RNA-Seq for Gene Expression Profiling of Human Necrotizing Enterocolitis: a Pilot Study

Necrotizing enterocolitis (NEC) characterized by inflammatory intestinal necrosis is a major cause of mortality and morbidity in newborns. Deep RNA sequencing (RNA-Seq) has recently emerged as a powerful technology enabling better quantification of gene expression than microarrays with a lower background signal. A total of 10 transcriptomes from 5 pairs of NEC lesions and adjacent normal tissues obtained from preterm infants with NEC were analyzed. As a result, a total of 65 genes (57 down-regulated and 8 up-regulated) revealed significantly different expression levels in the NEC lesion compared to the adjacent normal region, based on a significance at fold change ≥ 1.5 and P ≤ 0.05. The most significant gene, DPF3 (P < 0.001), has recently been reported to have differential expressions in colon segments. Our gene ontology analysis between NEC lesion and adjacent normal tissues showed that down-regulated genes were included in nervous system development with the most significance (P = 9.3 × 10⁻⁷; P(corr) = 0.0003). In further pathway analysis using Pathway Express based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, genes involved in thyroid cancer and axon guidance were predicted to be associated with different expression (P(corr) = 0.008 and 0.020, respectively). Although further replications using a larger sample size and functional evaluations are needed, our results suggest that altered gene expression and the genes' involved functional pathways and categories may provide insight into NEC development and aid in future research.

The association of 5-alpha reductase type 2 (SRD5A2) gene polymorphisms with prostate cancer in a Korean population

PURPOSE: Steroid 5-alpha reductase type 2 (SRD5A2) modifies testosterone to dihydrotestosterone (DHT) in the prostate. Single-nucleotide polymorphisms (SNPs) of the SRD5A2 gene might affect DHT. We sought to understand the relationship of SRD5A2 SNPs to prostate cancer in the Korean population. MATERIALS AND METHODS: Twenty-six common SNPs in the SRD5A2 gene were assessed in 272 prostate cancer cases and 173 controls. Single-locus analyses were conducted by using conditional logistic regression. Additionally, we performed a haplotype analysis for the SRD5A2 SNPs tested. RESULTS: Among the 20 SNPs and 4 haplotypes, there were no statistically significant results in the prostate cancer patients and the controls. In the logistic analysis of SRD5A2 polymorphisms with prostate-specific antigen (PSA) criteria, two SNPs (rs508562, rs11675297) and haplotype 1 displayed significant results (odds ratio [OR], 1.76; p=0.05; OR, 1.88-2.02; p=0.01-0.04; OR, 0.59; p=0.02, respectively). rs508562, rs11675297, rs2208532, and haplotype 1 (OR, 1.49; p=0.05; OR, 2.02; p=0.05; OR, 2.01; p=0.04; OR, 0.56-0.64, p=0.03-0.04, respectively) had significant associations with Gleason score. rs508562, rs11675297, and haplotype 1 (OR, 1.41-2.34; p=0.004-0.05; OR, 1.74-1.82; p=0.03-0.05; OR, 0.42-0.67; p=0.0005-0.03, respectively) were significantly associated with clinical stage. CONCLUSIONS: We conclude that there was no significant association between SRD5A2 SNPs and the risk of prostate cancer in the Korean population. However, we found that some SNPs and 1 haplotype influenced PSA level, Gleason score, and clinical stage.

Association Analysis of TEC Polymorphisms with Aspirin-Exacerbated Respiratory Disease in a Korean Population

