Nikoletta Nagy

MiR-125b, a MicroRNA Downregulated in Psoriasis, Modulates Keratinocyte Proliferation by Targeting FGFR2

MicroRNAs (miRNAs) are short, single-stranded, noncoding RNAs that play important roles in the regulation of gene expression. We previously identified a characteristic miRNA expression profile in psoriasis, distinct from that of healthy skin. One of the most downregulated miRNAs in psoriasis skin was microRNA-125b (miR-125b). In this study, we aimed to identify the potential role(s) of miR-125b in psoriasis pathogenesis. In situ hybridization results showed that the major cell type responsible for decreased miR-125b levels in psoriasis lesions was the keratinocyte. Overexpression of miR-125b in primary human keratinocytes suppressed proliferation and induced the expression of several known differentiation markers. Conversely, inhibition of endogenous miR-125b promoted cell proliferation and delayed differentiation. Fibroblast growth factor receptor 2 (FGFR2) was identified as one of the direct targets for suppression by miR-125b by luciferase reporter assay. The expression of miR-125b and FGFR2 was inversely correlated in both transfected keratinocytes and in psoriatic skin. Knocking down FGFR2 expression by siRNA suppressed keratinocyte proliferation, but did not enhance differentiation. Altogether, our results demonstrate a role for miR-125b in the regulation of keratinocyte proliferation and differentiation, partially through the regulation of FGFR2. Loss of miR-125b in psoriasis skin may contribute to hyperproliferation and aberrant differentiation of keratinocytes.

Mechanisms of IFN-γ–induced apoptosis of human skin keratinocytes in patients with atopic dermatitis

BACKGROUND Enhanced apoptosis of keratinocytes is the main cause of eczema and spongiosis in patients with the common inflammatory skin disease atopic dermatitis (AD). OBJECTIVE The aim of the study was to investigate molecular mechanisms of AD-related apoptosis of keratinocytes. METHODS Primary keratinocytes isolated from patients with AD and healthy donors were used to study apoptosis by using annexin V/7-aminoactinomycin D staining. Illumina mRNA Expression BeadChips, quantitative RT-PCR, and immunofluorescence were used to study gene expression. In silico analysis of candidate genes was performed on genome-wide single nucleotide polymorphism data. RESULTS We demonstrate that keratinocytes of patients with AD exhibit increased IFN-γ-induced apoptosis compared with keratinocytes from healthy subjects. Further mRNA expression analyses revealed differential expression of apoptosis-related genes in AD keratinocytes and skin and the upregulation of immune system-related genes in skin biopsy specimens of chronic AD lesions. Three apoptosis-related genes (NOD2, DUSP1, and ADM) and 8 genes overexpressed in AD skin lesions (CCDC109B, CCL5, CCL8, IFI35, LYN, RAB31, IFITM1, and IFITM2) were induced by IFN-γ in primary keratinocytes. The protein expression of IFITM1, CCL5, and CCL8 was verified in AD skin. In line with the functional studies and AD-related mRNA expression changes, in silico analysis of genome-wide single nucleotide polymorphism data revealed evidence of an association between AD and genetic markers close to or within the IFITM cluster or RAB31, DUSP1, and ADM genes. CONCLUSION Our results demonstrate increased IFN-γ responses in skin of patients with AD and suggest involvement of multiple new apoptosis- and inflammation-related factors in the development of AD.

The anti-apoptotic protein G1P3 is overexpressed in psoriasis and regulated by the non-coding RNA, PRINS

Please cite this paper as: The anti‐apoptotic protein G1P3 is overexpressed in psoriasis and regulated by the non‐coding RNA, PRINS. Experimental Dermatology 2010; 19: 269–278.

Schöpf-Schulz-Passarge syndrome resulting from a homozygous nonsense mutation in WNT10A

