Assessment of functional effects of unclassified genetic variants

Abstract

Inherited predisposition to disease is often linked to reduced activity of a disease associated gene product. Thus, quantitation of the influence of inherited variants on gene function can potentially be used to predict the disease relevance of these variants. While many disease genes have been extensively characterized at the functional level, few assays based on functional properties of the encoded proteins have been established for the purpose of predicting the contribution of rare inherited variants to disease. Much of the difficulty in establishing predictive functional assays stems from the technical complexity of the assays. However, perhaps the most challenging aspect of functional assay development for clinical testing purposes is the absolute requirement for validation of the sensitivity and specificity of the assays and the determination of positive predictive values (PPVs) and negative predictive values (NPVs) of the assays relative to a "gold standard" measure of disease predisposition. In this commentary, we provide examples of some of the functional assays under development for several cancer predisposition genes (BRCA1, BRCA2, CDKN2A, and mismatch repair [MMR] genes MLH1, MSH2, MSH6, and PMS2) and present a detailed review of the issues associated with functional assay development. We conclude that validation is paramount for all assays that will be used for clinical interpretation of inherited variants of any gene, but note that in certain circumstances information derived from incompletely validated assays may be valuable for classification of variants for clinical purposes when used to supplement data derived from other sources.

Figure 1

A. Diagram of human BRCA1 depicting defined domains and motifs (Green box, RING domain; Blue circles, BRCT domains; Black bars, nuclear localization signals). Regions showing conservation above 0.75 in a multiple sequence alignment (from human to puffer fish) are shown as dark blue boxes. Regions analyzed by domain-specific functional assays (ubiquitin ligase for the RING region; transcription assay for the C-terminal region) are depicted as red empty boxes. Graph aligned to the top diagram shows the number of entries in the BIC database for each missense variant recorded to date. Variants are shown as black (unclassified), red (pathogenic), and blue (neutral) bars. All pathogenic and neutral variants as classified in the BIC database are shown. B. Diagram for BRCA2 depicting the BRC repeats (arrows) and DNA binding domain. Regions showing conservation above 0.75 in a multiple sequence alignment (from human to puffer fish) are shown as dark blue boxes. Aligned graph shows position and frequency of all missense mutations in the BIC database. Variants are shown as black (unclassified), red (pathogenic), and blue (neutral) bars. All pathogenic and neutral variants as classified in the BIC database are shown.

Figure 2

Proposed decision tree for the in vitro analysis of variants in MMR genes in suspected Lynch syndrome cases.

Figure 3

Confounding factors in the interpretation of functional assays.