Analysis of non-synonymous-coding variants of Parkinson's disease-related pathogenic and susceptibility genes in East Asian populations.

To evaluate the contribution of non-synonymous-coding variants of known familial and genome-wide association studies (GWAS)-linked genes for Parkinson's disease (PD) to PD risk in the East Asian population, we sequenced all the coding exons of 39 PD-related disease genes and evaluated the accumulation of rare non-synonymous-coding variants in 375 early-onset PD cases and 399 controls. We also genotyped 782 non-synonymous-coding variants of these genes in 710 late-onset PD cases and 9046 population controls. Significant enrichment of LRRK2 variants was observed in both early- and late-onset PD (odds ratio = 1.58; 95% confidence interval = 1.29-1.93; P = 8.05 × 10(-6)). Moderate enrichment was also observed in FGF20, MCCC1, GBA and ITGA8. Half of the rare variants anticipated to cause loss of function of these genes were present in healthy controls. Overall, non-synonymous-coding variants of known familial and GWAS-linked genes appear to make a limited contribution to PD risk, suggesting that clinical sequencing of these genes will provide limited information for risk prediction and molecular diagnosis.

Validation study of candidate single nucleotide polymorphisms associated with left ventricular hypertrophy in the Korean population.

BACKGROUND: Left ventricular hypertrophy (LVH) is a valid predictor for cardiovascular mortality and morbidity regardless of age, gender, and race. The HyperGEN study conducted a genome-wide association study and identified twelve single nucleotide polymorphisms (SNPs) associated with LVH. The aim of this study was to validate these candidate SNPs in the Korean population. METHODS: Among 1637 individuals from the Korean Multi-Rural Communities Cohort Study (MRCohort) of the Korean Genome Epidemiology Study (KoGES), we carried out a linear regression analysis with left ventricular mass index (LVMI) and a logistic regression analysis for LVH status. RESULTS: The rs4129218 on chromosome 12 tended to be associated with LVM/body surface area (adjusted ß = -0.023; p = 0.036) and LVM/height(2.7) (adjusted ß = -0.027; p = 0.016), and was marginally protective against LVH after adjustment for age, sex, body mass index, serum creatinine, systolic blood pressure, heart rate and antihypertensive medication (adjusted odds ratio = 0.766 and 0.731; p = 0.027 and 0.007 according to indexation by BSA and height(2.7), respectively). CONCLUSIONS: In the Korean population, the minor allele of rs4129218 had borderline association with lower LVM. This study suggests that rs4129218 on chromosome 12 showed consistent tendency of possibly related loci for LVH independent of ethnic background.

Genome-wide association study of Crohn's disease in Koreans revealed three new susceptibility loci and common attributes of genetic susceptibility across ethnic populations.

OBJECTIVE: Crohn's disease (CD) is an intractable inflammatory bowel disease (IBD) of unknown cause. Recent meta-analysis of the genome-wide association studies (GWAS) and Immunochip data identified 163 susceptibility loci to IBD in Caucasians, however there are limited studies in other populations. METHODS: We performed a GWAS and two validation studies in the Korean population comprising a total of 2311 patients with CD and 2442 controls. RESULTS: We confirmed four previously reported loci: TNFSF15, IL23R, the major histocompatibility complex region, and the RNASET2-FGFR1OP-CCR6 region. We identified three new susceptibility loci at genome-wide significance: rs6856616 at 4p14 (OR=1.43, combined p=3.60×10(-14)), rs11195128 at 10q25 (OR=1.42, combined p=1.55×10(-10)) and rs11235667 at 11q13 (OR=1.46, combined p=7.15×10(-9)), implicating ATG16L2 and/or FCHSD2 as novel susceptibility genes for CD. Further analysis of the 11q13 locus revealed a non-synonymous single nucleotide polymorphism (SNP) (R220W/rs11235604) in the evolutionarily conserved region of ATG16L2 with stronger association (OR=1.61, combined p=2.44×10(-12)) than rs11235667, suggesting ATG16L2 as a novel susceptibility gene for CD and rs11235604 to be a potential causal variant of the association. Two of the three SNPs (rs6856616 (p=0.00024) and rs11195128 (p=5.32×10(-5))) showed consistent patterns of association in the International IBD Genetics Consortium dataset. Together, the novel and replicated loci accounted for 5.31% of the total genetic variance for CD risk in Koreans. CONCLUSIONS: Our study provides new biological insight to CD and supports the complementary value of genetic studies in different populations.

