Excel file detailing case-control populations

Supplementary Text for manuscript.

Excel file detailing single WHR SNP associations with EC risk

Excel file detailing single BMI SNP associations with EC risk and BMI

Excel file detailing average BMI per study population

BACKGROUND: Double-stranded DNA in plasma is known to carry single-stranded ends, called jagged ends. Plasma DNA jagged ends are biomarkers for pathophysiologic states such as pregnancy and cancer. It remains unknown whether urinary cell-free DNA (cfDNA) molecules have jagged ends. METHODS: Jagged ends of cfDNA were detected by incorporating unmethylated cytosines during a DNA endrepair process, followed by bisulfite sequencing. Incorporation of unmethylated cytosines during the repair of the jagged ends lowered the apparent methylation levels measured by bisulfite sequencing and were used to calculate a jagged end index. This approach is called jagged end analysis by sequencing. RESULTS: The jagged end index of urinary cfDNA was higher than that of plasma DNA. The jagged end index profile of plasma DNA displayed several strongly oscillating major peaks at intervals of approximately 165 bp (i.e., nucleosome size) and weakly oscillating minor peaks with periodicities of approximately 10 bp. In contrast, the urinary DNA jagged end index profile showed weakly oscillating major peaks but strongly oscillating minor peaks. The jagged end index was generally higher in nucleosomal linker DNA regions. Patients with bladder cancer (n = 46) had lower jagged end indexed of urinary DNA than participants without bladder cancer (n = 39). The area under the curve for differentiating between patients with and without bladder cancer was 0.83. CONCLUSIONS: Jagged ends represent a property of urinary cfDNA. The generation of jagged ends might be related to nucleosomal structures, with enrichment in linker DNA regions. Jagged ends of urinary DNA could potentially serve as a new biomarker for bladder cancer detection.

Blood lipids have been associated with the development of a range of cancers, including breast, lung and colorectal cancer. For endometrial cancer, observational studies have reported inconsistent associations between blood lipids and cancer risk. To reduce biases from unmeasured confounding, we performed a bidirectional, two-sample Mendelian randomization analysis to investigate the relationship between levels of three blood lipids (low-density lipoprotein [LDL] and high-density lipoprotein [HDL] cholesterol, and triglycerides) and endometrial cancer risk. Genetic variants associated with each of these blood lipid levels (P < 5 x 10(-8)) were identified as instrumental variables, and assessed using genome-wide association study data from the Endometrial Cancer Association Consortium (12 906 cases and 108 979 controls) and the Global Lipids Genetic Consortium (n = 188 578). Mendelian randomization analyses found genetically raised LDL cholesterol levels to be associated with lower risks of endometrial cancer of all histologies combined, and of endometrioid and non-endometrioid subtypes. Conversely, higher genetically predicted HDL cholesterol levels were associated with increased risk of non-endometrioid endometrial cancer. After accounting for the potential confounding role of obesity (as measured by genetic variants associated with body mass index), the association between genetically predicted increased LDL cholesterol levels and lower endometrial cancer risk remained significant, especially for non-endometrioid endometrial cancer. There was no evidence to support a role for triglycerides in endometrial cancer development. Our study supports a role for LDL and HDL cholesterol in the development of non-endometrioid endometrial cancer. Further studies are required to understand the mechanisms underlying these findings.

Background: Eltrombopag is a thrombopoietin receptor agonist that is increasingly being prescribed for the treatment of chronic thrombocytopenic conditions. Hepatotoxicity occurring in ∼15% of patients necessitates regular monitoring of liver function. Depending in which hospital a patient is followed-up, serum bilirubin concentration for a single sample can vary by more than 8-fold, or 64 μmol/L, with the true concentration unknown. Methods: Bilirubin was measured using up to nine assays including the Doumas Reference method and HPLC, for 66 samples from patients on 25–150 mg daily. Plasma/serum eltrombopag concentrations were determined using LC-MS/MS. Results: There was a positive correlation between inter-analyser total bilirubin difference and plasma eltrombopag concentration (r=0.679). Spike-in experiments demonstrated that methods including the Doumas reference method were susceptible to positive interference. HPLC quantified bilirubin after separating eltrombopag, and results show that different analysers are affected to varying degrees by eltrombopag and its metabolites. Conclusions: We are the first to demonstrate which bilirubin assays can accurately quantitate bilirubin concentration in patients on eltrombopag therapy. We are also the first to provide evidence that metabolites likely contribute to the interference. Resolution of inter-analyser discrepancies may prevent misdiagnosis of hepatotoxicity and improve our understanding of the prevalence of hyperbilirubinaemia in patients on eltrombopag therapy.

