scholarly journals Microsatellite instability as a tool to diagnose genome instability in CHO cell culture

2019 ◽  
Author(s):  
Dylan G. Chitwood ◽  
Qinghua Wang ◽  
Kathryn Elliott ◽  
Aiyana Bullock ◽  
Dwon Jordana ◽  
...  
Keyword(s):  
Microsatellite Instability ◽  
Genome Stability ◽  
De Novo ◽  
Genome Instability ◽  
Chinese Hamster ◽  
Loss Of Function ◽  
Cell Numbers ◽  
Functional Regions ◽  
Candidate Loci ◽  
Waste Accumulation

AbstractAs bioprocess intensification has increased over the last 30 years, yields from mammalian cell processes have increased from 10’s of milligrams to over 10’s of grams per liter. Most of these gains in productivity have been due to increasing cell numbers in the bioreactors, and with those increases in cell numbers, strategies have been developed to minimize metabolite waste accumulation, such as lactate and ammonia. Unfortunately, cell growth cannot occur without some waste metabolite accumulation, as central metabolism is required to produce the biopharmaceutical. Inevitably, metabolic waste accumulation leads to decline and termination of the culture. While it is understood that the accumulation of these unwanted compounds imparts a less than optimal culture environment, little is known about the genotoxic properties and the influence of these compounds on global genome instability. In this study, we examined the effects on Chinese hamster ovary (CHO) cells’ genome sequences and physiology due to exposure to elevated ammonia levels. We identified genome-wide de novo mutations, in addition to variants in functional regions of certain genes involved in the mismatch repair (MMR) pathway, such as DNA2, BRCA1 and RAD52, which led to loss-of-function and eventual genome instability. Additionally, we characterized the presence of microsatellites against the most recent Chinese Hamster genome assembly and discovered certain loci are not replicated faithfully in the presence of elevated ammonia, which represents microsatellite instability (MSI). Furthermore, we found 124 candidate loci that may be suitable biomarkers to gauge genome stability in CHO cultures.

scholarly journals Characterization of metabolic responses, genetic variations, and microsatellite instability in ammonia-stressed CHO cells grown in fed-batch cultures

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Dylan G. Chitwood ◽  
Qinghua Wang ◽  
Kathryn Elliott ◽  
Aiyana Bullock ◽  
Dwon Jordana ◽  
...  
Keyword(s):  
Microsatellite Instability ◽  
Microsatellite Loci ◽  
Cho Cells ◽  
Genome Stability ◽  
De Novo ◽  
Genome Instability ◽  
Chinese Hamster ◽  
Functional Genes ◽  
Nucleotide Polymorphisms ◽  
De Novo Mutations

Abstract Background As bioprocess intensification has increased over the last 30 years, yields from mammalian cell processes have increased from 10’s of milligrams to over 10’s of grams per liter. Most of these gains in productivity can be attributed to increasing cell densities within bioreactors. As such, strategies have been developed to minimize accumulation of metabolic wastes, such as lactate and ammonia. Unfortunately, neither cell growth nor biopharmaceutical production can occur without some waste metabolite accumulation. Inevitably, metabolic waste accumulation leads to decline and termination of the culture. While it is understood that the accumulation of these unwanted compounds imparts a suboptimal culture environment, little is known about the genotoxic properties of these compounds that may lead to global genome instability. In this study, we examined the effects of high and moderate extracellular ammonia on the physiology and genomic integrity of Chinese hamster ovary (CHO) cells. Results Through whole genome sequencing, we discovered 2394 variant sites within functional genes comprised of both single nucleotide polymorphisms and insertion/deletion mutations as a result of ammonia stress with high or moderate impact on functional genes. Furthermore, several of these de novo mutations were found in genes whose functions are to maintain genome stability, such as Tp53, Tnfsf11, Brca1, as well as Nfkb1. Furthermore, we characterized microsatellite content of the cultures using the CriGri-PICR Chinese hamster genome assembly and discovered an abundance of microsatellite loci that are not replicated faithfully in the ammonia-stressed cultures. Unfaithful replication of these loci is a signature of microsatellite instability. With rigorous filtering, we found 124 candidate microsatellite loci that may be suitable for further investigation to determine whether these loci may be reliable biomarkers to predict genome instability in CHO cultures. Conclusion This study advances our knowledge with regards to the effects of ammonia accumulation on CHO cell culture performance by identifying ammonia-sensitive genes linked to genome stability and lays the foundation for the development of a new diagnostic tool for assessing genome stability.

