• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.105-105
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• 2002

염색체의 구조적 이상으로 인한 습관성 유산과 기형아의 출산을 예방하기 위해 착상전 배아에서 할구를 분석하여 정상적인 핵형을 가진 배아만을 이식하는 착상전 유전자 진단 (preimplantation genetic diagnosis, PGD)의 성공적인 임상 적용이 보고되고 있으며, 그 적용 범위가 확대되고 있다. 그러나 일반적인 간기의 핵상을 이용한 PGD에서는 형광직접보합법 probe의 제약으로 보인자와 정상적인 핵형을 구분할 수 없는 단점이 있다. 따라서 본 연구에서는 보다 정확한 PGD를 위해 생쥐 배아를 이용하여 분리한 할구에서 중기 염색체상을 획득하기 위해 미세소관 (microtubule) 형성 저해제를 처리하였으며, 이를 통해 확립된 방법을 인간의 PGD에 적용하고자 하였다. 과배란이 유도된 ICR 생쥐에서 4- 또는 8-세포기 배아를 수획하여 colcemid, nocodazole, vinblastine을 각각 0.1, 0.5, 1.0, 5.0$\mu$M을 처리하고, hoechst 33342로 염색하여 핵상을 관찰하여 최적의 농도를 결정하였다. 또한 각 미세소관 형성 저해제를 혼합 처리하여 가장 높은 중기 염색체상을 획득할 수 있는 혼합 처리를 결정하였다. 이렇게 결정된 혼합 처리 방법을 인간의 체외 수정 및 배아 이식술에서 획득된 3PN 배아에 처리하여 중기 염색체를 획득하였다. Colcemid, nocodazole, vinblastine 모두 1 $\mu$M이 최적 농도임을 확인할 수 있었다 (각각 96.3%, 92.0%, 98,4%). 미세소관 형성저해제를 혼합 처리하였을 경우 nocodazole과 vinblastine (각각 1$\mu$M)을 혼합 처리했을 때 중기 염색체 획득률(97.3%)이 가장 높았다. 인간의 3PN 배아에 1$\mu$M의 nocodazole과 vinblastine을 혼합 처리한 후, 113개의 할구를 분석하여 44개(38.9%)의 할구에서 중기 염색체를 확인할 수 있었다. 본 실험 결과를 통해 중기 염색체를 획득하기 위하여 미세소관 형성 저해제를 처리하는 방법은 생쥐의 배아에서는 효과적이지만, 인간의 배아에서는 그 효율이 다소 낮음을 알 수 있었다. 그러나 이 방법을 개선하여 인간의 할구에서 중기 염색체의 획득률을 높이고, 이를 염색체의 구조적 이상에 대한 착상전 유전자 진단에 적용한다면, 보인자와 정상의 핵상을 구분하여 정상의 핵상만을 갖는 배아의 이식을 통하여 더욱 정확한 착상전 유전자 진단을 시행할 수 있으리라 사료된다.

• Journal of Genetic Medicine
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• v.6 no.2
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• pp.131-145
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• 2009

Preimplantation genetic diagnosis (PGD) has become an assisted reproductive technique for couples who are at risk that enables them to have unaffected baby without facing the risk of pregnancy termination after invasive prenatal diagnosis. The molecular biology and technology for single-cell genetics has reached an extremely high level of accuracy, and has enabled the possibility of performing multiple diagnoses on one cell using whole genome amplification. These technological advances have contributed to the avoidance of misdiagnosis in PGD for single gene disorders. Polymerase chain reaction (PCR)-based PGD will lead to a significant increase in the number of disorders diagnosed and will find more widespread use, benefiting many more couples who are at risk of transmitting an inherited disease to their baby. In this review, we will focus on the molecular biological techniques that are currently in use in the most advanced centers for PGD for single gene disorders, including biopsy procedure, multiplex PCR and post-PCR diagnostic methods, and multiple displacement amplification (MDA) and the problems in the single cell genetic analysis.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.2
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• pp.99-110
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• 2008

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.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.4
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• pp.293-300
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• 2005

연구목적: 단일 유전자 이상에 대한 착상전 유전진단을 성공적으로 시행하기 위해서는 효과적이고 신뢰도가 높은 PCR 방법의 확립이 중요하다. 본 연구에서는 alkaline lysis와 duplex nested PCR 방법을 단일 림프구와 할구의 유전자 분석에 적용하여 그 효용성을 확인하고자 하였다. 재료 및 방법: 단일 유전자의 이상이 확인된 Duchenne muscular dystrophy (DMD), ornithine transcarbamylase (OTC) 결핍증과 epidermolysis bullosa (EB) 가계의 대상자들에서 채취한 단일 림프구와 공여 받은 배아의 할구를 이용하여 각각 PCR, restriction fragment length polymorphism (RFLP)와 direct DNA sequencing 분석을 시행하였다. 이러한 분석에서 유전자 증폭률 (amplification rate)과 두개의 allele 중에서 하나의 allele이 증폭되지 않는 allele drop-out (ADO) 빈도에 대해 살펴보았다. 결 과: 단일 림프구와 할구를 이용한 PCR 방법의 유전자 증폭률은 DMD에서 91.1%와 81.8%, OTC 결핍증에서 96.0%와 78.1%, EB에서 91.3%와 90.0%를 각각 나타냈으며, ADO 빈도는 OTC 결핍증에서 13.3%, EB에서 16.8%로 관찰되었다. 결 론: 본 연구에서 적용한 alkaline lysis와 duplex nested PCR 방법은 단일 유전자에 대한 착상전 유전진단에 성공적으로 적용할 수 있는 방법으로 생각되며, ADO 빈도를 최소화할 수 있는 효율적인 방법의 개발에 대한 지속적인 연구가 필요하다.

• Clinical and Experimental Reproductive Medicine
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• v.34 no.3
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• pp.179-188
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• 2007

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.

• 대한생식의학회:학술대회논문집
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• 2003.12a
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• pp.83-84
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• 2003

• Journal of Embryo Transfer
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• v.14 no.1
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• pp.3-5
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• 1999

이식 전 수정란의 성판정은 많은 축산인의 소망이었다. 많은 방법이 제기되었지만, 세포유전학적 분석방법은 ET의 상용화에 거의 무의미할 정도로 한계가 많았고, 이후 개발된 응성 특이적 항원이나 X-염색체에서 발현되는 효소의 활동을 통한 성판정 방법은 흥미롭기는 하나 산업적 적용에 제약이 많았다. 80년대 중반 Y-염색체 특이적인 DNA 탐침자의 개발과 PCR을 통한 DNA증폭기술의 개발은 수정란 성판정을 획기적으로 개선시켰다. DNA기술을 통한 수정란의 성판정은 분자생물학을 현장으로 진출시킨 첫 경우이기도 했다. 90년대에 세포유전학적 분석에서 FISH가 소개되었으며, 염색체 특이적 library는 다른 기초적이고 응용세포유전학적 연구에 유용한 도구가 되었다. 최근에는 사람에서는 착상전 수정란의 성판별 및 유전진단을 위해 실시되고 있는 형광직접접합법(fluorescent in situ hybridization, FISH)은 효소적 유전자 증폭(polymerase chain reaction; PCR)에 비해 높은 민감도와 정확성을 보이고 있으나 hybridization 및 washing 과정에 매우 긴 시간이 소요되고, 절차도 까다로워 현장에서의 적용이 용이치 않았다. 그러나 direct labelled probe의 이용, heat programmable instrument의 개발, denaturing chemical의 사용배제 등을 통해 소요시간 및 절차의 대폭적인 간소화를 이루어 현장의 적용 가능성을 한층 높이고 있다. 현재 사람의 세포유전학 및 종양학에서는 FISH의 다양한 기술이 많이 이용하고 있으나 소에서는 탐침자(probe)가 개발되어 있지 않아 그 이용이 미미하다. 본 연구는 FISH를 이용하기 위한 탐침자의 개발을 궁극적인 목표로 삼았으며, 이를 위해 접근이 용이한 방식을 개발하여 기존의 방식과는 다른 소 배아세포의 성을 판별 할 수 있는 접근방법을 소개 하고자한다.

• Journal of Genetic Medicine
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• v.4 no.2
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• pp.186-189
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• 2007

