Genome Studio Research Articles

MIR4435-2HG as a possible novel predictive biomarker of chemotherapy response and death in pediatric B-cell ALL.

Introduction: Although B-cell acute lymphoblastic leukemia (B-cell ALL) survival rates have improved in recent years, Hispanic children continue to have poorer survival rates. There are few tools available to identify at the time of diagnosis whether the patient will respond to induction therapy. Our goal was to identify predictive biomarkers of treatment response, which could also serve as prognostic biomarkers of death, by identifying methylated and differentially expressed genes between patients with positive minimal residual disease (MRD+) and negative minimal residual disease (MRD-). Methods: DNA and RNA were extracted from tumor blasts separated by immunomagnetic columns. Illumina MethlationEPIC and mRNA sequencing assays were performed on 13 bone marrows from Hispanic children with B-cell ALL. Partek Flow was used for transcript mapping and quantification, followed by differential expression analysis using DEseq2. DNA methylation analyses were performed with Partek Genomic Suite and Genome Studio. Gene expression and differential methylation were compared between patients with MRD-/- and MRD+/+ at the end of induction chemotherapy. Overexpressed and hypomethylated genes were selected and validated by RT-qPCR in samples of an independent validation cohort. The predictive ability of the genes was assessed by logistic regression. Survival and Cox regression analyses were performed to determine the association of genes with death. Results: DAPK1, BOC, CNKSR3, MIR4435-2HG, CTHRC1, NPDC1, SLC45A3, ITGA6, and ASCL2 were overexpressed and hypomethylated in MRD+/+ patients. Overexpression was also validated by RT-qPCR. DAPK1, BOC, ASCL2, and CNKSR3 can predict refractoriness, but MIR4435-2HG is the best predictor. Additionally, higher expression of MIR4435-2HG increases the probability of non-response, death, and the risk of death. Finally, MIR4435-2HG overexpression, together with MRD+, are associated with poorer survival, and together with overexpression of DAPK1 and ASCL2, it could improve the risk classification of patients with normal karyotype. Conclusion: MIR4435-2HG is a potential predictive biomarker of treatment response and death in children with B-cell ALL.

Risk of adverse cardiovascular events based on common genetic variants in 8-year follow-up of the LIPIDOGEN2015 population using the polygenic risk score (PRS): study design and methodology.

Classical risk factors such as hypertension, hypercholesterolemia, pre-diabetes, diabetes and obesity can predict adverse cardiovascular events, but they are less prognostic in patients aged < 60 years. Polygenic risk scores (PRS) can be effective in predicting adverse coronary events in younger and middle-aged patients. Our main aim is to assess the utility of a new PRS created for the Polish population in predicting mortality during an 8-year follow-up in the nationwide LIPIDOGEN2015 population. All DNA samples of 1779 patients were genotyped using Infinium Global Screening Array-24+ v3.0 Kit microarrays. The samples were amplified, fragmented, and hybridized to BeadChips. The BeadChips were scanned using iScan and converted to genotypes using Genome Studio 2.0. We will develop a PRS based on the identified single nucleotide polymorphisms (SNPs) in the LIPIDOGEN2015 project's studied population and determine the analyzed group's risk of death due to cardiovascular diseases (CVD) based on data obtained from 8 years of patient-follow-up. Using the developed PRS scale and biochemical analyses, we will assess the effectiveness of lipid-lowering therapy with statins in patients with high and low genetic risk of sudden CVD events (secondary endpoints). The developed PRS scale, combined with clinical covariates, will facilitate the creation of an algorithm to predict long-term mortality. This will enable us to stratify CVD risk more precisely, which may result in earlier implementation of lifestyle changes and dietary adjustments and potentially initiate earlier pharmacotherapy for at-risk individuals.

Analyzes In Silico Indicate the lncRNAs MIR31HG and LINC00939 as Possible Epigenetic Inhibitors of the Osteogenic Differentiation in PDLCs.

Chromatin conformation, DNA methylation pattern, transcriptional profile, and non-coding RNAs (ncRNAs) interactions constitute an epigenetic pattern that influences the cellular phenotypic commitment and impacts the clinical outcomes in regenerative therapies. Here, we investigated the epigenetic landscape of the SP7 transcriptor factor (SP7) and Distal-Less Homeobox 4 (DLX4) osteoblastic transcription factors (TFs), in human periodontal ligament mesenchymal cells (PDLCs) with low (l-PDLCs) and high (h-PDLCs) osteogenic potential. Chromatin accessibility (ATAC-seq), genome DNA methylation (Methylome), and RNA sequencing (RNA-seq) assays were performed in l- and h-PDLCs, cultured at 10 days in non-induced (DMEM) and osteogenic (OM) medium in vitro. Data were processed in HOMER, Genome Studio, and edgeR programs, and metadata was analyzed by online bioinformatics tools and in R and Python environments. ATAC-seq analyses showed the TFs genomic regions are more accessible in l-PDLCs than in h-PDLCs. In Methylome analyses, the TFs presented similar average methylation intensities (AMIs), without differently methylated probes (DMPs) between l- and h-PDLCs; in addition, there were no differences in the expression profiles of TFs signaling pathways. Interestingly, we identified the long non-coding RNAs (lncRNAs), MIR31HG and LINC00939, as upregulated in l-PDLCs, in both DMEM and OM. In the following analysis, the web-based prediction tool LncRRIsearch predicted RNA:RNA base-pairing interactions between SP7, DLX4, MIR31HG, and LINC00939 transcripts. The machine learning program TriplexFPP predicted DNA:RNA triplex-forming potential for the SP7 DNA site and for one of the LINC00939 transcripts (ENST00000502479). PCR data confirmed the upregulation of MIR31HG and LINC00939 transcripts in l-PDLCs (× h-PDLCs) in both DMEM and OM (p < 0.05); conversely, SP7 and DLX4 were downregulated, confirming those results observed in the RNA-Seq analysis. Together, these results indicate the lncRNAs MIR31HG and LINC00939 as possible epigenetic inhibitors of the osteogenic differentiation in PDLCs by (post)transcriptional and translational repression of the SP7 and DLX4 TFs.

