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Vitamin D plays a central role in calcium homeostasis and bone metabolism.1 Vitamin D supplementation or food fortification for the prevention of rickets is advocated routinely for all infants. Although vitamin D is potentially dangerous in very high doses, the margin of safety between the daily requirements of vitamin D and levels that produce toxic effects is considered to be quite large.2 However, in the early 1950s, there were reports about a number of infants with unexplained hypercalcemia who presented with failure to thrive, vomiting, dehydration, spikes of fever, and nephrocalcinosis.3,4 Laboratory evaluation of these infants revealed severe hypercalcemia and suppressed parathyroid hormone levels. Approximately 200 cases occurred in Great Britain within only 2 years.5 Some of the affected children had a complex phenotype that was later identified as the Williams–Beuren syndrome.6,7 However, most affected infants did not have syndromic features and were considered to be affected by a milder variant of the syndrome, which was termed idiopathic infantile hypercalcemia or Lightwood type (Online Mendelian Inheritance in Man number, 143880).4,8
Although this disorder was originally considered to be relatively benign, during the acute phase of hypercalcemia, a substantial number of children died.9,10 The relation between the epidemic occurrence of idiopathic infantile hypercalcemia and increased doses of vitamin D (up to 4000 IU per day) in infant formula and fortified milk in Great Britain at that time implicated nutritional vitamin D intake in the pathogenesis of this disorder.5 However, it was obvious that vitamin D was not the only contributing factor, since most infants receiving this prophylaxis remained unaffected. Therefore, it was proposed that an intrinsic hypersensitivity to vitamin D might be implicated in the pathogenesis.11 It remained unclear whether the underlying defect involved excessive activation of vitamin D or defective inactivation.
During activation, vitamin D first undergoes hydroxylation by 25-hydroxylase (CYP2R1) in the liver, which leads to the formation of 25-hydroxyvitamin D3.12 A second hydroxylation by 1α-hydroxylase (CYP27B1) in the kidney then generates the active form 1,25-dihydroxyvitamin D3, which exerts its biologic effects by binding to the vitamin D receptor. This active form is inactivated by 24-hydroxylase (CYP24A1), an enzyme that is responsible for the five-step 24-oxidation pathway from 1,25-dihydroxyvitamin D3 to calcitroic acid.13,14 CYP24A1 can also break down the precursor, 25-hydroxyvitamin D3, to the inactive metabolite, 24,25-dihydroxyvitamin D3. The activity of both CYP27B1 and CYP24A1 is predominantly controlled by levels of 1,25-dihydroxyvitamin D3, serum calcium, and parathyroid hormone. In addition, CYP27B1 is negatively regulated by the concerted action of fibroblast growth factor 23 (FGF23) and klotho, a process that closely links vitamin D metabolism to phosphate homeostasis (Figure 1Figure 1Vitamin D Metabolism with Selected Candidate Genes.).15
Here we describe how inactivating mutations in CYP24A1 provide a probable molecular basis for idiopathic infantile hypercalcemia, which is inherited as an autosomal recessive trait.
We studied a cohort of six patients from four families with idiopathic infantile hypercalcemia with suspected autosomal recessive inheritance. A second cohort consisted of four patients with suspected vitamin D intoxication in whom severe hypercalcemia had developed after bolus prophylaxis with vitamin D. Data on clinical symptoms and biochemical measures at the time of disease manifestation were collected retrospectively from medical charts. We clinically reevaluated all patients during follow-up and obtained biochemical data. All genetic studies were approved by the ethics committee of the Westfälische Wilhelms University, Muenster. Patients or their parents provided written informed consent.
