1,25-(OH)2D-24 Hydroxylase (CYP24A1) Deficiency as a Cause of Nephrolithiasis

Abstract

Background and objectives: Elevated serum vitamin D with hypercalciuria can result in nephrocalcinosis and nephrolithiasis. This study evaluated the cause of excess 1,25-dihydroxycholecalciferol (1α,25(OH)2D3) in the development of those disorders in two individuals.

Design, setting, participants, & measurements: Two patients with elevated vitamin D levels and nephrocalcinosis or nephrolithiasis were investigated at the National Institutes of Health (NIH) Clinical Center and the NIH Undiagnosed Diseases Program, by measuring calcium, phosphate, and vitamin D metabolites, and by performing CYP24A1 mutation analysis.

Results: Both patients exhibited hypercalciuria, hypercalcemia, low parathyroid hormone, elevated vitamin D (1α,25(OH)2D3), normal 25-OHD3, decreased 24,25(OH)2D, and undetectable activity of 1,25(OH)2D-24-hydroxylase (CYP24A1), the enzyme that inactivates 1α,25(OH)2D3. Both patients had bi-allelic mutations in CYP24A1 leading to loss of function of this enzyme. On the basis of dbSNP data, the frequency of predicted deleterious bi-allelic CYP24A1 variants in the general population is estimated to be as high as 4%-20%.

Conclusions: The results of this study show that 1,25(OH)2D-24-hydroxylase deficiency due to bi-allelic mutations in CYP24A1 causes elevated serum vitamin D, hypercalciuria, nephrocalcinosis, and renal stones.

Figure 1.

Vitamin D metabolism. The first step of activation, 25-hydroxylation by CYP2R1 and CYP27A1, occurs in the liver. The second step, 1α-hydroxylation by CYP27B1 to yield active vitamin D (i.e., 1α,25(OH)₂D₃), occurs in the kidney. Inactivation of vitamin D occurs via the C-23 and C-24 oxidation pathways, catalyzed by CYP24A1 in the kidney. Calcitroic acid has no biologic activity. Larger type emphasizes important metabolites.

Figure 2.

Effect of ketoconazole treatment in patient 2 on 1,25 vitamin D levels, hypercalcemia, hypercalciuria, and parathyroid hormone. Patient 2 was treated with ketoconazole and the dosage was escalated to 800 mg per day in divided doses. The effects monitored before the initiation of therapy, approximately 1 month after the initiation of therapy, and approximately 1 month after discontinuation. The following levels are displayed: (A) 1α,25(OH)₂D₃, (B) serum ionized calcium, (C) 24-hour urine calcium, and (D) serum parathyroid hormone.

Figure 3.

CYP24A1 activity in patient 1 and patient 2. CYP24A1 activity was measured in fibroblasts from patients 1 and 2 and compared with values for normal non-stone formers. (A) CYP24A1 activity was measured in fibroblasts from patients 1 and 2, and compared with that of normal non-stone formers (all fibroblasts are the same passage number 3). Fibroblasts from patient 1 (P1) and patient 2 (P2) produced no metabolites of 1α,25(OH)₂D₃. In contrast, normal fibroblasts produced metabolites that have the same retention time (18.4 minutes) of metabolites using recombinant CYP24A1 (as indicated by the arrow). Our previous study revealed that this peak contained 1-α,24R,25(OH)₂D₃ and 1-α,23S,25-(OH)₂D₃ in the ratio of 4:1 (19). Although we found no other metabolites in normal fibroblasts, further metabolites may be observed with increasing reaction time. The shoulder on the main substrate peak is the 6-s-cis form of 1- α,25(OH)₂D₃ generated by rotation around the 6,7 carbon bond. The interconversion between the 6-s-trans- and 6-s-cis-forms has a low energy barrier and therefore occurs rapidly in solution at room temperature. (B) CYP24A1 protein amount is reduced in patient 1 and patient 2. Western blot of whole fibroblast lysates probed with CYP24A1 antibody shows reduced protein amount in patients compared with normal. (C) β-actin serves as loading control quantification by densitometry.

Figure 4.

Family pedigree and molecular analysis. The pedigrees of the two families are illustrated. Affected members are shown in black squares, whereas unaffected members are denoted in white solid objects. Patient 1 (I-2.2) harbored a 3-bp deletion, p.E143del, inherited from his mother (I-1.2), and a p.L148P missense mutation that was inherited from his father (I-1.1). Patient 2 (II-2.2) also had the same p.E143del inherited from his father (II-1.1), and a p.L409S inherited from his mother (II-1.2).