The tyrosine-protein kinase Tec (TEC) is a member of non-receptor tyrosine kinases and has critical roles in cell signaling transmission, calcium mobilization, gene expression, and transformation. TEC is also involved in various immune responses, such as mast cell activation. Therefore, we hypothesized that TEC polymorphisms might be involved in aspirin-exacerbated respiratory disease (AERD) pathogenesis. We genotyped 38 TEC single nucleotide polymorphisms in a total of 592 subjects, which comprised 163 AERD cases and 429 aspirin-tolerant asthma controls. Logistic regression analysis was performed to examine the associations between TEC polymorphisms and the risk of AERD in a Korean population. The results revealed that TEC polymorphisms and major haplotypes were not associated with the risk of AERD. In another regression analysis for the fall rate of forced expiratory volume in 1 second (FEV1) by aspirin provocation, two variations (rs7664091 and rs12500534) and one haplotype (TEC_BL2_ht4) showed nominal associations with FEV1 decline (p = 0.03-0.04). However, the association signals were not retained after performing corrections for multiple testing. Despite TEC playing an important role in immune responses, the results from the present study suggest that TEC polymorphisms do not affect AERD susceptibility. Findings from the present study might contribute to the genetic etiology of AERD pathogenesis.

Comprehensive Variant Screening of the UGT Gene Family

PURPOSE: UGT1A1, UGT2B7, and UGT2B15 are well-known pharmacogenes that belong to the uridine diphosphate glucuronyltransferase gene family. For personalized drug treatment, it is important to study differences in the frequency of core markers across various ethnic groups. Accordingly, we screened single nucleotide polymorphisms (SNPs) of these three genes and analyzed differences in their frequency among five ethnic groups, as well as attempted to predict the function of novel SNPs. MATERIALS AND METHODS: We directly sequenced 288 subjects consisting of 96 Korean, 48 Japanese, 48 Han Chinese, 48 African American, and 48 European American subjects. Subsequently, we analyzed genetic variability, linkage disequilibrium (LD) structures and ethnic differences for each gene. We also conducted in silico analysis to predict the function of novel SNPs. RESULTS: A total of 87 SNPs were detected, with seven pharmacogenetic core SNPs and 31 novel SNPs. We observed that the frequencies of UGT1A1 *6 (rs4148323), UGT1A1 *60 (rs4124874), UGT1A1 *93 (rs10929302), UGT2B7 *2 (rs7439366), a part of UGT2B7 *3 (rs12233719), and UGT2B15 *2 (rs1902023) were different between Asian and other ethnic groups. Additional in silico analysis results showed that two novel promoter SNPs of UGT1A1 -690G>A and -689A>C were found to potentially change transcription factor binding sites. Moreover, 673G>A (UGT2B7), 2552T>C, and 23269C>T (both SNPs from UGT2B15) changed amino acid properties, which could cause structural deformation. CONCLUSION: Findings from the present study would be valuable for further studies on pharmacogenetic studies of personalized medicine and drug response.

Screening of Dihydropyrimidine Dehydrogenase Genetic Variants by Direct Sequencing in Different Ethnic Groups

Dihydropyrimidine dehydrogenase (DPYD) is an enzyme that regulates the rate-limiting step in pyrimidine metabolism, especially catabolism of fluorouracil, a chemotherapeutic agent for cancer. In order to determine the genetic distribution of DPYD, we directly sequenced 288 subjects from five ethnic groups (96 Koreans, 48 Japanese, 48 Han Chinese, 48 African Americans, and 48 European Americans). As a result, 56 polymorphisms were observed, including 6 core polymorphisms and 18 novel polymorphisms. Allele frequencies were nearly the same across the Asian populations, Korean, Han Chinese and Japanese, whereas several SNPs showed different genetic distributions between Asians and other ethnic populations (African American and European American). Additional in silico analysis was performed to predict the function of novel SNPs. One nonsynonymous SNP (+199381A > G, Asn151Asp) was predicted to change its polarity of amino acid (Asn, neutral to Asp, negative). These findings would be valuable for further research, including pharmacogenetic and drug responses studies.

