- Human genomic variations can directly cause human diseases or affect them at various stages including disease susceptibility, pathological pathway, disease progression and severity, respond to treatment and recovery. It has been known that same human disease usually occurs from diverse causes and pathological processes attributable to marked genetic heterogeneity in different populations and individuals. Recently, compelling evidence suggests that rare mutations with severe effect are responsible for a substantial portion of complex human disease, and genetic heterogeneity is important at multiple levels of disease causation. Based on this information, we propose that complex human diseases in Thais are caused by rare mutations with severe effect of the responsible genes that possess genetic heterogeneity. Thus, it is necessary to directly investigate into etiologies and pathogeneses of these diseases in Thai patients to understand their natures and to design appropriate interventions. The availabilities of sequence information of human genome and powerful molecular biology techniques make it possible to conduct this investigation into the diseases that cause health and economic burdens to Thais. Our group has long-term experiences in the study of genetics and molecular biology of diabetes, kidney diseases, and dengue virus infection in Thais. We therefore take this advantage to focus our research interest in the investigation of these diseases by applying advanced technologies in genetics, genomics, and molecular biology. The results of five projects studied are reported. Project I: Genetics/Genomics and Molecular Biology of Diabetes Mellitus Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia. If it is untreated, DM will result in complications including retinopathy, nephropathy, neuropathy, and cardiovascular disease. Type 2 diabetes (T2D) is the most common, accounting for 90% of all DM patients. The monogenic T2D is classified as maturity-onset diabetes of the young (MODY), characterized by young age at onset with autosomal dominant inheritance. Identification of genes causing MODY and that influencing individual susceptibility to T2D leads to a better understanding of pathophysiology of diabetes. Our research group has shown that the six reported MODY genes account for a small proportion of MODY (19%) and early-onset T2D patients (10%) in Thais. We are the first group who identified the mutations and single nucleotide polymorphisms (SNPs) of PAX4 caused MODY9 and T2D susceptibility, respectively. In the continuing studies, our group aimed to examine whether genetic variations of such strong candidate genes as TCF7L2, CAPN10, AdipoQ, and PAX4 genes are associated with T2D in Thais or not. We found that these genes, especially PAX4, are associated with T2D in Thai patients. We studied into details of AdipoQ and PAX4 variants identified in Thai patients and reported the abnormal structures and functions of mutant adiponectin and PAX4 proteins expressed in cultured cell lines. We also employed genomic approaches using DNA microarrays and exome sequencing to identify novel genes causing MODY and early-onset T2D in Thai families. We have discovered novel genes, which will be soon reported. In addition, we also investigated the protective mechanism of sex hormones (estrogen and testosterone) against glucotoxicity on β-cells to understand the roles of these hormones in protection of DM and to search for an alternative strategy for prevention and treatment of DM, especially in elderly. We demonstrated that estrogen significantly decreases not only oxidative stress but also endoplasmic reticulum (ER) stress to protect against high glucose-induced pancreatic β-cell death. Similarly, testosterone can protect against male pancreatic β-cell apoptosis from glucotoxicity via reduction of both oxidative stress and ER stresses. Project II: Genetics/Genomics and Molecular Biology of Kidney Stone Disease Kidney stone disease (KSD) is an important public health problem in the Northeastern (NE) population of Thailand. Its prevalence is 5-10% with several thousands of new cases hospitalized for treatment each year. The etiology and pathogenesis of KSD in the NE Thai population are unknown but the disease in this population seems to be unique from what has been reported in other ethnic groups because it is not associated with the conditions of increased urinary stone promoters, such as hypercalciuria, hyperoxaluria, and hyperuricosuria. Our group has recently reported an initial evidence suggesting