CloudATAC: a cloud-based framework for ATAC-Seq data analysis.

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) generates genome-wide chromatin accessibility profiles, providing valuable insights into epigenetic gene regulation at both pooled-cell and single-cell population levels. Comprehensive analysis of ATAC-seq data involves the use of various interdependent programs. Learning the correct sequence of steps needed to process the data can represent a major hurdle. Selecting appropriate parameters at each stage, including pre-analysis, core analysis, and advanced downstream analysis, is important to ensure accurate analysis and interpretation of ATAC-seq data. Additionally, obtaining and working within a limited computational environment presents a significant challenge to non-bioinformatic researchers. Therefore, we present Cloud ATAC, an open-source, cloud-based interactive framework with a scalable, flexible, and streamlined analysis framework based on the best practices approach for pooled-cell and single-cell ATAC-seq data. These frameworks use on-demand computational power and memory, scalability, and a secure and compliant environment provided by the Google Cloud. Additionally, we leverage Jupyter Notebook's interactive computing platform that combines live code, tutorials, narrative text, flashcards, quizzes, and custom visualizations to enhance learning and analysis. Further, leveraging GPU instances has significantly improved the run-time of the single-cell framework. The source codes and data are publicly available through NIH Cloud lab https://github.com/NIGMS/ATAC-Seq-and-Single-Cell-ATAC-Seq-Analysis. This manuscript describes the development of a resource module that is part of a learning platform named ``NIGMS Sandbox for Cloud-based Learning'' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox [1] at the beginning of this Supplement. This module delivers learning materials on the analysis of bulk and single-cell ATAC-seq data in an interactive format that uses appropriate cloud resources for data access and analyses.

Identifying the risk of producing aneuploids using meiotic recombination genes as biomarkers: A copy number variation approach.

BACKGROUND & OBJECTIVES: Aneuploids are the most common chromosomal abnormality in liveborns and are usually the result of non-disjunction (NDJ) in meiosis. Copy number variations (CNVs) are large structural variations affecting the human genome. CNVs influence critical genes involved in causing NDJ by altering their copy number which affects the clinical outcome. In this study influence of CNVs on critical meiotic recombination was examined using new computational technologies to assess their role in causing aneuploidy. METHODS: This investigation was based on the analysis of 12 random normal populations consisting of 1714 individuals for aneuploid causing genes under CNV effect. To examine the effect of CNVs on genes causing aneuploidy, meiotic recombination genes were analyzed using EnrichR, WebGestalt and Ingenuity Pathway Analysis (IPA). RESULTS: Forty three NDJ genes were found under CNV burden; IPA (Ingenuity Pathway Analysis) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of CNV in meiotic recombination genes revealed a significant role of breast cancer gene 1, amyloid protein precursor, mitogen-activated protein kinase and nerve growth factor as key molecular players involved in causing aneuploidy. Interaction between these genes with other CNV-overlapping genes involved in cell cycle, recombination and meiosis might lead to increased incidences of aneuploidy. INTERPRETATION & CONCLUSIONS: The findings of this study implied that the effect of CNVs on normal genome contributed in amplifying the occurrences of chromosomal aneuploidies. The normal individuals consisting of variations in the susceptible genes causing aneuploids in the population remain undetected until the disorder genes express in the succeeding generations.

Global patterns of large copy number variations in the human genome reveal complexity in chromosome organization.

Global patterns of copy number variations (CNVs) in chromosomes are required to understand the dynamics of genome organization and complexity. For this study, analysis was performed using the Affymetrix Genome-Wide Human SNP Array 6.0 chip and CytoScan High-Density arrays. We identified a total of 44 109 CNVs from 1715 genomes with a mean of 25 CNVs in an individual, which established the first drafts of population-specific CNV maps providing a rationale for prioritizing chromosomal regions. About 19 905 ancient CNVs were identified across all chromosomes and populations at varying frequencies. CNV count, and sometimes CNV size, contributed to the bulk CNV size of the chromosome. Population specific lengthening and shortening of chromosomal length was observed. Sex bias for CNV presence was largely dependent on ethnicity. Lower CNV inheritance rate was observed for India, compared to YRI and CEU. A total of 33 candidate CNV hotspots from 5382 copy number (CN) variable region (CNVR) clusters were identified. Population specific CNV distribution patterns in p and q arms disturbed the assumption that CNV counts in the p arm are less common compared to long arms, and the CNV occurrence and distribution in chromosomes is length independent. This study unraveled the force of independent evolutionary dynamics on genome organization and complexity across chromosomes and populations.

