DNA Methylation Changes Separate Allergic Patients from Healthy Controls and May Reflect Altered CD4+ T-Cell Population Structure

Altered DNA methylation patterns in CD4⁺ T-cells indicate the importance of epigenetic mechanisms in inflammatory diseases. However, the identification of these alterations is complicated by the heterogeneity of most inflammatory diseases. Seasonal allergic rhinitis (SAR) is an optimal disease model for the study of DNA methylation because of its well-defined phenotype and etiology. We generated genome-wide DNA methylation (N_patients = 8, N_controls = 8) and gene expression (N_patients = 9, N_controls = 10) profiles of CD4⁺ T-cells from SAR patients and healthy controls using Illumina''s HumanMethylation450 and HT-12 microarrays, respectively. DNA methylation profiles clearly and robustly distinguished SAR patients from controls, during and outside the pollen season. In agreement with previously published studies, gene expression profiles of the same samples failed to separate patients and controls. Separation by methylation (N_patients = 12, N_controls = 12), but not by gene expression (N_patients = 21, N_controls = 21) was also observed in an in vitro model system in which purified PBMCs from patients and healthy controls were challenged with allergen. We observed changes in the proportions of memory T-cell populations between patients (N_patients = 35) and controls (N_controls = 12), which could explain the observed difference in DNA methylation. Our data highlight the potential of epigenomics in the stratification of immune disease and represents the first successful molecular classification of SAR using CD4⁺ T cells.     

A Genome-Wide Methylation Study on Essential Hypertension in Young African American Males

Objective

There is emerging evidence from animal studies suggesting a key role for methylation in the pathogenesis of essential hypertension. However, to date, very few studies have investigated the role of methylation in the development of human hypertension, and none has taken a genome-wide approach. Based on the recent studies that highlight the involvement of inflammation in the development of hypertension, we hypothesize that changes in DNA methylation of leukocytes are involved in the pathogenesis of hypertension.

Method & Results

We conducted a genome-wide methylation analysis on 8 hypertensive cases and 8 normotensive age-matched controls aged 14–23 years and performed validation of the most significant CpG sites in 2 genes in an independent sample of 36 hypertensive cases and 60 normotensive controls aged 14–30 years. Validation of the CpG sites in the SULF1 gene was further conducted in a second replication sample of 36 hypertensive cases and 34 controls aged 15.8–40 years. A CpG site in the SULF1 gene showed higher methylation levels in cases than in healthy controls in the genome-wide step (p = 6.2×10⁻⁵), which was confirmed in the validation step (p = 0.011) for subjects ≤30 years old but was not significant for subjects of all ages combined (p = 0.095).

Conclusion

The identification of a difference in a blood leukocyte DNA methylation site between hypertensive cases and normotensive controls suggests that changes in DNA methylation may play an important role in the pathogenesis of hypertension. The age dependency of the effect further suggests complexity of epigenetic regulation in this age-related disease.     

Selection of Suitable Housekeeping Genes for Real-Time Quantitative PCR in CD4+ Lymphocytes from Asthmatics with or without Depression

Objective

No optimal housekeeping genes (HKGs) have been identified for CD4⁺ T cells from non-depressive asthmatic and depressive asthmatic adults for normalizing quantitative real-time PCR (qPCR) assays. The aim of present study was to select appropriate HKGs for gene expression analysis in purified CD4⁺ T cells from these asthmatics.

Methods

Three groups of subjects (Non-depressive asthmatic, NDA, n = 10, Depressive asthmatic, DA, n = 11, and Healthy control, HC, n = 10 respectively) were studied. qPCR for 9 potential HKGs, namely RNA, 28S ribosomal 1 (RN28S1), ribosomal protein, large, P0 (RPLP0), actin, beta (ACTB), cyclophilin A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase 1 (PGK1), beta-2-microglobulin (B2M), glucuronidase, beta (GUSB) and ribosomal protein L13a (RPL13A), was performed. Then the data were analyzed with three different applications namely BestKeeper, geNorm, and NormFinder.

Results

The analysis of gene expression data identified B2M and RPLP0 as the most stable reference genes and showed that the level of PPIA was significantly different among subjects of three groups when the two best HKGs identified were applied. Post-hoc analysis by Student-Newman-Keuls correction shows that depressive asthmatics and non-depressive asthmatics exhibited lower expression level of PPIA than healthy controls (p<0.05).

