Biology subjects

de Soysa, T. Y.

Publications and source records attributed to de Soysa, T. Y..

3 recordsLinked to original sources

bioRxiv2018

Short hairpin RNAs artifactually impair cell growth and suppress clustered microRNA expression.

Functional gene disruption is a central tenet of cancer research, where novel drug targets are often identified and validated through cell-growth based knockdown studies or screens. Short hairpin RNA (shRNA)-mediated mRNA knockdown is widely used in both academic and pharmaceutical settings. However, off-target effects of shRNAs as well as interference with endogenous small RNA processing have been reported. We show here that lentiviral delivery of both gene-specific and non-targeting control shRNAs impair in vitro cell growth in a sequence independent manner. In addition, exogenous shRNAs induce a depressed cell-cycle-gene expression signature that is also shRNA-sequence independent and present across several studies. Further, we observe an shRNA mediated general repression of microRNAs belonging to polycistronic genetic clusters, including microRNAs from established oncogenic microRNA clusters. The collective impact of these observations is particularly relevant for cancer research, given the widespread historical use of shRNAs and the common goal of interrogating genes that regulate proliferation. We therefore recommend that when employing shRNA for target validation, care be taken to titrate shRNA dose, use hairpin-expressing controls, perform gene-of-interest rescue experiments and/or corroborate shRNA-derived results by small interfering RNA (siRNA) knockdown or CRISPR/Cas9-mediated genetic knockout. Minimizing these deleterious sequence independent effects will improve research fidelity and help address reported challenges in experimental reproducibility.

cancer biology↗

bioRxiv2018

Single-cell transcriptome analysis during cardiogenesis reveals basis for organ level developmental anomalies

Organogenesis involves integration of myriad cell types with reciprocal interactions, each progressing through successive stages of lineage specification and differentiation. Establishment of unique gene networks within each cell dictates fate determination, and mutations of transcription factors that drive such networks can result in birth defects. Congenital heart defects are the most common malformations and are caused by disruption of discrete subsets of progenitors1-3, however, determining the transcriptional changes in individual cells that lead to organ-level defects in the heart, or other organs, has not been tractable. Here, we employed single-cell RNA sequencing to interrogate early cardiac progenitor cells as they become specified during normal and abnormal cardiogenesis, revealing how dysregulation of specific cellular sub-populations can have catastrophic consequences. A network-based computational method for single-cell RNA-sequencing that predicts lineage specifying transcription factors4,5 identified Hand2 as a specifier of outflow tract cells but not right ventricular cells, despite failure of right ventricular formation in Hand2-null mice6. Temporal single-cell transcriptome analysis of Hand2-null embryos revealed failure of outflow tract myocardium specification, whereas right ventricular myocardium differentiated but failed to migrate into the anterior pole of the developing heart. Dysregulation of retinoic acid signaling, responsible for anterior-posterior patterning7, was associated with posteriorization of anterior cardiac progenitors in Hand2-null mutant hearts and ectopic atrial gene expression in outflow tract and right ventricle precursors. This work reveals transcriptional determinants in individual cells that specify cardiac progenitor cell fate and differentiation and exposes mechanisms of disrupted cardiac development at single-cell resolution, providing a framework to investigate congenital heart defects.

developmental biology↗

bioRxiv2018

Oligogenic inheritance of congenital heart disease involving a NKX2-5 modifier

Complex genetic inheritance is thought to underlie many human diseases, yet experimental proof of this model has been elusive. Here, we show that a human congenital heart defect, left ventricular non-compaction (LVNC), can be caused by a combination of rare, inherited heterozygous missense single nucleotide variants. Whole exome sequencing of a nuclear family revealed novel single nucleotide variants of MYH7 and MKL2 in an asymptomatic father while the offspring with severe childhood-onset LVNC harbored an additional missense variant in the cardiac transcription factor, NKX2-5, inherited from an unaffected mother. Mice bred to compound heterozygosity for the orthologous missense variants in Myh7 and Mkl2 had mild cardiac pathology; the additional inheritance of the Nkx2-5 variant yielded a more severe LVNC-like phenotype in triple compound heterozygotes. RNA sequencing identified genes associated with endothelial and myocardial development that were dysregulated in hearts from triple heterozygote mice and human induced pluripotent stem cell-derived cardiomyocytes harboring the three variants, with evidence for NKX2-5s contribution as a modifier on the molecular level. These studies demonstrate that the deployment of efficient gene editing tools can provide experimental evidence for complex inheritance of human disease.\n\nOne sentence summaryA combination of three inherited heterozygous missense single nucleotide variants underlying a familial congenital heart defect.

genetics↗