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

在大多数CAG重复序列疾病中，常见的现象是致病基因GAG重复序列扩增在亲代和后代之间不稳定。为了研究齿状管-白斑萎缩（DRPLA）基因中扩增CAG重复序列减数分裂不稳定性的机制，我们首先分析了来自2个DRPLA个体的316个单精子的CAG重复序列大小。结果如下：1)扩增等位基因单精子与正常等位基因单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) 168例精子中未检测到携带8和16个CAG重复序列的正常等位基因突变。3)患者1单精子DRPLA基因CAG重复序列大小变化的方差显著大于患者2单精子DRPLA基因CAG重复序列大小变化的方差（f检验，P<0.0001）。更重要的是，患者1在CAG重复疾病中表现出精子重复大小变化的最大差异。这些在单精子中的发现证实了DRPLA基因中CAG重复序列的显著不稳定性，这在DRPLA的父系传播中被观察到很大的预期。对于DRPLA基因中CAG重复序列的不稳定性是否存在顺式或反式因素，以及CAG重复序列疾病中是否存在三联体重复序列不稳定性的共同机制，还需要进一步研究。其次，我们使用激光捕获显微解剖方法（LCM）分析了DRPLA尸体解剖组织中精子发生和卵子发生的生殖系细胞的GAG重复大小。在无涂层玻璃载玻片上制备福尔马林固定和石蜡包埋睾丸和卵巢的标准5-10 mm切片。切片去蜡，在LCM前用苏木精和伊红染色。用LCM - 100 （ARCTURUS）在30 mm直径的斑点处收集玻璃片上的种系细胞。使用巢式PGR策略进行两轮PCR扩增CAG重复病变。PCR产物在全自动ABI PRISM 310遗传分析仪上电泳，测定生殖系细胞中DRPIA基因CAG重复数。结果如下：1)在福尔马林固定的睾丸和卵巢生殖系细胞中可以检测到DRPLA基因CAG重复序列的大小。2) LCM 100直径30 μ m的激光斑点太大，无法从睾丸上取下单个细胞。3) LCM 200（新系统，可在美国获得）的激光光斑直径为7.5微米。我们发现，使用LCM 200系统可以提取来自DRPLA睾丸的单个细胞。我们使用LCM - 100系统在福尔马林固定的睾丸和卵巢生殖系细胞中检测到了DRPLA基因的CAG重复序列大小。为了进一步阐明CAG扩增重复序列在精子发生和卵子发生过程中减数分裂不稳定的机制，需要进一步利用LCM - 200系统对单细胞进行研究。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