To assess the activity of wild-type and W317X mutant HDC proteins, constructs were expressed as N-terminal glutathione S-transferase fusion products, isolated under denaturing conditions, refolded, and subjected to a fluorescence quenching–based assay. Assessment for dominant negative activity (i.e., determination of whether the altered gene product derived from the mutant allele successfully antagonizes the protein product of the wild-type allele) was performed by means of in vitro transcription and translation. We evaluated the effect of mutant proteins on enzymatic activity by using a fixed amount of wild-type DNA template along with increasing amounts of W317X mutant DNA template. The production of histamine from its precursor l-histidine was measured by means of liquid chromatography–mass spectrometry (Table S10 in the Supplementary Appendix).
