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XLS file, 198K, Revised class assignment obtained in this work.

XLS file, 457K, List of 651 genes chosen as top markers for distinguising the three non-Proneural subtypes.

XLS file, 193K, Selected gene expression features distinguishing non-Proneural GBM classes.

PDF file, 6233K, S1: Inference of Aneuploid Genome Proportion and its goodness-of-fit measures; Figure S2: Histolopathological estimates of tumor purities versus AGP; S3: Relationship between AGP and gene expression pattern in ovarian cancer (OV); S4: PCA plots for CNA and CNA-MicroRNA joint analysis; S5: Principal component analyses of gene expression and CNA data for GBM2; S6: Clustering pattern of three data types: PC1 of copy number data, PC1 of expression data, and PC2 of methylation data; S7: Classification of Non-Proneural GBM tumors; S8: Cross-correlation analysis of GBM1-GBM2 at K=3 and 4; S9: Comparison between the revised and the previous GBM classification systems; S10: Cross-correlation analysis between GBM1 and Phillips' dataset at K=2, 3 and 4; S11: PCA plots for 46 Non-Proneural GBM samples in Phillips' dataset; S12: Survival time differences between GBM subtypes, compared between the current and previous classification systems; S13: Cox proportional hazard regression analysis with GBM subtypes as covariates, along with age and Karnofsky performance scores (KPS); S14: Four clusters formed by the 38 reference samples in the Cahoy's dataset.

PDF file, 323K.

XLS file, 535K, Genomic features and QC measures for GBM1 and GBM2 (n=284).

Purpose: Accurate classification of glioblastoma multiforme (GBM) is crucial for understanding its biologic diversity and informing diagnosis and treatment. The Cancer Genome Atlas (TCGA) project identified four GBM classes using gene expression data and separately identified three classes using methylation data. We sought to integrate multiple data types in GBM classification, understand biologic features of the newly defined subtypes, and reconcile with prior studies.Experimental Design: We used allele-specific copy number data to estimate the aneuploid content of each tumor and incorporated this measure of intratumor heterogeneity in class discovery. We estimated the potential cell of origin of individual subtypes and the euploid and aneuploid fractions using reference datasets of known neuronal cell types.Results: There exists an unexpected correlation between aneuploid content and the observed among-tumor diversity of expression patterns. Joint use of DNA and mRNA data in ab initio class discovery revealed a distinct group that resembles the Proneural subtype described in a separate study and the glioma-CpG island methylator phenotype (G-CIMP+) class based on methylation data. Three additional subtypes, Classical, Proliferative, and Mesenchymal, were also identified and revised the assignment for many samples. The revision showed stronger differences in patient outcome and clearer cell type–specific signatures. Mesenchymal GBMs had higher euploid content, potentially contributed by microglia/macrophage infiltration.Conclusion: We clarified the confusion about the “Proneural” subtype that was defined differently in different prior studies. The ability to infer within-tumor heterogeneity improved class discovery, leading to new subtypes that are closer to the fundamental biology of GBM. Clin Cancer Res; 18(20); 5595–605. ©2012 AACR.

Spontaneous coronary artery dissection (SCAD) is a potential precipitant of myocardial infarction and sudden death for which the etiology is poorly understood. Mendelian vascular and connective tissue disorders underlying thoracic aortic disease (TAD), have been reported in ~5% of individuals with SCAD. We therefore hypothesized that patients with TAD are at elevated risk for SCAD. We queried registries enrolling patients with TAD to define the incidence of SCAD. Of 7568 individuals enrolled, 11 (0.15%) were found to have SCAD. Of the sequenced cases (9/11), pathogenic variants were identified (N = 9), including COL3A1 (N = 3), FBN1 (N = 2), TGFBR2 (N = 2), TGFBR1 (N = 1), and PRKG1 (N = 1). Individuals with SCAD had an increased frequency of iliac artery dissection (25.0% vs. 5.1%, p = 0.047). The prevalence of SCAD among individuals with TAD is low. The identification of pathogenic variants in genes previously described in individuals with SCAD, particularly those underlying vascular Ehlers–Danlos, Marfan syndrome, and Loeys–Dietz syndrome, is consistent with prior reports from clinical SCAD series. Further research is needed to identify specific genetic influences on SCAD risk.

