Point mutations in the Human Vitamin D Receptor Gene Associated with Hypocalcemic Rickets

TLDR

Hypocalcemic vitamin D‑resistant rickets is a human genetic disease caused by target‑organ resistance to 1,25‑dihydroxyvitamin D₃. The study examined two affected families, sequenced all nine VDR exons, identified point mutations in the DNA‑binding domain, and recreated these mutations in vitro by site‑directed mutagenesis of wild‑type VDR cDNA followed by transfection into COS‑1 cells. Patients’ VDRs bind 1,25‑dihydroxyvitamin D₃ normally but have reduced DNA affinity due to distinct point mutations in the DNA‑binding domain, and the mutant proteins produced in vitro are biochemically identical to the patient receptors.

Abstract

Hypocalcemic vitamin D-resistant rickets is a human genetic disease resulting from target organ resistance to the action of 1,25-dihydroxyvitamin D3. Two families with affected children homozygous for this autosomal recessive disorder were studied for abnormalities in the intracellular vitamin D receptor (VDR) and its gene. Although the receptor displays normal binding of 1,25-dihydroxyvitamin D3 hormone, VDR from affected family members has a decreased affinity for DNA. Genomic DNA isolated from these families was subjected to oligonucleotide-primed DNA amplification, and each of the nine exons encoding the receptor protein was sequenced for a genetic mutation. In each family, a different single nucleotide mutation was found in the DNA binding domain of the protein; one family near the tip of the first zinc finger (Gly→Asp) and one at the tip of the second zinc finger (Arg→Gly). The mutant residues were created in vitro by oligonucleotide directed point mutagenesis of wild-type VDR complementary DNA and this cDNA was transfected into COS-1 cells. The produced protein is biochemically indistinguishable from the receptor isolated from patients.