Cancer-specific functional profiling in microsatellite-unstable (MSI) colon and endometrial cancers using combined differentially expressed genes and biclustering analysis.

Microsatellite-unstable (MSI) cancers have distinct genetic and clinical features from microsatellite-stable cancers, but the molecular functional differences between MSI cancers originating from different tissues or organs have not been well studied because the application of usual differentially expressed gene (DEG) analysis is error-prone, producing too many noncancer-specific normally functioning genes. To maximize therapeutic efficacy, biomarkers reflecting cancer-specific differences between MSI cancers of different tissue origins should be identified. To identify functional differences between MSI colon and endometrial cancers, we combined DEG analysis and biclustering instead of DEG analysis alone and refined functionally relevant biclusters reflecting genuine functional differences between the 2 tumors. Specifically, using The Cancer Genome Atlas and genome-tissue expression as data sources, gene ontology (GO) enrichment tests were performed after routinely identifying DEGs between the 2 tumors with the exclusion of DEGs identified in their normal counterparts. Cancer-specific biclusters and associated enriched GO terms were obtained by biclustering with enrichment tests for the preferences for cancer type (either colon or endometrium) and GO enrichment tests for each cancer-specific bicluster, respectively. A novel childness score was developed to select functionally relevant biclusters among cancer-specific biclusters based on the extent to which the enriched GO terms of the biclusters tended to be child terms of the enriched GO terms in DEGs. The selected biclusters were tested using survival analysis to validate their clinical significance. We performed multiple sequential analyses to produce functionally relevant biclusters from the RNA sequencing data of MSI colon and endometrial cancer samples and their normal counterparts. We identified 3066 cancer-specific DEGs. Biclustering analysis revealed 153 biclusters and 41 cancer-specific biclusters were selected using Fisher exact test. A mean childness score over 0.6 was applied as the threshold and yielded 8 functionally relevant biclusters from cancer-specific biclusters. Functional differences appear to include gland cavitation and the TGF-ß receptor, G protein, and cytokine pathways. In the survival analysis, 6 of the 8 functionally relevant biclusters were statistically significant. By attenuating noise and applying a synergistic contribution of DEG results, we refined candidate biomarkers to complement tissue-specific features of MSI tumors.

Idiopathic hypereosinophilia is clonal disorder? Clonality identified by targeted sequencing.

Idiopathic hypereosinophilia (IHE)/idiopathic hypereosinophilic syndrome (IHES) has been defined by a persistent elevation of the blood eosinophil count exceeding 1.5×103/µL, without evidence of reactive or clonal causes. While T-cell clonality assessment has been recommended for unexplained hypereosinophilia, this approach is not often applied to routine practice in the clinic. We hypothesized that the clonality would exist in a subset of IHE/IHES patients. We aimed to investigate the candidate mutations and T-cell clonality in IHE/IHES and to explore the role of mutations in eosinophil proliferation. We performed targeted capture sequencing for 88 genes using next-generation sequencing, T-cell receptor (TCR) gene rearrangement assays, and pathway network analysis in relation to eosinophil proliferation. By targeted sequencing, 140 variants in 59 genes were identified. Sixteen out of 30 patients (53.3%) harbored at least one candidate mutation. The most frequently affected genes were NOTCH1 (26.7%), SCRIB and STAG2 (16.7%), and SH2B3 (13.3%). Network analysis revealed that our 21 candidate genes (BIRC3, BRD4, CSF3R, DNMT3A, EGR2, EZH2, FAT4, FLT3, GATA2, IKZF, JAK2, MAPK1, MPL, NF1, NOTCH1, PTEN, RB1, RUNX1, TET2, TP53 and WT1) are functionally linked to the eosinophilopoietic pathway. Among the 21 candidate genes, five genes (MAPK1, RUNX1, GATA2, NOTCH1 and TP53) with the highest number of linkages were considered major genes. A TCR assay revealed that four patients (13.3%) had a clonal TCR rearrangement. NOTCH1 was the most frequently mutated gene and was shown to be a common node for eosinophilopoiesis in our network analysis, while the possibility of hidden T cell malignancy was indwelling in the presence of NOTCH1 mutation, though not revealed by aberrant T cell study. Collectively, these results provide new evidence that mutations affecting eosinophilopoiesis underlie a subset of IHE/IHES, and the candidate genes are inferred to act their potential roles in the eosinophilopoietic pathway.

