Skip to main content
The Journal of Clinical Endocrinology and Metabolism logoLink to The Journal of Clinical Endocrinology and Metabolism
. 2015 May 5;100(7):E1005–E1013. doi: 10.1210/jc.2015-1746

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

Tom D Thacher 1,, Philip R Fischer 1, Ravinder J Singh 1, Jeffrey Roizen 1, Michael A Levine 1
PMCID: PMC4490307  PMID: 25942481

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.


Vitamin D deficiency is prevalent worldwide (13) and remains the most common cause of rickets in children and osteomalacia in adults. Moreover, low vitamin D status is associated with reduced bone density and increased fracture risk in the elderly (4, 5). A growing number of association studies have also implicated low vitamin D status as a potential risk factor for diabetes mellitus, hypertension, malignancy, infection, and immune diseases (3, 610), and optimal vitamin D status is a topic of active investigation and ardent controversy (11, 12). Vitamin D, produced in the skin as vitamin D3 (cholecalciferol) after exposure to UV light, or supplied in the diet as vitamin D3 or vitamin D2 (ergocalciferol), must undergo 25-hydroxylation in the liver (13) by CYP2R1, to generate 25-hydroxyvitamin D [25(OH)D]. Further hydroxylation in the kidneys by the 25(OH)D-1α-hydroxylase enzyme CYP27B1 generates 1,25-dihydroxyvitamin D [1,25(OH)2D], the active hormone responsible for most of the physiological actions of vitamin D.

Because few foods naturally contain or are fortified with vitamin D, the principle source of vitamin D for most populations is cutaneous production of vitamin D3 from sunlight. Thus, it has been surprising that vitamin D deficiency remains prevalent in sun-enriched parts of the world (1417), and that rickets occurs in children who live in tropical countries (1). We sought to identify potential genetic causes of rickets in a cohort of children in central Nigeria (latitude 10°N) that responds more effectively to calcium supplementation than to conventional doses of vitamin D (18, 19). These subjects had low levels of 25(OH)D, which suggested a potential defect in 25-hydroxylation of vitamin D. Hence, we examined the CYP2R1 gene, which encodes the principle 25-hydroxylase, as recessive mutations in the human CYP2R1 gene (2022) and disruption of murine Cyp2r1 (23) by conventional gene targeting result in markedly reduced serum levels of 25(OH)D.

Materials and Methods

Subjects

Our cohort of rachitic Nigerian children included 27 children with sporadic rickets (serum concentration of 25[OH]D mean ± SD, 12.1 ± 4.8 ng/mL) and 12 subjects from families (serum concentration of 25[OH]D 11.4 ± 4.5 ng/mL) with more than one first-degree relative with rickets. Twenty-two first-degree relatives of 12 index cases had a history of leg deformities consistent with rickets, and 14 were included in this study. We confirmed rickets in all index cases and most siblings with rickets using a rickets radiographic score of greater than 1.5 on a 10-point severity scale (24). The control group consisted of 21 normal children between 19 and 59 months of age (mean ± SD, 35.7 ± 11.9 mo) who had been previously characterized (25). We collected medical and demographic data from each subject, with particular emphasis on skeletal deformities consistent with rickets, and measured biochemical parameters of bone and mineral metabolism. All studies were approved by the appropriate institutional review boards, and written informed consent was received from all patients or their parents prior to inclusion in the study.

Laboratory analyses and gene sequencing

We measured serum electrolytes and creatinine using routine methods. For measurements of vitamin D metabolites, blood samples were centrifuged within 30 minutes after collection, and serum samples were stored at −70°C until shipped frozen to the Mayo Clinic for analysis. Measurements of vitamin D3, vitamin D2, 25(OH)D3, and 25(OH)D2 were performed by isotope-dilution liquid chromatography tandem mass spectrometry using an API 4000 instrument (Applied Biosystems). We measured serum concentrations of 1,25(OH)2D by RIA (DiaSorin). Intact PTH was measured using the Immulite 2000 PTH assay (Diagnostics Product Corporation).

We extracted genomic DNA from dried blood spots or saliva using standard methods. All five exons containing the coding region, the exon-intron junctions, and the promoter of CYP2R1 (11p15.2) were sequenced as previously described (26). Nucleotide position was numbered according to the starting point of the ATG codon in the cDNA (sequence accession number NM_024514.4). The predicted effect of the variants was determined by in silico analyses using a web-based tool, Condel (27), which assesses the outcome of amino acid changes using a consensus deleteriousness score that combines various tools (eg, SIFT, Polyphen2, MutationAssessor) (2830). We used the HOPE tool (31) to determine the effect of variation on the 3D structure of the CYP2R1 protein.

We performed linkage analysis to determine potential associations of CYP2R1 alleles with rickets by genotyping members of all families for single nucleotide polymorphisms located within the CYP2R1 gene (rs12794714, rs7936142, rs1993116, rs1740157, rs10500804, rs11023373) (32). Genomic DNA from 59 unrelated subjects of Nigerian origin was genotyped for mutations and used as a population control. In addition, we analyzed CYP2R1 sequence data from 628 unrelated subjects in the 1000 Genomes Project.

