杉浦 一充

DNA mismatch repair (MMR) is one of the several DNA repair pathways conserved from bacteria to humans. The primary function of MMR is to eliminate the mismatch of base-base insertions and deletions that appear as a consequence of DNA polymerase errors at DNA synthesis. The genes encoding the DNA MMR enzymes (MMREs) are highly conserved throughout evolution. In humans, there are two sets of MMREs, corresponding to homologues of the bacterial MutLS systems. The human MutS enzymes consist of MSH2, MSH3 and MSH6, and the human MutL enzymes include MLH1, MLH3, PMS1 and PMS2. Since the beginning of this century, a few reports on autoantibodies to some MMREs have been reported in autoimmune inflammatory myopathy, cancer and hematological disorders. This review charts the functional structures of MMREs, their genetic defects and associated disorders, and autoimmunity to MMREs, including our recent data that was the first to analyze autoantibodies against all seven kinds of MMREs in systemic autoimmune diseases, including idiopathic inflammatory myopathies. (C) 2015 Elsevier B.V. All rights reserved.

Introduction: Anti-PM/Scl antibodies are associated with polymyositis (PM)/systemic scleroderma (SSc) overlap syndromes and are also found in other systemic autoimmune diseases. Although anti-PM/Scl reactivity is found in 3-11% of PM or SSc patients and in approximately 25% of PM/SSc overlap patients, previous large studies of Japanese patients with scleroderma reported that anti-PM/Scl are not found in Japanese patients at all. The PM/Scl autoantigen complex comprises 11-16 different polypeptides; ELISA with PM1-alpha peptide, which is a major epitope of the PM/Scl complex, has frequently been used for the detection of these antibodies in recent studies. However, no ELISA kit is commercially available in Japan. Methods: In this study, we developed an immunoassay for measuring antibodies against recombinant PM/Scl-100 and PM/Scl-75 polypeptides, which are the two major targets of the complex, and we investigated their presence in 600 Japanese patients with various systemic autoimmune conditions. Immunoprecipitation analysis using the recombinants in addition to traditional radiolabeled cell extracts were also applied to ELISA-positive sera. Results: In ELISA, 11 patients were positive for anti-PM/Scl-100 antibodies and 7 of these 11 patients were also positive for anti-PM/Scl-75 antibodies. Immunoprecipitation analysis using the recombinants in addition to traditional radiolabeled cell extracts confirmed that 9 out of these 11 patients immunoprecipitated the typical sets of PM/Scl proteins. In total, 4/16 (25%) undifferentiated connective tissue disease (UCTD) patients, 3/126 (2.4%) dermatomyositis patients, 1/223 (0.4%) SSc patients, 1/88 (1.1%) Sjogren's syndrome patients, 0/123 patients with systemic lupus erythematosus, 0/17 patients with overlap syndrome and 0/7 patients with PM were judged to be positive for anti-PM/Scl antibodies. Conclusions: This is the first report of Japanese autoimmune patients with anti-PM/Scl antibodies. In Japanese patients, anti-PM/Scl antibodies are only very rarely found, and they are not always specific for dermatomyositis (DM) or SSc; they are also present in various autoimmune conditions with the highest prevalence being in UCTD. All anti-PM/Scl-positive DM cases are complicated with interstitial lung disease and/or cancer, while no life-threatening involvement was found in other anti-PM/Scl-positive cases. Further studies on larger cohorts are necessary to define the clinical significance of anti-PM/Scl antibodies in autoimmune diseases.

