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Epigenetics alternation of non-genetic variation and genome-wide association study proven allelic variants may associate with insulin secretion in type 2 diabetes (T2D) development. We analyzed promoter DNA methylation array to evaluate the associated with increased susceptibility to T2D (30 cases, 10 controls) and found 1,091 gene hypermethylated in promoter regions. We performed the association study of T2D and found 698 single nucleotide polymorphisms in exon and promoter sites by using 2,270 subjects (560 cases, 1,710 controls). A comparison of DNA hypermethylation and gene silencing of mouse T2D results in our T2D patients' results showed that the 5'-nucleotidase, cytosolic II (NT5C2) and fucosyltransferase 8 (FUT8) genes were strongly associated with increased susceptibility to T2D. DNA hypermethylation in promoter regions reduced NT5C2 gene expression, but not FUT8 in T2D patients. NT5C2 protein expression was decreased in pancreatic β-cells from T2D mice. Transient transfection NT5C2 into RIN-m5F cells down-regulated DNA methyltransferase I (DNMT1) expression and up-regulation of the insulin receptor. Moreover, NT5C2 knockdown induced in DNMT1 overexpression and insulin receptor inhibition. Taken together, these results showed that NT5C2 epigenetically regulated insulin receptor in patients and mice with T2D, and maybe provide for T2D therapy strategy.

// Ming-Yu Lien 1, 2 , Chiao-Wen Lin 3, 4 , Hsiao-Chi Tsai 1 , Yng-Tay Chen 5 , Ming-Hsui Tsai 6 , Chun-Hung Hua 7 , Shun-Fa Yang 8, 9 and Chih-Hsin Tang 1, 10 1 Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan 2 Division of Hematology and Oncology, Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan 3 Institute of Oral Sciences, Chung Shan Medical University, Taichung, Taiwan 4 Department of Dentistry, Chung Shan Medical University Hospital, Taichung, Taiwan 5 Department of Pediatrics, Medical Research and Medical Genetics, China Medical College Hospital, Taichung, Taiwan 6 Department of Otolaryngology, China Medical University Hospital, Taichung, Taiwan 7 Department of Otorhinolaryngology, China Medical University Hospital, Taichung, Taiwan 8 Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan 9 Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan 10 Department of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan Correspondence to: Chih-Hsin Tang, email: chtang@mail.cmu.edu.tw Shun-Fa Yang, email: ysf@csmu.edu.tw Keywords: oral cancer, CCL4, single-nucleotide polymorphism Received: October 20, 2016 Accepted: January 22, 2017 Published: February 22, 2017 ABSTRACT In Taiwan, oral cancer has causally been associated with environmental carcinogens. CCL4 (C-C chemokine ligand 4), a macrophage inflammatory protein with a key role in inflammation and immune-regulation, was implicated in carcinogenesis by facilitating instability in the tumor environment. The purpose of this study was to identify gene polymorphisms of CCL4 specific to patients with oral squamous cell carcinoma (OSCC) susceptibility and clinicopathological characteristics. A total of 2,053 participants, including 1192 healthy people and 861 patients with oral cancer, were recruited for this study. Three single-nucleotide polymorphisms (SNPs) of the CCL4 gene were analyzed by a real-time PCR. We found that the T/T homozygotes of CCL4 rs1634507 G/T polymorphism and the GG haplotype of 2 CCL4 SNPs (rs1634507 and rs10491121) combined were associated with oral-cancer susceptibility. In addition, TA haplotype significantly decreased the risks for oral cancer by 0.118 fold. Among 1420 smokers, CCL4 polymorphisms carriers with the betel-nut chewing habit had a 15.476–20.247-fold greater risk of having oral cancer compared to CCL4 wild-type (WT) carriers without the betel-nut chewing habit. Finally, patients with oral cancer who had A/G heterozygotes of CCL4 rs10491121 A/G polymorphism showed a lower risk for an advanced tumor size (> T2) ( p =0.046), compared to those patients with AA homozygotes. Our results suggest that the CCL4 rs1634507 SNP have potential predictive significance in oral carcinogenesis. Gene-environment interactions of CCL4 polymorphisms might influence oral-cancer susceptibility. CCL4 rs10491121 may be a factor to predict the tumor size in OSCC patients.

