Biology subjects

Kirillova, A.

Publications and source records attributed to Kirillova, A..

5 recordsLinked to original sources

bioRxiv2026

Damaging RBM20 E-rich domain variants are not rescued by gene replacement

The promise of precision therapeutics in genetic cardiomyopathies relies on linking specific therapies to variant mechanisms. Missense variants in the cardiac splice regulator RBM20 cause a highly penetrant and arrhythmogenic dilated cardiomyopathy. Disease-causing variants in RBM20s arginine-serine rich (RS) domain act via formation of toxic gain of function cytoplasmic granules, but this is not true for a small number of clinically adjudicated pathogenic variants in its glutamate(E)-rich domain. To better define the effects of E-rich domain variants, we developed a scalable screen based on induced pluripotent stem cell (iPSC) cardiomyocyte differentiation that identified several additional damaging variants. Several of these reduced RBM20 protein abundance and stability. We therefore hypothesized that, unlike RS domain variants, these E-rich variants might be rescued by RBM20 overexpression. To test this hypothesis, we generated induced pluripotent stem cells (iPSCs) from a patient with a pathogenic E-rich domain variant (p.E913K), and confirmed reduced RBM20 protein expression in these RBM20+/p.E913K cells after differentiation to iPSC-derived cardiomyocytes (iPSC-CM, vs. engineered isogenic RBM20+/+). These iPSC-CMs also displayed aberrant transcriptional splicing, reduced contractility, increased calcium-induced calcium release, and nuclear localization of RBM20 protein, often with more than the two expected RBM20-centric splice factories. AAV-based overexpression of RBM20 reversed some, but not all of the mis-splicing events identified in RBM20+/p.E913K iPSC-CMs, and did not improve their abnormal contractility, calcium handling or supernumerary RBM20 nuclear granules. In summary, our data indicate that pathogenic E-rich domain variants reduce RBM20 protein abundance, but that their mechanism is unlikely to be explained by haploinsufficiency alone.

genetics↗

bioRxiv2025

Scaled multidimensional assays of variant effect identify sequence-function relationships in hypertrophic cardiomyopathy

BackgroundAn estimated 1 in 500 people live with hypertrophic cardiomyopathy (HCM), a disease for which genetic diagnosis can identify family members at risk, and increasingly guide therapy. Mutations in the myosin binding protein C3 (MYBPC3) gene account for a significant proportion of HCM cases. However, many of these variants are classified as variants of uncertain significance (VUS), complicating clinical decision-making. Scalable methods for variant interpretation in disease-specific cell types are crucial for understanding variant impact and uncovering disease mechanisms. MethodsWe developed a scaled multidimensional mapping strategy to evaluate the functional impact of variants across a critical domain of MYBPC3. We incorporate saturation base editing at the native MYBPC3 locus, a long-read RNA sequencing-enabled assay of variant splice effects, and measurements of HCM-relevant phenotypes, including MYBPC3 abundance, hypertrophic signaling, and ubiquitin-proteasome function in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). ResultsOur multidimensional mapping strategy enabled high-resolution functional analysis of MYBPC3 variants in iPSC-CMs. Targeted transient base editing generated a comprehensive variant library at the native locus, capturing diverse variant effects on cellular HCM-relevant phenotypes. Our massively parallel splicing assay identified novel splice-disrupting variants. Integration of functional assays revealed that decreased MYBPC3 abundance is a key driver of HCM-related phenotypes. In parallel, downregulation of protein degradation was observed as a compensatory response to MYBPC3 loss of function, and novel disease mechanisms were identified for missense variants near a critical binding domain, underscoring their contribution to pathogenesis. Bayesian estimates of variant effects enable the reclassification of clinical variants. ConclusionsThis work provides a platform for extending genome engineering in iPSCs to multiplexed assays of variant effects across diverse disease-relevant cellular phenotypes, enhancing the understanding of variant pathogenicity and uncovering novel biological mechanisms that could inform therapeutic strategies.

genetics↗

bioRxiv2022

The mitochondrial component of human infertility- an increased mtDNA content and an excess of ultra-rare mtDNA variants in aneuploid versus euploid embryos.

Using low-coverage, whole-genome sequences of trophectoderm biopsies from 11,610 human blastocyst-stage embryos, we analyzed the relationship between chromosomal abnormalities and mitochondrial (mt) DNA dynamics. Comparing 6,208 aneuploid and 5,402 euploid embryos in cohort studies, we found that mtDNA content in aneuploid embryos was significantly higher than that in euploid embryos. This outcome was confirmed through intrafamilial analyses of embryos with matched parents and in vitro fertilization cycles, and it occurred independent of maternal age. Additional human population-based studies uncovered a higher abundance of ultra-rare mtDNA variants located in never-altered positions in the human population in aneuploid compared to euploid embryos in both cohort- and family-based analyses. This maternal age-independent association of increased mtDNA content and aneuploidy in human embryos may reflect a novel mechanism of purifying selection against potentially deleterious mtDNA variants, which arise from germline or early developmental mtDNA damaging events, that occurs in human embryos prior to implantation.

bioinformatics↗

bioRxiv2022

Machine learning-based classification of binary dynamic fluorescence signals reveals muscle stem cell fate transitions in response to pro-regenerative niche factors

The proper regulation of muscle stem cell (MuSC) fate by cues from the niche is essential for regeneration of skeletal muscle. How pro-regenerative niche factors control the dynamics of MuSC fate decisions remains unknown due to limitations of population-level endpoint assays. To address this knowledge gap, we developed a novel binary dynamic fluorescence time lapse imaging analysis (BDFA) approach that leverages machine learning classification strategies to track single cell fate decisions with high temporal resolution. Using two fluorescent reporters that read out maintenance of stemness and myogenic commitment, we constructed detailed lineage trees for individual MuSCs and their progeny, classifying each division event as symmetric self-renewing, asymmetric, or symmetric committed. Our analysis reveals that treatment with the lipid metabolite, prostaglandin E2 (PGE2), accelerates the rate of MuSC proliferation over time, while biasing division events toward symmetric self-renewal. In contrast, the IL6 family member, Oncostatin M (OSM) decreases the proliferation rate after the first generation, while blocking myogenic commitment. These insights into the dynamics of MuSC regulation by niche cues were uniquely enabled by our BDFA approach. We anticipate that similar binary live cell readouts derived from BDFA will markedly expand our understanding of how niche factors control tissue regeneration in real time.

cell biology↗

bioRxiv2018

Cadherin switch marks germ layer formation in the diploblastic sea anemone Nematostella vectensis

Morphogenesis is a shape-building process during development of multicellular organisms. During this process the establishment and modulation of cell-cell contacts play an important role. Cadherins, the major cell adhesion molecules, form adherens junctions connecting ephithelial cells. Numerous studies in Bilateria have shown that cadherins are associated with the regulation of cell differentiation, cell shape changes, cell migration and tissue morphogenesis. To date, the role of Cadherins in non-bilaterians is unknown. Here, we study the expression and the function of two paralogous classical cadherins, cadherin1 and cadherin3, in the diploblastic animal, the sea anemone Nematostella vectensis. We show that a cadherin switch is accompanying the formation of germ layers. Using specific antibodies, we show that both cadherins are localized to adherens junctions at apical and basal positions in ectoderm and endoderm. During gastrulation, partial EMT of endodermal cells is marked by a step-wise downregulation of cadherin3 and upregulation of cadherin1. Knockdown experiments show that both cadherins are required for maintenance of tissue integrity and tissue morphogenesis. This demonstrates that cnidarians convergently use cadherins to differentially control morphogenetic events during development.

developmental biology↗