GENOME DIFFERENCE ANALYSIS BETWEEN MONOZYGOTIC TWINS DISCORDANT FOR NEUROPSYCHIATRIC DISORDERS

在1997年，我们得到了以下结果。我们将RLGS应用于颞叶癫痫、双相情感障碍和恐慌障碍的同卵双胞胎的基因组。然而，我们没有发现可重复的RLGS斑点差异在受影响和未受影响的双胞胎。今年，我们发现了一个非常罕见的同卵双胞胎自闭症不一致的病例，并在知情同意的情况下采集了血样。我们试图克隆去年在实验中发现的精神分裂症同卵双胞胎患病和未患病的RLGS斑点中包含的DNA片段（Tsujita et al., 1998）。我们面临着原始RLGS方法在RLGS点间克隆人类DNA片段的困难。我们面临克隆问题的原因是，原来的RLGS需要大量的人血，80ml，在一个RLGS点中平均只含有0.5 satto mol（5*10^<-19>）的DNA，比通常用于PCR的DNA少1/10^5。此外，NotI、PvuII、PstI组合酶切的DNA片段有5′和3′悬垂端，不便于PCR。因此，今年我们努力开发一种RLGS的修饰，包括PCR过程，增加了RLGS点中含有的DNA量，减少了克隆所需的人类血液量。我们几乎完成了改进的RLGS的开发，并获得了一些RLGS模式。如果我们能在同卵双胞胎之间找到不一致点，这种新的改良RLGS一定会使克隆变得更容易。该方法将基因组扫描与不一致的同卵双生子相结合，可用于研究各种具有复杂遗传基础的疾病的基因组机制，以避免同卵双生子中的一个成员患这种疾病，而不是由孪生兄弟避免。

In 1997, we obtained the following results.1. We applied RLGS to genomes from the monozygotic twins discordant for temporal lobe epilepsy, bipolar disorder and panic disorder. However, we found no reproducible RLGS spot difference between affected and non-affected twins. This year, we found a very rare case of monozygotic twin discordant for autism and collected blood samples with informed consents.2. We tried to cloning DNA fragments included in RLGS spots different between affected and non-affected monozygotic twins with schizophrenia that was found in the experiments last year (Tsujita et al., 1998). We faced with difficulties of the original RLGS method in the cloning of human DNA fragment among a RLGS spot. The reasons why we faced with the cloning problems were that the original RLGS required a large amount of human blood, 80ml, and contained only 0.5 satto moles(5*10^<-19>) DNA in average among a RLGS spot that was 1/10^5 less amount than that of DNA usually available for PCR.Moreover, the DNA fragments digested by the combination of NotI, PvuII, PstI had 5' and 3' overhanging ends that were inconvenient for PCR.Therefore, this year we made an effort to develop a modification of RLGS that included PCR process which increased an amount of DNA contained in a RLGS spot and decreased a required volume of human blood for thc cloning. We have almost completed to develop a modified RLGS and got a couple of RLGS patterns. This new modified RLGS must make the cloning easier if we find discordant spots between identical twins discordant for neuropsychiatric disorders.The method developed by principal investigator that combines genome scanning with discordant monozygotic twins may be available for various kind s of disorders with complex genetic basis in the investigation of genomic mechanisms to be avoided suffering from such disorders by one member of a monozygotic twin pair and not to be avoided by the co-twin.