A scalable Drosophila assay for clinical interpretation of human PTEN variants in suppression of PI3K/AKT induced cellular proliferation

Gene variant discovery is becoming routine, but it remains difficult to usefully interpret the functional consequence or disease relevance of most variants. To fill this interpretation gap, experimental assays of variant function are becoming common place. Yet, it remains challenging to make these assays reproducible, scalable to high numbers of variants, and capable of assessing defined gene-disease mechanism for clinical interpretation aligned to the ClinGen Sequence Variant Interpretation (SVI) Working Group guidelines for ‘well-established assays’. Drosophila melanogaster offers great potential as an assay platform, but was untested for high numbers of human variants adherent to these guidelines. Here, we wished to test the utility of Drosophila as a platform for scalable well-established assays. We took a genetic interaction approach to test the function of ~100 human PTEN variants in cancer-relevant suppression of PI3K/AKT signaling in cellular growth and proliferation. We validated the assay using biochemically characterized PTEN mutants as well as 23 total known pathogenic and benign PTEN variants, all of which the assay correctly assigned into predicted functional categories. Additionally, function calls for these variants correlated very well with our recent published data from a human cell line. Finally, using these pathogenic and benign variants to calibrate the assay, we could set readout thresholds for clinical interpretation of the pathogenicity of 70 other PTEN variants. Overall, we demonstrate that Drosophila offers a powerful assay platform for clinical variant interpretation, that can be used in conjunction with other well-established assays, to increase confidence in the accurate assessment of variant function and pathogenicity.

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Tailoring the ACMG/AMP sequence variant interpretation guidelines to the unique aspects of germline ACADVL variants

Molecular Genetics and Metabolism ◽

10.1016/s1096-7192(21)00472-8 ◽

2021 ◽

Vol 132 ◽

pp. S254

Author(s):

May Flowers ◽

Meredith Weaver ◽

Heather Baudet ◽

Marzia Pasquali ◽

Gregory Enns ◽

...

Keyword(s):

Sequence Variant ◽

Variant Interpretation

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Ki-67/MIB-1 and Recurrence in Pituitary Adenoma

Journal of Neurological Surgery Part B Skull Base ◽

10.1055/s-0041-1735874 ◽

2021 ◽

Author(s):

Kent Tadokoro ◽

Colten Wolf ◽

Joseph Toth ◽

Cara Joyce ◽

Meharvan Singh ◽

...

Keyword(s):

Systematic Review ◽

Pituitary Adenoma ◽

Recurrence Rate ◽

Pituitary Adenomas ◽

Cellular Proliferation ◽

Published Data ◽

Ki 67 ◽

Institutional Data ◽

Mib 1

Abstract Objectives Ki-67/MIB-1 is a marker of cellular proliferation used as a pathological parameter in the clinical assessment of pituitary adenomas, where its expression has shown utility in predicting the invasiveness of these tumors. However, studies have shown variable results when using Ki-67/MIB-1 association with recurrence. The purpose of this study is to determine if a high Ki-67/MIB-1 labeling index (LI) is predictive of recurrence in pituitary adenomas. Methods A retrospective chart review was performed for patients undergoing pituitary adenoma resection with at least 1 year of follow-up. Additionally, systematic data searches were performed and included studies that correlated recurrence rate to Ki-67/MIB-1 LI. Our institutional data were included in a synthesis with previously published data. Results Our institutional review included 79 patients with a recurrence rate of 26.6%. We found that 8.8% of our patients had a high Ki-67/MIB-1 LI (>3%); however, high Ki-67/MIB-1 was not associated with recurrence. The systematic review identified 244 articles and 49 full-text articles that were assessed for eligibility. Quantitative analysis was performed on 30 articles including our institutional data and 18 studies reported recurrence by level of Ki-67/MIB-1 LI. Among studies that compared Ki-67/MIB-1 ≥3 vs. <3%, 10 studies reported odds ratios (OR) greater than 1 of which 6 were statistically significant. A high Ki-67/MIB-1 had higher odds of recurrence via the pooled odds ratio (OR = 4.15, 95% confidence interval [CI]: 2.31–7.42). Conclusion This systematic review suggests that a high Ki-67/MIB-1 should prompt an increased duration of follow-up due to the higher odds of recurrence of pituitary adenoma.

