Hsin-Fu Liu

Molecular epidemiology of HCV genotypes among injection drug users in Taiwan: Full-length sequences of two new subtype 6w strains and a recombinant form_2b6w.

Human immunodeficiency virus type 1 (HIV‐1) circulating recombinant form (CRF) 07_BC strain has caused serious outbreaks among injection drug users in Taiwan since 2004. The objective of this study was to conduct a molecular epidemiological study of HCV genotypes in intravenous drug users in Taiwan. Blood samples and questionnaires from 591 intravenous drug users infected with HIV‐1 were collected nationwide. In total, 180 samples were selected for HCV genotyping using multiplex PCR and phylogenetic analysis of the core, E1 and NS5B regions. The Inno‐Lipa assay was used to confirm multiple infections with different genotypes. Eighty percent had a single infection with subtype 1b being the most common subtype (24%), 12% had double infections and two had triple infections. In addition, three recombinant forms (RFs)‐2a1a, 3a1b, and 2b6w were identified. Phylogenetic analyses showed that the 3a, 6a, and 6n strains were clustered with strains present in Thailand and mainland China. Full‐length sequence analysis showed that two 6w strains shared 89.4–90.2% sequence homology with the 6(r) strain from the Guangdong Province, China. Bootscan analysis revealed that the recombination breakpoint of RF_2b6w was located at the NS2‐NS3 junction. In summary, the distribution of HCV genotypes among Taiwanese intravenous drug users was complex and more than 12% of the drug users were infected with more than one genotype of HCV. J. Med. Virol. 82:57–68, 2010.

Association of CTLA4 gene A-G polymorphism with rheumatoid arthritis in Chinese.

The aim of this study was to investigate the allelic association of a single nucleotide polymorphism in exon 1 of the cytotoxic T-lymphocyte antigen-4 (CTLA4) gene with rheumatoid arthritis (RA) in Chinese people. One hundred and eighty-six unrelated adults with RA and 203 randomly selected normal adults were studied. All were ethnic Chinese living in Taiwan. The CTLA4 A-G polymorphism was genotyped with a polymerase chain reaction (PCR) and digestion with the restriction enzyme BstEII. The genotype and allele frequencies of CTLA4 in patients with rheumatoid arthritis differed significantly from those of adult controls (#E5/E5#=0.022 and #E5/E5#=0.037, respectively). Genotype CTLA4 49 G/G and allele G were associated with an increased risk of RA (RR=1.72, 95% CI=1.15–2.57, #E5/E5#=0.008; RR=1.39, 95% CI=1.02–1.89, #E5/E5#=0.037, respectively), whereas genotype A/G and allele A were associated with protection against RA (RR=0.58, 95% CI=0.39–0.87, #E5/E5#=0.008 and RR=0.72, 95% CI=0.53–0.98, #E5/E5#=0.037, respectively). We concluded that, the CTLA4 49 A-G polymorphism is associated with RA in Chinese patients from Taiwan.

Genetic Variation in the Vascular Endothelial Growth Factor Gene is Associated With Biliary Atresia

Background and Goals: Biliary atresia (BA) is a chronic inflammatory disease of the bile ducts resulting in biliary cirrhosis. Vascular endothelial growth factor (VEGF) has been implicated in cell-mediated inflammatory reactions. We aimed to study the relationship between genetic variations of the VEGF gene and susceptibility to BA using both case-control and family-based methodologies. Study: A total of 45 Taiwanese children with BA, 160 ethnically matched healthy controls, and 40 families (consisting of parents, affected children, and unaffected siblings) were studied. Three functional VEGF polymorphisms (−2578 A/C, −634 G/C, and +936 C/T) were assessed by using TaqMan assay. Results: The +936 CC genotype [odds ratio (OR) 3.51, 95% confidence interval 1.54-8.01, Pc=0.006] and C allele (OR 3.19, 95% confidence interval 1.48-6.90, Pc=0.004) were significantly associated with increased risk of BA. The association of the +936 C allele with BA was also confirmed in a family-based association study (OR 5.7, χ2=9.8, Pc=0.005). None of the haplotypes studied significantly influenced the risk to BA in either the case-control or family data sets. Conclusions: The VEGF +936 C/T polymorphism and particularly the C allele are associated with BA, possibly conferring increased susceptibility to the disease.