The etiology of renal artery stenosis (RAS) and abdominal aortic coarctation (AAC) causing the midaortic syndrome (MAS), often resulting in renovascular hypertension (RVH), remains ill-defined. Neurofibromatosis type 1 (NF-1) is frequently observed in children with RVH. Consecutive pediatric patients (N = 102) presenting with RVH secondary to RAS with and without concurrent AAC were prospectively enrolled in a clinical data base, and blood, saliva and operative tissue, when available, were collected. Among the 102 children, 13 were having a concurrent clinical diagnosis of NF-1 (12.5%). Whole exome sequencing was performed for germline variant detection, and RNA-Seq analysis of NF1, MAPK pathway genes and MCP1 levels were undertaken in five NF-1 stenotic renal arteries, as well as control renal and mesenteric arteries from children with no known vasculopathy or NF-1. In 11 unrelated children with sequencing data, 11 NF1 genetic variants were identified, of which 10 had not been reported in gnomAD. Histologic analysis of NF-1 RAS specimens consistently revealed intimal thickening, disruption of the internal elastic lamina and medial thinning. Analysis of transcript expression in arterial lesions documented an approximately 5-fold reduction in NF1 expression, confirming heterozygosity, MAPK pathway activation and increased MCP1 expression. In summary, NF-1-related RVH in children is rare but often severe and progressive and, as such, important to recognize. It is associated with histologic and molecular features consistent with an aggressive adverse vascular remodeling process. Further research is necessary to define the mechanisms underlying these findings.

Previous genome-wide association studies (GWAS) have identified and validated variants at more than 1000 loci with modest effects on population blood pressure (BP), explaining in aggregate ~6% of the trait variance and highlighting genetic differences in BP regulation in ethnically diverse populations. We performed an exome-wide association study (EWAS) in Han Chinese population (N=5,954) from Beijing and identified 4 EWAS single nucleotide polymorphism (SNP)-trait associations with 3 SNPs, including two novel associations (rs2165468-peripheral systolic BP and rs33975708-central systolic BP (cSBP). rs33975708 is a coding variant in the coiled-coil domain containing 93 ( CCDC93 ) gene, c.535C>T, p.Arg179Cys (MAF=0.15%) and was associated with substantially increased cSBP (β=29.3 mmHg, P =1.23E-7) in Han Chinese population. CRISPR/Cas9 was used to introduce this variant in mice to investigate vascular molecular mechanisms. While this did not yield a viable mouse with the specific SNP, we did generate mice with an out of frame deletion making a gene knock-out. Ccdc93 deletion ( Ccdc93 -/- ) in its homozygous form was embryonic lethal (Ccdc93 +/- heterozygous mice were viable and morphologically normal and displayed 1.31-fold lower aortic Ccdc93 protein expression (P=0.0041, N=6). Systolic BP was higher in Ccdc93 +/- mice (110±8 mmHg vs 125±10 mmHg, P=0.016, N=5-7) and showed impaired acetylcholine (Ach)-induced relaxation (max Ach 86% vs 68%, Pex vivo . Transcriptome analysis by RNA-seq of thoracic aorta identified significantly enriched biological processes in Ccdc93 +/- among the pathways altered in fatty acid metabolic process (adjusted P=2.9E-9) together with mitochondrial metabolism (P=1.3E-14). Plasma free fatty acid levels were higher in Ccdc93 +/- mice (96±7 μM vs 124±13 μM, P=0.0031, N=6) and aortic parkin protein expression, a marker of mitochondrial dysfunction, was 1.63-fold higher (P=0.028, N=6). In conclusion, our genetic and functional studies indicate a potential novel role of CCDC93 on blood pressure regulation through its effects on vascular mitochondrial function.

Genomic discovery efforts for hematological traits have been successfully conducted through genome-wide association study on samples of predominantly European ancestry. We sought to conduct unbiased genetic discovery for coding variants that influence hematological traits in a Han Chinese population. A total of 5257 Han Chinese subjects from Beijing, China were included in the discovery cohort and analyzed by an Illumina ExomeChip array. Replication analyses were conducted in 3827 independent Chinese subjects. We analyzed 12 hematological traits and identified 22 exome-wide significant single-nucleotide polymorphisms (SNP)–trait associations with 15 independent SNPs. Our study provides replication for two associations previously reported but not replicated. Further, one association was identified and replicated in the current study, of a coding variant in the myeloproliferative leukemia (MPL) gene, c.793C > T, p.Leu265Phe (L265F) with increased platelet count (β = 20.6 109 cells/l, Pmeta-analysis = 2.6 × 10−13). This variant is observed at ~2% population frequency in East Asians, whereas it has not been reported in gnomAD European or African populations. Functional analysis demonstrated that expression of MPL L265F in Ba/F3 cells resulted in enhanced phosphorylation of Stat3 and ERK1/2 as compared with the reference MPL allele, supporting altered activation of the JAK–STAT signal transduction pathway as the mechanism underlying the novel association between MPL L265F and platelet count.