Identification of copy number alternation profiles in metastatic oral squamous carcinoma patients by using microarray-based comparative genomic hybridization: A study on Turkish patients

ObjectiveOral squamous cell carcinoma (OSCC) is a severe form of cancer affecting different anatomic sites of the oral cavity. OSCC ranks as the sixth most common cancer type with an increasing prevalence globally. However, the mechanisms of OSCC process at later stages are not well understood. In this study, we aimed to determine genetic alternations in metastatic OSCC patients to identify genomic changes occurred at metastatic phase of the disease. Material and MethodsThe Illumina CytoSNP-12 Array was used to determine copy number variations in OSCC cancer genome. Hybridization procedures were performed according to the manufacturer procedures (Illumina). Arrays were scanned on iScan System (Illumina). Data were analyzed using Illumina Genotyping module of Genome Studio software (version 1.2, Illumina). Multiple CNV algorithms and copy number alternations were accessed by Genome Studio. CNVs in whole genome were investigated by using a chromosomal heat map. ResultsWe reported that gains in 8q21.11-ter, 9p21.3, 13q14.11-ter, 13q13.3-ter and losses in 5q14.3-ter, 5q35 and 17p13.3–12 were associated with the development of OSCC. In addition, we also detected that deletion in 2q33.2-ter and 2q35–37.3 regions were also associated with OSCC metastasis process. ConclusionsOur results were also showed that gains in 11q13.3-q13.4 and 2q13.2 chromosomal regions could promote the metastatic OSCC process. We believe that results of the study will help to find new biomarkers for diagnosis at later stage of OSCC.

The coefficients of inbreeding revealed by ROH study among inbred individuals belonging to each type of the first cousin marriage: A preliminary report from North India.

Genome-wide runs of homozygosity (ROH) are appropriate to estimate genomic inbreeding, determine population history, unravel the genetic architecture of complex traits and disorders. The study sought to investigate and compare the actual proportion of homozygosity or autozygosity in the genomes of progeny of four subtypes of first cousin mating in humans, using both pedigree and genomic measures for autosomes and sex chromosomes. For this purpose, Illumina Global Screening Array-24 v1.0 BeadChip followed by cyto-ROH analysis through Illumina Genome Studio was used to characterise the homozygosity in five participants from North Indian state (Uttar Pradesh). PLINK v.1.9 software was used to estimate the genomic inbreeding coefficients viz. ROH-based inbreeding estimate (FROH) and homozygous loci-based inbreeding estimate (FHOM). A total of 133 ROH segments were detected with maximum number and genomic coverage in Matrilateral Parallel (MP) type and minimum in outbred individual. ROH pattern revealed that MP type has a higher degree of homozygosity than other subtypes. The comparison of FROH, FHOM, and pedigree-based inbreeding estimate (FPED) showed some difference in theoretical and realised proportion of homozygosity for sex-chromosomal loci but not for autosome for each type of consanguinity. This is the very first study to compare and estimate the pattern of homozygosity among the kindreds of first cousin unions. However, a greater number of individuals from each type of marriage is required for statistical inference of no difference between theoretical and realized homozygosity among different degrees of inbreeding prevalent in humans worldwide.

Alzheimer's Disease Genotype Frequencies Differ in Top Risk Alleles Between Mexican Americans and Non‐Hispanic Whites in HABLE Cohort

AbstractBackgroundAlzheimer’s Disease (AD) and/or other related dementias remain a significant burden on our aging population. Here we evaluate the top 10 AD risk alleles previously reported by Kunkle et al. (2018) in Mexican Americans and non‐Hispanic whites enrolled in the Healthy Aging Brain in Latino Elders Study (HABLE) cohort to see if allele frequencies vary based on ethnicity.MethodDNA was extracted from buffy coat samples (n = 1635) on the Hamilton robotic system with the Mag‐Bind Blood &amp; Tissue DNA HDQ 96 Kit. Genotyping was performed per manufacturer’s protocol using the Illumina Infinium Global Screening Array (GSA) and analyzed with Genome Studio 2.0. Samples with call rates less than 98% were repeated or excluded.Allele and genotype frequencies were calculated using standard statistics by compiling the top ten AD risk alleles from Kunkle et al. (2018) and measuring their frequencies in the HABLE cohort.ResultOur data suggest varying degrees of allele and genotype frequencies among the top 10 risk conferring SNPs between Mexican Americans and Non‐Hispanic Whites. In particular, we show some instances (BIN1, PTK2B) where the heterozygotes are in higher frequency than homozygotes. 8 of our evaluated SNPs show a difference greater than 5% between the two ethnicitiesConclusionIt may be beneficial to further study the top AD risk alleles among different ethnicities to determine if there are variable frequencies in those populations. We plan to expand and continue this work in other ethnicities and further elaborate on these differences to promote ethnicity targeted diagnostics and help reduce health disparities in medicine and science.

