TPX2 Amplification-Driven Aberrant Mitosis in Culture Adapted Human Embryonic Stem Cells with gain of 20q11.21.

BACKGROUND: Despite highly effective machinery for the maintenance of genome integrity in human embryonic stem cells (hESCs), the frequency of genetic aberrations during in-vitro culture has been a serious issue for future clinical applications. METHOD: By passaging hESCs over a broad range of timepoints (up to 6 years), the isogenic hESC lines with different passage numbers with distinct cellular characteristics, were established. RESULT: We found that mitotic aberrations, such as the delay of mitosis, multipolar centrosomes, and chromosome mis-segregation, were increased in parallel with polyploidy compared to early-passaged hESCs (EP-hESCs) with normal copy number. Through high-resolution genome-wide approaches and transcriptome analysis, we found that culture adapted-hESCs with a minimal amplicon in chromosome 20q11.21 highly expressed TPX2, a key protein for governing spindle assembly and cancer malignancy. Consistent with these findings, the inducible expression of TPX2 in EP-hESCs reproduced aberrant mitotic events, such as the delay of mitotic progression, spindle stabilization, misaligned chromosomes, and polyploidy. CONCLUSION: These studies suggest that the increased transcription of TPX2 in culture adapted hESCs could contribute to an increase in aberrant mitosis due to altered spindle dynamics.

A non-synonymous variant rs12614 of complement factor B associated with risk of chronic hepatitis B in a Korean population.

BACKGROUND: Hepatitis B is known to cause several forms of liver diseases including chronic hepatitis B (CHB), and hepatocellular carcinoma. Previous genome-wide association study of CHB risk has demonstrated that rs12614 of complement factor B (CFB) was significantly associated with CHB risk. In this study, fine-mapping study of previously reported GWAS single nucleotide polymorphism (SNP; CFB rs12614) was performed to validate genetic effect of rs12614 on CHB susceptibility and identify possible additional causal variants around rs12614 in a Korean population. This association study was conducted in order to identify genetic effects of CFB single nucleotide polymorphisms (SNPs) and to identify additional independent CHB susceptible causal markers within a Korean population. METHODS: A total of 10 CFB genetic polymorphisms were selected and genotyped in 1716 study subjects comprised of 955 CHB patients and 761 population controls. RESULTS: A non-synonymous variant, rs12614 (Arg32Trp) in exon2 of CFB, had significant associations with risk of CHB (odds ratio = 0.43, P = 5.91 × 10- 10). Additional linkage disequilibrium and conditional analysis confirmed that rs12614 had independent genetic effect on CHB susceptibility with previously identified CHB markers. The genetic risk scores (GRSs) were calculated and the CHB patients had higher GRSs than the population controls. Moreover, OR was found to increase significantly with cumulative GRS. CONCLUSIONS: rs12614 showed significant genetic effect on CHB risk within the Korean population. As such rs12614 may be used as a possible causal genetic variant for CHB susceptibility.

Associations of BRAP polymorphisms with the risk of alcohol dependence and scores on the Alcohol Use Disorders Identification Test.

BACKGROUND: Alcohol dependence (AD) is a common disorder that is influenced by genetic as well as environmental factors. A previous genome-wide association study (GWAS) of the Korean population performed by our research group identified a number of genes, including BRCA1-associated protein (BRAP) and protein arginine methyltransferase 8 (PRMT8), as novel genetic markers of AD. METHODS: The present investigation was a fine-mapping follow-up study of 459 AD and 455 non-AD subjects of Korean descent to determine the associations between BRAP and PRMT8 polymorphisms and AD. The Alcohol Use Disorders Identification Test (AUDIT) was administered to screen for the degree of AD risk in the subjects and 58 genetic variants, 5 for BRAP and 53 for PRMT8, were genotyped for subsequent association analyses. RESULTS: In the present case-control analysis, BRAP rs3782886 showed the most significant association signal with a risk of AD (P=1.29×10-16, Pcorr =7.74×10-16, OR =0.19). There were also significant differences in the overall and subcategory scores for the BRAP genetic variants, including rs3782886 (P=9.94×10-31, Pcorr =5.96×10-30 at rs3782886 for the overall AUDIT score). However, the genetic effects of PRMT8 polymorphisms observed in our previous GWAS were not replicated in the present study (minimum P=0.0005, Pcorr >0.05, OR =0.30 at rs4766139 in the recessive model). Furthermore, the single-nucleotide polymorphisms of PRMT8 were not associated with the overall and subcategory AUDIT scores. CONCLUSION: The present findings suggest that the genetic variants of BRAP may contribute to a predisposition for an alcohol use disorder.

