title: LC-MS/MS for Identifying Patients with CYP24A1 Mutations.
abstract: BACKGROUND
  Patients have been described with loss-of-function CYP24A1 (cytochrome P450, family 24, subfamily A, polypeptide 1) mutations that cause a high ratio of 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D [25(OH)D/24,25(OH)2D], increased serum 1,25-dihydroxyvitamin D, and resulting hypercalcemia, hypercalciuria and nephrolithiasis. A 25(OH)D/24,25(OH)2D ratio that can identify patients who are candidates for confirmatory CYP24A1 genetic testing would be valuable. We validated an LC-MS/MS assay for 24,25(OH)2D (D3 and D2) and determined a 25(OH)D/24,25(OH)2D cutoff to identify candidates for confirmatory genetic testing.
  
  
  METHODS
  After addition of isotope-labeled internal standard, serum samples were extracted by solid-phase extraction, derivatized with 4-phenyl-1,2,4,-triazoline-3,5-dione, and quantified by LC-MS/MS. We measured 25(OH)D/24,25(OH)2D in 91 healthy patients and 34 patients with clinically suspected CYP24A1-mediated hypercalcemia.
  
  
  RESULTS
  The limits of detection and quantification were 0.03 (0.2) and 0.1 (0.24) nmol/L, respectively, for 24,25(OH)2D3, and 0.1 (0.23) and 0.5 (1.16) nmol/L for 24,25(OH)2D2. Intra- and interassay imprecision was 4%-15% across the analytical measurement range of 0.1-25 ng/mL (0.2-60 nmol/L). No interference was observed with 25(OH)D and 1,25(OH)2D. 25(OH)D/24,25(OH)2D of 7-35 was observed in healthy patients, whereas in 2 patients with CYP24A1 mutations, 25(OH)D/24,25(OH)2D was significantly increased (99-467; P < 0.001). A 25(OH)D/24,25(OH)2D ratio ≥99 identified patients who were candidates for CYP24A1 genetic testing.
  
  
  CONCLUSIONS
  Increased 25(OH)D/24,25(OH)2D supports the diagnosis of reduced CYP24A1 activity due to mutations in CYP24A1. Measurement of 25(OH)D/24,25(OH)2D should be considered a part of the clinical workup in patients with hypercalcemia of otherwise unknown etiology.
authors.affiliation: Department of Pathology, University of Michigan Health System, Ann Arbor, MI;
authors.forename: Hemamalini
authors.initials: H
authors.lastname: Ketha
authors.affiliation: Division of Nephrology and Hypertension, Department of Internal Medicine, and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN.
authors.forename: Rajiv
authors.initials: R
authors.lastname: Kumar
authors.affiliation: Department of Pathology, University of Michigan Health System, Ann Arbor, MI; singh.ravinder@mayo.edu.
authors.forename: Ravinder J
authors.initials: RJ
authors.lastname: Singh
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004252:DNA Mutational Analysis; D005260:Female; D006801:Humans; D006934:Hypercalcemia / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D053719:Tandem Mass Spectrometry; D065668:Vitamin D3 24-Hydroxylase / Q000097:blood / Q000235:genetics*
chemical_list: C585549:CYP24A1 protein, human; D065668:Vitamin D3 24-Hydroxylase