A homozygous loss-of-function variant in the MPO gene is associated with generalized pustular psoriasis.

Generalized pustular psoriasis (GPP) is a rare form of psoriasis, which is characterized by sudden onset of repeated erythema and pustule formation with generalized inflammation. Recent advances in molecular genetics have led to the identification of several genes associated with GPP, including IL36RN, CARD14, AP1S3, SERPINA3, and MPO. Of these, only limited cases of GPP have been reported to carry mutations in the AP1S3, SERPINA3, or MPO to date. In the present study, we investigated a Japanese patient with GPP and found a homozygous missense mutation c.1769G>T (p.Arg590Leu) in the MPO gene. Structural analysis predicted that the mutant MPO protein would abolish its ability to bind with heme protein. In vitro studies using cultured cells revealed that the mutant MPO was stably expressed, but completely lost its myeloperoxidase activity. Immunohistochemistry (IHC) using an anti-MPO antibody showed markedly reduced expression of MPO protein in the patient's skin, suggesting that the mutation would lead to an instability of the MPO protein in vivo. Finally, IHC with an anti-citrullinated Histone H3 antibody demonstrated a sparse formation of neutrophil extracellular traps within a Kogoj's spongiform pustule of the patient's skin. Collectively, we conclude that the c.1769G>T (p.Arg590Leu) in the MPO is a complete loss-of-function mutation associated with GPP in the patient. Our data further underscore critical roles of the MPO gene in the pathogenesis of GPP.

Novel compound heterozygous mutations in the PLEC gene in a neonate with epidermolysis bullosa simplex with pyloric atresia.

Epidermolysis bullosa (EB) is a heterogeneous group of inherited disorders characterized by the blistering of the skin and mucous membranes. Although the molecular basis of EB has been significantly elucidated, the precise phenotypes of the lethal types of EB have not been completely characterized. Herein, we report a severe case of EB with pyloric atresia (PA). The patient was a Japanese boy who not only had skin lesions but also various complications such as PA, dysphagia, hypotonia, infectious keratitis with corneal ulcer, obstructive uropathy and protein-losing enteropathy. Genetic analysis led to the identification of two novel compound heterozygous mutations in the last exon of the plectin (PLEC) gene. Based on this finding, EB simplex with PA was diagnosed. Immunostaining with anti-plectin antibodies revealed truncated plectin proteins lacking the C-terminus in the patient's skin. We also conducted a prenatal diagnosis in subsequent pregnancy. Our report further highlights the crucial role of plectin in many organs and provides valuable information regarding the phenotypes resulting from mutations in the PLEC gene.

Different degree of loss-of-function among four missense mutations in the EDAR gene responsible for autosomal recessive hypohidrotic ectodermal dysplasia may be associated with the phenotypic severity.

Hypohidrotic ectodermal dysplasia is a rare condition characterized by hypohidrosis, hypodontia, and hypotrichosis. The disease can show X-linked recessive, autosomal dominant or autosomal recessive inheritance trait. Of these, the autosomal forms are caused by mutations in either EDAR or EDARADD. To date, the underlying pathomechanisms or genotype-phenotype correlations for autosomal forms have not completely been disclosed. In this study, we performed a series of in vitro studies for four missense mutations in the death domain of EDAR protein: p.R358Q, p.G382S, p.I388T, and p.T403M. The results revealed that p.R358Q- and p.T403M-mutant EDAR showed different expression patterns from wild-type EDAR in both western blots and immunostainings. NF-κB reporter assays demonstrated that all the mutant EDAR showed reduced activation of NF-κB, but the reduction by p.G382S- and p.I388T-mutant EDAR was moderate. Co-immunoprecipitation assays showed that p.R358Q- and p.T403M-mutant EDAR did not bind with EDARADD at all, whereas p.G382S- and p.I388T-mutant EDAR maintained the affinity to some extent. Furthermore, we demonstrated that all the mutant EDAR proteins analyzed aberrantly bound with TRAF6. Sum of the data suggest that the degree of loss-of-function is different among the mutant EDAR proteins, which may be associated with the severity of the disease.