Objective: While rare variants in the COL5A1 gene have been associated with classical Ehlers-Danlos syndrome and rarely with arterial dissections, recurrent variants in COL5A1 underlying a systemic arteriopathy have not been described. Monogenic forms of multifocal fibromuscular dysplasia (mFMD) have not been previously defined. Approach and Results: We studied 4 independent probands with the COL5A1 pathogenic variant c.1540G>A, p.(Gly514Ser) who presented with arterial aneurysms, dissections, tortuosity, and mFMD affecting multiple arteries. Arterial medial fibroplasia and smooth muscle cell disorganization were confirmed histologically. The COL5A1 c.1540G>A variant is predicted to be pathogenic in silico and absent in gnomAD. The c.1540G>A variant is on a shared 160.1 kb haplotype with 0.4% frequency in Europeans. Furthermore, exome sequencing data from a cohort of 264 individuals with mFMD were examined for COL5A1 variants. In this mFMD cohort, COL5A1 c.1540G>A and 6 additional relatively rare COL5A1 variants predicted to be deleterious in silico were identified and were associated with arterial dissections ( P =0.005). Conclusions: COL5A1 c.1540G>A is the first recurring variant recognized to be associated with arterial dissections and mFMD. This variant presents with a phenotype reminiscent of vascular Ehlers-Danlos syndrome. A shared haplotype among probands supports the existence of a common founder. Relatively rare COL5A1 genetic variants predicted to be deleterious by in silico analysis were identified in ≈2.7% of mFMD cases, and as they were enriched in patients with arterial dissections, may act as disease modifiers. Molecular testing for COL5A1 should be considered in patients with a phenotype overlapping with vascular Ehlers-Danlos syndrome and mFMD.

Spontaneous coronary artery dissection (SCAD) is a non-atherosclerotic cause of myocardial infarction (MI), typically in young women. We undertook a genome-wide association study of SCAD (Ncases = 270/Ncontrols = 5,263) and identified and replicated an association of rs12740679 at chromosome 1q21.2 (Pdiscovery+replication = 2.19 × 10−12, OR = 1.8) influencing ADAMTSL4 expression. Meta-analysis of discovery and replication samples identified associations with P, Spontaneous coronary artery dissection (SCAD) is a cause of myocardial infarction Here, the authors present a genome-wide association study of SCAD, finding an association at 1q21.2 which potentially affects expression of ADAMTSL4.

While exogenous administration of recombinant erythropoietin has been associated with increased risk of hypertension, coronary artery disease, and mortality, it is not known if endogenous circulating erythropoietin level is associated with coronary artery disease and its risk factors. We measured and analyzed epidemiological and genetic associations of circulating plasma erythropoietin levels in 2 population cohorts, from China (N=4329) and the United States (N=3671). In vitro smooth muscle cell responses and in vivo murine studies of erythropoietin exposure were performed. Erythropoietin levels were positively and linearly associated with blood pressure traits and inversely associated with cholesterol levels and red cell indices. Higher erythropoietin level was associated with higher prevalence of hypertension (odds ratio, 1.20 [95% CI, 1.12–1.29], P =4.41×10 −7 ) and coronary artery disease (odds ratio, 1.16 [95% CI, 1.00–1.34], P =0.046). In a discovery stage genetic association study of erythropoietin level, we identified a previously reported locus on chromosome 6 (rs7776054 near HBS1L-MYB , P =4.86×10 −25 ) and a new locus on chromosome 4 (rs172629 near PDGFRA-KIT , P =2.1×10 −8 ), which was independently replicated. Meta-analysis of discovery and replication genetic association results identified a locus on chromosome 22 (rs855791 near TMPRSS6 , P =3.60×10 −9 ). Erythropoietin administration, within a physiological range of hematocrit achieved, induced hypertension in vivo and increased contraction of vascular smooth muscle cells in vitro. In conclusion, endogenous circulating erythropoietin level is influenced by common genetic variation and is associated with blood pressure traits, hypertension, and coronary artery disease. Vascular effects of erythropoietin demonstrated in vitro and in vivo support a newly discovered mechanism of hypertension and cardiovascular risk with potential implications for erythropoietic support in the clinic. Graphic Abstract: An online graphic abstract is available for this article.

Fibromuscular dysplasia (FMD) is an arteriopathy that presents clinically by hypertension and stroke, mostly in early middle-aged women. We report results from the first genome-wide association meta-analysis of FMD including 1962 FMD cases and 7100 controls. We confirmedPHACTR1and identified three new loci (LRP1, ATP2B1, andLIMA1)associated with FMD. Transcriptome-wide association analysis in arteries identified one additional locus (SLC24A3). FMD associated variants were located in arterial-specific enhancers active in vascular smooth muscle cells and fibroblasts. Target genes are broadly involved in mechanisms related to actin cytoskeleton and intracellular calcium homeostasis, central to vascular contraction. Cross-trait linkage disequilibrium analyses identified positive genetic correlations with blood pressure, migraine and intracranial aneurysm, and an inverse correlation with coronary artery disease, independent from the genetics of blood pressure.

