Search

Established genetic risk factors for Alzheimer’s disease (AD) account for only a portion of AD heritability. The aim of this study was to identify novel associations between genetic variants and AD-specific brain atrophy. We conducted genome-wide association studies for brain magnetic resonance imaging measures of hippocampal volume and entorhinal cortical thickness in 2643 Koreans meeting the clinical criteria for AD (n = 209), mild cognitive impairment (n = 1449) or normal cognition (n = 985). A missense variant, rs77359862 (R274W), in the SHANK-associated RH Domain Interactor (SHARPIN) gene was associated with entorhinal cortical thickness (p = 5.0 × 10−9) and hippocampal volume (p = 5.1 × 10−12). It revealed an increased risk of developing AD in the mediation analyses. This variant was also associated with amyloid-β accumulation (p = 0.03) and measures of memory (p = 1.0 × 10−4) and executive function (p = 0.04). We also found significant association of other SHARPIN variants with hippocampal volume in the Alzheimer’s Disease Neuroimaging Initiative (rs3417062, p = 4.1 × 10−6) and AddNeuroMed (rs138412600, p = 5.9 × 10−5) cohorts. Further, molecular dynamics simulations and co-immunoprecipitation indicated that the variant significantly reduced the binding of linear ubiquitination assembly complex proteins, SHPARIN and HOIL-1 Interacting Protein (HOIP), altering the downstream NF-κB signaling pathway. These findings suggest that SHARPIN plays an important role in the pathogenesis of AD.

Neuroblastomas (NBs) have heterogeneous clinical behavior, from spontaneous regression or differentiation to relentless progression. Evidence from our laboratory and others suggests that neurotrophin receptors contribute to these disparate behaviors. Previously, the role of TRK receptors in NB pathogenesis was investigated. In the present study, the expression of RET and its co‑receptors in a panel of NB cell lines was investigated and responses to cognate ligands GDNF, NRTN, and ARTN with GFRα1‑3 co‑receptor expression, respectively were found to be correlated. RET expression was high in NBLS, moderate in SY5Y, low/absent in NBEBc1 and NLF cells. All cell lines expressed at least one of GFRα co‑receptors. In addition, NBLS, SY5Y, NBEBc1 and NLF cells showed different morphological changes in response to ligands. As expected, activation of RET/GFRα3 by ARTN resulted in RET phosphorylation. Interestingly, activation of TrkA by its cognate ligand NGF resulted in RET phosphorylation at Y905, Y1015, and Y1062, and this was inhibited in a dose‑dependent manner by the TRK inhibitor (CEP‑701). Conversely, RET activation by ARTN in NBLS cells led to phosphorylation of TrkA. This suggests a physical association between RET and TRK proteins, and cross‑talk between these two receptor pathways. Finally, RET, GFR and TRK expression in primary tumors was investigated and a significant association between RET, its co‑receptors and TRK expression was demonstrated. Thus, the present data support a complex model of interacting neurotrophin receptor pathways in the regulation of cell growth and differentiation in NBs.

Background High-risk type human papilloma virus (HPV) is the most important cause of cervical cancer. Recently, real-time polymerase chain reaction and reverse blot hybridization assay-based HPV DNA genotyping kits are developed. So, we compared the performances of different three HPV genotyping kits using different analytical principles and methods. Methods Two hundred positive and 100 negative cervical swab specimens were used. DNA was extracted and all samples were tested by the MolecuTech REBA HPV-ID, Anyplex II HPV28 Detection, and HPVDNAChip. Direct sequencing was performed as a reference method for confirming high-risk HPV genotypes 16, 18, 45, 52, and 58. Results Although high-level agreement results were observed in negative samples, three kits showed decreased interassay agreement as screening setting in positive samples. Comparing the genotyping results, three assays showed acceptable sensitivity and specificity for the detection of HPV 16 and 18. Otherwise, various sensitivities showed in the detection of HPV 45, 52, and 58. Conclusions The three assays had dissimilar performance of HPV screening capacity and exhibited moderate level of concordance in HPV genotyping. These discrepant results were unavoidable due to difference in type-specific analytical sensitivity and lack of standardization; therefore, we suggested that the efforts to standardization of HPV genotyping kits and adjusting analytical sensitivity would be important for the best clinical performance.