Hinfp is a guardian of the somatic genome by repressing transposable elements

2021 ◽  
Vol 118 (41) ◽  
pp. e2100839118
Author(s):  
Niraj K. Nirala ◽  
Qi Li ◽  
Prachi N. Ghule ◽  
Hsi-Ju Chen ◽  
Rui Li ◽  
...  
Keyword(s):  
Dna Damage ◽  
Transposable Elements ◽  
Transposable Element ◽  
Cancer Progression ◽  
Genome Stability ◽  
Genome Instability ◽  
Tissue Growth ◽  
Loss Of Function ◽  
Element Mobilization ◽  
Host Genes

Germ cells possess the Piwi-interacting RNA pathway to repress transposable elements and maintain genome stability across generations. Transposable element mobilization in somatic cells does not affect future generations, but nonetheless can lead to pathological outcomes in host tissues. We show here that loss of function of the conserved zinc-finger transcription factor Hinfp causes dysregulation of many host genes and derepression of most transposable elements. There is also substantial DNA damage in somatic tissues of Drosophila after loss of Hinfp. Interference of transposable element mobilization by reverse-transcriptase inhibitors can suppress some of the DNA damage phenotypes. The key cell-autonomous target of Hinfp in this process is Histone1, which encodes linker histones essential for higher-order chromatin assembly. Transgenic expression of Hinfp or Histone1, but not Histone4 of core nucleosome, is sufficient to rescue the defects in repressing transposable elements and host genes. Loss of Hinfp enhances Ras-induced tissue growth and aging-related phenotypes. Therefore, Hinfp is a physiological regulator of Histone1-dependent silencing of most transposable elements, as well as many host genes, and serves as a venue for studying genome instability, cancer progression, neurodegeneration, and aging.

Nuclear Folate Metabolism

2018 ◽  
Vol 38 (1) ◽  
pp. 219-243 ◽  
Author(s):  
Martha S. Field ◽  
Elena Kamynina ◽  
James Chon ◽  
Patrick J. Stover
Keyword(s):  
Mammalian Cells ◽  
Genome Stability ◽  
De Novo ◽  
Genome Instability ◽  
Childbearing Age ◽  
Definitive Evidence ◽  
Dna Replication And Repair ◽  
One Carbon Metabolism ◽  
Salvage Pathways ◽  
Thymidylate Synthesis

Despite unequivocal evidence that folate deficiency increases risk for human pathologies, and that folic acid intake among women of childbearing age markedly decreases risk for birth defects, definitive evidence for a causal biochemical pathway linking folate to disease and birth defect etiology remains elusive. The de novo and salvage pathways for thymidylate synthesis translocate to the nucleus of mammalian cells during S- and G2/M-phases of the cell cycle and associate with the DNA replication and repair machinery, which limits uracil misincorporation into DNA and genome instability. There is increasing evidence that impairments in nuclear de novo thymidylate synthesis occur in many pathologies resulting from impairments in one-carbon metabolism. Understanding the roles and regulation of nuclear de novo thymidylate synthesis and its relationship to genome stability will increase our understanding of the fundamental mechanisms underlying folate- and vitamin B12–associated pathologies.

scholarly journals Gain-of-function GABRB3 variants identified in vigabatrin-hypersensitive epileptic encephalopathies