RNA-Seq for Gene Expression Profiling of Human Necrotizing Enterocolitis: a Pilot Study.

Necrotizing enterocolitis (NEC) characterized by inflammatory intestinal necrosis is a major cause of mortality and morbidity in newborns. Deep RNA sequencing (RNA-Seq) has recently emerged as a powerful technology enabling better quantification of gene expression than microarrays with a lower background signal. A total of 10 transcriptomes from 5 pairs of NEC lesions and adjacent normal tissues obtained from preterm infants with NEC were analyzed. As a result, a total of 65 genes (57 down-regulated and 8 up-regulated) revealed significantly different expression levels in the NEC lesion compared to the adjacent normal region, based on a significance at fold change ≥ 1.5 and P ≤ 0.05. The most significant gene, DPF3 (P < 0.001), has recently been reported to have differential expressions in colon segments. Our gene ontology analysis between NEC lesion and adjacent normal tissues showed that down-regulated genes were included in nervous system development with the most significance (P = 9.3 × 10⁻7; P(corr) = 0.0003). In further pathway analysis using Pathway Express based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, genes involved in thyroid cancer and axon guidance were predicted to be associated with different expression (P(corr) = 0.008 and 0.020, respectively). Although further replications using a larger sample size and functional evaluations are needed, our results suggest that altered gene expression and the genes' involved functional pathways and categories may provide insight into NEC development and aid in future research.

[Corrigendum] Promoter methylation of RASSF1A modulates the effect of the microtubule-targeting agent docetaxel in breast cancer.

Following the publication of this article, an interested reader drew to our attention that there were possible anomalies in the presentation of Fig. 5B in the above article. After having examined the figure, we recognized that several errors had indeed occurred during the process of compiling the figure. A corrected version of Fig. 5 is shown below, containing new data for Fig. 5B, after our having re-performed the western blot experiment according to the identical procedure detailed in the paper. We obtained broadly similar results to those featured originally in the article; therefore, the revision of this figure does not affect the conclusions reported in the study. We thank the reader of our article who drew this matter to our attention. [the original article was published in the International Journal of Oncology 41: 611-620, 2012; DOI: 10.3892/ijo.2012.1470].

A genome-wide association analysis of chromosomal aberrations and Hirschsprung disease.

Hirschsprung disease (HSCR) is a neurocristopathy characterized by the absence of intramural ganglion cells along variable lengths of the gastrointestinal tract. Although the RET proto-oncogene is considered to be the main risk factor for HSCR, only about 30% of the HSCR cases can be explained by variations in previously known genes including RET. Recently, copy number variation (CNV) and loss of heterozygosity (LOH) have emerged as new ways to understand human genomic variation. The goal of this present study is to identify new HSCR genetic factors related to CNV in Korean patients. In the genome-wide genotyping, using Illumina's HumanOmni1-Quad BeadChip (1,140,419 markers), of 123 HSCR patients and 432 unaffected subjects (total n = 555), a total of 8,188 CNVs (1 kb âˆ¼ 1 mb) were identified by CNVpartition. As a result, 16 CNV regions and 13 LOH regions were identified as associated with HSCR (minimum P = 0.0005). Two top CNV regions (deletions at chr6:32675155-32680480 and chr22:20733495-21607293) were successfully validated by additional real-time quantitative polymerase chain reaction analysis. In addition, 2 CNV regions (6p21.32 and 22q11.21) and 2 LOH regions (3p22.2 and 14q23.3) were discovered to be unique to the HSCR patients group. Regarding the large-scale chromosomal aberrations (>1 mb), 11 large aberrations in the HSCR patients group were identified, which suggests that they may be a risk factor for HSCR. Although further replication in a larger cohort is needed, our findings may contribute to the understanding of the etiology of HSCR.