OBJECTIVE: While rare variants in the COL5A1 gene have been associated with classical Ehlers-Danlos Syndrome (EDS) and rarely with arterial dissections, recurrent variants in COL5A1 underlying a systemic arteriopathy have not been described. Monogenic forms of multifocal fibromuscular dysplasia (mFMD) have not been previously defined. APPROACH AND RESULTS: We studied four independent probands with the COL5A1 pathogenic variant c.1540G>A, p.(Gly514Ser) who presented with arterial aneurysms, dissections, tortuosity, and mFMD affecting multiple arteries. Arterial medial fibroplasia and smooth muscle cell disorganization were confirmed histologically. The COL5A1 c.1540G>A variant is predicted to be pathogenic in silico and absent in gnomAD. The c.1540G>A variant is on a shared 160.1kb haplotype with 0.4% frequency in Europeans. Further, exome sequencing data from a cohort of 264 individuals with mFMD were examined for COL5A1 variants. In this mFMD cohort, COL5A1 c.1540G>A and six additional relatively rare COL5A1 variants predicted to be deleterious in silico were identified and were associated with arterial dissections (p=0.005). CONCLUSIONS: COL5A1 c.1540G>A is the first recurring variant recognized to be associated with arterial dissections and mFMD. This variant presents with a phenotype reminiscent of vascular EDS. A shared haplotype among probands supports the existence of a common founder. Relatively rare COL5A1 genetic variants predicted to be deleterious by in silico analysis were identified in ~2.7% of mFMD cases, and as they were enriched in patients with arterial dissections, may act as disease modifiers. Molecular testing for COL5A1 should be considered in patients with a phenotype overlapping with vascular EDS and mFMD.

Introduction Fibromuscular dysplasia (FMD) is a non-atherosclerotic arteriopathy of unknown etiology, affecting mostly middle-aged women. It is characterized by stenotic lesions of the vascular wall in middle-size arteries, sometimes associated with dissection, aneurysm or tortuosity. Objective We aim at identifying genetic causes of FMD to better understand its mechanism and relation with other vasculopathies. Methods We report results from the first genome-wide association meta-analysis of six studies including 1962 FMD cases and 7100 controls. We integrated genetic association with arterial gene expression using transcriptome-wide association analysis. To identify FMD associated variants located in regulatory elements, we determined open chromatin regions in artery-derived primary cells using ATAC-Seq and estimated heritability and genetic correlation of FMD with other vascular traits and diseases using LD Score regression. Results We found an estimate of SNP-based heritability compatible with a polygenic feature for FMD and report four robustly associated loci (PHACTR1, LRP1, ATP2B1, and LIMA1). Transcriptome-wide association analysis in arteries identified one additional locus (SLC24A3). We found that FMD associated variants were located in arterial-specific regulatory elements. Target genes were broadly involved in mechanisms related to actin cytoskeleton and intracellular calcium homeostasis, central to vascular contraction. We found significant genetic overlap between FMD and blood pressure, although hypertension was not confounding the genetic association with FMD. We also report an important genetic overlap with migraine, intracranial aneurysm, coronary artery disease and LDL cholesterol. Conclusion We identified several loci related to vascular contraction, suggesting that altered vascular tonicity may play a role in the pathogenesis of FMD. We find that FMD is genetically close to several vascular diseases where vascular integrity is impaired.

Contains fulltext : 169736.pdf (Publisher’s version ) (Closed access) Fibromuscular dysplasia (FMD) is a heterogeneous group of non-atherosclerotic and non-inflammatory arterial diseases that primarily involves the renal and cerebrovascular arteries. Grange syndrome is an autosomal-recessive condition characterized by severe and early-onset vascular disease similar to FMD and variable penetrance of brachydactyly, syndactyly, bone fragility, and learning disabilities. Exome-sequencing analysis of DNA from three affected siblings with Grange syndrome identified compound heterozygous nonsense variants in YY1AP1, and homozygous nonsense or frameshift YY1AP1 variants were subsequently identified in additional unrelated probands with Grange syndrome. YY1AP1 encodes yin yang 1 (YY1)-associated protein 1 and is an activator of the YY1 transcription factor. We determined that YY1AP1 localizes to the nucleus and is a component of the INO80 chromatin remodeling complex, which is responsible for transcriptional regulation, DNA repair, and replication. Molecular studies revealed that loss of YY1AP1 in vascular smooth muscle cells leads to cell cycle arrest with decreased proliferation and increased levels of the cell cycle regulator p21/WAF/CDKN1A and disrupts TGF-beta-driven differentiation of smooth muscle cells. Identification of YY1AP1 mutations as a cause of FMD indicates that this condition can result from underlying genetic variants that significantly alter the phenotype of vascular smooth muscle cells.

Hematologic measures such as hematocrit and white blood cell (WBC) count are heritable and clinically relevant. We analyzed erythrocyte and WBC phenotypes in 52,531 individuals (37,775 of European ancestry, 11,589 African Americans, and 3,167 Hispanic Americans) from 16 population-based cohorts with Illumina HumanExome BeadChip genotypes. We then performed replication analyses of new discoveries in 18,018 European-American women and 5,261 Han Chinese. We identified and replicated four new erythrocyte trait-locus associations (CEP89, SHROOM3, FADS2, and APOE) and six new WBC loci for neutrophil count (S1PR4), monocyte count (BTBD8, NLRP12, and IL17RA), eosinophil count (IRF1), and total WBC count (MYB). The association of a rare missense variant in S1PR4 supports the role of sphingosine-1-phosphate signaling in leukocyte trafficking and circulating neutrophil counts. Loss-of-function experiments for S1pr4 in mouse and s1pr4 in zebrafish demonstrated phenotypes consistent with the association observed in humans and altered kinetics of neutrophil recruitment and resolution in response to tissue injury.

