Hiroki Fujikawa

Mutation Analysis of the IL36RN Gene in 14 Japanese Patients with Generalized Pustular Psoriasis

Generalized pustular psoriasis (GPP) is a rare, potentially life threatening, and aggressive form of psoriasis, which is characterized by sudden onset with repeated episodic skin inflammation leading to pustule formation. Familial GPP is known to be caused by recessively inherited mutations in the IL36RN gene, which encodes interleukin 36 receptor antagonist (IL‐36Ra). In this article, we performed mutation analysis of the IL36RN gene in 14 Japanese patients with GPP, and identified mutations in two of these patients analyzed. One patient was compound heterozygous for mutations c.115+6T>C and c.368C>G (p.Thr123Arg), whereas the other carried compound heterozygous mutations c.28C>T (p.Arg10*) and c.115+6T>C in the IL36RN gene. Expression studies using total RNA from the patients’ skin revealed that the mutation c.115+6T>C resulted in skipping of exon 3, leading to a frameshift and a premature termination codon (p.Arg10Argfs*1). The protein structure analysis suggested that the missense mutation p.Thr123Arg caused misfolding and instability of IL‐36Ra protein. In vitro studies in cultured cells showed impaired expression of the p.Thr123Arg mutant IL‐36Ra protein, which failed to antagonize the IL‐36 signaling pathway. Our data further underscore the critical role of IL36RN in pathogenesis of GPP.

A missense mutation within the helix initiation motif of the keratin K71 gene underlies autosomal dominant woolly hair/hypotrichosis

Woolly hair (WH) is an abnormal variant of tightly curled hair, which is frequently associated with hypotrichosis. Non-syndromic forms of WH can show either autosomal-dominant WH (ADWH) or autosomal-recessive WH (ARWH) inheritance patterns. ARWH has recently been shown to be caused by mutations in either the lysophosphatidic acid receptor 6 (LPAR6) or lipase H (LIPH) gene. More recently, a mutation in the keratin K74 (KRT74) gene has been reported to underlie ADWH. Importantly, all of these genes are abundantly expressed in the inner root sheath (IRS) of human hair follicles. Besides these findings, the molecular mechanisms underlying hereditary WH have not been fully disclosed. In this study, we identified a Japanese family with ADWH and associated hypotrichosis. After exclusion of known causative genes, we discovered the heterozygous mutation c.422T>G (p.Phe141Cys) within the helix initiation motif of the IRS-specific keratin K71 (KRT71) gene in affected family members. We demonstrated that the mutant K71 protein led to disruption of keratin intermediate filament formation in cultured cells. To our knowledge, it is previously unreported that the KRT71 mutation is associated with a hereditary hair disorder in humans. Our findings further underscore the crucial role of the IRS-specific keratins in hair follicle development and hair growth in humans.

A Homozygous Frameshift Mutation in the HOXC13 Gene Underlies Pure Hair and Nail Ectodermal Dysplasia in a Syrian Family

Pure hair and nail ectodermal dysplasia (PHNED) is a rare genetic disorder characterized by hypotrichosis or complete alopecia, as well as nail dystrophy. Mutations in the type II hair keratin gene KRT85 and the HOXC13 gene on chromosome 12q have recently been identified in families with autosomal‐recessive PHNED. In the present study, we have analyzed a consanguineous Syrian family with an affected girl having complete alopecia and nail dystrophy since birth. The family clearly showed linkage to chromosome 12q13.13–12q14.3, which excluded the KRT85 gene. Sequencing of another candidate gene HOXC13 within the linkage interval identified a homozygous frameshift mutation (c.355delC; p.Leu119Trpfs*20). Expression studies in cultured cells revealed that the mutant HOXC13 protein mislocalized within the cytoplasm, and failed to upregulate the promoter activities of its target genes. Our results strongly suggest crucial roles of the HOXC13 gene in the development of hair and nails in humans.

GJB6, of which mutations underlie Clouston syndrome, is a potential direct target gene of p63

BACKGROUND Clouston syndrome is a rare autosomal dominant condition characterized by hypotrichosis, nail dystrophy, and occasionally palmoplantar keratoderma. The disease is caused by mutations in GJB6 gene, which encodes a gap junction protein connexin 30 (Cx30). OBJECTIVE To disclose the molecular basis of Clouston syndrome in a Lebanese-German family, and also to determine precise expression of Cx30 in normal skin of humans and mice, as well as transcriptional regulation for the GJB6 expression. METHODS We searched for mutations in the GJB6 gene using DNA of the family members with Clouston syndrome. We performed immunostaining to localize the Cx30 expression in normal human skin and mouse embryos. In addition, we did a series of in vitro studies to investigate if the GJB6 could be a direct transcriptional target gene of p63. RESULTS We identified a recurrent heterozygous mutation c.31G>C (p.Gly11Arg) in the GJB6 gene in the Lebanese-German family with Clouston syndrome. Immunostaining in normal human skin sections demonstrated predominant expression of Cx30 in hair follicles, nails, and palmoplantar epidermis, which partially overlapped with p63 expression. We also showed co-expression of Cx30 and p63 in developing mouse hair follicles and nail units. In cultured cells, the GJB6 expression was significantly upregulated by ΔNp63α isoform. Further in vitro analyses suggested that ΔNp63α was potentially involved in the GJB6 expression via binding to the sequences in intron 1 of the GJB6 gene. CONCLUSION Our data further underscore the crucial roles of Cx30 in morphogenesis and development of skin and its appendages.

