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Chopra, P.

Publications and source records attributed to Chopra, P..

18 recordsLinked to original sources

Real Science Is Harder Than Benchmarks: Evaluating Advanced AI Frameworks on Published Studies. I. Uncertainty Quantification, ML on Therapeutic Data Commons, and Agent-Based Modeling

Artificial Intelligence (AI) frameworks for automating scientific research have shown strong performance on benchmarks, but their capacity to routinely reproduce results from multiple real-life published studies remains largely untested. We evaluated five advanced AI research frameworks (Kosmos, K-Dense, ToolUniverse, BioAgents from bio.xyz, and the AI Scientist-v2 from Sakana AI) on three real-life tasks (including two recently published papers) spanning uncertainty quantification for molecular property predictions, machine learning on Therapeutic Data Commons benchmarks, and agent-based modeling. AI frameworks demonstrated genuine strengths: generating original hypotheses, competently executing routine data acquisition and coding tasks, providing statistical measures of confidence often absent from the original papers, and producing well-formatted final reports. At the same time, our experiments revealed that real-world scientific tasks remain considerably harder than current benchmarks suggest. No AI framework matched the scope or depth of the original studies, results varied across multiple runs of the same framework with the same prompt, and we documented cases of severe hallucinations in final reports, gaps in literature coverage, and overconfident conclusions. Verification of AI outputs required substantial domain expertise. While these three tasks are only partially representative of the broader scientific landscape, they offer a starting point for developing a more rigorous methodology for evaluation of AI performance than what is currently practiced. We conclude that AI frameworks are already valuable for prototyping research directions and stress-testing completed studies, and some of the limitations documented here appear largely tractable through infrastructure improvements and continued development.

bioinformatics↗

A long-chain heparan sulfate capture mechanism directs paracrine GDNF-GFRα1 signalling through RET

The receptor tyrosine kinase RET is activated by GDNF and GFR1 together as a bipartite ligand, driving receptor activation and signalling in developmental and neuroprotective contexts. Evidence from developmental and cell models has suggested that heparan sulfate (HS) functions as a fourth component in RET signalling by binding to GDNF, but the molecular details remain unclear. Here, we present the cryo-EM structure of the heterohexameric RET:GDNF:GFR1 complex with a fully resolved heparin ligand, revealing an unexpected extended HS binding site spanning all three proteins. The architecture of the complex and binding mode of the HS chain in this complex enables the formation of a higher order 4:4:4 assembly bound to a single 30-saccharide HS chain which bridges two intimately bound complexes. This multi-protein interface selectively binds the highly sulfated domains of HS over other GAG classes, and is essential for RET activation in trans with soluble GFR1, but not in cis when GFR1 is membrane-bound. Our data suggest that HS shapes the dynamics of RET signalling at every stage, from ligand diffusion to signalling complex formation. Thus, GDNF-GFRa1 paracrine signalling reveals a surprising dependence for long-chain GAG function in which the glycan engages in both receptor complex assembly and clustering.

biochemistry↗

Structural insights into antibody responses against influenza A virus in its natural reservoir

While influenza A virus undergoes rapid antigenic drift in humans, at least some subtypes, such as H3, have relatively stable antigenicity in natural waterfowl reservoirs, despite the presence of immune pressure. However, the underlying mechanisms remain poorly understood. This study identified and characterized 187 antibodies to H3 hemagglutinin from experimentally infected mallard ducks, 18 of which were further analyzed by cryo-EM. Compared with human H3 antibodies, duck H3 antibodies exhibited higher glycan-binding propensity, more balanced immunodominance hierarchy, and targeted distinct epitopes. Other unique features of duck H3 antibodies included a convergent CDR H3-independent heavy chain-only binding mode and an N-glycosylated CDR H3 as decoy receptor. By annotating duck immunoglobulin germline genes, we also demonstrated the importance of gene conversion in duck H3 antibodies. Overall, our findings provide insights into how millennia of coevolution have shaped the interplay between influenza A virus antigenic drift and antibody responses in the natural reservoir.

