Thermal shift assay (TSA) is an increasingly popular technique used for identifying protein stabilizing conditions or interacting ligands in X-ray crystallography and drug discovery applications. Although the setting up and running of TSA reactions is a relatively simple process, the subsequent analysis of TSA data, especially in high-throughput format, requires substantial amount of effort if conducted manually. We therefore developed the Thermal Shift Assay–Curve Rapid and Automatic Fitting Tool (TSA-CRAFT), a freely available software that enable automatic analysis of TSA data of any throughput. TSA-CRAFT directly reads real-time PCR instrument data files and displays the analyzed results in a web browser. This software features streamlined data processing and Boltzmann equation fitting, which is demonstrated in this study to provide more accurate data analysis than the commonly used first-derivative method. TSA-CRAFT is freely available as a cross-operating system-compatible standalone tool ( https://sourceforge.net/projects/tsa-craft/ ) and also as a freely accessible web server ( http://tbtlab.org/tsacraft.html ).

We have identified six patients harbouring distinct germline BAP1 mutations. In this study, we functionally characterise known BAP1 pathogenic and likely benign germline variants out of these six patients to aid in the evaluation and classification of unknown BAP1 germline variants. We found that pathogenic germline variants tend to encode truncated proteins, show diminished expression of epithelial-mesenchymal transition (EMT) markers, are localised in the cytosol and have reduced deubiquitinase capabilities. We show that these functional assays are useful for BAP1 variant curation and may be added in the American College of Medical Genetics and Genomics (ACMG) criteria for BAP1 variant classification. This will allow clinicians to distinguish between BAP1 pathogenic and likely benign variants reliably and may aid to quickly benchmark newly identified BAP1 germline variants. Classification of novel BAP1 germline variants allows clinicians to inform predisposed patients and relevant family members regarding potential cancer risks, with appropriate clinical interventions implemented if required.