Clinical and Experimental Reproductive Medicine

The Korean Society for Reproductive Medicine (대한생식의학회)
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Optimized Methods of Preimplantation Genetic Diagnosis for Trinucleotide Repeat Diseases of Huntington's Disease, Spinocerebellar Ataxia 3 and Fragile X Syndrome

삼핵산 반복서열 질환인 헌팅톤병, 척수소뇌성 운동실조증, X-염색체 취약 증후군의 착상전 유전진단 방법에 대한 연구

  • Kim, Min-Jee (Laboratory of Reproductive Biology and Infertility, Cheil General Hospital and Women's Healthcare Center, Kwandong University School of Medicine) ;
  • Lee, Hyoung-Song (Laboratory of Reproductive Biology and Infertility, Cheil General Hospital and Women's Healthcare Center, Kwandong University School of Medicine) ;
  • Lim, Chun-Kyu (Laboratory of Reproductive Biology and Infertility, Cheil General Hospital and Women's Healthcare Center, Kwandong University School of Medicine) ;
  • Cho, Jae-Won (Laboratory of Reproductive Biology and Infertility, Cheil General Hospital and Women's Healthcare Center, Kwandong University School of Medicine) ;
  • Kim, Jin-Young (Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Kwandong University School of Medicine) ;
  • Koong, Mi-Kyoung (Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Kwandong University School of Medicine) ;
  • Son, In-Ok (Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Kwandong University School of Medicine) ;
  • Kang, Inn-Soo (Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Kwandong University School of Medicine) ;
  • Jun, Jin-Hyon (Laboratory of Reproductive Biology and Infertility, Cheil General Hospital and Women's Healthcare Center, Kwandong University School of Medicine)
  • 김민지 (관동대학교 의과대학 제일병원 생식생물학 및 불임연구실) ;
  • 이형송 (관동대학교 의과대학 제일병원 생식생물학 및 불임연구실) ;
  • 임천규 (관동대학교 의과대학 제일병원 생식생물학 및 불임연구실) ;
  • 조재원 (관동대학교 의과대학 제일병원 생식생물학 및 불임연구실) ;
  • 김진영 (관동대학교 의과대학 제일병원 생식생물학 및 산부인과학교실) ;
  • 궁미경 (관동대학교 의과대학 제일병원 생식생물학 및 산부인과학교실) ;
  • 송인옥 (관동대학교 의과대학 제일병원 생식생물학 및 산부인과학교실) ;
  • 강인수 (관동대학교 의과대학 제일병원 생식생물학 및 산부인과학교실) ;
  • 전진현 (관동대학교 의과대학 제일병원 생식생물학 및 불임연구실)
  • Published : 2007.09.30
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Abstract

Objectives: Many neurological diseases are known to be caused by expansion of trinucleotide repeats (TNRs). It is hard to diagnose the alteration of TNRs with single cell level for preimplantation genetic diagnosis (PGD). In this study, we describe methods optimized for PGD of TNRs related diseases such as Huntington's disease (HD), spinocerebellar ataxia 3 (SCA3) and fragile X syndrome (FXS). Methods: We performed the preclinical assays with heterozygous patient's lymphocytes by single cell PCR strategy. Fluorescent semi-nested PCR and fragment analysis using automatic genetic analyzer were applied for HD and SCA 3. Whole genome amplification with multiple displacement amplification (MDA) method and fluorescent PCR were carried out for FXS. Amplification and allele drop-out (ADO) rate were evaluated in each case. Results: The fluorescent semi-nested PCR of single lymphocyte showed 100.0% of amplification and 14.0% of ADO rate in HD, and 94.7% of amplification and 5.6% of ADO rate in SCA3, respectively. We could not detect the PCR product of CGG repeats in FXS using the fluorescent semi-nested PCR alone. After applying the MDA method in FXS, 84.2% of amplification and 31.3% of ADO rate were achieved. Conclusions: Fluorescent semi-nested PCR is a reliable method for PGD of HD and SCA3. The advanced MDA method overcomes the problem of amplification failure in CGG repeats of FXS case. Optimization of methods for single cell analysis could improve the sensitivity and reliability of PGD for complicated single gene disorders of TNRs.

목 적: 본 연구에서는 삼핵산 반복서열 확장에 의해 발병하는 헌팅톤병, 척수소뇌성 운동실조증과 X-염색체 취약 증후군 등에 대한 착상전 유전진단을 시행하기 위한 전임상 검사에서 진단 방법을 최적화하는 과정을 통해 얻은 결과들에 대해 기술하고자 한다. 연구방법: 단일 림프구를 이용한 임상전 검사에서는 서로 다른 allele를 갖고 있는 환자의 단일 세포를 사용하였으며, 헌팅톤병과 척수소뇌성 운동실조증에서는 fluorescent semi-nested PCR 시행 후 fragment analysis를 수행하였다. X-염색체 취약 증후군의 경우 multiple displacement amplification (MDA) 방법을 이용한 whole genome amplification에서 얻어진 MDA 산물로 fluorescent PCR을 시도하였다. 결 과: 헌팅톤병의 경우 단일 림프구 시료 모두에서 CAG repeats 증폭에 성공하여 100.0%의 증폭성공률과 14.0% allele drop-out (ADO) rate를, 척수소뇌성 운동실조증의 경우 94.7%의 증폭성공률과 5.6%의 ADO rate을 나타내었다. X-염색체 취약 증후군의 경우 fluorescent semi-nested PCR 방법만으로는 단일 림프구 시료에서 CGG repeats이 증폭되지 않았지만, MDA 산물을 이용한 fluorescent PCR 결과 84.2%의 증폭성공률과 31.3%의 ADO rate을 얻을 수 있었다. 결 론: 본 연구를 통해 헌팅톤병과 척수소뇌성 운동실조증의 착상전 유전진단에는 fluorescent semi-nested PCR 방법의 적용이 가능함을 확인하였으며, X-염색체 취약 증후군의 경우에는 MDA를 이용한 fluorescent PCR 방법을 사용해야 함을 알 수 있었다. 유전자의 변이에 대한 분석이 쉽지 않은 단일 유전자 이상에 대한 착상전 유전진단의 경우 다양한 유전자 분석 방법을 이용한 단일 세포에서의 진단 방법의 최적화 연구가 필수적으로 선행되어야 할 것으로 사료된다.

Keywords

References

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