With regard to skin differentiation and barrier formation, selected alterations in gene expression are presented in Table S11. We also verified potential changes by performing quantitative PCR (qPCR) with RNA from whole skin of three individuals from the two pedigrees, as well as immortalized keratinocytes and primary fibroblasts from one affected person (see Supplemental Data for methods and controls). We observed reduced expression of GRHL2 for all templates and a contrasting increase in GRHL1 (MIM 609786) and GRHL3 (MIM 608317) expression: unique and cooperative roles for this transcription factor family have been previously documented.24 The most marked skin-barrier-associated gene changes were upregulation of aquaporin-encoding genes AQP5 (MIM 600442) and AQP7 (MIM 602974), the latter of which was expressed 50×–100× more in affected skin than in control skin. Gain-of-function mutations in AQP5 have previously been associated with a form of autosomal-dominant nonepidermolytic palmoplantar keratoderma (MIM 600962).25 Two-fold or greater reduction in gene expression was noted for S100A8 (MIM 123885) and S100A9 (MIM 123886), known targets for GRHL1. Previously, it has also been shown that GRHL2 enhances skin-barrier function by upregulating the tight-junction components claudins 3 and 4 and also Rab25, which localizes claudin 4 to tight junctions.26 In affected people, we noted increases in CLDN3 (MIM 602910), CLDN4 (MIM 602909), and RAB25 (MIM 612942) expression in whole skin (transcriptome and qPCR) and cultured keratinocytes (qPCR). Increased claudin 4 immunolabeling was also noted in the skin of two affected individuals (Figure S5).