Gene Expression Differences In Three-dimensional Myobundles Compared To Two-dimensional Myocultures

PURPOSE: Traditional two-dimensional (2D) in vitro models of human skeletal muscle are limited in their ability to fully mimic in vivo muscle, as in vivo muscle exists in a complex three-dimensional (3D) structure. We have developed a novel engineered three-dimensional (3D) myobundle in vitro model that we believe more closely models skeletal muscle behavior. Here, we determined baseline gene expression differences among three models: the 3D myobundles, 2D cell cultures, and explant biopsies. METHODS: Previously collected skeletal muscle (vastus lateralis) biopsy samples from adult men and women (n = 6) were used. Each sample was used to generate the following groups: explant (RNA from biopsy), 2D (RNA from differentiated myotubes) and 3D (RNA from 3D myobundles seeded from each primary sample). 200ng of isolated RNA for each sample was used to generate global gene expression profiles (HumanHT-12 v4.0 Gene Expression BeadChip Arrays). Data were processed using Illumina Genome Studio and imported into Partek Genomics Suite for statistical analysis. Differential gene expression was assessed via 2-way ANOVA (group*ID) with the following post-hoc comparisons: 2D/biopsy, 3D/biopsy and 3D/2D. Resultant lists were filtered at p<0.01 and fold change >|1.5|. Biological pathway analyses of differentially regulated gene sets were done using Ingenuity Pathway Analysis. RESULTS: ANOVA detected 3754 genes different between 2D/biopsy, 3273 genes different between 3D/biopsy and 488 genes different between 3D/2D cultures. Biological pathway analysis identified representation of the following canonical pathways in our gene set: calcium signaling (26 genes; z-score=1.508, -log p-value=13.9), integrin signaling (28 genes; z-score=-2.711, -log p-value=13.5), and actin cytoskeleton signaling (29 genes; -log p-value=12.3). CONCLUSION: The 3D cell myobundle system produced relatively fewer differences from biopsies as compared to 2D cell cultures, but some significant differences from biopsy samples remain. Comparison of 3D to 2D culture systems shows transcriptional changes that align with increases in calcium signaling, while downregulations in the actin cytoskeleton and integrin signaling demonstrate significant structural differences between the two in vitro models tested.

MON-LB129 A Pilot Genome Wide Association Study (GWAS) on Primary Aldosteronism Patients in a Multi-Ethnic Malaysian Cohort

Studies on excised aldosterone-producing lesions have found somatic mutations in five genes (KCNJ5, CACNA1D, ATP1A1, ATP2B3, and CTNNB1) commonly causes the excess aldosterone production. Interestingly, Oriental cohorts had the highest frequency of KCNJ5 mutations whereas CACNA1D mutations were most common in Black African Caribbean patients, suggesting that genetic background affects the prevalence and distribution of aldosterone-driving somatic mutation. We therefore aimed to identify the common germline variants that associates with excess aldosterone production through performing a pilot genome wide association study (GWAS) on primary aldosteronism (PA) patients. GWAS was performed using the Human Infinium OmniExpressExome-8 v1.4 BeadChip containing 960,919 markers to compare gDNA of 154 PA patients with 78 healthy controls. Samples were checked for sex discordance, heterozygosity rate, missing rate and the degree of recent shared ancestry for each pair of individuals using the PLINK program and Genome Studio (Illumina). In total, 150 patients and 75 controls (112 males and 113 females) were included in the downstream analysis. 630,749 markers that passed quality control steps (missing call rate <95% and minor allele frequency in controls >1%) were used to perform association analysis using the Chi-square Test which was then subjected to multiple testing corrections (Bonferroni correction). As expected with a pilot sample size, no variants passed the suggestive significant threshold of Bonferroni corrected P-value < 5 x 10-6 (-log10 P = 5.3). However, 27 SNPs had the uncorrected P-value<0.0002, odds ratio >2, and differences of frequencies in cases compared to control >0.1 or <-0.2, of which 3 genes (SRGAP3, AUTS2, and RORA) associated with these SNPs were also highlighted in the UK Biobank database of 72 patients with primary aldosteronism (https://biobankengine.stanford. edu/coding/HC189). Of these, RORA has recently been found to be down-regulated in adrenals from PA patients and spontaneously hypertensive rat adrenals compared to control adrenalsa,b. RORA encodes for the protein retinoic acid receptor (RAR)-related orphan receptor alpha, a member of the NR1 subfamily of nuclear hormone receptors (NR1F1). Interestingly, adrenal is the second organ to skin with the highest expression of RORA and treatment of angiotensin II in the adrenocortical cell line H295R increases RORA expressionc,d. Taken together, this pilot GWAS highlights RORA as a potential nuclear hormone receptor that regulates aldosterone production.References aChu et al., Int J Clin Exp Pathol 2017;10(9):10009-10018. bTanaka et al., Hypertens Res 2019;42(2):165-173. cNogueira et al., Mol Cell Endocrinol 2009; 302(2): 230–236. dGTEx Analysis Release V7 (dbGaP Accession phs000424.v7.p2)AcknowledgementsThis research was supported by the Malaysian Ministry of Higher Education Grant (FRGS/1/2015/SKK08/UKM/02/3), The National University of Malaysia (UKM) University Grant (GUP-2016-083), and The UKM Medical Center Fundamental Grant (FF-2016-302).

