2R1 mutations cause a novel form of genetic vitamin D deficiency with semidominant inheritance.

Figure 1.

Pedigrees and single nucleotide polymorphism haplotype analysis. A, Family 1; B, Family 2. The presence of a CYP2R1 mutation is noted under each subject, and the genotypes for single nucleotide polymorphisms in and around the CYP2R1 gene are shown in the haplotype boxes at the bottom of each pedigree. The CYP2R1 allele carrying the L99P mutation is shaded in gray. Probands are indicated by the arrows.

Figure 2.

Skeletal deformities and mutation analysis in affected members of family 2. A, The proband and her affected sister from family 2, who are heterozygous for the p.L99P mutation, both had marked genu varum deformity. B, A portion of the DNA sequence chromatogram of exon 2, which demonstrates heterozygous replacement of T by C at position c.296 (arrow) that leads to replacement of amino acid leucine at codon 99 with proline (p.99L>P) on one allele. C, A portion of the DNA sequence chromatogram of exon 3 from their mother, with the arrow denoting heterozygous replacement of adenine (A) by cytosine (C) at c.726 (A>C) that leads to replacement of amino acid lysine at codon 242 by asparagine (p.242K>N) on one allele.

Figure 3.

Skeletal deformities and mutation analysis in affected members of family 1. A and B, The proband of family 1 shows marked residual anterior tibial bowing at age 20 years. C, His brother shows residual genu valgum at age 18 years. D, A portion of the DNA sequence chromatogram of exon 2 from the proband. The nucleotide sequence of CYP2R1 is noted above each chromatogram. This reveals a homozygous thymidine (T) to cystosine (C) substitution at c.296 (arrow) that leads to replacement of amino acid leucine at codon 99 with proline (p.99L>P).

Figure 4.

Effect of CYP2R1 mutations on 25-hydroxylase activity. A, Peak serum vitamin D₂ and D₃ levels 24 h after administration of vitamin D₂ or D₃. B, Serum 25(OH)D₃ concentrations after administration of 50 000 IU of vitamin D₃. C, Serum 25(OH)D₂ concentrations after administration of 50 000 IU of vitamin D₂. D, Immunoblot of HEK293T cell extracts. Cells were transfected with plasmids containing vector sequences only (lane 1) or cDNAs corresponding to wild type (lane 2), L99P (lane 3), or K242N (lane 4) Cyp2r1 with FLAG epitope tag. Blots were sequentially incubated with anti-FLAG antibody an anti-J-actin antibody to assess protein loading of each lane. E, Response of normal and mutant Cyp2r1 enzymes to increasing concentrations of 1α-hydroxyvitamin D₃. The expression plasmids were introduced into HEK 293 cells with DNAs constituting the VDR-Gal4/GAL4-UAS-Luciferase hybrid reporter gene system. Relative activity is expressed as the ratio of induced firefly luciferase to control (constitutive) Renilla luciferase. Points on the graphs represent means of triplicate values established at each concentration of secosteroid. In all figures, error bars represent SE of the mean, and stars represent significant (P < .05) differences between subjects homozygous for the L99P mutation and control subjects, using the Kruskal-Wallis test with Dunn's post-test analysis. One subject in Family 1 (II-4) did not participate in the in vivo vitamin D response assessment.

Figure 5.

Relationship of Peak 25-hydroxyvitamin D with Peak Vitamin D Concentrations. A, Relationship of peak serum 25(OH)D₂ value at Day 3 with peak vitamin D₂ concentrations on Day 1 after vitamin D₂ administration. B, Relationship of peak serum 25(OH)D₃ value at Day 3 with peak vitamin D₃ concentrations on Day 1 after vitamin D₃ administration.