Racial differences in genome-wide methylation profiling and gene expression in breast tissues from healthy women

Breast cancer is more common in European Americans (EAs) than in African Americans (AAs) but mortality from breast cancer is higher among AAs. While there are racial differences in DNA methylation and gene expression in breast tumors, little is known whether such racial differences exist in breast tissues of healthy women. Genome-wide DNA methylation and gene expression profiling was performed in histologically normal breast tissues of healthy women. Linear regression models were used to identify differentially-methylated CpG sites (CpGs) between EAs (n = 61) and AAs (n = 22). Correlations for methylation and expression were assessed. Biological functions of the differentially-methylated genes were assigned using the Ingenuity Pathway Analysis. Among 485 differentially-methylated CpGs by race, 203 were hypermethylated in EAs, and 282 were hypermethylated in AAs. Promoter-related differentially-methylated CpGs were more frequently hypermethylated in EAs (52%) than AAs (27%) while gene body and intergenic CpGs were more frequently hypermethylated in AAs. The differentially-methylated CpGs were enriched for cancer-associated genes with roles in cell death and survival, cellular development, and cell-to-cell signaling. In a separate analysis for correlation in EAs and AAs, different patterns of correlation were found between EAs and AAs. The correlated genes showed different biological networks between EAs and AAs; networks were connected by Ubiquitin C. To our knowledge, this is the first comprehensive genome-wide study to identify differences in methylation and gene expression between EAs and AAs in breast tissues from healthy women. These findings may provide further insights regarding the contribution of epigenetic differences to racial disparities in breast cancer.     

Identification of POMC Exonic Variants Associated with Substance Dependence and Body Mass Index

Background

Risk of substance dependence (SD) and obesity has been linked to the function of melanocortin peptides encoded by the proopiomelanocortin gene (POMC).

Methods and Results

POMC exons were Sanger sequenced in 280 African Americans (AAs) and 308 European Americans (EAs). Among them, 311 (167 AAs and 114 EAs) were affected with substance (alcohol, cocaine, opioid and/or marijuana) dependence and 277 (113 AAs and164 EAs) were screened controls. We identified 23 variants, including two common polymorphisms (rs10654394 and rs1042571) and 21 rare variants; 12 of which were novel. We used logistic regression to analyze the association between the two common variants and SD or body mass index (BMI), with sex, age, and ancestry proportion as covariates. The common variant rs1042571 in the 3′UTR was significantly associated with BMI in EAs (Overweight: P _adj = 0.005; Obese: P _adj = 0.018; Overweight+Obese: P _adj = 0.002) but not in AAs. The common variant, rs10654394, was not associated with BMI and neither common variant was associated with SD in either population. To evaluate the association between the rare variants and SD or BMI, we collapsed rare variants and tested their prevalence using Fisher’s exact test. In AAs, rare variants were nominally associated with SD overall and with specific SD traits (SD: P _FET,1df = 0.026; alcohol dependence: P _FET,1df = 0.027; cocaine dependence: P _FET,1df = 0.007; marijuana dependence: P _FET,1df = 0.050) (the P-value from cocaine dependence analysis survived Bonferroni correction). There was no such effect in EAs. Although the frequency of the rare variants did not differ significantly between the normal-weight group and the overweight or obese group in either population, certain rare exonic variants occurred only in overweight or obese subjects without SD.

Conclusion

These findings suggest that POMC exonic variants may influence risk for both SD and elevated BMI, in a population-specific manner. However, common and rare variants in this gene may exert different effects on these two phenotypes.     

Aberrant Gene Promoter Methylation Associated with Sporadic Multiple Colorectal Cancer

Background

Colorectal cancer (CRC) multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect.

Methodology/Principal Findings

We examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals) and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2), RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008) and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047) as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006). Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17), SFRP1 (r = 0.83, 0.06), HPP1 (r = 0.64, p = 0.17), 3OST2 (r = 0.83, p = 0.06) and GATA4 (r = 0.6, p = 0.24). Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant difference in any evaluated gene.

Conclusions

These results provide a proof-of-concept that gene promoter methylation is associated with tumor multiplicity. This underlying epigenetic defect may have noteworthy implications in the prevention of patients with sporadic CRC.     