Laboratory Analyses and Sequencing
We measured levels of serum and urine electrolytes and creatinine in samples obtained from all patients using routine methods. (Detailed descriptions of the analyses of serum parathyroid hormone, 25-hydroxyvitamin D3, and 1,25-dihydroxyvitamin D3 are provided in the Supplementary Appendix, available with the full text of this article at NEJM.org.) We extracted genomic DNA from the whole blood of affected patients and available family members using standard methods. The entire coding regions and splice sites of CYP24A1, CYP27B1, FGF23, and KL (the latter encoding klotho) were sequenced from both strands. The presence of newly identified CYP24A1 sequence variations was tested in at least 204 ethnically matched control alleles, whereas the presence of the two previously reported sequence variations R396W (rs114368325) and L409S (rs6068812) was analyzed in 1024 control alleles.
Preparation of Plasmid Constructs
Full-length human CYP24A1 16-18 was subcloned into a pcDNA5/FRT construct (Invitrogen). Site-directed mutagenesis was conducted with the use of a QuickChange kit (Stratagene). CYP24A1 mutants were generated (E143del, R159Q, E322K, R396W, L409S, and A475fsX490), and the presence of the desired mutations was confirmed by DNA sequencing.
Human wild-type and mutant CYP24A1 constructs were transiently or stably transfected into V79-4 Chinese hamster lung fibroblast cells. For stable transfections, a targeted integration method mediated by Flippase recombination enzyme (Flp) was used (Flp-In system, Invitrogen); for transient transfections, CYP24A1 constructs containing pcDNA5/FRT were transfected directly into native cells, and enzyme activity was assayed. Experimental details are provided in the Supplementary Appendix.
Cell Culture and Analysis of CYP24A1 Activity
Details of cell-culture experiments and analyses of CYP24A1 activity are provided in the Supplementary Appendix. Transfected cells were incubated in medium containing [1β-3H]1,25-dihydroxyvitamin D3. The incubation mediums were extracted and analyzed by high-performance liquid chromatography, as described previously.17,18
The four index patients in the four families with idiopathic infantile hypercalcemia (Patients 1.1, 2.1, 3.1, and 4.1) presented between the ages of 6 and 8 months with typical symptoms (Figure 2AFigure 2Family Pedigrees of Patients with Idiopathic Infantile Hypercalcemia, Laboratory Values for Patient 1.1, and Family Pedigrees of Patients with Suspected Vitamin D Intoxication.). Laboratory evaluation revealed profound hypercalcemia, suppressed intact parathyroid hormone, and hypercalciuria (Table 1Table 1Characteristics of the Patients.). Of note, all four infants had received oral vitamin D supplementation (500 IU per day) from birth. Medullary nephrocalcinosis was seen in all four infants on renal ultrasonography. Short-term treatment included intravenous rehydration and the use of furosemide, glucocorticoids, and pamidronate. Vitamin D prophylaxis was stopped, and a low-calcium diet was initiated. Serum calcium levels normalized within days to weeks. However, as shown in Patient 1.1 as an example, serum calcium levels tended to be continuously elevated during follow-up, whereas intact parathyroid hormone levels remained suppressed (Figure 2B).
After the diagnosis in the index cases, we evaluated two asymptomatic siblings of index patients. Biochemical analysis in Patient 2.2, the monozygotic twin of Patient 2.1, revealed a similar serum calcium level (3.7 mmol per liter [14.8 mg per deciliter]), a suppressed intact parathyroid hormone level, and hypercalciuria. Medullary nephrocalcinosis was seen on renal ultrasonography. On the basis of the hypercalcemia, Patient 2.2 was treated accordingly. Patient 3.2, the asymptomatic younger brother of Patient 3.1, had serum calcium levels in the upper limit of the normal range during the neonatal period. Given the history of Patient 3.1, the boys' parents decided against vitamin D prophylaxis for Patient 3.2. The diagnosis was established in Patient 3.2 at 18 months of age after the family's workup. Notably, his serum calcium level at that time was within the normal range, and intact parathyroid hormone levels were suppressed. Medullary hyperechogenicity was seen on renal ultrasonography. No additional treatment was initiated. All patients except for Patient 3.2 had received regular vitamin D3 supplementation.