// Yng-Tay Chen 1 , Jiunn-Wang Liao 2 , Ya-Ching Tsai 1 and Fuu-Jen Tsai 1,3,4,5,6 1 Human Genetic Center, Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan 2 Graduate Institute of Veterinary Pathobiology, Research Center for Animal Medicine, Animal Disease Diagnostic Center, National Chung Hsing University, Taichung, Taiwan 3 Graduate Institute of China Medical Science, China Medical University, Taichung, Taiwan 4 Department of Medical Genetics, China Medical University Hospital, Taichung, Taiwan 5 School of Chinese Medicine, China Medical University, Taichung, Taiwan 6 Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan Correspondence to: Fuu-Jen Tsai, email: // Keywords : type 2 diabetes, DNA methylation, NR4A1, DNMT1, epigenetics, Gerotarget Received : March 01, 2016 Accepted : May 29, 2016 Published : June 14, 2016 Abstract Our previous genome-wide association studies showed that DNA methyltransferase 1 (DNMT1) is associated with increased susceptibility to type 2 diabetes (T2D) in Han Chinese individuals. Here, we aimed to further evaluate the role of DNMT1 in T2D. We performed a genome-wide DNA methylation array and found that the nuclear receptor subfamily 4 group A member 1 ( NR4A1 ) promoter was hypermethylated in patients with T2D and in a mouse model of T2D. Moreover, DNA hypermethylation of the NR4A1 promoter reduced NR4A1 mRNA expression. Transient transfection of human NR4A1 into RIN-m5F and 293T cells caused DNMT1 inhibition and induced insulin receptor activation. NR4A1knockdown by shRNA resulted in overexpression of DNMT1 and inhibition of insulin receptor, suggesting that the NR4A1 gene is involved in the epigenetics pathway. Furthermore, T2D model mice treated with the DNMT1 inhibitor aurintricarboxylic acid (ATA) showed reduced activation of DNMT1 in pancreatic β cells; this effect reversed the changes in NR4A1 expression and decreased blood glucose in T2D model mice. Thus, our results showed for the first time that DNMT1 caused NR4A1 DNA hypermethylation and blocked insulin signaling in patients with T2D. Importantly, ATA therapy may be useful for decreasing blood glucose levels by reversing NR4A1-dependent insulin signaling. These findings improve our understanding of the crucial roles of these regulatory elements in human T2D.

Genome-wide association study (GWAS) data showed that the protein tyrosine phosphatase receptor type delta (PTPRD) is associated with increased susceptibility to type 2 diabetes (T2D) in Han Chinese. A replication study indicated that PTPRD is involved in the insulin signaling pathway; however, the underlying mechanism remains unclear. We evaluated PTPRD expression in patients with T2D and controls. PTPRD expression levels were lower in patients and were correlated with the duration of the disease. Overexpression of the human insulin receptor PPARγ2 in HepG2 cells induced overexpression of PTPRD and the insulin receptor. PTPRD knockdown, using a shRNA, resulted in down-regulation of the insulin receptor. These results indicate that PTPRD activates PPARγ2 in the insulin signaling pathway. Similar results for PTPRD expression were found using a T2D mouse model. Silencing of PTPRD was caused by DNA methylation in T2D mice and patients, and correlated with DNMT1 expression. Furthermore, we showed that a DNMT1 SNP (rs78789647) was correlated with susceptibility to T2D. This study shows for the first time that DNMT1 caused PTPRD DNA hypermethylation and induced insulin signaling silencing in T2D patients. Our findings contribute to a better understanding of the crucial roles of these regulatory elements in human T2D.

