Seon-Hee Yim

Copy number variations in East-Asian population and their evolutionary and functional implications

Recent discovery of the copy number variation (CNV) in normal individuals has widened our understanding of genomic variation. However, most of the reported CNVs have been identified in Caucasians, which may not be directly applicable to people of different ethnicities. To profile CNV in East-Asian population, we screened CNVs in 3578 healthy, unrelated Korean individuals, using the Affymetrix Genome-Wide Human SNP array 5.0. We identified 144 207 CNVs using a pooled data set of 100 randomly chosen Korean females as a reference. The average number of CNVs per genome was 40.3, which is higher than that of CNVs previously reported using lower resolution platforms. The median size of CNVs was 18.9 kb (range 0.2–5406 kb). Copy number losses were 4.7 times more frequent than copy number gains. CNV regions (CNVRs) were defined by merging overlapping CNVs identified in two or more samples. In total, 4003 CNVRs were defined encompassing 241.9 Mb accounting for ∼8% of the human genome. A total of 2077 CNVRs (51.9%) were potentially novel. Known CNVRs were larger and more frequent than novel CNVRs. Sixteen percent of the CNVRs were observed in ≥1% of study subjects and 24% overlapped with the OMIM genes. A total of 476 (11.9%) CNVRs were associated with segmental duplications. CNVS/CNVRs identified in this study will be valuable resources for studying human genome diversity and its association with disease.

Clinical implication of recurrent copy number alterations in hepatocellular carcinoma and putative oncogenes in recurrent gains on 1q

To elucidate the pathogenesis of hepatocellular carcinoma (HCC) and develop useful prognosis predictors, it is necessary to identify biologically relevant genomic alterations in HCC. In our study, we defined recurrently altered regions (RARs) common to many cases of HCCs, which may contain tumor‐related genes, using whole‐genome array‐CGH and explored their associations with the clinicopathologic features. Gene set enrichment analysis was performed to investigate functional implication of RARs. On an average, 23.1% of the total probes were altered per case. Mean numbers of altered probes are significantly higher in high‐grade, bigger and microvascular invasion (MVI) positive tumors. In total, 32 RARs (14 gains and 18 losses) were defined and 4 most frequent RARs are gains in 1q21.1‐q32.1 (64.5%), 1q32.1‐q44 (59.2%), 8q11.21‐q24.3 (48.7%) and a loss in 17p13.3‐p12 (51.3%). Through focusing on RARs, we identified genes and functional pathways likely to be involved in hepatocarcinogenesis. Among genes in the recurrently gained regions on 1q, expression of KIF14 and TPM3 was significantly increased, suggesting their oncogenic potential in HCC. Some RARs showed the significant associations with the clinical features. Especially, the recurrent loss in 9p24.2‐p21.1 and gain in 8q11.21‐q24.3 are associated with the high tumor grade and MVI, respectively. Functional analysis showed that cytokine receptor binding and defense response to virus pathways are significantly enriched in high grade‐related RARs. Taken together, our results and the strategy of analysis will help to elucidate pathogenesis of HCC and to develop biomarkers for predicting behaviors of HCC.

Genome-Wide Screening of Genomic Alterations and Their Clinicopathologic Implications in Non–Small Cell Lung Cancers

Purpose: Although many genomic alterations have been observed in lung cancer, their clinicopathologic significance has not been thoroughly investigated. This study screened the genomic aberrations across the whole genome of non–small cell lung cancer cells with high-resolution and investigated their clinicopathologic implications. Experimental Design: One-megabase resolution array comparative genomic hybridization was applied to 29 squamous cell carcinomas and 21 adenocarcinomas of the lung. Tumor and normal tissues were microdissected and the extracted DNA was used directly for hybridization without genomic amplification. The recurrent genomic alterations were analyzed for their association with the clinicopathologic features of lung cancer. Results: Overall, 36 amplicons, 3 homozygous deletions, and 17 minimally altered regions common to many lung cancers were identified. Among them, genomic changes on 13q21, 1p32, Xq, and Yp were found to be significantly associated with clinical features such as age, stage, and disease recurrence. Kaplan-Meier survival analysis revealed that genomic changes on 10p, 16q, 9p, 13q, 6p21, and 19q13 were associated with poor survival. Multivariate analysis showed that alterations on 6p21, 7p, 9q, and 9p remained as independent predictors of poor outcome. In addition, significant correlations were observed for three pairs of minimally altered regions (19q13 and 6p21, 19p13 and 19q13, and 8p12 and 8q11), which indicated their possible collaborative roles. Conclusions: These results show that our approach is robust for high-resolution mapping of genomic alterations. The novel genomic alterations identified in this study, along with their clinicopathologic implications, would be useful to elucidate the molecular mechanisms of lung cancer and to identify reliable biomarkers for clinical application.