2020 ◽  
Vol 2 (2) ◽  
Author(s):  
Nathan L Absalom ◽  
Vivian W Y Liao ◽  
Kavitha Kothur ◽  
Dinesh C Indurthi ◽  
Bruce Bennetts ◽  
...  
Keyword(s):  
De Novo ◽  
Drug Effects ◽  
Receptor Expression ◽  
Aminobutyric Acid ◽  
Loss Of Function ◽  
Molecular Phenotype ◽  
Gain Of Function ◽  
Gene Encoding ◽  
Epileptic Encephalopathies ◽  
Receptor Phenotype

Abstract Variants in the GABRB3 gene encoding the β3-subunit of the γ-aminobutyric acid type A ( receptor are associated with various developmental and epileptic encephalopathies. Typically, these variants cause a loss-of-function molecular phenotype whereby γ-aminobutyric acid has reduced inhibitory effectiveness leading to seizures. Drugs that potentiate inhibitory GABAergic activity, such as nitrazepam, phenobarbital or vigabatrin, are expected to compensate for this and thereby reduce seizure frequency. However, vigabatrin, a drug that inhibits γ-aminobutyric acid transaminase to increase tonic γ-aminobutyric acid currents, has mixed success in treating seizures in patients with GABRB3 variants: some patients experience seizure cessation, but there is hypersensitivity in some patients associated with hypotonia, sedation and respiratory suppression. A GABRB3 variant that responds well to vigabatrin involves a truncation variant (p.Arg194*) resulting in a clear loss-of-function. We hypothesized that patients with a hypersensitive response to vigabatrin may exhibit a different γ-aminobutyric acid A receptor phenotype. To test this hypothesis, we evaluated the phenotype of de novo variants in GABRB3 (p.Glu77Lys and p.Thr287Ile) associated with patients who are clinically hypersensitive to vigabatrin. We introduced the GABRB3 p.Glu77Lys and p.Thr287Ile variants into a concatenated synaptic and extrasynaptic γ-aminobutyric acid A receptor construct, to resemble the γ-aminobutyric acid A receptor expression by a patient heterozygous for the GABRB3 variant. The mRNA of these constructs was injected into Xenopus oocytes and activation properties of each receptor measured by two-electrode voltage clamp electrophysiology. Results showed an atypical gain-of-function molecular phenotype in the GABRB3 p.Glu77Lys and p.Thr287Ile variants characterized by increased potency of γ-aminobutyric acid A without change to the estimated maximum open channel probability, deactivation kinetics or absolute currents. Modelling of the activation properties of the receptors indicated that either variant caused increased chloride flux in response to low concentrations of γ-aminobutyric acid that mediate tonic currents. We therefore propose that the hypersensitivity reaction to vigabatrin is a result of GABRB3 variants that exacerbate GABAergic tonic currents and caution is required when prescribing vigabatrin. In contrast, drug strategies increasing tonic currents in loss-of-function variants are likely to be a safe and effective therapy. This study demonstrates that functional genomics can explain beneficial and adverse anti-epileptic drug effects, and propose that vigabatrin should be considered in patients with clear loss-of-function GABRB3 variants.

scholarly journals Targeting Genome Stability in Melanoma—A New Approach to an Old Field

2021 ◽  
Vol 22 (7) ◽  
pp. 3485
Author(s):  
Marta Osrodek ◽  
Michal Wozniak
Keyword(s):  
Genome Stability ◽  
Genome Instability ◽  
Checkpoint Inhibitors ◽  
Mapk Pathway ◽  
Rapid Development ◽  
Melanoma Cells ◽  
Old Field ◽  
Number Of Patients ◽  
Recent Developments ◽  
Mapk Inhibitors

Despite recent groundbreaking advances in the treatment of cutaneous melanoma, it remains one of the most treatment-resistant malignancies. Due to resistance to conventional chemotherapy, the therapeutic focus has shifted away from aiming at melanoma genome stability in favor of molecularly targeted therapies. Inhibitors of the RAS/RAF/MEK/ERK (MAPK) pathway significantly slow disease progression. However, long-term clinical benefit is rare due to rapid development of drug resistance. In contrast, immune checkpoint inhibitors provide exceptionally durable responses, but only in a limited number of patients. It has been increasingly recognized that melanoma cells rely on efficient DNA repair for survival upon drug treatment, and that genome instability increases the efficacy of both MAPK inhibitors and immunotherapy. In this review, we discuss recent developments in the field of melanoma research which indicate that targeting genome stability of melanoma cells may serve as a powerful strategy to maximize the efficacy of currently available therapeutics.

