Quantitative DNA Methylation Analysis of Candidate Genes in Cervical Cancer

Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97–1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated.     

Eleven Candidate Susceptibility Genes for Common Familial Colorectal Cancer

Hereditary factors are presumed to play a role in one third of colorectal cancer (CRC) cases. However, in the majority of familial CRC cases the genetic basis of predisposition remains unexplained. This is particularly true for families with few affected individuals. To identify susceptibility genes for this common phenotype, we examined familial cases derived from a consecutive series of 1514 Finnish CRC patients. Ninety-six familial CRC patients with no previous diagnosis of a hereditary CRC syndrome were included in the analysis. Eighty-six patients had one affected first-degree relative, and ten patients had two or more. Exome sequencing was utilized to search for genes harboring putative loss-of-function variants, because such alterations are likely candidates for disease-causing mutations. Eleven genes with rare truncating variants in two or three familial CRC cases were identified: UACA, SFXN4, TWSG1, PSPH, NUDT7, ZNF490, PRSS37, CCDC18, PRADC1, MRPL3, and AKR1C4. Loss of heterozygosity was examined in all respective cancer samples, and was detected in seven occasions involving four of the candidate genes. In all seven occasions the wild-type allele was lost (P = 0.0078) providing additional evidence that these eleven genes are likely to include true culprits. The study provides a set of candidate predisposition genes which may explain a subset of common familial CRC. Additional genetic validation in other populations is required to provide firm evidence for causality, as well as to characterize the natural history of the respective phenotypes.     

Identification of Novel Candidate Genes for Early-Onset Colorectal Cancer Susceptibility

Approximately 25–30% of colorectal cancer (CRC) cases are expected to result from a genetic predisposition, but in only 5–10% of these cases highly penetrant germline mutations are found. The remaining CRC heritability is still unexplained, and may be caused by a hitherto-undefined set of rare variants with a moderately penetrant risk. Here we aimed to identify novel risk factors for early-onset CRC using whole-exome sequencing, which was performed on a cohort of CRC individuals (n = 55) with a disease onset before 45 years of age. We searched for genes that were recurrently affected by rare variants (minor allele frequency ≤0.001) with potentially damaging effects and, subsequently, re-sequenced the candidate genes in a replication cohort of 174 early-onset or familial CRC individuals. Two functionally relevant genes with low frequency variants with potentially damaging effects, PTPN12 and LRP6, were found in at least three individuals. The protein tyrosine phosphatase PTP-PEST, encoded by PTPN12, is a regulator of cell motility and LRP6 is a component of the WNT-FZD-LRP5-LRP6 complex that triggers WNT signaling. All variants in LRP6 were identified in individuals with an extremely early-onset of the disease (≤30 years of age), and two of the three variants showed increased WNT signaling activity in vitro. In conclusion, we present PTPN12 and LRP6 as novel candidates contributing to the heterogeneous susceptibility to CRC.     

Oncogene Panel Sequencing Analysis Identifies Candidate Actionable Genes in Advanced Well-Differentiated Gastroenteropancreatic Neuroendocrine Tumors

Objective: To report the rate of candidate actionable somatic mutations in patients with locally advanced and metastatic gastro-enteropancreatic (GEP) neuroendocrine tumors (NET) and of other genetic alterations that may be associated with tumorigenesis.Methods: A phase II mutation targeted therapy trial was conducted in patients with advanced well-differentiated G1/G2 GEP-NET. Mutations found in the mTOR pathway-associated genes led to treatment with the mTOR inhibitor everolimus, and were defined as actionable. Tumor deoxyribonucleic acid (DNA) from GEP-NET were sequenced and compared with germline DNA, using the OncoVAR-NET assay, designed for hybrid capture sequencing of 500 tumor suppressor genes and oncogenes. Somatic variants were called and copy-number (CN) variant analysis was performed.Results: Thirty patients (14 small-intestine, 8 pancreatic, 3 unknown primary NET, and 5 of other primary sites) harbored 37 lesions (4 patients had DNA of multiple lesions sequenced). Only 2 patients with sporadic NET (n = 26) had an actionable mutation leading to treatment with everolimus. Driver somatic mutations were detected in 18 of 30 patients (21/37 lesions sequenced). In the remaining samples without a driver mutation, CN alterations were found in 11/16 tumors (10/12 patients), including CN loss of chromosome (Chr) 18 (P<.05), CN gain of Chr 5, and loss of Chr 13. CN losses in Chr 18 were more common in patients without driver mutations detected. Pronounced genetic heterogeneity was detected in patients with multiple lesions sequenced.Conclusion: Genome-wide DNA sequencing may identify candidate actionable genes and lead to the identification of novel target genes for advanced well-differentiated GEP-NET.Abbreviations: Chr = chromosome; CN = copy number; DNA = deoxyribonucleic acid; FDA = Food and Drug Administration; GEP = gastro-enteropancreatic; MEN-1 = multiple endocrine neoplasia syndrome type 1; mTOR = mammalian target of rapamycin; NET = neuroendocrine tumor; PFS = progression-free survival; PNET = pancreatic neuroendocrine tumors; SINET = small-intestine neuroendocrine tumor     