Conclusions

B2M and RPLP0 were identified as the most optimal HKGs in gene expression studies involving human blood CD4⁺ T cells derived from normal, depressive asthmatics and non-depressive asthmatics. The suitability of using the PPIA gene as the HKG for such studies was questioned due to its low expression in asthmatics.     

A Novel MMP12 Locus Is Associated with Large Artery Atherosclerotic Stroke Using a Genome-Wide Age-at-Onset Informed Approach

Genome-wide association studies (GWAS) have begun to identify the common genetic component to ischaemic stroke (IS). However, IS has considerable phenotypic heterogeneity. Where clinical covariates explain a large fraction of disease risk, covariate informed designs can increase power to detect associations. As prevalence rates in IS are markedly affected by age, and younger onset cases may have higher genetic predisposition, we investigated whether an age-at-onset informed approach could detect novel associations with IS and its subtypes; cardioembolic (CE), large artery atherosclerosis (LAA) and small vessel disease (SVD) in 6,778 cases of European ancestry and 12,095 ancestry-matched controls. Regression analysis to identify SNP associations was performed on posterior liabilities after conditioning on age-at-onset and affection status. We sought further evidence of an association with LAA in 1,881 cases and 50,817 controls, and examined mRNA expression levels of the nearby genes in atherosclerotic carotid artery plaques. Secondly, we performed permutation analyses to evaluate the extent to which age-at-onset informed analysis improves significance for novel loci. We identified a novel association with an MMP12 locus in LAA (rs660599; p = 2.5×10⁻⁷), with independent replication in a second population (p = 0.0048, OR(95% CI) = 1.18(1.05–1.32); meta-analysis p = 2.6×10⁻⁸). The nearby gene, MMP12, was significantly overexpressed in carotid plaques compared to atherosclerosis-free control arteries (p = 1.2×10⁻¹⁵; fold change = 335.6). Permutation analyses demonstrated improved significance for associations when accounting for age-at-onset in all four stroke phenotypes (p<0.001). Our results show that a covariate-informed design, by adjusting for age-at-onset of stroke, can detect variants not identified by conventional GWAS.     

Pharmacogenomics of Interferon-? Therapy in Multiple Sclerosis: Baseline IFN Signature Determines Pharmacological Differences between Patients

Background

Multiple sclerosis (MS) is a heterogeneous disease. In order to understand the partial responsiveness to IFNß in Relapsing Remitting MS (RRMS) we studied the pharmacological effects of IFNß therapy.

Methodology

Large scale gene expression profiling was performed on peripheral blood of 16 RRMS patients at baseline and one month after the start of IFNß therapy. Differential gene expression was analyzed by Significance Analysis of Microarrays. Subsequent expression analyses on specific genes were performed after three and six months of treatment. Peripheral blood mononuclear cells (PBMC) were isolated and stimulated in vitro with IFNß. Genes of interest were measured and validated by quantitative realtime PCR. An independent group of 30 RRMS patients was used for validation.

Principal Findings

Pharmacogenomics revealed a marked variation in the pharmacological response to IFNß between patients. A total of 126 genes were upregulated in a subset of patients whereas in other patients these genes were downregulated or unchanged after one month of IFNß therapy. Most interestingly, we observed that the extent of the pharmacological response correlates negatively with the baseline expression of a specific set of 15 IFN response genes (R = −0.7208; p = 0.0016). The negative correlation was maintained after three (R = −0.7363; p = 0.0027) and six (R = −0.8154; p = 0.0004) months of treatment, as determined by gene expression levels of the most significant correlating gene. Similar results were obtained in an independent group of patients (n = 30; R = −0.4719; p = 0.0085). Moreover, the ex vivo results could be confirmed by in vitro stimulation of purified PBMCs at baseline with IFNß indicating that differential responsiveness to IFNß is an intrinsic feature of peripheral blood cells at baseline.

Conclusion

These data imply that the expression levels of IFN response genes in the peripheral blood of MS patients prior to treatment could serve a role as biomarker for the differential clinical response to IFNß.     