(-)-Epigallocatechin-3-gallate (EGCG) induces apoptosis in cancer cells without adversely affecting normal cells. Understanding the cancer-specific cytotoxic activity of EGCG is very important in defining the mechanism of tumorigenesis and identifying superb chemotherapeutic agents against cancer. We comparatively assayed human telomerase reverse transcriptase (hTERT)-mediated apoptosis by EGCG-induced reactive oxygen species (ROS) in normal cells and cancer cells. EGCG showed differential levels of ROS induction between the cell types; ROS, especially hydrogen peroxide, was highly induced in cancer cells, while it was not in normal cells. In addition, the higher level of ROS down-regulated hTERT via binding of CCCTC binding factor (CTCF) to the core promoter region of hTERT, which repressed hTERT expression. CTCF binding was epigenetically controlled by the demethylation of the previously hypermethylated site for CTCF, which was induced by down-regulation of DNA methyltransferase 1 (DNMT1). In contrast, hTERT down-regulation was not observed in normal cells. These results suggest that preferential death of cancer cells by EGCG could be caused by the cancer-specific induction of ROS and epigenetic modulation of expression of apoptosis-related genes, such as hTERT.

A common epitope region of enteroviruses was identified by sequence-independent single-primer amplification (SISPA), followed by immunoscreening of 11 cDNA libraries from two Korean enterovirus isolates (echoviruses 7 and 30) and a coxsackievirus B3 (ATCC-VR 30). The putative common epitope region was localized in the N terminus of VP1 when the displayed recombinant proteins from the phages were chased by the convalescent-phase sera. The genomic region encoding the common epitope region was amplified and then expressed by using the vector pGEX-5X-1. The antigenicity of the expressed recombinant protein was identified by Western blotting with guinea pig antisera for six different serotypes of enteroviruses. After successive immunization of mice with the recombinant common epitope protein, splenocytes were extracted and hybridized with P3X63-Ag8-653 cells. A total of 24 hybridomas that produced monoclonal antibodies (MAbs) against the putative common epitope of enteroviruses were selected. Four of these were immunoglobulin G1 isotypes with a kappa light chain. These MAbs recognized 15 Korean endemic serotypes and prototypes of enteroviruses in an indirect immunofluorescence assay. These results suggest that the expressed protein might be a useful antigen for producing group common antibodies and that the use of the MAbs against the putative common epitope of enteroviruses might be a valuable diagnostic tool for rapidly identifying a broad range of enteroviruses.

Background To prevent the recurrence of genitourinary infections and to reduce the risks of their complications, accurate and rapid diagnosis are required. STDetect® Chip is a DNA chip which allows for the simultaneous detection of 13 major genitourinary pathogens in a single vaginal swab or urine specimen. We evaluated the analytical performance of the STDetect® Chip for detecting target pathogens that commonly cause genitourinary infections. Methods The target pathogens of the STDetect® Chip are Chlamydia trachomatis , Candida albicans , Enterococcus faecalis , Gardnerella vaginalis , Mycoplasma hominis , Neisseria gonorrhoeae , Mycoplasma genitalium , Ureaplasma urealyticum , Staphylococcus aureus , Klebsiella pneumoniae , Trichomonas vaginalis , and Herpes simplex virus types 1 and 2. Performance of the STDetect® Chip for the detection of target pathogens was evaluated comparing with the result of direct sequencing and conventional multiplex PCR assay. And precision tests for STDetect® Chip were performed with quality control materials. Results The STDetect® Chip showed high sensitivities (95.1%–100%), specificities (93.4% to 100%), concordance rates (95.0%–100%), positive predictive values (69.8%–100%), and negative predictive values (93.1%–100%) in its identification of 13 target pathogens. The STDetect® Chip had a particularly excellent concordance rate (96.5%) for the 4 major pathogens, C. albicans , G. vaginalis , M. hominis , and U. urealyticum , compared with direct sequencing. Comparing to multiplex PCR assay, STDetect® Chip showed better sensitivity for detecting M. hominis (97.0% vs. 54.5%) and U. urealyticum (93.2% vs. 65.9%). In precision tests, coefficients of variations for signal intensities were ranged from 11.2% to 26.2%. Conclusion The STDetect® Chip showed excellent analytical performance, and its result was in good agreement with that obtained by direct sequencing.