a genetic contribution to KSD in the NE Thai population because it was found to have familial aggregation with a high relative risk (λR = 3.18) among members of the affected families. To investigate into the role of genetic factor in pathogenesis of KSD in the NE Thai population, we have employed multiple genetic/genomic approaches, such as candidate-gene association study, genome-wide association study (GWAS), and genome-wide linkage analysis using DNA microarrays to identify disease-susceptibility and disease-causing genes. We firstly conducted a candidate gene association study and found the association between KSD in Northeastern Thai patients and prothrombin (F2) gene. After sequencing the entire coding regions of F2, we identified one exonic non-synonymous single nucleotide polymorphism (nsSNP; rs5896; c.494 C>T) in exon 6 resulting in a T165M substitution. Our results indicate that prothrombin variant (T165M) is associated with KSD risk in the NE Thai female patients. The genome-wide association study (GWAS) by using DNA microarrays was also conducted. A SNP rs759330, located at a predicted microRNA binding site at 3’UTR of PAQR6 – a gene encoding progestin and adipoQ receptor family member VI, was found to be associated with KSD, suggesting that PAQR6 is a modifying gene for KSD. We also selected a large family with KSD (UBRS082) to perform a genome-wide linkage and exome sequencing. KSD phenotype was inherited as autosomal dominant model in this family. Chromosomal regions with high logarithm of odd scores (LOD >2.80) were initially identified by genome-wide linkage and genetic variations in these regions were examined by exome sequencing. Two novel variations (p.N909K and p.K1809R) of SCN10A on chromosome 3, encoding Nav1.8α subunit of voltage-gated sodium channel, were co-segregated with KSD in this family without its presence in the normal control subjects. As these two variations were co-inherited in the same allele, they might have combined effects in causing KSD. An additional variation (p.V1149M) of SCN10A was identified in another affected family. Nav1.8α subunit mRNA and protein are expressed in human kidney tissues. All these findings provide evidences supporting that the mutations of SCN10A cause KSD in these families. Project III: Molecular Mechanism of Human Kidney Anion Exchanger 1 Trafficking Associated with Distal Renal Tubular Acidosis Human kidney anion exchanger 1 (kAE1) is a basolateral anion (Cl-/HCO3-) exchanger of the acid-secreting type A intercalated cells in the distal nephron, involved in maintaining acid-base homeostasis in the human body. Several mutations in the SLC4A1 gene encoding kAE1 result in autosomal dominant or autosomal recessive distal renal tubular acidosis (dRTA). This disease is characterized by an inability of the kidney to secrete H+ into urine resulting in systemic metabolic acidosis often accompanied by several clinical manifestations including muscle weakness, growth retardation, metabolic bone disease, nephrocalcinosis, nephrolithiasis, chronic pyelonephritis, and renal failure. The SLC4A1 mutations associated with dRTA usually do not cause defect in the anion exchange function of kAE1 but result in impaired trafficking or mistargeting of the mutant kAE1 proteins. However, it has yet been unknown how the protein trafficking fails or why mistargeting of kAE1 protein occurs. To understand the pathogenesis of v- dRTA caused by SLC4A1 mutations, it is necessary to investigate the trafficking and targeting process of kAE1 protein from its biosynthesis site to the cell surface in both normal and abnormal conditions. Our group is interested in identifying the protein that interacts with kAE1 that plays a role in its basolateral trafficking. We identified kAE1-interacting proteins by using yeast two hybrid (Y2H) screening. The interaction between kAE1 and adaptor-related protein complex 1 mu1A (AP-1 mu1A), were identified. We have also discovered that kAE1 interacts with kinesin family member 3B (KIF3B) – a motor protein, in kidney cells, suggesting that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney alpha-intercalated cells. However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occur. We thus studied the role of basolateral-related sorting proteins, including mu1 subunit of adaptor protein (AP) complexes, clathrin, and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. We found that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated co-localization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1, and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting alpha-intercalated cells. Project IV: Molecular Pathogenesis of Dengue Virus Infected Liver Cells Dengue virus (DENV) infection is one of the most important mosquito-borne viral diseases, affecting many million people worldwide. DENV particle contains a single positive-stranded RNA genome, encoding a single precursor polypeptide, which is cleaved by host and viral proteases into three structural proteins, including capsid (C), membrane (M), and envelope (E), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). Clinical symptoms of DENV infection range from a predominantly febrile disease, dengue fever (DF), to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), which usually occurs in cases with subsequent infection with a different serotype of DENV. The patients with DHF generally present with hemorrhagic tendencies, plasma leakage, thrombocytopenia, and hemoconcentration. Liver cell injury is commonly observed in patients with DHF/DSS, as evident by elevation of aminotransferases, reactive hepatitis and fulminant hepatic failure. The cause of hepatocyte injury during DENV infection, which may lead to fulminant hepatic failure, remains unclear. In this project, we determined cell death responses and inflammatory cytokine production induced by DENV infection in cultured hepatic cells. And, the roles and mechanisms of DENV C and DENV NS5 proteins in cell death responses and induction of inflammatory cytokine were also investigated in the cultured hepatic cells. The results of these studies would provide insight into molecular pathogenesis of DENV infection causing liver cell injury and will facilitate the development of new therapeutic modalities for DENV infection. We examined the expression of cell death genes during DENV-infection of HepG2 cells by using real-time PCR arrays. The expression changes were consistent with activation of apoptosis and autophagy, including the up-regulation of RIPK2, HRK, TGF-β, PERK, and LC3B. RIPK2 – receptor-interacting serine/threonine protein kinase 2 is a crucial mediator of multiple stress responses that leads to the activation of caspase, NF-κB and MAP kinases including JNK and p38. The inhibition of RIPK2 expression by SB203580 significantly reduced apoptosis and suppression of endogenous RIPK2 in DENV-infected HepG2 cells by small interfering RNA (siRNA) significantly decreased apoptosis, suggesting for the first time that RIPK2 plays a role in DENV-mediated apoptosis. From real-time PCR arrays, we also found the up-regulation of cathepsin gene expression in DENV-infected HepG2 cells. Cathepsins – cysteine proteases inside the lysosome were previously reported to be up-regulated in patients with DHF. We showed for the first time that DENV induces lysosomal membrane permeabilization. The resulting cytosolic cathepsin B and S contributed to apoptosis via caspase-9 and caspase-3 activation, which was significantly reduced by cathepsin B or S inhibitors and cathepsin B-siRNA. We have previously described the translocation of DENV C into nucleus and its interaction with death-domain-associate (DAXX) protein to induce apoptosis. Expression of CD137, which is a member of the tumor necrosis factor receptor family, increased significantly in HepG2 cells expressing DENV C. CD137 recruits TNF receptor associated factor 2 (TRAF2) and activates apoptosis signal regulating kinase 1 (ASK1), resulting in activation of cJun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). p38 MAPK participates in both apoptosis-related signaling and pro-inflammatory cytokine production. The role of p38 MAPK in DENV-infected HepG2 cells was examined using siRNA, which showed that DENV infection activated p38 MAPK and induced apoptosis. Thus, DENV induces CD137 signaling to enhance apoptosis by increasing TNFα production via activation of p38 MAPK. The in vivo role of ERK1/2, a member of the MAPK family, in a mouse model of DENV infection was also examined. Our results showed that DENV induces phosphorylation of ERK1/2 and increases apoptosis. Inhibition of phosphorylated ERK1/2 by the selective ERK1/2 inhibitor, FR180204, limits hepatocyte apoptosis and reduces DENV-induced liver injury. Clinical parameters, including leucopenia, thrombocytopenia, transaminases and histology, show improvements after FR180204 treatment. Caspase-3 was significantly decreased in FR180204 treated DENV-infected mice compared to the levels of untreated DENV-infected mice, suggesting the role of ERK1/2 signaling in immune-mediated liver