A concept to detect a subgingival finish line using an intraoral optical coherence tomography system: A clinical report.

This clinical report introduces an approach for detecting the supragingival finish line by penetrating the teeth and gingival tissue using optical coherence tomography (OCT) technology. This approach was used in 3 patients who underwent tooth preparation with a subgingival finish line. Consequently, the subgingival finish line, typically challenging to discern clearly in intraoral scans, was identifiable in the OCT image.

Massive parallel sequencing of short tandem repeats in the Korean population.

STR analysis using capillary electrophoresis has been the most widely used method for forensic DNA typing. Recently, massive parallel sequencing (MPS) technique has been emerging as an innovative tool to supplement or replace the conventional CE process. In this study, we evaluated the application of commercial MiSeqFGx™ forensic signature kit (Illumina Inc., San Diego, CA, USA) in the Korean population, including performance comparison with CE-based STR profiling kits. The genotyping results of 209 unrelated random Korean individuals were summarized according to the International Society for Forensic Genetics guideline. The study revealed that 26 novel sequence variations in autosomal STR were newly found that had not been previously reported in other forensic literature. This indicates that MPS may be an effective supplementary tool for forensic DNA typing and the database to increase the discriminatory power of individual identification.

Identification and evaluation of age-correlated DNA methylation markers for forensic use.

In forensics, age prediction is useful to narrow down the number of potential suspects because it can provide some general characteristics for predicting appearance. Previous genome-wide studies based on DNA methylation have reported age prediction algorithms using a penalized multivariate regression method known as elastic net and a few dozen to hundreds of CpG sites. Although more CpG sites may provide better accuracy than fewer CpG sites, this approach is not applicable to forensics because the amounts of crime-scene DNA are usually limited. In this study, we selected three age-correlated CpG sites, namely cg16867657 (ELOVL2), which is known to be an excellent age predictor, cg04208403 (ZNF423), and cg19283806 (CCDC102B), from HumanMethylation450 BeadChip datasets of 1415 individuals. Furthermore, we evaluated these markers in a 535-sample training set and a 230-sample validation set from Korean individuals using a pyrosequencing platform. From the training set, an age prediction model using the multiple linear regression method explained 91.44% of age-correlated variation in DNA methylation patterns. The standard error of estimate and mean absolute deviation were 6.320 and 3.156 years, respectively. In the validation set, the standard error of estimate and mean absolute deviation were estimated as 6.853 and 3.346 years, respectively. For the validation set, the model explained 91.08% of the variation in methylation and predicted age (±6years) with accuracy of 77.30% in the <60years age group and 57.30% in the older group (≥60 years). These results suggest that our three DNA methylation markers may be useful for age prediction in samples from Asian populations.

Identification of body fluid-specific DNA methylation markers for use in forensic science.

DNA methylation, which occurs at the 5'-position of the cytosine in CpG dinucleotides, has great potential for forensic identification of body fluids, because tissue-specific patterns of DNA methylation have been demonstrated, and DNA is less prone to degradation than proteins or RNA. Previous studies have reported several body fluid-specific DNA methylation markers, but DNA methylation differences are sometimes low in saliva and vaginal secretions. Moreover, specific DNA methylation markers in four types of body fluids (blood, saliva, semen, and vaginal secretions) have not been investigated with genome-wide profiling. Here, we investigated novel DNA methylation markers for identification of body fluids for use in forensic science using the Illumina HumanMethylation 450K bead array, which contains over 450,000 CpG sites. Using methylome data from 16 samples of blood, saliva, semen, and vaginal secretions, we first selected 2986 hypermethylated or hypomethylated regions that were specific for each type of body fluid. We then selected eight CpG sites as novel, forensically relevant DNA methylation markers: cg06379435 and cg08792630 for blood, cg26107890 and cg20691722 for saliva, cg23521140 and cg17610929 for semen, and cg01774894 and cg14991487 for vaginal secretions. These eight selected markers were evaluated in 80 body fluid samples using pyrosequencing, and all showed high sensitivity and specificity for identification of the target body fluid. We suggest that these eight DNA methylation markers may be good candidates for developing an effective molecular assay for identification of body fluids in forensic science.

Microarray screening and qRT-PCR evaluation of microRNA markers for forensic body fluid identification.

