Supplementary Figure S1; Supplementary Tables S1-S2.

Epiplakin, a cytolinker protein expressed in the epithelium that interacts with intermediate filaments [ [1] Fujiwara S. Takeo N. Otani Y. Parry D.A.D. Kunimatsu M. Lu R. et al. Epiplakin, a novel member of the plakin family originally identified as a 450-kDa human epidermal autoantigen : Structure and tissue localization. J. Biol. Chem. 2001; 276: 13340-13347 Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar , [2] Wang W. Sumiyoshi H. Yoshioka H. Fujiwara S. Interactions between epiplakin and intermediate filaments. J. Dermatol. 2006; 33: 518-527 Crossref PubMed Google Scholar ], contains domains that are homologous to the B domain in the carboxy-terminal region of desmoplakin. The last five B domains in human epiplakin, together with their associated linker regions, are highly conserved. Our previous study showed that there is copy number variation (CNV) in the 3′ terminal half of the epiplakin gene (EPPK1) in human individuals and that CNV reflects a variation of molecular size. Indeed, epiplakin isoforms in humans contain between 11 and 17 B domains, although 13 B domains predominate [ [3] Ishikawa K. Furuhashi M. Sasaki T. Kudoh J. Tsuchisaka A. Hashimoto T. et al. Intragenic copy number variation within human epiplakin 1 (EPPK1) generates variation of molecular size of epiplakin. J. Dermatol. Sci. 2018; 91: 228-231 Abstract Full Text Full Text PDF Google Scholar ].

Epiplakin is a versatile cytolinker protein that is expressed in the epithelium, including the epidermis, several glands, and digestive organs [ [1] Fujiwara S. Takeo N. Otani Y. Parry D.A.D. Kunimatsu M. Lu R. et al. Epiplakin, a novel member of the plakin family originally identified as a 450-kDa human epidermal autoantigen: Structure and tissue localizatio. J. Biol. Chem. 2001; 276: 13340-13347 Crossref PubMed Scopus (50) Google Scholar ]. Human epiplakin consists of 13 B domains that are homologous to the B domain found in the carboxy-terminal region of desmoplakin; the last five B domains at the carboxy-terminal end, together with their associated linker regions, are highly conserved [ [1] Fujiwara S. Takeo N. Otani Y. Parry D.A.D. Kunimatsu M. Lu R. et al. Epiplakin, a novel member of the plakin family originally identified as a 450-kDa human epidermal autoantigen: Structure and tissue localizatio. J. Biol. Chem. 2001; 276: 13340-13347 Crossref PubMed Scopus (50) Google Scholar ] (Fig. 1A). The mRNA of epiplakin is transcribed from the third exon of the epiplakin gene (EPPK1) [ 2 https://www.ncbi.nlm.nih.gov/gene/83481 updated on 20-Apr-2017. Google Scholar , 3 Takeo N. Wang W. Matsuo N. Sumiyoshi H. Yoshioka H. Fujiwara S. Structure and heterogeneity of the human gene for epiplakin (EPPK1). J. Invest. Dermatol. 2003; 121: 1224-1226 Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar ] (Fig. 1B). The molecular size of epiplakin is estimated to be about 555 kDa, which is 25 amino acids longer than previously reported [ 1 Fujiwara S. Takeo N. Otani Y. Parry D.A.D. Kunimatsu M. Lu R. et al. Epiplakin, a novel member of the plakin family originally identified as a 450-kDa human epidermal autoantigen: Structure and tissue localizatio. J. Biol. Chem. 2001; 276: 13340-13347 Crossref PubMed Scopus (50) Google Scholar , 2 https://www.ncbi.nlm.nih.gov/gene/83481 updated on 20-Apr-2017. Google Scholar ]. However, in previous studies, immunoblot analysis of epidermal extracts and cell lysates revealed additional protein bands above and below the primary epiplakin band at 555 kDa [ 4 Fujiwara S. Shinkai H. Takayasu S. Owaribe K. Tsukita S. Kageshita T. A case of subepidermal blister disease associated with autoantibody against 450 kD protein. J. Dermatol. 1992; 19: 610-613 Crossref PubMed Scopus (20) Google Scholar , 5 Jang S.I. Kalinin A. Takahashi K. Marekov L.N. Steinert P.M. Characterization of human epiplakin: RNAi-mediated epiplakin depletion leads to the disruption of keratin and vimentin IF networks. J. Cell Sci. 2005; 118: 781-793 Crossref PubMed Scopus (42) Google Scholar , 6 Tsuchisaka A. Numata S. Teye K. Natsuaki Y. Kawakami T. Takeda Y. et al. Epiplakin is a paraneoplastic pemphigus autoantigen and related to bronchiolitis obliterans in Japanese patients. J. Invest. Dermatol. 2016; 136: 399-408 Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar ]. Spazierer et al. estimated the size of human epiplakin to be 725 kDa, which is similar to mouse epiplakin that contains 16 B domains [ [7] Spazierer D. Fuchs P. Pröll V. Janda L. Oehler S. Fischer I. et al. Epiplakin gene analysis in mouse reveals a single exon encoding a 725-kDa protein with expression restricted to epithelial tissues. J. Biol. Chem. 2003; 278: 31657-31666 Crossref PubMed Scopus (31) Google Scholar ].

