Novel gene mutations in patients with 1α-hydroxylase deficiency that confer partial enzyme activity in vitro

被引:49
作者
Wang, XM [1 ]
Zhang, MYH [1 ]
Miller, WL [1 ]
Portale, AA [1 ]
机构
[1] Univ Calif San Francisco, Dept Pediat, San Francisco, CA 94143 USA
关键词
D O I
10.1210/jc.87.6.2424
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
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 la-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 la-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.
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页码:2424 / 2430
页数:7
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