Molecular and gentic analysis of receptor for 1, 25-dihydroxyvitamin D in patients with vitamin D-dependent rickets type II

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Vitamin D-dependent rickets type II is a hereditary disease resulting from a defective vitamin D receptor. We suggested that this disease was an autosomal recessive disorder by showing intermediate levels of 25-hydroxyvitamin D-24-hydroxylase induction in the parents. Therefore, it would be important to identify genetic defects causing the disease and to detect heterozygous carriers at the molecular level. In six patients with vitamin D-d. ependent rickets type II whose fibroblasts displayed normal cytosol binding and impaired nuclear uptake of 1, 25-dihydroxyvitamin D_3, western, Southern, and northern analyses failed to disclose any abnormalities in vitamin D_3 receptor protein and its gene. Exons 2 and 3 of the vitamin D receptor CDNA, which er), code the DNA-binding domain consisting of two zinc fingers, -were amplified by PCR and sequenced to identify the specific mutation in the vitamin D receptor gene. In the three patients and one normal control a T-to-C transition was found in the putative initiation codon, while this transition was not observed in another normal control. This finding suggested that an original initiation codon was located at position 10-12 in the human vitamin D receptor CDNA sequence reported previously. In contrast, a unique G-to-A transition at position 140 in exon 3, resulting in substitution of arginine by glutamine at residue 47, was-revealed only-in these three patients. The arginine at 47 is located between two zinc fingers and is conserved within all steroid hormone receptors. Therefore, it is highly conceivable that this amino acid substitution is responsible for the defect of vitamin D receptor in the patients. Single-strand conformation polymorphism analysis of amplified DNA confirmed that all patients were homozygous and that parents from one family were heterozygous carriers for this mutation.