Background: Vascular smooth muscle cells (vSMCs) are essential for maintaining blood vessel tone and vascular remodeling. Although they play an important role in cardiovascular pathologies, studying peripheral artery vSMCs in vivo has been complicated given their interspersed anatomic distribution and technical difficulties with isolating vSMCs from arteries and arterioles while preserving transcriptome profiles. Methods and Results: Combining the murine Rpl22 fl Ribotag model with a vSMC-specific Transgelin-Cre ( Tagln-Cre ) Recombinase knock-in, we performed translating ribosome affinity purification and isolation of mRNA derived specifically from Cre -expressing + cells only. Subsequent high-throughput sequencing of both whole tissue mRNA and the vSMC-selected mRNA of the brain, kidney and liver was followed by differential expression analysis to identify genes enriched in the vSMC compartment. Genes significantly enriched in all three tissues (FDR Acta2 ) and Calponin-1 ( Cnn1 ) in addition to Tagln. Furthermore, transcripts involved in biological processes associated with extracellular matrix and structure organization were enriched and significant overrepresentation of these genes was also seen by gene ontology analysis. Genes with roles in blood pressure regulation, such as Regulator of G protein signaling 5 ( Rgs5 ) and Sphingosine-1-phosphate receptor 3 ( S1pr3 ) were also significantly enriched in the vSMC transcriptome, and the expression and vSMC-specific localization of these genes were confirmed by fluorescent single-molecule in situ hybridization assays. Further, we performed gene set enrichment analysis of genes associated with blood pressure and hypertension based on human genome-wide association studies, and a significant number of these genes ( P =0.003) showed vSMC-specific expression patterns in our in vivo data. Conclusion: Our data demonstrate the feasibility of profiling vSMC gene expression in vivo. Furthermore, we identified enrichment of blood pressure-associated genes in the vSMC transcriptome. This model has the potential to provide new insights into the role of vSMCs in physiology and a variety of cardiovascular pathologies.

Genome-wide association studies (GWASs) have identified loci for erythrocyte traits in primarily European ancestry populations. We conducted GWAS meta-analyses of six erythrocyte traits in 71,638 individuals from European, East Asian, and African ancestries using a Bayesian approach to account for heterogeneity in allelic effects and variation in the structure of linkage disequilibrium between ethnicities. We identified seven loci for erythrocyte traits including a locus (RBPMS/GTF2E2) associated with mean corpuscular hemoglobin and mean corpuscular volume. Statistical fine-mapping at this locus pointed to RBPMS at this locus and excluded nearby GTF2E2. Using zebrafish morpholino to evaluate loss of function, we observed a strong invivo erythropoietic effect for RBPMS but not for GTF2E2, supporting the statistical fine-mapping at this locus and demonstrating that RBPMS is a regulator of erythropoiesis. Our findings show the utility of trans-ethnic GWASs for discovery and characterization of genetic loci influencing hematologic traits.

Fibromuscular dysplasia (FMD) is a nonatherosclerotic vascular disease leading to stenosis, dissection and aneurysm affecting mainly the renal and cerebrovascular arteries. FMD is often an underdiagnosed cause of hypertension and stroke, has higher prevalence in females (~80%) but its pathophysiology is unclear. We analyzed ~26K common variants (MAF>0.05) generated by exome-chip arrays in 249 FMD patients and 689 controls. We replicated 13 loci (P, Author Summary Fibromuscular Dysplasia (FMD) is a vascular disease characterized by a succession of occlusions and dilatation of medium-sized arteries (e.g renal, carotid or coronary arteries) with important health consequences, mainly resistant hypertension and stroke. FMD is an atypical vascular disease because it is not associated with overweight or dyslipidemia and 80% of patients are early middle aged women. Our genetic study conducted in >1100 patients and >3800 controls demonstrate that a common variant rs9349379 located on chromosome 6 in the phosphatase and actin regulator 1 gene (PHACTR1) increases by ~40% the risk of FMD. This is the first time a genetic risk factor is reported for FMD because it has been longtime considered rare and potentially under a Mendelian mode of inheritance. We also show that rs9349379 correlates with the expression of PHACTR1 in fibroblasts from FMD patients and controls. Interestingly, the same allele that increases the risk of FMD is at risk for cervical artery dissection and migraine, often reported in FMD patients but protective from myocardial infarction and coronary disease, where atherosclerosis is more common. The clear role of PHACTR1 in maintaining vascular well integrity is not fully elucidated. Using a specific antibody we detected PHACTR1 both on endothelial and smooth muscle cells of human FMD and control carotids, which suggests that PHACTR1 may have multiple functions depending on the cell type and the degree of atherosclerosis of the arteries.

