Advances in identification methods of alien genomic components in plants / 生物工程学报

Plants with alien genomic components (alien chromosomes / chromosomal fragments / genes) are important materials for genomic research and crop improvement. To date, four strategies based on trait observation, chromosome analysis, specific proteins, and DNA sequences have been developed for the identification of alien genomic components. Among them, DNA sequence-based molecular markers are mainly used to identify alien genomic components. This review summarized several molecular markers for identification of alien genomic components in wheat, cabbage and other important crops. We also compared the characteristics of nine common molecular markers, such as simple sequence repeat (SSR), insertion-deletion (InDel) and single nucleotide polymorphism (SNP). In general, the accuracy of using a combination of different identification methods is higher than using a single identification method. We analyzed the application of different combination of identification methods, and provided the best combination for wheat, brassica and other crops. High-throughput detection can be easily achieved by using the new generation molecular markers such as InDel and SNP, which can be used to determine the precise localization of alien introgression genes. To increase the identification efficiency, other new identification methods, such as microarray comparative genomic hybridization (array-CGH) and suppression subtractive hybridization (SSH), may also be included.

Diagnosis of an uncertain karyotype and mentally retarded child using the whole genome microarray scanning technique / 临床儿科杂志

Objective To investigate the possibility and feasibility of the whole genome microarray scanning technique in clinical cytogenetic diagnosis of an uncertain karyotype and mentally retarded child. Methods The karyotype analysis of the mental development delayed child was 47, XY+mar. Genomic DNA was extracted from the peripheral blood and the whole genome microarray scanning technique was used to analyze the derivative chromosome. Results The whole genome microar-ray scanning technique indicated the derivative chromosome fragment had originated from 9p13.1-p24.3. Conclusions Com-paring to conventional cytogenetic analysis methods, the whole genome microarray scanning technique is of high resolution, high-throughput and high accuracy, which can detect the submicroscopic chromosomal aberrations and replace the conven-tional karyotype analysis.

Detection and analysis of genomic copy number variations in a 46,X0, +der(?) fetus by array-based comparative genomic hybridization / 基础医学与临床

Objective To understand genomic copy number variations (CNVs) and ascertain karyotype for a 46,X0, + der(?) fetus, and investigate possibility and superiority of array-based comparative genomic hybridization (array-CGH ) in clinical cytogenetic diagnosis. Methods G-banded chromosome analysis was carried out. The whole genome of the fetus was scanned and analysed by array-CGH. The results of array-CGH were confirmed by RT-qPCR. Results G-banded chromosome analysis showed that the fetal karyotype was 46,X0, +der(?). Array-CGH revealed the derivative chromosome as Y chromosome without CNVs. A total number of 118 submicroscopic CNVs were identified. Comparable results between array-CGH and RT-qPCR were obtained for 9 novel CNVs. Conclusion Comparing with conventional cytogenetic analysis, array-CGH is of high resolution, high-throughput and high accuracy, which provides a technical platform for accurate detection of submicroscopic chromosomal aberrations.

Transcervical embryoscopy in missed abortion / 대한산부인과학회지

OBJECTIVE: Approximately 15-20% of all clinically recognized pregnancies result in spontaneous abortion and 60-70% of these are attributable to detectable chromosome abnormalities. Although the incidence of first trimester losses is high, spontaneous abortion material is often poorly described from a development perspective. The purpose of this study was to determine the usefulness of transcervical embryoscopy in diagnosing localized and generalized defects in the embryonic morphogenesis of missed abortions. The embryoscopic findings are supplemented by the results of cytogenetic analysis in all cases. METHODS: In this study, consisted of 26 women with the final diagnosis of missed abortion between August 1, 2003 and October 31, 2003 in Samsung Cheil Hospital. Prior to the instrumental evacuation of the uterus a rigid hysteroscope was passed transcervically into the amniotic cavity to obtain a detail view of the embryo. Karyotyping was done in all cases included in this study. RESULTS: Visualization of embryo or early fetus was successful in 24 cases. Among 24 examined cases, 21 cases had successful karyotype and as a result 11 (11/21, 52.4%) had abnormal karyotype. Among 21 cases which had successful karyope, 4 (4/21, 19.0%) had normal external features, 10 (10/21, 47.6%) had classified as growth-disorganized and 7 (7/21, 33.3%) had either isolated or multiple defects, including facial dysplasia, delayed limb development, facial fusion to chest, umbilical cyst, brownish discoloration of ventral part and increased nuchal thickness. Of the morphologically normal and growth-disorganized embryo in embryoscopic examination, only 1 and 4 (1/4, 25.0% and 4/10, 40.0%) had a abnormal karyotype. In contrast, of the morphologically abnormal embryo in embryoscopic examination, 5 (5/7, 71.4%) had a abnormal karyotype. CONCLUSION: Transcervical embryoscopy permits visualization of the embryo in utero, unaffected by the damage usually caused by its instrumental evacuation or spontaneous passage. This technique can be a helpful tool for understanding human embryonic malformations and genetic counseling for parents. Futhermore, correlation of morphological and cytogenetic findings in spontaneous abortion specimens could provide the need of further evaluation for future pregnancies in couples which had fear of repeated abortions. But, more larger scaled controlled study is needed for widely use of transcervical embryoscopy in missed abortion.