Genome-wide copy number scan identifies IRF6 involvement in Van der Woude syndrome in an Indian family.

Summary Van der Woude syndrome (VWS) is an autosomal dominant developmental malformation presenting with bilateral lower lip pits related to cleft lip, cleft palate and other malformations. We performed a whole-genome copy number variations (CNVs) scan in an Indian family with members suffering from VWS using 2·6 million combined SNP and CNV markers. We found CNVs affecting IRF6, a known candidate gene for VWS, in all three cases, while none of the non-VWS members showed any CNVs in the IRF6 region. The duplications and deletions of the chromosomal critical region in 1q32-q41 confirm the involvement of CNVs in IRF6 in South Indian VWS patients. Molecular network analysis of these and other cleft lip/palate related module genes suggests that they are associated with cytokine-mediated signalling pathways and response to interferon-gamma mediated signalling pathways. This is a maiden study indicating the involvement of CNVs in IRF6 in causing VWS in the Indian population.

Impact of copy number variations burden on coding genome in humans using integrated high resolution arrays.

Copy number variations (CNVs) alter the transcriptional and translational levels of genes by disrupting the coding structure and this burden of CNVs seems to be a significant contributor to phenotypic variations. Therefore it was necessary to assess the complexities of CNV burden on the coding genome. A total of 1715 individuals from 12 populations were used for CNV analysis in the present investigation. Analysis was performed using Affymetrix Genome-Wide Human SNP Array 6·0 chip and CytoScan High-Density arrays. CNVs were more frequently observed in the coding region than in the non-coding region. CNVs were observed vastly more frequently in the coding region than the non-coding region. CNVs were found to be enriched in the regions containing functional genes (83-96%) compared with the regions containing pseudogenes (4-17%). CNVs across the genome of an individual showed multiple hits across many genes, whose proteins interact physically and function under the same pathway. We identified varying numbers of proteins and degrees of interactions within protein complexes of single individual genomes. This study represents the first draft of a population-specific CNV genes map as well as a cross-populational map. The complex relationship of CNVs on genes and their physically interacting partners unravels many complexities involved in phenotype expression. This study identifies four mechanisms contributing to the complexities caused by the presence of multiple CNVs across many genes in the coding part of the genome.

Molecular interaction network and pathway studies of ADAM33 potentially relevant to asthma.

BACKGROUND: Asthma is a complex disease caused by gene-gene, gene-protein, and protein-protein interactions and the influence of environment, which plays a significant role in causing asthma pathogenesis. ADAM33 is known to be an important gene involved in asthma pathogenesis. No one single gene is a causal factor of asthma; rather, asthma is caused by a complex interaction of multiple genes having pathogenetic and protective effects. OBJECTIVE: To identify and understand the interacting genes and proteins of ADAM33. METHODS: The Ingenuity Pathway Analysis and GeneMANIA tools and a literature survey were used to identify the interacting candidates of ADAM33 and the WEB-based GEne SeT AnaLysis Toolkit was used to perform enrichment analysis of the proteins identified. RESULTS: Keeping ADAM33 as a major hub, the authors identified some proteins whose interaction with ADAM33 had been associated with asthma and they recognized some proteins, such as amyloid ß (A4) precursor protein, ataxin-7, α4-integrin, α5-integrin, α9-integrin, tissue inhibitor of metalloproteinase-4, and ubiquilin-4, that had not been previously associated with asthma. CONCLUSION: The proteins identified in this study were enriched for various mechanisms that are involved in airway hyperresponsiveness, and through the interaction with ADAM33, they may have potential relevance in asthma.

Insertion-deletions burden in copy number polymorphisms of the Tibetan population.

BACKGROUND: Many studies have been conducted to identify either insertions-deletions (inDels) or copy number variations (CNVs) in humans, but few studies have been conducted to identify both of these forms coexisting in the same region. AIMS AND OBJECTIVES: To map the functionally significant sites within human genes that are likely to influence human traits and diseases. MATERIALS AND METHODS: In this report, we describe an inDel map in the 1051 Tibetan CNV regions obtained through CNV genotyping using Affymetrix Genome-wide single nucleotide polymorphism 6.0 chip. InDel polymorphisms in these copy number polymorphism regions were identified with a computational approach using the 2500 deoxyribonucleic acid sequences obtained from the 1000 Genome Project. RESULTS: The study identified a total of 95935 inDels that range from 1 bp to several bps in length which were found scattered across regulatory regions, exons and in introns of genes underlying the CNVs. A study on the distribution of inDels revealed that the majority of inDels were found in coding regions of the genome than the noncoding, while within the genes, inDels in intron regions were more followed by exonic regions and finally the regulatory regions. CONCLUSION: Study of inDels in CNV regions contribute to the enhanced understanding of the role played by the two variations and their collective influence on the genome. Further, a collection of these inDel genetic markers will aid in genetic mapping, further understanding of the phenotypic variability, identification of disease genes and in detecting novel CNVs.