Only two homozygous nonsense mutations in the epidermal isoform of the dystonin gene, DST-e, have been reported previously in autosomal recessive epidermolysis bullosa simplex (EBS); the affected pedigrees were Kuwaiti and Iranian. This subtype of EBS is therefore considered to be a rare clinicopathological entity. In this study, we identified four seemingly unrelated Kuwaiti families in which a total of seven individuals had predominantly acral trauma-induced blistering since infancy. All affected individuals were homozygous for the mutation p.Gln1124* in DST-e, the same mutation that was identified in the originally reported family from Kuwait. Haplotype analysis in the five pedigrees (including the previous case) revealed a shared block of similar to 60 kb of genomic DNA across the site of the mutation, consistent with a founder effect. Most heterozygotes had no clinical abnormalities although one subject had mild transient skin fragility during childhood, an observation noted in the previously reported Iranian pedigree, suggesting that the condition may also be semidominant in some pedigrees rather than purely autosomal recessive. Our study reveals propagation of a mutant ancestral allele in DST-e throughout Kuwait, indicating that this subtype of EBS may be more common in Kuwait, and perhaps other Middle Eastern countries, than is currently appreciated.

Objective. Myositis-specific autoantibodies (MSAs) are useful tools for identifying clinical subsets of patients with idiopathic inflammatory myopathies (IIMs). There have been few reports on antibodies to some DNA mismatch repair enzymes (MMREs) in patients with IIMs. This study was undertaken to determine the frequencies and clinical associations of antibodies to 7 types of MMREs (MLH1, MLH3, MSH2, MSH3, MSH6, PMS1, and PMS2) in patients with IIMs and other systemic autoimmune diseases. Methods. Clinical data and serum samples were collected from 239 Japanese patients with IIMs (147 with adult dermatomyositis, 13 with juvenile dermatomyositis, 57 with polymyositis, and 22 with myositis overlap syndrome). One hundred patients with other diseases, including 40 with systemic lupus erythematosus (SLE), were assessed as disease controls. The presence of anti-MMRE antibodies in serum was examined by immunoprecipitation, enzyme-linked immunosorbent assay, and immunoprecipitation/Western blotting. Results. Anti-MMRE antibodies were found in 15 patients with IIMs and 3 patients with SLE. They were restricted to MLH1, PMS1, MSH2, and PMS2, with simultaneous positivity for more than one of these antibodies occurring in some patients. Nine IIM pa-tients with anti-MMREs also had other MSAs and their associated clinical features. All patients with antiMMREs were still living at the time of the present analysis. Conclusion. Anti-MMRE antibodies, which often coexist with other MSAs, may be serologic markers for good prognosis in IIMs.

ATP-binding cassette transporter family A member 12 (ABCA12) is a keratinocyte transmembrane lipid transporter that plays a critical role in preserving the skin permeability barrier. Biallelic loss of function of the ABCA12 gene is causative of some forms of recessive congenital ichthyosis, an intractable disease marked by dry, thickened and scaly skin on the whole body. Genetic diagnosis is essential, although the results may occasionally be inconclusive, because some patients with low ABCA12 expression have one mutant allele and one apparently intact allele. Aside from aberrant splicing or deletion mutations, one possible explanation for such discrepancy is loss of promoter function. This study aims to elucidate the promoter region of ABCA12 and to locate the essential elements therein, thus providing the necessary information for genetic diagnostic screening of congenital ichthyosis. Close examination of the 2980-bp upstream regions of the ABCA12 gene revealed that a palindromic motif (tgagtca) at -2084 to -2078 is essential for the promoter function, and a short fragment of -2200/-1934 alone has potent promoter activity. Identification of the key promoter element of ABCA12 in this study may provide relevant information for genetic diagnosis of recessive congenital ichthyosis.

BackgroundNagashima-type palmoplantar keratosis (NPPK) is a distinct autosomal recessive genodermatosis characterized by diffuse transgressive palmoplantar keratoderma (PPK). Very recently, putative loss-of-function mutations in SERPINB7, which encodes a member of the serine protease inhibitor superfamily and is abundantly expressed in the epidermis, have been identified as a cause of NPPK. ObjectivesTo confirm further the role of SERPINB7 mutations in the pathogenesis of NPPK. MethodsWe analysed 10 Japanese families with NPPK using Sanger and/or whole-exome sequencing. ResultsWe identified one novel and three recurrent null mutations in SERPINB7. In all the families, the NPPK trait was inherited in an autosomal recessive manner; in one of the families, there was pseudodominant inheritance, which had not been described in NPPK. ConclusionsThese data clearly provide further evidence that NPPK is caused by loss-of-function mutations in SERPINB7.