Association analysis of NOX5 polymorphisms with Hirschsprung disease.

BACKGROUND/PURPOSE: Hirschsprung disease (HSCR) is a developmental disease characterized by the absence of ganglion cells in the intestinal region. NADPH oxidase5 (NOX5) has been identified as one of the possible candidate genes for risk of Hirschsprung disease in our recent genome wide association study (GWAS). In this study, we performed a replication study to analyze the association of NOX5 polymorphisms with HSCR risk and conducted an extended analysis to investigate further associations for sub-groups and haplotypes. METHODS: A total of 23 NOX5 single nucleotide polymorphisms (SNPs) were genotyped in 187 HSCR patients and 283 unaffected controls. Statistical analysis was performed to examine the effects of genotype on risk of HSCR and HSCR subtype. RESULTS: Logistic regression analyses revealed that six SNPs (rs59355559, rs62010828, rs34990910, rs11856030, rs311905, and rs8024894) were associated with risk of HSCR (minimum p = 0.007 at rs62010828). Moreover, three SNPs (rs59355559, rs62010828, and rs8024894) were significantly associated with risk of long-segment HSCR (L-HSCR) subtype and 5 SNPs (rs59355559, rs62010828, rs34990910, rs11856030, and rs8024894) were found to be associated with risk of TCA subtype. CONCLUSION: Our results demonstrate that genetic variants in NOX5 have genetic effects on risk of HSCR, which may serve as useful preliminary information for further study. LEVELS OF EVIDENCE: Level III of prognosis study.

Identification of additional EHMT2 variant associated with the risk of chronic hepatitis B by GWAS follow-up study.

Chronic hepatitis B (CHB) is a precursor to liver cirrhosis and hepatocellular carcinoma, caused by a Hepatitis B viral infection. Genome-wide association studies (GWASs) have been conducted to find genes associated with CHB risk. In previous GWAS, EHMT2 was identified as one of the susceptibility genes for CHB. To further characterize this association and discover possible causal variants, we conducted an additional association study. A total of 11 EHMT2 single-nucleotide polymorphisms (SNP) were selected and genotyped in 3902 subjects (1046 CHB patients and 2856 controls). An additional eight imputed SNPs were also included in further analysis. As a result, rs35875104 showed a strong association with the CHB, along with the previously reported genetic marker for CHB risk, rs652888 (odds ratio (OR) = 0.53, P = 2.20 × 10-8 at rs35875104 and OR = 1.58, P = 9.90 × 10-12 at rs652888). In addition, linkage disequilibrium and conditional analysis identified one SNP (rs35875104) as a novel genetic marker for CHB susceptibility. The GRSs (genetic risk scores) were calculated to visualize the combined genetic effects of all known CHB-associated loci, including EHMT2 rs35875104, which was additionally identified in this study. The findings from the present study may be useful for further understanding of the genetic etiology of CHB.

Selective Elimination of Culture-Adapted Human Embryonic Stem Cells with BH3 Mimetics.

The selective survival advantage of culture-adapted human embryonic stem cells (hESCs) is a serious safety concern for their clinical application. With a set of hESCs with various passage numbers, we observed that a subpopulation of hESCs at late passage numbers was highly resistant to various cell death stimuli, such as YM155, a survivin inhibitor. Transcriptome analysis from YM155-sensitive (YM155S) and YM155-resistant (YM155R) hESCs demonstrated that BCL2L1 was highly expressed in YM155R hESCs. By matching the gene signature of YM155R hESCs with the Cancer Therapeutics Response Portal dataset, BH3 mimetics were predicted to selectively ablate these cells. Indeed, short-course treatment with a sub-optimal dose of BH3 mimetics induced the spontaneous death of YM155R, but not YM155S hESCs by disrupting the mitochondrial membrane potential. YM155S hESCs remained pluripotent following BH3 mimetics treatment. Therefore, the use of BH3 mimetics is a promising strategy to specifically eliminate hESCs with a selective survival advantage.

