Dysregulated epigenome in perinatally HIV-infected children on antiretrovirals 1 1 2 2 1 Stephanie Shiau, MPH Stephanie Shiau , Stephen M. Arpadi , Renate Strehlau , Avy Violari , Shuang Wang , Columbia University 2 1 2 1 1 G.H. Sergievsky Center Faeezah Patel , Marc Foca , Ashraf Coovadia , Elaine J. Abrams , Louise Kuhn , 622 W 168th Street PH 19-110 New York, NY 10032 CHANGES Study Team [email protected] 1Columbia University, New York, NY, USA, 2University of the Witwatersrand, Johannesburg, South Africa 851 Pre-Processing and Normalization Table 3. Characteristics of 119 HIV-infected and Gene Function Introduction 60 HIV-uninfected children ages 5-9 years Pre-processing and normalization was performed with the Comparing HIV-infected and HIV-uninfected children, functional Early initiation of antiretroviral therapy (ART) has improved R/Bioconductor RnBeads package (2). HIV+ HIV- analysis identified 33 enriched GO terms for the 83 gene regions and survival for perinatally HIV-infected children. Characteristic (N = 119) (N = 60) 68 enriched GO terms for the 184 promoter regions. The top 15 GO A total of 85049 probes were removed in pre-processing (Table 1), Site, N (%) terms for both are presented in Table 5. A major focus is now on the increased risk of complications leaving a total of 400528 sites suitable for analysis. ESRU 62 (52.1) 20 (33.3) related to long-term HIV infection and its treatment, including PHRU 57 (47.9) 40 (66.7) Table 5. Top 15 enriched gene ontology terms identified in cardiovascular disease, central nervous system abnormalities, and Background subtraction was performed using methylumi (3) and the Male, % 46 50 functional analysis using DAVID musculoskeletal complications. BMIQ method was used to normalize the values (4). Age, Mean (SD) 6.4 ± 1.4 6.4 ± 1.4 Primary Caregiver Mother, N (%) 109 (90.8) 57 (95.0) GO Terms (Genes) Count GO Terms (Promoter Regions) Count Table 1. Detailed on sites filtered in pre-processing immune response 11 second-messenger-mediated signaling 10 DNA methylation is an important epigenetic marker and widely Lives in house, N (%) 74 (62.2) 38 (63.3) positive regulation of cell communication 8 behavior 13 studied in the context of biological processes and diseases. Total sites 485577 Someone in home smokes, N (%) 41 (34.5) 22 (36.7) G-protein coupled receptor protein regulation of adenylate cyclase activity 6 signaling pathway 13 Studying these epigenetic markers may identify novel biological SNP-enriched probes removed 47555 Highest caregiver grade, N (%) regulation of cyclase activity 6 cell surface receptor linked signal regulation of lyase activity 6 No School 0 (0.0) 1 (1.7) transduction 17 pathways affected by HIV and ART. Cross-reactive probes removed 25673 regulation of cAMP biosynthetic process 6 1-11 66 (55.5) 36 (60.0) positive regulation of signal transduction 7 Probes with detection p-value ≥ 0.05 removed 1855 regulation of cAMP metabolic process 6 12 (finished high school) 53 (44.5) 23 (38.3) immune system process 12 Context-specific probes removed 65 regulation of cyclic nucleotide biosynthetic It plausible that the epigenome is affected, as perinatal acquisition taxis 5 Probes on sex chromosomes removed 9901 Caregiver has paid job, N (%) 52 (43.7) 22 (36.7) process 6 of HIV and early initiation of ART takes place in a critical chemotaxis 5 regulation of nucleotide biosynthetic process 6 developmental period. Studies of in utero tobacco exposure and Total probes removed 85049 locomotory behavior 6 cell surface receptor linked signal transduction 30 Characteristics between HIV-infected and HIV-uninfected children prenatal exposure to famine have detected epigenetic changes. Total sites remaining for analysis 400528 defense response 8 G-protein coupled receptor protein signaling were similar when stratified by sex. inflammatory response 6 pathway 21 Analysis regulation of cell communication 10 regulation of cyclic nucleotide metabolic process 6 The aim of this study is to investigate the cumulative impact of positive regulation of multicellular G-protein signaling, coupled to cyclic nucleotide Differentially Methylated Loci (DML) organismal process 5 second messenger 6 perinatal HIV infection and ART on the epigenetic profile of Analysis was performed using the RnBeads package (2). regulation of signal transduction 9 regulation of hydrolase activity 10 school-aged children and identify affected biological pathways by signal transduction 18 regulation of nucleotide metabolic process 6 conducting an epigenome-wide association study. Differentially methylated loci (DML) meeting an adjusted FDR p- Manhattan plots of the DMLs are shown in Figure 1. value <0.05 were identified using limma (5). Manhattan plots were For boys, GO terms for genes and promoters were similar to above. generated to provide a visual representation of the DML. 161241 DMLs were identified for HIV-infected vs. HIV-uninfected; For girls, although GO terms for promoters were similar to above, 100216 for boys and 3091 for girls. DMRs mapped to only 3 named