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Heritability in the immune tumor microenvironment (iTME) has been widely observed yet remains largely uncharacterized. Here, we developed a machine learning approach to map iTME modifiers within loci from genome-wide association studies (GWASs) for breast cancer (BrCa) incidence. A random forest model was trained on a positive set of immune-oncology (I-O) targets, and then used to assign I-O target probability scores to 1,362 candidate genes in linkage disequilibrium with 155 BrCa GWAS loci. Cluster analysis of the most probable candidates revealed two subfamilies of genes related to effector functions and adaptive immune responses, suggesting that iTME modifiers impact multiple aspects of anticancer immunity. Two of the top ranking BrCa candidates, LSP1 and TLR1 , were orthogonally validated as iTME modifiers using BrCa patient biopsies and comparative mapping studies, respectively. Collectively, these data demonstrate a robust and flexible framework for functionally fine-mapping GWAS risk loci to identify translatable therapeutic targets., Competing Interests: B.R.G., E.K., S.W., J.R., Z.D., and M.J.F. are employees of AbbVie. All animal studies and histological analysis of human breast cancer specimens were conducted at the Medical College of Wisconsin (MCW), at which time M.J.F. was a full-time faculty member of MCW. S.W.T., A.R.P., K.W., A.L., and H.R. are employees of MCW and have no financial relationship with AbbVie to disclose. The design, study conduct, and financial support for all other research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication., (© 2023 The Author(s).)

Background: The remarkable growth of genome-wide association studies (GWAS) has created a critical need to experimentally validate the disease-associated variants, 90% of which involve non-coding variants., Methods: To determine how the field is addressing this urgent need, we performed a comprehensive literature review identifying 36,676 articles. These were reduced to 1454 articles through a set of filters using natural language processing and ontology-based text-mining. This was followed by manual curation and cross-referencing against the GWAS catalog, yielding a final set of 286 articles., Results: We identified 309 experimentally validated non-coding GWAS variants, regulating 252 genes across 130 human disease traits. These variants covered a variety of regulatory mechanisms. Interestingly, 70% (215/309) acted through cis-regulatory elements, with the remaining through promoters (22%, 70/309) or non-coding RNAs (8%, 24/309). Several validation approaches were utilized in these studies, including gene expression (n = 272), transcription factor binding (n = 175), reporter assays (n = 171), in vivo models (n = 104), genome editing (n = 96) and chromatin interaction (n = 33)., Conclusions: This review of the literature is the first to systematically evaluate the status and the landscape of experimentation being used to validate non-coding GWAS-identified variants. Our results clearly underscore the multifaceted approach needed for experimental validation, have practical implications on variant prioritization and considerations of target gene nomination. While the field has a long way to go to validate the thousands of GWAS associations, we show that progress is being made and provide exemplars of validation studies covering a wide variety of mechanisms, target genes, and disease areas., (© 2022. The Author(s).)

Background: Deep mutational scanning (DMS) studies exploit the mutational landscape of sequence variation by systematically and comprehensively assaying the effect of single amino acid variants (SAVs; also referred to as missense mutations, or non-synonymous Single Nucleotide Variants - missense SNVs or nsSNVs) for particular proteins. We assembled SAV annotations from 22 different DMS experiments and normalized the effect scores to evaluate variant effect prediction methods. Three trained on traditional variant effect data (PolyPhen-2, SIFT, SNAP2), a regression method optimized on DMS data (Envision), and a naïve prediction using conservation information from homologs., Results: On a set of 32,981 SAVs, all methods captured some aspects of the experimental effect scores, albeit not the same. Traditional methods such as SNAP2 correlated slightly more with measurements and better classified binary states (effect or neutral). Envision appeared to better estimate the precise degree of effect. Most surprising was that the simple naïve conservation approach using PSI-BLAST in many cases outperformed other methods. All methods captured beneficial effects (gain-of-function) significantly worse than deleterious (loss-of-function). For the few proteins with multiple independent experimental measurements, experiments differed substantially, but agreed more with each other than with predictions., Conclusions: DMS provides a new powerful experimental means of understanding the dynamics of the protein sequence space. As always, promising new beginnings have to overcome challenges. While our results demonstrated that DMS will be crucial to improve variant effect prediction methods, data diversity hindered simplification and generalization.

Background: The Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function., Results: Here, we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes. Specifically, we performed experimental whole-genome mutation screening in Candida albicans and Pseudomonas aureginosa genomes, which provided us with genome-wide experimental data for genes associated with biofilm formation and motility. We further performed targeted assays on selected genes in Drosophila melanogaster, which we suspected of being involved in long-term memory., Conclusion: We conclude that while predictions of the molecular function and biological process annotations have slightly improved over time, those of the cellular component have not. Term-centric prediction of experimental annotations remains equally challenging; although the performance of the top methods is significantly better than the expectations set by baseline methods in C. albicans and D. melanogaster, it leaves considerable room and need for improvement. Finally, we report that the CAFA community now involves a broad range of participants with expertise in bioinformatics, biological experimentation, biocuration, and bio-ontologies, working together to improve functional annotation, computational function prediction, and our ability to manage big data in the era of large experimental screens.