Screening of Dihydropyrimidine Dehydrogenase Genetic Variants by Direct Sequencing in Different Ethnic Groups

Dihydropyrimidine dehydrogenase (DPYD) is an enzyme that regulates the rate-limiting step in pyrimidine metabolism, especially catabolism of fluorouracil, a chemotherapeutic agent for cancer. In order to determine the genetic distribution of DPYD, we directly sequenced 288 subjects from five ethnic groups (96 Koreans, 48 Japanese, 48 Han Chinese, 48 African Americans, and 48 European Americans). As a result, 56 polymorphisms were observed, including 6 core polymorphisms and 18 novel polymorphisms. Allele frequencies were nearly the same across the Asian populations, Korean, Han Chinese and Japanese, whereas several SNPs showed different genetic distributions between Asians and other ethnic populations (African American and European American). Additional in silico analysis was performed to predict the function of novel SNPs. One nonsynonymous SNP (+199381A > G, Asn151Asp) was predicted to change its polarity of amino acid (Asn, neutral to Asp, negative). These findings would be valuable for further research, including pharmacogenetic and drug responses studies.

Screening of Genetic Polymorphisms of CYP3A4 and CYP3A5 Genes

Given the CYP3A4 and CYP3A5's impact on the efficacy of drugs, the genetic backgrounds of individuals and populations are regarded as an important factor to be considered in the prescription of personalized medicine. However, genetic studies with Korean population are relatively scarce compared to those with other populations. In this study, we aimed to identify CYP3A4/5 polymorphisms and compare the genotype distributions among five ethnicities. To identify CYP3A4/5 SNPs, we first performed direct sequencing with 288 DNA samples which consisted of 96 Koreans, 48 European-Americans, 48 African-Americans, 48 Han Chinese, and 48 Japanese. The direct sequencing identified 15 novel SNPs, as well as 42 known polymorphisms. We defined the genotype distributions, and compared the allele frequencies among five ethnicities. The results showed that minor allele frequencies of Korean population were similar with those of the Japanese and Han Chinese populations, whereas there were distinct differences from European-Americans or African-Americans. Among the pharmacogenetic markers, frequencies of CYP3A4*1B (rs2740574) and CYP3A5*3C (rs776742) in Asian groups were different from those in other populations. In addition, minor allele frequency of CYP3A4*18 (rs28371759) was the highest in Korean population. Additional in silico analysis predicted that two novel non-synonymous SNPs in CYP3A5 (+27256C>T, P389S and +31546T>G, I488S) could alter protein structure. The frequency distributions of the identified polymorphisms in the present study may contribute to the expansion of pharmacogenetic knowledge.

WDR46 is a Genetic Risk Factor for Aspirin-Exacerbated Respiratory Disease in a Korean Population

PURPOSE: The human WD repeat-containing protein 46 (WDR46; also known as C6orf11), located at the disease-relevant centromere side of the class II major histocompatibility complex region, is hypothesized to be associated with risk of aspirin-exacerbated respiratory disease (AERD) as well as a decline in forced expiratory volume in the first second (FEV1), an important diagnostic marker of asthma. METHODS: To investigate the association between WDR46 and AERD, five single-nucleotide polymorphisms (SNPs) were genotyped in 93 AERD cases and 96 aspirin-tolerant asthma controls of Korean ethnicity. Three major haplotypes were inferred from pairwise comparison of the SNPs, and one was included in the association analysis. Differences in the frequency distribution of WDR46 SNPs and haplotype were analyzed using logistic and regression models via various modes of genetic inheritance. RESULTS: Depending on the genetic model, the logistic and regression analyses revealed significant associations between rs463260, rs446735, rs455567, rs469064, and WDR46_ht2 and the risk of AERD (P=0.007-0.04, Pcorr=0.01-0.04) and FEV1 decline after aspirin provocation (P=0.006-0.03, Pcorr=0.01-0.03). Furthermore, functional analysis in silico showed that the G>A allele of rs463260 located in the 5' untranslated region potentially matched a nucleotide sequence within an upstream open reading frame of WDR46. CONCLUSIONS: These findings show for the first time that WDR46 is an important genetic marker of aspirin-induced airway inflammation and may be useful for formulating new disease-management strategies.

