First Korean case of factor V Leiden mutation in pregnant woman with a history of recurrent pregnancy loss

Thrombophilia refers to inherited or acquired hemostatic disorders that result in a predisposition to blood clot formation. When combined with the hypercoagulable state that is characteristic of pregnancy, there is an increased risk of severe and recurrent pregnancy complications. Activated protein C resistance caused by factor V Leiden (FVL) mutation is known to be the most common cause of inherited thrombophilia in Caucasian population. FVL mutation has been related to pregnancy complications associated with hypercoagulation, e.g. miscarriage, intrauterine fetal demise, placental abruption, and intrauterine growth retardation. Although the FVL mutation is easily detected using molecular DNA techniques, patients who are heterozygous for this disorder often remain asymptomatic until they develop a concurrent prothrombotic condition. Because there are potentially serious effects of FVL mutation for pregnancy, and because effective treatment strategies exist, early detection and treatment of this condition might be considered.

Genetic Screening for Chromosomal Abnormalities and Y Chromosome Microdeletions in 846 Infertile Korean Men

BACKGROUND: Chromosomal abnormalities are confirmed as one of the frequent causes of male infertility. The microdeletion of the azoospermia factor (AZF) region in the Y chromosome was discovered as another frequent genetic cause associated with male infertility. The aim of this study was to evaluate the frequency and type of chromosomal abnormalities and Y chromosome microdeletions in Korean infertile men. METHODS: A total of 846 infertile men with azoospermia and severe oligozoospermia were included for genetic screening. Cytogenetic analyses using G-banding and screening for Y chromosome microdeletions by multiplex PCR for AZF genes were performed. RESULTS: Chromosomal abnormalities were detected in 112 infertile men (13.2%). Of these, Klinefelter's syndrome was the most common (55.4%, 62/112), followed by balanced translocation including translocation between sex chromosome and autosome (14.3%), Yq deletion (13.4%), X/XY mosaicism with Yq deletion (12.5%), and XX male (4.5%). The overall prevalence of Y chromosome microdeletions was 9.2% (78/846). Most microdeletions were in the AZFc region (51.3%) with a low incidence in AZFa (7.7 %) and AZFb (6.4 %). Combined deletions involving the AZFbc and AZFabc regions were detected in 26.9 % and 7.7 % of men, respectively. Among the infertile men with Y chromosome microdeletions, the incidence of chromosomal abnormality was 25.6% (20/78). CONCLUSIONS: There was a high incidence (20.1%) of chromosomal abnormalities and Y chromosome microdeletions in Korean infertile men. These findings strongly suggest that genetic screening for chromosomal abnormalities and Y chromosome microdeletions should be performed, and genetic counseling should be provided before starting assisted reproductive techniques.

Application of Diagnostic Microarray Technique in Subtyping and Pathotyping of Avian Influenza Viruses Isolated in Mongolia

Asian-lineage H5 highly pathogenic avian influenza (HPAI) viruses have caused continuous outbreaks in poultry and wild birds. Development of rapid and accurate diagnostic methods is needed for preventing further spread of the virus and reducing the time required for eradication of the virus. We developed a low-density microarray for the rapid detection and identification of avian influenza virus subtypes H5, H7, and H9 and their pathotypes in a previous study. In the present study, we evaluated previously developed diagnostic microarray using avian influenza viruses isolated in Mongolia, including H5 HPAI viruses. All H5 HPAI viruses isolated in Mongolia were shown as H5-specific and highly pathogenic pattern in the microarray. H2, H3 and H12 viruses isolated in Mongolia used in this study did not show any H5, H7 and H9 patterns. These results indicated that this diagnostic microarray has enormous potential for the rapid subtyping and pathotyping of influenza viruses, including viruses isolated in Mongolia.

Application of digital polymerase chain reaction technology for noninvasive prenatal test

Recently, noninvasive prenatal test (NIPT) has been adopted as a primary screening tool for fetal chromosomal aneuploidy. The principle of NIPT lies in isolating the fetal fraction of cell-free DNA in maternal plasma and analyzing it with bioinformatic tools to measure the amount of gene from the target chromosome, such as chromosomes 21, 18, and 13. NIPT will contribute to decreasing the need for unnecessary invasive procedures, including amniocentesis and chorionic villi sampling, for confirming fetal aneuploidy because of its higher positive predictive value than that of the conventional prenatal screening method. However, its greater cost than that of the current antenatal screening protocol may be an obstacle to the adoption of this innovative technique in clinical practice. Digital polymerase chain reaction (dPCR) is a novel approach for detecting and quantifying nucleic acid. dPCR provides real-time diagnostic advantages with higher sensitivity, accuracy, and absolute quantification than conventional quantitative PCR. Since the groundbreaking discovery that fetal cell-free nucleic acid exists in maternal plasma was reported, dPCR has been used for the quantification of fetal DNA and for screening for fetal aneuploidy. It has been suggested that dPCR will decrease the cost by targeting specific sequences in the target chromosome, and dPCR-based noninvasive testing will facilitate progress toward the implementation of a noninvasive approach for screening for trisomy 21, 18, and 13. In this review, we highlight the principle of dPCR and discuss its future implications in clinical practice.

