CpG Islands Undermethylation in Human Genomic Regions under Selective Pressure

DNA methylation at CpG islands (CGIs) is one of the most intensively studied epigenetic mechanisms. It is fundamental for cellular differentiation and control of transcriptional potential. DNA methylation is involved also in several processes that are central to evolutionary biology, including phenotypic plasticity and evolvability. In this study, we explored the relationship between CpG islands methylation and signatures of selective pressure in Homo Sapiens, using a computational biology approach. By analyzing methylation data of 25 cell lines from the Encyclopedia of DNA Elements (ENCODE) Consortium, we compared the DNA methylation of CpG islands in genomic regions under selective pressure with the methylation of CpG islands in the remaining part of the genome. To define genomic regions under selective pressure, we used three different methods, each oriented to provide distinct information about selective events. Independently of the method and of the cell type used, we found evidences of undermethylation of CGIs in human genomic regions under selective pressure. Additionally, by analyzing SNP frequency in CpG islands, we demonstrated that CpG islands in regions under selective pressure show lower genetic variation. Our findings suggest that the CpG islands in regions under selective pressure seem to be somehow more “protected” from methylation when compared with other regions of the genome

Promoter Methylation in Head and Neck Squamous Cell Carcinoma Cell Lines Is Significantly Different than Methylation in Primary Tumors and Xenografts

Studies designed to identify novel methylation events related to cancer often employ cancer cell lines in the discovery phase of the experiments and have a relatively low rate of discovery of cancer-related methylation events. An alternative algorithm for discovery of novel methylation in cancer uses primary tumor-derived xenografts instead of cell lines as the primary source of nucleic acid for evaluation. We evaluated DNA extracted from primary head and neck squamous cell carcinomas (HNSCC), xenografts grown from these primary tumors in nude mice, HNSCC-derived cell lines, normal oral mucosal samples, and minimally transformed oral keratinocyte-derived cell lines using Illumina Infinum Humanmethylation 27 genome-wide methylation microarrays. We found >2,200 statistically significant methylation differences between cancer cell lines and primary tumors and when comparing normal oral mucosa to keratinocyte cell lines. We found no statistically significant promoter methylation differences between primary tumor xenografts and primary tumors. This study demonstrates that tumor-derived xenografts are highly accurate representations of promoter methylation in primary tumors and that cancer derived cell lines have significant drawbacks for discovery of promoter methylation alterations in primary tumors. These findings also support use of primary tumor xenografts for the study of methylation in cancer, drug discovery, and the development of personalized cancer treatments

Interaction between genetic and epigenetic variation defines gene expression patterns at the asthma-associated locus 17q12-q21 in lymphoblastoid cell lines

Phenotypic variation results from variation in gene expression, which is modulated by genetic and/or epigenetic factors. To understand the molecular basis of human disease, interaction between genetic and epigenetic factors needs to be taken into account. The asthma-associated region 17q12-q21 harbors three genes, the zona pellucida binding protein 2 (ZPBP2), gasdermin B (GSDMB) and ORM1-like 3 (ORMDL3), that show allele-specific differences in expression levels in lymphoblastoid cell lines (LCLs) and CD4+ T cells. Here, we report a molecular dissection of allele-specific transcriptional regulation of the genes within the chromosomal region 17q12-q21 combining in vitro transfection, formaldehyde-assisted isolation of regulatory elements, chromatin immunoprecipitation and DNA methylation assays in LCLs. We found that a single nucleotide polymorphism rs4795397 influences the activity of ZPBP2 promoter in vitro in an allele-dependent fashion, and also leads to nucleosome repositioning on the asthma-associated allele. However, variable methylation of exon 1 of ZPBP2 masks the strong genetic effect on ZPBP2 promoter activity in LCLs. In contrast, the ORMDL3 promoter is fully unmethylated, which allows detection of genetic effects on its transcription. We conclude that the cis-regulatory effects on 17q12-q21 gene expression result from interaction between several regulatory polymorphisms and epigenetic factors within the cis-regulatory haplotype region

Variation in Array Size, Monomer Composition and Expression of the Macrosatellite DXZ4