Replication of genome wide association studies on hepatocellular carcinoma susceptibility loci of STAT4 and HLA-DQ in a Korean population.

A recent genome-wide association study (GWAS) for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) identified two loci (rs7574865 in STAT4 and rs9275319 in HLA-DQ) in a Chinese population. We attempted to replicate the associations between the two SNP loci and the risk of HCC in a Korean population. The rs7574865 in STAT4 and rs9275319 in HLA-DQ were genotyped in a total of 3838 Korean subjects composed of 287 HBV-related hepatocellular carcinoma patients, 671 chronic hepatitis B virus (CHB) patients, and 2880 population controls using TaqMan genotyping assay. Gene expression was measured by microarray. A logistic regression analysis revealed that rs7574865 in STAT4 and rs9275319 in HLA-DQ were associated with the risk of CHB (OR = 1.25, P = 0.0002 and OR = 1.57, P= 1.44 × 10(-10), respectively). However, these loci were no association with the risk of HBV-related HCC among CHB patients. In the gene expression analyses, although no significant differences in mRNA expression of nearby genes according to genotypes were detected, a significantly decreased mRNA expression in HCC subjects was observed in STAT4, HLA-DQA1, and HLA-DQB1. Although the genetic effects of two HCC susceptibility loci were not replicated, the two loci were found to exert susceptibility effects on the risk of CHB in a Korean population. In addition, the decreased mRNA expression of STAT4, HLA-DQA1, and HLA-DQB1 in HCC tissue might provide a clue to understanding their role in the progression to HCC.

Interaction Effects of Lipoprotein Lipase Polymorphisms with Lifestyle on Lipid Levels in a Korean Population: A Cross-sectional Study.

Lipoprotein lipase (LPL) plays an essential role in the regulation of high-density lipoprotein cholesterol (HDLC) and triglyceride levels, which have been closely associated with cardiovascular diseases. Genetic studies in European have shown that LPL single-nucleotide polymorphisms (SNPs) are strongly associated with lipid levels. However, studies about the influence of interactions between LPL SNPs and lifestyle factors have not been sufficiently performed. Here, we examine if LPL polymorphisms, as well as their interaction with lifestyle factors, influence lipid concentrations in a Korean population. A two-stage association study was performed using genotype data for SNPs on the LPL gene, including the 3' flanking region from 7,536 (stage 1) and 3,703 (stage 2) individuals. The association study showed that 15 SNPs and 4 haplotypes were strongly associated with HDLC (lowest p = 2.86 × 10(-22)) and triglyceride levels (lowest p = 3.0 × 10(-15)). Interactions between LPL polymorphisms and lifestyle factors (lowest p = 9.6 × 10(-4)) were also observed on lipid concentrations. These findings suggest that there are interaction effects of LPL polymorphisms with lifestyle variables, including energy intake, fat intake, smoking, and alcohol consumption, as well as effects of LPL polymorphisms themselves, on lipid concentrations in a Korean population.

Promoter methylation of RASSF1A modulates the effect of the microtubule-targeting agent docetaxel in breast cancer.

