Case report: A novel PTCH1 frameshift mutation leading to nevoid basal cell carcinoma syndrome.

A patient presenting with several basal cell carcinomas, pigmented nevi, and developmental defects was diagnosed with nevoid basal cell carcinoma syndrome. Gene panel sequencing and Sanger sequencing were used to identify a novel heterozygous frameshift mutation, c.1312dupA:p.Ser438Lysfs, in exon 9 of PTCH1. I-Tasser and PyMol analyses indicated that the mutated protein patched homolog 1 (PTCH1) lacked 12 transmembrane domains and the intracellular and extracellular rings of ECD2 compared with the wild-type protein, resulting in a remarkably different structure from that of the wild-type protein. This case extends our knowledge of the mutation spectrum of NBCCS.

METTL3-mediated HPV vaccine enhances the effect of anti PD-1 immunotherapy to alleviate the development of cutaneous squamous cell carcinoma.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) develops from epithelial keratinocytes by dysregulation of self-renewal and differentiation. Recent studies have found that the size and number of cSCC tumors gradually decrease or even disappear after HPV vaccination. However, the role of the HPV vaccine in the cSCC mechanism is poorly understood. OBJECTIVE: The aim of this study is to investigate the effect and mechanism of the HPV vaccine in cSCC. METHODS: Immunofluorescence was used to study the immune infiltrating cells in the tumor tissues of patients with cSCC. The effects of the HPV vaccine on cSCC cells and tissues were studied by Cell Culture, Real-time PCR, Western Blot, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, m6A Blotting, CCK-8 Assay, m6A Ribonucleic acid Methylation Quantification and tumor transplantation. RESULTS: The HPV vaccine enhanced the toxic effect of CD8+T cells on cSCC cells and promoted the secretion of multiple cytokines by CD8+T cells. In addition, HPV vaccines can increase tumor sensitivity to anti-PD-1 therapy by downregulating METTL3 in tumor tissue, with the combination of HPV vaccine and PD-1 monoclonal antibodies producing enhanced immune cell infiltration compared to PD-1 blockade alone. STUDY LIMITATIONS: It is important to note the limitations of this study, including the small sample size, the construction of the mouse model, and the choice of HPV vaccine and PD-1 monoclonal antibody, which may limit the generalization of our findings to a wider population. CONCLUSIONS: It is hoped that this research will contribute to a deeper understanding of the role of the HPV vaccine in the treatment of cSCC. HPV vaccine is expected to become an important approach to alleviate the development of cSCC.

Interleukin-7 regulates CD127 expression and promotes CD8+ T cell activity in patients with primary cutaneous melanoma.

BACKGROUND: Interleukin (IL)-7 signaling through CD127 is impaired in lymphocytes in cancers and chronic infections, resulting in CD8+ T cell exhaustion. The mechanisms underlying CD8+ T cell responses to IL-7 in melanoma remain not completely elucidated. We previously showed reduced IL-7 level in melanoma patients. Thus, the aim of this study was to investigate the effect of IL-7 regulation on CD127 expression and CD8+ T cell responses in melanoma. METHODS: Healthy controls and primary cutaneous melanoma patients were enrolled. Membrane-bound CD127 (mCD127) expression on CD8+ T cells was determined by flow cytometry. Soluble CD127 (sCD127) protein level was measured by ELISA. Total CD127 and sCD127 mRNA level was measured by real-time PCR. CD8+ T cells were stimulated with recombinant human IL-7, along with signaling pathway inhibitors. CD8+ T cells were co-cultured with melanoma cell line, and the cytotoxicity of CD8+ T cells was assessed by measurement of lactate dehydrogenase expression. RESULTS: Plasma sCD127 was lower in melanoma patients compared with controls. The percentage of CD8+ T cells expressing mCD127 was higher, while sCD127 mRNA level was lower in peripheral and tumor-infiltrating CD8+ T cells from melanoma patients. There was no significant difference of total CD127 mRNA expression in CD8+ T cells between groups. IL-7 stimulation enhanced total CD127 and sCD127 mRNA expression and sCD127 release by CD8+ T cells. However, mCD127 mRNA expression on CD8+ T cells was not affected. This process was mainly mediated by phosphatidylinositol 3-kinase (PI3K) pathway. CD8+ T cells from melanoma patients exhibited decreased cytotoxicity. IL-7 stimulation promoted CD8+ T cell cytotoxicity, while inhibition of PI3K dampened IL-7-induced elevation of CD8+ T cell cytotoxicity. CONCLUSION: The current data suggested that insufficient IL-7 secretion might contribute to CD8+ T cell exhaustion and CD127 dysregulation in patients with primary cutaneous melanoma.

