14 From Phenotype to Genotype: Using the 25-hydroxyvitamin D/24, 25-Dihydroxyvitamin D Ratio As a Surrogate for CYP24A1 Mutation Status During Hypercalcemia Workup. (11th January 2018)

• Record Type: Journal Article
• Authors: Sepiashvili, Lusia; Milosevic, Dragana; Fahse, Matthew; Singh, Ravinder
• Is Part Of: American journal of clinical pathology. Volume 149(2018)Supplement 1
• Page Start: S170
• Page End: S171
• Publication Date: 2018-01-11
• Subjects: Diagnosis, Laboratory -- Periodicals; Pathology -- Periodicals; 616.07
• DOI: 10.1093/ajcp/aqx149.383
• Languages: English
• ISSNs: 0002-9173

Abstract

Abstract: Introduction: Vitamin D plays a key role in calcium homeostasis. The active vitamin D metabolite, 1, 25-dihydroxyvitamin D (1, 25OH2D), and the inactive metabolite, 24, 25-hydroxyvitamin D (24, 25OH2D), are respectively synthesized from 25-hydroxyvitamin D (25OHD) by cytochrome P450 enzymes, 1-hydroxylase and 24-hydroxylase. Deficiency in 24-hydroxylase caused by pathogenic CYP24A1 gene mutations abrogates 24, 25OH2D synthesis, leading to hypercalcemia and predisposing patients to hypercalciuria, nephrolithiasis, and nephrocalcinosis. An inappropriately high (>80) 25OHD/24, 25OH2D ratio (r25/24) determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) has been suggested to serve as a rapid biochemical screen for 24-hydroxylase deficiency, which can precede availability of genetic testing results by weeks (Molin et al, 2015, JCEM ). Conversely, normal 24-hydroxylase activity has been described in conjunction with r25/24<80. However, the evidence of correlation between r25/24 and CYP24A1 genotype has been largely limited to familial studies or case reports. Objectives: The objective of this study was to explore the relationship between the r25/24 determined in a clinical laboratory setting and CYP24A1 genotype to better define the ratio that is a reliable surrogate for 24-hydroxylase deficiency. Methods: Total 25OHD and total 24, 25OH2D concentrations were measured in patient sera (n = 97) by LC-MS/MS using previously reported methods (Netzel et al, 2011, Clinical Chemistry ; Ketha et al, 2016, Clinical Chemistry ). DNA was purified from residual sera using QIAamp circulating nucleic acid extraction kit (Qiagen). The mutation status of 11 exons and 3'-UTR region of the CYP24A1 gene was evaluated with Sanger sequencing using BigDye Terminator (ABI BigDyeTerminator TM v1.1). Sequencing traces were analyzed manually with Mutation Surveyor software. A comprehensive literature search was conducted for reported CYP24A1 genotypes and associated 25OHD/24, 25OH2D ratios. Results: Of the 97 patient sera, 22 (23%) had an r25/24>80. Among the 23 sera with CYP24A1 genotyping, 18 (78%) had r25/24 >80, and five (22%) had r25/24 <80. Eight unique pathogenic variations were found across six of 11 exons, with mutations on exons 2 and 9 being most common, in line with previous reports. Cases with r25/24 >80 (range 103–700) were found to harbor two pathogenic mutations (n = 10), homozygous mutations (n = 1), heterozygous mutations (n = 4), and no pathogenic mutations (n = 3). The average 24, 25OHD2D concentrations and r25/24 in patients with at least two pathogenic variants were 0.2 ng/mL (IQR 0.1–0.3) and 287 (IQR 159–357). All other patients had significantly higher 24, 25OH2D concentrations (2.3 ng/mL [IQR 1.2–3.8]), and significantly lower r25/24 (14 [IQR 10–21]) ( P values < .001, Mann Whitney U test), respectively. Specimens with r25/24 <80 were found to harbor heterozygous pathogenic mutations (n = 3), or no pathogenic variants (n = 2). Conclusions: The relationship between r25/24 and CYP24A1 genotype is complex and multifactorial. Two pathogenic mutations were found in 61% of patients with elevated r25/24 (>103), supporting 24-hydroxylase deficiency diagnosis.