Cytogenetic Analysis in 785 Cases of Midtrimester Amniocentesis Using In Situ Coverslip Culture / 대한산부인과학회잡지

OBJECTIVE: Amniocentesis is the most commonly used invasive method for prenatal diagnosis of genetic disorders. We performed this study to analyze the indications, distributions of maternal age and cytogenetic results of midtrimester amniocentesis. METHODS: We retrospectively analyzed 785 cases of midtrimester prenatal genetic amniocentesis which were performed in the cytogenetics laboratory using in situ coverslip culture at Dong-A University Hospital from January 1995 to March 2003. RESULTS: Amniocentesis was practiced mostly from 15 weeks to 20 weeks of gestational ages. Requested indications of amniocentesis were abnormal maternal serum screening (421, 53.7%), advanced maternal age (233, 29.7%) and abnormal ultrasonographic finding (61, 7.8%) in the order of decrease. The overall incidence of chromosome abnormalities was 5.1% (40 cases), and it contains 27 cases (3.4%) of numerical abnormalities and 13 cases (1.7%) of structural abnormalities. Among autosomal abnormalities Down syndrome was most common (13 cases) and followed by Edward syndrome (2 cases). Of the sex chromosomal abnormalities, three cases of Turner syndrome and three cases of Kleinefelter syndrome were found. Chromosomal abnormalities were most frequently noted in the maternal age of 30 to 34 years old (14 cases, 35.0%), 25 to 29 years old (12 cases, 30.0%), followed by 35 to 39 years old (7 cases, 17.5%). The frequency of pseudomosaicism were 5 cases (0.6%). CONCLUSION: Maternal serum screening, advanced maternal age and antenatal ultrasonographic finding must be important screening methods for amniocentesis which is considered to the most effective diagnostic procecdure for prenatal cytogenetic studies. I conclude that the karyotyping analysis of midtrimester amniocentesis is efficacious method for detection of chromosomal aberration and genetic counselling for parents.

Chromosomal Abnormality of Spontaneous Abortus in Relation to Transvaginal Ultrasonographic Finding / 대한산부인과학회잡지

OBJECTIVE: To investigate the importance of transvaginal ultrasonographic findings of spontaneous abortion and the relation of transvaginal ultrasonographic finding and the karyotype of abortus. MATERIALS AND METHODS: Transvaginal sonography was undergone to 48 subjects with spontaneous abortion in their early pregnancies who had visited Ewha Womans University Mokdong Hospital, from March 1996 to June 1999. A chromosome analysis of their aborted products was done. RESULTS: The frequency of chromosome abnormalities of abortus was 47.9%. Among the chromosomal abnormalities, trisomy was 31.2%(simple trisomy 27.0%, double trisomy 4.2%), monosomy X 8.4%, poliploidy 8.4%(tripoidy 4.2%, tetraploidy 4.2%). The average maternal age for trisomy was 30.2+/-4.9, and their past number of spontaneous abortions was 1.0+/-0.9, with trisomy 16 most frequent with 23.0%. In the case with trisomy 5, 22, and double trisomy, the average maternal age was increased with 38.0, 36.0+/-2.8, 35.5+/-5.0, respectively. However, no significant relevance could be found. The average maternal age for polyploidy was 30.5+/-3.7, and their past number of spontaneous abortions was 1.0+/-2.0. No significant relevance could be found compared with normal karyotypes. The average maternal age for monosomy X was 27.5+/-1.7, which is lower than the average maternal age for normal karyotypes, but not significantly so. The mean number of past abortions was no different with 1.0+/-0.8. No significant relevance could be found between ultrasonographic findings and chromosomal abnormalities. The measured/expected crown-rump length ratio of the aborted group was smaller than those of the control group. CONCLUSION: Serial ultrasonographic findings in early pregnancies help to diagnose spontaneous abortion, but not efficient to diagnose chromosomal abnormalities in the aborted group.

