Consultation informs strategies to improve functional evidence use in variant classification

Abstract

To determine if a variant identified by diagnostic genetic testing is causal for disease, applied genetics professionals evaluate all available evidence to assign a clinical classification. Experimental assay data can provide strong functional evidence for or against pathogenicity in variant classification, but appears to be underutilised. We surveyed genetic diagnostic professionals in Australasia to assess their application of functional evidence in clinical practice. Results indicated that survey respondents are not confident to apply functional evidence, mainly due to uncertainty around practice recommendations. Respondents also identified need for support resources, educational opportunities, and in particular requested expert recommendations and updated practice guidelines to improve translation of experimental data to curation evidence. As an initial step, we have collated a list of functional assays recommended by 19 ClinGen Variant Curation Expert Panels as a source of international expert opinion on functional evidence evaluation. Additional support resources for diagnostic practice are in development.

Keywords: Functional evidence; assay; diagnostic genetics; education; variant classification.

Figure 1.. Comfort and familiarity of survey participants with the ACMG/AMP framework and associated recommendations and tools for functional evidence application.

A) Confidence level of participants (n=33) according to the rating scale ‘1 = not at all comfortable’ to ‘5 = very comfortable’ for each of the indicated functional evidence types: biochemical assays (e.g. enzyme assays), transcript assays (e.g. splicing assays or transcriptome data), cell models (for e.g. in vitro cell assays), animal models (e.g. mouse models) and high throughput functional assays (including multiplexed assays of variant effect). B) – E) shows number of participants according to awareness level category (‘use’, ‘are aware or’, or ‘do not know of’) for the indicated resources for functional evidence evaluation (33 participants each, unless otherwise noted). Note: awareness level was assessed independent of the standard operating procedure used by their practice/institution. B) Awareness of ClinGen Sequence Variant Interpretation (SVI) Working Group functional evidence recommendations (2). C) Awareness of ClinGen SVI Splicing Subgroup recommendations (20), n = 32 (one “Not Applicable” result excluded). D) Awareness of ClinGen SVI Functional Assay Documentation Worksheet (16). E) Awareness of VCEP specific guidance on functional evidence use (4).

Figure 2.. Participant views related to functional evidence application.

Viewpoints were framed around three questions (A-C) on barriers, enablers and resources for future use, with option to select all that apply, and to provide free text answers to an “other” option. A) Which of the following might prevent your use of functional evidence in variant classification? B) What would improve your interaction with and use of functional evidence (existing or not)? C) Which of the following training and/or additional resources would you use to increase/improve your use/understanding of applying functional evidence?

Figure 3.. Summary of functional assays specifically mentioned within VCEP CSpecs.

A) Number of assays identified for each of the 33 genes covered by the VCEP CSpecs (total n=226). B) Number of variants per assay across all VCEP CSpecs. C) Distribution of pathogenic evidence strength (applicable under ACMG/AMP code PS3) recommended across all the specific assays listed in CSpecs. D) Distribution of benign evidence strength (applicable under ACMG/AMP code BS3) recommended across all the specific assays listed in CSpecs. A full list of specific assays collated can be found in Supplementary Table 1. Darker bar regions indicate where VCEPs denote additional considerations and/or modifications for application of the given evidence level.