PORCN Mutations Research Articles

Porcn is essential for growth and invagination of the mammalian optic cup.

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 2.5days 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.

Novel uses of laser therapy in Goltz syndrome

Goltz syndrome is an X-linked dominant, multisystem birth defect due to PORCN mutation. The skin findings follow Blaschko's lines and often show epidermal atrophy and herniation of subcutaneous fatty tissue. Regarding treatment, light sources can offer a good therapeutic option for some manifestations of this rare disease and improve the aesthetic appearance of the skin lesions. We report two new cases of Goltz syndrome in which the cutaneous findings remarkably improved with pulsed dye laser and carbon dioxide laser. This article is protected by copyright. All rights reserved.

Novel insights into PORCN mutations, associated phenotypes and pathophysiological aspects

BackgroundGoltz syndrome (GS) is a X-linked disorder defined by defects of mesodermal- and ectodermal-derived structures and caused by PORCN mutations. Features include striated skin-pigmentation, ocular and skeletal malformations and supernumerary or hypoplastic nipples. Generally, GS is associated with in utero lethality in males and most of the reported male patients show mosaicism (only three non-mosaic surviving males have been described so far). Also, precise descriptions of neurological deficits in GS are rare and less severe phenotypes might not only be caused by mosaicism but also by less pathogenic mutations suggesting the need of a molecular genetics and functional work-up of these rare variants.ResultsWe report two cases: one girl suffering from typical skin and skeletal abnormalities, developmental delay, microcephaly, thin corpus callosum, periventricular gliosis and drug-resistant epilepsy caused by a PORCN nonsense-mutation (c.283C > T, p.Arg95Ter). Presence of these combined neurological features indicates that CNS-vulnerability might be a guiding symptom in the diagnosis of GS patients. The other patient is a boy with a supernumerary nipple and skeletal anomalies but also, developmental delay, microcephaly, cerebral atrophy with delayed myelination and drug-resistant epilepsy as predominant features. Skin abnormalities were not observed. Genotyping revealed a novel PORCN missense-mutation (c.847G > C, p.Asp283His) absent in the Genome Aggregation Database (gnomAD) but also identified in his asymptomatic mother. Given that non-random X-chromosome inactivation was excluded in the mother, fibroblasts of the index had been analyzed for PORCN protein-abundance and -distribution, vulnerability against additional ER-stress burden as well as for protein secretion revealing changes.ConclusionsOur combined findings may suggest incomplete penetrance for the p.Asp283His variant and provide novel insights into the molecular etiology of GS by adding impaired ER-function and altered protein secretion to the list of pathophysiological processes resulting in the clinical manifestation of GS.

Two new patients with focal dermal hypoplasia: A novel PORCN variant and insights on the diagnostic considerations.

Mutations in the PORCN gene cause an X-linked dominant condition; focal dermal hypoplasia (FDH), characterized by atrophic skin, pigmented skin lesions in addition to several ocular and skeletal malformations. FDH is rare with around 275 cases reported so far from diverse ethnic groups. Herein, we provide a report of two new patients with FDH from Egypt. In addition to the typical clinical manifestations of the disease, infrequently reported clinical findings in the form of broad metaphysis, bilateral short broad femurs, and dermal sinus over the sacrum were seen in Patient 1 and partial fusion of labia majora, ventral hernia, and bladder extrophy were present in Patient 2. Two heterozygous protein-truncating PORCN mutations were identified in our patients, a known nonsense c.370C>T p.(Arg124Ter) and a novel frameshift c.375delG p.(Ala126HisfsTer3). Segregation analyses confirmed that the two mutations were "de novo" and not inherited from any of the parents. Our study expands the clinical and mutational spectrum of focal dermal hypoplasia and emphasizes the importance of investigating the different body systems and organs for the early management of patients.

Structural model of human PORCN illuminates disease-associated variants and drug-binding sites.

