Dysregulation of NEUROG2 plays a key role in focal cortical dysplasia.

OBJECTIVE: Focal cortical dysplasias (FCDs) are an important cause of drug-resistant epilepsy. In this work, we aimed to investigate whether abnormal gene regulation, mediated by microRNA, could be involved in FCD type II. METHODS: We used total RNA from the brain tissue of 16 patients with FCD type II and 28 controls. MicroRNA expression was initially assessed by microarray. Quantitative polymerase chain reaction, in situ hybridization, luciferase reporter assays, and deep sequencing for genes in the mTOR pathway were performed to validate and further explore our initial study. RESULTS: hsa-let-7f (p = 0.039), hsa-miR-31 (p = 0.0078), and hsa-miR34a (p = 0.021) were downregulated in FCD type II, whereas a transcription factor involved in neuronal and glial fate specification, NEUROG2 (p < 0.05), was upregulated. We also found that the RND2 gene, a NEUROG2-target, is upregulated (p < 0.001). In vitro experiments showed that hsa-miR-34a downregulates NEUROG2 by binding to its 5'-untranslated region. Moreover, we observed strong nuclear expression of NEUROG2 in balloon cells and dysmorphic neurons and found that 28.5% of our patients presented brain somatic mutations in genes of the mTOR pathway. INTERPRETATION: Our findings suggest a new molecular mechanism, in which NEUROG2 has a pivotal and central role in the pathogenesis of FCD type II. In this way, we found that the downregulation of hsa-miR-34a leads to upregulation of NEUROG2, and consequently to overexpression of the RND2 gene. These findings indicate that a faulty coupling in neuronal differentiation and migration mechanisms may explain the presence of aberrant cells and complete dyslamination in FCD type II. Ann Neurol 2018;83:623-635.

An in vitro fatty acylation assay reveals a mechanism for Wnt recognition by the acyltransferase Porcupine.

Wnt proteins are a family of secreted signaling proteins that play key roles in regulating cell proliferation in both embryonic and adult tissues. Production of active Wnt depends on attachment of palmitoleate, a monounsaturated fatty acid, to a conserved serine by the acyltransferase Porcupine (PORCN). Studies of PORCN activity relied on cell-based fatty acylation and signaling assays as no direct enzyme assay had yet been developed. Here, we present the first in vitro assay that accurately recapitulates PORCN-mediated fatty acylation of a Wnt substrate. The critical feature is the use of a double disulfide-bonded Wnt peptide that mimics the two-dimensional structure surrounding the Wnt acylation site. PORCN-mediated Wnt acylation was abolished when the Wnt peptide was treated with DTT, and did not occur with a linear (non-disulfide-bonded) peptide, or when the double disulfide-bonded Wnt peptide contained Ala substituted for the Ser acylation site. We exploited this in vitro Wnt acylation assay to provide direct evidence that the small molecule LGK974, which is in clinical trials for managing Wnt-driven tumors, is a bona fide PORCN inhibitor whose IC50 for inhibition of Wnt fatty acylation in vitro closely matches that for inhibition of Wnt signaling. Side-by-side comparison of PORCN and Hedgehog acyltransferase (HHAT), two enzymes that attach 16-carbon fatty acids to secreted proteins, revealed that neither enzyme will accept the other's fatty acyl-CoA or peptide substrates. These findings illustrate the unique enzyme-substrate selectivity exhibited by members of the membrane-bound O-acyl transferase family.

Revisiting histopathologic findings in Goltz syndrome.

Goltz syndrome (focal dermal hypoplasia) is an X-linked dominant disorder that is classically associated with yellowish papules representing fat herniation (superficial adipocytes). We report a series of three cases, with clinicopathologic correlation of biopsies from Blaschkoid streaks. A range of histopathologic features, including some underreported findings (increased papillary dermal blood vessels, decreased thickness of the dermis, and adipocytes high in the dermis), are reproducible and can strongly point to the correct diagnosis of Goltz syndrome.