Generalized pustular psoriasis (GPP) is often present in patients with existing or prior psoriasis vulgaris (PV; "GPP with PV"). However, cases of GPP have been known to arise without a history of PV ("GPP alone"). There has long been debate over whether GPP alone and GPP with PV are distinct subtypes that are etiologically different from each other. We recently reported that the majority of GPP alone cases is caused by recessive mutations of IL36RN. In contrast, only a few exceptional cases of GPP with PV were found to have recessive IL36RN mutations. Very recently, we also reported that CARD14 p.Asp176His, a gain-of-function variant, is a predisposing factor for GPP with PV; in contrast, the variant is not associated with GPP alone in the Japanese population. These results suggest that GPP alone is genetically different from GPP with PV. IL36RN mutations are also found in some patients with severe acute generalized exanthematous pustulosis, palmar-plantar pustulosis, and acrodermatitis continua of hallopeau. CARD14 mutations and variants are causal or disease susceptibility factors of PV, GPP, or pityriasis rubra pilaris, depending on the mutation or variant position of CARD14. It is clinically important to analyze IL36RN mutations in patients with sterile pustulosis. For example, identifying recessive IL36RN mutations leads to early diagnosis of GPP, even at the first episode of pustulosis. In addition, individuals with IL36RN mutations are very susceptible to GPP or GPP-related generalized pustulosis induced by drugs (e.g., amoxicillin), infections, pregnancy, or menstruation. (C) 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

When two mutations, one dominant pathogenic and the other "confining'' nonsense, coexist in the same allele, theoretically, reversion of the latter may elicit a disease, like the opening of Pandora's box. However, cases of this hypothetical pathogenic mechanism have never been reported. We describe a lethal form of keratitis-ichthyosis-deafness (KID) syndrome caused by the reversion of the GJB2 nonsense mutation p. Tyr136X that would otherwise have confined the effect of another dominant lethal mutation, p. Gly45Glu, in the same allele. The patient's mother had the identical misssense mutation which was confined by the nonsense mutation. The biological relationship between the parents and the child was confirmed by genotyping of 15 short tandem repeat loci. Haplotype analysis using 40 SNPs spanning the >39 kbp region surrounding the GJB2 gene and an extended SNP microarray analysis spanning 83,483 SNPs throughout chromosome 13 in the family showed that an allelic recombination event involving the maternal allele carrying the mutations generated the pathogenic allele unique to the patient, although the possibility of coincidental accumulation of spontaneous point mutations cannot be completely excluded. Previous reports and our mutation screening support that p. Gly45Glu is in complete linkage disequilibrium with p. Tyr136X in the Japanese population. Estimated from statisitics in the literature, there may be approximately 11,000 p. Gly45Glu carriers in the Japanese population who have this second-site confining mutation, which acts as natural genetic protection from the lethal disease. The reversion-triggered onset of the disesase shown in this study is a previously unreported genetic pathogenesis based on Mendelian inheritance.

Background: The aminoacyl transfer RNA synthetases (ARSs) are a group of enzymes that charge amino acids to the cognate transfer RNA during the translation process. Previous reports demonstrated autoantibodies to 8 different ARS. Although anti-glycyl-tRNA synthetase antibodies (anti-EJ) are mainly found in patients with inflammatory myopathy, information on their clinical significances is limited, partly due to a lack of commercially available tests. Methods: We developed an ELISA and immunoprecipitation method by using recombinant EJ protein to detect the anti-EJ of 453 patients with various autoimmune connective tissue diseases (ACTDs). We also studied the influence of 3 cytokines-IL-1 beta, IFN-gamma and IFN-alpha-on the level of EJ mRNA and protein expressed by human fetal lung fibroblasts. Results: Five patients were positive for anti-EJ. Although 3 of these patients had dermatomyositis/polymyositis, the other 2 patients did not have myositis. The three patients with high levels of anti-EJ antibodies in ELISA were complicated with interstitial lung disease. There was no significant change in the level of EJ protein expressed by human fetal lung fibroblasts stimulated by the cytokines. Conclusion: We developed an ELISA to detect anti-EJ by using recombinant protein. This easy-to-use ELISA could help clarify the clinical significance of anti-EJ in ACTDs. (C) 2014 Elsevier B.V. All rights reserved.

