Monogenic diseases are due to mutations that affect 7,000 different genes. One major roadblock in the diagnosis of rare diseases is the number of gene variants of uncertain significance. Novel methodologies need to be developed specifically to determine the pathogenicity of variants in the coding sequence. This project focuses on the MEFV gene mutated in familial Mediterranean fever. Thanks to synthetic biology and DNA bar-coding, in vitro assays combined to deep sequencing, large scale in silico analyses and validation using primary cells from patients, we will determine the comprehensive repertoire of pathogenic MEFV variants. Finally, we will develop a web-based platform to provide the information on each MEFV variants to clinicians and geneticists. This project should provide a proof of concept that this innovative approach, can identify simultaneously all pathogenic variants in the coding sequence of a given gene with implications for numerous monogenic diseases.