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Novel Gene Mutations in Patients with 1-Hydroxylase Deficiency That Confer Partial Enzyme Activity in Vitro

Department of Pediatrics, University of California, San Francisco, California 94143

Address all correspondence and requests for reprints to: Anthony A. Portale, M.D., University of California, 533 Parnassus Avenue, Room U-585, Box 0748, San Francisco, California 94143-0748. E-mail: . aportale{at}peds.ucsf.edu

Abstract

The rate-limiting, hormonally regulated step in the biological activation of vitamin D is its 1-hydroxylation to 1,25-dihydroxyvitamin D [1,25-(OH)₂D] in the kidney, catalyzed by the mitochondrial cytochrome P450 enzyme, P450c1. We previously cloned the human P450c1 cDNA and gene, and identified 14 different mutations, including 7 missense, in 19 patients with 1-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 1-hydroxylase deficiency, but in 2 they were unusually mild [mild hypocalcemia and normal serum 1,25-(OH)₂D concentration]. Direct sequencing revealed that all patients had P450c1 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 1-hydroxylase deficiency.

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