Crystallin gene mutations are responsible for about half of the congenital cataract caused by genetic disorders. L45P and Y46D mutations of gamma C-crystallin have been reported in patients with nuclear congenital cataract. In this study, we explored the thermal stability of wild type (WT), L45P, and Y46D mutants of gamma C-crystallin at low and high concentrations, as well as the effect of alpha A-crystallin on the thermal stability of mutants. Spectroscopic experiments were used to monitor the structural changes on temperature-gradient and time-course heating process. Intermediate morphologies were determined through cryo-electron microscopy. The thermal stability of WT and mutants at concentrations ranging up to hundreds of milligrams were assessed via the UNcle multifunctional protein stability analysis system. The results showed that L45P and Y46D mutations impaired the thermal stability of gamma C-crystallin at low (0.2 mg/mL) and high concentrations (up to 200 mg/mL). Notably, with increase in protein concentration, the thermal stability of L45P and Y46D mutants of gamma C-crystallin simultaneously decreased. Thermal stability of L45P and Y46D mutants could be rescued by alpha A-crystallin in a concentration-dependent manner. The dramatic decrease in thermal stability of gamma C-crystallin caused by L45P and Y46D mutations contributed to congenital cataract in the mature human lens. (C) 2021 Elsevier Inc. All rights reserved.