Intratumoral human epidermal growth factor receptor 2 (HER2) heterogeneity has been reported in 16–36% of HER2-positive breast cancer and its clinical impact is under discussion. We examined the biological effects of HER2-heterogeneity on mouse models and analyzed metastatic brains by RNA sequence analysis. A metastatic mouse model was developed using 231-Luc (triple negative cells) and 2 HER2-positive cell lines, namely, HER2-60 and HER2-90 which showed heterogeneous and monotonous HER2 expressions, respectively. Metastatic lesions developed in 3 weeks in all the mice injected with HER2-60 cells, and in 69% of the mice injected with HER2-90 and 87.5% of the mice injected with 231-Luc. The median survival days of mice injected with 231-Luc, HER2-60, and HER2-90 cells were 29 (n = 24), 24 (n = 22) and 30 (n = 13) days, respectively. RNA sequence analysis showed that CASP-1 and its related genes were significantly downregulated in metastatic brain tumors with HER2-60 cells. The low expression of caspase-1 could be a new prognostic biomarker for early relapse in HER2-positive breast cancer.

Metastasis is the main cause of treatment failure and death in cancer patients. Metastasis of tumor cells to the brain occurs frequently in individuals with breast cancer, non–small cell lung cancer, or melanoma. Despite recent advances in our understanding of the causes and in the treatment of primary tumors, the biological and molecular mechanisms underlying the metastasis of cancer cells to the brain have remained unclear. Metastasizing cancer cells interact with their microenvironment in the brain to establish metastases. We have now developed mouse models of brain metastasis based on intracardiac injection of human breast cancer or melanoma cell lines, and we have performed RNA sequencing analysis to identify genes in mouse brain tissue and the human cancer cells whose expression is associated specifically with metastasis. We found that the expressions of the mouse genes Tph2, Sspo, Ptprq, and Pole as well as those of the human genes CXCR4, PLLP, TNFSF4, VCAM1, SLC8A2, and SLC7A11 were upregulated in brain tissue harboring metastases. Further characterization of such genes that contribute to the establishment of brain metastases may provide a basis for the development of new therapeutic strategies and consequent improvement in the prognosis of cancer patients.

Adrenal hypoplasia is a rare, life-threatening congenital disorder. Here we define a new form of syndromic adrenal hypoplasia, which we propose to term MIRAGE (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy) syndrome. By exome sequencing and follow-up studies, we identified 11 patients with adrenal hypoplasia and common extra-adrenal features harboring mutations in SAMD9. Expression of the wild-type SAMD9 protein, a facilitator of endosome fusion, caused mild growth restriction in cultured cells, whereas expression of mutants caused profound growth inhibition. Patient-derived fibroblasts had restricted growth, decreased plasma membrane EGFR expression, increased size of early endosomes, and intracellular accumulation of giant vesicles carrying a late endosome marker. Of interest, two patients developed myelodysplasitc syndrome (MDS) that was accompanied by loss of the chromosome 7 carrying the SAMD9 mutation. Considering the potent growth-restricting activity of the SAMD9 mutants, the loss of chromosome 7 presumably occurred as an adaptation to the growth-restricting condition.

