Re-evaluating the driving force behind mutations.

Experiments on tropical trees suggest that new mutations in plants are driven by age rather than number of cell divisions during growth.

Molecular Dynamics-Derived Descriptor Informs the Impact of Mutation on the Catalytic Turnover Number in Lactonase Across Substrates.

Molecular dynamics simulations have been extensively employed to reveal the roles of protein dynamics in mediating enzyme catalysis. However, simulation-derived predictive descriptors that inform the impacts of mutations on catalytic turnover numbers remain largely unexplored. In this work, we report the identification of molecular modeling-derived descriptors to predict mutation effect on the turnover number of lactonase SsoPox with both native and non-native substrates. The study consists of 10 enzyme-substrate complexes resulting from a combination of five enzyme variants with two substrates. For each complex, we derived 15 descriptors from molecular dynamics simulations and applied principal component analysis to rank the predictive capability of the descriptors. A top-ranked descriptor was identified, which is the solvent-accessible surface area (SASA) ratio of the substrate to the active site pocket. A uniform volcano-shaped plot was observed in the distribution of experimental activation free energy against the SASA ratio. To achieve efficient lactonase hydrolysis, a non-native substrate-bound enzyme variant needs to involve a similar range of the SASA ratio to the native substrate-bound wild-type enzyme. The descriptor reflects how well the enzyme active site pocket accommodates a substrate for reaction, which has the potential of guiding optimization of enzyme reaction turnover for non-native chemical transformations.

Predicting and interpreting large-scale mutagenesis data using analyses of protein stability and conservation.

Understanding and predicting the functional consequences of single amino acid changes is central in many areas of protein science. Here, we collect and analyze experimental measurements of effects of >150,000 variants in 29 proteins. We use biophysical calculations to predict changes in stability for each variant and assess them in light of sequence conservation. We find that the sequence analyses give more accurate prediction of variant effects than predictions of stability and that about half of the variants that show loss of function do so due to stability effects. We construct a machine learning model to predict variant effects from protein structure and sequence alignments and show how the two sources of information support one another and enable mechanistic interpretations. Together, our results show how one can leverage large-scale experimental assessments of variant effects to gain deeper and general insights into the mechanisms that cause loss of function.

Fibrodysplasia Ossificans Progressiva: What Have We Achieved and Where Are We Now? Follow-up to the 2015 Lorentz Workshop.

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare progressive genetic disease effecting one in a million individuals. During their life, patients with FOP progressively develop bone in the soft tissues resulting in increasing immobility and early death. A mutation in the ACVR1 gene was identified as the causative mutation of FOP in 2006. After this, the pathophysiology of FOP has been further elucidated through the efforts of research groups worldwide. In 2015, a workshop was held to gather these groups and discuss the new challenges in FOP research. Here we present an overview and update on these topics.

GH Resistance Is a Component of Idiopathic Short Stature: Implications for rhGH Therapy.

Idiopathic short stature (ISS) is a term used to describe a selection of short children for whom no precise aetiology has been identified. Molecular investigations have made notable discoveries in children with ISS, thus removing them from this category. However, many, if not the majority of children referred with short stature, are designated ISS. Our interest in defects of GH action, i.e. GH resistance, has led to a study of children with mild GH resistance, who we believe can be mis-categorised as ISS leading to potential inappropriate management. Approval of ISS by the FDA for hGH therapy has resulted in many short children receiving this treatment. The results are extremely variable. It is therefore important to correctly assess and investigate all ISS subjects in order to identify those with mild but unequivocal GH resistance, as in cases of PAPP-A2 deficiency. The correct identification of GH resistance defects will direct therapy towards rhIGF-I rather than rhGH. This example illustrates the importance of recognition of GH resistance among the very large number patients referred with short stature who are labelled as 'ISS'.

Germline Testing Data Validate Inferences of Mutational Status for Variants Detected From Tumor-Only Sequencing.

