title: Novel gene mutations in patients with 1alpha-hydroxylase deficiency that confer partial enzyme activity in vitro.
abstract: The rate-limiting, hormonally regulated step in the biological activation of vitamin D is its 1alpha-hydroxylation to 1,25-dihydroxyvitamin D [1,25-(OH)(2)D] in the kidney, catalyzed by the mitochondrial cytochrome P450 enzyme, P450c1alpha. We previously cloned the human P450c1alpha cDNA and gene, and identified 14 different mutations, including 7 missense, in 19 patients with 1alpha-hydroxylase deficiency, also known as vitamin D-dependent rickets type 1. None of the missense mutations encoded a protein with detectable enzymatic activity in vitro. Although there is phenotypic variation among such patients, the molecular basis of this variation is unknown. We analyzed 6 additional patients with clinical and radiographic features of rickets; in 4 patients the laboratory abnormalities were typical of 1alpha-hydroxylase deficiency, but in 2 they were unusually mild [mild hypocalcemia and normal serum 1,25-(OH)(2)D concentration]. Direct sequencing revealed that all patients had P450c1alpha mutations on both alleles. Five new and 2 known mutations were identified. The new mutations included a 5-bp deletion with a 6-bp novel insertion causing a frameshift in exon 2, and a G to A change at +1 of intron 2; a minigene experiment proved that this intronic mutation prevented proper splicing. Three new missense mutations were found and tested by expressing the mutant cDNA in mouse Leydig MA-10 cells. The R389G mutant was totally inactive, but mutant L343F retained 2.3% of wild-type activity, and mutant E189G retained 22% of wild-type activity. The two mutations that confer partial enzyme activity in vitro were found in the 2 patents with mild laboratory abnormalities, suggesting that such mutations contribute to the phenotypic variation observed in patients with 1alpha-hydroxylase deficiency.
authors.affiliation: Department of Pediatrics, University of California-San Francisco, 533 Parnassus Avenue, San Francisco, CA 94143, USA.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Wang
authors.forename: Martin Y H
authors.initials: MY
authors.lastname: Zhang
authors.forename: Walter L
authors.initials: WL
authors.lastname: Miller
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Portale
mesh_terms: D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology*; D020125:Mutation, Missense / Q000502:physiology; D014808:Vitamin D Deficiency / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase