한국 남성 불임환자에서 Protamine 1과 2 유전자의 Single Nucleotide Polymorphism에 관한 연구

이형송,최혜원,박용석,서주태,궁미경,전진현,Lee, Hyoung-Song,Choi, Hye Won,Park, Yong-Seog,Seo, Ju Tae,Koong, Mi Kyoung,Jun, Jin Hyun 대한생식의학회 2005 Clinical and Experimental Reproductive Medicine Vol.32 No.3

Objective: Although several genetic factors have been associated with defects in human spermatogenesis, the unambiguous causative genes have not been elucidated. The male infertility by haploinsufficiency of PRM1 or PRM2 has been reported in mouse model. The aim of this study was to identify the single nucleotide polymorphisms (SNPs) of PRM1 and PRM2, related to the genotype of Korean infertile men. Methods: Genomic DNAs were extracted from peripheral bloods of infertile men with oligozoospermia or azoospermia, and analyzed using polymerase chain reaction (PCR) and direct sequencing. We carried out the direct sequencing analysis of amplified fragments in PRM1 (557 nucleotides from -42 to 515) and PRM2 (599 nucleotides from 49 to 648) genes, respectively. Results: Three SNPs of coding region in the PRM1 gene was found in the analysis of 130 infertile men. However, the SNPs at a133g (aa 96.9%, ag 3.1% and gg 0.0%), c160a (cc 99.2%, ca 0.8% and aa 0.0%) and c321a (cc 56.9%, ca 35.4% and aa 7.7%) coded the same amino acids, in terms of silence phenotypes. On the other hand, as results of the PRM2 gene sequencing in 164 infertile men, only two SNPs, g398c (gg 62.2%, gc 31.1% and ga 6.7%) and a473c (aa 63.4%, ac 29.9% and cc 6.7%), were identified in the intron of the PRM2 gene. Conclusions: There was no mutation and significant SNPs on PRM1 and PRM2 gene in Korean infertile men. These results suggest that the PRM1 and PRM2 genes are highly conserved and essential for normal fertility of men.

근이영양증에 대한 착상전 유전진단에서 Duplex-nested PCR과 Fluorescent PCR 방법의 효용성

이형송,최혜원,임천규,박소연,김진영,궁미경,전진현,강인수,Lee, Hyoung-Song,Choi, Hye Won,Lim, Chun Kyu,Park, So Yeon,Kim, Jin Young,Koong, Mi Kyoung,Jun, Jin Hyun,Kang, Inn Soo 대한생식의학회 2005 Clinical and Experimental Reproductive Medicine Vol.32 No.1

Objective: Preimplantation genetic diagnosis (PGD) is reserved for couples with a risk of transmitting a serious and incurable disease, and hence avoids the undesirable therapeutic abortion. In this study, we evaluated the efficacy of PGD for Duchenne muscular dystrophy (DMD) cases by the fluorescent PCR with polymorphic linked markers and the conventional duplex-nested PCR methods. Methods: Biopsy of one or two blastomeres was done from the embryos fertilized by ICSI on the third day after fertilization. We performed two cases of PGD-DMD by the duplex-nested PCR for the causative mutation loci and the SRY gene on Y chromosome. The triplex fluorescent PCR for the mutation loci, the SRY gene and the polymorphic microsatellite marker on X chromosome was applied for two cases of PGD-DMD. Results: By the duplex-nested PCR, successful diagnosis rate was 95.5% (21/22), but we could not discriminate the female embryos whether normal or carrier in this X-linked recessive disease. However, the triplex fluorescent PCR method showed 100% (27/27) of successful diagnosis rate, and all female embryos (n=17) were distinguished normal (n=10) from carrier (n=7) embryos. Unaffected and normal embryos were transferred into mother's uterus after diagnosis. A healthy normal male was achieved after PGD with the duplex-nested PCR method and a twin, a male and a female, were delivered with triplex fluorescent PCR method. The normality of dystrophin gene was confirmed by amniocentesis and postnatal genetic analysis in all offsprings. Conclusion: The fluorescent PCR with polymorphic marker might be useful in improving the specificity and reliability of PGD for single gene disorders.

