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Lee M.,Peking University | Lee M.,Beijing Key Laboratory of Molecular Diagnosis on Dermatoses | Xu G.,Peking University | Xu G.,Beijing Key Laboratory of Molecular Diagnosis on Dermatoses | And 13 more authors.
Clinical and Experimental Dermatology | Year: 2016

Background Recessive dystrophic epidermolysis bullosa (RDEB) is a rare heritable blistering skin condition caused by loss-of-function mutations in the COL7A1 gene. Incongruent gene transmission is occasionally reported in recessive diseases, and its underlying mechanism is often uniparental disomy (UPD). Aim To understand the genetic basis of incongruent gene transmission in a Chinese family with RDEB, in which a discrepancy of COL7A1 genotyping was encountered during our mutation analysis. Methods We used a pCAS2 minigene-based in vitro splicing assay to confirm the pathogenicity of the splicing variant we identified in the proband. Next, a combination of genetic tools, including whole-genome SNP array analysis and multiplex ligation-dependent probe amplification copy number analysis, was used to unravel the cause of the discrepancy in the COL7A1 genotyping. Results Sanger sequencing identified a novel, single-peak mutation, c.4980+5G>C, in COL7A1 in the proband, which was heterozygous in his father and wild type in his mother. In vitro splicing assay showed that c.4980+5G>C was pathogenic and led to skipping of COL7A1 exon 53. SNP array analysis and multiplex ligation-dependent probe amplification of the proband's DNA revealed a maternally derived, de novo, interstitial deletion on chromosome 3p21.31, which removed COL7A1 and 15 flanking genes, excluding the possibility of UPD. Conclusion Our findings favour an exceptionally rare event, namely a de novo COL7A1 microdeletion in concurrence with an inherited mutation in trans. This study should aid molecular diagnosis and genetic counselling of RDEB and possibly other recessive diseases in which genotyping discrepancy is encountered. © 2016 British Association of Dermatologists.


Wang Y.M.,Peking University | Wang Y.M.,Beijing Key Laboratory of Molecular Diagnosis on Dermatoses | Huang Y.S.,University of British Columbia | Ma Z.H.,Heilongjiang University | And 6 more authors.
Clinical and Experimental Dermatology | Year: 2014

Background. Basal cell carcinoma (BCC) is a prevalent form of nonmelanoma skin cancer. Although numerous studies in white populations suggest that mutations in the TP53 gene play an important role in the development of BCC, it is not clear whether this is also the case in East Asian populations such as in China. Aim. To investigate the frequency and the features of TP53 mutation in sporadic BCC in a Chinese population. Methods. In total, 30 patients with sporadic BCC, who had previously taken part in a study on PTCH1 mutations, were enrolled. BCC and control cells were obtained by laser-capture microdissection, and DNA was amplified and sequenced for analysis of TP53 mutations. Results. In the 30 BCC samples, 6 TP53 point mutations were found (frequency of 20%), and 4 of these 6 mutations had ultraviolet (UV)-specific alterations. Combining these results with those of the previous study on PTCH1 mutations, we found that two patients with had three types of genetic alterations (each had two PTCH1 mutations and one TP53 point mutation). A further two patients each had one PTCH1 mutation and one UV signature TP53 mutation. In addition, the total number of UV-specific mutations of PTCH1 and TP53 accounted for 20% of the total patient group. Conclusions. The incidence of TP53 mutation in BCC in our Chinese subjects was lower than that reported for white populations. Many of the patients carried mutations of other genes in addition to of TP53. The majority of TP53 mutations were UV-induced specific alterations. However, the results of the two studies on TP53 and PTCH1 indicated that the incidence of UV-specific mutations is much lower in Chinese than in white populations. © 2014 British Association of Dermatologists.


Wang H.J.,Peking University | Wang H.J.,Beijing Key Laboratory of Molecular Diagnosis on Dermatoses | Lin Z.M.,Peking University | Lin Z.M.,Beijing Key Laboratory of Molecular Diagnosis on Dermatoses | And 6 more authors.
Clinical and Experimental Dermatology | Year: 2014

Diffuse cutaneous mastocytosis (DCM) is an extremely rare disease characterized by massive proliferation of mast cells infiltrating the entire skin. We report a Chinese family with indolent DCM, and detection of a new germline KIT mutation located in the fifth immunoglobulin-like loop of the KIT protein, which probably results in a gain-of-function effect and consequent overactivation of mast cells. Our report expands the knowledge of correlations between the genotype of KIT mutations and the phenotype of DCM. © 2013 British Association of Dermatologists.