The present study was performed to identify the susceptible single nucleotide polymorphisms (SNPs) for the prediction of Korean type 2 diabetes mellitus (T2DM) and to clarify the matrilineal origin of Korean T2DM‑specific SNPs. Fourteen SNPs from the adiponectin (ADIPOQ), hepatocyte nuclear factor 4α, phosphoenolpyruvate carboxykinase 1 and glucokinase genes in the Korean population were analyzed. Only one SNP, ‑11,377 C/G on the ADIPOQ gene, was finally determined to be responsible for the incidence of Korean T2DM (P=0.028). The G‑T‑T‑A haplotype at positions ‑11,377, +45, +276 and +349 on the ADIPOQ gene was also associated with a high incidence of Korean T2DM (P=0.023). In addition, the susceptibility of Korean individuals to T2DM appears to be affected by their matrilineal origin. Of note, the group of Southern origin, consisting of mitochondrial DNA macrohaplogroups F and R, was predisposed to T2DM, whereas the group of Northern origin, consisting of haplogroups A and Y, was resistant to T2DM. This implied that the differential genetics between the two groups, which were formed from the initial peopling of the proto‑Korean population via Southern and Northern routes to the present time, may explain their differing susceptibility to T2DM. In conclusion, from Southern Asia Northward, a matrilineal origin of Korean individuals appears to be responsible for the prevalence of Korean T2DM caused by the ‑11,377 G allele.

IL-6 and TNFαwere significantly increased in the bone marrow aspirate samples of patients with active multiple myeloma (MM) compared to those of normal controls. Furthermore, MM patients with advanced aggressive disease had significantly higher levels of IL-6 and TNFαthan those with MM in plateau phase. TNFαincreased interleukin-6 (IL-6) production from MM cells. However, the detailed mechanisms involved in signaling pathways by which TNFαpromotes IL-6 secretion from MM cells are largely unknown. In our study, we found that TNFαtreatments induce MEK and AKT phosphorylation. TNFα-stimulated IL-6 production was abolished by inhibition of JAK2 and IKKβor by small interfering RNA (siRNA) targeting TNF receptors (TNFR) but not by MEK, p38, and PI3K inhibitors. Also, TNFαincreased phosphorylation of STAT3 (ser727) including c-Myc and cyclin D1. Three different types of JAK inhibitors decreased the activation of the previously mentioned pathways. In conclusion, blockage of JAK/STAT-mediated NF-κB activation was highly effective in controlling the growth of MM cells and, consequently, an inhibitor of TNFα-mediated IL-6 secretion would be a potential new therapeutic agent for patients with multiple myeloma.

A novel method of ancient DNA (aDNA) purification was developed using ion-exchange columns to improve PCR-amplifiable DNA extraction from ancient bone samples. Thirteen PCR-resistant ancient bone samples aged 500-3,300 years were tested to extract aDNA using a recently reported, silica-based aDNA extraction method and an ion-exchange column method for the further purification. The PCR success rates of the aDNA extracts were evaluated for the amplification ability of the fragments of mitochondrial DNA, a high-copy DNA, and amelogenin, a low-copy DNA. The results demonstrate that the further purification of silica-based aDNA extracts using ion-exchange columns considerably improved PCR amplification. We suggest that the ion-exchange column-based method will be useful for the improvement of PCR-amplifiable aDNA extraction, particularly from the poorly preserved, PCR-resistant, ancient samples.