Wnt5a activates THP-1 monocytic cells via a ß-catenin-independent pathway involving JNK and NF-κB activation.

Wnt5a has been implicated in the activation of macrophages. However, the profile and mechanism of downstream regulation has not been characterized. In this study, we have investigated the regulation of Wnt5a-induced activation in monocytic THP-1 cells. Wnt5a activated THP-1 cells, enhancing adhesion to endothelial cells. Hypoxia induced the production of Wnt5a, suggesting a role in the hypoxia-induced activation of macrophages. Wnt5a induced the expression of various pro-inflammatory cytokines and inflammatory mediators, particularly IL8 and CXCL2, suggesting a major role in the secretion of CXC chemokines by macrophages. Wnt5a induced JNK phosphorylation and NF-κB activation via ß-catenin-independent signaling. Interestingly, SP600125, a specific inhibitor of JNK, inhibited Wnt5a-induced activation of NF-κB, supporting JNK-dependent NF-κB activation. Our data suggest that Wnt5a activates monocytic cells via JNK and NF-κB activation.

No association between TNFSF15 and IL23R with ulcerative colitis in Koreans.

Recent genome-wide association studies have shown that some Crohn's disease (CD)-associated loci also contribute to ulcerative colitis (UC) susceptibility. To understand the genetic relationship between the two forms of inflammatory bowel disease, we investigated the well-established CD-susceptibility genes TNFSF15 (tumor necrosis factor ligand superfamily, member 15) and IL23R in Korean patients with UC. Eight TNFSF15 and five IL23R single-nucleotide polymorphisms were genotyped in 654 patients with UC and in 601 healthy controls. There was no association between TNFSF15 or IL23R variants and UC in Korean individuals, in contrast to previous reports of a shared association of the IL23R gene with both CD and UC in Caucasian individuals. Our data suggest that neither TNFSF15 nor IL23R variants contribute to UC susceptibility in Koreans.

Serum Sorbitol Dehydrogenase as a Novel Prognostic Factor for Hepatocellular Carcinoma after Surgical Resection.

The majority of patients with hepatocellular carcinoma (HCC) undergoing curative resection experience tumor recurrence. To examine the association between preoperative serum sorbitol dehydrogenase (SORD), a liver-derived enzyme that reflects liver damage, and recurrence of HCC after curative resection, 92 patients were randomly selected who underwent curative resection for HCC between 2011 and 2012 from a prospective registry. Recurrence-free survival (RFS) was compared based on serum SORD levels. Cox proportional hazard models were used to investigate prognostic factors for RFS. During a median follow-up duration of 57.1 months, 43 patients experienced HCC recurrence. Patients with serum SORD ≥15 ng/mL (HR, 3.46; 95% CI, 1.76-6.81; p < 0.001) had worse RFS compared with patients with serum SORD <15 ng/mL. Serum AFP and SORD levels were two independent prognostic factors for RFS. When patients were stratified by baseline serum SORD and AFP levels, patients with serum AFP levels ≥400 ng/mL and serum SORD levels ≥15 ng/mL had a distinctly poor prognosis with the lowest RFS rates (HR, 22.08; 95% CI, 6.91-70.50; p < 0.001). Baseline serum SORD is an effective prognostic factor for HCC after resection. It may help guide patient selection for surgery, especially when combined with serum AFP levels.

Structural insight into the distinct properties of copper transport by the Helicobacter pylori CopP protein.