DNA methylation signature is prognostic of choroid plexus tumor aggressiveness

BackgroundHistological grading of choroid plexus tumors (CPTs) remains the best prognostic tool to distinguish between aggressive choroid plexus carcinoma (CPC) and the more benign choroid plexus papilloma (CPP) or atypical choroid plexus papilloma (aCPP); however, these distinctions can be challenging. Standard treatment of CPC is very aggressive and often leads to severe damage to the young child’s brain. Therefore, it is crucial to distinguish between CPC and less aggressive entities (CPP or aCPP) to avoid unnecessary exposure of the young patient to neurotoxic therapy. To better stratify CPTs, we utilized DNA methylation (DNAm) to identify prognostic epigenetic biomarkers for CPCs.MethodsWe obtained DNA methylation profiles of 34 CPTs using the HumanMethylation450 BeadChip from Illumina, and the data was analyzed using the Illumina Genome Studio analysis software. Validation of differentially methylated CpG sites chosen as biomarkers was performed using pyrosequencing analysis on additional 22 CPTs. Sensitivity testing of the CPC DNAm signature was performed on a replication cohort of 61 CPT tumors obtained from Neuropathology, University Hospital Münster, Germany.ResultsGenerated genome-wide DNAm profiles of CPTs showed significant differences in DNAm between CPCs and the CPPs or aCPPs. The prediction of clinical outcome could be improved by combining the DNAm profile with the mutational status of TP53. CPCs with homozygous TP53 mutations clustered as a group separate from those carrying a heterozygous TP53 mutation or CPCs with wild type TP53 (TP53-wt) and showed the worst survival outcome. Specific DNAm signatures for CPCs revealed AK1, PER2, and PLSCR4 as potential biomarkers for CPC that can be used to improve molecular stratification for diagnosis and treatment.ConclusionsWe demonstrate that combining specific DNAm signature for CPCs with histological approaches better differentiate aggressive tumors from those that are not life threatening. These findings have important implications for future prognostic risk prediction in clinical disease management.

Cytokine Gene Polymorphisms Are Associated with Disease Response to Blinatumomab in Patients with B-Cell Acute Lymphoblastic Leukemia

Blinatumomab (BT), a CD19/CD3 bispecific T-cell engager (BiTE) antibody recently approved for treatment of relapsed/refractory acute lymphoblastic leukemia (r/r ALL), has resulted in a 40-50% complete response (CR)/CR with incomplete count recovery (CRi) rate and frequent cytokine release syndrome (CRS) events. While extent of disease burden has been identified as a key predictor of disease response, there have been no studies to date evaluating the impact of single nucleotide polymorphisms (SNPs) in cytokine genes on disease response or CRS after treatment with BT. Here, we evaluated the possible association between cytokine SNPs, disease response, and/or CRS in r/r ALL patients with receiving BT.A total of 82 patients (consecutive case-series) received BT between 2012 and 2017 at City of Hope. Of these, 68 patients had archived DNA samples available for study and analysis. Genotyping was done using Illumina's Omni 5-4 bead array, which interrogates more than 4.2 million SNPs. Array processing was done following manufacturer's recommendations and arrays were scanned by Illumina's iScan instrument. Beeline software and Genome Studio (Illumina) were used for analysis of scanned images and to generate the genotyping calls and quality control reports, respectively. Our analysis focused on the following cytokine genes of interest (known to be associated with autoimmune diseases/graft-versus-host disease): IL-1B, IL-2, IL-6, IL-7R, IL-10, IL-11, IL-12, IL-17A, IL-23R, TNF-α, TGF-β, and IFN-γ. Two modes of inheritance (dominant and recessive) were considered whenever appropriate. Univariate analyses of SNPs, response and CRS were performed using Fisher's exact test. P values were not adjusted for multiple hypothesis testing due to the exploratory nature of the analyses.In our cohort, median age of patients at BT treatment was 34 years old (range: 7-85), with 32 females (39%) and 50 males (61%). Patients' race/ethnicity included 49 Hispanic (60%), 20 non-Hispanic Caucasians (24%), 9 Asians (11%), and 3 African Americans (3%). All patients had primary refractory (n=16) or relapsed disease status (1st relapse: n=38, 2nd or subsequent relapse: n=27) with median lines of prior therapy of 2 (range: 0-5). Cytogenetic abnormalities included Ph+ALL in 11 patients, while 50 patients had other cytogenetic abnormalities and 15 had normal cytogenetics. Bone marrow blasts of >50% at BT treatment was detected in 45 patients, and 22 patients had a history of or active extra-medullary involvement. After BT treatment, 37 patients (45%) achieved CR/CRi with a median duration of 9.5 months (range: 1-37), 54 patients (66%) experienced CRS (grade 1: n=35, grade 2: n=14, and grade 3: n=5), and 9 patients developed neurotoxicity. Lastly, the peak C-reactive protein level (median/range) was 17 mg/L (range: 1-392).By univariate analysis, disease response (CR/CRi) to BT was significantly associated with the following cytokine SNPs: IL2 rs2069762 [48.3% (T/T-G/T: n=60) Vs. 0% (G/G; n=7), p=0.016], IL17A rs3819024 [30.8% (A/A; n=39) Vs. 58.6% (A/G-G/G; n=29), p=0.027], and IL17A rs4711998 [54.5% (A/A: n=33) Vs. 29.4% (A/G-G/G: n=34), p=0.049]. (Table 1) These three cytokine SNPs and two additional SNPs with p-values between 0.05 and 0.1 (IL7R rs1494555, IL17A rs8193036) were evaluated by multivariable analysis, adjusted for >50% bone marrow blasts, which was associated with lower rate of CR/CRi (33% vs. 63% with <50%, p=0.02). By multivariable analysis, IL17A rs3819024 remained significant while IL17A rs4711998 was at borderline significance (p=0.06). (IL2 rs2069762 could not be tested due to no CR/CRi occurrence). None of the cytokine SNPs analyzed were associated with CRS (0 vs. any grade, or grades 0-1 vs. 2-3).In conclusion, to our knowledge this is the first study to demonstrate a possible association between treatment response to BT and cytokine genetic polymorphisms. Our hypothesis-generating data suggest a potential role of IL17 and IL2 in BT response and justify a larger confirmatory study, which may lead to personalized BT immunotherapy for B-ALL. [Display omitted] DisclosuresStein:Amgen Inc.: Speakers Bureau; Celgene: Speakers Bureau. Forman:Mustang Therapeutics: Other: Licensing Agreement, Patents & Royalties, Research Funding.

