Hye-Young Nam

A genome-wide association study of a coronary artery disease risk variant

Although over 30 common genetic susceptibility loci have been identified to be independently associated with coronary artery disease (CAD) risk through genome-wide association studies (GWAS), genetic risk variants reported to date explain only a small fraction of heritability. To identify novel susceptibility variants for CAD and confirm those previously identified in European population, GWAS and a replication study were performed in the Koreans and Japanese. In the discovery stage, we genotyped 2123 cases and 3591 controls with 521 786 SNPs using the Affymetrix SNP Array 6.0 chips in Korean. In the replication, direct genotyping was performed using 3052 cases and 4976 controls from the KItaNagoya Genome study of Japan with 14 selected SNPs. To maximize the coverage of the genome, imputation was performed based on 1000 Genome JPT+CHB and 5.1 million SNPs were retained. CAD association was replicated for three GWAS-identified loci (1p13.3/SORT1 (rs599839), 9p21.3/CDKN2A/2B (rs4977574), and 11q22.3/ PDGFD (rs974819)) in Koreans. From GWAS and a replication, SNP rs3782889 showed a strong association (combined P=3.95 × 10−14), although the association of SNP rs3782889 doesn’t remain statistically significant after adjusting for SNP rs11066015 (proxy SNP with BRAP (r2=1)). But new possible CAD-associated variant was observed for rs9508025 (FLT1), even though its statistical significance did marginally reach at the genome-wide a significance level (combined P=6.07 × 10−7). This study shows that three CAD susceptibility loci, which were previously identified in European can be directly replicated in Koreans and also provides additional evidences implicating suggestive loci as risk variants for CAD in East Asian.

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.

A common intronic variant of CXCR3 is functionally associated with gene expression levels and the polymorphic immune cell responses to stimuli

BACKGROUND CXCR3 is a chemokine receptor that plays important roles in mediating chemotactic signals and modulating the activation of lymphocytes. We have previously conducted a case-control study by using a candidate gene approach to investigate the association of CXCR3 polymorphisms with the risk of asthma. Results from the epidemiologic study showed that a common nucleotide variant in the CXCR3 intron (rs2280964G>A) was associated with disease susceptibility (1006 cases and 384 control subjects; odds ratio, 0.81; 95% CI, 0.69-0.94; P = .007). OBJECTIVE The aim of our study was to evaluate the epidemiologic study and provide functional evidence for the association of rs2280964G>A with asthma by investigating the effects of intronic variant on chemokine-mediated phenotypes of human-derived T cells. METHODS We used cell line-based in vitro and human primary T cell-based ex vivo studies to examine the functional consequences of the intronic polymorphism, focusing on the regulation of gene expression, splicing, and immune responsiveness toward activating signals. RESULTS We present functional evidence indicating that the rs2280964A allele significantly correlates with decreased CXCR3 gene expression, which would lead to variation in immune cell responses to chemokine-cytokine signals in vitro and ex vivo that includes a decrease in chemotactic activity. CONCLUSION These findings, in conjunction with those of our previous epidemiologic studies, might implicate a functional link between a common nucleotide variant of a chemokine receptor gene, CXCR3, and a cause for a complex-trait disease, asthma.

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.

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.

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.

Human transcriptome analysis of acute responses to glucose ingestion reveals the role of leukocytes in hyperglycemia-induced inflammation

Glucose ingestion-induced hyperglycemia has been known to induce inflammation, which is related to the pathogenesis of diabetic complications. To examine acute gene expression responses to physiological oral glucose ingestion in human circulating leukocytes, we conducted a microarray study of human circulating leukocytes sampled before, 1 h after, and 2 h after glucose ingestion in community-based participants without previous histories of diabetes (n = 60). Ingestion of 75 g glucose successfully induced acute hyperglycemia (glucose concentration 91.6 ± 5.3 mg/dl for fasting and 180.7 ± 48.5 mg/dl for 1 h after glucose ingestion). Oral glucose ingestion significantly increased the expressions of 23 genes and decreased the expressions of 13 genes [false discovery rate (FDR) P value <0.05]. These genes are significantly involved in immunity by way of natural killer cell-mediated immunity, granulocyte-mediated immunity, and the cytokine-mediated signaling pathway (FDR P value <0.05). The present study demonstrated 36 genes that showed acute gene expression change in human leukocytes within 1 h after glucose ingestion, suggesting that leukocytes participate in the inflammatory process induced by acute hyperglycemia. We believe that these results will provide some basic insight into the role of leukocytes in hyperglycemia-induced inflammation and the pathogenesis of diabetic complications.

National Biobank of Korea: Quality control Programs of Collected-human Biospecimens

Personalized medicine is emerging as a main paradigm for risk prediction, pre-diagnosis, and effective prevention and treatment of disease. A large number of human biospecimens and their clinical data are essential resources for the success of personalized medicine as well as other biomedical research. The National Biobank of Korea (NBK) has collected well-annotated and high quality human biospecimens, and distributes them to the Korean biomedical scientists, through the Korea Biobank Project (KBP). The ultimate goal of NBK activities is to promote biomedical research and public health. As of December- 2011, the NBK has collected various human biospecimens from 525,416 participants including 325,952 Korean populations and 199,464 patients. The purpose of this paper is to introduce the KBP and quality control programs for collection of human biospecimens with high quality of NBK.