Consensus Definition of Blood Samples from the Subcategorized Normal Controls in the Korea Biobank Network.

A control group is defined as a group of people used for comparison. Depending on the type of study, it can be a group of healthy people or a group not exposed to risk factors. It is important to allow researchers to select the appropriate control participants. The Korea Biobank Project-sponsored biobanks are affiliated with the Korea Biobank Network (KBN), for which the National Biobank of Korea plays a central coordinating role among KBN biobanks. KBN organized several working groups to address new challenges and needs in biobanking. The "Normal Healthy Control Working Group" developed standardized criteria for three defined control groups, namely, normal, normal-plus, and disease-specific controls. Based on the consensus on the definition of a normal control, we applied the criteria for normal control participants to retrospective data. The main reason for exclusion from the "Normal-plus" group was blood test results beyond 5% of the reference range, including hypercholesterolemia. Subclassification of samples of normal controls by detailed criteria will help researchers select optimal normal controls for their studies.

Inorganic Phosphorus and Potassium Are Putative Indicators of Delayed Separation of Whole Blood.

OBJECTIVES: The delayed separation of whole blood can influence the concentrations of circulating blood components, including metabolites and cytokines. The aim of this study was to determine whether clinical-biochemistry analytes can be used to assess the delayed separation of whole blood. METHODS: We investigated the plasma and serum concentrations of five clinical-biochemistry analytes and free hemoglobin when the centrifugation of whole blood stored at 4°C or room temperature was delayed for 4 hours, 6 hours, 24 hours, or 48 hours, and compared the values with those of matched samples that had been centrifuged within 2 hours after whole-blood collection. RESULTS: The inorganic phosphorus (IP) levels in the plasma and serum samples were elevated ≥ 1.5-fold when whole-blood centrifugation was delayed at room temperature for 48 hours. Furthermore, the IP levels in the plasma samples showed excellent assessment accuracy [area under the receiver-operating-characteristic curve (AUC) > 0.9] after a 48-hour delay in whole-blood separation, and high sensitivity (100%) and specificity (95%) at an optimal cutoff point. The IP levels in the serum samples also exhibited good assessment accuracy (AUC > 0.8), and high sensitivity (81%) and specificity (100%). The potassium (K(+)) levels were elevated 1.4-fold in the serum samples following a 48-hour delay in whole-blood separation. The K(+) levels showed excellent assessment accuracy (AUC > 0.9) following a 48-hour delay in whole-blood separation, and high sensitivity (95%) and specificity (91%) at an optimal cutoff point. CONCLUSION: Our study showed that the IP and K(+) levels in the plasma or serum samples could be considered as putative indicators to determine whether whole-blood separation had been delayed for extended periods.

Instability of Plasma and Serum Progastrin-Releasing Peptide During Repeated Freezing and Thawing.

OBJECTIVES: Progastrin-releasing peptide (proGRP) is a promising biomarker for small cell lung cancer. However, not much is known about how sample processing and storage conditions affect the stability of proGRP. Here, we examined the effects of repeated freeze-thaw cycles on the stability of proGRP in plasma and serum. METHODS: Concentrations of proGRP were measured in plasma and serum samples exposed to two, three, or four freeze-thaw cycles and these were compared with values of corresponding samples exposed to one cycle (baseline). We also performed the area under the receiver-operating-characteristic curve (AUC) analysis to determine whether the differences of proGRP concentrations between each paired plasma and serum sample (ΔproGRP) can be used for identifying the samples that have been exposed to multiple freeze-thaw cycles. RESULTS: Concentrations of proGRP gradually decreased in both plasma and serum samples with increasing numbers of freeze-thaw cycles. Reduction rates of proGRP concentrations were greater in serum than in plasma samples and serum proGRP levels declined with statistical significance (p < 0.001) up to 10.1% after four freeze-thaw cycles. The ΔproGRP measurement showed fair accuracy (AUC = 0.741) for identifying samples that had been through four freeze-thaw cycles. The sensitivity was 82.8% and specificity was 62.1% at an optimal cut-off point of > 4.9. CONCLUSION: Our study shows that the stability of circulating proGRP is affected in both plasma and serum samples by repeated freezing and thawing. We also show that ΔproGRP could be used for identifying paired plasma and serum samples subjected to multiple freeze-thaw cycles.