The Association Study between Twenty One Polymorphisms in Seven Candidate Genes and Coronary Heart Diseases in Chinese Han Population

Previous genome-wide association studies (GWAS) in multiple populations identified several genetic loci for coronary heart diseases (CHD). Here we utilized a 2-stage candidate gene association strategy in Chinese Han population to shed light on the putative association between several metabolic-related candidate genes and CHD. At the 1^st stage, 190 patients with CHD and 190 controls were genotyped through the MassARRAY platform. At the 2^nd stage, a larger sample including 400 patients and 392 controls was genotyped by the High Resolution Melt (HRM) method to confirm or rule out the associations with CHD. MLXIP expression level was quantified by the real time PCR in 65 peripheral blood samples. From the 21 studied single nucleotide polymorphisms (SNPs) of seven candidate genes: MLXIPL, MLXIP, MLX, ADIPOR1, VDR, SREBF1 and NR1H3, only one tag SNP rs4758685 (T→C) was found to be statistically associated with CHD (P-value = 0.02, Odds ratio (OR) of 0.83). After adjustment for the age, sex, lipid levels and diabetes, the association remained significant (P-value = 0.03). After adjustment for the hypertension, P-value became 0.20 although there was a significant difference in the allele distribution between the CHD patients with hypertension and the controls (P-value = 0.04, 406 vs 582). In conclusion, among the 21 tested SNPs, we identified a novel association between rs4758685 of MLXIP gene and CHD. The C allele of common variant rs4758685 interacted with hypertension, and was found to be protective against CHD in both allelic and genotypic models in Chinese Han population.     

Assessment of the Association of Matrix Metalloproteinases with Myopia,Refractive Error and Ocular Biometric Measures in an Australian Cohort

Extracellular matrix proteins have been implicated in protein remodelling of the sclera in refractive error. The matrix metalloproteinases (MMPs) falling into the collagenase (MMP1, MMP8, MMP13), gelatinase (MMP2, MMP9) and stromelysin (MMP3, MMP10, MMP11) functional groups are particularly important. We wished to assess their association with myopia, refractive error and ocular biometric measures in an Australian cohort. A total of 543 unrelated individuals of Caucasian ethnicity were genotyped including 269 myopes (≤−1.0D) and 274 controls (>−1.0D). Tag single nucleotide polymorphisms (SNPs) (n = 53) were chosen to encompass these eight MMPs. Association tests were performed using linear and logistic regression analysis with age and gender as covariates. Spherical equivalent, myopia, axial length, anterior chamber depth and corneal curvature were the phenotypes of interest. Initial findings indicated that the best p values for each trait were 0.02 for myopia at rs2274755 (MMP9), 0.02 for SE at both rs3740938 (MMP8) and rs131451 (MMP11), 0.01 for axial length at rs11225395 (MMP8), 0.01 for anterior chamber depth at rs498186 (MMP1) and 0.02 at rs10488 (MMP1). However, following correction for multiple testing, none of these SNPs remained statistically significant. Our data suggests that the MMPs in the collagenase, gelatinase and stromelysin categories do not appear to be associated with myopia, refractive error or ocular biometric measures in this cohort.     

MicroRNA expression aberration as potential peripheral blood biomarkers for schizophrenia

Since brain tissue is not readily accessible, a new focus in search of biomarkers for schizophrenia is blood-based expression profiling of non-protein coding genes such as microRNAs (miRNAs), which regulate gene expression by inhibiting the translation of messenger RNAs. This study aimed to identify potential miRNA signature for schizophrenia by comparing genome-wide miRNA expression profiles in patients with schizophrenia vs. healthy controls. A genome-wide miRNA expression profiling was performed using a Taqman array of 365 human miRNAs in the mononuclear leukocytes of a learning set of 30 cases and 30 controls. The discriminating performance of potential biomarkers was validated in an independent testing set of 60 cases and 30 controls. The expression levels of the miRNA signature were then evaluated for their correlation with the patients'' clinical symptoms, neurocognitive performances, and neurophysiological functions. A seven-miRNA signature (hsa-miR-34a, miR-449a, miR-564, miR-432, miR-548d, miR-572 and miR-652) was derived from a supervised classification with internal cross-validation, with an area under the curve (AUC) of receiver operating characteristics of 93%. The putative signature was then validated in the testing set, with an AUC of 85%. Among these miRNAs, miR-34a was differentially expressed between cases and controls in both the learning (P = 0.005) and the testing set (P = 0.002). These miRNAs were differentially correlated with patients'' negative symptoms, neurocognitive performance scores, and event-related potentials. The results indicated that the mononuclear leukocyte-based miRNA profiling is a feasible way to identify biomarkers for schizophrenia, and the seven-miRNA signature warrants further investigation.     