Gong J.,Peking University | Gong J.,Beijing Key Laboratory of Molecular Diagnosis on Dermatoses | Ran M.,Peking University | Wang X.,Peking University | And 5 more authors.
Mycopathologia | Year: 2016

An accurate diagnosis of tinea unguium is necessary for the selection of antimycotics and successful treatment. To rapidly and accurately identify the aetiological agents causing tinea unguium, we improved upon the conventional boiling method for DNA extraction and developed a novel real-time PCR detection system that includes two assays. The two assays, based on the amplification of ribosomal internal transcribed spacer regions and 28S rDNA, were designed to detect pan-dermatophyte and Trichophyton rubrum, respectively. The analytical sensitivities of both assays permitted the detection of ten copies of plasmid DNA template. The analytical specificity of the detection system was confirmed using 11 dermatophyte strains and 25 non-dermatophyte strains. In total, 165 nail specimens were examined by microscopy, culture, conventional PCR, and the novel real-time PCR method. Real-time PCR gave positive results in 47.3 % of the specimens (78), a rate exceeding those obtained using microscopy (72, 43.6 %), conventional PCR (69, 41.8 %), and culture (49, 29.7 %). All conventional PCR-positive specimens were detected by real-time PCR, and real-time PCR detected nine specimens that were missed by conventional PCR. The results from latent class analysis, and further calculations, showed that real-time PCR was the most sensitive method, but the diagnostic specificity of the four approaches was equivalent. In particular, molecular approaches may be more effective than microscopy and culture when the clinical symptoms of tinea unguium are not evident. © 2015, Springer Science+Business Media Dordrecht.


Wu W.,Peking University | Wu W.,Beijing Key Laboratory of Molecular Diagnosis on Dermatoses | Zhang R.,Peking University | Zhang R.,Beijing Key Laboratory of Molecular Diagnosis on Dermatoses | And 10 more authors.
Mycopathologia | Year: 2016

Dematiaceous fungi are a large group of pathogens that can cause a wide range of diseases in both immunocompetent and immunocompromised hosts. Based on our previous finding of caspase recruitment domain-containing protein 9 (CARD9) mutations in patients with subcutaneous phaeohyphomycosis caused by Phialophora verrucosa (P. verrucosa), we further investigated the exact role of CARD9 in the pathogenesis of phaeohyphomycosis using Card9 knockout (Card9 KO) mice. We showed that Card9 KO mice are profoundly susceptible to P. verrucosa infection compared with wild-type mice, reflected by significantly more severe footpad swelling, higher fungal burden, lower survival, and systemic dissemination. The inability of Card9 KO mice to control P. verrucosa infection was associated with lack of Th17 differentiation and reduction of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-17A levels in footpad homogenates. In vitro experiments showed a defect of fungal conidia killing and pro-inflammatory cytokine production in Card9 KO bone marrow-derived macrophages and dendritic cells. Furthermore, ex vivo coculture and in vitro T cell differentiation assay demonstrated that Card9 signaling pathway acts indispensably on differentiation of Th17 cells. In conclusion, our findings suggest that CARD9 mediate the innate immune and Th17-mediated adaptive immune responses against dematiaceous fungal infections at the early stage of infection. © 2016 Springer Science+Business Media Dordrecht


Gong J.,Peking University | Gong J.,Beijing Key Laboratory of Molecular Diagnosis on Dermatoses | Wu W.,Peking University | Wu W.,Beijing Key Laboratory of Molecular Diagnosis on Dermatoses | And 11 more authors.
Fungal Genetics and Biology | Year: 2016

Little is known regarding the population information of Trichophyton rubrum due to a lack of strains with clear sampling information and molecular markers with high discriminatory power. In the present study, we developed a set of microsatellite markers that have a cumulative discriminatory power was 0.993. Using these microsatellites loci, 243 strains T. rubrum that had clear sampling information were analysed. Three genetic diversity indices (Shannon's Information Index, Nei's unbiased gene diversity and allelic richness) were shown to be related to the human population size of the sampling city rather than mean annual temperature or humidity. Population structure analyses revealed that T. rubrum can be separated into two clusters. AMOVA results indicated that genetic variation was more significant between these two clusters than among geographical populations. Our work is the first to reveal population information of T. rubrum using highly discriminatory molecular markers, and suggest that T. rubrum populations in cities with larger population size might have better adaptability due to higher genetic diversity under selective pressures (such as antifungal agents). © 2016


Zhang J.,Shandong University | Zhang J.,Peking University | Zhang J.,Beijing Key Laboratory of Molecular Diagnosis on Dermatoses | Duo L.,Peking Tsinghua Center for Life science | And 20 more authors.
Gene | Year: 2015

As a powerful tool to identify the molecular pathogenesis of Mendelian disorders, exome sequencing was used to identify the genetic basis of two siblings with hearing loss and hypotrichosis and clarify the diagnosis. No pathogenic mutations in GJB2, GJB3 and GJB6 genes were found in the siblings. By analysis of exome of the proband, we identified a novel missense (p.R306C) mutation and a nonsense (p.R186*) mutation in the BCS1L gene. Mutations were confirmed by Sanger sequencing. The siblings were compound heterozygotes, and the inheritance mode of autosomal recessive was postulated. BCS1L is the causative gene of Björnstad syndrome, which is characterized by sensorineural hearing loss and pili torti. The longitudinal gutters along the hair shaft were found by scanning electron microscopy in our patient. Therefore the diagnosis of Björnstad syndrome was eventually made for the patients. Our study extends the phenotypic spectrum of Björnstad syndrome and highlights the clinical applicability of exome sequencing as a diagnostic tool for atypical Mendelian disorders. © 2015 Elsevier B.V.