Assessment of 25-hydroxylase activity in vivo

To assess the functional capacity of 25-hydroxylase in vivo, we administered 50 000 IU orally of vitamin D2 (Pliva, Inc.) or vitamin D3 (Bio-Tech) on separate occasions at least 3 months apart to patients with CYP2R1 mutations, their unaffected first-degree relatives, and control subjects (25).

Cell culture, analysis of Cyp2r1 in HEK293T cells, and statistical methods

See Supplemental Methods.

Results

Molecular genetic studies

CYP2R1 sequences were normal in all 27 children with sporadic rickets (data not shown). By contrast, probands from two of 12 Nigerian families with more than one member affected by rickets carried CYP2R1 mutations. In a micro linkage analysis, there was concordance between CYP2R1 alleles and rickets and/or low serum levels of 25(OH)D in affected members of both of these two families (Figure 1). Table 1 lists relevant characteristics of the patients in these two families. By contrast, probands from the other 10 families did not carry CYP2R1 mutations, and there was discordance between CYP2R1 haplotypes and transmission of rickets in these kindreds (data not shown).

Figure 1.

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.

Table 1.

Biochemical and Radiographic Characteristics of Five Subjects With CYP2R1 Mutations

Subject Subject II-1 (Family 1) Subject II-2 (Family 1) Subject I-1 (Family 1) Subject II-1 (Family 2) Subject I-2 (Family 2)
Mutation Homozygous L99P/L99P Homozygous L99P/L99P Homozygous L99P/L99P Heterozygous L99P/wt Heterozygous K242N/wt

Age, y 12.5 13 13.5 20 11 11.5 18 49 5 5.5 24
Characteristic Before After calciuma After vitamin D3b Before After calciuma Before After calcium Reference Ranges

Calcium, mg/dL 5.9 5.7 6.7 6.1 6.3 7.8 9.6 9.6 9.9 9.2 8.5–10.6
Phosphorus. mg/dL 2.6 3.4 4.0 3.9 4.3 5.5 3.2 2.0 4.6 3 2.5–5.4; 5–14 y
2.5–4.5 adults
Albumin, g/dL 3.7 3.9 3.7 3.4 4.4 3.9 4.3 3.5–5.0
Alkaline phosphatase, U/L 4866 5029 551 2391 2131 413 109 714 275 172 162–587; 5–14 y
45–115 adults
25(OH)D, ng/mL 8.0 2.2 1.5 4.1 3.7 3.1 4.9 16.4 25.9 18.7 >20
1,25(OH)2D. pg/mL 18 22 17 26 180 241 22–67
PTH, pg/mL 123 382 339 208 199 182 60 107 42 85 11–67
Radiographic score 10 8 0.5 9 1 5 1 0

To convert values for calcium to millimoles per L, multiply by 0.25. To convert values for phosphorus to millimoles per L, multiply by 0.32. To convert values for 25(OH)D to nanomoles per L, multiply by 2.50. To convert values for 1,25(OH)2D to picomoles per L, multiply by 2.40. To convert the values for PTH to picomoles per L, multiply by 0.11.

a

Calcium was given as ground fish including bones 10 g twice daily, providing approximately 952 mg of elemental calcium daily for 6 mo.

b

Vitamin D3 600 000 IU (15 mg) was given im twice at 3-month intervals.

We identified the previously described c.296T>C (L99P) CYP2R1 mutation (21) in eight subjects in two generations in both Families 1 and 2. The mutation was in linkage disequilibrium with a consistent haplotype across 45 kb of gDNA on the CYP2R1 locus. This haplotype was not present in any of the 59 subjects in the 10 other families that we analyzed and hence is rare. Accordingly, these two apparently unrelated Nigerian pedigrees show identity by state at the CYP2R1 locus on the mutant chromosome, and likely share a common origin with the L99P allele we previously identified in an affected member of a third family with rickets that originated in Nigeria (21).

The mother of the proband in family 2 was heterozygous for a substitution of cytosine for adenine at nucleotide position 726 (c.726A>C) in exon 3 of the CYP2R1 gene (Figure 2C). This missense mutation replaces lysine with asparagine at amino acid 242 of the CYP2R1 protein (p.K242N).

Figure 2.

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.

Both the L99P and K242N mutations were absent in CYP2R1 alleles from 59 unrelated subjects of Nigerian origin and 628 unrelated subjects in the 1000 Genomes Project, thereby excluding the possibility that the identified mutations represent population-specific sequence variants. Moreover, Leucine 99 and Lysine 242 are both conserved in the CYP2R1 enzymes of mammals, chickens, and fish, suggesting that these residues play important roles in protein function or structure. The effects of the two point mutations were analyzed using the bioinformatics software Condel, which predicted that the L99P and K242N amino acid replacements were both deleterious. Replacement of Leucine 99 has been proposed to impair CYP2R1 folding (33), whereas our in silico analysis of the K242N mutation predicts that this amino acid substitution disturbs the interaction surface (31). Replacement of leucine99 by proline, a known helix breaker, disrupts the hydrogen bond network and sterics of the helix. Replacement of lysine242 by asparagine is predicted to destabilize positioning of Phe240 with consequent decreased interaction of CYP2R1 with its substrate, parent vitamin D.