Genetic association of complement component 2 variants with chronic hepatitis B in a Korean population.

BACKGROUND & AIMS: Numerous single nucleotide polymorphisms associated with an increased risk of liver diseases, chronic hepatitis B and chronic hepatitis B-related hepatocellular carcinoma have been identified. In this study, we scrutinized the genetic effects of C2 variants, which were conflicting in previous results, on the risk of chronic hepatitis B in a Korean population. METHODS: We genotyped 22 common C2 genetic variants of 977 chronic hepatitis B cases including 302 chronic hepatitis B-related hepatocellular carcinoma cases and 785 population controls. Statistical analysis was performed to examine the effects of genotype on the risk of chronic hepatitis B and chronic hepatitis B-related hepatocellular carcinoma. RESULTS: Logistic regression analyses showed that six C2 single nucleotide polymorphisms had significant associations with the risk of chronic hepatitis B and chronic hepatitis B-related hepatocellular carcinoma among the Korean subjects. Stepwise analysis revealed that causal markers (rs9267665 and rs10947223) were identified among the C2 variants (stepwise P = 3.32 × 10-9 and 2.04 × 10-5 respectively). In further conditional analysis with previous chronic hepatitis B-associated loci, these two single nucleotide polymorphisms were independently associated with the risk of chronic hepatitis B. In addition, we investigated the ability of genetic risk scores combining 12 multi-chronic hepatitis B loci to predict the risk of chronic hepatitis B. Individuals with higher genetic risk scores showed increased risk for chronic hepatitis B. CONCLUSIONS: Our results suggested that the C2 gene might be a susceptibility locus for chronic hepatitis B in Korean populations. The cumulative genetic effects may contribute to future etiological explanations for chronic hepatitis B.

PRMT8 Controls the Pluripotency and Mesodermal Fate of Human Embryonic Stem Cells By Enhancing the PI3K/AKT/SOX2 Axis.

Basic fibroblast growth factor (bFGF) supplementation is critical to maintain the pluripotency of human pluripotent stem cells (hPSCs) through activation of PI3K/AKT, rather than MEK/ERK pathway. Thus, elaborate molecular mechanisms that preserve PI3K/AKT signaling upon bFGF stimulation may exist in hPSCs. Protein arginine methyltransferase 8 (PRMT8) was expressed and then its level gradually decreased during spontaneous differentiation of human embryonic stem cells (hESCs). PRMT8 loss- or gain-of-function studies demonstrated that PRMT8 contributed to longer maintenance of hESC pluripotency, even under bFGF-deprived conditions. Direct interaction of membrane-localized PRMT8 with p85, a regulatory subunit of PI3K, was associated with accumulation of phosphoinositol 3-phosphate and consequently high AKT activity. Furthermore, the SOX2 induction, which was controlled by the PRMT8/PI3K/AKT axis, was linked to mesodermal lineage differentiation. Thus, we propose that PRMT8 in hESCs plays an important role not only in maintaining pluripotency but also in controlling mesodermal differentiation through bFGF signaling toward the PI3K/AKT/SOX2 axis. Stem Cells 2017;35:2037-2049.

Identification of novel OCT4 genetic variant associated with the risk of chronic hepatitis B in a Korean population.

BACKGROUND & AIMS: Hepatitis B viral infection is a serious risk factor for chronic hepatitis B (CHB), cirrhosis and hepatocellular carcinoma. Recently, several genome-wide association studies (GWASs) have been conducted to identify important genetic variant associated with the risk of CHB. In our previous GWAS, TCF19 was identified as one of the susceptibility genes for CHB risk (P=4.2×10-9 at rs1419881). In order to discover possible additional causal variants around TCF19, we performed an association study by genotyping single nucleotide polymorphisms (SNPs) in OCT4, a nearby gene to TCF19. METHODS: Nineteen OCT4 genetic variants were selected and genotyped in 3902 subjects (1046 CHB patients and 2856 population controls). RESULTS: Logistic regression analysis revealed that OCT4 rs1265163 showed the most significant association signal for the risk of CHB (OR=1.46, P=4.78×10-12 ). Linkage disequilibrium and conditional analysis confirmed rs1265163 in OCT4 as a novel genetic marker for CHB susceptibility. The genetic risk scores (GRSs) were calculated to visualize the combined genetic effects of all known CHB-associated loci, including OCT4 rs1265163, which had been identified in this study. Individuals with higher cumulative GRSs showed significantly increased ORs. The luciferase activity of rs885952, a tagging SNP of rs1265163, showed that OCT4 promoter activity was significantly different between the wild-type and SNP mutant form (P<.05). CONCLUSIONS: This follow-up study to our previous GWAS identified a possible causal genetic variant associated with the risk of CHB, and findings from this study may prove useful in the understanding of genetic susceptibility to CHB.