Epidemiology of urticaria in China: a population-based study.

BACKGROUND: Urticaria is a common skin disease characterized by episodes of wheals, and it has a negative effect on patients' quality of life. Large-scale population-based epidemiological studies of urticaria are scarce in China. The aim of this survey was to determine the prevalence, clinical forms, and risk factors of urticaria in the Chinese population. METHODS: This survey was conducted in 35 cities from 31 provinces, autonomous regions, and municipalities of China. Two to three communities in each city were selected in this investigation. Participants completed questionnaires and received dermatological examinations. We analyzed the prevalence, clinical forms, and risk factors of urticaria. RESULTS: In total, 44,875 questionnaires were distributed and 41,041 valid questionnaires were collected (17,563 male and 23,478 female participants). The lifetime prevalence of urticaria was 7.30%, with 8.26% in female and 6.34% in male individuals ( P  < 0.05). The point prevalence of urticaria was 0.75%, with 0.79% in female and 0.71% in male individuals ( P  < 0.05). Concomitant angioedema was found in 6.16% of patients. Adults had a higher prevalence of urticaria than adolescents and children. Living in urban areas, exposure to pollutants, an anxious or depressed psychological status, a personal and family history of allergy, thyroid diseases, and Helicobacter pylori infection were associated with a higher prevalence of urticaria. Smoking was correlated with a reduced risk of urticaria. CONCLUSION: This study demonstrated that the lifetime prevalence of urticaria was 7.30% and the point prevalence was 0.75% in the Chinese population; women had a higher prevalence of urticaria than men. Various factors were correlated with urticaria.

Induction of T helper 17 cell response by interleukin-7 in patients with primary cutaneous melanoma.

Interleukin (IL)-7 plays a vital role in proliferation and activation of T cells, however, its signaling through CD127 is impaired in T cells in cancers and chronic infections. The mechanisms underlying T helper 17 (Th17) cell responses by IL-7 in melanoma remain not fully understood. The aim of this study was to assess the effect of IL-7 signaling on Th17 responses in patients with primary cutaneous melanoma. Healthy and primary cutaneous melanoma donors were selected for this study of Th17 cell function. IL-17+CD4+ Th17 cells and CD127 expression on Th17 cells were determined by flow cytometry. Cytokine level was measured by ELISA. Peripheral and tissue-infiltrating CD4+ T cells were isolated using magnetic beads, and then stimulated with IL-7 and/or signal transducer and activator of transcription 5 inhibitor. Activated signaling molecules were analyzed by flow cytometry. Peripheral and tumor-infiltrating Th17 cells percentage was decreased, while peripheral IL-7 level was also reduced in melanoma patients. There was no significant difference of CD127 expression on Th17 cells between melanoma patients and controls. Antiapoptotic protein Bcl-2 was downregulated, whereas proapoptotic protein-activated caspase-3 was upregulated in peripheral and tissue-infiltrating Th17 cells in melanoma patients. Higher concentration of IL-7 (10 ng/mL), but not lower IL-7 concentration (1 ng/mL), promoted Bcl-2 expression and decreased caspase-3 expression in Th17 cells in melanoma patients. Inhibition of signal transducer and activator of transcription 5 resulted in the downregulation of Bcl-2 while upregulation of caspase-3 in Th17 cells. The present data suggested that reduced IL-7 responsiveness might be insufficient for Th17 activation in patients with primary cutaneous melanoma.