Detection of copy number variations in brown and white layers based on genotyping panels with different densities

BackgroundCopy number variations (CNV) are an important source of genetic variation that has gained increasing attention over the last couple of years. In this study, we performed CNV detection and functional analysis for 18,719 individuals from four pure lines and one commercial cross of layer chickens. Samples were genotyped on four single nucleotide polymorphism (SNP) genotyping platforms, i.e. the Illumina 42K, Affymetrix 600K, and two different customized Affymetrix 50K chips. CNV recovered from the Affymetrix chips were identified by using the Axiom® CNV Summary Tools and PennCNV software and those from the Illumina chip were identified by using the cnvPartition in the Genome Studio software.ResultsThe mean number of CNV per individual varied from 0.50 to 4.87 according to line or cross and size of the SNP genotyping set. The length of the detected CNV across all datasets ranged from 1.2 kb to 3.2 Mb. The number of duplications exceeded the number of deletions for most lines. Between the lines, there were considerable differences in the number of detected CNV and their distribution. Most of the detected CNV had a low frequency, but 19 CNV were identified with a frequency higher than 5% in birds that were genotyped on the 600K panel, with the most common CNV being detected in 734 birds from three lines.ConclusionsCommonly used SNP genotyping platforms can be used to detect segregating CNV in chicken layer lines. The sample sizes for this study enabled a detailed characterization of the CNV landscape within commercially relevant lines. The size of the SNP panel used affected detection efficiency, with more CNV detected per individual on the higher density 600K panel. In spite of the high level of inter-individual diversity and a large number of CNV observed within individuals, we were able to detect 19 frequent CNV, of which, 57.9% overlapped with annotated genes and 89% overlapped with known quantitative trait loci.

Identification of an Epigenetic Signature of Osteoporosis in Blood DNA of Postmenopausal Women.

Osteoporosis is one of the most common age-related progressive bone diseases in elderly people. Approximately one in three women and one in five men are predisposed to developing osteoporosis. In postmenopausal women, a reduction in BMD leads to an increased risk of fractures. In the current study, we delineated the DNA methylation signatures in whole blood samples of postmenopausal osteoporotic women. We obtained whole blood DNA from 22 normal women and 22 postmenopausal osteoporotic women (51 to 89 years old) from the Canadian Multicenter Osteoporosis Study (CaMos) cohort. These DNA samples were subjected to Illumina Infinium human methylation 450 K analysis. Illumina 450K raw data were analyzed by Genome Studio software. Analysis of the female participants with early and advanced osteoporosis resulted in the generation of a list of 1233 differentially methylated CpG sites when compared with age-matched normal women. T test, ANOVA, and post hoc statistical analyses were performed, and 77 significantly differentially methylated CpG sites were identified. From the 13 most significant genes, ZNF267, ABLIM2, RHOJ, CDKL5, and PDCD1 were selected for their potential role in bone biology. A weighted polygenic DNA methylation score of these genes predicted osteoporosis at an early stage with high sensitivity and specificity and correlated with measures of bone density. Pyrosequencing analysis of these genes was performed to validate the results obtained from Illumina 450 K methylation analysis. The current study provides proof of principal for the role of DNA methylation in osteoporosis. Using whole blood DNA methylation analysis, women at risk of developing osteoporosis can be identified before a diagnosis of osteoporosis is made using BMD as a screening method. Early diagnosis will help to select patients who might benefit from early therapeutic intervention. © 2018 American Society for Bone and Mineral Research.

