Biology subjects

Sharp, A. J.

Publications and source records attributed to Sharp, A. J..

12 recordsLinked to original sources

bioRxiv2024

TRGT-denovo: accurate detection of de novo tandem repeat mutations

MotivationIdentifying de novo tandem repeat (TR) mutations on a genome-wide scale is essential for understanding genetic variability and its implications in rare diseases. While PacBio HiFi sequencing data enhances the accessibility of the genomes TR regions for genotyping, simple de novo calling strategies often generate an excess of likely false positives, which can obscure true positive findings, particularly as the number of surveyed genomic regions increases. ResultsWe developed TRGT-denovo, a computational method designed to accurately identify all types of de novo TR mutations--including expansions, contractions, and compositional changes-- within family trios. TRGT-denovo directly interrogates read evidence, allowing for the detection of subtle variations often overlooked in variant call format (VCF) files. TRGT-denovo improves the precision and specificity of de novo mutation (DNM) identification, reducing the number of de novo candidates by an order of magnitude compared to genotype-based approaches. In our experiments involving eight rare disease trios previously studied TRGT-denovo correctly reclassified all false positive DNM candidates as true negatives. Using an expanded repeat catalog, it identified new candidates, of which 95% (19/20) were experimentally validated, demonstrating its effectiveness in minimizing likely false positives while maintaining high sensitivity for true discoveries. Availability and implementationBuilt in Rust, TRGT-denovo is available as source code and a pre-compiled Linux binary along with a user guide at: https://github.com/PacificBiosciences/trgt-denovo.

bioinformatics↗

bioRxiv2023

Feeder-Free Generation of Endocardial and Cardiac Valve Cells from Human Pluripotent Stem Cells

Valvular heart disease presents a significant health burden, yet advancements in valve biology and novel therapeutics have been hindered by the lack of accessibility to human valve cells. In this study, we have developed a scalable and feeder-free method to differentiate human induced pluripotent stem cells (iPSCs) into endocardial cells. Importantly, we show that these endocardial cells are transcriptionally and phenotypically distinct from vascular endothelial cells and can be directed to undergo endothelial-to-mesenchymal transition (EndMT) to generate cardiac valve cell populations. Following this, we identified two distinct populations--one population undergoes EndMT to become valvular interstitial cells (VICs), while the other population reinforces their endothelial identity to become valvular endothelial cells (VECs). Lastly, we confirmed the identities of our iPSC-derived cell populations and identified putative markers through transcriptomic analyses. By increasing the accessibility to these cell populations, we aim to accelerate discoveries for cardiac valve biology and disease.

cell biology↗

bioRxiv2023

Assessing the burden of rare DNA methylation outliers in schizophrenia

Along with case-control group differences in DNA methylation (DNAm) identified in epigenomewide association studies (EWAS), multiple rare DNAm outliers may exist in subsets of cases, underlying the etiological heterogeneity of some disorders. This creates an impetus for novel approaches focused on detecting rare/private outliers in the individual methylomes. Here, we present a novel, data-driven method - Outlier Methylation Analysis (OMA) - which through optimization detects genomic regions with strongly deviating DNAm levels, which we call outlier methylation regions (OMRs). Focusing on schizophrenia (SCZ) - a neuropsychiatric disorder with a heterogeneous etiology - we applied the OMA method in two independent, publicly available SCZ case-control samples with DNAm array information. We found SCZ cases had an increased burden of OMRs compared to controls (IRR=1.22, p=1.8x10-8), and case OMRs were enriched in regions relevant to cellular differentiation and development (i.e. polycomb repressed elements in the Gm12878 differentiated cell line, p=1.9x10-5, and poised promoters in the H1hesc stem cell line, p=5.4x10-4). Furthermore, SCZ cases were ~2.5-fold enriched (p=1.1x10-3) for OMRs overlapping genesets associated with developmental processes. The OMR burden was reduced in clozapine-treated, compared to untreated, SCZ cases (IRR=0.88, p=9.5x10-3), and also associated with increased chronological age (IRR=1.01, p= 2.7x10-16). Our findings demonstrate an elevated burden of OMRs in SCZ, implying methylomic dysregulation in SCZ which could correspond to the etiological heterogeneity among cases. These results remain to be causally examined and replicated in other cohorts and tissues. For this, and applications in other traits, we offer the OMA method to the scientific community.