Helicobacter pylori CopP (HpCopP) is a putative copper binding regulatory protein composed of 66 amino acid residues. The small HpCopP protein is homologous to CopZ, encoded by the E. hirae and B. subtilis cop operons. To clarify the role of HpCopP in copper metabolism in H. pylori, we studied the structural and copper binding characteristics by NMR spectroscopy. Based on the resonance assignments, the tertiary structure of HpCopP was determined. Unlike the betaalphabetabetaalphabeta fold of the homologous CopZ, HpCopP adopts the betaalphabetabetaalpha fold. The superposition with structures of other bacterial copper binding proteins showed that the global structure of HpCopP follows the general topology of the family, regardless of absence of the C-terminal beta-strand. The Cu(I) binding property of HpCopP was well conserved like CopZs: the structural changes due to Cu(I) and Ag(I) bindings were primarily restricted to the metal binding motif (CXXC motif). On the other hand, the Cu(II) binding property of CopP was different with that of CopZ: in the absence of reducing agent, Cu(II) ion oxidized a mutant HpCopP, resulting in disulfide bond formation in the CXXC motif. The Cu(II) ion binding property was evaluated using the mutant HpCopP, in which two amino acids were artificially introduced at the C-terminus, since the reduced state of the CXXC motif was more stabile in the mutant HpCopP without a reducing agent. Here, the structure and copper binding property of HpCopP are discussed in detail.

Immunomodulating activities of water-soluble exopolysaccharides obtained from submerged culture of Lentinus lepideus.

Immunomodulating activities of water-soluble exopolysaccharides (LL-EX) obtained from submerged mycelial culture of Lentinus lepideus were studied and their effectiveness was compared with lipopolysaccharide (LPS). The influence of the LL-EX on macrophage cellular lysosomal enzyme activity was to stimulate up to 267%, 392%, and 464% at the level of 10, 50, and 100 microg/ml, respectively. When the LL-EX was further fractionated into LL-Fr.I and Fr.II by Sepharose CL-6B gel chromatography, the cellular lysosomal enzyme activity of LL-Fr.II (2.1- fold) was higher than Fr.I (1.2-fold). Moreover, both LL-Fr.I and Fr.II stimulated the cytokines IL-1beta, TNF-alpha, and IL-6 in macrophages. In mixed lymphocyte reaction, LL-Fr.I and Fr.II enhanced the splenocyte proliferation up to 1.2-fold and 1.4-fold (50 microg/ml), respectively, stimulating only T lymphocytes. The fractions of LL-EX not show any direct toxicity against human gastric adenocarcinoma cell (AGS). The molecular masses of LL-Fr.I and Fr.II were estimated to be about 1,986 kDa and 21 kDa, respectively. The total sugar and protein contents of the two fractions were 84.97% and 69.88% and 15.03% and 30.12%, respectively. The sugar and amino acid compositions of the LL-Fr.I and Fr.II were also analyzed in detail.

TC1 (C8orf4) enhances the Wnt/beta-catenin pathway by relieving antagonistic activity of Chibby.

The Wnt/beta-catenin pathway has been implicated in human cancers. Here, we show that TC1 (C8orf4), a small protein present in vertebrates, functions as a positive regulator of the pathway. TC1 interacts with Chibby (Cby) and thereby enhances the signaling pathway by relieving the antagonistic function of Cby on the beta-catenin-mediated transcription. Upon coexpression in mammalian cells, TC1 redistributes from nucleolus to nuclear speckles, where it colocalizes with Cby. TC1 up-regulates the expression of beta-catenin target genes that are implicated in invasiveness and aggressive behavior of cancers, such as metalloproteinases, laminin gamma2, and others. Our data indicate that TC1 is a novel upstream regulator of the Wnt/beta-catenin pathway that enhances aggressive behavior of cancers.

Downregulation of Raf-1 kinase inhibitory protein as a sorafenib resistance mechanism in hepatocellular carcinoma cell lines.

PURPOSE: Although sorafenib enhances overall survival, sorafenib resistance has been reported to be a significant limiting factor for improved prognosis in patients with hepatocellular carcinoma (HCC). Therefore, it is important to identify the mechanism of sorafenib resistance. This study aimed to identify the causative factor of sorafenib resistance and suggest methods for overcoming it. METHODS: The sensitivity to sorafenib was compared in human HCC cell lines and patient-derived HCC primary cells. Based on its cytotoxicity, signaling pathways altered by sorafenib and the causative factors were examined through assays. The mechanism by which sorafenib modified the sorafenib-resistance inducer through gene or protein expression or stability was also investigated. We also designed a treatment option to overcome sorafenib resistance. RESULTS: Sorafenib activated the Raf/MEK/ERK pathway and caused sorafenib resistance in HCC cell lines and patient-derived HCC primary cells. Sorafenib reactivated the MAPK pathway by down-regulating RKIP at the post-translational level. Knockdown of RKIP increased phosphorylated ERK and thus suppressed sorafenib-mediated cell death. We also found that sorafenib-reactivated ERK maybe an attractive target for second-line therapy for patients with sorafenib resistance. Sequential combination treatment with sorafenib and PD98059 significantly reduced the viability and proliferation of sorafenib-resistant cells, while their increasing apoptosis efficacy. CONCLUSION: Reactivation of the Raf/MEK/ERK pathway through aberrant expression of RKIP is one of the mechanisms behind sorafenib resistance in HCC. Sequential combination treatment with sorafenib and PD98059 could provide a new strategy to overcome sorafenib resistance in future clinical studies.

