Ratio of 25(OH)D-to-24,25(OH)2D: A new test to confirm 24-hydroxylase (CYP24A1) deficiency as the cause of hypercalcemia

June 12, 2018

Exogenous and endogenous vitamin D are rapidly converted to 25-hydroxyvitamin D (25[OH]D) in the liver by vitamin D 25-hydroxylase.

Peter J. Tebben, M.D., with Endocrinology, Diabetes, Metabolism, and Nutrition and Pediatric Endocrinology and Metabolism at Mayo Clinic's campus in Rochester, Minnesota, says: "Additional hydroxylation of 25(OH)D takes place in the kidney via 1-alpha hydroxylase (CYP27B1), producing the bioactive hormone 1,25-dihydroxyvitamin D (1,25[OH]₂D), which plays a critical role in calcium absorption through interaction with the vitamin D receptor (VDR). Production of 1,25(OH)₂D is tightly regulated through the concentrations of serum calcium, phosphorus and parathyroid hormone (PTH).

"The abundant precursor metabolite and storage form of vitamin D, 25(OH)D, can also be removed from circulation and converted to inactive 24,25-dihydroxyvitamin D (24,25[OH]₂D) — an enzymatic step catalyzed by 24-hydroxylase.

"The CYP24A1 gene, encoding the vitamin D 24-hydroxylase, is of major clinical and physiologic importance because it also serves to regulate the catabolism of 1,25(OH)₂D. Thus, excess 25(OH)D and 1,25(OH)₂D are catabolized by vitamin D 24-hydroxylase to the inert metabolites 24,25(OH)₂D and 1,24,25-trihydroxyvitamin D, respectively."

Rajiv Kumar, M.D., with Nephrology and Hypertension at Mayo Clinic's campus in Minnesota, explains: "CYP24A1 can be stimulated hormonally by 1,25(OH)₂D and by FGF23, whereas CYP27B1, encoding the vitamin D 1-alpha hydroxylase, is stimulated hormonally by PTH and downregulated by FGF23.

"Thus CYP24A1 and CYP27B1, together, provide for alternate and regulated fates of 25(OH)D and control the availability of the active metabolite, 1,25(OH)₂D, depending upon physiological needs. These two enzymes are therefore central to the homeostatic control of vitamin D metabolism, and as a result affect calcium metabolism in critical ways."

Robert A. Wermers, M.D., with Endocrinology, Diabetes, Metabolism, and Nutrition at Mayo Clinic's campus in Minnesota, notes: "Loss-of-function mutations in the CYP24A1 gene can lead to inactive or no vitamin D 24-hydroxylase enzyme protein, resulting in increasing concentration of vitamin D metabolites and a clinical phenotype characterized by suppressed serum PTH, increased serum 1,25(OH)₂D concentrations, hypercalcemia, and hypercalciuria or nephrolithiasis or both.

"In a New England Journal of Medicine report in 2011, biallelic loss-of-function mutations (autosomal recessive) in CYP24A1 were reported in a population of children presenting with idiopathic infantile hypercalcemia (IIH).

Dr. Tebben adds: "Subsequent publications in the Journal of Clinical Endocrinology and Metabolism in 2012 and Osteoporosis International in 2016 showed that deficiency in 24-hydroxylase caused by pathogenic CYP24A1 gene mutations abrogates 24,25(OH)₂D synthesis, leading to hypercalcemia and predisposing patients to hypercalciuria, nephrolithiasis and nephrocalcinosis."

Dr. Kumar notes: "As highlighted in Endocrine Reviews in 2016, although the prevalence of CYP24A1-associated hypercalcemia is rare — estimated to be in the order of 1 in 40,000 — the testing of 24,25(OH)₂D should be considered when other causes of hypercalcemia have been excluded. This is especially true for patients with non-PTH mediated hypercalcemia due to 1,25-dihydroxyvitamin D excess in association with a strong family history of nephrolithiasis or hypercalcemia or both.

"The clinical manifestations of hypercalcemia depend largely on the age at diagnosis. Infants present with weight loss or failure to thrive, vomiting, dehydration, lethargy, and hypotonia, whereas adults with mutations in CYP24A1 most frequently present with renal manifestations such as nephrolithiasis or nephrocalcinosis or both, and may experience polyuria. The degree of hypercalcemia and symptoms can vary from mild and intermittent to severe but in general are less pronounced compared with those who manifest disease during infancy. As with other causes of vitamin D-mediated hypercalcemia, adults may develop neuropsychiatric symptoms such as lethargy, confusion and irritability with severe hypercalcemia."

Ravinder J. Singh, Ph.D., with Laboratory Medicine and Pathology at Mayo Clinic's campus in Minnesota, explains: "Formation of 24,25(OH)₂D is dependent in part on the concentration of 25(OH)D. In healthy individuals, the circulating concentration of 24,25(OH)₂D is 10 to 25 percent of the total 25(OH)D. Patients who are homozygous for CYP24A1 mutations with no enzyme activity develop undetectable levels of 24,25(OH)₂D.

"Importantly, testing and reporting the 24,25(OH)₂D result alone can mislead the interpretation of the test. Hence, the ratio of 25(OH)D-to-24,25(OH)₂D is necessary to estimate the calculation of vitamin D 24-hydroxylase enzyme activity, and this ratio is anticipated to be available as an orderable test in midyear 2017 (Mayo Test ID 2425D, 63416).

"The sample requirement is similar to a 25(OH)D measurement (Mayo Test ID 25HDN, 83670) where serum can be collected in a red top tube and shipped to the lab in refrigerated packing. In patients with homozygous or compound heterozygous CYP24A1 mutations (no 24-hydroxylase activity), 24,25(OH)₂D levels should be close to undetectable despite the presence of adequate amounts of the substrate 25(OH)D.

"Nearly all patients described to date with biallelic CYP24A1 mutations or deletions have had a ratio of 25(OH)D-to-24,25(OH)₂D greater than 80. The ratio results should be interpreted in the context of other biochemical findings including serum calcium, PTH and 1,25-dihydroxyvitamin D concentrations. If the ratio of 25(OH)D-to-24,25(OH)₂D is less than 25, the result would be considered normal, but such findings can be observed in heterozygous carriers of CYP24A1 mutations who may have an attenuated clinical phenotype compared with individuals with biallelic mutations. A ratio of 25(OH)D-to-24,25-(OH)₂D between 25 and 80 may be seen in patients with low serum vitamin D concentrations or in individuals with heterozygous CYP24A1 mutations. Confirmation with molecular testing is recommended."