TY - JOUR
T1 - Improved Screening Test for Idiopathic Infantile Hypercalcemia Confirms Residual Levels of Serum 24,25-(OH)2D3 in Affected Patients
AU - Kaufmann, Martin
AU - Morse, Nicole
AU - Molloy, Billy Joe
AU - Cooper, Donald P.
AU - Schlingmann, Karl Peter
AU - Molin, Arnaud
AU - Kottler, Marie Laure
AU - Gallagher, J. Christopher
AU - Armas, Laura
AU - Jones, Glenville
N1 - Funding Information:
This work was supported by grants from the National Institute of Standards and Technology (NIST) and NIH-ODS as part of the Vitamin D Standardization Program (VDSP) (Grant 60NANB13D203) and the European Rare Diseases Consortium (E-Rare-2)/Canadian Institutes for Health Research, Grant #ERA-132931) to G Jones. Through a Queen's University/Waters Corporation agreement, Waters Corporation generously provided the LC-MS/MS instrumentation used in these studies. We gratefully acknowledge the provision of individual IIH serum samples from Celia Rodd (University of Manitoba, Winnipeg, MB, Canada), Tom Jacobs (Columbia University, New York, NY, USA), David Saxon (University of Colorado, Denver, CO, USA). We also thank J. Wesley Pike (University of Wisconsin?Madison, Madison, WI, USA) and Ren? St-Arnaud (McGill University and Shriner's Hospital, Montr?al, QC, Canada) for the generous gift of serum from Vdr-null and Cyp24a1-null mice. Authors? roles: Study design: MK, NM, BJM, DPC, and GJ. Study conduct: MK, NM, BJM, DPC, KPS, AM, MLK, JCG, LA, and GJ. Provision of patient samples and or genetic analysis: KPS, AM, MLK, JCG, and LA. Data collection: MK and NM. Data analysis: MK, NM, and GJ. Data interpretation: MK, NM, BJM, DPC, and GJ. Drafting and revising the manuscript: MK and GJ.
PY - 2017/7
Y1 - 2017/7
N2 - CYP24A1 mutations are now accepted as a cause of idiopathic infantile hypercalcemia (IIH). A rapid liquid-chromatography tandem mass spectrometry (LC-MS/MS)-based blood test enabling measurement of the 25-OH-D3:24,25-(OH)2D3 ratio (R) can identify IIH patients on the basis of reduced C24-hydroxylation of 25-OH-D3 by CYP24A1 in vivo. Although values of this ratio are significantly elevated in IIH, somewhat surprisingly, serum 24,25-(OH)2D3 remains detectable. The current study explores possible explanations for this including: residual CYP24A1 enzyme activity in individuals with certain CYP24A1 genotypes, expression of alternative C24-hydroxylases, and the possibility of isobaric contamination of the 24,25-(OH)2D3 peak on LC-MS/MS. We employed an extended 20-min run time on LC-MS/MS to study serum vitamin D metabolites in patients with IIH due to mutations of CYP24A1 or SLC34A1; in unaffected heterozygotes and dialysis patients; in patients with vitamin D deficiency; as well as in normal subjects exhibiting a broad range of 25-OH-D levels. We identified 25,26-(OH)2D3 as a contaminant of the 24,25-(OH)2D3 peak. In normals, the concentration of 24,25-(OH)2D3 greatly exceeds 25,26-(OH)2D3; however, 25,26-(OH)2D3 becomes more significant in IIH with CYP24A1 mutations and in dialysis patients, where 24,25-(OH)2D3 levels are low when CYP24A1 function is compromised. Mean R in 30 IIH-CYP24A1 patients was 700 (range, 166 to 2168; cutoff = 140) as compared with 31 in 163 controls. Furthermore, patients possessing CYP24A1 L409S alleles exhibited higher 24,25-(OH)2D3 levels and lower R (mean R = 268; n = 8) than patients with other mutations. We conclude that a chromatographic approach which resolves 24,25-(OH)2D3 from 25,26-(OH)2D3 produces a more accurate R that can be used to differentiate pathological states where CYP24A1 activity is altered. The origin of the residual serum 24,25-(OH)2D3 in IIH patients appears to be multifactorial.
AB - CYP24A1 mutations are now accepted as a cause of idiopathic infantile hypercalcemia (IIH). A rapid liquid-chromatography tandem mass spectrometry (LC-MS/MS)-based blood test enabling measurement of the 25-OH-D3:24,25-(OH)2D3 ratio (R) can identify IIH patients on the basis of reduced C24-hydroxylation of 25-OH-D3 by CYP24A1 in vivo. Although values of this ratio are significantly elevated in IIH, somewhat surprisingly, serum 24,25-(OH)2D3 remains detectable. The current study explores possible explanations for this including: residual CYP24A1 enzyme activity in individuals with certain CYP24A1 genotypes, expression of alternative C24-hydroxylases, and the possibility of isobaric contamination of the 24,25-(OH)2D3 peak on LC-MS/MS. We employed an extended 20-min run time on LC-MS/MS to study serum vitamin D metabolites in patients with IIH due to mutations of CYP24A1 or SLC34A1; in unaffected heterozygotes and dialysis patients; in patients with vitamin D deficiency; as well as in normal subjects exhibiting a broad range of 25-OH-D levels. We identified 25,26-(OH)2D3 as a contaminant of the 24,25-(OH)2D3 peak. In normals, the concentration of 24,25-(OH)2D3 greatly exceeds 25,26-(OH)2D3; however, 25,26-(OH)2D3 becomes more significant in IIH with CYP24A1 mutations and in dialysis patients, where 24,25-(OH)2D3 levels are low when CYP24A1 function is compromised. Mean R in 30 IIH-CYP24A1 patients was 700 (range, 166 to 2168; cutoff = 140) as compared with 31 in 163 controls. Furthermore, patients possessing CYP24A1 L409S alleles exhibited higher 24,25-(OH)2D3 levels and lower R (mean R = 268; n = 8) than patients with other mutations. We conclude that a chromatographic approach which resolves 24,25-(OH)2D3 from 25,26-(OH)2D3 produces a more accurate R that can be used to differentiate pathological states where CYP24A1 activity is altered. The origin of the residual serum 24,25-(OH)2D3 in IIH patients appears to be multifactorial.
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U2 - 10.1002/jbmr.3135
DO - 10.1002/jbmr.3135
M3 - Article
C2 - 28304097
AN - SCOPUS:85018631394
VL - 32
SP - 1589
EP - 1596
JO - Journal of Bone and Mineral Research
JF - Journal of Bone and Mineral Research
SN - 0884-0431
IS - 7
ER -