Potential Association of DCBLD2 Polymorphisms with Fall Rates of FEV1 by Aspirin Provocation in Korean Asthmatics

Aspirin exacerbated respiratory disease (AERD) is a clinical syndrome characterized by chronic rhinosinusitis with nasal polyposis and aspirin hypersensitivity. The aspirin-induced bronchospasm is mediated by mast cell and eosinophilic inflammation. Recently, it has been reported that the expression of discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2) is up-regulated in lung cancers and is regulated by transcription factor AP-2 alpha (TFAP2A), a component of activator protein-2 (AP-2) that is known to regulate IL-8 production in human lung fibroblasts and epithelial cells. To investigate the associations between AERD and DCBLD2 polymorphisms, 12 common variants were genotyped in 163 AERD subjects and 429 aspirin tolerant asthma (ATA) controls. Among these variants, seven SNPs (rs1371687, rs7615856, rs828621, rs828618, rs828616, rs1062196, and rs8833) and one haplotype (DCBLD2-ht1) show associations with susceptibility to AERD. In further analysis, this study reveals significant associations between the SNPs or haplotypes and the percentage of forced expiratory volume in one second (FEV1) decline following aspirin challenge using multiple linear regression analysis. Furthermore, a non-synonymous SNP rs16840208 (Asp723Asn) shows a strong association with FEV1 decline in AERD patients. Although further studies for the non-synonymous Asp723Asn variation are needed, our findings suggest that DCBLD2 could be related to FEV1-related phenotypes in asthmatics.

Potential Association of DCBLD2 Polymorphisms with Fall Rates of FEV1 by Aspirin Provocation in Korean Asthmatics

Aspirin exacerbated respiratory disease (AERD) is a clinical syndrome characterized by chronic rhinosinusitis with nasal polyposis and aspirin hypersensitivity. The aspirin-induced bronchospasm is mediated by mast cell and eosinophilic inflammation. Recently, it has been reported that the expression of discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2) is up-regulated in lung cancers and is regulated by transcription factor AP-2 alpha (TFAP2A), a component of activator protein-2 (AP-2) that is known to regulate IL-8 production in human lung fibroblasts and epithelial cells. To investigate the associations between AERD and DCBLD2 polymorphisms, 12 common variants were genotyped in 163 AERD subjects and 429 aspirin tolerant asthma (ATA) controls. Among these variants, seven SNPs (rs1371687, rs7615856, rs828621, rs828618, rs828616, rs1062196, and rs8833) and one haplotype (DCBLD2-ht1) show associations with susceptibility to AERD. In further analysis, this study reveals significant associations between the SNPs or haplotypes and the percentage of forced expiratory volume in one second (FEV1) decline following aspirin challenge using multiple linear regression analysis. Furthermore, a non-synonymous SNP rs16840208 (Asp723Asn) shows a strong association with FEV1 decline in AERD patients. Although further studies for the non-synonymous Asp723Asn variation are needed, our findings suggest that DCBLD2 could be related to FEV1-related phenotypes in asthmatics.

Polymorphisms of the Reg1alpha Gene and Early Onset Type 2 Diabetes in the Korean Population / 당뇨병

BACKGROUND: The Reg gene has been reported to be expressed in regenerating islets and Reg1 protein to be up-regulated at an early stage of diabetes in mice. As human Reg1alpha is homologous with murine Reg1, we investigated whether common variants in Reg1alpha are associated with type 2 diabetes in the Korean population. METHODS: We sequenced the Reg1alpha gene to identify common polymorphisms using 24 Korean DNA samples. Of 11 polymorphisms found, five common ones (g.-385T>C [rs10165462], g.-36T>G [rs25689789], g.209G>T [rs2070707], g.1385C>G [novel], and g.2199G>A [novel]) were genotyped in 752 type 2 diabetic patients and 642 non-diabetic subjects. RESULTS: No polymorphism was associated with the risk of type 2 diabetes. However, g.-385C and g.2199A lowered the risk of early-onset type 2 diabetes, defined as a diagnosis in subjects whose age at diagnosis was 25 years or more but less than 40 years (odds ratio [OR], 0.721 [0.535 to 0.971] and 0.731 [0.546 to 0.977] for g.-385C and g.2199A, respectively) and g.1385G increased the risk of early-onset diabetes (OR, 1.398 [1.055 to 1.854]). Although adjusting for errors in multiple hypotheses-testing showed no statistically significant association between the three individual polymorphisms and early-onset diabetes, the haplotype H1, composed of g.-385C, g.1385C, and g.2199A, was associated with a reduced risk of early-onset diabetes (OR, 0.590 [0.396 to 0.877], P = 0.009). CONCLUSION: Polymorphisms in the Reg1alpha were not found to be associated with overall susceptibility to type 2 diabetes, though some showed modest associations with early-onset type 2 diabetes in the Korean population.