Clinical characterization of family 1

The index case in Family 1 (Figure 1A) was homozygous for a missense mutation c.296T>C in exon 2 of the CYP2R1 gene (Figure 3D), which results in substitution of proline for leucine at position 99 (p.L99P). In addition to the proband (II-1), his brother with rickets (II-2), and their father (I-1) were also homozygous for the L99P mutation, and two sisters and the mother were heterozygous for the L99P allele. Both brothers who were homozygous for the L99P mutation had severe rickets.

Figure 3.

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).

The proband (Figure 1A, II-1; Figure 3, A and B; Table 1) presented at age 12.5 years with leg pain, and had marked anterior tibial bowing that was first noted at age 2 years. He had begun to walk at age 9 months but stopped at age 3 years. His typical daily dairy product calcium intake was 50 mg. Rib beading and wrist enlargement were prominent, and radiographs and biochemistries were consistent with severe rickets. The serum 25(OH)D concentration was 8 ng/mL. As part of a clinical trial (34), he had a minimal response to 6 months of supplemental calcium (reduction in radiographic severity score from 10.0 to 8.0), and 3 months after additional treatment with im vitamin D3, 600 000 IU (Hamexmedica Ltd) his radiographic score declined from 8 to 3.5. A second dose of vitamin D3 was given, and 3 months later the radiographic score was 0.5, indicating the rickets was healed. He began walking unaided, but his deformities persisted.

The proband's brother (Figure 1A, II-2; Figure 3C; Table 1) presented simultaneously at age 11 years with leg pain and genu valgum that had been first noted at age 6.5 years. Walking had been delayed until age 3 years. His typical daily dairy product calcium intake was only 15 mg and his serum level of 25(OH)D was 4.1 ng/mL. Radiographs showed severe rickets, and the biochemical features were similar to those of his brother. In the same clinical trial as the proband (34), he had a favorable clinical response to treatment with calcium supplementation with reduction in the radiographic severity score from 9.0 to 1.0 (near complete healing), resolution of leg pain and improvement in genu valgum. The serum 25(OH)D concentration remained low, and other biochemical features of rickets remained unchanged, however. He developed scoliosis in adolescence.

The 49-year-old father (Table 1 and Figure 1A, I-1) had no history of childhood skeletal deformities. At age 24 years, he developed spastic paraparesis due to tuberculosis of the spine. He recovered after antituberculosis treatment, but he had a relapse of paraparesis at age 44 years and has remained unable to walk. A spine radiograph at age 46 years showed reduced mineralization and multiple thoracic vertebral compression fractures; radiographs of the pelvis, hips, femurs, and scapulae were normal, without pseudofractures of osteomalacia. His serum 25(OH)D concentration was 4.9 ng/mL and PTH was modestly elevated.

One of the proband's sisters (Figure 1A, II-3) had a history of mild genu varum that had resolved spontaneously as a toddler, and the mother and the other sister had no history of bone deformity or pain

Clinical characterization of family 2

The proband of family 2 (Figure 1B, II-1) was heterozygous for the c.296T>C missense mutation, p.L99P, as was her affected sister (Figure 1B, II-2) and father (Figure 1B and Figure 2B). Both children had clinical (Figure 2A), biochemical (Table 1), and radiographic (Figure 3, A and B) evidence of rickets. Their father (I-1) had a history compatible with childhood rickets. A younger sister (II-3) was clinically unaffected. The proband and her affected sister had presented with bowed legs at ages 62 and 32 months with radiographic scores of 5.0 and 2.0, respectively. The typical diet of the proband provided a daily calcium intake of 175 mg. Her serum 25(OH)D concentration was 16.4 ng/mL. Both affected sisters were treated with calcium and the proband had also received oral vitamin D2, 50 000 IU monthly for 6 months in a clinical trial (35). After treatment, rickets had resolved in both subjects (radiographic scores of 1.0 and 1.5, respectively). The mother had no history of bone deformity, but she had an elevated serum PTH concentration and a low serum 25(OH)D concentration.

