A calibrated functional patch clamp assay to enhance clinical variant interpretation in KCNH2-related long QT syndrome

Purpose: Modern sequencing technologies have revolutionised our detection of gene variants. In most genes, including KCNH2, the majority of missense variants are currently classified as variants of uncertain significance (VUS). The aim of this study is to investigate the utility of an automated patch-clamp assay for aiding clinical variant classification in the KCNH2 gene. Methods: The assay was designed according to recommendations of the ClinGen sequence variant interpretation framework. Thirty-one control variants of known clinical significance (17 pathogenic/likely pathogenic, 14 benign/likely benign) were heterozygously expressed in Flp-In HEK293 cells. Variants were analysed for effects on current density and channel gating. A panel of 44 VUS was then assessed for reclassification. Results: All 17 pathogenic variant controls had reduced current density and 13/14 benign variant controls had normal current density, which enabled determination of normal and abnormal ranges for applying moderate or supporting evidence strength for variant classification. Inclusion of KCNH2 functional assay evidence enabled us to reclassify 6 out of 44 VUS as likely pathogenic. Conclusion: The high-throughput patch clamp assay can provide moderate strength evidence for clinical interpretation of clinical KCNH2 variants and demonstrates the value proposition for developing automated patch clamp assays for other ion channel genes.

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Comparative analysis of functional assay evidence use by ClinGen Variant Curation Expert Panels

Genome Medicine ◽

10.1186/s13073-019-0683-1 ◽

2019 ◽

Vol 11 (1) ◽

Cited By ~ 7

Author(s):

Dona M. Kanavy ◽

Shannon M. McNulty ◽

Meera K. Jairath ◽

Sarah E. Brnich ◽

Chris Bizon ◽

...

Keyword(s):

Functional Data ◽

Functional Assay ◽

Sequence Variant ◽

Variant Classification ◽

Variant Interpretation ◽

Functional Studies ◽

Functional Assays ◽

Starting Point ◽

Validation Parameters ◽

The Individual

Abstract Background The 2015 American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) guidelines for clinical sequence variant interpretation state that “well-established” functional studies can be used as evidence in variant classification. These guidelines articulated key attributes of functional data, including that assays should reflect the biological environment and be analytically sound; however, details of how to evaluate these attributes were left to expert judgment. The Clinical Genome Resource (ClinGen) designates Variant Curation Expert Panels (VCEPs) in specific disease areas to make gene-centric specifications to the ACMG/AMP guidelines, including more specific definitions of appropriate functional assays. We set out to evaluate the existing VCEP guidelines for functional assays. Methods We evaluated the functional criteria (PS3/BS3) of six VCEPs (CDH1, Hearing Loss, Inherited Cardiomyopathy-MYH7, PAH, PTEN, RASopathy). We then established criteria for evaluating functional studies based on disease mechanism, general class of assay, and the characteristics of specific assay instances described in the primary literature. Using these criteria, we extensively curated assay instances cited by each VCEP in their pilot variant classification to analyze VCEP recommendations and their use in the interpretation of functional studies. Results Unsurprisingly, our analysis highlighted the breadth of VCEP-approved assays, reflecting the diversity of disease mechanisms among VCEPs. We also noted substantial variability between VCEPs in the method used to select these assays and in the approach used to specify strength modifications, as well as differences in suggested validation parameters. Importantly, we observed discrepancies between the parameters VCEPs specified as required for approved assay instances and the fulfillment of these requirements in the individual assays cited in pilot variant interpretation. Conclusions Interpretation of the intricacies of functional assays often requires expert-level knowledge of the gene and disease, and current VCEP recommendations for functional assay evidence are a useful tool to improve the accessibility of functional data by providing a starting point for curators to identify approved functional assays and key metrics. However, our analysis suggests that further guidance is needed to standardize this process and ensure consistency in the application of functional evidence.

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Comparative analysis of functional assay evidence use by ClinGen Variant Curation Expert Panels

10.1101/19000661 ◽

2019 ◽

Author(s):

Dona M. Kanavy ◽

Shannon M. McNulty ◽

Meera K. Jairath ◽

Sarah E. Brnich ◽

Chris Bizon ◽

...