A KRT6A and a Novel KRT16 Gene Mutations in Chinese Patients with Pachyonychia Congenita.

BACKGROUND: Pachyonychia congenita (PC) is a rare, autosomal dominant genodermatosis characterized by palmoplantar keratoderma, nail dystrophy, cystic lesions, follicular hyperkeratosis, mucosal leukokeratoses, hyperhidrosis, hoarseness, and, rarely, natal teeth. Five keratin genes, KRT6A, KRT6B, KRT6C, KRT16 and KRT17, have been found to be associated with PC. METHODS: Using polymerase chain reaction and Sanger sequencing techniques, the purpose of the present study was to investigate the clinical features associated with PC and discover disease-associated variants. The KRT6A, KRT16, KRT17, and KRT6B exonic and flanking region sequences were amplified and directly sequenced to detect mutations. RESULTS: Across two independent instances of PC, we identified a previously reported c.1393T>C (p.Tyr465His) mutation in exon 7 of KRT6A, and a novel c.1237G>C (p.Glu413Gln) heterozygous missense mutation in exon 6 of the KRT16 gene. CONCLUSION: Through phenotype-genotype analysis among PC pedigrees, confirmed diagnoses of PC-K6a and PC-K16 were made in the two patients who presented with symptoms of PC. A new pathogenic mutation site in PC-K16 was potentially discovered.

The effective role of sodium copper chlorophyllin on the dysfunction of bone marrow mesenchymal stem cells in multiple myeloma via regulating TGF-ß1.

BACKGROUND: The osteoblast differentiation of bone marrow-derived stem cells (BMSCs) is impaired in multiple myeloma (MM). We investigated the effects of sodium copper chlorophyllin (SCC) on osteoblast differentiation ability of BMSCs from MM. METHODS: Clinical bone marrow samples were collected. Fluorescence Activated Cell Sorter (FACS) was used to identify surface markers of BMSCs. BMSCs were treated with different concentrations of SCC and cell viability was detected by MTT assay. Relative mRNA and protein expressions of transforming growth factor-ß1 (TGF-ß1), SMAD2/3, osteogenic differentiation indicators (RUNX2 and OCN) were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Alkaline phosphatase (ALP) was stained for activity detection. Formation of calcium nodus of BMSCs was examined by Alizarin Red S staining. RESULTS: CD90 and CD105 were high-expressed, but CD34 and CD45 were not expressed in BMSCs. BMSCs in MM group showed a lower expression of TGF-ß1 and a lower degree of osteogenic differentiation. SCC enhanced activities of BMSCs, ALP activity, and formation of calcium nodus, activated TGF-ß1, SMAD2/3 pathway and increased RUNX2 and OCN expressions in BMSCs. Silencing TGF-ß1 reversed the effects of SCC on BMSCs in MM. CONCLUSION: SCC could effectively improve the proliferation and osteogenic differentiation of BMSCs in MM through regulating TGF-ß1.

SASH1 promotes melanin synthesis and migration via suppression of TGF-ß1 secretion in melanocytes resulting in pathologic hyperpigmentation.