genes with no GO terms were Pre-defined regions (Table 2) were assigned a rank (lower rank = included EGFLAM-AS1 (chr5), LINC00615 (chr12), MIR1914 (chr20). Methods more evidence for differential methylation) based on these criteria: Figure 1. Manhattan plots Table 6. Exploratory selection of genes with biological function Study Sample 1. Mean difference in mean methylation potentially relevant to HIV infection and its complications 2. Log2 of the mean quotient in mean methylation HIV+ vs. HIV- Boys HIV+ vs. Boys HIV- Girls HIV+ vs. Girls HIV- The study included 120 HIV-infected and 60 frequency age-matched 3. Combined adjusted FDR p-value <0.05 from all sites in the region Category Genes (Chromosome) HIV-uninfected children aged 4-9 years participating in a Table 2. Pre-defined regions Immune response; FCER1G (chr1), LURAP1 (chr1), ICOS (chr2), IL1B (chr2), longitudinal observational study (CHANGES) at two sites (Empilweni TRAT1 (chr3), NKAPL (chr6), CD163 (chr12), CLEC4A inflammation; Services and Research Unit and Perinatal HIV Research Unit) in Region Maximum Definition (chr12), CLEC4E (chr12), ENDOU (chr12), CEBPE (chr14), cytokines Johannesburg, South Africa. Genes 27862 Whole locus from transcription start site to end CCL16 (chr17), CCL2 (chr17), CCL23 (chr17), CCL8 (chr17) site Fat metabolism; AGPAT2 (chr9); LIPF (chr10), SLCO1B1 (chr12) Promoters 28116 Span 1500 bases upstream and 500 bases Lipids; Lipodystrophy downstream of transcription start sites of genes Bone: CTSK (chr1), DKK4 (chr8), LNX2 (chr13) Musculoskeletal Muscle: MYF5 (chr12) We assessed differentially methylated regions (DMRs) that met a HTR2B (chr2), TACR3 (chr4), TBATA (chr10), MTNR1B Central nervous rank cut-off selected by the software. (chr11), P2RX2 (chr 12), SKOR2 (chr18), MC5R (chr18), Differentially Methylated Regions (DMRs) system CBLN4 (chr20) HIV-infected children were initiated on ART ≤24 months of age, Functional analysis of DMRs mapping to genes and promoter regions Special senses Vision: MTNR1B (chr11), PDE6H (chr12); HSPB6 (chr19) consistently on treatment, and suppressed (HIV RNA <400 was explored using the Database for Annotation, Visualization, and The number of DMRs at genes and promoters meeting the rank cut- (vision and hearing) Hearing: OR8B12 (chr11), OR8B8 (chr11), P2RX2 (chr 12) copies/mL) on a LPV/r-based regimen. Integrated Discovery (DAVID) (6). The software produced lists of off and with gene ontology information for all children and stratified enriched gene ontology (GO) terms. In an exploratory analysis, we by sex was determined (Table 4). At the enrollment visit, detailed demographic and clinical data was selected genes with biological functions relevant to HIV infection collected. Bloods were drawn and processed for storage. CD4 and and its complications and placed them in categories. Table 4. Differentially methylated regions meeting rank cut-off HIV-1 RNA levels were measured for HIV-infected children. The analysis was repeated stratified by sex. HIV+ HIV+ Boys HIV+ Girls Conclusions Infinium HumanMethylation450 BeadChip Region Type Group vs. vs. vs. HIV- HIV- Boys HIV- Girls We detected a dysregulated epigenome in school-aged children Extraction of genomic DNA (50ng/µl) from total WBC was Results Meeting rank cut-off 202 81 13 perinatally infected with HIV and initiated on ART <2 years of age. performed via picogreen quantitation (Lifetechnologies) by Genes Has gene name 106 42 3 Biomarkers Shared Resource at Columbia University. One sample did Has gene ontology info 83 32 0 Characteristics Functional analysis identified genes potentially affected by HIV not meet adequate DNA extraction leaving 119 samples for analysis. Meeting rank cut-off 352 125 256 and ART. A sub-set of these genes may be relevant to HIV-related Characteristics of 119 HIV-infected children and 60 HIV-uninfected Promoters Has gene name 232 80 167 complications. DNA methylation analysis was performed at Roswell Park Cancer children included in this analysis are shown in Table 3. Has gene ontology info 184 61 127 Institute (Buffalo, NY) Core Facility with the Infinium HumanMethylation450 BeadChip Kit (Illumina, Inc.). Mean age was similar between HIV-infected and uninfected children (6.4 ± 1.4 vs. 6.4 ± 1.4, NS), as was the proportion of males (46.2 vs The OSAT tool was used to allocate randomly assign samples (by 50.0%, NS). group, sex, and age) to specific positions to minimize the impact of References Acknowledgements All HIV-infected children were on LPV/r-based ART and had a viral (1) Yan et al. BMC Genomics 2012. (2) Assenov et al. Nat Methods 2014; “batch” effects across chips (1). This study was supported by funding from the National Institute (3) Davis et al. R: methylumi 2015; (4) Teschendorff et al. Bioinformatics 2013; load <400 copies/ml. Mean CD4% was 33.4 ± 6.4. Mean duration on of Child Health & Human Development (HD 073977, HD 073952). Raw intensity data (IDAT) files were obtained for analysis. treatment was 5.7 ± 1.4 years. (5) Ritchie et al. Nuc Acid Research 2015; (6) Huang et al.