The second cohort consisted of four children (Patients 5.1, 6.1, 7.1, and 8.1) with suspected vitamin D toxic effects in whom symptomatic hypercalcemia had developed 1 to 3 weeks after receiving an oral dose of 600,000 IU of vitamin D2 (Table 1 and Figure 2C). The administration of vitamin D given five times during the first 2 years of life (known as pulse therapy) was the preferred mode of prophylaxis in the German Democratic Republic for several decades.19 All four children received no additional daily vitamin D supplementation. Clinical details regarding Patients 5.1 and 6.1 have been reported previously.20 Serum 25-hydroxyvitamin D3 levels were increased in Patients 5.1 and 7.1. Serum 1,25-dihydroxyvitamin D3 was measured only in Patient 5.1 and was elevated. In these patients, levels of whole parathyroid hormone that were measured at presentation were normal; no assay for intact parathyroid hormone was available at that time in East Germany. Treatment with parenteral fluid and glucocorticoids resulted in a rapid normalization of serum calcium levels. The clinical course beyond infancy was favorable in all patients, without recurrence of hypercalcemic symptoms. Other causes of hypercalcemia were ruled out in all 10 patients.
The parental consanguinity (in Family 1) and familial occurrence (in Families 2 and 3) pointed to an inherited basis of idiopathic infantile hypercalcemia. Therefore, we performed a candidate-gene analysis, including the genes of the key enzymes involved in vitamin D metabolism (Figure 1). Although conventional sequencing of the coding regions of CYP27B1, FGF23, and KL did not reveal pathogenic mutations in patients from both cohorts, the sequence analysis of CYP24A1 yielded nonsense and missense mutations in the homozygous or compound-heterozygous state in Patients 1.1 to 7.1 (Table 1 and Figure 3AFigure 3 CYP24A1 Mutations, Protein Sequence Alignments, and Secondary Structure of CYP24A1.). Cosegregation analysis was compatible with autosomal recessive inheritance in all families. In Patient 8.1, only one pathogenic mutation was identified, which raised the possibility of a second pathogenic mutation outside the coding region or a heterozygous deletion that was not detected by sequence analysis.
Besides different missense mutations, we identified one premature stop mutation as well as two frameshift mutations leading to truncated CYP24A1 proteins. Furthermore, we identified an in-frame deletion of E143. All mutations were ruled out in at least 204 control alleles. For the two mutations, R396W and L409S, that had previously been annotated as putative polymorphisms in the Single Nucleotide Polymorphism Database (dbSNP), we tested a larger sample of 1024 control alleles. Although we did not detect L409S in any control allele, R396W was identified in 4 of the control alleles.
In Vitro Analysis of CYP24A1 Activity
In order to determine the consequence of the identified mutations to human CYP24A1 function in vitro, we stably and transiently transfected human CYP24A1 constructs containing the mutations into V79-4 host cells and compared the catabolism of 1,25-dihydroxyvitamin D3 with wild-type and nontransfected control cells. Transient and stable transfection protocols allowed us to test mutants at both nonsaturating substrate concentrations (0.003 μM for transient transfection and 0.3 μM for stable transfection) and saturating substrate concentrations (0.75 μM for transient transfection and 9.0 μM for stable transfection), respectively, in which stable transfection ensured high and reproducible levels of expression.