Wnt signaling is essential for normal development and is a therapeutic target in cancer. The enzyme PORCN, or porcupine, is a membrane-bound O-acyltransferase (MBOAT) that is required for the post-translational modification of all Wnts, adding an essential mono-unsaturated palmitoleic acid to a serine on the tip of Wnt hairpin 2. Inherited mutations in PORCN cause focal dermal hypoplasia, and therapeutic inhibition of PORCN slows the growth of Wnt-dependent cancers. Based on homology to mammalian MBOAT proteins, we developed and validated a structural model of human PORCN. The model accommodates palmitoleoyl-CoA and Wnt hairpin 2 in two tunnels in the conserved catalytic core, shedding light on the catalytic mechanism. The model predicts how previously uncharacterized human variants of uncertain significance can alter PORCN function. Drugs including ETC-159, IWP-L6 and LGK-974 dock in the PORCN catalytic site, providing insights into PORCN pharmacologic inhibition. This structural model enhances our mechanistic understanding of PORCN substrate recognition and catalysis, as well as the inhibition of its enzymatic activity, and can facilitate the development of improved inhibitors and the understanding of disease-relevant PORCN mutants. This article has an associated First Person interview with the joint first authors of the paper.

The Role of Genetics Mutations in Genes PORCN, TWIST2, HCCS in Goltz Syndrome

The Role of Genetics Mutations in Genes PORCN, TWIST2, HCCS in Goltz Syndrome

A non-mosaic PORCN mutation in a male with severe congenital anomalies overlapping focal dermal hypoplasia

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 8years 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.

Dermatologic findings of focal dermal hypoplasia (Goltz syndrome).

Goltz syndrome, caused by mutations in PORCN, is an X-linked dominant ectodermal dysplasia which is also known as focal dermal hypoplasia. This name is derived from the predominant pathologic skin findings of the syndrome. Nineteen Goltz-affected participants attended a multidisciplinary scientific and clinical conference convened by the National Foundation for Ectodermal Dysplasia which allowed further characterization of the features of this very rare condition. At birth, the affected areas of skin are typically erythematous and fragile. The hallmark cutaneous features, which vary widely due to mosacism and X-inactivation, include the previously described skin changes of asymmetric Blaschko-linear and reticulated atrophy, pigmentary changes, and telangectasias. Lipomatous changes and papillomas as characteristically defined were reported in the majority of patients. A newly recognized skin finding was progressive hyperpigmented freckling that occurred within the hypopigmented areas which were noted to be photosensitive. Many patients also had a pebbly texture to the central face, dorsal hands and feet. Punctate erosions within the atrophic areas and hypohidrosis were also common. Most had patchy alopecia and many had diffusely thin hair. Scanning electron microscopy of the hair shafts revealed abnormalities in the majority of participants with several different features identified, including atrophic hairs with reduced diameters, markedly flattened hairs as noted in cross-sectional views, trichorrhexis nodosa, pili torti, and pili trianguli et canaliculi. Nail changes included V-nicking and longitudinal ridging of the nail plate, in addition to micronychia. Early recognition of the dermatologic features, in addition to the variable but universal limb anomalies, of Goltz syndrome will allow early and accurate diagnosis without the need for extensive diagnostic studies, while also allowing for accurate prognosis and appropriate genetic counseling.

Identification of Key Residues and Regions Important for Porcupine-mediated Wnt Acylation

Wnts comprise a family of lipid-modified, secreted signaling proteins that control embryogenesis, as well as tissue homeostasis in adults. Post-translational attachment of palmitoleate (C16:1) to a conserved Ser in Wnt proteins is catalyzed by Porcupine (Porcn), a member of the membrane bound O-acyltransferase (MBOAT) family, and is required for Wnt secretion and signaling. Moreover, genetic alterations in the PORCN gene lead to focal dermal hypoplasia, an X-linked developmental disorder. Despite its physiological importance, the biochemical mechanism governing Wnt acylation by Porcn is poorly understood. Here, we use a cell-based fatty acylation assay that is a direct readout of Porcn acyltransferase activity to perform structure-function analysis of highly conserved residues in Porcn and Wnt3a. In total, 16-point mutations in Porcn and 13 mutations in Wnt3a were generated and analyzed. We identified key residues within Porcn required for enzymatic activity, stability, and Wnt3a binding and mapped these active site residues to predicted transmembrane domain 9. Analysis of focal dermal hypoplasia-associated mutations in Porcn revealed that loss of enzymatic activity arises from altered stability. A consensus sequence within Wnt3a was identified (CXCHGXSXXCXXKXC) that contains residues that mediate Porcn binding, fatty acid transfer, and Wnt signaling. We also showed that Ser or Thr, but not Cys, can serve as a fatty acylation site in Wnt, establishing Porcn as an O-acyltransferase. This analysis sheds light into the mechanism by which Porcn transfers fatty acids to Wnt proteins and provides insight into the mechanisms of fatty acid transfer by MBOAT family members.