Elevation of Peripheral BDNF Promoter Methylation Links to the Risk of Alzheimer's Disease

Brain derived neurotrophic factor (BDNF) has been known to play an important role in various mental disorders or diseases such as Alzheimer''s disease (AD). The aim of our study was to assess whether BDNF promoter methylation in peripheral blood was able to predict the risk of AD. A total of 44 AD patients and 62 age- and gender-matched controls were recruited in the current case-control study. Using the bisulphite pyrosequencing technology, we evaluated four CpG sites in the promoter of the BDNF. Our results showed that BDNF methylation was significantly higher in AD cases than in the controls (CpG1: p = 10.021; CpG2: p = 0.002; CpG3: p = 0.007; CpG4: p = 0.005; average methylation: p = 0.004). In addition, BDNF promoter methylation was shown to be significantly correlated with the levels of alkaline phosphatase (ALP), glucose, Lp(a), ApoE and ApoA in males (ALP: r = −0.308, p = 0.042; glucose: r = −0.383, p = 0.010; Lp(a): r = 0.333, p = 0.027; ApoE: r = −0.345, p = 0.032;), ApoA levels in females (r = 0.362, p = 0.033), and C Reactive Protein (CRP) levels in both genders (males: r = −0.373, p = 0.016; females: r = −0.399, p = 0.021). Our work suggested that peripheral BDNF promoter methylation might be a diagnostic marker of AD risk, although its underlying function remains to be elaborated in the future.     

Identification of a DNA Methylome Profile of Esophageal Squamous Cell Carcinoma and Potential Plasma Epigenetic Biomarkers for Early Diagnosis

DNA methylation is a critical epigenetic mechanism involved in key cellular processes. Its deregulation has been linked to many human cancers including esophageal squamous cell carcinoma (ESCC). This study was designed to explore the whole methylation status of ESCC and to identify potential plasma biomarkers for early diagnosis. We used Infinium Methylation 450k array to analyze ESCC tissues (n = 4), paired normal surrounding tissues (n = 4) and normal mucosa from healthy individuals (n = 4), and combined these with gene expression data from the GEO database. One hundred and sixty eight genes had differentially methylated CpG sites in their promoter region and a gene expression pattern inverse to the direction of change in DNA methylation. These genes were involved in several cancer-related pathways. Three genes were validated in additional 42 ESCC tissues and paired normal surrounding tissues. The methylation frequency of EPB41L3, GPX3, and COL14A1 were higher in tumor tissues than in normal surrounding tissues (P<0.017). The higher methylation frequency of EPB41l3 was correlated with large tumor size (P = 0.044) and advanced pT tumor stage (P = 0.001). The higher methylation frequency of GPX3 and COL14A1 were correlated with advanced pN tumor stage (P = 0.001 and P<0.001). The methylation of EPB41L3, GPX3, and COL14A1 genes were only found in ESCC patients'' plasma, but not in normal individuals upon testing 42 ESCC patients and 50 healthy individuals. Diagnostic sensitivity was increased when methylation of any of the 3 genes were counted (64.3% sensitivity and 100% specificity). These differentially methylated genes in plasma may be used as biomarkers for early diagnosis of ESCC.     

CpG Methylation Changes within the IL2RA Promoter in Type 1 Diabetes of Childhood Onset

None of the polymorphic variants of the IL2RA gene found associated with Type 1 Diabetes (T1D) was shown to have a functional effect. To test if the epigenetic variation could play a role at this locus, we studied the methylation of 6 CpGs located within the proximal promoter of IL2RA gene in 252 T1D patients compared with 286 age-matched controls. We found that DNA methylation at CpGs −373 and −456 was slightly but significantly higher in patients than in controls (40.4±4.6 vs 38.3±5.4, p = 1.4E4; 91.4±2.8 vs 89.5±5.3, p = 1.8E-6), while other CpG showed a strictly comparable methylation. Among 106 single nucleotide polymorphisms (SNPs) located in the neighboring 180kb region, we found that 28 SNPs were associated with DNA methylation at CpG −373. Sixteen of these SNPs were known to be associated with T1D. Our findings suggest that the effect of IL2RA risk alleles on T1D may be partially mediated through epigenetic changes.     