Although expression of gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for ganglioside synthesis during evolution of melanomas from melanocytes are very important to understand roles of gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like ganglioside profiles in melanocytes. © 2014 Elsevier Inc. All rights reserved.

The nuclear localization signal (NLS)-containing proteins LEDGF and STAT3 localize to the nucleus in both the spinous and basal layers of the epidermis in psoriatic skin, where they function as transcription factors or co-factors to activate epidermal keratinocytes (KCs). However, the mechanism underlying the localization of these proteins remains to be elucidated. We investigated the differential nucleocytoplasmic transport of NLS-containing proteins as a potential pathogenic mechanism for psoriasis vulgaris. Nucleoporins play an important role in the Ran-GTP-dependent nucleocytoplasmic transport of NLS-containing proteins. We showed, using immunohistochemical staining, that the nucleoporins Ran-binding protein 2 (RanBP2) and Ran-GTPase-activating protein 1 (RanGAP1) have greater expression on the nuclear envelope in psoriatic epidermal KCs than in KCs from healthy controls. We then studied the signalling pathways involved in the regulation of these proteins in HaCaT cells. The two major downstream pathways of epidermal growth factor receptor (EGFR) signalling activated in psoriatic KCs are the MAPK/Erk/1/2 and the phosphatidylinositol-3-kinase/Akt pathways. Therefore, we treated HaCaT cells with inhibitors to disrupt the MAP kinase kinase 1 (MEK1), PI3-kinase, or mTOR pathways. RanBP2 and RanGAP1 protein expression levels were significantly greater in the nuclear envelope of HaCaT cells that were not treated with inhibitors than in cells treated with a combination of PI3-kinase and MEK1 inhibitors or mTOR and MEK1 inhibitors. These results suggest that adequate nuclear envelope expression of RanBP2 and RanGAP1 could be a prerequisite for nucleocytoplasmic transport in KCs in psoriatic epidermis.

Background: Paraneoplastic pemphigus (PNP) serum preferentially reacts with periplakin and envoplakin, which are plakin family proteins localized to desmosomes and intermediate filaments. Recently, anti-periplakin antibodies were also detected in patients with idiopathic pulmonary fibrosis (IPF). Although previous epitope-mapping studies showed multiple epitopes in each protein, enzyme-linked immunosorbent assays have used several truncated, but not full-length, recombinant proteins. Methods: This study aimed to produce full-length biotinylated recombinant proteins of periplakin and envoplakin for detection of autoantibodies by immunoprecipitation and ELISA. Serum from a PNP patient who had been confirmed as carrying anti-periplakin and anti-envoplakin antibodies in our previous study was used as a positive control. Sera from 15 patients with IPF were analyzed for both antibodies by immunoprecipitation and by ELISA. Results: The PNP serum reacted strongly with the full-length recombinant proteins in immunoprecipitation and ELISA. Longitudinal serum samples from the PNP patient showed a clear decline of autoantibodies to both periplakin and envoplakin. None of the IPF sera showed both autoantibodies. Conclusions: We found that the detection of anti-periplakin and anti-envoplakin antibodies using full-length recombinant proteins is useful immunoprecipitation and ELISA. (C) 2013 Elsevier B.V. All rights reserved.