Response to oral vitamin D challenge

Affected members of Families 1 and 2 were given a single dose of 50 000 IU orally of vitamin D2 or vitamin D3 on separate occasions. Serum concentrations of parent vitamin D2 and vitamin D3 were less than 5 ng/mL in all study and control subjects at baseline, and showed robust increases 1 day after administration of the respective vitamin D compound (Figure 4A). The three subjects who were homozygous for L99P had lower baseline 25(OH)D3 concentrations (3.1 ± 1.7 ng/mL; P < .001) and showed significantly blunted responses to oral vitamin D3 compared with individuals who were heterozygous for the L99P or K242N mutations or normal subjects (Figure 4B). Subjects who were heterozygous for the L99P mutations had lower baseline 25(OH)D3 concentrations than control children (13.4 ± 2.3 ng/mL vs 25.9 ± 6.1 ng/mL, respectively; P < .001), and showed subnormal responses to administration of vitamin D3. The baseline 25(OH)D3 concentration for the subject who was heterozygous for the K242N mutation was 15.6 ng/mL. The peak 25(OH)D3 values were observed 3 days after oral vitamin D3, and represented incremental increases in serum 25(OH)D3 of 9.8 ± 2.7, 19.9 ± 9.6, and 30.6 ± 16.0 ng/mL in L99P homozygous, L99P heterozygous, and control subjects, respectively (P = .03). The incremental increase in serum 25(OH)D3 in the K242N heterozygous subject was 10.5 ng/mL.

Figure 4.

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.

Serum levels of 25(OH)D2 were undetectable prior to administration of vitamin D2, and peaked at Day 3 with incremental responses that were lower in individuals who were homozygous (9.0 ± 0.5 ng/mL) than those who were heterozygous (19.0 ± 7.8 ng/mL) for the L99P mutation (P = .025) (Figure 4C). Moreover, heterozygous individuals had lower peak values than control children (38.2 ± 13.3 ng/mL; P = .008) (Figure 4C). In the K242N heterozygous subject, the peak 25(OH)D2 concentration was 12.4 ng/mL.

Peak serum levels of 25(OH)D on Day 3 positively correlated with peak serum concentrations of vitamin D on Day 1 in control and heterozygous subjects (Figure 5) but not in subjects who were homozygous for the L99P mutation. The relatively lower vitamin D levels achieved in homozygous and heterozygous subjects generally resulted in greater 25(OH)D concentrations at similar vitamin D levels in healthy controls. Among the L99P homozygous subjects, the relatively low concentrations of 1,25(OH)2D at baseline increased in response to administration of vitamin D2 or D3, consistent with a functional vitamin D deficiency (Supplemental Figure 1).

Figure 5.

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.

Expression and functional analyses

The abundance of both mutant and wild-type CYP2R1 recombinant proteins in transiently transfected HEK293T cells was similar (Figure 4D), indicating that neither mutation affected expression of the protein. To determine the consequence of the identified mutations on CYP2R1 enzyme function, we transiently transfected HEK293T host cells with empty vector, wild-type, or mutant CYP2R1 cDNAs and compared the generation of 1,25(OH)D3 from substrate 1α-hydroxyvitamin D3 using a luciferase reporter gene that is under the control of the vitamin D receptor (VDR). In the absence of 1α-hydroxyvitamin D3 or recombinant CYP2R1, very little luciferase activity was detected (Figure 4E). Cells expressing wild-type CYP2R1 showed robust increases in luciferase activity compared with cells transfected with vector DNA, indicating that synthesis of 1,25(OH)2D3 was dependent upon forced expression of 25-hydroxylase activity. Moreover, the catalytic properties of the recombinant CYP2R1 enzyme with 1α-hydroxyvitamin D as a substrate (Km 9.6μM) compared favorably with the properties of the purified enzyme (Km 11.3μM) (33). By contrast, the p.L99P CYP2R1 did not induce activity of the reporter gene, indicating that this mutation abolished 25-hydroxylase activity. The K242N mutant induced a blunted response to 1α-hydroxyvitamin D3 (Km, 10.4 ± 7.7μM; Vmax, 0.55 ± 0.12 relative units) compared with the wild-type enzyme (Km, 9.6 ± 2.8μM; Vmax, 0.93 ± 0.08 relative units), indicating a significant reduction in 25-hydroxylase activity. Because recent studies show that the closely related CYP2C8 exists as a dimer when bound to mammalian membranes, and that this structure has functional significance (36), we found it conceivable that the dimeric structure of CYP2R1 that was observed after crystallization of solubilized protein might also exist in the membrane-bound form of CYP2R1 (33). Hence, we assessed whether the L99P and K242N proteins might inhibit activity of the wild-type enzyme. However, when coexpressed with equal amount of wild-type CYP2R1, neither mutant enzyme showed a dominant negative effect (data not shown).