Identification of the polymorphisms in IFITM1 gene and their association in a Korean population with ulcerative colitis.

Interferon inducible transmembrane protein (IFITM) family genes have been implicated in several cellular processes such as the homotypic cell adhesion functions of IFNs and cellular anti-proliferative activities. We previously showed that the IFITM3 single nucleotide polymorphisms (SNPs) associated with susceptibility to ulcerative colitis (UC). The present study aimed to investigate whether the polymorphisms in the IFITM1 gene are associated with susceptibility to UC. We also evaluated the expression levels in the putative functional promoter polymorphisms to determine the change of their activity. Gene expression profiles in the tissues obtained from human digestive tracts by RT-PCR, and the possible variation sites and SNPs of IFITM1 were identified by direct sequencing method. Genotype analysis in the IFITM1 SNPs was performed by high resolution melting and TaqMan probe analysis, and the haplotype frequencies of IFITM1 SNPs for multiple loci were estimated using the expectation maximization (EM) algorithm. The expression levels in the putative functional promoter polymorphisms were evaluated by performing a luciferase reporter assay. We identified two SNPs and two variation sites, g.-1920G>A (rs77537847), g.-1547delA (novel) and g.-416C>G (rs11246062) in the promoter region, and g.364delA (rs200576757) in intron 1. The genotype and allele frequencies of the g.-1920G>A polymorphism of IFITM1 gene in the UC patients were significantly different from those of the healthy controls (P=0.002 and 0.042, respectively). These results suggest that the g.-1920G>A polymorphism in IFITM1 may be associated with susceptibility to UC.

EED gene polymorphism in patients with colorectal cancer.

BACKGROUND: Our previous work indicated that, first, the embryonic ectoderm development (EED) gene is a candidate gene associated with the pathogenesis of ulcerative colitis (UC) and, second, that the haplotypes of the EED polymorphism are one of the markers for UC susceptibility. The risk of developing colorectal cancer (CRC) increases in patients with inflammatory bowel disease. 
 AIM: The present study aimed at determining the association between polymorphisms in the EED gene and CRC. 
 METHODS: Genotype analysis of EED single nucleotide polymorphisms (SNPs) was performed with high-resolution melting analysis, and the genotype and allele frequencies of the EED SNPs were compared between CRC patients and healthy controls. The haplotype frequencies of EED for multiple loci were estimated using the expectation maximization (EM) algorithm. 
 RESULTS: Our study had a power of 76.6% at a 0.05 significance level. Genotype and allele frequencies of the SNPs and haplotype frequencies of the EED gene in CRC patients were not significantly different from those in healthy controls. Only the allele frequency of g.-1850G>C in the rectal cancer (RC) patient group was significantly different from that of the control group (p=0.04). Similarly, the genotype and allelic frequencies of the EED SNPs for either tumor site (left or right) or tumor stage were not significantly different from those in healthy controls. However, our data show an association between the g.-993G>C polymorphism in the EED gene and the presence of lymph node metastasis in CRC.
 CONCLUSIONS: These results suggest that the SNPs of the EED gene might not be associated with susceptibility to CRC. However, this study shows that the allele frequency of g.-1850G>C in the RC patient group was significantly different from that in the control group (p=0.04) and that g.-993G>C may play a role in the lymph node metastatic process of CRC.

Identifying Polymorphisms in IL-31 and Their Association with Susceptibility to Asthma.