Genetically engineered mouse models to evaluate the role of Wnt secretion in bone development and homeostasis.

Alterations in components of the Wnt signaling pathway are associated with altered bone development and homeostasis in several human diseases. We created genetically engineered mouse models (GEMMs) that mimic the cellular defect associated with the Porcupine mutations in patients with Goltz Syndrome/Focal Dermal Hypoplasia. These GEMMs were established by utilizing mice containing a conditionally inactivatable allele of Wntless/GPR177 (a gene encoding a protein required for the transport of Porcupine-modified ligand to the plasma membrane for secretion). We crossed this strain to another which drives cre-mediated gene deletion in mature osteoblasts (Osteocalcin-cre) resulted in mice lacking the ability to secrete Wnt ligands in this cell type. These mice displayed severely reduced bone mass and provide a model to understand the effects of disrupting the ability to secrete Wnt ligands on the skeletal system.

Multiple requirements of the focal dermal hypoplasia gene porcupine during ocular morphogenesis.

Wnt glycoproteins control key processes during development and disease by activating various downstream pathways. Wnt secretion requires post-translational modification mediated by the O-acyltransferase encoded by the Drosophila porcupine homolog gene (PORCN). In humans, PORCN mutations cause focal dermal hypoplasia (FDH, or Goltz syndrome), an X-linked dominant multisystem birth defect that is frequently accompanied by ocular abnormalities such as coloboma, microphthalmia, or even anophthalmia. Although genetic ablation of Porcn in mouse has provided insight into the etiology of defects caused by ectomesodermal dysplasia in FDH, the requirement for Porcn and the actual Wnt ligands during eye development have been unknown. In this study, Porcn hemizygosity occasionally caused ocular defects reminiscent of FDH. Conditional inactivation of Porcn in periocular mesenchyme led to defects in mid- and hindbrain and in craniofacial development, but was insufficient to cause ocular abnormalities. However, a combination of conditional Porcn depletion in optic vesicle neuroectoderm, lens, and neural crest-derived periocular mesenchyme induced severe eye abnormalities with high penetrance. In particular, we observed coloboma, transdifferentiation of the dorsal and ventral retinal pigment epithelium, defective optic cup periphery, and closure defects of the eyelid, as well as defective corneal morphogenesis. Thus, Porcn is required in both extraocular and neuroectodermal tissues to regulate distinct Wnt-dependent processes during morphogenesis of the posterior and anterior segments of the eye.

Histologic and immunohistochemical characteristics of cutaneous cysts in Goltz-Gorlin syndrome: clues for differentiation of nonsyndromic cysts.

Goltz-Gorlin syndrome presents with multiple basal cell carcinomas, odontogenic keratocysts, and cutaneous cysts, among other manifestations. The cutaneous cysts have been described as both epidermoid cysts and keratocysts but were not further characterized. Light microscopic examinations were made on 23 cutaneous cysts in 4 patients associated with Goltz-Gorlin syndrome located on extremities, face, trunk, palms, and soles and compared with nonsyndromic vellus hair cysts, steatocystomas, and hybrid cysts. Twenty-one of the syndromic cysts revealed alternating infundibular-like and steatocystoma-like squamous epitheliums in varying proportions. The cysts were lined by both smooth and corrugated squamous epithelium. The horny layer was composed by alternating areas of thin, lamellate, and compact eosinophilic keratin. Only 2 cases showed an exclusive steatocystoma-like type of epithelium very similar to odontogenic keratocysts. Sebaceous glands and follicular structures were absent. There were no differences between palmar and plantar cysts and other anatomic locations. The ultrastructural findings in syndromatic cysts confirmed variable expression of keratohyalin granules. Only 3 of 6 cases of nonsyndromic hybrid cysts showed overlapping features with syndromic cysts. Immunohistochemical profiling of keratin, involucrin, filaggrin, loricrin, and BCL-2 expression in syndromatic cysts showed exclusive positivity of K19 and continuous staining for BCL-2. In summary, 2 types of cutaneous cysts are characteristic of Goltz-Gorlin, irrelevant of their anatomic location, namely steatocystoma-like and more frequently hybrid-like. The diagnosis of syndromic hybrid-like cysts should be considered whenever infundibular and steatocystoma differentiation alternate and overlap. Altogether, these findings in epithelial cysts may raise the suspicion of Goltz-Gorlin as an underlying cause.

