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

维生素D依赖性佝偻病II型是由维生素D受体缺陷引起的遗传性疾病。我们认为，这种疾病是一种常染色体隐性遗传疾病，显示中间水平的25-羟维生素D-24-羟化酶诱导的父母。因此，重要的是要确定引起疾病的遗传缺陷，并在分子水平上检测杂合携带者。6例患者服用维生素D-d。2型佝偻病的成纤维细胞胞浆结合正常，核摄取1，25-二羟维生素D_3受损，Western、Southern和北方分析均未发现维生素D_3受体蛋白及其基因的异常。通过PCR扩增维生素D受体cDNA的外显子2和3，编码由两个锌指组成的DNA结合结构域，并测序以鉴定维生素D受体基因中的特异性突变。在三名患者和一名正常对照中，在假定的起始密码子中发现了T到C的转变，而在另一名正常对照中未观察到这种转变。这一发现表明，一个原始的起始密码子位于10-12位的人维生素D受体cDNA序列中以前报道的。相比之下，仅在这三名患者中发现了外显子3第140位的独特G至A转变，导致残基47处的精氨酸被谷氨酰胺取代。第47位的精氨酸位于两个锌指之间，在所有类固醇激素受体中是保守的。因此，很可能这种氨基酸取代是导致患者维生素D受体缺陷的原因。扩增DNA的单链构象多态性分析证实所有患者均为纯合子，且来自一个家庭的父母为该突变的杂合子携带者。

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.