ITPKC gene SNP rs28493229 and Kawasaki disease in Taiwanese children

Kawasaki disease (KD) is a systemic vasculitis caused by unknown infectious agents, host immune dysregulation and genetic susceptibility in children. Coronary artery lesions (CALs) complicate 15-25% of cases of untreated KD. The aim of this study was to investigate if the single-nucleotide polymorphism (SNP) rs28493229 of the ITPKC gene is associated with susceptibility to KD or with CALs in Taiwanese children. A total of 385 unrelated Taiwanese children (222 boys and 163 girls) with KD were included, 140 of whom had CALs. Mean age at diagnosis was 1.9 +/- 1.7 (0.1-10.2) years. Rs28493229 was genotyped in children with KD and 1158 ethnically matched healthy controls using the TaqMan Allelic Discrimination Assay. In 184 families with KD, both biological parents were available, constituting 184 trios with their children. They were assessed in a family-based study by means of a transmission/disequilibrium test (TDT). No significant differences in genotype (P = 0.29 and P = 0.29, respectively), allele (P = 0.14 and P = 0.22, respectively) and carrier (P = 0.22 and P = 0.25, respectively) frequencies of the SNP were found between healthy controls and children with KD or those with CALs. TDT in the 184 family trios and in 69 trios where the child had CALs did not reveal significant overtransmittion of the C allele. In conclusion, we did not find a statistically significant association between the ITPKC gene SNP rs28493229 and KD or CALs in Taiwanese children.

The promoter region of the CTLA4 gene is associated with type 1 diabetes mellitus.

The CTLA4 (cytotoxic T lymphocyte associated antigen-4) gene encodes the T cell receptor involved in the control of T cell proliferation and mediates T cell apoptosis. C-T polymorphism is present at position -318 from the ATG start codon in the promoter region of the gene. We report a study on the polymorphism in 347 unrelated children with type 1 diabetes mellitus (DM) (age at diagnosis 7.2+/-3.8 years) and their 260 healthy siblings as controls. Genotype C/C conferred a risk of type 1 DM (RR = 2.02, 95% CI 1.32-3.10, pc = 0.0033). The gene frequency of the C allele was higher in patients (RR = 1.91, 95% CI 1.28-2.84, pc = 0.0026). The gene frequency and phenotype frequency of the T allele were negatively associated with type 1 DM (RR = 0.52, 95% CI 0.35-0.78, pc = 0.0026 and RR = 0.49, 95% CI 0.32-0.76, pc = 0.0022, respectively). The frequency of genotype C/T was lower in patients (RR = 0.50, 95% CI 0.32-0.78, pc = 0.0051). This study demonstrates that nucleotide -318 C-T polymorphism of the CTLA4 gene is associated with type 1 DM. The promoter allele -318 C confers a risk of type 1 DM but allele -318 T confers protection against this disease.

Molecular Epidemiology of Enterovirus 71 Infection in the Central Region of Taiwan from 2002 to 2012

Enterovirus 71 (EV71), a causative agent of hand, foot, and mouth disease can be classified into three genotypes and many subtypes. The objectives of this study were to conduct a molecular epidemiological study of EV71 in the central region of Taiwan from 2002–2012 and to test the hypothesis that whether the alternative appearance of different EV71 subtypes in Taiwan is due to transmission from neighboring countries or from re-emergence of pre-existing local strains. We selected 174 EV71 isolates and used reverse transcription-polymerase chain reaction to amplify their VP1 region for DNA sequencing. Phylogenetic analyses were conducted using Neighbor-Joining, Maximum Likelihood and Bayesian methods. We found that the major subtypes of EV71 in Taiwan were B4 for 2002 epidemic, C4 for 2004–2005 epidemic, B5 for 2008–2009 epidemic, C4 for 2010 epidemic and B5 for 2011–2012 epidemic. Phylogenetic analysis demonstrated that the 2002 and 2008 epidemics were associated with EV71 from Malaysia and Singapore; while both 2010 and 2011–2012 epidemics originated from different regions of mainland China including Shanghai, Henan, Xiamen and Gong-Dong. Furthermore, minor strains have been identified in each epidemic and some of them were correlated with the subsequent outbreaks. Therefore, the EV71 infection in Taiwan may originate from pre-existing minor strains or from other regions in Asia including mainland China. In addition, 101 EV71 isolates were selected for the detection of new recombinant strains using the nucleotide sequences spanning the VP1-2A-2B region. No new recombinant strain was found. Analysis of clinical manifestations showed that patients infected with C4 had significantly higher rates of pharyngeal vesicles or ulcers than patients infected with B5. This is the first study demonstrating that different EV 71 genotypes may have different clinical manifestations and the association of EV71 infections between Taiwan and mainland China.

Simvastatin prevents proliferation and bone metastases of lung adenocarcinoma in vitro and in vivo.