TC1(C8orf4) correlates with Wnt/beta-catenin target genes and aggressive biological behavior in gastric cancer.

PURPOSE: We have recently reported that TC1(C8orf4), a small protein present in vertebrates, functions as a novel regulator of the Wnt/beta-catenin pathway. TC1 up-regulates beta-catenin target genes that are implicated in the aggressive behavior of cancers. Our aim was to investigate the clinical and pathobiological relevance of TC1 in gastric cancer. EXPERIMENTAL DESIGN: The expression of TC1 was analyzed using tissue microarray in correlation with clinicopathologic variables and beta-catenin target genes in 299 gastric cancers. The biological effects of TC1 on Matrigel invasiveness and the proliferation of cancer cells were analyzed. TC1 expression was analyzed in gastric cancer cells after serial peritoneal implantation in nude mice. RESULTS: TC1 expression was present in 111 carcinomas (37.1%), correlating with tumor stage (P < 0.002), poor differentiation (P < 0.001), lymphatic infiltration (P < 0.005), and lymph node metastasis (P < 0.006). TC1 also correlated with poor survival in diffuse type carcinomas (P < 0.0001), and even in patients with lymph node metastasis (P < 0.0014). TC1 also correlated with the expression of beta-catenin target genes including laminin gamma2, metalloproteinase-7 and metalloproteinase-14, cyclin D1, c-Met, and CD44. TC1 enhanced Matrigel invasiveness and proliferation, supporting its role in the aggressive biological behavior of cancers. The expression of TC1 increased in MKN45 cells after serial peritoneal seeding in nude mice. CONCLUSIONS: Our data suggests that TC1 coordinates the up-regulation of Wnt/beta-catenin target genes that are implicated in the aggressive biological behavior of cancers. The strong clinical relevance, even in patients with lymph node metastasis, suggested that TC1 could be a potential therapeutic target of advanced gastric cancers.

TC1(C8orf4) is upregulated by IL-1beta/TNF-alpha and enhances proliferation of human follicular dendritic cells.

Follicular dendritic cells (FDC) play crucial roles in immune regulation. TNF-alpha has been shown to be essential to the FDC network. However, the molecular regulation of FDC proliferation has not been characterized. Here, we show that TC1(C8orf4), a novel positive regulator of the Wnt/beta-catenin pathway in vertebrates, is upregulated by IL-1beta and TNF-alpha in the human FDC-like line HK. TC1 enhances HK cell proliferation, while TC1-knockdown inhibits the proliferation induced by IL-1beta, suggesting a role of TC1 as a regulator of FDC proliferation. The regulation by pro-inflammatory cytokines suggests that TC1 might be implicated in linking local inflammation to immune response by stimulating FDC.

Expression profiling and subtype-specific expression of stomach cancer.

The expression profiling and molecular grouping of stomach cancers has been a challenging task because of their complexity and variation. We have analyzed gene expression profiles of 22 gastric cancer/nontumor mucosa couples using 14K cDNA microarray chips designed for gastric cancer analysis. Upon pairwise analysis of the individual couples at the false significance rate 0.91%, 79 and 398 genes were reported to be up-regulated and down-regulated in tumors, respectively. Tumors were clustered into two groups having high and low inflammatory infiltration, respectively. The latter consisted of three subgroups, including diffuse type carcinomas and intestinal types with distinct pathological characteristics of aggressive behavior. When the pooled tumor was hybridized against the pooled nontumor mucosa samples, more genes were detected to express differentially than those detected by the pairwise analysis at the same threshold level. However, they did not render satisfactory clustering of individual tumors. Our data showed that stomach cancers could be clustered effectively using stomach-specific microarrays and pairwise analysis of tumor/nontumor mucosa couples. It is suggested that the application of specific goal-oriented experimental designing would be advantageous for efficient analysis of expression profiles of such a complex disease as gastric cancer.