CHRNA7 Polymorphisms and Response to Cholinesterase Inhibitors in Alzheimer's Disease

Background

CHRNA7 encodes the α7 nicotinic acetylcholine receptor subunit, which is important to Alzheimer''s disease (AD) pathogenesis and cholinergic neurotransmission. Previously, CHRNA7 polymorphisms have not been related to cholinesterase inhibitors (ChEI) response.

Methods

Mild to moderate AD patients received ChEIs were recruited from the neurology clinics of three teaching hospitals from 2007 to 2010 (n = 204). Nine haplotype-tagging single nucleotide polymorphisms of CHRNA7 were genotyped. Cognitive responders were those showing improvement in the Mini-Mental State Examination score ≧2 between baseline and 6 months after ChEI treatment.

Results

AD women carrying rs8024987 variants [GG+GC vs. CC: adjusted odds ratio (AOR) = 3.62, 95% confidence interval (CI) = 1.47–8.89] and GG haplotype in block1 (AOR = 3.34, 95% CI = 1.38–8.06) had significantly better response to ChEIs (false discovery rate <0.05). These variant carriers using galantamine were 11 times more likely to be responders than female non-carriers using donepezil or rivastigmine.

Conclusion

For the first time, this study found a significant association between CHRNA7 polymorphisms and better ChEI response. If confirmed by further studies, CHRNA7 polymorphisms may aid in predicting ChEI response and refining treatment choice.     

Inhibin Alpha-Subunit (INHA) Expression in Adrenocortical Cancer Is Linked to Genetic and Epigenetic INHA Promoter Variation

Adrenocortical carcinoma (ACC) is a rare, but highly malignant tumor of unknown origin. Inhibin α-subunit (Inha) knockout mice develop ACCs following gonadectomy. In man, INHA expression varies widely within ACC tissues and its circulating peptide inhibin pro-αC has been described as a novel tumor marker for ACC. We investigated whether genetic and epigenetic changes of the INHA gene in human ACC cause loss or variation of INHA expression. To this end, analyses of INHA sequence, promoter methylation and mRNA expression were performed in human adrenocortical tissues. Serum inhibin pro-αC levels were also measured in ACC patients. INHA genetic analysis in 37 unique ACCs revealed 10 novel, heterozygous rare variants. Of the 3 coding bases affected, one variant was synonymous and two were missense variants: S72F and S184F. The minor allele of rs11893842 at −124 bp was observed at a low frequency (24%) in ACC samples and was associated with decreased INHA mRNA levels: 4.7±1.9 arbitrary units for AA, compared to 26±11 for AG/GG genotypes (P = 0.034). The methylation of four proximal INHA promoter CpGs was aberrantly increased in five ACCs (47.7±3.9%), compared to normal adrenals (18.4±0.6%, P = 0.0052), whereas the other 14 ACCs studied showed diminished promoter methylation (9.8±1.1%, P = 0.020). CpG methylation was inversely correlated to INHA mRNA levels in ACCs (r = −0.701, p = 0.0036), but not associated with serum inhibin pro-αC levels. In conclusion, aberrant methylation and common genetic variation in the INHA promoter occur in human ACCs and are associated with decreased INHA expression.     

A Complex Interplay between Personality Domains,Marital Status and a Variant in CHRNA5 on the Risks of Cocaine,Nicotine Dependences and Cocaine-Induced Paranoia

Background

Personality correlates highly with both cocaine and nicotine dependencies (CD, ND), and their co-morbid psychopathologies. However, little is known about the nature of these relationships. This study examined if environment (marriage) or genetics (a single SNP, CHRNA5*rs16969968) would moderate the correlation of personality with CD, ND and cocaine-induced paranoia (CIP) in African and European Americans (AAs, EAs).

