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Sample records for hpv16 gene copy

  1. The DNA damage response activates HPV16 late gene expression at the level of RNA processing.

    PubMed

    Nilsson, Kersti; Wu, Chengjun; Kajitani, Naoko; Yu, Haoran; Tsimtsirakis, Efthymios; Gong, Lijing; Winquist, Ellenor B; Glahder, Jacob; Ekblad, Lars; Wennerberg, Johan; Schwartz, Stefan

    2018-06-01

    We show that the alkylating cancer drug melphalan activated the DNA damage response and induced human papillomavirus type 16 (HPV16) late gene expression in an ATM- and Chk1/2-dependent manner. Activation of HPV16 late gene expression included inhibition of the HPV16 early polyadenylation signal that resulted in read-through into the late region of HPV16. This was followed by activation of the exclusively late, HPV16 splice sites SD3632 and SA5639 and production of spliced late L1 mRNAs. Altered HPV16 mRNA processing was paralleled by increased association of phosphorylated BRCA1, BARD1, BCLAF1 and TRAP150 with HPV16 DNA, and increased association of RNA processing factors U2AF65 and hnRNP C with HPV16 mRNAs. These RNA processing factors inhibited HPV16 early polyadenylation and enhanced HPV16 late mRNA splicing, thereby activating HPV16 late gene expression.

  2. HPV16 early gene E5 specifically reduces miRNA-196a in cervical cancer cells

    PubMed Central

    Liu, Chanzhen; Lin, Jianfei; Li, Lianqin; Zhang, Yonggang; Chen, Weiling; Cao, Zeyi; Zuo, Huancong; Chen, Chunling; Kee, Kehkooi

    2015-01-01

    High-risk human papillomavirus (HPV) type 16, which is responsible for greater than 50% of cervical cancer cases, is the most prevalent and lethal HPV type. However, the molecular mechanisms of cervical carcinogenesis remain elusive, particularly the early steps of HPV infection that may transform normal cervical epithelium into a pre-neoplastic state. Here, we report that a group of microRNAs (microRNAs) were aberrantly decreased in HPV16-positive normal cervical tissues, and these groups of microRNAs are further reduced in cervical carcinoma. Among these miRNAs, miR196a expression is the most reduced in HPV16-infected tissues. Interestingly, miR196a expression is low in HPV16-positive cervical cancer cell lines but high in HPV16-negative cervical cancer cell lines. Furthermore, we found that only HPV16 early gene E5 specifically down-regulated miRNA196a in the cervical cancer cell lines. In addition, HoxB8, a known miR196a target gene, is up-regulated in the HPV16 cervical carcinoma cell line but not in HPV18 cervical cancer cell lines. Various doses of miR196a affected cervical cancer cell proliferation and apoptosis. Altogether, these results suggested that HPV16 E5 specifically down-regulates miR196a upon infection of the human cervix and initiates the transformation of normal cervix cells to cervical carcinoma. PMID:25563170

  3. Analysis of E2 gene integrity in HPV16 and HPV58 viruses isolated from women with cervical pathology

    PubMed Central

    González-Losa, María del R; Puerto-Solis, Marylin; Tenorio Ruiz, Juan; Rosado-López, Ariel I; Hau-Aviles, Oscar; Ayora-Talavera, Guadalupe; Cisneros-Cutz, Isidro; Conde-Ferráez, Laura

    2016-01-01

    Integration of human papillomavirus (HPV) DNA into human cells accompanied by the disruption of the viral genome has been described as a prerequisite for cancer development. This study aimed to investigate E2 gene integrity of HPV16 and HPV58 viruses isolated from infected women with cervical lesions. Forty-two HPV16- and 31 HPV58-positive samples were analysed. E2 integrity was assumed when all fragments covering the E2 gene were amplified with specific polymerase chain reaction primers. Overall, in 59% of the samples, at least one fragment was not amplified in HPV16- (57%) and HPV58-positive samples (61%). Samples from high-grade squamous intraepithelial lesions had the highest frequency of E2 gene disruptions (73%), followed by samples from low-grade squamous intraepithelial lesions (63%) and, finally, samples from invasive cervical cancer (35%). Association between the integrity status of the E2 gene, and lesion grade was assessed by the chi-squared test applied to the combined set of viruses (p = 0.6555) or to populations of the same virus type (HPV58, p = 0.3101; HPV16, p = 0.3024). In conclusion, in this study, no association was found between the presence of E2 gene disruptions and the grade of cervical lesions caused by HPV16 and HPV58. PMID:27812600

  4. HPV16 integration probably contributes to cervical oncogenesis through interrupting tumor suppressor genes and inducing chromosome instability.

    PubMed

    Zhao, Jun-Wei; Fang, Fang; Guo, Yi; Zhu, Tai-Lin; Yu, Yun-Yun; Kong, Fan-Fei; Han, Ling-Fei; Chen, Dong-Sheng; Li, Fang

    2016-11-25

    The integration of human papilloma virus (HPV) into host genome is one of the critical steps that lead to the progression of precancerous lesion into cancer. However, the mechanisms and consequences of such integration events are poorly understood. This study aims to explore those questions by studying high risk HPV16 integration in women with cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (SCC). Specifically, HPV integration status of 13 HPV16-infected patients were investigated by ligation-mediated PCR (DIPS-PCR) followed by DNA sequencing. In total, 8 HPV16 integration sites were identified inside or around genes associated with cancer development. In particular, the well-studied tumor suppressor genes SCAI was found to be integrated by HPV16, which would likely disrupt its expression and therefore facilitate the migration of tumor. On top of that, we observed several cases of chromosome translocation events coincide with HPV integration, which suggests the existence of chromosome instability. Additionally, short overlapping sequences were observed between viral derived and host derived fragments in viral-cellular junctions, indicating that integration was mediated by micro homology-mediated DNA repair pathway. Overall, our study suggests a model in which HPV16 might contribute to oncogenesis not only by disrupting tumor suppressor genes, but also by inducing chromosome instability.

  5. Targeting of HPV-16+ Epithelial Cancer Cells by TCR Gene Engineered T Cells Directed against E6.

    PubMed

    Draper, Lindsey M; Kwong, Mei Li M; Gros, Alena; Stevanović, Sanja; Tran, Eric; Kerkar, Sid; Raffeld, Mark; Rosenberg, Steven A; Hinrichs, Christian S

    2015-10-01

    The E6 and E7 oncoproteins of HPV-associated epithelial cancers are in principle ideal immunotherapeutic targets, but evidence that T cells specific for these antigens can recognize and kill HPV(+) tumor cells is limited. We sought to determine whether TCR gene engineered T cells directed against an HPV oncoprotein can successfully target HPV(+) tumor cells. T-cell responses against the HPV-16 oncoproteins were investigated in a patient with an ongoing 22-month disease-free interval after her second resection of distant metastatic anal cancer. T cells genetically engineered to express an oncoprotein-specific TCR from this patient's tumor-infiltrating T cells were tested for specific reactivity against HPV(+) epithelial tumor cells. We identified, from an excised metastatic anal cancer tumor, T cells that recognized an HLA-A*02:01-restricted epitope of HPV-16 E6. The frequency of the dominant T-cell clonotype from these cells was approximately 400-fold greater in the patient's tumor than in her peripheral blood. T cells genetically engineered to express the TCR from this clonotype displayed high avidity for an HLA-A*02:01-restricted epitope of HPV-16, and they showed specific recognition and killing of HPV-16(+) cervical, and head and neck cancer cell lines. These findings demonstrate that HPV-16(+) tumors can be targeted by E6-specific TCR gene engineered T cells, and they provide the foundation for a novel cellular therapy directed against HPV-16(+) malignancies, including cervical, oropharyngeal, anal, vulvar, vaginal, and penile cancers. ©2015 American Association for Cancer Research.

  6. Depletion of HPV16 early genes induces autophagy and senescence in a cervical carcinogenesis model, regardless of viral physical state.

    PubMed

    Hanning, Jennifer E; Saini, Harpreet K; Murray, Matthew J; Caffarel, Maria M; van Dongen, Stijn; Ward, Dawn; Barker, Emily M; Scarpini, Cinzia G; Groves, Ian J; Stanley, Margaret A; Enright, Anton J; Pett, Mark R; Coleman, Nicholas

    2013-11-01

    In cervical carcinomas, high-risk human papillomavirus (HR-HPV) may be integrated into host chromosomes or remain extra-chromosomal (episomal). We used the W12 cervical keratinocyte model to investigate the effects of HPV16 early gene depletion on in vitro cervical carcinogenesis pathways, particularly effects shared by cells with episomal versus integrated HPV16 DNA. Importantly, we were able to study the specific cellular consequences of viral gene depletion by using short interfering RNAs known not to cause phenotypic or transcriptional off-target effects in keratinocytes. We found that while cervical neoplastic progression in vitro was characterized by dynamic changes in HPV16 transcript levels, viral early gene expression was required for cell survival at all stages of carcinogenesis, regardless of viral physical state, levels of early gene expression or histology in organotypic tissue culture. Moreover, HPV16 early gene depletion induced changes in host gene expression that were common to both episome-containing and integrant-containing cells. In particular, we observed up-regulation of autophagy genes, associated with enrichment of senescence and innate immune-response pathways, including the senescence-associated secretory phenotype (SASP). In keeping with these observations, HPV16 early gene depletion induced autophagy in both episome-containing and integrant-containing W12 cells, as evidenced by the appearance of autophagosomes, punctate expression of the autophagy marker LC3, conversion of LC3B-I to LC3B-II, and reduced levels of the autophagy substrate p62. Consistent with the reported association between autophagy and senescence pathways, HPV16 early gene depletion induced expression of the senescence marker beta-galactosidase and increased secretion of the SASP-related protein IGFBP3. Together, these data indicate that depleting HR-HPV early genes would be of potential therapeutic benefit in all cervical carcinogenesis pathways, regardless of viral

  7. Changes in global gene expression profiles induced by HPV 16 E6 oncoprotein variants in cervical carcinoma C33-A cells

    SciTech Connect

    Zacapala-Gómez, Ana Elvira, E-mail: zak_ana@yahoo.com.mx; Del Moral-Hernández, Oscar, E-mail: odelmoralh@gmail.com; Villegas-Sepúlveda, Nicolás, E-mail: nvillega@cinvestav.mx

    We analyzed the effects of the expression of HPV 16 E6 oncoprotein variants (AA-a, AA-c, E-A176/G350, E-C188/G350, E-G350), and the E-Prototype in global gene expression profiles in an in vitro model. E6 gene was cloned into an expression vector fused to GFP and was transfected in C33-A cells. Affymetrix GeneChip Human Transcriptome Array 2.0 platform was used to analyze the expression of over 245,000 coding transcripts. We found that HPV16 E6 variants altered the expression of 387 different genes in comparison with E-Prototype. The altered genes are involved in cellular processes related to the development of cervical carcinoma, such asmore » adhesion, angiogenesis, apoptosis, differentiation, cell cycle, proliferation, transcription and protein translation. Our results show that polymorphic changes in HPV16 E6 natural variants are sufficient to alter the overall gene expression profile in C33-A cells, explaining in part the observed differences in oncogenic potential of HPV16 variants. - Highlights: • Amino acid changes in HPV16 E6 variants modulate the transciption of specific genes. • This is the first comparison of global gene expression profile of HPV 16 E6 variants. • Each HPV 16 E6 variant appears to have its own molecular signature.« less

  8. Multifocal Epithelial Hyperplasia of Oral Cavity Expressing HPV 16 Gene: A Rare Entity

    PubMed Central

    Prabhat, M. P. V.; Raja Lakshmi, Chintamaneni; Sai Madhavi, N.; Bhavana, Sujana Mulk; Sarat, Gummadapu; Ramamohan, Kodali

    2013-01-01

    Focal epithelial hyperplasia is a rare contagious disease caused by human papilloma virus. Usually HPV involves either cutaneous or mucosal surfaces, whereas concomitant mucocutaneous involvement is extremely rare. We report such a unique case of multifocal epithelial hyperplasia involving multiple sites of oral cavity along with skin lesions in a 65-year-old female. We also discuss the probable multifactorial etiology and variable clinical presentations of the lesions, including evidence of HPV 16 expression, as detected by polymerase chain reaction. The present report illustrates the need for careful examination and prompt diagnosis of the disease, as it might be associated with high risk genotypes such as HPV 16 and 18. PMID:24455323

  9. Sites of disruption within E1 and E2 genes of HPV16 and association with cervical dysplasia.

    PubMed

    Tsakogiannis, D; Gortsilas, P; Kyriakopoulou, Z; Ruether, I G A; Dimitriou, T G; Orfanoudakis, G; Markoulatos, P

    2015-11-01

    Integration of HPV16 DNA into the host chromosome usually disrupts the E1 and/or E2 genes. The present study investigated the disruption of E1, E2 genes in a total of eighty four HPV16-positive precancerous and cervical cancer specimens derived from Greek women (seventeen paraffin-embedded cervical biopsies and sixty seven Thin Prep samples). Complete E2 and E1 genes were amplified using three and nine overlapping primer sets respectively, in order to define the sites of disruption. Extensive mapping analysis revealed that disruption/deletion events within E2 gene occurred in high grade and cervical cancer samples (x(2) test, P < 0.01), while no evidence of E2 gene disruption was documented among low grade cervical intraepithelial neoplasias. In addition, disruptions within the E1 gene occur both in high and low grade cervical intraepithelial neoplasia. This leads to the assumption that in low grade cervical intraepithelial neoplasias only E1 gene disruption was involved (Fisher's exact test, P < 0.05), while in high grade malignancies and cervical cancer cases deletions in both E1 and E2 genes occurred. Furthermore, the most prevalent site of disruption of E1 gene was located between nucleotides 1059 and 1323, while the most prevalent deleted region of the E2 gene was located between nucleotides 3172 and 3649 (E2 hinge region). Therefore, it is proposed that each population has its own profile of frequencies and sites of disruptions and extensive mapping analysis of E1 and E2 genes is mandatory in order to determine suitable markers for HPV16 DNA integration analysis in distinct populations. © 2015 Wiley Periodicals, Inc.

  10. Mutation detection of E6 and LCR genes from HPV 16 associated with carcinogenesis.

    PubMed

    Mosmann, Jessica P; Monetti, Marina S; Frutos, Maria C; Kiguen, Ana X; Venezuela, Raul F; Cuffini, Cecilia G

    2015-01-01

    Human papillomavirus (HPV) is responsible for one of the most frequent sexually transmitted infections. The first phylogenetic analysis was based on a LCR region fragment. Nowadays, 4 variants are known: African (Af-1, Af-2), Asian-American (AA) and European (E). However the existence of sub-lineages of the European variant havs been proposed, specific mutations in the E6 and LCR sequences being possibly related to persistent viral infections. The aim of this study was a phylogenetic study of HPV16 sequences of endocervical samples from Cordoba, in order to detect the circulating lineages and analyze the presence of mutations that could be correlated with malignant disease. The phylogenetic analysis determined that 86% of the samples belonged to the E variant, 7% to AF-1 and the remaining 7% to AF-2. The most frequent mutation in LCR sequences was G7521A, in 80% of the analyzed samples; it affects the binding site of a transcription factor that could contribute to carcinogenesis. In the E6 sequences, the most common mutation was T350G (L83V), detected in 67% of the samples, associated with increased risk of persistent infection. The high detection rate of the European lineage correlated with patterns of human migration. This study emphasizes the importance of recognizing circulating lineages, as well as the detection of mutations associated with high-grade neoplastic lesions that could be correlated to the development of carcinogenic lesions.

  11. Real-time PCR assays using internal controls for quantitation of HPV-16 and beta-globin DNA in cervicovaginal lavages.

    PubMed

    Lefevre, Jonas; Hankins, Catherine; Pourreaux, Karina; Voyer, Hélène; Coutlée, François

    2003-12-01

    High-risk human papillomavirus 16 (HPV-16) DNA viral load has been measured with real-time PCR assays by amplifying HPV-16 and a human gene. However, these assays have not used internal controls (ICs) to screen for the presence of inhibitors contained in samples. To quantitate HPV-16 DNA and cell content with real-time PCR, ICs for HPV-16 DNA and beta-globin were synthesised and used to control for inhibition. The assays were sensitive and linear over 5 logs. Good reproducibility was achieved with inter-run coefficients of variation of 23% (10(2) HPV-16 copies), 12% (10(4) HPV-16 copies), 17% (274 beta-globin DNA copies) and 7% (27,400 beta-globin DNA copies). Samples containing 56,800,000, 306,000, 18,000, and 4,070 HPV-16 copies/microg of cellular DNA were tested blindly and estimated to contain 48,800,000, 479,000, 20,300, and 6,620 HPV-16 copies/microg of DNA (mean ratio of measured to expected viral load of 1.27+/-0.32). Inhibition of amplification of HPV-16 and beta-globin ICs by six samples known to contain PCR inhibitors was variable: four inhibited both ICs while two inhibited only the HPV-16 IC. The use of internal controls with real-time PCR for HPV-16 quantitation allows to screen for the presence of inhibitors that do not affect equally primer-driven genomic amplification.

  12. The HPV16 E7 oncoprotein increases the expression of Oct3/4 and stemness-related genes and augments cell self-renewal

    SciTech Connect

    Organista-Nava, Jorge; Gómez-Gómez, Yazmín

    Oct3/4 is a transcription factor involved in maintenance of the pluripotency and self-renewal of stem cells. The E7 oncoprotein and 17β-estradiol (E{sub 2}) are key factors in cervical carcinogenesis. In the present study, we aimed to investigate the effect of the HPV16 E7 oncoprotein and E{sub 2} on the expression pattern of Oct3/4, Sox2, Nanog and Fgf4. We also determined whether the E7 oncoprotein is associated with cell self-renewal. The results showed that Oct3/4, Sox2, Nanog and Fgf4 were upregulated by the E7 oncoprotein in vivo and in vitro and implicate E{sub 2} in the upregulation of these factors inmore » vivo. We also demonstrated that E7 is involved in cell self-renewal, suggesting that the HPV16 E7 oncoprotein upregulates Oct3/4, Sox2, Nanog and Fgf4 expression to maintain the self-renewal capacity of cancer stem cells. -- Graphical abstract: The HPV16 E7 oncoprotein and 17β-estradiol are involved in the upregulation of Oct3/4, Sox2, Nanog and Fgf4 expression to maintain the self-renewal ability of cancer stem cells in cervical cancer. - Highlights: •The HPV16 E7 oncoprotein enhances cellular proliferation and dedifferentiation. •The E7 oncoprotein induces stemness-related genes expression in vivo and in vitro. •The 17β-estradiol induces stemness-related genes expression in vivo. •The HPV16 E7 oncoprotein is involved in the cell self-renewal of cancer cells.« less

  13. HPV frequency in penile carcinoma of Mexican patients: important contribution of HPV16 European variant.

    PubMed

    López-Romero, Ricardo; Iglesias-Chiesa, Candela; Alatorre, Brenda; Vázquez, Karla; Piña-Sánchez, Patricia; Alvarado, Isabel; Lazos, Minerva; Peralta, Raúl; González-Yebra, Beatriz; Romero, Anae; Salcedo, Mauricio

    2013-01-01

    The role of human papillomavirus (HPV) infection in penile carcinoma (PeC) is currently reported and about half of the PeC is associated with HPV16 and 18. We used a PCR-based strategy by using HPV general primers to analyze 86 penile carcinomas paraffin-embedded tissues. Some clinical data, the histological subtype, growth pattern, and differentiation degree were also collected. The amplified fragments were then sequenced to confirm the HPV type and for HPV16/18 variants. DNA samples were also subjected to relative real time PCR for hTERC gene copy number. Some clinical data were also collected. Global HPV frequency was 77.9%. Relative contributions was for HPV16 (85%), 31 (4.4%), 11 (4.4%), 58, 33, 18, and 59 (1.4% each one). Sequence analysis of HPV16 identified European variants and Asian-American (AAb-c) variants in 92% and in 8% of the samples, respectively. Furthermore hTERC gene amplification was observed in only 17% of the cases. Our results suggest that some members of HPV A9 group (represented by HPV16, 58, and 31) are the most frequent among PeC patients studied with an important contribution from HPV16 European variant. The hTERC gene amplification could be poorly related to penile epithelial tissue.

  14. HPV frequency in penile carcinoma of Mexican patients: important contribution of HPV16 European variant

    PubMed Central

    López-Romero, Ricardo; Iglesias-Chiesa, Candela; Alatorre, Brenda; Vázquez, Karla; Piña-Sánchez, Patricia; Alvarado, Isabel; Lazos, Minerva; Peralta, Raúl; González-Yebra, Beatriz; Romero, AnaE; Salcedo, Mauricio

    2013-01-01

    The role of human papillomavirus (HPV) infection in penile carcinoma (PeC) is currently reported and about half of the PeC is associated with HPV16 and 18. We used a PCR-based strategy by using HPV general primers to analyze 86 penile carcinomas paraffin-embedded tissues. Some clinical data, the histological subtype, growth pattern, and differentiation degree were also collected. The amplified fragments were then sequenced to confirm the HPV type and for HPV16/18 variants. DNA samples were also subjected to relative real time PCR for hTERC gene copy number. Some clinical data were also collected. Global HPV frequency was 77.9%. Relative contributions was for HPV16 (85%), 31 (4.4%), 11 (4.4%), 58, 33, 18, and 59 (1.4% each one). Sequence analysis of HPV16 identified European variants and Asian-American (AAb-c) variants in 92% and in 8% of the samples, respectively. Furthermore hTERC gene amplification was observed in only 17% of the cases. Our results suggest that some members of HPV A9 group (represented by HPV16, 58, and 31) are the most frequent among PeC patients studied with an important contribution from HPV16 European variant. The hTERC gene amplification could be poorly related to penile epithelial tissue. PMID:23826423

  15. HPV16 E2 gene disruption and polymorphisms of E2 and LCR: some significant associations with cervical cancer in Indian women.

    PubMed

    Bhattacharjee, Bornali; Sengupta, Sharmila

    2006-02-01

    We evaluated the status of the HPV16 E2 gene (disrupted or intact), nucleotide sequence alterations within intact E2 genes and LCR of HPV16 isolates in a group of CaCx cases (invasive squamous cell carcinomas, n = 81) and population controls (normal cervical scrapes, n = 27) from Indian women. E2 disruption was detected by amplifying the entire E2 gene with single set of primers, while overlapping primers were used to determine if any particular region got selectively disrupted. Nucleotide variations in E2 and LCR were analyzed by PCR amplification followed by bi-directional sequencing. The associations between the viral factors and CaCx were analyzed using Fisher's Exact or Chi-squared test and interpreted as OR (95% CI) and P values. E2 disruption was significantly higher among the cases [3.38 (1.07-10.72); P = 0.02], which was maximum in the region between nucleotides 3650 and 3872 (DNA-binding region). The European (E) variant was found to be the prevalent subgroup (87.76% among cases and 96.30% among the controls), and the remaining samples were Asian-American variants. Among the E subgroup, variation at position 7450 (T > C) within the E2-binding site-IV was found to be significantly higher among the E2 undisrupted cases (21/37; 56.76%), compared to controls (5/18; 27.78%) [3.41 (1.01-11.55); P = 0.03]. Besides HPV16 E2 disruption, LCR 7450T > C variation within undisrupted E2 of E subgroup appears to be a major factor contributing to the risk of CaCx development in Indian women. Furthermore, polymorphisms in the E2 gene of HPV16 may not be significant for disease risk.

  16. Rapid identification of HPV 16 and 18 by multiplex nested PCR-immunochromatographic test.

    PubMed

    Kuo, Yung-Bin; Li, Yi-Shuan; Chan, Err-Cheng

    2015-02-01

    Human papillomavirus (HPV) types 16 and 18 are known to be high-risk viruses that cause cervical cancer. An HPV rapid testing kit that could help physicians to make early and more informed decisions regarding patient care is needed urgently but not yet available. This study aimed to develop a multiplex nested polymerase chain reaction-immunochromatographic test (PCR-ICT) for the rapid identification of HPV 16 and 18. A multiplex nested PCR was constructed to amplify the HPV 16 and 18 genotype-specific L1 gene fragments and followed by ICT which coated with antibodies to identify rapidly the different PCR products. The type-specific gene regions of high-risk HPV 16 and 18 could be amplified successfully by multiplex nested PCR at molecular sizes of approximately 99 and 101bp, respectively. The capture antibodies raised specifically against the moleculars labeled on the PCR products could be detected simultaneously both HPV 16 and 18 in one strip. Under optimal conditions, this PCR-ICT assay had the capability to detect HPV in a sample with as low as 100 copies of HPV viral DNA. The PCR-ICT system has the advantage of direct and simultaneous detection of two high-risk HPV 16 and 18 DNA targets in one sample, which suggested a significant potential of this assay for clinical application. Copyright © 2014. Published by Elsevier B.V.

  17. Viral load and genomic integration of HPV 16 in cervical samples from HIV-1-infected and uninfected women in Burkina Faso.

    PubMed

    Rousseau, Marie-Noelle Didelot; Costes, Valérie; Konate, Issouf; Nagot, Nicolas; Foulongne, Vincent; Ouedraogo, Abdoulaye; Van de Perre, Philippe; Mayaud, Philippe; Segondy, Michel

    2007-06-01

    The relationships between human papillomavirus type 16 (HPV 16) viral load, HPV 16 integration status, human immunodeficiency virus type 1 (HIV-1) status, and cervical cytology were studied among women enrolled in a cohort of female sex workers in Burkina Faso. The study focused on 24 HPV 16-infected women. The HPV 16 viral load in cervical samples was determined by real-time PCR. Integration ratio was estimated as the ratio between E2 and E6 genes DNA copy numbers. Integrated HPV16 viral load was defined as the product of HPV 16 viral load by the integration ratio. High HPV 16 viral load and high integration ratio were more frequent among women with squamous intraepithelial lesions compared with women with normal cytology (33% vs. 11%, and 33% vs. 0%, respectively), and among women with high-grade squamous intraepithelial lesions compared with women without high-grade squamous intraepithelial lesions (50% vs. 17%, and 50% vs. 11%, respectively). High HPV 16 DNA load, but not high integration ratio, was also more frequent among HIV-1-positive women (39% vs. 9%; and 23% vs. 18%, respectively). The absence of statistical significance of these differences might be explained by the small study sample size. High-integrated HPV 16 DNA load was significantly associated with the presence of high-grade squamous intraepithelial lesions (50% vs. 5%, P = 0.03) in univariate and multivariate analysis (adjusted odds-ratio: 19.05; 95% confidence interval (CI), 1.11-328.3, P = 0.03), but not with HIV-1 or other high-risk HPV types (HR-HPV). Integrated HPV 16 DNA load may be considered as a useful marker of high-grade cervical lesions in HPV 16-infected women. (c) 2007 Wiley-Liss, Inc.