Cell-free DNA (cfDNA) in human plasma is a class of biomarkers with many current and potential future diagnostic applications. Recent studies have shown that cfDNA molecules are not randomly fragmented and possess information related to their tissues of origin. Pathologies causing death of cells from particular tissues result in perturbations in the relative distribution of DNA from the affected tissues. Such tissue-of-origin analysis is particularly useful in the development of liquid biopsies for cancer. It is therefore of value to accurately determine the relative contributions of the tissues to the plasma DNA pool in a simultaneous manner. In this work, we report that in open chromatin regions, cfDNA molecules show characteristic fragmentation patterns reflected by sequencing coverage imbalance and differentially phased fragment end signals. The latter refers to differences in the read densities of sequences corresponding to the orientation of the upstream and downstream ends of cfDNA molecules in relation to the reference genome. Such cfDNA fragmentation patterns preferentially occur in tissue-specific open chromatin regions where the corresponding tissues contributed DNA into the plasma. Quantitative analyses of such signals allow measurement of the relative contributions of various tissues toward the plasma DNA pool. These findings were validated by plasma DNA sequencing data obtained from pregnant women, organ transplantation recipients, and cancer patients. Orientation-aware plasma DNA fragmentation analysis therefore has potential diagnostic applications in noninvasive prenatal testing, organ transplantation monitoring, and cancer liquid biopsy.

Background: Eltrombopag is a thrombopoietin receptor agonist used for the treatment of thrombocytopenic conditions. It can cause pH-dependent discoloration of plasma/serum. Eltrombopag is potentially hepatotoxic. It can affect the assessment of hyperbilirubinemia because of its (i) absorbance at similar to 450 nm (bilirubin), (ii) absorbance at similar to 550 nm (diazo-bilirubin) and (iii) it can cause yellowish discoloration of the eyes at normal circulating bilirubin levels. Methods: We collected 66 samples from patients on a range of eltrombopag dosages up to 150 mg daily. Bilirubin was measured using multiple routine spectrophotometric analyzers, the Doumas reference method and high-performance liquid chromatography (HPLC). Plasma/serum eltrombopag concentrations were determined using liquid chromatography tandem mass spectrometry (LC-MS/MS). Spike-in and admixture experiments delineated the effects of eltrombopag and its metabolites. Results: Forty-nine of 52 samples from patients on >= 50 mg daily eltrombopag therapy showed significantly discrepant inter-analyzer total bilirubin results, a difference up to 64 mu mol/L (3.7 mg/dL). In one sample, total bilirubin varied from 8 to 65 mu mol/L (0.4-3.8 mg/dL) by different routine analyzers, with direct bilirubin <= 4 mu mol/L (0.2 mg/dL). There was a positive correlation between total bilirubin difference and plasma eltrombopag concentration (r= 0.679), and spike-in experiments demonstrated that Beckman AU and Doumas reference methods were susceptible to positive interference. HPLC can quantify bilirubin after separating eltrombopag, and results suggest different analyzers are affected to varying degrees by eltrombopag and its metabolites. Conclusions: Eltrombopag and its metabolites can cause positive interference to the spectrophotometric measurements of total bilirubin. Accurate measurements of total bilirubin may improve our understanding of the prevalence of hyperbilirubinemia in patients on eltrombopag therapy.