Is Whole Exome Sequencing Clinically Practical in the Management of Pediatric Crohn's Disease?

BACKGROUND/AIMS: The aim of this study was to identify the profile of rare variants associated with Crohn's disease (CD) using whole exome sequencing (WES) analysis of Korean children with CD and to evaluate whether genetic profiles could provide information during medical decision making. METHODS: DNA samples from 18 control individuals and 22 patients with infantile, very-early and early onset CD of severe phenotype were used for WES. Genes were filtered using panels of inflammatory bowel disease (IBD)-associated genes and genes of primary immunodeficiency (PID) and monogenic IBD. RESULTS: Eighty-one IBD-associated variants and 35 variants in PID genes were revealed by WES. The most frequently occurring variants were carried by nine (41%) and four (18.2%) CD probands and were ATG16L2 (rs11235604) and IL17REL (rs142430606), respectively. Twenty-four IBD-associated variants and 10 PID variants were predicted to be deleterious and were identified in the heterozygous state. However, their functions were unknown with the exception of a novel p.Q111X variant in XIAP (X chromosome) of a male proband. CONCLUSIONS: The presence of many rare variants of unknown significance limits the clinical applicability of WES for individual CD patients. However, WES in children may be beneficial for distinguishing CD secondary to PID.

Immunochip analysis identification of 6 additional susceptibility loci for Crohn's disease in Koreans.

BACKGROUND: Crohn's disease (CD) is an intractable inflammatory bowel disease of unknown cause. Recent genome-wide association studies of CD in Korean and Japanese populations suggested marginal sharing of susceptibility loci between Caucasian and Asian populations. As the 7 identified loci altogether explain 5.31% of the risk for CD, the objective of this study was to identify additional CD susceptibility loci in the Korean population. METHODS: Using the ImmunoChip custom single-nucleotide polymorphism array designed for dense genotyping of 186 loci identified through GWAS, we analyzed 722 individuals with CD and 461 controls for 96,048 SNP markers in the discovery stage, followed by validation in an additional 948 affected individuals and 977 controls. RESULTS: We confirmed 6 previously reported loci in Caucasian: GPR35 at 2q37 (rs3749172; P = 5.30 × 10, odds ratio [OR] = 1.45), ZNF365 at 10q21 (rs224143; P = 2.20 × 10, OR = 1.38), ZMIZ1 at 10q22 (rs1250569; P = 3.05 × 10, OR = 1.30), NKX2-3 at 10q24 (rs4409764; P = 7.93 × 10, OR = 1.32), PTPN2 at 18p11 (rs514000; P = 9.00 × 10, OR = 1.33), and USP25 at 21q11 (rs2823256; P = 2.49 × 10, OR = 1.35), bringing the number of known CD loci (including 3 in the HLA) in Koreans to 15. The 6 additional loci increased the total genetic variance for CD risk from 5.31% to 7.27% in Koreans. CONCLUSIONS: Although the different genetic backgrounds of CD between Asian and Western countries has been well established for the major susceptibility genes, our findings of overlapping associations offer new insights into the genetic architecture of CD.

TRF2 is in neuroglial cytoplasm and induces neurite-like processes.

TRF2 is a ubiquitous protein that protects telomeres in the nucleus. We found that TRF2 was present at the peripheral nerve axons and the brain neuroglial cell processes extensively. It was in the cytoplasmic membrane as well as nuclear fractions, but not in the soluble cytoplasmic fraction of SH-SY5Y neuroblastoma cells. TRF2 was up-regulated in P19 embryonal carcinoma cells at the early stage of induced neural differentiation with retinoic acid treatment. Upon transfection, TRF2-expressing COS cells often produced neurite-like long cytoplasmic processes. TRF2 is a component of neuroglial cells and appears to be involved in the cytoplasmic process formation that is necessary for neural differentiation.