scholarly journals The de novo CACNA1A pathogenic variant Y1384C associated with hemiplegic migraine, early onset cerebellar atrophy and developmental delay leads to a loss of Cav2.1 channel function

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Maria A. Gandini ◽  
Ivana A. Souza ◽  
Laurent Ferron ◽  
A. Micheil Innes ◽  
Gerald W. Zamponi
Keyword(s):  
Developmental Delay ◽  
Early Onset ◽  
Structural Damage ◽  
De Novo ◽  
Splice Variants ◽  
Cerebellar Atrophy ◽  
Familial Hemiplegic Migraine ◽  
Significant Loss ◽  
Loss Of Function ◽  
Hemiplegic Migraine

AbstractCACNA1A pathogenic variants have been linked to several neurological disorders including familial hemiplegic migraine and cerebellar conditions. More recently, de novo variants have been associated with severe early onset developmental encephalopathies. CACNA1A is highly expressed in the central nervous system and encodes the pore-forming CaVα1 subunit of P/Q-type (Cav2.1) calcium channels. We have previously identified a patient with a de novo missense mutation in CACNA1A (p.Y1384C), characterized by hemiplegic migraine, cerebellar atrophy and developmental delay. The mutation is located at the transmembrane S5 segment of the third domain. Functional analysis in two predominant splice variants of the neuronal Cav2.1 channel showed a significant loss of function in current density and changes in gating properties. Moreover, Y1384 variants exhibit differential splice variant-specific effects on recovery from inactivation. Finally, structural analysis revealed structural damage caused by the tyrosine substitution and changes in electrostatic potentials.

scholarly journals NME3 Regulates Mitochondria to Reduce ROS-Mediated Genome Instability

2020 ◽  
Vol 21 (14) ◽  
pp. 5048
Author(s):  
Chih-Wei Chen ◽  
Ning Tsao ◽  
Wei Zhang ◽  
Zee-Fen Chang
Keyword(s):  
Oxidative Stress ◽  
Dna Repair ◽  
Genome Stability ◽  
Nuclear Dna ◽  
Genome Instability ◽  
Mitochondrial Fission ◽  
Nucleoside Diphosphate Kinase ◽  
Mitochondrial Fusion ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Oxidative Stress

NME3 is a member of the nucleoside diphosphate kinase (NDPK) family that binds to the mitochondrial outer membrane to stimulate mitochondrial fusion. In this study, we showed that NME3 knockdown delayed DNA repair without reducing the cellular levels of nucleotide triphosphates. Further analyses revealed that NME3 knockdown increased fragmentation of mitochondria, which in turn led to mitochondrial oxidative stress-mediated DNA single-strand breaks (SSBs) in nuclear DNA. Re-expression of wild-type NME3 or inhibition of mitochondrial fission markedly reduced SSBs and facilitated DNA repair in NME3 knockdown cells, while expression of N-terminal deleted mutant defective in mitochondrial binding had no rescue effect. We further showed that disruption of mitochondrial fusion by knockdown of NME4 or MFN1 also caused mitochondrial oxidative stress-mediated genome instability. In conclusion, the contribution of NME3 to redox-regulated genome stability lies in its function in mitochondrial fusion.

