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.

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.

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.

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.

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.

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.

Mouse thymic epithelial cell lines expressing "Aire" and peripheral tissue-specific antigens reproduce in vitro negative selection of T cells.

In the human thymus, AIRE (autoimmune regulator) gene is expressed in a very limited type of medullary thymic epithelial cells (mTECs) and no cognate cell lines are available, hence the molecular analysis of AIRE gene function has been difficult. To improve this situation, we attempted to isolate Aire-expressing cells and established three cell lines (Aire⁺TEC1, Aire⁺TEC2, Aire⁺DC) from the abnormally enlarged thymus, which was developed in the transgenic mice expressing SV40 T-antigen driven by the mouse Aire gene promoter. When these Aire⁺ cell lines were co-cultured with fresh thymocytes, they adhered to the majority of thymocytes and induced apoptosis as if negative selection of T-cells in the thymus is occurring in vitro. Further analysis revealed that these Aire⁺ cell lines are derived from mTECs and exhibit characteristic natures of "antigen presenting cells" including several distinct abilities: to express a variety of peripheral tissue-specific antigens, to produce immunoproteasome and immunological synapse, and to express some of TNFSFs (tumor necrosis factor super families). Thus, the newly established Aire⁺ cell lines will be invaluable for the further detailed analysis of AIRE gene function in the central tolerance of immunity and autoimmune disease.

DNA sequence and analysis of human chromosome 8.

The International Human Genome Sequencing Consortium (IHGSC) recently completed a sequence of the human genome. As part of this project, we have focused on chromosome 8. Although some chromosomes exhibit extreme characteristics in terms of length, gene content, repeat content and fraction segmentally duplicated, chromosome 8 is distinctly typical in character, being very close to the genome median in each of these aspects. This work describes a finished sequence and gene catalogue for the chromosome, which represents just over 5% of the euchromatic human genome. A unique feature of the chromosome is a vast region of approximately 15 megabases on distal 8p that appears to have a strikingly high mutation rate, which has accelerated in the hominids relative to other sequenced mammals. This fast-evolving region contains a number of genes related to innate immunity and the nervous system, including loci that appear to be under positive selection--these include the major defensin (DEF) gene cluster and MCPH1, a gene that may have contributed to the evolution of expanded brain size in the great apes. The data from chromosome 8 should allow a better understanding of both normal and disease biology and genome evolution.

AIRE functions as an E3 ubiquitin ligase.

Autoimmune regulator (AIRE) gene mutation is responsible for the development of autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy, an organ-specific autoimmune disease with monogenic autosomal recessive inheritance. AIRE is predominantly expressed in medullary epithelial cells of the thymus and is considered to play important roles in the establishment of self-tolerance. AIRE contains two plant homeodomain (PHD) domains, and the novel role of PHD as an E3 ubiquitin (Ub) ligase has just emerged. Here we show that the first PHD (PHD1) of AIRE mediates E3 ligase activity. The significance of this finding was underscored by the fact that disease-causing missense mutations in the PHD1 (C311Y and P326Q) abolished its E3 ligase activity. These results add a novel enzymatic function for AIRE and suggest an indispensable role of the Ub proteasome pathway in the establishment of self-tolerance, in which AIRE is involved.

Simple PCR-based DNA microarray system to identify human pathogenic fungi in skin.

Fungal diseases in immunocompromised hosts pose significant threats to their prognoses. An accurate diagnosis and identification of the fungal pathogens causing the infection are critical to determine the proper therapeutic interventions, but these are often not achieved, due to difficulties with isolation and morphological identification. In an effort to ultimately carry out the simultaneous detection of all human pathogenic microbes, we developed a simple system to identify 26 clinically important fungi by using a combination of PCR amplification and DNA microarray assay (designated PCR-DM), in which PCR-amplified DNA from the internal transcribed spacer region of the rRNA gene was hybridized to a DNA microarray fabricated with species-specific probes sets using the Bubble Jet technology. PCR-DM reliably identified all 26 reference strains; hence, we applied it to cases of onychomycosis, taking advantage of the accessibility of tissue from skin. PCR-DM detected fungal DNA and identified pathogens in 92% of 106 microscopy-confirmed onychomycosis specimens. In contrast, culture was successful for only 36 specimens (34%), 3 of which had results inconsistent with the results of PCR-DM, but sequence analysis of the isolates proved that the PCR-DM result was correct. Thus, PCR-DM provides a powerful method to identify pathogenic fungi with high sensitivity and speed directly from tissue specimens, and this concept could be applied to other fungal or nonfungal infectious human diseases in less accessible anatomical sites.

Microdeletion of the Down syndrome critical region at 21q22.

The concept of the Down syndrome critical region implies the existence of several dosage-sensitive genes that result in an abnormal phenotype when duplicated. Among the genes in the presumed Down syndrome critical region, DYRK1A and SIM2 are thought to be particularly important because of their critical roles in the development of the central nervous system in model organisms. Considering that regulatory imbalances resulting in an altered amount of expression from crucial target genes tend to produce phenotypic effects in both monosomics and trisomics, haploinsufficiency for the Down syndrome critical region is expected to be associated with an abnormal phenotype. We report on a patient with severe microcephaly, a developmental delay, hypospadias, and corneal opacity who had a microdeletion spanning the Down syndrome critical region, including DYRK1A and SIM2. He presented with intrauterine growth retardation, hypospadias, corneal clouding, arched eyebrows, upslanting and narrow palpebral fissures, bifid uvula, prominent nasal root, short columella, prominent central incisors, pegged shaped teeth, retrognathia, hypoplastic nipples, and severe developmental delay. His G-banded karyotype was normal, but array comparative genomic hybridization showed a de novo deletion of 3.97 Mb at chromosome 21q22. The extreme degree of microcephaly in this patient may be ascribed to the haploinsufficiency of DYRK1A, since brain size is severely reduced in heterozygotes for the Dyrk1a null mutation in mice.