OBJECTIVESnTo explore the mechanism about how HMG-CoA reductase (HMGR) inhibitor inhibit proliferation and bone metastases of lung adenocarcinoma in vitro and in vivo.nnnMETHODSnThe HMGR inhibitor simvastatin, human lung cancer cell line A549 and Balb/c nude mouse were used in this study. The mice were randomly divided into 2 groups: control group (0.9% NaCl solution, i.v.) and simvastatin group (5mg/kg simvastatin, i.v.). A scratch assay using A549 cell monolayer was also tested. An invasion assay using collagen-coated membrane in trans-wells was applied to evaluate the effect of simvastatin on the metastatic potential of A549 cells in vitro. The expressions of CD44, PUMA, P53, MMP2 and MMP9 were determined by real-time PCR and western blotting; the phosphorylation status of MAPK/ERK signaling parthway was investigated by western blot. .nnnRESULTSnCompared with the control group, the migration of A549 cells in simvastatin-treated group was markedly inhibited (p ≤ 0.01). Untreated A549 cells showed marked invasion, while simvastatin significantly inhibited the invasion of tumor cells (p ≤ 0.001). Incubation of A549 cells with simvastatin significantly reduced the levels of CD44, MMP2 and MMP9 (p <0.01), while significantly increased p53 (p < 0.01). Simvastatin significantly inhibits tumor growth and bone metastasis in lung cancer xenograft mouse model, simvastatin can inhibit the kinase phosphorylation inMAPK/ERK signaling parthway.nnnCONCLUSIONSnThe HMGR inhibitor simvastatin prevents proliferation and osteolytic bone metastases of lung adenocarcinoma cells in vitro and vivo. Its mechanism may be associated with regulation of CD44, P53, MMP family and inactivation of MAPK/ERK signaling parthway.

High prevalence of GB virus C/hepatitis G virus in Kinshasa, Democratic Republic of Congo: a phylogenetic analysis.

A prevalence of 10.3% of GB virus C (GBV‐C)/hepatitis G virus (HGV) carriers was found in 97 pregnant women from Kinshasa, Congo (formerly Zaire), while prevalences of 1%, 4.1%, and 0% were found for hepatitis C virus, human immunodeficiency virus, and human T‐lymphotropic virus respectively. Phylogenetic analysis of the ten GBV‐C/HGV positives based on the 5′ non‐coding region using three different methods identified consistently three GBV‐C/HGV genotypes. Four main clades were found within the type 1 sequences. All the Congolese isolates are GBV‐C/HGV type 1 in two different clades. The clustering of seven Congolese isolates was inconsistent in different methods. Further likelihood‐mapping analysis showed a well‐resolved phylogeny, confirming the clustering of the seven Congolese isolates with a Belgian strain representing a new clade in the GBV‐C/HGV type 1 sequences. J. Med Virol. 60:159–165, 2000.

Genetic Polymorphisms in the CD40 Ligand Gene and Kawasaki Disease

BackgroundAlthough some previous studies have reported that genetic and immunological factors play important roles in the pathogenesis of Kawasaki disease (KD), the etiological factors of this enigmatical pediatric disease are still poorly understood.PurposeThis study aims to investigate whether polymorphisms of the CD40 ligand (CD40L) gene are associated with KD and the development of coronary artery lesions (CAL) in the Taiwanese children.Materials and methodsThe CD40L −3459 A/G and IVS4+121 A/G single nucleotide polymorphisms (SNPs) were genotyped in 167 children with KD and 1,010 ethnically matched healthy controls by TaqMan assay.ResultsNone of the CD40L polymorphisms was associated with susceptibility or CAL development of KD, and this finding was supported by the haplotype analysis.ConclusionIn summary, these results provide little support for specific CD40L SNPs in the susceptibility or CAL development of KD in Taiwanese children. However, it will be necessary to validate or replicate this association in other independent large-size ethnic groups.

Molecular analysis of HLA-DRB1 allelic associations with systemic lupus erythematous and lupus nephritis in Taiwan

To evaluate the association of human leukocyte antigen (HLA)–DRB1 alleles with systemic lupus erythematosus (SLE) and lupus nephritis (LN) in the Taiwanese population, and to investigate the possible association of HLA-DRB1 alleles with disease severity in LN. HLA-DRB1 alleles were studied in 105 SLE patients (82 patients with LN, 23 patients without LN) and 855 healthy controls by polymerase chain reaction and sequence-based typing assays. The frequency of the HLA class II alleles DRB1*0301 (Odds ratio [OR] = 2.01, 95% confidence interval [CI] = 1.31–3.10, Pc = 0.02) and DRB1*1501 (OR = 2.06, 95% CI = 1.36–3.13, Pc = 0.01) were both increased in SLE patients, compared to healthy controls. The frequency of DRB1*1202 was significantly lower in LN patients than in SLE patients without nephritis (OR = 0.23, 95% CI = 0.09–0.57, Pc = 0.01). No specific allele was significantly associated with an increased or decreased risk for severity of LN in this sample. In Taiwanese people, the DRB1*0301 and DRB1*1501 alleles are significant risk factors for SLE, while the DRB1*1202 allele is protective for LN.