Jae-Pil Jeon

Copy number variation at leptin receptor gene locus associated with metabolic traits and the risk of type 2 diabetes mellitus

BackgroundRecent efforts have been made to link complex human traits and disease susceptibility to DNA copy numbers. The leptin receptor (LEPR) has been implicated in obesity and diabetes. Mutations and genetic variations of LEPR gene have been discovered in rodents and humans. However, the association of DNA copy number variations at the LEPR gene locus with human complex diseases has not been reported. In an attempt to study DNA copy number variations associated with metabolic traits and type 2 diabetes mellitus (T2DM), we targeted the LEPR gene locus in DNA copy number analyses.ResultsWe identified DNA copy number variations at the LEPR gene locus among a Korean population using genome-wide SNP chip data, and then quantified copy numbers of the E2 DNA sequence in the first two exons overlapped between LEPR and LEPROT genes by the quantitative multiplex PCR of short fluorescent fragment (QMPSF) method. Among the non-diabetic subjects (n = 1,067), lower E2 DNA copy numbers were associated with higher fasting glucose levels in men (p = 1.24 × 10-7) and women (p = 9.45 × 10-5), as well as higher total cholesterol levels in men (p = 9.96 × 10-7). In addition, the significant association between lower E2 DNA copy numbers and lower level of postprandial 2hr insulin was evident only in non-diabetic women, whereas some obesity-related phenotypes and total cholesterol level exhibited significant associations only in non-diabetic men. Logistic regression analysis indicated that lower E2 DNA copy numbers were associated with T2DM (odds ratio, 1.92; 95% CI, 1.26~2.96; p < 0.003) in our nested case-control study. Interestingly, the E2 DNA copy number exhibited a negative correlation with LEPR gene expression, but a positive correlation with LEPROT gene expression.ConclusionsThis work suggests that a structural variation at the LEPR gene locus is functionally associated with complex metabolic traits and the risk of T2DM.

Expression phenotype changes of EBV-transformed lymphoblastoid cell lines during long-term subculture and its clinical significance

Objectives:  The EBV‐transformed lymphoblastoid cell line (LCL) is a useful resource for population‐based human genetic and pharmacogenetic studies. The principal objective here was to assess expression phenotype changes during long‐term subculture of LCLs, and its clinical significance.

Isoliquiritigenin suppresses cocaine-induced extracellular dopamine release in rat brain through GABAB receptor

Glycyrrhizae radix (licorice) comprises a variety of flavonoids as major constituents including isoliquiritigenin, liquiritin, liquiritigenin, and glycyrrihizin. It has shown various biological activities such as anti-inflammatory, anti-carcinogenic and antihistamic. As very little is known in regard to drug addiction, we carried out a study on the effect of G. radix and its active component, isoliquiritigenin, on acute cocaine-induced extracellular dopamine release in moving rats. Male Sprague-Dawley rats were orally administered with methanolic extracts of G. radix or isoliquiritigenin 1 h prior to an injection of cocaine (20 mg/kg, intraperitoneal (i.p.)). Extracellular dopamine was measured by in vivo microdialysis. Extract of G. radix and isoliquiritigenin inhibited cocaine-induced extracellular dopamine level in the nucleus accumbens by dose-dependent manner. Inhibition of dopamine release by isoliquiritigenin resulted in attenuation of the expression of c-Fos, an immediately early gene induced by cocaine. Effect of isoliquiritigenin was completely prevented by a GABA(B) receptor antagonist. Thus, these results showed that G. radix and isoliquiritigenin inhibit cocaine-induced dopamine release by modulating GABA(B) receptor, suggesting that isoliquiritigenin might be effective in blocking the reinforcing effects of cocaine.

Sustained viral activity of epstein-Barr virus contributes to cellular immortalization of lymphoblastoid cell lines

EBV-transformed lymphoblastoid cell lines (LCLs) are used as a resource for human genetic, immunological, and pharmacogenomic studies. We investigated the biological activity of 20 LCL strains during continuous long-term subculture up to a passage number of 160. Out of 20 LCL strains, 17 proliferated up to a passage number of 160, at which point LCLs are generally considered as “immortalized”. The other three LCL strains lost the ability to proliferate at an average passage number of 41, during which these LCLs may have undergone cellular crisis. These non-immortal LCL strains exhibited no telomerase activity, decreased EBV gene expression, and a lower copy number of the EBV genome and mitochondrial DNA when compared with immortal LCLs. Thus, this study suggests that sustained EBV viral activity as well as telomerase activity may be required for complete LCL immortalization.

MicroRNA signatures associated with immortalization of EBV-transformed lymphoblastoid cell lines and their clinical traits

Objective:  MicroRNAs (miRNAs) are negative regulators of gene expression that play important roles in cell processes such as proliferation, development and differentiation. Recently, it has been reported that miRNAs are related to development of carcinogenesis. The aim of this study was to identify miRNAs associated with terminal immortalization of Epstein–Barr virus (EBV)‐transformed lymphoblastoid cell line (LCL) and associated clinical traits.

Genotype instability during long-term subculture of lymphoblastoid cell lines.

Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) promise to address the challenge posed by the limited availability of primary cells needed as a source of genomic DNA for genetic studies. However, the genetic stability of LCLs following prolonged culture has never been rigorously investigated. To evaluate genotypic errors caused by EBV integration into human chromosomes, we isolated genomic DNA from human peripheral blood mononuclear cells and LCLs collected from 20 individuals and genotyped the DNA samples using the Affymetrix 500K SNP array set. Genotype concordance measurements between two sources of DNA from the same individual indicated that genotypic discordance is negligible in early-passage LCLs (<20 passages) but substantial in late-passage LCLs (>50 passages). Analysis of concordance on a chromosome-by-chromosome basis identified genomic regions with a high frequency of genotypic errors resulting from the loss of heterozygosity observed in late-passage LCLs. Our findings suggest that, although LCLs harvested during early stages of propagation are a reliable source of genomic DNA for genetic studies, investigations that involve genotyping of the entire genome should not use DNA from late-passage LCLs.

Identification of stathmin 1 expression induced by Epstein-Barr virus in human B lymphocytes.

Abstract.  Introduction: The Epstein–Barr virus transforms resting B cells into proliferating lymphoblastoid cells, the origin of cell lines. Method and results: Our cDNA microarray analyses led to the identification of 232 up‐regulated and 112 down‐regulated genes with more than a 3‐fold difference in lymphoblastoid cell lines compared to resting B cells. The functional classification of these genes exhibited the distinct expression signature for cell proliferation, cell cycle and an immune response. Among them, we verified the differential expression of several oncogenes such as stathmin 1 (STMN1), RAB27A, RAB9A, BACH1 and BACH2 using quantitative real‐time reverse transcriptase‐polymerase chain reactions or Western blot analysis. Expression of STMN1 (which is involved in regulation of the microtubule filament system, cell growth and S‐phase of cell cycle) was increased in lymphoblastoid cell line as well as in 7‐day post‐Epstein–Barr virus infection B cells, compared to resting B cells. Conclusion: Thus, this study suggests that Epstein–Barr virus infection induces STMN1 expression, which play a role in cell cycle progression and proliferation in the human B lymphocyte.

Proteomic and behavioral analysis of response to isoliquiritigenin in brains of acute cocaine treated rats.

Isoliquiritigenin (ISL) is a licorice flavonoid with chalcone structure and is an active component of the root of the plant genus Glycyrrhiza. In addition to anti-inflammatory and antioxidant effects, ISL was previously reported to antagonize increased striatal dopamine release. In the present study, we aimed to investigate whether ISL has an effect on hyperlocomotion in animals subjected to acute cocaine administration and whether ISL modulates acute cocaine-induced molecular changes. To achieve our goals, we analyzed behavior and differential proteomic changes between ISL and vehicle in acute cocaine treated rats. Locomoter activity was reduced in ISL treated animals compared to vehicle in acute cocaine treated rats. Two dimensional electrophoresis (2-DE) revealed that 56 proteins were differentially expressed in response to ISL. Further proteomic analyses using mass spectroscopy, and subsequent validation experiments confirmed that ISL induced changes in proteins related to metabolism, signal transduction, protein folding and transport, oxidative stress, and neural toxicity. Furthermore, cocaine-induced neuronal toxicity was attenuated by ISL treatment, suggesting a neuroprotective role of ISL.

A comprehensive profile of DNA copy number variations in a Korean population: identification of copy number invariant regions among Koreans

To examine copy number variations among the Korean population, we compared individual genomes with the Korean reference genome assembly using the publicly available Korean HapMap SNP 50 k chip data from 90 individuals. Korean individuals exhibited 123 copy number variation regions (CNVRs) covering 27.2 mb, equivalent to 1.0% of the genome in the copy number variation (CNV) analysis using the combined criteria of P value (P < 0.01) and standard deviation of copy numbers (SD ≥ 0.25) among study subjects. In contrast, when compared to the Affymetrix reference genome assembly from multiple ethnic groups, considerably more CNVRs (n = 643) were detected in larger proportions (5.0%) of the genome covering 135.1 mb even by more stringent criteria (P < 0.001 and SD ≥ 0.25), reflecting ethnic diversity of structural variations between Korean and other populations. Some CNVRs were validated by the quantitative multiplex PCR of short fluorescent fragment (QMPSF) method, and then copy number invariant regions were detected among the study subjects. These copy number invariant regions would be used as good internal controls for further CNV studies. Lastly, we demonstrated that the CNV information could stratify even a single ethnic population with a proper reference genome assembly from multiple heterogeneous populations.

Expressed sequence tag analysis of the diapausing queen of the bumblebee Bombus ignitus

We constructed a full‐length cDNA library from diapausing queens of the bumblebee Bombus ignitus. A total of 480 randomly selected clones was sequenced by single‐run 5′‐end sequencing. Of these, there were 437 high quality clones, 23 poor quality clones and 20 read‐fail clones. Each high quality clone sequence was searched against a public protein database. The most frequently found matching genes were ribosomal proteins (12.5%), p10 (3.58%), cytochrome P450 monooxygenase (3.13%) and sensory appendage protein (2.9%). Sequence similarity analysis between bumblebees and other insect species showed that 72 out of 437 (16.5%) bumblebee expressed sequence tags (EST) matched sequences of Apis mellifera, with matches to Drosophila melanogaster (6.6%), Caenorhabditis briggsae (6.2%), Lysiphlebus testaceipes (4.8%), Periplaneta americana (3.7%) and Anopheles gambiae (3.4%) following, suggesting that sequence similarity of bumblebee EST is closest to that of A. mellifera. Functional classification of EST based on Gene Ontology showed that most genes found by sequencing are associated with physiological processes in the bumblebee. The results of sequencing and analysis of our 437 cDNA demonstrated that high‐throughput EST sequencing and data analysis are powerful means for identifying novel genes and for expression profiling. Our bumblebee EST collection could be a useful platform for further studies of gene expression in diapausing bumblebees.