CLONING AND CHARACTERIZATION OF HUMAN CYP25

描述（改编自申请人摘要）：维生素D必须经历 通过维生素D酶羟基化为25-羟基维生素D [25（OH）D] D-25-羟化酶（CYP 25）变得具有生物活性。猪CYP 25 cDNA是 克隆并鉴定。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的转化， D3至25（OH）D3。孤立性25（OH）D缺乏症患者， 对药理剂量和生理剂量的维生素D有反应的佝偻病 25（OH）D3、1 α（OH）D3或相关药物中的一种被推定为具有CYP 25 缺陷该提案的具体目标是使用猪的部分 CYP 25 cDNA，分离和表征人CYP 25 cDNA， 表征人CYP 25基因，以确定CYP 25 DNA的染色体位点 和组织表达，利用基因定位，连锁分析， 定位克隆，以表征家族谱系和确定分子 孤立性25（OH）D缺乏症和佝偻病患者的机制， 表征受影响人群中的CYP 25基因多态性，并将其与 基因型与表型。事实上，猪CYP 25 cDNA具有83%的 与人CYP 2D 6 cDNA同源，这是一种细胞色素P450微粒体酶， 在药物代谢中的作用，干扰了人CYP 25 cDNA的克隆。猪 据报道肝提取物没有CYP 2DK活性。使用辐射 杂交细胞面板（ImpRH），我们发现猪CYP 25基因定位到猪 染色体5 p14 -15与人类同线的ACO 2基因相邻 染色体22 q12 -13，其中人类CYP 2D 6被定位。我们用单倍型分析 孤立性CYP 25缺乏症和佝偻病患者的家族谱系， 发现这种疾病既不映射到CYP 2D 6基因位点， 22号染色体。拟议研究的结果应提供重要的新的 关于CYP 25的分子结构和染色体位点的信息 基因和孤立的25（OH）D和佝偻病的分子原因。

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.