Previously, we described the safety and therapeutic potential of neurospheres (NSs) derived from a human induced pluripotent stem cell (iPSC) clone, 201B7, in a spinal cord injury (SCI) mouse model. However, several safety issues concerning iPSC-based cell therapy remain unresolved. Here, we investigated another iPSC clone, 253G1, that we established by transducing OCT4, SOX2, and KLF4 into adult human dermal fibroblasts collected from the same donor who provided the 201B7 clone. The grafted 253G1-NSs survived, differentiated into three neural lineages, and promoted functional recovery accompanied by stimulated synapse formation 47 days after transplantation. However, long-term observation (for up to 103 days) revealed deteriorated motor function accompanied by tumor formation. The tumors consisted of Nestin(+) undifferentiated neural cells and exhibited activation of the OCT4 transgene. Transcriptome analysis revealed that a heightened mesenchymal transition may have contributed to the progression of tumors derived from grafted cells.

Pachydermoperiostosis is a rare hereditary disease, which presents with the cutaneous manifestations of pachydermia and cutis verticis gyrata. Histological findings in pachydermia frequently include dermal edema, mucin deposition, elastic fiber degeneration, dermal fibrosis and adnexal hyperplasia. However, the severity of these findings varies between clinical reports, and a systematic multiple-case clinicopathological correlative analysis has not been performed to date. In the present study, we reviewed the skin biopsy specimens obtained from the pachydermia of six pachydermoperiostosis patients. The severity of the characteristic histological features was semiquantitatively evaluated and correlated with the grade of pachydermia. Dermal edema, mucin deposition and elastic fiber degeneration were observed in all cases. Patients with severe pachydermia had sebaceous gland hyperplasia and fibrosis. These results suggest that the triad of mucin deposition, dermal edema and elastic fiber degeneration are found from very early stage pachydermia, and could be considered diagnostic findings. To ensure an earlier diagnosis of pachydermoperiostosis, a biopsy should be taken when a patient has grade 1 pachydermia to determine the presence of this histological triad.

Improvement in de novo assembly of large genomes is still to be desired. Here, we improved draft genome sequence quality by employing doubled-haploid individuals. We sequenced wildtype and doubled-haploid Takifugu rubripes genomes, under the same conditions, using the Illumina platform and assembled contigs with SOAPdenovo2. We observed 5.4-fold and 2.6-fold improvement in the sizes of the N50 contig and scaffold of doubled-haploid individuals, respectively, compared to the wildtype, indicating that the use of a doubled-haploid genome aids in accurate genome analysis.

Down syndrome (DS), also known as Trisomy 21, is the most common chromosome aneuploidy in live-born children and displays a complicated symptom. To date, several kinds of mouse models have been generated to understand the molecular pathology of DS, yet the gene dosage effects and gene(s)-phenotype(s) correlation are not well understood. In this study, we established a novel method to generate a partial trisomy mice using the mouse ES cells that harbor a single copy of human artificial chromosome (HAC), into which a small human DNA segment containing human chromosome 21 genes cloned in a bacterial artificial chromosome (BAC) was recombined. The produced mice were found to maintain the HAC carrying human genes as a mini-chromosome, hence termed as a Trans-Mini-Chromosomal (TMC) mouse, and HAC was transmitted for more than twenty generations independent from endogenous mouse chromosomes. The three human transgenes including cystathionine β-synthase, U2 auxiliary factor and crystalline alpha A were expressed in several mouse tissues with various expression levels relative to mouse endogenous genes. The novel system is applicable to any of human and/or mouse BAC clones. Thus, the TMC mouse carrying a HAC with a limited number of genes would provide a novel tool for studying gene dosage effects involved in the DS molecular pathogenesis and the gene(s)-phenotype(s) correlation.

