The molecular etiology of pseudoxanthoma elasticum (PXE), an autosomal recessive connective tissue disorder, has become increasingly complex as not only mutations in ABCC6 but also ENPP1 and GGCX can cause resembling phenotypes. Identification of modifier genes, such as VEGFA, has further contributed to the molecular heterogeneity of PXE. In such heterogeneous diseases, Next Generation Sequencing allows to perform mutation screening of several genes in a single reaction. We explored whole exome sequencing (WES) as an efficient diagnostic tool to identify the causal mutations in ABCC6, GGCX, ENPP1 and VKORC1, in 16 PXE patients. WES identified a causal ABCC6 mutation in 30 out of 32 alleles and one GGCX mutation, whereas no causal mutations in ENPP1 or VKORC1 were detected. Exomes with insufficient reads (≤20 depth) for the 4 genes and patients with single mutations were further evaluated by Sanger sequencing (SS) but no additional mutations were found. The potential of WES compared to targeted NGS, is the ease to examine target genes and the opportunity to search for novel genes when targeted analysis is negative. Together with low cost, rapid and less laborious workflow, we conclude that WES complemented with SS can provide a tiered approach to molecular diagnostics of PXE.Journal of Investigative Dermatology accepted article preview online, 29 September 2014; doi:10.1038/jid.2014.421.