Characterization of EDARADD gene mutations responsible for hypohidrotic ectodermal dysplasia.

Hypohidrotic ectodermal dysplasia (HED) is a genetic disorder characterized by hypohidrosis, hypodontia, and hypotrichosis. Autosomal forms of the disease are caused by mutations in either EDAR or EDARADD. To date, the underlying pathomechanisms for HED resulting from EDARADD mutations have not fully been disclosed. In this study, we performed detailed in vitro analyses in order to characterize three dominantly inherited missense mutations, p.D120Y, p.L122R, and p.D123N, and one recessively inherited missense mutation, p.E152K, in the EDARADD gene. Nuclear factor (NF)-κB reporter assays demonstrated that all the mutant EDARADD showed reduction in activation of NF-κB. Importantly, p.D120Y-, p.L122R-, and p.D123N-mutant EDARADD slightly reduced the NF-κB activity induced by wild-type EDARADD in a dominant negative manner. Co-immunoprecipitation assays showed that all of the mutant EDARADD were capable of binding to EDAR and wild-type EDARADD. Additional co-immunoprecipitation assays revealed that p.D120Y-, p.L122R-, and p.D123N-mutant EDARADD markedly prevented the interaction between EDAR and wild-type EDARADD, which further indicated a dominant negative effect by these mutations. Finally, we found that p.D120Y-, p.L122R-, and p.D123N-mutant EDARADD completely lost the ability to bind with TRAF6, while p.E152K-mutant EDARADD showed a mild reduction in the affinity. Our findings will provide crucial information toward unraveling the molecular mechanisms how EDARADD gene mutations cause the disease.

Evidence for a dominant-negative effect of a missense mutation in the SERPING1 gene responsible for hereditary angioedema type I.

Hereditary angioedema (HAE) is a rare condition characterized by episodic local edema involving various organs, which can be life-threatening in some cases. Among the three subtypes of the disease, HAE types I and II are known to be caused by heterozygous mutations in the SERPING1 gene encoding C1 inhibitor (C1INH). Although a number of mutations in the SERPING1 gene have been identified to date, the mechanisms how these mutations cause HAE are not completely understood. We herein performed detailed in vitro studies for a missense SERPING1 gene mutation p.S150F which we recently identified in a Japanese patient with HAE type I. We showed that the p.S150F-mutant C1INH was stably expressed within the cultured cells, while it was not secreted into the medium at all. Furthermore, we demonstrated that the mutant C1INH significantly prevented secretion of wild-type C1INH. Finally, the results suggested that the wild-type protein was not only retained but also degraded within the cytoplasm through interacting with the mutant protein. Our study clearly revealed a dominant-negative effect of the p.S150F-mutant C1INH against the wild-type C1INH.

Functional studies for a dominant mutation in the EDAR gene responsible for hypohidrotic ectodermal dysplasia.

Hypohidrotic ectodermal dysplasia (HED) is a rare genetic disorder characterized by hypotrichosis, hypohidrosis and hypodontia. The disease shows X-linked recessive, autosomal dominant or autosomal recessive inheritance traits. The X-linked form of HED is caused by mutations in the EDA gene, while autosomal forms result from mutations in either EDAR or EDARADD genes. Regarding recessive mutations in the EDAR gene, the pathomechanisms have been well characterized. However, it has remained largely unknown how dominant mutations in the EDAR cause HED. In this study, we performed in vitro analyses for a dominant EDAR gene mutation, p.F398*, as a representative. We showed that the p.F398* mutant EDAR completely lost its affinity to EDARADD, and suppressed the downstream nuclear factor-κB activation induced by wild-type EDAR in a dominant-negative manner. Furthermore, we demonstrated that the mutant EDAR was capable of binding with the wild-type EDAR, which led to reduced interaction between the wild-type EDAR and EDARADD. Our findings not only underscore an essential role of the interaction between EDAR and EDARADD in ectodermal development, but also disclose, in part, the molecular basis of autosomal dominant HED.