Macrosatellites are some of the most polymorphic regions of the human genome, yet many remain uncharacterized despite the association of some arrays with disease susceptibility. This study sought to explore the polymorphic nature of the X-linked macrosatellite DXZ4. Four aspects of DXZ4 were explored in detail, including tandem repeat copy number variation, array instability, monomer sequence polymorphism and array expression. DXZ4 arrays contained between 12 and 100 3.0 kb repeat units with an average array containing 57. Monomers were confirmed to be arranged in uninterrupted tandem arrays by restriction digest analysis and extended fiber FISH, and therefore DXZ4 encompasses 36–288 kb of Xq23. Transmission of DXZ4 through three generations in three families displayed a high degree of meiotic instability (8.3%), consistent with other macrosatellite arrays, further highlighting the unstable nature of these sequences in the human genome. Subcloning and sequencing of complete DXZ4 monomers identified numerous single nucleotide polymorphisms and alleles for the three microsatellite repeats located within each monomer. Pairwise comparisons of DXZ4 monomer sequences revealed that repeat units from an array are more similar to one another than those originating from different arrays. RNA fluorescence in situ hybridization revealed significant variation in DXZ4 expression both within and between cell lines. DXZ4 transcripts could be detected originiating from both the active and inactive X chromosome. Expression levels of DXZ4 varied significantly between males, but did not relate to the size of the array, nor did inheritance of the same array result in similar expression levels. Collectively, these studies provide considerable insight into the polymorphic nature of DXZ4, further highlighting the instability and variation potential of macrosatellites in the human genome

Adhesion molecule gene variants and plasma protein levels in patients with suspected obstructive sleep apnea.

Study objectivesUntreated obstructive sleep apnea (OSA) patients have an increased risk of cardiovascular disease (CVD). Adhesion molecules, including soluble E-selectin (sE-selectin), intercellular adhesion molecule-1 (ICAM-1), and vascular adhesion molecule-1 (VCAM-1), are associated with incident CVD. We hypothesized that specific genetic variants will be associated with plasma levels of adhesion molecules in suspected OSA patients. We also hypothesized that there may be an interaction between these variants and OSA.MethodsWe measured levels of sE-selectin, sICAM-1 and sVCAM-1 in 491 patients with suspected OSA and genotyped them for 20 polymorphisms.ResultsThe most significant association was between the ABO rs579459 polymorphism and sE-selectin levels (P = 7×10-21), with the major allele T associated with higher levels. The direction of effect and proportion of the variance in sE-selectin levels accounted for by rs579459 (16%) was consistent with estimates from non-OSA cohorts. In a multivariate regression analysis, addition of rs579459 improved the model performance in predicting sE-selectin levels. Three polymorphisms were nominally associated with sICAM-1 levels but none with sVCAM-1 levels. The combination of severe OSA and two rs579459 T alleles identified a group of patients with high sE-selectin levels; however, the increase in sE-selectin levels associated with severe OSA was greater in patients without two T alleles (P = 0.05 test for interaction).ConclusionsThese genetic polymorphisms may help to identify patients at greatest risk of incident CVD and may help in developing a more precision-based approach to OSA care

Nitric oxide synthase polymorphisms, gene expression and lung function in chronic obstructive pulmonary disease

