Hughes M R, Malloy P J, Kieback D G, Kesterson R A, Pike J W, Feldman D, O'Malley B W
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.
Science. 1988 Dec 23;242(4886):1702-5. doi: 10.1126/science.2849209.
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.
低钙血症性维生素D抵抗性佝偻病是一种人类遗传病，由靶器官对1,25 - 二羟维生素D3作用的抵抗引起。对两个患有这种常染色体隐性疾病的纯合子患儿家庭的细胞内维生素D受体（VDR）及其基因异常情况进行了研究。尽管该受体对1,25 - 二羟维生素D3激素表现出正常结合，但来自患病家庭成员的VDR对DNA的亲和力降低。从这些家庭分离出的基因组DNA进行了寡核苷酸引物介导的DNA扩增，并对编码该受体蛋白的九个外显子中的每一个进行测序以寻找基因突变。在每个家庭中，在该蛋白的DNA结合域发现了不同的单核苷酸突变；一个家庭的突变发生在第一个锌指末端附近（甘氨酸变为天冬氨酸），另一个家庭的突变发生在第二个锌指末端（精氨酸变为甘氨酸）。通过对野生型VDR互补DNA进行寡核苷酸定向点突变在体外产生突变残基，并将该cDNA转染到COS - 1细胞中。所产生的蛋白在生化性质上与从患者分离出的受体没有区别。