  18. hnRNP L controls HPV16 RNA polyadenylation and splicing in an Akt kinase-dependent manner

    PubMed Central

    Kajitani, Naoko; Glahder, Jacob; Wu, Chengjun; Yu, Haoran; Nilsson, Kersti

    2017-01-01

    Abstract Inhibition of the Akt kinase activates HPV16 late gene expression by reducing HPV16 early polyadenylation and by activating HPV16 late L1 mRNA splicing. We identified ‘hot spots’ for RNA binding proteins at the early polyA signal and at splice sites on HPV16 late mRNAs. We observed that hnRNP L was associated with sequences at all HPV16 late splice sites and at the early polyA signal. Akt kinase inhibition resulted in hnRNP L dephosphorylation and reduced association of hnRNP L with HPV16 mRNAs. This was accompanied by an increased binding of U2AF65 and Sam68 to HPV16 mRNAs. Furthermore, siRNA knock-down of hnRNP L or Akt induced HPV16 gene expression. Treatment of HPV16 immortalized keratinocytes with Akt kinase inhibitor reduced hnRNP L binding to HPV16 mRNAs and induced HPV16 L1 mRNA production. Finally, deletion of the hnRNP L binding sites in HPV16 subgenomic expression plasmids resulted in activation of HPV16 late gene expression. In conclusion, the Akt kinase inhibits HPV16 late gene expression at the level of RNA processing by controlling the RNA-binding protein hnRNP L. We speculate that Akt kinase activity upholds an intracellular milieu that favours HPV16 early gene expression and suppresses HPV16 late gene expression. PMID:28934469

  19. Specific interaction between hnRNP H and HPV16 L1 proteins: Implications for late gene auto-regulation enabling rapid viral capsid protein production

    SciTech Connect

    Zheng, Zi-Zheng; Sun, Yuan-Yuan; Zhao, Min

    2013-01-18

    Highlights: ► The RNA-binding hnRNP H regulates late viral gene expression. ► hnRNP H activity was inhibited by a late viral protein. ► Specific interaction between HPV L1 and hnRNP H was demonstrated. ► Co-localization of HPV L1 and hnRNP H inside cells was observed. ► Viral capsid protein production, enabling rapid capsid assembly, was implicated. -- Abstract: Heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP H, are RNA-binding proteins that function as splicing factors and are involved in downstream gene regulation. hnRNP H, which binds to G triplet regions in RNA, has been shown to play an important role in regulatingmore » the staged expression of late proteins in viral systems. Here, we report that the specific association between hnRNP H and a late viral capsid protein, human papillomavirus (HPV) L1 protein, leads to the suppressed function of hnRNP H in the presence of the L1 protein. The direct interaction between the L1 protein and hnRNP H was demonstrated by complex formation in solution and intracellularly using a variety of biochemical and immunochemical methods, including peptide mapping, specific co-immunoprecipitation and confocal fluorescence microscopy. These results support a working hypothesis that a late viral protein HPV16 L1, which is down regulated by hnRNP H early in the viral life cycle may provide an auto-regulatory positive feedback loop that allows the rapid production of HPV capsid proteins through suppression of the function of hnRNP H at the late stage of the viral life cycle. In this positive feedback loop, the late viral gene products that were down regulated earlier themselves disable their suppressors, and this feedback mechanism could facilitate the rapid production of capsid proteins, allowing staged and efficient viral capsid assembly.« less

  20. In Vitro Progression of HPV16 Episome-Associated Cervical Neoplasia Displays Fundamental Similarities to Integrant-Associated Carcinogenesis

    PubMed Central

    Gray, Elizabeth; Pett, Mark R.; Ward, Dawn; Winder, David M.; Stanley, Margaret A.; Roberts, Ian; Scarpini, Cinzia G.; Coleman, Nicholas

    2010-01-01

    An important event in the development of cervical squamous cell carcinoma (SCC) is deregulated expression of high-risk human papillomavirus (HR-HPV) oncogenes, most commonly related to viral integration into host DNA. Mechanisms of development of the ~15% of SCCs that contain extra-chromosomal (episomal) HR-HPV are poorly understood, due to limited longitudinal data. We therefore employed the W12 model to study mechanisms of cervical carcinogenesis associated with episomal HPV16. In vitro progression of W12 normally occurs through selection of cells containing integrated HPV16. However, in one long-term culture, keratinocytes developed a selective growth advantage and invasive phenotype, while retaining HPV16 episomes at increased copy number in the absence of transcriptionally active integrants. Longitudinal investigations revealed similarities between the episome- and integrant-associated routes of neoplastic progression. Most notable were dynamic changes in viral early gene expression in episome-retaining cells, consistent with continually changing selective pressures. An early increase in viral transcription preceded elevated episome copy number and was followed by a reduction to near baseline after the development of invasiveness. Episomal transcriptional deregulation did not require selection of a specific sequence variant of the HPV16 upstream regulatory region, although increased levels of acetylated histone H4 around the late promoter implicated a role for altered chromatin structure. Interestingly, invasive episome-retaining cells demonstrated high levels of HPV16-E2/E6 proteins (despite decreased transcript levels) and reduced expression of interferon-stimulated genes, adaptations that support viral persistence and cell survival. Our findings suggest a unified working model for events important in cervical neoplastic progression, regardless of HR-HPV physical state. PMID:20442284

  1. Monitoring HPV-16 E7 phosphorylation events

    SciTech Connect

    Nogueira, Marcela O.; Hošek, Tomáš; Calçada, Eduar

    HPV-16 E7 is one of the key proteins that, by interfering with the host metabolism through many protein-protein interactions, hijacks cell regulation and contributes to malignancy. Here we report the high resolution investigation of the CR3 region of HPV-16 E7, both as an isolated domain and in the full-length protein. This opens the way to the atomic level study of the many interactions in which HPV-16 E7 is involved. Along these lines we show here the effect of one of the key post-translational modifications of HPV-16 E7, the phosphorylation by casein kinase II.

  2. The HPV16 E7 oncoprotein increases the expression of Oct3/4 and stemness-related genes and augments cell self-renewal.

    PubMed

    Organista-Nava, Jorge; Gómez-Gómez, Yazmín; Ocadiz-Delgado, Rodolfo; García-Villa, Enrique; Bonilla-Delgado, José; Lagunas-Martínez, Alfredo; Tapia, Jesús Santa-Olalla; Lambert, Paul F; García-Carrancá, Alejandro; Gariglio, Patricio

    2016-12-01

    Oct3/4 is a transcription factor involved in maintenance of the pluripotency and self-renewal of stem cells. The E7 oncoprotein and 17β-estradiol (E 2 ) are key factors in cervical carcinogenesis. In the present study, we aimed to investigate the effect of the HPV16 E7 oncoprotein and E 2 on the expression pattern of Oct3/4, Sox2, Nanog and Fgf4. We also determined whether the E7 oncoprotein is associated with cell self-renewal. The results showed that Oct3/4, Sox2, Nanog and Fgf4 were upregulated by the E7 oncoprotein in vivo and in vitro and implicate E 2 in the upregulation of these factors in vivo. We also demonstrated that E7 is involved in cell self-renewal, suggesting that the HPV16 E7 oncoprotein upregulates Oct3/4, Sox2, Nanog and Fgf4 expression to maintain the self-renewal capacity of cancer stem cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. HPV-16 viral load in oropharyngeal squamous cell carcinoma using digital PCR.

    PubMed

    Antonsson, Annika; Knight, Lani; Panizza, Benedict J; Porceddu, Sandro V; Emmett, Sarah; Whiteman, David C

    2018-05-09

    We did not identify any strong associations between HPV-16 viral load and any of the clinical or lifestyle factors. The epidemiology of oropharyngeal SCC is changing, with an increasing proportion of HPV-positive cases seen in the last decade. It is known that a high viral load is linked to the development of cervical cancer, the relation between viral load and oropharyngeal SCC is less clear. We sought to determine HPV-16 viral load in HPV-positive oropharyngeal SCCs using highly sensitive digital PCR and to identify clinical and lifestyle factors associated with viral load. We analysed 45 HPV-16 positive oropharyngeal SCCs diagnosed between 2013 and 2015. All patients completed a lifestyle questionnaire and clinical data were extracted from medical charts. Viral load was determined using digital PCR assays for HPV-L1 and RNAseP. We found large variations in HPV-16 viral load from 1 to 930 copies per cell (median 34 copies per cell).

  4. Eight Nucleotide Substitutions Inhibit Splicing to HPV-16 3′-Splice Site SA3358 and Reduce the Efficiency by which HPV-16 Increases the Life Span of Primary Human Keratinocytes

    PubMed Central

    Li, Xiaoze; Johansson, Cecilia; Cardoso Palacios, Carlos; Mossberg, Anki; Dhanjal, Soniya; Bergvall, Monika; Schwartz, Stefan

    2013-01-01

    The most commonly used 3′-splice site on the human papillomavirus type 16 (HPV-16) genome named SA3358 is used to produce HPV-16 early mRNAs encoding E4, E5, E6 and E7, and late mRNAs encoding L1 and L2. We have previously shown that SA3358 is suboptimal and is totally dependent on a downstream splicing enhancer containingmultiple potential ASF/SF2 binding sites. Here weshow that only one of the predicted ASF/SF2 sites accounts for the majority of the enhancer activity. We demonstrate that single nucleotide substitutions in this predicted ASF/SF2 site impair enhancer function and that this correlates with less efficient binding to ASF/SF2 in vitro. We provide evidence that HPV-16 mRNAs that arespliced to SA3358 interact with ASF/SF2 in living cells. In addition,mutational inactivation of the ASF/SF2 site weakened the enhancer at SA3358 in episomal forms of the HPV-16 genome, indicating that the enhancer is active in the context of the full HPV-16 genome.This resulted in induction of HPV-16 late gene expression as a result of competition from late splice site SA5639. Furthermore, inactivation of the ASF/SF2 site of the SA3358 splicing enhancer reduced the ability of E6- and E7-encoding HPV-16 plasmids to increase the life span of primary keratinocytes in vitro, demonstrating arequirement for an intact splicing enhancer of SA3358 forefficient production of the E6 and E7 mRNAs. These results link the strength of the HPV-16 SA3358 splicing enhancer to expression of E6 and E7 and to the pathogenic properties of HPV-16. PMID:24039800

  5. HPV16 synthetic long peptide (HPV16-SLP) vaccination therapy of patients with advanced or recurrent HPV16-induced gynecological carcinoma, a phase II trial.

    PubMed

    van Poelgeest, Mariette I E; Welters, Marij J P; van Esch, Edith M G; Stynenbosch, Linda F M; Kerpershoek, Gijs; van Persijn van Meerten, Els L; van den Hende, Muriel; Löwik, Margriet J G; Berends-van der Meer, Dorien M A; Fathers, Lorraine M; Valentijn, A Rob P M; Oostendorp, Jaap; Fleuren, Gert Jan; Melief, Cornelis J M; Kenter, Gemma G; van der Burg, Sjoerd H

    2013-04-04

    Human papilloma virus type 16 (HPV16)-induced gynecological cancers, in particular cervical cancers, are found in many women worldwide. The HPV16 encoded oncoproteins E6 and E7 are tumor-specific targets for the adaptive immune system permitting the development of an HPV16-synthetic long peptide (SLP) vaccine with an excellent treatment profile in animal models. Here, we determined the toxicity, safety, immunogenicity and efficacy of the HPV16 SLP vaccine in patients with advanced or recurrent HPV16-induced gynecological carcinoma. Patients with HPV16-positive advanced or recurrent gynecological carcinoma (n = 20) were subcutaneously vaccinated with an HPV16-SLP vaccine consisting of a mix of 13 HPV16 E6 and HPV16 E7 overlapping long peptides in Montanide ISA-51 adjuvant. The primary endpoints were safety, toxicity and tumor regression as determined by RECIST. In addition, the vaccine-induced T-cell response was assessed by proliferation and associated cytokine production as well as IFNγ-ELISPOT. No systemic toxicity beyond CTCAE grade II was observed. In a few patients transient flu-like symptoms were observed. In 9 out of 16 tested patients vaccine-induced HPV16-specific proliferative responses were detected which were associated with the production of IFNγ, TNFα, IL-5 and/or IL-10. ELISPOT analysis revealed a vaccine-induced immune response in 11 of the 13 tested patients. The capacity to respond to the vaccine was positively correlated to the patient's immune status as reflected by their response to common recall antigens at the start of the trial. Median survival was 12.6 ± 9.1 months. No regression of tumors was observed among the 12 evaluable patients. Nineteen patients died of progressive disease. The HPV16-SLP vaccine was well tolerated and induced a broad IFNγ-associated T-cell response in patients with advanced or recurrent HPV16-induced gynecological carcinoma but neither induced tumor regression nor prevented progressive disease. We, therefore

  6. Cyclin A1 shows age-related expression in benign tonsils, HPV16-dependent overexpression in HNSCC and predicts lower recurrence rate in HNSCC independently of HPV16

    PubMed Central

    2012-01-01

    Background Promoter methylation of the tumor suppressor gene Cyclin A1 could be associated with Human Papillomavirus 16 (HPV16) induced Head and Neck Squamous Cell Carcinoma (HNSCC) and Cervical Carcinoma. There is disagreement about the impact of this epigenetic event on protein expression of Cyclin A1 in malignant and non-malignant tissue and there hardly exists any information about possible relationships between Cyclin A1 expression and clinicopathological characteristics in HNSCC. Methods We analyzed protein expression of Cyclin A1 in 81 HNSCC and 74 benign tonsils by immunohistochemistry and correlated it to Cyclin A1 methylation status, presence of HPV16 infection and other clinicopathological characteristics. Results Overexpression of Cyclin A1 was more present in HNSCC than in tonsils (p < 0.001). In both entities, HNSCC and benign tonsils, expression of Cyclin A1 significantly correlated with the expression of Cyclin-dependent kinase-inhibitor p16 (p = 0.000672 and 0.00495). In tonsils, expression of Cyclin A1 was inversely proportional to age (p = 0.00000396), and further correlated with expression of tumor suppressor gene p53 (p = 0.000228). In HNSCC Cyclin A1 expression was associated with the presence of HPV16 DNA (p = 0.0014) and a lower recurrence rate in univariate and multivariate analysis (p = 0.002 and 0.013). Neither in HNSCC nor in tonsils Cyclin A1 expression correlated with promoter methylation. Conclusions Cyclin A1 is an important cell cycle regulator with age-related increased expression in tonsils of children. HPV16 induces overexpression of Cyclin A1 in HNSCC despite promoter methylation. Overexpression of Cyclin A1 predicts a lower recurrence rate in HNSCC independently of HPV16. PMID:22712549

  7. HPV16 genetic variation and the development of cervical cancer worldwide

    PubMed Central

    Cornet, I; Gheit, T; Iannacone, M R; Vignat, J; Sylla, B S; Del Mistro, A; Franceschi, S; Tommasino, M; Clifford, G M

    2013-01-01

    Background: Factors that favour a small proportion of HPV16 infections to progress to cancer are still poorly understood, but several studies have implicated a role of HPV16 genetic variation. Methods: To evaluate the association between HPV16 genetic variants and cervical cancer risk, we designed a multicentre case–control study based on HPV16-positive cervical samples (1121 cervical cancer cases and 400 controls) from the International Agency for Research on Cancer biobank. By sequencing the E6 gene, HPV16 isolates were classified into variant lineages and the European (EUR)-lineage isolates were subclassified by the common polymorphism T350G. Results: Incidence of variant lineages differed between cases and controls in Europe/Central Asia (P=0.006, driven by an underrepresentation of African lineages in cases), and South/Central America (P=0.056, driven by an overrepresentation of Asian American/North American lineages in cases). EUR-350G isolates were significantly underrepresented in cervical cancer in East Asia (odds ratio (OR)=0.02 vs EUR-350T; 95% confidence interval (CI)=0.00–0.37) and Europe/Central Asia (OR=0.42; 95% CI=0.27–0.64), whereas the opposite was true in South/Central America (OR=4.69; 95% CI=2.07–10.66). Conclusion: We observed that the distribution of HPV16 variants worldwide, and their relative risks for cervical cancer appear to be population-dependent. PMID:23169278

  8. Radioimmunotherapy with an antibody to HPV16 E6 oncoprotein is effective in experimental cervical tumor expressing low levels of E6

    PubMed Central

    Jiang, Zewei; Wang, Xing Guo; Einstein, Mark H; Goldberg, Gary L; Casadevall, Arturo

    2010-01-01

    Purpose HPV16 is associated with ∼50% of all cervical cancers worldwide. The E6 and E7 genes of oncogenic HPV types, such as HPV16, are necessary for the HPV transforming function and tumorogenesis making them ideal targets for novel treatments. Radioimmunotherapy employs systemically administered radiolabeled monoclonal antibodies (mAbs) that bind to tumor-associated antigens. Previously we demonstrated in mice that radioimmunotherapy targeting viral antigens with mAb to HPV16 E6 suppressed CasKi cervical tumors expressing high levels of E6 (∼600 copies of HPV per cell). However, that study opened the question whether radioimmunotherapy can suppress the growth of cervical tumors with low E6 and E7 expression, such as may be seen in patients. Experimental Design We evaluated the expression of E6 in patients' tumors and in the SiHa cell line expressing low levels of E6 and E7 (1–2 copies of HPV per cell) and found them comparable. We initiated SiHa tumors in nude mice, radiolabeled C1P5 mAb to E6 with a beta-emitter 188-Rhenium (188Re) and treated tumor-bearing mice with: (1) 200 µCi 188Re-C1P5 alone; (2) proteasome inhibitor MG132 alone; (3) MG132 followed by 200 µCi 188Re-C1P5; (4) unlabeled C1P5; (5) 200 µCi 188Re-18B7 (isotype-matching control mAb); (6) no treatment. 188Re-C1P5 alone and in combination with MG-132 significantly retarded tumor growth compared to all control groups. Conclusions Our data demonstrate the possibility to suppress tumor growth by targeting viral antigens even in cervical tumors with low E6 expression and provide additional evidence for the potential usefulness of radioimmunotherapy targeting HPV-related antigens in the clinic. PMID:20861673

  9. HPV16 seropositivity and subsequent HPV16 infection risk in a naturally infected population: comparison of serological assays.

    PubMed

    Lin, Shih-Wen; Ghosh, Arpita; Porras, Carolina; Markt, Sarah C; Rodriguez, Ana Cecilia; Schiffman, Mark; Wacholder, Sholom; Kemp, Troy J; Pinto, Ligia A; Gonzalez, Paula; Wentzensen, Nicolas; Esser, Mark T; Matys, Katie; Meuree, Ariane; Quint, Wim; van Doorn, Leen-Jan; Herrero, Rolando; Hildesheim, Allan; Safaeian, Mahboobeh

    2013-01-01

    Several serological assays have been developed to detect antibodies elicited against infections with oncogenic human papillomavirus (HPV) type 16. The association between antibody levels measured by various assays and subsequent HPV infection risk may differ. We compared HPV16-specific antibody levels previously measured by a virus-like particle (VLP)-based direct enzyme-linked immunoassay (ELISA) with levels measured by additional assays and evaluated the protection against HPV16 infection conferred at different levels of the assays. Replicate enrollment serum aliquots from 388 unvaccinated women in the control arm of the Costa Rica HPV vaccine trial were measured for HPV16 seropositivity using three serological assays: a VLP-based direct ELISA; a VLP-based competitive Luminex immunoassay (cLIA); and a secreted alkaline phosphatase protein neutralization assay (SEAP-NA). We assessed the association of assay seropositivity and risk of subsequent HPV16 infection over four years of follow-up by calculating sampling-adjusted odds ratios (OR) and HPV16 seropositivity based on standard cutoff from the cLIA was significantly associated with protection from subsequent HPV16 infection (OR = 0.48, CI = 0.27-0.86, compared with seronegatives). Compared with seronegatives, the highest seropositive tertile antibody levels from the direct ELISA (OR = 0.53, CI = 0.28-0.90) as well as the SEAP-NA (OR = 0.20, CI = 0.06, 0.64) were also significantly associated with protection from HPV16 infection. Enrollment HPV16 seropositivity by any of the three serological assays evaluated was associated with protection from subsequent infection, although cutoffs for immune protection were different. We defined the assays and seropositivity levels after natural infection that better measure and translate to protective immunity.

  10. Combined effects of smoking and HPV16 in oropharyngeal cancer

    PubMed Central

    Anantharaman, Devasena; Muller, David C; Lagiou, Pagona; Ahrens, Wolfgang; Holcátová, Ivana; Merletti, Franco; Kjærheim, Kristina; Polesel, Jerry; Simonato, Lorenzo; Canova, Cristina; Castellsague, Xavier; Macfarlane, Tatiana V; Znaor, Ariana; Thomson, Peter; Robinson, Max; Conway, David I; Healy, Claire M; Tjønneland, Anne; Westin, Ulla; Ekström, Johanna; Chang-Claude, Jenny; Kaaks, Rudolf; Overvad, Kim; Drogan, Dagmar; Hallmans, Göran; Laurell, Göran; Bueno-de-Mesquita, HB; Peeters, Petra H; Agudo, Antonio; Larrañaga, Nerea; Travis, Ruth C; Palli, Domenico; Barricarte, Aurelio; Trichopoulou, Antonia; George, Saitakis; Trichopoulos, Dimitrios; Quirós, J Ramón; Grioni, Sara; Sacerdote, Carlotta; Navarro, Carmen; Sánchez, María-José; Tumino, Rosario; Severi, Gianluca; Boutron-Ruault, Marie-Christine; Clavel-Chapelon, Francoise; Panico, Salvatore; Weiderpass, Elisabete; Lund, Eiliv; Gram, Inger T; Riboli, Elio; Pawlita, Michael; Waterboer, Tim; Kreimer, Aimée R; Johansson, Mattias; Brennan, Paul

    2016-01-01

    Abstract Background: Although smoking and HPV infection are recognized as important risk factors for oropharyngeal cancer, how their joint exposure impacts on oropharyngeal cancer risk is unclear. Specifically, whether smoking confers any additional risk to HPV-positive oropharyngeal cancer is not understood. Methods: Using HPV serology as a marker of HPV-related cancer, we examined the interaction between smoking and HPV16 in 459 oropharyngeal (and 1445 oral cavity and laryngeal) cancer patients and 3024 control participants from two large European multi-centre studies. Odds ratios and credible intervals [CrI], adjusted for potential confounders, were estimated using Bayesian logistic regression. Results: Both smoking [odds ratio (OR [CrI]: 6.82 [4.52, 10.29]) and HPV seropositivity (OR [CrI]: 235.69 [99.95, 555.74]) were independently associated with oropharyngeal cancer. The joint association of smoking and HPV seropositivity was consistent with that expected on the additive scale (synergy index [CrI]: 1.32 [0.51, 3.45]), suggesting they act as independent risk factors for oropharyngeal cancer. Conclusions: Smoking was consistently associated with increase in oropharyngeal cancer risk in models stratified by HPV16 seropositivity. In addition, we report that the prevalence of oropharyngeal cancer increases with smoking for both HPV16-positive and HPV16-negative persons. The impact of smoking on HPV16-positive oropharyngeal cancer highlights the continued need for smoking cessation programmes for primary prevention of head and neck cancer. PMID:27197530

  11. Some novel insights on HPV16 related cervical cancer pathogenesis based on analyses of LCR methylation, viral load, E7 and E2/E4 expressions.

    PubMed

    Das Ghosh, Damayanti; Bhattacharjee, Bornali; Sen, Shrinka; Premi, Laikangbam; Mukhopadhyay, Indranil; Chowdhury, Rahul Roy; Roy, Sudipta; Sengupta, Sharmila

    2012-01-01

    This study was undertaken to decipher the interdependent roles of (i) methylation within E2 binding site I and II (E2BS-I/II) and replication origin (nt 7862) in the long control region (LCR), (ii) expression of viral oncogene E7, (iii) expression of the transcript (E7-E1/E4) that encodes E2 repressor protein and (iv) viral load, in human papillomavirus 16 (HPV16) related cervical cancer (CaCx) pathogenesis. The results revealed over-representation (p<0.001) of methylation at nucleotide 58 of E2BS-I among E2-intact CaCx cases compared to E2-disrupted cases. Bisulphite sequencing of LCR revealed overrepresentation of methylation at nucleotide 58 or other CpGs in E2BS-I/II, among E2-intact cases than E2-disrupted cases and lack of methylation at replication origin in case of both. The viral transcript (E7-E1/E4) that produces the repressor E2 was analyzed by APOT (amplification of papillomavirus oncogenic transcript)-coupled-quantitative-RT-PCR (of E7 and E4 genes) to distinguish episomal (pure or concomitant with integrated) from purely integrated viral genomes based on the ratio, E7 C(T)/E4 C(T). Relative quantification based on comparative C(T) (threshold cycle) method revealed 75.087 folds higher E7 mRNA expression in episomal cases over purely integrated cases. Viral load and E2 gene copy numbers were negatively correlated with E7 C(T) (p = 0.007) and E2 C(T) (p<0.0001), respectively, each normalized with ACTB C(T), among episomal cases only. The k-means clustering analysis considering E7 C(T) from APOT-coupled-quantitative-RT-PCR assay, in conjunction with viral load, revealed immense heterogeneity among the HPV16 positive CaCx cases portraying integrated viral genomes. The findings provide novel insights into HPV16 related CaCx pathogenesis and highlight that CaCx cases that harbour episomal HPV16 genomes with intact E2 are likely to be distinct biologically, from the purely integrated viral genomes in terms of host genes and/or pathways involved in cervical

  12. Immunogenicity of an HPV-16 L2 DNA vaccine

    PubMed Central

    Hitzeroth, Inga I.; Passmore, Jo-Ann S.; Shephard, Enid; Stewart, Debbie; Müller, Martin; Williamson, Anna-Lise; Rybicki, Edward P.; Kast, W. Martin

    2009-01-01

    The ability to elicit cross-neutralizing antibodies makes human papillomavirus (HPV) L2 capsid protein a possible HPV vaccine. We examined and compared the humoral response of mice immunised with a HPV-16 L2 DNA vaccine or with HPV-16 L2 protein. The L2 DNA vaccine elicited a non-neutralising antibody response unlike the L2 protein. L2 DNA vaccination suppressed the growth of L2-expressing C3 tumor cells, which is a T cell mediated effect, demonstrating that the lack of non-neutralizing antibody induction by L2 DNA was not caused by lack of T cell immunogenicity of the construct. PMID:19559114

  13. Isolation and characterization of an RNA aptamer for the HPV-16 E7 oncoprotein.

    PubMed

    Toscano-Garibay, Julia D; Benítez-Hess, María L; Alvarez-Salas, Luis M

    2011-02-01

    Cervical cancer is a common neoplastic disease affecting women worldwide. Expression of human papillomavirus type 16 (HPV-16) E6/E7 genes is frequently associated with cervical cancer, representing ideal targets for diagnostic and therapeutic strategies. Aptamers are oligonucleotide ligands capable of binding with high affinity and specificity to relevant markers in therapeutics and disease detection. The aim of the study was to isolate an RNA aptamer specific for the HPV-16 E7 protein. Aptamers were selected from a randomized oligonucleotide library using a modified SELEX method and recombinant HPV-16 E7 protein. Isolated aptamers were cloned and sequenced for in silico analysis. Interaction and electromobility shift assays (EMSA) were performed to establish aptamer specificity and affinity for E7. RNase footprinting and serial deletions of the aptamer and the E7 protein were made to characterize the aptamer-protein complex. Sandwich slot-blot assays were used for K(D) determination. After several rounds of SELEX, an aptamer (G5α3N.4) exhibited specificity for E7 using cell-free and protein extracts. G5α3N.4 binding yielded a K(D) comparable to aptamers directed to other small targets. Enzymatic and genetic analysis of G5α3N.4 binding showed a secondary structure with two stem-loop domains joined by single-stranded region contacting E7 in a clamp-like manner. The G5α3N.4 aptamer also produced specific complexes in HPV-positive cervical carcinoma cells. The affinity and specificity of G5α3N.4 binding domains for the HPV-16 E7 protein may be used for the detection of papillomavirus infection and cervical cancer. Copyright © 2011 IMSS. Published by Elsevier Inc. All rights reserved.

  14. Patients with integrated HPV16 in head and neck cancer show poor survival.

    PubMed

    Nulton, Tara J; Kim, Nak-Kyeong; DiNardo, Laurence J; Morgan, Iain M; Windle, Brad

    2018-05-01

    We previously reported identifying three categories of HPV16-positive head and neck tumors based on The Cancer Genome Atlas (TCGA) RNA and DNA sequence data. Category 1 had truly integrated HPV16 genomes, category 2 had simple episomal genomes, and category 3 had novel episomes that were a hybrid between viral and human DNA. Using our categorization, we investigated in this study survival of patients with integrated HPV16 tumors versus patients with episomal HPV16 tumors. The TCGA RNA-Seq sequence reads were used to quantify HPV E2 and E7 gene expression, which was used as a marker for HPV integration. The results demonstrate that integration is associated with poor survival; those patients with integrated HPV tumors fared no better than non-HPV tumors in their five-year survival. Integrated HPV in tumors was found strikingly to be prevalent in patients born earlier while episomal HPV was prevalent in patients born later. We also observed a fairly constant incidence of all HPV forms among head and neck cancer patients over the last eight years of this study (2006-2013). We propose our characterization of HPV integrated and episomal state is more accurate than previous studies that may have mischaracterized the hybrid HPV-human DNA episomes as integrated. The state of integrated HPV is associated with a poor clinical outcome. Results suggest that the incidence of integrated HPV among all HPV forms peaked and is decreasing. We discuss the importance of our findings for the management of HPV positive head and neck cancer. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration.

    PubMed

    Bojilova, Ekaterina Dimitrova; Weyn, Christine; Antoine, Marie-Hélène; Fontaine, Véronique

    2016-11-15

    Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk.

  16. Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration

    PubMed Central

    Bojilova, Ekaterina Dimitrova; Weyn, Christine; Antoine, Marie-Hélène; Fontaine, Véronique

    2016-01-01

    Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk. PMID:27705914

  17. Expression and characterization of HPV-16 L1 capsid protein in Pichia pastoris

    PubMed Central

    Bazan, Silvia Boschi; de Alencar Muniz Chaves, Agtha; Aires, Karina Araújo; Cianciarullo, Aurora Marques; Garcea, Robert L.; Ho, Paulo Lee

    2013-01-01

    Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin–sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines. PMID:19756360

  18. SOX2 as a New Regulator of HPV16 Transcription.

    PubMed

    Martínez-Ramírez, Imelda; Del-Castillo-Falconi, Víctor; Mitre-Aguilar, Irma B; Amador-Molina, Alfredo; Carrillo-García, Adela; Langley, Elizabeth; Zentella-Dehesa, Alejandro; Soto-Reyes, Ernesto; García-Carrancá, Alejandro; Herrera, Luis A; Lizano, Marcela

    2017-07-05

    Persistent infections with high-risk human papillomavirus (HPV) constitute the main risk factor for cervical cancer development. HPV16 is the most frequent type associated to squamous cell carcinomas (SCC), followed by HPV18. The long control region (LCR) in the HPV genome contains the replication origin and sequences recognized by cellular transcription factors (TFs) controlling viral transcription. Altered expression of E6 and E7 viral oncogenes, modulated by the LCR, causes modifications in cellular pathways such as proliferation, leading to malignant transformation. The aim of this study was to identify specific TFs that could contribute to the modulation of high-risk HPV transcriptional activity, related to the cellular histological origin. We identified sex determining region Y (SRY)-box 2 (SOX2) response elements present in HPV16-LCR. SOX2 binding to the LCR was demonstrated by in vivo and in vitro assays. The overexpression of this TF repressed HPV16-LCR transcriptional activity, as shown through reporter plasmid assays and by the down-regulation of endogenous HPV oncogenes. Site-directed mutagenesis revealed that three putative SOX2 binding sites are involved in the repression of the LCR activity. We propose that SOX2 acts as a transcriptional repressor of HPV16-LCR, decreasing the expression of E6 and E7 oncogenes in a SCC context.