Lin Z.,Peking University | Lin Z.,Beijing Key Laboratory of Molecular Diagnosis on Dermatoses | Zhao J.,Peking University | Zhao J.,Beijing Key Laboratory of Molecular Diagnosis on Dermatoses | And 28 more authors.
American Journal of Human Genetics | Year: 2015

Calpastatin is an endogenous specific inhibitor of calpain, a calcium-dependent cysteine protease. Here we show that loss-of-function mutations in calpastatin (CAST) are the genetic causes of an autosomal-recessive condition characterized by generalized peeling skin, leukonychia, acral punctate keratoses, cheilitis, and knuckle pads, which we propose to be given the acronym PLACK syndrome. In affected individuals with PLACK syndrome from three families of different ethnicities, we identified homozygous mutations (c.607dup, c.424A>T, and c.1750delG) in CAST, all of which were predicted to encode truncated proteins (p.Ile203Asnfs∗8, p.Lys142∗, and p.Val584Trpfs∗37). Immunohistochemistry shows that staining of calpastatin is reduced in skin from affected individuals. Transmission electron microscopy revealed widening of intercellular spaces with chromatin condensation and margination in the upper stratum spinosum in lesional skin, suggesting impaired intercellular adhesion as well as keratinocyte apoptosis. A significant increase of apoptotic keratinocytes was also observed in TUNEL assays. In vitro studies utilizing siRNA-mediated CAST knockdown revealed a role for calpastatin in keratinocyte adhesion. In summary, we describe PLACK syndrome, as a clinical entity of defective epidermal adhesion, caused by loss-of-function mutations in CAST. © 2015 The American Society of Human Genetics.


Wang H.,Peking University | Wang H.,Beijing Key Laboratory of Molecular Diagnosis on Dermatoses | Wang H.,Peking Tsinghua Center for Life science | Cao X.,Peking University | And 28 more authors.
Human Molecular Genetics | Year: 2015

Keratoderma-hypotrichosis-leukonychia totalis syndrome (KHLS) is an extremely rare, autosomal-dominant disorder characterized by severe skin hyperkeratosis, congenital alopecia and leukonychia totalis. The genetic defect underlying KHLS remained undetermined. By performing whole-exome sequencing in a family with KHLS, we identified a heterozygous mutation (c.23G>T [p.Gly8Val]) in GJA1, which cosegregated with the phenotype in the family. In an additional affected individual, we also found the identical de novo mutation which was absent in his unaffected family members. GJA1 encodes a gap junction protein connexin 43 (Cx43) which is ubiquitously expressed in various organs, including the epidermis and hair follicles. In vitro studies on HEK293 cells expressing Cx43Gly8Val found that the protein formed gap junction plaques between adjacent transfected cells, as observed in the wild-type. Dye-transfer experiments by microinjection of Lucifer yellow displayed functional gap junction of the Cx43Gly8Val mutant. Using patch clamp and Ca2+ imaging methods, we observed that the Cx43Gly8Val hemichannel had significantly more openings than Cx43WT, facilitating Ca2+ influx at resting potential. Such gain-of-function effect might result in cytoplasmic Ca2+ overload, accelerated apoptosis of keratinocytes and subsequent skin hyperkeratosis. Taken together, our results demonstrated that, with probably enhanced hemichannel activities, a mutation in GJA1 is linked to KHLS without extracutaneous involvement. © The Author 2014. Published by Oxford University Press. All rights reserved.


Liang P.,Peking University | Liang P.,Beijing Key Laboratory of Molecular Diagnosis on Dermatoses | Wang X.,Peking University | Wang X.,Beijing Key Laboratory of Molecular Diagnosis on Dermatoses | And 7 more authors.
Mycopathologia | Year: 2015

Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor molecule that is critical for NF-κB activation and forms a complex with B cell lymphoma 10 and mucosa-associated lymphoid tissue lymphoma translocation gene 1 that mediates C-type lectin receptors (CLRs)-triggered intracellular signaling during antifungal immunity. However, the role of CARD9 in the host defense against Phialophora verrucosa (P. verrucosa) infection remains to be elucidated. In the present study, we investigated the functions of polymorphonuclear neutrophils (PMNs) from patients with CARD9 deficiencies against P. verrucosa. By isolating PMNs from patients and healthy blood donors and subsequently challenging the cells with P. verrucosa, we demonstrated that, compared with healthy donors, CARD9-deficient PMNs exhibited defects in P. verrucosa killing and pro-inflammatory cytokine productions, which can be rescued in the presence of serum; however, the CARD9-deficient PMNs exhibited normal reactive oxygen species generation and phagocytotic ability. In conclusion, our results indicate that CARD9 is indispensable for P. verrucosa killing by PMNs, and serum opsonization acts as a CARD9-independent way, which could be a promising immunotherapy in the future. © 2015, Springer Science+Business Media Dordrecht.

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