Methods

1432 EAs and 1513 AAs were examined using logistic regression. Personality was assessed by NEO-PI-R, while CD, ND and CIP were diagnosed according to DSM-IV. ND and CD were examined as binary traits and for the analysis of CIP, subjects were divided into 3 groups: (A) Controls with no CIP; (B) CD cases without CIP; and (C) CD cases with CIP. Multiple testing was Bonferroni-corrected.

Results

For CD and ND in the EA population, marital status proved to be a significant moderator in their relationship with openness only (OR = 1.90, 95%CI = 1.36–2.64, p = 1.54e-04 and OR = 2.12, 95%CI = 1.52–2.90, p = 4.65e-06 respectively). For CIP, marriage was observed to moderate its correlation with openness and neuroticism (OR = 1.39, 95%CI = 1.18–1.63, p = 7.64e-04 and OR = 1.26, 95%CI = 1.12–1.42, p = 1.27e-03 respectively). The correlations moderated by rs16969968 were those of conscientiousness and CD (OR = 1.62, 95%CI: 1.23–2.12, p = 8.94e-04) as well as CIP (OR = 1.21, 95%CI: 1.11–1.32, p = 4.93e-04 when comparing group A versus group C). No significant interactions were observed in AA population. The Bonferroni-corrected significance threshold was set to be 1.67e-03.

Conclusion

The role of personality in CD and CIP may be interceded by both environment and genetics, while in ND by environment only.     

Heterogeneity in White Blood Cells Has Potential to Confound DNA Methylation Measurements

Epigenetic studies are commonly conducted on DNA from tissue samples. However, tissues are ensembles of cells that may each have their own epigenetic profile, and therefore inter-individual cellular heterogeneity may compromise these studies. Here, we explore the potential for such confounding on DNA methylation measurement outcomes when using DNA from whole blood. DNA methylation was measured using pyrosequencing-based methodology in whole blood (n = 50–179) and in two white blood cell fractions (n = 20), isolated using density gradient centrifugation, in four CGIs (CpG Islands) located in genes HHEX (10 CpG sites assayed), KCNJ11 (8 CpGs), KCNQ1 (4 CpGs) and PM20D1 (7 CpGs). Cellular heterogeneity (variation in proportional white blood cell counts of neutrophils, lymphocytes, monocytes, eosinophils and basophils, counted by an automated cell counter) explained up to 40% (p<0.0001) of the inter-individual variation in whole blood DNA methylation levels in the HHEX CGI, but not a significant proportion of the variation in the other three CGIs tested. DNA methylation levels in the two cell fractions, polymorphonuclear and mononuclear cells, differed significantly in the HHEX CGI; specifically the average absolute difference ranged between 3.4–15.7 percentage points per CpG site. In the other three CGIs tested, methylation levels in the two fractions did not differ significantly, and/or the difference was more moderate. In the examined CGIs, methylation levels were highly correlated between cell fractions. In summary, our analysis detects region-specific differential DNA methylation between white blood cell subtypes, which can confound the outcome of whole blood DNA methylation measurements. Finally, by demonstrating the high correlation between methylation levels in cell fractions, our results suggest a possibility to use a proportional number of a single white blood cell type to correct for this confounding effect in analyses.     

Multiple cholinergic nicotinic receptor genes affect nicotine dependence risk in African and European Americans

Several independent studies show that the chromosome 15q25.1 region, which contains the CHRNA5–CHRNA3–CHRNB4 gene cluster, harbors variants strongly associated with nicotine dependence, other smoking behaviors, lung cancer and chronic obstructive pulmonary disease. We investigated whether variants in other cholinergic nicotinic receptor subunit (CHRN) genes affect the risk of nicotine dependence in a new sample of African Americans (AAs) (N = 710). We also analyzed this AA sample together with a European American (EA) sample (N = 2062, 1608 of which have been previously studied), allowing for differing effects in the two populations. Cases are current nicotine‐dependent smokers and controls are non‐dependent smokers. Variants in or near CHRND–CHRNG, CHRNA7 and CHRNA10 show modest association with nicotine dependence risk in the AA sample. In addition, CHRNA4, CHRNB3–CHRNA6 and CHRNB1 show association in at least one population. CHRNG and CHRNA4 harbor single nucleotide polymorphisms (SNPs) that have opposite directions of effect in the two populations. In each of the population samples, these loci substantially increase the trait variation explained, although no loci meet Bonferroni‐corrected significance in the AA sample alone. The trait variation explained by three key associated SNPs in CHRNA5–CHRNA3–CHRNB4 is 1.9% in EAs and also 1.9% in AAs; this increases to 4.5% in EAs and 7.3% in AAs when we add six variants representing associations at other CHRN genes. Multiple nicotinic receptor subunit genes outside chromosome 15q25 are likely to be important in the biological processes and development of nicotine dependence, and some of these risks may be shared across diverse populations.     

