Shift of paradigm in prenatal diagnosis

No abstract available.

Characterization of Clones of Human Cell Line Infected with Porcine Endogenous Retrovirus (PERV) from Porcine Cell Line, PK-15 / 감염과화학요법

BACKGROUND: Porcine endogenous retroviruses (PERVs) form part of the chromosomes of all pigs. Since they can be produced as infectious virion and infect human cells, safety issues on PERVs infection to human are still controversial and is one of main hurdles of xenotransplantation using pig cells or organs. It has been reported that the established porcine cell line, PK-15, produces PERVs and can infect the human cell lines. Therefore, clonal analysis on human cell line infected with PERV is a prerequisite to characterize the infectivity to human cells and to investigate the harmfulness of PERVs to human. MATERIALS AND METHODS: For the characterization of PERV that originates from porcine cell line, PK-15, full length PERV cloning from genomic DNA of PK-15 was performed and partial sequences of both ends were achieved. Cell clones from human cell line, 293, persistently infected with PERVs from PK-15 were established by the method of limiting dilution. Nested PCR and direct sequencing of PCR products in each clone were carried out so as to confirm the PERV genomes in each clone. The growth rate of each clone was checked using cell counting and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, the infectivity by reverse transcriptase (RT) assay, and genetic analysis by karyotyping. RESULTS: A total of 12 genomic PERV clones could be retrieved; 1 with full length, 4 with defective forms, and others with irrelevant sequences. Intact PERV was thought to be able to infect 293 and the PERV-infected cell clones were selected by limiting dilution. PCR results confirmed that nine cell clones were infected with PERV, and sequence alignment data on PCR products of pol region from PK-15 and human cell clones with PERV showed very similar results. Cell counting and MTT assay for growth kinetics of each clone indicated that two clones showed reduced growth rate. However, it was difficult to verify the effect of PERV infection on the cell growth because of the presence of many genetic alterations in 293 parental cells. No RT activities were detected in the culture supernatant from PERV-infected 293 cell clones. CONCLUSION: The sequences of PERVs were detected in human cell clones after PERV infection, but PERV virions could not be detected from the culture supernatant by RT assay.

Characterization of Clones of Human Cell Line Infected with Porcine Endogenous Retrovirus (PERV) from Porcine Cell Line, PK-15 / 감염과화학요법

BACKGROUND: Porcine endogenous retroviruses (PERVs) form part of the chromosomes of all pigs. Since they can be produced as infectious virion and infect human cells, safety issues on PERVs infection to human are still controversial and is one of main hurdles of xenotransplantation using pig cells or organs. It has been reported that the established porcine cell line, PK-15, produces PERVs and can infect the human cell lines. Therefore, clonal analysis on human cell line infected with PERV is a prerequisite to characterize the infectivity to human cells and to investigate the harmfulness of PERVs to human. MATERIALS AND METHODS: For the characterization of PERV that originates from porcine cell line, PK-15, full length PERV cloning from genomic DNA of PK-15 was performed and partial sequences of both ends were achieved. Cell clones from human cell line, 293, persistently infected with PERVs from PK-15 were established by the method of limiting dilution. Nested PCR and direct sequencing of PCR products in each clone were carried out so as to confirm the PERV genomes in each clone. The growth rate of each clone was checked using cell counting and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, the infectivity by reverse transcriptase (RT) assay, and genetic analysis by karyotyping. RESULTS: A total of 12 genomic PERV clones could be retrieved; 1 with full length, 4 with defective forms, and others with irrelevant sequences. Intact PERV was thought to be able to infect 293 and the PERV-infected cell clones were selected by limiting dilution. PCR results confirmed that nine cell clones were infected with PERV, and sequence alignment data on PCR products of pol region from PK-15 and human cell clones with PERV showed very similar results. Cell counting and MTT assay for growth kinetics of each clone indicated that two clones showed reduced growth rate. However, it was difficult to verify the effect of PERV infection on the cell growth because of the presence of many genetic alterations in 293 parental cells. No RT activities were detected in the culture supernatant from PERV-infected 293 cell clones. CONCLUSION: The sequences of PERVs were detected in human cell clones after PERV infection, but PERV virions could not be detected from the culture supernatant by RT assay.