Characterizing the effect of porcupine on neural tube closure (979.3)

Incomplete closure of the embryonic neural tube is responsible for common birth defects such as spina bifida. Wnts, a group of secreted lipid‐modified signaling proteins, are known to play a critical role in embryonic spinal cord development. Wnt proteins are palymitoylated by the enzyme Porcupine (Porcn), a transmembrane protein localized to the endoplasmic reticulum and Golgi apparatus. The proposed function of palmitoyation is to facilitate the secretion of Wnt proteins. Mutations in human Porcn can result in Focal Dermal Hypoplasia that can manifest multiple abnormalities including neural tube defects. Thus it is hypothesized that inhibition of Porcn in early stage chick embryos will cause abnormal neural tube closure. To characterize Porcn's role in neural tube closure, we developed an in vitro culture method that permitted addition of small molecule inhibitors of Porcn IWP‐1, IWP‐12 and C‐59. Surprisingly, although all of the compounds tested inhibited human Porcn, only IWP‐12 appeared to inhibit chick Porcn. Treatment with IWP‐12 revealed neural tube defects and decreased cell elongation which is consistent with a model in which IWP‐12 inhibits Wnt signaling via the planar cell polarity pathway. To test whether IWP‐1 and C‐59 are species specific inhibitors, we are currently comparing the ability of all three compounds to inhibit human and chick Porcn using an assay that measures Wnt palmitoylation.Grant Funding Source: Supported by NSF RUI MCB‐1244602, NIH‐RIMI P20MD000262, Area Grant NIH #1R15HD070206‐01A1

Focal dermal hypoplasia without focal dermal hypoplasia

Focal dermal hypoplasia (FDH; Goltz-Gorlin syndrome) is an X-linked dominant disorder affecting mainly tissues of ectodermal and mesodermal origin. The phenotype is characterized by hypoplastic linear skin lesions, eye malformations, hair and teeth anomalies, and multiple limbs malformations. The disorder is caused by PORCN mutations. Here we describe a mother and daughter with FDH in whom a c.938T>G in PORCN was detected. Neither of the two had FDH, but otherwise the phenotype was classical. Focal skin hypoplasia is a hallmark of FDH but the present family indicates that FDH should also be considered in absence of this skin manifestation.

Zygotic Porcn Paternal Allele Deletion in Mice to Model Human Focal Dermal Hypoplasia

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.

Novel and recurrent PORCN gene mutations in almost unilateral and typical focal dermal hypoplasia patients

Goltz syndrome or Focal dermal hypoplasia (FDH) is an X-linked dominant disorder characterized by malformations affecting the skin, eyes, central nervous system, and skeletonC:\GetARef\Refs\focal dermal hypoplasia (2007-).re. Mutations in the PORCN gene were identified as the molecular basis of FDH. We report two cases, one caused by a current mutation c.129G>A, which leads to a nonsense mutation W43X, and the other one caused by a novel mutation, c.386delT. The female patient with the recurrent mutation presented with typical cutaneous symptoms and skeletal abnormality, but the female patient with the novel mutation manifested only cutaneous symptoms, with hypo-pigmentation along Blaschko's lines, mainly on her right hemibody. In the latter case, DNA was isolated from peripheral blood cells, lesional skin, and non-lesional skin. The percentage of cells carrying the mutation estimated by subcloning and sequencing of the PCR products was 3.1% in peripheral blood cells, 21% in lesional skin, and 16% in non-lesional skin. X-chromosome inactivation assay showed a slightly skewed pattern in lesional skin, but a random pattern in non-lesional skin and blood. RT-PCR analysis from skin samples showed that PORCN mRNA of the mutated allele had a 13bp nucleotide insertion created by an alternative splicing site. This resulted in abnormal PORCN protein with in-frame insertion of eight amino acids, TTHRGTDD, instead of the original four amino acids, AQMI (126-129). We report a typical FDH patient with a recurrent PORCN mutation, which was previously identified in a male Japanese FDH patient, and a second female, an almost unilateral FDH patient with a postzygotic PORCN mutation.

