Schlingmann, et al.Jun Ki Kim

Mutations in CYP24A1 and Idiopathic Infantile

Hypercalcemia

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

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

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

Patients- Candidate-gene approach of two cohorts of subjects1) 6 patients from 4 families with IIH with suspected autosomal recessive inheritance2) 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

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

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