injury during DENV infection. DAXX was also identified to interact with DENV NS5 by yeast two-hybrid (Y2H) assay. The in vivo relevance of this interaction was suggested by co-immunoprecipitation and nuclear co-localization of these two proteins in HEK293 cells expressing DENV NS5. HEK293 cells expressing DENV NS5-K/A, which were mutated at the nuclear localization sequences (NLS), were created to assess its functional roles in nuclear translocation, DAXX interaction, and cytokine production. In the absence of NLS, DENV NS5 could neither translocate into the nucleus nor interact with DAXX to increase the DHF-associated cytokine, RANTES (CCL5) production. This demonstrates the interaction between DENV NS5 and DAXX and the role of the interaction on the modulation of RANTES production. Increased levels of cytokines - the so-called 'cytokine storm', contribute to the pathogenesis of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). We therefore compared the expression of cytokine genes between mock-infected and DENV-infected HepG2 cells using a real-time PCR array and revealed several up-regulated chemokines and cytokines, including CXCL10 and TNF-α. In this study, we also used compound A (CpdA), a plant-derived phenyl aziridine precursor containing anti-inflammatory action and acting as a dissociated nonsteroidal glucocorticoid receptor modulator, as a candidate agent to modulate secretion of DENV-induced cytokines. CpdA is not a glucocorticoid but has an anti-inflammatory effect with no metabolic side effects as steroidal ligands. CpdA significantly reduced DENV-induced CXCL10 and TNF-α secretion and decreased leukocyte migration, indicating for the first time the therapeutic potential of CpdA in decreasing massive immune activation during DENV infection. Project V: Production of Human Single Chain Antibody Variable Fragments and Peptide Inhibitors Specific to Dengue Virus Proteins Nowadays, a licensed vaccine and anti-viral agent for DENV infection have not been available and only supportive treatment is given to the patients. We proposed that blocking or inhibiting functions of viral proteins could reduce disease severity and symptoms in the DENV-infected patients. In this project, we produced human single chain antibody variable fragments (HuScFv) specific to dengue virus proteins and test their binding and inhibiting activities to the corresponding antigens with an ultimate objective to produce therapeutic biomolecules for treatment of dengue virus infection. HuScFv molecules were screened and selected from the human antibody phage display library by using purified recombinant DENV NS1 (rNS1), full-length envelope (rFL-E) and its domain III (rEDIII) proteins as target antigens for bio-panning. HuScFv from two phagemid transformed E. coli clones, i.e., clones 11 and 13, bound to the rNS1 as well as native NS1 in both secreted and intracellular forms. Culture fluids of the HuScFv11/HuScFv13 exposed DENV2 infected cells had significant reduction of the infectious viral particles, implying that the antibody fragments affected the virus morphogenesis or release. rEDIII-specific HuScFv15A exhibited neutralizing effect to DENV infection in Vero cells in a dose-dependent manner as determined by plaque formation and cell ELISA. Epitope mapping and molecular docking results concordantly revealed interaction of HuScFv15A to functional loop structure in EDIII of the DENV E protein. Although the functions of the epitopes and the molecular mechanism of the HuScFvs further investigations, these small antibodies have high potential for development as an effective anti-DENV biomolecules. Furthermore, we used molecular docking to search for a safe anti-DENV drug. The short peptides targeting to the hydrophobic pocket on DENV E protein; a structural transition in the membrane fusion in DENV infection process, were identified. The information of predicted ligand-binding site of reported active compounds to DENV2 hydrophobic pocket was also used for peptide inhibitor selection. The di-peptide, EF, was the most effective on DENV2 infection inhibition in vitro with a half maximal inhibition concentration (IC50) of 96 μM. Treatment of DENV2 with EF at the concentration of 200 μM resulted in 83.47% and 84.15% reduction of viral genome and intracellular E protein, respectively. Among four DENV serotypes, DENV2 was the most effective for the inhibition. Our results provide the proof-of-concept for development of therapeutic peptide inhibitors against DENV infection by the computer-aided molecular design.