Pathogenic germline variants (PGVs) in cancer susceptibility genes are usually identified through germline testing of DNA from blood or saliva: their detection can affect treatment options and potential risk-reduction strategies for patient relatives. PGV can also be identified in tumor sequencing assays, which, when performed without patient-matched normal specimens, render determination of variants' germline or somatic origin critical. METHODS: Tumor-only sequencing data from 1,608 patients were retrospectively analyzed to infer germline versus somatic status of variants using an information-theoretic, gene-independent approach. Loss of heterozygosity was also determined. Predicted mutational models were compared with clinical germline testing results. Statistical measures were computed to evaluate performance. RESULTS: Tumor-only sequencing detected 3,988 variants across 70 cancer susceptibility genes for which germline testing data were available. We imputed germline versus somatic status for > 75% of all detected variants, with a sensitivity of 65%, specificity of 88%, and overall accuracy of 86% for pathogenic variants. False omission rate was 3%, signifying minimal error in misclassifying true PGV. A higher portion of PGV in known hereditary tumor suppressors were found to be retained with loss of heterozygosity in the tumor specimens (72%) compared with variants of uncertain significance (58%). CONCLUSION: Analyzing tumor-only data in the context of specimens' tumor cell content allows precise, systematic exclusion of somatic variants and suggests a balance between type 1 and 2 errors for identification of patients with candidate PGV for standard germline testing. Although technical or systematic errors in measuring variant allele frequency could result in incorrect inference, misestimation of specimen purity could result in inferring somatic variants as germline in somatically mutated tumor suppressor genes. A user-friendly bioinformatics application facilitates objective analysis of tumor-only data in clinical settings.

Gaining Insight into Mitochondrial Genetic Variation and Downstream Pathophysiology: What Can i(PSCs) Do?

Mitochondria are specialized organelles involved in energy production that have retained their own genome throughout evolutionary history. The mitochondrial genome (mtDNA) is maternally inherited and requires coordinated regulation with nuclear genes to produce functional enzyme complexes that drive energy production. Each mitochondrion contains 5-10 copies of mtDNA and consequently, each cell has several hundreds to thousands of mtDNAs. Due to the presence of multiple copies of mtDNA in a mitochondrion, mtDNAs with different variants may co-exist, a condition called heteroplasmy. Heteroplasmic variants can be clonally expanded, even in post-mitotic cells, as replication of mtDNA is not tied to the cell-division cycle. Heteroplasmic variants can also segregate during germ cell formation, underlying the inheritance of some mitochondrial mutations. Moreover, the uneven segregation of heteroplasmic variants is thought to underlie the heterogeneity of mitochondrial variation across adult tissues and resultant differences in the clinical presentation of mitochondrial disease. Until recently, however, the mechanisms mediating the relation between mitochondrial genetic variation and disease remained a mystery, largely due to difficulties in modeling human mitochondrial genetic variation and diseases. The advent of induced pluripotent stem cells (iPSCs) and targeted gene editing of the nuclear, and more recently mitochondrial, genomes now provides the ability to dissect how genetic variation in mitochondrial genes alter cellular function across a variety of human tissue types. This review will examine the origins of mitochondrial heteroplasmic variation and propagation, and the tools used to model mitochondrial genetic diseases. Additionally, we discuss how iPSC technologies represent an opportunity to advance our understanding of human mitochondrial genetics in disease.

Beyond Ca2+ signalling: the role of TRPV3 in the transport of NH4.

Mutations of TRPV3 lead to severe dermal hyperkeratosis in Olmsted syndrome, but whether the mutants are trafficked to the cell membrane or not is controversial. Even less is known about TRPV3 function in intestinal epithelia, although research on ruminants and pigs suggests an involvement in the uptake of NH4+. It was the purpose of this study to measure the permeability of the human homologue (hTRPV3) to NH4+, to localize hTRPV3 in human skin equivalents, and to investigate trafficking of the Olmsted mutant G573S. Immunoblotting and immunostaining verified the successful expression of hTRPV3 in HEK-293 cells and Xenopus oocytes with trafficking to the cell membrane. Human skin equivalents showed distinct staining of the apical membrane of the top layer of keratinocytes with cytosolic staining in the middle layers. Experiments with pH-sensitive microelectrodes on Xenopus oocytes demonstrated that acidification by NH4+ was significantly greater when hTRPV3 was expressed. Single-channel measurements showed larger conductances in overexpressing Xenopus oocytes than in controls. In whole-cell experiments on HEK-293 cells, both enantiomers of menthol stimulated influx of NH4+ in hTRPV3 expressing cells, but not in controls. Expression of the mutant G573S greatly reduced cell viability with partial rescue via ruthenium red. Immunofluorescence confirmed cytosolic expression, with membrane staining observed in a very small number of cells. We suggest that expression of TRPV3 by epithelia may have implications not just for Ca2+ signalling, but also for nitrogen metabolism. Models suggesting how influx of NH4+ via TRPV3 might stimulate skin cornification or intestinal NH4+ transport are discussed.