Carrier detection and prenatal diagnosis of hemophilia A in a Korean population by fluorescent pcr analysis of dinucleotide repeats in intron 13 and intron 22 of the factor VIII gene / 대한산부인과학회지

OBJECTIVE: To set up the methodology for fluorescent PCR analysis of intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene, and to identify the usefulness of intron 13 and intron 22 microsatellite polymorphism for the carrier detection and prenatal diagnosis of hemophilia A in the Korean population. METHODS: Intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene were analyzed in 30 unrelated Korean mothers of patients with severe hemophilia A using fluorescent PCR. RESULTS: Analysis of intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene was feasible by the fluorescent-PCR method. The expected heterozygosity rates of intron 13 and intron 22 polymorphisms of the factor VIII gene were 67% and 34%, respectively. Combined analysis of intron 13 and intron 22 polymorphisms revealed heterozygous patterns in 16 (53%) of 30 mothers studied. Using linkage analysis with intron 13 and intron 22 polymorphisms, we have attempted three cases of carrier detection and one cases of prenatal diagnosis in two families of patients with severe hemophilia A. CONCLUSION: These results suggest that flourescent-PCR analysis of the intron 13 and intron 22 microsatellite polymorphisms within the factor VIII gene is very useful in the carrier detection and prenatal diagnosis of hemophilia A in the Korean population.

Carrier detection and prenatal diagnosis of hemophilia A in a Korean population by fluorescent pcr analysis of dinucleotide repeats in intron 13 and intron 22 of the factor VIII gene / 대한산부인과학회지

OBJECTIVE: To set up the methodology for fluorescent PCR analysis of intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene, and to identify the usefulness of intron 13 and intron 22 microsatellite polymorphism for the carrier detection and prenatal diagnosis of hemophilia A in the Korean population. METHODS: Intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene were analyzed in 30 unrelated Korean mothers of patients with severe hemophilia A using fluorescent PCR. RESULTS: Analysis of intron 13 and intron 22 microsatellite polymorphisms of the factor VIII gene was feasible by the fluorescent-PCR method. The expected heterozygosity rates of intron 13 and intron 22 polymorphisms of the factor VIII gene were 67% and 34%, respectively. Combined analysis of intron 13 and intron 22 polymorphisms revealed heterozygous patterns in 16 (53%) of 30 mothers studied. Using linkage analysis with intron 13 and intron 22 polymorphisms, we have attempted three cases of carrier detection and one cases of prenatal diagnosis in two families of patients with severe hemophilia A. CONCLUSION: These results suggest that flourescent-PCR analysis of the intron 13 and intron 22 microsatellite polymorphisms within the factor VIII gene is very useful in the carrier detection and prenatal diagnosis of hemophilia A in the Korean population.

Molecular Genetic Diagnosis of Hemophilia A by Linkage Analysis of XbaI/intron 22 DNA Polymorphism Using PCR / 대한산부인과학회잡지

OBJECTIVE: To set up the methodology for PCR analysis of XbaI/intron 22 polymorphism of the factor VIII gene, and to identify the usefulness of XbaI/intron 22 polymorphism analysis for carrier detection and prenatal diagnosis of hemophilia A in the Korean population. DESIGN: A laboratory analysis. MATERIALS AND METHODS: A XbaI/intron 22 polymorphism of the factor VIII gene was analyzed in 56 unrelated Korean mothers of patients with severe hemophilia A, using polymerase chain reaction. RESULTS: Analysis of XbaI/intron 22 polymorphisms of the factor VIII gene were feasible by PCR method. The expected heterozygosity rates of XbaI/intron 22 polymorphism of the factor VIII gene were 44.8%. Analysis of XbaI/intron 22 polymorphism revealed heterozygous patterns in 22 (39.3%) of 56 mothers studied. Using linkage analysis with XbaI/intron 22 polymorphism, we have attempted one case of carrier detection and two cases of prenatal diagnosis in two families of patients with severe hemophilia A. CONCLUSION: These results suggest that PCR analysis of the XbaI/intron 22 polymorphism within the factor VIII gene is very useful in the carrier detection and prenatal diagnosis of hemophilia A in the Korean population.

