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

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.

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

2019 ◽  
Vol 11 (1) ◽  
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.

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

2021 ◽  
Author(s):  
Connie Jiang ◽  
Ebony Richardson ◽  
Jessica Farr ◽  
Adam P Hill ◽  
Rizwan Ullah ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Current Density ◽  
Hek293 Cells ◽  
Functional Assay ◽  
Sequence Variant ◽  
Supporting Evidence ◽  
Variant Classification ◽  
Variant Interpretation ◽  
Clinical Variant ◽  
Automated Patch Clamp

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.

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

2019 ◽  
Vol 12 (1) ◽  
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.

Assessing concordance among human, in silico predictions and functional assays on genetic variant classification

2019 ◽  
Vol 35 (24) ◽  
pp. 5163-5170
Author(s):  
Jiaqi Luo ◽  
Tianliangwen Zhou ◽  
Xiaobin You ◽  
Yi Zi ◽  
Xiaoting Li ◽  
...  
Keyword(s):  
Neural Network ◽  
Network Model ◽  
Neural Network Model ◽  
In Silico ◽  
Supplementary Information ◽  
Functional Assay ◽  
Variant Classification ◽  
Validation Data ◽  
Functional Assays ◽  
Nucleotide Resolution

Abstract Motivation A variety of in silico tools have been developed and frequently used to aid high-throughput rapid variant classification, but their performances vary, and their ability to classify variants of uncertain significance were not systemically assessed previously due to lack of validation data. This has been changed recently by advances of functional assays, where functional impact of genetic changes can be measured in single-nucleotide resolution using saturation genome editing (SGE) assay. Results We demonstrated the neural network model AIVAR (Artificial Intelligent VARiant classifier) was highly comparable to human experts on multiple verified datasets. Although highly accurate on known variants, AIVAR together with CADD and PhyloP showed non-significant concordance with SGE function scores. Moreover, our results indicated that neural network model trained from functional assay data may not produce accurate prediction on known variants. Availability and implementation All source code of AIVAR is deposited and freely available at https://github.com/TopGene/AIvar. Supplementary information Supplementary data are available at Bioinformatics online.

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

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.

Development of a data model and programmatic tooling for annotation of functional assay evidence used in sequence variant interpretation

2021 ◽  
Vol 132 ◽  
pp. S255
Author(s):  
Shannon McNulty ◽  
Dona Kanavy ◽  
Meera Jairath ◽  
Courtney Thaxton ◽  
Jack Ward ◽  
...  
Keyword(s):  
Data Model ◽  
Functional Assay ◽  
Sequence Variant ◽  
Variant Interpretation

Impact of proactive high-throughput functional assay data on BRCA1 variant interpretation in 3684 patients with breast or ovarian cancer

2020 ◽  
Vol 65 (3) ◽  
pp. 209-220 ◽  
Author(s):  
Hyun-Ki Kim ◽  
Eun Jin Lee ◽  
Young-Jae Lee ◽  
Jisun Kim ◽  
Yongsub Kim ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
High Throughput ◽  
Functional Assay ◽  
Variant Interpretation

scholarly journals Recombination within the HLA-D region. Correlation of molecular genotyping with functional data.

1984 ◽  
Vol 160 (1) ◽  
pp. 222-238 ◽  
Author(s):  
M A Robinson ◽  
E O Long ◽  
A H Johnson ◽  
R J Hartzman ◽  
B Mach ◽  
...  
Keyword(s):  
Functional Data ◽  
Southern Blot Analysis ◽  
Hla Typing ◽  
Subtle Difference ◽  
Molecular Genotyping ◽  
Recombinant Haplotype ◽  
Allelic Differences ◽  
D Region ◽  
The Individual ◽  
Hla Haplotypes

Molecular genotyping of the HLA-D/DR region in a family correlated with serologic and cellular typing data. It was further possible to predict a subtle difference in SB region-related functions from such molecular studies. A family that included an individual who inherited an HLA haplotype with a paternal recombination between HLA-B and the HLA-D/DR region was identified by classic HLA typing techniques. Segregation of HLA-D/DR region genes in this family was studied by Southern blot analysis using cDNA probes for DR alpha, DR beta, DC alpha, DC beta, and SB beta. Restriction enzyme fragment polymorphisms observed for every gene tested were in concordance with assigned HLA haplotypes (including the individual known to have inherited a paternal recombinant haplotype) with one exception: two HLA identical siblings were observed to have different SB beta restriction fragment patterns. Further testing revealed that one individual inherited a maternal HLA haplotype recombinant between the HLA-D/DR region and SB beta. Although both maternal SB alleles typed as SB4, allelic differences could be detected cellularly by primed lymphocytes and by the differential expression of a class II cell surface antigen using monoclonal antibody. Therefore, predicted and nonpredicted recombinant haplotypes were detected in a family by molecular genotyping.

Tailoring the ACMG/AMP sequence variant interpretation guidelines to the unique aspects of germline ACADVL variants

2021 ◽  
Vol 132 ◽  
pp. S254
Author(s):  
May Flowers ◽  
Meredith Weaver ◽  
Heather Baudet ◽  
Marzia Pasquali ◽  
Gregory Enns ◽  
...  
Keyword(s):  
Sequence Variant ◽  
Variant Interpretation
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