Basic Research of pre-implantation genetic diagnosis of hypophosphatasia

低磷酸酯酶症着床前遗传学诊断的基础研究

基本信息

批准号：
15591788
负责人：
HORIUCHU Isao
金额：
$ 2.3万
依托单位：
Hyogo College of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and deficiency of tissue nonspecific alkaline phosphatase (TNSALP) activity. This disorder is caused by various mutations in the TNSALP gene. We reported hypophosphatasia in two siblings, both of them severely affected by the perinatal (lethal) type. We diagnosed the first infant by clinical and radiologic manifestations, and laboratory findings. Laboratory findings were characterized by deficiency of serum alkaline phosphatase. Both parents and the second infant were then analyzed by molecular techniques. The radiograph of the first infant showed severe hypomineralization of the skeleton. Molecular analysis of the second infant showed that this condition was caused by a homozygous single T nucleotide deletion at cDNA number 1559 (1559delT). Both parents were heterozygous carriers for this mutation, although they were not consanguineous. This mutation has been frequently found in Japanese hypophosp … More hatasia patients, but this is the first observation of a homozygous deletion. This report shows that homozygosity for the 1559delT mutation of the TNSALP gene results in a severe lethal phenotype. To perform pre-implantation diagnosis first we set up a mouse system for pre-implantation diagnosis. We pick up a blastomeret of mouse embryo, then one blastomere was subjected for sexing and the remaining blastomere was transferred in utero. We calculated the accuracy of the sexing, when the futus of mice were born. The accuracy was more than 90%. Next we set up single cells (leukocyte) amplification system from the carrier individuals of hypophosphatasia. The amplification was successful in more than 90% cells. However the problem of allele drop out still remains. Overall approximately half of the samples were successful for both allele amplification. However if the normal allele is amplified, the individual is normal or carrier, then the clinically. Then our strategy should be useful for pre-implantation diagnosis of hypophosphatasia. Less

下磷酸是一种遗传性疾病，其特征是骨矿化缺陷和组织非特异性酒精线磷酸酶（TNSALP）活性的缺乏。该疾病是由TNSALP基因中各种突变引起的。我们报道了两个兄弟姐妹中的下磷酸，两者都受到围产期（致命）类型的严重影响。我们通过临床和放射学表现以及实验室发现来诊断第一个婴儿。实验室发现的特征是血清醇磷酸酶的缺乏。然后通过分子技术分析父母和第二个婴儿。第一个婴儿的X光片显示骨骼严重的低矿物化。第二婴儿的分子分析表明，这种情况是由cDNA数字1559（1559delt）的纯合单T核苷酸缺失引起的。父母双方都是这种突变的杂合载体，尽管它们不是近亲。这种突变经常在日本的Hatophosp…更多的Hatasia患者中发现，但这是对纯合缺失的首次观察。该报告表明，TNSALP基因1559DELT突变的纯合性导致严重的致命表型。首先要进行植入前诊断，我们建立了一个小鼠系统以进行植入前诊断。我们拿起一个小鼠胚胎的囊泡组，然后对一个胚胎进行性爱，并在子宫内转移剩余的卵母子。当小鼠的未来诞生时，我们计算了性别的准确性。准确性超过90％。接下来，我们从次磷酸载体的载体个体设置了单细胞（白细胞）扩增系统。扩增在90％以上的细胞中成功。但是，等位基因退出静止图。总体而言，大约一半的样品成功进行了等位基因扩增。但是，如果正常的等位基因被放大，则个体是正常或载体，则在诊所中。那么我们的策略应有助于植入前诊断次磷酸症。较少的