Expression of hTERT has been recognized an important factor in cellular aging and immortalization. Therefore, to analyze regulatory mechanism of hTERT expression, we investigated the CpG methylation pattern of the hTERT promoter as an epigenetic mechanism and its implication in transcriptional regulation of hTERT using tissues of colorectal carcinoma. As a result, we were able to observe an increased pattern of hTERT expression according to the malignant progression of colorectal carcinoma. Additionally, we could find that hTERT expression was induced when the P1 and P2 region of hTERT were sufficiently hypermethylated and, oppositely, the G1 region of hTERT was hypomethylated. Importantly, we could find three specific CpG sites (7th CpG of P2 and 11th and 2nd–10th CpGs of P1) closely related with the increasing of hTERT expression. These findings may provide important clues to deducing the expression mechanisms of hTERT .

Since nonsense mutations are closely associated with severe conditions of genetic disorders, including familial cancers, rapid and precise detection of those mutations is very important for research purposes and molecular diagnosis. Currently, screening methods such as the FASAY (functional analysis of separated alleles in yeast) and the Y-SC (stop codon assay in yeast) are used for functional detection of nonsense mutations in genes of interest. But these yeast-based approaches are time-consuming, expensive and complicated. In order to circumvent these problems, we, in the present study, devised a novel Escherichia coli-based screening method, the E-SC (E. coli stop codon assay) for the detection of heterozygous nonsense mutations in genes of interest. Our strategy was based on the fact that the plasmid replicating with a low copy number in E. coli allows an effective separation of normal and mutant alleles. Moreover, it relies on the expression vector, resulting in the formation of white and blue colonies for mutant and normal alleles through the expression of PCR-amplified fragment/lacZ fusion protein respectively. To evaluate the applicability of the E-SC method for the detection of the heterozygous truncating mutation, PCR-amplified exon 7 of the StAR [steroidogenic acute regulatory protein; causative gene of the CAH (congenital lipoid adrenal hyperplasia)] and RT (reverse transcription)-PCR-amplified full-length cDNA of MeCP2 (methyl-CpG-binding protein 2; causative gene of Rett syndrome) were used. The E-SC showed an almost 1:1 ratio of blue/white colonies in all patients examined, whereas the control samples produced blue colonies only. These results demonstrate that the E-SC system is useful for rapid and precise detection of known and unknown heterozygous truncation mutations in genes that cause genetic disorders and familial cancers.

Background: Although many single nucleotide polymorphisms (SNPs) of mtDNA h ave been found to be associated with type 2 diabetes mellitus, the results of studies using different population samples and different methods are mixed. Therefore, we conducted a genetic association study of mtDNA SNPs and type 2 diabetes mellitus in a Korean sample and compared our results with those of studies co nducted in other human populations. Methods: A total of 298 blood samples from 147 type 2 diabetic patients and 151 normal controls were surveyed for SNPs via PCR directed sequencing. Sequencing analy ses were performed using the SeqMan module of the DNASTAR program. The identified SNPs were compared to previously reported SNP lists on NCBI and V-mitoSNP. Results: A total of 24 SNPs were identified in the MT-RNR2, MR-TL1 and MT-ND1 mtDNA genes in Korean type 2 diabetes mellitus patients and normal controls. The SNPs identified in the Korean sample were not closely associated with the type 2 diabetes mellitus phenotype, a significantly different result from those previously observed in European, Chinese and Japanese samples. Additionally, a haplotype and prevalence analysis could not detect any differences between the type 2 diabetes mellitus patients and normal controls. Conclusion: The 24 mtDNA SNPs were not associ ated with type 2 diabetes mellitus risk in our Korean sample. The results of the present study support the possibilit y that mtDNA SNPs have a differential effect on the risk of type 2 diabetes mellitus according to geographical origin. (Korean Diabetes J 33:382-391, 2009)