Homozygous deletion of CDKN2A (p16, p14) and CDKN2B (p15) genes is a poor prognostic factor in adult but not in childhood B-lineage acute lymphoblastic leukemia: a comparative deletion and hypermethylation study

The biological behavior of childhood B-lineage acute lymphoblastic leukemia (B-ALL) is different from that of adults. We performed a comprehensive analysis of the deletion and the methylation profile of CDKN2A (hereafter identified separately as p16 and p14, for the different proteins encoded) and CDKN2B (hereafter p15) in 91 newly diagnosed B-ALL patients (61 children, 30 adults). The prognostic significance of the profiles of these genes and the association between alterations in these genes and known cytogenetic prognostic factors (BCR/ABL; ETV6/RUNX1, formerly TEL/AML1; MLL rearrangement; and ploidy changes of chromosomes) were also assessed. The prevalence of homozygous deletion, hemizygous deletion, and no deletion of the 9p21 region was 11.5%, 16.4%, and 72.1%, respectively, in children and 30.0%, 20.0%, and 50.0%, respectively, in adults; the higher incidence of homozygous deletion in adults was significant (P=0.029). Homozygous deletion was associated with poor overall survival in adults (P=0.019), but not in children. The incidence of promoter methylation of p16, p14, and p15 was 34.4%, 14.8%, and 34.4%, respectively, in children and 26.7%, 10.0%, and 40.0%, respectively, in adults, with no significant difference between the two groups. No significant association was observed between deletion and methylation or with known cytogenetic prognostic factors. The difference in incidence, distribution, and prognostic effect of homozygous deletion in children and adults may explain the prognostic disparity.

Trends in cervical cancer mortality in Korea 1993-2002: corrected mortality using national death certification data and national cancer incidence data.

Cervical cancer is a major health problem for Korean women, accounting for 9.8% of new female cancer cases, even though incidence rates have been decreasing. The Korean cervical cancer mortality rate for 1993–2002 based on National Statistical Office data shows an increasing trend, but the actual rates are thought to have decreased by epidemiologists, clinicians and other cancer experts. To explain this gap and solve this problem, we corrected the number of cervical cancer deaths by comparing death certificate cases of unspecified uterine cancer data with the national cancer incidence databases of entire cancer registries in Korea. We used 2 different methods to make a correction. First, we considered “uterus, unspecified” deaths previously registered as “cervix, uterine” cases misclassified and added them to the cervical cancer deaths. Alternatively, we multiplied the total number of registered unspecified uterine cancer deaths by age‐specific proportions of registered incident cervical cancer cases among all cancers and added the product to cervical cancer deaths. The overall corrected age‐standardized cervical cancer mortality rates per 100,000 women decreased from 5.2 in 1993 to 3.9 in 2002 (estimated annual percentage change (EAPC): −4.05%, 95% CI: −4.88, −3.22). While cervical cancer mortality showed a decreasing tendency in women aged 30–69 years, it increased substantially in women aged ≥70 years (EAPC: 3.62%, 95% CI: 1.92–5.35). Results of this study will provide evidence‐based foundation for the evaluation of the existing cervical cancer‐screening programs.

Copy number variations associated with idiopathic autism identified by whole-genome microarray-based comparative genomic hybridization.