CDK19-related disorder results from both loss-of-function and gain-of-function de novo missense variants

2021 ◽  
Author(s):  
Yuri A. Zarate ◽  
Tomoko Uehara ◽  
Kota Abe ◽  
Masayuki Oginuma ◽  
Sora Harako ◽  
...  
Keyword(s):  
De Novo ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Related Disorder ◽  
Missense Variants

Carcinogen-induced DNA amplification in vitro: overreplication of the simian virus 40 origin region in extracts from carcinogen-treated CO60 cells

1990 ◽  
Vol 10 (1) ◽  
pp. 75-83
Author(s):  
Y Berko-Flint ◽  
S Karby ◽  
D Hassin ◽  
S Lavi
Keyword(s):  
De Novo ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Dna Molecules ◽  
Viral Origin ◽  
Cell Extracts ◽  
Origin Region

An in vitro system to study carcinogen-induced amplification in simian virus 40 (SV40)-transformed Chinese hamster (CO60) cells is described. SV40 amplification in this system resembled in many aspects the viral overreplication observed in drug-treated CO60 cells. Cytosolic extracts from N-methyl-N'-nitro-N-nitrosoguanidine-treated cells supported de novo DNA synthesis in the presence of excess exogenous T antigen and the SV40-containing plasmid pSVK1. The pattern of viral replication in these extracts was unique, since only the 2.4-kilobase-pair region spanning the origin was overreplicated, whereas distal sequences were not replicated significantly. Extracts from control cells supported only marginal levels of replication. In HeLa extracts, complete SV40 DNA molecules were replicated efficiently. The overreplication of the origin region in CO60 cell extracts was bidirectional and symmetrical. A fraction of the newly synthesized DNA molecules underwent a second round of replication, yielding MboI-sensitive fragments representing the 2.4-kilobase-pair region around the origin. The mechanisms controlling the amplification of the viral origin region, the nature of the cellular factors induced in the carcinogen-treated cells, and their putative association with general drug-induced SOS-like responses are discussed.

scholarly journals De novo mutations in inhibitors of Wnt, BMP, and Ras/ERK signaling pathways in non-syndromic midline craniosynostosis

2017 ◽  
Vol 114 (35) ◽  
pp. E7341-E7347 ◽  
Author(s):  
Andrew T. Timberlake ◽  
Charuta G. Furey ◽  
Jungmin Choi ◽  
Carol Nelson-Williams ◽  
Erin Loring ◽  
...  
Keyword(s):  
De Novo ◽  
Erk Signaling ◽  
Loss Of Function ◽  
De Novo Mutations ◽  
Neurodevelopmental Outcomes ◽  
Locus Heterogeneity ◽  
Syndromic Craniosynostosis ◽  
New Genes ◽  
Morphogenetic Protein ◽  
Nuclear Targets

Non-syndromic craniosynostosis (NSC) is a frequent congenital malformation in which one or more cranial sutures fuse prematurely. Mutations causing rare syndromic craniosynostoses in humans and engineered mouse models commonly increase signaling of the Wnt, bone morphogenetic protein (BMP), or Ras/ERK pathways, converging on shared nuclear targets that promote bone formation. In contrast, the genetics of NSC is largely unexplored. More than 95% of NSC is sporadic, suggesting a role for de novo mutations. Exome sequencing of 291 parent–offspring trios with midline NSC revealed 15 probands with heterozygous damaging de novo mutations in 12 negative regulators of Wnt, BMP, and Ras/ERK signaling (10.9-fold enrichment, P = 2.4 × 10−11). SMAD6 had 4 de novo and 14 transmitted mutations; no other gene had more than 1. Four familial NSC kindreds had mutations in genes previously implicated in syndromic disease. Collectively, these mutations contribute to 10% of probands. Mutations are predominantly loss-of-function, implicating haploinsufficiency as a frequent mechanism. A common risk variant near BMP2 increased the penetrance of SMAD6 mutations and was overtransmitted to patients with de novo mutations in other genes in these pathways, supporting a frequent two-locus pathogenesis. These findings implicate new genes in NSC and demonstrate related pathophysiology of common non-syndromic and rare syndromic craniosynostoses. These findings have implications for diagnosis, risk of recurrence, and risk of adverse neurodevelopmental outcomes. Finally, the use of pathways identified in rare syndromic disease to find genes accounting for non-syndromic cases may prove broadly relevant to understanding other congenital disorders featuring high locus heterogeneity.

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