BACKGROUND: Interleukin 31 (IL-31) is a T helper type 2 effector cytokine that plays an important role in the pathogenesis of atopic and allergic diseases. IL-31 may be involved in promoting allergic inflammation and in inducing airway epithelial responses such as allergic asthma. METHODS: Single-base extension analysis was used to detect the genotypes of IL-31 single nucleotide polymorphisms (SNPs), and we compared the genotype and allele frequencies of the IL-31 SNPs between patients with asthma and healthy controls. RESULTS: There were no significant differences in the genotype and allele frequencies of the IL-31 SNPs between patients with asthma and healthy controls. Furthermore we compared the genotype and allele frequencies of IL-31 SNPs between patients with atopic asthma, those with non-atopic asthma and healthy controls. This showed that the SNPs were not associated with the susceptibility to atopic asthma. There were no significant differences in the haplotype frequencies of IL-31 SNPs between patients with asthma and healthy controls. In patients with asthma, the IL-31 SNPs were significantly correlated with total serum levels of IgE (p=0.035). CONCLUSIONS: Our results indicate that, the IL-31 SNPs may be associated with IgE production in patients with asthma.

Promoter polymorphism of the EED gene is associated with the susceptibility to ulcerative colitis.

BACKGROUND: Embryonic ectoderm development (EED) protein is involved in multiple cellular protein complexes. EED mediates the repression of gene activity through histone deacetylation, and it may act as a specific regulator of integrin's function. This gene was identified as a candidate gene for the susceptibility to IBD by our previous cDNA microarray analysis. AIM: The present study aimed to validate the expression level of the EED gene in patients with IBD by performing RT-PCR, and we investigated whether the polymorphisms in the EED gene are associated with the susceptibility to UC, and whether a functional EED promoter polymorphism is related to UC. METHODS: Genotype analysis of the EED SNPs was performed by single-base extension analysis. The haplotype frequencies of the EED gene for multiple loci were estimated using the expectation maximization algorithm. The promoter region of the human EED gene, including the g.-1850G>C allele, was isolated by PCR. The amplified PCR products were inserted into the pGL3-basic vector and the luciferase activity was analyzed. RESULTS: The expression level of the EED gene was significantly decreased in both the UC and CD patients and it was significantly higher in the liver and ileum than in the other tissues of the human digestive system. The genotype and allele frequencies of the g.-1850G>C polymorphism of the EED gene in the UC patients were significantly different from those of the healthy controls (p = 0.018 and 0.017, respectively). The luciferase activity assay showed that the promoter activity was decreased about twofold in the construct containing the g.-1850G allele compared to that of the construct containing the g.-1850C allele, which means that the allele G could produce less EED mRNA. CONCLUSIONS: These results suggest that the g.-1850G>C polymorphism in the EED gene might be associated with the susceptibility to UC by the change of the EED expression level.

The haplotypes of TNFRSF17 polymorphisms are associated with colon cancer in a Korean population.

PURPOSE: We previously found that the haplotypes of TNFRSF17 single nucleotide polymorphisms (SNPs) were associated with the susceptibility to inflammatory bowel disease on Korean population. The present study aimed to investigate whether the polymorphisms in the TNFRSF17 gene are associated with susceptibility to colorectal cancer (CRC). METHODS: Genotype analysis in the TNFRSF17 SNPs was performed by high-resolution melting and TaqMan probe analysis, and the genotype and allele frequencies of TNFRSF17 SNPs were compared between the CRC patients and the healthy controls. The haplotype frequencies of TNFRSF17 for multiple loci were estimated using the expectation maximization algorithm. RESULTS: Although, the genotype and allelic frequencies of these SNPs, in the colon cancer and rectal cancer patients, were not significantly different from those in the healthy controls, the genotype and allele frequency of g.2493G>A was significantly different between the healthy controls and the right colon cancer patients (P = 0.014 and 0.004, respectively). Moreover, the haplotypes frequencies in the healthy controls were significantly different from those in the colon cancer patients. CONCLUSION: Our results suggest that TNFRSF17 may be a candidate gene associated with the pathogenesis of colon cancer, and the haplotypes of the TNFRSF17 polymorphisms might be one of the markers for colon cancer susceptibility.

Identifying the polymorphisms in the thymic stromal lymphopoietin receptor (TSLPR) and their association with asthma.