Keyword(s):

Functional Data ◽

Functional Assay ◽

Sequence Variant ◽

Variant Classification ◽

Variant Interpretation ◽

Functional Studies ◽

Functional Assays ◽

Inherited Cardiomyopathy ◽

Validation Parameters ◽

The Individual

ABSTRACTBackgroundThe 2015 American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) guidelines for clinical sequence variant interpretation state that “well-established” functional studies can be used as evidence in variant classification. These guidelines articulated key attributes of functional data, including that assays should reflect the biological environment and be analytically sound; however, details of how to evaluate these attributes were left to expert judgment. The Clinical Genome Resource (ClinGen) designates Variant Curation Expert Panels (VCEPs) in specific disease areas to make gene-centric specifications to the ACMG/AMP guidelines, including more specific definitions of appropriate functional assays. We set out to evaluate the existing VCEP guidelines for functional assays.MethodsWe evaluated the functional criteria (PS3/BS3) of six VCEPs (CDH1, Hearing Loss, Inherited Cardiomyopathy-MYH7, PAH, PTEN, RASopathy). We then established criteria for evaluating functional studies based on disease mechanism, general class of assay, and the characteristics of specific assay instances described in primary literature. Using these criteria, we extensively curated assay instances cited by each VCEP in their pilot variant classification to analyze VCEP recommendations and their use in the interpretation of functional studies.ResultsUnsurprisingly, our analysis highlighted the breadth of VCEP-approved assays, reflecting the diversity of disease mechanisms among VCEPs. We also noted substantial variability between VCEPs in the method used to select these assays and in the approach used to specify strength modifications, as well as differences in suggested validation parameters. Importantly, we observed discrepancies between the parameters VCEPs specified as required for approved assay instances and the fulfillment of these requirements in the individual assays cited in pilot variant interpretation.ConclusionsInterpretation of the intricacies of functional assays often requires expert-level knowledge of the gene and disease and current VCEP recommendations for functional assay evidence are a useful tool to improve the accessibility of functional data. However, our analysis suggests that further guidance is needed to standardize this process and ensure consistency in the application of functional evidence.

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Evaluation of ACMG Rules for In silico Evidence Strength Using An Independent Computational Tool Absent of Circularities on ATM and CHEK2 Breast Cancer Cases and Controls

10.1101/835264 ◽

2019 ◽

Author(s):

Colin C. Young ◽

Bing-Jian Feng ◽

Colin B. Mackenzie ◽

Elodie Girard ◽

Donglei Hu ◽

...

Keyword(s):

Breast Cancer ◽

Mutation Screening ◽

Case Control ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Cancer Case ◽

Sequence Variant ◽

Supporting Evidence ◽

Variant Classification ◽

Computational Tool

AbstractThe American College of Medical Genetics and Genomics (ACMG) guidelines for sequence variant classification include two criteria, PP3 and BP4, for combining computational data with other evidence types contributing to sequence variant classification. PP3 and BP4 assert that computational modeling can provide “Supporting” evidence for or against pathogenicity within the ACMG framework. Here, leveraging a meta-analysis of ATM and CHEK2 breast cancer case-control mutation screening data, we evaluate the strength of evidence determined from the relatively simple computational tool Align-GVGD. Importantly, application of Align-GVGD to these ATM and CHEK2 data is free of logical circularities, hidden multiple testing, and use of other ACMG evidence types. For both genes, rare missense substitutions that are assigned the most severe Align-GVGD grade exceed a “Moderate pathogenic” evidence threshold when analyzed in a Bayesian framework; accordingly, we argue that the ACMG classification rules be updated for well-calibrated computational tools. Additionally, congruent with previous analyses of ATM and CHEK2 case-control mutation screening data, we find that both genes have a considerable burden of pathogenic missense substitutions, and that severe ATM rare missense have increased odds ratios compared to truncating and splice junction variants, indicative of a potential dominant-negative effect for those missense substitutions.

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Recommendations for application of the functional evidence PS3/BS3 criterion using the ACMG/AMP sequence variant interpretation framework

Genome Medicine ◽

10.1186/s13073-019-0690-2 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 39

Author(s):

Sarah E. Brnich ◽

◽

Ahmad N. Abou Tayoun ◽

Fergus J. Couch ◽

Garry R. Cutting ◽

...

Keyword(s):