BackgroundFilaggrin (FLG) is a major protein component of the stratum corneum (SC) layer, and FLG loss-of-function mutations are a predisposing factor for atopic dermatitis (AD). Previous cohort studies of children from northern and western Europe have reported FLG loss-of-function mutation frequencies of 15.1–20.9% and 5.8–13.0% in AD and non-AD groups, respectively.ObjectiveTo elucidate the association between AD prevalence of FLG loss-of-function mutation carriers and climate conditions, we determined the AD prevalence and FLG loss-of-function mutation frequencies in a cohort of children from Ishigaki Island. Ishigaki Island has a subtropical climate with high humidity (monthly average, 60.8–78.7%) and high temperature (monthly average, 18.5–29.4°C) throughout the year.MethodsWe diagnosed AD prevalence and analyzed eight FLG loss-of-function mutations in the Japanese population against a cohort of 721 children from the Kyushu University Ishigaki Atopic Dermatitis Study (KIDS) cohort. Parents gave consent for the mutation analysis during their medical examinations from 2001 to 2006.ResultsAverage AD prevalence was 7.3% per year, and a total of 127 children (17.6%) were diagnosed with AD at least once between 2001 and 2006. The average total serum IgE level differed significantly between the AD and non-AD groups (199.0 and 69.0IU/ml, respectively). Although five kinds of FLG loss-of-function mutations isolated in previous Japanese FLG mutation studies were identified, the FLG loss-of-function mutation frequency in children of the KIDS cohort was not significantly different between the AD and non-AD groups (7.9% and 6.1%, respectively; P=0.174).ConclusionThe FLG loss-of-function mutation frequency was not significantly different between the AD and non-AD groups in a cohort of children from Ishigaki Island, which has a subtropical climate, suggesting that FLG loss-of-function mutations are not always a predisposing factor for AD prevalence.

We previously established three mouse cell lines (Aire(+)TEC1, Aire(+)TEC2 and Aire(+)DC) from the medullary thymic epithelial cells (mTECs) and dendritic cells (mDCs). These cells constitutively expressed "autoimmune regulator (Aire) gene'' and they exhibited various features of self antigen-presenting cells (self-APCs) present in the thymic medullary region.Here, we confirmed our previous observation that Aire(+) thymic epithelial cells adhere to fresh thymocytes and kill them by inducing apoptosis, thus potentially reproducing in vitro some aspects of the negative selection of Tcells in vivo. In this system, a single Aire(+) cell appeared able to kill similar to 30 thymocytes within 24 hrs. Moreover, we observed that ectopic expression of peripheral tissue-specific antigens (TSAs), and expression of several surface markers involved in mTEC development, increased as Aire(+) cell density increases toward confluency. Thus, these Aire(+) cells appear to behave like differentiating mTECs as if they pass through the developmental stages from intermediate state toward mature state. Surprisingly, an in vitro co-culture system consisting of Aire(+) cells and fractionated sub-populations of fresh thymocytes implied the possible existence of two distinct subtypes of thymocytes (named as CD4(+) killer and CD4(-) rescuer) that may determine the fate (dead or alive) of the differentiating Aire(+) mTECs. Thus, our in vitro co-culture system appears to mimic a part of "in vivo thymic crosstalk''.

Spinal cord injury (SCI) is a devastating condition with no established treatment. To better understand the pathology and develop a treatment modality for SCI, an understanding of the physiological changes following SCI at the molecular level is essential. However, studies on SCI have primarily used rodent models, and few studies have examined SCI in non-human primates. In this study, we analyzed the temporal changes in gene expression patterns following SCI in common marmosets (Callithrix jacchus) using microarray analysis and mRNA deep sequencing. This analysis revealed that, although the sequence of events is comparable between primates and rodents, the inflammatory response following SCI is significantly prolonged and the onset of glial scar formation is temporally delayed in primates compared with rodents. These observations indicate that the optimal time window to treat SCI significantly differs among different species. This study provides the first extensive analysis of gene expression following SCI in non-human primates and will serve as a valuable resource in understanding the pathology of SCI.

