Classification of Mutations by Functional Impact Type: Gain of Function, Loss of Function, and Switch of Function

AbstractWhile advancements in genome sequencing have identified millions of somatic mutations in cancer, their functional impact is poorly understood. We previously developed the expression-based variant impact phenotyping (eVIP) method to use gene expression data to characterize the function of gene variants. The eVIP method uses a decision tree-based algorithm to predict the functional impact of somatic variants by comparing gene expression signatures induced by introduction of wild-type versus mutant cDNAs in cell lines. The method distinguishes between variants that are gain-of-function, loss-of-function, change-of-function, or neutral. We present eVIP2, software that allows for pathway analysis (eVIP Pathways) and usage with RNA-seq data. To demonstrate the eVIP2 software and approach, we characterized two recurrent frameshift variants in RNF43, a negative regulator of Wnt signaling, frequently mutated in colorectal, gastric and endometrial cancer. RNF43 WT, RNF43 R117fs, RNF43 G659fs, or GFP control cDNA were overexpressed in HEK293T cells. Analysis with eVIP2 predicted that the frameshift at position 117 was a loss-of-function mutation, as expected. The second frameshift at position 659, was, surprisingly, predicted to be a gain-of-function mutation. Additional eVIP Pathways analysis of RNF43 G659fs predicted 10 pathways to be significantly altered, including TNF alpha via NFKB signaling, KRAS signaling, and hypoxia. To validate these predictions, we performed reporter assays and found that all eVIP2 impactful pathways tested in the assay were activated by expression of RNF43 G659fs, but not by expression of RNF43 WT, supporting that RNF43 G659fs is a gain-of-function mutation and its effect on the identified pathways. The eVIP2 method is an important step towards overcoming the current challenge of variant interpretation in the implementation of precision medicine. eVIP2 is available at https://github.com/BrooksLabUCSC/eVIP2.

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Massively parallel phenotyping of variant impact in cancer with Perturb-seq reveals a shift in the spectrum of cell states induced by somatic mutations

10.1101/2020.11.16.383307 ◽

2020 ◽

Author(s):

Oana Ursu ◽

James T. Neal ◽

Emily Shea ◽

Pratiksha I. Thakore ◽

Livnat Jerby-Arnon ◽

...

Keyword(s):

Single Cells ◽

Dominant Negative ◽

Loss Of Function ◽

Functional Categories ◽

Cell Level ◽

Gain Of Function ◽

Function Loss ◽

Sequencing Studies ◽

Experimental Approaches ◽

The Impact

AbstractGenome sequencing studies have identified millions of somatic variants in cancer, but their phenotypic impact remains challenging to predict. Current experimental approaches to distinguish between functionally impactful and neutral variants require customized phenotypic assays that often report on average effects, and are not easily scaled. Here, we develop a generalizable, high-dimensional, and scalable approach to functionally assess variant impact in single cells by pooled Perturb-seq. Specifically, we assessed the impact of 200 TP53 and KRAS variants in >300,000 single lung cancer cells, and used the profiles to categorize variants into phenotypic subsets to distinguish gain-of-function, loss-of-function and dominant negative variants, which we validated by comparison to orthogonal assays. Surprisingly, KRAS variants did not merely fit into discrete functional categories, but rather spanned a continuum of gain-of-function phenotypes driven by quantitative shifts in cell composition at the single cell level. We further discovered novel gain-of-function KRAS variants whose impact could not have been predicted solely by their occurrence in patient samples. Our work provides a scalable, gene-agnostic method for coding variant impact phenotyping, which can be applied in cancer and other diseases driven by somatic or germline coding mutations.

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Faculty Opinions recommendation of Loss-of-Function and Gain-of-Function Mutations in KCNQ5 Cause Intellectual Disability or Epileptic Encephalopathy.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727772380.793535929 ◽

2017 ◽

Author(s):

Mitsuhiro Kato

Keyword(s):

Intellectual Disability ◽

Epileptic Encephalopathy ◽

Loss Of Function ◽

Gain Of Function

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

Brain Communications ◽

10.1093/braincomms/fcaa162 ◽

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.