Insights into agonist-elicited activation of the human glucose-dependent insulinotropic polypeptide receptor.

Glucose-dependent insulinotropic polypeptide (GIP) and its receptor (GIPR) are part of the incretin system that regulates glucose homeostasis. A series of GIPR residues putatively important for ligand binding and receptor activation were mutated and pharmacologically evaluated using GIPR selective agonists in cAMP accumulation, ERK1/2 phosphorylation (pERK1/2) and ß-arrestin 2 recruitment assays. The impact of mutation on ligand efficacy was determined by operational modelling of experimental data for each mutant, with results mapped onto the full-length, active-state GIPR structure. Two interaction networks, comprising transmembrane helix (TM) 7, TM1 and TM2, and extracellular loop (ECL) 2, TM5 and ECL3 were revealed, respectively. Both networks were critical for Gαs-mediated cAMP accumulation and the recruitment of ß-arrestin 2, however, cAMP response was more sensitive to alanine substitution, with most mutated residues displaying reduced signaling. Unlike the other two assays, activation of ERK1/2 was largely independent of the network involving ECL2, TM5 and ECL3, indicating that pERK1/2 is at least partially distinct from Gαs or ß-arrestin pathways and this network is also crucial for potential biased agonism at GIPR. Collectively, our work advances understanding of the structure-function relationship of GIPR and provides a framework for the design and/or interpretation of GIP analogues with unique signaling profiles.

Dopaminergic Dysregulation in Syndromic Autism Spectrum Disorders: Insights From Genetic Mouse Models.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder defined by altered social interaction and communication, and repetitive, restricted, inflexible behaviors. Approximately 1.5-2% of the general population meet the diagnostic criteria for ASD and several brain regions including the cortex, amygdala, cerebellum and basal ganglia have been implicated in ASD pathophysiology. The midbrain dopamine system is an important modulator of cellular and synaptic function in multiple ASD-implicated brain regions via anatomically and functionally distinct dopaminergic projections. The dopamine hypothesis of ASD postulates that dysregulation of dopaminergic projection pathways could contribute to the behavioral manifestations of ASD, including altered reward value of social stimuli, changes in sensorimotor processing, and motor stereotypies. In this review, we examine the support for the idea that cell-autonomous changes in dopaminergic function are a core component of ASD pathophysiology. We discuss the human literature supporting the involvement of altered dopamine signaling in ASD including genetic, brain imaging and pharmacologic studies. We then focus on genetic mouse models of syndromic neurodevelopmental disorders in which single gene mutations lead to increased risk for ASD. We highlight studies that have directly examined dopamine neuron number, morphology, physiology, or output in these models. Overall, we find considerable support for the idea that the dopamine system may be dysregulated in syndromic ASDs; however, there does not appear to be a consistent signature and some models show increased dopaminergic function, while others have deficient dopamine signaling. We conclude that dopamine dysregulation is common in syndromic forms of ASD but that the specific changes may be unique to each genetic disorder and may not account for the full spectrum of ASD-related manifestations.

Cav1.4 dysfunction and congenital stationary night blindness type 2.

Cav1.4 L-type Ca2+ channels are predominantly expressed in retinal neurons, particularly at the photoreceptor terminals where they mediate sustained Ca2+ entry needed for continuous neurotransmitter release at their ribbon synapses. Cav1.4 channel gating properties are controlled by accessory subunits, associated regulatory proteins, and also alternative splicing. In humans, mutations in the CACNA1F gene encoding for Cav1.4 channels are associated with X-linked retinal disorders such as congenital stationary night blindness type 2. Mutations in the Cav1.4 protein result in a spectrum of altered functional channel activity. Several mouse models broadened our understanding of the role of Cav1.4 channels not only as Ca2+ source at retinal synapses but also as synaptic organizers. In this review, we highlight different structural and functional phenotypes of Cav1.4 mutations that might also occur in patients with congenital stationary night blindness type 2. A further important yet mostly neglected aspect that we discuss is the influence of alternative splicing on channel dysfunction. We conclude that currently available functional phenotyping strategies should be refined and summarize potential specific therapeutic options for patients carrying Cav1.4 mutations. Importantly, the development of new therapeutic approaches will permit a deeper understanding of not only the disease pathophysiology but also the physiological function of Cav1.4 channels in the retina.