[1] Tomita Y, Suzuki T. Genetics of pigmentary disorders. Am J Med Genet C Semin Med Genet 2004;131C:75–81. [2] Miyamura Y, Suzuki T, Kono M, Inagaki K, Ito S, Suzuki N, et al. Mutations of the RNA-specific adenosine deaminase gene (DSRAD) are involved in dyschromatosis symmetrica hereditaria. Am J Hum Genet 2003;73:693–9. [3] Zhang F, Liu H, Jiang D, Tian H, Wang C, Yu L. Six novel mutations of the ADAR1 gene in Chinese patients with dyschromatosis symmetrica hereditaria. J Dermatol Sci 2008;50:109–14. [4] Kondo T, Suzuki T, Mitsuhashi Y, Ito S, Kono M, Komine M, et al. Six novel mutations of the ADAR1 gene in patients with dyschromatosis symmetrica hereditaria: Histological observation and comparison of genotypes and clinical phenotypes. J Dermatol 2008;35:395–406. [5] Hayashi M, Suzuki T. A missense mutation c.G2747A (p.R916Q) of ADAR1 gene in dyschromatosis symmetrica hereditaria is not a novel mutation. Arch Dermatol Res 2010. doi: 10.1007/s00403-010r-r1036-5. [6] Bass BL, Weintraub H. An unwinding activity that covalently modifies its double-stranded RNA substrate. Cell 1988;55:1089–98. [7] Wagner RW, Smith JE, Cooperman BS, Nishikura KA. Double-stranded RNA unwinding activity introduces structural alterations by means of adenosine to inosine conversions in mammalian cells and Xenopus eggs. Proc Natl Acad Sci USA 1989;86:2647–51. [8] Liu Q, Jiang L, Liu WL, Kang XJ, Ao Y, Sun M, et al. Two novel mutations and evidence for haploinsufficiency of the ADAR gene in dyschromatosis symmetrica hereditaria. Br J Dermatol 2006;154:636–42. [9] Murata I, HozumiY, Kawaguchi M, Katagiri Y, Yasumoto S, Kubo Y, et al. Four Novel Mutations of the ADAR1 Gene in dyschromatosis symmetrica hereditaria. J Dermatol Sci 2009;53:76–7. [10] Suzuki N, Suzuki T, Inagaki K, Ito S, Kono M, Fukai K, et al. Mutation analysis of the ADAR1 gene in dyschromatosis symmetrica hereditaria and genetic differentiation from both dyschromatosis universalis hereditaria and acropigmentatio reticularis. J Invest Dermatol 2005;124:1186–92.

CTSC and Papillon-Lefèvre syndrome: detection of recurrent mutations in Hungarian patients, a review of published variants and database update.

Papillon–Lefèvre syndrome (PLS; OMIM 245000) is an autosomal recessive condition characterized by palmoplantar hyperkeratosis and periodontitis. In 1997, the gene locus for PLS was mapped to 11q14‐21, and in 1999, variants in the cathepsin C gene (CTSC) were identified as causing PLS. To date, a total of 75 different disease‐causing mutations have been published for the CTSC gene. A summary of recurrent mutations identified in Hungarian patients and a review of published mutations is presented in this update. Comparison of clinical features in affected families with the same mutation strongly confirm that identical mutations of the CTSC gene can give rise to multiple different phenotypes, making genotype–phenotype correlations difficult. Variable expression of the phenotype associated with the same CTSC mutation may reflect the influence of other genetic and/or environmental factors. Most mutations are missense (53%), nonsense (23%), or frameshift (17%); however, in‐frame deletions, one splicing variant, and one 5′ untranslated region (UTR) mutation have also been reported. The majority of the mutations are located in exons 5–7, which encodes the heavy chain of the cathepsin C protein, suggesting that tetramerization is important for cathepsin C enzymatic activity. All the data reviewed here have been submitted to the CTSC base, a mutation registry for PLS at http://bioinf.uta.fi/CTSCbase/.

The Arg160Trp allele of melanocortin-1 receptor gene might protect against vitiligo.

Melanocortin‐1 receptor (MC1R) and agouti signaling protein (ASIP) play pivotal roles in the regulation of human pigmentation. We aimed to study whether single nucleotide polymorphisms (SNPs) of the MC1R and ASIP genes contribute to the pathogenesis of the polygenic pigment skin disorder, vitiligo. The PCR‐amplified, full‐length MC1R gene was studied with sequence analysis, and the 3′ untranslated region (3′ UTR) SNP of ASIP was detected using restriction fragment length polymorphism. The allele frequency of the ASIP SNP did not show any difference between the skin type, hair color and eye color‐matched 97 vitiligo patients and the 59 healthy control individuals. As one of the MC1R polymorphisms showed significantly higher incidence among fair‐skinned individuals (Fitzpatrick I + II, n = 140) than among dark‐skinned individuals (Fitzpatrick III + IV, n = 90), both vitiligo patients and controls were divided into two groups and the frequency of the MC1R alleles was studied separately in fair‐skinned and dark‐skinned subgroups of diseased and healthy groups. C478T, one of the MC1R SNPs studied in 108 fair‐skinned vitiligo patients and in 70 fair‐skinned healthy control individuals, showed a significant difference (P = 0.0262, odds ratio [95% confidence interval] = 3.6 [0.0046–0.1003]) in allele frequency between the two groups: the allele frequency was higher in the control group, suggesting protection against vitiligo. Computer prediction of antigenicity has revealed that the Arg160Trp amino acid change caused by this SNP results in a decrease in antigenicity of the affected peptide epitope.

Phenotype–genotype correlations for clinical variants caused by CYLD mutations

Brooke-Spiegler syndrome (BSS; OMIM 605041) is an autosomal dominant condition characterized by skin appendageal neoplasms including cylindromas, trichoepitheliomas, and/or spiradenomas. In 1996, the gene locus for BSS was mapped to 16q12-13, and, in 2000, mutations in the cylindromatosis (CYLD) gene were determined to cause BSS, familial cylindromatosis (FC; OMIM 132700) and multiple familial trichoepithelioma type 1 (MFT1; OMIM 601606). The CYLD gene encodes an enzyme with deubiquitinase activity. To date, a total of 95 different diseases-causing mutations have been published for the CYLD gene. A summary of mutations identified in Hungarian patients and a review of previously published mutations are presented in this update. The majority of the sequence changes are frameshift (48%), nonsense (27%), missense (12%) and splice-site (11%) mutations; however, two in-frame deletions have also been reported. Most mutations are located in exons 9-20. Analysis of the identified CYLD gene mutations and the observed BSS, FC and MFT1 clinical phenotypes of the patients revealed significant genotype-phenotype correlations. Elucidation of these genotype-phenotype correlations is critical for the diagnosis of these rare monogenic skin diseases. In addition, characterizing these correlations may promote the understanding of their mechanisms and may hopefully contribute to the development of future therapeutic modalities.