The analysis of fetal gender and BclI polymorphism with fetal cells in maternal blood / 대한산부인과학회잡지

OBJECTIVE: We used nucleated erythrocytes in maternal blood for prenatal determination of the fetal gender as the preliminary experiment for the screening of fetal genetic status and the BclI DNA polymorphism in an attempt to clarify the origin of erythrocytes in maternal blood. METHODS: In seventeen pregnant women, venous blood was withdrawn and the nucleated erythrocytes were recovered by magnetic activated cell sorting (MACS) and immunostaining. After isolation of nucleated erythrocytes by micromanipulation, we performed nested PCR for amelogenin gene to identify the fetal gender and performed BclI DNA polymorphism to clarify the origin of erythrocytes. RESULTS: We could amplify the minute DNA in a single cell by primer extension preamplification and nested PCR of amelogenin gene in 94 (48.7%) cells and could identify the fetal gender by 58.8%. BclI DNA polymorphism revealed that the several cells, which did not reveal the specific band of Y chromosome in spite of the pregnancy of male fetuses, must be the cells from mother. CONCLUSION: Through this study, we could conclude that several nucleated erythrocytes in maternal blood circulation can originate from mother, therefore we must develop the new method to identify the nucleated erythrocyte of fetal origin. Considering that we must apply for the larger number of pregnant women to screen, the procedure was multi-step and complex. Therefore, we must design the new scheme to utilize the nucleated erythrocytes in maternal blood.

Multicenter Trial of Aberrations of Chromosomes in Abortuses, Chorionic villi, Amniocytes, and Fetal Blood Cells / 대한산부인과학회잡지

OBJECTIVES: Chromosomal anomalies are common disease entity among genetic diseases. But there are scanty reports about the status of chromosomal abnormalities in Korean. In addition, the comprehensive multi-center study of chromosomal abnormalities in Korean has never been performed. METHODS: We have collected 1,793 cases (394 abortuses; 198 chorionic villi; 1,060 amniotic fluid cells; 141 fetal blood), which showed abnormal karyotype results from twenty three cytogenetic centers in Korea from the year of 1977 to 1999. RESULTS: In abortuses, numerical chromosomal abnormalities was 85%, and structural abnormalities was 13% and the most frequent anomaly was trisomy 16. In chorionic villi, numerical anomaly was 60.0% and structural anomaly was 31.3% and the most frequent karyotype was Down syndrome. In amniotic fluid cells, structural anomaly exceeded the number of numerical anomaly and the most frequent anomaly was Down syndrome. In fetal blood, numerical anomaly was 63.8% and the most frequent karyotype was trisomy 18 (23.4%) CONCLUSIONS: This may be the first comprehensive and multi-center study in Korea. The proportion of the abnormal karyotypes in each specimens was different from each other. Based on this study, the more comprehensive study should be performed to all the Korean population.