Accumulation of Endogenous Mutant Huntingtin in Astrocytes Exacerbates Neuropathology of Huntington Disease in Mice.

Selective neuronal accumulation of misfolded proteins is a key step toward neurodegeneration in a wide range of neurodegenerative diseases, including Huntington's (HD) diseases. Our recent studies suggest that Hsp70-binding protein 1 (HspBP1), an Hsp70/CHIP inhibitor that reduces protein folding, is highly expressed in neuronal cells and accounts for the accumulation of the HD protein huntingtin (HTT) in neuronal cells. To further determine the role of HspBP1 in regulation of mutant protein accumulation, we investigated whether increasing expression of HspBP1 in glial cells can also induce the accumulation of endogenous mutant HTT in glial cells and yield non-cell-autonomous toxic effects. We performed stereotaxic injection of AAV to selectively express HspBP1 in astrocytes in the brains of HD140Q knock-in (KI) mice that express mutant HTT ubiquitously but do not display obvious neurodegeneration. However, HspBP1 expression in HD140Q astrocytes led to the increased accumulation of endogenous mutant HTT and robust neuronal loss in the striatum of HD140Q KI mice. In transgenic HD mice that selectively express mutant HTT in astrocytes, increased accumulation of mutant HTT in astrocytes via HspBP1 expression did not elicit neurodegeneration but could exacerbate neurological symptoms. Consistently, suppressing the expression of endogenous HspBp1 in the striatum of HD140Q KI mice via CRISPR/Cas9 led to a significant reduction of mutant HTT accumulation. Our findings suggest that although endogenous mutant HTT in astrocytes can exacerbate neurological symptoms, it mediates neurodegeneration only when mutant HTT is also accumulated in neuronal cells.

Lewy Body-like Inclusions in Human Midbrain Organoids Carrying Glucocerebrosidase and α-Synuclein Mutations.

OBJECTIVE: We utilized human midbrain-like organoids (hMLOs) generated from human pluripotent stem cells carrying glucocerebrosidase gene (GBA1) and α-synuclein (α-syn; SNCA) perturbations to investigate genotype-to-phenotype relationships in Parkinson disease, with the particular aim of recapitulating α-syn- and Lewy body-related pathologies and the process of neurodegeneration in the hMLO model. METHODS: We generated and characterized hMLOs from GBA1-/- and SNCA overexpressing isogenic embryonic stem cells and also generated Lewy body-like inclusions in GBA1/SNCA dual perturbation hMLOs and conduritol-b-epoxide-treated SNCA triplication hMLOs. RESULTS: We identified for the first time that the loss of glucocerebrosidase, coupled with wild-type α-syn overexpression, results in a substantial accumulation of detergent-resistant, ß-sheet-rich α-syn aggregates and Lewy body-like inclusions in hMLOs. These Lewy body-like inclusions exhibit a spherically symmetric morphology with an eosinophilic core, containing α-syn with ubiquitin, and can also be formed in Parkinson disease patient-derived hMLOs. We also demonstrate that impaired glucocerebrosidase function promotes the formation of Lewy body-like inclusions in hMLOs derived from patients carrying the SNCA triplication. INTERPRETATION: Taken together, the data indicate that our hMLOs harboring 2 major risk factors (glucocerebrosidase deficiency and wild-type α-syn overproduction) of Parkinson disease provide a tractable model to further elucidate the underlying mechanisms for progressive Lewy body formation. ANN NEUROL 2021;90:490-505.

Glycine substitution of α1F64 residue at the loop D of GABAA receptor impairs gating - Implications for importance of binding site-channel gate linker rigidity.

