Variants of uncertain clinical significance in hereditary breast and ovarian cancer genes: best practices in functional analysis for clinical annotation

Abstract

Germline DNA tests to identify pathogenic variants in genes linked to hereditary breast and ovarian cancer susceptibility have become widely available. However, the clinical utility of genetic testing depends on reliable evidence-based classification of sequence variants. Determination of pathogenicity traditionally relies on painstaking pedigree-based segregation analyses. However, the rapid increase in usage of germline DNA tests has led to the discovery of a large number of variants of uncertain clinical significance (VUS). For most VUS there is insufficient information for segregation analysis and therefore assessment of functional consequences is increasingly being used to support clinical annotation. Functional assays need to be accurate, robust, and reproducible to be used for clinical purposes. Here we use the lessons learned from BRCA1 and BRCA2 to identify best practices for the use of functional assays for clinical annotation of germline VUS in breast and ovarian cancer genes. We provide recommendations for the interpretation and use of established functional assays as well as for the development of new assays.

Keywords: cancer: breast; clinical genetics; genetic screening/counselling; getting research into practice; molecular genetics.

Figure 1.. Circos plot illustrating the concordance between AlignGVGD predictions and experimental data derived from analysis of BRCA1 C-terminal variants (aa 1396–1863).

Variants were analyzed using the transcription activation assay [29] and assigned to functional classes by VarCall [57]. Blue ribbons show that all variants scoring C0 and C15 in Align GVGD and predicted to have no or little functional impact score as non-pathogenic or likely non-pathogenic (fClass1 or 2) in a validated functional assay. Conversely, most variants scoring as C65 and predicted to have a functional score as fClass5 (thick purple ribbon). Despite the strong concordance between alignGVGD and the transcriptional assay, a small but significant fraction of variants scoring as fClass1 were incorrectly predicted (C45-C65).