Clinical and Cytogenetic Features in Turner Syndrome / 대한산부인과학회잡지

OBJECTIVE: To identify cytogenetic diversity of Turner syndrome, and its correlation with clinical manifestation including fertility. METHOD: From 1986 to 1996, we reviewed medical records of 137 patients, cytogenetically and clinically diagnosed as Turner syndrome. Cytogenetic study was performed using peripheral lymphocytes with GTG banding. Marker chromosomes were re-evaluated by quinacrine staining. RESULTS: Overall incidence of mosaic pattern was 57.7% in Turner syndrome, showing 45,X line combined with structural anomalies of X chromosome(n=39), marker chromosome(n=22), numerical abnormalities of sex chromosome(n=6), and 46,XX(n=5). With comparison of clinical manifestation in 45,X/46,XX(n=5) and four most common pattern, 45,X(n=42), 45,X/46,X,i(Xq)(n=26), 45,X/46,X,+mar(n=22), 46,X,i(Xq)(n=9), there was no significant differences statistically, except short stature and diabetes. Incidence of short stature was significantly higher in 45,X or 46,X,i(Xq) than 45,X/46,XX(78.6%, 88.9%, 20.0%), and significant difference existed in incidence of diabetes between 45,X and 46,X,i(Xq)(0%, 22.2%). Sixteen patients had a history of marriage, but only one had a history of spontaneous conception and delivery with mosaic 45,X[2]/51,XXXXXXX[1]/46,XX[47]. CONCLUSION: The karyotypes of Turner syndrome was very variable, and about 60% had mosaicism. Patients with 45,X/46,XX had a significant lower incidence of short stature than in 45,X or 46,X,i(Xq), and 46,X,i(Xq) had a significant higher incidence of diabetes than in 45,X. We identified a woman with mosaic 45,X/51,XXXXXXX/46,XX was fertile.

Carrier Detection and Prenatal Diagnosis of Hemophilia A in a Korean Population by Analysis of Two Variable Dinucleotide Tandem Repeats within the Factor VIII Gene / 대한산부인과학회잡지

We have undertaken this study to identify the usefulness of two variable dinucleotide tandem repeats within the factor VIII gene for carrier detection and prenatal diagnosis of hemophilia A in the Korean population. We have analyzed these polymorphisms in 50 unrelated Korean mothers of patients with severe hemophilia A, using polymerase chain reaction. The expected heterozygosity rates of the intron 13 and intron 22 dinucleotide repeats were 56% and 40%, respectively. Analysis of the intron 13 and intron 22 dinucleotide repeats revealed heterozygous patterns in 29(58%) and 17(34%) of 50 mothers studied, respectively. The combined overall informativity of the intron 13 and intron 22 dinucleotide repeats was 68%. Using linkage analysis with the intron 13 dinucleotide repeats, we have attempted three cases of carrier detection and two cases of prenatal diagnosis in two families of patients with severe hemophilia A. Two pregnant women were diagnosed as carriers, and the other patients as non-carrier Prenatal diagnosis revealed an unaffected male in one fetus, and an unaffected female in another fetus. This data demonstrated that the analysis of the intron 13 and intron 22 dinucleotide repeats very useful in the carrier detection and prenatal diagnosis of hemophilia A in the Korean population.

Application of FISH(Fluorescence In Situ Hybridization) in uncultured chorionic villus cells / 대한산부인과학회잡지

OBJECTIVE: The cytogenetic analysis for earlier detection of fetal chromosome aneuploidies is performed from chorionic villus using either long-term culture or direct chromosome preparation. To analyze the cause of pregnancy loss, we also attempt the cytogenetic study in product of conception(POC) using chorionic villi or fetal tissue. But the failure of analysis often occurs in direct preparation of villus cells and product of conception(POC). We studied to evaluate the clinical usefulness of FISH in uncultured chorionic villus cells of culture-failed cases. METHODS: According to the patient's indication, we performed FISH for chromosome 18, 21, X and Y in chorionic villi as well as POC and compared FISH results with their chromosomal studies. RESULTS: We found one trisomy 18 and one trisomy 21 in Chorionic Villus Sampling and one trisomy 18 and one monosomy X(45, X) in POC. The averages for accuracy of FISH were 83-91% and all cases are represented consistent results with their chromosomal studies. Among them, we could analyze using FISH only in 5 cases of culture failure including one case of monosomy X in POC. CONCLUSION: We could detect aneuploidy with uncultured chorionic villus cells in case of culture failure, using FISH, it may be the potential method to assist the cytogenetic study.

