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1.
Nucleic Acid Ther ; 2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34388351

RESUMO

Steric-blocking oligonucleotides (SBOs) are short, single-stranded nucleic acids designed to modulate gene expression by binding to RNA transcripts and blocking access from cellular machinery such as splicing factors. SBOs have the potential to bind to near-complementary sites in the transcriptome, causing off-target effects. In this study, we used RNA-seq to evaluate the off-target differential splicing events of 81 SBOs and differential expression events of 46 SBOs. Our results suggest that differential splicing events are predominantly hybridization driven, whereas differential expression events are more common and driven by other mechanisms (including spurious experimental variation). We further evaluated the performance of in silico screens for off-target splicing events, and found an edit distance cutoff of three to result in a sensitivity of 14% and false discovery rate (FDR) of 99%. A machine learning model incorporating splicing predictions substantially improved the ability to prioritize low edit distance hits, increasing sensitivity from 4% to 26% at a fixed FDR of 90%. Despite these large improvements in performance, this approach does not detect the majority of events at an FDR <99%. Our results suggest that in silico methods are currently of limited use for predicting the off-target effects of SBOs, and experimental screening by RNA-seq should be the preferred approach.

2.
Am J Hum Genet ; 108(7): 1283-1300, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34214447

RESUMO

Most rare clinical missense variants cannot currently be classified as pathogenic or benign. Deficiency in human 5,10-methylenetetrahydrofolate reductase (MTHFR), the most common inherited disorder of folate metabolism, is caused primarily by rare missense variants. Further complicating variant interpretation, variant impacts often depend on environment. An important example of this phenomenon is the MTHFR variant p.Ala222Val (c.665C>T), which is carried by half of all humans and has a phenotypic impact that depends on dietary folate. Here we describe the results of 98,336 variant functional-impact assays, covering nearly all possible MTHFR amino acid substitutions in four folinate environments, each in the presence and absence of p.Ala222Val. The resulting atlas of MTHFR variant effects reveals many complex dependencies on both folinate and p.Ala222Val. MTHFR atlas scores can distinguish pathogenic from benign variants and, among individuals with severe MTHFR deficiency, correlate with age of disease onset. Providing a powerful tool for understanding structure-function relationships, the atlas suggests a role for a disordered loop in retaining cofactor at the active site and identifies variants that enable escape of inhibition by S-adenosylmethionine. Thus, a model based on eight MTHFR variant effect maps illustrates how shifting landscapes of environment- and genetic-background-dependent missense variation can inform our clinical, structural, and functional understanding of MTHFR deficiency.


Assuntos
Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Análise Mutacional de DNA , Diploide , Biblioteca Gênica , Genótipo , Humanos , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Metilenotetra-Hidrofolato Redutase (NADPH2)/fisiologia , Saccharomyces cerevisiae/genética
3.
Elife ; 92020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32870157

RESUMO

Vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin. Despite VKOR's pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency or alter warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells. The large-scale functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. Functionally constrained regions of the protein define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. Finally, 25% of human VKOR missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease.


Assuntos
Domínio Catalítico , Variação Genética , Mutação de Sentido Incorreto , Vitamina K Epóxido Redutases/química , Vitamina K Epóxido Redutases/genética , Cisteína/química , Resistência a Medicamentos , Células HEK293 , Humanos , Erros Inatos do Metabolismo , Modelos Moleculares , Análise de Sequência de DNA , Varfarina/farmacologia
4.
NPJ Genom Med ; 5: 16, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32284880

RESUMO

Wilson disease is a recessive genetic disorder caused by pathogenic loss-of-function variants in the ATP7B gene. It is characterized by disrupted copper homeostasis resulting in liver disease and/or neurological abnormalities. The variant NM_000053.3:c.1934T > G (Met645Arg) has been reported as compound heterozygous, and is highly prevalent among Wilson disease patients of Spanish descent. Accordingly, it is classified as pathogenic by leading molecular diagnostic centers. However, functional studies suggest that the amino acid change does not alter protein function, leading one ClinVar submitter to question its pathogenicity. Here, we used a minigene system and gene-edited HepG2 cells to demonstrate that c.1934T > G causes ~70% skipping of exon 6. Exon 6 skipping results in frameshift and stop-gain, leading to loss of ATP7B function. The elucidation of the mechanistic effect for this variant resolves any doubt about its pathogenicity and enables the development of genetic medicines for restoring correct splicing.

5.
Genome Med ; 12(1): 13, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32000841

RESUMO

BACKGROUND: For the majority of rare clinical missense variants, pathogenicity status cannot currently be classified. Classical homocystinuria, characterized by elevated homocysteine in plasma and urine, is caused by variants in the cystathionine beta-synthase (CBS) gene, most of which are rare. With early detection, existing therapies are highly effective. METHODS: Damaging CBS variants can be detected based on their failure to restore growth in yeast cells lacking the yeast ortholog CYS4. This assay has only been applied reactively, after first observing a variant in patients. Using saturation codon-mutagenesis, en masse growth selection, and sequencing, we generated a comprehensive, proactive map of CBS missense variant function. RESULTS: Our CBS variant effect map far exceeds the performance of computational predictors of disease variants. Map scores correlated strongly with both disease severity (Spearman's ϱ = 0.9) and human clinical response to vitamin B6 (ϱ = 0.93). CONCLUSIONS: We demonstrate that highly multiplexed cell-based assays can yield proactive maps of variant function and patient response to therapy, even for rare variants not previously seen in the clinic.