More than 6,000 rare disorders including genetic diseases have been reported. Of them, 1,500 diseases (1,211 for clinical diagnosis and 289 for research only) are technically possible for genetic testing. In Korea, since 2005, only 63 genetic diseases is permitted for prenatal genetic testing by the "Bioethics and Biosafety Law". The article 25 in the law prescribes 63 genetic diseases without clear indication for its selection and inclusion criteria. In EU, USA, and other foreign countries, however, there is no provision in the statute on prenatal genetic testing; it is not restricted by a law. Recently, a woman (Mrs. L, 38y) who is a carrier for Menkes disease made an appeal to a government for an amendment of the "Bioethics and Biosafety Law" prohibiting the prenatal diagnosis of her pregnancy at risk for Menkes disease. Menkes disease (MNK) is an X-linked recessive disorder characterized by neurodegeneration, connective tissue defects and hair abnormalities, and no effective treatment is available yet. The prevalence rate of MNK is one in about 250,000 live births. Menkes syndrome patients fail to absorb copper from the gastrointestinal tract in quantities adequate for meeting nutritional needs. These needs seem particularly acute during the initial 12 month of life, when the velocity of brain growth and motor neurodevelopment. Most of pts. die around 3yrs. of age. Mrs. L had a boy with Menkes disease who died at 2y.o. in 2001. Subsequent pregnancy in 2003, she was able to have prenatal genetic testing for mutation of the Menkes (ATP7A) gene and delivered a healthy baby boy. Now, She is pregnant again and wants to have prenatal diagnosis. however, this time, she was not allowed to have any more because Menkes disease is not included in 63 genetic diseases permitted by the law for prenatal genetic testing, in spite of the fact that she is a Menkes disease carrier and her pregnancy is at risk to have an affected baby. This case shows the practical problem of the legal restriction for prenatal genetic testing in Korea. In this study, we report a arguable case and discuss the controversial issues in the legal restriction for prenatal genetic testing in Korea.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.109-114
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• 1996

General PCR technique alone has a limitation for preimplantation genetic diagnosis(PGD) using single blastomere. Recntly developed primer extension preamplification(PEP) technology amplifies the whole genome and thus, simultaneous multiple locus analysis became possible. In this study, we report the efficacy of PEP-PCR for PGD in three muscular dystrophy carriers undergoing IVF-ET. A total of 37 blastomeres were obtained from 40 embryos at six to eight cell stage in three IVF cycles in two DMD and one BMD carriers. Whole genome from single blastomeres were amplified using I5-base oligonucleotide random primers. PCR amplified products of exon 45 in the dystrophin gene and alphoid X/Y loci for gender determination were analysed by 2% metaphor gel electrophoresis. A total of 37 PEP-PCR replicates from 37 single blastomeres from 40 embryos and 37 blanks were performed. We obtained the reliable results for exon 45 and alphoid X/Y. Transfer of female embryos and unaffected male embryo was attempted in three couples. Unfortunately, pregnancy was not achieved in these cases. PEP-PCR is a reliable and efficient PGD method in multiple locus analysis using single blastomere.

• Journal of Genetic Medicine
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• v.7 no.2
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• pp.125-132
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• 2010

Purpose: Preimplantation genetic diagnosis (PGD), also known as embryo screening, is a pre-pregnancy technique used to identify genetic defects in embryos created through in vitro fertilization. PGD is considered a means of prenatal diagnosis of genetic abnormalities. PGD is used when one or both genetic parents has a known genetic abnormality; testing is performed on an embryo to determine if it also carries the genetic abnormality. The main advantage of PGD is the avoidance of selective pregnancy termination as it imparts a high likelihood that the baby will be free of the disease under consideration. The application of PGD to genetic practices, reproductive medicine, and genetic counseling is becoming the key component of fertility practice because of the need to develop a custom PGD design for each couple. Materials and Methods: In this study, a survey on the contents of genetic counseling in PGD was carried out via direct contact or e-mail with the patients and specialists who had experienced PGD during the three months from February to April 2010. Results: A total of 91 persons including 60 patients, 49 of whom had a chromosomal disorder and 11 of whom had a single gene disorder, and 31 PGD specialists responded to the survey. Analysis of the survey results revealed that all respondents were well aware of the importance of genetic counseling in all steps of PGD including planning, operation, and follow-up. The patient group responded that the possibility of unexpected results (51.7%), genetic risk assessment and recurrence risk (46.7%), the reproduction options (46.7%), the procedure and limitation of PGD (43.3%) and the information of PGD technology (35.0%) should be included as a genetic counseling information. In detail, 51.7% of patients wanted to be counseled for the possibility of unexpected results and the recurrence risk, while 46.7% wanted to know their reproduction options (46.7%). Approximately 96.7% of specialists replied that a non-M.D. genetic counselor is necessary for effective and systematic genetic counseling in PGD because it is difficult for physicians to offer satisfying information to patients due to lack of counseling time and specific knowledge of the disorders. Conclusions: The information from the survey provides important insight into the overall present situation of genetic counseling for PGD in Korea. The survey results demonstrated that there is a general awareness that genetic counseling is essential for PGD, suggesting that appropriate genetic counseling may play a important role in the success of PGD. The establishment of genetic counseling guidelines for PGD may contribute to better planning and management strategies for PGD.