Rapid detection of aneuploidy in uncultured fetal cord blood cells by FISH ( Fluorescence In Situ Hybridization ) / 대한산부인과학회잡지

OBJECTIVE: To determine the fetal aneuploidy in fetal blood cells from cordocentesis. METHODS: We analyzed their karyotype and performed fluorescence in situ hybridization(FISH) for chromosome 18, 21, X, and Y in 14 cases of fetal blood cells from cordocentesis at Department of Obstetrics & Gynecology, College of Medicine, Seoul National University and Hamchoon Women's Clinic. RESULTS: In all cases we obtained the consistent results in both methods and were able to rapidly detect aneuploidy in uncultured fetal blood cells using FISH before karyotyping with culture for 48 hr. The averages for accuracy of FISH were from 84.6 % to 93.9%. CONCLUSION: In this study we suggest that the rapid detection in uncultured fetal blood using FISH is possible and that this diagnostic method will be clinically useful when rapid result would be demanded.

Usefulness of HhaI and MseI DNA Polymorphism of Factor IX Gene in the Molecular Genetic Diagnosis of Hemophilia B in Korean Population / 대한산부인과학회잡지

OBJECTIVES: Hemophilia B has been known to result from more than 500 kinds of mutations. And it is difficult to find out a mutation specific for each family. Therefore, linkage analysis of DNA polymorphism within or near the factor IX gene has been frequently used in the clinical practice for molecular genetic diagnosis of hemophilia B. But the ethnic variation makes more difficult to apply useful markers in Caucasian population. To investigate the usefulness of the MseI and HhaI polymorphism in Korean population, we analysed the MseI and HhaI polymorphism. METHODS: Forty-five normal Korean and thirteen parents of the hemophilia B patients, using PCR and restriction enzyme analysis. RESULTS: The heterozygosity rate of MseI polymorphism was 49.7% and that of HhaI polymorphism was 25.5%. CONCLUSION: These data indicated that PCR-based analysis of MseI and HhaI polymorphism of factor IX was useful in molecular genetic diagnosis of hemophilia B in Korean population.

Incidence of Fragile X Syndrome in Korean Patients with Mental Retardation / 대한산부인과학회잡지

Fragile X syndrome is the most common cause of inherited mental retardation. It accounts for 0.2% - 2.7% of patients with mental retardation, based upon the molecular genetic diagnosis. However, the exact prevalence of fragile X syndrome in Korean patients with mental retardation is unknown. We have performed cytogenetic and molecular analysis for fragile X syndrome in 212 Korean patients with mental retardation. Among them, six patients (2.8%) was identified as carrying fragile X syndrome by both cytogenetic and molecular analysis. The results by cytogenetic analysis was identical to those by molecular analysis. Cytogenetic analysis of 6 carriers (mothers of patients with proven fragile X syndrome) showed a fragile X chromosome in one patients (16.7%) while molecular analysis revealed premutation in all patients. PCR method using Klentaq1 Pfu polymerase showed the same results as those by PCR method using Exo(-) Pfu polymerase, but the former method is recommended because of its simplicity in technical aspect. These data suggest that the prevalence of fragile X syndrome in Korean patients with mental retardation is 2.8%, not significantly different from those in Caucasians.