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VIP-HL: Semi-automated ACMG/AMP variant interpretation platform for genetic hearing loss

10.22541/au.160682407.73344037/v1 ◽

2020 ◽

Author(s):

Jiguang Peng ◽

Jiale Xiang ◽

Xiangqian Jin ◽

Junhua Meng ◽

Nana Song ◽

...

Keyword(s):

Hearing Loss ◽

Expert Panel ◽

Sequence Variant ◽

Variant Interpretation ◽

Web Interface ◽

Online Tool ◽

Genetic Hearing Loss ◽

Genetics And Genomics ◽

Evidence Based Guidelines ◽

User Friendly

The American College of Medical Genetics and Genomics, and the Association for Molecular Pathology (ACMG/AMP) have proposed a set of evidence-based guidelines to support sequence variant interpretation. The ClinGen hearing loss expert panel (HL-EP) introduced further specifications into the ACMG/AMP framework for genetic hearing loss. This study developed a tool named VIP-HL, aiming to semi-automate the HL ACMG/AMP rules. VIP-HL aggregates information from external databases to automate 13 out of 24 ACMG/AMP rules specified by HL-EP, namely PVS1, PS1, PM1, PM2, PM4, PM5, PP3, BA1, BS1, BS2, BP3, BP4, and BP7. We benchmarked VIP-HL using 50 variants where 83 rules were activated by the ClinGen HL-EP. VIP-HL concordantly activated 96% (80/83) rules, significantly higher than that of by InterVar (47%; 39/83). Of 4948 ClinVar star 2+ variants from 142 deafness-related genes, VIP-HL achieved an overall variant interpretation concordance in 88.0% (4353/4948). VIP-HL is an integrated online tool for reliable automated variant classification in hearing loss genes. It assists curators in variant interpretation and provides a platform for users to share classifications with each other. VIP-HL is available with a user-friendly web interface at http://hearing.genetics.bgi.com/.

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KNOX HOMOLOGS SHOOT MERISTEMLESS (STM) and KNAT6 are epistatic to CLAVATA3 (CLV3) during embryonic meristem development in Arabidopsis thaliana

10.1101/2020.11.11.378539 ◽

2020 ◽

Author(s):

Sharma Nidhi ◽

Liu Tie

Keyword(s):

Arabidopsis Thaliana ◽

Shoot Apex ◽

Double Mutant ◽

Genetic Interaction ◽

Homeobox Gene ◽

The Other ◽

Shoot Meristem ◽

Published Data ◽

Meristem Development ◽

Shoot Meristemless

AbstractIn Arabidopsis, the genes SHOOT MERISTEMLESS (STM) and CLAVATA3 (CLV3) antagonistically regulate shoot meristem development. STM is essential for both development and maintenance of the meristem, as stm mutants fail to develop a shoot meristem during embryogenesis. CLV3, on the other hand, negatively regulates meristem proliferation, and clv3 mutants possess an enlarged shoot meristem. Genetic interaction studies revealed that stm and clv3 dominantly suppress each other’s phenotypes. STM works in conjunction with its closely related homologue KNOTTED1-LIKE HOMEOBOX GENE 6 (KNAT6) to promote meristem development and organ separation, as stm knat6 double mutants fail to form a meristem and produce a fused cotyledon. In this study, we show that clv3 fails to promote post-embryonic meristem formation in stm-1 background if we also remove KNAT6. stm-1 knat6 clv3 triple mutants result in early meristem termination and produce fused cotyledons similar to stm knat6 double mutant. Notably, the stm-1 knat6 and stm-1 knat6 clv3 alleles lack tissue in the presumed region of SAM. stm knat6 clv3 also showed reduced inflorescence size and shoot apex size as compared to clv3 single or stm clv3 double mutants. In contrast to previously published data, these data suggest that stm is epistatic to clv3 in postembryonic meristem development.HighlightSTM and KNAT6 genes determine post-embryonic meristem formation and activity in Arabidopsis. clv3 mutation is unable to rescue the stm knat6 meristemless phenotype.