Association Analysis of SERPINB5 Polymorphisms with HBV Clearance and HCC Occurrence in a Korean Population

Serpin peptidase inhibitor, Clade B (ovalbumin), Member 5 (SERPINB5), also known as maspin, is a potent tumor suppressor gene. It has correlations with many tumor cells, from pancreas cancer to breast cancer, so it is possible that it may also affect liver cancer. There has also been a report that SERPINB12, a gene placed right next to SERPINB5, is expressed in liver. For this study, 32 polymorphisms were identified in SERPINB5 by direct DNA sequencing, and 11 of them were selected to be tested with a larger scale subjects. The association of the 11 SERPINB5 polymorphisms with Hepatitis B virus (HBV) clearance, hepatocellular carcinoma (HCC) occurrence and the onset age of HCC were analyzed. There were no significant associations found between 11 SERPINB5 polymorphisms and HBV clearance. In the case of HCC occurrence, one of the haplotypes (ht) showed association with HCC occurrence (OR=2.26, p=0.005, P(Cor)=0.05), albeit with a low statistical power (40.8%) and haplotype frequency (0.052). Further study with a bigger sample size will be needed to clearly verify the association between ht5 and HCC occurrence.

Putative Association of ITGB1 Haplotype with the Clearance of HBV Infection

Integrins are transmembrane receptor proteins that mediate cell-cell adhesion and cell-extracellular matrix (ECM) adhesion. The deregulation of cell-ECM adhesion and the abnormal expression of beta1 (beta1) integrins (ITGB1s) are involved in tumor development and metastasis. In the liver, the expression of integrins and ECM proteins can be a cause of hepatocellular carcinoma (HCC) development. We performed direct DNA sequencing of 24 individuals, and identified 23 sequence variants of ITGB1 polymorphisms. Among these 23 variants, 7 common variants were selected based on frequencies and linkage disequilibrium, and then genotyped in a larger-scale group of subjects (n=1,103). The genetic associations of ITGB1 polymorphisms with the clearance of HBV and HCC outcome of HBV patients were analyzed using logistic regression models and Cox relative hazard models. Although there was no significant association observed between the polymorphisms and the HCC outcome of HBV patients, the second most common haplotype (ITGB1 haplotype-2 [C-C-C-C-T-C-T]) was putatively associated with HBV clearance (OR=0.75, p=0.008 and P(corr)=0.05). The minor allele frequency (MAF) of ITGB1 haplotype-2 of the spontaneously recovered (SR) group was significantly higher than that of the chronic carrier group (CC) (freq. = 0.248 vs. 0.199). The information derived from this study could be valuable for understanding the genetic factors involved in the clearance of HBV.

Identification of Polymorphisms in CYP2E1 Gene and Association Analysis among Chronic HBV Patients

Cytochrome P450 2E1 (CYP2E1) is a member of the cytochrome P450 superfamily, and it is a key enzyme responsible for the metabolic activation of many smallmolecular-weight compounds such as alcohol, which is classified as a human carcinogen. In this study, we identified 19 single nucleotide polymorphisms (SNPs) in CYP2E1 in Korean population. In these SNPs, we examined possible genetic association of CYP2E1 polymorphisms with HBV clearance and the risk of hepatocellular carcinoma (HCC). Five common polymorphic sites were selected, CYP2E1 polymorphisms at rs381-3867, rs3813870, rs2070673, rs2515641 and rs2480257 , considering their allele frequencies, haplotype-tagging status and LDs for genotyping in larger-scale subjects (n=1,092). Statistical analysis demonstrated that CYP2E1 polymorphisms and haplotypes show no significant association with HBV clearance, HCC occurrence and onset age of HCC (p>0.05). Previous studies, however, have shown contradictory findings on associations of CYP2E1 polymorphisms with CYP2E1 activities and HCC risk. Comparing the contrasting results of previous researches suggest that CYP2E1 polymorphism is associated with CYP2E1 activity induced by ethanol, but is not directly associated with HCC risk. CYP2E1 variation/haploype information identified in this study will provide valuable information for future studies on CYP2E1.