Dyschromatosis universalis hereditaria (DUH) is an autosomal dominant pigmentary genodermatosis characterized by the presence of patches of hyperpigmentation and hypopigmented macules distributed over the body, with most cases reported in Asia. DUH is a heterogeneous disease and a small portion of patients carry the ABCB6 variant. In the present study, exome sequencing of four generations of a Chinese family with DUH identified a c.1761C>G (p.Ser587Arg) mutation in exon 15 of SAM and SH3 domain containing 1 (SASH1) that was found to co-segregate in some family members. Immunohistological analysis of biopsy specimens showed that SASH1 was diffusely distributed in all layers of the epidermis, suggesting increased transepithelial migration of melanocytes (MCs). The point mutation c.1761C>G of SASH1 was successfully induced in immortalized human melanocyte (PIG1) cells, which resulted in the downregulation of SASH1 expression. Bioinformatics analysis showed that mutated SASH1 downregulated thrombospondin 1 (THBS1) expression and inactivated transforming growth factor beta 1 (TGF-ß1) signaling. TGF-ß1 expression by PIG1cells was found to negatively regulate SASH1 protein expression. Transwell migration and wound-healing assays showed an increase in the migration and invasion capabilities of the cells carrying the mutation. Further, SASH1 mutations induced downregulation of melanin content. The study results suggest cross-talking between SASH1-TGF-ß1 signaling, demonstrating the proposed MC migration modulation models and affecting melanin trafficking in the epithelium.

Effects of triptolide on bone marrow-derived mesenchymal stem cells from patients with multiple myeloma.

Triptolide (TPL), an extract of the Chinese herb Tripterygium wilfordii Hook F, is a potent anti-inflammatory agent that further possesses anticancer activity. Its antiproliferative effects are well established. Only few studies have focused on TPL as a potential treatment in multiple myeloma (MM). In the current study, bone marrow-derived mesenchymal stem cells (BMMSCs) from patients with MM were isolated and treated with TPL at varying concentrations. Thalidomide is currently used as a positive control drug in the treatment of MM. Cell Counting kit-8 assays were performed to assess proliferation activity and flow cytometry with Annexin V-fluorescein/propidium iodide was used to detect cell apoptosis of TPL-treated BMMSCs. Reverse transcription-quantitative polymerase chain reaction assays were applied to measure interleukin (IL)-6, IL-1ß and stem cell factor (SCF or Kit ligand) mRNA expression and western blot assays were performed to analyze transcription factor p65 (P65) expression in TPL-treated BMMSCs. ELISA was applied to measure vascular endothelial growth factor (VEGF) levels in the supernatant of the cultured and treated BMMSCs. TPL treatment significantly inhibited BMMSC proliferation compared with the untreated control (P<0.05). At 48 h following TPL treatment, a Cell Counting kit-8 study was performed and the IC50 value was determined at 101.55±2.45 ng/ml. Apoptotic rates were observed to increase with increasing concentrations of TPL (P<0.001), and IL-6, IL-1ß and SCF mRNA expression was significantly decreased with increasing TPL (P<0.001). P65 expression following TPL treatment was significantly decreased compared with the untreated control (P<0.05). VEGF levels were significantly reduced in the presence of increasing amounts of TPL (P<0.05). These findings suggest that TPL inhibited BMMSC growth and improved the bone marrow hematopoietic microenvironment by decreasing IL-6, IL-1ß and SCF mRNA expression, subsequently inhibiting the proliferation of MM cells. Therefore, TPL may be used in the future to treat patients with MM.

Analysis of differential ß variable region of T cell receptor expression and NAV3/TNFRSF1B gene mutation in mycosis fungoides.

OBJECTIVE: This study aimed to analyze the predominant expression of the variable region of T cell receptor (TRBV) and determine whether NAV3 or TNFRSF1B gene mutation involved in the pathogenesis of MF. RESULTS: TRBV5-7 expression increased from the normal, early-stage to advanced-stage lesion in MF patient. By contrast, TRBV2 decreased with the lesion developed. We found no mutations of NAV3 or TNFRSF1B in the lesions from this study. MATERIALS AND METHODS: Real-time PCR were used to screen differential expression of TRBV in different lesions. Mutational analyses were used to validate genetic alterations in the skin lesions. CONCLUSIONS: The identification of TRBV gene expression differences between normal and different stages of MF lesions provide insight into promising new diagnostic and prognostic biomarkers. None of the reported genetic abnormalities suggests complexity of progress from a primary cytogenetic event to an advanced stage with poor prognosis in MF.