Background: Long intergenic non-coding RNAs (lincRNAs) are emerging as a novel class of non-coding RNAs and potent gene regulators. High-throughput RNA-sequencing combined with de novo assembly promises quantity discovery of novel transcripts. However, the identification of lincRNAs from thousands of assembled transcripts is still challenging due to the difficulties of separating them from protein coding transcripts (PCTs). Results: We have implemented iSeeRNA, a support vector machine (SVM)-based classifier for the identification of lincRNAs. iSeeRNA shows better performance compared to other software. A public available webserver for iSeeRNA is also provided for small size dataset. Conclusions: iSeeRNA demonstrates high prediction accuracy and runs several magnitudes faster than other similar programs. It can be integrated into the transcriptome data analysis pipelines or run as a web server, thus offering a valuable tool for lincRNA study. Background Over the past decade, evidence from numerous highthroughput genomic platforms reveals that even though less than 2% of the mammalian genome encodes proteins, a significant fraction can be transcribed into different complex families of non-coding RNAs (ncRNAs) [1-4]. Other than microRNAs and other families of small noncoding RNAs, long non-coding RNAs (lncRNAs, >200nt) are emerging as potent regulators of gene expression [5]. Originally identified by Guttman et al. [6] from four mouse cell types using chromatin state maps as a subtype of lncRNAs, long intergenic non-coding RNAs (lincRNAs), are discrete transcriptional unit intervening known protein-coding loci. Recent studies demonstrate the functional significance of lincRNAs. However, it remains a daunting task to identify all the lincRNAs existent in various biological processes and systems. Whole transcriptome sequencing, known as RNA-Seq, offers the promise of rapid comprehensive discovery of novel genes and transcripts [7]. With the de novo assembly software such as Cufflinks [8] and Scripture [6], a large set of novel assemblies can be obtained from RNA-Seq data. Several programs have been used to facilitate the cataloging of lincRNAs from RNA-Seq assemblies. For example, Li et al. [9] used Codon Substitution Frequency (CSF) score [10] to identify lincRNAs from de novo assembled transcripts in chicken skeletal muscle. Pauli et al. [11] took advantage of PhyloCSF score [12] followed by other filtering steps to identify lincRNAs expressed during zebrafish embryogenesis. Cabili et al. [13] also used PhyloCSF program to eliminate the de novo assembled transcripts with positive coding potential and identified ~8200 lincRNA loci in 24 human tissues. However, the extremely high computational times demanded by PhyloCSF, may become the bottleneck for handling millions of assemblies generated from high throughput sequencing. Furthermore, * Correspondence: xfsong@nuaa.edu.cn; huating.wang@cuhk.edu.hk; haosun@cuhk.edu.hk Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China Department of Biomedical Engineering, Nanjing University of Aeronautics and Astronautics, Nanjing 210016, China Full list of author information is available at the end of the article Sun et al. BMC Genomics 2013, 14(Suppl 2):S7 http://www.biomedcentral.com/1471-2164/14/S2/S7 © 2013 Sun et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Genome-wide survey by ChIP-seq reveals YY1 regulation of lincRNAs in skeletal myogenesis Leina Lu, Kun Sun, Xiaona Chen, Yu Zhao, Lijun Wang, Liang Zhou, Hao Sun* and Huating Wang* Department of Chemical Pathology, Li Ka Shing Institute of Health Sciences, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China and Department of Obstetrics and Gynaecology, Li Ka Shing Institute of Health Sciences, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China Skeletal muscle differentiation is orchestrated by a network of transcription factors, epigenetic regulators, and noncoding RNAs. The transcription factor Yin Yang 1 (YY1) silences multiple target genes in myoblasts (MBs) by recruiting Ezh2 (Enhancer of Zeste Homologue2). To elucidate genome-wide YY1 binding in MBs, we performed chromatin immunoprecipitation (ChIP)-seq and found 1820 specific binding sites in MBs with a large portion residing in intergenic regions. Detailed analysis demonstrated that YY1 acts as an activator for many loci in addition to its known repressor function. No significant co-occupancy was found between YY1 and Ezh2, suggesting an additional Ezh2-independent function for YY1 in MBs. Further analysis of intergenic binding sites showed that YY1 potentially regulates dozens of large intergenic non-coding RNAs (lincRNAs), whose function in myogenesis is underexplored. We characterized a novel muscle-associated lincRNA (Yam-1) that is positively regulated by YY1. Yam-1 is downregulated upon differentiation and acts as an inhibitor of myogenesis. We demonstrated that Yam-1 functions through in cis regulation of miR-715, which in turn targets Wnt7b. Our findings not only provide the first genome-wide picture of YY1 association in muscle cells, but also uncover the functional role of lincRNAYam-1. The EMBO Journal (2013) 32, 2575–2588. doi:10.1038/ emboj.2013.182; Published online 13 August 2013 Subject Categories: chromatin & transcription; RNA; differentiation & death