Deletion of Porcn in Mice Leads to Multiple Developmental Defects and Models Human Focal Dermal Hypoplasia (Goltz Syndrome)

BackgroundFocal 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.ResultsWe introduced intronic loxP sites and a neomycin gene in the mouse Porcn locus for conditional inactivation. Porcn-ex3-7flox mice have no apparent developmental defects, but chimeric mice retaining the neomycin gene (Porcn-ex3-7Neo-flox) have limb, skin, and urogenital abnormalities. Conditional Porcn inactivation by EIIa-driven or Hprt-driven Cre recombinase results in increased early embryonic lethality. Mesenchyme-specific Prx-Cre-driven inactivation of Porcn produces FDH-like limb defects, while ectodermal Krt14-Cre-driven inactivation produces thin skin, alopecia, and abnormal dentition. Furthermore, cell-based assays confirm that human PORCN mutations reduce WNT3A secretion.ConclusionsThese data indicate that Porcn inactivation in the mouse produces a model for human FDH and that phenotypic features result from defective WNT signaling in ectodermal- and mesenchymal-derived structures.

Focal dermal hypoplasia: report of a Taiwanese case

Focal dermal hypoplasia (FDH), or Goltz syndrome, is a rare X-linked dominant ectodermal and mesodermal disease involving the skin, distal limbs, and eyes. About 95% of the cases appear de novo , and 90% are females. Recent studies reveal that FDH is caused by mutations in the PORCN gene. Female patients are either heterozygous or mosaic for PORCN mutations, whereas all male patients are mosaic. About 5% of the female patients analyzed have no detectable mutations or microdeletions of PORCN gene. We report a sporadic case of FDH in a 16-year-old girl presenting with atrophic or erythematous macules and patches distributed along the lines of Blaschko over the trunk and extremities with prominent soft yellowish fat herniation over the left axilla and left groin, and papillomas in the oral and genital areas. Multiple developmental anomalies of the digits and ear were also noted. Histopathology of the skin lesion revealed severe dermal hypoplasia. Mutation analysis of all coding regions and flanking intron boundaries of the genomic DNA revealed no detectable mutation of the PORCN gene. Our case manifested mucocutaneous and multiple developmental anomalies typical of FDH, but no mutation in the PORCN gene was detected by mutation analysis.

Porcupine homolog is required for canonical Wnt signaling and gastrulation in mouse embryos

Wnt signaling plays important roles in development and disease. The X-chromosomal Porcupine homolog gene (Porcn) encodes an evolutionary conserved member of the membrane bound O-acyl transferase (MBOAT) superfamily that has been shown to be required for the palmitoylation and secretion of Wnt3a, a mechanism that has been suggested to be conserved for all mammalian Wnt ligands. PORCN mutations in humans cause Focal Dermal Hypoplasia (FDH), a disorder causing developmental defects in heterozygous females and embryonic lethality in hemizygous males. In this study, Porcn mutant mouse embryonic stem (ES) cells were used to analyze the role of Porcn in mammalian embryonic development. In vitro, we show an exclusive requirement for Porcn in Wnt secreting cells and further, that any of the four Porcn isoforms is sufficient to allow for the secretion of functional Wnt3a. Embryos generated by aggregation of Porcn mutant ES cells with wildtype embryos fail to complete gastrulation in vivo, but remain in an epiblast-like state, similar to Wnt3 and Gpr177/Wls mutants. Consistent with this phenotype, in vitro differentiated mutant ES cells fail to generate endoderm and mesoderm derivatives. Taken together, these data confirm the importance of Porcn for Wnt secretion and gastrulation and suggest that disruption of early development underlies the male lethality of human PORCN mutants.