immunology↗

Vaccine-induced antigenic drift of a human-origin H3N2 Influenza A virus in swine alters glycan binding and sialic acid avidity

Interspecies transmission of human influenza A viruses (FLUAV) to swine occurs frequently, yet the molecular factors driving adaptation remain poorly understood. Here we investigated how vaccine-induced immunity shapes the evolution of a human-origin H3N2 virus in pigs using an in vivo sustained transmission model. Pigs (seeders) were vaccinated with a commercial inactivated swine vaccine and then infected with an antigenically distinct FLUAV containing human-origin HA/NA. Contact pigs were introduced two days later. After 3 days, seeder pigs were removed, and new contacts introduced. This was repeated for a total of 4 contacts. Sequencing of nasal swab samples showed the emergence of mutations clustered near the HA receptor binding site, enabling immune escape and abolishing binding to N-glycolylneuraminic acid. Mutant viruses recognized 2,6-sialosides with 3 N-acetyllactosamine repeats, which are rare in swine lungs, while the parental virus bound structures with a minimum of 2 repeats. Adaptative HA mutations enhanced avidity for 2,6-linked sialic acid, likely compensating for the low abundance of extended glycans. Notably, residues outside the canonical HA binding pocket contribute to glycan binding, suggesting a trade-off between receptor breadth and avidity. These findings show that non-neutralizing immunity promotes viral adaptation by fine-tuning receptor engagement and immune evasion. SIGNIFICANCEUnderstanding how vaccination shapes influenza A virus (FLUAV) evolution across species barriers is critical for predicting and preventing zoonotic and reverse-zoonotic events. Our study demonstrates that vaccine-induced immune pressure can drive antigenic drift in a human-origin H3N2 virus, altering HA receptor binding properties that could inadvertently facilitate adaptation to swine. These changes shifted glycan specificity toward extended poly-LacNAc structures and enhanced 2,6-linked sialoglycans binding while abolishing Neu5Gc recognition. By revealing how non-neutralizing immunity fine-tunes HA-glycan interactions by engaging antigenically relevant residues in glycan binding, this work highlights vaccination as an underappreciated driver of host adaptation and viral evolution.

microbiology↗

Chemoenzymatic Synthesis of Asymmetric Bisecting Bi-, Tri-, and Tetra-Antennary N-Glycans

N-Acetylglucosaminyltransferase-III (GnT-III) is a glycosyltransferase that can install a {beta}1,4-linked N-acetylglucosamine (GlcNAc) residue at the central {beta}-mannoside of N-glycans. The resulting so-called bisecting GlcNAc is not further extended by glycosyl transferases and has been implicated a wide range of biological processes. The molecular mechanisms by which bisection modulates the biosynthesis of N-glycans and influences molecular recognition is not well understood, which is due to a lack of well-defined N-glycans with and without bisection. We describe a chemoenzymatic methodology that can readily provide a wide range of asymmetrical bisecting bi-, tri- and tetra-antennary N-glycans. It was found GnT-III can act on bi-, tri- and tetra-antennary N-glycans and can also accepts N-glycans having a {beta}1,2GlcNTFA or GlcN3 moiety at the 1,2Man- or 1,6Man-antenna making it possible to prepare panels of asymmetrical N-glycans with and without bisection and having different patterns of sialylation and fucosylation. Kinetic experiments showed GnT-III preferentially modifies bi-antennary glycans. The compounds were printed as a glycan microarray, which was screened for lectin binding. It was found that some lectins preferentially bind to bisecting glycans, whereas others do not tolerate or are not affected by this modification. We investigated receptor specificities of human H1N1 and H2N3 influenza viruses and animal H5N1 viruses that pose a pandemic threat including a virus that has become endemic in cattle. The H1N1 and H2N3 viruses did not tolerate bisection whereas it did not affect H5N1 viruses. A/bovine had the broadest receptor specificity providing a rationale for its wide host range.