Precise regulation of porcupine activity is required for physiological Wnt signaling.

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.

Deletion of Porcn in mice leads to multiple developmental defects and models human focal dermal hypoplasia (Goltz syndrome).

BACKGROUND: 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. RESULTS: We 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. CONCLUSIONS: These 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.

Nonsense mutations of the bHLH transcription factor TWIST2 found in Setleis Syndrome patients cause dysregulation of periostin.

Setleis Syndrome (OMIM ID: 227260) is a rare autosomal recessive disease characterized by abnormal facial development. Recently, we have reported that two nonsense mutations (c.486C>T [Q119X] and c.324C>T [Q65X]) of the basic helix-loop-helix (bHLH) transcription factor TWIST2 cause Setleis Syndrome. Here we show that periostin, a cell adhesion protein involved in connective tissue development and maintenance, is down-regulated in Setleis Syndrome patient fibroblast cells and that periostin positively responds to manipulations in TWIST2 levels, suggesting that TWIST2 is a transactivator of periostin. Functional analysis of the TWIST2 mutant form (Q119X) revealed that it maintains the ability to localize to the nucleus, forms homo and heterodimers with the ubiquitous bHLH protein E12, and binds to dsDNA. Reporter gene assays using deletion constructs of the human periostin promoter also reveal that TWIST2 can activate this gene more specifically than Twist1, while the Q119X mutant results in no significant transactivation. Chromatin immunoprecipitation assays show that both wild-type TWIST2 and the Q119X mutant bind the periostin promoter, however only wild-type TWIST2 is associated with higher levels of histone acetylation across the 5'-regulatory region of periostin. Taken together, these data suggest that the C-terminal domain of TWIST2, which is missing in the Q119X mutant form of TWIST2, is responsible for proper transactivation of the periostin gene. Improper regulation of periostin by the mutant form of TWIST2 could help explain some of the soft tissue abnormalities seen in these patients therefore providing a genotype-phenotype relationship for Setleis Syndrome.

Deletion of mouse Porcn blocks Wnt ligand secretion and reveals an ectodermal etiology of human focal dermal hypoplasia/Goltz syndrome.

The Drosophila porcupine gene is required for secretion of wingless and other Wnt proteins, and sporadic mutations in its unique human ortholog, PORCN, cause a pleiotropic X-linked dominant disorder, focal dermal hypoplasia (FDH, also known as Goltz syndrome). We generated a conditional allele of the X-linked mouse Porcn gene and analyzed its requirement in Wnt signaling and embryonic development. We find that Porcn-deficient cells exhibit a cell-autonomous defect in Wnt ligand secretion but remain responsive to exogenous Wnts. Consistent with the female-specific inheritance pattern of FDH, Porcn hemizygous male embryos arrest during early embryogenesis and fail to generate mesoderm, a phenotype previously associated with loss of Wnt activity. Heterozygous Porcn mutant females exhibit a spectrum of limb, skin, and body patterning abnormalities resembling those observed in human patients with FDH. Many of these defects are recapitulated by ectoderm-specific deletion of Porcn, substantiating a long-standing hypothesis regarding the etiology of human FDH and extending previous studies that have focused on downstream elements of Wnt signaling, such as ß-catenin. Conditional deletion of Porcn thus provides an experimental model of FDH, as well as a valuable tool to probe Wnt ligand function in vivo.

