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对各种遗传疾病进行基于dna的诊断的概念。在特定人群中普遍存在的突变是这种方法的良好候选者。在这种情况下，开发一种适合临床应用的可靠的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种致病突变的可能性。我们测试的方法是采用荧光标记的dNTP进行微型测序。含有突变位点的DNA片段通过多重PCR扩增，变性，并与固定在玻璃板上的寡核苷酸杂交。引物延伸反应在cy5标记的dNTP存在下进行。清洗后，用激光束扫描阵列以检测合并的荧光信号。然而，该方法的信噪比较差，阻碍了基因分型的可重复性和可靠性。因此，我们采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，该方法可以很容易地应用于进一步修改的微阵列格式。此外，我们能够鉴定出糖原储存病la型和lb型、连接蛋白26突变引起的遗传性耳聋和全新羧化酶合成酶缺乏症中普遍存在的基因突变，以帮助开发一种适用于日本人群的突变面板。

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.