LC-MS/MS for Identifying Patients with CYP24A1 Mutations

Area of Science:

• Biochemistry
• Endocrinology
• Genetics

Background:

• Loss-of-function mutations in CYP24A1 (cytochrome P450, family 24, subfamily A, polypeptide 1) lead to elevated 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D levels.
• This imbalance causes hypercalcemia, hypercalciuria, and nephrolithiasis, posing diagnostic challenges.

Purpose of the Study:

• To validate an LC-MS/MS assay for quantifying 24,25-dihydroxyvitamin D (D3 and D2).
• To establish a cutoff 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D ratio for identifying patients requiring CYP24A1 genetic testing.

Main Methods:

• Serum samples were processed using solid-phase extraction and derivatization.
• Quantification of 24,25-dihydroxyvitamin D was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
• The 25-hydroxyvitamin D to 24,25-dihydroxyvitamin D ratio was measured in healthy individuals and patients with suspected CYP24A1-mediated hypercalcemia.

Main Results:

• The LC-MS/MS assay demonstrated reliable quantification of 24,25-dihydroxyvitamin D with low imprecision.
• Healthy individuals exhibited a 25(OH)D/24,25(OH)2D ratio of 7-35.
• Patients with CYP24A1 mutations showed significantly elevated ratios (99-467), with a ratio ≥99 identifying candidates for genetic testing.

Conclusions:

• An elevated 25(OH)D/24,25(OH)2D ratio supports the diagnosis of reduced CYP24A1 activity.
• Measuring this ratio should be part of the clinical workup for hypercalcemia of unknown origin.
• This biomarker aids in identifying patients for confirmatory CYP24A1 genetic analysis.