An ACACB Variant Implicated in Diabetic Nephropathy Associates with Body Mass Index and Gene Expression in Obese Subjects

Acetyl coenzyme A carboxylase B gene (ACACB) single nucleotide polymorphism (SNP) rs2268388 is reproducibly associated with type 2 diabetes (T2DM)-associated nephropathy (DN). ACACB knock-out mice are also protected from obesity. This study assessed relationships between rs2268388, body mass index (BMI) and gene expression in multiple populations, with and without T2DM. Among subjects without T2DM, rs2268388 DN risk allele (T) associated with higher BMI in Pima Indian children (n = 2021; p-additive = 0.029) and African Americans (AAs) (n = 177; p-additive = 0.05), with a trend in European Americans (EAs) (n = 512; p-additive = 0.09), but not Germans (n = 858; p-additive = 0.765). Association with BMI was seen in a meta-analysis including all non-T2DM subjects (n = 3568; p-additive = 0.02). Among subjects with T2DM, rs2268388 was not associated with BMI in Japanese (n = 2912) or EAs (n = 1149); however, the T allele associated with higher BMI in the subset with BMI≥30 kg/m² (n = 568 EAs; p-additive = 0.049, n = 196 Japanese; p-additive = 0.049). Association with BMI was strengthened in a T2DM meta-analysis that included an additional 756 AAs (p-additive = 0.080) and 48 Hong Kong Chinese (p-additive = 0.81) with BMI≥30 kg/m² (n = 1575; p-additive = 0.0033). The effect of rs2268388 on gene expression revealed that the T risk allele associated with higher ACACB messenger levels in adipose tissue (41 EAs and 20 AAs with BMI>30 kg/m²; p-additive = 0.018) and ACACB protein levels in the liver tissue (mixed model p-additive = 0.03, in 25 EA bariatric surgery patients with BMI>30 kg/m² for 75 exams). The T allele also associated with higher hepatic triglyceride levels. These data support a role for ACACB in obesity and potential roles for altered lipid metabolism in susceptibility to DN.     

A Preliminary Study of the Relationship between Promoter Methylation of the ABCG1, GALNT2 and HMGCR Genes and Coronary Heart Disease

Aims

To investigate the association of ABCG1, GALNT2 and HMGCR genes promoter DNA methylation with coronary heart disease (CHD) and explore the interaction between their methylation status and the CHD patients'' clinical characteristics in Han Chinese population.

Methods and results

Methylation-specific polymerase chain reaction (MSP) technology was used to examine the role of the aberrant gene promoter methylation in CHD in Han Chinese population. A total of 85 CHD patients and 54 participants without CHD confirmed by angiography were recruited. 82.8% of the participants with ABCG1 gene promoter hypermethylation have CHD, while only 17.4% of the participants without hypermethylation have it. The average age of the participants with GALNT2 gene promoter hypermethylation is 62.10±8.21, while that of the participants without hypermethylation is 57.28±9.87; in the former group, 75.4% of the participants have CHD, compared to only 50% in the latter group. As for the HMGCR gene, the average age of the participants with promoter hypermethylation is 63.24±8.10 and that of the participants without hypermethylation is 57.79±9.55; its promoter hypermethylation is likely to be related to smoking. Our results indicated a significant statistical association of promoter methylation of the ABCG1 gene with increased risk of CHD (OR = 19.966; 95% CI, 7.319–54.468; P ^*<0.001; P ^*: adjusted for age, gender, smoking, lipid level, hypertension, and diabetes). Similar results were obtained for that of the GALNT2 gene (OR = 2.978; 95% CI, 1.335–6.646; P ^* = 0.008), but not of HMGCR gene (OR = 1.388; 95% CI, 0.572–3.371; P ^* = 0.469).