Genetic testing for neurodegenerative diseases (NDs) is highly challenging because of genetic heterogeneity and overlapping manifestations. Targeted-gene panels (TGPs), coupled with next-generation sequencing (NGS), can facilitate the profiling of a large repertoire of ND-related genes. Due to the technical limitations inherent in NGS and TGPs, short tandem repeat (STR) variations are often ignored. However, STR expansions are known to cause such NDs as Huntington's disease and spinocerebellar ataxias type 3 (SCA3). Here, we studied the clinical utility of a custom-made TGP that targets 199 NDs and 311 ND-associated genes on 118 undiagnosed patients. At least one known or likely pathogenic variation was found in 54 patients; 27 patients demonstrated clinical profiles that matched the variants; and 16 patients whose original diagnosis were refined. A high concordance of variant calling were observed when comparing the results from TGP and whole-exome sequencing of four patients. Our in-house STR detection algorithm has reached a specificity of 0.88 and a sensitivity of 0.82 in our SCA3 cohort. This study also uncovered a trove of novel and recurrent variants that may enrich the repertoire of ND-related genetic markers. We propose that a combined comprehensive TGPs-bioinformatics pipeline can improve the clinical diagnosis of NDs.

BACKGROUNDThe current diagnosis and monitoring of bladder cancer are heavily reliant on cystoscopy, an invasive and costly procedure. Previous efforts in urine-based detection of bladder cancer focused on targeted approaches that are predicated on the tumor expressing specific aberrations. We aimed to noninvasively detect bladder cancer by the genome-wide assessment of methylomic and copy number aberrations (CNAs). We also investigated the size of tumor cell-free (cf)DNA fragments.METHODSShallow-depth paired-end genome-wide bisulfite sequencing of urinary cfDNA was done for 46 bladder cancer patients and 39 cancer-free controls with hematuria. We assessed (a) proportional contribution from different tissues by methylation deconvolution, (b) global hypomethylation, (c) CNA, and (d) cfDNA size profile.RESULTSMethylomic and copy number approaches were synergistically combined to detect bladder cancer with a sensitivity of 93.5% (84.2% for low-grade nonmuscle-invasive disease) and a specificity of 95.8%. The prevalence of methylomic and CNAs reflected disease stage and tumor size. Sampling over multiple time points could assess residual disease and changes in tumor load. Muscle-invasive bladder cancer was associated with a higher proportion of long cfDNA, as well as longer cfDNA fragments originating from genomic regions enriched for tumor DNA.CONCLUSIONSBladder cancer can be detected noninvasively in urinary cfDNA by methylomic and copy number analysis without previous knowledge or assumptions of specific aberrations. Such analysis could be used as a liquid biopsy to aid diagnosis and for potential longitudinal monitoring of tumor load. Further understanding of the differential size and fragmentation of cfDNA could improve the detection of bladder cancer.