Family based genome-wide copy number scan identifies complex rearrangements at 17q21.31 in dyslexics.

Developmental dyslexia (DD) is a complex heritable disorder with unexpected difficulty in learning to read and spell despite adequate intelligence, education, environment, and normal senses. We performed genome-wide screening for copy number variations (CNVs) in 10 large Indian dyslexic families using Affymetrix Genome-Wide Human SNP Array 6.0. Results revealed the complex genomic rearrangements due to one non-contiguous deletion and five contiguous micro duplications and micro deletions at 17q21.31 region in three dyslexic families. CNVs in this region harbor the genes KIAA1267, LRRC37A, ARL17A/B, NSFP1, and NSF. The CNVs in case 1 and case 2 at this locus were found to be in homozygous state and case 3 was a de novo CNV. These CNVs were found with at least one CNV having a common break and end points in the parents. This cluster of genes containing NSF is implicated in learning, cognition, and memory, though not formally associated with dyslexia. Molecular network analysis of these and other dyslexia related module genes suggests NSF and other genes to be associated with cellular/vesicular membrane fusion and synaptic transmission. Thus, we suggest that NSF in this cluster would be the nearest gene responsible for the learning disability phenotype.

Coordination among frequent genetic variants imparts substance use susceptibility and pathogenesis.

Determining the key genetic variants is a crucial step to comprehensively understand substance use disorders (SUDs). In this study, utilizing whole exome sequences of five multi-generational pedigrees with SUDs, we used an integrative omics-based approach to uncover candidate genetic variants that impart susceptibility to SUDs and influence addition traits. We identified several SNPs and rare, protein-function altering variants in genes, GRIA3, NCOR1, and SHANK1; compound heterozygous variants in LNPEP, LRP1, and TBX2, that play a significant role in the neurotransmitter-neuropeptide axis, specifically in the dopaminergic circuits. We also noted a greater frequency of heterozygous and recessive variants in genes involved in the structural and functional integrity of synapse receptors, CHRNA4, CNR2, GABBR1, DRD4, NPAS4, ADH1B, ADH1C, OPRM1, and GABBR2. Variant analysis in upstream promoter regions revealed regulatory variants in NEK9, PRRX1, PRPF4B, CELA2A, RABGEF1, and CRBN, crucial for dopamine regulation. Using family-and pedigree-based data, we identified heterozygous recessive alleles in LNPEP, LRP1 (4 frameshift deletions), and TBX2 (2 frameshift deletions) linked to SUDs. GWAS overlap identified several SNPs associated with SUD susceptibility, including rs324420 and rs1229984. Furthermore, miRNA variant analysis revealed notable variants in mir-548 U and mir-532. Pathway studies identified the presence of extensive coordination among these genetic variants to impart substance use susceptibility and pathogenesis. This study identified variants that were found to be overrepresented among genes of dopaminergic circuits participating in the neurotransmitter-neuropeptide axis, suggesting pleiotropic influences in the development and sustenance of chronic substance use. The presence of a diverse set of haploinsufficient variants in varying frequencies demonstrates the existence of extraordinary coordination among them in attributing risk and modulating severity to SUDs.

Copy number variation-based polymorphism in a new pseudoautosomal region 3 (PAR3) of a human X-chromosome-transposed region (XTR) in the Y chromosome.

A 3.5-Mb region of the X chromosome underwent duplication and transposition to the Y chromosome ~5-6 Mya. This X-transposed-region (XTR) originated at Xq21.3 and was inserted at Yp11.2. The two locations have 98.78 % homology and a high concentration of tandem repeats. In whole-genome scans of ten large families with dyslexic members, we identified transposed blocks comprising >102 kb of the Yp11.2 region in its homologous region at Xq21.3 in three females from three different families. Although recombination is known to be limited only to the pseudoautosomal regions (PARs) of the X and Y chromosomes, we report allelic unequal recombination between the XTR region Yp11.2 and Xq21.3, indicating the presence of a new PAR, which we named PAR3. This PAR3 region was also found in 2 % of the general population. An additional layer of justification could be provided from six other dyslexic cases which harbored duplications and deletions in the same Xq21.3 and Yp11.2 regions through allelic unequal recombination.