Y 염색체 미세결실과 정자형성장애의 연관성에 대한 연구

이형송,최혜원,박용석,궁미경,강인수,윤종민,이유식,서주태,전진현,Lee, Hyoung-Song,Choi, Hye-Won,Park, Yong-Seog,Koong, Mi-Kyoung,Kang, Inn-Soo,Yun, Jong-Min,Lee, You-Sik,Seo, Ju-Tae,Jun, Jin-Hyun 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.4

Objective s: To estimate the frequency of Y chromosome microdeletions in the Korean population of infertile men and to evaluate the relationship between microdeletion on the Y chromosome and clinical phenotypes of infertile men with idiopathic azoospermia and oligozoospermia. Materials and Methods: Genomic DNA was extracted from blood samples collected from 330 infertile men attending the Infertility Clinic at Samsung Cheil Hospital, Korea. Six sequence tagged sites (STSs) spanning the azoospermia factor (AZF) regions of the Y chromosome were amplified by polymerase chain reactions (PCRs). Results: Microdeletions on Y chromosome were detected in 35 (10.6%) of the 330 infertile men. Most of the microdeletions (91.4%) involved AZFb or AZFc. The high incidence of microdeletions were found in AZFc region (57.1%), but the low in AZFa (8.6%) and AZFb (5.7%). Larger microdeletions involving two or three AZF regions were detected in 28.6% of cases. All patients (6 patients) with deletion of AZFa region showed no germ cell phenotypes, Sertoli cell only syndrome or Leydig cell hyperplasia in histopathologic examinations. Conclusion: Microdeletions on the Y chromosome, especially, at AZFc/DAZ regions may be the major cause of azoospermia and severe oligozoospermia. We suggest that idiopathic infertile men have genetic counselling and microdeletion analysis on the Y chromosome before IVF-ET and ART program.

Ornithine transcarbamylase (OTC) 효소결핍증, 수포성 표피박리증 및 lactic acidosis 가계에서 duplex nested PCR 방법을 이용한 착상전 유전진단: OTC 효소결핍증 가계에서의 정상아 임신 및 출산

이형송 ( Lee Hyeong Song ),최혜원 ( Choe Hye Won ),임천규 ( Im Cheon Gyu ),민동미 ( Min Dong Mi ),변혜경 ( Byeon Hye Gyeong ),김진영 ( Kim Jin Yeong ),궁미경 ( Gung Mi Gyeong ),유한욱 ( Yu Han Ug ),김수찬 ( Kim Su Chan ),전진현 ( 대한산부인과학회 2004 Obstetrics & Gynecology Science Vol.47 No.4

목적: 착상전 유전진단 (preimplantation genetic diagnosis: PGD)은 체외수정에서 획득한 수정란으로부터 할구를 분리한 후 염색체 또는 유전자 검사를 통해 정상적인 배아만을 선별적으로 자궁에 이식함으로써 유전적으로 이상이 있는 환아를 출산할 확률이 높은 부부들에서 환아의 임신과 출산을 예방할 수 있다. 본 연구에서는 체외수정을 통해 얻어진 배아에서 하나 혹은 두개의 할구를 분리하고, duplex nested PCR 방법을 사용 Objective: Preimplantation genetic diagnosis (PGD) is reserved for couples with a risk of transmitting a serious and incurable disease, and hence avoids the undesirable therapeutic abortion. Herein, we report the result of PGD to carriers at risk of trans

Congenital Adrenal Hyperplasia 환자에서 21 - hydroxylase ( CYP21 ) 유전자 돌연변이의 분자 진단에 관한 연구

이형송(Hyoung Song Lee),천강우(Kang Woo Cheon),박용석(Yong Seok Park),한인권(In Kwon Han),강인수(Inn Soo Kang) 대한산부인과학회 2001 Obstetrics & Gynecology Science Vol.44 No.6