  19. Sensitivity and specificity of antibodies against HPV16 E6 and other early proteins for the detection of HPV16-driven oropharyngeal squamous cell carcinoma.

    PubMed

    Holzinger, Dana; Wichmann, Gunnar; Baboci, Lorena; Michel, Angelika; Höfler, Daniela; Wiesenfarth, Manuel; Schroeder, Lea; Boscolo-Rizzo, Paolo; Herold-Mende, Christel; Dyckhoff, Gerhard; Boehm, Andreas; Del Mistro, Annarosa; Bosch, Franz X; Dietz, Andreas; Pawlita, Michael; Waterboer, Tim

    2017-06-15

    To determine the sensitivity and specificity of HPV16 serology as diagnostic marker for HPV16-driven oropharyngeal squamous cell carcinoma (OPSCC), 214 HNSCC patients from Germany and Italy with fresh-frozen tumor tissues and sera collected before treatment were included in this study. Hundred and twenty cancer cases were from the oropharynx and 94 were from head and neck cancer regions outside the oropharynx (45 oral cavity, 12 hypopharynx and 35 larynx). Serum antibodies to early (E1, E2, E6 and E7) and late (L1) HPV16 proteins were analyzed by multiplex serology and were compared to tumor HPV RNA status as the gold standard. A tumor was defined as HPV-driven in the presence of HPV16 DNA and HPV16 transformation-specific RNA transcript patterns (E6*I, E1 ∧ E4 and E1C). Of 120 OPSCC, 66 (55%) were HPV16-driven. HPV16 E6 seropositivity was the best predictor of HPV16-driven OPSCC (diagnostic accuracy 97% [95%CI 92-99%], Cohen's kappa 0.93 [95%CI 0.8-1.0]). Of the 66 HPV-driven OPSCC, 63 were HPV16 E6 seropositive, compared to only one (1.8%) among the 54 non-HPV-driven OPSCC, resulting in a sensitivity of 96% (95%CI 88-98) and a specificity of 98% (95%CI 90-100). Of 94 HNSCC outside the oropharynx, six (6%) were HPV16-driven. In these patients, HPV16 E6 seropositivity had lower sensitivity (50%, 95%CI 19-81), but was highly specific (100%, 95%CI 96-100). In conclusion, HPV16 E6 seropositivity appears to be a highly reliable diagnostic marker for HPV16-driven OPSCC with very high sensitivity and specificity, but might be less sensitive for HPV16-driven HNSCC outside the oropharynx. © 2017 UICC.

  20. C3-Luc Cells Are an Excellent Model for Evaluation of Cellular Immunity following HPV16L1 Vaccination.

    PubMed

    Li, Li-Li; Wang, He-Rong; Zhou, Zhi-Yi; Luo, Jing; Wang, Xiao-Li; Xiao, Xiang-Qian; Zhou, Yu-Bai; Zeng, Yi

    2016-01-01

    C3 and TC-1 are the two model cell lines most commonly used in studies of vaccines and drugs against human papillomavirus (HPV) infection. Because C3 cells contain both the HPV16 E and L genes, but TC-1 cells contain only the HPV16 E genes, C3 cells are usually used as the model cell line in studies targeting the HPV16 L protein. However, expression of the L1 protein is difficult to detect in C3 cells using common methods. In our study, Short tandem repeat analysis (STR) was used to demonstrate that C3 cells are indeed derived from mice, PCR results show that HPV16 L1, E6 and E7 genes were detected in C3 genomic DNA, and RT-PCR results demonstrated that L1 transcription had occurred in C3 cells. However, the expression of C3 protein was not found in the results of western blot and immunohistochemistry (IHC). Growth and proliferation of C3 were inhibited by mice spleen lymphocytes that had been immunized with a vaccine against HPV16L1. The luciferase gene was integrated into C3 cells, and it was confirmed that addition of the exogenous gene had no effect on C3 cells by comparing cell growth and tumor formation with untransformed cells. Cells stably expressing luciferase (C3-luc) were screened and subcutaneously injected into the mice. Tumors became established and were observed using a Spectrum Pre-clinical in Vivo Imaging System. Tumor size of mice in the different groups at various time points was calculated by counting photons. The sensitivity of the animals to the vaccine was quantified by statistical comparison. Ten or 30 days following injection of the C3-luc cells, tumor size differed significantly between the PBS and vaccine groups, indicating that C3 cells were susceptible to vaccination even after tumors were formed in vivo.

  1. HPV16 E6 Promotes Breast Cancer Proliferation via Upregulation of COX-2 Expression

    PubMed Central

    Li, Y. Z.; Zhang, Z. Y.; Wang, J. Q.

    2017-01-01

    Background. Breast cancer remains the leading cause of cancer-related mortality worldwide. It has been indicated that human papillomaviruses 16 (HPV16) might participate in the pathogenesis and development of breast cancer. However, the detected rate of HPV16 varies with region. We will investigate HPV16 E6 expression in North China and explore the effects and mechanism of HPV16 E6 on breast cancer proliferation in this study. Methods. The expressions of HPV16 E6 and COX-2 in paraffin-embedded tissues of the invasive ductal breast cancer were detected by qPCR and IHC. The effects of HPV16 E6 on breast cancer proliferation were determined by function studies. The mechanism of HPV16 E6 in promoting breast cancer proliferation was explored by Western blot and Dual-Luciferase Reporter Assay. Results. HPV16 E6 was positive in 28% invasive ductal breast carcinoma in North China; HPV16 E6 promoted breast cancer proliferation. Inhibition of COX-2 by siCOX-2 or Celecoxib attenuated the proliferation of breast cancer cells with HPV16 E6 expression; and the upregulation of COX-2 could be suppressed by the inhibition of NF-κB activity. Conclusion. HPV16 E6 promotes breast cancer proliferation by activation of NF-κB signaling pathway and increase of COX-2 expression. COX-2 will be a potential target for HPV16 E6-associated breast cancer. PMID:29250535

  2. Sensitive molecular detection of small nodal metastasis in uterine cervical cancer using HPV16-E6/CK19/MUC1 cancer biomarkers

    PubMed Central

    Samouëlian, Vanessa; Mechtouf, Nawel; Leblanc, Eric; Cardin, Guillaume B.; Lhotellier, Valérie; Querleu, Denis; Révillion, Françoise; Rodier, Francis

    2018-01-01

    Metastatic nodal involvement is a critical prognostic factor in uterine cervical cancer (UCC). To improve current methods of detecting UCC metastases in lymph nodes (LNs), we used quantitative PCR (qPCR) to assess mRNA expression of potential metastatic biomarkers. We found that expression of HPV16-E6, cytokeratin19 (CK19), and mucin1 (MUC1) is consistently upregulated in tumors and metastatic tissues, supporting a role for these genes in UCC progression. These putative biomarkers were able to predict the presence of histologically positive metastatic LNs with respective sensitivities and specificities of 82% and 99% (CK19), 76% and 95% (HPV16-E6), and 76% and 78% (MUC1). While the biomarkers failed to detect 1.7% to 2.2% of the histologically positive LNs when used individually, combining CK19 and HPV16-E6 enhanced sensitivity and specificity to 100% and 94%, respectively. To explore the sensitivity of qPCR-based detection of varying proportions of invading HPV16-positive UCC cells, we designed a LN metastasis model that achieved a fresh cell detection limit of 0.008% (1:12500 HPV16-positive to HPV16-negative cells), and a paraffin-embedded, formalin-fixed (PEFF) detection limit of 0.02% (1:5000 HPV16-positive to HPV16-negative cells), both of which are within the theoretical detection limit for micrometastasis. Thus, HPV E6/E7 oncogenes may be useful targets for the ultrasensitive detection of nodal involvements like micrometastases in fresh or archived tissue samples. Moreover, our results suggest that the biomarker combination of CK19/HPV-E6 could support a real-time intraoperative strategy for the detection of small, but potentially lethal, metastatic nodal involvements in fresh UCC tissues. PMID:29774091

  3. Sensitive molecular detection of small nodal metastasis in uterine cervical cancer using HPV16-E6/CK19/MUC1 cancer biomarkers.

    PubMed

    Samouëlian, Vanessa; Mechtouf, Nawel; Leblanc, Eric; Cardin, Guillaume B; Lhotellier, Valérie; Querleu, Denis; Révillion, Françoise; Rodier, Francis

    2018-04-24

    Metastatic nodal involvement is a critical prognostic factor in uterine cervical cancer (UCC). To improve current methods of detecting UCC metastases in lymph nodes (LNs), we used quantitative PCR (qPCR) to assess mRNA expression of potential metastatic biomarkers. We found that expression of HPV16-E6, cytokeratin19 (CK19), and mucin1 (MUC1) is consistently upregulated in tumors and metastatic tissues, supporting a role for these genes in UCC progression. These putative biomarkers were able to predict the presence of histologically positive metastatic LNs with respective sensitivities and specificities of 82% and 99% (CK19), 76% and 95% (HPV16-E6), and 76% and 78% (MUC1). While the biomarkers failed to detect 1.7% to 2.2% of the histologically positive LNs when used individually, combining CK19 and HPV16-E6 enhanced sensitivity and specificity to 100% and 94%, respectively. To explore the sensitivity of qPCR-based detection of varying proportions of invading HPV16-positive UCC cells, we designed a LN metastasis model that achieved a fresh cell detection limit of 0.008% (1:12500 HPV16-positive to HPV16-negative cells), and a paraffin-embedded, formalin-fixed (PEFF) detection limit of 0.02% (1:5000 HPV16-positive to HPV16-negative cells), both of which are within the theoretical detection limit for micrometastasis. Thus, HPV E6/E7 oncogenes may be useful targets for the ultrasensitive detection of nodal involvements like micrometastases in fresh or archived tissue samples. Moreover, our results suggest that the biomarker combination of CK19/HPV-E6 could support a real-time intraoperative strategy for the detection of small, but potentially lethal, metastatic nodal involvements in fresh UCC tissues.

  4. Impact of HPV 16/18 infection on clinical outcomes in locally advanced cervical cancers treated with radical radio (chemo) therapy - A prospective observational study.

    PubMed

    Mahantshetty, Umesh; Teni, Tanuja; Naga, Pushpa; Hotwani, Chandni; Umesh, Sumuki; Kannan, Sadhana; Hande, Vinod; Pawar, Sagar; Engineer, Reena; Chopra, Supriya; Deodhar, Kedar; Maheshwari, Amita; Gurram, Lavanya; Gupta, Sudeep; Shrivastava, Shyam Kishore

    2018-02-01

    With an aim to investigate the impact of Human Papilloma Virus (HPV) 16/18 infection on clinical outcomes in locally advanced cervical cancers treated with radical radio (chemo) therapy, we undertook this prospective study. Between May 2010 and April 2012, 150 histologically proven cervical cancer patients treated with radio (chemo) therapy were accrued. Cervical biopsies/brushings were collected at pre-treatment, end of treatment and at 3 monthly intervals up to 24months. Quantitative estimation of HPV 16/18 was done using real-time polymerase chain reaction (RT-PCR) and correlated with various clinical end-points. Out of 150 patients accrued, 135 patients were considered for final analysis. Pre-treatment HPV16/18 DNA was detected in 126 (93%) patients, with HPV-16 present in 91%. The mean log (±SD) HPV-16 and HPV-18 viral load at pre-treatment was 4.76 (±2.5) and 0.14 (±2.1) copies/10ng of DNA, respectively. Though significant decline in viral load was observed on follow-ups (p<0.0001); by 9-month follow-up, 89 (66%) patients had persistence of HPV infection. Patients with persistent HPV 16/18 infection had a significantly higher overall and loco-regional relapses [44/89 (49%) and 29/89 (32%)] as compared to HPV clearance by 9months [12/43 (28%) and 5/43 (11%)] with p=0.024 and p=0.02, respectively. Also, persistent HPV infection by 24-month showed a significant impact on loco-regional control (LRC) and recurrence-free survival (RFS). In locally advanced cervical cancers treated with radical radio (chemo) therapy, persistent HPV 16/18 infection is significantly high in immediate post-treatment period and correlated with higher loco-regional, overall relapses and was also associated with early relapses. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Chromosomal abnormalities in HPV-16-immortalized oral epithelial cells.

    PubMed

    Oda, D; Bigler, L; Mao, E J; Disteche, C M

    1996-09-01

    Human papilloma virus (HPV) type 16 has an established association with anogenital carcinoma, and to some extent with human oral squamous cell carcinoma. We hypothesize that HPV type 16 is capable of inducing chromosomal and cell cycle changes in cultured oral epithelial cells. Normal human oral epithelia] cells were immortalized with recombinant retrovirus containing the E6/E7 open reading frames of HPV type 16. These cells have been in culture for more than 350 passages and over 4 years. Flow cytometry demonstrated an average of 42% nuclear aneuploidy in HPV 16-immortalized cells; 16% in normal controls (probably tetrasomy). Cytogenetic analysis demonstrated significant progression of chromosomal abnormalities. Cells at early passage (p10) showed trisomy 20, with no other major changes. At passage 18, trisomy 1q and monosomy 13 were seen in addition to trisomy 20. At passage 61 there were two distinct cell populations ('a' and 'b'), with multiple chromosomal changes including trisomy 5q,14,20 in one line and 7p,9q,llq in the other. Both populations had monosomy 3p, with monosomy 8p in one population and monosomy 13 in the other. At passage 136, the cells were essentially identical to population 'b' of passage 61. At this passage, mutation of the p53 gene was detected at codon 273 of exon 8, with G to T conversion (Arg to Leu). This was absent in the normal cells from which this line was developed. Passage 262 contained the two major cell populations, each with a sub-group with additional chromosomal changes such as 10p monosomy. Cells from passages 217 and 305 were injected into nude mice a year apart. Both failed to produce tumors, as did normal cells. In conclusion, we present an HPV type 16-immortalized oral epithelial cell line (IHGK) with extensive and progressive chromosomal abnormalities, invasive growth in culture and yet no tumor formation in nude mice. We suggest that the question as to whether HPV alone can induce transformation is still open.

  6. [Human Papilloma virus in Quechua women from Jujuy with high frequency of cervical cancer: viral types and HPV-16 variants].

    PubMed

    Picconi, Maria Alejandra; Gronda, Jorge; Alonio, Lidia V; Villa, Luisa L; Sichero, Laura; Miranda, Sergio; Barcena, Martin; Teyssie, Angelica

    2002-01-01

    Human Papillomaviruses (HPVs) are etiologically associated to cervical carcinoma. In order to evaluate HPV infection and its relationship with the high frequency of this neoplasia in Quechua women from Jujuy (Argentina), 271 cervical samples from preneoplastic and neoplastic lesions (biopsies) and normal controls (cytologies) were studied. Detection and typing were performed using PCR-RFLP or PCR-hybridization and the HPV-16 variability in L1 and E6 genes (by PCR-hybridization) was analysed. HPV was detected in 52% of controls, 91% of low-grade lesions, 97% of high-grade lesions and 100% of invasive carcinomas, corresponding 55% to HPV-16. HPV-16 European variants were predominant, most of them being non-prototypic strains. The high frequency of high risk infection types and the raised proportion of HPV-16 non-prototypic variants related to a greater oncogenic potential could explain, in part, the high cervical cancer frequency of this native population. These data may contribute to disease control and vaccinal formulation.

  7. Unexpected inverse correlation between Native American ancestry and Asian American variants of HPV16 in admixed Colombian cervical cancer cases.

    PubMed

    Lopera, Esteban A; Baena, Armando; Florez, Victor; Montiel, Jehidys; Duque, Constanza; Ramirez, Tatiana; Borrero, Mauricio; Cordoba, Carlos M; Rojas, Fredy; Pareja, Rene; Bedoya, Astrid M; Bedoya, Gabriel; Sanchez, Gloria I

    2014-12-01

    European (E) variants of HPV 16 are evenly distributed among world regions, meanwhile Non-European variants such as European-Asian (EAs), Asian American (AA) and African (Af) are mostly confined to Eastern Asia, The Americas and African regions respectively. Several studies have shown that genetic variation of HPV 16 is associated with the risk of cervical cancer, which also seems to be dependent on the population. This relationship between ethnicity and variants have led to the suggestion that there is co-evolution of variants with humankind. Our aim was to evaluate the relationship between the individual ancestry proportion and infection with HPV 16 variants in cervical cancer. We examined the association between ancestry and HPV 16 variants in samples of 82 cervical cancer cases from different regions of Colombia. Individual ancestry proportions (European, African and Native American) were estimated by genotyping 106 ancestry informative markers. Variants were identified by PCR amplification of the E6 gene, followed by reverse line blot hybridization (RLB) with variants specific probes. Overall European (E) and Asian American (AA) variants frequency was 66.5% and 33.5% respectively. Similar distribution was observed in cases with higher proportions of European or African ancestry. A higher Native American ancestry was significantly associated with higher frequency of E variants (median ancestry>23.6%, Age and place of birth adjusted OR: 3.55, 95% CI: 1.26-10.03, p=0.01). Even further, an inverse geographic correlation between Native American ancestry and frequency of infections with AA variants was observed (ρ=-0.825, p=0.008). Regions with higher proportion of Native American ancestry had a lower frequency of AA variants of HPV 16. This study suggests replacement of AA variants by E variants of human papillomavirus 16 in cervical cancer cases with high Native American ancestry. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. A role for HPV16 E5 in cervical carcinogenesis.

    PubMed

    Maufort, John P; Shai, Anny; Pitot, Henry C; Lambert, Paul F

    2010-04-01

    A subset of the mucosotropic human papillomaviruses (HPV), including HPV16, are etiologic agents for the vast majority of cervical cancers, other anogenital cancers, and a subset of head and neck squamous cell carcinomas. HPV16 encodes three oncogenes: E5, E6, and E7. Although E6 and E7 have been well-studied and clearly shown to be important contributors to these cancers, less is known about E5. In this study, we used E5 transgenic mice to investigate the role of E5 in cervical cancer. When treated for 6 months with estrogen, a cofactor for cervical carcinogenesis, E5 transgenic mice developed more severe neoplastic cervical disease than similarly treated nontransgenic mice, although no frank cancers were detected. In addition, E5 when combined with either E6 or E7 induced more severe neoplastic disease than seen in mice expressing only one viral oncogene. Prolonged treatment of E5 transgenic mice with exogenous estrogen uncovered an ability of E5 to cause frank cancer. These data indicate that E5 acts as an oncogene in the reproductive tracts of female mice.

  9. Enhanced immunogenicity of HPV 16 E7 fusion proteins in DNA vaccination.

    PubMed

    Michel, Nico; Osen, Wolfram; Gissmann, Lutz; Schumacher, Ton N M; Zentgraf, Hanswalter; Müller, Martin

    2002-03-01

    DNA vaccination is a promising approach for inducing both humoral and cellular immune responses. For immunotherapy of HPV-16-associated diseases the E7 protein is considered a prime candidate, as it is expressed in all HPV-16-positive tumors. Unfortunately, the E7 protein is a very poor inducer of a cytotoxic T-cell response, when being used as antigen in DNA vaccination. Here we demonstrate that after fusion to protein export/import signals such as the herpes simplex virus ferry protein VP22, E7 can translocate in vitro from VP22-E7-expressing cells to neighboring cells that do not carry the VP22-E7 gene. In vivo, the VP22-E7 fusion shows significantly increased efficiency in inducing a cytotoxic T-cell response. Our data suggest that the export function of VP22 plays a major role in this phenomenon, since VP22 can be replaced by classical protein export signals, without impairing the induction of the E7-specific cellular immune response. However, all E7 fusion constructs showed significantly elevated protein steady-state levels, which might also account for the observed boost in immunogenicity. (C)2002 Elsevier Science (USA).

  10. A novel multiplex real-time PCR assay for the detection and quantification of HPV16/18 and HSV1/2 in cervical cancer screening.

    PubMed

    Zhao, Youyun; Cao, Xuan; Tang, Jingfeng; Zhou, Li; Gao, Yinglin; Wang, Jiangping; Zheng, Yi; Yin, Shanshan; Wang, Yefu

    2012-04-01

    Infection with human papillomavirus (HPV), particularly HPV16 and HPV18, is the main cause of invasive cervical cancer, although other factors such as herpes simplex virus (HSV) may act in conjunction with HPV in this context. To explore the possibility of developing a system for rapid diagnosis and clinical screening of cervical cancer, we developed a multiplex real-time PCR assay that can simultaneously detect and quantify HPV16/18 and HSV1/2. To evaluate its possibilities and practical uses, 177 samples collected from patients with suspected HPV and HSV infection in exfoliated cervical cells, genital herpes or labial herpes were tested by multiplex real-time PCR and compared with results obtained by DNA sequencing. Each virus was detected over a range from 1.0 × 10(1) to 1.0 × 10(7) copies/reaction. The clinical sensitivity was 100% for HPV16/18 and HSV1/2. The clinical specificity was 97.1% for HPV16, 98.1% for HPV18, 97.0% for HSV1 and 96.0% for HSV2. The kappa value was 0.96 for HPV16, 0.92 for HPV18, 0.94 for HSV1 and 0.93 for HSV2, when DNA sequencing was used as the reference standard. In summary, this novel multiplex real-time PCR allows the rapid and specific detection of HPV16/18 and HSV1/2, as well as coinfection with HPV and HSV, in clinical samples. In the future, this multiplex real-time PCR assay will assist in cervical cancer screening, viral treatment evaluation and epidemiological studies in which high throughput analysis is required. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Human papillomavirus hpv-16 DNA as an epitheliotropic virus that induces hyperproliferation in squamous penile tissue.

    PubMed

    Salazar, Edith L; Mercado, E; Calzada, L

    2005-01-01

    The prevalence of human papillomavirus HPV-16DNA sequences in 57 penile carcinoma biopsies was examined using the polymerase chain reaction (PCR) with type specific internal probes, employing HPV consensus primers from the L1 region. The cases comprised 39 typical squamous cell carcinoma and 18 specimens with different subtype. PCR products were analyzed and HPV-16DNA was detected in a high percentage of specimens. Thirty-eight biopsies were HPV-16DNA positive. This determination was correlated with cellular differentiation and growth pattern. Our data corroborates that squamous cell carcinoma was invariably associated with HPV-16DNA.

  12. Cleavage of HPV-16 E6/E7 mRNA mediated by modified 10-23 deoxyribozymes.

    PubMed

    Reyes-Gutiérrez, Pablo; Alvarez-Salas, Luis M

    2009-09-01

    Deoxyribozymes (DXZs) are small oligodeoxynucleotides capable of mediating phosphodiester bond cleavage of a target RNA in a sequence-specific manner. These molecules are a new generation of artificial catalytic nucleic acids currently used to silence many disease-related genes. The present study describes a DXZ (Dz1023-434) directed against the polycistronic mRNA from the E6 and E7 genes of human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer. Dz1023-434 showed efficient cleavage against a bona fide antisense window at nt 410-445 within HPV-16 E6/E7 mRNA even in low [Mg(2+)] conditions. Using a genetic analysis as guidance, we introduced diverse chemical modifications within Dz1023-434 catalytic core to produce a stable locked nucleic acid (LNA)-modified DXZ (Dz434-LNA) with significant cleavage activity of full E6/E7 transcripts. Cell culture testing of Dz434-LNA produced a sharp decrement of E6/E7 mRNA levels in HPV-16-positive cells resulting in decreased proliferation and considerable cell death in a specific and dose-dependent manner. No significant effects were observed with inactive or scrambled control DXZs nor from using HPV-negative cells, suggesting catalysis-dependent effect and high specificity. The biological effects of Dz434-LNA suggest a potential use for the treatment of cervical cancer.

