A new look at vitamin D metabolism and "idiopathic" hypercalcemia.

The differential diagnosis of endogenous hypercalcemia typically involves an assessment of the hormones and paracrine factors, such as PTH, PTH-related protein, and 1,25-dihydroxyvitamin D, whose oversecretion can pathologically activate bone resorption and/or intestinal absorption of calcium to an extent that overrides the usual homeostatic mechanisms. With our focus thus on investigating hormone oversecretion, we endocrinologists typically give little thought to disorders of hormone catabolism that might also result in hypercalcemia. It is in this context that an elegant clinical study in this issue by Dauber et al. (1) is both thought-provoking and illuminating. Dauber et al. (1) define a genetic defect and characterize a critical pathophysiology in a patient with severe infantile hypercalcemia. The patient, a product of a consanguineous marriage in which the parents were fifth-degree relatives, presented at 10 months of age with failure to thrive, increased urination, and refusal of solid foods. At presentation, the patient was markedly hypercalcemic, with a serum calcium of 15.3 mg/dl (3.8 mmol/liter), and inpatient evaluation revealed suppressed PTH, undetectable PTH-related protein, normal levels of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, and normal thyroid and renal function. The infant was found to have severe hypercalciuria and bilateral nephrocalcinosis but no evidence of rickets or bone demineralization on imaging studies. Furthermore, no chromosome 7 deletion was found on genetic analysis to support a diagnosis of Williams syndrome, a rare cause of infantile hypercalcemia. With suppression of PTH, the investigators suspected an absorptive pathophysiology for the hypercalcemia, and indeed metabolic studies employing oral and parenteral administration of stable calcium isotopes along with a defined calcium diet demonstrated a marked intestinal hyperabsorption of calcium in this infant. Dauber et al. (1) then pursued an unbiased genome-wide investigation for the cause of the patient's hypercalcemia which, given the history of parental consanguinity, was suspected to be an autosomal recessive inherited genetic disorder. Recent advances in exome sequencing, in which the subset of the human genome that carries the protein-coding information is targeted for sequencing, make it an efficient and cost-effective way to elucidate the genetic basis of metabolic and other Mendelian disorders (2, 3). Whole exome sequencing of the patient's DNA revealed 1277 single nucleotide variants and 367 indels (alterations in DNA sequence arising from insertion or deletion of nucleotides) not included in the latest compendium of human nucleotide polymorphisms (1). Of these novel variants, seven single nucleotide changes encoding missense alterations at the protein level and seven indels mapped to one of three large blocks of genetic homozygosity on chromosomes 7, 16, and 20. Within the patient's DNA sequence on chromosome 20, a homozygous 3-bp in-frame deletion in the CYP24A1 gene was discovered. Both parents were proven to be heterozygous mutation carriers. This deletion in CYP24A1 is predicted to excise an evolutionarily highly conserved glutamate residue at position 143 in the vitamin D 24-hydroxylase enzyme (E143del). This finding captured the attention of Dauber et al. (1) because biallelic germline inactivation of the orthologous mouse gene, Cyp24a1, produces a phenotype of hypercalcemia and nephrocalcinosis reminiscent of the findings in the human infant (4–6). That vitamin D 24-hydroxylase is critically responsible for oxidation and degradation of bioactive vitamin D is supported by studies of such Cyp24a1 null mice demonstrating markedly impaired catabolism and clearance of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D (5, 6). The present study by Dauber et al. (1) complements and extends other recent clinical reports linking germline CYP24A1 loss of function mutation with clinically relevant hypercalcemia (7, 8). Using a candidate gene approach, Schlingmann et al. (7) demonstrated germline biallelic inactivation of CYP24A1 in four infant probands with idiopathic infantile hypercalcemia, medullary nephrocalcinosis, and suspected autosomal recessive inheritance. One of the probands described by Schlingmann et al. (7) was the product of a consanguineous marriage and was homozygous for a frameshift mutation in CYP24A1, whereas the remaining three were compound heterozygotes for mutations in CYP24A1, including two who carried the E143del mutant allele (7). The case of a patient who presented in young adulthood with nephrolithiasis and mild hypercalcemia, subsequently became asymptomatic, then re-presented 20 yr later with unexplained non-parathyroid-mediated hypercalcemia was described by Streeten et al. (8). The patient was found at age 47 to be homozygous for the CYP24A1 E143del mutant allele (8). In the present case described