Conclusions

The present work provides evidence to support the association of promoter DNA methylation status with the risk profile of CHD. Our data indicates that promoter DNA hypermethylation of the ABCG1 and GALNT2 genes, but not the HMGCR gene, is associated with an increased risk of CHD. CHD, smoking and aging are likely to be the important factors influencing DNA hypermethylation.     

Genome-Wide Analysis of DNA Methylation Differences in Muscle and Fat from Monozygotic Twins Discordant for Type 2 Diabetes

Background

Monozygotic twins discordant for type 2 diabetes constitute an ideal model to study environmental contributions to type 2 diabetic traits. We aimed to examine whether global DNA methylation differences exist in major glucose metabolic tissues from these twins.

Methodology/Principal Findings

Skeletal muscle (n = 11 pairs) and subcutaneous adipose tissue (n = 5 pairs) biopsies were collected from 53–80 year-old monozygotic twin pairs discordant for type 2 diabetes. DNA methylation was measured by microarrays at 26,850 cytosine-guanine dinucleotide (CpG) sites in the promoters of 14,279 genes. Bisulfite sequencing was applied to validate array data and to quantify methylation of intergenic repetitive DNA sequences. The overall intra-pair variation in DNA methylation was large in repetitive (LINE1, D4Z4 and NBL2) regions compared to gene promoters (standard deviation of intra-pair differences: 10% points vs. 4% points, P<0.001). Increased variation of LINE1 sequence methylation was associated with more phenotypic dissimilarity measured as body mass index (r = 0.77, P = 0.007) and 2-hour plasma glucose (r = 0.66, P = 0.03) whereas the variation in promoter methylation did not associate with phenotypic differences. Validated methylation changes were identified in the promoters of known type 2 diabetes-related genes, including PPARGC1A in muscle (13.9±6.2% vs. 9.0±4.5%, P = 0.03) and HNF4A in adipose tissue (75.2±3.8% vs. 70.5±3.7%, P<0.001) which had increased methylation in type 2 diabetic individuals. A hypothesis-free genome-wide exploration of differential methylation without correction for multiple testing identified 789 and 1,458 CpG sites in skeletal muscle and adipose tissue, respectively. These methylation changes only reached some percentage points, and few sites passed correction for multiple testing.

Conclusions/Significance

Our study suggests that likely acquired DNA methylation changes in skeletal muscle or adipose tissue gene promoters are quantitatively small between type 2 diabetic and non-diabetic twins. The importance of methylation changes in candidate genes such as PPARGC1A and HNF4A should be examined further by replication in larger samples.     

Role of Nicotine Dependence on the Relationship between Variants in the Nicotinic Receptor Genes and Risk of Lung Adenocarcinoma

Several variations in the nicotinic receptor genes have been identified to be associated with both lung cancer risk and smoking in the genome-wide association (GWA) studies. However, the relationships among these three factors (genetic variants, nicotine dependence, and lung cancer) remain unclear. In an attempt to elucidate these relationships, we applied mediation analysis to quantify the impact of nicotine dependence on the association between the nicotinic receptor genetic variants and lung adenocarcinoma risk. We evaluated 23 single nucleotide polymorphisms (SNPs) in the five nicotinic receptor related genes (CHRNB3, CHRNA6, and CHRNA5/A3/B4) previously reported to be associated with lung cancer risk and smoking behavior and 14 SNPs in the four ‘control’ genes (TERT, CLPTM1L, CYP1A1, and TP53), which were not reported in the smoking GWA studies. A total of 661 lung adenocarcinoma cases and 1,347 controls with a smoking history, obtained from the Environment and Genetics in Lung Cancer Etiology case-control study, were included in the study. Results show that nicotine dependence is a mediator of the association between lung adenocarcinoma and gene variations in the regions of CHRNA5/A3/B4 and accounts for approximately 15% of this relationship. The top two CHRNA3 SNPs associated with the risk for lung adenocarcinoma were rs1051730 and rs12914385 (p-value = 1.9×10⁻¹⁰ and 1.1×10⁻¹⁰, respectively). Also, these two SNPs had significant indirect effects on lung adenocarcinoma risk through nicotine dependence (p = 0.003 and 0.007). Gene variations rs2736100 and rs2853676 in TERT and rs401681 and rs31489 in CLPTM1L had significant direct associations on lung adenocarcinoma without indirect effects through nicotine dependence. Our findings suggest that nicotine dependence plays an important role between genetic variants in the CHRNA5/A3/B4 region, especially CHRNA3, and lung adenocarcinoma. This may provide valuable information for understanding the pathogenesis of lung adenocarcinoma and for conducting personalized smoking cessation interventions.     