Discussion

Rickets generally results from inadequate cutaneous synthesis or insufficient dietary supply of vitamin D. Less commonly, rickets arises from pseudovitamin D deficiency, in which severe hypocalcemia and rickets develop within the first year of life and do not respond to treatment with standard doses of vitamin D and calcium. The initial characterization of this hereditary form of pseudovitamin D deficiency rickets noted that to maintain health, intake of vitamin D had to be consistently in vast excess of the recommended daily allowance, hence, the term vitamin D dependency was proposed to describe the new syndrome (37). Later reports characterized a second form of pseudovitamin D deficiency that was not responsive to even high doses of vitamin D (38). The mechanisms and responsible genes for these two types of pseudovitamin D deficiency have been characterized. Mutations in the CYP27b gene encoding 25(OH)D 1α-hydroxylase are the basis for vitamin D-dependent rickets type 1A (VDDR1A, MIM 264700) (39, 40), and explain the inability to synthesize the fully active form of vitamin D, 1,25(OH)2D. By contrast, mutations in the VDR gene encoding the vitamin D receptor are the basis for vitamin D-dependent rickets type 2A (VDDR2A, MIM 277440) (41, 42), and account for resistance to high doses of vitamin D or even 1,25(OH)2D. More recently, overexpression of a heterogeneous nuclear ribonucleoprotein–like dominant-negative protein that binds to the VDR response element in vitamin D target genes has been described as a second mechanism for VDDR2 (VDDR2B, MIM 600785), although the precise molecular defect is unknown (43). In this report we describe another mechanism for pseudovitamin D deficiency that is uniquely associated with low serum concentrations of 25(OH)D, vitamin D–dependent rickets type 1B (VDDR1B, MIM 600081).

We identified two different CYP2R1 missense mutations in affected members of two families with VDDR1B. Linkage analysis excluded an association between the CYP2R1 locus and rickets in 10 additional families. Moreover, we did not identify CYP2R1 mutations in 27 unrelated patients with sporadic rickets. Although our mutation-detection strategy may have missed some mutations in CYP2R1, our results are consistent with genetic heterogeneity in VDDR1B and/or undisclosed environmental effects.

Our data support a gene dosage effect of CYP2R1 on conversion of vitamin D to 25(OH)D in humans. Semidominant inheritance is signified by a more severe disease phenotype when the mutant allele is homozygous and a less severe phenotype when the allele is heterozygous. Although VDDR1A is an autosomal-recessive condition, VDDR1B is a more complex disorder in which the phenotype is dependent upon the number of defective alleles. Patients with one defective CYP2R1 allele showed a mild form of VDDR1B. These patients produce less 25(OH)D than control subjects after administration of either vitamin D2 or vitamin D3, but are able to maintain near normal mineral homeostasis as adults. By contrast, patients who are homozygous for CYP2R1 mutations have a more severe form of VDDR1B. These patients show minimal increases in serum 25(OH)D after vitamin D, and clinical improvement is effected only with very high doses of vitamin D or calcium (21, 44). Similarly, two Saudi siblings with low serum concentrations of 25(OH)D due to compound heterozygosity for two other CYP2R1 mutations showed very modest responses to high-dose vitamin D supplementation (20). Interestingly, subject I-1 in family 1, an adult, showed near-normal serum levels of PTH, calcium, and phosphorus despite homozygosity for the nonfunctional L99P allele. This is similar to the natural history of many patients with VDDR2A, who, during and after puberty and into adulthood are able to maintain normal mineral metabolism with modest oral calcium supplements (45), in accordance with a proposed mechanism for vitamin D–independent calcium absorption from the intestine (46). Nutritional rickets in Nigerian children commonly results from the interaction of very low dietary calcium intakes and suboptimal vitamin D status (35). The susceptibility of children to develop rickets with the CYP2R1 mutation may be augmented by dietary calcium deprivation or by vitamin D deficiency. The clinical response to calcium supplementation in some of the subjects with CYP2R1 mutations suggests the critical role of providing adequate calcium.

We demonstrated in one L99P homozygous subject that radiographic healing of rickets could be achieved with two large doses of IM vitamin D (600 000 IU), indicating that the vitamin D deficiency could be overcome. The small but significant increases in serum 25(OH)D in patients who have biallelic loss of function mutations in CYP2R1 may reflect the activity of other cytochrome P450 enzymes that can also serve as 25-hydroxylases (26, 47, 48). These data are similar to observations in mice in which Cyp2r1 alleles have been genetically ablated, and which show both a gene-dosage effect on circulating levels of 25(OH)D and markedly reduced but detectable levels of 25(OH)D in homozygous Cyp2r1 knockout mice (23, 49). The identity of the auxiliary 25-hydroxylase(s) is unknown. The increase in 1,25-dihydroxyvitamin D after oral administration of 50 000 IU of vitamin D2 or D3 in the L99P homozygous subjects was also consistent with functional vitamin D deficiency, characterized by induction of renal CYP27B1 by elevated PTH (25). Whether lower doses of vitamin D would raise the substrate concentration sufficiently to overcome the 25-hydroxylation deficit is unknown.

This report has several limitations. The two kindreds whose responses to vitamin D2 and vitamin D3 were studied included adult parents and older children, whereas control subjects were all children enrolled in a study of vitamin D. It is possible that vitamin D absorption is greater in children than in adults, possibly accounting for some of the observed differences. The blunted increase in 25(OH)D observed in subjects possessing the CYP2R1 mutation may, in part, reflect impaired absorption of vitamin D rather than impaired CYP2R1 25-hydroxylation. However, even in subjects with the CYP2R1 mutation, the peak vitamin D concentrations achieved should have been sufficient to produce a greater increase in 25(OH)D values than we observed. Although consistent with a model of semidominant inheritance, we concede that our findings are not definitive, and that the dosage effects that we have observed may be due to differences in age or genetic background of the subjects we studied.

