Take another CYP: confirming a novel mechanism for "idiopathic" hypercalcemia.

Three recent studies have described a novel mechanism of hypercalcemia related to the effects of vitamin D on calcium homeostasis. These studies collectively confirm that loss of function of the vitamin D 24-hydroxylase enzyme may result in significant clinical hypercalcemia. The initial studies of this disorder, by Schlingmann et al. (1) and Dauber et al. (2), identify homozygous loss-of-function mutations in CYP24A1, the gene encoding the 24-hydroxylase, in the setting of otherwise unexplained infantile hypercalcemia. The report by Schlingmann et al. (1) identified several loss-of-function mutations in eight hypercalcemic infants who were investigated at 6–8 months of age. Their data are most consistent with recessive transmission of the syndrome. Transfection experiments document impaired in vitro 24-hydroxylase activity using constructs of six mutations identified clinically; clinical evidence of impaired enzyme activity was not reported. The Dauber et al. (2) study identified a hypercalcemic infant with a homozygous loss-of-function mutation identical to one of the mutations observed in a compound heterozygote presented by the Schlingmann group (1) and provided strong in vivo evidence relating this mutation to disease. The 10-month-old proband had low circulating 24,25-dihydroxyvitamin D [24,25(OH)2D] levels compared with age-matched normal children and markedly elevated fractional intestinal absorption of calcium (∼90% vs. ∼45% in age-matched controls). The absence of a coding region mutation in 27 other hypercalcemic infants examined by the Dauber team would suggest that loss of function of CYP24A1 is probably an uncommon cause of this syndrome. In the current issue of the JCEM, Tebben et al. (3) extend these observations, reporting a similar syndrome in adults with presumed loss-of-function mutations in the same enzyme. Several interesting features in the current report differ from the pediatric studies. First, the severity of the hypercalcemia in the infants decreased or corrected with age. Secondly, the Tebben et al. (3) study reports an overt clinical phenotype in the heterozygous state. Of interest, the possibility of a partial or mild phenotype in heterozygotes is raised by the maternal history of nephrolithiasis in the Dauber et al. (2) report, and the identification of a (deletion) abnormality of only one allele in a proband is reported by Schlingmann et al. (1). Finally, the current report describes a clinical response to therapy with the cytochrome inhibitor, ketoconazole, an agent also used in a report of 20 infants with idiopathic hypercalcemia by Nguyen et al. (4). Decreased enzymatic function of the 24-hydroxylase was identified in skin fibroblasts from one of the patients in the Nguyen et al. (4) study. Interest in the 24-hydroxylase enzyme previously focused on its provision of an alternative pathway for the precursor vitamin D metabolite, 25-hydroxyvitamin D (25-OHD). The 24-hydroxylase provides an energy-favorable conversion of 25-OHD to 24,25(OH)2D and, when up-regulated, allows a relative enhancement of the pathway to 24,25(OH)2D rather than to 1,25-dihydroxyvitamin D [1,25(OH)2D]. Many have considered that this alternative to 1-hydroxylation is primarily of importance in providing the initial step by which abundant stores of 25-OHD can be metabolized to an inactive product, thus leading to eventual catabolism and accelerated clearance of the major circulating vitamin D metabolite. Numerous conditions up-regulate 1-hydroxylase in concert with down-regulation of the 24-hydroxylase, and vice versa (see Ref. 5 for review). Thus, many (but not all) physiological conditions calling for vitamin D activation would limit 24,25(OH)2D production, and under conditions where vitamin D functions were well met, synthesis of 24,25(OH)2D would be increased to limit further 1,25(OH)2D production. The 1,25(OH)2D metabolite is the most systemically active form of vitamin D, and the physiological activity of 24,25(OH)2D is controversial. A body of evidence suggests that 24,25(OH)2D has distinct physiological actions itself, including inhibition of PTH secretion (6, 7), enhancement of fracture healing (8), and various functions of growth plate cartilage (9). 1,25(OH)2D serves as a substrate for the 24-hydroxylase as well, thereby generating 1,24,25-trihydroxyvitamin D. Thus, the enzyme is a 25-OHD/1,25(OH)2D 24-hydroxylase, and it serves as a key enzyme in catabolism of the most active vitamin D metabolite. Indeed, an argument has been made that this is the primary function of the enzyme because the preferred substrate for the 24-hydroxylase is 1,25(OH)2D (and not 25-OHD) (10). An informative tool in the study of the in vivo physiology of this enzyme has been the Cyp24a1 knockout (KO) mouse (11). Early survival of KO mice is limited and appears to be favorable in pups where down-regulation of 1,25(OH)2D production occurs. The KO animals have normal 1,25(OH)2D levels at baseline, but clearance of administered 1,25(OH)2D is impaired, and levels increase excessively with addition of a vitamin D-supplemented diet. A reduction in cortical bone occurs in the KO mouse, and this finding is a consequence of 1,25(OH)2D-induced bone resorption. Thus initial studies appear to relate the observed skeletal pathology to limited clearance of 1,25(OH)2D as opposed to the inability to generate 24,25(OH)2D. Likewise, the mutant phenotype does not include abnormal growth plate cartilage, suggesting that in vitro effects on the growth plate may be compensated for by redundant mechanisms in vivo. The KO mouse has been shown to have impaired fracture healing, suggesting that the increased CYP24A1 expression and increased 24,25(OH)2D production in forming callus (see Refs. 8, 12, and 13 for review) may have important physiological consequences. The current case report (3) also highlights the use of ketoconazole in blocking hormone synthesis dependent upon enzyme complexes with a cytochrome component. Ketoconazole is a cytochrome inhibitor primarily used as an antifungal agent, but it has been used widely to treat other causes of hypercalcemia due to excess 1,25(OH)2D production. Paradoxically, it has been used to inhibit CYP24 itself, specifically for the purpose of allowing 1,25(OH)2D to accumulate by decreasing its degradation. This strategy has been employed to optimize antitumor effects of 1,25(OH)2D in peripheral tissues (14). With no functional 24-hydroxylase (as in the cases at hand), the relevant clinical effect is presumably on the 1α-hydroxylase and its inhibition of 1,25(OH)2D synthesis. Nevertheless, ketoconazole-induced inhibition of CYP in general also affects testosterone and cortisol metabolism, so that potential complications of hypogonadism and adrenal insufficiency must be watched for, along with monitoring hepatic function because of well-known toxicity to the liver. It does appear to be generally safe in infancy (4). Of particular interest are the many important issues that the current study (3) raises with respect to vitamin D metabolism and the relationships of the reported mutations to disease: