Assessment of the Impact of Preanalytical DNA Integrity on the Genome Data Quality.

Many molecular approaches have been employed for the quality control (QC) of biobanked DNA samples. Since 2003, the National Biobank of Korea (NBK) has provided various studies with over half a million quality-controlled genomic DNA samples using conventional agarose gel electrophoresis and spectrophotometry. We assessed the postanalytical genomic data quality of DNA samples (n = 41) with a different range of the DNA quality index such as genomic quality number (GQN) for developing an evidence-based best practice for DNA quality criteria. We examined the quality indices of three different platforms, including single nucleotide polymorphism arrays, methylation arrays, and next-generation sequencing, using the same DNA samples (n = 41) of different quality, ranging from 4.0 to 10.0 values of the GQN. Our data analysis revealed that higher GQN value and/or double-stranded DNA concentration resulted in higher quality genomic data. In addition, all the analyzed DNA samples successfully generated good-quality genomic data. This study provides a guide for the QC of biobanked DNA samples for genomic analysis platforms.

A combination of multiple autoantibodies is associated with the risk of Alzheimer's disease and cognitive impairment.

Autoantibodies are self-antigen reactive antibodies that play diverse roles in the normal immune system, tissue homeostasis, and autoimmune and neurodegenerative diseases. Anti-neuronal autoantibodies have been detected in neurodegenerative disease serum, with unclear significance. To identify diagnostic biomarkers of Alzheimer's disease (AD), we analyzed serum autoantibody profiles of the HuProt proteome microarray using the discovery set of cognitively normal control (NC, n = 5) and AD (n = 5) subjects. Approximately 1.5-fold higher numbers of autoantibodies were detected in the AD group (98.0 ± 39.9/person) than the NC group (66.0 ± 39.6/person). Of the autoantigen candidates detected in the HuProt microarray, five autoantigens were finally selected for the ELISA-based validation experiment using the validation set including age- and gender-matched normal (NC, n = 44), mild cognitive impairment (MCI, n = 44) and AD (n = 44) subjects. The serum levels of four autoantibodies including anti-ATCAY, HIST1H3F, NME7 and PAIP2 IgG were significantly different among NC, MCI and/or AD groups. Specifically, the anti-ATCAY autoantibody level was significantly higher in the AD (p = 0.003) and MCI (p = 0.015) groups compared to the NC group. The anti-ATCAY autoantibody level was also significantly correlated with neuropsychological scores of MMSE (rs = - 0.229, p = 0.012), K-MoCA (rs = - 0.270, p = 0.003), and CDR scores (rs = 0.218, p = 0.016). In addition, a single or combined occurrence frequency of anti-ATCAY and anti-PAIP2 autoantibodies was significantly associated with the risk of MCI and AD. This study indicates that anti-ATCAY and anti-PAIP2 autoantibodies could be a potential diagnostic biomarker of AD.

The Brain Donation Program in South Korea.

PURPOSE: Obtaining brain tissue is critical to definite diagnosis and to furthering understanding of neurodegenerative diseases. The present authors have maintained the National Neuropathology Reference and Diagnostic Laboratories for Dementia in South Korea since 2016. We have built a nationwide brain bank network and are collecting brain tissues from patients with neurodegenerative diseases. We are aiming to facilitate analyses of clinic-pathological and image-pathological correlations of neurodegenerative disease and to broaden understanding thereof. MATERIALS AND METHODS: We recruited participants through two routes: from memory clinics and the community. As a baseline evaluation, clinical interviews, a neurological examination, laboratory tests, neuropsychological tests, and MRI were undertaken. Some patients also underwent amyloid PET. RESULTS: We recruited 105 participants, 70 from clinics and 35 from the community. Among them, 11 died and were autopsied. The clinical diagnoses of the autopsied patients included four with Alzheimer's disease (AD), two with subcortical vascular dementia, two with non-fluent variant primary progressive aphasia, one with leukoencephalopathy, one with frontotemporal dementia (FTD), and one with Creutzfeldt-Jakob disease (CJD). Five patients underwent amyloid PET: two with AD, one with mixed dementia, one with FTD, and one with CJD. CONCLUSION: The clinical and neuropathological information to be obtained from this cohort in the future will provide a deeper understanding of the neuropathological mechanisms of cognitive impairment in Asia, especially Korea.

Effective litmus gene test for monitoring the quality of blood samples: Application to Alzheimer's disease diagnostics.

Gene expression profiles reflect the biologically diverse activities of cells under specific cell environments. Using the transcriptional response of cultured cells to blood composition, we developed a litmus gene assay to discriminate blood samples reflecting different sample qualities or disease conditions. This cell-based litmus gene assay identified six genes (CCL20, CEMIP, IL1B, IL8, PRG2, PTGS2) as potential biomarkers of plasma quality control and the SPC25 gene as a diagnostic biomarker of Alzheimer's disease (AD). In addition, the SPC25 gene expression level was significantly increased in the cell-based assay using serum samples from patients with mild cognitive impairment (MCI). In conclusion, we demonstrated the effectiveness and potential of a litmus gene assay to detect the orchestrated effects of circulating systemic factors, leading to the successful diagnosis of AD and MCI. This method is broadly applicable to the diagnosis of disease subtypes or patho-physiological stages of complex diseases and tumors.

Proposal Guidelines for Standardized Operating Procedures of Brain Autopsy: Brain Bank in South Korea.

To obtain an in-depth understanding of brain diseases, including neurodegenerative diseases, psychiatric illnesses, and neoplasms, scientific approach and verification using postmortem human brain tissue with or without disease are essential. Compared to other countries that have run brain banks for decades, South Korea has limited experience with brain banking; nationwide brain banks started only recently. The goal of this study is to provide provisional guidelines for brain autopsy for hospitals and institutes that have not accumulated sufficient expertise. We hope that these provisional guidelines will serve as a useful reference for pathologists and clinicians who are involved and interested in the brain bank system. Also, we anticipate updating the provisional guidelines in the future based on collected data and further experience with the practice of brain autopsy in South Korea.

Elevated Epstein-Barr Virus Antibody Level is Associated with Cognitive Decline in the Korean Elderly.

Chronic viral infection is implicated in cognitive decline and Alzheimer's disease (AD). Our goal was to identify biomarkers for the development of amnestic mild cognitive impairment (aMCI) from cognitively normal state. To accomplish this, we analyzed plasma IgG levels against Epstein-Barr virus (EBV) and herpes simplex virus 1 (HSV-1) in study subjects with incident aMCI (Converter) and normal cognitive function (NC Control) who did or did not convert from cognitively normal state to aMCI during the 2-year follow-up period, respectively. The Converter group exhibited elevated levels of anti-EBV IgG antibodies in the post-follow-up phase (aMCI state) compared to the pre-follow-up phase (cognitively normal state), but not the NC Control group. In contrast, the total IgG level was not significantly changed over the follow-up period. Moreover, elevated anti-EBV IgG levels were significantly associated with CDR scales and total CERAD scores in the Converter group. These results suggest that EBV infection or its related host immune response is linked to cognitive decline. Thus, an EBV antibody level may be used as a potential biomarker for assessing the risk of aMCI development, implying a role for chronic EBV infection in AD pathogenesis.

Conversion pattern and predictive factor of mild cognitive impairment in elderly Koreans.

OBJECTIVE: We aimed to understand conversion characteristics of mild cognitive impairment (MCI) in elderly Koreans. METHODS: We analyzed clinical data of 760 individuals who participated in a two-year follow-up study. Neuropsychological assessments and clinical examination were conducted in the follow-ups. Logistic regression model was used to estimate predictive risk factors of MCI conversion. RESULT: The participants at baseline (n=760) represented 462 cognitively normal individuals (60.8%), 286 individuals with MCI (37.6%), and 12 individuals with dementia (1.6%). Among the cognitively normal individuals (n=462), 108 (23.4%) progressed to MCI during the two-year follow-up period, including 92 with amnestic mild cognitive impairment (aMCI; 19.9%) and 16 with non-amnestic mild cognitive impairment (non-aMCI; 3.5%). Interestingly, 3.7% of participants with aMCI converted to non-aMCI, while 45.5% of participants with non-aMCI converted to aMCI. Moreover, a higher proportion of non-aMCI (27.3%) reverted to a cognitively normal state, compared to aMCI participants (18.6%), indicating that non-amnestic cognitive impairment is more unstable than amnestic cognitive impairment, and probably converges toward aMCI. Additionally, we found that weight loss was associated with incident MCI and future MCI. Weight loss was negatively correlated with Clinical Dementia Rating (p=0.005), and significantly associated with a higher risk of MCI conversion from a cognitively normal state (OR=1.10, 95% CI: 1.00-1.21, p=0.042). CONCLUSION: This study supports that non-amnestic MCI is prone to converge toward amnestic MCI, and the elderly people with weight loss are at risk for developing cognitive decline.

An epigenomic signature of postprandial hyperglycemia in peripheral blood leukocytes.

Postprandial hyperglycemia is known to be one of the earliest signs of abnormal glucose homeostasis associated with type 2 diabetes. This study aimed to assess clinical significance of a 1-h postprandial glucose level for the development of diabetes, and identify epigenetic biomarkers of postprandial hyperglycemia. We analyzed clinical data from the oral glucose tolerance tests for healthy subjects (n=4502). The ratio (Glu60/Glu0) of 1-h glucose levels to fasting glucose levels was significantly associated with an insulin sensitive index (QUICKI, quantitative insulin sensitivity check index) (ß=0.055, P=1.25E-04) as well as a risk of future pre-diabetic and diabetic conversion. Next, DNA methylation profile analyses of 24 matched pairs of the high and low Glu60/Glu0 ratio subjects showed that specific DNA methylation levels in the promoter region of an olfactory receptor gene (olfactory receptor gene family10 member A4, OR10A4) were associated with the Glu60/Glu0 ratios (ß=0.337, P=0.03). Moreover, acute oral glucose challenges decreased the DNA methylation levels of OR10A4 but not the global DNA methylation in peripheral leukocytes of healthy subjects (n=7), indicating that OR10A4 is a specific epigenomic target of postprandial hyperglycemia. This work suggests possible relevance of olfactory receptor genes to an earlier molecular biomarker of peripheral hyperglycemia and diabetic conversion.

Network signatures of cellular immortalization in human lymphoblastoid cell lines.

Human lymphoblastoid cell line (LCL) has been used as an in vitro cell model in genetic and pharmacogenomic studies, as well as a good model for studying gene expression regulatory machinery using integrated genomic analyses. In this study, we aimed to identify biological networks of LCL immortalization from transcriptomic profiles of microRNAs and their target genes in LCLs. We first selected differentially expressed genes (DEGs) and microRNAs (DEmiRs) between early passage LCLs (eLCLs) and terminally differentiated late passage LCLs (tLCLs). The in silico and correlation analysis of these DEGs and DEmiRs revealed that 1098 DEG-DEmiR pairs were found to be positively (n=591 pairs) or negatively (n=507 pairs) correlated with each other. More than 41% of DEGs are possibly regulated by miRNAs in LCL immortalizations. The target DEGs of DEmiRs were enriched for cellular functions associated with apoptosis, immune response, cell death, JAK-STAT cascade and lymphocyte activation while non-miRNA target DEGs were over-represented for basic cell metabolisms. The target DEGs correlated negatively with miR-548a-3p and miR-219-5p were significantly associated with protein kinase cascade, and the lymphocyte proliferation and apoptosis, respectively. In addition, the miR-106a and miR-424 clusters located in the X chromosome were enriched in DEmiR-mRNA pairs for LCL immortalization. In this study, the integrated transcriptomic analysis of LCLs could identify functional networks of biologically active microRNAs and their target genes involved in LCL immortalization.

MicroRNAs in human lymphoblastoid cell lines.

Human lymphoblastoid cell lines (LCLs) are generated by EBV-mediated B-cell transformation to provide unlimited genomic resources for human genetics and immunological studies. The LCL is a good in vitro cell model for assessing population differences in the basal expression of genes and miRNAs as well as in cellular responses to various stimulators. Recently, the utility of LCLs was extended to pharmacogenomic studies to discover genetic factors underlying individual variations in response to chemicals and environmental stresses. Although LCLs represent generally lymphoid tissue-specific biological characteristics, genomic signatures of LCLs can distinguish patients with brain-related diseases and nonlymphoid tumors from normal controls. MicroRNA is known to be an epigenetic transcriptional regulator, and its expression is induced in abnormal conditions such as perturbagen-stimulated, virus-infected, or cancer cells. The epigenetic regulation of gene expression mediated by microRNA and DNA methylation is important for understanding the pathogenesis of cancers and complex diseases as well as discovering for therapeutic targets. For integrative genomic analyses, LCLs can be utilized to generate cellular phenotypes and various genomic data (e.g., SNP, CNV, transcriptome, methylome, etc.), which can be linked to clinical information of donors. Here, we discuss miRNA-mediated gene expression in LCLs and its application to disease genomics and global transcriptional regulatory machinery studies.

Antitumor and Immunostimulating Activities of Elfvingia applanata Hot Water Extract on Sarcoma 180 Tumor-bearing ICR Mice.

Elfvingia applanata, a medicinal mushroom belonging to Basidiomycota, has been used in the effort to cure cancers of the esophagus and stomach, and is also known to have inhibitory effects on hepatitis B virus infection. The hot water soluble fraction (as Fr. HW) was extracted from fruiting bodies of the mushroom. In vitro cytotoxicity tests showed that hot water extract was not cytotoxic against cancer cell lines such as Sarcoma 180, HT-29, HepG2, and TR at concentrations of 10~2,000 µg/mL. Intraperitoneal injection with Fr. HW resulted in a life prolongation effect of 45.2% in mice previously inoculated with Sarcoma 180. Treatment of Fr. HW resulted in a 2.53-fold increase in the numbers of murine spleen cells at a concentration of 50 µg/mL, compared with control. Incubation of murine spleen cells with Fr. HW at a concentration of 500 µg/mL resulted in improved immune-potwntiating activity of B lymphocytes through an 8.3-folds increase in alkaline phosphatase activity, compared with control. Fr. HW generated 12.5 µM of nitric oxide (NO) when cultured with RAW 264.7, a mouse macrophage cell line, at the concentration of 50 µg/mL, while lipopolysaccharide, a positive control, produced 15.2 µM of NO. Therefore, the results suggested that antitumor activities of Fr. HW from E. applanata might, in part, be due to host mediated immunostimulating activity.

Human lymphoblastoid cell lines: a goldmine for the biobankomics era.

Biobanking became a necessity for translating genetic discoveries into clinical practice. Approaches to personalized medicine require a new model system for functional and pharmacogenomic studies of a variety of accumulating genetic variations, as well as new research environments such as biobankomics. Human lymphoblastoid cell lines (LCLs) will provide a valuable tool to meet such new demands in the biobankomics era. The National Biobank of Korea (NBK), which is leading the Korea Biobank Project, has a large collection of LCLs derived mostly from population-based cohort samples. Using a special long-term subculture collection of NBK LCLs, biological characteristics of early passage LCLs and terminally immortalized LCLs have been investigated to promote the utilization of LCLs and provide well quality-controlled LCLs for genetic and pharmacogenomic studies. As LCLs have been successfully phenotyped for cytotoxicity in response to various stimulators, including chemotherapeutic agents, environmental chemicals and irradiation, the utility of LCLs will increase in the future. Here, we discuss current and future applications of NBK LCLs for the biobankomics era.

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

BACKGROUND: Recent 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. RESULTS: We 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 x 10(-7)) and women (p = 9.45 x 10(-5)), as well as higher total cholesterol levels in men (p = 9.96 x 10(-7)). In addition, the significant association between lower E2 DNA copy numbers and lower level of postprandial 2 hr 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. CONCLUSIONS: This work suggests that a structural variation at the LEPR gene locus is functionally associated with complex metabolic traits and the risk of T2DM.

Bacterial Contamination of Blood DNA Samples is Associated with Donor's Health Condition.