Epigenome analysis of colorectal cancer: a genome wide approach

Event Abstract Back to Event Epigenome analysis of colorectal cancer: a genome wide approach Muhiddin Ishak1, Nurul-Syakima Ab Mutalib1*, Najwa F. Md Yusof1, Sazuita Saidin1, Isa Mohamed Rose2, Luqman Mazlan3, Ismail Sagap3 and Rahman Jamal1 1 UKM Medical Molecular Biology Institute (UMBI), Malaysia 2 Department of Pathology, Faculty of Medicine, National University of Malaysia, Malaysia 3 Department of Surgery, Faculty of medicine, University of Malaya, Malaysia Background Colorectal cancer (CRC) contributes to around 1.36 million of the total cases worldwide. In Malaysia, the incidence rate of CRC is 21.3 cases per 100,000 population. Many molecular pathways are involved in carcinogenesis which includes DNA methylation. Genes with high levels of 5-methylcytosine (hypermethylation) in the promoter regions are associated with gene silencing and may alter the signalling pathway that contribute to CRC tumourigenesis. Subsequently, genes with low levels of 5-methylcytosine (hypomethylation) have also been implicated in CRC progression through tumour-suppressor genes or act as oncogenes. Many studies have also revealed the involvement of enhancer regions in the regulation of gene expression, hence the demand for a more comprehensive tool for methylation profiling. Methods Genomic DNA was extracted from 12 paired matched samples collected from UKM Medical Centre (UKMMC). Bisulfite conversion was performed and the bisulfite converted DNA was subjected to microarray using Human Infinium Epic Beadchip Array. The data was analyzed using Bioconductor-ChAMP V2.8.1 and Genome Studio V1.8. Results There were 25170 significantly differentially methylated probes (p≤ 0.05), with 7254 being hypermethylated and 17916 being hypomethylated. ADHFE1, C1orf70 and CMTM3 were the top three hypermethylated genes whereas AMPH, CHST10 and MYBPC3 were the top three hypomethylated genes. Pathway enrichment analysis revealed that majority of the hypermethylated genes were involved in neuroactive ligand receptor, cancer pathways, calcium pathway, and cAMP signalling, amongst others. Meanwhile, the hypomethylated genes were involved in olfactory transduction, neuroactive ligand receptor, PI3K-Akt signalling and focal adhesion. Out of this differentially methylated genes, 715 genes were found in Phantom4 enhancers, 355 genes in Phantom5 enhancers and 2214 genes at the open chromatin. Conclusion This is the first study that investigated the methylation profile of local CRC patients using the latest platform assay. The new knowledge from this study can be utilized to increase our understanding of CRC methylomics further. Keywords: colorectal cancer, epigenetics, MethylationEPIC, DNA Methylation, Open chromatin, Enhancer Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019. Presentation Type: Oral Presentation Topic: Cancer Citation: Ishak M, Ab Mutalib N, Md Yusof NF, Saidin S, Mohamed Rose I, Mazlan L, Sagap I and Jamal R (2019). Epigenome analysis of colorectal cancer: a genome wide approach. Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2018.63.00036 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2018; Published Online: 17 Jan 2019. * Correspondence: PhD. Nurul-Syakima Ab Mutalib, UKM Medical Molecular Biology Institute (UMBI), Kuala Lumpur, Malaysia, syakima@ppukm.ukm.edu.my Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Muhiddin Ishak Nurul-Syakima Ab Mutalib Najwa F Md Yusof Sazuita Saidin Isa Mohamed Rose Luqman Mazlan Ismail Sagap Rahman Jamal Google Muhiddin Ishak Nurul-Syakima Ab Mutalib Najwa F Md Yusof Sazuita Saidin Isa Mohamed Rose Luqman Mazlan Ismail Sagap Rahman Jamal Google Scholar Muhiddin Ishak Nurul-Syakima Ab Mutalib Najwa F Md Yusof Sazuita Saidin Isa Mohamed Rose Luqman Mazlan Ismail Sagap Rahman Jamal PubMed Muhiddin Ishak Nurul-Syakima Ab Mutalib Najwa F Md Yusof Sazuita Saidin Isa Mohamed Rose Luqman Mazlan Ismail Sagap Rahman Jamal Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

114: Effects of maternal oxytocin on gene expression in the brains of perinatal and neonatal mice

The hormone/neuropeptide oxytocin (OXT) facilitates childbirth, and promotes maternal social bonding and lactation. However, little is known about the effects of prenatal OXT exposure on the developing brain. Given the behavioral effects of synthetic OXT, we investigated the effect of maternal OXT administration on gene expression in peri- and neonatal mouse brains. Pregnant C57BL/6 mice (10 weeks old, gestational day 18) were given saline or OXT (1U/mouse) subcutaneously every 30 minutes for 2 hours. 4 hours later, dams (7 saline, 7 oxytocin) and pups (delivered by C-section) were euthanized. Other dams (6 saline, 7 oxytocin) delivered naturally and pups were euthanized on postnatal day 9 (PND9). DNA and RNA were isolated from pup tails and brains, respectively. Gender was determined by PCR. Brain RNA (12F, 12M, with RIN >9.3) was converted to ds-cDNA and hybridized to MouseWG6v2.0 BeadChip (Illumina), scanned (via Illumina HiScan) and analyzed using Genome Studio v2011.1 (Illumina). Differential expression analysis was done with the moderated t-test in the R limma package. Findings were confirmed by RT-qPCR. Pathway analysis was performed with the R gage package and the mm_GO, mm_metabolic, and mm_pathway annotated gene sets from the R gskb package. GSEA was performed on the clustered pathways. We identified distinct gene expression patterns in perinatal mouse brains following in utero OXT exposure (Fig 1A). Female perinatal brains exposed to OXT showed significantly more differential expression patterns than male brains, with changes in several genes implicated in neurodevelopment and neuropsychiatric disorders. Although OXT-exposed PND9 brains showed no significant differences vs saline-brains, when both timepoints were analyzed together >1700 genes were differentially expressed (Fig 1B), including Sfrs11 (pre-mRNA processing), Jak1 (promotes astrogliosis), and Tardbp (implicated in dementia). Sex-specific differences were found in gene expression and in pathways, including DNA repair, DNA replication, myelin sheath, and INCEP. Prenatal OXT exposure leads to differential regulation of several nervous system-related genes and pathways in peri- and postnatal brains. Ongoing studies are linking gene expression patterns to neurodevelopment, social behaviors, and disease states.

Genomic profiling of non-small cell lung cancer: A pilot study from South India

&lt;p&gt;Introduction: Lung cancer accounts for the highest mortality globally, and there is a need to find new strategies and novel treatment options. Lung cancer is genetically heterogeneous across various ethnic populations, and therefore identifying the mutational landscape in Indian patients is important. Methods: The aim of the pilot study is to identify the prevalence and temporal sequence of molecular lesions in lung cancer patients from a regional cancer institute in South India using the circulating tumor DNA technique. Genotyping was performed on 26 newly diagnosed metastatic lung cancer patients using Illumina Omni Express Exome. Chip data were inspected using Genome Studio and subsequently exported for use with PLINK and further annotation was performed. Results: Seven genes including TP53 (61.5%), MUC16 (57.6%), KRAS (38.4%), STK11 (19.2%), Epidermal growth factor receptor (15.3%), ataxia telangiectasia mutated (15.3%), and nuclear factor-κβ (7.6%) demonstrated high incidence of mutations in patients. The other genes identified were observed in &amp;lt;5% of patients. Several types of mutations including missense, silent, nonsense, and frameshift mutations were observed in these genes. Conclusion: Integration of mutational profiling to identify gene mutations is required to facilitate personalized lung cancer therapy.&lt;/p&gt;