Candidate Luminal B Breast Cancer Genes Identified by Genome,Gene Expression and DNA Methylation Profiling

Breast cancers (BCs) of the luminal B subtype are estrogen receptor-positive (ER+), highly proliferative, resistant to standard therapies and have a poor prognosis. To better understand this subtype we compared DNA copy number aberrations (CNAs), DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes, in 32 luminal B tumors with those observed in 156 BCs of the other molecular subtypes. Frequent CNAs included 8p11-p12 and 11q13.1-q13.2 amplifications, 7q11.22-q34, 8q21.12-q24.23, 12p12.3-p13.1, 12q13.11-q24.11, 14q21.1-q23.1, 17q11.1-q25.1, 20q11.23-q13.33 gains and 6q14.1-q24.2, 9p21.3-p24,3, 9q21.2, 18p11.31-p11.32 losses. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs) presented a deregulated expression in relation with their CNAs, including 11 genes previously reported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. A total of 100 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 of these was associated with poor survival in luminal tumors. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, KCNB2 mutations were associated with luminal B cases while candidate TSGs MDN1 (6q15) and UTRN (6q24), were mutated in this subtype. In conclusion, we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype.     

An Integrative Analysis to Identify Driver Genes in Esophageal Squamous Cell Carcinoma

BackgroundFew driver genes have been well established in esophageal squamous cell carcinoma (ESCC). Identification of the genomic aberrations that contribute to changes in gene expression profiles can be used to predict driver genes.MethodsWe searched for driver genes in ESCC by integrative analysis of gene expression microarray profiles and copy number data. To narrow down candidate genes, we performed survival analysis on expression data and tested the genetic vulnerability of each genes using public RNAi screening data. We confirmed the results by performing RNAi experiments and evaluating the clinical relevance of candidate genes in an independent ESCC cohort.ResultsWe found 10 significantly recurrent copy number alterations accompanying gene expression changes, including loci 11q13.2, 7p11.2, 3q26.33, and 17q12, which harbored CCND1, EGFR, SOX2, and ERBB2, respectively. Analysis of survival data and RNAi screening data suggested that GRB7, located on 17q12, was a driver gene in ESCC. In ESCC cell lines harboring 17q12 amplification, knockdown of GRB7 reduced the proliferation, migration, and invasion capacities of cells. Moreover, siRNA targeting GRB7 had a synergistic inhibitory effect when combined with trastuzumab, an anti-ERBB2 antibody. Survival analysis of the independent cohort also showed that high GRB7 expression was associated with poor prognosis in ESCC.ConclusionOur integrative analysis provided important insights into ESCC pathogenesis. We identified GRB7 as a novel ESCC driver gene and potential new therapeutic target.     

DNA Methyltransferase Candidate Polymorphisms,Imprinting Methylation,and Birth Outcome

Background

Birth weight and prematurity are important obstetric outcomes linked to lifelong health. We studied a large birth cohort to look for evidence of epigenetic involvement in birth outcomes.

Methods

We investigated the association between birth weight, length, placental weight and duration of gestation and four candidate variants in 1,236 mothers and 1,073 newborns; DNMT1 (rs2162560), DNMT3A (rs734693), DNMT3B (rs2424913) and DNMT3L (rs7354779). We measured methylation of LINE1 and the imprinted genes, PEG3, SNRPN, and IGF2, in cord blood.