genetics↗

bioRxiv2021

Limitations of lymphoblastoid cell lines for establishing genetic reference datasets in the immunoglobulin loci

Lymphoblastoid cell lines (LCLs) have been critical to establishing genetic resources for biomedical science. They have been used extensively to study human genetic diversity, genome function, and inform the development of tools and methodologies for augmenting disease genetics research. While the validity of variant callsets from LCLs has been demonstrated for most of the genome, previous work has shown that DNA extracted from LCLs is modified by V(D)J recombination within the immunoglobulin (IG) loci, regions that harbor antibody genes critical to immune system function. However, the impacts of V(D)J on data generated from LCLs has not been extensively investigated. In this study, we used LCL-derived short read sequencing data from the 1000 Genomes Project (n=2,504) to identify signatures of V(D)J recombination. Our analyses revealed sample-level impacts of V(D)J recombination that varied depending on the degree of inferred monoclonality. We showed that V(D)J associated somatic deletions impacted genotyping accuracy, leading to adulterated population-level estimates of allele frequency and linkage disequilibrium. These findings illuminate limitations of using LCLs for building genetic resources in the IG loci, with implications for interpreting previous disease association studies in these regions. Author summaryLymphoblastoid cell lines (LCLs) are cells that have been manipulated to proliferate indefinitely in order to provide a replenishable source of DNA. However, because these cell lines are derived from B cells which have undergone V(D)J recombination they contain somatic deletions within regions of the genome that encode antibody genes. Although several large collaborative projects have utilized DNA from LCLs to generate invaluable genomic resources for the scientific community, the negative impacts of cell line artifacts in these regions of the genome have not been fully appreciated. In this study, we used newly released sequencing data from a large collection of LCLs to determine that the non-inherited artificial deletions within the antibody gene loci can have detrimental effects on downstream genetic analyses.

genomics↗

bioRxiv2020

Pervasive cis effects of variation in copy number of large tandem repeats on local epigenetics and gene expression

Variable Number Tandem Repeats (VNTRs) are composed of large tandemly repeated motifs, many of which are highly polymorphic in copy number. However, due to their large size and repetitive nature, they remain poorly studied. To investigate the regulatory potential of VNTRs, we used read-depth data from Illumina whole genome sequencing to perform association analysis between copy number of ~70,000 VNTRs (motif size [≥]10bp) with both gene expression (404 samples in 48 tissues) and DNA methylation (235 samples in peripheral blood), identifying thousands of VNTRs that are associated with local gene expression (eVNTRs) and DNA methylation levels (mVNTRs). Using large-scale replication analysis in an independent cohort we validated 73-80% of signals observed in the two discovery cohorts, providing robust evidence to support that these represent genuine associations. Further, conditional analysis indicated that many eVNTRs and mVNTRs act as QTLs independently of other local variation. We also observed strong enrichments of eVNTRs and mVNTRs for regulatory features such as enhancers and promoters. Using the Human Genome Diversity Panel, we defined sets of VNTRs that show highly divergent copy numbers among human populations, show that these are enriched for regulatory effects on gene expression and epigenetics, and preferentially associate with genes that have been linked with human phenotypes through GWAS. Our study provides strong evidence supporting functional variation at thousands of VNTRs, and defines candidate sets of VNTRs, copy number variation of which potentially plays a role in numerous human phenotypes.

genomics↗

bioRxiv2020

Integrating tracking and resight data from breeding Painted Bunting populations enables unbiased inferences about migratory connectivity and winter range survival