  13. Multisite HPV16/18 Vaccine Efficacy Against Cervical, Anal, and Oral HPV Infection

    PubMed Central

    Kreimer, Aimée R.; Schiffman, Mark; Herrero, Rolando; Wacholder, Sholom; Rodriguez, Ana Cecilia; Lowy, Douglas R.; Porras, Carolina; Schiller, John T.; Quint, Wim; Jimenez, Silvia; Safaeian, Mahboobeh; Struijk, Linda; Schussler, John; Hildesheim, Allan; Gonzalez, Paula

    2016-01-01

    Background: Previous Costa Rica Vaccine Trial (CVT) reports separately demonstrated vaccine efficacy against HPV16 and HPV18 (HPV16/18) infections at the cervical, anal, and oral regions; however, the combined overall multisite efficacy (protection at all three sites) and vaccine efficacy among women infected with HPV16 or HPV18 prior to vaccination are less known. Methods: Women age 18 to 25 years from the CVT were randomly assigned to the HPV16/18 vaccine (Cervarix) or a hepatitis A vaccine. Cervical, oral, and anal specimens were collected at the four-year follow-up visit from 4186 women. Multisite and single-site vaccine efficacies (VEs) and 95% confidence intervals (CIs) were computed for one-time detection of point prevalent HPV16/18 in the cervical, anal, and oral regions four years after vaccination. All statistical tests were two-sided. Results: The multisite woman-level vaccine efficacy was highest among “naïve” women (HPV16/18 seronegative and cervical HPV high-risk DNA negative at vaccination) (vaccine efficacy = 83.5%, 95% CI = 72.1% to 90.8%). Multisite woman-level vaccine efficacy was also demonstrated among women with evidence of a pre-enrollment HPV16 or HPV18 infection (seropositive for HPV16 and/or HPV18 but cervical HPV16/18 DNA negative at vaccination) (vaccine efficacy = 57.8%, 95% CI = 34.4% to 73.4%), but not in those with cervical HPV16 and/or HPV18 DNA at vaccination (anal/oral HPV16/18 VE = 25.3%, 95% CI = -40.4% to 61.1%). Concordant HPV16/18 infections at two or three sites were also less common in HPV16/18-infected women in the HPV vaccine vs control arm (7.4% vs 30.4%, P < .001). Conclusions: This study found high multisite vaccine efficacy among “naïve” women and also suggests the vaccine may provide protection against HPV16/18 infections at one or more anatomic sites among some women infected with these types prior to HPV16/18 vaccination. PMID:26467666

  14. Efficacy of a bivalent HPV 16/18 vaccine against anal HPV16/18 infection among young women: a nested analysis within the Costa Rica Vaccine Trial

    PubMed Central

    Kreimer, Aimée R.; Gonzalèz, Paula; Katki, Hormuzd A.; Porras, Carolina; Schiffman, Mark; Rodriguez, Ana Cecilia; Solomon, Diane; Jimenez, Silvia; Schiller, John T.; Lowy, Douglas R.; van Doorn, Leen-Jan; Struijk, Linda; Quint, Wim; Chen, Sabrina; Wacholder, Sholom; Hildesheim, Allan; Herrero, Rolando

    2011-01-01

    Background Anal cancer remains rare (incidence of ∼1.5 per 100,000 women annually) but rates are increasing in many countries. Human papillomavirus-16 (HPV16) infection causes most cases. We evaluated vaccine efficacy (VE) of an ASO4-adjuvanted HPV16/18 vaccine against anal HPV16/18 infection. Methods In a randomized double-blind controlled trial designed to evaluate VE against persistent cervical HPV16/18 infections and associated precancerous lesions in Costa Rica, 4210 healthy women underwent anal specimen collection (4224 of 5968= 70.8% of eligible women) at the final blinded study visit 4 years after vaccination to evaluate anal HPV16/18 VE. Cervical HPV16/18 VE among the same women at the same visit was calculated as a comparator. For this ancillary work, analyses were conducted in a restricted cohort of women both cervical HPV16/18 DNA negative and HPV 16/18 seronegative prior at enrollment (N=1989), and in the full cohort (all women with an anal specimen). Findings In the restricted cohort, VE against prevalent HPV16/18 anal infection measured one-time, four-years post-vaccination was 83.6% (95%CI 66.7% to 92.8%), which was comparable to cervical HPV16/18 VE (87.9%, 95%CI 77.4% to 94.0%). In the full cohort, HPV16/18 VE was statistically lower at the anus (62.0%, 95%CI 47.1% to 73.1%) compared to the cervix (76.4%, 95%CI 67.0% to 83.5%) (p for anatomic-site interaction =0.03). Significant and comparable VE estimates against a composite endpoint of HPV31/33/45 (i.e.: cross-protection) was observed at the anus and cervix. Interpretation The ASO4-adjuvanted vaccine affords strong protection against anal HPV, particularly among women more likely to be HPV naïve at vaccination. Funding. The Costa Rica HPV Vaccine Trial is sponsored and funded by the NCI (contract N01-CP-11005), with funding support from the National Institutes of Health Office of Research on Women's Health, and conducted with support from the Ministry of Health of Costa Rica. Vaccine was

  15. HPV16 variants distribution in invasive cancers of the cervix, vulva, vagina, penis, and anus.

    PubMed

    Nicolás-Párraga, Sara; Gandini, Carolina; Pimenoff, Ville N; Alemany, Laia; de Sanjosé, Silvia; Xavier Bosch, F; Bravo, Ignacio G

    2016-10-01

    Human papillomavirus (HPV)16 is the most oncogenic human papillomavirus, responsible for most papillomavirus-induced anogenital cancers. We have explored by sequencing and phylogenetic analysis the viral variant lineages present in 692 HPV16-monoinfected invasive anogenital cancers from Europe, Asia, and Central/South America. We have assessed the contribution of geography and anatomy to the differential prevalence of HPV16 variants and to the nonsynonymous E6 T350G polymorphism. Most (68%) of the variance in the distribution of HPV16 variants was accounted for by the differential abundance of the different viral lineages. The most prevalent variant (above 70% prevalence) in all regions and in all locations was HPV16_A1-3, except in Asia, where HPV16_A4 predominated in anal cancers. The differential prevalence of variants as a function of geographical origin explained 9% of the variance, and the differential prevalence of variants as a function of anatomical location accounted for less than 3% of the variance. Despite containing similar repertoires of HPV16 variants, we confirm the worldwide trend of cervical cancers being diagnosed significantly earlier than other anogenital cancers (early fifties vs. early sixties). Frequencies for alleles in the HPV16 E6 T350G polymorphism were similar across anogenital cancers from the same geographical origin. Interestingly, anogenital cancers from Central/South America displayed higher 350G allele frequencies also within HPV16_A1-3 lineage compared with Europe. Our results demonstrate ample variation in HPV16 variants prevalence in anogenital cancers, which is partly explained by the geographical origin of the sample and only marginally explained by the anatomical location of the lesion, suggesting that tissue specialization is not essential evolutionary forces shaping HPV16 diversity in anogenital cancers. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  16. Gene copy number variation and its significance in cyanobacterial phylogeny

    PubMed Central

    2012-01-01

    Background In eukaryotes, variation in gene copy numbers is often associated with deleterious effects, but may also have positive effects. For prokaryotes, studies on gene copy number variation are rare. Previous studies have suggested that high numbers of rRNA gene copies can be advantageous in environments with changing resource availability, but further association of gene copies and phenotypic traits are not documented. We used one of the morphologically most diverse prokaryotic phyla to test whether numbers of gene copies are associated with levels of cell differentiation. Results We implemented a search algorithm that identified 44 genes with highly conserved copies across 22 fully sequenced cyanobacterial taxa. For two very basal cyanobacterial species, Gloeobacter violaceus and a thermophilic Synechococcus species, distinct phylogenetic positions previously found were supported by identical protein coding gene copy numbers. Furthermore, we found that increased ribosomal gene copy numbers showed a strong correlation to cyanobacteria capable of terminal cell differentiation. Additionally, we detected extremely low variation of 16S rRNA sequence copies within the cyanobacteria. We compared our results for 16S rRNA to three other eubacterial phyla (Chroroflexi, Spirochaetes and Bacteroidetes). Based on Bayesian phylogenetic inference and the comparisons of genetic distances, we could confirm that cyanobacterial 16S rRNA paralogs and orthologs show significantly stronger conservation than found in other eubacterial phyla. Conclusions A higher number of ribosomal operons could potentially provide an advantage to terminally differentiated cyanobacteria. Furthermore, we suggest that 16S rRNA gene copies in cyanobacteria are homogenized by both concerted evolution and purifying selection. In addition, the small ribosomal subunit in cyanobacteria appears to evolve at extraordinary slow evolutionary rates, an observation that has been made previously for morphological

  17. Diversity of human copy number variation and multicopy genes.

    PubMed

    Sudmant, Peter H; Kitzman, Jacob O; Antonacci, Francesca; Alkan, Can; Malig, Maika; Tsalenko, Anya; Sampas, Nick; Bruhn, Laurakay; Shendure, Jay; Eichler, Evan E

    2010-10-29

    Copy number variants affect both disease and normal phenotypic variation, but those lying within heavily duplicated, highly identical sequence have been difficult to assay. By analyzing short-read mapping depth for 159 human genomes, we demonstrated accurate estimation of absolute copy number for duplications as small as 1.9 kilobase pairs, ranging from 0 to 48 copies. We identified 4.1 million "singly unique nucleotide" positions informative in distinguishing specific copies and used them to genotype the copy and content of specific paralogs within highly duplicated gene families. These data identify human-specific expansions in genes associated with brain development, reveal extensive population genetic diversity, and detect signatures consistent with gene conversion in the human species. Our approach makes ~1000 genes accessible to genetic studies of disease association.

  18. A few nucleotide polymorphisms are sufficient to recruit nuclear factors differentially to the intron 1 of HPV-16 intratypic variants.

    PubMed

    López-Urrutia, Eduardo; Valdés, Jesús; Bonilla-Moreno, Raúl; Martínez-Salazar, Martha; Martínez-Garcia, Martha; Berumen, Jaime; Villegas-Sepúlveda, Nicolás

    2012-06-01

    The HPV-16 E6/E7 genes, which contain intron 1, are processed by alternative splicing and its transcripts are detected with a heterogeneous profile in tumours cells. Frequently, the HPV-16 positive carcinoma cells bear viral variants that contain single nucleotide polymorphisms into its DNA sequence. We were interested in analysing the contribution of this polymorphism to the heterogeneity in the pattern of the E6/E7 spliced transcripts. Using the E6/E7 sequences from three closely related HPV-16 variants, we have shown that a few nucleotide changes are sufficient to produce heterogeneity in the splicing profile. Furthermore, using mutants that contained a single SNP, we also showed that one nucleotide change was sufficient to reproduce the heterogeneous splicing profile. Additionally, a difference of two or three SNPs among these viral sequences was sufficient to recruit differentially several splicing factors to the polymorphic E6/E7 transcripts. Moreover, only one SNP was sufficient to alter the binding site of at least one splicing factor, changing the ability of splicing factors to bind the transcript. Finally, the factors that were differentially bound to the short form of intron 1 of one of these E6/E7 variants were identified as TIA1 and/or TIAR and U1-70k, while U2AF65, U5-52k and PTB were preferentially bound to the transcript of the other variants. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Identification of host transcriptional networks showing concentration-dependent regulation by HPV16 E6 and E7 proteins in basal cervical squamous epithelial cells

    PubMed Central

    Smith, Stephen P.; Scarpini, Cinzia G.; Groves, Ian J.; Odle, Richard I.; Coleman, Nicholas

    2016-01-01

    Development of cervical squamous cell carcinoma requires increased expression of the major high-risk human-papillomavirus (HPV) oncogenes E6 and E7 in basal cervical epithelial cells. We used a systems biology approach to identify host transcriptional networks in such cells and study the concentration-dependent changes produced by HPV16-E6 and -E7 oncoproteins. We investigated sample sets derived from the W12 model of cervical neoplastic progression, for which high quality phenotype/genotype data were available. We defined a gene co-expression matrix containing a small number of highly-connected hub nodes that controlled large numbers of downstream genes (regulons), indicating the scale-free nature of host gene co-expression in W12. We identified a small number of ‘master regulators’ for which downstream effector genes were significantly associated with protein levels of HPV16 E6 (n = 7) or HPV16 E7 (n = 5). We validated our data by depleting E6/E7 in relevant cells and by functional analysis of selected genes in vitro. We conclude that the network of transcriptional interactions in HPV16-infected basal-type cervical epithelium is regulated in a concentration-dependent manner by E6/E7, via a limited number of central master-regulators. These effects are likely to be significant in cervical carcinogenesis, where there is competitive selection of cells with elevated expression of virus oncoproteins. PMID:27457222

  20. Immunization with mutant HPV16 E7 protein inhibits the growth of TC-1 cells in tumor-bearing mice.

    PubMed

    Li, Yan-Li; Ma, Zhong-Liang; Zhao, Yue; Zhang, Jing

    2015-04-01

    Two human papillomavirus (HPV) 16 oncogenic proteins, E6 and E7, are co-expressed in the majority of HPV16-induced cervical cancer cells. Thus, the E6 and E7 proteins are good targets for developing therapeutic vaccines for cervical cancer. In the present study, immunization with the mutant non-transforming HPV16 E7 (mE7) protein was demonstrated to inhibit the growth of TC-1 cells in the TC-1 mouse model. The HPV16 mE7 gene was amplified by splicing overlap extension polymerase chain reaction using pET-28a(+)-E7 as a template, and the gene was cloned into pET-28a(+) to form pET-28a(+)-mE7. Compared with the E7 protein, mE7 lacks amino acid residues 94-98, and at residue 24, there is a Cys to Gly substitution. pET-28a(+)-mE7 was then introduced into Escherichia coli Rosetta. The expression of mE7 was induced by isopropyl β-D-1-thiogalactopyranoside. The mE7 protein was purified using Ni-NTA agarose and detected by SDS-PAGE and western blot analysis. In the tumor prevention model, no tumor was detected in the mice vaccinated with the mE7 protein. After 40 days, the tumor-free mice and control mice were challenged with 2×10 5 TC-1 cells. All control mice developed tumors six days later, but mE7 immunized mice were tumor free until 90 days. In the tumor therapy model, the TC-1 cells were initially injected subcutaneously, and the mice were subsequently vaccinated. Vaccination against the mE7 protein may significantly inhibit TC-1 cell growth compared to the control. These results demonstrated that immunization with the HPV16 mE7 protein elicited a long-term protective immunity against TC-1 tumor growth and generated a significant inhibition of TC-1 growth in a TC-1 mouse model.

  1. HPV-16 E6/E7 promotes cell migration and invasion in cervical cancer via regulating cadherin switch in vitro and in vivo.

    PubMed

    Hu, Dongxiao; Zhou, Jiansong; Wang, Fenfen; Shi, Haiyan; Li, Yang; Li, Baohua

    2015-12-01

    Cadherin switch, as a key hallmark of epithelial-mesenchymal transition (EMT), is characterized by reduced E-cadherin expression and increased N-cadherin or P-cadherin expression, and has been implicated in many aggressive tumors, but the importance and regulatory mechanism of cadherin switch in cervical cancer have not been investigated. Our study aimed to explore the role of cadherin switch by regulation of HPV-16 E6/E7 in progression and metastasis of cervical cancer. The expressions of E-cadherin and P-cadherin were examined by immunohistochemical staining in 40 cases of high-grade cervical lesions with HPV-16 infection only in which HPV-16 E6 and E7 expression had been detected using qRT-PCR method. Through modulating E6 and E7 expression using HPV-16 E6/E7 promoter-targeting siRNAs or expressed vector in vitro, cell growth, migration, and invasion were separately tested by MTT, wound-healing and transwell invasion assays, as well as the expressions of these cadherins by western blot analyses. Finally, the expressions of these cadherins in cancerous tissues of BALB/c-nu mouse model inoculated with the stable HPV-16 E6/E7 gene silencing Siha and Caski cells were also measured by immunohistochemical staining. Pearson correlation coefficient analyses showed the strongly inverse correlation of E-cadherin expression and strongly positive correlation of P-cadherin expression with E6/E7 level in 40 cases of high-grade cervical lesions. Furthermore, the modulation of HPV-16 E6/E7 expression remarkably influenced cell proliferation, migration, and invasion, as well as the protein levels of E-cadherin and P-cadherin in cervical cell lines. Finally, the reduction of HPV-16 E6/E7 expression led to up-regulated expression of E-cadherin and down-regulated expression of P-cadherin in BALB/c-nu mouse model in vivo assay. Our results unraveled the possibility that HPV-16 E6/E7 could promote cell invasive potential via regulating cadherin switching, and consequently contribute

  2. Intramuscular Therapeutic Vaccination Targeting HPV16 Induces T Cell Responses That Localize in Mucosal Lesions

    PubMed Central

    Jotova, Iveta; Wu, T. C.; Wang, Chenguang; Desmarais, Cindy; Boyer, Jean D.; Tycko, Benjamin; Robins, Harlan S.; Clark, Rachael A.; Trimble, Cornelia L.

    2014-01-01

    About 25% of high-grade cervical intraepithelial neoplasias (CIN2/3) caused by human papillomavirus serotype 16 (HPV16) undergo complete spontaneous regression. However, to date, therapeutic vaccination strategies for HPV disease have yielded limited success when measured by their ability to induce robust peripheral blood T cell responses to vaccine antigen. We report marked immunologic changes in the target lesion microenvironment after intramuscular therapeutic vaccination targeting HPV16 E6/E7 antigens, in subjects with CIN2/3 who had modest detectable responses in circulating T lymphocytes. Histologic and molecular changes, including markedly (average threefold) increased intensity of CD8+ T cell infiltrates in both the stromal and epithelial compartments, suggest an effector response to vaccination. Postvaccination cervical tissue immune infiltrates included organized tertiary lymphoid-like structures in the stroma subjacent to residual intraepithelial lesions and, unlike infiltrates in unvaccinated lesions, showed evidence of proliferation induced by recognition of cognate antigen. At a molecular level, these histologic changes in the stroma were characterized by increased expression of genes associated with immune activation (CXCR3) and effector function (Tbet and IFNβ), and were also associated with an immunologic signature in the overlying dysplastic epithelium. High-throughput T cell receptor sequencing of unmanipulated specimens identified clonal expansions in the tissue that were not readily detectable in peripheral blood. Together, these findings indicate that peripheral therapeutic vaccination to HPV antigens can induce a robust tissue-localized effector immune response, and that analyses of immune responses at sites of antigen are likely to be much more informative than analyses of cells that remain in the circulation. PMID:24477000

  3. Gene copy number evolution during tetraploid cotton radiation.

    PubMed

    Rong, J; Feltus, F A; Liu, L; Lin, L; Paterson, A H

    2010-11-01

    After polyploid formation, retention or loss of duplicated genes is not random. Genes with some functional domains are convergently restored to 'singleton' state after many independent genome duplications, and have been referred to as 'duplication-resistant' (DR) genes. To further explore the timeframe for their restoration to the singleton state, 27 cotton homologs of genes found to be 'DR' in Arabidopsis were selected based on diagnostic Pfam domains. Their copy numbers were studied using southern hybridization and sequence analysis in five tetraploid species and their ancestral A and D genome diploids. DR genes had significantly lower copy number than gene families hybridizing to randomly selected cotton ESTs. Three DR genes showed complete loss of D genome-derived homoeologs in some or all tetraploid species. Prior analysis has shown gene loss in polyploid cotton to be rare, and herein only one randomly selected gene showed loss of a homoeolog in only one of the five tetraploid species (Gossypium mustelinum). BAC sequencing confirmed two cases of gene loss in tetraploid cotton. Divergence among 5' sequences of DR genes amplified from G. arboreum, G. raimondii, and Gossypioides kirkii was correlated with gene copy number. These results show that genes containing Pfam domains associated with duplication resistance in Arabidopsis have also been preferentially restored to low copy number after a more recent polyploidization event in cotton. In tetraploid cotton, genes from the progenitor D genome seem to experience more gene copy number divergence than genes from the A genome. Together with D subgenome-biased alterations in gene expression, perhaps gene loss may contribute to the relatively larger portion of quantitative trait variation attributable to D than A subgenome chromosomes of tetraploid cotton.

  4. HPV16-E2 protein modifies self-renewal and differentiation rate in progenitor cells of human immortalized keratinocytes.

    PubMed

    Domínguez-Catzín, Victoria; Reveles-Espinoza, Alicia-María; Sánchez-Ramos, Janet; Cruz-Cadena, Raúl; Lemus-Hernández, Diana; Garrido, Efraín

    2017-04-03

    Cervical cancer is the fourth cause of death worldwide by cancer in women and is a disease associated to persistent infection with human papillomavirus (HPV), particularly from two high-risk types HPV16 and 18. The virus initiates its replicative cycle infecting cells located in the basal layer of the epithelium, where a small population of epithelial stem cells is located performing important functions of renewal and maintenance of the tissue. Viral E2 gene is one of the first expressed after infection and plays relevant roles in the replicative cycle of the virus, modifying fundamental processes in the infected cells. Thus, the aim of the present study was to demonstrate the presence of hierarchic subpopulations in HaCaT cell line and evaluate the effect of HPV16-E2 expression, on their biological processes. HaCaT-HPV16-E2 cells were generated by transduction of HaCaT cell line with a lentiviral vector. The α6-integrin-CD71 expression profile was established by immunostaining and flow cytometric analysis. After sorting, cell subpopulations were analyzed in biological assays for self-renewal, clonogenicity and expression of stemness factors (RT-qPCR). We identified in HaCaT cell line three different subpopulations that correspond to early differentiated cells (α6-integrin dim ), transitory amplifying cells (α6-integrin bri /CD71 bri ) and progenitor cells (α6-integrin bri /CD71 dim ). The last subpopulation showed stem cell characteristics, such as self-renewal ability, clonogenicity and expression of the well-known stem cell factors SOX2, OCT4 and NANOG, suggesting they are stem-like cells. Interestingly, the expression of HPV16-E2 in HaCaT cells changed its α6-integrin-CD71 immunophenotype modifying the relative abundance of the cell subpopulations, reducing significantly the percentage of α6-integrin bri /CD71 dim cells. Moreover, the expression of the stem cell markers was also modified, increasing the expression of SOX2 and NANOG, but decreasing notably

  5. Efficacy of the HPV-16/18 Vaccine: Final according to protocol results from the blinded phase of the randomized Costa Rica HPV-16/18 Vaccine Trial

    PubMed Central

    Hildesheim, Allan; Wacholder, Sholom; Catteau, Gregory; Struyf, Frank; Dubin, Gary; Herrero, Rolando

    2014-01-01

    Background A community-based randomized trial was conducted in Costa Rica to evaluate the HPV-16/18 AS04-adjuvanted vaccine (NCT00128661). The primary objective was to evaluate efficacy of the vaccine to prevent cervical intraepithelial neoplasia 2 or more severe disease (CIN2+) associated with incident HPV-16/18 cervical infections. Secondary objectives were to evaluate efficacy against CIN2+ associated with incident cervical infection by any oncogenic HPVs and to evaluate duration of protection against incident cervical infection with HPV-16/18. Vaccine safety and immunogenicity over the 4-year follow-up were also evaluated. Methods We randomized (3,727 HPV arm; 3,739 Control arm), vaccinated (HPV-16/18 or Hepatitis A) and followed (median 53.8 months) 7,466 healthy women aged 18-25 years. 5,312 women (2,635 HPV arm; 2,677 Control arm) were included in the according to protocol analysis for efficacy. The full cohort was evaluated for safety. Immunogenicity was considered on a subset of 354 (HPV-16) and 379 (HPV-18) women. HPV type was assessed by PCR on cytology specimens. Immunogenicity was assessed using ELISA and inhibition enzyme immunoassays. Disease outcomes were histologically confirmed. Vaccine efficacy and 95% confidence intervals (95%CI) were computed. Results Vaccine efficacy was 89.8% (95% CI: 39.5 - 99.5; N=11 events total) against HPV-16/18 associated CIN2+, 59.9% (95% CI: 20.7 - 80.8; N=39 events total) against CIN2+ associated with non-HPV-16/18 oncogenic HPVs and 61.4% (95% CI: 29.5-79.8; N=51 events total) against CIN2+ irrespective of HPV type. The vaccine had an acceptable safety profile and induced robust and long-lasting antibody responses. Conclusions Our findings confirm the high efficacy and immunogenicity of the HPV-16/18 vaccine against incident HPV infections and cervical disease associated with HPV-16/18 and other oncogenic HPV types. These results will serve as a benchmark to which we can compare future findings from ongoing extended

  6. HPV16 E6 regulates annexin 1 (ANXA1) protein expression in cervical carcinoma cell lines

    SciTech Connect

    Calmon, Marilia Freitas; Sichero, Laura; Boccardo, Enrique

    Annexin 1 (ANXA1) is a substrate for E6AP mediated ubiquitylation. It has been hypothesized that HPV 16 E6 protein redirects E6AP away from ANXA1, increasing its stability and possibly contributing to viral pathogenesis. We analyzed ANXA1 expression in HPV-positive and negative cervical carcinoma-derived cells, in cells expressing HPV-16 oncogenes and in cells transduced with shRNA targeting E6AP. We observed that ANXA1 protein expression increased in HPV-16-positive tumor cells, in keratinocytes expressing HPV-16 E6wt (wild-type) or E6/E7 and C33 cells expressing HPV-16 E6wt. ANXA1 protein expression decreased in cells transfected with E6 Dicer-substrate RNAs (DsiRNA) and C33 cells cotransduced with HPV-16more » E6wt and E6AP shRNA. Moreover, colony number and proliferation rate decreased in HPV16-positive cells transduced with ANXA1 shRNA. We observed that in cells infected with HPV16, the E6 binds to E6AP to degrade p53 and upregulate ANXA1. We suggest that ANXA1 may play a role in HPV-mediated carcinogenesis. - Highlights: • ANXA1 upregulation requires the presence of E6 and E6AP and is dependent on E6 integrity. • E6 binds to E6AP to degrade p53 and upregulate ANXA1 in cells infected with HPV16. • ANXA1 plays a role in cell proliferation in HPV-positive cervical cells.« less

  7. Digital droplet PCR (ddPCR) for the detection and quantification of HPV 16, 18, 33 and 45 - a short report.

    PubMed

    Lillsunde Larsson, Gabriella; Helenius, Gisela

    2017-10-01

    Human papilloma virus (HPV) infection is associated with several anogenital malignancies. Here, we set out to evaluate digital droplet PCR (ddPCR) as a tool for HPV 16, 18, 33 and 45 viral load quantification and, in addition, to compare the efficacy of the ddPCR assay for HPV 16 detection with that of quantitative real-time PCR (qPCR). Clinical samples, positive for HPV genotypes 16, 18, 33 and 45 were analyzed for viral load using ddPCR. Sample DNA was cleaved before droplet generation and PCR. Droplets positive for VIC and FAM fluorescence were read in a QX200 Droplet reader™ (BIO-RAD) after which the viral load was calculated using Quantasoft software. We found that DNAs extracted from formalin fixed paraffin embedded (FFPE) tissue samples yielded lower amplification signals compared to those obtained from liquid based cytology (LBC) samples, but they were clearly distinguishable from negative background signals. The viral limit of detection was 1.6 copies of HPV 16, 2.8 copies of HPV 18, 4.6 copies of HPV 33 and 1.6 copies of HPV 45. The mean inter-assay coefficients of variability (CV) for the assays ranged from 3.4 to 7.0%, and the mean intra-assay CV from 2.6 to 8.2%. The viral load in the different cohorts of tumor samples ranged from 154 to 340,200 copies for HPV 16, 244 to 31,300 copies for HPV 18 and 738 to 69,100 copies for HPV 33. One sample positive for HPV 45 contained 1331 viral copies. When comparing qPCR data with ddPCR copy number data, the qPCR values were found to be 1 to 31 times higher. Separation of fragments in nanodroplets may facilitate the amplification of fragmented human and viral DNA. The method of digital droplet PCR may, thus, provide a new and promising tool for evaluating the HPV viral load in clinical samples.

  8. Detection of HPV16 in Esophageal Cancer in a High-Incidence Region of Malawi

    PubMed Central

    Geßner, Anja Lidwina; Borkowetz, Angelika; Baier, Michael; Göhlert, Angela; Wilhelm, Torsten J.; Thumbs, Alexander; Borgstein, Eric; Jansen, Lars; Beer, Katrin; Mothes, Henning

    2018-01-01

    This study was designed to explore the role of human papillomavirus (HPV) in esophageal squamous cell carcinoma (ESCC). Fifty-five patients receiving diagnostic upper gastrointestinal endoscopy at Zomba Central Hospital or Queen Elizabeth Hospital in Blantyre (Malawi) in 2010, were included in our study. Formalin-fixed paraffin-embedded biopsies were collected for histopathological diagnosis. HPV DNA was detected using multiplex Quantitative PCR (qPCR) and in situ hybridization (ISH). p16INK4a staining served as a surrogate marker for HPV oncogene activity. Cell proliferation was determined by Ki-67 staining. Human immunodeficiency virus (HIV) status was evaluated by serology. Data on the consumption of alcohol and tobacco, and history of tuberculosis (TBC), oral thrush, and Herpes zoster, were obtained by questionnaire. Forty patients displayed ESCC, three displayed dysplastic epithelium, and 12 displayed normal epithelium. HPV16 was detected in six ESCC specimens and in one dysplastic lesion. Among HPV-positive patients, viral load varied from 0.001 to 2.5 copies per tumor cell. HPV DNA presence could not be confirmed by ISH. p16INK4a positivity correlated with the presence of HPV DNA (p = 0.03). Of particular note is that the Ki-67 proliferation index, in areas with diffuse nuclear or cytoplasmatic p16INK4a staining ≥50%, was significantly higher in HPV-positive tumors compared to the corresponding p16INK4a stained areas of HPV-negative tumors (p = 0.004). HPV infection in ESCC was not associated with the consumption of tobacco or alcohol, but there were significantly more patients drinking locally brewed alcohol among HPV-positive tumor patients compared to non-tumor patients (p = 0.02) and compared to HPV-negative tumor patients (p = 0.047). There was no association between HIV infection, history of TBC, Herpes zoster, oral thrush, or HPV infection, in ESCC patients. Our indirect evidence for viral oncogene activity is restricted to single tumor cell areas

  9. Effects of Methylation Status of CpG Sites within the HPV16 Long Control Region on HPV16-Positive Head and Neck Cancer Cells.

    PubMed

    Zhang, Chunlin; Deng, Zeyi; Pan, Xiaoli; Uehara, Takayuki; Suzuki, Mikio; Xie, Minqiang

    2015-01-01

    To map comprehensively the methylation status of the CpG sites within the HPV16 long control region (LCR) in HPV-positive cancer cells, and to explore further the effects of methylation status of HPV16 LCR on cell bioactivity and E6 and E7 expression. In addition, to analyze the methylation status of the LCR in HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) patients. Methylation patterns of HPV16 LCR in UM-SCC47, CaSki, and SiHa cells and HPV16-positiive OPSCC specimens were detected by bisulfite-sequencing PCR and TA cloning. For cells treated with 5-aza-2'-deoxycytidine and E6 and E7 knockdown, MTS and trypan blue staining, annexin-V and 7-AAD staining, and prodidium iodide were used to evaluate cell growth and cell proliferation, cell apoptosis, and cell cycle arrest, respectively. E6 and E7 mRNA and protein expression were analyzed by quantitative real-time PCR and immunocytochemistry, respectively. Hypermethylation status of the LCR in UM-SCC47 (79.8%) and CaSki cells (90.0%) and unmethylation status of the LCR in SiHa cells (0%) were observed. Upon demethylation, the cells with different methylation levels responded differently during growth, apoptosis, and cell cycle arrest, as well as in terms of their E6 and E7 expression. In HPV16-positive OPSCC patients, the methylation rates were 9.5% in the entire LCR region, 13.9% in the 5'-LCR, 6.0% in the E6 enhancer, and 9.5% in the p97 promoter, and hypermethylation of p97 promoter was found in a subset of cases (20.0%, 2/10). Our study revealed two different methylation levels of the LCR in HPV16-positive cancer cells and OPSCC patients, which may represent different carcinogenesis mechanisms of HPV-positive cancers cells. Demethylating the meCpGs in HPV16 LCR might be a potential target for a subgroup of HPV16-positive patients with head and neck squamous cell carcinoma.