Determination of DPYD enzyme activity in Korean population.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPYD) is an enzyme that regulates the rate-limiting step in pyrimidine metabolism, especially catabolism of fluorouracil. This study was performed to analyze the association between DPYD genetic variants and DPYD enzyme activity in the Korean population. METHODS: We screened the genetic variants and analyzed the enzyme activity in 73 healthy Korean subjects (69 men and 4 women; mean age, 22.6 years). Direct sequencing was conducted using the ABI 3730XL system, and enzyme activity was determined using high-performance liquid chromatography. RESULTS: A total of 83 genetic variants were observed. Among the identified genetic variants, 32 were polymorphic including 3 core and 11 novel genetic variants. Association analysis between each genetic variant and enzyme activity in Korean subjects showed that 2 novel genetic variants, -832 G>A and -131 C>A, induced a significant difference in enzyme activity (P < 0.05). CONCLUSIONS: To our knowledge, this is the first study that has examined the association between enzyme activity and DPYD genetic variants in the Korean population. In this study, we identified novel genetic variants that are associated with the enzyme activity. These findings will be valuable for further pharmacogenetic studies and especially useful for personalized medicine.

Screening of dihydropyrimidine dehydrogenase genetic variants by direct sequencing in different ethnic groups.

Dihydropyrimidine dehydrogenase (DPYD) is an enzyme that regulates the rate-limiting step in pyrimidine metabolism, especially catabolism of fluorouracil, a chemotherapeutic agent for cancer. In order to determine the genetic distribution of DPYD, we directly sequenced 288 subjects from five ethnic groups (96 Koreans, 48 Japanese, 48 Han Chinese, 48 African Americans, and 48 European Americans). As a result, 56 polymorphisms were observed, including 6 core polymorphisms and 18 novel polymorphisms. Allele frequencies were nearly the same across the Asian populations, Korean, Han Chinese and Japanese, whereas several SNPs showed different genetic distributions between Asians and other ethnic populations (African American and European American). Additional in silico analysis was performed to predict the function of novel SNPs. One nonsynonymous SNP (+199381A > G, Asn151Asp) was predicted to change its polarity of amino acid (Asn, neutral to Asp, negative). These findings would be valuable for further research, including pharmacogenetic and drug responses studies.

Genetic association analysis of ERBB4 polymorphisms with the risk of schizophrenia and SPEM abnormality in a Korean population.

The human receptor tyrosine-protein kinase erbB-4 (ERBB4) gene mediates neuregulin 1 (NRG1) signaling, and is involved in neuronal migration and differentiation. Despite the potential significance of ERBB4 in the development of schizophrenia, relatively few genetic studies for the association of ERBB4 with schizophrenia were performed in the populations including Ashkenazi Jews, Americans including Caucasians and African Americans, and Han Chinese. In this study, differences in ERBB4 variations were investigated to determine association with schizophrenia and smooth pursuit eye movement (SPEM) abnormality in a Korean population. Seven polymorphisms in ERBB4 gene were genotyped in 435 schizophrenia cases and 390 unrelated healthy controls. In order to investigate the relationship between ERBB4 and the risk of schizophrenia and SPEM abnormality, differences in SNP and haplotype distribution were analyzed using logistic and multiple regression analyses. However, we failed to replicate the associations reported by previous studies in other populations. Although statistically not significant, the tendency towards associations between ERBB4 polymorphisms and the risk of schizophrenia and SPEM abnormality in this study from a Korean population would be helpful for further genetic etiology studies in schizophrenia.