HER2 Heterogeneity Is Associated with Poor Survival in HER2-Positive Breast Cancer.

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.

RNA Sequencing Analysis Reveals Interactions between Breast Cancer or Melanoma Cells and the Tissue Microenvironment during Brain Metastasis.

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.

SAMD9 mutations cause a novel multisystem disorder, MIRAGE syndrome, and are associated with loss of chromosome 7.

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.

Pathological characterization of pachydermia in pachydermoperiostosis.

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.

Long-term safety issues of iPSC-based cell therapy in a spinal cord injury model: oncogenic transformation with epithelial-mesenchymal transition.

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.

In vitro co-culture systems for studying molecular basis of cellular interaction between Aire-expressing medullary thymic epithelial cells and fresh thymocytes.

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 T cells in vivo. In this system, a single Aire(+) cell appeared able to kill ∼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".

Dramatic improvement in genome assembly achieved using doubled-haploid genomes.

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.

Filaggrin loss-of-function mutations are not a predisposing factor for atopic dermatitis in an Ishigaki Island under subtropical climate.

BACKGROUND: Filaggrin (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. OBJECTIVE: To 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. METHODS: We 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. RESULTS: Average 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). CONCLUSION: The 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.

Global gene expression analysis following spinal cord injury in non-human primates.

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.

A novel mouse model for Down syndrome that harbor a single copy of human artificial chromosome (HAC) carrying a limited number of genes from human chromosome 21.

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 ß-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.

Mutations in SERPINB7, encoding a member of the serine protease inhibitor superfamily, cause Nagashima-type palmoplantar keratosis.

"Nagashima-type" palmoplantar keratosis (NPPK) is an autosomal recessive nonsyndromic diffuse palmoplantar keratosis characterized by well-demarcated diffuse hyperkeratosis with redness, expanding on to the dorsal surfaces of the palms and feet and the Achilles tendon area. Hyperkeratosis in NPPK is mild and nonprogressive, differentiating NPPK clinically from Mal de Meleda. We performed whole-exome and/or Sanger sequencing analyses of 13 unrelated NPPK individuals and identified biallelic putative loss-of-function mutations in SERPINB7, which encodes a cytoplasmic member of the serine protease inhibitor superfamily. We identified a major causative mutation of c.796C>T (p.Arg266(∗)) as a founder mutation in Japanese and Chinese populations. SERPINB7 was specifically present in the cytoplasm of the stratum granulosum and the stratum corneum (SC) of the epidermis. All of the identified mutants are predicted to cause premature termination upstream of the reactive site, which inhibits the proteases, suggesting a complete loss of the protease inhibitory activity of SERPINB7 in NPPK skin. On exposure of NPPK lesional skin to water, we observed a whitish spongy change in the SC, suggesting enhanced water permeation into the SC due to overactivation of proteases and a resultant loss of integrity of the SC structure. These findings provide an important framework for developing pathogenesis-based therapies for NPPK.

Diverse spectrum of rare deafness genes underlies early-childhood hearing loss in Japanese patients: a cross-sectional, multi-center next-generation sequencing study.

BACKGROUND: Genetic tests for hereditary hearing loss inform clinical management of patients and can provide the first step in the development of therapeutics. However, comprehensive genetic tests for deafness genes by Sanger sequencing is extremely expensive and time-consuming. Next-generation sequencing (NGS) technology is advantageous for genetic diagnosis of heterogeneous diseases that involve numerous causative genes. METHODS: Genomic DNA samples from 58 subjects with hearing loss from 15 unrelated Japanese families were subjected to NGS to identify the genetic causes of hearing loss. Subjects did not have pathogenic GJB2 mutations (the gene most often associated with inherited hearing loss), mitochondrial m.1555A>G or 3243A>G mutations, enlarged vestibular aqueduct, or auditory neuropathy. Clinical features of subjects were obtained from medical records. Genomic DNA was subjected to a custom-designed SureSelect Target Enrichment System to capture coding exons and proximal flanking intronic sequences of 84 genes responsible for nonsyndromic or syndromic hearing loss, and DNA was sequenced by Illumina GAIIx (paired-end read). The sequences were mapped and quality-checked using the programs BWA, Novoalign, Picard, and GATK, and analyzed by Avadis NGS. RESULTS: Candidate genes were identified in 7 of the 15 families. These genes were ACTG1, DFNA5, POU4F3, SLC26A5, SIX1, MYO7A, CDH23, PCDH15, and USH2A, suggesting that a variety of genes underlie early-childhood hearing loss in Japanese patients. Mutations in Usher syndrome-related genes were detected in three families, including one double heterozygous mutation of CDH23 and PCDH15. CONCLUSION: Targeted NGS analysis revealed a diverse spectrum of rare deafness genes in Japanese subjects and underscores implications for efficient genetic testing.