Evaluation of whole genome amplified DNA to decrease material expenditure and increase quality

AimThe overall aim of this study is to evaluate whole genome amplification of DNA extracted from dried blood spot samples. We wish to explore ways of optimizing the amplification process, while decreasing the amount of input material and inherently the cost. Our primary focus of optimization is on the amount of input material, the amplification reaction volume, the number of replicates and amplification time and temperature. Increasing the quality of the amplified DNA and the subsequent results of array genotyping is a secondary aim of this project. MethodsThis study is based on DNA extracted from dried blood spot samples. The extracted DNA was subsequently whole genome amplified using the REPLIg kit and genotyped on the PsychArray BeadChip (assessing>570,000 SNPs genome wide). We used Genome Studio to evaluate the quality of the genotype data by call rates and log R ratios. ResultsThe whole genome amplification process is robust and does not vary between replicates. Altering amplification time, temperature or number of replicates did not affect our results. We found that spot size i.e. amount of input material could be reduced without compromising the quality of the array genotyping data. We also showed that whole genome amplification reaction volumes can be reduced by a factor of 4, without compromising the DNA quality. DiscussionWhole genome amplified DNA samples from dried blood spots is well suited for array genotyping and produces robust and reliable genotype data. However, the amplification process introduces additional noise to the data, making detection of structural variants such as copy number variants difficult. With this study, we explore ways of optimizing the amplification protocol in order to reduce noise and increase data quality. We found, that the amplification process was very robust, and that changes in amplification time or temperature did not alter the genotyping calls or quality of the array data. Adding additional replicates of each sample also lead to insignificant changes in the array data. Thus, the amount of noise introduced by the amplification process was consistent regardless of changes made to the amplification protocol. We also explored ways of decreasing material expenditure by reducing the spot size or the amplification reaction volume. The reduction did not affect the quality of the genotyping data.

Successful Replication of GWAS Hits for Multiple Sclerosis in 10,000 Germans Using the Exome Array.

Genome-wide association studies (GWAS) successfully identified various chromosomal regions to be associated with multiple sclerosis (MS). The primary aim of this study was to replicate reported associations from GWAS using an exome array in a large German study. German MS cases (n = 4,476) and German controls (n = 5,714) were genotyped using the Illumina HumanExome v1-Chip. Genotype calling was performed with the Illumina Genome Studio(TM) Genotyping Module, followed by zCall. Single-nucleotide polymorphisms (SNPs) in seven regions outside the human leukocyte antigen (HLA) region showed genome-wide significant associations with MS (P values < 5 × 10(-8) ). These associations have been reported previously. In addition, SNPs in three previously reported regions outside the HLA region yielded P values < 10(-5) . The effect of nine SNPs in the HLA region remained (P < 10(-5) ) after adjustment for other significant SNPs in the HLA region. All of these findings have been reported before or are driven by known risk loci. In summary, findings from previous GWAS for MS could be successfully replicated. We conclude that the regions identified in previous GWAS are also associated in the German population. This reassures the need for detailed investigations of the functional mechanisms underlying the replicated associations.

Abstract 1056: Impact of host methylome on cervical cancer pathogenesis

Abstract Identification of episomal HPV16 in cervical cancers (CaCx) besides the integrated forms, prompted us to investigate the global CpG methylation signatures among the different categories of CaCx samples, since alterations of CpG methylation in host genomes, common in many cancers, are likely to influence host pathogen interactions and the disease pathogenesis. We determined global DNA methylation patterns of the host genomes, in HPV16 (+) CaCx cases (11 episomal and 12 integrated) and controls (12 HPV16 (-) and 12 HPV16 (+), employing the Illumina Infinium 450K Bead Chip assay. Using Genome Studio software, we extracted the raw signal intensities of each probe. With statistical methods developed in-house, we performed color adjustment and normalization of the data using control probes. Differentially methylated CpG loci were identified by comparing methylation estimates (M-values) using a two-sample t-test followed by adjustment of p-values based on False Discovery Rate threshold of 0.01 among (i) CaCx cases, irrespective of HPV16 physical status, (ii) CaCx cases with episomal HPV16 and (iii) CaCx cases with integrated HPV16, compared to controls irrespective of HPV, since no difference was recorded among these two groups. Differentially methylated CpGs were identified and mapped using Illumina annotation to various genic regions (TSS1500, TSS200, 1st Exon, Gene Body, 5′ UTR and 3′ UTR). Irrespective of viral physical status, hypermethylation was more prominent across all genic regions than hypomethylation, albeit hypermethylation being lower in Gene Body and 3′-UTR regions only. Subsequently employing a Δβ threshold of 0.3 i.e. difference of β between two groups as 30%, differentially methylated CpGs mapped to 2536, 3222 and 3003 genes among total CaCx set, episomal and integrated CaCx cases, respectively. Ingenuity Pathway Analysis revealed a number of pathways that were significantly enriched among total CaCx cases, episomal or integrated, while some enriched pathways were recorded exclusively among episomal (Gap Junction Signaling, FGF Signaling, Huntington's Disease Signaling) and integrated (Chemokine Signaling etc.) CaCx cases. Expression of FGF2 gene (FGF Signaling pathway), PIK3R1 gene (Gap Junction Signaling pathway) and CXCL12 gene (Chemokine Signaling pathway) was found to be down regulated, based on Illumina -HT12_V4 array analysis on same set of samples. Significant negative correlation between hypermethylation in the promoter region, involving 5 CpGs of FGF and 1 CpG of CXCL12, were recorded (Pearson Correlation p-value&amp;lt;0.001). In case of PIK3R1, downregulated expression was negatively correlated with hypermethylation of 2 CpGs in the Gene Body. The study highlights the relevance of host methylation in cervical cancer pathogenesis involving Gene Body methylation in addition to promoters, and provides novel insights into HPV16 related CaCx pathogenesis involving episomal and integrated viral genomes. Citation Format: Shrinka Sen, Samsiddhi Bhattacharjee, Indranil Mukhopadhyay, Paramita Mandal, Sweta Sharma, Rahul Roy Chowdhury, Sharmila Sengupta. Impact of host methylome on cervical cancer pathogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1056. doi:10.1158/1538-7445.AM2015-1056

