Clinical implications of copy number variations in autoimmune disorders

Human genetic variation is represented by the genetic differences both within and among populations, and most genetic variants do not cause overt diseases but contribute to disease susceptibility and influence drug response. During the last century, various genetic variants, such as copy number variations (CNVs), have been associated with diverse human disorders. Here, we review studies on the associations between CNVs and autoimmune diseases to gain some insight. First, some CNV loci are commonly implicated in various autoimmune diseases, such as Fcgamma receptors in patients with systemic lupus erythemoatosus or idiopathic thrombocytopenic purpura and beta-defensin genes in patients with psoriasis or Crohn's disease. This means that when a CNV locus is associated with a particular autoimmune disease, we should examine its potential associations with other diseases. Second, interpopulation or interethnic differences in the effects of CNVs on phenotypes exist, including disease susceptibility, and evidence suggests that CNVs are important to understand susceptibility to and pathogenesis of autoimmune diseases. However, many findings need to be replicated in independent populations and different ethnic groups. The validity and reliability of detecting CNVs will improve quickly as genotyping technology advances, which will support the required replication.

Silencing of KIF14 interferes with cell cycle progression and cytokinesis by blocking the p27(Kip1) ubiquitination pathway in hepatocellular carcinoma

Although it has been suggested that kinesin family member 14 (KIF14) has oncogenic potential in various cancers, including hepatocellular carcinoma (HCC), the molecular mechanism of this potential remains unknown. We aimed to elucidate the role of KIF14 in hepatocarcinogenesis by knocking down KIF14 in HCC cells that overexpressed KIF14. After KIF14 knockdown, changes in tumor cell growth, cell cycle and cytokinesis were examined. We also examined cell cycle regulatory molecules and upstream Skp1/Cul1/F-box (SCF) complex molecules. Knockdown of KIF14 resulted in suppression of cell proliferation and failure of cytokinesis, whereas KIF14 overexpression increased cell proliferation. In KIF14-silenced cells, the levels of cyclins E1, D1 and B1 were profoundly decreased compared with control cells. Of the cyclin-dependent kinase inhibitors, the p27Kip1 protein level specifically increased after KIF14 knockdown. The increase in p27Kip1 was not due to elevation of its mRNA level, but was due to inhibition of the proteasome-dependent degradation pathway. To explore the pathway upstream of this event, we measured the levels of SCF complex molecules, including Skp1, Skp2, Cul1, Roc1 and Cks1. The levels of Skp2 and its cofactor Cks1 decreased in the KIF14 knockdown cells where p27Kip1 accumulated. Overexpression of Skp2 in the KIF14 knockdown cells attenuated the failure of cytokinesis. On the basis of these results, we postulate that KIF14 knockdown downregulates the expression of Skp2 and Cks1, which target p27Kip1 for degradation by the 26S proteasome, leading to accumulation of p27Kip1. The downregulation of Skp2 and Cks1 also resulted in cytokinesis failure, which may inhibit tumor growth. To the best of our knowledge, this is the first report that has identified the molecular target and oncogenic effect of KIF14 in HCC.

Reflections on the US FDA's Warning on Direct-to-Consumer Genetic Testing

In November 2013, the US Food and Drug Administration (FDA) sent a warning letter to 23andMe, Inc. and ordered the company to discontinue marketing of the 23andMe Personal Genome Service (PGS) until it receives FDA marketing authorization for the device. The FDA considers the PGS as an unclassified medical device, which requires premarket approval or de novo classification. Opponents of the FDA's action expressed their concerns, saying that the FDA is overcautious and paternalistic, which violates consumers' rights and might stifle the consumer genomics field itself, and insisted that the agency should not restrict direct-to-consumer (DTC) genomic testing without empirical evidence of harm. Proponents support the agency's action as protection of consumers from potentially invalid and almost useless information. This action was also significant, since it reflected the FDA's attitude towards medical application of next-generation sequencing techniques. In this review, we followed up on the FDA-23andMe incident and evaluated the problems and prospects for DTC genetic testing.

Introduction to International Ethical Standards Related to Genetics and Genomics

The rapid advances in genetic knowledge and technology raise various, sometimes unprecedented, ethical dilemmas in the scientific community as well as the public realm. To deal with these dilemmas, the international community has prepared and issued ethical standards in various formats. In this review, seven international standards regarding genetics and genomics will be briefly introduced in chronological order. Critical reflections on them will not be provided in this review, and naturally, they have their own problems and shortcomings. However, a common set of the principles expressed in them will be highlighted here, because they are still relevant, and many of them will be more relevant in the future. Some of the interesting contents will be selected and described. After that, the morality of one recent event related to whole-genome sequencing and person-identifiable genetic data will be explored based on those international standards.

Genome-Wide Association Study of Lung Cancer in Korean Non-Smoking Women

Lung cancer in never-smokers ranks as the seventh most common cause of cancer death worldwide, and the incidence of lung cancer in non-smoking Korean women appears to be steadily increasing. To identify the effect of genetic polymorphisms on lung cancer risk in non-smoking Korean women, we conducted a genome-wide association study of Korean female non-smokers with lung cancer. We analyzed 440,794 genotype data of 285 cases and 1,455 controls, and nineteen SNPs were associated with lung cancer development (P < 0.001). For external validation, nineteen SNPs were replicated in another sample set composed of 293 cases and 495 controls, and only rs10187911 on 2p16.3 was significantly associated with lung cancer development (dominant model, OR of TG or GG, 1.58, P = 0.025). We confirmed this SNP again in another replication set composed of 546 cases and 744 controls (recessive model, OR of GG, 1.32, P = 0.027). OR and P value in combined set were 1.37 and < 0.001 in additive model, 1.51 and < 0.001 in dominant model, and 1.54 and < 0.001 in recessive model. The effect of this SNP was found to be consistent only in adenocarcinoma patients (1.36 and < 0.001 in additive model, 1.49 and < 0.001 in dominant model, and 1.54 and < 0.001 in recessive model). Furthermore, after imputation with HapMap data, we found regional significance near rs10187911, and five SNPs showed P value less than that of rs10187911 (rs12478012, rs4377361, rs13005521, rs12475464, and rs7564130). Therefore, we concluded that a region on chromosome 2 is significantly associated with lung cancer risk in Korean non-smoking women.

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 x 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 x 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.

No Association between Copy Number Variation of the TCRB Gene and the Risk of Autism Spectrum Disorder in the Korean Population

Although autism spectrum disorder (ASD) has been thought to have a substantial genetic background, major contributing genes have yet to be identified or successfully replicated. Immunological dysfunction has been suggested to be associated with ASD, and T cell-mediated immunity was considered important for the development of ASD. In this study, we analyzed 163 ASD subjects and 97 normal controls by genomic quantitative PCR to evaluate the association between the copy number variation of the 7q34 locus, harboring the TCRB gene, and ASDs. As a result, there was no significant difference of the frequency distribution of TCRB copy numbers between ASD cases and normal controls. TCRB gene copy numbers ranged from 0 to 5 copies, and the frequency distribution of each copy number was similar between the two groups. The proportion of the individuals with 2 copies of TCRB was 11.7% (19/163) in ASD cases and 12.1% (11/91) in the control group (p=0.68). After the effects of sex were adjusted by logistic regression, ORs for individuals with 2 copies showed no significant difference compared with the diploid copy number as reference (n=2). Although we could not see the positive association, our results will be valuable information for mining ASD-associated genes and for exploring the role of T cell immunity further in the pathogenesis of ASD.