This chapter contains sections titled: Introduction Materials and methods Results Discussion Acknowledgements

Although mutations of autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), presenting a wide spectrum of many characteristic and non-characteristic clinical features, some patients lack AIRE gene mutations. Therefore, something other than a mutation, such as dysregulation of AIRE gene, may be a causal factor for APECED or its related diseases. However, regulatory mechanisms for AIRE gene expression and/or translation have still remained elusive. We found that IL-2-stimulated CD4(+) T (IL-2T) cells showed a high expression of AIRE gene, but very low AIRE protein production, while Epstein-Barr virus-transformed B (EBV-B) cells express both AIRE gene and AIRE protein. By using microarray analysis, we could identify miR-220b as a possible regulatory mechanism for AIRE gene translation in IL-2T cells. Here we report that miR-220b significantly reduced the expression of AIRE protein in AIRE gene with 3'UTR region transfected 293T cells, whereas no alteration of AIRE protein production was observed in the open reading frame of AIRE gene alone transfected cells. In addition, anti-miR-220b reversed the inhibitory function of miR-220b for the expression of AIRE protein in AIRE gene with 3'UTR region transfected cells. Moreover, when AIRE gene transfected cells with mutated 3'UTR were transfected with miR-220b, no reduction of AIRE protein production was observed. Taken together, it was concluded that miR-220b inhibited the AIRE gene translation through the 3'UTR region of AIRE gene, indicating that miR-220b could serve as a regulator for human AIRE gene translation.

The compact genome of torafugu (Takifugu rubripes) is a useful model for annotating the vertebrate genomes. However, the currently incomplete assembly of the torafugu genome has decreased its value for genetic and genomic analyses. This is partly due to the usage of naturally heterozygous diploid genome as a starting material. Therefore, we generated mito-gynogenetic torafugu using the eggs fertilized by UV-irradiated sperm, followed by cold shock treatment. After a preliminary confirmation of homozygosity by microsatellite genotyping in one mito-gynogenetic torafugu, genome-wide single nucleotide polymorphism (SNP) analyses were employed for an accurate assessment of whole-genome homozygosity levels. SNP genotyping results indicated that the torafugu obtained through mitotic gynogenesis induction was doubled haploid. We report the first instance of assessing homozygosity levels by genome-wide SNP analyses in mito-gynogenetic fishes.

BACKGROUND:Flaky tail (ma/ma Flg(ft/ft)) mice have a frameshift mutation in the filaggrin (Flg(ft)) gene and are widely used as a model of human atopic dermatitis associated with FLG mutations. These mice possess another recessive hair mutation, matted (ma), and develop spontaneous dermatitis under specific pathogen-free conditions, whereas genetically engineered Flg(-/-) mice do not.OBJECTIVE:We identified and characterized the gene responsible for the matted hair and dermatitis phenotype in flaky tail mice.METHODS:We narrowed down the responsible region by backcrossing ma/ma mice with wild-type mice and identified the mutation using next-generation DNA sequencing. We attempted to rescue the matted phenotype by introducing the wild-type matted transgene. We characterized the responsible gene product by using whole-mount immunostaining of epidermal sheets.RESULTS:We demonstrated that ma, but not Flg(ft), was responsible for the dermatitis phenotype and corresponded to a Tmem79 gene nonsense mutation (c.840C>G, p.Y280*), which encoded a 5-transmembrane protein. Exogenous Tmem79 expression rescued the matted hair and dermatitis phenotype of Tmem79(ma/ma) mice. Tmem79 was mainly expressed in the trans-Golgi network in stratum granulosum cells in the epidermis in both mice and humans. The Tmem79(ma/ma) mutation impaired the lamellar granule secretory system, which resulted in altered stratum corneum formation and a subsequent spontaneous dermatitis phenotype.CONCLUSIONS:The Tmem79(ma/ma) mutation is responsible for the spontaneous dermatitis phenotype in matted mice, probably as a result of impaired lamellar granule secretory system and altered stratum corneum barrier function.