The instability of expanded CAG repeats in the genes for CAG repeat Diseases

在大多数CAG重复病中，普遍存在致病基因扩增的GAG重复大小在亲代和子代之间不稳定的现象。为探讨齿状核苍白质萎缩(DRLA)基因CAG重复序列扩增后减数分裂不稳定的机制，我们首先分析了2名DRLA患者的316个单个精子的CAG重复序列大小，结果如下：1)扩增等位基因的单个精子与正常等位基因的单个精子的分离比与预期的1：1分离比无显著差异(P=0.26)。我们未能证明DRPLA等位基因在雄性减数分裂中的分离扭曲。2)在168例精子中未检测到携带8次和16次CAG重复的正常等位基因突变。(3)患者1单精子的CAG重复序列大小变化显著大于患者2的单精子CAG重复序列大小的变化(F检验，P&lt；0.0001)。Mo…更重要的是，在CAG重复疾病中，患者1显示的精子重复大小变化最大。在单个精子中的这些发现证实了DRLA基因中CAG重复序列的显著不稳定性，这被观察到在DRLA的父系传递中被观察到很大程度上的预期。进一步的研究需要确定DRLA基因中CAG重复序列的不稳定性是否存在顺式或反式因子，以及CAG重复序列疾病中是否存在共同的机制。第二，我们利用激光捕获显微切片方法(LCM)分析了DRLA尸检组织中生殖系细胞在精子发生和卵子发生中的GAG重复序列大小。取福尔马林固定、石蜡包埋的睾丸和卵巢5~10微米的标准切片，在非镀膜玻片上制作。切片脱蜡后行苏木精-伊红染色，用LCM-100(Arcturus)收集直径30微米处的胚系细胞。采用套式PGR策略进行两轮聚合酶链式反应扩增CAG重复序列。扩增产物在ABI PRISM 310全自动遗传分析仪上进行电泳，测定生殖细胞中DRPIA基因的CAG重复数。结果如下：1)在福尔马林固定的睾丸和卵巢生殖细胞中可以检测到DRPLA基因CAG重复序列的大小。2)LCM-100直径30微米的激光光斑太大，无法从睾丸中拾取单个细胞。3)LCM 200(新系统)的激光光斑直径为7.5微米，可在美国获得。结果表明，用LCM-200系统可以从DRPLA睾丸中提取单个细胞。结果表明，用LCM-100系统可以在福尔马林固定的睾丸和卵巢生殖细胞中检测到DRLA基因的CAG重复大小。利用LCM 200系统对单细胞进行进一步研究，以阐明精子发生和卵子发生过程中扩增的CAG重复序列减数分裂不稳定的机制。较少

In the majority of CAG repeat diseases, there is a common phenomenon that expanded GAG repeat size in the causative gene is unstable between parents and offsprings. To investigate the mechanism of the meiotic instability of expanded CAG repeats in the gene for dentatorubral-pallidoluysian atrophy (DRPLA), we at first analyzed CAG repeat sizes of 316 single sperm from 2 individuals with DRPLA.Results are as follows ; 1) The segregation ratio between single sperm with an expanded allele and ones with a normal allele was not significantly different (P=O.26) from the expected 1 : 1 segregation ratio. We failed to demonstrate the segregation distortions of DRPLA alleles in male meiosis. 2) No mutations in the normal alleles carrying 8 and 16 CAG repeats were detected in 168 sperm. 3) The variance of the change in size of the CAG repeats in the DRPLA gene of single sperm from Patient I was significantly greater than that in the DRPLA gene of single sperm from Patient 2 (F-test, P<0.0001). Mo … More reover, Patient 1 showed the largest variance of the change in repeat size in sperm among CAG repeat diseases. These findings in single sperm confirmed the marked instability of the CAG repeats in the DRPLA gene, which was observed as large anticipationi in paternal transmission in DRPLA.Further study is required to determine whether there is a cis or trans factor on the instability of the CAG repeats in the DRPLA gene and whether there is a common mechanism underlying the instability of the triplet repeats in CAG repeat diseases.Second, we analyzed GAG repeat sizes of the germ line cells in spermatogenesis and oogenesis from DRPLA autopsy tissues using Laser Capture Microdissection method (LCM). Standard 5-10 mum sections from formalin-fixed and paraffin-embedded testis and ovary were prepared on non coated glass slides. Sections were deparaffinized, stained with hematoxylin and eosin before LCM.The germ line cells from the glass slides were collected in the spots of 30 mum diameter using LCM 100 (ARCTURUS). Two rounds of PCR were performed to amplify the CAG repeat lesion using the nested PGR strategy. The PCR products were then electrophoresed on an automated ABI PRISM 310 genetic analyzer, and the numbers of CAG repeats of the DRPIA gene in germ line cells were determined. Results are as follows : 1) We could detect the CAG repeat sizes of the DRPLA gene in the germ line cells from formalin-fixed testis and ovary. 2) The laser spots of 30 mum diameter of LCM 100 is too large to pick up a single cell from the testis. 3) The laser spots of LCM 200 (new system), which can be obtained in USA) is 7.5 mum diameter. We showed that a single cell from the DRPLA testis can be picked up using LCM 200 system. We showed that the CAG repeat sizes of the DRPLA gene can be detected in the germ line cells from formalin-fixed testis and ovary using LCM 100 system. Further study of single cell using LCM 200 system is required to elucidate the mechanism of meiotic instability of expanded CAG repeats during spermatogenesis and oogenesis. Less