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

维生素D依赖型软骨病II型是一种由维生素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病，在双亲中表现出中等水平的25-羟基维生素D-24-羟基酶诱导。因此，在分子水平上识别引起该病的遗传缺陷和检测杂合子携带者将是非常重要的。在6名服用维生素D-d的患者中。成纤维细胞胞浆结合正常，核摄取1，25-二羟基维生素D_3受损的II型患者，Western、Southern和Northern分析未发现维生素D_3受体蛋白及其基因的任何异常。用聚合酶链式反应扩增维生素D受体基因的外显子2和外显子3，该外显子编码由两个锌指组成的DNA结合域，并对其进行测序，以确定维生素D受体基因的特异性突变。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-C转换，而在另一名正常对照中没有观察到这种转换。这一发现表明，在先前报道的人类维生素D受体CDNA序列中，原始起始密码子位于第10-12位。相比之下，仅在这三个患者中发现了外显子3第140位的独特的G-A转换，导致第47位的谷氨酰胺取代精氨酸。精氨酸位于两个锌指之间，在所有类固醇激素受体中都是保守的。因此，这种氨基酸替代很可能是导致患者维生素D受体缺陷的原因。扩增DNA单链构象多态性分析证实，所有患者均为纯合子，父母均为杂合子携带者。

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.