N/A Objective: Congenital adrenal hyperplasia (CAH) is an autosomal recessive diseav which is most often caused by a deficiency in steroid 21-hydroxylase (21-0H), a microsomal enzyme encoded by the CYP21 gene. Although several CAH causing mutations have been identified in the CYP21 gene of patients with 21-OH deficiency, genotyping of the 21-OH locus is quite complex because of the high frequency of gene conversion and the presence of multiple mutations on single CAH alleles. This study was aimed to analyze the complete characterization of the CYP21 gene coding region in a Korean CAH patient and to conform the PCR-based single strand conformation polymorphism (SSCP) and heteroduplex analysis as a diagnostic tool. Methods: We used a highly sensitive, non-radioactive method allowing PCR-based single strand c<formation polymorphism (SSCP) analysis. This method was applied to the characterization of all the exons and intron-exon junctions of the CYP21 gene in one patients affected by the salt wasting form and 4 normal controls. Results: In all samples showing SSCP abnormal band pattems, sequence analysis showed the presence of sequence variants. In particular, one mutation (I172N) which is already known to cause the disease and 3 silent mutations were detected. Conclusion: PCR-based single strand conformation polymorphism (SSCP) and heteroduplex analysis should be useful for the clinical application as a diagnostic tool for the detection of 21-hydroxylase gene mutations.

단일 유전자 질환에 대한 착상전 유전진단

이형송(Hyoung-Song Lee),김민지(Min Jee Kim),강인수(Inn Soo Kang) 대한의학유전학회 2009 대한의학유전학회지 Vol.6 No.2

착상전 유전진단은 유전질환이 이환될 가능성이 있는 부부들을 대상으로 산전진단을 통한 임신중절의 위험성 없이 정상적인 아이를 가질 수 있게 도와주는 보조생식술의 한 방법으로 확립되었다 . 단일 할구를 대상으로 하는 분자생물학 및 분자생물학적 기술의 발전은 착상전 유전진단의 정확성을 높은 수준에 이르게 하였고 whole genome amplification 방법을 이용함으로써 단일세포로부터 여러 가지 다양한 진단을 동시에 수행 가능케 하였으며 단일 유전자 질환에 대한 착상전 유전진단에서의 오진을 감소시킬 수 있었다. 따라서 PCR을 이용한 단일 유전자 질환에 대한 착상전 유전진단의 적용가능 유전질환은 더욱 확대될 것이며 건강한 아이의 출산을 원하는 더 많은 부부들에게 기회를 제공해 줄 것이다. 본 종설에서는 현재 단일유전자 질환에 대한 착상전 유전진단을 시행하는 대부분의 센터에서 시행하고 있는 생검 방법과 multiplex PCR, PCR 후 진단 방법, 그리고 multiple displacement amplification 등의 분자생물학적 방법과 단일 세포 분석에서의 문제점 등을 포함한 단일 유전자 질환에 대한 착상전 유전진단 전반에 관하여 논의할 것이다. 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.

착상 전 유전진단

김진영,이형송,강인수 대한의사협회 2015 대한의사협회지 Vol.58 No.11

Preimplantation genetic diagnosis (PGD) is a technique to examine genetic disease or chromosome abnormalities in single cell biopsied from embryos before implantation to uterus. It allows achieving normal pregnancy by transfer of unaffected embryos. The main indications are single gene disorders and recurrent miscarriage related to chromosome aberration and it has advantages to avoid termination of pregnancy or miscarriages in couples with high risk. PGD is also widely applied for aneuploidy screening in assisted reproduction to improve the outcome in infertile patients such as advanced maternal age, although its efficacy still needs to be established. Furthermore, the application of PGD has expanded to other indications, such as late onset-diseases with genetic predisposition and human leukocyte antigen typing for stem cell transplantation. With the advances of molecular diagnostic technologies using single cells, such as fluorescent in situ hybridization, multiplex polymerase chain reaction, fluorescent polymerase chain reaction, linkage analysis, whole genome amplification, array comparative genomic hybridization (array comparative genomic hybridization), and next generation sequencing, PGD can provide more comprehensive and reliable diagnosis.

한국 남성 불임환자에서 Y 염색체상의 AZF Gene에 대한 분석 및 DAZ Gene의 발현 양상

이호준,이형송,송견지,변혜경,서주태,김종현,이유식,Lee, Ho-Joon,Lee, Hyoung-Song,Song, Gyun-Jee,Byun, Hye-Kyung,Seo, Ju-Tae,Kim, Jong-Hyun,Lee, You-Sik 대한생식의학회 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.1