Associations of ABCB1 and XPC genetic polymorphisms with susceptibility to colorectal cancer and therapeutic prognosis in a Chinese population.

Associations between ABCB1 and XPC genetic polymorphisms and risk of developing colorectal cancer (CRC) as well as clinical outcomes in CRCs with chemotherapy were investigated. A case-control study was performed on the ABCB1 C3435T, G2677T/A and XPC Lys939Gln polymorphisms in 428 CRC cases and 450 hospital- based, age and sex frequency-matched controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays. We observed that the ABCB1 3435CT or CC+CT variants were significantly linked with increasing risk of developing CRC (adjusted OR (95% CI): 1.814 (1.237-2.660), P=0.0022; adjusted OR (95% CI): 1.605 (1.117-2.306), P=0.0102, respectively). Moreover, the distribution frequency of XPC AC genotype or AC+CC genotypes also showed a tendency towards increasing the suscepbility for CRC (P=0.0759 and P=0.0903, respectively). Kaplan-Meier curves showed that the ABCB1 C3435T variant was associated with a tendency toward longer progression-free survival (PFS) (n=343, Log-rank test: P=0.063), and the G2677T/A variant genotypes (GT+TT+GA+AA) with a tendency for longer OS in postoperative oxaliplatin-based patients (n=343, Log-rank test: P=0.082). However, no correlation of the XPC Lys939Gln polymorphism was found with PFS and OS in patients with postoperative oxaliplatin-based chemotherapy (n=343). Our study indicated that ABCB1 polymorphisms might be candidate pharmacogenomic factors for the prediction of CRC susceptibility, but not for prognosis with oxaliplatin chemosensitivity in CRC patients.

Implication of polymorphisms in DNA repair genes in prognosis of hepatocellular carcinoma.

XRCC1 genetic polymorphisms could be associated with increased risk of various cancer, including hepatocellular carcinoma (HCC), the fifth most common cancer. We here conducted a study to explore the role of selective SNPs of the XRCC1 and XPD genes in the prognosis of HCC. A total of 231 cases were collected, and genotyping of XRCC1 Arg194Trp, XRCC1 Arg399Gln, XPD Lys751Gln and XPD Asp312Asn was performed by duplex polymerase-chain-reaction with the confronting-two-pair primer method. Our findings indicated XRCC1 399Gln/Gln genotype was associated with a significant difference in the median survival time compared with patients carrying Arg/Trp and Arg/Arg genotypes, and individuals with XPD 751 Gln/ Gln genotype had a significantly greater survival time than patients carrying Lys/Lys and Lys/Gln genotypes. The Cox's regression analysis showed individuals carrying XRCC1 399Trp/Trp genotype had 0.55 fold risk of death from HCC than Arg/Arg genotype. Similarly, XPD 751Gln/Gln had a strong decreasein comparison to XPD Lys/Lys carriers with an HR of 0.34. These results suggest that polymorphisms in XRCC1 and XPD may have functional significance in the prognosis of HCC.

[Over-expression of osteopontin in non-small cell lung cancers: its clinical significance].

