CLONING AND CHARACTERIZATION OF HUMAN CYP25

描述(摘自申请者的摘要)：维生素D必须 维生素酶羟化合成25-羟基维生素D[25(OH)D] D-25-羟基酶(CYP25)具有生物活性。猪细胞色素P25基因(CyP25) 进行了克隆和鉴定。该基因编码细胞色素P450微粒体酶。 在肝脏和肾脏中表达，催化维生素D2和维生素D2的转化 D3~25(OH)D3。孤立性25(OH)D缺乏症和 对药物剂量的维生素D和生理剂量的维生素D有反应的软骨病 25个(OH)D3、1个α(OH)D3或相关药物被推定为具有CYP25 缺乏症。这项提议的具体目的是使用部分猪 目的：分离和鉴定人细胞色素P25基因，克隆和鉴定 鉴定人类细胞色素P25基因，确定细胞色素P25 DNA的染色体定位 和组织表达的细胞色素P25基因，使用基因作图、连锁分析和 定位克隆，以确定家系特征和确定分子 孤立性25(OH)D缺乏症和软骨病患者的发病机制 研究受影响人群中的细胞色素P25基因的多态性，并进行相关性分析 具有表型的基因。猪细胞色素P25基因的cDNA83% 与人细胞色素P450微粒体酶的同源性 在药物代谢中的作用，干扰了人细胞色素P25基因的克隆。猪 据报道，肝脏提取物没有CYP2DK活性。使用辐射 杂交细胞板(ImpRH)，我们发现猪的CYP25基因定位于猪 染色体5p14-15与ACO2基因相近，ACO2基因是人类的一个区域共轭基因 染色体22q12-13，人类细胞色素P4502D6被定位的位置。我们使用了单倍型分析 单纯性细胞色素P25缺乏症和软骨病患者的家系 发现这种疾病既没有映射到CYP2D6基因位点，也没有映射到 22号染色体。拟议研究的结果应提供重要的新的 关于CYP25的分子结构和染色体位置的信息 基因和分离的25(OH)D和软骨病的分子病因。

DESCRIPTION (Adapted from the Applicant's Abstract): Vitamin D must undergo hydroxylation to 25-hydroxyvitamin D [25(OH)D] by the enzyme vitamin D-25-hydroxylase (CYP25) to become biologically active. Porcine CYP25 cDNA was cloned and characterized. The gene encodes a cytochrome P450 microsomal enzyme expressed in liver and kidney that catalyzes the conversion of vitamins D2 and D3 to 25(OH)D3, respectively. Patients with isolated 25(OH)D deficiency and rickets who respond to pharmacologic doses of vitamin D and physiologic doses of 25(OH)D3, 1alpha(OH)D3, or related drugs are presumed to have CYP25 deficiency. The specific aims of this proposal are to use portions of porcine CYP25 cDNA to isolate and characterize human CYP25 cDNA, to clone and characterize the human CYP25 gene, to determine chromosome locus of CYP25 DNA and tissue expression of CYP25 mRNA, to use gene mapping, linkage analysis and positional cloning, to characterize family pedigrees and determine molecular mechanisms of patients with isolated 25(OH)D deficiency and rickets, to characterize CYP25 gene polymorphisms in affected populations, and to correlate genotype with phenotype. The fact that porcine CYP25 cDNA has 83 percent homology with human CYP2D6 cDNA, a cytochrome P450 microsomal enzyme that plays a role in drug metabolism, has interfered with cloning of human CYP25 cDNA. Pig liver extracts are reported to have no CYP2DK activity. Using a radiation hybrid cell panel (ImpRH), we found that the porcine CYP25 gene maps to pig chromosome 5p14-15 close to the ACO2 gene, a region syntenic ot human chromosome 22q12-13 where human CYP2D6 was mapped. We used haplotype analysis of family pedigrees of patients with isolated CYP25 deficiency and rickets and found that the disease does not map to either the CYP2D6 gene locus or chromosome 22. The results of the proposed studies should provide important new information about the molecular structure and chromosomal locus of the CYP25 gene and the molecular causes of isolated 25(OH)D and rickets.