Purpose: Accurate classification of glioblastoma multiforme (GBM) is crucial for understanding its biologic diversity and informing diagnosis and treatment. The Cancer Genome Atlas (TCGA) project identified four GBM classes using gene expression data and separately identified three classes using methylation data. We sought to integrate multiple data types in GBM classification, understand biologic features of the newly defined subtypes, and reconcile with prior studies. Experimental Design: We used allele-specific copy number data to estimate the aneuploid content of each tumor and incorporated this measure of intratumor heterogeneity in class discovery. We estimated the potential cell of origin of individual subtypes and the euploid and aneuploid fractions using reference datasets of known neuronal cell types. Results: There exists an unexpected correlation between aneuploid content and the observed among-tumor diversity of expression patterns. Joint use of DNA and mRNA data in ab initio class discovery revealed a distinct group that resembles the Proneural subtype described in a separate study and the glioma-CpG island methylator phenotype (G-CIMP+) class based on methylation data. Three additional subtypes, Classical, Proliferative, and Mesenchymal, were also identified and revised the assignment for many samples. The revision showed stronger differences in patient outcome and clearer cell type–specific signatures. Mesenchymal GBMs had higher euploid content, potentially contributed by microglia/macrophage infiltration. Conclusion: We clarified the confusion about the “Proneural” subtype that was defined differently in different prior studies. The ability to infer within-tumor heterogeneity improved class discovery, leading to new subtypes that are closer to the fundamental biology of GBM. Clin Cancer Res; 18(20); 5595–605. ©2012 AACR.

Copy number variations (CNVs), a major source of human genetic polymorphism, have been suggested to have an important role in genetic susceptibility to common diseases such as cancer, immune diseases and neurological disorders. Nasopharyngeal carcinoma (NPC) is a multifactorial tumor closely associated with genetic background and with a male preponderance over female (3:1). Previous genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) that are associated with NPC susceptibility. Here, we sought to explore the possible association of CNVs with NPC predisposition. Utilizing genome-wide SNP-based arrays and five CNV-prediction algorithms, we identified eight regions with CNV that were significantly overrepresented in NPC patients compared with healthy controls. These CNVs included six deletions (on chromosomes 3, 6, 7, 8 and 19), and two duplications (on chromosomes 7 and 12). Among them, the CNV located at chromosome 6p21.3, with single-copy deletion of the MICA and HCP5 genes, showed the highest association with NPC. Interestingly, it was more specifically associated with an increased NPC risk among males. This gender-specific association was replicated in an independent case–control sample using a self-established deletion-specific polymerase chain reaction strategy. To the best of our knowledge, this is the first study to explore the role of constitutional CNVs in NPC, using a genome-wide platform. Moreover, we identified eight novel candidate regions with CNV that merit future investigation, and our results suggest that similar to neuroblastoma and prostate cancer, genetic structural variations might contribute to NPC predisposition.