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SAMHD1 Regulates Human Papillomavirus 16-Induced Cell Proliferation and Viral Replication during Differentiation of Keratinocytes

mSphere ◽

10.1128/msphere.00448-19 ◽

2019 ◽

Vol 4 (4) ◽

Cited By ~ 5

Author(s):

Claire D. James ◽

Apurva T. Prabhakar ◽

Raymonde Otoa ◽

Michael R. Evans ◽

Xu Wang ◽

...

Keyword(s):

Cell Proliferation ◽

Human Papillomavirus ◽

Life Cycle ◽

Viral Replication ◽

Cellular Proliferation ◽

Specific Interaction ◽

Viral Life Cycle ◽

Human Papillomaviruses ◽

Cellular Growth ◽

Human Papillomavirus 16

ABSTRACT Human papillomaviruses induce a host of anogenital cancers, as well as oropharyngeal cancer (HPV+OPC); human papillomavirus 16 (HPV16) is causative in around 90% of HPV+OPC cases. Using telomerase reverse transcriptase (TERT) immortalized foreskin keratinocytes (N/Tert-1), we have identified significant host gene reprogramming by HPV16 (N/Tert-1+HPV16) and demonstrated that N/Tert-1+HPV16 support late stages of the viral life cycle. Expression of the cellular dNTPase and homologous recombination factor sterile alpha motif and histidine-aspartic domain HD-containing protein 1 (SAMHD1) is transcriptionally regulated by HPV16 in N/Tert-1. CRISPR/Cas9 removal of SAMHD1 from N/Tert-1 and N/Tert-1+HPV16 demonstrates that SAMHD1 controls cell proliferation of N/Tert-1 only in the presence of HPV16; the deletion of SAMHD1 promotes hyperproliferation of N/Tert-1+HPV16 cells in organotypic raft cultures but has no effect on N/Tert-1. Viral replication is also elevated in the absence of SAMHD1. This new system has allowed us to identify a specific interaction between SAMHD1 and HPV16 that regulates host cell proliferation and viral replication; such studies are problematic in nonimmortalized primary keratinocytes due to their limited life span. To confirm the relevance of our results, we repeated the analysis with human tonsil keratinocytes (HTK) immortalized by HPV16 (HTK+HPV16) and observed the same hyperproliferative phenotype following CRISPR/Cas9 editing of SAMHD1. Identical results were obtained with three independent CRISPR/Cas9 guide RNAs. The isogenic pairing of N/Tert-1 with N/Tert-1+HPV16, combined with HTK+HPV16, presents a unique system to identify host genes whose products functionally interact with HPV16 to regulate host cellular growth in keratinocytes. IMPORTANCE HPVs are causative agents in human cancers and are responsible for around of 5% of all cancers. A better understanding of the viral life cycle in keratinocytes will facilitate the development of novel therapeutics to combat HPV-positive cancers. Here, we present a unique keratinocyte model to identify host proteins that specifically interact with HPV16. Using this system, we report that a cellular gene, SAMHD1, is regulated by HPV16 at the RNA and protein levels in keratinocytes. Elimination of SAMHD1 from these cells using CRISPR/Cas9 editing promotes enhanced cellular proliferation by HPV16 in keratinocytes and elevated viral replication but not in keratinocytes that do not have HPV16. Our study demonstrates a specific intricate interplay between HPV16 and SAMHD1 during the viral life cycle and establishes a unique model system to assist exploring host factors critical for HPV pathogenesis.