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Emerging Role of ODC1 in Neurodevelopmental Disorders and Brain Development

Genes ◽

10.3390/genes12040470 ◽

2021 ◽

Vol 12 (4) ◽

pp. 470

Author(s):

Jeremy W. Prokop ◽

Caleb P. Bupp ◽

Austin Frisch ◽

Stephanie M. Bilinovich ◽

Daniel B. Campbell ◽

...

Keyword(s):

Brain Development ◽

Neural Progenitor Cells ◽

Neurodevelopmental Disorder ◽

Fetal Brain ◽

Missense Variant ◽

Neural Progenitor ◽

Loss Of Function ◽

Gain Of Function ◽

Systematic Analysis ◽

Function Expression

Ornithine decarboxylase 1 (ODC1 gene) has been linked through gain-of-function variants to a rare disease featuring developmental delay, alopecia, macrocephaly, and structural brain anomalies. ODC1 has been linked to additional diseases like cancer, with growing evidence for neurological contributions to schizophrenia, mood disorders, anxiety, epilepsy, learning, and suicidal behavior. The evidence of ODC1 connection to neural disorders highlights the need for a systematic analysis of ODC1 genotype-to-phenotype associations. An analysis of variants from ClinVar, Geno2MP, TOPMed, gnomAD, and COSMIC revealed an intellectual disability and seizure connected loss-of-function variant, ODC G84R (rs138359527, NC_000002.12:g.10444500C > T). The missense variant is found in ~1% of South Asian individuals and results in 2.5-fold decrease in enzyme function. Expression quantitative trait loci (eQTLs) reveal multiple functionally annotated, non-coding variants regulating ODC1 that associate with psychiatric/neurological phenotypes. Further dissection of RNA-Seq during fetal brain development and within cerebral organoids showed an association of ODC1 expression with cell proliferation of neural progenitor cells, suggesting gain-of-function variants with neural over-proliferation and loss-of-function variants with neural depletion. The linkage from the expression data of ODC1 in early neural progenitor proliferation to phenotypes of neurodevelopmental delay and to the connection of polyamine metabolites in brain function establish ODC1 as a bona fide neurodevelopmental disorder gene.

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Identification and Characterization of Genes That Interact With lin-12 in Caenorhabditis elegans

Genetics ◽

10.1093/genetics/147.4.1675 ◽

1997 ◽

Vol 147 (4) ◽

pp. 1675-1695 ◽

Cited By ~ 2

Author(s):

Frans E Tax ◽

James H Thomas ◽

Edwin L Ferguson ◽

H Robert Horvitzt

Keyword(s):

Cell Fate ◽

Low Frequency ◽

Signal Transduction Pathway ◽

Temperature Shift ◽

Egg Laying ◽

Null Alleles ◽

Temperature Sensitive ◽

Loss Of Function ◽

Gain Of Function ◽

Suppressor Mutations

Abstract We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-l7, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup1 7 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup1 7and lag-2, suggest that both genes act at approximately the same time as lin-12in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1.

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Rare Gain-of-Function KCND3 Variant Associated with Cerebellar Ataxia, Parkinsonism, Cognitive Dysfunction, and Brain Iron Accumulation

International Journal of Molecular Sciences ◽

10.3390/ijms22158247 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8247

Author(s):

Cheng-Tsung Hsiao ◽

Thomas F. Tropea ◽

Ssu-Ju Fu ◽

Tanya M. Bardakjian ◽

Pedro Gonzalez-Alegre ◽

...