Family-based genome-wide copy number scan identifies five new genes of dyslexia involved in dendritic spinal plasticity.

Genome-wide screening for copy number variations (CNVs) in ten Indian dyslexic families revealed the presence of five de novo CNVs in regions harboring GABARAP, NEGR1, ACCN1, DCDC5, and one in already known candidate gene CNTNAP2. These genes are located on regions of chromosomes 17p13.1, 1p31.1, 17q11.21, 11p14.1 and 7q35, respectively, and are implicated in learning, cognition and memory processes through dendritic spinal plasticity, though not formally associated with dyslexia. Molecular network analysis of these and other dyslexia-related module genes suggests them to be associated with synaptic transmission, axon guidance and cell adhesion. Thus, we suggest that dyslexia may also be caused by neuronal disconnection in addition to the earlier view that it is due to neuronal migrational disorder.

Mutation analysis of the SLC4A11 gene in Indian families with congenital hereditary endothelial dystrophy 2 and a review of the literature.

PURPOSE: Congenital hereditary endothelial dystrophy 2 (CHED2) is an autosomal recessive disorder caused by mutations in the solute carrier family 4, sodium borate transporter, member 11 (SLC4A11) gene. The purpose of this study was to identify the genetic cause of CHED2 in six Indian families and catalog all known mutations in the SLC4A11 gene. METHODS: Peripheral blood samples were collected from individuals of the families with CHED2 and used in genomic DNA isolation. PCR primers were used to amplify the entire coding region including intron-exon junctions of SLC4A11. Amplicons were subsequently sequenced to identify the mutations. RESULTS: DNA sequence analysis of the six families identified four novel (viz., p.Thr262Ile, p.Gly417Arg, p.Cys611Arg, and p.His724Asp) mutations and one known p.Arg869His homozygous mutation in the SLC4A11 gene. The mutation p.Gly417Arg was identified in two families. CONCLUSIONS: This study increases the mutation spectrum of the SLC4A11 gene. A review of the literature showed that the total number of mutations in the SLC4A11 gene described to date is 78. Most of the mutations are missense, followed by insertions-deletions. The present study will be helpful in genetic diagnosis of the families reported here.

Genome-wide copy number scan identifies disruption of PCDH11X in developmental dyslexia.

Developmental dyslexia (DD) is a complex heritable disorder with unexpected difficulty in learning to read and spell despite adequate intelligence, education, environment, and normal senses. We performed a whole genome copy number variations (CNV) scan on 11 dyslexic families consisting of 14 dyslexic subjects and 24 non dyslexic members using 1.8 million combined SNP and CNV markers. We found CNVs affecting protocadherin genes in six dyslexics from three families, while none among the non-dyslexic control members showed any CNV in protocadherins. We identified duplications in five cases and a deletion in one case in Xq21.3 region bearing PCDH11X. Unequal recombination between the X-transposed region (XTR) of Yp11.2 and the X chromosome might be causing these structural changes. PCDH11X, expressed in brain is implicated in cell-cell communication, verbal ability, cerebral asymmetry, and dendritic synaptic plasticity, may be regarded as a new candidate gene for dyslexia.

Placental microRNA methylome signatures may serve as biomarkers and therapeutic targets for prenatally opioid-exposed infants with neonatal opioid withdrawal syndrome.

Introduction: The neonate exposed to opioids in utero faces a constellation of withdrawal symptoms postpartum commonly called neonatal opioid withdrawal syndrome (NOWS). The incidence of NOWS has increased in recent years due to the opioid epidemic. MicroRNAs (miRNAs) are small non-coding RNA molecules that play a crucial role in gene regulation. Epigenetic variations in microRNAs (miRNAs) and their impact on addiction-related processes is a rapidly evolving area of research. Methods: The Illumina Infinium Methylation EPIC BeadChip was used to analyze DNA methylation levels of miRNA-encoding genes in 96 human placental tissues to identify miRNA gene methylation profiles as-sociated with NOWS: 32 from mothers whose prenatally opioid-exposed infants required pharmacologic management for NOWS, 32 from mothers whose prenatally opioid-exposed infants did not require treat-ment for NOWS, and 32 unexposed controls. Results: The study identified 46 significantly differentially methylated (FDR p-value ≤ 0.05) CpGs associated with 47 unique miRNAs, with a receiver operating characteristic (ROC) area under the curve (AUC) ≥0.75 including 28 hypomethylated and 18 hypermethylated CpGs as potentially associated with NOWS. These dysregulated microRNA methylation patterns may be a contributing factor to NOWS pathogenesis. Conclusion: This is the first study to analyze miRNA methylation profiles in NOWS infants and illustrates the unique role miRNAs might have in diagnosing and treating the disease. Furthermore, these data may provide a step toward feasible precision medicine for NOWS babies as well.