DNA Methylation Status of Epithelial-Mesenchymal Transition (EMT) - Related Genes Is Associated with Severe Clinical Phenotypes in Ulcerative Colitis (UC)

Background

Epithelial-to-mesenchymal transition (EMT) is a phenomenon that allows the conversion of adherent epithelial cells to a mesenchymal cell phenotype, which enhances migratory capacity and invasiveness. Recent studies have suggested that EMT contributes to the pathogenesis of ulcerative colitis (UC). We investigated the promoter DNA methylation status of EMT-related genes in the colonic mucosa in UC.

Methods

Colonic biopsies were obtained from the rectal inflammatory mucosa of 86 UC patients and the non-inflammatory proximal colonic mucosa of 10 paired patients. Bisulfite pyrosequencing was used to quantify the methylation of 5 candidate CpG island promoters (NEUROG1, CDX1, miR-1247, CDH1, and CDH13) and LINE1.

Results

Using an unsupervised hierarchical clustering analysis, inflamed rectal mucosa was well separated from mucosa that appeared normal. The CDH1 and CDH13 promoters were significantly associated with patient age (p = 0.04, 0.03, respectively). A similar trend was found between those genes and the duration of disease (CDH1: p = 0.07, CDH13: p = 0.0002, mean of both: p<0.00001). Several positive associations were found between hypermethylation and severe clinical phenotypes (CDX1 and miR-1247 and a refractory phenotype: p = 0.04 and 0.006, respectively. miR-1247 and CDH1 hyper methylation and a more severe Mayo endoscopic subscore: miR-1247: p = 0.0008, CDH1: p = 0.03, mean of both: p = 0.003). When the severe clinical phenotype was defined as having any of five phenotypes (hospitalized more than twice, highest Mayo endoscopic subscore, steroid dependence, refractory, or a history of surgery) miR-1247 hypermethylation was associated with the same phenotype (p = 0.008).

Conclusions

Our data suggest that variability in the methylation status of EMT-related genes is associated with more severe clinical phenotypes in UC.     

Association of CHRNA5-A3-B4 Variation with Esophageal Squamous Cell Carcinoma Risk and Smoking Behaviors in a Chinese Population

Background

CHRNA5-A3-B4, the gene cluster encoding nicotinic acetylcholine receptor subunits, is associated with lung cancer risk and smoking behaviors in people of European descent. Because cigarette smoking is also a major risk factor for esophageal squamous cell carcinoma (ESCC), we investigated the associations between variants in CHRNA5-A3-B4 and ESCC risk, as well as smoking behaviors, in a Chinese population.

Methods

A case-control study of 866 ESCC patients and 952 healthy controls was performed to study the association of polymorphisms (rs667282 and rs3743073) in CHRNA5-A3-B4 with cancer risk using logistic regression models. The relationships between CHRNA5-A3-B4 polymorphisms and smoking behaviors that can be quantified by cigarettes smoked per day (CPD) and pack-years of smoking were separately estimated with Kruskal-Wallis tests among all 840 smokers.

Results

CHRNA5-A3-B4 rs667282 TT/TG genotypes were associated with significantly increased risk of ESCC [adjusted odds ratio (OR) = 1.32, 95% confidence interval (CI) = 1.03 – 1.69, P = 0.029]. The increased ESCC risk was even higher among younger subjects (≤60 years) (OR = 1.44, 95% CI = 1.04 – 1.98, P = 0.024). These effects were not found in another polymorphism rs3743073. No evident association between the two polymorphisms and smoking behaviors was observed.