Comparison of the Affymetrix SNP Array 5.0 and Oligoarray Platforms for Defining CNV

Together with single nucleotide polymorphism (SNP), copy number variations (CNV) are recognized to be the major component of human genetic diversity and used as a genetic marker in many disease association studies. Affymetrix Genome-wide SNP 5.0 is one of the commonly used SNP array platforms for SNP-GWAS as well as CNV analysis. However, there has been no report that validated the accuracy and reproducibility of CNVs identified by Affymetrix SNP array 5.0. In this study, we compared the characteristics of CNVs from the same set of genomic DNAs detected by three different array platforms; Affymetrix SNP array 5.0, Agilent 2X244K CNV array and NimbleGen 2.1M CNV array. In our analysis, Affymetrix SNP array 5.0 seems to detect CNVs in a reliable manner, which can be applied for association studies. However, for the purpose of defining CNVs in detail, Affymetrix Genome-wide SNP 5.0 might be relatively less ideal than NimbleGen 2.1M CNV array and Agilent 2X244K CNV array, which outperform Affymetrix array for defining the small-sized single copy variants. This result will help researchers to select a suitable array platform for CNV analysis.

Replication of the Association between Copy Number Variation on 8p23.1 and Autism by Using ASD-specific BAC Array

To discover genetic markers for autism spectrum disorder (ASD), we previously applied genome-wide BAC array comparative genomic hybridization (array-CGH) to 28 autistic patients and 62 normal controls in Korean population, and identified that chromosomal losses on 8p23.1 and on 17p11.2 are significantly associated with autism. In this study, we developed an 8.5K ASD-specific BAC array covering 27 previously reported ASD-associated CNV loci including ours and examined whether the associations would be replicated in 8 ASD patient cell lines of four different ethnic groups and 10 Korean normal controls. As a result, a CNV-loss on 8p23.1 was found to be significantly more frequent in patients regardless of ethnicity (p<0.0001). This CNV region contains two coding genes, DEFA1 and DEFA3, which are members of DEFENSIN gene family. Two other CNVs on 17p11.2 and Xp22.31 were also distributed differently between ASDs and controls, but not significant (p=0.069 and 0.092, respectively). All the other loci did not show significant association. When these evidences are considered, the association between ASD and CNV of DEFENSIN gene seems worthy of further exploration to elucidate the pathogenesis of ASD. Validation studies with a larger sample size will be required to verify its biological implication.

Integrated analysis of copy number alteration and RNA expression profiles of cancer using a high-resolution whole-genome oligonucleotide array

Recently, microarray-based comparative genomic hybridization (array-CGH) has emerged as a very efficient technology with higher resolution for the genome-wide identification of copy number alterations (CNA). Although CNAs are thought to affect gene expression, there is no platform currently available for the integrated CNA-expression analysis. To achieve high-resolution copy number analysis integrated with expression profiles, we established human 30k oligoarray-based genome-wide copy number analysis system and explored the applicability of this system for integrated genome and transcriptome analysis using MDA-MB-231 cell line. We compared the CNAs detected by the oligoarray with those detected by the 3k BAC array for validation. The oligoarray identified the single copy difference more accurately and sensitively than the BAC array. Seventeen CNAs detected by both platforms in MDA-MB-231 such as gains of 5p15.33-13.1, 8q11.22-8q21.13, 17p11.2, and losses of 1p32.3, 8p23.3-8p11.21, and 9p21 were consistently identified in previous studies on breast cancer. There were 122 other small CNAs (mean size 1.79 mb) that were detected by oligoarray only, not by BAC-array. We performed genomic qPCR targeting 7 CNA regions, detected by oligoarray only, and one non-CNA region to validate the oligoarray CNA detection. All qPCR results were consistent with the oligoarray-CGH results. When we explored the possibility of combined interpretation of both DNA copy number and RNA expression profiles, mean DNA copy number and RNA expression levels showed a significant correlation. In conclusion, this 30k oligoarray-CGH system can be a reasonable choice for analyzing whole genome CNAs and RNA expression profiles at a lower cost.