Circulating tumor-derived cell-free DNA (ctDNA) analysis offers an attractive noninvasive means for detection and monitoring of cancers. Evidence for the presence of cancer is dependent on the ability to detect features in the peripheral circulation that are deemed as cancer-associated. We explored approaches to improve the chance of detecting the presence of cancer based on sequence information present on ctDNA molecules. We developed an approach to detect the total pool of somatic mutations. We then investigated if there existed a class of ctDNA signature in the form of preferred plasma DNA end coordinates. Cell-free DNA fragmentation is a nonrandom process. Using plasma samples obtained from liver transplant recipients, we showed that liver contributed cell-free DNA molecules ended more frequently at certain genomic coordinates than the nonliver-derived molecules. The abundance of plasma DNA molecules with these liver-associated ends correlated with the liver DNA fractions in the plasma samples. Studying the DNA end characteristics in plasma of patients with hepatocellular carcinoma and chronic hepatitis B, we showed that there were millions of tumor-associated plasma DNA end coordinates in the genome. Abundance of plasma DNA molecules with tumor-associated DNA ends correlated with the tumor DNA fractions even in plasma samples of hepatocellular carcinoma patients that were subjected to shallow-depth sequencing analysis. Plasma DNA end coordinates may therefore serve as hallmarks of ctDNA that could be sampled readily and, hence, may improve the cost-effectiveness of liquid biopsy assessment.

Endometrial cancer is the most commonly diagnosed cancer of the female reproductive tract in developed countries. Through genome-wide association studies (GWAS), we have previously identified eight risk loci for endometrial cancer. Here, we present an expanded meta-analysis of 12,906 endometrial cancer cases and 108,979 controls (including new genotype data for 5624 cases) and identify nine novel genome-wide significant loci, including a locus on 12q24.12 previously identified by meta-GWAS of endometrial and colorectal cancer. At five loci, expression quantitative trait locus (eQTL) analyses identify candidate causal genes; risk alleles at two of these loci associate with decreased expression of genes, which encode negative regulators of oncogenic signal transduction proteins ( SH2B3 (12q24.12) and NF1 (17q11.2)). In summary, this study has doubled the number of known endometrial cancer risk loci and revealed candidate causal genes for future study.

Epidemiological, biological, and molecular data suggest links between endometriosis and endometrial cancer, with recent epidemiological studies providing evidence for an association between a previous diagnosis of endometriosis and risk of endometrial cancer. We used genetic data as an alternative approach to investigate shared biological etiology of these two diseases. Genetic correlation analysis of summary level statistics from genomewide association studies (GWAS) using LD Score regression revealed moderate but significant genetic correlation ( r g = 0.23, P = 9.3 × 10 −3 ), and SNP effect concordance analysis provided evidence for significant SNP pleiotropy ( P = 6.0 × 10 −3 ) and concordance in effect direction ( P = 2.0 × 10 −3 ) between the two diseases. Cross‐disease GWAS meta‐analysis highlighted 13 distinct loci associated at P ≤ 10 −5 with both endometriosis and endometrial cancer, with one locus (SNP rs2475335) located within PTPRD associated at a genomewide significant level ( P = 4.9 × 10 −8 , OR = 1.11, 95% CI = 1.07–1.15). PTPRD acts in the STAT3 pathway, which has been implicated in both endometriosis and endometrial cancer. This study demonstrates the value of cross‐disease genetic analysis to support epidemiological observations and to identify biological pathways of relevance to multiple diseases.