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Effect of Combined Leukocyte-Poor Platelet-Rich Plasma and Hyaluronic Acid on Bone Marrow–Derived Mesenchymal Stem Cell and Chondrocyte Metabolism

Cartilage ◽

10.1177/1947603519858739 ◽

2019 ◽

pp. 194760351985873 ◽

Cited By ~ 2

Author(s):

Alexander M. Satin ◽

Jolanta B. Norelli ◽

Nicholas A. Sgaglione ◽

Daniel A. Grande

Keyword(s):

Bone Marrow ◽

Hyaluronic Acid ◽

Cellular Proliferation ◽

Culture Media ◽

Platelet Rich Plasma ◽

Interleukin 1 ◽

Cellular Growth ◽

Sprague Dawley ◽

Vitro Effect

ObjectiveGiven the potential applications of combined biologics, the authors sought to evaluate the in vitro effect of combined platelet-rich plasma (PRP) and hyaluronic acid (HA) on cellular metabolism.DesignBone marrow–derived mesenchymal stem cells (BMSCs) and chondrocytes were obtained from the femurs of Sprague-Dawley rats. An inflammatory model was created by adding 10 ng/mL interleukin-1-beta to culture media. Non-crosslinked high-molecular-weight HA, activated-PRP (aPRP), and unactivated-PRP (uPRP) were tested. Cellular proliferation and gene expression were measured at 1 week. Genes of interest included aggrecan, matrix metalloproteinase (MMP)-9, and MMP-13.ResultsCombined uPRP-HA was associated with a significant increase in chondrocyte and BMSC proliferation at numerous preparations. There was a trend of increased chondrocyte aggrecan expression with combined PRP-HA. The greatest and only significant decrease in BMSC MMP-9 expression was observed with combined PRP-HA. While a significant reduction of BMSC MMP-13 expression was seen with PRP and HA-alone, a greater reduction was observed with PRP-HA. MMP-9 chondrocyte expression was significantly reduced in cells treated with PRP-HA. PRP-alone and HA-alone at identical concentrations did not result in a significant reduction. The greatest reduction of MMP-13 chondrocyte expression was observed in chondrocytes plus combined PRP-HA.ConclusionsWe demonstrated a statistically significant increase in BMSC and chondrocyte proliferation and decreased expression of catabolic enzymes with combined PRP-HA. These results demonstrate the additive in vitro effect of combined PRP-HA to stimulate cellular growth, restore components of the articular extracellular matrix, and reduce inflammation.

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c-Kit-Mediated Overlapping and Unique Functional and Biochemical Outcomes via Diverse Signaling Pathways

Molecular and Cellular Biology ◽

10.1128/mcb.24.3.1401-1410.2004 ◽

2004 ◽

Vol 24 (3) ◽

pp. 1401-1410 ◽

Cited By ~ 38

Author(s):

Li Hong ◽

Veerendra Munugalavadla ◽

Reuben Kapur

Keyword(s):

Signaling Pathways ◽

Binding Sites ◽

Cellular Proliferation ◽

Critical Issue ◽

Cellular Growth ◽

Mitogen Activated Protein Kinase ◽

Downstream Signaling ◽

Kit Receptor ◽

And Migration ◽

The Individual

ABSTRACT A critical issue in understanding receptor tyrosine kinase signaling is the individual contribution of diverse signaling pathways in regulating cellular growth, survival, and migration. We generated a functionally and biochemically inert c-Kit receptor that lacked the binding sites for seven early signaling pathways. Restoring the Src family kinase (SFK) binding sites in the mutated c-Kit receptor restored cellular survival and migration but only partially rescued proliferation and was associated with the rescue of the Ras/mitogen-activated protein kinase, Rac/JNK kinase, and phosphatidylinositol 3-kinase (PI-3 kinase)/Akt pathways. In contrast, restoring the PI-3 kinase binding site in the mutated receptor did not affect cellular proliferation but resulted in a modest correction in cell survival and migration, despite a complete rescue in the activation of the PI-3 kinase/Akt pathway. Surprisingly, restoring the binding sites for Grb2, Grb7, or phospholipase C-γ had no effect on cellular growth or survival, migration, or activation of any of the downstream signaling pathways. These results argue that SFKs play a unique role in the control of multiple cellular functions and in the activation of distinct biochemical pathways via c-Kit.