Docetaxel is one of the most commonly used chemotherapeutic agents in breast cancer. To avert from significant toxicities with no clinical benefit, identification of predictive markers for response is one of the most important unsolved clinical needs. Therefore, the potential associations of RASSF1A hypermethylation and response to docetaxel-based chemotherapy were evaluated, and the underlying mechanism was studied. The expression of RASSF1A in breast cancer cell lines and tissues of normal breast, ductal carcinoma in situ (DCIS), and breast cancer (n=45) was analyzed by immunohistochemistry and western blot analysis. Immunohistochemical staining showed that the expression of RASSF1A was frequently lost in primary breast cancers and human breast cancer cell lines, while normal breast tissues or DCIS displayed moderate to strong expression. Furthermore, quantitative methylation analysis of the RASSF1A promoter region in 45 primary breast cancers revealed that RASSF1A was frequently methylated in primary breast cancers (≥20% methylation in 53% of the patients), and prospective analysis in patients with locally advanced or recurrent breast cancer showed that the mean level of methylation of RASSF1A was significantly higher in patients who did not respond to docetaxel-based chemotherapy (30.6±8.5%) than patients with partial or complete response (20.1±11.2%, p=0.042). Finally, in vitro studies showed that RASSF1A had cooperative activity in suppression of cancer cell growth and proliferation by enhancing docetaxel-induced cell cycle arrest. Our results suggest that hypermethylated RASSF1A is an important modulating factor for the efficacy of docetaxel-based chemotherapy in breast cancer.

Lack of association of RAD51 genetic variations with hepatitis B virus clearance and occurrence of hepatocellular carcinoma in a Korean population.

The RecA homolog, E. coli (S. cerevisiae) (RAD51) may modulate hepatitis B virus (HBV) infection by maintaining genome integrity and mediating homologous DNA repairs. In this study, 16 sequence variations were detected by resequencing all exons, the exon-intron boundary, and promoter regions of the human RAD51 gene in DNA samples of 24 unrelated individuals. To investigate the association of common variations in the RAD51 locus with HBV infection and hepatocellular carcinoma (HCC) occurrence, six common polymorphisms were genotyped in a total of 1,103 Korean HBV cohort, composed of 433 spontaneously recovered patients as controls and 670 chronic carriers of HBV, who were stratified further into 327 cirrhosis/chronic hepatitis patients and 343 patients with HCC infected with HBV. Logistic analyses revealed no significant association of RAD51 polymorphisms and haplotypes with HBV clearance and HCC occurrence (P > 0.05). Furthermore, with age of infection as an important factor in disease progression to HCC, results from the Cox proportional hazards analysis showed no significant associations between any of the tested RAD51 variants and the age of onset of HCC (P > 0.05), suggesting that genetic polymorphisms of RAD51 may not play an important role in clearance of HBV and disease progression to HCC. Although studies in other populations are needed to confirm these findings, this preliminary data may contribute to the current knowledge on the pathogenesis of hepatitis.

No associations of polymorphisms in ADPRT with hepatitis B virus clearance and hepatocellular carcinoma occurrence in a Korean population.

AIM: The human adenosine diphosphate ribosyl transferase (ADPRT) gene might significantly affect cancer by encoding poly(ADP-ribose) polymerase 1 enzyme (PARP-1) and promoting an important role in cellular responses to DNA damage, genomic stabilization and regulation of tumor suppressor genes. We explored whether polymorphisms of ADPRT affect clearance of hepatitis B virus (HBV) infection or risk of hepatocellular carcinoma (HCC) occurrence in a Korean HBV cohort. METHODS: Genotyping was performed in a total of 1066 subjects composed of 434 spontaneously recovered (SR) subjects as normal controls and 632 chronic carriers (CC) of HBV who were further classified into 325 patients with liver cirrhosis (LC)/chronic hepatitis (CH) and 307 patients with HCC. RESULTS: Logistic analyses of six common single nucleotide polymorphisms (SNP) and their haplotypes revealed that none of the polymorphisms were significantly associated with clearance of HBV infection and HCC occurrence, except for nominal evidence of association between haplotype 2 (ht2) with HBV clearance (P = 0.05). In the analysis of age of HCC occurrence which is an important factor in disease progression to HCC, results from Cox proportional hazards showed that none of the variants were significantly associated with onset age of HCC occurrence, although a nominal signal in ht4 (P = 0.03, but P(corr) > 0.05) was initially detected. CONCLUSION: Although ADPRT is an important gene for cellular responses and tumor regulations, our study provides evidence that ADPRT variations do not affect HBV clearance and HCC occurrence.