Because it is often a more sensitive indicator for a mild PBD,25,28 we also analyzed the peroxisomal phenotype in the cultured fibroblasts at 37°C from individuals F1-II:3 and F5-II:2 by immunofluorescence (IF) microscopy by using antibodies against the membrane protein PMP70 (ABCD3) and the peroxisomal matrix protein catalase (Figure 3). For both cell lines, we observed a so-called “mosaic” peroxisomal pattern. We saw different types of cells, including cells with normal peroxisomal staining, cells with a reduced number of peroxisomes, and cells with only peroxisomal membrane remnants, referred to as “ghosts,” but no import of matrix protein. Peroxisomal mosaicism has been described previously for hypomorphic variants in PEX1 and PEX6 and is typically associated with mild PBDs.29,30 Consistent with cells displaying peroxisomal mosaicism, the cells from individuals F1-II:3 and F5-II:2 showed a more severe peroxisomal phenotype when cultured at an elevated temperature (40°C), and the vast majority of cells lacked catalase-positive peroxisomes (Figure 3). Electron microscopy of fibroblasts cultured at normal temperature revealed no striking ultra-structural abnormalities of peroxisomes (Figure S3).