Archival geolocators have transformed the study of small, migratory organisms but analysis of data from these devices requires bias correction because tags are only recovered from individuals that survive and are re-captured at their tagging location. We show that integrating geolocator recovery data and mark-resight data enables unbiased estimates of both migratory connectivity between breeding and non-breeding populations and region-specific survival probabilities for wintering locations. Using simulations, we first demonstrate that an integrated Bayesian model returns unbiased estimates of transition probabilities between seasonal ranges and to determine how different sampling designs influence estimability of transition probabilities. We then parameterize the model with tracking data and mark-resight data from declining Painted Bunting (Passerina ciris) populations breeding in the eastern United States, hypothesized to be threatened by the illegal pet trade in parts of their Caribbean, non-breeding range. Consistent with this hypothesis, we found that male buntings wintering in Cuba were 20% less likely to return to the breeding grounds than birds wintering elsewhere in their range. Improving inferences from archival tags through proper data collection and further development of integrated models will advance our understanding of full annual cycle ecology of migratory species.

ecology↗

bioRxiv2020

A novel framework for characterizing genomic haplotype diversity in the human immunoglobulin heavy chain locus

An incomplete ascertainment of genetic variation within the highly polymorphic immunoglobulin heavy chain locus (IGH) has hindered our ability to define genetic factors that influence antibody and B cell mediated processes. To date, methods for locus-wide genotyping of all IGH variant types do not exist. Here, we combine targeted long-read sequencing with a novel bioinformatics tool, IGenotyper, to fully characterize genetic variation within IGH in a haplotype-specific manner. We apply this approach to eight human samples, including a haploid cell line and two mother-father-child trios, and demonstrate the ability to generate high-quality assemblies (>98% complete and >99% accurate), genotypes, and gene annotations, including 2 novel structural variants and 16 novel gene alleles. We show that multiplexing allows for scaling of the approach without impacting data quality, and that our genotype call sets are more accurate than short-read (>35% increase in true positives and >97% decrease in false-positives) and array/imputation-based datasets. This framework establishes a foundation for leveraging IG genomic data to study population-level variation in the antibody response.

genomics↗

bioRxiv2020

A survey of rare epigenetic variation in 23,116 human genomes identifies disease-relevant epivariations and novel CGG expansions

There is growing recognition that epivariations, most often recognized as promoter hypermethylation events that lead to gene silencing, are associated with a number of human diseases. However, little information exists on the prevalence and distribution of rare epigenetic variation in the human population. In order to address this, we performed a survey of methylation profiles from 23,116 individuals using the Illumina 450k array. Using a robust outlier approach, we identified 4,452 unique autosomal epivariations, including potentially inactivating promoter methylation events at 384 genes linked to human disease. For example, we observed promoter hypermethylation of BRCA1 and LDLR at population frequencies of ~1 in 3,000 and ~1 in 6,000 respectively, suggesting that epivariations may underlie a fraction of human disease which would be missed by purely sequence-based approaches. Using expression data, we confirmed that many epivariations are associated with outlier gene expression. Analysis of SNV data and monozygous twin pairs suggests that approximately two thirds of epivariations segregate in the population secondary to underlying sequence mutations, while one third are likely sproradic events that occur post-zygotically. We identified 25 loci where rare hypermethylation coincided with the presence of an unstable CGG tandem repeat, and validated the presence of novel CGG expansions at several of these, identifying the molecular defect underlying most of the known folate-sensitive fragile sites in the genome. Our study provides a catalog of rare epigenetic changes in the human genome, gives insight into the underlying origins and consequences of epivariations, and identifies many novel hypermethylated CGG repeat expansions.

genomics↗

bioRxiv2020

Meta-analysis of epigenome-wide association studies in Alzheime's disease highlights 220 differentially methylated loci across cortex

Epigenome-wide association studies of Alzheimers disease have highlighted neuropathology-associated DNA methylation differences, although existing studies have been limited in sample size and utilized different brain regions. Here, we combine data from six DNA methylomic studies of Alzheimers disease (N=1,453 unique individuals) to identify differential methylation associated with Braak stage in different brain regions and across cortex. We identify 236 CpGs in the prefrontal cortex, 95 CpGs in the temporal gyrus and ten CpGs in the entorhinal cortex at Bonferroni significance, with none in the cerebellum. Our cross-cortex meta-analysis (N=1,408 donors) identifies 220 CpGs associated with neuropathology, annotated to 121 genes, of which 84 genes have not been previously reported at this significance threshold. We have replicated our findings using two further DNA methylomic datasets consisting of a further > 600 unique donors. The meta-analysis summary statistics are available in our online data resource (www.epigenomicslab.com/ad-meta-analysis/).