Conclusions

These results support the hypothesis that CHRNA5-A3-B4 is a susceptibility gene cluster for ESCC. The relationship between CHRNA5-A3-B4 and smoking behaviors in a Chinese population needs further investigation.     

Association between DAPK1 Promoter Methylation and Cervical Cancer: A Meta-Analysis

Background

Death-associated protein kinase1 (DAPK1) is an important tumor suppressor gene. DNA methylation can inactivate genes, which has often been observed in the carcinogenesis of cervical cancer. During the past several decades, many studies have explored the association between DAPK1 promoter methylation and cervical cancer. However, many studies were limited by the small samples size and the findings were inconsistent among them. Thus, we conducted a meta-analysis to assess the association between DAPK1 promoter methylation and cervical cancer.

Methods

We systematically searched eligible studies in the PubMed, Web of Science, EMBASE and CNKI databases. Using meta-regression, subgroup analysis and sensitivity analysis, we explored the potential sources of heterogeneity. The odds ratio (OR) and 95% confidence interval (95% CI) were calculated by Meta-Analysis in R.

Results

A total of 15 studies from 2001 to 2012, comprising 818 tumor tissues samples and 671 normal tissues samples, were analyzed in this meta-analysis. The frequencies of DAPK1 promoter methylation ranged from 30.0% to 78.6% (median, 59.3%) in cervical cancer tissue and 0.0% to 46.7% (median, 7.8%) in normal cervical tissue. The pooled OR was 19.66 (95%CI = 8.72–44.31) with the random effects model, and heterogeneity was found through the sensitivity analysis. The I² = 60% (P = 0.002) decreased to I² = 29.2% (P = 0.144) when one heterogeneous study was excluded, and the pooled OR increased to 21.80 (95%CI = 13.44–35.36) with the fixed effects model.

Conclusion

The results suggested a strong association between DAPK1 promoter methylation and cervical cancer. This study also indicated that DAPK1 promoter methylation may be a biomarker during cervical carcinogenesis that might serve as an early indication of cervical cancer.     

MLH1 Promoter Methylation Frequency in Colorectal Cancer Patients and Related Clinicopathological and Molecular Features

Purpose

To describe the frequency of MLH1 promoter methylation in colorectal cancer (CRC); to explore the associations between MLH1 promoter methylation and clinicopathological and molecular factors using a systematic review and meta-analysis.

Methods

A literature search of the PubMed and Embase databases was conducted to identify relevant articles published up to September 7, 2012 that described the frequency of MLH1 promoter methylation or its associations with clinicopathological and molecular factors in CRC. The pooled frequency, odds ratio (OR) and 95% confidence intervals (95% CI) were calculated.

Results

The pooled frequency of MLH1 promoter methylation in unselected CRC was 20.3% (95% CI: 16.8–24.1%). They were 18.7% (95% CI: 14.7–23.6%) and 16.4% (95% CI: 11.9–22.0%) in sporadic and Lynch syndrome (LS) CRC, respectively. Significant associations were observed between MLH1 promoter methylation and gender (pooled OR = 1.641, 95% CI: 1.215–2.215; P = 0.001), tumor location (pooled OR = 3.804, 95% CI: 2.715–5.329; P<0.001), tumor differentiation (pooled OR = 2.131, 95% CI: 1.464–3.102; P<0.001), MSI (OR: 27.096, 95% CI: 13.717–53.526; P<0.001). Significant associations were also observed between MLH1 promoter methylation and MLH1 protein expression, BRAF mutation (OR = 14.919 (95% CI: 6.427–34.631; P<0.001) and 9.419 (95% CI: 2.613–33.953; P = 0.001), respectively).

Conclusion

The frequency of MLH1 promoter methylation in unselected CRC was 20.3%. They were 18.7% in sporadic CRC and 16.4% in LS CRC, respectively. MLH1 promoter methylation may be significantly associated with gender, tumor location, tumor differentiation, MSI, MLH1 protein expression, and BRAF mutation.