Single Nucleotide Polymorphisms (SNPs) identified through genome-wide association studies (GWAS) provide insight into the mechanism of human genetic diseases, and majority of functional GWAS mutations target genomic regulatory elements. During erythroid differentiation of human CD34+ cells, we mapped regulatory DNA elements (enhancers and open chromatin regions) by H3K27Ac ChIP-seq and ATAC-seq, and studied the SNPs that reside within these DNA regulatory elements. We followed genomic binding of lineage restricted GATA transcription factors and also chose to examine the binding of the BMP signal responsive transcription factor SMAD1 in CD34+ cells during erythropoiesis. By overlapping their genomic occupancy with stage-matched RNA-seq, we found that SMAD1, in association with GATA-factors, serves as marker of genes responsible for differentiation at every step of erythropoiesis. ChIP-seq for other crucial signaling transcription factors, such as WNT-responsive and TGFb-responsive factors (TCF7L2 and SMAD2, respectively) demonstrated a remarkable co-existence of such factors at GATA+SMAD1 co-bound regions nearby stage-specific genes. We defined such regions as "Transcriptional Signaling Centers (TSC)" where multiple signaling transcription factors converge with master transcription factors to determine optimum stage-specific gene expression in response to growth factors. Our bioinformatics-algorithms demonstrated that PU1 and FLI1 binding sites were present in progenitor-specific TSCs whereas KLF1 and NFE2 sites were enriched in TSCs of red blood cells. We performed CRISPR-CAS9 mediated perturbations of each of the PU1, GATA and SMAD1 motifs separately in a representative progenitor TSC in K562 and HUDEP2 cells. Similar to loss of PU1 and GATA motifs, loss of SMAD1 motif selectively inhibited expression of the associated gene and showed defects in erythroid differentiation, demonstrating that TSCs are important to provide optimum gene expression and proper erythroid differentiation. To determine if such TSCs are targeted by GWAS mutations, we have studied 1270 lead and additional 27,799 SNPs in linkage disequilibrium with lead SNPs that are associated with six critical red blood cell traits - hemoglobin concentration (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and red blood cell count (RBC). Surprisingly, we observed that, out of the 3831 "functional" SNPs that fall within non-exonic H3K27Ac enhancers, while only 5% (188) of RBC-SNPs target only blood-master-transcription-factor motifs, at least 48% (1821) of them reside on various signaling pathway associated transcription factor motifs including SMADs (BMP/TGFb signaling), RXR/ROR (nuclear receptor signaling), FOXO/FOXA (FOX signaling), CREBs (cAMP signaling) and TCF7L2 (WNT signaling). Additionally, these RBC-trait-SNPs are specifically enriched in GATA+SMAD1 co-bound TSCs and fall within signaling factor binding sites. We validated such SNPs that target SMAD-motifs. The SNP rs9467664 is associated with the MCV-trait near HIST1H4A, a gene that increases in expression during differentiation. Using gel-shift assay, we found that SMAD1 binding is compromised when the major allele T changes to minor allele A under MCV-trait. Remarkably, eQTL analysis using microarray gene expression profiles of peripheral blood obtained from the Framingham Heart Studies revealed that expression of HIST1H4A is significantly more in a population with T-allele than that with A-allele. This demonstrates that inhibition of SMAD1 binding by the SNP causes a loss of allele-specific HIST1H4A expression. Another MCV-associated SNP rs737092 targets a SMAD motif within an erythroid-specific TSC near RBM38 gene. T-allele, in comparison with C-allele, that retains SMAD1 binding showed more expression in luciferase-based reporter assays specifically under BMP stimulation suggesting that rs737092 compromise BMP-responsiveness. Taken together, our study provides the first evidence that naturally occurring GWAS variations directly impact gene expression from signaling centers by modulating binding of signaling transcription factors under stimulation. Such aberrant signaling events over time could lead to "signalopathies", ultimately resulting in phenotypic variations of RBC traits. Disclosures Abraham: Syros Pharmaceuticals: Equity Ownership. Young:Omega Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Camp4 Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Single Nucleotide Polymorphisms (SNPs) identified through genome-wide association studies (GWAS) could provide insight into the mechanism of human genetic diseases. Here we have studied SNPs that are associated with six critical red blood cell traits - hemoglobin concentration (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and red blood cell count (RBC). During erythroid differentiation of human CD34+ cells, we mapped enhancers and open chromatin regions by H3K27Ac ChIPseq and ATACseq, and studied the SNPs that reside within these DNA regulatory elements. We followed genomic binding of lineage restricted GATA transcription factors and BMP signal responsive transcription factor SMAD1 in CD34+ cells during erythropoiesis. By overlapping their genomic occupancy with stage-matched RNAseq, we found that SMAD1, in association with GATA-factors, serves as marker of genes responsible for differentiation at every step of differentiation. ATACseq and H3K27Ac patterns demonstrated that GATA+SMAD1 co-occupied regions correlate with open chromatin and super enhancers at every stage, whereas GATA-only regions are associated with genes with low/basal level of expression during differentiation. ChIPseq for other crucial signaling transcription factors, such as cAMP-responsive and TGFb-responsive factors (CREB and SMAD2, respectively) demonstrated a remarkable co-existence of such factors at GATA+SMAD1 co-bound regions nearby stage-specific genes. We defined such regions as "signaling centers" where multiple signaling transcription factors converge with master transcription factors to determine optimum stage-specific gene expression in response to growth factors. Surprisingly, we observed that while only 15% of RBC-SNPs target blood-master-transcription-factor motifs, at least 70% of them reside on various signaling pathway associated transcription factor motifs including SMADs (BMP/TGFβ signaling), RXR/ROR (nuclear receptor signaling), FOXO/FOXA (FOX signaling), CREBs (cAMP signaling) and TCF7L2 (WNT signaling). Our bioinformatics-algorithms demonstrated that, in contrast to GATA-only sites, SMAD1+GATA co-bound signaling centers harbor cis -acting motifs and display enriched binding of cell-type specific transcription factors (e.g. PU1 and FLI1 in progenitor vs. KLF1 and NFE2 in differentiated cells). Such distinct identities of signaling centers could serve as codes to distinguish progenitor-specific genes from erythroid-specific genes, and govern their stage-specific expression. We performed CRISPR-CAS9 mediated perturbations of each of the PU1, GATA and SMAD1 motifs separately in a representative progenitor signaling center in K562 cells. Similar to loss of PU1 and GATA motifs, loss of SMAD1 motif selectively inhibited expression of the associated gene. This suggests a signaling factor SMAD1 is important within signaling centers to obtain optimum gene expression. Moreover, a progenitor factor PU1 direct binding of SMAD1 to progenitor-specific signaling centers since with overexpression of PU1 in K562 cells, SMAD1 occupancy was concomitantly increased in selective genomic regions where PU1 binding was increased. More than 80% of the RBC-trait-SNPs are enriched within SMAD1-bound signaling centers. Such SNPs either destroy or create new signaling factor binding sites, e.g. SMAD motifs. We validated one such SNP associated with the MCV-trait near HIST1H4A, agene that increases in expression during differentiation. Using gel-shift assay, we found that SMAD1 binding is compromised when the major allele T changes to minor allele A under MCV-trait. Remarkably, eQTL analysis using microarray gene expression profiles of peripheral blood obtained from the Framingham Heart Studies revealed that expression of HIST1H4A is significantly more in a population with T-allele than that with A-allele. This demonstrates that inhibition of SMAD1 binding by the SNP causes a loss of allele-specific HIST1H4A expression. Taken together, our study provides the first evidence that naturally occurring GWAS variations directly impact gene expression from signaling centers by modulating binding of signaling transcription factors. Such aberrant signaling events over time could lead to "signalopathies", ultimately resulting in phenotypic variations of RBC traits. Disclosures Zon: Fate, Inc.: Consultancy, Equity Ownership; Marauder, Inc.: Consultancy, Equity Ownership; Scholar Rock, Inc: Consultancy, Equity Ownership; Stemgent: Consultancy.