  10. Six1 overexpression at early stages of HPV16-mediated transformation of human keratinocytes promotes differentiation resistance and EMT

    SciTech Connect

    Xu, Hanwen; Pirisi, Lucia; Creek, Kim E., E-mail: creekk@sccp.sc.edu

    Previous studies in our laboratory discovered that SIX1 mRNA expression increased during in vitro progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we explored the role of Six1 at early stages of HPV16-mediated transformation by overexpressing Six1 in HKc/HPV16. We found that Six1 overexpression in HKc/HPV16 increased cell proliferation and promoted cell migration and invasion by inducing epithelial–mesenchymal transition (EMT). Moreover, the overexpression of Six1 in HKc/HPV16 resulted in resistance to serum and calcium-induced differentiation, which is the hallmark of the HKc/DR phenotype. Activation of MAPK in HKc/HPV16 overexpressing Six1 is linked to resistancemore » to calcium-induced differentiation. In conclusion, this study determined that Six1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation of human keratinocytes. - Highlights: • Six1 expression increases during HPV16-mediated transformation. • Six1 overexpression causes differentiation resistance in HPV16-immortalized cells. • Six1 overexpression in HPV16-immortalized keratinocytes activates MAPK. • Activation of MAPK promotes EMT and differentiation resistance. • Six1 overexpression reduces Smad-dependent TGF-β signaling.« less

  11. A1E reduces stemness and self-renewal in HPV 16-positive cervical cancer stem cells.

    PubMed

    Kwon, Taeho; Bak, Yesol; Ham, Sun-Young; Yu, Dae-Yeul; Yoon, Do-Young

    2016-02-02

    Cervical cancer is the second most common cancer in females. Recent reports have revealed the critical role of cervical cancer stem cells (CSCs) in tumorigenicity and metastasis. Previously we demonstrated that A1E exerts an anti-proliferative action, which inhibits the growth of cervical cancer cells. A1E is composed of 11 oriental medicinal herbs. Cervical cancer cell culture, wund healing and invasion assay, flow cytometry, sheroid formation assay, and wstern blot assays were performed in HPV 16-positive SiHa cell and HPV 16-negative C33A cells. A1E targets the E6 and E7 oncogenes; thus, A1E significantly inhibited proliferation of human papilloma virus (HPV) 16-positive SiHa cells, it did not inhibit the proliferation of HPV-negative C33A cells. Accordingly, we investigated whether A1E can regulate epithelial-to-mesenchymal transition (EMT), CSC self-renewal, and stemness-related gene expression in cervical cancer cells. Down rgulation of cell migration, cell invasion, and EMT was observed in A1E-treated SiHa cells. Specifically, A1E-treated SiHa cells showed significant decreases in OCT-3/4 and Sox2 expression levels and in sphere formation. Moreover, CSCs makers ALDH+ and ALDH, CD133 double positive cell were significantly decreased in A1E-treated SiHa cells. However, A1E treatment did not down regulate ALDH+ expression and the number of ALDH/CD133 double positive cells in C33A cells. Taken together, A1E can inhibit CSCs and reduce the expression of stemness markers. Treating CSCs with A1E may be a potential therapy for cervical cancer.

  12. Viral load, integration and methylation of E2BS3 and 4 in human papilloma virus (HPV) 16-positive vaginal and vulvar carcinomas.

    PubMed

    Lillsunde Larsson, Gabriella; Helenius, Gisela; Sorbe, Bengt; Karlsson, Mats G

    2014-01-01

    To investigate if viral load, integration and methylation of E2BS3 and 4 represent different ways of tumor transformation in vaginal and vulvar carcinoma and to elucidate its clinical impact. Fifty-seven samples, positive for HPV16, were selected for the study. Detection of viral load was made with realtime-PCR using copy numbers of E6 and integration was calculated from comparing E2 to E6-copies. Methylation of E2BS3 and 4 was analysed using bisulphite treatment of tumor DNA, followed by PCR and pyrosequencing. Vaginal tumors were found to have a higher viral load (p = 0.024) compared to vulvar tumors but a high copy number (> median value, 15,000) as well as high methylation (>50%) was significantly (p = 0.010 and p = 0.045) associated with a worse cancer-specific survival rate in vulvar carcinoma, but not in vaginal carcinoma. Four groups could be defined for the complete series using a Cluster Two step analysis; (1) tumors holding episomal viral DNA, viral load below 150,000 copies not highly methylated (n = 25, 46.3%); (2) tumors harboring episomal viral DNA and being highly methylated (>50%; n = 6, 11.1%); (3) tumors with viral DNA fully integrated (n = 11, 20.4%), and (4) tumors harboring episomal viral DNA and being medium- or unmethylated (<50%) and having a high viral load (> total mean value 150,000; n = 12, 22.2%). The completely integrated tumors were found to be distinct group, whilst some overlap between the groups with high methylation and high viral load was observed. HPV16- related integration, methylation in E2BS3 and 4 and viral load may represent different viral characteristics driving vaginal and vulvar carcinogenesis. HPV16- related parameters were found to be of clinical importance in the vulvar series only.

  13. [Experimental studies of using real-time fluorescence quantitative PCR and RT-PCR to detect E6 and E7 genes of human papillomavirus type 16 in cervical carcinoma cell lines].

    PubMed

    Chen, Yue-yue; Peng, Zhi-lan; Liu, Shan-ling; He, Bing; Hu, Min

    2007-06-01

    To establish a method of using real-time fluorescence quantitative PCR and RT-PCR to detect the E6 and E7 genes of human papillomavirus type 16 (HPV-16). Plasmids containing HPV-16 E6 or E7 were used to generate absolute standard curves. Three cervical carcinoma cell lines CaSki, SiHa and HeLa were tested by real-time fluorescence quantitative PCR and RT-PCR analyses for the expressions of HPV-16 E6 and E7. The correlation coefficients of standard curves were larger than 0. 99, and the PCR efficiency was more than 90%. The relative levels of HPV-16 E6 and E7 DNA and RNA were CaSki>SiHa>HeLa cell. HPV-16 E6 and E7 quantum by real-time fluorescence quantitative PCR and RT-PCR analyses may serve as a reliable and sensitive tool. This study provides the possibility of further researches on the relationship between HPV-16 E6 or E7 copy number and cervical carcinoma.

  14. Screening for common copy-number variants in cancer genes.

    PubMed

    Tyson, Jess; Majerus, Tamsin M O; Walker, Susan; Armour, John A L

    2010-12-01

    For most cases of colorectal cancer that arise without a family history of the disease, it is proposed that an appreciable heritable component of predisposition is the result of contributions from many loci. Although progress has been made in identifying single nucleotide variants associated with colorectal cancer risk, the involvement of low-penetrance copy number variants is relatively unexplored. We have used multiplex amplifiable probe hybridization (MAPH) in a fourfold multiplex (QuadMAPH), positioned at an average resolution of one probe per 2 kb, to screen a total of 1.56 Mb of genomic DNA for copy number variants around the genes APC, AXIN1, BRCA1, BRCA2, CTNNB1, HRAS, MLH1, MSH2, and TP53. Two deletion events were detected, one upstream of MLH1 in a control individual and the other in APC in a colorectal cancer patient, but these do not seem to correspond to copy number polymorphisms with measurably high population frequencies. In summary, by means of our QuadMAPH assay, copy number measurement data were of sufficient resolution and accuracy to detect any copy number variants with high probability. However, this study has demonstrated a very low incidence of deletion and duplication variants within intronic and flanking regions of these nine genes, in both control individuals and colorectal cancer patients. Copyright © 2010 Elsevier Inc. All rights reserved.

  15. Characterization of an RNA aptamer against HPV-16 L1 virus-like particles.

    PubMed

    Leija-Montoya, Ana Gabriela; Benítez-Hess, María Luisa; Toscano-Garibay, Julia Dolores; Alvarez-Salas, Luis Marat

    2014-10-01

    The human papillomavirus (HPV) capsid is mainly composed of the L1 protein that can self-assemble into virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. We report here the characterization of RNA aptamers that recognize baculovirus-produced HPV-16 L1 VLPs. Interaction and slot-blot binding assays showed that all isolated aptamers efficiently bound HPV-16 VLPs, although the Sc5-c3 aptamer showed the highest specificity and affinity (Kd=0.05 pM). Sc5-c3 secondary structure consisted of a hairpin with a symmetric bubble and an unstructured 3'end. Biochemical and genetic analyses showed that the Sc5-c3 main loop is directly involved on VLPs binding. In particular, binding specificity appeared mediated by five non-consecutive nucleotide positions. Experiments using bacterial-produced HPV-16 L1 resulted in low Sc5-c3 binding, suggesting that recognition of HPV-16 L1 VLPs relies on quaternary structure features not present in bacteria-produced L1 protein. Sc5-c3 produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection.

  16. Characterization of an RNA Aptamer Against HPV-16 L1 Virus-Like Particles

    PubMed Central

    Leija-Montoya, Ana Gabriela; Benítez-Hess, María Luisa; Toscano-Garibay, Julia Dolores

    2014-01-01

    The human papillomavirus (HPV) capsid is mainly composed of the L1 protein that can self-assemble into virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. We report here the characterization of RNA aptamers that recognize baculovirus-produced HPV-16 L1 VLPs. Interaction and slot-blot binding assays showed that all isolated aptamers efficiently bound HPV-16 VLPs, although the Sc5-c3 aptamer showed the highest specificity and affinity (Kd=0.05 pM). Sc5-c3 secondary structure consisted of a hairpin with a symmetric bubble and an unstructured 3′end. Biochemical and genetic analyses showed that the Sc5-c3 main loop is directly involved on VLPs binding. In particular, binding specificity appeared mediated by five non-consecutive nucleotide positions. Experiments using bacterial-produced HPV-16 L1 resulted in low Sc5-c3 binding, suggesting that recognition of HPV-16 L1 VLPs relies on quaternary structure features not present in bacteria-produced L1 protein. Sc5-c3 produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection. PMID:25111024

  17. tRNA gene copy number variation in humans

    PubMed Central

    Iben, James R.; Maraia, Richard J.

    2014-01-01

    The human tRNAome consists of more than 500 interspersed tRNA genes comprising 51 anticodon families of largely unequal copy number. We examined tRNA gene copy number variation (tgCNV) in six individuals; two kindreds of two parents and a child, using high coverage whole genome sequence data. Such differences may be important because translation of some mRNAs is sensitive to the relative amounts of tRNAs and because tRNA competition determines translational efficiency vs. fidelity and production of native vs. misfolded proteins. We identified several tRNA gene clusters with CNV, which in some cases were part of larger iterations. In addition there was an isolated tRNALysCUU gene that was absent as a homozygous deletion in one of the parents. When assessed by semiquantitative PCR in 98 DNA samples representing a wide variety of ethnicities, this allele was found deleted in hetero- or homozygosity in all groups at ~50% frequency. This is the first report of copy number variation of human tRNA genes. We conclude that tgCNV exists at significant levels among individual humans and discuss the results in terms of genetic diversity and prior genome wide association studies (GWAS) that suggest the importance of the ratio of tRNALys isoacceptors in Type-2 diabetes. PMID:24342656

  18. E6D25E, HPV16 Asian variant shows specific proteomic pattern correlating in cells transformation and suppressive innate immune response

    SciTech Connect

    Chopjitt, Peechanika; Pientong, Chamsai; Sunthamala, Nuchsupha

    HPV16 Asian variant (HPV16As) containing E6D25E oncogene, is commonly associated with cervical cancers of Asian populations. To explore a mechanism of E6D25E oncoprotein in carcinogenesis, we compared protein profiles in human keratinocytes expressing E6D25E with E6 of HPV16 prototype (E6Pro). A human cervical keratinocyte cell line, HCK1T, was transduced with retroviruses containing E6D25E or E6Pro genes. Biological properties of E6D25E or E6Pro transduced HCK1T cells were characterized. Protein profiles of the transduced HCK1T cells were analyzed using 2D-PAGE and characterized by mass spectrometry and western blotting. Reactomes of modulated proteins were analyzed by using the Reactome Knowledgebase. The E6D25E andmore » E6Pro oncoproteins were comparable for their abilities to degrade p53 and suppress the induction of p21, and induce cell proliferation. Interestingly, the protein profiles of the HCK1T cells transduced with E6D25E showed specific proteomic patterns different from those with E6Pro. Among altered proteins, more than 1.5-fold up- or down- regulation was observed in E6D25E-expressing cells for gp96 and keratin7 which involved in activation of TLR signaling and transformation of squamocolumnar junction cells, respectively. This report describes new cellular proteins specifically targeted by E6D25E oncoprotein that may contribute to impair immune response against viral infection and cell transformation associated with oncogenic property of HPV16As variant. - Highlights: • E6D25E HPV16 specifically modulates protein profile of human keratinocytes. • E6D25E HPV16 modulates protein profile which involves in TLR signalling and transformation of squamocolumnar junction cells. • E6D25E oncoprotein may correlate to impair of immune response against viral infection and cells transformation.« less

  19. Activation of Wnt signaling pathway by human papillomavirus E6 and E7 oncogenes in HPV16-positive oropharyngeal squamous carcinoma cells.

    PubMed

    Rampias, Theodore; Boutati, Eleni; Pectasides, Eirini; Sasaki, Clarence; Kountourakis, Panteleimon; Weinberger, Paul; Psyrri, Amanda

    2010-03-01

    We sought to determine the role of human papillomavirus (HPV) E6 and E7 oncogenes in nuclear beta-catenin accumulation, a hallmark of activated canonical Wnt signaling pathway. We used HPV16-positive oropharyngeal cancer cell lines 147T and 090, HPV-negative cell line 040T, and cervical cell lines SiHa (bearing integrated HPV16) and HeLa (bearing integrated HPV18) to measure the cytoplasmic and nuclear beta-catenin levels and the beta-catenin/Tcf transcriptional activity before and after E6/E7 gene silencing. Repression of HPV E6 and E7 genes induced a substantial reduction in nuclear beta-catenin levels. Luciferase assay showed that transcriptional activation of Tcf promoter by beta-catenin was lower after silencing. The protein levels of beta-catenin are tightly regulated by the ubiquitin/proteasome system. We therefore performed expression analysis of regulators of beta-catenin degradation and nuclear transport and showed that seven in absentia homologue (Siah-1) mRNA and protein levels were substantially upregulated after E6/E7 repression. Siah-1 protein promotes the degradation of beta-catenin through the ubiquitin/proteasome system. To determine whether Siah-1 is important for the proteasomal degradation of beta-catenin in HPV16-positive oropharyngeal cancer cells, we introduced a Siah-1 expression vector into 147T and 090 cells and found substantial reduction of endogenous beta-catenin in these cells. Thus, E6 and E7 are involved in beta-catenin nuclear accumulation and activation of Wnt signaling in HPV-induced cancers. In addition, we show the significance of the endogenous Siah-1-dependent ubiquitin/proteasome pathway for beta-catenin degradation and its regulation by E6/E7 viral oncoproteins in HPV16-positive oropharyngeal cancer cells.

  20. The intron 1 of HPV 16 has a suboptimal branch point at a guanosine.

    PubMed

    De la Rosa-Rios, Marco Antonio; Martínez-Salazar, Martha; Martínez-Garcia, Martha; González-Bonilla, César; Villegas-Sepúlveda, Nicolás

    2006-06-01

    The branch point sequence (BPS) of intron 1 of the HPV-16 was determined via RT-PCR in a cell free system, using lariat intermediates obtained by in vitro splicing reactions. We used synthetic E6/E7 transcripts and HeLa nuclear protein extracts to obtain the splicing intermediates. Then, a divergent oligonucleotide primer set, pairing on the lariat RNA that encompassed the 2'-5' phosphodiester bond formed between the 5' end of the intron and the BPS, was used for cDNA synthesis and PCR amplification. Subsequent RT-PCR assays revealed four splicing intermediates, made up of a major intermediary corresponding to the BPS and four cryptic branched sequences. Only intermediates bound at the 5' end of the intron are probably the authentic branch point sequence, and all of them branch at guanosine 328 instead of the typical adenosine. Unusually, the BPS of intron 1 of HPV-16 is a suboptimal sequence (AGUGAGU) that differs from the eukaryotic consensus BPS, which correlates with the splicing profile observed for early transcripts of HPV-16 in tumors and tumor derived cell lines. The implications of this unusual branch point sequence for splicing of the HPV-16 pre-mRNA are discussed.

  1. Sustained efficacy, immunogenicity, and safety of the HPV-16/18 AS04-adjuvanted vaccine

    PubMed Central

    Naud, Paulo S; Roteli-Martins, Cecilia M; De Carvalho, Newton S; Teixeira, Julio C; de Borba, Paola C; Sanchez, Nervo; Zahaf, Toufik; Catteau, Gregory; Geeraerts, Brecht; Descamps, Dominique

    2014-01-01

    HPV-023 (NCT00518336; ClinicalTrial.gov) is a long-term follow-up of an initial double-blind, randomized (1:1), placebo-controlled study (HPV-001, NCT00689741) evaluating the efficacy against human papillomavirus (HPV)-16/18 infection and associated cyto-histopathological abnormalities, persistence of immunogenicity, and safety of the HPV-16/18 AS04-adjuvanted vaccine. Among the women, aged 15–25 years, enrolled in HPV-001 and who participated in the follow-up study HPV-007 (NCT00120848), a subset of 437 women from five Brazilian centers participated in this 36-month long-term follow-up (HPV-023) for a total of 113 months (9.4 years). During HPV-023, anti-HPV-16/18 antibodies were measured annually by enzyme-linked immunosorbent assay (ELISA) and pseudovirion-based neutralisation assay (PBNA). Cervical samples were tested for HPV DNA every 6 months, and cyto-pathological examinations were performed annually. During HPV-023, no new HPV-16/18-associated infections and cyto-histopathological abnormalities occurred in the vaccine group. Vaccine efficacy (VE) against HPV-16/18 incident infection was 100% (95%CI: 66.1, 100). Over the 113 months (9.4 years), VE was 95.6% (86.2, 99.1; 3/50 cases in vaccine and placebo groups, respectively) against incident infection, 100% (84·1, 100; 0/21) against 6-month persistent infection (PI); 100% (61·4, 100; 0/10) against 12-month PI; 97·1% (82.5, 99.9; 1/30) against ≥ ASC-US; 95·0% (68.0, 99.9; 1/18) against ≥ LSIL; 100% (45.2, 100; 0/8) against CIN1+; and 100% (–128.1, 100; 0/3) against CIN2+ associated with HPV-16/18. All vaccinees remained seropositive to HPV-16/18, with antibody titers remaining several folds above natural infection levels, as measured by ELISA and PBNA. There were no safety concerns. To date, these data represent the longest follow-up reported for a licensed HPV vaccine. PMID:25424918

  2. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

    SciTech Connect

    Chen, Yi; Pirisi, Lucia; Creek, Kim E., E-mail: creekk@sccp.sc.edu

    2013-09-15

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cellmore » proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski.« less

  3. Single-copy gene detection using branched DNA (bDNA) in situ hybridization.

    PubMed

    Player, A N; Shen, L P; Kenny, D; Antao, V P; Kolberg, J A

    2001-05-01

    We have developed a branched DNA in situ hybridization (bDNA ISH) method for detection of human papillomavirus (HPV) DNA in whole cells. Using human cervical cancer cell lines with known copies of HPV DNA, we show that the bDNA ISH method is highly sensitive, detecting as few as one or two copies of HPV DNA per cell. By modifying sample pretreatment, viral mRNA or DNA sequences can be detected using the same set of oligonucleotide probes. In experiments performed on mixed populations of cells, the bDNA ISH method is highly specific and can distinguish cells with HPV-16 from cells with HPV-18 DNA. Furthermore, we demonstrate that the bDNA ISH method provides precise localization, yielding positive signals retained within the subcellular compartments in which the target nucleic acid sequences are localized. As an effective and convenient means for nucleic acid detection, the bDNA ISH method is applicable to the detection of cancers and infectious agents. (J Histochem Cytochem 49:603-611, 2001)

  4. Plasmodium copy number variation scan: gene copy numbers evaluation in haploid genomes.

    PubMed

    Beghain, Johann; Langlois, Anne-Claire; Legrand, Eric; Grange, Laura; Khim, Nimol; Witkowski, Benoit; Duru, Valentine; Ma, Laurence; Bouchier, Christiane; Ménard, Didier; Paul, Richard E; Ariey, Frédéric

    2016-04-12

    In eukaryotic genomes, deletion or amplification rates have been estimated to be a thousand more frequent than single nucleotide variation. In Plasmodium falciparum, relatively few transcription factors have been identified, and the regulation of transcription is seemingly largely influenced by gene amplification events. Thus copy number variation (CNV) is a major mechanism enabling parasite genomes to adapt to new environmental changes. Currently, the detection of CNVs is based on quantitative PCR (qPCR), which is significantly limited by the relatively small number of genes that can be analysed at any one time. Technological advances that facilitate whole-genome sequencing, such as next generation sequencing (NGS) enable deeper analyses of the genomic variation to be performed. Because the characteristics of Plasmodium CNVs need special consideration in algorithms and strategies for which classical CNV detection programs are not suited a dedicated algorithm to detect CNVs across the entire exome of P. falciparum was developed. This algorithm is based on a custom read depth strategy through NGS data and called PlasmoCNVScan. The analysis of CNV identification on three genes known to have different levels of amplification and which are located either in the nuclear, apicoplast or mitochondrial genomes is presented. The results are correlated with the qPCR experiments, usually used for identification of locus specific amplification/deletion. This tool will facilitate the study of P. falciparum genomic adaptation in response to ecological changes: drug pressure, decreased transmission, reduction of the parasite population size (transition to pre-elimination endemic area).

  5. Multiple copies of genes coding for electron transport proteins in the bacterium Nitrosomonas europaea.

    PubMed

    McTavish, H; LaQuier, F; Arciero, D; Logan, M; Mundfrom, G; Fuchs, J A; Hooper, A B

    1993-04-01

    The genome of Nitrosomonas europaea contains at least three copies each of the genes coding for hydroxylamine oxidoreductase (HAO) and cytochrome c554. A copy of an HAO gene is always located within 2.7 kb of a copy of a cytochrome c554 gene. Cytochrome P-460, a protein that shares very unusual spectral features with HAO, was found to be encoded by a gene separate from the HAO genes.

  6. Immunogold-agglutination assay for direct detection of HPV-16 E6 and L1 proteins from clinical specimens.

    PubMed

    Bhattarakosol, Parvapan; Plaignam, Kamolwan; Sereemaspun, Amornpun

    2018-05-01

    HPV-16 infection is the most common cause of cervical cancer. As HPV-16 transforms the cell, E6 oncoprotein is over-expressed. Therefore, molecular detection of HPV-16 E6 mRNA is now being used for diagnosis and prediction of cancer development. Besides detecting E6 mRNA, a rapid lateral flow detecting the E6 protein using enzyme immunoassay is also now on market with a sensitivity of 53.5% for cervical intraepithelial neoplasia (CIN)-3 or more severe (CIN-3+). Here, an immunogold-agglutination assay was developed to detect not only HPV-16 E6 protein but also L1, a major capsid protein found in the productive stage of the virus. Evaluation of this test using HPV-16 DNA positive cervical samples showed that the HPV-16 E6 immunogold-agglutination assay results correlated well with the progression of the cervical lesions, i.e., 10.34% of CIN-1, 68.75% of CIN-3 and 80% of cancer (CaCx) and none for healthy normal samples. Interestingly, the HPV-16 L1 protein was found in most of the cases with cancer indicating the possibility of virion production. Immunogold-agglutination assay for E6 protein is simpler, easier to be performed with a sensitivity of 73.1% for CIN-3+ suggesting a good method for laboratory diagnostic use. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Usage of heparan sulfate, integrins, and FAK in HPV16 infection

    PubMed Central

    Abban, Cynthia Y.; Meneses, Patricio I.

    2010-01-01

    Human Papillomavirus Type 16 (HPV16) is the major causative agent of cervical cancer. Studies regarding the early binding and signaling molecules that play a significant role in infection are still lacking. The current study analyses the role of heparan sulfate, integrins, and the signaling molecule FAK in HPV16 infection of human adult keratinocytes cell line (HaCaTs). Our data demonstrate that infection requires the binding of viral particles to heparan sulfate followed by activation of focal adhesion kinase through an integrin. Infections were reduced in the presence of the FAK inhibitor, TAE226. TAE226 was observed to inhibit viral entry to the early endosome a known infectious route. These findings suggest that FAK can serve as a novel target for antiviral therapy. PMID:20441998

  8. HPV16 variant lineage, clinical stage, and survival in women with invasive cervical cancer

    PubMed Central

    2011-01-01

    Background HPV16 variants are associated with different risks for development of CIN3 and invasive cancer, although all are carcinogenic. The relationship of HPV 16 variants to cancer survival has not been studied. Methods 155 HPV16-positive cervical cancers were categorized according to European and non-European variant patterns by DNA sequencing of the E6 open reading frame. Clinico-pathologic parameters and clinical outcome were collected by chart review and death registry data. Results Of the 155 women (mean age 44.7 years; median follow-up 26.7 months), 85.2% harbored European variants while 14.8% had non-European sequences. HPV16 variants differed by histologic cell type (p = 0.03) and stage (1 vs. 2+; p = 0.03). Overall, 107 women (68.0%) were alive with no evidence of cancer, 42 (27.1%) died from cervical cancer, 2 (1.3%) were alive with cervical cancer, and 4 (2.6%) died of other causes. Death due to cervical cancer was associated with European variant status (p < 0.01). While 31% of women harboring tumors with European variants died from cervical cancer during follow-up, only 1 of 23 (4.4%) non-European cases died of cancer. The better survival for non-European cases was partly mediated by lower stage at diagnosis. Conclusions Overall, invasive cervical cancers with non-European variants showed a less aggressive behavior than those with European variants. These findings should be replicated in a population with more non-European cases. PMID:22035468

  9. HPV16/18 genotyping for the triage of HPV positive women in primary cervical cancer screening in Chile.

    PubMed

    Lagos, Marcela; Van De Wyngard, Vanessa; Poggi, Helena; Cook, Paz; Viviani, Paola; Barriga, María Isabel; Pruyas, Martha; Ferreccio, Catterina

    2015-01-01

    We previously conducted a population-based screening trial of high-risk human papillomavirus (hrHPV) testing and conventional cytology, demonstrating higher sensitivity (92.7 % vs 22.1 % for CIN2+) but lower positive predictive value (10.5 % vs 23.9 %) of hrHPV testing. Here we report the performance of HPV16/18 genotyping to triage the hrHPV positive participants. Women aged 25 years and older received hrHPV (Hybrid Capture 2) and Papanicolaou testing; positives by either test underwent colposcopy and directed biopsy, as did a sample of double-negatives. hrHPV positive women were reflex-tested with HPV16/18 genotyping (Digene HPV Genotyping PS Test). Among the 8,265 participants, 10.7 % were hrHPV positive, 1.7 % had ASCUS+ cytology, 1.2 % had CIN2+; 776 (88 %) hrHPV positive women had complete results, of whom 38.8 % were positive for HPV16 (24.0 %), HPV18 (9.7 %) or both (5.1 %). CIN2+ prevalence in HPV16/18 positive women (16.3 %, 95 % CI 12.3-20.9) was twice that of HPV16/18 negative women (8.0 %, 95 % CI 5.7-10.8). HPV16/18 genotyping identified 40.5 % of CIN2, 66.7 % of CIN3 and 75.0 % of cancers. Compared to hrHPV screening alone, HPV16/18 triage significantly reduced the referral rate (10.7 % vs 3.7 %) and the number of colposcopies required to detect one CIN2+ (9 vs 6). When HPV16/18 negative women with baseline ASCUS+ cytology were also colposcopied, an additional 14 % of CIN2+ was identified; referral increased slightly to 4.2 %. HPV16/18 triage effectively stratified hrHPV positive women by their risk of high-grade lesions. HPV16/18 positive women must be referred immediately; referral could be deferred in HPV16/18 negative women given the slower progression of non-HPV16/18 lesions, however, they will require active follow-up.