biochemistry↗

Indigenous gut microbes modulate neural cell state and neurodegenerative disease susceptibility

The native microbiome influences a plethora of host processes, including neurological function. However, its impacts on diverse brain cell types remains poorly understood. Here, we performed single nucleus RNA sequencing on hippocampi from wildtype, germ-free mice and reveal the microbiome-dependent transcriptional landscape across all major neural cell types. We found conserved impacts on key adaptive immune and neurodegenerative transcriptional pathways, underscoring the microbiomes contributions to disease-relevant processes. Mono-colonization with select indigenous microbes identified species-specific effects on the transcriptional state of brain myeloid cells. Colonization by Escherichia coli induced a distinct adaptive immune and neurogenerative disease-associated cell state, suggesting increased disease susceptibility. Indeed, E. coli exposure in the 5xFAD mouse model resulted in exacerbated cognitive decline and amyloid pathology, demonstrating its sufficiency to worsen Alzheimers disease-relevant outcomes. Together, these results emphasize the broad, species-specific, microbiome-dependent consequences on neurological transcriptional state and highlight the capacity of specific microbes to modulate disease susceptibility. HighlightsO_LIThe microbiome impacts the transcriptional landscape of all major brain cell types. C_LIO_LIDiscrete microbes specifically modulate resident myeloid cell status. C_LIO_LIGut E. coli triggers dynamic transcriptional responses across neural cell types. C_LIO_LIExposure to E. coli exacerbates behavioral and cellular pathologies in 5xFAD mice. C_LI

neuroscience↗

A Light Sensitive Hyaluronic Acid-based Hydrogel for 3D Culturing, Differentiation, and Harvesting of Mesenchymal Stromal Cells

Hydrogels derived from natural sources are commonly used for 3D cell culturing due to their favourable interactions with cells. However, these biomaterials (e.g. Matrigel) suffer from variations in composition, limited mechanical tunability, the presence of xenogenic components and difficulties of cell retrieval. Semi-synthetic hydrogels are emerging to address these limitations, making these attractive for drug delivery and tissue engineering. Here, we describe a hydrogel platform based on hyaluronic acid (HA) modified by (1R,8S,9S)-bicycle[6.1.0]non-4-yn-9-ylmethanol (BCN) and a cross-linker composed of light sensitive o-nitrobenzyl moieties and polyethylene glycol (PEG) chains terminating in azides. The two components can undergo strain-promoted azide-alkyne cycloaddition (SPAAC) resulting in rapid gel formation. The stiffness of the hydrogel can be modulated by varying the crosslinker ratio and multi-functionalizing is possible by the incorporation of chemical cues modified by an azide. The fast hydrogelation enables easy encapsulation of cells and harvesting with preservation of viability is possible be short exposure to UV light. Moreover, it is demonstrated that visible light (405 nm) can soften the hydrogel resulting in phenotypical changes of human Mesenchymal Stromal Cells (hMSCs) co-cultured with endothelial colony-forming cells (ECFCs) with high viability. Our findings highlight that the photosensitive HA-based hydrogel provides a versatile and biocompatible platform for cell culture and tissue engineering applications, offering advantages over traditional 3D cell culturing platforms.