We did not treat any subjects with the CYP2R1 mutation with 25(OH)D, to bypass the enzymatic defect of 25-hydroxylation. This would merit further study and potentially strengthen our conclusion regarding the functional significance of CYP2R1 mutations in vivo and the benefit of treatment with 25(OH)D.

In the context of recent reports of inactivating mutations of CYP24A1 (5052), which encodes an enzyme that degrades 25(OH)D and 1α,25(OH)2D, this study provides new insights into the contribution of genetic variability to human vitamin D homeostasis. The L99P allele prevalence is nearly 1% in subjects of African descent (53). Given the abundance of other nonsynonymous polymorphisms distributed throughout the coding region of the human CYP2R1 gene (28), and recent genome wide association studies showing an association between circulating 25(OH)D concentrations and the CYP2R1 locus (54), we suggest that variation in CYP2R1 expression may have an important role in determining vitamin D requirements. Hence, our study raises important questions regarding the effectiveness of standardized doses of supplemental vitamin D for maintaining optimal vitamin D status and encourages additional study of the relationship between CYP2R1 genotype and vitamin D status.

Acknowledgments

We acknowledge the expert technical assistance of Dr Anna Dang and Dr Changlin Ding in performing the molecular and biochemical studies. We are grateful to Brian Netzel for determination of serum concentrations of vitamin D2 and vitamin D3 and their metabolites.

This work was supported by the National Institutes of Health through NIH T32-HD043021, NIH R01DK079970 as well as the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant UL1TR000003; the Lerner Research Institute of the Cleveland Clinic Foundation (to M.A.L.); and the Friedman and Sadikoglu families.

Disclosure Summary: T.D.T. is a consultant for Biomedical Systems. P.R.F., R.J.S., J.R., and M.A.L. have nothing to disclose.

Footnotes

Abbreviations:
25(OH)D
25-hydroxyvitamin D
VDDR
vitamin D-dependent rickets
VDR
vitamin D receptor.