Deficiency of PORCN, a regulator of Wnt signaling, is associated with focal dermal hypoplasia.

Focal dermal hypoplasia (FDH) is an X-linked dominant multisystem birth defect affecting tissues of ectodermal and mesodermal origin. Using a stepwise approach of (i) genetic mapping of FDH, (ii) high-resolution comparative genome hybridization to seek deletions in candidate chromosome areas and (iii) point mutation analysis in candidate genes, we identified PORCN, encoding a putative O-acyltransferase and potentially crucial for cellular export of Wnt signaling proteins, as the gene mutated in FDH. The findings implicate FDH as a developmental disorder caused by a deficiency in PORCN.

Focal dermal hypoplasia: ultrastructural abnormalities of the connective tissue.

We followed over 10 years three girls with focal dermal hypoplasia syndrome. The histopathological changes demonstrated at the optical level an hypoplastic dermis with thin and scarce collagen bundles and a marked diminution of elastic fibers. Mature adipose tissue was found scattered within the papillary and reticular dermis. No alterations in the basal membrane were observed by immunocytochemical or ultrastructural techniques. Ultrastructurally, in the skin-affected areas, loosely arranged collagen bundles composed of few fibrils were seen scattered in the extracellular matrix. Scarce elastic fibers of normal morphology were also observed. Fibroblasts were smaller, oval-shaped, and diminished in number with a poorly developed cytoplasm. In these fibroblasts, the most conspicuous feature was a remarkable and irregular thickening of the nuclear fibrous lamina. Taking into account that a common link between all laminopaties may be a failure of stem cells to regenerate mesenchymal tissue, this failure would induce the dermal hypoplasia observed in our patients presenting Goltz syndrome.

Integrin-mediated signal transduction in cells lacking focal adhesion kinase p125FAK.

We have previously shown that integrin-dependent tyrosine phosphorylation of p130Cas (Cas) could be induced in a mouse fibroblast cell line that does not express focal adhesion kinase p125FAK (FAK). By analyzing FAK-deficient (FAK-/-) cells transiently expressing Cas mutant proteins, we demonstrate here that the Src homology 3 (SH3) domain of Cas is indispensable for adhesion-mediated Cas phosphorylation in this mutant cell line. While the FAK directly binds to Cas-SH3, our findings imply that SH3-binding molecule(s) other than FAK might regulate Cas phosphorylation, at least in FAK-/- cells. In this regard, we observed that FAK-/- cells expressed cell adhesion kinase beta (CAKbeta), a protein tyrosine kinase of the FAK subfamily. CAKbeta expressed by FAK-/- cells was associated in vivo with Cas in a Cas-SH3-dependent manner. Moreover, integrin stimulation induces tyrosine phosphorylation of CAKbeta in FAK-/- cells. Thus, our results suggest that CAKbeta contributes to integrin-mediated signal transduction in place of FAK in FAK-deficient cells.

Focal dermal hypoplasia (Goltz syndrome): a decreased accumulation of hyaluronic acid in three-dimensional culture.

We report a 28-year-old female with focal dermal hypoplasia, (Goltz syndrome). We compared the growth kinetics and the production of type I collagen and glycosaminoglycans by fibroblasts from affected and unaffected skin. Fibroblasts were grown in conventional medium supplemented with ascorbic acid 2-phosphate, which makes fibroblasts from a tissue-like structure in vitro. The population doubling time of fibroblasts in affected skin was slightly shorter than that of unaffected skin. There was no difference in the levels of the carboxyterminal propeptide of type I procollagen liberated into the media between affected skin and unaffected skin. However, the cell layer of affected skin fibroblasts revealed a decreased amount of hyaluronic acid-derived disaccharide unit (delta Di-HA). The abnormal metabolism of glycosaminoglycan in the affected dermis might be involved in the development of skin changes in focal dermal hypoplasia.