Objectives Autism spectrum disorder (ASD) has been thought to have strong genetic background, but major contributing genes or associated molecular–genetic pathways are yet to be identified. To explore the idiopathic ASD-associated copy number variations (CNVs), we conducted case–control study using whole-genome copy number analysis. Methods Whole-genome microarray-based comparative genomic hybridization was carried out on 28 children (24 boys and four girls) diagnosed as ASD and 62 Korean adults (45 males and 17 females) without any signs of abnormalities and family history of genetic disorders as normal controls. Fluorescence in-situ hybridization and capillary electrophoresis-single-strand conformational polymorphism were used for quantitative verification of the ASD-associated CNVs. Results Thirty-eight CNVs were identified. Among them, the distributions of copy number loss CNVs on 8p23.1 (odds ratio: 5.1, 95% confidence interval: 1.7–14.5, P=0.003) and on 17p11.2 (odds ratio: uncalculable because of zero cell, P=0.008) were found to be significantly different between ASD and control groups. DEFENSIN family occurs in a cluster at 8p23.1 region. Fluorescence in-situ hybridization and capillary electrophoresis-single-strand conformational polymorphism coherently showed reduced copy number of DEFENSIN in cases with 8p23.1 copy number loss CNV, which validated microarray-based comparative genomic hybridization results; but there are no known coding genes in the CNV on 17p11.2. Conclusion Our approach as well as results can help to elucidate the genetic mechanism of idiopathic ASD.

Tropomyosin3 overexpression and a potential link to epithelial-mesenchymal transition in human hepatocellular carcinoma

BackgroundSince hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, it is still important to understand hepatocarcinogenesis mechanisms and identify effective markers for tumor progression to improve prognosis. Amplification and overexpression of Tropomyosin3 (TPM3) are frequently observed in HCC, but its biological meanings have not been properly defined. In this study, we aimed to elucidate the roles of TPM3 and related molecular mechanisms.MethodsTPM3-siRNA was transfected into 2 HCC cell lines, HepG2 and SNU-475, which had shown overexpression of TPM3. Knockdown of TPM3 was verified by real-time qRT-PCR and western blotting targeting TPM3. Migration and invasion potentials were examined using transwell membrane assays. Cell growth capacity was examined by colony formation and soft agar assays.ResultsSilencing TPM3 resulted in significant suppression of migration and invasion capacities in both HCC cell lines. To elucidate the mechanisms behind suppressed migration and invasiveness, we examined expression levels of Snail and E-cadherin known to be related to epithelial-mesenchymal transition (EMT) after TPM3 knockdown. In the TPM3 knockdown cells, E-cadherin expression was significantly upregulated and Snail downregulated compared with negative control. TPM3 knockdown also inhibited colony formation and anchorage independent growth of HCC cells.ConclusionsBased on our findings, we formulate a hypothesis that overexpression of TPM3 activates Snail mediated EMT, which will repress E-cadherin expression and that it confers migration or invasion potentials to HCC cells during hepatocarcinogenesis. To our knowledge, this is the first evidence that TPM3 gets involved in migration and invasion of HCCs by modifying EMT pathway.

Deletion variants of RABGAP1L, 10q21.3, and C4 are associated with the risk of systemic lupus erythematosus in Korean women.

OBJECTIVE Several copy number variations (CNVs) have been found to be associated with systemic lupus erythematosus (SLE) through the target gene approach. However, genome-wide features of CNVs and their role in the risk of SLE remain unknown. The aim of this study was to identify SLE-associated CNVs in Korean women. METHODS Genome-wide assessments of CNVs were performed in 382 SLE patients and 191 control subjects, using an Illumina HumanHap610 BeadChip genotyping platform. SLE-associated CNV regions that were identified by genome-wide association study (GWAS) were replicated in quantitative polymerase chain reaction (PCR) and deletion-typing PCR analyses in an independent sample set comprising 564 SLE patients and 511 control subjects. RESULTS Of 144 common CNV regions, 3 deletion-type CNV regions in 1q25.1, 8q23.3, and 10q21.3 were found to be significantly associated with SLE by GWAS analysis. In the independent replication, the CNV regions in 1q25.1 (RABGAP1L) and 10q21.3 were successfully replicated (odds ratio [OR] 1.30, P=0.038 and OR 1.90, P=3.6×10(-5), respectively), and the associations were confirmed again by deletion-typing PCR. The CNV region in the C4 gene, which showed a potential association in the discovery stage, was included in the replication analysis and was found to be significantly associated with the risk of SLE (OR 1.88, P=0.01). Through deletion-typing PCR, the exact sizes and breakpoint sequences of the deletions were defined. Individuals with the deletions in all 3 loci (RABGAP1L, 10q21.3, and C4) had a much higher risk of SLE than did those without any deletions in the 3 loci (OR 5.52, P=3.9×10(-4)). CONCLUSION These CNV regions can be useful to identify the pathogenic mechanisms of SLE, and might be used to more accurately predict the risk of SLE by taking into consideration their synergistic effects on disease susceptibility.