MicroRNAs (miRNA) are a class of small (∼22 nucleotides) noncoding RNAs that regulate diverse biological processes at the post-transcriptional level. MiRNAs have great potential for forensic body fluid identification because they are expressed in a tissue specific manner and are less prone to degradation. Previous studies reported several miRNAs as body fluid specific, but there are few overlaps among them. Here, we used a genome-wide miRNA microarray containing over 1700 miRNAs to assay 20 body fluid samples and identify novel miRNAs useful for forensic body fluid identification. Based on Shannon Entropy and Q-statistics, 203 miRNAs specifically expressed in each body fluid were first selected. Eight miRNAs were then selected as novel forensically relevant miRNA markers: miR-484 and miR-182 for blood, miR-223 and miR-145 for saliva, miR-2392 and miR-3197 for semen, and miR-1260b and miR-654-5p for vaginal secretions. When the eight selected miRNAs were evaluated in 40 additional body fluid samples by qRT-PCR, they showed high sensitivity and specificity for the identification of the target body fluid. We suggest that the eight miRNAs may be candidates for developing an effective molecular assay for forensic body fluid identification.

High-throughput STR analysis for DNA database using direct PCR.

Since the Korean criminal DNA database was launched in 2010, we have focused on establishing an automated DNA database profiling system that analyzes short tandem repeat loci in a high-throughput and cost-effective manner. We established a DNA database profiling system without DNA purification using a direct PCR buffer system. The quality of direct PCR procedures was compared with that of conventional PCR system under their respective optimized conditions. The results revealed not only perfect concordance but also an excellent PCR success rate, good electropherogram quality, and an optimal intra/inter-loci peak height ratio. In particular, the proportion of DNA extraction required due to direct PCR failure could be minimized to <3%. In conclusion, the newly developed direct PCR system can be adopted for automated DNA database profiling systems to replace or supplement conventional PCR system in a time- and cost-saving manner.

Genome-wide mRNA profiling and multiplex quantitative RT-PCR for forensic body fluid identification.

In forensic science, identifying a tissue where a forensic specimen was originated is one of the principal challenges. Messenger RNA (mRNA) profile clearly reveals tissue-specific gene expression patterns that many attempts have been made to use RNA for forensic tissue identification. To systematically investigate the body-fluid-specific expression of mRNAs and find novel mRNA markers for forensic body fluid identification, we performed DNA microarray experiment with 24 Korean body fluid samples. Shannon entropy and Q-values were calculated for each gene, and 137 body-fluid-specific candidate genes were selected. By applying more stringent criteria, we further selected 28 candidate genes and validated them by RT-PCR and qRT-PCR. As a result, we suggest a novel combination of four body-fluid-specific mRNA makers: PPBP for blood, FDCSP for saliva, MSMB for semen and MSLN for vaginal secretion. Multiplex qRT-PCR assay was designed using the four mRNA markers and DNA/RNA co-extraction method was tested for forensic use. This study will provide a thorough examination of body-fluid-specifically expressed mRNAs, which will enlarge the possibility of practical use of RNA for forensic purpose.

Forensic Body Fluid Identification by Analysis of Multiple RNA Markers Using NanoString Technology.

RNA analysis has become a reliable method of body fluid identification for forensic use. Previously, we developed a combination of four multiplex quantitative PCR (qRT-PCR) probes to discriminate four different body fluids (blood, semen, saliva, and vaginal secretion). While those makers successfully identified most body fluid samples, there were some cases of false positive and negative identification. To improve the accuracy of the identification further, we tried to use multiple markers per body fluid and adopted the NanoString nCounter system instead of a multiplex qRT-PCR system. After measuring tens of RNA markers, we evaluated the accuracy of each marker for body fluid identification. For body fluids, such as blood and semen, each body fluid-specific marker was accurate enough for perfect identification. However, for saliva and vaginal secretion, no single marker was perfect. Thus, we designed a logistic regression model with multiple markers for saliva and vaginal secretion and achieved almost perfect identification. In conclusion, the NanoString nCounter is an efficient platform for measuring multiple RNA markers per body fluid and will be useful for forensic RNA analysis.

Quantitative analysis of cell-free DNA in the plasma of gastric cancer patients.

In the present study, an accurate and reproducible method for quantifying cell-free DNA (cfDNA) in human blood was established and tested for its ability to predict gastric cancer in patients. Using 'Alu81-qPCR' to amplify 81-bp Alu DNA sequences, we first estimated the amount of cfDNA in the serum or plasma of 130 patients with gastric cancer to identify which source of cfDNA is more suitable for the biomarker screening of these patients. The results of Alu81-qPCR revealed that the amount of cfDNA in the plasma was low compared with that in the serum, but was found at similar levels among the samples, indicating that the plasma may be a more suitable source of cfDNA for biomarker screening. For the 54 patients with gastric cancer and the 59 age-matched healthy controls, the mean levels of plasma cfDNA were 2.4-fold higher in the patient group compared with the control group, indicating that plasma cfDNA levels may be useful for predicting patients with gastric cancer. The results of our study suggest that Alu81-qPCR is a more reliable method than other techniques, such as the PicoGreen assay, for quantifying cfDNA in human blood, demonstrating the potential to complement current diagnostic procedures for the management of gastric cancer patients.