Functional Variants in NFKBIE and RTKN2 Involved in Activation of the NF-κB Pathway Are Associated with Rheumatoid Arthritis in Japanese

Rheumatoid arthritis is an autoimmune disease with a complex etiology, leading to inflammation of synovial tissue and joint destruction. Through a genome-wide association study (GWAS) and two replication studies in the Japanese population (7,907 cases and 35,362 controls), we identified two gene loci associated with rheumatoid arthritis susceptibility (NFKBIE at 6p21.1, rs2233434, odds ratio (OR) = 1.20, P = 1.3×10⁻¹⁵; RTKN2 at 10q21.2, rs3125734, OR = 1.20, P = 4.6×10⁻⁹). In addition to two functional non-synonymous SNPs in NFKBIE, we identified candidate causal SNPs with regulatory potential in NFKBIE and RTKN2 gene regions by integrating in silico analysis using public genome databases and subsequent in vitro analysis. Both of these genes are known to regulate the NF-κB pathway, and the risk alleles of the genes were implicated in the enhancement of NF-κB activity in our analyses. These results suggest that the NF-κB pathway plays a role in pathogenesis and would be a rational target for treatment of rheumatoid arthritis.     

Genetically Dependent ERBB3 Expression Modulates Antigen Presenting Cell Function and Type 1 Diabetes Risk

Type 1 diabetes (T1D) is an autoimmune disease resulting from the complex interaction between multiple susceptibility genes, environmental factors and the immune system. Over 40 T1D susceptibility regions have been suggested by recent genome-wide association studies; however, the specific genes and their role in the disease remain elusive. The objective of this study is to identify the susceptibility gene(s) in the 12q13 region and investigate the functional link to the disease pathogenesis. A total of 19 SNPs in the 12q13 region were analyzed by the TaqMan assay for 1,434 T1D patients and 1,865 controls. Thirteen of the SNPs are associated with T1D (best p = 4×10⁻¹¹), thus providing confirmatory evidence for at least one susceptibility gene in this region. To identify candidate genes, expression of six genes in the region was analyzed by real-time RT-PCR for PBMCs from 192 T1D patients and 192 controls. SNP genotypes in the 12q13 region are the main factors that determine ERBB3 mRNA levels in PBMCs. The protective genotypes for T1D are associated with higher ERBB3 mRNA level (p<10⁻¹⁰). Furthermore, ERBB3 protein is expressed on the surface of CD11c⁺ cells (dendritic cells and monocytes) in peripheral blood after stimulation with LPS, polyI:C or CpG. Subjects with protective genotypes have significantly higher percentages of ERBB3⁺ monocytes and dendritic cells (p = 1.1×10⁻⁹); and the percentages of ERBB3⁺ cells positively correlate with the ability of APC to stimulate T cell proliferation (R² = 0.90, p<0.0001). Our results indicate that ERBB3 plays a critical role in determining APC function and potentially T1D pathogenesis.     

A Genome-Wide Investigation of Copy Number Variation in Patients with Sporadic Brain Arteriovenous Malformation

Background

Brain arteriovenous malformations (BAVM) are clusters of abnormal blood vessels, with shunting of blood from the arterial to venous circulation and a high risk of rupture and intracranial hemorrhage. Most BAVMs are sporadic, but also occur in patients with Hereditary Hemorrhagic Telangiectasia, a Mendelian disorder caused by mutations in genes in the transforming growth factor beta (TGFβ) signaling pathway.

Methods

To investigate whether copy number variations (CNVs) contribute to risk of sporadic BAVM, we performed a genome-wide association study in 371 sporadic BAVM cases and 563 healthy controls, all Caucasian. Cases and controls were genotyped using the Affymetrix 6.0 array. CNVs were called using the PennCNV and Birdsuite algorithms and analyzed via segment-based and gene-based approaches. Common and rare CNVs were evaluated for association with BAVM.

Results

A CNV region on 1p36.13, containing the neuroblastoma breakpoint family, member 1 gene (NBPF1), was significantly enriched with duplications in BAVM cases compared to controls (P = 2.2×10⁻⁹); NBPF1 was also significantly associated with BAVM in gene-based analysis using both PennCNV and Birdsuite. We experimentally validated the 1p36.13 duplication; however, the association did not replicate in an independent cohort of 184 sporadic BAVM cases and 182 controls (OR = 0.81, P = 0.8). Rare CNV analysis did not identify genes significantly associated with BAVM.

