Mutational analysis of 23 autosomal short tandem repeats based on trio paternity testing in the Korean population.

This study aimed to estimate A-STR mutation rates in 2,317 Korean parent-child trios by examining 20 Combined DNA Index System (CODIS) core loci (D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, vWA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, and D22S1045) and three non-CODIS loci (Penta E, Penta D, and SE33). Locus-specific mutation rate estimates varied from 0.00 to 8.63 × 10-3 per generation, with an average mutation rate of 1.62 × 10-3 (95 % CI, 1.39-1.88 × 10-3). We also combined data from previous studies to obtain comprehensive genetic values for the Korean population, and the average mutation rate was 1.59 × 10-3 (95 % CI, 1.38-1.82 × 10-3). Single-step mutations (95.69 %) and double-step mutations (3.35 %) were observed in the mutation pattern analysis, and cases expected to have multi-step mutations (0.96 %) were also observed. Large-sized alleles exhibited more loss mutations than gain mutations, and paternal mutations (62.68 %) were more frequently observed than maternal mutations (19.62 %). The calculated values and features of the 23 A-STRs explored in this study are expected to play a crucial role in establishing criteria for forensic genetic interpretation.

Analysis of mutation rates and haplotypes of 23 Y-chromosomal STRs in Korean father-son pairs.

Y-chromosomal short tandem repeats (Y-STRs) have been widely used in forensic genetics, and accurate knowledge of mutation rates at Y-STR loci is essential in kinship analysis. The main aim of this study was to estimate Y-STR mutation rates in Korean males. To obtain locus-specific mutations and haplotypes at 23 Y-STRs, we analyzed samples from 620 Korean father-son pairs. In addition, we also analyzed 476 unrelated individuals using the PowerPlex® Y23 System, with the aim of augmenting the available data for the Korean population. The PowerPlex® Y23 system facilitates analysis of the 23 Y-STR loci (DYS576, DYS570, DYS458, DYS635, DYS389 II, DYS549, DYS385, DYS481, DYS439, DYS456, DYS389 I, DYS19, DYS393, DYS391, DYS533, DYS437, DYS390, Y GATA H4, DYS448, DYS438, DYS392, and DYS643). Locus-specific mutation rate estimates varied from 0.00 to 8.06 × 10-3 per generation, with an average mutation rate of 2.17 × 10-3 (95% CI, 1.5-3.1 × 10-3). To obtain comprehensive genetic values for the Korean population, we combined data obtained in this study with previously reported values, thereby enabling us to estimate the locus-specific mutation rates regarding 22,711 allele transmissions. By combining these data, we obtained an overall average mutation rate of 2.91 × 10-3 (95% CI, 2.3-3.7 × 10-3). In addition, among the 476 unrelated Korean males, we detected 467 different haplotypes, with an overall haplotype diversity value of 0.9999. By extracting haplotypes of Y-STRs described in previous literature on 23 Y-STR reported in Korea, we obtained gene diversities for 1133 Korean individuals. We believe that the values and characteristics of the 23 Y-STRs analyzed in this study will contribute to establishing criteria for forensic genetic interpretation, including kinship analysis.

Identification of novel SNP markers for kinship analysis in the Korean population.

Kinship testing using genetic markers such as short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) is crucial for forensic analysis. Although STR markers have superior discriminatory power due to their highly polymorphic properties, they have several weak points in determining extended distant or complex relationships because of high mutation rates and low success rates in degraded samples. Therefore, SNPs are regarded as promising tools in forensic science because they have low mutation rates and small amplicon sizes. Herein, we propose an SNP panel consisting of 1400 autosomal SNPs obtained from the Korean National Standard Reference Variome (KoVariome) database. To evaluate its performance, in-silico analysis was performed using whole-genome sequencing (WGS) data from 21 Korean families. Subsequently, to estimate pairwise relatedness, kinship coefficients were calculated using PLINK, and Welch's one-way ANOVA test with Games-Howell's pairwise comparison test was performed. As a result, the average kinship coefficients of first- (parent-offspring and full siblings), second- (grandparent-grandchildren and aunt/uncle-niece/nephew), and third- (first cousin and grandniece/grandnephew) degree relatives, and unrelated were 0.24, 0.11, - 0.054, and - 0.0082, respectively. Consequently, relatives (first and second degree) were distinguished from non-relatives; however, further studies are required to investigate more effective SNP markers for discriminating extended kinship. Nevertheless, the results of this study go beyond the scope of screening using the discovered 1400 SNPs in Korean families and suggest the applicability of kinship analysis in the Korean population.

Validation and assessment of the Investigator® 24plex QS kit for forensic casework application: Comparison with the PowerPlex® fusion system and GlobalFiler™ PCR amplification kits.

Casework evidence samples are likely to be placed under diverse and harsh environments as compared to quantified DNA samples including serial-diluted standard DNA samples. Internal validation of a novel STR kit using casework evidence sample, which is conducted according to various conditions such as DNA contamination and degradation, is crucial before being used as a forensic application. Therefore, this study aimed to elucidate the reliability of the Investigator® 24plex QS kit through DNA derived from casework evidence and to assess whether it is applicable to STR analysis together with PowerPlex® Fusion System and GlobalFiler™ PCR Amplification Kit. DNA was extracted from 189 casework evidence samples in a total of 77 cases. The mismatch of the allelic size of this kit through allelic sizing precision test, was suitable according to ENFSI guidelines. All heterozygous balance of the three kits were above 0.6 recommended value of ENFSI guideline. The number of allele drop-in was most frequent in the GlobalFiler™ PCR Amplification Kit. In addition, the number of allele drop-out was most frequent in the Investigator® 24plex QS kit. The cutoff concentration of DNA detected in three kits of one complete STR was approximately 45 pg/µL on average. Despite of several limitations, the Investigator® 24plex QS kit is considered to have the capability to be used for STR analysis of casework evidence samples.