GABAA receptors (GABAARs) play a crucial role in mediating inhibition in adult mammalian brains. In the recent years, an impressive progress in revealing the static structure of GABAARs was achieved but the molecular mechanisms underlying their conformational transitions remain elusive. Phenylalanine 64 (α1F64) is located at the loop D of the orthosteric binding site of GABAAR and was found to directly interact with GABA molecule. Mutations of α1F64 were demonstrated to affect not only binding but also some gating properties. Loop D is a rigid ß strand which seems to be particularly suitable to convey activatory signaling from the ligand binding site (LBS) to the gate at the channel pore. To test this scenario, we have investigated the substitution of α1F64 with glycine, the smallest amino acid, widely recognized as a rigidity "reducer" of protein structures. To this end, we assessed the impact of the α1F64G mutation in the α1ß2γ2L type of GABAARs on gating properties by analyzing both macroscopic responses to rapid agonist applications and single-channel currents. We found that this substitution dramatically altered all gating features of the receptor (opening/closing, preactivation and desensitization) which contrasts with markedly weaker effects of previously considered substitutions (α1F64L and α1F64A). In particular, α1F64G mutation practically abolished the desensitization process. At the same time, the α1F64G mutant maintained gating integrity manifested as single-channel activity in the form of clusters. We conclude that rigidity of the loop D plays a crucial role in conveying the activation signal from the LBS to the channel gate.

Arc1 and the microbiota together modulate growth and metabolic traits in Drosophila.

Perturbations to animal-associated microbial communities (the microbiota) have deleterious effects on various aspects of host fitness, but the molecular processes underlying these impacts are poorly understood. Here, we identify a connection between the microbiota and the neuronal factor Arc1 that affects growth and metabolism in Drosophila. We find that Arc1 exhibits tissue-specific microbiota-dependent expression changes, and that germ-free flies bearing a null mutation of Arc1 exhibit delayed and stunted larval growth, along with a variety of molecular, cellular and organismal traits indicative of metabolic dysregulation. Remarkably, we show that the majority of these phenotypes can be fully suppressed by mono-association with a single Acetobacter sp. isolate, through mechanisms involving both bacterial diet modification and live bacteria. Additionally, we provide evidence that Arc1 function in key neuroendocrine cells of the larval brain modulates growth and metabolic homeostasis under germ-free conditions. Our results reveal a role for Arc1 in modulating physiological responses to the microbial environment, and highlight how host-microbe interactions can profoundly impact the phenotypic consequences of genetic mutations in an animal host.

Potential crosstalk between sonic hedgehog-WNT signaling and neurovascular molecules: Implications for blood-brain barrier integrity in autism spectrum disorder.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disease originating from combined genetic and environmental factors. Post-mortem human studies and some animal ASD models have shown brain neuroinflammation, oxidative stress, and changes in blood-brain barrier (BBB) integrity. However, the signaling pathways leading to these inflammatory findings and vascular alterations are currently unclear. The BBB plays a critical role in controlling brain homeostasis and immune response. Its dysfunction can result from developmental genetic abnormalities or neuroinflammatory processes. In this review, we explore the role of the Sonic Hedgehog/Wingless-related integration site (Shh/Wnt) pathways in neurodevelopment, neuroinflammation, and BBB development. The balance between Wnt-ß-catenin and Shh pathways controls angiogenesis, barriergenesis, neurodevelopment, central nervous system (CNS) morphogenesis, and neuronal guidance. These interactions are critical to maintain BBB function in the mature CNS to prevent the influx of pathogens and inflammatory cells. Genetic mutations of key components of these pathways have been identified in ASD patients and animal models, which correlate with the severity of ASD symptoms. Disruption of the Shh/Wnt crosstalk may therefore compromise BBB development and function. In turn, impaired Shh signaling and glial activation may cause neuroinflammation that could disrupt the BBB. Elucidating how ASD-related mutations of Shh/Wnt signaling could cause BBB leaks and neuroinflammation will contribute to our understanding of the role of their interactions in ASD pathophysiology. These observations may provide novel targeted therapeutic strategies to prevent or alleviate ASD symptoms while preserving normal developmental processes. Cover Image for this issue: https://doi.org/10.1111/jnc.15081.

ALS-linked mutations impair UBQLN2 stress-induced biomolecular condensate assembly in cells.