The nonsense mutations of the hepatitis B virus (HBV) surface (S) gene have been reported to have oncogenic potential. We have previously identified several transforming nonsense mutations of the HBV S gene from hepatocarcinoma (HCC) patients. Among them, the sW182* mutant (the stop codon for tryptophan 182) showed the most potent oncogenicity in a mouse xenograft model using stably transfected mouse fibroblast cells. This study is aimed at understanding the molecular mechanisms leading to the oncogenic activity of the sW182* mutant.A gene expression microarray in combination with gene set enrichment analysis (GSEA) revealed differentially expressed gene sets in the sW182* cells, including those related to cell-cycle regulation, deoxyribonucleic acid repair, and genome instability. Of the differentially expressed genes, the transforming growth factor-β-induced (TGFBI) gene was further validated to be dysregulated in the sW182* cells. This dysregulation was accompanied by hypermethylation of the TGFBI promoter.The level of cyclin D1, a negatively regulated TGFBI target, was highly elevated in the sW182* mutant cells, which is consistent with the potent oncogenicity. Furthermore, frequent abnormal mitosis and multinucleation were observed in the mutant cells. Exogenous expression of TGFBI alleviated the oncogenic activity of the sW182* cells. In human HBV-related HCC cancerous tissue, expression of TGFBI was downregulated in 25 of the 55 (45%) patients examined, suggesting that TGFBI dysregulation could occur in HBV-related HCC development in some cases.These results suggest that dysregulation of TGFBI is involved in the oncogenic activity of the sW182* mutant of the hepatitis B virus S gene.

The purpose of this study was to determine whether toll-like receptors 9 (TLR9) gene polymorphisms (rs352139 and rs352140) were markers of susceptibility to the development and progression of membranous nephropathy (MGN) in Taiwanese patients. The polymorphisms were investigated by polymerase chain reaction in 397 Taiwanese individuals (134 MGN patients and 263 controls). Patients with malignancy, chronic infectious diseases, lupus nephritis, or drug-induced secondary MGN were excluded from the study. Data showed AA genotype at rs352139 SNP or GG genotype at rs352140 SNP may indicate higher risk for MGN (odds ratio [OR] = 1.55; 95% confidence interval [CI] = 1.02-2.35, at rs352139 SNP; OR = 1.57; 95% CI = 1.03-2.39, at rs352140 SNP). However, MGN patients with A-G haplotype were susceptible for decreased creatinine clearance rate and for seriously tubule-interstitial fibrosis. The result suggests for the first time that TLR9 (rs352139 and rs352140) polymorphisms may contribute to the development and progression of MGN.

Background : Collagen structure is a key element in mitral valves. Collagen defects were proposed as the primary events causing floppy mitral valves (FMV). The role of collagen genetic variant in floppy mitral valve/mitral valve prolapse (FMV/MVP) has not been well studied. The purpose of this study is to investigate the possible relationship between the collagen gene polymorphisms and risk of FMV/MVP among the Chinese population in Taiwan. Methods : We studied 100 patients with FMV/MVP diagnosed by echocardiography and 243 age- and sex-matched normal control subjects. The polymorphisms of exon 31 and exon 52 of the collagen type III-α1 gene ( COL3A1 ) were identified by polymerase chain reaction (PCR)-based restriction analysis. Results : There was a significant difference in either the genotype distribution ( P P COL3A1 exon 31 polymorphism. An odds ratio for risk of FMV/MVP associated with COL3A1 exon 31 GG genotype was 7.42 (95% confidence interval 4.40–12.52). An odds ratio for risk of FMV/MVP associated with COL3A1 exon 31 G allele was 2.28 (95% confidence interval 1.57–3.29). There was no significant difference in the distribution of COL3A1 exon 52 genotypes ( P =0.31) and allelic frequencies ( P =0.32) between FMV/MVP cases and controls. Further categorization of the FMV/MVP patients into mild and severe subgroups revealed no statistical difference from the controls for exon 31 or exon 52 polymorphism. Conclusions : This study shows that patients with FMV/MVP have higher frequency of COL3A1 exon 31 GG genotype that supports a role of the COL3A1 exon 31 polymorphism in determining the risk of FMV/MVP among the Chinese population in Taiwan.