OBJECTIVE: Data obtained from a differentially expressed cDNA library constructed previously in this laboratory demonstrated that the extracellular matrix molecule osteopontin (OPN) is one of most considerably over-expressed genes in non-small cell lung cancers (NSCLCs). The purpose of the present study was to explore the expression status of OPN in a large scale NSCLC tissue samples, and estimate its significance in progression of the malignant disease. METHODS: RT-PCR was performed with the tumor and adjacent normal tissues from 35 patients with NSCLC, at transcriptional levels of OPN. To determine the expression of OPN protein in the tumor tissues, immunohistochemical (IHC) staining was subsequently carried out on paraffin-embedded sections in tissue microarrays containing 662 samples derived from NSCLC cases. The correlation between the expression level of OPN and clinical characteristics was analyzed statistically. RESULTS: Comparing with the paired normal lung tissue, high level RNA of OPN was detected in 80.0% (28/35) of the NSCLC tumor tissues by RT-PCR, which confirmed the information obtained previously by our differentially expressed cDNA library. The results of IHC analysis showed that positively stained OPN protein was observed in 59.6% (331/555) of the tumor tissues, which was remarkably higher than that (25.2%, 27/107) detected in the normal control tissues (P < 0.001). Among the NSCLCs investigated, over-expressed OPN was more frequently found in squamous cell carcinomas (SCCs) than in adenocarcinomas. A further analysis on SCCs demonstrated that the rate of over-expressed OPN was significantly different between the primary tumors with and without lymphatic metastases (68.6% vs. 49.7%, P = 0.001), but similar in the primary tumors and their corresponding metastases in lymph nodes (68.6% vs. 75.5%, P = 0.171). CONCLUSION: Expression of OPN protein is distinctly increased in NSCLCs, particularly in SCCs. OPN over-expression is considerably correlated with lymph node metastasis, increasing the risk of tumor metastasis (OR = 2.212). The resulting data suggest that OPN facilitates the progression of NSCLCs.

[Study on the relationship between serum adiponectin and insulin resistance in women with polycystic ovary syndrome].

OBJECTIVE: To study the relationship between serum adiponectin and insulin resistance in women with polycystic ovary syndrome (PCOS). METHODS: Forty women with PCOS and twenty five healthy women were divided into PCOS obese group [body weight index (BMI) > or = 25kg/m(2)], PCOS non-obese group (BMI < 25 kg/m(2)) and control group. There are 19 cases in PCOS obese group and 21 cases in PCOS non-obese group, 9 cases in obese control group and 16 in non-obese control group. Serum adiponectin levels of the four groups were detected by enzyme linked immunosorbent assay (ELISA) method, insulin by electrochemiluminescence immunoassay method, blood sugar by glucose oxidation enzyme method, tumor necrosis factor-alpha (TNF-alpha) by radioimmunoassay. Insulin sensitivity index (ISI) was calculated. RESULTS: (1) Serum adiponectin levels of PCOS obese group was (1.6 +/- 0.5) mg/L, of PCOS non-obese group was (3.0 +/- 0.6) mg/L. Their values were lower than obese control group (3.2 +/- 0.3) mg/L, and non-obese control group (4.9 +/- 0.5) mg/L (P < 0.05). (2) Fasting insulin levels of PCOS obese group was (17 +/- 6) mU/L, PCOS non-obese group was (14 +/- 6) mU/L. They were higher than obese control group (10 +/- 3) mU/L, and non-obese control group (7 +/- 3) mU/L (P < 0.05). (3) Fasting blood sugar level was (5.2 +/- 0.7) mmol/L in PCOS obese group, in PCOS non-obese group was (5.1 +/- 0.6) mmol/L, in obese control group was (5.4 +/- 0.5) mmol/L, and non-obese control group (4.8 +/- 0.6) mmol/L, without marked difference among four groups. (4) TNF-alpha levels of PCOS obese group was (1.32 +/- 0.14) microg/L, of PCOS non-obese group was (1.02 +/- 0.12) microg/L. They were higher than obese control group (0.93 +/- 0.15) microg/L, and non-obese control group (0.63 +/- 0.18) microg/L (P < 0.05). (5) ISI of PCOS obese group was -4.5 +/- 0.3, PCOS non-obese group was -4.1 +/- 0.4. Their values were lower than obese control group -3.6 +/- 0.3, and non-obese control group (-3.1 +/- 0.4) (P < 0.05). Serum adiponectin levels of the women with PCOS were correlated negatively with BMI (r = -0.56, P < 0.05), and correlated positively with ISI (r = 0.49, P < 0.05). CONCLUSION: Serum adiponectin levels of women with PCOS is decreased compared with healthy women, particularly in obese women with PCOS. The decrease is correlated with ISI.