Any two unrelated individuals differ by about 10,000 single amino acid variants (SAVs). Do these impact molecular function? Experimental answers cannot answer comprehensively, while state-of-the-art prediction methods can. We predicted the functional impacts of SAVs within human and for variants between human and other species. Several surprising results stood out. Firstly, four methods (CADD, PolyPhen-2, SIFT, and SNAP2) agreed within 10 percentage points on the percentage of rare SAVs predicted with effect. However, they differed substantially for the common SAVs: SNAP2 predicted, on average, more effect for common than for rare SAVs. Given the large ExAC data sets sampling 60,706 individuals, the differences were extremely significant (p-value < 2.2e-16). We provided evidence that SNAP2 might be closer to reality for common SAVs than the other methods, due to its different focus in development. Secondly, we predicted significantly higher fractions of SAVs with effect between healthy individuals than between species; the difference increased for more distantly related species. The same trends were maintained for subsets of only housekeeping proteins and when moving from exomes of 1,000 to 60,000 individuals. SAVs frozen at speciation might maintain protein function, while many variants within a species might bring about crucial changes, for better or worse.

Transmembrane proteins (TMPs) are important drug targets because they are essential for signaling, regulation, and transport. Despite important breakthroughs, experimental structure determination remains challenging for TMPs. Various methods have bridged the gap by predicting transmembrane helices (TMHs), but room for improvement remains. Here, we present TMSEG, a novel method identifying TMPs and accurately predicting their TMHs and their topology. The method combines machine learning with empirical filters. Testing it on a non-redundant dataset of 41 TMPs and 285 soluble proteins, and applying strict performance measures, TMSEG outperformed the state-of-the-art in our hands. TMSEG correctly distinguished helical TMPs from other proteins with a sensitivity of 98 ± 2% and a false positive rate as low as 3 ± 1%. Individual TMHs were predicted with a precision of 87 ± 3% and recall of 84 ± 3%. Furthermore, in 63 ± 6% of helical TMPs the placement of all TMHs and their inside/outside topology was correctly predicted. There are two main features that distinguish TMSEG from other methods. First, the errors in finding all helical TMPs in an organism are significantly reduced. For example, in human this leads to 200 and 1600 fewer misclassifications compared to the second and third best method available, and 4400 fewer mistakes than by a simple hydrophobicity-based method. Second, TMSEG provides an add-on improvement for any existing method to benefit from. Proteins 2016; 84:1706-1716. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Developments in experimental and computational biology are advancing our understanding of how protein sequence variation impacts molecular protein function. However, the leap from the micro level of molecular function to the macro level of the whole organism, e.g. disease, remains barred. Here, we present new results emphasizing earlier work that suggested some links from molecular function to disease. We focused on non-synonymous single nucleotide variants, also referred to as single amino acid variants (SAVs). Building upon OMIA (Online Mendelian Inheritance in Animals), we introduced a curated set of 117 disease-causing SAVs in animals. Methods optimized to capture effects upon molecular function often correctly predict human (OMIM) and animal (OMIA) Mendelian disease-causing variants. We also predicted effects of human disease-causing variants in the mouse model, i.e. we put OMIM SAVs into mouse orthologs. Overall, fewer variants were predicted with effect in the model organism than in the original organism. Our results, along with other recent studies, demonstrate that predictions of molecular effects capture some important aspects of disease. Thus, in silico methods focusing on the micro level of molecular function can help to understand the macro system level of disease., Competing Interests: The authors have declared that no competing interests exist.

Experimental structure determination continues to be challenging for membrane proteins. Computational prediction methods are therefore needed and widely used to supplement experimental data. Here, we re-examined the state of the art in transmembrane helix prediction based on a nonredundant dataset with 190 high-resolution structures. Analyzing 12 widely-used and well-known methods using a stringent performance measure, we largely confirmed the expected high level of performance. On the other hand, all methods performed worse for proteins that could not have been used for development. A few results stood out: First, all methods predicted proteins in eukaryotes better than those in bacteria. Second, methods worked less well for proteins with many transmembrane helices. Third, most methods correctly discriminated between soluble and transmembrane proteins. However, several older methods often mistook signal peptides for transmembrane helices. Some newer methods have overcome this shortcoming. In our hands, PolyPhobius and MEMSAT-SVM outperformed other methods., (© 2014 Wiley Periodicals, Inc.)

The prediction of protein sub-cellular localization is an important step toward elucidating protein function. For each query protein sequence, LocTree2 applies machine learning (profile kernel SVM) to predict the native sub-cellular localization in 18 classes for eukaryotes, in six for bacteria and in three for archaea. The method outputs a score that reflects the reliability of each prediction. LocTree2 has performed on par with or better than any other state-of-the-art method. Here, we report the availability of LocTree3 as a public web server. The server includes the machine learning-based LocTree2 and improves over it through the addition of homology-based inference. Assessed on sequence-unique data, LocTree3 reached an 18-state accuracy Q18=80±3% for eukaryotes and a six-state accuracy Q6=89±4% for bacteria. The server accepts submissions ranging from single protein sequences to entire proteomes. Response time of the unloaded server is about 90 s for a 300-residue eukaryotic protein and a few hours for an entire eukaryotic proteome not considering the generation of the alignments. For over 1000 entirely sequenced organisms, the predictions are directly available as downloads. The web server is available at http://www.rostlab.org/services/loctree3., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)