Abnormalities of proteoglycan, collagen, and elastic fibers were found in floppy mitral valves. Perlecan is one of the three major classes of heparan sulfate proteoglycans within the cardiovascular system. The role of perlecan genetic variant in mitral valve prolapse (MVP) has not been studied. We therefore performed a case-controlled study investigating the possible relation between the perlecan gene intron 6 BamHI polymorphism and MVP among the Chinese population in Taiwan. We studied 100 patients with MVP diagnosed by echocardiography and 100 age- and sex-matched normal control subjects. The perlecan gene intron 6 BamHI polymorphism was identified by polymerase chain reaction-based restriction analysis. There were no significant differences in either the genotype distribution or allelic frequencies between MVP cases and controls for perlecan gene intron 6 BamHI polymorphism (P = 0.20 and 0.76, respectively). Further categorization of the MVP patients into mild and severe subgroups also revealed no statistical difference from controls for perlecan gene intron 6 BamHI polymorphism. It is concluded that perlecan gene intron 6 BamHI polymorphism is not a suitable genetic marker of MVP in Taiwan Chinese.

Background Some studies have reported that patients with mitral valve prolapse syndrome (MVPS) also have a disorder in the autonomic or neuroendocrine function, which can cause a host of related symptoms. A potential role of the renin-angiotensin system in the pathogenesis of MVPS has been addressed. However, the role of angiotensin I-converting enzyme (ACE) genetic variant in MVPS has not been studied. We therefore performed a case-control study investigating the possible relation between ACE gene polymorphisms and MVPS in Taiwan Chinese. Methods We studied 100 patients with MVPS diagnosed by echocardiography and 100 age- and sex-matched normal control patients. ACE gene insertion/deletion (I/D), A-240T, and G2350A polymorphisms were identified by polymerase chain reaction-based restriction analysis. Results There was a significant difference in the distribution of ACE I/D genotypes ( P = .003) and allelic frequencies ( P = .001) between MVPS cases and control patients. An odds ratio for the risk of MVPS associated with the ACE II genotype was 2.14 (95% CI 1.20-3.80 ). An odds ratio for the risk of MVPS associated with ACE I allele was 1.96 (95% CI 1.30-2.97). The A-240T and G2350A polymorphisms of the ACE gene showed no association with MVPS ( P = .20, P = .13, respectively). Conclusions This study showed that patients with MVPS have a higher frequency of ACE II genotype, which supports a role of the ACE I/D gene polymorphism in determining the risk of MVPS among the Chinese population in Taiwan. (Am Heart J 2003;145:169-73.)

The formation of urinary stones is presumed to be associated with polymorphism of the osteocalcin gene. The most frequently seen polymorphism is the Hind III type located at the promoter region. This polymorphism has been used as a genetic marker in the search for a correlation between urolithiasis and normal subjects. In our study, a normal control group of 105 healthy people and 102 patients with calcium oxalate stones were examined. The polymorphism was seen following polymerase chain reaction-based restriction analysis. The results revealed no significant differences between normal individuals and stone patients (P = 0.978), and distribution of the TT homozygote in the control group (42.9%) was similar to that in the patient group (42.2%). Further categorization of the stone patients into normocalciuric and hypercalciuric groups also revealed no statistical differences from controls. We conclude that Hind III polymorphism of the osteocalcin gene is not a suitable genetic marker of urinary stone disease. Further searches for other polymorphisms on this gene correlated with stone disease are suggested.