  10. Comparative immunogenicity and safety of human papillomavirus (HPV)-16/18 vaccine and HPV-6/11/16/18 vaccine

    PubMed Central

    Baron, Mira; Levin, Myron J; Chatterjee, Archana; Fox, Bradley; Scholar, Sofia; Rosen, Jeffrey; Chakhtoura, Nahida; Meric, Dorothée; Dessy, Francis J; Datta, Sanjoy K; Descamps, Dominique; Dubin, Gary

    2011-01-01

    In this observer-blind study (NCT00423046), women (N = 1,106), stratified by age (18–26, 27–35, 36–45 y), were randomized (1:1) to receive the HPV-16/18 vaccine (Cervarix®, GlaxoSmithKline Biologicals, Months 0, 1, 6) or the HPV-6/11/16/18 vaccine (Gardasil® Merck and Co., Inc., Months 0, 2, 6). Month 7 results were previously reported; we now report Month 24 results. In the according-to-protocol cohort for immunogenicity (seronegative and DNA-negative at baseline for HPV type analyzed), seropositivity rates of neutralizing antibodies (nAbs) [pseudovirion-based neutralization assay] were, across all age strata, 100% (HPV-16/18 vaccine) and 97.5–100% (HPV-6/11/16/18 vaccine) for HPV-16, and 99.0–100% (HPV-16/18 vaccine) and 72.3–84.4% (HPV-6/11/16/18 vaccine) for HPV-18. Corresponding geometric mean titers (GMTs) were 2.4–5.8-fold higher for HPV-16 and 7.7–9.4-fold higher for HPV-18 with the HPV-16/18 vaccine vs. the HPV-6/11/16/18 vaccine; HPV-16 and HPV-18 GMTs were significantly higher with the HPV-16/18 vaccine than the HPV-6/11/16/18 vaccine (p < 0.0001) in the total vaccinated cohort (received ≥1 vaccine dose, irrespective of baseline sero/DNA-status). Similar results were obtained using enzyme-linked immunosorbent assay (ELISA ). Positivity rates and GMTs of antigen-specific IgG antibodies in cervicovaginal secretions (ELISA) were not significantly different between vaccines. At Month 24, CD4+ T-cell responses for HPV-16 and HPV-18 were higher with the HPV-16/18 vaccine; memory B-cell response was higher for HPV-18 with the HPV-16/18 vaccine and similar between vaccines for HPV-16. Both vaccines were generally well tolerated. Although an immunological correlate of protection has not been defined, differences in the magnitude of immune response between vaccines may represent determinants of duration of protection. PMID:22048173

  11. An Examination of HPV16 Natural Immunity in Men Who Have Sex with Men (MSM) in the HPV in Men (HIM) Study.

    PubMed

    Beachler, Daniel C; Pinto, Ligia A; Kemp, Troy J; Nyitray, Alan G; Hildesheim, Allan; Viscidi, Raphael; Schussler, John; Kreimer, Aimée R; Giuliano, Anna R

    2018-04-01

    Background: Evidence suggests that natural antibodies developed after HPV16 infection may protect some women but not men against subsequent HPV16 reacquisition. Less is known whether antibodies developed following HPV16 infection are protective among men who have sex with men (MSM). Methods: Four hundred seventy-five MSM from the Human Papillomavirus Infection in Men (HIM) study were tested for serum antibodies to HPV16 L1 using enzyme-linked immunosorbent assays, and for anal and genital HPV16 DNA using PCR consensus primer system (PGMY 09/11). Adjusted Cox regression was used to evaluate whether baseline HPV16 seropositivity impacts subsequent genital or anal HPV16 DNA. Results: The risk of subsequent genital HPV16 [aHR = 1.05, 95% confidence interval (CI) = 0.66-1.68] and anal HPV16 infections among MSM (aHR = 2.34, 95% CI = 0.92-5.98) was similar or nonsignificantly higher in HPV16-seropositive than HPV16-seronegative MSM. The risk of genital HPV16 was also similar between HPV16-seronegative and HPV16-seropositive MSM in the highest tertile of HPV16 antibody levels and when restricting to those with new sex partners during follow-up ( P > 0.20). Among the 118 MSM who were HPV16 seropositive, 90% remained HPV16 seropositive up to 4 years later. When tested together, MSM with the highest antibody titers (top tertile) had similar levels to females (mean = 130.3 vs. 134.5 EU/mL, P = 0.84). Conclusions: Despite years of HPV16 seropositivity persistence and antibody titers comparable with females, this study suggested no evidence of HPV16 natural antibodies protecting against subsequent genital or anal HPV16 infection in MSM. Impact: This could help partially explain the high incidence of genital and anal HPV16 infection and related anal cancer seen in middle-aged and older MSM. Cancer Epidemiol Biomarkers Prev; 27(4); 496-502. ©2018 AACR . ©2018 American Association for Cancer Research.

  12. Copy-number and gene dependency analysis reveals partial copy loss of wild-type SF3B1 as a novel cancer vulnerability. | Office of Cancer Genomics

    Cancer.gov

    Genomic instability is a hallmark of human cancer, and results in widespread somatic copy number alterations. We used a genome-scale shRNA viability screen in human cancer cell lines to systematically identify genes that are essential in the context of particular copy-number alterations (copy-number associated gene dependencies). The most enriched class of copy-number associated gene dependencies was CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) genes, and spliceosome components were the most prevalent.

  13. Application of droplet digital PCR to determine copy number of endogenous genes and transgenes in sugarcane.

    PubMed

    Sun, Yue; Joyce, Priya Aiyar

    2017-11-01

    Droplet digital PCR combined with the low copy ACT allele as endogenous reference gene, makes accurate and rapid estimation of gene copy number in Q208 A and Q240 A attainable. Sugarcane is an important cultivated crop with both high polyploidy and aneuploidy in its 10 Gb genome. Without a known copy number reference gene, it is difficult to accurately estimate the copy number of any gene of interest by PCR-based methods in sugarcane. Recently, a new technology, known as droplet digital PCR (ddPCR) has been developed which can measure the absolute amount of the target DNA in a given sample. In this study, we deduced the true copy number of three endogenous genes, actin depolymerizing factor (ADF), adenine phosphoribosyltransferase (APRT) and actin (ACT) in three Australian sugarcane varieties, using ddPCR by comparing the absolute amounts of the above genes with a transgene of known copy number. A single copy of the ACT allele was detected in Q208 A , two copies in Q240 A , but was absent in Q117. Copy number variation was also observed for both APRT and ADF, and ranged from 9 to 11 in the three tested varieties. Using this newly developed ddPCR method, transgene copy number was successfully determined in 19 transgenic Q208 A and Q240 A events using ACT as the reference endogenous gene. Our study demonstrates that ddPCR can be used for high-throughput genetic analysis and is a quick, accurate and reliable alternative method for gene copy number determination in sugarcane. This discovered ACT allele would be a suitable endogenous reference gene for future gene copy number variation and dosage studies of functional genes in Q208 A and Q240 A .

  14. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

    PubMed Central

    Chen, Yi; Pirisi, Lucia; Creek, Kim E.

    2013-01-01

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. PMID:23809940

  15. A bacterial reporter system for the evaluation of antisense oligodeoxynucleotides directed against human papillomavirus type 16 (HPV-16).

    PubMed

    Guapillo, Mario R; Márquez, Miguel A; Benítez-Hess, María L; Alvarez-Salas, Luis M

    2006-07-01

    Antisense oligodeoxynucleotides (AS-ODNs) are a promising alternative for the cure of many diseases because of their in vivo specificity and stability. However, AS-ODNs have a strong dependence on the target mRNA structure making necessary extensive in vivo testing. There is, therefore, a need to develop assays to rapidly evaluate in vivo ODN performance. We report a simple and inexpensive bacterial reporter system for the rapid in vivo evaluation of AS-ODNs directed against human papillomavirus type 16 (HPV-16) based on the destruction of a chimeric CFP mRNA using the reported HPV-16 nt 410-445 target. In vitro RNaseH assays confirmed target RNA accessibility after AS-ODN treatment. Expression of CFP in Escherichia coli BL21(DE3) with pGST-TSd2-CFP plasmid containing HPV-16 nt 410-445 target linked to CFP was blocked by transformed antisense PS-ODNs but not by two different scrambled ODN controls. A correlation was observed between bacterial CFP downregulation with the HPV-16 E6/E7 mRNA downregulation and the inhibition of anchorage-independent growth of HPV-16 containing cells suggesting that inhibition of HPV-16 E6/E7 expression by AS-ODNs directed against 410-445 target in cervical tumor cells can be tested in bacterial models.

  16. The Transcription Factors TBX2 and TBX3 Interact with Human Papillomavirus 16 (HPV16) L2 and Repress the Long Control Region of HPVs

    PubMed Central

    Schneider, Marc A.; Scheffer, Konstanze D.; Bund, Timo; Boukhallouk, Fatima; Lambert, Carsten; Cotarelo, Cristina; Pflugfelder, Gert O.

    2013-01-01

    The minor capsid protein L2 of human papillomaviruses (HPVs) has multiple functions during the viral life cycle. Although L2 is required for effective invasion and morphogenesis, only a few cellular interaction partners are known so far. Using yeast two-hybrid screening, we identified the transcription factor TBX2 as a novel interaction partner of HPV type 16 (HPV16) L2. Coimmunoprecipitations and immunofluorescence analyses confirmed the L2-TBX2 interaction and revealed that L2 also interacts with TBX3, another member of the T-box family. Transcription of the early genes during HPV infection is under the control of an upstream enhancer and early promoter region, the long control region (LCR). In promoter-reporter gene assays, we observed that TBX2 and TBX3 repress transcription from the LCR and that this effect is enhanced by L2. Repression of the HPV LCR by TBX2/3 seems to be a conserved mechanism, as it was also observed with the LCRs of different HPV types. Finally, interaction of TBX2 with the LCR was detected by chromatin immunoprecipitation, and we found a strong colocalization of L2 and TBX2 in HPV16-positive cervical intraepithelial neoplasia (CIN) I-II tissue sections. These results suggest that TBX2/3 might play a role in the regulation of HPV gene expression during the viral life cycle. PMID:23388722

  17. Human papillomavirus detection with genotyping by the cobas and Aptima assays: Significant differences in HPV 16 detection?

    PubMed

    Chorny, Joseph A; Frye, Teresa C; Fisher, Beth L; Remmers, Carol L

    2018-03-23

    The primary high-risk human papillomavirus (hrHPV) assays in the United States are the cobas (Roche) and the Aptima (Hologic). The cobas assay detects hrHPV by DNA analysis while the Aptima detects messenger RNA (mRNA) oncogenic transcripts. As the Aptima assay identifies oncogenic expression, it should have a lower rate of hrHPV and genotype detection. The Kaiser Permanente Regional Reference Laboratory in Denver, Colorado changed its hrHPV assay from the cobas to the Aptima assay. The rates of hrHPV detection and genotyping were compared over successive six-month periods. The overall hrHPV detection rates by the two platforms were similar (9.5% versus 9.1%) and not statistically different. For genotyping, the HPV 16 rate by the cobas was 1.6% and by the Aptima it was 1.1%. These differences were statistically different with the Aptima detecting nearly one-third less HPV 16 infections. With the HPV 18 and HPV 18/45, there was a slightly higher detection rate of HPV 18/45 by the Aptima platform (0.5% versus 0.9%) and this was statistically significant. While HPV 16 represents a low percentage of hrHPV infections, it was detected significantly less by the Aptima assay compared to the cobas assay. This has been previously reported, although not highlighted. Given the test methodologies, one would expect the Aptima to detect less HPV 16. This difference appears to be mainly due to a significantly increased number of non-oncogenic HPV 16 infections detected by the cobas test as there were no differences in HPV 16 detection rates in the high-grade squamous intraepithelial lesions indicating that the two tests have similar sensitivities for oncogenic HPV 16. © 2018 Wiley Periodicals, Inc.

  18. The HPV-16 E7 oncoprotein induces centriole multiplication through deregulation of Polo-like kinase 4 expression

    PubMed Central

    2011-01-01

    Background Infection with high-risk human papillomaviruses (HPVs) such as HPV-16 is intimately associated with squamous cell carcinomas (SCCs) of the anogenital tract and a subset of oropharyngeal carcinomas. Such lesions, including pre-invasive precursors, frequently show multipolar mitoses and aneuploidy. The high-risk HPV-16-encoded E7 oncoprotein has been shown to rapidly induce centrosome abnormalities thereby causing the formation of supernumerary mitotic spindle poles and increasing the risk for chromosome missegregation. HPV-16 E7 has been found to rapidly induce centriole overduplication, in part, through the simultaneous formation of more than one daughter centriole at single maternal centrioles (centriole multiplication). The precise molecular mechanism that underlies HPV-16 E7-induced centriole multiplication, however, remains poorly understood. Findings Here, we show that human keratinocytes engineered to stably express the HPV-16 E7 oncoprotein exhibit aberrant Polo-like kinase 4 (PLK4) protein expression at maternal centrioles. Real-time quantitative reverse transcriptase (qRT-PCR) analysis of these cells revealed an increase of PLK4 mRNA levels compared to control cells. Importantly, the ability of the HPV-16 E7 oncoprotein to induce centriole multiplication was found to correlate with its ability to activate the PLK4 promoter and to up-regulate PLK4 mRNA. Conclusions These results highlight the critical role of PLK4 transcriptional deregulation in centriole multiplication in HPV-16 E7-expressing cells. Our findings encourage further experiments to test transcriptional inhibitors or small molecules targeting PLK4 to prevent centriole abnormalities, mitotic infidelity and malignant progression in HPV-associated neoplasms and other tumors in which PLK4 regulation is disrupted. PMID:21609466

  19. Establishment of immunoassay for detecting HPV16 E6 and E7 RNA

    PubMed Central

    Ding, Sen; Qian, Steven Y.; Zhang, Yang; Wu, Wenlei; Lu, Gensheng; Lu, Yan; Feng, Xiujing; Li, Li; Shen, Pingping

    2015-01-01

    Cervical carcinoma is the most prevalent malignancy second only to breast cancer among women worldwide. Since more than 99% of cervical cancers are caused by human papilloma virus (HPV), measurement of HPV (HPV test) was commonly used in screening risk and/or early stage of cervical cancer as well as assessing the efficacies of the treatments that can decrease the incidence of cervical cancer. Many approaches that diagnose HPV infections have been developed, while most of them have distinct shortcomings. We here established a novel immunoassay method in which the pairs of unlabeled DNA probes firstly bind to HPV16 E6 and E7 RNAs to form the DNA-RNA hybrids, and the hybrids will subsequently be identified by S9.6 antibody. The sensitivity of this highly specific method can reach ~0.923 pg/mL and ~0.424 pg/mL of in vitro transcribed HPV16 E6 and E7 RNA, respectively, and reverse transcription and polymerase chain reaction (PCR) amplification were no longer needed. Thus, our immunoassay approaches can precisely reflect the actually viral load that is related to the course of HPV infection. In addition, it has also fast and low cost characteristic feature. PMID:26333509

  20. GeneBreak: detection of recurrent DNA copy number aberration-associated chromosomal breakpoints within genes.

    PubMed

    van den Broek, Evert; van Lieshout, Stef; Rausch, Christian; Ylstra, Bauke; van de Wiel, Mark A; Meijer, Gerrit A; Fijneman, Remond J A; Abeln, Sanne

    2016-01-01

    Development of cancer is driven by somatic alterations, including numerical and structural chromosomal aberrations. Currently, several computational methods are available and are widely applied to detect numerical copy number aberrations (CNAs) of chromosomal segments in tumor genomes. However, there is lack of computational methods that systematically detect structural chromosomal aberrations by virtue of the genomic location of CNA-associated chromosomal breaks and identify genes that appear non-randomly affected by chromosomal breakpoints across (large) series of tumor samples. 'GeneBreak' is developed to systematically identify genes recurrently affected by the genomic location of chromosomal CNA-associated breaks by a genome-wide approach, which can be applied to DNA copy number data obtained by array-Comparative Genomic Hybridization (CGH) or by (low-pass) whole genome sequencing (WGS). First, 'GeneBreak' collects the genomic locations of chromosomal CNA-associated breaks that were previously pinpointed by the segmentation algorithm that was applied to obtain CNA profiles. Next, a tailored annotation approach for breakpoint-to-gene mapping is implemented. Finally, dedicated cohort-based statistics is incorporated with correction for covariates that influence the probability to be a breakpoint gene. In addition, multiple testing correction is integrated to reveal recurrent breakpoint events. This easy-to-use algorithm, 'GeneBreak', is implemented in R ( www.cran.r-project.org ) and is available from Bioconductor ( www.bioconductor.org/packages/release/bioc/html/GeneBreak.html ).

  1. Investigating Reports of Complex Regional Pain Syndrome: An Analysis of HPV-16/18-Adjuvanted Vaccine Post-Licensure Data

    PubMed Central

    Huygen, Frank; Verschueren, Kristin; McCabe, Candida; Stegmann, Jens-Ulrich; Zima, Julia; Mahaux, Olivia; Van Holle, Lionel; Angelo, Maria-Genalin

    2015-01-01

    Complex regional pain syndrome (CRPS) is a chronic pain disorder that typically follows trauma or surgery. Suspected CRPS reported after vaccination with human papillomavirus (HPV) vaccines led to temporary suspension of proactive recommendation of HPV vaccination in Japan. We investigated the potential CRPS signal in relation to HPV-16/18-adjuvanted vaccine (Cervarix®) by database review of CRPS cases with independent expert confirmation; a disproportionality analysis and analyses of temporality; an observed versus expected analysis using published background incidence rates; systematic reviews of aggregate safety data, and a literature review. The analysis included 17 case reports of CRPS: 10 from Japan (0.14/100,000 doses distributed) and seven from the United Kingdom (0.08/100,000). Five cases were considered by independent experts to be confirmed CRPS. Quantitative analyses did not suggest an association between CRPS and HPV-16/18-adjuvanted vaccine. Observed CRPS incidence after HPV-16/18 vaccination was statistically significantly below expected rates. Systematic database reviews using search terms varying in specificity and sensitivity did not identify new cases. No CRPS was reported during clinical development and no unexpected results found in the literature. There is not sufficient evidence to suggest an increased risk of developing CRPS following vaccination with HPV-16/18-adjuvanted vaccine. Post-licensure safety surveillance confirms the acceptable benefit-risk of HPV-16/18 vaccination. PMID:26501109

  2. Low copy number of the salivary amylase gene predisposes to obesity.

    PubMed

    Falchi, Mario; El-Sayed Moustafa, Julia Sarah; Takousis, Petros; Pesce, Francesco; Bonnefond, Amélie; Andersson-Assarsson, Johanna C; Sudmant, Peter H; Dorajoo, Rajkumar; Al-Shafai, Mashael Nedham; Bottolo, Leonardo; Ozdemir, Erdal; So, Hon-Cheong; Davies, Robert W; Patrice, Alexandre; Dent, Robert; Mangino, Massimo; Hysi, Pirro G; Dechaume, Aurélie; Huyvaert, Marlène; Skinner, Jane; Pigeyre, Marie; Caiazzo, Robert; Raverdy, Violeta; Vaillant, Emmanuel; Field, Sarah; Balkau, Beverley; Marre, Michel; Visvikis-Siest, Sophie; Weill, Jacques; Poulain-Godefroy, Odile; Jacobson, Peter; Sjostrom, Lars; Hammond, Christopher J; Deloukas, Panos; Sham, Pak Chung; McPherson, Ruth; Lee, Jeannette; Tai, E Shyong; Sladek, Robert; Carlsson, Lena M S; Walley, Andrew; Eichler, Evan E; Pattou, Francois; Spector, Timothy D; Froguel, Philippe

    2014-05-01

    Common multi-allelic copy number variants (CNVs) appear enriched for phenotypic associations compared to their biallelic counterparts. Here we investigated the influence of gene dosage effects on adiposity through a CNV association study of gene expression levels in adipose tissue. We identified significant association of a multi-allelic CNV encompassing the salivary amylase gene (AMY1) with body mass index (BMI) and obesity, and we replicated this finding in 6,200 subjects. Increased AMY1 copy number was positively associated with both amylase gene expression (P = 2.31 × 10(-14)) and serum enzyme levels (P < 2.20 × 10(-16)), whereas reduced AMY1 copy number was associated with increased BMI (change in BMI per estimated copy = -0.15 (0.02) kg/m(2); P = 6.93 × 10(-10)) and obesity risk (odds ratio (OR) per estimated copy = 1.19, 95% confidence interval (CI) = 1.13-1.26; P = 1.46 × 10(-10)). The OR value of 1.19 per copy of AMY1 translates into about an eightfold difference in risk of obesity between subjects in the top (copy number > 9) and bottom (copy number < 4) 10% of the copy number distribution. Our study provides a first genetic link between carbohydrate metabolism and BMI and demonstrates the power of integrated genomic approaches beyond genome-wide association studies.

  3. Strategies for continuous evaluation of the benefit-risk profile of HPV-16/18-AS04-adjuvanted vaccine.

    PubMed

    Angelo, Maria-Genalin; Taylor, Sylvia; Struyf, Frank; Tavares Da Silva, Fernanda; Arellano, Felix; David, Marie-Pierre; Dubin, Gary; Rosillon, Dominique; Baril, Laurence

    2014-11-01

    The HPV types 16/18-AS04-adjuvanted cervical cancer vaccine, Cervarix(®) (HPV-16/18-vaccine, GlaxoSmithKline, Belgium) was first approved in 2007 and is licensed in 134 countries for the prevention of persistent infection, premalignant cervical lesions and cervical cancer caused by oncogenic HPV. Benefit-risk status requires continual re-evaluation as vaccine uptake increases, as the epidemiology of the disease evolves and as new information becomes available. This paper provides an example of benefit-risk considerations and risk-management planning. Evaluation of the benefit-risk of HPV-16/18-vaccine post-licensure includes studies with a range of designs in many countries and in collaboration with national public agencies and regulatory authorities. The strategy to assess benefit versus risk will continue to evolve and adapt to the changing HPV-16/18-vaccine market.

  4. Health and economic outcomes of HPV 16,18 vaccination in 72 GAVI-eligible countries.

    PubMed

    Goldie, Sue J; O'Shea, Meredith; Campos, Nicole Gastineau; Diaz, Mireia; Sweet, Steven; Kim, Sun-Young

    2008-07-29

    The risk of dying from cervical cancer is disproportionately borne by women in developing countries. Two new vaccines are highly effective in preventing HPV 16,18 infection, responsible for approximately 70% of cervical cancer, in girls not previously infected. The GAVI Alliance (GAVI) provides technical assistance and financial support for immunization in the world's poorest countries. Using population-based and epidemiologic data for 72 GAVI-eligible countries we estimate averted cervical cancer cases and deaths, disability-adjusted years of life (DALYs) averted and incremental cost-effectiveness ratios (I$/DALY averted) associated with HPV 16,18 vaccination of young adolescent girls. In addition to vaccine coverage and efficacy, relative and absolute cancer reduction depended on underlying incidence, proportion attributable to HPV types 16 and 18, population age-structure and competing mortality. With 70% coverage, mean reduction in the lifetime risk of cancer is below 40% in some countries (e.g., Nigeria, Ghana) and above 50% in others (e.g., India, Uganda, Kenya). At I$10 per vaccinated girl (approximately $2.00 per dose assuming three doses, plus wastage, administration, program support) vaccination was cost-effective in all countries using a per capita GDP threshold; for 49 of 72 countries, the cost per DALY averted was less than I$100 and for 59 countries, it was less than I$200. Taking into account country-specific assumptions (per capita GNI, DPT3 coverage, percentage of girls who are enrolled in fifth grade) for the year of introduction, percent coverage achieved in the first year, and years to maximum coverage, a 10-year modeled scenario prevented the future deaths of approximately 2 million women vaccinated as adolescents. Despite favorable cost-effectiveness, assessment of financial costs raised concerns about affordability; as the cost per vaccinated girl was increased from I$10 to I$25 (approximately $2 to $5 per dose), the financial costs for the

  5. Seroepidemiology of Human Papillomavirus 16 (HPV16) L2 and Generation of L2-Specific Human Chimeric Monoclonal Antibodies

    PubMed Central

    Wang, Joshua W.; Jagu, Subhashini; Wu, Wai-Hong; Viscidi, Raphael P.; Macgregor-Das, Anne; Fogel, Jessica M.; Kwak, Kihyuck; Daayana, Sai; Kitchener, Henry; Stern, Peter L.; Gravitt, Patti E.; Trimble, Cornelia L.

    2015-01-01

    Presently, the seroprevalence of human papillomavirus (HPV) minor capsid antigen L2-reactive antibody is not well understood, and no serologic standard exists for L2-specific neutralizing antibodies. Therefore, we screened a total of 1,078 serum samples for HPV16 L2 reactivity, and these were obtained from four prior clinical studies: a population-based (n = 880) surveillance study with a high-risk HPV DNA prevalence of 10.8%, a cohort study of women (n = 160) with high-grade cervical intraepithelial neoplasia (CIN), and two phase II trials in women with high-grade vulvar intraepithelial neoplasia (VIN) receiving imiquimod therapy combined with either photodynamic therapy (PDT) (n = 19) or vaccination with a fusion protein comprising HPV16 L2, E7, and E6 (TA-CIN) (n = 19). Sera were screened sequentially by HPV16 L2 enzyme-linked immunosorbent assay (ELISA) and then Western blot. Seven of the 1,078 serum samples tested had L2-specific antibodies, but none were detectably neutralizing for HPV16. To develop a standard, we substituted human IgG1 sequences into conserved regions of two rodent monoclonal antibodies (MAbs) specific for neutralizing epitopes at HPV16 L2 residues 17 to 36 and 58 to 64, creating JWW-1 and JWW-2, respectively. These chimeric MAbs retained neutralizing activity and together reacted with 33/34 clinically relevant HPV types tested. In conclusion, our inability to identify an HPV16 L2-specific neutralizing antibody response even in the sera of patients with active genital HPV disease suggests the subdominance of L2 protective epitopes and the value of the chimeric MAbs JWW-1 and JWW-2 as standards for immunoassays to measure L2-specific human antibodies. PMID:25972404

  6. Curcumin modulates cellular AP-1, NF-kB, and HPV16 E6 proteins in oral cancer.

    PubMed

    Mishra, Alok; Kumar, Rakesh; Tyagi, Abhishek; Kohaar, Indu; Hedau, Suresh; Bharti, Alok C; Sarker, Subhodeep; Dey, Dipankar; Saluja, Daman; Das, Bhudev

    2015-01-01

    In this study, we investigated the effects of the natural antioxidant curcumin on the HPV16-positive oral carcinoma cell line 93VU147T and demonstrated that curcumin is not only a potent inhibitor for the activity of host nuclear transcription factors AP-1 and NF-kB but it also selectively suppresses transcription of the HPV16/E6 oncogene during the carcinogenic process in oral cancer cells. This study suggests a therapeutic potential of curcumin for high-risk human papilloma virus (HPV)-infected oral cancers.

  7. Curcumin modulates cellular AP-1, NF-kB, and HPV16 E6 proteins in oral cancer

    PubMed Central

    Mishra, Alok; Kumar, Rakesh; Tyagi, Abhishek; Kohaar, Indu; Hedau, Suresh; Bharti, Alok C; Sarker, Subhodeep; Dey, Dipankar; Saluja, Daman; Das, Bhudev

    2015-01-01

    In this study, we investigated the effects of the natural antioxidant curcumin on the HPV16-positive oral carcinoma cell line 93VU147T and demonstrated that curcumin is not only a potent inhibitor for the activity of host nuclear transcription factors AP-1 and NF-kB but it also selectively suppresses transcription of the HPV16/E6 oncogene during the carcinogenic process in oral cancer cells. This study suggests a therapeutic potential of curcumin for high-risk human papilloma virus (HPV)-infected oral cancers. PMID:25932049

  8. Adenovirus-mediated transfer of HPV 16 E6/E7 antisense RNA combined with cisplatin inhibits cellular growth and induces apoptosis in HPV-positive head and neck cancer cells.

    PubMed

    Kojima, Yasutaka; Otsuki, Naoki; Kubo, Mie; Kitamoto, Junko; Takata, Eri; Saito, Hiroki; Kosaka, Kyoko; Morishita, Naoya; Uehara, Natsumi; Shirakawa, Toshiro; Nibu, Ken-Ich

    2018-05-24

    Human papillomavirus (HPV) infection has been identified as an etiologic factor of head and neck cancers (HNCs). We explored the potential use of antisense HPV RNA transcripts for gene therapy and its effect in combination with cisplatin (CDDP) for HPV-positive HNCs. We introduced the antisense RNA transcripts of the E6 and E7 genes of HPV type 16 into UM-SCC-47 cells harboring HPV 16 and YCU-T892 cells that were HPV-negative using a recombinant adenoviral vector, Ad-E6/E7-AS. We then analyzed the effects of the introduction of Ad-E7-AS on cell and tumor growth and the synergistic effect with CDDP in vitro and in vivo. After infection of Ad-E6/E7-AS, the cellular growth of UM-SCC-47 cells were suppressed, but not that of YCU-T892 cells. E7 protein expression was suppressed, and p53 and pRb protein expression increased after infection of Ad-E7-AS. Cell growth and tumorigenicity were greatly suppressed in combination with CDDP compared with Ad-E7-AS or CDDP treatment alone in vitro. Ad-E7-AS combined with CDDP treatment significantly reduced the volumes of established subcutaneous tumors. Transfection with HPV 16 E7 antisense RNA combined with CDDP treatment might be a potentially useful approach to the therapy of HPV 16-positive HNC.

  9. p16INK4A expression as biomarker for HPV 16-related vulvar neoplasias.