Analysis of the promoter region of human placenta-specific DSCR4 gene.

The gene DSCR4 locates in the band q22.2 of human chromosome 21 and encodes a protein of 118 amino acids. Expression of DSCR4 is restricted to human placenta and placental choriocarcinoma cell lines BeWo and JEG3. The 5'-RACE method using RNA from human placenta indicated the major transcription start site at 93 nt upstream (nt -93) of the initiation codon. Transfection assay using a series of deletion constructs of the 5'-flanking region fused to the luciferase reporter gene identified three positive regions nt -2200 to -2088, nt -2064 to -1924, nt -810 to -632 and two negative regions nt -1923 to -1740, nt -631 to -425. The computer analysis predicted the presence of several cis-elements in these regions and the promoter assay using various mutants of consensus sequence identified two distinct cis-elements for OLF-1 and E47. The electrophoretic mobility shift assay (EMSA) using the extracts of DSCR4-expressing cells confirmed the binding of certain protein factors to these cis-elements. In fact, OLF-1-like transcription factor, EBF-3 and EBF-4 were detected in the DSCR4-expressing cell lines and human placenta. Based on these data, we postulated that transcription of DSCR4 gene is regulated positively by binding of OLF-1-like transcription factor and negatively by binding of E47-like transcription factor.

Theoretical study of phase transitions in Kr and Ar clathrate hydrates from structure II to structure I under pressure.

The theory developed in our earlier papers is extended to predict dynamical and thermodynamic properties of clathrate structures by accounting for the possibility of multiple filling of cavities by guest molecules. The method is applied to the thermodynamic properties of argon and krypton hydrates, considering both structures I (sI) and II (sII), in which the small cages can be singly occupied and large cages of sII can be singly or doubly occupied. It was confirmed that the structure of the clathrate hydrate is determined by two main factors: intermolecular interaction between guest and host molecules and the configurational entropy. It is shown that for guests weakly interacting with water molecules, such as argon or krypton, the free energy of host lattices without the contribution of entropy is the main structure-determining factor for clathrate hydrates, and it is a cause of hydrate sII formation at low pressure with these guests. Explicit account of the entropy contribution in the Gibbs free energy allows one to determine the stability of hydrate phases and to estimate the line of structural transition from sII to sI in P-T plane. The structural transition between sII and sI in argon and krypton hydrates at high pressure is shown to be the consequence of increasing intermolecular interaction and the degree of occupancy of the large cavities.

WITHDRAWN: Analysis of the promoter region of human placenta-specific DSCR4 gene.

The Publisher regrets that this article is an accidental duplication of an article that has already been published in Biochem. Biophys. Acta, doi:10.1016/j.bbagrm.2007.09.005. The duplicate article has therefore been withdrawn.

Sequence analysis of filaggrin gene by novel shotgun method in Japanese atopic dermatitis.

BACKGROUND: Recent reports indicated that nonsense mutations in filaggrin (FLG) found in ichthyosis vulgaris (IV) patients are predisposing factors for atopic dermatitis (AD) with asthma. The exon 3 of FLG contains tandemly repeated, highly homologous, 11-13 sequence units of 972 or 975 bp, each of which corresponds to the coding sequence of the processed filaggrin with slight sequence difference. This unique gene structure has hampered the precise DNA sequence determination. OBJECTIVE: We developed a novel DNA sequencing method "FLG-shotgun" to directly characterize the mutations in Japanese AD patients. METHODS: We examined 24 Japanese AD patients with "FLG-shotgun" method. RESULTS: Multiple units of FLG were amplified by PCR using several sets of common primers for the conserved regions, and DNA sequences of each cloned PCR product were determined. Multiple reads of DNA sequences in both alleles were aligned and re-constructed to cover the entire coding regions. We found three major genotypes (A, B, and C) which represent different numbers (11-13) of homologous sequence units. Furthermore, we found two novel nonsense mutations; one mutation 8666-8667CC>GA on the unit 9 of allele B that causes a nonsense mutation S2899X in two patients and the other mutation 9887C>A on the unit 10 of allele B that causes a nonsense mutation S3296X in two patients. CONCLUSION: We found two novel FLG mutations by directly analyzing Japanese patients with AD. FLG-shotgun will provide a valuable tool to further define the nature of the AD phenotype associated with FLG mutations.

Binding sites on tau proteins as components for antimicrobial peptides.

To design new antimicrobial peptides, we have focused on various proteins which are not essential for self-defense but carry important responsibilities for biosystems. Previously, we reported that highly efficient antimicrobial properties or antiviral properties are inherent in the nuclear translocation signals and binding sites on laminin receptors. Here we introduce microtubule binding sites on tau proteins as new components for antimicrobial peptides. Strong antimicrobial activities against Staphylococcus aureus and Escherichia coli were found in tandem sequences of the binding sites on tau proteins. Moreover, the binding sites obtained significantly strong antimicrobial activities against bacteria and fungi when combined with a nuclear localization signal (NLS) and/or a peptide derived from a binding site of a laminin receptor. The antimicrobial activities of some of the tau-derived peptides were not affected by salt, cations, or serum that simulate the natural environment present in blood. Tau proteins so far have only been known as one of the microtubule-associated proteins (MAPs) which are especially abundant in the central nervous system within the brain. Our finding demonstrates that the binding sites on tau proteins possess high potential for becoming components in antimicrobial peptides. Designs based on binding sites of various proteins could become a useful method in peptide antibiotic research.

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.