The present study aimed to investigate whether the polymorphisms in the TSLPR gene are associated with atopic and asthmatic disease in the Korean population. We identified eleven single nucleotide polymorphisms (SNPs) and two variation sites in the TSLPR gene, including the promoter region. The genotype and allele frequencies of g.33G>C of the TSLPR gene in asthma patients were significantly different from the respective frequencies of the control group (P =0.006 and 0.003, respectively). Our additional analysis showed that the genotype and allele frequencies of the g.33G>C and g.19646A>G of the TSLPR gene were significantly associated in the atopic asthma patients rather than in the non-atopic asthma patients (genotype frequencies; P =0.0001 and 0.0003 respectively, allele frequencies; P =0.0005 and 0.0001 in that order). Our results suggest that the SNPs of the TSLPR gene could be associated with the susceptibility to atopic asthma in the Korean population.

Identification of the polymorphisms in IFITM3 gene and their association in a Korean population with ulcerative colitis.

Interferons play critical roles in tumor pathogenesis by controlling apoptosis and through cellular anti-proliferative and differentiation activities. Interferon inducible transmembrane protein (IFITM) family genes have been implicated in several cellular processes such as the homotypic cell adhesion functions of IFN and cellular anti-proliferative activities. Expression levels of IFITM genes have been found to be up-regulated in gastric cancer cells and colorectal tumors. IFITM3 (also known as 1-8U) is a member of the IFITM family, and has been described as a key player in specification of germ cell fate. IFITM3 was first isolated from a genetic screen aimed at identifying genes involved in acquisition of germ cell competence. It has been proposed that epiblast cells have the highest expression of IFITM3 initiated germ cell specification and that homotypic association can discriminate germ cells from their somatic neighbors. In an attempt to better understand the genetic influences of IFITM3 on ulcerative colitis, we have identified possible variation sites and single nucleotide polymorphisms (SNPs) through two exons and their boundary IFITM3 intron sequences including the approximately 2.1 kb promoter regions. To determine whether or not these IFITM3 SNPs are associated with susceptibility to ulcerative colitis, frequencies of the genotype and allele of IFITM3 polymorphisms were analyzed on genomic DNAs isolated from patients with ulcerative colitis and from healthy controls. We also investigated the haplotype frequencies constructed by these SNPs in both groups. In this study, we also showed that expression level of IFITM3 mRNA was significantly higher in tissues of the ileum and cecum of the digestive system. We identified a total of seven SNPs and multiple variation regions in the IFITM3 gene. The genotype frequency of the g.-204T>G polymorphism in patients with ulcerative colitis was significantly different from that of the control group. Our results strongly suggest that polymorphisms of the IFITM3 gene may be associated with susceptibility to ulcerative colitis.

Identification of single nucleotide polymorphisms in the TNFRSF17 gene and their association with gastrointestinal disorders.

TNFRSF17 is preferentially expressed in mature B lymphocytes, and may be important for the development of B cells. TNFRSF17 is selected as a candidate susceptibility gene to IBD pathogenesis by our cDNA microarray analysis, and we showed the specific expression of TNFRSF17 in resting and activated CD19(+) cells obtained from human blood. We identified four SNPs (g-1729G>A, g.2295T>C, g.2445G>A and g.2493G>A) and one variation site (g.894delT) in the TNFRSF17 gene using direct sequencing analysis. In addition, the association of the genotype and allelic frequencies of these SNPs was studied in healthy controls and in patients with ulcerative colitis (UC) or irritable bowel syndrome (IBS). Although, the genotype and allelic frequencies of these SNPs, in the UC and IBS patients, were not significantly different from those in the healthy controls, the distribution of the AAG, GGA, AGG and AAA haplotypes, of the SNPs (g.-1729G>A, g.2445G> A and g.2493G>A) associated with the TNFRSF17 gene, in the UC patients, were notably different from those of the healthy controls (P = 0.002, 0.002, 4.7E-4 and 3.3E-6, respectively). Moreover, the frequencies of the AAG, AGG, GAG and GAA haplotypes were significantly different in the IBS patients compared to the healthy controls (P = 4.2E-5, 4.4E-17, 1.8E-22 and 1.6E-10, respectively). These results suggest that the haplotypes of the TNFRSF17 polymorphisms might be associated with UC and IBS susceptibility.