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대한생식의학회지 제35권 제2호 2010년
골형성부전증의 착상전 유전진단을 위한 분자유전학적 방법의 조건 확립과 적용
관동대학교 의과대학 제일병원 생식생물학 및 불임연구실1, 산부인과학교실2
김민지1, 이형송1, 최혜원1, 임천규1, 조재원1, 김진영2, 송인옥2, 강인수2*,
Establishment and Application of Molecular Genetic Techniques for Preimplantation Genetic Diagnosis of Osteogenesis Imperfecta
Min Jee Kim1, Hyoung-Song Lee1, Hye Won Choi1, Chun Kyu Lim1, Jae Won Cho1, Jin Young Kim2, In Ok Song2, Inn Soo Kang2*
1Laboratory of Reproductive Biology and Infertility, 2Department of Obstetrics and Gynecology, Cheil General Hospital and Women's Healthcare Center, Kwandong University College of Medicine, Seoul, Korea
.
Objectives: Preimplantation genetic diagnosis (PGD) has become an assisted reproductive technique for couples carrying genetic conditions that may affect their offspring. Osteogenesis imperfecta (OI) is an autosomal dominant disorder of connective tissue characterized by bone fragility and low bone mass. At least 95% of cases are caused by dominant mutations in the COL1A1 or COL1A2. In this study, we report on our experience clinical outcomes with 5 PGD cycles for OI in two couples. Methods: Before clinical PGD, we assessed the amplification rate and allele drop-out (ADO) rate of alkaline lysis and nested PCR protocol using heterozygous patient's single lymphocytes in the pre-clinical diagnostic tests for OI. We performed 5 cycles of PGD for OI by nested PCR for the causative mutation loci, COL1A1 c.2452G>A and c.3226G>A, in case 1 and case 2, respectively. The PCR products were analyzed by agarose gel electrophoresis, restriction fragment length polymorphism (RFLP) analysis with HaeIII restriction enzyme in the case 1 and direct DNA sequencing. Results: We confirmed the causative mutation loci, COL1A1 c.2452G>A in case 1 and c.3226G>A in case 2. In the pre-clinical tests, the amplification rate was 94.2% and ADO rate was 22.5% in case 1, while 98.1% and 1.9% in case 2, respectively. In case 1, a total of 34 embryos were analyzed and 31 embryos (91.2%) were successfully diagnosed in 3 PGD cycles. Eight out of 19 embryos diagnosed as unaffected embryos were transferred in all 3 cycles, and in the third cycle, pregnancy was achieved and a healthy baby was delivered without any complications in July, 2005. In case 2, all 19 embryos (100.0%) were successfully diagnosed and 4 out of 11 unaffected embryos were transferred in 2 cycles. Pregnancy was achieved in the second cycle and the healthy baby was delivered in March, 2008. The causative locus was confirmed as a normal by amniocentesis and postnatal diagnosis. Conclusions: To our knowledge, these two cases are the first successful PGD for OI in Korea. Our experience provides a further demonstration that PGD is a reliable and effective clinical techniques and a useful option for many couples with a high risk of transmitting a genetic disease.
키워드 : Preimplantation genetic diagnosis (PGD), Osteogenesis imperfecta (OI), COL1A1, COL1A2, Allele drop-out (ADO)
교신저자 : ikang67@yahoo.co.kr
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