Cascade of interactions between candidate genes reveals convergent mechanisms in keratoconus disease pathogenesis.

Keratoconus is a progressive thinning, steepening and distortion of the cornea which can lead to loss of vision if left untreated. Keratoconus has a complex multifactorial etiology, with genetic and environmental components contributing to the disease pathophysiology. Studies have observed high concordance between monozygotic twins, discordance between dizygotic twins, and high familial segregation indicating the presence of a very strong genetic component in the pathogenesis of keratoconus. The use of genome-wide linkage studies on families and twins, genome-wide association studies (GWAS) on case-controls, next-generation sequencing (NGS)-based genomic screens on both familial and non-familial cohorts have led to the identification of keratoconus candidate genes with much greater success and increased resproducibility of genetic findings. This review focuses on candidate genes identified till date and attempts to understand their role in biological processes underlying keratoconus pathogenesis. In addition, using these genes I propose molecular pathways that could contribute to keratoconus pathogenesis. The pathways identified the presence of direct cross-talk between known candidate genes of keratoconus and remarkably, 28 known candidate genes have a direct relationship among themselves that involves direct protein-protein binding, regulatory activities such as activation and inhibition, chaperone, transcriptional activation/co-activation, and enzyme catalysis. This review attempts to describe these relationships and cross-talks in the context of keratoconus pathogenesis.

A systems omics-based approach to decode substance use disorders and neuroadaptations.

Substance use disorders (SUDs) are a group of neuropsychiatric conditions manifesting due to excessive dependence on potential drugs of abuse such as psychostimulants, opioids including prescription opioids, alcohol, inhalants, etc. Experimental studies have generated enormous data in the area of SUDs, but outcomes from such data have remained largely fragmented. In this review, we attempt to coalesce these data points providing an important first step towards our understanding of the etiology of SUDs. We propose and describe a 'core addictome' pathway that behaves central to all SUDs. Besides, we also have made some notable observations paving way for several hypotheses; MECP2 behaves as a master switch during substance use; five distinct gene clusters were identified based on respective substance addiction; a central cluster of genes serves as a hub of the addiction pathway connecting all other substance addiction clusters. In addition to describing these findings, we have emphasized the importance of some candidate genes that are of substantial interest for further investigation and serve as high-value targets for translational efforts.

Placental epigenetics for evaluation of fetal congenital heart defects: Ventricular Septal Defect (VSD).

Ventricular Septal Defect (VSD), the most common congenital heart defect, is characterized by a hole in the septum between the right and left ventricles. The pathogenesis of VSD is unknown in most clinical cases. There is a paucity of data relevant to epigenetic changes in VSD. The placenta is a fetal tissue crucial in cardiac development and a potentially useful surrogate for evaluating the development of heart tissue. To understand epigenetic mechanisms that may play a role in the development of VSD, genome-wide DNA methylation assay on placentas of 8 term subjects with isolated VSD and no known or suspected genetic syndromes and 10 unaffected controls was performed using the Illumina HumanMethylation450 BeadChip assay. We identified a total of 80 highly accurate potential CpGs in 80 genes for detection of VSD; area under the receiver operating characteristic curve (AUC ROC) 1.0 with significant 95% CI (FDR) p-values < 0.05 for each individual locus. The biological processes and functions for many of these differentially methylated genes are previously known to be associated with heart development or disease, including cardiac ventricle development (HEY2, ISL1), heart looping (SRF), cardiac muscle cell differentiation (ACTC1, HEY2), cardiac septum development (ISL1), heart morphogenesis (SRF, HEY2, ISL1, HEYL), Notch signaling pathway (HEY2, HEYL), cardiac chamber development (ISL1), and cardiac muscle tissue development (ACTC1, ISL1). In addition, we identified 8 microRNAs that have the potential to be biomarkers for the detection of VSD including: miR-191, miR-548F1, miR-148A, miR-423, miR-92B, miR-611, miR-2110, and miR-548H4. To our knowledge this is the first report in which placental analysis has been used for determining the pathogenesis of and predicting VSD.