Identification of two novel mutations in SLC29A3 encoding an equilibrative nucleoside transporter (hENT3) in two distinct Syrian families with H syndrome: expression studies of SLC29A3 (hENT3) in human skin.

Background: H syndrome is a rare autosomal recessive genetic disorder which involves the skin and other systemic organs and is caused by mutations in the SLC29A3 gene. Objectives: To disclose the molecular basis of H syndrome in two Syrian families, and to determine the localization of hENT3 in human skin. Methods: DNA from two Syrian families with H syndrome was analyzed through direct sequencing, and the expression of hENT3 in normal human skin was investigated by in situ hybridization and immunostaining. Results: We identified two novel mutations in the SLC29A3 gene: a homozygous splice site mutation IVS1+2T>G predicted to cause a splicing error, and a homozygous missense mutation c.1157G>A (p.R386Q) which substituted highly conserved amino acid residue in a transmembrane domain of hENT3. Furthermore, we demonstrate that hENT3 is expressed in histiocytes as well as in endothelium of blood and lymphatic vessels in normal human skin. Conclusions: Our results further enhance the mutation spectrum of the SLC29A3 gene for this rare genetic disorder, and also suggest potential pathomechanisms for the skin lesions resulting from SLC29A3 mutations.

A novel mutation in the PLCD1 gene, which leads to an aberrant splicing event, underlies autosomal recessive leuconychia

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Characterization of a novel missense mutation in the prodomain of GDF5, which underlies brachydactyly type C and mild Grebe type chondrodysplasia in a large Pakistani family

All TGF-beta family members have a prodomain that is important for secretion. Lack of secretion of a TGF-beta family member GDF5 is known to underlie some skeletal abnormalities, such as brachydactyly type C that is characterized by a huge and unexplained phenotypic variability. To search for potential phenotypic modifiers regulating secretion of GDF5, we compared cells overexpressing wild type (Wt) GDF5 and GDF5 with a novel mutation in the prodomain identified in a large Pakistani family with Brachydactyly type C and mild Grebe type chondrodyslplasia (c527T>C; p.Leu176Pro). Initial in vitro expression studies revealed that the p.Leu176Pro mutant (Mut) GDF5 was not secreted outside the cells. We subsequently showed that GDF5 was capable of forming a complex with latent transforming growth factor binding proteins, LTBP1 and LTBP2. Furthermore, secretion of LTBP1 and LTBP2 was severely impaired in cells expressing the Mut-GDF5 compared to Wt-GDF5. Finally, we demonstrated that secretion of Wt-GDF5 was inhibited by the Mut-GDF5, but only when LTBP (LTBP1 or LTBP2) was co-expressed. Based on these findings, we suggest a novel model, where the dosage of secretory co-factors or stabilizing proteins like LTBP1 and LTBP2 in the microenvironment may affect the extent of GDF5 secretion and thereby function as modifiers in phenotypes caused by GDF5 mutations.

Case of Netherton syndrome with an elevated serum thymus and activation‐regulated chemokine level

Dear Editor, A 5-month-old girl, who was born at the 35th week of gestation after an uneventful pregnancy to healthy unrelated parents, showed generalized scaly erythema from birth (Fig. 1a). Erythema and thick yellowish seborrheic scales were pronounced on her head and face. At 16 months of age, scaly erythema was reduced but persisted in the axillae and groin. Light microscopic hair analysis showed trichorrhexis invaginata, one of the characteristics of Netherton syndrome (NS). At 4 years of age, the skin lesions worsened (Fig. 1b). In addition, she developed severe fish allergy with laryngospasm and facial swelling, which was accompanied by increased total immunoglobulin (Ig)E (19 000 IU/mL) and specific IgE to multiple food allergens including peanuts, cow’s milk, chicken’s egg and various types of fish and shellfish. At 7 years of age, erythematous scaly patches persisted on the scalp, face and extremities (Fig. 1c). Enzyme immunoassay revealed that her serum thymus and activation-regulated chemokine (TARC) level was elevated at 1664 pg/mL, and further increased to 2300 pg/mL 4 months later without exacerbation of skin lesions. The patient did not develop allergic asthma, allergic rhinitis or angioedema other than multiple food allergies at the age of 7 years. Using the genomic DNA of the patient, all exons and exon– intron boundaries of the SPINK5 gene were amplified by polymerase chain reaction (PCR) as described previously. Direct sequencing analysis of the SPINK5 gene identified a nonsense mutation c.1732C>T (p.R578X), a known pathogenic mutation for NS, in exon 19 of SPINK5 but no wild-type exon 19 sequence (Fig. 2a). The presence of this mutation was confirmed by PCR restriction fragment length polymorphism assay (Fig. 2b). These studies led to the definite diagnosis of NS. Fur-

A novel splice‐site ECM1 gene mutation in a Lebanese girl with lipoid proteinosis

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Netherton syndrome showing a large clinical overlap with generalized inflammatory peeling skin syndrome.

ejd.2012.1720 Auteur(s) : Muhammad Farooq1, Mazen Kurban2,3,a, Ossama Abbas2, Abdul-Ghani Kibbi2, Atsushi Fujimoto1, Hiroki Fujikawa1, Yutaka Shimomura1,a yshimo@med.niigata-u.ac.j 1 Laboratory of Genetic Skin Diseases, Niigata University Medical School, 1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan 2 Department of Dermatology, 3 Department of Biochemistry and molecular genetics, American University of Beirut, Beirut, Lebanon a These authors equally contributed to this work. Netherton [...]