Serum cancer biomarker discovery through analysis of gene expression data sets across multiple tumor and normal tissues.

The development of convenient serum bioassays for cancer screening, diagnosis, prognosis, and monitoring of treatment is one of top priorities in cancer research community. Although numerous biomarker candidates have been generated by applying high-throughput technologies such as transcriptomics, proteomics, and metabolomics, few of them have been successfully validated in the clinic. Better strategies to mine omics data for successful biomarker discovery are needed. Using a data set of 22,794 tumor and normal samples across 23 tissues, we systematically analyzed current problems and challenges of serum biomarker discovery from gene expression data. We first performed tissue specificity analysis to identify genes that are both tissue-specific and up-regulated in tumors compared to controls, but identified few novel candidates. Then, we designed a novel computation method, the multiple normal tissues corrected differential analysis (MNTDA), to identify genes that are expected to be significantly up-regulated even after their expressions in other normal tissues are considered, and, in a simulation study, showed that the multiple normal tissues corrected differential analysis outperformed the single tissue differential analysis combined with tissue specificity analysis. By applying the multiple normal tissues corrected differential analysis, we identified some genes as novel biomarker candidates. However, the number of potential candidates was disappointingly small, exemplifying the difficulty of finding serum cancer biomarkers. We discussed a few important points that should be considered during biomarker discovery from omics data.

Epigenetic regulation of microRNA-10b and targeting of oncogenic MAPRE1 in gastric cancer.

MicroRNAs act as negative regulators of gene expression, and the altered expression of microRNAs by epigenetic mechanisms is strongly implicated in carcinogenesis. Here we report that the microRNA-10b gene (miR-10b) was silenced in gastric cancer cells by promoter methylation. In this study, using a methylation array and bisulfate pyrosequencing analysis, we found that miR-10b promoter CpGs were heavily methylated in gastric cancers. Clinicopathologic data showed that miR-10b methylation increased with patient age and occurred significantly more frequently in intestinal-type (28/44, 64%) than in diffuse-type (22/56, 39%) gastric cancers (P = 0.016). In addition, miR-10b methylation was also associated with an increase in expression of the oncogene that encodes microtubule-associated protein, RP/EB family, member 1 (MAPRE1; P = 0.004), which was identified as a potential miR-10b target. After 5-aza-2'-deoxycytidine treatment of gastric cancer cells, miR-10b methylation was significantly decreased, and expression of miR-10b and HOXD4, which is 1 kb downstream of miR-10b, was greatly restored. Moreover, decreased MAPRE1 expression coincided with increased miR-10b expression, suggesting that miR-10b targets MAPRE1 transcription. We also found that transfection with precursor miR-10b into gastric cancer cells dramatically decreased MAPRE1 mRNA and protein, resulting in a significant decrease in colony formation and cell growth rates. Thus, we show a tumor-suppressive role for miR-10b in gastric carcinogenesis. miR-10b methylation may be a useful molecular biomarker for assessing the risk of gastric cancer development, and modulation of miR-10b may represent a therapeutic approach for treating gastric cancer.

Identification of DNA methylation markers for lineage commitment of in vitro hepatogenesis.

Hepatocytes that have differentiated from human embryonic stem cells (hESCs) have great potential for the treatment of liver disease as well as for drug testing. Moreover, in vitro hepatogenesis is a powerful model system for studying the molecular mechanisms underlying liver development. DNA methylation is an important epigenetic mechanism that influences differential gene expression during embryonic development. We profiled gene expression and DNA methylation of three cell states of in vitro hepatogenesis-hESC, definitive endoderm and hepatocyte-using microarray analysis. Among 525 state-specific expressed genes, 67 showed significant negative correlation between gene expression and DNA methylation. State-specific expression and methylation of target genes were validated by quantitative reverse transcription-polymerase chain reaction and pyrosequencing, respectively. To elucidate genome-scale methylation changes beyond the promoter, we also performed high-throughput sequencing of methylated DNA captured by the MBD2 protein. We found dynamic methylation changes in intergenic regions of the human genome during differentiation. This study provides valuable methylation markers for the lineage commitment of in vitro hepatogenesis and should help elucidate the molecular mechanisms underlying stem cell differentiation and liver development.

Factors influencing the detection of the BRAF T1799A mutation in papillary thyroid carcinoma.