However, the cTnT increase is probably not a sign of AMI, as the patients have neither a relative dynamic change in cTnT nor significantly higher proportions of ECG manifestations of myocardial ischemia.Given that the proportion of females in patients without cancer was higher than in patients with cancer (47.4% vs 35.1%), we assessed the potential confounding effect of sex both in a multivariate linear regression analysis with cTnT on day 0 as the dependent variable and in a multivariate logistic regression analysis with ⌬ cTnT Ͼ20% as the dependent variable.The associations between sex and baseline/⌬ cTnT were nonsignificant in both models.In all, 11.4% of ischemic stroke patients had a cancer diagnosis, which is in line with a previous Norwegian study (4 ), and the 3 most common cancer types were the same.cTn may be increased in cancer patients on chemotherapy due to a cardiotoxic effect, but none of the patients in the present study were on chemotherapy.Neutrophil extracellular traps are associated with stroke, AMI, and cancer.In a recent study, the authors suggested that troponin increase in ischemic stroke could be associated with cancer-associated arterial microthrombosis promoted by neutrophil extracellular traposis (5 ).We acknowledge that this is a single-center, retrospective study with a small sample size, and the results must therefore be interpreted with caution until verified in larger prospective studies.For instance, the nonsignificant differences in the risk factor profile presented in this study may become significant once tested in a larger study sample.The definition of a relevant change in cTnT is still a matter of debate (sex-specific upper reference limit, absolute vs relative, cutoff level of relative change, time interval between 2 measurements) and should be explored more thoroughly in future studies.The findings may be helpful in a clinical setting when evaluating the significance of cTnT increase in patients presenting with acute stroke symptoms and with an underlying cancer disease.

Urinary cell-free (cf) DNA holds great potential as a completely noninvasive form of liquid biopsy. Knowledge of the composition of cfDNA by tissue of origin is useful for guiding its clinical uses. We conducted a global survey of urinary cfDNA composition using genomewide bisulfite sequencing. While previous studies focused on detecting cfDNA from a single source at a time, genomewide tissue specific methylation signatures allow us to simultaneously deduce the proportional contribution from each contributing tissue. The proportional contributions derived from methylation deconvolution are highly correlated with those calculated using allograft-derived donor-specific genetic markers in the urine of hematopoetic stem cell and renal transplant recipients. We found a large variation of proportional contributions from different tissues. We then assessed if cfDNA undergoes time-dependent fragmentation in urine by conducting in vitro incubation experiments. In vitro incubation at 37°C showed that urinary cfDNA concentration decreased under first order kinetics with a half-life of 2.6 to 5.1h. This is reflected in parallel by a decrease in the proportion of long fragments and increase in amplitude of 10bp periodicity seen in the cfDNA size profile. This global survey of urinary cfDNA has deepened our understanding of the composition, degradation and variation of cfDNA in the urinary tract and has laid a foundation for the use of genomewide urinary cfDNA sequencing as a molecular diagnostics tool.

Endometrial cancer is the most common gynecological malignancy in the developed world. Although there is evidence of genetic predisposition to the disease, most of the genetic risk remains unexplained. We present the meta-analysis results of four genome-wide association studies (4907 cases and 11 945 controls total) in women of European ancestry. We describe one new locus reaching genome-wide significance (P < 5 × 10 -8) at 6p22.3 (rs1740828; P = 2.29 × 10 -8, OR = 1.20), providing evidence of an additional region of interest for genetic susceptibility to endometrial cancer.

A recent meta-analysis of multiple genome-wide association and follow-up endometrial cancer case-control datasets identified a novel genetic risk locus for this disease at chromosome 14q32.33. To prioritize the functional SNP(s) and target gene(s) at this locus, we employed an in silico fine-mapping approach using genotyped and imputed SNP data for 6,608 endometrial cancer cases and 37,925 controls of European ancestry. Association and functional analyses provide evidence that the best candidate causal SNP is rs2494737. Multiple experimental analyses show that SNP rs2494737 maps to a silencer element located within AKT1, a member of the PI3K/AKT/MTOR intracellular signaling pathway activated in endometrial tumors. The rs2494737 risk A allele creates a YY1 transcription factor-binding site and abrogates the silencer activity in luciferase assays, an effect mimicked by transfection of YY1 siRNA. Our findings suggest YY1 is a positive regulator of AKT1, mediating the stimulatory effects of rs2494737 increasing endometrial cancer risk. Identification of an endometrial cancer risk allele within a member of the PI3K/AKT signaling pathway, more commonly activated in tumors by somatic alterations, raises the possibility that well tolerated inhibitors targeting this pathway could be candidates for evaluation as chemopreventive agents in individuals at high risk of developing endometrial cancer.