Bacterial contamination often occurs in human blood DNA samples, possibly due to bacteremia or an inappropriate procedure during sample preparation. This study aimed at analyzing the clinical significance of bacterial DNA contamination in human blood DNA samples and to assess its influence on experimental data. DNA samples (N = 1359) were randomly selected from population-based cohort samples to determine bacterial DNA contamination by polymerase chain reaction and direct DNA sequencing. Bacterial DNA contaminated samples (N = 150) were then assessed for experimental quality of single nucleotide polymorphism (SNP) chip data, compared with uncontaminated DNA samples (N = 1209). DNA sequencing data showed that a major source of bacterial contaminants was derived from Alcaligenes species. The occurrence of bacterial DNA contaminations was significantly associated with some clinical variables including a postprandial glucose level at 60 min, % body fat, and waist-to-hip ratio. It was also found that there was no difference of SNP call rates between bacterial DNA contaminated samples and uncontaminated DNA samples. This study showed that bacterial DNA contamination in human blood samples was related to donor's health condition, suggesting that the occurrence of bacterial DNA contamination may provide useful health information of blood donors and a potential tool for human disease genomics.

Multilaboratory assessment of variations in spectrophotometry-based DNA quantity and purity indexes.

Human genetic studies are using an increasing number of biobanked DNA samples, which require consistent measurements of DNA quantity and purity between multicenters or multilaboratories. In an attempt to standardize DNA quantitation protocols, a DNA quantitation project was performed, in which 16 technicians from 11 laboratories participated in measuring optical density (A260, A280, A230) of multiple DNA samples (N = 35) of known concentrations. We analyzed variations in the measurement of DNA quantity and purity and found that the mean interoperator coefficients of variation percentage for A260, A260/A280, and A260/A230 values among individuals were 21.9%, 7.4%, and 24.7%, respectively. In contrast, the mean intra-operator coefficients of variation percentage for A260, A260/A280, and A260/A230 values were 9.9%, 1.7%, and 8.3%, respectively. The variability in A260/A230 determination was much more sensitive to the method of DNA quantitation and the technical skill of the individual than those of A260 and A260/A280. In addition, a concentration of DNA of >100 ng/µL was found to reduce the variability of DNA quantity (A260) and purity (A260/A280 and A260/A230 ratios) indexes. This work emphasizes the need for standardization of DNA quantitation protocols for multicenter DNA work, as well as the importance of training and education of technicians at these centers.

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>or= 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>or=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.

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.

Novel promoter polymorphism in RUNX2 is associated with serum triglyceride level.

Much research evidence supports the hypothesis that chronic, low-grade inflammation related to innate immunity may play an important role in the pathophysiology of type 2 diabetes mellitus (T2DM). Runt-related transcription factor 2 (RUNX2; MIM# 600211) acts as a scaffold that controls the integration, organization, and assembly of nucleic acids. To examine whether the novel promoter variant in RUNX2 is associated with the risk of T2DM and related phenotypes, RUNX2-742G > T was genotyped in 378 T2DM patients and 382 normal controls recruited in the Korean T2DM Study. Statistical analysis revealed that RUNX2-742G > T was associated with serum triglyceride level (TG) in nondiabetic controls, although it was not associated with the risk of T2DM. Individuals who carry T/T, T/G, and G/G genotypes had the highest (2.061 +/- 0.20), intermediate (2.01 +/- 0.19), and the lowest (1.97 +/- 0.18) levels of log [TG (mmol/l)] (P = 0.007), respectively. Our data on this important variant of RUNX2 suggest that lipid metabolism might be affected by genetic polymorphisms in the promoter region.

Wp specific methylation of highly proliferated LCLs.

The epigenetic regulation of viral genes may be important for the life cycle of EBV. We determined the methylation status of three viral promoters (Wp, Cp, Qp) from EBV B-lymphoblastoid cell lines (LCLs) by pyrosequencing. Our pyrosequencing data showed that the CpG region of Wp was methylated, but the others were not. Interestingly, Wp methylation was increased with proliferation of LCLs. Wp methylation was as high as 74.9% in late-passage LCLs, but 25.6% in early-passage LCLs. From two Burkitt's lymphoma cell lines, Wp specific hypermethylation was also found (>80%). Interestingly, the expression of EBNA2 gene which located directly next to Wp was associated with its methylation. Our data suggested that Wp specific methylation may be important for the indicator of the proliferation status of LCLs, and the epigenetic viral gene regulation of EBNA2 gene by Wp should be further defined possibly with other biological processes.