Mutations in ubiquilin-2 (UBQLN2), a ubiquitin-binding shuttle protein involved in several protein quality control processes, can lead to amyotrophic lateral sclerosis (ALS). We previously found that wild-type UBQLN2 forms dynamic, membraneless biomolecular condensates upon cellular stress, and undergoes liquid-liquid phase separation in vitro. However, the impact of ALS-linked mutations on UBQLN2 condensate formation in cells remains unknown. Here, we overexpress mCherry-fused UBQLN2 with five patient-derived ALS-linked mutations and employ live-cell imaging and photokinetic analysis to investigate how each of these mutations impact stress-induced UBQLN2 condensate assembly and condensate material properties. Unlike endogenous UBQLN2, exogenously introduced UBQLN2 forms condensates distinct from stress granules. Both wild-type and mutant UBQLN2 condensates are generally cytoplasmic and liquid-like. However, mutant UBQLN2 forms fewer stress-induced UBQLN2 condensates than wild-type UBQLN2. Exogenously expressed P506T UBQLN2 forms the lowest number of stress-induced condensates of all UBQLN2 mutants, and these condensates are significantly smaller than those of wild-type UBQLN2. Fluorescence recovery after photobleaching (FRAP) analysis of UBQLN2 condensates revealed higher immobile fractions for UBQLN2 mutants, especially P506T. P497S and P497H mutations differentially impact condensate properties, demonstrating that the effects of ALS-linked mutations are both position- and amino acid-dependent. Collectively, our data show that disease mutations hinder assembly and alter viscoelastic properties of stress-induced UBQLN2 condensates, potentially leading to aggregates commonly observed in ALS.

Bioinformatics analysis and identification of genes and molecular pathways involved in Parkinson's disease in patients with mutations in the glucocerebrosidase gene.

Glucocerebrosidase (GBA) mutations occur frequently in Parkinson's disease (PD) patients. This study aims to identify potential crucial genes and pathways associated with GBA mutations in patients with PD and to further analyze new molecular mechanisms related to the occurrence of gene mutations from the perspective of bioinformatics. Gene expression profiles of datasets GSE53424 and GSE99142 were acquired from the Gene Expression Ominibus database. Differentially expressed genes (DEGs) were detected, using the 'limma' package in R, comparing IDI-PD 1 (idiopathic PD patients) and GBA-PD 1 [PD patients with heterozygous GBA mutations (GBA N370S)] group samples. The functions of top modules were assessed using the DAVID, whereas gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed. Protein-protein interaction networks were assembled with Cytoscape software and separated into subnetworks using the Molecular Complex Detection Algorithm. Data from GSE53424 and GSE99142 were also extracted to verify our findings. There were 283 DEGs identified in PD patients heterozygous for GBA mutations. Module analysis revealed that GBA mutations in PD patients were associated with significant pathways, including Calcium signaling pathway, Rap1 signaling pathway and Cytokine-cytokine receptor interaction. Hub genes of the two modules were corticotropin-releasing hormone (CRH) and Melatonin receptor 1B (MTNR1B). The expression of CRH was downregulated, whereas that of MTNR1B was upregulated in PD patients with GBA mutations. The expression of CRH and MTNR1B has diagnostic value for PD patients with heterozygous GBA mutations. Novel DEGs and pathways identified herein might provide new insights into the underlying molecular mechanisms of heterozygous GBA mutations in PD patients.

Transcriptomic heterogeneity of driver gene mutations reveals novel mutual exclusivity and improves exploration of functional associations.

BACKGROUND: Lung adenocarcinoma (LUAD), as the most common subtype of lung cancer, is the leading cause of cancer deaths in the world. The accumulation of driver gene mutations enables cancer cells to gradually acquire growth advantage. Therefore, it is important to understand the functions and interactions of driver gene mutations in cancer progression. METHODS: We obtained gene mutation data and gene expression profile of 506 LUAD tumors from The Cancer Genome Atlas (TCGA). The subtypes of tumors with driver gene mutations were identified by consensus cluster analysis. RESULTS: We found 21 significantly mutually exclusive pairs consisting of 20 genes among 506 LUAD patients. Because of the increased transcriptomic heterogeneity of mutations, we identified subtypes among tumors with non-silent mutations in driver genes. There were 494 mutually exclusive pairs found among driver gene mutations within different subtypes. Furthermore, we identified functions of mutually exclusive pairs based on the hypothesis of functional redundancy of mutual exclusivity. These mutually exclusive pairs were significantly enriched in nuclear division and humoral immune response, which played crucial roles in cancer initiation and progression. We also found 79 mutually exclusive triples among subtypes of tumors with driver gene mutations, which were key roles in cell motility and cellular chemical homeostasis. In addition, two mutually exclusive triples and one mutually exclusive triple were associated with the overall survival and disease-specific survival of LUAD patients, respectively. CONCLUSIONS: We revealed novel mutual exclusivity and generated a comprehensive functional landscape of driver gene mutations, which could offer a new perspective to understand the mechanisms of cancer development and identify potential biomarkers for LUAD therapy.