The BRAF T1799A mutation is a heterozygous point mutation and its reported prevalence in papillary thyroid carcinoma (PTC) has varied from 29 to 83%, with an overall mean of 44%. In Korea, the reported mutation rate reached 83% in PTC and 52% in micropapillary carcinoma. We hypothesized that the differences in prevalence may be influenced by the methods of mutation analysis, the sizes of tumor and ethnic differences. Three types of DNA samples from the same PTC mass (0.4-1.6 cm, mean 0.83 cm sized) per each patient (n=17) were isolated. The first type was obtained from frozen PTC tissues using laser-captured microdissection (Frozen-laser, n=17), the second was obtained from frozen tissue by manual tumor margin dissection using a blade (Frozen-blade, n=17) and the third was obtained from formalin-fixed, paraffin-embedded tissue by manual margin dissection (Paraffin-blade, n=15, 2 failed). The mutation rates of the three-matched DNA samples were compared by the SNP mode and AQ mode of pyrosequencing, and direct DNA sequencing. Both the AQ mode of pyrosequencing and the direct DNA sequencing detected the BRAF T1799A mutation in 100% of the 'Frozen-laser' samples, but the mutation was omitted in 1/17 of the 'Frozen-blade' samples and in 5/15 of the 'Paraffin-blade' samples, while the former was more rapid and objective than the latter. The SNP mode of pyrosequencing variably detected the mutation from 40 to 100%, and it showed the lowest sensitivity. Our results indicate that the reported prevalence of the BRAF T1799A mutation in PTC can be underestimated due the mutation analysis methods, and especially in small PTCs. The BRAF T1799A mutation may be an early and essential carcinogenic event in nearly all Korean PTCs, and even in micro-PTCs. For the accurate detection of the BRAF T1799A mutation from small PTCs, fresh or frozen tissues and more cautious microdissection are required, and the AQ mode of pyrosequencing assay is preferred.

Aberrant up-regulation of LAMB3 and LAMC2 by promoter demethylation in gastric cancer.

The LAMB3 and LAMC2 genes encode the laminin-5 ß3 and γ2 chains, respectively, which are parts of laminin-5, one of the major components of the basement membrane zone. Here, we report the frequent up-regulation of LAMB3 and LAMC2 by promoter demethylation in gastric cancer. Gene expression data analysis showed that LAMB3 and LAMC2 were up-regulated in various tumor tissues. Combined analyses of DNA methylation and gene expression of both genes in gastric cancer cell lines and tissues showed that DNA hypomethylation was associated with the up-regulation of both genes. Treatment with a methylation inhibitor induced LAMB3 and LAMC2 expression in gastric cancer cell lines in which both genes were silenced. By chromatin immunoprecipitation assay, we showed the activation histone mark H3K4me3 was associated with the expression of both genes. The expression level of LAMB3 affected multiple malignant phenotypes in gastric cancer cell lines. These results suggest that epigenetic activation of LAMB3 and LAMC2 may play an important role in gastric carcinogenesis.

Human ZNF312b oncogene is regulated by Sp1 binding to its promoter region through DNA demethylation and histone acetylation in gastric cancer.

In a previous study, human ZNF312b was identified as a cell proliferation-associated oncogene via the K-ras/extracellular signal-regulated kinase cascade in gastric cancer. However, the mechanism concerning its transcriptional activation remains unknown. Here, we show that DNA methylation and histone acetylation of the ZNF312b promoter function as a switch for ZNF312b transcriptional activation in gastric cancer. The transcription level of ZNF312b was increased by treatment with a demethylating agent, 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor sodium butyrate, in several human cancer cell lines including gastric cancer. Consistent with these results, epigenetic analysis, such as pyrosequencing, bisulfate sequencing and methyl-specific polymerase chain reaction (MSP), showed that the expression level of ZNF312b is highly dependent on the degree of DNA methylation in gastric cancer cell lines. In addition, by ChIP assay using anti-acetyl/methyl H3K9 antibodies, histone acetylation was shown to mediate the expression of the ZNF312b gene. Interestingly, ChIP assay using the Sp1 antibody revealed that the binding of transcription factor Sp1 to the ZNF312b promoter for its transcriptional activation requires DNA demethylation and histone acetylation. Moreover, a knockdown of Sp1 resulted in a decrease in ERK-mediated proliferation via downregulation of the ZNF312b gene in gastric cancer cells. Taken together, these results suggest that the aberrant expression of ZNF312b promotes gastric tumorigenesis through epigenetic modification of its promoter region and provides a molecular mechanism for ZNF312b expression to contribute to the progression of gastric cancer.