References

  • 1. Thacher TD, Fischer PR, Strand MA, Pettifor JM. Nutritional rickets around the world: Causes and future directions. Ann Trop Paediatr. 2006;26:1–16. [DOI] [PubMed] [Google Scholar]
  • 2. Holick MF, Chen TC. Vitamin D deficiency: A worldwide problem with health consequences. Am J Clin Nutr. 2008;87:1080S–1086S. [DOI] [PubMed] [Google Scholar]
  • 3. Bendik I, Friedel A, Roos FF, Weber P, Eggersdorfer M. Vitamin D: A critical and essential micronutrient for human health. Front Physiol. 2014;5:248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Cranney A, Horsley T, O'Donnell S, et al. Effectiveness and safety of vitamin D in relation to bone health. Evidence Report/Technology Assessment No. 158 (Prepared by the University of Ottawa Evidence-based Practice Center under Contract No. 290-02-0021) AHRQ Publication No 07-E013. Rockville, MD: Agency for Healthcare Research and Quality; 2007. [Google Scholar]
  • 5. Bischoff-Ferrari HA, Willett WC, Wong JB, et al. Prevention of nonvertebral fractures with oral vitamin D and dose dependency: A meta-analysis of randomized controlled trials. Arch Intern Med. 2009;169:551–561. [DOI] [PubMed] [Google Scholar]
  • 6. Reis JP, von Mühlen D, Miller ER, 3rd, Michos ED, Appel LJ. Vitamin D status and cardiometabolic risk factors in the United States adolescent population. Pediatrics. 2009;124:e371–e379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Zipitis CS, Akobeng AK. Vitamin D supplementation in early childhood and risk of type 1 diabetes: A systematic review and meta-analysis. Arch Dis Child. 2008;93:512–517. [DOI] [PubMed] [Google Scholar]
  • 8. Bolland MJ, Grey A, Gamble GD, Reid IR. Calcium and vitamin D supplements and health outcomes: A reanalysis of the Women's Health Initiative (WHI) limited-access data set. Am J Clin Nutr. 2011;94:1144–1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Urashima M, Segawa T, Okazaki M, Kurihara M, Wada Y, Ida H. Randomized trial of vitamin D supplementation to prevent seasonal influenza A in schoolchildren. Am J Clin Nutr. 2010;91:1255–1260. [DOI] [PubMed] [Google Scholar]
  • 10. Camargo CA, Jr, Ingham T, Wickens K, et al. Cord-blood 25-hydroxyvitamin D levels and risk of respiratory infection, wheezing, and asthma. Pediatrics. 2011;127:e180–e187. [DOI] [PubMed] [Google Scholar]
  • 11. Bischoff-Ferrari HA. Optimal serum 25-hydroxyvitamin D levels for multiple health outcomes. Adv Exp Med Biol. 2014;810:500–525. [DOI] [PubMed] [Google Scholar]
  • 12. Rosen CJ, Abrams SA, Aloia JF, et al. IOM committee members respond to Endocrine Society vitamin D guideline. J Clin Endocrinol Metab. 2012;97:1146–1152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Henry HL. Regulation of vitamin D metabolism. Best Pract Res Clin Endocrinol Metab. 2011;25:531–541. [DOI] [PubMed] [Google Scholar]
  • 14. van Schoor NM, Lips P. Worldwide vitamin D status. Best Pract Res Clin Endocrinol Metab. 2011;25:671–680. [DOI] [PubMed] [Google Scholar]
  • 15. Arabi A, El Rassi R, El-Hajj Fuleihan G. Hypovitaminosis D in developing countries-prevalence, risk factors and outcomes. Nat Rev Endocrinol. 2010;6:550–561. [DOI] [PubMed] [Google Scholar]
  • 16. Wahl DA, Cooper C, Ebeling PR, et al. A global representation of vitamin D status in healthy populations: Reply to comment by Saadi. Arch Osteoporos. 2013;8:122. [DOI] [PubMed] [Google Scholar]
  • 17. Hilger J, Friedel A, Herr R, et al. A systematic review of vitamin D status in populations worldwide. Br J Nutr. 2014;111:23–45. [DOI] [PubMed] [Google Scholar]
  • 18. Thacher TD, Fischer PR, Pettifor JM, et al. A comparison of calcium, vitamin D, or both for nutritional rickets in Nigerian children. N Engl J Med. 1999;341:563–568. [DOI] [PubMed] [Google Scholar]
  • 19. Thacher TD, Fischer PR, Pettifor JM, Lawson JO, Isichei CO, Chan GM. Case-control study of factors associated with nutritional rickets in Nigerian children. J Pediatr. 2000;137:367–373. [DOI] [PubMed] [Google Scholar]
  • 20. Al Mutair AN, Nasrat GH, Russell DW. Mutation of the CYP2R1 vitamin D 25-hydroxylase in a Saudi Arabian family with severe vitamin D deficiency. J Clin Endocrinol Metab. 2012;97:E2022–E2025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Cheng JB, Levine MA, Bell NH, Mangelsdorf DJ, Russell DW. Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase. Proc Natl Acad Sci USA. 2004;101:7711–7715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Dong Q, Miller WL. Vitamin D 25-hydroxylase deficiency. Mol Genet Metab. 2004;83:197–198. [DOI] [PubMed] [Google Scholar]
  • 23. Zhu JG, Ochalek JT, Kaufmann M, Jones G, Deluca HF. CYP2R1 is a major, but not exclusive, contributor to 25-hydroxyvitamin D production in vivo. Proc Natl Acad Sci USA. 2013;110:15650–15655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Thacher TD, Fischer PR, Pettifor JM, Lawson JO, Manaster BJ, Reading JC. Radiographic scoring method for the assessment of the severity of nutritional rickets. J Trop Pediatr. 2000;46:132–139. [DOI] [PubMed] [Google Scholar]
  • 25. Thacher TD, Fischer PR, Obadofin MO, Levine MA, Singh RJ, Pettifor JM. Comparison of metabolism of vitamins D2 and D3 in children with nutritional rickets. J Bone Miner Res. 2010;25:1988–1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Cheng JB, Motola DL, Mangelsdorf DJ, Russell DW. De-orphanization of cytochrome P450 2R1: A microsomal vitamin D 25-hydroxilase. J Biol Chem. 2003;278:38084–38093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. González-Pérez A, López-Bigas N. Improving the assessment of the outcome of nonsynonymous SNVs with a consensus deleteriousness score, Condel. Am J Hum Genet. 2011;88:440–449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc. 2009;4:1073–1081. [DOI] [PubMed] [Google Scholar]