biochemistry↗

Receptor Binding Specificity of a Bovine A(H5N1) Influenza Virus

Outbreaks in the US of highly pathogenic avian influenza virus (H5N1) in dairy cows have been occurring for months creating new possibilities for direct contact between the virus and humans. Eisfeld et al. examined the pathogenicity and transmissibility of a bovine HPAI H5N1 virus isolated from New Mexico in a series of in vitro and in vivo assays. They found the virus has a dual human- and avian virus-like receptor-binding specificity as measured in a solid phase glycan binding assay. Here, we examined the receptor specificity of a bovine HPAI H5N1 virus (A/bovine/OH/B24OSU-432/2024, H5N1, clade 2.3.4.4b) employing four different assays including glycan array technology, bio-layer interferometry (BLI), a solid phase capture assay and hemagglutination of glycan remodeled erythrocytes. As controls, well characterized avian (A/Vietnam/1203/2004, H5N1, clade 1) and human (A/CA/04/2009, H1N1) IAVs were included that bind 2,3- and 2,6-sialosides, respectively. We found that A/bovine/OH/B24OSU-432/2024 preferentially binds to "avian type" receptors (2,3-sialosides). Furthermore, sequence alignments showed that A/bovine has maintained amino acids in its HA associated with 2,3-sialoside (avian) receptor specificity. We conclude that while we find no evidence that A/bovine has acquired human virus receptor binding specificity, ongoing efforts must be placed on monitoring for this trait.

biochemistry↗

Cross-species transcriptomic analysis identifies mitochondrial dysregulation as a functional consequence of the schizophrenia-associated 3q29 deletion

Recent advances in the genetics of schizophrenia (SCZ) have identified rare variants that confer high disease risk, including a 1.6 Mb deletion at chromosome 3q29 with a staggeringly large effect size (O.R. > 40). Understanding the impact of the 3q29 deletion (3q29Del) on the developing CNS may therefore lead to insights about the pathobiology of schizophrenia. To gain clues about the molecular and cellular perturbations caused by the 3q29 deletion, we interrogated transcriptomic effects in two experimental model systems with complementary advantages: isogenic human forebrain cortical organoids and isocortex from the 3q29Del mouse model. We first created isogenic lines by engineering the full 3q29Del into an induced pluripotent stem cell line from a neurotypical individual. We profiled transcriptomes from isogenic cortical organoids that were aged for 2 months and 12 months, as well as day p7 perinatal mouse isocortex, all at single cell resolution. Differential expression analysis by genotype in each cell-type cluster revealed that more than half of the differentially expressed genes identified in mouse cortex were also differentially expressed in human cortical organoids, and strong correlations were observed in mouse-human differential gene expression across most major cell-types. We systematically filtered differentially expressed genes to identify changes occurring in both model systems. Pathway analysis on this filtered gene set implicated dysregulation of mitochondrial function and energy metabolism, although the direction of the effect was dependent on developmental timepoint. Transcriptomic changes were validated at the protein level by analysis of oxidative phosphorylation protein complexes in mouse brain tissue. Assays of mitochondrial function in human heterologous cells further confirmed robust mitochondrial dysregulation in 3q29Del cells, and these effects are partially recapitulated by ablation of the 3q29Del gene PAK2. Taken together these data indicate that metabolic disruption is associated with 3q29Del and is conserved across species. These results converge with data from other rare SCZ-associated variants as well as idiopathic schizophrenia, suggesting that mitochondrial dysfunction may be a significant but overlooked contributing factor to the development of psychotic disorders. This cross-species scRNA-seq analysis of the SCZ-associated 3q29 deletion reveals that this copy number variant may produce early and persistent changes in cellular metabolism that are relevant to human neurodevelopment.

genomics↗

Chemoenzymatic Synthesis of Heparan Sulfate Oligosaccharides having a Domain Structure

Heparan sulfate (HS) have domain structures in which regions that are substantially modified by epimerization and sulfation (NS domains) are interspersed by unmodified fragments (NA domains). There is data to support that the domain structure of HS can regulate protein binding, however, such a binding mode has been difficult to probe. Here, we report a chemoenzymatic methodology that can provide HS oligosaccharides composed of two or more NS domains separated by NA domains of different length. It is based on the chemical synthesis of a sulfated HS oligosaccharide that enzymatically could be extended by various GlcA-GlcNAc units and terminated in a GlcNAc-6N3 moiety. HS oligosaccharides having an azide and alkyne moiety could assembled by copper catalyzed alkyne-azide cycloaddition (CuAAC) to give compounds having various NS domains separated by unsulfated regions. Competition binding studies showed that the length of an NA domain modulates the binding of the chemokines CCL5 and CXCL8.

