In-depth characterization of HINT1 pathogenic variants

Abstract

ABSTRACTLoss-of-function variants in HINT1 were identified to cause axonal recessive peripheral neuropathy with neuromyotonia (NMAN). Patients suffer from motor-greater-than-sensory polyneuropathy with an age of onset, mainly within the first decade of life. Currently, nearly 30 NMAN disease-causing variants have been described, predominantly in sporadic cases and small families, most of them with limited functional evidence of pathogenicity. We systematically characterized all reported pathogenic missense mutations in HINT1, aiming to dissect their underlying loss-of-function mechanism. Individual variants were mapped onto the crystal structure of the HINT1 protein, and their potential effect on protein stability was computed. These variants were grouped into three main clusters: around the catalytic pocket, at the dimer interface, and in the β-sheet behind the catalytic pocket of the protein. The stability and functionality of the corresponding altered proteins were testedin vivousing HINT1 KO cells and a yeast model deficient for the orthologous gene (HNT1), providing insights into the structure-function relations. Our findings support the pathogenic character of most of the variants and uncover their differential effect on HINT1 function. Classifying the variants in three clusters sets the basis for patient stratification strategies for future therapeutic development.