Focal dermal hypoplasia in a male patient due to mosaicism for a novel PORCN single nucleotide deletion

Focal dermal hypoplasia (FDH) is an X-linked dominant disorder caused by nonsense mutations and deletions in the PORCN gene coding for a transmembrane endoplasmic reticulum protein required for Wingless signalling. Symptoms consist mainly of linear atrophic skin defects, skeletal deformities and, in many cases, mental retardation. Osteopathia striata is a nearly constant feature. Approximately 90% of patients are women. A few instances of father-to-daughter transmission and a number of sporadic male cases presumably as a result of somatic mosaicism have been recorded. The aim of this study was to demonstrate the presence of somatic mosaicism for PORCN mutations in a male patient. We sequenced the PORCN gene in different tissues from a boy with symptoms of FDH. We demonstrate post-zygotic mosaicism for a novel deletion in the PORCN gene. A novel PORCN deletion, present in a post-zygotic mosaic, causes focal dermal hyplasia in a male patient.

Co‐occurrence of severe Goltz–Gorlin syndrome and pentalogy of Cantrell – Case report and review of the literature

Goltz-Gorlin syndrome is a highly variable disorder affecting many body parts of meso-ectodermal origin. Mutations in X-linked PORCN have been identified in almost all patients with a classical Goltz-Gorlin phenotype. The pentalogy of Cantrell is an infrequently described congenital disorder characterized by the combination of five anomalies: a midline supra-umbilical abdominal wall defect; absent or cleft lower part of the sternum; deficiency of the diaphragmatic pericardium; deficiency of the anterior diaphragm; and congenital heart anomalies. Etiology and pathogenesis are unknown. We report on an infant with findings fitting both Goltz-Gorlin syndrome (sparse hair; anophthalmia; clefting; bifid nose; irregular vermillion of both lips; asymmetrical limb malformations; caudal appendage; linear aplastic skin defects; unilateral hearing loss) and the pentalogy of Cantrell (absent lower sternum; anterior diaphragmatic hernia; ectopia cordis; omphalocele). The clinical diagnosis Goltz-Gorlin syndrome was confirmed molecularly by a point mutation in PORCN (c.727C>T). The presence of molecularly confirmed Goltz-Gorlin syndrome and pentalogy of Cantrell in a single patient has been reported twice before. The present patient confirms that the pentalogy of Cantrell can be caused in some patients by a PORCN mutation. It remains at present uncertain whether this can be explained by the type or localization of the mutation within PORCN, or whether the co-occurrence of the two entities is additionally determined by mutations or polymorphisms in other genes, environmental factors, and/or epigenetic influences.

A novel PORCN mutation in focal dermal hypoplasia

A novel PORCN mutation in focal dermal hypoplasia

Survival of a Male Mosaic for PORCN Mutation with Mild Focal Dermal Hypoplasia Phenotype

We report a 46,XY boy with a mild focal dermal hypoplasia phenotype who had both wild-type and mutated copies of the PORCN gene and was, therefore, mosaic for the mutation. He had cutaneous syndactyly, hydronephrosis, and nail dystrophy. Small whitish depigmented spots, which were slightly depressed from the skin surface, were distributed linearly on the trunk and arms. Aside from these findings, streaks of brown-pigmented macules were seen on the dorsal aspect of the legs. Both the linear arrangement of the whitish spots and the streaks of pigmented macules followed the lines of Blaschko. The phenotype of the patient, who did not exhibit cribriform atrophy, telangiectasia or fat herniation, seemed to be much milder than that of typical female patients with focal dermal hypoplasia. Analysis of the genomic DNA extracted from the peripheral lymphocytes revealed a transition 129G>A within exon 1 of PORCN, which leads to a nonsense mutation W43X. The percentage of peripheral lymphocytes carrying a mutation was estimated to be 50% by the subcloning and sequencing of individual clones of the PCR product amplified across the mutation. This patient's case history provides further molecular evidence supporting the concept that "male focal dermal hypoplasia" does exist and that typical features such as telangiectasia and fat herniation are sometimes absent.