Biology subjects

Manshaei, R.

Publications and source records attributed to Manshaei, R..

3 recordsLinked to original sources

bioRxiv2024

Modelling DNA replication fork stability and collapse using chromatin fiber analysis and the R-ODD-BLOBS program

We describe the anatomy of replication forks by comparing the lengths of synthesized BrdU-labelled DNA in wild type, mrc1{Delta} and cds1{Delta} Schizoasaccharomyces pombe. We correlated Rad51 and Cdc45 proteins relative to their positions on the fork, replicated tract, or unreplicated regions. We did this by using chromatin fiber images. These fibers track pixel intensity data, which is analyzed using our program: R-ODD-BLOBS. We compared the lengths of BrdU tracts and proteins, as well as the percentage of Rad51 and Cdc45 colocalization, and compared our results with literature findings. We measured average BrdU lengths consistent with current literature; cds1{Delta} was the longest at [~]2.9 kb (8.6 pixels, px), wild type was [~] 2.5 kb (7.5 px), and mrc1{Delta} was the shortest at [~]1.7 kb (5.1 px). Intriguingly, Rad51 was found at 22% more replicated areas in mrc1{Delta} than in wild type. This suggests that homologous recombination repair may be more common at mrc1{Delta} forks. In this study, we summarize the usefulness of a computational modeling tool to assess large datasets of chromatin spread data. In turn, we find patterns of DNA replication length and protein components at replication forks, to describe the anatomy of a fork and how structures change with checkpoint loss. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/621594v2_figa1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1fd59forg.highwire.dtl.DTLVardef@1f234org.highwire.dtl.DTLVardef@1c46b51org.highwire.dtl.DTLVardef@61ce37_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACT:C_FLOATNO R-ODD-BLOBS uses chromatin fiber data to rigorously model replication fork structures. DNA replication forks are multi-subunit structures that must pair and regulate DNA copying activity of the polymerases with unwinding activity of helicase. Chromatin fiber data retains proteins, and can be used to detect DNA synthesis (blue) and associated DNA replication fork proteins such as MCM4 helicase (MCM4) and replication protein A (RPA). In our work, we have used homologous recombination protein Rad51 and helicase factor Cdc45 to understand how DNA replication fork structures are destabilized during hydroxyurea treatment, and how they fail to recover because of Cdc45/helicase mis-localization. C_FIG

cell biology↗

bioRxiv2020

GeneTerpret: a customizable multilayer approach to genomic variant prioritization and interpretation

Variant interpretation is the main bottleneck in medical genomic sequencing efforts. This usually involves genome analysts manually scouring through a multitude of independent databases, often with the aid of several and mostly independent computational tools. To streamline the variant interpretation process, we developed GeneTerpret platform that collates data from current interpretation tools and databases, and applies a phenotype-driven query to categorize the variants identified in a given genome. The platform assigns quantitative validity scores to genes by query and assembly of the current genotype-phenotype data, sequence homology, molecular interactions, expression data, and animal models. The platform uses the American College of Medical Genetics (ACMG) criteria to categorize variants into five tiers (from benign to pathogenic). The platform then outputs a prioritized list of potentially causal variants/genes in a given genome for a specific case. GeneTerpret is a flexible and free platform designed to streamline the variant interpretation process through a unique interface, with improved ease, speed and accuracy. This unique integrated system provides effective validity and pathogenicity modules to assess genetic variant data and allows the user to decide which output and impact level should be considered in this process. The platform can be accessed and used online at https://geneterpret.com.

bioinformatics↗

bioRxiv2020

Genes and pathways implicated in tetralogy of Fallot revealed by ultra-rare variant burden analysis in 231 genome sequences

Recent genome-wide studies of rare genetic variants have begun to implicate novel mechanisms for tetralogy of Fallot (TOF), a severe congenital heart defect (CHD). To provide statistical support for case-only data without parental genomes, we re-analyzed genome sequences of 231 individuals with TOF or related CHD. We adapted a burden test originally developed for de novo variants to assess singleton variant burden in individual genes, and in gene-sets corresponding to functional pathways and mouse phenotypes, accounting for highly correlated gene-sets, and for multiple testing. The gene burden test identified a significant burden of deleterious missense variants in NOTCH1 (Bonferroni-corrected p-value <0.01). These NOTCH1 variants showed significant enrichment for those affecting the extracellular domain, and especially for disruption of cysteine residues forming disulfide bonds (OR 39.8 vs gnomAD). Individuals with NOTCH1 variants, all with TOF, were enriched for positive family history of CHD. Other genes not previously implicated in TOF had more modest statistical support and singleton missense variant results were non-significant for gene-set burden. For singleton truncating variants, the gene burden test confirmed significant burden in FLT4. Gene-set burden tests identified a cluster of pathways corresponding to VEGF signaling (FDR=0%), and of mouse phenotypes corresponding to abnormal vasculature (FDR=0.8%), that suggested additional candidate genes not previously identified (e.g., WNT5A and ZFAND5). Analyses using unrelated sequencing datasets supported specificity of the findings for CHD. The findings support the importance of ultra-rare variants disrupting genes involved in VEGF and NOTCH signaling in the genetic architecture of TOF. These proof-of-principle data indicate that this statistical methodology could assist in analyzing case-only sequencing data in which ultra-rare variants, whether de novo or inherited, contribute to the genetic etiopathogenesis of a complex disorder. Author summaryWe analyzed the ultra-rare nonsynonymous variant burden for genome sequencing data from 231 individuals with congenital heart defects, most with tetralogy of Fallot. We adapted a burden test originally developed for de novo variants. In line with other studies, we identified a significant truncating variant burden for FLT4 and deleterious missense burden for NOTCH1, both passing a stringent Bonferroni multiple-test correction. For NOTCH1, we observed frequent disruption of cysteine residues establishing disulfide bonds in the extracellular domain. We also identified genes with BH-FDR <10% that were not previously implicated. To overcome limited power for individual genes, we tested gene-sets corresponding to functional pathways and mouse phenotypes. Gene-set burden of truncating variants was significant for vascular endothelial growth factor signaling and abnormal vasculature phenotypes. These results confirmed previous findings and suggested additional candidate genes for experimental validation in future studies. This methodology can be extended to other case-only sequencing data in which ultra-rare variants make a substantial contribution to genetic etiology.

genomics↗