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Mutations in CYP24A1 and Idiopathic Infantile Hypercalcemia

Mutations in CYP24A1 and Idiopathic Infantile Hypercalcemia

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Mutations in CYP24A1 and Idiopathic Infantile Hypercalcemia

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  1. Mutations in CYP24A1 and Idiopathic Infantile Hypercalcemia Schlingmann, et al. Jun Ki Kim

  2. Vitamin D supplementation to prevent rickets- led to idiopathic infantile hypercalcemia (in Britain during the 1950s) • Hypercalcemia- too much calcium in blood • Symptoms- severe hypercalcemia, failure to thrive, vomiting, dehydration, and nephrocalcinosis Idiopathic Infantile Hypercalcemia

  3. Vitamin D activation- 25-hydroxylation in the liver= 25-hydroxyvitamin D3 (25-OH-D3) • 1α- hydroxylation in the kidney= 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) • 1,25-(OH)2D3= exerts biologic effects by binding to the vitamin D receptor • CYP24A1 catabolizes 1,25-(OH)2D3 (end product= calcitroic acid) • Selected candidate genes= CYP27B1, CYP24A1, FGF23, and KL Vitamin D Metabolism

  4. Vitamin D not the only contributing factor since most infants receiving prophylaxis remained unaffected. • Intrinsic hypersensitivity to vitamin D- infantile hypercalcemia • Mutations in CYP24A1 contributing/ providing a molecular basis for idiopathic infantile hypercalcemia (inherited as an autosomal recessive trait). Hypothesis- Mutations in CYP24A1

  5. Patients- Candidate-gene approach of two cohorts of subjects 1) 6 patients from 4 families with IIH with suspected autosomal recessive inheritance 2) 4 patients with suspected vitamin D intoxication; developed hypercalcemia after prophylaxis with vitamin D. • Lab Analyses and sequencing- Serum calcium, serum iPTH, and Urinary calcium excretion levels measured; DNA from patients extracted and entire coding regions and splice sites of the 4 candidate genes sequenced from both strands. • Preparation of Plasmid constructs- Full-length human CYP24A1 subcloned into a pcDNA5/FRT. Mutagenesis conducted (E143del, R159Q, E322K, R396W, L409S, and A475fsX490) • Transfection- Human wild-type and mutant CYP24A1 constructs transfected into V79-4 cells (hamster) Experiment

  6. Clinical findings- 1st cohort of 4 patients from 4 families with IIH. All received vitamin D supplementation. After vitamin D prophylaxis was stopped, serum calcium levels normalized at first but continuously elevated during the follow-up. 2nd cohort of 4 children were suspected of developing hypercalcemia after receiving vitamin D supplementation. They displayed elevated levels of 25-hydroxyvitamin D3 also. • Mutational Analysis- conventional sequencing of the coding regions of CYP27B1, FGF23, and KL did not reveal any mutations; nonsense and missense mutations found in CYP24A1 in all patients. One premature stop mutation and two frameshift mutations also detected. R396W mutation detected in control alleles. • In Vitro analysis of CYP24A1 Activity- Human CYP24A1 constructs (containing mutations) transfected into V79-4 host cells, compared with wild-type and nontransfected control cells on catabolism of 1,25-dihydroxyvitamin D3 to determine the consequence of CYP24A1 mutations. 1,25-dihydroxyvitamin D3 completely broken down by wild-type CYP24A1 to calcitroic acid (radioactivity detectors and photodiode-array detectors used). On the other hand, the CYP24A1 mutations resulted in complete loss of CYP24A1 enzymatic activity. Results

  7. Loss-of-function mutations found in both cohorts. In vitro data showed decreased levels of 24-hydroxylated metabolites (Vitamin D inactivation initiated by CYP24A1) in cells with CYP24A1 mutations- suggest loss of enzyme activity caused by mutations. • CYP24A1 mutations found in healthy individuals who developed vitamin D intoxication after prophylaxis. • Careful administration of prophylactic vitamin D needed to avoid vitamin D toxicity. Conclusion