Results

The minor DNMT3L allele in the baby was associated with higher birth weight (+54 95% CI 10,99 g; p = 0.016), birth length (+0.23 95% CI 0.04,0.42 cm; p = 0.017), placental weight, (+18 95% CI 3,33 g; p = 0.017), and reduced risk of being in the lowest birth weight decile (p = 0.018) or requiring neonatal care (p = 0.039). The DNMT3B minor allele in the mother was associated with an increased risk of prematurity (p = 0.001). Placental size was related to PEG3 (p<0.001) and IGF2 (p<0.001) methylation. Birth weight was related to LINE1 and IGF2 methylation but only at p = 0.052. The risk of requiring neonatal treatment was related to LINE1 (p = 0.010) and SNRPN (p = 0.001) methylation. PEG3 methylation was influenced by baby DNMT3A genotype (p = 0.012) and LINE1 by baby 3B genotype (p = 0.044). Maternal DNMT3L genotype was related to IGF2 methylation in the cord blood but this effect was only seen in carriers of the minor frequency allele (p = 0.050).

Conclusions

The results here suggest that epigenetic processes are linked birth outcome and health in early life. Our emerging understanding of the role of epigenetics in health and biological function across the lifecourse suggests that these early epigenetic events could have longer term implications.     

烟草FIE和MSI1基因的克隆及序列分析

采用电子克隆与实验克隆结合的方法获得了烟草胚乳发育相关基因NTFIE和NTMSI1的cDNA序列,序列号分别为EU375458和EU375459.序列分析结果表明,这两个cDNA序列均含有完整的开放读码框,分别编码370和424个氨基酸,含有保守的WD基序.氨基酸序列比对和系统发育分析结果显示,不同物种之间FIE和MSI1基因编码氨基酸序列同源性都较高.组织表达分析结果表明,这两个基因均具有一定程度的组织表达特异性,NTFIE cDNA基因在花中的表达量最多,但在根和茎中未检测到表达,而NTMSI1 cDNA基因只在离体培养的细胞和根中特异性表达.     

Identification of Candidate Driver Genes in Common Focal Chromosomal Aberrations of Microsatellite Stable Colorectal Cancer

Colorectal cancer (CRC) is a leading cause of cancer deaths worldwide. Chromosomal instability (CIN) is a major driving force of microsatellite stable (MSS) sporadic CRC. CIN tumours are characterised by a large number of somatic chromosomal copy number aberrations (SCNA) that frequently affect oncogenes and tumour suppressor genes. The main aim of this work was to identify novel candidate CRC driver genes affected by recurrent and focal SCNA. High resolution genome-wide comparative genome hybridisation (CGH) arrays were used to compare tumour and normal DNA for 53 sporadic CRC cases. Context corrected common aberration (COCA) analysis and custom algorithms identified 64 deletions and 32 gains of focal minimal common regions (FMCR) at high frequency (>10%). Comparison of these FMCR with published genomic profiles from CRC revealed common overlap (42.2% of deletions and 34.4% of copy gains). Pathway analysis showed that apoptosis and p53 signalling pathways were commonly affected by deleted FMCR, and MAPK and potassium channel pathways by gains of FMCR. Candidate tumour suppressor genes in deleted FMCR included RASSF3, IFNAR1, IFNAR2 and NFKBIA and candidate oncogenes in gained FMCR included PRDM16, TNS1, RPA3 and KCNMA1. In conclusion, this study confirms some previously identified aberrations in MSS CRC and provides in silico evidence for some novel candidate driver genes.     

Comprehensive DNA Methylation Analysis Reveals a Common Ten-Gene Methylation Signature in Colorectal Adenomas and Carcinomas

Microarray analysis of promoter hypermethylation provides insight into the role and extent of DNA methylation in the development of colorectal cancer (CRC) and may be co-monitored with the appearance of driver mutations. Colonic biopsy samples were obtained endoscopically from 10 normal, 23 adenoma (17 low-grade (LGD) and 6 high-grade dysplasia (HGD)), and 8 ulcerative colitis (UC) patients (4 active and 4 inactive). CRC samples were obtained from 24 patients (17 primary, 7 metastatic (MCRC)), 7 of them with synchronous LGD. Field effects were analyzed in tissues 1 cm (n = 5) and 10 cm (n = 5) from the margin of CRC. Tissue materials were studied for DNA methylation status using a 96 gene panel and for KRAS and BRAF mutations. Expression levels were assayed using whole genomic mRNA arrays. SFRP1 was further examined by immunohistochemistry. HT29 cells were treated with 5-aza-2’ deoxycytidine to analyze the reversal possibility of DNA methylation. More than 85% of tumor samples showed hypermethylation in 10 genes (SFRP1, SST, BNC1, MAL, SLIT2, SFRP2, SLIT3, ALDH1A3, TMEFF2, WIF1), whereas the frequency of examined mutations were below 25%. These genes distinguished precancerous and cancerous lesions from inflamed and healthy tissue. The mRNA alterations that might be caused by systematic methylation could be partly reversed by demethylation treatment. Systematic changes in methylation patterns were observed early in CRC carcinogenesis, occuring in precursor lesions and CRC. Thus we conclude that DNA hypermethylation is an early and systematic event in colorectal carcinogenesis, and it could be potentially reversed by systematic demethylation therapy, but it would need more in vitro and in vivo experiments to support this theory.     

