Association of macular corneal dystrophy with excessive cell senescence and apoptosis induced by the novel mutant CHST6

Macular corneal dystrophy (MCD) is a rare form of hereditary corneal dystrophy caused by CHST6 mutations. Owing to the genetic heterogeneity and population differences among patients with MCD, the genetic cause of MCD has not been fully elucidated, and the pathogenesis underlying the genetic mutation is still unclear. In this study, Chinese families and sporadic patients were included as subjects, and clinical and genetic analyses were performed to detect novel CHST6 mutations. In addition, the underlying pathogenic mechanisms of MCD were investigated by in vitro cell experiments. Two consanguineously married families and 10 sporadic patients with MCD were enrolled. Direct sequencing of the CHST6 gene was performed in all the patients to identify novel mutations. Wild-type and mutant overexpression cell lines were constructed to study the effects of the mutation in vitro. The expressions of endoplasmic reticulum (ER) stress markers and apoptotic factors, cell senescence, and migration levels tests were performed in different overexpression cell lines. As a result, four novel mutations (R155Afs*66, S84Cfs*17, E71G, and E71Q) and 10 previously reported mutations in the CHST6 gene were identified. Among the reported mutations, the most frequent mutations detected in the patients were L21Rfs*88 (4/14) and L21H (4/14). All the novel mutations were absent in the 50 healthy controls and were predicted to alter highly conserved amino acids across the different species and considered to be “disease causing” by function prediction. The results of the in vitro cell experiment further demonstrated that the novel homozygous frameshift mutations (S84Cfs*17 and R155Afs*66) of CHST6 detected in the consanguineously married families could lead to truncated proteins with defect functions, higher ER stress and apoptotic levels, decreased cell migration, and excessive cell senescence in corneal stromal cells, thereby affecting the normal functions of corneal stromal cells. These changes might play important roles in corneal opacity, which is characteristic of corneas with MCD. Our study extended the existing spectrum of disease-causing mutations and further elucidated the underlying pathogenic mechanisms of MCD.