Mutations in LIPH cause of autosomal recessive woolly hair/hypotrichosis (ARWH), and the 2 missense mutations c.736T>A (p.Cys246Ser) and c.742C>A (p.His248Asn) are considered prevalent founder mutations for ARWH in the Japanese population. To reveal genotype/phenotype correlations in ARWH cases in Japan and the haplotypes in 14 Japanese patients from 14 unrelated Japanese families. 13 patients had woolly hair, and 1 patient had complete baldness since birth. An LIPH mutation search revealed homozygous c.736T>A mutations in 10 of the patients. Compound heterozygous c.736T>A and c.742C>A mutations were found in 3 of the patients, and homozygous c.742C>A mutation in 1 patient. The phenotype of mild hypotrichosis with woolly hair was restricted to the patients with the homozygous c.736T>A mutation. The severe phenotype of complete baldness was seen in only 1 patient with homozygous c.742C>A. Haplotype analysis revealed that the alleles containing the LIPH c.736T>A mutation had a haplotype identical to that reported previously, although 4 alleles out of 5 chromosomes containing the LIPH c.742C>A mutation had a different haplotype from the previously reported founder allele. These alleles with c.742C>A are thought to be the third founder LIPH mutation causing ARWH. To accurately determine the prevalence of the founder mutations, we investigated allele frequencies of those mutations in 819 Japanese controls. Heterozygous c.736T>A mutations were found in 13 controls (allele frequency: 0.0079; carrier rate: 0.016), and heterozygous c.742C>A mutations were found in 2 controls (allele frequency: 0.0012; carrier rate: 0.0024). In conclusion, this study confirms the more accurate allele frequencies of the pathogenic founder mutations of LIPH and shows that there is a third founder mutation in Japan. In addition, the present findings suggest that the mutation patterns of LIPH might be associated with hypotrichosis severity in ARWH.

Juvenile xanthogranuloma (JXG) is a non-Langerhans histiocytosis occurring predominantly in infancy and early childhood. Resolution usually occurs over a period of months to several years, and it is rare for the condition to persist beyond late childhood (1). The symmetrical giant facial plaque variant of JXG (SGFP-JXG) is very rare. It was originally reported by Gunson & Birchall. (2). Herein we report a case of SGFP-JXG that persisted beyond 10 years of age.

Hydroxyurea (HU) is a chemotherapeutic agent used for the treatment of myeloproliferative disorders such as chronic myeloid leukemia, polycythemia vera, and essential thrombocytosis. We describe a 69-year-old man who had essential thrombocytosis and developed amyopathic dermatomyositis after long-term HU therapy. He presented with Gottron papules and heliotrope erythema. The former has been described in all cases of HU-induced dermatomyositis the latter has been seen in a few cases of that disorder. © 2014 by the article author(s).

Generalized pustular psoriasis (GPP) is a rare inflammatory skin disease that can be life-threatening. Recently, it has been reported that familial GPP is caused by homozygous or compound heterozygous mutations of IL36RN. However, the majority of GPP cases are sporadic and it is controversial whether IL36RN mutations are a causative/predisposing factor for sporadic GPP. We searched for IL36RN mutations in two groups of GPP patients in the Japanese population in this study: GPP without psoriasis vulgaris (PV), and GPP with PV. Eleven cases of GPP without PV (GPP alone) and 20 cases of GPP accompanied by PV (GPP with PV) were analyzed. Surprisingly, 9 out of 11 cases of GPP alone had homozygous or compound heterozygous mutations in IL36RN. In contrast, only 2 of 20 cases of GPP with PV had compound heterozygous mutations in IL36RN. The two cases of GPP with PV who had compound heterozygous mutations in IL36RN are siblings, and both cases had PV-susceptible HLA-A*0206. We determined that GPP alone is a distinct subtype of GPP and is etiologically distinguished from GPP with PV, and that the majority of GPP alone is caused by deficiency of the interleukin-36 receptor antagonist due to IL36RN mutations.

