Closing the gap: Systematic integration of multiplexed functional data resolves variants of uncertain significance in BRCA1, TP53, and PTEN

Fayer, Shawn; Horton, Carolyn; Dines, Jennifer N.; Rubin, Alan F.; Richardson, Marcy E.; McGoldrick, Kelly; Hernandez, Felicia; Pesaran, Tina; Karam, Rachid; Shirts, Brian H.; Fowler, Douglas M.

doi:10.1016/j.ajhg.2021.11.001

Cited by 69 publications

(81 citation statements)

References 42 publications

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“…Importantly, because variants are introduced into the endogenous locus, genomic and regulatory context is preserved, therefore non-coding and synonymous variants which alter splicing or transcriptional control can also be detected 7 . SGE has been demonstrated to effectively resolve most VUS in BRCA1 7,8 , outperforms in silico prediction algorithms 9 , and has proven diagnostic utility 8,10 . Pathogenic variation in DDX3X, an X-linked gene encoding an RNA helicase of the DEADbox protein family, has been robustly associated with both NDD (germline variation) and cancer (somatic mutations).…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Saturation genome editing of DDX3X clarifies pathogenicity of germline and somatic variation

Radford

Tan

Andersson

et al. 2022

Preprint

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Loss-of-function of DDX3X is a leading cause of neurodevelopmental disorders (NDD) in females. DDX3X is also a somatically mutated cancer driver gene proposed to have tumour promoting and suppressing effects. We performed saturation genome editing of DDX3X, testing in vitro the functional impact of 12,776 nucleotide variants. We identified 3,432 functionally abnormal variants, in three distinct classes. We trained a machine learning classifier to identify functionally abnormal variants of NDD-relevance. This classifier has at least 97% sensitivity and 99% specificity to detect variants pathogenic for NDD, substantially out-performing in silico predictors, and resolving up to 93% of variants of uncertain significance. Moreover, functionally-abnormal variants could account for almost all of the excess nonsynonymous DDX3X somatic mutations seen in DDX3X-driven cancers. Systematic maps of variant effects generated in experimentally tractable cell types have the potential to transform clinical interpretation of both germline and somatic disease-associated variation.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Saturation genome editing of DDX3X clarifies pathogenicity of germline and somatic variation

Radford

Tan

Andersson

et al. 2022

Preprint

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“…As many VUSs are identified in diagnostic testing, many studies are focusing on VUS assessment and reclassification 50,51 . For instance, Dines et al reclassified BRCA1 exon 11 as a coldspot, suggesting a strong benign reinterpretation of variants located within that region.…”

Section: Discussionmentioning

confidence: 99%

DeMAG predicts the effects of variants in clinically actionable genes by integrating structural and evolutionary epistatic features

Luppino

Adzhubei

Cassa

et al. 2022

Preprint

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Despite an increasing use of genomic sequencing in clinical practice, interpretation of rare genetic variants remains challenging even in well-studied disease genes, resulting in many patients with Variants of Uncertain Significance (VUSs). Computational Variant Effect Predictors (VEPs) are currently used to provide valuable evidence in variant classifications, but they often misclassify benign variants, contributing to potential misdiagnoses. Here, we developed Deciphering Mutations in Actionable Genes (DeMAG), a supervised classifier for interpreting missense variants in actionable disease genes with improved performance over existing VEPs (20% decrease of false positive rate). Our tool has balanced specificity (82%) and sensitivity (94%) on clinical data, and the lowest misclassification rate on putatively benign variants among evaluated tools. DeMAG takes advantage of a novel epistatic feature, the partners score, which is based on evolutionary and structural partnerships of residues as estimated by evolutionary information and AlphaFold2 structural models. The partners score as a general framework of epistatic interactions, can integrate not only clinical but functional information. We anticipate that our tool (demag.org) will facilitate the interpretation of variants and improve clinical decision-making.

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“…For many genes, culling the training set to remove such variants may not be practical -- obtaining a sufficient number of control variants is emerging as a key rate limiting step for many MAVEs; at least 11 control variants are needed to reach a ‘moderate’ strength of evidence [27]. This challenge is highlighted by a recent effort to reclassify variants in PTEN using MAVE data [21], which was hampered by the limited number (n=2) of known benign variants. In many cases, filtering variants for this or other criteria may not even be possible: public-facing databases such as ClinVar are often the primary source for these controls, and to protect privacy they do not provide individual-level clinical or demographic data.…”

Section: Discussionmentioning

confidence: 99%

Saturation-scale functional evidence supports clinical variant interpretation in Lynch Syndrome

Scott

Hernandez

Chamberlin

et al. 2022

Preprint

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Lynch Syndrome (LS) is a cancer predisposition syndrome affecting more than 1 in every 300 individuals worldwide. Clinical genetic testing for LS can be life-saving but is complicated by the heavy burden of variants of uncertain significance (VUS), especially missense changes. To address this challenge, we leveraged a multiplex analysis of variant effect (MAVE) map covering >94% of the 17,746 possible missense variants in the key LS gene MSH2. Here, to establish the clinical validity of these functional data, and to demonstrate their utility in large-scale variant reclassification, we overlaid them on clinical databases comprising >15,000 individuals with an MMR gene variant uncovered during clinical genetic testing. Our functional measurements agreed with the clinical interpretation for every one of 47 control variants with available classifications, satisfying accepted thresholds for "strong" evidence for or against pathogenicity. We then used these scores to attempt reclassification for 682 unique missense VUS, among which 34 (5.0%) scored as deleterious in our function map, in line with previously published rates among other cancer predisposition genes. Consistent with their pathogenicity, functionally abnormal missense variants were associated with significantly elevated risk for LS-related cancers. Combining functional data and other lines of evidence, ten variants were reclassified as pathogenic/likely pathogenic, and 497 could be moved to benign/likely benign. Finally, we applied these functional scores to paired tumor-normal genetic tests, and identified a subset of patients with biallelic somatic loss of function, reflecting a sporadic Lynch-like Syndrome with distinct implications for treatment and relatives' risk. This study demonstrates how high-throughput functional assays can empower scalable VUS resolution and prospectively generate strong evidence for variant classification.

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Closing the gap: Systematic integration of multiplexed functional data resolves variants of uncertain significance in BRCA1, TP53, and PTEN

Cited by 69 publications

References 42 publications

Saturation genome editing of DDX3X clarifies pathogenicity of germline and somatic variation

Saturation genome editing of DDX3X clarifies pathogenicity of germline and somatic variation

DeMAG predicts the effects of variants in clinically actionable genes by integrating structural and evolutionary epistatic features

Saturation-scale functional evidence supports clinical variant interpretation in Lynch Syndrome

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