Molecular cloning of (25-OH D)-1 alpha-hydroxylase: an approach to the understanding of vitamin D pseudo-deficiency.

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.