We found that 1,25-dihydroxyvitamin D3 was almost completely metabolized by wild-type CYP24A1 through the C-24 oxidation pathway intermediates (Figure 4AFigure 4Enzyme Activity for Mutant CYP24A1 in Patients with Idiopathic Infantile Hypercalcemia.) into the water-soluble metabolite, calcitroic acid, which was quantified as radioactivity in the aqueous phase (Figure 4B), as well as the terminal lipid-soluble metabolites, including tetranor-1,23-dihydroxyvitamin D3 (Figure 4D). When saturating substrate concentrations were used, almost all the intermediates in the C24-hydroxylation pathway (in addition to 1,23,25-trihydroxyvitamin D3) were observed with the use of both radioactivity detectors (as measured in millivolts) and photodiode-array detectors (as measured at a wavelength of 265 nm in the ultraviolet spectrum), representing characteristic human CYP24A1 activity, as reported previously.17,18 Under each transfection system and incubation condition used, the CYP24A1 mutations that were identified in patients with idiopathic infantile hypercalcemia resulted in the ablation of CYP24A1 catabolic activity (Figure 4B and Figure 4E through 4J), reminiscent of similar studies conducted on primary keratinocytes isolated from CYP24A1 knockout mice.21 Only L409S retained small but measurable levels of activity (5.3±0.3% of wild-type activity) (Figure 4I). Data were essentially the same for cells that had been either transiently or stably transfected.
In most studies of engineered mutations at substrate-contact residues in CYP24A1, there have been alterations in regioselectivity or relatively subtle changes in enzyme activity.17,18,22,23 In our study, however, the mutations in patients with idiopathic infantile hypercalcemia affected residues of critical structural importance (Figure 3B, and Figure S1 in the Supplementary Appendix) and resulted in complete loss of enzyme activity in most cases.
In a cohort of infants with idiopathic infantile hypercalcemia, we found loss-of-function mutations in CYP24A1 that appeared to lead to the disease development. CYP24A1 mutations were also detected in a second cohort of patients who presented with clinical symptoms of vitamin D intoxication 2 to 3 weeks after receiving intermittent high-dose vitamin D prophylaxis. Cosegregation analysis indicated autosomal recessive inheritance. Overexpression of the mutant CYP24A1 enzymes in a eukaryotic cell line revealed a complete loss of function for all identified mutations.
The physiologic importance of CYP24A1 in the catabolism of 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 has already been shown in CYP24A1 knockout (–/–) mice, which have severe hypercalcemia leading to perinatal death in approximately 50% of the animals.21,24,25 Long-term vitamin D treatment in CYP24A1–/– mice results in renal calcium deposition compatible with nephrocalcinosis.25 The administration of exogenous 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 to CYP24A1–/– mice leads to a significant increase in 1,25-dihydroxyvitamin D3 levels, indicating an inability to clear the active vitamin D hormone from the bloodstream.21,25 As expected, CYP24A1 /– mice lack 24-hydroxylated vitamin D metabolites.21
Our data provide evidence for a crucial biologic role for CYP24A1 in humans. Analyses of vitamin D metabolites in healthy persons who are receiving high doses of vitamin D have shown that in contrast to sharp increases in levels of serum 25-hydroxyvitamin D3 and its inactive products, serum 1,25-dihydroxyvitamin D3 levels remain within the normal reference range, indicating tight regulative mechanisms.19,26-28 In contrast, patients with idiopathic infantile hypercalcemia have an exaggerated and prolonged increase in levels of active 1,25-dihydroxyvitamin D3 after receiving prophylactic vitamin D, reflecting the impaired catabolism.29,30 Previously reported measurements of serum 24-hydroxylated metabolites in patients with idiopathic infantile hypercalcemia have had inconclusive results.30 However, in vitro data that were obtained after the incubation of skin fibroblasts obtained from a patient with idiopathic infantile hypercalcemia with 1,25-dihydroxyvitamin D3 showed decreased 24-hydroxylated metabolites.30 This result is confirmed by the results of our overexpression studies, which showed a lack of 24-hydroxylated vitamin D metabolites after incubation with [1β-3H]1,25-dihydroxyvitamin D3, indicating a complete loss of enzyme activity caused by a number of mutations in human CYP24A1.
Surprisingly, we also identified CYP24A1 mutations in four previously healthy children (Families 5 through 8) in whom symptoms of vitamin D intoxication developed after intermittent high-dose vitamin D prophylaxis that was regularly used in East Germany until 1989. Levels of 25-hydroxyvitamin D3 in these patients, as far as available, were still below values that are generally considered to lead to acute toxic effects (>200 to 240 ng per milliliter).27,28 Nevertheless, 1,25-dihydroxyvitamin D3 levels were found to be elevated in a single patient (Patient 5.1).
