Wang J T, Lin C J, Burridge S M, Fu G K, Labuda M, Portale A A, Miller W L
Department of Pediatrics, University of California, San Francisco, USA.
Am J Hum Genet. 1998 Dec;63(6):1694-702. doi: 10.1086/302156.
Vitamin D-dependent rickets type I (VDDR-I), also known as pseudo-vitamin D-deficiency rickets, appears to result from deficiency of renal vitamin D 1alpha-hydroxylase activity. Prior work has shown that the affected gene lies on 12q13.3. We recently cloned the cDNA and gene for this enzyme, mitochondrial P450c1alpha, and we and others have found mutations in its gene in a few patients. To determine whether all patients with VDDR-I have mutations in P450c1alpha, we have analyzed the P450c1alpha gene in 19 individuals from 17 families representing various ethnic groups. The whole gene was PCR amplified and subjected to direct sequencing; candidate mutations were confirmed by repeat PCR of the relevant exon from genomic DNA from the patients and their parents. Microsatellite haplotyping with the markers D12S90, D12S305, and D12S104 was also done in all families. All patients had P450c1alpha mutations on both alleles. In the French Canadian population, among whom VDDR-I is common, 9 of 10 alleles bore the haplotype 4-7-1 and carried the mutation 958DeltaG. This haplotype and mutation were also seen in two other families and are easily identified because the mutation ablates a TaiI/MaeII site. Six families of widely divergent ethnic backgrounds carried a 7-bp duplication in association with four different microsatellite haplotypes, indicating a mutational hot spot. We found 14 different mutations, including 7 amino acid replacement mutations. When these missense mutations were analyzed by expressing the mutant enzyme in mouse Leydig MA-10 cells and assaying 1alpha-hydroxylase activity, none retained detectable 1alpha-hydroxylase activity. These studies show that most if not all patients with VDDR-I have severe mutations in P450c1alpha, and hence the disease should be referred to as "1alpha-hydroxylase deficiency."
I型维生素D依赖性佝偻病（VDDR-I），也称为假性维生素D缺乏性佝偻病，似乎是由于肾脏维生素D 1α-羟化酶活性缺乏所致。先前的研究表明，相关基因位于12q13.3。我们最近克隆了该酶（线粒体P450c1α）的cDNA和基因，并且我们和其他人在一些患者中发现了其基因中的突变。为了确定所有VDDR-I患者的P450c1α是否存在突变，我们分析了来自17个家庭的19名个体的P450c1α基因，这些家庭代表了不同的种族。对整个基因进行PCR扩增并直接测序；通过对患者及其父母的基因组DNA中相关外显子进行重复PCR来确认候选突变。所有家庭还进行了D12S90、D12S305和D12S104标记的微卫星单倍型分析。所有患者的两个等位基因均存在P450c1α突变。在法裔加拿大人中，VDDR-I很常见，10个等位基因中有9个带有单倍型4-7-1并携带958DeltaG突变。在另外两个家庭中也发现了这种单倍型和突变，并且很容易识别，因为该突变消除了一个TaiI/MaeII位点。六个不同种族背景的家庭携带一个7 bp的重复，与四种不同的微卫星单倍型相关，表明存在一个突变热点。我们发现了14种不同的突变，包括7种氨基酸替代突变。当通过在小鼠睾丸间质MA-10细胞中表达突变酶并检测1α-羟化酶活性来分析这些错义突变时，没有一个保留可检测到的1α-羟化酶活性。这些研究表明，大多数（如果不是全部）VDDR-I患者的P450c1α存在严重突变，因此该疾病应称为“1α-羟化酶缺乏症”。