Dysregulation of NRXN1 by mutant MIR8485 leads to calcium overload in pre-synapses inducing neurodegeneration in Multiple sclerosis.

OBJECTIVES: To identify Damaging mutations in microRNAs (miRNAs) and 3' untranslated regions (UTRs) of target genes to establish Multiple sclerosis (MS) disease pathway. METHODS: Female aged 16, with Relapsing Remitting Multiple sclerosis (RRMS) was reported with initial symptoms of blurred vision, severe immobility, upper and lower limb numbness and backache. Whole Exome Sequencing (WES) and disease pathway analysis was performed to identify mutations in miRNAs and UTRs. RESULTS: We identified Deleterious/Damaging multibase mutations in MIR8485 and NRXN1. miR-8485 was found carrying frameshift homozygous deletion of bases CA, while NRXN1 was found carrying nonframeshift homozygous substitution of bases CT to TC in exon 8 replacing Serine with Leucine. CONCLUSIONS: Mutations in miR-8485 and NRXN1 was found to alter calcium homeostasis and NRXN1/NLGN1 cell adhesion molecule binding affinities. The miR-8485 mutation leads to overexpression of NRXN1 altering pre-synaptic Ca2+ homeostasis, inducing neurodegeneration.

Complex interaction between mutant HNRNPA1 and gE of varicella zoster virus in pathogenesis of multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system causing axonal injury, neuronal loss, and atrophy of the central nervous system leading to permanent neurological and clinical disability. Presence of mutations in M9 domain of HNRNPA1 and detection of autoantibodies against this domain in HNRNPA1 qualifies it as a strong candidate for causing MS. These two aspects indicate the presence of a facilitator in associating them. Varicella zoster virus (VZV), known to cause chicken pox infection in humans, is a significant contender in sensitizing the infected people towards MS. Reactivation of latent herpes viruses by other infectious agents and cross-recognition of common viral antigens with antigens found in the myelin sheath induces molecular mimicry or superantigens. Mutations in HNRNPA1 cause mislocalization to the cytoplasm, and co-localize with stress granules (SG) causing cellular apoptosis, this creates the first step toward MS pathogenesis. Mutant HNRNPA1 accumulates in SG allowing the cells to display peptides of HNRNPA1 on surfaces of major histocompatibility complex (MHC) I triggering a cascade of immune reactions. Since glycoprotein E (gE) of VZV shares >62% amino acids sequence similarity with Prion-like domain (PrLD) of HNRNPA1, signifying the reason behind autoantibodies against M9 and PrLD of HNRNPA1. This review attempts to delineate the interactions of VZV, gE of VZV, with M9 domain and PrLD of HNRNPA1 in a step-by-step process. This supports the tripartite model that an environmental trigger in genetically susceptible individuals causes an autoimmune response to self-CNS antigens that result in the pathology observed in the brain and spinal cord of MS patients.

Newborn blood DNA epigenetic variations and signaling pathway genes associated with Tetralogy of Fallot (TOF).

Tetralogy of Fallot (TOF) is the most common Critical Congenital Heart Defect (CCHD). The etiology of TOF is unknown in most cases. Preliminary data from our group and others suggest that epigenetic changes may play an important role in CHD. Epidemiologically, a significant percentage of CHD including TOF fail to be diagnosed in the prenatal and early newborn period which can negatively affect health outcomes. We performed genome-wide methylation assay in newborn blood in 24 non-syndromic TOF cases and 24 unaffected matched controls using Illumina Infinium HumanMethylation450 BeadChips. We identified 64 significantly differentially methylated CpG sites in TOF cases, of which 25 CpG sites had high predictive accuracy for TOF, based on the area under the receiver operating characteristics curve (AUC ROC) ≥ 0.90). The CpG methylation difference between TOF and controls was ≥10% in 51 CpG targets suggesting biological significance. Gene ontology analysis identified significant biological processes and functions related to these differentially methylated genes, including: CHD development, cardiomyopathy, diabetes, immunological, inflammation and other plausible pathways in CHD development. Multiple genes known or plausibly linked to heart development and post-natal heart disease were found to be differentially methylated in the blood DNA of newborns with TOF including: ABCB1, PPP2R5C, TLR1, SELL, SCN3A, CREM, RUNX and LHX9. We generated novel and highly accurate putative molecular markers for TOF detection using leucocyte DNA and thus provided information on pathogenesis of TOF.