  • 29. Adzhubei IA, Schmidt S, Peshkin L, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7:248–249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Reva B, Antipin Y, Sander C. Predicting the functional impact of protein mutations: Application to cancer genomics. Nucleic Acids Res. 2011;39:e118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Venselaar H, Te Beek TA, Kuipers RK, Hekkelman ML, Vriend G. Protein structure analysis of mutations causing inheritable diseases. An e-Science approach with life scientist friendly interfaces. BMC Bioinformatics. 2010;11:548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. SNP linked to gene CYP2R1 (geneID:120227) via contig annotation. http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?geneId=120227 (accessed June 19, 2012).
  • 33. Strushkevich N, Usanov SA, Plotnikov AN, Jones G, Park HW. Structural analysis of CYP2R1 in complex with vitamin D3. J Mol Biol. 2008;380:95–106. [DOI] [PubMed] [Google Scholar]
  • 34. Thacher TD, Bommersbach TJ, Pettifor JM, Isichei CO, Fischer PR. Comparison of limestone and ground fish for treatment of nutritional rickets in Nigerian children [published online March 20, 2015]. J Pediatr. doi:10.1016/j.jpeds.2015.02.008. [DOI] [PubMed] [Google Scholar]
  • 35. Thacher TD, Fischer PR, Pettifor JM. Vitamin D treatment in calcium-deficiency rickets: A randomised controlled trial. Arch Dis Child. 2014;99:807–811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Hu G, Johnson EF, Kemper B. CYP2C8 exists as a dimer in natural membranes. Drug Metab Dispos. 2010;38:1976–1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Scriver CR. Vitamin D dependency. Pediatrics. 1970;45:361–363. [PubMed] [Google Scholar]
  • 38. Marx SJ, Spiegel AM, Brown EM, et al. A familial syndrome of decrease in sensitivity to 1,25-dihydroxyvitamin D. J Clin Endocrinol Metab. 1978;47:1303–1310. [DOI] [PubMed] [Google Scholar]
  • 39. Fu GK, Lin D, Zhang MY, et al. Cloning of human 25-hydroxyvitamin D-1 alpha-hydroxylase and mutations causing vitamin D-dependent rickets type 1. Mol Endocrinol. 1997;11:1961–1970. [DOI] [PubMed] [Google Scholar]
  • 40. Wang X, Zhang MY, Miller WL, Portale AA. Novel gene mutations in patients with 1alpha-hydroxylase deficiency that confer partial enzyme activity in vitro. J Clin Endocrinol Metab. 2002;87:2424–2430. [DOI] [PubMed] [Google Scholar]
  • 41. Malloy PJ, Pike JW, Feldman D. The vitamin D receptor and the syndrome of hereditary 1,25-dihydroxyvitamin D-resistant rickets. Endocr Rev. 1999;20:156–188. [DOI] [PubMed] [Google Scholar]
  • 42. Malloy PJ, Wang J, Peng L, et al. A unique insertion/duplication in the VDR gene that truncates the VDR causing hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia. Arch Biochem Biophys. 2007;460:285–292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Chen H, Hewison M, Hu B, Adams JS. Heterogeneous nuclear ribonucleoprotein (hnRNP) binding to hormone response elements: A cause of vitamin D resistance. Proc Natl Acad Sci USA. 2003;100:6109–6114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44. Casella SJ, Reiner BJ, Chen TC, Holick MF, Harrison HE. A possible genetic defect in 25-hydroxylation as a cause of rickets. J Pediatr. 1994;124:929–932. [DOI] [PubMed] [Google Scholar]
  • 45. Tiosano D, Hadad S, Chen Z, et al. Calcium absorption, kinetics, bone density, and bone structure in patients with hereditary vitamin D-resistant rickets. J Clin Endocrinol Metab. 2011;96:3701–3709. [DOI] [PubMed] [Google Scholar]
  • 46. Van Cromphaut SJ, Rummens K, Stockmans I, et al. Intestinal calcium transporter genes are upregulated by estrogens and the reproductive cycle through vitamin D receptor-independent mechanisms. J Bone Miner Res. 2003;18:1725–1736. [DOI] [PubMed] [Google Scholar]
  • 47. Gupta RP, Hollis BW, Patel SB, Patrick KS, Bell NH. CYP3A4 is a human microsomal vitamin D 25-hydroxylase. J Bone Miner Res. 2004;19:680–688. [DOI] [PubMed] [Google Scholar]
  • 48. Yamasaki T, Izumi S, Ide H, Ohyama Y. Identification of a novel rat microsomal vitamin D3 25-hydroxylase. J Biol Chem. 2004;279:22848–22856. [DOI] [PubMed] [Google Scholar]
  • 49. Wang Y, Marling SJ, McKnight SM, Danielson AL, Severson KS, Deluca HF. Suppression of experimental autoimmune encephalomyelitis by 300–315nm ultraviolet light. Arch Biochem Biophys. 2013;536:81–86. [DOI] [PubMed] [Google Scholar]
  • 50. Schlingmann KP, Kaufmann M, Weber S, et al. Mutations in CYP24A1 and idiopathic infantile hypercalcemia. N Engl J Med. 2011;365:410–421. [DOI] [PubMed] [Google Scholar]
  • 51. Dauber A, Nguyen TT, Sochett E, et al. Genetic defect in CYP24A1, the vitamin D 24-hydroxylase gene, in a patient with severe infantile hypercalcemia. J Clin Endocrinol Metab. 2012;97:E268–E274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52. Tebben PJ, Milliner DS, Horst RL, et al. Hypercalcemia, hypercalciuria, and elevated calcitriol concentrations with autosomal dominant transmission due to CYP24A1 mutations: Effects of ketoconazole therapy. J Clin Endocrinol Metab. 2012;97:E423–E427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53. Sherry ST, Ward MH, Kholodov M, et al. dbSNP: The NCBI database of genetic variation. Nucleic Acids Res. 2001;29:308–311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54. Wang TJ, Zhang F, Richards JB, et al. Common genetic determinants of vitamin D insufficiency: A genome-wide association study. Lancet. 2010;376:180–188. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Clinical Endocrinology and Metabolism are provided here courtesy of The Endocrine Society

RESOURCES