Detection of Down Syndrome & Edward Syndrome in uncultured amniocytes using FISH ( Fluorescence In Situ Hybridization / 대한산부인과학회잡지

FISH is suggested as a possible method to detect the numerical and structural abnormalities of chromosomes in interphase nucleus. We performed this study to discuss the clinical usefulness of FISH in uncultured amniocytes and to set up the cut-off value for further study. We collected amniotic fluid samples from patients whose chromosome studies were recommended due to screen positive for Down and Edword syndrome in triple marker test using maternal serum. The centromeric probe for chromosome 18 and the locus-specific probe for chromosome 21 were used and the results were compared to their karyotypes. We could find 2 cases of trisony 21 and 2 cases of trisony 18 and the other cases represented normal karyotypes. The accuracies were 91% for disomy 18, 89% for trisomy 18, 92% for disomy 21 and 88% for trisomy 21. Therefore FISH technique is a possible method to detect the chromosomal abnormalities in uncultured amniocytes and the use of locus-specific probe for chromosome 21 would be more useful for detecting the aneuploidy of chromosome 21 than 13/21 centromeric probe.

Study on the Abnormal Karyotyping Detected by Cordocentesis in Korean Women / 대한산부인과학회잡지

Fetal umbilical blood was obtained by cordocentesis guided by transabdominal USG in 276 fetuses at more than 14 weeks gestation. Fetal daryotyping was carried out in 7 cases of cordocentesis-failed group by cardiocentesis (3 cases) and amniocntesis (4 cases). The 46 cases in 263 fetuses revealed abnormal karyotypes (17.5%). There were 24 cases of numerical abnormality, 14 cases of structural abnormality, 5 cases of mosaicism, and 3 miscellaneous abnormalities. In the numerical abnormalities, trisomies and sex chromosomal abnormalities were 22 and 2 case, respectively. In 22 trisomy cases, there were 16 cases of trisomy of 18 chromosome which is the most common single cytogenetic abnormality in cordocentesis. The most common associated anomalies in trisomy 18 was congenital heart anomaly, in which vetricular septal defect was the most common. Pregnancy outcome is as follows: 22 cases of termination of pregnancy, 9 cases of still-birth, 5 cases of neonatal death, three living babies and 7 cases lost to follow-up.

Carrier Dectection and Prenatal Diagnosis of Hemophilia Ain Korean Populations Using PCR Analysis of DNA Polymorphismin St14 VNTR Locus / 대한산부인과학회잡지

At present, because of enormous variety of mutations in hemophilia A, carrier detection and prenatal diagnosis by DNA analysis has been relied almost always on indirect detection using linkage analysis of DNA polymorphisms withim or near to the factor VIII gene. However, there is marked ethnic variation in the incidence of heterozygosity for a given DNA polymorphism. So it is very important to find out which DNA polymorphism pattern is useful in Korean families with hemophilia A for carrier detection and prenatal diagnosis. To identify the usefulness of DNA polymorphism in St14 VNTR locus for carrier detection and prenatal diagnosis of hemophilia A in Korean populations, we have analysed the DNA polymorphism in St14 VNTR locus in 80 Korean families with hemophilia A using polymerase chain reaction. We could identify 14 alleles in subjects studied, which ranges from 620 bp to 2830 bp. Expected heterozygosity rate, calculated from the allele frequencies, was 78.7%, and observed heterozygosity rate was 71.3% (57/80). Carrier detection was performed in 43 women from families informative with St14 VNTR : Seventeen women were diagnosed as non-carriers, 11 women as carriers. And 15 women were suspected to be carriers since they were from families of sporadic cases of hemophilia A. And prenatal diagnosis was done in 4 pregnant carrier women : noe fetus proved to be normal males, two fetuses to be normal females, and one to be a carrier. And five pregnant women, suspected to be carrier since they were from families of sporadic cases of hemophilia A, underwent prenatal diagnosis : One fetus was diagnosed as a normal mali, one as a normal female, two as possible carriers, and one as a possible affeted mali, whom the analysis of factor VIII level in fetal blood by cordocentesis revealed to be affected by hemophilia A. These data indicate that PCR-based analysis of St14 VNTR is very useful for the carrier detection and prenatal diagnosis of hemophilia A in Korea.

Treatment with EMA-CO regimen in the management of high risk gestational trophoblastic tumor / 대한산부인과학회잡지

No abstract available.