DNA DIAGNOSIS OF RARE GENETIC DISEASES USING JAPANESE MICROARRAY

快速增长的突变数据库提出了通过筛选已知突变来诊断各种遗传疾病的基于DNA的概念。在特定群体中流行的突变是这种方法的良好候选者。在此背景下，重要的是开发一种适合临床应用的稳健的DNA诊断方法。在当前的研究中，我们探索了应用DNA微阵列方法同时检测多种致病突变的可能性。我们测试的方法是使用荧光标记的dNTP的微型测序。通过多重PCR扩增含有突变位点的DNA片段，变性，并与固定在玻璃板上的寡核苷酸杂交。在Cy5标记的dNTP存在下进行引物延伸反应。洗涤后，用激光束扫描阵列以检测掺入的荧光信号。然而，这种方法的信噪比差，阻碍了可重复性和可靠的基因分型。因此，我们应用了一种新的基因分型方法“DNA棒”检测各种突变。成功的基因分型表明，这种方法可以很容易地应用到微阵列格式与进一步的修改。此外，我们能够确定流行的遗传突变，糖原累积病型Ia和Ib型，遗传性耳聋由于连接蛋白26突变，和全羧化酶合成酶缺乏症，以帮助开发一个突变面板在日本人口有用。

The rapidly growing mutation database raises the notion of a DNA-based diagnosis of various genetic diseases by screening known mutations. A mutation prevalent in a defined population is a good candidate for this type of approach. Within this context, it is important to develop a robust DNA diagnostic method that is suitable for clinical application. In the current study, we have explored the possibility of applying a DNA microarray method for the simultaneous detection of multiple disease-causing mutations. The method we tested was mini-sequencing employing fluorescence-labeled dNTP. DNA fragments containing mutation-sites were amplified by multiplex PCR, denatured, and hybridized with oligonucleotides immobilized on a glass plate. Primer extension reaction was performed in the presence of Cy5-labeled dNTP. After washing, the array was scanned with laser-beam to detect incorporated fluorescent signals. The poor signal-to-noise ratio in this method, however, hampered reproducible and reliable genotyping. We therefore applied a new genotyping method "DNA stick" for the detection of various mutations. Successful genotyping indicated that this method may be readily applied to a microarray format with further modification. In addition, we were able to identify prevalent genetic mutations in glycogen storage disease type la and type lb, hereditary deafness due to connexin26 mutations, and holocarboxylase synthetase deficiency to aid developing a mutation-panel useful in Japanese population.