The genetic findings in both cohorts pose a critical question regarding the effect of dose and mode of administration of supplemental vitamin D for the manifestation of infantile hypercalcemia. The epidemic of idiopathic infantile hypercalcemia occurred in the United Kingdom in the 1950s after the implementation of an increased dose of vitamin D supplementation (up to 4000 IU per day).5,31,32 Concomitantly, less than 10 such cases were reported in the United States, where vitamin D supplementation was approximately 10 to 25% of the dose used in the United Kingdom.32 After the U.K. epidemic, the British Ministry of Health reduced daily allowances of vitamin D to approximately 400 IU, resulting in a significant decline in infantile hypercalcemia.33 The identification of patients with idiopathic infantile hypercalcemia as an at-risk group may bring a new aspect to the debate concerning vitamin D supplementation.
The strongest argument for the critical role of vitamin D in idiopathic infantile hypercalcemia is the time course in our second cohort, in which clinical symptoms developed rapidly after vitamin D bolus prophylaxis. In the first cohort, all index patients with idiopathic infantile hypercalcemia had received supplementation with 500 IU of vitamin D per day, which is in the range of currently advocated daily vitamin D doses in most Western European countries, Canada, and the United States.34 Symptomatic hypercalcemia developed in these patients after several months of vitamin D prophylaxis. Of note, two siblings (from Families 2 and 3) remained asymptomatic and were identified only retrospectively by laboratory testing and genetic screening. In this context, Patient 3.2, the brother of Patient 3.1, is of special interest, since despite an uneventful medical history, laboratory analysis showed normocalcemia but suppressed parathyroid hormone levels, and only discrete hyperechogenicity of the medullary pyramids was seen on renal ultrasonography. Importantly, the parents had decided against regular vitamin D prophylaxis in this child because of his brother's illness. This observation supports the hypothesis that a substantial number of genetically affected persons may remain asymptomatic as long as the dose of prophylactic vitamin D is restricted. Such an incomplete penetrance of phenotype is consistent with the reduction of disease incidence after limitation of vitamin D supplementation.
Taken together, our findings indicate that defects in CYP24A1 are causative for idiopathic infantile hypercalcemia and serve as a genetic risk factor for the development of a serious adverse effect of generally advocated vitamin D prophylaxis. There is no doubt about the value and adequacy of daily vitamin D prophylaxis for the prevention of vitamin D deficiency and rickets in infants. Our findings, however, renew the demand for the careful administration of prophylactic vitamin D to avoid vitamin D toxicity.
Supported by grants from the Canadian Institutes of Health Research and Cytochroma (to Dr. Jones).
Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.
Drs. Jones and Konrad contributed equally to this article.
This article (10.1056/NEJMoa1103864) was published on June 15, 2011, at NEJM.org.
We thank the patients and their parents for participating in this study, Alexandra Wassmuth and Susanne Kipp for their technical assistance, and Andrew Annalora for his helpful discussions.
From University Children's Hospital, Muenster (K.P.S., E.K.-B., M. Konrad); University Children's Hospital, Marburg (K.P.S., C.G.); University Children's Hospital, Essen (S.W., A.M.W.); University Children's Hospital, Jena (U.J., J.M.); Kuratorium für Heimdialyse Pediatric Kidney Center, Marburg (G.K.); and Children's Hospital, Memmingen (H.F.) — all in Germany; Queen's University, Kingston, ON, Canada (M. Kaufmann, A.I., D.E.P., G.J.); University Children's Hospital, Istanbul, Turkey (T.G.); and Radboud University, Nijmegen, the Netherlands (J.G.H., R.J.B.).
Address reprint requests to Dr. Konrad at the Department of General Pediatrics, University Children's Hospital, Waldeyerstr. 22, D-48149 Muenster, Germany, or at firstname.lastname@example.org.
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