The potential role of VPREB1 gene copy number variation in susceptibility to rheumatoid arthritis

Although the etiology of rheumatoid arthritis (RA) remains unknown, it has been widely suggested that RA has a genetic background. In humans, a copy number loss of 22q11.2, a region harboring the VPREB1 gene, has been suggested to be associated with several immunologic disorders, but there has been no study on the copy number variation (CNV) of the VPREB1 and its potential association with RA. Here, we explored the association between the RA and the CNV of the VPREB1 gene by performing genomic quantitative PCR and quantification of B cell subsets in RA patients and controls. The proportion of the individuals with <2 copies of the VPREB1 gene was significantly higher in the patient group than that in the controls (12.9% vs 0.9%, p<0.0001), while that of the individuals with >2 copies was lower in the patient group than that in the controls (1.7% vs 18.9%, p<0.0001). The odds ratio (OR) of the individuals with <2 copies was significantly higher compared with the odds ratio of those individuals with 2 copies (OR=12.1, 95% confidence interval (CI) 2.8-51.6). Likewise, the OR of the individuals with >2 copies was significantly lower than the OR of those individuals with 2 copies (OR=0.09, 95% CI 0.03-0.3). We also found that the proportion of CD21⁻CD23⁻ B cells was significantly higher in the RA patients compared with that of the controls (11.9% vs 5.7%, p=0.002), but the proportion of CD21+CD23+ cells was significantly lower in the RA patients (26.2% in RA vs 34.9% in the controls, p=0.005). To the best of our knowledge, this is the first evidence showing the association between a low copy number of the VPREB1 gene and RA, and this may help understanding the pathogenesis of RA and other autoimmune disorders.

Common variants at the promoter region of the APOM confer a risk of rheumatoid arthritis

Although the genetic component in the etiology of rheumatoid arthritis (RA) has been consistently suggested, many novel genetic loci remain to uncover. To identify RA risk loci, we performed a genome-wide association study (GWAS) with 100 RA cases and 600 controls using Affymetrix SNP array 5.0. The candidate risk locus (APOM gene) was re-sequenced to discover novel promoter and coding variants in a group of the subjects. Replication was performed with the independent case-control set comprising of 578 RAs and 711 controls. Through GWAS, we identified a novel SNP associated with RA at the APOM gene in the MHC class III region on 6p21.33 (rs805297, odds ratio (OR) = 2.28, P = 5.20 × 10-7). Three more polymorphisms were identified at the promoter region of the APOM by the re-sequencing. For the replication, we genotyped the four SNP loci in the independent case-control set. The association of rs805297 identified by GWAS was successfully replicated (OR = 1.40, P = 6.65 × 10-5). The association became more significant in the combined analysis of discovery and replication sets (OR = 1.56, P = 2.73 ± 10-10). The individuals with the rs805297 risk allele (A) at the promoter region showed a significantly lower level of APOM expression compared with those with the protective allele (C) homozygote. In the logistic regressions by the phenotype status, the homozygote risk genotype (A/A) consistently showed higher ORs than the heterozygote one (A/C) for the phenotype-positive RAs. These results indicate that APOM promoter polymorphisms are significantly associated with the susceptibility to RA.