biochemistry↗

Structure, immunogenicity, and conformation-dependent receptor binding of the post-fusion human metapneumovirus F protein

Human metapneumovirus (hMPV) is an important cause of acute viral respiratory infection. As the only target of neutralizing antibodies, the hMPV fusion (F) protein has been a major focus for vaccine development and targeting by drugs and monoclonal antibodies (mAbs). While X-ray structures of trimeric pre-fusion and post-fusion hMPV F proteins from genotype A, and monomeric pre-fusion hMPV F protein from genotype B have been determined, structural data for the post-fusion conformation for genotype B is lacking. We determined the crystal structure of this protein and compared the structural differences of post-fusion hMPV F between hMPV A and B genotypes. We also assessed the receptor binding properties of the hMPV F protein to heparan sulfate. A library of heparan sulfate (HS) oligomers was used to verify the HS binding activity of hMPV F, and several compounds showed binding to predominantly pre-fusion hMPV F, but had limited binding to post-fusion hMPV F. Furthermore, mAbs to antigenic sites III and the 66-87 intratrimeric epitope block heparan binding. In addition, we evaluated the efficacy of post-fusion hMPV B2 F protein as a vaccine candidate in BALB/c mice. Mice immunized with hMPV B2 post-fusion F protein showed a balanced Th1/Th2 immune response and generated neutralizing antibodies against both subgroup A2 and B2 hMPV strains, which protected the mice from hMPV challenge. Antibody competition analysis revealed the antibodies generated by immunization target two known antigenic sites (III and IV) on hMPV F. Overall, this study provides new characteristics of the hMPV F protein informative for vaccine and therapy development. ImportanceHuman metapneumovirus (hMPV) is an important cause of viral respiratory disease. In this paper, we report the X-ray crystal structure of the hMPV fusion (F) protein in the post-fusion conformation from genotype B. We also assessed binding of the hMPV F protein to heparin and heparan sulfate, a previously reported receptor for the hMPV F protein. Furthermore, we determined the immunogenicity and protective efficacy of post-fusion hMPV B2 F protein, which is the first study using a homogenous conformation of the protein. Antibodies generated in response to vaccination give a balanced TH1/TH2 response and target two previously discovered neutralizing epitopes.

microbiology↗

Sialic acid-Dependent Binding and Viral Entry of SARS-CoV-2

Emerging evidence suggests that host glycans influence infection by SARS-CoV-2. Here, we reveal that the receptor-binding domain (RBD) of the spike (S)-protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (SA), with preference for the oligosaccharide of monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind the RBD. The monomeric affinities (Kd = 100-200 M) of gangliosides for the RBD are similar to heparan sulfate, another negatively charged glycan ligand of the RBD proposed as a viral coreceptor. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to ACE2-expressing cells is decreased upon depleting cell surface SA level using three approaches: sialyltransferase inhibition, genetic knock-out of SA biosynthesis, or neuraminidase treatment. These effects on RBD binding and pseudotyped viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.

biochemistry↗

SARS-CoV-2 spike protein binds heparan sulfate in a length- and sequence-dependent manner

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing an unprecedented global pandemic demanding the urgent development of therapeutic strategies. Microarray binding experiments using an extensive heparan sulfate (HS) oligosaccharide library showed that the receptor binding domain (RBD) of the spike of SARS-CoV-2 can bind HS in a length-and sequence-dependent manner. Hexa- and octa-saccharides composed of IdoA2S-GlcNS6S repeating units were identified as optimal ligands. Surface plasma resonance (SPR) showed the SARS-CoV-2 spike protein binds with much higher affinity to heparin (KD = 55 nM) compared to the RBD (KD = 1 M) alone. We also found that heparin does not interfere in angiotensin-converting enzyme 2 (ACE2) binding or proteolytic processing of the spike. Our data supports a model in which HS functions as the point of initial attachment for SARS-CoV-2 infection. Tissue staining studies using biologically relevant tissues indicate that heparan sulfate proteoglycan (HSPG) is a critical attachment factor for the virus. Collectively, our results highlight the potential of using HS oligosaccharides as a therapeutic agent by inhibiting SARS-CoV-2 binding to target cells.