TC1(C8orf4) regulates hematopoietic stem/progenitor cells and hematopoiesis.

Hematopoiesis is a complex process requiring multiple regulators for hematopoietic stem/progenitor cells (HSPC) and differentiation to multi-lineage blood cells. TC1(C8orf4) is implicated in cancers, hematological malignancies and inflammatory activation. Here, we report that Tc1 regulates hematopoiesis in mice. Myeloid and lymphoid cells are increased markedly in peripheral blood of Tc1-deleted mice compared to wild type controls. Red blood cells are small-sized but increased in number. The bone marrow of Tc1-/- mice is normocellular histologically. However, Lin-Sca-1+c-Kit+ (LSK) cells are expanded in Tc1-/- mice compared to wild type controls. The expanded population mostly consists of CD150-CD48+ cells, suggesting the expansion of lineage-restricted hematopoietic progenitor cells. Colony forming units (CFU) are increased in Tc1-/- mice bone marrow cells compared to controls. In wild type mice bone marrow, Tc1 is expressed in a limited population of HSPC but not in differentiated cells. Major myeloid transcriptional regulators such as Pu.1 and Cebpα are not up-regulated in Tc1-/- mice bone marrow. Our findings indicate that TC1 is a novel hematopoietic regulator. The mechanisms of TC1-dependent HSPC regulation and lineage determination are unknown.

A common missense variant in NUDT15 confers susceptibility to thiopurine-induced leukopenia.

Thiopurine therapy, commonly used in autoimmune conditions, can be complicated by life-threatening leukopenia. This leukopenia is associated with genetic variation in TPMT (encoding thiopurine S-methyltransferase). Despite a lower frequency of TPMT mutations in Asians, the incidence of thiopurine-induced leukopenia is higher in Asians than in individuals of European descent. Here we performed an Immunochip-based 2-stage association study in 978 Korean subjects with Crohn's disease treated with thiopurines. We identified a nonsynonymous SNP in NUDT15 (encoding p.Arg139Cys) that was strongly associated with thiopurine-induced early leukopenia (odds ratio (OR) = 35.6; P(combined) = 4.88 × 10(-94)). In Koreans, this variant demonstrated sensitivity and specificity of 89.4% and 93.2%, respectively, for thiopurine-induced early leukopenia (in comparison to 12.1% and 97.6% for TPMT variants). Although rare, this SNP was also strongly associated with thiopurine-induced leukopenia in subjects with inflammatory bowel disease of European descent (OR = 9.50; P = 4.64 × 10(-4)). Thus, NUDT15 is a pharmacogenetic determinant for thiopurine-induced leukopenia in diverse populations.

Alzheimer's disease and Parkinson's disease genome-wide association study top hits and risk of Parkinson's disease in Korean population.

Alzheimer's disease (AD) and Parkinson's disease (PD) have overlapping clinical and pathological features, suggesting a common pathway for these 2 neurodegenerative disorders. Here we investigated the association of both AD and PD GWAS top hits with PD susceptibility. We selected 25 single nucleotide polymorphisms (SNPs) in 9 genes (ABCA7, APOE, BST1, CLU, CR1, LRRK2, PARK16, PICALM, and SNCA) that were genotyped in 1036 PD case patients and 1208 controls. Case patients and controls were all ethnic Koreans. Logistic regression analysis was performed to calculate age- and sex-adjusted odds ratios. None of the AD-susceptibility loci (ABCA7, APOE, CLU, CR1, and PICALM) showed statistically significant association with PD susceptibility. In contrast, we replicated associations of SNCA, LRRK2, BST1, and PARK16 with PD susceptibility in Koreans. Of those, the SNCA SNP rs11931074 showed the most significant association with PD susceptibility (adjusted odds ratio = 1.48; 95% confidence interval = 1.31-1.67; p = 2.20E-10). In a logistic regression analysis with SNPs coded under an additive model, there was no significant genetic interaction between the LRRK2 and the PARK16 locus gene RAB7L1 in PD risk. Our results confirm the associations of SNCA, LRRK2, BST1, and PARK16 with PD susceptibility and fail to show significant associations of AD genome-wide association study (GWAS) top hits with PD susceptibility in a Korean population.