Methylation of the Promoter Region of the RASSF1A Candidate Tumor Suppressor Gene in Primary Epithelial Tumors

The methylation of the promoter CpG island of the RASSF1A tumor suppressor gene in primary tumors of 172 Muscovites with renal cell carcinoma (RCC), breast cancer (BC), or ovarian epithelial tumors (OET) was assayed by means of methylation-specific PCR (MSP) and PCR-based methylation-sensitive restriction enzyme analysis (MSRA). The MSP, MSRA, and previous bisulfite sequencing data correlated significantly with each other (P 10^–6 for Spearman's rank correlation coefficients). By MSP and MSRA, the respective methylation frequencies of the RASSF1A promoter were 86% (25/29) and 94% (50/53) in RCC, 64% (18/28) and 78% (32/41) in BC, and 59% (17/29) and 73% (33/45) in OET. Methylation-sensitive restriction enzymes (HpaII, HhaI, Bsh1236I, AciI) increased the analysis sensitivity and made it possible to establish the methylation status for 18 CpG dinucleotides of the RASSF1A promoter region. With the MSRA data, the density of methylation of the CpG island was estimated at 72% in RCC, 63% in BC, and 58% in OET (the product of the number of CpG dinucleotides and the number of specimens with RASSF1A methylation was taken as 100%). Methylation of the RASSF1A promoter region was observed in 11–35% of the DNA specimens from the histologically normal tissue adjacent to the tumor but not in the peripheral blood DNA of 15 healthy subjects. The RASSF1A methylation frequency showed no significant correlation with the stage, grade, and metastatic potential of the tumor. On the other hand, epigenetic modification of RASSF1A was considerably more frequent than hemizygous or homozygous deletions from the RASSF1A region. These results testify that methylation of the RASSF1A promoter region takes place early in carcinogenesis and is a major mechanism inactivating RASSF1A in epithelial tumors.     

Pathways and Networks-Based Analysis of Candidate Genes Associated with Nicotine Addiction

Nicotine is the addictive substance in tobacco and it has a broad impact on both the central and peripheral nervous systems. Over the past decades, an increasing number of genes potentially involved in nicotine addiction have been identified by different technical approaches. However, the molecular mechanisms underlying nicotine addiction remain largely unclear. Under such situation, a comprehensive analysis focusing on the overall functional characteristics of these genes, as well as how they interact with each other will provide us valuable information to understand nicotine addiction. In this study, we presented a systematic analysis on nicotine addiction-related genes to identify the major underlying biological themes. Functional analysis revealed that biological processes and biochemical pathways related to neurodevelopment, immune system and metabolism were significantly enriched in the nicotine addiction-related genes. By extracting the nicotine addiction-specific subnetwork, a number of novel genes associated with addiction were identified. Moreover, we constructed a schematic molecular network for nicotine addiction via integrating the pathways and network, providing an intuitional view to understand the development of nicotine addiction. Pathway and network analysis indicated that the biological processes related to nicotine addiction were complex. Results from our work may have important implications for understanding the molecular mechanism underlying nicotine addiction.     

Gastrointestinal Stromal Tumors,Somatic Mutations and Candidate Genetic Risk Variants

