Novel gene mutations in patients with 1alpha-hydroxylase deficiency that confer partial enzyme activity in vitro.
Novel gene mutations in patients with 1alpha-hydroxylase deficiency that confer partial enzyme activity in vitro.
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DOI：
10.1210/jcem.87.6.8534
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发表时间：
2002-06
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影响因子：
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通讯作者：
Xuemei Wang;Martin Y. H. Zhang;W. Miller;A. Portale
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文献类型：
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作者：
Xuemei Wang;Martin Y. H. Zhang;W. Miller;A. Portale
维生素D生物活化中的限速、肾脏调节步骤是其在肾脏中由线粒体细胞色素P450酶P450 c1 α催化1 α-羟基化为1，25-二羟基维生素D [1，25-（OH）（2）D]。我们先前克隆了人P450 c1 α cDNA和基因，并在19例1 α-羟化酶缺乏症（也称为维生素D依赖性佝偻病1型）患者中鉴定出14种不同的突变，包括7种错义突变。没有一个错义突变编码的蛋白质在体外具有可检测的酶活性。虽然在这些患者中存在表型变异，但这种变异的分子基础尚不清楚。我们分析了另外6例具有佝偻病临床和影像学特征的患者，其中4例患者的实验室异常为典型的1 α-羟化酶缺乏症，但2例患者的异常轻微[轻度低钙血症和正常血清1，25-（OH）（2）D浓度]。直接测序显示所有患者在两个等位基因上都有P450 c1 α突变。发现5个新突变和2个已知突变。新的突变包括一个5-bp的缺失和一个6-bp的新插入，导致外显子2的移码，以及内含子2的+1处G到A的变化;一个小基因实验证明，这种内含子突变阻止了正确的剪接。通过在小鼠Leydig MA-10细胞中表达突变体cDNA，发现并检测了三个新的错义突变。R389 G突变体完全失活，但突变体L343 F保留了野生型活性的2.3%，突变体E189 G保留了野生型活性的22%。在2例轻度实验室异常患者中发现了两种体外赋予部分酶活性的突变，表明此类突变有助于在1 α-羟化酶缺乏症患者中观察到的表型变异。
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.