In mouse and humans, the X-chromosomal Porcupine homolog (Porcn) gene is required for the acylation and secretion of all 19 Wnt ligands, thus representing a bottleneck in the secretion of Wnt ligands. In humans, mutations in PORCN cause the X-linked dominant syndrome Focal Dermal Hypoplasia (FDH, OMIM#305600). This disorder is characterized by ecto-mesodermal dysplasias and shows a highly variable phenotype, potentially due to individual X chromosome inactivation patterns. To improve the understanding of human FDH, we have established a mouse model by generation of Porcn heterozygous animals carrying a zygotic deletion of the paternal allele. We show that heterozygous female fetuses display variable defects that do not significantly affect survival in the uterus, but lead to perinatal lethality in more than 95% of females. Rare survivors develop to adulthood and display variable skeletal and skin defects, representing an adult zygotic mouse model for human FDH. Although not frequently reported in humans, we also observed bronchopneumonia, rhinitis, and otitis media in these animals, suggesting a potential link between Porcn function and the normal development of ciliated cells in these tissues.

We encountered three women from three generations of the same family with features of focal dermal hypoplasia (FDH). Two of the patients, the proposita and her mother, demonstrated severe manifestations, including skin, dental, skeletal, and visceral abnormalities. The proposita's grandmother, the first family member affected, had very mild expression, with aplasia cutis congenita and dental caries as the only features expressed. This family illustrates both the marked variability of expression and the proposed X-linked dominant mode of inheritance of FDH. We postulate that early embryologic random inactivation of the X chromosome bearing the mutant gene responsible for FDH is the cause of the variable expression.

Mutations in the PORCN gene cause the X-linked dominant condition focal dermal hypoplasia (FDH). Features of FDH include striated pigmentation of the skin, ocular and skeletal malformations. FDH is generally associated with in utero lethality in non-mosaic males and most of the currently reported male patients show mosaicism due to de novo post-zygotic mutations in the PORCN gene. There is only one previous report of a surviving male with an inherited mutation in the PORCN gene. Here, we report two male siblings with multiple malformations including skeletal, ocular and renal defects overlapping with FDH. A novel PORCN mutation (p.Ser250Phe) was identified in a non-mosaic, hemizygous state in one of the siblings who survived to 8 years of age. The mother is a heterozygous carrier, has a random X-inactivation pattern and is asymptomatic. Findings unusual for FDH include dysplastic clavicles and bilateral Tessier IV facial clefts. This is the second case report of a non-mosaic PORCN mutation in a male individual with multiple congenital anomalies. While the pathogenicity of this mutation remains to be further investigated, the survival of a male with a non-mosaic mutation in PORCN is suggestive of a functionally mild mutation leading to an X-linked recessive mode of inheritance.

Focal Dermal Hypoplasia (FDH) is a genetic disorder characterized by developmental defects in skin, skeleton and ectodermal appendages. FDH is caused by dominant loss-of-function mutations in X-linked PORCN. PORCN orthologues in Drosophila and mice encode endoplasmic reticulum proteins required for secretion and function of Wnt proteins. Wnt proteins play important roles in embryo development, tissue homeostasis and stem cell maintenance. Since features of FDH overlap with those seen in mouse Wnt pathway mutants, FDH likely results from defective Wnt signaling but molecular mechanisms by which inactivation of PORCN affects Wnt signaling and manifestations of FDH remain to be elucidated.

Gradients of diverse Wnt proteins regulate development, renewal, and differentiation. Porcupine (PORCN) is a membrane-bound O-acyltransferase that is required for post-translational modification of all Wnts to enable their transport, secretion, and activity. Mutations in PORCN are associated with focal dermal hypoplasia (FDH), whereas gene deletion causes embryonic lethality in mice. To study the protein in more detail, zinc finger nucleases were used to edit the PORCN genomic locus, establishing two HT1080 fibrosarcoma clones null for PORCN activity that facilitate the study of PORCN structure and function. We establish that PORCN is a key non-redundant node for the regulation of global Wnt signaling because PORCN null cells are completely incapable of autocrine Wnt signaling. The strength of Wnt signaling is exquisitely sensitive to PORCN expression, with a dynamic range of at least 3 orders of magnitude, suggesting that PORCN activity is a key modulator of all Wnt ligand activity. Consistent with this, we find that multiple FDH-associated mutants have only subtle alterations in enzyme activity yet are associated with a severe FDH phenotype. These studies support an essential regulatory role of PORCN in shaping Wnt signaling gradients.

Wnt gradients elicit distinct cellular responses, such as proliferation, specification, differentiation and survival in a dose-dependent manner. Porcupine (PORCN), a membrane-bound O-acyl transferase (MBOAT) that resides in the endoplasmic reticulum, catalyses the addition of monounsaturated palmitate to Wnt proteins and is required for Wnt gradient formation and signalling. In humans, PORCN mutations are causal for focal dermal hypoplasia (FDH), an X-linked dominant syndrome characterized by defects in mesodermal and endodermal tissues. PORCN is also an emerging target for cancer therapeutics. Despite the importance of this enzyme, its structure remains poorly understood. Recently, the crystal structure of DltB, an MBOAT family member from bacteria, was solved. In this report, we use experimental data along with homology modelling to DltB to determine the membrane topology of PORCN. Our studies reveal that PORCN has 11 membrane domains, comprising nine transmembrane spanning domains and two reentrant domains. The N-terminus is oriented towards the lumen while the C-terminus is oriented towards the cytosol. Like DltB, PORCN has a funnel-like structure that is encapsulated by multiple membrane-spanning helices. This new model for PORCN topology allows us to map residues that are important for biological activity (and implicated in FDH) onto its three-dimensional structure.

Microphthalmia, anophthalmia, and coloboma (MAC) are congenital ocular malformations causing 25% of childhood blindness. The X-linked disorder Focal Dermal Hypoplasia (FDH) is frequently associated with MAC and results from mutations in Porcn, a membrane bound O-acyl transferase required for palmitoylation of Wnts to activate multiple Wnt-dependent pathways. Wnt/β-catenin signaling is suppressed in the anterior neural plate for initiation of eye formation and is subsequently required during differentiation of the retinal pigment epithelium (RPE). Non-canonical Wnts are critical for early eye formation in frog and zebrafish. However, it is unclear whether this also applies to mammals. We performed ubiquitous conditional inactivation of Porcn in mouse around the eye field stage. In Porcn CKO , optic vesicles (OV) arrest in growth and fail to form an optic cup. Ventral proliferation is significantly decreased in the mutant OV, with a concomitant increase in apoptotic cell death. While pan-ocular transcription factors such as PAX6, SIX3, LHX2, and PAX2 are present, indicative of maintenance of OV identity, regional expression of VSX2, MITF, OTX2, and NR2F2 is downregulated. Failure of RPE differentiation in Porcn CKO is consistent with downregulation of the Wnt/β-catenin effector LEF1, starting around 2.5 days after inactivation. This suggests that Porcn inactivation affects signaling later than a potential requirement for Wnts to promote eye field formation. Altogether, our data shows a novel requirement for Porcn in regulating growth and morphogenesis of the OV, likely by controlling proliferation and survival. In FDH patients with ocular manifestations, growth deficiency during early ocular morphogenesis may be the underlying cause for microphthalmia.