Epigenetic modification of retinoic acid-treated human embryonic stem cells.

Epigenetic modification of the genome through DNA methylation is the key to maintaining the differentiated state of human embryonic stem cells (hESCs), and it must be reset during differentiation by retinoic acid (RA) treatment. A genome-wide methylation/gene expression assay was performed in order to identify epigenetic modifications of RA-treated hESCs. Between undifferentiated and RA-treated hESCs, 166 differentially methylated CpG sites and 2,013 differentially expressed genes were discovered. Combined analysis of methylation and expression data revealed that 19 genes (STAP2, VAMP8, C10orf26, WFIKKN1, ELF3, C1QTNF6, C10orf10, MRGPRF, ARSE, LSAMP, CENTD3, LDB2, POU5F1, GSPT2, THY1, ZNF574, MSX1, SCMH1, and RARB) were highly correlated with each other. The results provided in this study will facilitate future investigations into the interplay between DNA methylation and gene expression through further functional and biological studies.

Genome-wide association of serum bilirubin levels in Korean population.

A large-scale, genome-wide association study was performed to identify genetic variations influencing serum bilirubin levels using 8841 Korean individuals. Significant associations were observed at UGT1A1 (rs11891311, P = 4.78 x 10(-148)) and SLCO1B3 (rs2417940, P = 1.03 x 10(-17)), which are two previously identified loci. The two single-nucleotide polymorphisms (SNPs) were replicated (rs11891311, P = 3.18 x 10(-15)) or marginally significant (rs2417940, P = 8.56 x 10(-4)) in an independent cohort of 1096 individuals. In a conditional analysis adjusted for the top UGT1A1 variant (rs11891311), another variant in UGT1A1 (rs4148323, P = 1.22 x 10(-121)) remained significant; this suggests that in UGT1A1 at least two independent genetic variations influence the bilirubin levels in the Korean population. The protein coding variant rs4148323, which is monomorphic in European-derived populations, may be specifically associated with serum bilirubin levels in Asians (P = 2.56 x 10(-70)). The SLCO1B3 variant (rs2417940, P = 1.67 x 10(-18)) remained significant in a conditional analysis for the top UGT1A1 variant. Interestingly, there were significant differences in the associated variations of SLCO1B3 between Koreans and European-derived populations. While the variant rs2417940 at intron 7 of SLCO1B3 was more significantly associated in Koreans, variants rs17680137 (P = 0.584) and rs2117032 (P = 2.76 x 10(-5)), two of the top-ranked SNPs in European-derived populations, did not reach the genome-wide significance level. Also, variants in SLCO1B1 did not reach genome-wide significance in Koreans. Our result supports the idea that there are considerable ethnic differences in genetic association of bilirubin levels between Koreans and European-derived populations.

Preliminary analysis of the G178A polymorphism of insulin-like factor 3 in male infertility.

We investigated the association of nonsynonymous insulin-like factor 3 (INSL3) G178A polymorphism with nonobstructive male infertility. The common INSL3 G178A polymorphism was not statistically significantly associated with male infertility (P>.05), even though INSL3 is essential for the occurrence of bilateral cryptorchidism.

Association study of four polymorphisms in three folate-related enzyme genes with non-obstructive male infertility.

BACKGROUND: Three typical folate metabolism enzymes-i.e. methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MS) and MS reductase (MTRR) in the folate cycle-play a critical role in DNA synthesis and methylation reactions. We evaluated whether polymorphisms of these three enzymes are associated with non-obstructive male infertility. METHOD: Three hundred and sixty patients with non-obstructive infertility and 325 fertile men without any chromosomal abnormalities were included in this study. The single-nucleotide polymorphism (SNP) analysis was performed by pyrosequencing and PCR-restriction fragment length polymorphism (RFLP) analysis RESULTS: The frequencies of MTHFR 677TT and MTRR 66GG genotypes were higher in non-obstructive infertile men compared with those in fertile men. By classifying 360 infertile patients into 174 azoospermia and 186 oligoasthenoteratozoospermia (OAT) subjects, the MTHFR 677TT and MS 2756GG types were significantly associated with the azoospermia group (P = 0.0227 and 0.0063, respectively). The frequency of MTRR 66GG was significant in the OAT group (P = 0.0014 versus fertile males). CONCLUSIONS: By analysis of a large number of subjects and a more specific patient selection, we showed the first genetic evidence that MTHFR C677T, MS A2756G and MTRR A66G genotypes were independently associated with male infertility. Each SNP of the three enzymes may have a different impact on the folate cycle during spermatogenesis.