    PubMed

    Riethdorf, Sabine; Neffen, Eduardo F; Cviko, Aida; Löning, Thomas; Crum, Christopher P; Riethdorf, Lutz

    2004-12-01

    Up-regulation of p16INK4A is associated with high-risk human papillomavirus (HPV) in preinvasive and invasive cervical neoplasia. However, its expression in vulvar carcinomas, which have a diverse pathogenesis, has not been extensively studied. One hundred seventy-seven vulvar intraepithelial neoplasms (VIN), squamous cell carcinomas (SCC), and benign squamous epithelia were analyzed for p16 expression. RNA/RNA in situ hybridization was used to detect HPV 16 E6/E7 transcripts in 112. Ninety-five percent of VIN 3 and basaloid or warty SCCs (76/80) and 4% of keratinizing SCC (2/48) were moderately to strongly immunopositive for p16, which localized to nucleus and cytoplasm; 52/58 analyzed (90%) contained HPV 16 transcripts. The positive predictive value (PPV) of moderate to strong diffuse p16 immunostaining and HPV positivity for the diagnosis of VIN 3 and of basaloid or warty SCC was 97% and 95%, respectively. Conversely, 94% of keratinizing SCC contained heterogeneous staining, and when present, it was strictly cytoplasmic and frequently localized to the cells at the epithelial-stromal interface. Benign squamous epithelia were p16 negative, with the exception of lichen sclerosus, which contained focal and heterogeneously p16 positive in 42%. As in the cervix, intense diffuse p16 expression supports an HPV-related neoplastic process in vulvar neoplasia, irrespective of the level of differentiation. Up-regulation of p16 at the epithelial-stromal interface in HPV negative keratinizing SCCs is consistent with an HPV-independent response to alterations associated with invasion. These disparate patterns of p16 expression underscore 2 different mechanisms for p16 expression in HPV-related and HPV-unrelated vulvar carcinomas.

  10. HPV16 E7 Protein and hTERT Proteins Defective for Telomere Maintenance Cooperate to Immortalize Human Keratinocytes

    PubMed Central

    Miller, Jonathan; Dakic, Aleksandra; Chen, Renxiang; Palechor-Ceron, Nancy; Dai, Yuhai; Kallakury, Bhaskar; Schlegel, Richard; Liu, Xuefeng

    2013-01-01

    Previous studies have shown that wild-type human telomerase reverse transcriptase (hTERT) protein can functionally replace the human papillomavirus type 16 (HPV-16) E6 protein, which cooperates with the viral E7 protein in the immortalization of primary keratinocytes. In the current study, we made the surprising finding that catalytically inactive hTERT (hTERT-D868A), elongation-defective hTERT (hTERT-HA), and telomere recruitment-defective hTERT (hTERT N+T) also cooperate with E7 in mediating bypass of the senescence blockade and effecting cell immortalization. This suggests that hTERT has activities independent of its telomere maintenance functions that mediate transit across this restriction point. Since hTERT has been shown to have a role in gene activation, we performed microarray studies and discovered that E6, hTERT and mutant hTERT proteins altered the expression of highly overlapping sets of cellular genes. Most important, the E6 and hTERT proteins induced mRNA and protein levels of Bmi1, the core subunit of the Polycomb Group (PcG) complex 1. We show further that Bmi1 substitutes for E6 or hTERT in cell immortalization. Finally, tissue array studies demonstrated that expression of Bmi1 increased with the severity of cervical dysplasia, suggesting a potential role in the progression of cervical cancer. Together, these data demonstrate that hTERT has extra-telomeric activities that facilitate cell immortalization and that its induction of Bmi1 is one potential mechanism for mediating this activity. PMID:23592995

  11. Significantly Reduced Genoprevalence of Vaccine-Type HPV-16/18 Infections among Vaccinated Compared to Non-Vaccinated Young Women 5.5 Years after a Bivalent HPV-16/18 Vaccine (Cervarix®) Pilot Project in Uganda

    PubMed Central

    Berggren, Vanja; Wabinga, Henry; Lillsunde-Larsson, Gabriella; Helenius, Gisela; Kaliff, Malin; Karlsson, Mats; Kirimunda, Samuel; Musubika, Caroline; Andersson, Sören

    2016-01-01

    The objective of this study was to determine the prevalence and some predictors for vaccine and non-vaccine types of HPV infections among bivalent HPV vaccinated and non-vaccinated young women in Uganda. This was a comparative cross sectional study 5.5 years after a bivalent HPV 16/18 vaccination (Cervarix®, GlaxoSmithKline, Belgium) pilot project in western Uganda. Cervical swabs were collected between July 2014-August 2014 and analyzed with a HPV genotyping test, CLART® HPV2 assay (Genomica, Madrid Spain) which is based on PCR followed by microarray for determination of genotype. Blood samples were also tested for HIV and syphilis infections as well as CD4 and CD8 lymphocyte levels. The age range of the participants was 15–24 years and mean age was 18.6(SD 1.4). Vaccine-type HPV-16/18 strains were significantly less prevalent among vaccinated women compared to non-vaccinated women (0.5% vs 5.6%, p 0.006, OR 95% CI 0.08(0.01–0.64). At type-specific level, significant difference was observed for HPV16 only. Other STIs (HIV/syphilis) were important risk factors for HPV infections including both vaccine types and non-vaccine types. In addition, for non-vaccine HPV types, living in an urban area, having a low BMI, low CD4 count and having had a high number of life time sexual partners were also significant risk factors. Our data concurs with the existing literature from other parts of the world regarding the effectiveness of bivalent HPV-16/18 vaccine in reducing the prevalence of HPV infections particularly vaccine HPV- 16/18 strains among vaccinated women. This study reinforces the recommendation to vaccinate young girls before sexual debut and integrate other STI particularly HIV and syphilis interventions into HPV vaccination packages. PMID:27482705

  12. Selection of suitable endogenous reference genes for relative copy number detection in sugarcane.

    PubMed

    Xue, Bantong; Guo, Jinlong; Que, Youxiong; Fu, Zhiwei; Wu, Luguang; Xu, Liping

    2014-05-19

    Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential "single copy" genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3--high copy number group, TST-1 and PRR-1--medium copy number group, P4H-1, APRT-2 and CYC-2--low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.

  13. Modulation of basal cell fate during productive and transforming HPV-16 infection is mediated by progressive E6-driven depletion of Notch.

    PubMed

    Kranjec, Christian; Holleywood, Christina; Libert, Diane; Griffin, Heather; Mahmood, Radma; Isaacson, Erin; Doorbar, John

    2017-08-01

    In stratified epithelia such as the epidermis, homeostasis is maintained by the proliferation of cells in the lower epithelial layers and the concomitant loss of differentiated cells from the epithelial surface. These differentiating keratinocytes progressively stratify and form a self-regenerating multi-layered barrier that protects the underlying dermis. In such tissue, the continual loss and replacement of differentiated cells also limits the accumulation of oncogenic mutations within the tissue. Inactivating mutations in key driver genes, such as TP53 and NOTCH1, reduce the proportion of differentiating cells allowing for the long-term persistence of expanding mutant clones in the tissue. Here we show that through the expression of E6, HPV-16 prevents the early fate commitment of human keratinocytes towards differentiation and confers a strong growth advantage to human keratinocytes. When E6 is expressed either alone or with E7, it promotes keratinocyte proliferation at high cell densities, through the combined inactivation of p53 and Notch1. In organotypic raft culture, the activity of E6 is restricted to the basal layer of the epithelium and is enhanced during the progression from productive to abortive or transforming HPV-16 infection. Consistent with this, the expression of p53 and cleaved Notch1 becomes progressively more disrupted, and is associated with increased basal cell density and reduced commitment to differentiation. The expression of cleaved Notch1 is similarly disrupted also in HPV-16-positive cervical lesions, depending on neoplastic grade. When taken together, these data depict an important role of high-risk E6 in promoting the persistence of infected keratinocytes in the basal and parabasal layers through the inactivation of gene products that are commonly mutated in non-HPV-associated neoplastic squamous epithelia. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great

  14. Identification of RNA Aptamers that Internalize into HPV-16 E6/E7 Transformed Tonsillar Epithelial Cells

    PubMed Central

    Gourronc, Francoise A.; Rockey, William M.; Thiel, William H.; Giangrande, Paloma H.; Klingelhutz, Aloysius J.

    2013-01-01

    Human papillomavirus type 16 (HPV-16) associated oropharyngeal cancers are on a significant increase and better therapeutic strategies are needed. The HPV-16 oncogenes E6 and E7 are expressed in HPV-associated cancers and are able to transform human tonsillar epithelial cells (HTECs). We used cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to select for RNA aptamers that entered into HPV-16 E6/E7-HTECs. After 12 rounds of cell-SELEX, a pool of aptamers was obtained that had significantly greater internalization capacity (~5-fold) into E6/E7-HTECs as compared to primary HTECs or fibroblasts. Analysis of individual aptamers from the pool indicated variable internalization into E6/E7-HTECs (1 to 8-fold as compared to a negative control). Most of the individual aptamers internalized into E6/E7 and primary HTECs with similar efficiency, while one aptamer exhibited ~3-fold better internalization into E6/E7-HTECs. Aptamers that internalize into cells may be useful for delivering therapeutic agents to HPV-16 associated malignancies. PMID:24074596

  15. Human papillomavirus genotypes and phylogenetic analysis of HPV-16 variants in HIV-1 infected subjects in Italy.

    PubMed

    Tanzi, Elisabetta; Amendola, Antonella; Bianchi, Silvia; Fasolo, M Michela; Beretta, Rosangela; Pariani, Elena; Zappa, Alessandra; Frati, Elena; Orlando, Giovanna

    2009-05-29

    A cross-sectional study was carried out to improve the state of evidence regarding the spectrum of HPV types and HPV-16 LCR variants circulating among men and women infected with HIV-1 in Italy. This study, conducted in 518 HIV-positive subjects (346 males and 172 females), showed a high prevalence of HPV anal infections (88.7%) in men and of cervical infections (65.1%) in women. A wide spectrum of HPV genotypes has been observed, as both single and multiple infections. Low-risk HPV types 6, 11 and 61 were frequently detected. HPV-16 was the prevalent high-risk type. Fourteen different HPV-16 LCR variants were found. Ten belonged to the European lineage (78.7% were detected in Italian subjects and 21.3% in foreign-born, all homo/bisexual men), two to the Asiatic lineage and two to the African-2 lineage. This study underlines the great genotypic heterogeneity characterizing anal and cervical HPV infections and the marked polymorphism of the predominant HPV-16 in this high-risk population in Italy.

  16. DNA methylation regulated microRNAs in HPV-16-induced head and neck squamous cell carcinoma (HNSCC).

    PubMed

    Sannigrahi, M K; Sharma, Rajni; Singh, Varinder; Panda, Naresh K; Rattan, Vidya; Khullar, Madhu

    2018-02-17

    Epigenetic modifications have been reported to play an important role in regulating gene expression and these modifications become critical when they have a role in controlling another important layer of epigenetic regulation namely microRNAs. In the present study, we have identified the microRNAs that may be regulated by promoter DNA methylation and histone acetylation in Human papilloma virus-positive head and neck squamous cell carcinoma. HPV-negative cell line (UPCI:SCC-116) and HPV-16 +ve cell line (UPCI:SCC-090) were treated with methylation inhibitor (5-aza-2'-deoxycytidine, AZA) and acetylation inhibitor (Trichostatin-A, TSA), followed by micro-array analysis. The differentially expressed miRNAs were validated in control (n = 10), HPV-16 +ve (n = 30), and HPV -ve (n = 30) HNC, TCGA (n = 529) tissue samples, and two HPV -ve (SCC116 and Hacat) and two HPV +ve (SCC090 and SiHa) cell lines. Methylation-specific PCR (MSP) and chromatin immunoprecipitation assay (CHIP) were performed to validate their regulation. In silico and in vitro analyses of identified miRNAs were done to study putative pathways they target and their possible role in carcinogenesis. Among 10 miRNAs specifically up-regulated in microarray analysis of AZA-treated SCC090 cells, we observed significantly decreased expression of hsa-miR-181c-5p, hsa-miR-132-5p, hsa-miR-658 in HPV +ve HNC cohort, TCGA tissue samples, and cell lines as compared to their HPV -ve counterpart, and their promoter region also possesses CpG islands. MSP and analysis of TCGA data (MethHC) revealed increased frequency of methylation at the promoter of hsa-miR-132-5p that is negatively correlated with its expression. In TSA-treated SCC090 cells, out of 7 miRNAs, two namely Hsa-miR-129-2-3p and Hsa-miR-449a were found to be up-regulated as compared to HPV -ve cells. However, the levels of enrichment by anti-acetyl-H3 and anti-acetyl-H4 were significantly low in cell lines compared to respective controls

  17. Integration of Oncogenes via Sleeping Beauty as a Mouse Model of HPV16+ Oral Tumors and Immunologic Control.

    PubMed

    Lin, Yi-Hsin; Yang, Ming-Chieh; Tseng, Ssu-Hsueh; Jiang, Rosie; Yang, Andrew; Farmer, Emily; Peng, Shiwen; Henkle, Talia; Chang, Yung-Nien; Hung, Chien-Fu; Wu, T-C

    2018-01-23

    Human papillomavirus type 16 (HPV16) is the etiologic factor for cervical cancer and a subset of oropharyngeal cancers. Although several prophylactic HPV vaccines are available, no effective therapeutic strategies to control active HPV diseases exist. Tumor implantation models are traditionally used to study HPV-associated buccal tumors. However, they fail to address precancerous phases of disease progression and display tumor microenvironments distinct from those observed in patients. Previously, K14-E6/E7 transgenic mouse models have been used to generate spontaneous tumors. However, the rate of tumor formation is inconsistent, and the host often develops immune tolerance to the viral oncoproteins. We developed a preclinical, spontaneous, HPV16 + buccal tumor model using submucosal injection of oncogenic plasmids expressing HPV16-E6/E7, NRas G12V , luciferase, and sleeping beauty (SB) transposase, followed by electroporation in the buccal mucosa. We evaluated responses to immunization with a pNGVL4a-CRT/E7(detox) therapeutic HPV DNA vaccine and tumor cell migration to distant locations. Mice transfected with plasmids encoding HPV16-E6/E7, NRas G12V , luciferase, and SB transposase developed tumors within 3 weeks. We also found transient anti-CD3 administration is required to generate tumors in immunocompetent mice. Bioluminescence signals from luciferase correlated strongly with tumor growth, and tumors expressed HPV16-associated markers. We showed that pNGVL4a-CRT/E7(detox) administration resulted in antitumor immunity in tumor-bearing mice. Lastly, we demonstrated that the generated tumor could migrate to tumor-draining lymph nodes. Our model provides an efficient method to induce spontaneous HPV + tumor formation, which can be used to identify effective therapeutic interventions, analyze tumor migration, and conduct tumor biology research. Cancer Immunol Res; 6(3); 1-15. ©2018 AACR. ©2018 American Association for Cancer Research.

  18. HPV-16 and HLA-DRB1 alleles are associated with cervical carcinoma in Mexican Mestizo women.

    PubMed

    Alaez-Verson, Carmen; Berumen-Campos, Jaime; Munguía-Saldaña, Andrea; Flores-Aguilar, Hilario; Guardado-Estrada, Mariano; Rodríguez-Gomez, Araceli; Gorodezky-Lauferman, Clara

    2011-07-01

    The aim of this report was to investigate the contribution of HLA-DRB1/DQB1 alleles to the expression of cervical cancer (CC) and squamous intraepithelial lesion (SIL) in Mexican patients. A total of 257 women were included in the study: 61with low-grade squamous intraepithelial lesions (LSIL), 30 with high-grade (HSIL), 73 with CC and 93 healthy females. All were Mexican Mestizos. For HLA class II typing, PCR-SSOP methodology was used. HPV-16 viral DNA was detected by PCR with specific primers for E6-E7 region. HPV-16 was found in 52% of the patients with CC as well as in 19% of women with HSIL and in 12.5% of females with LSIL. HLA-DRB1∗04:03 (OR = 5.88) was found increased in patients with HSIL as compared with controls, although significance (p = 0.04) was lost after correction (pc =NS). HLA-DRB1∗04:03 seems to influence the risk for developing HSIL, disregarding the presence of HPV-16. HLA-DRB1∗01:01 (OR = 0.12; p = 0.01) may confer protection to the development of CC. An analysis performed stratifying by the presence of HPV-16 infection showed that the frequency of HLA-DRB1∗04:07 (OR = 2.71) was increased in CC patients infected with HPV-16, confirming that the HLA association is HPV dependent. These results shed light on the influence that this virus may have in the expression of CC in the susceptible host. Genetic background is, therefore, a crucial factor in understanding the etiopathogenesis of CC in HPV-positive patients. Copyright © 2011 IMSS. Published by Elsevier Inc. All rights reserved.

  19. Aluminum tolerance in maize is associated with higher MATE1 gene copy number

    PubMed Central

    Maron, Lyza G.; Guimarães, Claudia T.; Kirst, Matias; Albert, Patrice S.; Birchler, James A.; Bradbury, Peter J.; Buckler, Edward S.; Coluccio, Alison E.; Danilova, Tatiana V.; Kudrna, David; Magalhaes, Jurandir V.; Piñeros, Miguel A.; Schatz, Michael C.; Wing, Rod A.; Kochian, Leon V.

    2013-01-01

    Genome structure variation, including copy number variation and presence/absence variation, comprises a large extent of maize genetic diversity; however, its effect on phenotypes remains largely unexplored. Here, we describe how copy number variation underlies a rare allele that contributes to maize aluminum (Al) tolerance. Al toxicity is the primary limitation for crop production on acid soils, which make up 50% of the world’s potentially arable lands. In a recombinant inbred line mapping population, copy number variation of the Al tolerance gene multidrug and toxic compound extrusion 1 (MATE1) is the basis for the quantitative trait locus of largest effect on phenotypic variation. This expansion in MATE1 copy number is associated with higher MATE1 expression, which in turn results in superior Al tolerance. The three MATE1 copies are identical and are part of a tandem triplication. Only three maize inbred lines carrying the three-copy allele were identified from maize and teosinte diversity panels, indicating that copy number variation for MATE1 is a rare, and quite likely recent, event. These maize lines with higher MATE1 copy number are also Al-tolerant, have high MATE1 expression, and originate from regions of highly acidic soils. Our findings show a role for copy number variation in the adaptation of maize to acidic soils in the tropics and suggest that genome structural changes may be a rapid evolutionary response to new environments. PMID:23479633

  20. DR-Integrator: a new analytic tool for integrating DNA copy number and gene expression data.

    PubMed

    Salari, Keyan; Tibshirani, Robert; Pollack, Jonathan R

    2010-02-01

    DNA copy number alterations (CNA) frequently underlie gene expression changes by increasing or decreasing gene dosage. However, only a subset of genes with altered dosage exhibit concordant changes in gene expression. This subset is likely to be enriched for oncogenes and tumor suppressor genes, and can be identified by integrating these two layers of genome-scale data. We introduce DNA/RNA-Integrator (DR-Integrator), a statistical software tool to perform integrative analyses on paired DNA copy number and gene expression data. DR-Integrator identifies genes with significant correlations between DNA copy number and gene expression, and implements a supervised analysis that captures genes with significant alterations in both DNA copy number and gene expression between two sample classes. DR-Integrator is freely available for non-commercial use from the Pollack Lab at http://pollacklab.stanford.edu/ and can be downloaded as a plug-in application to Microsoft Excel and as a package for the R statistical computing environment. The R package is available under the name 'DRI' at http://cran.r-project.org/. An example analysis using DR-Integrator is included as supplemental material. Supplementary data are available at Bioinformatics online.

  1. Copy Number Variation of KIR Genes Influences HIV-1 Control

    PubMed Central

    Shianna, Kevin V.; Feng, Sheng; Urban, Thomas J.; Ge, Dongliang; De Luca, Andrea; Martinez-Picado, Javier; Wolinsky, Steven M.; Martinson, Jeremy J.; Jamieson, Beth D.; Bream, Jay H.; Martin, Maureen P.; Borrow, Persephone; Letvin, Norman L.; McMichael, Andrew J.; Haynes, Barton F.; Telenti, Amalio; Carrington, Mary; Goldstein, David B.; Alter, Galit

    2011-01-01

    A genome-wide screen for large structural variants showed that a copy number variant (CNV) in the region encoding killer cell immunoglobulin-like receptors (KIR) associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses the KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK) cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3DS1 count associates with a lower viral set point if its putative ligand is present (p = 0.00028), as does an increase in KIR3DL1 count in the presence of KIR3DS1 and appropriate ligands for both receptors (p = 0.0015). We further provide functional data that demonstrate that NK cells from individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative amounts of these activating and inhibitory KIR play a role in regulating the peripheral expansion of highly antiviral KIR3DS1+ NK cells, which may determine differences in HIV-1 control following infection. PMID:22140359

  2. Engineered outer membrane vesicle is potent to elicit HPV16E7-specific cellular immunity in a mouse model of TC-1 graft tumor.

    PubMed

    Wang, Shijie; Huang, Weiwei; Li, Kui; Yao, Yufeng; Yang, Xu; Bai, Hongmei; Sun, Wenjia; Liu, Cunbao; Ma, Yanbing

    2017-01-01

    Currently, therapeutic tumor vaccines under development generally lack significant effects in human clinical trials. Exploring a powerful antigen delivery system is a potential approach to improve vaccine efficacy. We sought to explore engineered bacterial outer membrane vesicles (OMVs) as a new vaccine carrier for efficiently delivering tumor antigens and provoking robust antitumor immune responses. First, the tumoral antigen human papillomavirus type 16 early protein E7 (HPV16E7) was presented on Escherichia coli -derived OMVs by genetic engineering methods, acquiring the recombinant OMV vaccine. Second, the ability of recombinant OMVs delivering their components and the model antigen green fluorescent protein to antigen-presenting cells was investigated in the macrophage Raw264.7 cells and in bone marrow-derived dendritic cells in vitro. Third, it was evaluated in TC-1 graft tumor model in mice that the recombinant OMVs displaying HPV16E7 stimulated specific cellular immune response and intervened the growth of established tumor. E. coli DH5α-derived OMVs could be taken up rapidly by dendritic cells, for which vesicle structure has been proven to be important. OMVs significantly stimulated the expression of dendritic cellmaturation markers CD80, CD86, CD83 and CD40. The HPV16E7 was successfully embedded in engineered OMVs through gene recombinant techniques. Subcutaneous immunization with the engineered OMVs induced E7 antigen-specific cellular immune responses, as shown by the increased numbers of interferon-gamma-expressing splenocytes by enzyme-linked immunospot assay and interferon-gamma-expressing CD4 + and CD8 + cells by flow cytometry analyses. Furthermore, the growth of grafted TC-1 tumors in mice was significantly suppressed by therapeutic vaccination. The recombinant E7 proteins presented by OMVs were more potent than those mixed with wild-type OMVs or administered alone for inducing specific cellular immunity and suppressing tumor growth. The results

  3. CpG methylation of HPV 16 LCR at E2 binding site proximal to P97 is associated with cervical cancer in presence of intact E2.

    PubMed

    Bhattacharjee, Bornali; Sengupta, Sharmila

    2006-10-25

    Human papillomavirus type 16 (HPV-16) E2 protein negatively regulates transcription of the E6 and E7 genes. This study was done to test the hypothesis that methylation of the HPV 16 long control region (LCR) is overrepresented among cervical cancer (CaCx) cases compared to cytologically normal controls harboring intact E2 gene. Methylation of the E2 binding site (E2BS-I), proximal to the P97 promoter, was assessed by HpaII/ MspI restriction digestion while McrBC digestion was used to assess LCR-E6 (7289-540) for 57 CaCx samples and 15 normal controls. E2BS-I methylation was found to be significantly higher (56.14%) in cases compared to (20%) controls [OR(age-adjusted) (95% CI): 4.53 (1.05-19.43) p=0.042]. The difference between cases (54.39%) and controls (40%) with respect to LCR-E6 methylation status [OR(age-adjusted) (95% CI): 1.77(0.5-6.3); p=0.38] was not significant. Sequencing of a randomly selected set of 13 methylated malignant samples revealed absence or rare presence, of methylation at CpGs 7579, 7535, 7683 and 7862 respectively. Methylation was found to be more at CpGs within E2 binding sites proximal to the P97 promoter. These results indicate the involvement of E2 binding site methylation in presence of intact E2, leading to loss of E2 repressor activity in CaCx.

  4. Single-Copy Genes as Molecular Markers for Phylogenomic Studies in Seed Plants

    PubMed Central

    De La Torre, Amanda R.; Sterck, Lieven; Cánovas, Francisco M.; Avila, Concepción; Merino, Irene; Cabezas, José Antonio; Cervera, María Teresa; Ingvarsson, Pär K.

    2017-01-01

    Phylogenetic relationships among seed plant taxa, especially within the gymnosperms, remain contested. In contrast to angiosperms, for which several genomic, transcriptomic and phylogenetic resources are available, there are few, if any, molecular markers that allow broad comparisons among gymnosperm species. With few gymnosperm genomes available, recently obtained transcriptomes in gymnosperms are a great addition to identifying single-copy gene families as molecular markers for phylogenomic analysis in seed plants. Taking advantage of an increasing number of available genomes and transcriptomes, we identified single-copy genes in a broad collection of seed plants and used these to infer phylogenetic relationships between major seed plant taxa. This study aims at extending the current phylogenetic toolkit for seed plants, assessing its ability for resolving seed plant phylogeny, and discussing potential factors affecting phylogenetic reconstruction. In total, we identified 3,072 single-copy genes in 31 gymnosperms and 2,156 single-copy genes in 34 angiosperms. All studied seed plants shared 1,469 single-copy genes, which are generally involved in functions like DNA metabolism, cell cycle, and photosynthesis. A selected set of 106 single-copy genes provided good resolution for the seed plant phylogeny except for gnetophytes. Although some of our analyses support a sister relationship between gnetophytes and other gymnosperms, phylogenetic trees from concatenated alignments without 3rd codon positions and amino acid alignments under the CAT + GTR model, support gnetophytes as a sister group to Pinaceae. Our phylogenomic analyses demonstrate that, in general, single-copy genes can uncover both recent and deep divergences of seed plant phylogeny. PMID:28460034

  5. RUBIC identifies driver genes by detecting recurrent DNA copy number breaks

    PubMed Central

    van Dyk, Ewald; Hoogstraat, Marlous; ten Hoeve, Jelle; Reinders, Marcel J. T.; Wessels, Lodewyk F. A.

    2016-01-01

    The frequent recurrence of copy number aberrations across tumour samples is a reliable hallmark of certain cancer driver genes. However, state-of-the-art algorithms for detecting recurrent aberrations fail to detect several known drivers. In this study, we propose RUBIC, an approach that detects recurrent copy number breaks, rather than recurrently amplified or deleted regions. This change of perspective allows for a simplified approach as recursive peak splitting procedures and repeated re-estimation of the background model are avoided. Furthermore, we control the false discovery rate on the level of called regions, rather than at the probe level, as in competing algorithms. We benchmark RUBIC against GISTIC2 (a state-of-the-art approach) and RAIG (a recently proposed approach) on simulated copy number data and on three SNP6 and NGS copy number data sets from TCGA. We show that RUBIC calls more focal recurrent regions and identifies a much larger fraction of known cancer genes. PMID:27396759

  6. EPSPS Gene Copy Number and Whole-Plant Glyphosate Resistance Level in Kochia scoparia

    PubMed Central

    Gaines, Todd A.; Barker, Abigail L.; Patterson, Eric L.; Westra, Philip; Westra, Eric P.; Wilson, Robert G.; Jha, Prashant; Kumar, Vipan

    2016-01-01

    Glyphosate-resistant (GR) Kochia scoparia has evolved in dryland chemical fallow systems throughout North America and the mechanism of resistance involves 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene duplication. Agricultural fields in four states were surveyed for K. scoparia in 2013 and tested for glyphosate-resistance level and EPSPS gene copy number. Glyphosate resistance was confirmed in K. scoparia populations collected from sugarbeet fields in Colorado, Wyoming, and Nebraska, and Montana. Glyphosate resistance was also confirmed in K. scoparia accessions collected from wheat-fallow fields in Montana. All GR samples had increased EPSPS gene copy number, with median population values up to 11 from sugarbeet fields and up to 13 in Montana wheat-fallow fields. The results indicate that glyphosate susceptibility can be accurately diagnosed using EPSPS gene copy number. PMID:27992501

  7. EPSPS Gene Copy Number and Whole-Plant Glyphosate Resistance Level in Kochia scoparia.

    PubMed

    Gaines, Todd A; Barker, Abigail L; Patterson, Eric L; Westra, Philip; Westra, Eric P; Wilson, Robert G; Jha, Prashant; Kumar, Vipan; Kniss, Andrew R

    2016-01-01

    Glyphosate-resistant (GR) Kochia scoparia has evolved in dryland chemical fallow systems throughout North America and the mechanism of resistance involves 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene duplication. Agricultural fields in four states were surveyed for K. scoparia in 2013 and tested for glyphosate-resistance level and EPSPS gene copy number. Glyphosate resistance was confirmed in K. scoparia populations collected from sugarbeet fields in Colorado, Wyoming, and Nebraska, and Montana. Glyphosate resistance was also confirmed in K. scoparia accessions collected from wheat-fallow fields in Montana. All GR samples had increased EPSPS gene copy number, with median population values up to 11 from sugarbeet fields and up to 13 in Montana wheat-fallow fields. The results indicate that glyphosate susceptibility can be accurately diagnosed using EPSPS gene copy number.