biochemistry↗

Coding de novo mutations identified by WGS reveal novel orofacial cleft genes

While de novo mutations (DNMs) are known to increase risk of congenital defects, DNMs have not been fully explored regarding orofacial clefts (OFCs), one of the most common human birth defects. Therefore, whole-genome sequencing of 756 case-parent trios of European, Colombian, and Taiwanese ancestry was performed to determine the contributions of coding DNMs to OFC risk. Overall, we identified a significant excess of loss-of-function DNMs in genes highly expressed in craniofacial tissues, as well as genes associated with known autosomal dominant OFC syndromes. This analysis also revealed roles for zinc-finger homeobox domain and SOX2-interacting genes in OFC etiology.

genetics↗

A novel redox-active switch in Fructosamine-3-kinases expands the regulatory repertoire of the protein kinase superfamily

Aberrant regulation of metabolic kinases by altered redox homeostasis is a major contributing factor in aging and disease such as diabetes. However, the biochemical mechanisms by which metabolic kinases are regulated under oxidative stress is poorly understood. In this study, we demonstrate that the catalytic activity of a conserved family of Fructosamine-3-kinases (FN3Ks), which are evolutionarily related to eukaryotic protein kinases (ePKs), are regulated by redox-active cysteines in the kinase domain. By solving the crystal structure of FN3K homolog from Arabidopsis thaliana (AtFN3K), we demonstrate that it forms an unexpected strand-exchange dimer in which the ATP binding P-loop and adjoining beta strands are swapped between two chains in the dimer. This dimeric configuration is characterized by strained inter-chain disulfide bonds that stabilize the P-loop in an extended conformation. Mutational analysis and solution studies confirm that the strained disulfides function as redox "switches" to reversibly regulate FN3K activity and dimerization. Consistently, we find that human FN3K (HsFN3K), which contains an equivalent P-loop Cys, is also redox-sensitive, whereas ancestral bacterial FN3K homologs, which lack a P-loop Cys, are not. Furthermore, CRISPR knockout of FN3K in human HepG2 cells results in significant upregulation of redox metabolites including glutathione. We propose that redox regulation evolved progressively in FN3Ks in response to changing cellular redox conditions. Our studies provide important new insights into the origin and evolution of redox regulation in the protein kinase superfamily and open new avenues for targeting HsFN3K in diabetic complications.

biochemistry↗

Salt-free fractionation of complex isomeric mixtures of glycosaminoglycan oligosaccharides compatible with ESI-MS and microarray analysis

Heparin and heparan sulfate (Hp/HS) are linear complex glycosaminoglycans which are involved in diverse biological processes. The structural complexity brings difficulties in separation, making the study of structure-function relationships challenging. Here we present a separation method for Hp/HS oligosaccharide fractionation with cross-compatible solvent and conditions, combining size exclusion chromatography (SEC), ion-pair reversed phase chromatography (IPRP), and hydrophilic interaction chromatography (HILIC) as three orthogonal separation methods that do not require desalting or extensive sample handling. With this method, the final eluent is suitable for structure-function relationship studies, including tandem mass spectrometry and microarray printing. Our data indicate that high resolution is achieved on both IPRP and HILIC for Hp/HS isomers. In addition, the fractions co-eluted in IPRP could be further separated by HILIC, with both separation dimensions capable of resolving some isomeric oligosaccharides. We demonstrate this method using both unpurified reaction products from isomeric synthetic hexasaccharides and an octasaccharide fraction from enoxaparin, identifying isomers resolved by this multi-dimensional separation method. We demonstrate both structural analysis by MS, as well as functional analysis by microarray printing and screening using a prototypical Hp/HS binding protein: basic-fibroblast growth factor (FGF2). Collectively, this method provides a strategy for efficient Hp/HS structure-function characterization.