Protein Function ◽

Working Group ◽

Steering Committee ◽

Moderate Level ◽

Sequence Variant ◽

Variant Interpretation ◽

Functional Assays ◽

Expert Opinions ◽

Clinical Variant ◽

Normal Gene

Abstract Background The American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) clinical variant interpretation guidelines established criteria for different types of evidence. This includes the strong evidence codes PS3 and BS3 for “well-established” functional assays demonstrating a variant has abnormal or normal gene/protein function, respectively. However, they did not provide detailed guidance on how functional evidence should be evaluated, and differences in the application of the PS3/BS3 codes are a contributor to variant interpretation discordance between laboratories. This recommendation seeks to provide a more structured approach to the assessment of functional assays for variant interpretation and guidance on the use of various levels of strength based on assay validation. Methods The Clinical Genome Resource (ClinGen) Sequence Variant Interpretation (SVI) Working Group used curated functional evidence from ClinGen Variant Curation Expert Panel-developed rule specifications and expert opinions to refine the PS3/BS3 criteria over multiple in-person and virtual meetings. We estimated the odds of pathogenicity for assays using various numbers of variant controls to determine the minimum controls required to reach moderate level evidence. Feedback from the ClinGen Steering Committee and outside experts were incorporated into the recommendations at multiple stages of development. Results The SVI Working Group developed recommendations for evaluators regarding the assessment of the clinical validity of functional data and a four-step provisional framework to determine the appropriate strength of evidence that can be applied in clinical variant interpretation. These steps are as follows: (1) define the disease mechanism, (2) evaluate the applicability of general classes of assays used in the field, (3) evaluate the validity of specific instances of assays, and (4) apply evidence to individual variant interpretation. We found that a minimum of 11 total pathogenic and benign variant controls are required to reach moderate-level evidence in the absence of rigorous statistical analysis. Conclusions The recommendations and approach to functional evidence evaluation described here should help clarify the clinical variant interpretation process for functional assays. Further, we hope that these recommendations will help develop productive partnerships with basic scientists who have developed functional assays that are useful for interrogating the function of a variety of genes.

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Sequence Variant Interpretation 2.0: Perspective on New Guidelines for Sequence Variant Classification

Clinical Chemistry ◽

10.1373/clinchem.2015.240812 ◽

2015 ◽

Vol 61 (11) ◽

pp. 1317-1319 ◽

Cited By ~ 5

Author(s):

Shaoke Chen ◽

Xuyun Hu ◽

Yiping Shen

Keyword(s):

Sequence Variant ◽

Variant Classification ◽

Variant Interpretation ◽

New Guidelines

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Late cardiac sodium current can be assessed using automated patch-clamp

F1000Research ◽

10.12688/f1000research.5544.1 ◽

2014 ◽

Vol 3 ◽

pp. 245 ◽

Cited By ~ 6

Author(s):

Morgan Chevalier ◽

Bogdan Amuzescu ◽

Vaibhavkumar Gawali ◽

Hannes Todt ◽

Thomas Knott ◽

...

Keyword(s):

Patch Clamp ◽

Prolonged Exposure ◽

Sodium Current ◽

Hek293 Cells ◽

Fluctuation Analysis ◽

Open Probability ◽

Drug Induced ◽

Experimental Conditions ◽

Current Increase ◽

Automated Patch Clamp

The cardiac late Na+ current is generated by a small fraction of voltage-dependent Na+ channels that undergo a conformational change to a burst-gating mode, with repeated openings and closures during the action potential (AP) plateau. Its magnitude can be augmented by inactivation-defective mutations, myocardial ischemia, or prolonged exposure to chemical compounds leading to drug-induced (di)-long QT syndrome, and results in an increased susceptibility to cardiac arrhythmias. Using CytoPatch™ 2 automated patch-clamp equipment, we performed whole-cell recordings in HEK293 cells stably expressing human Nav1.5, and measured the late Na+ component as average current over the last 100 ms of 300 ms depolarizing pulses to -10 mV from a holding potential of -100 mV, with a repetition frequency of 0.33 Hz. Averaged values in different steady-state experimental conditions were further corrected by the subtraction of current average during the application of tetrodotoxin (TTX) 30 μM. We show that ranolazine at 10 and 30 μM in 3 min applications reduced the late Na+ current to 75.0 ± 2.7% (mean ± SEM, n = 17) and 58.4 ± 3.5% (n = 18) of initial levels, respectively, while a 5 min application of veratridine 1 μM resulted in a reversible current increase to 269.1 ± 16.1% (n = 28) of initial values. Using fluctuation analysis, we observed that ranolazine 30 μM decreased mean open probability p from 0.6 to 0.38 without modifying the number of active channels n, while veratridine 1 μM increased n 2.5-fold without changing p. In human iPSC-derived cardiomyocytes, veratridine 1 μM reversibly increased APD90 2.12 ± 0.41-fold (mean ± SEM, n = 6). This effect is attributable to inactivation removal in Nav1.5 channels, since significant inhibitory effects on hERG current were detected at higher concentrations in hERG-expressing HEK293 cells, with a 28.9 ± 6.0% inhibition (mean ± SD, n = 10) with 50 μM veratridine.

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Evaluating the impact of in silico predictors on clinical variant classification

10.1101/2021.08.09.455612 ◽

2021 ◽

Author(s):

Emma Wilcox ◽

Mahdi Sarmady ◽

Bryan Wulf ◽

Matt Wright ◽

Heidi Rehm ◽

...