One Korean Patient with a Family History of BRCA1-associated Ovarian Cancer

Germline mutations in BRCA1 and BRCA2 confer high risks of breast and ovarian cancer. Among BRCA1- and BRCA2- mutation carriers, the average cumulative risks for ovarian cancer by age 70 years were 39% and 11%, respectively. There are other hereditary cancer syndromes such as Hereditary nonpolyposis colorectal cancer also confer a higher risk for developing ovarian cancer, but over 90% of all hereditary ovarian cancers are thought to be associated with BRCA1 or BRCA2 mutations. This report concerns a Korean woman diagnosed with ovarian cancer present with a family history of ovarian and various other cancers, in whom a germline BRCA1 mutation was identified and the same mutation was found in one of two daughters of her's. Since there could be more hereditary ovarian cancer patients in Korean than clinicians thought, both primary and secondary prevention of ovarian cancer based on family history and genetic information is important to reduce cancer incidence and mortality.

Comparison of Cancer Survival by Age Group for 1997 and for 2002: Application of Period Analysis using the National Cancer Incidence Database / 예방의학회지

OBJECTIVES: Period analysis estimates up-to-date survival rates of cancer patients. In this approach, analysis is restricted to recent time period by left-truncating all observations at the beginning of the period and rightcensoring at its end. Here, we applied period analysis to examine changes in 5-year relative survival (RS) by age group for 1997 and for 2002. METHODS: Using the National Cancer Incidence Database, 5-year RS was estimated for 1997 and 2002 in four age groups (15-54, 55-64, 65-74, and 75 years old and over) using period analysis. After excluding death certificate-only cases, patients with an unknown date of diagnosis or follow-up length, a total of 813,889 patients diagnosed with a first primary invasive cancer during 1992-2002 were included for analysis. Followup for vital status was included until 31 December 2002. RESULTS: Five-year RS increased from 41.7% for 1997 to 46.7% for 2002. Increases in survival occurred in all age groups except in the 75 and over group. CONCLSIONS: The age gradient in cancer prognosis seems to have widened between 1997 and 2002, a finding that requires further study of prognostic factors, including stage at diagnosis. Period analysis accurately estimates survival rates, especially for cancers with better prognosis.

Copy Number Variations in the Human Genome: Potential Source for Individual Diversity and Disease Association Studies

The widespread presence of large-scale genomic variations, termed copy number variation (CNVs), has been recently recognized in phenotypically normal individuals. Judging by the growing number of reports on CNVs, it is now evident that these variants contribute significantly to genetic diversity in the human genome. Like single nucleotide polymorphisms (SNPs), CNVs are expected to serve as potential biomarkers for disease susceptibility or drug responses. However, the technical and practical concerns still remain to be tackled. In this review, we examine the current status of CNV DBs and research, including the ongoing efforts of CNV screening in the human genome. We also discuss the characteristics of platforms that are available at the moment and suggest the potential of CNVs in clinical research and application.

Copy Number Variations in the Human Genome: Potential Source for Individual Diversity and Disease Association Studies

The widespread presence of large-scale genomic variations, termed copy number variation (CNVs), has been recently recognized in phenotypically normal individuals. Judging by the growing number of reports on CNVs, it is now evident that these variants contribute significantly to genetic diversity in the human genome. Like single nucleotide polymorphisms (SNPs), CNVs are expected to serve as potential biomarkers for disease susceptibility or drug responses. However, the technical and practical concerns still remain to be tackled. In this review, we examine the current status of CNV DBs and research, including the ongoing efforts of CNV screening in the human genome. We also discuss the characteristics of platforms that are available at the moment and suggest the potential of CNVs in clinical research and application.