A homozygous nonsense mutation in the gene for Tmem79, a component for the lamellar granule secretory system, produces spontaneous eczema in an experimental model of atopic dermatitis.

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.

Regulation of human autoimmune regulator (AIRE) gene translation by miR-220b.

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.

Characterization of canine filaggrin: gene structure and protein expression in dog skin.

BACKGROUND: Filaggrin (FLG) is a key protein for skin barrier formation and hydration of the stratum corneum. In humans, a strong association between FLG gene mutations and atopic dermatitis has been reported. Although similar pathogenesis and clinical manifestation have been argued in canine atopic dermatitis, our understanding of canine FLG is limited. HYPOTHESIS/OBJECTIVES: The aim of this study was to determine the structure of the canine FLG gene and to raise anti-dog FLG antibodies, which will be useful to detect FLG protein in dog skin. METHODS: The structure of the canine FLG gene was determined by analysing the publicly available canine genome DNA sequence. Polyclonal anti-dog FLG antibodies were raised based on the canine FLG sequence analysis and used for defining the FLG expression pattern in dog skin by western blotting and immunohistochemistry. RESULTS: Genomic DNA sequence analysis revealed that canine FLG contained four units of repeated sequences corresponding to FLG monomer protein. Western blots probed with anti-dog FLG monomer detected two bands at 59 and 54 kDa, which were estimated sizes. The results of immunohistochemistry showed that canine FLG was expressed in the stratum granulosum of the epidermis as a granular staining pattern in the cytoplasmic region. CONCLUSIONS AND CLINICAL IMPORTANCE: This study revealed the unique gene structure of canine FLG that results in production of FLG monomers larger than those of humans or mice. The anti-dog FLG antibodies raised in this study identified FLG in dog skin. These antibodies will enable us to screen FLG-deficient dogs with canine atopic dermatitis or ichthyosis.

LINE1 family member is negative regulator of HLA-G expression.

Class Ia molecules of human leucocyte antigen (HLA-A, -B and -C) are widely expressed and play a central role in the immune system by presenting peptides derived from the lumen of the endoplasmic reticulum. In contrast, class Ib molecules such as HLA-G serve novel functions. The distribution of HLA-G is mostly limited to foetal trophoblastic tissues and some tumour tissues. The mechanism required for the tissue-specific regulation of the HLA-G gene has not been well understood. Here, we investigated the genomic regulation of HLA-G by manipulating one copy of a genomic DNA fragment on a human artificial chromosome. We identified a potential negative regulator of gene expression in a sequence upstream of HLA-G that overlapped with the long interspersed element (LINE1); silencing of HLA-G involved a DNA secondary structure generated in LINE1. The presence of a LINE1 gene silencer may explain the limited expression of HLA-G compared with other class I genes.

Identification of mutations in the prostaglandin transporter gene SLCO2A1 and its phenotype-genotype correlation in Japanese patients with pachydermoperiostosis.

BACKGROUND: Pachydermoperiostosis (PDP) is a rare genetic disorder characterized by 3 major symptoms: pachydermia including cutis verticis gyrata (CVG), periostosis, and finger clubbing. Recently, a homozygous mutation in the gene HPGD, which encodes 15-hydroxyprostaglandin dehydrogenase (15-PGDH), was found to be associated with PDP. However, mutations in HPGD have not been identified in Japanese PDP patients. OBJECTIVE: We aimed to identify a novel responsible gene for PDP using whole exome sequencing by next-generation DNA sequencer (NGS). METHODS: Five patients, including 2 patient-parent trios were enrolled in this study. Entire coding regions were sequenced by NGS to identify candidate mutations associated with PDP. The candidate mutations were subsequently sequenced using the Sanger method. To determine clinical characteristics, we analyzed histological samples, as well as serum and urinary prostaglandin E2 (PGE2) levels for each of the 5 PDP patients, and 1 additional patient with idiopathic CVG. RESULTS: From initial analyses of whole exome sequencing data, we identified mutations in the solute carrier organic anion transporter family, member 2A1 (SLCO2A1) gene, encoding prostaglandin transporter, in 3 of the PDP patients. Follow-up Sanger sequencing showed 5 different SLCO2A1 mutations (c.940+1G>A, p.E427_P430del, p.G104*, p.T347I, p.Q556H) in 4 unrelated PDP patients. In addition, the splice-site mutation c.940+1G>A identified in 3 of 4 PDP patients was determined to be a founder mutation in the Japanese population. Furthermore, it is likely that the combination of these SLCO2A1 mutations in PDP patients is also associated with disease severity. CONCLUSION: We found that SLCO2A1 is a novel gene responsible for PDP. Although the SLCO2A1 gene is only the second gene discovered to be associated with PDP, it is likely to be a major cause of PDP in the Japanese population.