A Next-Generation Sequencing Approach to Study the Transcriptomic Changes during the Differentiation ofPhysarumat the Single-Cell Level

Germline mutations in the BRCA1 and BRCA2 genes are responsible for hereditary breast and ovarian cancer syndrome. Germline and somatic BRCA1/2 mutations may define therapeutic targets and refine cancer treatment options. However, routine BRCA diagnostic approaches cannot reveal the exact time and origin of BRCA1/2 mutation formation, and thus, the fine details of their contribution to tumor progression remain less clear. Here, we establish a diagnostic pipeline using high-resolution microscopy and laser microcapture microscopy to test for BRCA1/2 mutations in the tumor at the single-cell level, followed by deep next-generation sequencing of various tissues from the patient. To demonstrate the power of our approach, here, we describe a detailed single-cell-level analysis of an ovarian cancer patient we found to exhibit constitutional somatic mosaicism of a pathogenic BRCA2 mutation. Employing next-generation sequencing, BRCA2 c.7795G>T, p.(Glu2599Ter) was detected in 78% of reads in DNA extracted from ovarian cancer tissue and 25% of reads in DNA derived from peripheral blood, which differs significantly from the expected 50% of a hereditary mutation. The BRCA2 mutation was subsequently observed at 17–20% levels in the normal ovarian and buccal tissue of the patient. Together, our findings suggest that this mutation occurred early in embryonic development. Characterization of the mosaic mutation at the single-cell level contributes to a better understanding of BRCA mutation formation and supports the concept that the combination of single-cell and next-generation sequencing methods is advantageous over traditional mutational analysis methods. This study is the first to characterize constitutional mosaicism down to the single-cell level, and it demonstrates that BRCA2 mosaicism occurring early during embryogenesis can drive tumorigenesis in ovarian cancer.

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Cutting-Edge Technologies for Ovarian Cancer: An Overview of the Impact of Genetic Testing, Next-Generation Sequencing, and Single-Cell Analysis

10.1007/978-981-16-1873-4_7 ◽

2021 ◽

pp. 203-229

Author(s):

Alia Ghoneum ◽

Amal Tazzite ◽

Khalid El Bairi ◽

Neveen Said

Keyword(s):

Ovarian Cancer ◽

Genetic Testing ◽

Next Generation Sequencing ◽

Single Cell ◽

Single Cell Analysis ◽

Cutting Edge ◽

Cell Analysis ◽

Next Generation ◽

The Impact ◽

Generation Sequencing

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Divide and conquer: A perspective on biochips for single-cell and rare-molecule analysis by next-generation sequencing

APL Bioengineering ◽

10.1063/1.5095962 ◽

2019 ◽

Vol 3 (2) ◽

pp. 020901 ◽

Cited By ~ 2

Author(s):

A. C. Lee ◽

Y. Lee ◽

D. Lee ◽

S. Kwon

Keyword(s):

Next Generation Sequencing ◽

Single Cell ◽

Divide And Conquer ◽

Next Generation ◽

Generation Sequencing

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RGC-32 Acts as a Hub to Regulate the Transcriptomic Changes Associated With Astrocyte Development and Reactive Astrocytosis

Frontiers in Immunology ◽

10.3389/fimmu.2021.705308 ◽

2021 ◽

Vol 12 ◽

Author(s):

Alexandru Tatomir ◽

Austin Beltrand ◽

Vinh Nguyen ◽

Jean-Paul Courneya ◽

Dallas Boodhoo ◽

...

Keyword(s):

Next Generation Sequencing ◽

Radial Glia ◽

Axonal Guidance ◽

Molecular Networks ◽

Pathway Enrichment Analysis ◽

Next Generation ◽

Reactive Changes ◽

Transcriptomic Changes ◽

Generation Sequencing

Response Gene to Complement 32 (RGC-32) is an important mediator of the TGF-β signaling pathway, and an increasing amount of evidence implicates this protein in regulating astrocyte biology. We showed recently that spinal cord astrocytes in mice lacking RGC-32 display an immature phenotype reminiscent of progenitors and radial glia, with an overall elongated morphology, increased proliferative capacity, and increased expression of progenitor markers when compared to their wild-type (WT) counterparts that make them incapable of undergoing reactive changes during the acute phase of experimental autoimmune encephalomyelitis (EAE). Here, in order to decipher the molecular networks underlying RGC-32’s ability to regulate astrocytic maturation and reactivity, we performed next-generation sequencing of RNA from WT and RGC-32 knockout (KO) neonatal mouse brain astrocytes, either unstimulated or stimulated with the pleiotropic cytokine TGF-β. Pathway enrichment analysis showed that RGC-32 is critical for the TGF-β-induced up-regulation of transcripts encoding proteins involved in brain development and tissue remodeling, such as axonal guidance molecules, transcription factors, extracellular matrix (ECM)-related proteins, and proteoglycans. Our next-generation sequencing of RNA analysis also demonstrated that a lack of RGC-32 results in a significant induction of WD repeat and FYVE domain-containing protein 1 (Wdfy1) and stanniocalcin-1 (Stc1). Immunohistochemical analysis of spinal cords isolated from normal adult mice and mice with EAE at the peak of disease showed that RGC-32 is necessary for the in vivo expression of ephrin receptor type A7 in reactive astrocytes, and that the lack of RGC-32 results in a higher number of homeodomain-only protein homeobox (HOPX)+ and CD133+ radial glia cells. Collectively, these findings suggest that RGC-32 plays a major role in modulating the transcriptomic changes in astrocytes that ultimately lead to molecular programs involved in astrocytic differentiation and reactive changes during neuroinflammation.

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