by Dauber et al. (1), evidence for functional deficiency of vitamin D 24-hydroxylase activity was strongly supported by documentation of undetectable serum levels of 24,25-dihydroxyvitamin D, a major vitamin D metabolite whose levels in control subjects were proportionate to vitamin D stores as reflected in 25-hydroxyvitamin D levels (1). The adult CYP24A1 E143del homozygote described by Streeten et al. (8) also had low serum 24,25-dihydroxyvitamin D levels. The study by Schlingmann et al. (7) did not report serum 24,25-dihydroxyvitamin D levels in their patient cohort, but did analyze the function of CYP24A1 by transfection and documented a striking lack of 24-hydroxylated vitamin D metabolites when patient-derived CYP24A1 mutants were tested in vitro with 1,25-dihydroxyvitamin D as substrate. The determination of the crystal structure of rat Cyp24a1 has provided a structural template to allow molecular modeling studies that probe the effect of patient-derived mutations on the function of human CYP24A1 by computer simulation (9). On the basis of such molecular-modeling simulation, it was found that of four CYP24A1 missense mutations reported by Schlingmann et al. (7), only one was predicted to weaken substrate-enzyme binding, whereas three appeared to disrupt interactions between the CYP24A1 enzyme and its heme prosthetic moiety (10). Despite the convincing correlation of homozygous CYP24A1 mutation, hypercalcemia, and intestinal hyperabsorption of calcium in the present case, a number of questions remain when considering the findings of Dauber et al. (1) in the context of other recent reports linking CYP24A1 loss of function to hypercalcemia. A major question is the prevalence of CYP24A1 mutation among patients with apparently idiopathic infantile hypercalcemia. Although Dauber et al. (1) found no germline CYP24A1 missense mutation or deletions among 27 patients with infantile hypercalcemia referred to a pediatric calcium disorder specialty clinic, Schlingmann et al. (7) identified biallelic germline inactivating mutations in four of four kindreds with idiopathic infantile hypercalcemia. Whether this difference reflects occult clinical heterogeneity or ascertainment bias in the population of patients with apparently idiopathic infantile hypercalcemia under study, or whether some CYP24A1 mutation-negative patients nevertheless harbor defects in other molecular components that diminish vitamin D catabolism or enhance vitamin D activation (for example, activating mutations in the CYP27B1 25-hydroxyvitamin D 1-α-hydroxylase) remains to be determined. Another interesting question posed by these cases concerns the inconsistency of serum 1,25-dihydroxyvitamin D elevation in patients with biallelic CYP24A1 inactivation despite documented hypercalcemia. For example, the patient described by Dauber et al. (1) often had normal 1,25-dihydroxyvitamin D levels when tested over time, even while hypercalcemic. Three of the four index cases presented by Schlingmann et al. (7) also had normal circulating 1,25-dihydroxyvitamin D levels despite marked hypercalcemia. The patient described by Streeten et al. (8), in contrast, had clearly elevated 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D levels accompanying his hypercalcemia. A possibly related issue is the variability of hypercalcemia over time in patients with biallelic CYP24A1 inactivation, such as in the present case in which the patient was able to resume a normal calcium diet without recurrence of hypercalcemia after 3 yr of age (1). The observation that Cyp24a1 null mice exhibit an apparently compensatory down-regulation of renal Cyp27b1, the 25-hydroxyvitamin D 1-α-hydroxylase, and have lower circulating 1,25-dihydroxyvitamin D levels than control mice may be relevant to humans in this regard (5, 6). The possibility that serum levels of 1,25-dihydroxyvitamin D may not always reflect hormone activity in target tissues, such as intestine, bone, or parathyroid cells, for example, because of local variability in vitamin D homeostasis must also be considered (1, 8). The present work by Dauber et al. (1) and the related clinical studies cited here, as well as findings from the Cyp24a1-null mouse model (5, 6), underscore the importance of CYP24A1 and vitamin D catabolism in calcium homeostasis as well as the potential pathophysiological importance of this pathway in hypercalcemia. It remains to be seen whether nonsynonymous “benign polymorphisms” in the coding region or pathological variants in the intronic or promoter regions of CYP24A1 might also affect the catabolism of vitamin D and have the potential to induce hypercalcemia, either at baseline or upon bolus loading with vitamin D2 or D3. In the investigation of hypercalcemia, particularly in “idiopathic” cases with suppressed PTH, we endocrinologists clearly have a new mechanism to ponder and should consider adding a screening 24,25-dihydroxyvitamin D determination to our workup as clinically warranted.