Proteomic analysis of pancreas in miniature pigs according to developmental stages using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry / 한국실험동물학회지

Organ transplantation is limited by the shortage of human organs. Many studies have sought to overcome this hurdle by using animal organs. Porcine organs, especially from miniature pigs, have been used for organ xenotransplantation rather than nonhuman primates. While the molecular profiling for transplantation is well known in humans and rodents, the situation for pigs is almost completely unknown. The present study examined protein regulation of the developing stages of the pancreatic proteome (4 day-old miniature neonate, 19 day-old miniature piglet, and 14 month-old miniature adult pigs) using two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight mass spectrometry. Thirteen different expressed spots were observed and nine were identified. The data presented within this study provides critical direction relating to the development of pancreas of miniature pigs, which will assist future proteome analysis of the pancreas, and advance our understanding of the hurdles facing xenotransplantation.

Proteomic analysis of liver in miniature pigs according to developmental stages using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry / 한국실험동물학회지

Due to the shortage of human organ donors for transplant, various studies of xenotransplantation, or the use of animal organs instead of human organs, have been carried out. The organs of porcine are thought to be safer and of a more suitable size for xenotransplantationthan those of nonhuman primates. Understanding the levels of expression of proteins, and their post-translational regulation, would be very practical between different species and among developing stages, though the molecular profiling for xenotransplantation has been rarely studied for porcine, while that of human and rodent is well known. Here, in this present study, we report protein regulation of the developing stages of liver (4-day old neonate, 19-day old piglet and 14-month old adult miniature pigs) using 2-DE and MALDI-TOF. From images of the three different stages, a total of 8 spotswhich were differently regulated were identified, and 5 spots were identified with MALDI-TOF MS. The data presented within this study provides critical direction relating to the development of livers of miniature pigs, which will assist future proteome analysis of the liver, and advance our understanding of the hurdles facing xenotransplantaion.

DNA Microarray-Based Gene Expression Profiling in Porcine Keratocytes and Corneal Endothelial Cells and Comparative Analysis Associated with Xeno-related Rejection

Porcine to rat corneal xenotransplantation resulted in severe inflammation and rejection of the corneal stroma, whereas an allograft showed mainly endothelial cell-associated rejection. We, therefore, investigated and compared the gene expression between porcine keratocytes and corneal endothelial cells. RNA was isolated from primary cultured porcine or human keratocytes and porcine corneal endothelial cells. Gene expression was comparatively analyzed after normalization with microarray method using Platinum pig 13 K oligo chip (GenoCheck Co., Ltd., Ansan, Korea). Real-time polymerase chain reaction (PCR) was performed for C1R, CCL2, CXCL6, and HLA-A in porcine keratocytes and corneal endothelial cells. As a result, upregulated expression more than 2 folds was observed in 1,162 genes of porcine keratocytes versus porcine endothelial cells. Among the immune-regulatory genes, SEMA3C, CCL2, CXCL6, F3, HLA-A, CD97, IFI30, C1R, and G1P3 were highly expressed in porcine keratocytes, compared to porcine corneal endothelial cells or human keratocytes. When measured by real-time PCR, the expression of C1R, CCL2, and HLA-A was higher in porcine keratocytes compared to that in porcine corneal endothelial cells. In conclusion, the increased expression of C1R, CCL2, and HLA-A genes in porcine keratocytes might be responsible for the stromal rejection observed in a porcine to rat corneal xenotransplantation.

Application of DNA chip techniques for Yq microdeletion analysis in infertile males

Our aim was to apply DNA chip technology as a diagnostic tool in infertility research and clinics. Six loci, including a sex-determining region on the Y chromosome and five sequence-tagged sites in azoospermia-factor regions were investigated in infertile male patients. Our method produced a sensitive signal, which showed the presence or absence of the STS regions on the Y chromosome. The results from 93 patients with non- obstructive azoospermia, oligoathenoteratozoospermia, or oligozoospermia were identical when analyzed with either the DNA chip technique or conventional PCR-gel electrophoresis. We have demonstrated its application in the molecular diagnosis of male infertility. This system provides an economic and high-throughput method for detecting the deletion of genomic DNA sequences of large groups of infertile patients, and a completely new approach to male infertility screening. The application of DNA chip technology to identify Yq deletions can also facilitate our understanding of male infertility.

Basic Concept of Gene Microarray

The genome sequencing project has generated and will continue to generate enormous amounts of sequence data including 5 eukaryotic and about 60 prokaryotic genomes. Given this ever-increasing amounts of sequence information new strategies are necessary to efficiently pursue the next phase of the genome project-the elucidation of gene expression patterns and gene product function on a whole genome scale. In order to assign functional information to the genome sequence, DNA chip(or gene microarray) technology was developed to efficiently identify the differential expression pattern of independent biological samples. DNA chip provides a new tool for genome expression analysis that may revolutionize many aspects of biotechnology including new drug discovery and disease diagnostics.