Nasopharyngeal carcinoma (NPC) is a multifactorial malignancy closely associated with genetic factors and Epstein-Barr virus infection. To identify the common genetic variants linked to NPC susceptibility, we conducted a genome-wide association study (GWAS) in 277 NPC patients and 285 healthy controls within the Taiwanese population, analyzing 480,365 single-nucleotide polymorphisms (SNPs). Twelve statistically significant SNPs were identified and mapped to chromosome 6p21.3. Associations were replicated in two independent sets of case-control samples. Two of the most significant SNPs (rs2517713 and rs2975042; p(combined) = 3.9 x 10(-20) and 1.6 x 10(-19), respectively) were located in the HLA-A gene. Moreover, we detected significant associations between NPC and two genes: specifically, gamma aminobutyric acid b receptor 1 (GABBR1) (rs29232; p(combined) = 8.97 x 10(-17)) and HLA-F (rs3129055 and rs9258122; p(combined) = 7.36 x 10(-11) and 3.33 x 10(-10), respectively). Notably, the association of rs29232 remained significant (residual p5 x 10(-4)) after adjustment for age, gender, and HLA-related SNPs. Furthermore, higher GABA(B) receptor 1 expression levels can be found in the tumor cells in comparison to the adjacent epithelial cells (p0.001) in NPC biopsies, implying a biological role of GABBR1 in NPC carcinogenesis. To our knowledge, it is the first GWAS report of NPC showing that multiple loci (HLA-A, HLA-F, and GABBR1) within chromosome 6p21.3 are associated with NPC. Although some of these relationships may be attributed to linkage disequilibrium between the loci, the findings clearly provide a fresh direction for the study of NPC development.

Blood lipids are important risk factors for coronary artery disease (CAD). Here we perform an exome-wide association study by genotyping 12,685 Chinese, using a custom Illumina HumanExome BeadChip, to identify additional loci influencing lipid levels. Single-variant association analysis on 65,671 single nucleotide polymorphisms reveals 19 loci associated with lipids at exome-wide significance (P, An important risk factor for coronary artery disease is the level of blood lipids. Here the authors conduct an exome-wide association study in Chinese cohorts and identify three novel loci associated with lipid levels as well as three Asian-specific variants in known loci.

Nasopharyngeal carcinoma (NPC) is a multi-factorial malignancy closely associated with genetic factors and Epstein-Barr virus infection. Recently, we have conducted a genome-wide association study (GWAS) in 277 NPC patients and 285 healthy controls within the Taiwanese population, analyzing 480,365 single-nucleotide polymorphisms (SNPs). Twelve statistically significant SNPs were identified and mapped to chromosome 6p21.3 in the major histocompatibility complex region. In addition to HLA-A gene, we detected novel significant associations between NPC and two genes: specifically, gamma aminobutyric acid b receptor 1 and HLA-F. To our knowledge, it is the first GWAS report of NPC showing that multiple loci (HLA-A, HLA-F, and GABBR1) within chromosome 6p21.3 are associated with NPC. Following the GWAS study, we made an effort to search systematically for copy number variations (CNVs) that confer increased risk to NPC using the same dataset. We generated CNV calls using four different softwares and used 15 internal duplicated samples to look for best filtering criteria. CNVs were selected if same CNVs were identified from two independent softwares to increase credibility of prediction. We identified eight CNVs that overrepresented in NPC patients compared to healthy controls. Six deletions on chromosomes 3, 6, 7, 8 and 19, as well as 2 duplications on chromosomes 7 and 12 were found. One of the most significant CNV on chromosome 6p21.3 was further replicated in an independent case-control dataset, using a deletion-specific polymerase-chain reaction. To the best of our knowledge, it is the first study of genome-wide constitutional CNV in NPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 882. doi:10.1158/1538-7445.AM2011-882