Cell-Free DNA Analysis of Targeted Genomic Regions in Maternal Plasma for Non-Invasive Prenatal Testing of Trisomy 21, Trisomy 18, Trisomy 13, and Fetal Sex

BACKGROUND There is great need for the development of highly accurate cost effective technologies that could facilitate the widespread adoption of noninvasive prenatal testing (NIPT). METHODS We developed an assay based on the targeted analysis of cell-free DNA for the detection of fetal aneuploidies of chromosomes 21, 18, and 13. This method enabled the capture and analysis of selected genomic regions of interest. An advanced fetal fraction estimation and aneuploidy determination algorithm was also developed. This assay allowed for accurate counting and assessment of chromosomal regions of interest. The analytical performance of the assay was evaluated in a blind study of 631 samples derived from pregnancies of at least 10 weeks of gestation that had also undergone invasive testing. RESULTS Our blind study exhibited 100% diagnostic sensitivity and specificity and correctly classified 52/52 (95% CI, 93.2%-100%) cases of trisomy 21, 16/16 (95% CI, 79.4%-100%) cases of trisomy 18, 5/5 (95% CI, 47.8%-100%) cases of trisomy 13, and 538/538 (95% CI, 99.3%-100%) normal cases. The test also correctly identified fetal sex in all cases (95% CI, 99.4%-100%). One sample failed prespecified assay quality control criteria, and 19 samples were nonreportable because of low fetal fraction. CONCLUSIONS The extent to which free fetal DNA testing can be applied as a universal screening tool for trisomy 21, 18, and 13 depends mainly on assay accuracy and cost. Cell-free DNA analysis of targeted genomic regions in maternal plasma enables accurate and cost-effective noninvasive fetal aneuploidy detection, which is critical for widespread adoption of NIPT.

Successful treatment of multiple basaliomas with bleomycin-based electrochemotherapy: a case series of three patients with Gorlin-Goltz syndrome.

Gorlin-Goltz syndrome is a rare multisystemic disease, characterized by numerous basal cell carcinomas. The ideal approach for patients with the syndrome would be a treatment with a high cure rate, minimal scarring, short healing time and mild side-effects. Electrochemo-therapy is a novel therapeutic option that ablates tumours with electrical current and simultaneously administered anticancer drugs. Three patients with Gorlin-Goltz syndrome were treated with electrochemotherapy using intravenous bleomycin. Clinical response was obtained in 98 (99%) of the lesions, 86 (87%) of them showed complete response. In 2 tumours, regression was confirmed with histological examination. Long-term cosmetic results were excellent. We consider electrochemotherapy to be an additional tool in the therapeutic armamentarium for Gorlin-Goltz syndrome, and suggest using it as early as possible in selected patients to avoid disfiguring scarring.

The expression of keratinocyte growth factor receptor (FGFR2-IIIb) correlates with the high proliferative rate of HaCaT keratinocytes

Abstract:  Keratinocyte growth factor receptor (KGFR = FGFR2‐IIIb) is a tyrosine kinase receptor expressed by keratinocytes, which mediates the effects of fibroblast growth factors (FGF). There are contradictory data in the literature regarding the role of FGFR2‐IIIb during the proliferation/differentiation programme of keratinocytes. In this study, we aimed to investigate whether overexpression of FGFR2‐IIIb may have a role in the regulation of keratinocyte proliferation. We analysed the expression of FGFR2‐IIIb in an in vitro HaCaT model system representing different stages of proliferation and differentiation of keratinocytes. Real‐time RT‐PCR and Western blot analyses demonstrated a correlation between FGFR2‐IIIb mRNA and protein expression and the proportion of cells in S/G2/M phase in synchronized HaCaT keratinocytes and thus with proliferation activity (r = 0.96). After treatment with the antipsoriatic drug, dithranol, FGFR2‐IIIb is downregulated dose dependently both at mRNA and protein levels. Moreover, when the rate of proliferation is decreased by the lack of cell attachment to the culturing surface, FGFR2‐IIIb mRNA (P = 0.0315) and protein expressions were also reduced (P = 0.0242), while a differentiation marker, keratin 10, mRNA (P = 0.0003) and protein levels (P = 0.001) were increased (r = −0.92). Based on our results we conclude that FGFR2‐IIIb expression in HaCaT keratinocytes corresponds with the proliferative activation of the cells and is not related to the differentiation programme.