Keyword(s):

Cerebellar Ataxia ◽

Iron Accumulation ◽

Molecular Spectra ◽

Loss Of Function ◽

Brain Iron ◽

Gain Of Function ◽

Progressive Cerebellar Ataxia ◽

Voltage Dependent ◽

Window Current

Loss-of-function mutations in the KV4.3 channel-encoding KCND3 gene are linked to neurodegenerative cerebellar ataxia. Patients suffering from neurodegeneration associated with iron deposition may also present with cerebellar ataxia. The mechanism underlying brain iron accumulation remains unclear. Here, we aim to ascertain the potential pathogenic role of KCND3 variant in iron accumulation-related cerebellar ataxia. We presented a patient with slowly progressive cerebellar ataxia, parkinsonism, cognitive impairment, and iron accumulation in the basal ganglia and the cerebellum. Whole exome sequencing analyses identified in the patient a heterozygous KCND3 c.1256G>A (p.R419H) variant predicted to be disease-causing by multiple bioinformatic analyses. In vitro biochemical and immunofluorescence examinations revealed that, compared to the human KV4.3 wild-type channel, the p.R419H variant exhibited normal protein abundance and subcellular localization pattern. Electrophysiological investigation, however, demonstrated that the KV4.3 p.R419H variant was associated with a dominant increase in potassium current amplitudes, as well as notable changes in voltage-dependent gating properties leading to enhanced potassium window current. These observations indicate that, in direct contrast with the loss-of-function KCND3 mutations previously reported in cerebellar ataxia patients, we identified a rare gain-of-function KCND3 variant that may expand the clinical and molecular spectra of neurodegenerative cerebellar disorders associated with brain iron accumulation.

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Implementation and clinical outcomes of pharmacogenetic CYP2C19 testing for clopidogrel therapy in real-world clinical practice.

European Journal of Preventive Cardiology ◽

10.1093/eurjpc/zwab061.067 ◽

2021 ◽

Vol 28 (Supplement_1) ◽

Author(s):

POD O"drisceoil ◽

TK Kiernan ◽

SA Arnous

Keyword(s):

Real World ◽

Point Of Care ◽

Coronary Intervention ◽

Buccal Swab ◽

Loss Of Function ◽

Gain Of Function ◽

Funding Sources ◽

Patients At Risk ◽

Clopidogrel Therapy ◽

Stable Cad

Abstract Funding Acknowledgements Type of funding sources: None. BACKGROUNDCYP2C19 loss-of-function (LOF) polymorphisms are associated with adverse ischaemic events after PCI. The use of a point-of-care assay (POC) to routinely genotype patients immediately post PCI could rapidly identify patients at risk of adverse cardiac outcomes. PURPOSE To investigate the incidence of CYP2C19 polymorphisms (*2, *17) and 30-day MACE in patients presenting to catheter laboratory for PCI (See table 1).METHODS We performed a single centre prospective analysis of patients presenting to a cardiac catheterisation laboratory for percutaneous coronary intervention. Participants underwent prospective rapid point-of-care genotyping of CYP2C19 major alleles (2*,17*), using the SpartanRx PCR device via buccal swab sample. All patients provided written consent. RESULTS:A total of 120 tests were performed, 51 patients were normal allele carriers (*1), 31 patients were carriers of LOF alleles (*2) and 38 patients were carriers of gain of function alleles (*17). All tests results returned in one hour. Rate of dyslipidaemia was significantly different between three groups (55% vs. 63% vs. 36%; p = 0.050). A numerically higher proportion of LOF allele carriers received clopidogrel prior to undergoing pharmacogenetic testing but this was not statistically significant (52% vs. 35% vs. 34%; p = 0.09). Two cases of MACE at 30 day follow up occurred in the loss-of-function group. Both cases received clopidogrel.CONCLUSIONSWe have demonstrated that a rapid POC of CYP2C19 testing can take place in a real-world setting. Our incidence rate of LOF carriers is concordant with international published literature. We found 52% of LOF carriers were commenced on clopidogrel therapy prior to genetic analysis. Comparison of CPY2C19 Metabolisers genotype Loss of function normal Gain of function p values baseline characteristics age in years, median (range) 65 (43-82) 64 (43-85) 65 (42-89) 0.717 Male, N (%) 21 (68%) 43 (64%) 27 (71%) 0.198 Hypertensive, N (%) 16 (52%) 29 (57%) 24 (50%) 0.623 Dyslipidaemia. N (%) 17 (55%) 32 (63%) 14 (36%) 0.050 Indication, N (%) St-Elevation MI 12 (39%) 18 (35%) 11 (29%) 0.558 NSTEMI 5 (16%) 15 (29%) 14 (37%) 0.142 Unstable Angina 5 (16%) 7 (14%) 3 (8%) 0.518 Stable CAD 9 (29%) 11 (22%) 10 (26%) 0.731 Antiplatelet, N (%) Ticagrelor 15 (48%) 33 (65%) 25 (66%) 0.09 Clopidogrel 16 (52%) 18 (35%) 13 (34%) Complication, N (%) 30-day MACE 2 (6.5%) 0 0 0.01

