Download Mutations in CYP24A1 and Idiopathic Infantile Hypercalcemia

Schlingmann, et al.
Jun Ki Kim
 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
 Vitamin D activation- 25hydroxylation in the liver=
25-hydroxyvitamin D3 (25OH-D3)
 1α- hydroxylation in the
kidney= 1,25dihydroxyvitamin 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 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).
 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 constructsFull-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)
 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 25hydroxyvitamin 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.
 Loss-of-function mutations found
in both cohorts. In vitro data
showed decreased levels of 24hydroxylated 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.