1998 Fiscal Year Final Research Report Summary
The instability of expanded CAG repeats in the genes for CAG repeat Diseases
| Project/Area Number |
09670666
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurology
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| Research Institution | Jichi Medical School |
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Principal Investigator |
TAKIYAMA Yoshihisa Jichi Medical School, Department of Neurology, Assistant Professor, 医学部, 講師 (00245052)
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| Co-Investigator(Kenkyū-buntansha) |
NISHIZAWA Masatoyo Jichi Medical School, Department of Neurology, Associate Professor, 医学部, 助教授 (80198457)
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| Project Period (FY) |
1997 – 1998
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| Keywords | CAG repeat / DRPLA / instability / single sperm / laser capture microdissection / germ line cells |
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| Research Abstract |
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
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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
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