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基于DNA诊断各种遗传病的概念。在确定的人群中流行的突变是这种方法的一个很好的候选者。在这种背景下，开发一种适合临床应用的稳健的DNA诊断方法是很重要的。在目前的研究中，我们探索了应用DNA微阵列方法同时检测多个致病突变的可能性。我们测试的方法是使用荧光标记dNTP的微型测序。用多重聚合酶链式反应扩增含有突变位点的DNA片段，变性后与固定在玻片上的寡核苷酸杂交。在Cy5标记的dNTP存在的情况下进行引物延伸反应。清洗后，用激光束扫描阵列以检测合并的荧光信号。然而，这种方法的低信噪比阻碍了基因分型的重复性和可靠性。因此，我们应用了一种新的基因分型方法“DNA棒”来检测各种突变。成功的基因分型表明，经过进一步修改，该方法可以很容易地应用于微阵列形式。此外，我们能够确定糖原储存疾病1a和1b型的普遍基因突变，连接蛋白26突变所致的遗传性耳聋，以及全羧酶合成酶缺乏症，以帮助开发适用于日本人群的突变小组。

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.