Lack of Association of BIRC5 Polymorphisms with Clearance of HBV Infection and HCC Occurrence in a Korean Population

BIRC5 (Survivin) belongs to the inhibitor of apoptosis gene family. The BIRC5 protein inhibits caspases and consequently blocks apoptosis. Thus, BIRC5 contributes to the progression of cancer allowing for continued cell proliferation and survival. In this study, we identified eight sequence variants of BIRC5 through direct DNA sequencing. Among the eight single nucleotide polymorphisms (SNPs), six common variants with frequencies higher than 0.05 were selected for larger-scale genotyping (n=1,066). Results of the study did not show any association between the promoter region polymorphisms and the clearance of hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) occurrence. This is in line with a previous study in which polymorphisms in the promoter region does not influence the function of BIRC5. Initially, we were able to detect a signal with the +9194A>G, which disappeared after multiple corrections but led to a change in amino acid. Similarly, we were also able to detect an association signal between two haplotypes (haplotype-2 and haplotype- 5) on the onset age of HCC and/or HCC occurrence, but the signals also disappeared after multiple corrections. As a result, we concluded that there was no association between BIRC5 polymorphisms and the clearance HBV infection and/or HCC occurrence. However, our results might useful to future studies.

Association of common promoter polymorphisms of MCP1 with hepatitis B virus clearance

Hepatocellular carcinoma (HCC) is one of the most common malignant cancers closely associated with chronic infection by the hepatitis B virus (HBV) or the hepatitis C virus (HCV) throughout the world. In this study, the genetic associations of 20 known polymorphisms in eight candidate genes, including angiotensinogen (AGT), cadherin 1 (CDH1), cyclooxygenase 2 (COX2), monocyte chemotactic protein-1 (MCP1), multidrug resistance 1 (MDR1), chemokine ligand 5 (RANTES), thrombospondin 2 (THBS2), and thrombospondin 4 (THBS4), were analyzed in a large chronic hepatitis B cohort (n=1,095) recruited from the Korean population. In addition, three polymorphisms in chemokine receptor 4 (CXCR4) and vimentin (VIM) identified in this study were also genotyped. Using logistic regression analysis controlling possible confounding factors, one common (freq.=0.367) promoter polymorphism of MCP1 (MCP1-2518G>A) among analyzed polymorphisms was significantly associated with clearance of HBV infection. The frequency of homozygotes for the MCP1-2518A allele (MCP1-2518A/A) among chronic hepatitis B virus (HBV) carrier patients was significantly higher than that among spontaneously recovered (SR) subjects (17.7% vs. 10.4%)(OR=1.78, P=0.004). Our findings imply a plausible explanation for the contribution of host genetic determinants to the variable outcome of HBV infection, which might provide valuable information for future genetic study in this area.

Common interleukin 10 polymorphism associated with decreased risk of tuberculosis

Interleukin 10 (IL10) is a powerful TH2-cell cytokine that inhibits lymphocyte replication and secretion of inflammatory cytokines. The genetic associations of polymorphisms in IL10 with clinical manifestations of tuberculosis (TB) were examined in a large number of patients with clinical TB infection (n=459) and normal controls (n=871). One common promoter SNP (IL10 -592 A>C) was found to be significantly associated with decreased risk of TB manifestation. The frequency of the "C"-bearing genotype was higher in normal controls than in patients with clinical TB infection (P=0.005, OR=0.69). A summary of the genetic effect of IL10 -1082 A>G, the other nearby promoter SNP, in other ethnic groups is also presented.