Gastrointestinal stromal tumors (GISTs) are rare but treatable soft tissue sarcomas. Nearly all GISTs have somatic mutations in either the KIT or PDGFRA gene, but there are no known inherited genetic risk factors. We assessed the relationship between KIT/PDGFRA mutations and select deletions or single nucleotide polymorphisms (SNPs) in 279 participants from a clinical trial of adjuvant imatinib mesylate. Given previous evidence that certain susceptibility loci and carcinogens are associated with characteristic mutations, or “signatures” in other cancers, we hypothesized that the characteristic somatic mutations in the KIT and PDGFRA genes in GIST tumors may similarly be mutational signatures that are causally linked to specific mutagens or susceptibility loci. As previous epidemiologic studies suggest environmental risk factors such as dioxin and radiation exposure may be linked to sarcomas, we chose 208 variants in 39 candidate genes related to DNA repair and dioxin metabolism or response. We calculated adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for the association between each variant and 7 categories of tumor mutation using logistic regression. We also evaluated gene-level effects using the sequence kernel association test (SKAT). Although none of the association p-values were statistically significant after adjustment for multiple comparisons, SNPs in CYP1B1 were strongly associated with KIT exon 11 codon 557-8 deletions (OR = 1.9, 95% CI: 1.3-2.9 for rs2855658 and OR = 1.8, 95% CI: 1.2-2.7 for rs1056836) and wild type GISTs (OR = 2.7, 95% CI: 1.5-4.8 for rs1800440 and OR = 0.5, 95% CI: 0.3-0.9 for rs1056836). CYP1B1 was also associated with these mutations categories in the SKAT analysis (p = 0.002 and p = 0.003, respectively). Other potential risk variants included GSTM1, RAD23B and ERCC2. This preliminary analysis of inherited genetic risk factors for GIST offers some clues about the disease''s genetic origins and provides a starting point for future candidate gene or gene-environment research.     

家族性不宁腿综合征候选基因的连锁分析

不宁腿综合征（restless legs syndrome,RLS)是以下肢部出现蚁行样及酸、麻、胀等不适感而使肢体不得休息为特征的一组病症。由于症状常在晚间发作并导致运动不安,患者长期入睡困难,经受严重的继发性失眠。作为一种常见的神经系统疾病,RLS发病率高达5％,其中原发性RLS多呈阳性家族史,表现为单基因决定的常染色体显性遗传。现在,人们普遍认为RLS的发生很可能与神经系统内多巴胺能功能异常和脑内铁缺乏有关,并初步建立了脑铁－多巴胺能系统的致病模型。为了探求脑铁－多巴胺能系统在RLS中的作用,选择了与脑铁－多巴胺能系统相关的16个疾病侯选基因,在每个候选基因附近染色体区域内选取若干个微卫星多态标记,应用微卫星引物荧光标记－基因扫描技术,对一个汉族家族性不宁腿综合征家系进行了基因分型和常染色体显性遗传模式下的连锁分析,试图从分子遗传学层面上确认或排除一些可能与RLS相关的重要侯选基因。结果显示,当重组系数θ=0.00时,LOD值均小于－2.00,所选位点与家族性不宁腿综合征不连锁。由此得出结论,在本家系中,所有候选基因均与家族性不宁腿综合征的发病无关,家族性不宁腿综合征可能是由其他多巴胺传导和脑铁代谢相关基因所致,或是存在全新的致病机制参与RLS的发生。     

An Integrative -omics Approach to Identify Functional Sub-Networks in Human Colorectal Cancer

Emerging evidence indicates that gene products implicated in human cancers often cluster together in “hot spots” in protein-protein interaction (PPI) networks. Additionally, small sub-networks within PPI networks that demonstrate synergistic differential expression with respect to tumorigenic phenotypes were recently shown to be more accurate classifiers of disease progression when compared to single targets identified by traditional approaches. However, many of these studies rely exclusively on mRNA expression data, a useful but limited measure of cellular activity. Proteomic profiling experiments provide information at the post-translational level, yet they generally screen only a limited fraction of the proteome. Here, we demonstrate that integration of these complementary data sources with a “proteomics-first” approach can enhance the discovery of candidate sub-networks in cancer that are well-suited for mechanistic validation in disease. We propose that small changes in the mRNA expression of multiple genes in the neighborhood of a protein-hub can be synergistically associated with significant changes in the activity of that protein and its network neighbors. Further, we hypothesize that proteomic targets with significant fold change between phenotype and control may be used to “seed” a search for small PPI sub-networks that are functionally associated with these targets. To test this hypothesis, we select proteomic targets having significant expression changes in human colorectal cancer (CRC) from two independent 2-D gel-based screens. Then, we use random walk based models of network crosstalk and develop novel reference models to identify sub-networks that are statistically significant in terms of their functional association with these proteomic targets. Subsequently, using an information-theoretic measure, we evaluate synergistic changes in the activity of identified sub-networks based on genome-wide screens of mRNA expression in CRC. Cross-classification experiments to predict disease class show excellent performance using only a few sub-networks, underwriting the strength of the proposed approach in discovering relevant and reproducible sub-networks.