Glorieux F H, St-Arnaud R
Genetics Unit, Shriners Hospital for Children, Montréal, Québec, Canada.
Recent Prog Horm Res. 1998;53:341-9; discussion 350.
Pseudovitamin D-deficiency rickets (PDDR) is the first identified inborn error of vitamin D metabolism. Its clinical course is similar to that of nutritional rickets due to simple vitamin D deficiency. The treatment of choice is replacement therapy with calcitriol [1,25(OH)2D3]. PDDR is inherited as a simple autosomal recessive trait. The PDDR locus has been mapped to chromosome 12q13-q14. The molecular defect underlying the 25-hydroxyvitamin D-1 alpha-hydroxylase enzyme dysfunction has remained elusive due to the lack of sequence information for the gene encoding the cytochrome P450 moiety of the enzyme. We have used a probe derived from the rat 25-hydroxyvitamin D-24-hydroxylase sequence to identify and clone the 1 alpha-OHase cDNA. The candidate gene was transiently expressed in P19 embryonal carcinoma cells. Only those cells that were transfected with the candidate cDNA in the sense orientation were able to produce a compound that co-eluted with the 1 alpha, 25 vitamin D3 standard. Mass spectrometry analysis confirmed the identity of the produced metabolite. A human genomic clone was isolated from a chromosome 12 cosmid library and subsequently mapped to human chromosome 12q13.1-q13.3. To address the putative biological function of 24,25-dihydroxyvitamin I) 24,25(OH)2D, we also engineered a null mutation in the 24-OHase gene in embryonic stem cells (ES). Animals heterozygous for the engineered mutation are normal and fertile. One half of the homozygous animals die before weaning. Breeding of surviving females gives an F2 generation in which bone development is abnormal at sites of intramembranous ossification. Growthplate maturation and endochondral ossification appeared to proceed normally. The results show that a complete absence of vitamin D metabolites hydroxylated in position 24 during embryogenesis leads to abnormal bone structure and suggests a key role for 24,25(OH)2D in the developmental regulation of intramembranous ossification.
假性维生素D缺乏性佝偻病（PDDR）是最早被发现的维生素D代谢先天性缺陷。其临床病程与单纯维生素D缺乏所致的营养性佝偻病相似。治疗的首选方法是用骨化三醇[1,25(OH)₂D₃]进行替代治疗。PDDR以简单的常染色体隐性性状遗传。PDDR基因座已被定位到12号染色体q13 - q14区域。由于缺乏编码该酶细胞色素P450部分的基因的序列信息，导致25 - 羟维生素D - 1α - 羟化酶功能障碍的分子缺陷一直难以捉摸。我们使用了源自大鼠25 - 羟维生素D - 24 - 羟化酶序列的探针来鉴定和克隆1α - OHase cDNA。候选基因在P19胚胎癌细胞中瞬时表达。只有那些以正义方向转染了候选cDNA的细胞能够产生一种与1α,25 - 维生素D₃标准品共洗脱的化合物。质谱分析证实了所产生代谢物的身份。从12号染色体黏粒文库中分离出一个人类基因组克隆，随后将其定位到人类12号染色体q13.1 - q13.3区域。为了研究24,25 - 二羟维生素D [24,25(OH)₂D]的假定生物学功能，我们还在胚胎干细胞（ES）中构建了24 - OHase基因的无效突变。携带工程突变的杂合动物正常且可育。一半的纯合动物在断奶前死亡。存活雌性动物的繁殖产生了F2代，其中膜内骨化部位的骨骼发育异常。生长板成熟和软骨内骨化似乎正常进行。结果表明，胚胎发育过程中完全缺乏在24位羟化的维生素D代谢物会导致骨骼结构异常，并提示24,25(OH)₂D在膜内骨化的发育调控中起关键作用。