Chromosome Analysis of Ascitic Fluids from Patients with Malignant Tumor / 대한해부학회지

Detecting malignant cells in ascitic fluid from tumor patients is important since the existence of malignant cells in ascitic fluids is related to the prognosis of patients. Various laboratory methods are being used to obtain diagnosis in ascitic fluids, but some ascitic fluids can not be diagnosed reliably. Cytogenetic analysis of ascitic fluid is not used routinely as a laboratory tool. In this presentation a cytogenetic study of the ascitic fluids from 9 patients with malignant tumor was performed by a direct or short-term culture method. According to cytogenetic study, 5 cases had positive findings for malignant cells. One case had a inconclusive result. There were no malignant cells in the remaining 4 cases. On blind cytologic data, no informations could be obtained in 4 out of 9 cases and the remaining 5 cases had negative findings for detecting malignant cells. Among the 5 cases, cytogenetic findings were negative in 3 cases but in the remaining 2 cases, one was reported positive and the other inconclusive each other. In present study, even though the ascitic fluids from 5 patients were subjected to the comparison of the cytologic study with cytogenetic analysis, two different findings could be obtained. Therefore if further study of a large series of cancer patients with ascitic fluids is done, the value of cytogenetic analysis will be clearly shown. In addition, the cytogenetic study of cell present in ascitic fluids can be used as useful adjunct to cytologic study, and also it can indicate that more invasive diagnostic procedures are necessary.

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.

Assessment of Modified Chromosome Analysis Method Using Culture Tube for Peripheral Blood / 대한임상병리학회지

BACKGROUND: Peripheral blood is the most frequently used specimen for routine chromosome analysis. The classical method using flask for cell culture needs a lot of reagent and is cumbersome. To simplify the procedure, we tried using culture tube and modifying the fixation method. The purpose of this study is to assess the reliability of the modified method using culture tube for chromosome analysis of peripheral blood. METHODS: We tested peripheral blood of ten normal healthy persons. In the modified method, culture tube (110 mm 16 mm, Nunc, Roskilde, Denmark) containing 2.25 mL RPMI 1640 supplemented with fetal calf serum and phytohemagglutinin was used. Harvest was done in the culture tube. Fixative 2.0 mL was added without centrifugation after hypotonic treatment. After 10 min incubation at room temperature, the cells were pelleted and washed. This method was compared with the reference method using flask. We evaluated the quality of chromosome and calculated mitotic index. RESULTS: Chromosome quality of the modified method using culture tube was good and the same as that of the reference method. Mitotic index was higher in the modified method (1.0~4.3%, mean 2.5%) than in the reference method (0.4~2.2%, mean 1.2%) (P<0.01). CONCLUSIONS: The modified method needs less amount of reagent and is easy to do. The chromosome quality was good enough to evaluate the karyotype. So, this modification enable to improve the effectiveness of chromosome laboratory.

MALIGNANT TRANSFORMATION OF MIXED CULTURE CELLS OF LUNG AND LIVER OF NEW BORN MOUSE INDUCED BY~3H-TdR AND ITS CHROMOSOME ANALYSIS / 解剖学报

In this experiment, primary mixed culture cells of lung and liver derived from new born mouse was made use of target cells, 0.1 ?Ci 3~H-TdR per milliliter medium was added in the culture in order to induce malignant transformation of the cells in the culture. Results of the experiment was that the cells effected by 3~H-TdR had a unlimited growing and formed sarcoma after being inoculated into new born mice immunosuppressed with ATS. It suggested that they had became malignant transformation cells. Results of analysis of chromosome aberrations of the transformed cells, the long arm chromosome was observed in 5% of cells, the metacentric chromosome in 7% of cells, the acentric fregment in 8% of cells. It shows that DNA damage of the cells induced by 3~H-TdR causes their chromosome aberrations and, futhermore, development of malignant cells. The fact that unstable aberrations was Still in sight in the malignant transformation cells suggested that there have been a bit of 3~HTdR left in these cells which kept damaging DNA of the cells.