Comparison of Normalization Methods for Defining Copy Number Variation Using Whole-genome SNP Genotyping Data

Precise and reliable identification of CNV is still important to fully understand the effect of CNV on genetic diversity and background of complex diseases. SNP marker has been used frequently to detect CNVs, but the analysis of SNP chip data for identifying CNV has not been well established. We compared various normalization methods for CNV analysis and suggest optimal normalization procedure for reliable CNV call. Four normal Koreans and NA10851 HapMap male samples were genotyped using Affymetrix Genome-Wide Human SNP array 5.0. We evaluated the effect of median and quantile normalization to find the optimal normalization for CNV detection based on SNP array data. We also explored the effect of Robust Multichip Average (RMA) background correction for each normalization process. In total, the following 4 combinations of normalization were tried: 1) Median normalization without RMA background correction, 2) Quantile normalization without RMA background correction, 3) Median normalization with RMA background correction, and 4) Quantile ormalization with RMA background correction. CNV was called using SW-ARRAY algorithm. We applied 4 different combinations of normalization and compared the effect using intensity ratio profile, box plot, and MA plot. When we applied median and quantile normalizations without RMA background correction, both methods showed similar normalization effect and the final CNV calls were also similar in terms of number and size. In both median and quantile normalizations, RMA background correction resulted in widening the range of intensity ratio distribution, which may suggest that RMA background correction may help to detect more CNVs compared to no correction.

Introduction of Relative Survival Analysis Program: Using Sample of Cancer Registry Data with Stata Software / 한국역학회지

Patient survival is one of the most important measures for the evaluation of progress in cancer patient care across the wide spectrum from diagnosis to treatment. The optimal monitoring method for cancer patient survival is to estimate survival based on representative data from cancer patients in the population, which is only achievable through using population-based cancer registration data. Relative survival is used to compare the survival experience in a study cohort that expected to result from background population mortality rates. This technique is useful when the cause of death is not accurate or not available, since it provides a measure of excess mortality in a group of patients with a certain disease. The purpose of this article is to demonstrate the procedures for estimating relative survival using the statistical software Stata. For this survival analysis to show the procedure, the example data set was randomly selected from the National Cancer Incidence Database, which was used in a recent article reporting the overall relative survival of cancer patients diagnosed during 1993-2002 in Korea.

Cancer Survival in Korea 1993-2002: A Population-Based Study

Population-based survival reflect the average prognosis of unselected patients with a variety of natural histories as well as treatment patterns and are also useful for evaluating effectiveness and efficiency of cancer-directed health services in a given region. Although survival data have been reported based on hospital data, the survival data from population-based registry have been rarely reported in Korea. Based on the Korea National Cancer Incidence Database, we report the results from survival analysis for cancer patients diagnosed during 1993-2002 and followed up until 31 December 2005 at primary cancer sites. The five-year relative survival rates (RSR) were calculated using the Ederer II method. The Kaplan-Meier method was used to estimate median survival and the 95% confidence intervals. In males, the five-year RSR for all cancers was 32.5% during 1993-1997 and was 37.8% during 1998-2002. In females, the five-year RSR for all cancers was 53.7% during 1993-1997 and was 57.0% during 1998-2002. The largest improvement in survival was shown in prostate cancer in males and breast and stomach cancer in females. The median survival durations were 16.3 months in males and 81.6 months in females. This result will be useful for evaluation of cancer treatment outcomes in Korea.

Population-based Breast Cancer Statistics in Korea during 1993-2002: Incidence, Mortality, and Survival

In 2002, breast became the most common cancer site in Korean women. Using national breast cancer incidence data during 1993-2002, crude, age-standardized, and age-specific rates for incidence and mortality were calculated. Survival was examined for cases diagnosed during 1993-2002 and followed up to 2004. Observed survival was calculated using the life table method and relative survival using the Ederer II method. Age-standardized incidence rates in female increased from 14.5 in 1993 to 26.2 per 100,000 in 2002. Age-specific incidences showed peaks in women in their forties. Mortality rates increased from 3.7 in 1993 to 4.6 per 100,000 in 2002 and showed peaks in women in their fifties. Five-year relative survival for female breast cancer diagnosed during 1993-2002 was 82.2%. When we examined the secular trends using cases diagnosed 1993-1999 for complete 5-yr follow-up, the 5-yr relative survival increased from 75.2% in 1993 to 83.5% in 1999. The data from this study will provide valuable information to plan and evaluate actions against breast cancer including national breast cancer screening.