Assuntos
Cistationina beta-Sintase/genética , Teste de Complementação Genética/métodos , Testes Genéticos/métodos , Homocistinúria/genética , Mutação de Sentido Incorreto , Cistationina beta-Sintase/metabolismo , Genótipo , Humanos , Fenótipo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética
6.
Hum Mutat ; 40(9): 1463-1473, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31283071

RESUMO

This paper reports the evaluation of predictions for the "CALM1" challenge in the fifth round of the Critical Assessment of Genome Interpretation held in 2018. In the challenge, the participants were asked to predict effects on yeast growth caused by missense variants of human calmodulin, a highly conserved protein in eukaryotic cells sensing calcium concentration. The performance of predictors implementing different algorithms and methods is similar. Most predictors are able to identify the deleterious or tolerated variants with modest accuracy, with a baseline predictor based purely on sequence conservation slightly outperforming the submitted predictions. Nevertheless, we think that the accuracy of predictions remains far from satisfactory, and the field awaits substantial improvements. The most poorly predicted variants in this round surround functional CALM1 sites that bind calcium or peptide, which suggests that better incorporation of structural analysis may help improve predictions.


Assuntos
Calmodulina/química , Calmodulina/genética , Biologia Computacional/métodos , Mutação de Sentido Incorreto , Leveduras/crescimento & desenvolvimento , Algoritmos , Sítios de Ligação , Cálcio/metabolismo , Calmodulina/metabolismo , Evolução Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Aptidão Genética , Humanos , Modelos Genéticos , Modelos Moleculares , Conformação Proteica , Engenharia de Proteínas , Leveduras/genética
7.
Bioinformatics ; 35(17): 3191-3193, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30649215

RESUMO

SUMMARY: The promise of personalized genomic medicine depends on our ability to assess the functional impact of rare sequence variation. Multiplexed assays can experimentally measure the functional impact of missense variants on a massive scale. However, even after such assays, many missense variants remain poorly measured. Here we describe a software pipeline and application to impute missing information in experimentally determined variant effect maps. AVAILABILITY AND IMPLEMENTATION: http://impute.varianteffect.org source code: https://github.com/joewuca/imputation. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Software , Genoma , Genômica , Mutação de Sentido Incorreto
8.
Mol Syst Biol ; 14(5): e7985, 2018 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-29807908

RESUMO

Condition-dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State-of-the-art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double-mutant strains, does not scale readily to multi-condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG-GI), by which double-mutant strains generated via en masse "party" mating can also be monitored en masse for growth to detect genetic interactions. By using site-specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG-GI enables multiplexed quantitative tracking of double mutants via next-generation sequencing. We applied BFG-GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO), bleomycin, zeocin, and three other DNA-damaging environments. BFG-GI recapitulated known genetic interactions and yielded new condition-dependent genetic interactions. We validated and further explored a subnetwork of condition-dependent genetic interactions involving MAG1, SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53.


Assuntos
Mapeamento Cromossômico , Código de Barras de DNA Taxonômico , Dano ao DNA , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Reparo do DNA , Epistasia Genética , Deleção de Genes , Loci Gênicos , Sequenciamento de Nucleotídeos em Larga Escala , Metanossulfonato de Metila , Modelos Teóricos , Regiões Promotoras Genéticas , Reprodutibilidade dos Testes
9.
Mol Syst Biol ; 13(12): 957, 2017 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-29269382

RESUMO

Although we now routinely sequence human genomes, we can confidently identify only a fraction of the sequence variants that have a functional impact. Here, we developed a deep mutational scanning framework that produces exhaustive maps for human missense variants by combining random codon mutagenesis and multiplexed functional variation assays with computational imputation and refinement. We applied this framework to four proteins corresponding to six human genes: UBE2I (encoding SUMO E2 conjugase), SUMO1 (small ubiquitin-like modifier), TPK1 (thiamin pyrophosphokinase), and CALM1/2/3 (three genes encoding the protein calmodulin). The resulting maps recapitulate known protein features and confidently identify pathogenic variation. Assays potentially amenable to deep mutational scanning are already available for 57% of human disease genes, suggesting that DMS could ultimately map functional variation for all human disease genes.


Assuntos
Análise Mutacional de DNA/métodos , Mutação de Sentido Incorreto/genética , Calmodulina/genética , Doença/genética , Humanos , Aprendizado de Máquina , Fenótipo , Filogenia , Reprodutibilidade dos Testes , Proteína SUMO-1/genética , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
10.
Mol Syst Biol ; 12(4): 863, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-27107012

RESUMO

High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods.


Assuntos
Centrossomo/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteoma/metabolismo , Saccharomyces cerevisiae/genética , Cromossomos Humanos/metabolismo , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido
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