Phenotypes of X-linked Charcot-Marie-Tooth disease and altered trafficking of mutant connexin 32 (GJB1).

To clarify the pathomechanism in three patients with X-linked Charcot-Marie-Tooth disease (CMTX) and unique clinical features, we studied three connexin (Cx) 32 (GJB1) mutants with respect to cellular localization in cultured cells. Wild-type Cx32 and three Cx32 mutants (Va163Ile and Glu186Lys, obtained from CMTX patients with hearing impairment; and Arg22Gln, obtained from a CMTX patient with a fair number of onion-bulb formations) were transfected to rat pheochromocytoma cells (PC12). We investigated the expression of Cx32 protein in each clone by immunoblotting and immunohistochemical staining. While Cx32 protein with the Arg22Gln mutation was detectable immunohistochemically only in the cytoplasm, Cx32 protein with the Va163Ile or Glu186Lys mutation was detected in both the plasma membrane and the cytoplasm. Cx32 protein with the wild-type sequence was detected mostly in the plasma membrane, with plaques indicating the existence of active gap junction formation. These three Cx32 mutations associated with CMTX patients with unique clinical and pathological findings caused altered trafficking of the Cx32 protein. These altered expressions indicated loss of active gap junction formation with different expression abnormalities in these CMTX patients.