Conclusion

We did not identify common CNVs associated with sporadic BAVM that replicated in an independent cohort. Replication in larger cohorts is required to elucidate the possible role of common or rare CNVs in BAVM pathogenesis.     

Analysis of Germline Variants in CDH1, IGFBP3, MMP1, MMP3, STK15 and VEGF in Familial and Sporadic Renal Cell Carcinoma

Background

The investigation of rare familial forms of kidney cancer has provided important insights into the biology of sporadic renal cell carcinoma (RCC). In particular, the identification of the von Hippel Lindau (VHL) familial cancer syndrome gene (VHL) provided the basis for the discovery that VHL is somatically inactivated in most sporadic clear cell RCC. Many cases of familial RCC do not have mutations in known RCC susceptibility genes and there is evidence that genetic modifiers may influence the risk of RCC in VHL disease patients. Hence we hypothesised that low-penetrance functional genetic variants in pathways related to the VHL protein (pVHL) function might (a) modify the phenotypic expression of VHL disease and/or (b) predispose to sporadic RCC.

Methodology/Principal Findings

We tested this hypothesis for functional polymorphisms in CDH1 (rs16260), IGFBP3 (rs2854744), MMP1 (rs1799750), MMP3 (rs679620), STK15 (rs2273535) and VEGF (rs1570360). We observed that variants of MMP1 and MMP3 were significant modifiers of RCC risk (and risks of retinal angioma and cerebellar haemangioblastoma) in VHL disease patients. In addition, higher frequencies of the MMP1 rs1799750 2G allele (p = 0.017, OR 1.49, 95%CI 1.06–2.08) and the MMP1/MMP3 rs1799750/rs679620 2G/G haplotype (OR 1.45, 95%CI 1.01–2.10) were detected in sporadic RCC patients than in controls (n = 295).

Conclusions/Significance

These findings (a) represent the first example of genetic modifiers of RCC risk in VHL disease, (b) replicate a previous report of an association between MMP1/MMP3 variants and sporadic RCC and (c) further implicate MMP1/MMP3-related pathways in the pathogenesis of familial and sporadic RCC.     

Hybrid Models Identified a 12-Gene Signature for Lung Cancer Prognosis and Chemoresponse Prediction

Background

Lung cancer remains the leading cause of cancer-related deaths worldwide. The recurrence rate ranges from 35–50% among early stage non-small cell lung cancer patients. To date, there is no fully-validated and clinically applied prognostic gene signature for personalized treatment.

Methodology/Principal Findings

From genome-wide mRNA expression profiles generated on 256 lung adenocarcinoma patients, a 12-gene signature was identified using combinatorial gene selection methods, and a risk score algorithm was developed with Naïve Bayes. The 12-gene model generates significant patient stratification in the training cohort HLM & UM (n = 256; log-rank P = 6.96e-7) and two independent validation sets, MSK (n = 104; log-rank P = 9.88e-4) and DFCI (n = 82; log-rank P = 2.57e-4), using Kaplan-Meier analyses. This gene signature also stratifies stage I and IB lung adenocarcinoma patients into two distinct survival groups (log-rank P<0.04). The 12-gene risk score is more significant (hazard ratio = 4.19, 95% CI: [2.08, 8.46]) than other commonly used clinical factors except tumor stage (III vs. I) in multivariate Cox analyses. The 12-gene model is more accurate than previously published lung cancer gene signatures on the same datasets. Furthermore, this signature accurately predicts chemoresistance/chemosensitivity to Cisplatin, Carboplatin, Paclitaxel, Etoposide, Erlotinib, and Gefitinib in NCI-60 cancer cell lines (P<0.017). The identified 12 genes exhibit curated interactions with major lung cancer signaling hallmarks in functional pathway analysis. The expression patterns of the signature genes have been confirmed in RT-PCR analyses of independent tumor samples.

Conclusions/Significance

The results demonstrate the clinical utility of the identified gene signature in prognostic categorization. With this 12-gene risk score algorithm, early stage patients at high risk for tumor recurrence could be identified for adjuvant chemotherapy; whereas stage I and II patients at low risk could be spared the toxic side effects of chemotherapeutic drugs.