genomics↗

bioRxiv2020

Notch Signaling Commits Mesoderm to the Cardiac Lineage

During development multiple progenitor populations contribute to the formation of the four-chambered heart and its diverse lineages. However, the underlying mechanisms that result in the specification of these progenitor populations are not yet fully understood. We have previously identified a population of cells that gives rise selectively to the heart ventricles but not the atria. Here, we have used this knowledge to transcriptionally profile subsets of cardiac mesoderm from the mouse embryo and have identified an enrichment for Notch signaling components in ventricular progenitors. Using directed differentiation of human pluripotent stem cells, we next investigated the role of Notch in cardiac mesoderm specification in a temporally controlled manner. We show that transient Notch induction in mesoderm increases cardiomyocyte differentiation efficiency, while maintaining cardiomyocytes in an immature state. Finally, our data suggest that Notch interacts with WNT to enhance commitment to the cardiac lineage. Overall, our findings support the notion that key signaling events during early heart development are critical for proper lineage specification and provide evidence for early roles of Notch and WNT during mouse and human heart development. Summary statementEarly fate decisions are dictated by the embryonic signaling environment. We show that Notch signaling is active during early mouse development and that activating Notch in human cardiac mesoderm enhances cardiomyocyte differentiation efficiency.

developmental biology↗

bioRxiv2017

Robust identification of deletions in exome and genome sequence data based on clustering of Mendelian errors

Multiple tools have been developed to identify copy number variants (CNVs) from whole exome (WES) and whole genome sequencing (WGS) data. Current tools such as XHMM for WES and CNVnator for WGS identify CNVs based on changes in read depth. For WGS, other methods to identify CNVs include utilizing discordant read pairs and split reads and genome-wide local assembly with tools such as Lumpy and SvABA, respectively. Here, we introduce a new method to identify deletion CNVs from WES and WGS trio data based on the clustering of Mendelian errors (MEs). Using our Mendelian Error Method (MEM), we identified 127 deletions (inherited and de novo) in 2,601 WES trios from the Pediatric Cardiac Genomics Consortium, with a validation rate of 88% by digital droplet PCR. MEM identified additional de novo deletions compared to XHMM, and also identified sample switches, DNA contamination, a significant enrichment of 15q11.2 deletions compared to controls and eight cases of uniparental disomy. We applied MEM to WGS data from the Genome In A Bottle Ashkenazi trio and identified deletions with 97% specificity. MEM provides a robust, computationally inexpensive method for identifying deletions, and an orthogonal approach for verifying deletions called by other tools.

genetics↗

bioRxiv2017

A survey of DNA methylation polymorphism identifies environmentally responsive co-regulated networks of epigenetic variation in the human genome

While studies such as the 1000 Genomes Projects have resulted in detailed maps of genetic variation in humans, to date there are few robust maps of epigenetic variation. We defined sites of common epigenetic variation, termed Variably Methylated Regions (VMRs) in five purified cell types. We observed that VMRs occur preferentially at enhancers and 3 UTRs. While the majority of VMRs have high heritability, a subset of VMRs within the genome show highly correlated variation in trans, forming co-regulated networks that have low heritability, differ between cell types and are enriched for specific transcription factor binding sites and biological pathways of functional relevance to each tissue. For example, in T cells we defined a network of 72 co-regulated VMRs enriched for genes with roles in T-cell activation; in fibroblasts a network of 21 coregulated VMRs comprising all four HOX gene clusters enriched for control of tissue growth; and in neurons a network of 112 VMRs enriched for roles in learning and memory. By culturing genetically-identical fibroblasts under varying conditions of nutrient deprivation and cell density, we experimentally demonstrate that some VMR networks are responsive to environmental conditions, with methylation levels at these loci changing in a coordinated fashion in trans dependent on cellular growth. Intriguingly these environmentally-responsive VMRs showed a strong enrichment for imprinted loci (p<10-94), suggesting that these are particularly sensitive to environmental conditions. Our study provides a detailed map of common epigenetic variation in the human genome, showing that both genetic and environmental causes underlie this variation.

genomics↗