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Genetic and Molecular Characterization of the Caenorhabditis elegans Gene, mel-26, a Postmeiotic Negative Regulator of MEI-1, a Meiotic-Specific Spindle Component

Genetics ◽

10.1093/genetics/150.1.119 ◽

1998 ◽

Vol 150 (1) ◽

pp. 119-128

Author(s):

M Rhys Dow ◽

Paul E Mains

Keyword(s):

Caenorhabditis Elegans ◽

Protein Interactions ◽

Negative Regulator ◽

Meiotic Spindle ◽

Loss Of Function ◽

Protein Protein Interactions ◽

Gain Of Function ◽

Recessive Mutations ◽

Female Germline

Abstract We have previously described the gene mei-1, which encodes an essential component of the Caenorhabditis elegans meiotic spindle. When ectopically expressed after the completion of meiosis, mei-1 protein disrupts the function of the mitotic cleavage spindles. In this article, we describe the cloning and the further genetic characterization of mel-26, a postmeiotic negative regulator of mei-1. mel-26 was originally identified by a gain-of-function mutation. We have reverted this mutation to a loss-of-function allele, which has recessive phenotypes identical to the dominant defects of its gain-of-function parent. Both the dominant and recessive mutations of mel-26 result in mei-1 protein ectopically localized in mitotic spindles and centrosomes, leading to small and misoriented cleavage spindles. The loss-of-function mutation was used to clone mel-26 by transformation rescue. As suggested by genetic results indicating that mel-26 is required only maternally, mel-26 mRNA was expressed predominantly in the female germline. The gene encodes a protein that includes the BTB motif, which is thought to play a role in protein-protein interactions.

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Role of BMP receptor traffic in synaptic growth defects in an ALS model

Molecular Biology of the Cell ◽

10.1091/mbc.e16-07-0519 ◽

2016 ◽

Vol 27 (19) ◽

pp. 2898-2910 ◽

Cited By ~ 22

Author(s):

Mugdha Deshpande ◽

Zachary Feiger ◽

Amanda K. Shilton ◽

Christina C. Luo ◽

Ethan Silverman ◽

...

Keyword(s):

Neurological Impairment ◽

Bmp Signaling ◽

Loss Of Function ◽

Gain Of Function ◽

Synaptic Growth ◽

Recycling Endosome ◽

Recycling Endosomes ◽

Bmp Receptor ◽

Bmp Receptors ◽

Induced Defects

TAR DNA-binding protein 43 (TDP-43) is genetically and functionally linked to amyotrophic lateral sclerosis (ALS) and regulates transcription, splicing, and transport of thousands of RNA targets that function in diverse cellular pathways. In ALS, pathologically altered TDP-43 is believed to lead to disease by toxic gain-of-function effects on RNA metabolism, as well as by sequestering endogenous TDP-43 and causing its loss of function. However, it is unclear which of the numerous cellular processes disrupted downstream of TDP-43 dysfunction lead to neurodegeneration. Here we found that both loss and gain of function of TDP-43 in Drosophila cause a reduction of synaptic growth–promoting bone morphogenic protein (BMP) signaling at the neuromuscular junction (NMJ). Further, we observed a shift of BMP receptors from early to recycling endosomes and increased mobility of BMP receptor–containing compartments at the NMJ. Inhibition of the recycling endosome GTPase Rab11 partially rescued TDP-43–induced defects in BMP receptor dynamics and distribution and suppressed BMP signaling, synaptic growth, and larval crawling defects. Our results indicate that defects in receptor traffic lead to neuronal dysfunction downstream of TDP-43 misregulation and that rerouting receptor traffic may be a viable strategy for rescuing neurological impairment.

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