Abstract P3-06-24: Brief exposure to trastuzumab prior to preoperative chemotherapy confirms predictors of response to treatment

Abstract Background: HER2-positive (HER2+) breast cancer is biologically heterogeneous; no consistent biomarkers of response to trastuzumab (T) have been identified. Recent data suggest intrinsic subtypes applied to HER2 cohorts are able to predict response to T-based therapy. We designed a multicenter trial (DFCI 03-311) to determine if exposure to T-alone prior to combination T-based chemotherapy (C) could produce predictors of pathologic complete response (pCR). We previously reported that change in AKT and IGF signatures by gene expression could predict pCR. New gene expression pathways (immune and HER2 subtypes) were evaluated. Design : Fresh tumor core biopsies were taken at baseline and 2 weeks after a single dose of T (8mg/m2) from 80 HER2-overexpressing, stage II/III breast cancer patients enrolled on a clinical trial of T&amp;gt;T+C. A total of 122 samples (50 matched pairs and 22 single timepoints) produced useable gene expression data. Nucleic acids were extracted using Qiagen AllPrep and were analyzed with Illumina HT12v3. Data generated was normalized and median subtracted using Illumina's Genome Studio software; intrinsic subtyping was performed using median-subtracted PAM50 genes; ‘Estimation of STromal and Immune cells in Malignant Tumors using Expression data’ (ESTIMATE) algorithm was used to infer the fraction of stromal and immune cells in tumor samples. Results: ESTIMATE Immune score predicts pCR after one dose of treatment of T compared with patients with stable disease or progression as best response ,(NOR;p=0.01) PAM50 subtyping of baseline samples showed increase in pCR in the HER2-enriched cluster (7/17; 41%) versus the Luminal B (2/14; 14%) and HER2 basal clusters (1/8; 12.5% ; P=0.04). ). Change in subtype between baseline and 2 weeks appeared less common in HER2-Luminal B (4/13; 31%) compared with HER2 enriched (9/16; 56%) or HER2 Basal (5/9; 55%). Of note, HER2-Basal had the highest frequency of NOR (4/13; 31%; P=0.06), despite having greater subtype change than HER2-luminal tumors. Conclusions: Biomarkers of response to T-based regimens are greatly needed as no consistent predictors beyond HER2 are available. This data confirms (Carey et al, ASCO 2014) that the HER2 enriched subtype predicts pCR and (Harris et al, CCR 2007) that HER2 basal tumors are more likely to be non-responders. In this study, one dose of T is necessary to predict pCR using ESTIMATE immune signature. Validation of these findings a large cohort is needed. Citation Format: Aditi Shirish Vadodkar, Vinay Varadan, Kristy Miskimen, VV Bossuyt, MM Abu-khalaf, I Krop, E Winer, LN Harris. Brief exposure to trastuzumab prior to preoperative chemotherapy confirms predictors of response to treatment [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-24.

BT-01 * TENASCIN-C: A NOVEL THERAPEUTIC TARGET IN PEDIATRIC BRAINSTEM GLIOMA

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is the most deadly solid tumor of childhood. Histone 3 mutation occurs in 80% of DIPGs, causing global transcriptional changes. Tenascin-C (TNC) is an extracellular matrix protein expressed during normal brain development by oligodendroglial progenitor cells (OPCs), the purported DIPG cell of origin. TNC is highly expressed in adult glioma, contributing to local invasion and poor survival. We report increased TNC in tumor tissue and cerebrospinal fluid (CSF) from children with high-grade glioma, including DIPG, and characterize TNC expression in relation to H3K27M mutation and DNA methylation status. METHODS: Tumor tissue collected intraoperatively or post-mortem from children with brainstem (DIPG, n = 14), supratentorial (n = 7), and cerebellar astrocytoma (n = 2), and CSF from DIPG (n = 9) and supratentorial astrocytoma (n = 17) was subjected to MS/MS proteomic analysis. Tissue gene expression, DNA methylation, H3F3A and HIST1H3B sequencing was performed. TNC expression was validated via Western blot and immunohistochemistry. Data integration was performed with Genome Studio, Partek and Ingenuity Pathway Analysis. RESULTS: TNC expression was significantly increased in 75% of gliomas compared to normal tissue, including all DIPG specimens tested (fold change >2, p < 0.05). Secreted TNC was detected in 7/9 DIPG CSF specimens (77.8%). Tumor-specific TNC expression was confirmed with Western blot and tissue immunohistochemistry. TNC expression correlated with tumor grade and was associated with Notch pathway activation, H3K27M mutation, and TNC promoter hypomethylation. CONCLUSIONS: We report increased TNC expression in tissue and CSF of pediatric high-grade gliomas, including DIPGs, associated with promoter hypomethylation and H3K27M mutation. Given the effect of TNC on OPC proliferation, migration and differentiation, TNC could serve as a clinical biomarker of disease and rational therapeutic target for a substantial subgroup of DIPG patients. Studies exploring the mechanism of TNC overexpression and effects of targeting TNC expression in DIPG are currently underway.