Reticulate acropigmentation of Kitamura (RAK) is a rare genetic disorder of cutaneous pigmentation with an autosomal dominant pattern of inheritance and a high penetration rate. The characteristic skin lesions are reticulate, slightly depressed pigmented macules mainly affecting the dorsa of the hands and feet, which first appear before puberty and subsequently expand to the proximal limb and the trunk. To identify mutations that cause RAK, we performed exome sequencing of four family members in a pedigree with RAK. Fifty-three SNV/Indels were considered as candidate mutations after some condition narrowing. We confirmed the mutation status in each candidate gene of four other members in the same pedigree to find the gene that matched the mutation status and phenotype of each member. A mutation in ADAM10 encoding a zinc metalloprotease, a disintegrin and metalloprotease domain-containing protein 10 (ADAM10), was identified in the RAK family. ADAM10 is known to be involved in the ectodomain shedding of various substrates in the skin. Sanger sequencing of four additional unrelated RAK patients revealed four additional ADAM10 mutations. We identified a total of three truncating mutations, a splice site mutation and a missense mutation in ADAM10. We searched for mutations in the KRT5 gene, a causative gene for the similar pigmentation disorder Dowling-Degos disease (DDD), in all the patients and found no KRT5 mutation. These results reveal that mutations in ADAM10 are a cause of RAK and that RAK is an independent clinical entity distinct from DDD.

Background Mutations in LIPH are a cause of autosomal recessive woolly hair (ARWH). Homozygous c. 736T>A (p. Cys246Ser), and compound heterozygous c. 736T>A and c. 742C>A (p. His248Asn) have been reported in 5 and 7 Japanese children with ARWH respectively. The severity of hypotrichosis is known to be able to change in the clinical course, and the mutation patterns of LIPH do not always correlate with the severity of hypotrichosis in ARWH caused by other mutation sites of LIPH. However, all 12 Japanese children previously reported to have ARWH have shown similar severity of hypotrichosis. Objective In this study, we investigated the clinical features and molecular basis of ARWH in patients including three adults (three adults and two children) from five non-related Japanese families. Methods Five families of Japanese origin that presented with woolly hair were studied. The phenotype was confirmed by clinical examination. Direct automated DNA sequencing of the LIPH gene was performed to identify the mutations in our probands. Results All patients had had woolly hair since birth. Homozygous c. 736T>A mutations were found in four patients, including three adult cases, and compound heterozygous c. 736T>A and c. 742C>A mutations were found in one child patient. The two adults and two children had only sparse scalp hair, although one adult woman had mild hypotrichosis with long hairs. Conclusion Some patients with homozygous c. 736T>A can have a mild hypotrichosis phenotype with long hairs in adulthood.

Background: Barrier-to-autointegration factor 1 (BANF1) is an essential component of the nuclear lamina. Recent studies have clarified that BANF1 is a causative molecule of Nestor-Guillermo progeria syndrome. Despite recent progress in studies on BANF1, the role of BANF1 in keratinocytes has not been addressed at all. Objective: This study aims to determine the localization of BANF1 in psoriatic epidermal keratinocytes as well as in normal keratinocytes and to clarify its possible function in those keratinocytes. Methods: Immunohistochemistry of BANF1 was performed on 10 cases of psoriasis and 10 healthy control individuals. Expression of molecules associated with inflammation of the skin by HSC-1, a human skin squamous cell carcinoma cell line, stimulated by TPA and treated with siRNA to BANF1 were analyzed with quantitative PCR and Western blot. Results: Strong nuclear-dominant immunostaining of BANF1 was seen in the epidermal keratinocytes of psoriatic lesions, although in the normal epidermis, all the KCs in the upper epidermis showed cytoplasmic-dominant staining of BANF1. By BANF1 knockdown in TPA-stimulated HSC-1 cells, the mRNA levels of S100A9 were significantly elevated compared with those of control HSC-1 cells treated with siRNA to CD4. The protein expression level of S100A9 and phosphorylated c-Jun was elevated by BANF1 knockdown. Conclusion: BANF1 is translocated onto the nuclear envelope in the psoriatic epidermal keratinocytes, suggesting that BANF1 is associated with upregulated proliferation of keratinocytes in psoriatic lesions. Activation of BANF1 possibly suppresses S100A9 expression and inactivates c-Jun, resulting in suppression of cutaneous inflammation. (C) 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.