Background: Due to the pleiotropic effects of nitric oxide (NO) within the lungs, it is likely that NO is a significant factor in the pathogenesis of chronic obstructive pulmonary disease (COPD). The aim of this study was to test for association between single nucleotide polymorphisms (SNPs) in three NO synthase (NOS) genes and lung function, as well as to examine gene expression and protein levels in relation to the genetic variation. Methods: One SNP in each NOS gene (neuronal NOS (NOS1), inducible NOS (NOS2), and endothelial NOS (NOS3)) was genotyped in the Lung Health Study (LHS) and correlated with lung function. One SNP (rs1800779) was also analyzed for association with COPD and lung function in four COPD case–control populations. Lung tissue expression of NOS3 mRNA and protein was tested in individuals of known genotype for rs1800779. Immunohistochemistry of lung tissue was used to localize NOS3 expression. Results: For the NOS3 rs1800779 SNP, the baseline forced expiratory volume in one second in the LHS was significantly higher in the combined AG + GG genotypic groups compared with the AA genotypic group. Gene expression and protein levels in lung tissue were significantly lower in subjects with the AG + GG genotypes than in AA subjects. NOS3 protein was expressed in the airway epithelium and subjects with the AA genotype demonstrated higher NOS3 expression compared with AG and GG individuals. However, we were not able to replicate the associations with COPD or lung function in the other COPD study groups. Conclusions: Variants in the NOS genes were not associated with lung function or COPD status. However, the G allele of rs1800779 resulted in a decrease of NOS3 gene expression and protein levels and this has implications for the numerous disease states that have been associated with this polymorphism.Non UBCMedicine, Faculty ofMedicine, Department ofRespiratory Medicine, Division ofScience, Faculty ofMicrobiology and Immunology, Department ofAnesthesiology, Pharmacology and Therapeutics, Department ofReviewedFacult

Alpha-1 Antitrypsin MZ Heterozygosity Is an Endotype of Chronic Obstructive Pulmonary Disease

RATIONALE: Multiple studies have demonstrated an increased risk of chronic obstructive pulmonary disease (COPD) in heterozygous carriers of the AAT (alpha-1 antitrypsin) Z allele. However, it is not known if MZ subjects with COPD are phenotypically different from noncarriers (MM genotype) with COPD. OBJECTIVE: To assess if MZ subjects with COPD have different clinical features compared with MM subjects with COPD. METHODS: Genotypes of SERPINA1 were ascertained by using whole-genome sequencing data in three independent studies. We compared outcomes between MM subjects with COPD and MZ subjects with COPD in each study and combined the results in a meta-analysis. We performed longitudinal and survival analyses to compare outcomes in MM and MZ subjects with COPD over time. MEASUREMENTS AND MAIN RESULTS: We included 290 MZ subjects with COPD and 6,184 MM subjects with COPD across the three studies. MZ subjects had a lower FEV1% predicted and greater quantitative emphysema on chest computed tomography scans compared with MM subjects. In a meta-analysis, the FEV1 was 3.9% lower (95% confidence interval [CI], −6.55% to −1.26%) and emphysema (the percentage of lung attenuation areas <−950 HU) was 4.14% greater (95% CI, 1.44% to 6.84%) in MZ subjects. We found one gene, PGF (placental growth factor), to be differentially expressed in lung tissue from one study between MZ subjects and MM subjects. CONCLUSIONS: Carriers of the AAT Z allele (those who were MZ heterozygous) with COPD had lower lung function and more emphysema than MM subjects with COPD. Taken with the subtle differences in gene expression between the two groups, our findings suggest that MZ subjects represent an endotype of COPD

Methylome-wide comparison of human genomic DNA extracted from whole blood and from EBV-transformed lymphocyte cell lines

DNA from Epstein–Barr virus-transformed lymphocyte cell lines (LCLs) has proven useful for studies of genetic sequence polymorphisms. Whether LCL DNA is suitable for methylation studies is less clear. We conduct a genome-wide methylation investigation using an array set with 45 million probes to investigate the methylome of LCL DNA and technical duplicates of WB DNA from the same 10 individuals. We focus specifically on methylation sites that show variation between individuals and, therefore, are potentially useful as biomarkers. The sample correlations for the methylation variable probes ranged from 0.69 to 0.78 for the WB duplicates and from 0.27 to 0.72 for WB vs LCL. To compare the pattern of the methylation signals, we grouped adjacent probes based on their inter-correlations. These analyses showed ∼29 000 and ∼14 000 blocks in WB and LCL, respectively. Merely 31% of the methylated regions detected in WB were detectable in LCLs. Furthermore, we observed significant differences in mean difference between WB and LCL as compared with duplicates of WB (P-value =2.2 × 10(−16)). Our study shows that there are substantial differences in the DNA methylation patterns between LCL and WB. Thus, LCL DNA should not be used as a proxy for WB DNA in methylome-wide studies