Keyword(s):

In Silico ◽

Sequence Variant ◽

Variant Classification ◽

Likelihood Ratios ◽

Data Set ◽

Variant Of Uncertain Significance ◽

Missense Variants ◽

Clinical Variant ◽

The Impact ◽

In Silico Tools

Background: In silico evidence is important to consider when interpreting genetic variants. According to the ACMG/AMP, in silico evidence is applied at the supporting strength level using the PP3 and BP4 criteria, for pathogenic and benign evidence, respectively. While PP3 has been determined to be one of the most commonly applied criteria, less is known about the effect of these two criteria on variant classification outcomes. Methods: In this study, a total of 727 missense variants curated by Clinical Genome Resource (ClinGen) Variant Curation Expert Panels (VCEPs) were analyzed to determine how often PP3 and BP4 were applied and how often they influenced final variant classifications. The current categorical system of variant classification was compared with a point-based system being developed by the ClinGen Sequence Variant Interpretation Working Group. In addition, the performance of four in silico tools (REVEL, VEST, FATHMM, and MPC) was assessed by using a gold set of 237 variants (classified as benign or pathogenic independent of PP3 or BP4) to calculate pathogenicity likelihood ratios. Results: Collectively, the PP3 and BP4 criteria were applied by ClinGen VCEPs to 55% of missense variants in this data set. Removing in silico criteria from variants where they were originally applied caused variants to change classification from pathogenic to likely pathogenic (14%), likely pathogenic to variant of uncertain significance (VUS) (24%), or likely benign to VUS (64%). The proportion of downgrades with the categorical classification system was similar to that of the point-based system, though the latter resolved borderline classifications. REVEL and VEST performed at a level consistent with moderate strength towards either benign or pathogenic evidence, while FATHMM performed at the supporting level. Conclusions: Overall, this study demonstrates that in silico criteria PP3 and BP4 are commonly applied in variant classification and often affect the final classification. Our results suggest that when sufficient thresholds for in silico predictors are established, PP3 and BP4 may be appropriate to use at a moderate strength. However, further calibration with larger datasets is needed to optimize the performance of current in silico tools given the impact they have on clinical variant classification.

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Recommendations for application of the functional evidence PS3/BS3 criterion using the ACMG/AMP sequence variant interpretation framework

10.1101/709428 ◽

2019 ◽

Author(s):

Sarah E. Brnich ◽

Ahmad N. Abou Tayoun ◽

Fergus J. Couch ◽

Garry R. Cutting ◽

Marc S. Greenblatt ◽

...

Keyword(s):

Working Group ◽

Steering Committee ◽

Moderate Level ◽

Sequence Variant ◽

Variant Interpretation ◽

Level Of Evidence ◽

Functional Assays ◽

Expert Opinions ◽

Clinical Variant ◽

Interpretation Process

ABSTRACTBackgroundThe American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) clinical variant interpretation guidelines established criteria (PS3/BS3) for functional assays that specified a “strong” level of evidence. However, they did not provide detailed guidance on how functional evidence should be evaluated, and differences in the application of the PS3/BS3 codes is a contributor to variant interpretation discordance between laboratories. This recommendation seeks to provide a more structured approach to the assessment of functional assays for variant interpretation and guidance on the use of various levels of strength based on assay validation.MethodsThe Clinical Genome Resource (ClinGen) Sequence Variant Interpretation (SVI) Working Group used curated functional evidence from ClinGen Variant Curation Expert Panel-developed rule specifications and expert opinions to refine the PS3/BS3 criteria over multiple in-person and virtual meetings. We estimated odds of pathogenicity for assays using various numbers of variant controls to determine the minimum controls required to reach moderate level evidence. Feedback from the ClinGen Steering Committee and outside experts were incorporated into the recommendations at multiple stages of development.ResultsThe SVI Working Group developed recommendations for evaluators regarding the assessment of the clinical validity of functional data and a four-step provisional framework to determine the appropriate strength of evidence that can be applied in clinical variant interpretation. These steps are: 1. Define the disease mechanism; 2. Evaluate applicability of general classes of assays used in the field; 3. Evaluate validity of specific instances of assays; 4. Apply evidence to individual variant interpretation. We found that a minimum of eleven total pathogenic and benign variant controls are required to reach moderate-level evidence in the absence of rigorous statistical analysis.ConclusionsThe recommendations and approach to functional evidence evaluation described here should help clarify the clinical variant interpretation process for functional assays. Further, we hope that these recommendations will help develop productive partnerships with basic scientists who have developed functional assays that are useful for interrogating the function of a variety of genes.

Download Full-text