biochemistry↗

Whole genome sequencing of orofacial cleft trios from the Gabriella Miller Kids First Pediatric Research Consortium identifies a new locus on chromosome 21

Orofacial clefts (OFCs) are one of the most common birth defects worldwide and create a significant health burden. The majority of OFCs are non-syndromic, and the genetic component has been only partially determined. Here, we analyze whole genome sequence (WGS) data for association with risk of OFCs in European and Colombian families selected from a multicenter family-based OFC study. Part of the Gabriella Miller Kids First Pediatric Research Program, this is the first large-scale WGS study of OFC in parent-offspring trios. WGS provides deeper and more specific genetic data than currently available using imputation on single nucleotide polymorphic (SNP) marker panels. Here, association analysis of genome-wide single nucleotide variants (SNV) and short insertions and deletions (indels) identified a new locus on chromosome 21 in Colombian families, within a region known to be expressed during craniofacial development. This study reinforces the ancestry differences seen in the genetic etiology of OFCs, and the need for larger samples when for studying OFCs and other birth defects in admixed populations.

genetics↗

Peripheral blood DNA methylation and autism spectrum disorder

Background\n\nSeveral reports have suggested a role for epigenetic mechanisms in ASD etiology. Epigenome-wide association studies (EWAS) in autism spectrum disorder (ASD) may shed light on particular biological mechanisms. However, studies of ASD cases versus controls have been limited by post-mortem timing and severely small sample sizes. Reports from in-life sampling of blood or saliva have also been very limited in sample size, and/or genomic coverage. We present the largest case-control EWAS for ASD to date, combining data from population-based case-control and case-sibling pair studies.\n\nMethods\n\nDNA from 968 blood samples from children in the Study to Explore Early Development (SEED 1) was used to generate epigenome-wide array DNA methylation (DNAm) data at 485,512 CpG sites for 453 cases and 515 controls, using the Illumina 450K Beadchip. The Simons Simplex Collection (SSC) provided 450K array DNAm data on an additional 343 cases and their unaffected siblings. We performed EWAS meta-analysis across results from the two data sets, with adjustment for sex and surrogate variables that reflect major sources of biological variation and technical confounding such as cell type, batch, and ancestry. We compared top EWAS results to those from a previous brain-based analysis. We also tested for enrichment of ASD EWAS CpGs for being targets of meQTL associations using available SNP genotype data in the SEED sample.\n\nFindings\n\nIn this meta-analysis of blood-based DNA from 796 cases and 858 controls, no single CpG met a Bonferroni discovery threshold of p < 1.12x10-7. Seven CpGs showed differences at p < 1x10-5 and 48 at 1x10-4. Of the top 7, 5 showed brain-based ASD associations as well, often with larger effect sizes, and the top 48 overall showed modest concordance (r = 0.31) in direction of effect with cerebellum samples. Finally, we observed suggestive evidence for enrichment of CpG sites controlled by SNPs (meQTL targets) among the EWAS CpGs hits, which was consistent across EWAS and meQTL discovery p-value thresholds.\n\nConclusions\n\nWe report the largest case-control EWAS study of ASD to date. No single CpG site showed a large enough DNAm difference between cases and controls to achieve epigenome-wide significance in this sample size. However, our results suggest the potential to observe disease associations from blood-based samples. Among the 7 sites achieving suggestive statistical significance, we observed consistent, and stronger, effects at the same sites among brain samples. Discovery-oriented EWAS for ASD using blood samples will likely need even larger samples and unified genetic data to further understand DNAm differences in ASD.

genomics↗