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The Evolution of Constitutional Sequence Variant Interpretation

Advances in Molecular Pathology ◽

10.1016/j.yamp.2019.06.001 ◽

2019 ◽

Vol 2 (1) ◽

pp. 1-11

Author(s):

Jessica Mester ◽

Tina Pesaran

Keyword(s):

Sequence Variant ◽

Variant Interpretation

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VACM-1, a cul-5 gene, inhibits cellular growth by a mechanism that involves MAPK and p53 signaling pathways

AJP Cell Physiology ◽

10.1152/ajpcell.00338.2002 ◽

2003 ◽

Vol 285 (6) ◽

pp. C1386-C1396 ◽

Cited By ~ 24

Author(s):

C. Van Dort ◽

P. Zhao ◽

K. Parmelee ◽

B. Capps ◽

A. Poel ◽

...

Keyword(s):

Cell Lines ◽

Cellular Proliferation ◽

Phosphorylation Site ◽

Chinese Hamster ◽

Inhibitory Effect ◽

Cellular Growth ◽

P53 Expression ◽

Mapk Phosphorylation ◽

P53 Signaling

Vasopressin-activated Ca2+-mobilizing (VACM)-1 gene product is a 780-amino acid membrane protein that shares sequence homology with cullins, a family of genes involved in the regulation of cell cycle. However, when expressed in vitro, VACM-1 attenuates basal and vasopressin- and forskolin-induced cAMP production. Mutating the PKA-dependent phosphorylation site in the VACM-1 sequence (S730AVACM-1) prevents this inhibitory effect. To further examine the biological role of VACM-1, we studied the effect of VACM-1 and S730AVACM-1 proteins on cellular proliferation and gene expression in Chinese hamster ovary and COS-1 cells. Cellular proliferation of VACM-1-expressing cell lines was significantly lower compared with that of the vector-transfected cells, whereas it was significantly increased in S730AVACM-1-derived cell lines. Furthermore, expression of VACM-1 but not S730AVACM-1 protein retarded cytokinesis and prevented MAPK phosphorylation. Screening with the Human PathwayFinder-1 GEArray system and subsequent Western blot analysis demonstrated that VACM-1 induces p53 mRNA and protein expression. In summary, VACM-1 inhibits cellular growth by a mechanism that involves cAMP, MAPK phosphorylation, and p53 expression.

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Genetic interactions and functional analyses of the fission yeast gsk3 and amk2 single and double mutants defective in TORC1-dependent processes

Scientific Reports ◽

10.1038/srep44257 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 6

Author(s):

Charalampos Rallis ◽

StJohn Townsend ◽

Jürg Bähler

Keyword(s):

Fission Yeast ◽

Double Mutant ◽

Genetic Interaction ◽

Genetic Network ◽

Cellular Growth ◽

Regulatory Subunit ◽

Triple Mutant ◽

Network Information ◽

Damage Repair ◽

Dependent Processes

Abstract The Target of Rapamycin (TOR) signalling network plays important roles in aging and disease. The AMP-activated protein kinase (AMPK) and the Gsk3 kinase inhibit TOR during stress. We performed genetic interaction screens using synthetic genetic arrays (SGA) with gsk3 and amk2 as query mutants, the latter encoding the regulatory subunit of AMPK. We identified 69 negative and 82 positive common genetic interactors, with functions related to cellular growth and stress. The 120 gsk3-specific negative interactors included genes functioning in translation and ribosomes. The 215 amk2-specific negative interactors included genes functioning in chromatin silencing and DNA damage repair. Both amk2- and gsk3-specific interactors were enriched in phenotype categories related to abnormal cell size and shape. We also performed SGA screen with the amk2 gsk3 double mutant as a query. Mutants sensitive to 5-fluorouracil, an anticancer drug are under-represented within the 305 positive interactors specific for the amk2 gsk3 query. The triple-mutant SGA screen showed higher number of negative interactions than the double mutant SGA screens and uncovered additional genetic network information. These results reveal common and specialized roles of AMPK and Gsk3 in mediating TOR-dependent processes, indicating that AMPK and Gsk3 act in parallel to inhibit TOR function in fission yeast.

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