BACKGROUND: X‐ray repair cross‐complementing group 3 (XRCC3) plays a crucial role in mammalian DNA repair processes. The polymorphism of XRCC3, rs861539 (Thr > Met at codon 241), is common in populations worldwide. This study analyzed the relationship between this functional single nucleotide polymorphism and systemic lupus erythematosus (SLE) in the Han Chinese population in Taiwan (HC‐TW). METHODS: Genotyping was performed using polymerase chain reaction restriction fragment length polymorphism on 163 SLE patients and 191 healthy participants in the control group. RESULTS: The data showed that the genotype frequency at codon 241 did not differ significantly between the SLE patients and the healthy participants in the control group; however, the allele frequency analysis indicated a significant difference between these groups. In addition, we used the genotype and allele frequencies of 191 healthy HC‐TW participants for comparison with HapMap populations. The results indicated a significant difference of XRCC3 Thr241Met allele and genotype frequencies between the HC‐TW population and HapMap populations, except for the other Han Chinese populations. A prior study showed that Thr241 > Met substitution in XRCC3 protein was positive as damaging and functional consequences as well. CONCLUSION: This is the first study to demonstrate the difference of XRCC3 Thr241 > Met variant between the HC‐TW population and HapMap population.

Background : Abnormalities of collagen and elastic fibers were found in floppy mitral valves (FMV). Urokinase-plasminogen activator (PLAU) was suggested to be involved in the pathogenesis of elastin and collagen degradation in arterial aneurysm. The role of PLAU genetic variant in mitral valve prolapse (MVP) has not been studied. We, therefore, performed a case-controlled study investigating the possible relation between the PLAU gene polymorphisms and risk of MVP in Taiwan Chinese. Methods : We studied 100 patients with MVP diagnosed by echocardiography and 106 age- and sex-matched normal control subjects. The T4065C and T3995C polymorphisms of the PLAU gene were identified by polymerase chain reaction (PCR)-based restriction analysis. Results : There was a significant difference in either the genotype distribution or allelic frequencies between MVP cases and controls for PLAU gene T4065C polymorphism ( P =0.0001 and 0.0002, respectively). An odds ratio for risk of MVP associated with PLAU T4065C TC genotype was 6.03 (95% confidence interval 2.11–14.83). An odds ratio for risk of MVP associated with PLAU T4065C T allele was 4.99 (95% confidence interval 1.93–12.91). There was no significant difference in either the genotype distribution or allelic frequencies between MVP cases and controls for PLAU T3995C polymorphism. Further categorization of the MVP patients into mild and severe subgroups revealed no statistical difference between these two subgroups for PLAU T4065C and T3995C polymorphisms. Conclusions : This study shows that patients with MVP have a higher frequency of PLAU T4065C TC genotype and T allele that supports a role of the PLAU T4065C polymorphism in determining the risk of MVP among the Chinese population in Taiwan.

It has been reported that patients with mitral valve prolapse syndrome (MVPS) also have a disorder in autonomic or neuroendocrine function which can cause many related symptoms. Although a potential role of the reninangiotensin system in the pathogenesis of MVPS has been addressed, the role of the angiotensinogen (AGT) genetic variant in MVPS has not been studied. Thus, a case-controlled study was performed to investigate the possible relationship between AGT gene polymorphisms and MVPS.A total of 100 patients with MVP diagnosed by echocardiography and 100 age- and sex-matched normal control subjects was studied. AGT gene M235T and T174M polymorphisms were identified by polymerase chain reaction-based restriction analysis.There was a significant difference in the distribution of AGT gene M235T genotypes (p0.001) and allelic frequencies (p0.001) between MVPS cases and controls. An Odds Ratio (OR) for risk of MVPS associated with M235T TT genotype was 8.55 (95% CI 4.51-16.18). An OR for risk of MVPS associated with the T allele at the M235T locus of the AGT gene was 3.27 (95% CI 2.05-5.22). The T174M polymorphism of AGT gene showed no association with MVPS (p = 0.94).These findings suggest that the M235T polymorphism of the AGT gene is associated with MVPS in the Chinese population of Taiwan. The association of the TT genotype with MVPS is more noteworthy than an overall increase in the frequency of the T allele at the M235T locus.