National Cancer Incidence for the Year 2002 in Korea / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Since the revised Cancer Act of October 2006, cancer registration was reactivated, based on the Statistics Law. MATERIALS AND METHODS: The incidence of cancer during 2002 was calculated on the basis of the information available from the National Cancer Incidence Database. Crude and age-standardized rates were calculated by gender for 18 age groups (0~4, 5~9, 10~14, every five years, 85 years and over). RESULTS: The overall crude incidence rates (CRs) were 269.2 and 212.8 per 100,000 for males and females, and the overall age-standardized incidence rates (ASRs) were 287.8 and 172.9 per 100,000, respectively. Among males, the five leading primary cancer sites were stomach (CR 62.4, ASR 65.7), lung (CR 45.4, ASR 51.0), liver (CR 43.2, ASR 43.7), colon and rectum (CR 30.7, ASR 32.7), and prostate (CR 8.0, ASR 9.6). Among females, the most common cancer sites were breast (CR 33.1, ASR 26.9), followed by stomach (CR 32.8, ASR 26.0), colon and rectum (CR 23.1, ASR 18.5), thyroid (CR 19.1, ASR 15.7), and uterine cervix (CR 18.2, ASR 14.7). In the 0~14 age group, leukemia was the most common cancer for both genders. For males, stomach cancer was the most common cancer in the 15~64 age-group, but lung cancer was more frequent in men 65 or older. For females, thyroid cancer among the 15~34 age-group, breast cancer among 35~64 age-group and stomach cancer in women 65 years or older were the most common forms of cancer for each age group. The quality indices for the percentage of deaths, by death certificate only, were 4.7% for males and 4.5% for females. CONCLUSIONS: Since the National Cancer Incidence Database was started, the annual percent change of cancer cases increased by 4.8% (4.1% for males, 5.7% for females) during 1999~2002. This value reflects the increase in prostate cancer for males and breast and thyroid cancer in females during 2002. The timely reporting of improved quality of cancer registration is needed for evidence-based decisions regarding cancer control in Korea.

Nationwide Cancer Incidence in Korea, 1999~2001; First Result Using the National Cancer Incidence Database / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The first Korean national population- based cancer registry using nationwide hospital-based recording system and the regional cancer registries provided the source to obtain national cancer incidences for the period 1999~2001. MATERIALS AND METHODS: The incidence of cancer in Korea was calculated based on the Korea Central Cancer Registry database, data from additional medical record review survey, the Regional Cancer Registry databases, site-specific cancer registry databases, and cancer mortality data from the Korea National Statistical Office. Crude and age-standardized rates were calculated by sex for 18 age groups. RESULTS: The overall crude incidence rates (CR) were 247.3 and 188.3 per 100, 000 for men and women and the overall age-standardized incidence rates (ASR) were 281.2 and 160.3 per 100, 000, respectively. Among men, five leading primary cancer sites were stomach (CR 58.6, ASR 65.6), lung (CR 42.1, ASR 50.9), liver (CR 41.9, ASR 44.9), colon and rectum (CR 24.2, ASR 27.3) and bladder (CR 7.7, ASR 9.2). Among women, the most common cancers were stomach (CR 30.8, ASR 25.8), breast (CR 25.7, ASR 21.7), colon and rectum (CR 19.6, ASR 16.7), uterine cervix (CR 18.4, ASR 15.5), and lung cancer (CR 15.1, ASR 12.4). In 0~14 age group, leukemia was most common for both sexes. For men, stomach cancer was most common in 15~64 age group, but lung cancer was more frequent for over 65 age group. For women, thyroid cancer in 15~34 age group, breast cancer in 35~64 age group, and stomach cancer in over 65 age group were most common for each age group. The proportions of death certificate only were 7.5% for men and 7.4% for women. CONCLUSION: This is the first attempt to determine the national cancer incidence and this data will be useful to plan for research and national cancer control in Korea.