Cytogenetic observations of loss of the distal portion of the Y chromosome long arm were found to be associated with disrupted spermatogenesis. The existence of a gene involved in the regulation of spermatogenesis, the azoospermia factor (AZF), was postulated. In this study, we screened the AZF region including DAZ and DAZH genes and observed the expression pattern of DAZ and DAZH transcript in infertile men with azoospermia and oligospermia by using a sequence-tagged site (STS)-based PCR method. PCR primers were synthesized for 11 STSs that span Yq interval 6, SRY, DAZ, and DAZH, functional DAZ homologue on chromosome 3. Microdeletions were detected in 4/32 (12.5%) azoospermic men and 1/11 (9%) severe oligospermic men. Only 2 of 5 patients had microdeletions of Yq that contained the DAZ gene, whereas the other 3 patients had deletions extending from intervals 5L-6F proximal to the DAZ gene on Yq. Testis biopsies of the azoospermic patients revealed a variety from Sertoli cell-only syndrome to testicular maturation arrest. Of 4 men with clinical data available, average testis size was R: 13.8 cc, L: 13.8 cc, serum T was $4.0{\pm}1.25$ ng/ml, LH was $3.63{\pm}1.90$ mIU/ml, and FSH was $8.85{\pm}5.13$ mIU/ml. These values did not differ significantly from the remainder of the patients tested. We could not observed the DAZ transcript in 2 patients, who have no mature spermatozoa. In 11.6% of patients microdeletions of the AZF could be detected. These deletions in the AZF region seem to be involved causing spermatogenic failure. But the frequency of microdeletions proximal to DAZ suggests that DAZ is not the only gene associated with spermatogenic failure.

폐쇄성 무정자증 환자의 신선고환조직 정자와 동결고환조직 정자의 운동성이 임신율에 미치는 영향

박용석,이형송,변혜경,염혜원,송상진,임천규,이유식,윤종민,서주태,송지홍,강인수,궁미경,Park, Yong-Seog,Lee, Hyoung-Song,Byun, Hye-Kyung,Youm, Hye-Won,Song, Sang-Jin,Lim, Chun-Kyu,Lee, You-Sik,Yun, Jong-Min,Seo, Ju-Tae,Song, Ji-Hong,Kang, I 대한생식의학회 2001 Clinical and Experimental Reproductive Medicine Vol.28 No.2

Objective: ICSI with testicular sperm could achieve optimal fertilization and pregnancy. This study was performed to observe the influence on fertilization and pregnancy of motility of fresh testicular sperm and sperm extracted from frozen-thawed seminiferous tubules in obstructive azoospermia. Materials and Methods: We analysed clinical outcome of ICSI using fresh testicular sperm and sperm extracted from thawed seminiferous tubules. The presence of motility were compared to determine the factor for optimal fertilization and pregnancy rates. Results: In 316 cases of TESE-ICSI in obstructive azoospermia, ICSI with fresh testicular sperm (fresh sperm group) were 163 cases and ICSI with sperm testicular sperm extracted from frozen-thawed seminiferous tubule (thawed sperm group) were 153 cases. The fertilization rates were 71.3% and pregnancy rates were 32.5% in fresh sperm group, in thawed sperm group, 65.1% and 33.3% respectively. The fertilization and pregnancy rates of motile and non-motile testicular sperm were 72.9% and 33.6%, 50.0% and 18.2%, respectively (p<0.05). The fertilization and pregnancy rates of motile and non-motile sperm extracted from the thawed seminiferous tubule were 67.8% and 34.7%, 55.1% and 28.1%, respectively (p<0.05). The comparative of the results of ICSI using motile fresh testicular sperm and motile sperm extracted from thawed seminiferous tubule, fertilization and pregnancy rates were not significantly different (72.9% and 33.6%, 67.8% and 34.7%, respectively). Conclusion: These results suggest that successful pregnancy in TESE-ICSI treatment is influenced by the motility of fresh testicular sperm and sperm extracted from thawed seminiferous tubule in obstructive azoospennic patients.

Reliability of the Single Cell PCR analysis for Preimplantation Genetic Diagnosis of Single Gene Disorders

최혜원,이형송,임천규,궁미경,강인수,전진현,Choi, Hye Won,Lee, Hyoung-Song,Lim, Chun Kyu,Koong, Mi Kyoung,Kang, Inn Soo,Jun, Jin Hyun The Korean Society for Reproductive Medicine 2005 Clinical and Experimental Reproductive Medicine Vol.32 No.4

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.