Aiko Shiohama

A homozygous frameshift mutation in the mouse Flg gene facilitates enhanced percutaneous allergen priming

Loss-of-function mutations in the FLG (filaggrin) gene cause the semidominant keratinizing disorder ichthyosis vulgaris and convey major genetic risk for atopic dermatitis (eczema), eczema-associated asthma and other allergic phenotypes. Several low-frequency FLG null alleles occur in Europeans and Asians, with a cumulative frequency of ∼9% in Europe. Here we report a 1-bp deletion mutation, 5303delA, analogous to common human FLG mutations, within the murine Flg gene in the spontaneous mouse mutant flaky tail (ft). We demonstrate that topical application of allergen to mice homozygous for this mutation results in cutaneous inflammatory infiltrates and enhanced cutaneous allergen priming with development of allergen-specific antibody responses. These data validate flaky tail as a useful model of filaggrin deficiency and provide experimental evidence for the hypothesis that antigen transfer through a defective epidermal barrier is a key mechanism underlying elevated IgE sensitization and initiation of cutaneous inflammation in humans with filaggrin-related atopic disease.

Identification of eight members of the Argonaute family in the human genome.

A number of genes have been identified as members of the Argonaute family in various nonhuman organisms and these genes are considered to play important roles in the development and maintenance of germ-line stem cells. In this study, we identified the human Argonaute family, consisting of eight members. Proteins to be produced from these family members retain a common architecture with the PAZ motif in the middle and Piwi motif in the C-terminal region. Based on the sequence comparison, eight members of the Argonaute family were classified into two subfamilies: the PIWI subfamily (PIWIL1/HIWI, PIWIL2/HILI, PIWIL3, and PIWIL4/HIWI2) and the eIF2C/AGO subfamily (EIF2C1/hAGO1, EIF2C2/hAGO2, EIF2C3/hAGO3, and EIF2C4/hAGO4). PCR analysis using human multitissue cDNA panels indicated that all four members of the PIWI subfamily are expressed mainly in the testis, whereas all four members of the eIF2C/AGO subfamily are expressed in a variety of adult tissues. Immunoprecipitation and affinity binding experiments using human HEK293 cells cotransfected with cDNAs for FLAG-tagged DICER, a member of the ribonuclease III family, and the His-tagged members of the Argonaute family suggested that the proteins from members of both subfamilies are associated with DICER. We postulate that at least some members of the human Argonaute family may be involved in the development and maintenance of stem cells through the RNA-mediated gene-quelling mechanisms associated with DICER.

Molecular cloning and expression analysis of a novel gene DGCR8 located in the DiGeorge syndrome chromosomal region

We have identified and cloned a novel gene (DGCR8) from the human chromosome 22q11.2. This gene is located in the DiGeorge syndrome chromosomal region (DGCR). It consists of 14 exons spanning over 35kb and produces transcripts with ORF of 2322bp, encoding a protein of 773 amino acids. We also isolated a mouse ortholog Dgcr8 and found it has 95.3% identity with human DGCR8 at the amino acid sequence level. Northern blot analysis of human and mouse tissues from adult and fetus showed rather ubiquitous expression. However, the in situ hybridization of mouse embryos revealed that mouse Dgcr8 transcripts are localized in neuroepithelium of primary brain, limb bud, vessels, thymus, and around the palate during the developmental stages of embryos. The expression profile of Dgcr8 in developing mouse embryos is consistent with the clinical phenotypes including congenital heart defects and palate clefts associated with DiGeorge syndrome (DGS)/conotruncal anomaly face syndrome (CAFS)/velocardiofacial syndrome (VCFS), which are caused by monoallelic microdeletion of chromosome 22q11.2.

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.

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.

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.

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.

The novel SLCO2A1 heterozygous missense mutation p.E427K and nonsense mutation p.R603* in a female patient with pachydermoperiostosis with an atypical phenotype.