Exosomal miR-193a and let-7g accelerate cancer progression on primary colorectal cancer and paired peritoneal metastatic cancer.

A metastasis of colorectal cancer is difficult to diagnose, and has a poor prognosis. Therefore, we tried to elucidate the possibility of a diagnostic and prognostic marker. Exosomal miR-193a and let-7g were sorted by miRNA microarray. The expression of miR-193a in the PTM group was lower than that of the primary CRC group, and the expression of let-7g was higher than that of the primary CRC. MMP16 and CDKN1A expression was confirmed respectively for target genes of two miRNAs. When the mimics of these miRNAs were treated with cell lines, both MMP16 and CDKN1A decreased intracellular expression. Cell invasiveness and proliferation were decreased by miR-193a and increased by let-7g. The differences in expression of exosomal miR-193a and let-7g extracted from the plasma of patients were classified as cancer progression indicators. Furthermore, the survival rate decreased in the group with low miR-193a expression and high let-7g expression. Our study confirmed the possibility of using this as a diagnostic and prognostic marker for colorectal cancer by measuring the expression levels of exosomal miR-193a and let-7g in blood.

The role of novel fusion genes in human GIST cell lines derived from imatinib-resistant GIST patients: A therapeutic potential of fusion gene.

Gastrointestinal stromal tumor (GIST) is the most common sarcoma in the gastrointestinal (GI) tract. Approximately 85% of the GIST is associated with a c-KIT mutation. A few GISTs show mutations in the gene encoding platelet-derived growth factor receptor alpha (PDGFR α or PDGFRA) without c-KIT gene mutation. GIST without c-KIT or PDGFRA mutations, which called wild type GIST, is about 5-10% of the total GIST. Fusion genes were also reported as one of the factors associated with carcinogenesis and drug resistance. With five cell lines derived from imatinib-resistant patients, novel fusion genes were identified from RNA sequencing and both physiological role and therapeutic potential were elucidated. Next-generation sequencing (NGS) analysis and lentiviral transduction were used to effect of fusion gene on GISTs. All the GIST cell lines carried c-KIT-positivity. Three different fusion gene analysis methods were used to find candidate fusion genes, including EIF3K-ACTN4, SYNCRIP-SNX14 and EXOC2-AK7. A novel interchromosomal fusion gene of the candidates, especially EXOC2-AK7, was confirmed in both tissue and cell line. The transduction of fusion gene increased the proliferation compared with the control group. Additionally, the fusion gene increased wound coverage capability. The fusion gene-transduced cell lines were more sensitive than the control group in the treatment of imatinib. In conclusion, five different imatinib-resistant GIST cell lines including the EXOC2-AK7 fusion gene derived from GIST-R5 represent important research tools for the investigation of cancer cell mechanisms underlying drug resistance and genetic variation. Furthermore, our study may facilitate pre-clinical studies of new therapeutic strategies.

SORBS1 serves a metastatic role via suppression of AHNAK in colorectal cancer cell lines.

Cbl­associated protein (CAP) is encoded by the sorbin and SH3 domain­containing 1 (SORBS1) gene. CAP has been reported to be associated with the actin cytoskeleton, receptor tyrosine kinase signaling and cell adhesion through interactions with various proteins. It may be hypothesized that SORBS1 has numerous unknown functions, which may include providing a favorable condition for metastasis. Although CAP has been demonstrated to possess a number of functions, the role of this protein has only been reported in metabolic signaling pathways and its function in cancer remains to be elucidated. In the present study, SORBS1 expression was detected in colorectal cancer cell lines divided into the primary group and the metastatic group by reverse transcription­quantitative PCR and western blot analysis. In addition, SORBS1 expression was manipulated by vector transfection and lentivirus transduction. The metastatic role of SORBS1, as determined by assessing its effects on cell proliferation and migration, was determined by colony formation assay, cell cycle analysis and Boyden chamber assay. To elucidate the SORBS1­binding protein, immunoprecipitation was performed. Co­localization of SORBS1 and AHNAK nucleoprotein (AHNAK) was identified by confocal microscopy. Notably, the protein expression levels of CAP were higher in SNU­769A and SW480 cells than in SNU­769B and SW620 cells. In addition, the number of colonies in the SORBS1­overexpressing group was significantly increased compared with that of the control group, as determined using the colony formation assay; the SORBS1 overexpression group formed >8­fold more colonies than the control group. The proliferative ability of the SORBS1 overexpression group was also significantly increased compared with the control group over the entire incubation period. Cell migration assays revealed that the number of migrated SORBS1­knockdown cells was reduced compared with the control in both HCT­116 and SNU­C4 cell lines; migration area was decreased to 31 and 26% in HCT­116 and SNU­C4 cell lines, respectively. Consequently, it was confirmed that SORBS1 could form a complex with AHNAK, which functions as a tumor suppressor through inhibition of phosphorylated­ERK and Rho­associated coiled­coil containing protein kinase 1. In conclusion, SORBS1 may serve a crucial role in cancer growth and migration via inhibition of AHNAK expression.