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

CAG 重复疾病基因中扩展的 CAG 重复序列的不稳定性

基本信息

批准号：
09670666
负责人：
TAKIYAMA Yoshihisa
金额：
$ 1.86万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670666/
关键词：
CAG repeat DRPLA instability single sperm laser capture microdissection germ line cells germ line cell sperm

项目摘要

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

在大多数CAG重复疾病中，有一个共同的现象会在父母和后代之间扩大严重基因的插科打go重复大小是不稳定的。为了研究基因在基因中扩展的CAG重复序列的减数分裂不稳定性的机制，用于牙齿牙周膜 - 核糖 - 核糖萎缩症（DRPLA），我们首先分析了来自2个drpla的个体的316个单个精子的CAG重复大小，如下所示； 1）具有扩展等位基因的单个精子与具有正常等位基因的单个精子之间的隔离比与预期的1：1分离率没有显着差异（P = O.26）。我们未能证明男性减数分裂中的Drpla等位基因的隔离扭曲。 2）在168个精子中检测到携带8和16 CAG重复的正常等位基因中未发生突变。 3）从患者I的单个精子的Drpla基因中CAG重复的大小变化的方差明显大于患者2的单个精子的Drpla基因（F-Test，p-Test，p <0.0001）。 Mo…更多的重新覆盖，患者1显示了CAG重复疾病中精子重复大小变化的最大差异。单个精子中的这些发现证实了Drpla基因中CAG重复的明显不稳定性，在Drpla中，这是在父亲传播中大量预期的。需要确定在DRPLA基因中是否存在CAG重复序列的不稳定性以及是否存在drpra基因的不稳定性以及是否存在cag重复症的不稳定性，以及在三元中是否存在cag重复症的不稳定性。使用激光捕获显微解剖方法（LCM），插入生殖细胞的生物发生和卵子组织中的生殖线细胞重复大小。在非涂层载玻片上制备了来自福尔马林固定和石蜡包裹的睾丸和卵巢的标准5-10个妈妈切片。将切片脱蜡，在LCM之前用苏木精和曙红染色。使用LCM 100（Arcturus），将载玻片的生殖线细胞收集在30 MUM直径的斑点中。使用嵌套的PGR策略进行了两轮PCR以扩增CAG重复病变。然后将PCR产物在自动化的ABI PRISM 310遗传分析仪上电泳，并确定生殖系细胞中DrPIA基因的CAG重复序列。结果如下：1）我们可以从福尔马林固定睾丸和卵巢中检测到生殖线细胞中Drpla基因的CAG重复大小。 2）LCM 100直径30 MUM直径的激光斑太大，无法从睾丸中捡起单个细胞。 3）LCM 200（可以在美国获得的新系统）的激光点为7.5 MUM直径。我们表明，可以使用LCM 200系统拾取Drpla睾丸的单个单元。我们表明，使用LCM 100系统，可以在福尔马林固定睾丸和卵巢的种系细胞中检测到Drpla基因的CAG重复大小。需要使用LCM 200系统对单个细胞进行进一步研究，以阐明在精子发生和卵子发生过程中扩展的CAG重复的减数分裂不稳定机制。较少的