DEAR EDITOR, Pachydermoperiostosis (PDP), or primary hypertrophic osteoarthropathy (PHO: MIM 167100), is a rare genetic disease affecting the skin and bones. The major diagnostic criteria include finger clubbing, periostosis, pachydermia and cutis verticis gyrata (CVG). Additional symptoms, including sebaceous hyperplasia, hyperhidrosis and arthropathy, have been reported. Uppal et al. discovered that a homozygous mutation in HPGD, which encodes 15-hydroxyprostaglandin dehydrogenase (15-PGDH), causes PHO and PDP. However, PHO and PDP are genetically heterogeneous. Exome analysis of PDP in Japanese, Chinese, Caucasian and other races has revealed homozygous mutations in the solute carrier organic anion transporter family member 2A1 (SLCO2A1) gene, which encodes prostaglandin transporter (PGT). Increased levels of prostaglandin E2 (PGE2) resulting from defective degradation contribute to the pathogenesis of PHO and PDP. A genetic defect in either SLCO2A1 or HPGD can cause PHO and PDP. In this study, we describe the first observation of a SLCO2A1 mutation in a female patient. A 67-year-old woman was referred for SLCO2A1 mutation analysis. At the age of 43 years, she developed myelopathy of unknown aetiology. She received rehabilitation therapy without medication. A neurologist had examined her muscle weakness at the T-helper 7 level on the right side following a diagnosis of suspected multiple sclerosis. At the age of 64 years, she had multiple seronegative arthralgias but no serious problems. She was referred to Tohoku Kouseinenkin Hospital because of recurring arthralgia and was treated with methotrexate and prednisone. She responded favourably to the medication with alleviation of the pain and decreased serum levels of C-reactive protein. Physical examination revealed finger clubbing and swelling of the large joints, as seen in Figure 1b,c. No skin manifestations, including facial coarseness or greasiness, and no hyperhidrosis were observed. Marked thickening of the scalp (CVG) was not evident. Radiological examination showed the presence of periostosis of the diaphysis of the tibia and fibula (Fig. 1d). No hydrarthrosis was evident. A diagnosis of possible incomplete type of PDP or PHO was made because of minimal pachydermia. She had no history of peptic ulcers or anaemia. Diagnostic imaging and laboratory data revealed no evidence of secondary PDP. She has a healthy son and daughter. This study was approved by the ethics committee of the National Centre for Child Health and Development and Keio University School of Medicine. The participants provided written informed consent. All exons of HPGD and SLCO2A1 along with sequences adjacent to the exon–intron borders were amplified, sequenced and screened for mutations. Serum and urinary levels of PGE2 were measured with a commercial enzyme immunoassay kit (Cayman, Cayman Biochemical, Ann Arbor, MI, U.S.A.). We identified compound heterozygous novel mutations c.1279G>A/p.E427K and c.1807C>T/p.R603* in SLCO2A1 (Fig. 2). We also detected a heterozygous mutation c.1279G>A in her daughter, but she has not developed any triad of PDP. In her seventh decade, the patient’s atypical history showed minimal impact of pachydermia. Serum PGE2 was not detected and her urinary PGE2 was within normal limits (372 pg mL ). One of the mutations, c.1279G>A (p.E427K), is included within the region of a previously reported deletion, c.1279_1290del12 (p.E427_P430del). Another mutation, c.1807C>T/p.R603*, is detected close to the C-terminus of PGT, resulting in a shortened predicted protein. The loss of function in truncated PGT is consistent with the presence of the p.R603* mutation in another patient, who had the complete type of PDP (manuscript in preparation). This patient is the first woman with PDP who had an SLCO2A1 gene mutation. It is unlikely that the mild phenotype of P1 was due to the missense mutation p.E427K. A recent report on PDP in a Chinese family described a homozygous p.A286Qfs*35 frameshift mutation in a male proband who had PDP. Two of the proband’s sisters were also homozygous for p.A286Qfs*35, but at ages 42 years and 47 years, they had neither history nor findings suggestive of PDP. Diggle et al. reported that two women in two PDP families were homozygous for pathogenic SLCO2A1 mutations. One had mild finger clubbing but no musculoskeletal or skin symptoms at 34 years of age. The other was asymptomatic at 19 years of age. Taken together, we propose that PDP resulting from SLCO2A1 defects is a sex-dependent autosomal recessive disease, and women homozygous for pathogenic SLCO2A1 mutations may develop late-onset PDP symptoms. The explanation of mildand low-frequency disease in women remains unclear. Hatano et al. suggested that reactivity to prostaglandin was milder in women than in men. Ospina et al.

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.

Radiation hybrid maps of medaka chromosomes LG 12, 17, and 22

Abstract The Medaka is an excellent genetic system for studies of vertebrate development and disease and environmental and evolutionary biology studies. To facilitate the mapping of markers or the cloning of affected genes in Medaka mutants identified by forward-genetic screens, we have established a panel of whole-genome radiation hybrids (RHs) and RH maps for three Medaka chromosomes. RH mapping is useful, since markers to be mapped need not be polymorphic and one can establish the order of markers that are difficult to resolve by genetic mapping owing to low genetic recombination rates. RHs were generated by fusing the irradiated donor, OLF-136 Medaka cell line, with the host B78 mouse melanoma cells. Of 290 initial RH clones, we selected 93 on the basis of high retention of fragments of the Medaka genome to establish a panel that allows genotyping in the 96-well format. RH maps for linkage groups 12, 17, and 22 were generated using 159 markers. The average retention for the three chromosomes was 19% and the average break point frequency was ∼33 kb/cR. We estimate the potential resolution of the RH panel to be ∼186 kb, which is high enough for integrating RH data with bacterial artificial chromosome clones. Thus, this first RH panel will be a useful tool for mapping mutated genes in Medaka.