  8. Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

    EPA Science Inventory

    Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

  9. Clinicopathological Implications of Human Papilloma Virus (HPV) L1 Capsid Protein Immunoreactivity in HPV16-Positive Cervical Cytology

    PubMed Central

    Lee, Sung-Jong; Lee, Ah-Won; Kang, Chang-Suk; Park, Jong-Sup; Park, Dong-Choon; Ki, Eun-Young; Lee, Keun-Ho; Yoon, Joo-Hee; Hur, Soo-Young; Kim, Tae-Jung

    2014-01-01

    Background: The objective of this study was to investigate the expression of human papilloma virus (HPV) L1 capsid protein in abnormal cervical cytology with HPV16 infection and analyze its association with cervical histopathology in Korean women. Material and Methods: We performed immunocytochemistry for HPV L1 in 475 abnormal cervical cytology samples from patients with HPV16 infections using the Cytoactiv® HPV L1 screening set. We investigated the expression of HPV L1 in cervical cytology samples and compared it with the results of histopathological examination of surgical specimens. Results: Of a total of 475 cases, 188 (39.6%) were immunocytochemically positive and 287 (60.4%) negative for HPV L1. The immunocytochemical expression rates of HPV L1 in atypical squamous cells of unknown significance (ASCUS), low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL), and cancer were 21.8%, 59.7%, 19.1%, and 0.0%, respectively. LSIL exhibited the highest rate of HPV L1 positivity. Of a total of 475 cases, the multiple-type HPV infection rate, including HPV16, in HPV L1-negative cytology samples was 27.5%, which was significantly higher than that in HPV L1-positive cytology samples (p = 0.037). The absence of HPV L1 expression in ASCUS and LSIL was significantly associated with high-grade (≥cervical intraepithelial neoplasia [CIN] 2) than low-grade (≤CIN1) histopathology diagnoses (p < 0.05), but was not significantly different between HPV16 single and multiple-type HPV infections (p > 0.05). On the other hand, among 188 HPV L1-positive cases, 30.6% of multiple-type HPV infections showed high-grade histopathology diagnoses (≥CIN3), significantly higher than the percentage of HPV16 single infections (8.6%) (p = 0.0004) Conclusions: Our study demonstrates that the expression of HPV L1 is low in advanced dysplasia. Furthermore, the absence of HPV L1 in HPV16-positive low-grade cytology (i.e., ASCUS and LSIL) is

  10. Methylation-specific digital karyotyping of HPV16E6E7-expressing human keratinocytes identifies novel methylation events in cervical carcinogenesis.

    PubMed

    Steenbergen, Renske D M; Ongenaert, Maté; Snellenberg, Suzanne; Trooskens, Geert; van der Meide, Wendy F; Pandey, Deeksha; Bloushtain-Qimron, Noga; Polyak, Kornelia; Meijer, Chris J L M; Snijders, Peter J F; Van Criekinge, Wim

    2013-09-01

    Transformation of epithelial cells by high-risk human papillomavirus (hrHPV) types can lead to anogenital carcinomas, particularly cervical cancer, and oropharyngeal cancers. This process is associated with DNA methylation alterations, often affecting tumour suppressor gene expression. This study aimed to comprehensively unravel genome-wide DNA methylation events linked to a transforming hrHPV-infection, which is driven by deregulated expression of the viral oncogenes E6 and E7 in dividing cells. Primary human keratinocytes transduced with HPV16E6E7 and their untransduced counterparts were subjected to methylation-specific digital karyotyping (MSDK) to screen for genome-wide DNA-methylation changes at different stages of HPV-induced transformation. Integration of the obtained methylation profiles with genome-wide gene expression patterns of cervical carcinomas identified 34 genes with increased methylation in HPV-transformed cells and reduced expression in cervical carcinomas. For 12 genes (CLIC3, CREB3L1, FAM19A4, LFNG, LHX1, MRC2, NKX2-8, NPTX-1, PHACTR3, PRDM14, SOST and TNFSF13) specific methylation in HPV-containing cell lines was confirmed by semi-quantitative methylation-specific PCR. Subsequent analysis of FAM19A4, LHX1, NKX2-8, NPTX-1, PHACTR3 and PRDM14 in cervical tissue specimens showed increasing methylation levels for all genes with disease progression. All six genes were frequently methylated in cervical carcinomas, with highest frequencies (up to 100%) seen for FAM19A4, PHACTR3 and PRDM14. Analysis of hrHPV-positive cervical scrapes revealed significantly increased methylation levels of the latter three genes in women with high-grade cervical disease compared to controls. In conclusion, MSDK analysis of HPV16-transduced keratinocytes at different stages of HPV-induced transformation resulted in the identification of novel DNA methylation events, involving FAM19A4, LHX1, NKX2-8, PHACTR3 and PRDM14 genes in cervical carcinogenesis. These genes may

  11. Reduced Prevalence of Vulvar HPV16/18 Infection Among Women Who Received the HPV16/18 Bivalent Vaccine: A Nested Analysis Within the Costa Rica Vaccine Trial

    PubMed Central

    Lang Kuhs, Krystle A.; Gonzalez, Paula; Rodriguez, Ana Cecilia; van Doorn, Leen-Jan; Schiffman, Mark; Struijk, Linda; Chen, Sabrina; Quint, Wim; Lowy, Douglas R.; Porras, Carolina; DelVecchio, Corey; Jimenez, Silvia; Safaeian, Mahboobeh; Schiller, John T.; Wacholder, Sholom; Herrero, Rolando; Hildesheim, Allan; Kreimer, Aimée R.

    2014-01-01

    Background Vaccine efficacy (VE) against vulvar human papillomavirus (HPV) infection has not been reported and data regarding its epidemiology are sparse. Methods Women (n = 5404) age 22–29 present at the 4-year study visit of the Costa Rica Vaccine Trial provided vulvar and cervical samples. A subset (n = 1044) was tested for HPV DNA (SPF10/LiPA25 version 1). VE against 1-time detection of vulvar HPV16/18 among HPV vaccinated versus unvaccinated women was calculated and compared to the cervix. Prevalence of and risk factors for HPV were evaluated in the control arm (n = 536). Results Vulvar HPV16/18 VE (54.1%; 95% confidence interval [CI], 4.9%–79.1%) was comparable to cervix (45.8%; 95% CI, 6.4%–69.4%). Vulvar and cervical HPV16 prevalence within the control arm was 3.0% and 4.7%, respectively. Independent risk factors for vulvar HPV were similar to cervix and included: age (adjusted odds ratio [aOR] 0.5 [95% CI, .3–.9] ≥28 vs 22–23]); marital status (aOR 2.3 [95% CI, 1.5–3.5] single vs married/living-as-married); and number of sexual partners (aOR 3.6 [95% CI, 1.9–7.0] ≥6 vs 1). Conclusions In this intention-to-treat analysis, VE against vulvar and cervical HPV16/18 were comparable 4 years following vaccination. Risk factors for HPV were similar by anatomic site. Clinical Trials Registration NCT00128661. PMID:24958910

  12. Reduced prevalence of vulvar HPV16/18 infection among women who received the HPV16/18 bivalent vaccine: a nested analysis within the Costa Rica Vaccine Trial.

    PubMed

    Lang Kuhs, Krystle A; Gonzalez, Paula; Rodriguez, Ana Cecilia; van Doorn, Leen-Jan; Schiffman, Mark; Struijk, Linda; Chen, Sabrina; Quint, Wim; Lowy, Douglas R; Porras, Carolina; DelVecchio, Corey; Jimenez, Silvia; Safaeian, Mahboobeh; Schiller, John T; Wacholder, Sholom; Herrero, Rolando; Hildesheim, Allan; Kreimer, Aimée R

    2014-12-15

    Vaccine efficacy (VE) against vulvar human papillomavirus (HPV) infection has not been reported and data regarding its epidemiology are sparse. Women (n = 5404) age 22-29 present at the 4-year study visit of the Costa Rica Vaccine Trial provided vulvar and cervical samples. A subset (n = 1044) was tested for HPV DNA (SPF10/LiPA25 version 1). VE against 1-time detection of vulvar HPV16/18 among HPV vaccinated versus unvaccinated women was calculated and compared to the cervix. Prevalence of and risk factors for HPV were evaluated in the control arm (n = 536). Vulvar HPV16/18 VE (54.1%; 95% confidence interval [CI], 4.9%-79.1%) was comparable to cervix (45.8%; 95% CI, 6.4%-69.4%). Vulvar and cervical HPV16 prevalence within the control arm was 3.0% and 4.7%, respectively. Independent risk factors for vulvar HPV were similar to cervix and included: age (adjusted odds ratio [aOR] 0.5 [95% CI, .3-.9] ≥28 vs 22-23]); marital status (aOR 2.3 [95% CI, 1.5-3.5] single vs married/living-as-married); and number of sexual partners (aOR 3.6 [95% CI, 1.9-7.0] ≥6 vs 1). In this intention-to-treat analysis, VE against vulvar and cervical HPV16/18 were comparable 4 years following vaccination. Risk factors for HPV were similar by anatomic site. NCT00128661. Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  13. Extensive Copy-Number Variation of Young Genes across Stickleback Populations

    PubMed Central

    Eizaguirre, Christophe; Samonte, Irene E.; Kalbe, Martin; Lenz, Tobias L.; Stoll, Monika; Bornberg-Bauer, Erich; Milinski, Manfred; Reusch, Thorsten B. H.

    2014-01-01

    Duplicate genes emerge as copy-number variations (CNVs) at the population level, and remain copy-number polymorphic until they are fixed or lost. The successful establishment of such structural polymorphisms in the genome plays an important role in evolution by promoting genetic diversity, complexity and innovation. To characterize the early evolutionary stages of duplicate genes and their potential adaptive benefits, we combine comparative genomics with population genomics analyses to evaluate the distribution and impact of CNVs across natural populations of an eco-genomic model, the three-spined stickleback. With whole genome sequences of 66 individuals from populations inhabiting three distinct habitats, we find that CNVs generally occur at low frequencies and are often only found in one of the 11 populations surveyed. A subset of CNVs, however, displays copy-number differentiation between populations, showing elevated within-population frequencies consistent with local adaptation. By comparing teleost genomes to identify lineage-specific genes and duplications in sticklebacks, we highlight rampant gene content differences among individuals in which over 30% of young duplicate genes are CNVs. These CNV genes are evolving rapidly at the molecular level and are enriched with functional categories associated with environmental interactions, depicting the dynamic early copy-number polymorphic stage of genes during population differentiation. PMID:25474574

  14. Copy number polymorphism of the salivary amylase gene: implications in human nutrition research.

    PubMed

    Santos, J L; Saus, E; Smalley, S V; Cataldo, L R; Alberti, G; Parada, J; Gratacòs, M; Estivill, X

    2012-01-01

    The salivary α-amylase is a calcium-binding enzyme that initiates starch digestion in the oral cavity. The α-amylase genes are located in a cluster on the chromosome that includes salivary amylase genes (AMY1), two pancreatic α-amylase genes (AMY2A and AMY2B) and a related pseudogene. The AMY1 genes show extensive copy number variation which is directly proportional to the salivary α-amylase content in saliva. The α-amylase amount in saliva is also influenced by other factors, such as hydration status, psychosocial stress level, and short-term dietary habits. It has been shown that the average copy number of AMY1 gene is higher in populations that evolved under high-starch diets versus low-starch diets, reflecting an intense positive selection imposed by diet on amylase copy number during evolution. In this context, a number of different aspects can be considered in evaluating the possible impact of copy number variation of the AMY1 gene on nutrition research, such as issues related to human diet gene evolution, action on starch digestion, effect on glycemic response after starch consumption, modulation of the action of α-amylases inhibitors, effect on taste perception and satiety, influence on psychosocial stress and relation to oral health. Copyright © 2012 S. Karger AG, Basel.

  15. E6/E7 oncoproteins of high risk HPV-16 upregulate MT1-MMP, MMP-2 and MMP-9 and promote the migration of cervical cancer cells.

    PubMed

    Zhu, Dingjun; Ye, Mei; Zhang, Wei

    2015-01-01

    E6 and E7 of high risk human papillomavirus 16 (HPV16) were reported to correlate with the cervical cancer (CC). And the presence of matrix metalloproteinases (MMPs) has also been indicated to be associated with CC. The present study investigated the expression of MMPs (MT1-MMP, MMP-2 and MMP-9) in CC cells with HPV16-E6/E7 oncoprotein(s) negative or positive, and then determined the regulation of HPV16-E6/E7 oncoproteins on the expression of MMPs (MT1-MMP, MMP-2 and MMP-9) and the migration of cervical cancer Caski and SiHa cells with RNAi technology. It was demonstrated that the overexpression or the knockdown of HPV16-E6/E7 promoted or reduced MT1-MMP, MMP-2 and MMP-9 in CC cells. And the HPV16-E6, -E7 or -E6E7 influenced the migration of CC cells. The overexpression or the knockdown of them promoted or inhibited the migration of C33A or Caski/SiHa cells. Moreover, the chemical inhibition of MMP-2 or MMP-9 significantly reduced the migration of CC Caski or SiHa cells. Our results demonstrated that the E6-HPV16 or E7-HPV16 promoted the activity of MMP-2/9, and contributed to the migration of cervical cells.

  16. A Phase I Trial of a Human Papillomavirus (HPV) DNA Vaccine for HPV16+ Cervical Intraepithelial Neoplasia 2/3

    PubMed Central

    Trimble, Cornelia L.; Peng, Shiwen; Kos, Ferdynand; Gravitt, Patti; Viscidi, Raphael; Sugar, Elizabeth; Pardoll, Drew; Wu, TC

    2010-01-01

    Purpose: To evaluate the safety and immunogenicity of a therapeutic HPV16 DNA vaccine administered to women with HPV16+CIN2/3. Experimental Design: This phase I trial incorporated the standard ‘3+3” dose escalation design with an additional 6 patients allocated to the maximally tolerated dose (MTD). Healthy adult women with colposcopically-directed biopsy-proven HPV16+ CIN2/3 received three intramuscular (IM) vaccinations (0.5 mg, 1 mg, or 3mg) of a plasmid expressing a Sig-E7(detox)-HSP70 fusion protein on days 0, 28 and 56, and underwent standard therapeutic resection of the cervical squamocolumnar junction at day 105 (week 15). Safety and immunogenicity of the vaccine and histologic outcome based on resection at week 15 were assessed. Results: Fifteen patients were evaluable (3 each at 0.5 mg and 1mg, 9 at 3mg). The vaccine was well tolerated: most adverse events were mild transient injection-site discomfort; no dose-limiting toxicities were observed. Although HPVE7-specific T-cell responses to E7 detected by enzyme-linked immunospot assays (IFNγ) were of low frequency and magnitude, detectable increases in response subsequent to vaccination were identified in subjects in the second and third cohorts. Complete histologic regression occurred in 3/9 (33%, CI: 7%-70%)) individuals in the highest dose cohort, Although the difference is not significant, it is slightly higher than would be expected in an unvaccinated cohort (25%). Conclusions: This HPV16 DNA vaccine was safe and well tolerated. While it appears possible to elicit HPV-specific T cell responses in patients with established dysplastic lesions, other factors are likely to play a role in lesion regression. PMID:19118066

  17. Gefitinib and EGFR Gene Copy Number Aberrations in Esophageal Cancer.

    PubMed

    Petty, Russell D; Dahle-Smith, Asa; Stevenson, David A J; Osborne, Aileen; Massie, Doreen; Clark, Caroline; Murray, Graeme I; Dutton, Susan J; Roberts, Corran; Chong, Irene Y; Mansoor, Wasat; Thompson, Joyce; Harrison, Mark; Chatterjee, Anirban; Falk, Stephen J; Elyan, Sean; Garcia-Alonso, Angel; Fyfe, David Walter; Wadsley, Jonathan; Chau, Ian; Ferry, David R; Miedzybrodzka, Zosia

    2017-07-10

    Purpose The Cancer Esophagus Gefitinib trial demonstrated improved progression-free survival with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib relative to placebo in patients with advanced esophageal cancer who had disease progression after chemotherapy. Rapid and durable responses were observed in a minority of patients. We hypothesized that genetic aberration of the EGFR pathway would identify patients benefitting from gefitinib. Methods A prespecified, blinded molecular analysis of Cancer Esophagus Gefitinib trial tumors was conducted to compare efficacy of gefitinib with that of placebo according to EGFR copy number gain (CNG) and EGFR, KRAS, BRAF, and PIK3CA mutation status. EGFR CNG was determined by fluorescent in situ hybridization (FISH) using prespecified criteria and EGFR FISH-positive status was defined as high polysomy or amplification. Results Biomarker data were available for 340 patients. In EGFR FISH-positive tumors (20.2%), overall survival was improved with gefitinib compared with placebo (hazard ratio [HR] for death, 0.59; 95% CI, 0.35 to 1.00; P = .05). In EGFR FISH-negative tumors, there was no difference in overall survival with gefitinib compared with placebo (HR for death, 0.90; 95% CI, 0.69 to 1.18; P = .46). Patients with EGFR amplification (7.2%) gained greatest benefit from gefitinib (HR for death, 0.21; 95% CI, 0.07 to 0.64; P = .006). There was no difference in overall survival for gefitinib versus placebo for patients with EGFR, KRAS, BRAF, and PIK3CA mutations, or for any mutation versus none. Conclusion EGFR CNG assessed by FISH appears to identify a subgroup of patients with esophageal cancer who may benefit from gefitinib as a second-line treatment. Results of this study suggest that anti-EGFR therapies should be investigated in prospective clinical trials in different settings in EGFR FISH-positive and, in particular, EGFR-amplified esophageal cancer.

  18. Genotyping for Human Papillomavirus (HPV) 16/18/52/58 Has a Higher Performance than HPV16/18 Genotyping in Triaging Women with Positive High-risk HPV Test in Northern Thailand

    PubMed Central

    Khunamornpong, Surapan; Settakorn, Jongkolnee; Sukpan, Kornkanok; Suprasert, Prapaporn; Srisomboon, Jatupol; Intaraphet, Suthida; Siriaunkgul, Sumalee

    2016-01-01

    Background Testing for high-risk human papillomavirus DNA (HPV test) has gained increasing acceptance as an alternative method to cytology in cervical cancer screening. Compared to cytology, HPV test has a higher sensitivity for the detection of histologic high-grade squamous intraepithelial lesion or worse (HSIL+), but this could lead to a large colposcopy burden. Genotyping for HPV16/18 has been recommended in triaging HPV-positive women. This study was aimed to evaluate the screening performance of HPV testing and the role of genotyping triage in Northern Thailand. Methods A population-based cervical screening program was performed in Chiang Mai (Northern Thailand) using cytology (conventional Pap test) and HPV test (Hybrid Capture 2). Women who had abnormal cytology or were HPV-positive were referred for colposcopy. Cervical samples from these women were genotyped using the Linear Array assay. Results Of 5,456 women, 2.0% had abnormal Pap test results and 6.5% tested positive with Hybrid Capture 2. Of 5,433 women eligible for analysis, 355 with any positive test had histologic confirmation and 57 of these had histologic HSIL+. The sensitivity for histologic HSIL+ detection was 64.9% for Pap test and 100% for Hybrid Capture 2, but the ratio of colposcopy per detection of each HSIL+ was more than two-fold higher with Hybrid Capture 2 than Pap test (5.9 versus 2.8). Genotyping results were available in 316 samples. HPV52, HPV16, and HPV58 were the three most common genotypes among women with histologic HSIL+. Performance of genotyping triage using HPV16/18/52/58 was superior to that of HPV16/18, with a higher sensitivity (85.7% versus 28.6%) and negative predictive value (94.2% versus 83.9%). Conclusions In Northern Thailand, HPV testing with genotyping triage shows better screening performance than cervical cytology alone. In this region, the addition of genotyping for HPV52/58 to HPV16/18 is deemed necessary in triaging women with positive HPV test. PMID

  19. GeneCount: genome-wide calculation of absolute tumor DNA copy numbers from array comparative genomic hybridization data

    PubMed Central

    Lyng, Heidi; Lando, Malin; Brøvig, Runar S; Svendsrud, Debbie H; Johansen, Morten; Galteland, Eivind; Brustugun, Odd T; Meza-Zepeda, Leonardo A; Myklebost, Ola; Kristensen, Gunnar B; Hovig, Eivind; Stokke, Trond

    2008-01-01

    Absolute tumor DNA copy numbers can currently be achieved only on a single gene basis by using fluorescence in situ hybridization (FISH). We present GeneCount, a method for genome-wide calculation of absolute copy numbers from clinical array comparative genomic hybridization data. The tumor cell fraction is reliably estimated in the model. Data consistent with FISH results are achieved. We demonstrate significant improvements over existing methods for exploring gene dosages and intratumor copy number heterogeneity in cancers. PMID:18500990

  20. HPV16-E7 Expression in skin induces TSLP secretion, type 2 ILC infiltration and atopic dermatitis-like lesions

    PubMed Central

    Bergot, Anne-Sophie; Monnet, Nastasia; Tran, Le Son; Mittal, Deepak; Al-Kouba, Jane; Steptoe, Raymond J.; Grimbaldeston, Michele A.; Frazer, Ian H.; Wells, James W.

    2014-01-01

    Atopic dermatitis is a common pruritic and inflammatory skin disorder with unknown etiology. Most commonly occurring during early childhood, atopic dermatitis is associated with eczematous lesions and lichenification, in which the epidermis becomes hypertrophied resulting in thickening of the skin. In this study, we report an atopic dermatitis-like pathophysiology results in a murine model following the expression of the high-risk Human Papillomavirus (HPV) 16 oncoprotein E7 in keratinocytes under the Keratin 14 promoter. We show that HPV 16 E7 expression in the skin is associated with skin thickening, acanthosis and light spongiosis. Locally, HPV 16 E7 expressing skin secreted high levels of TSLP and contained increased numbers of ILCs. High levels of circulating IgE were associated with increased susceptibility to skin allergy in a model of cutaneous challenge, and to airway bronchiolar inflammation, enhanced airway goblet cell metaplasia and mucus production in a model of atopic march. Surprisingly, skin pathology occurred independently of T-cells and mast cells. Thus, our findings suggest that the expression of a single HPV oncogene in the skin can drive the onset of atopic dermatitis-like pathology through the induction of TSLP and type 2 ILC infiltration. PMID:25601274

  1. Studies towards the potential of poliovirus as a vector for the expression of HPV 16 virus-like-particles.

    PubMed

    van Kuppeveld, Frank J M; de Jong, Arjan; Dijkman, Henri B P M; Andino, Raul; Melchers, Willem J G

    2002-11-15

    Development of human cervical carcinomas is associated with infection by certain human papillomavirus (HPV) types. Thus, protection against HPV infection through vaccination may prevent development of cervical cancer. The purpose of this study was to investigate the possibility of using a poliovirus recombinant vector to induce immunity against HPV. A poliovirus recombinant was constructed which contained the complete coding sequence of the HPV 16 major capsid protein L1, between the P1 and P2 region of the poliovirus polyprotein. A replication-competent virus was obtained after transfection of the recombinant RNA into tissue culture cells. Electron microscopically examination of cells infected with the poliovirus-HPV L1 recombinant indicated that HPV 16 L1 self-assembles into virus-like particles. To investigate the immunological response in vivo, susceptible transgenic mice carrying the poliovirus receptor were infected with the recombinant poliovirus. In all mice a modest but consistent immune response against HPV 16 was observed. Based on these results, the potential for picornavirus-derived vectors in vaccine development against HPV infection is discussed.

  2. Clinical and epidemiological correlations between the infection with HPV 16 and HPV 18 and female cervical lesions.

    PubMed

    Stoian, M; Repanovici, R; Corniţescu, F

    1995-01-01

    A number of 66 specimens from female cervical lesions were examined for infection with human papillomavirus (HPV) types 6, 11, 16, and 18 by nucleic acid hybridization in dot-blot techniques and 35 sera were tested by the immunodot-blot technique, in order to detect the presence of anti E4 and E7 HPV protein antibodies. The findings were compared with the histologic diagnosis. Fifty-six per cent of specimens contained HPV DNA sequences. In 47% of specimens from cervical carcinoma, HPV 11 was detected in 4 cases, HPV 16 in 21 cases, and HPV 18 in 7 cases. Serum antibodies against HPV 16 E4 and HPV 16 E7 occurred in all the cases of uterine carcinoma, in 4 of 10 cases of CIN I-II, and in 3 of 5 sera obtained from apparently healthy women. The analysis of risk factors disclosed the early onset of sexual activity, a relatively high number of births and abortions before the age of 22 years, the use of oral oestroprogestative contraceptive agents, the presence in anamnesis of genital infections with bacterial flora--Candida albicans, Trichomonas vaginalis, Chlamydia trachomatis, Mycoplasma, etc. Our results showed that HPV typing by nucleic acid hybridization was useful for differentiating low- from high-risk cervical lesions and also tried to elucidate the risk factors associated with HPV infections and progression to malignancy.

  3. HPV16 induces a wasting syndrome in transgenic mice: Amelioration by dietary polyphenols via NF-κB inhibition.

    PubMed

    Gil da Costa, Rui M; Aragão, Sofia; Moutinho, Magda; Alvarado, Antonieta; Carmo, Diogo; Casaca, Fátima; Silva, Sandra; Ribeiro, Joana; Sousa, Hugo; Ferreira, Rita; Nogueira-Ferreira, Rita; Pires, Maria João; Colaço, Bruno; Medeiros, Rui; Venâncio, Carlos; Oliveira, Maria Manuel; Bastos, Margarida M S M; Lopes, Carlos; Oliveira, Paula A

    2017-01-15

    Cancer patients often show a wasting syndrome for which there are little therapeutic options. Dietary polyphenols have been proposed for treating this syndrome, but their usefulness in cases associated with human papillomavirus (HPV)-induced cancers is unknown. We characterized HPV16-transgenic mice as a model of cancer cachexia and tested the efficacy of long-term oral supplementation with polyphenols curcumin and rutin. Both compounds were orally administered to six weeks-old HPV16-transgenic mice showing characteristic multi-step skin carcinogenesis, for 24weeks. Skin lesions and blood, liver and spleen inflammatory changes were characterized histologically and hematologically. Hepatic oxidative stress, skeletal muscle mass and the levels of muscle pro-inflammatory transcription factor NF-κB were also assessed. Skin carcinogenesis was associated with progressive, severe, systemic inflammation (leukocytosis, hepatitis, splenitis), significant mortality and cachexia. Curcumin and rutin totally suppressed mortality while reducing white blood cells and the incidence of splenitis and hepatitis. Rutin prevented muscle wasting more effectively than curcumin. Preservation of muscle mass and reduced hepatic inflammation were associated with down-regulation of the NF-κB canonical pathway and with reduced oxidative stress, respectively. These results point out HPV16-transgenic mice as a useful model for studying the wasting syndrome associated with HPV-induced cancers. Dietary NF-κB inhibitors may be useful resources for treating this syndrome. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Health and economic impact of HPV 16 and 18 vaccination and cervical cancer screening in India

    PubMed Central

    Diaz, M; Kim, J J; Albero, G; de Sanjosé, S; Clifford, G; Bosch, F X; Goldie, S J

    2008-01-01

    Cervical cancer is a leading cause of cancer death among women in low-income countries, with ∼25% of cases worldwide occurring in India. We estimated the potential health and economic impact of different cervical cancer prevention strategies. After empirically calibrating a cervical cancer model to country-specific epidemiologic data, we projected cancer incidence, life expectancy, and lifetime costs (I$2005), and calculated incremental cost-effectiveness ratios (I$/YLS) for the following strategies: pre-adolescent vaccination of girls before age 12, screening of women over age 30, and combined vaccination and screening. Screening differed by test (cytology, visual inspection, HPV DNA testing), number of clinical visits (1, 2 or 3), frequency (1 × , 2 × , 3 × per lifetime), and age range (35–45). Vaccine efficacy, coverage, and costs were varied in sensitivity analyses. Assuming 70% coverage, mean reduction in lifetime cancer risk was 44% (range, 28–57%) with HPV 16,18 vaccination alone, and 21–33% with screening three times per lifetime. Combining vaccination and screening three times per lifetime provided a mean reduction of 56% (vaccination plus 3-visit conventional cytology) to 63% (vaccination plus 2-visit HPV DNA testing). At a cost per vaccinated girl of I$10 (per dose cost of $2), pre-adolescent vaccination followed by screening three times per lifetime using either VIA or HPV DNA testing, would be considered cost-effective using the country's per capita gross domestic product (I$3452) as a threshold. In India, if high coverage of pre-adolescent girls with a low-cost HPV vaccine that provides long-term protection is achievable, vaccination followed by screening three times per lifetime is expected to reduce cancer deaths by half, and be cost-effective. PMID:18612311

  5. Immortalization of human prostate epithelial cells by HPV 16 E6/E7 open reading frames.

    PubMed

    Choo, C K; Ling, M T; Chan, K W; Tsao, S W; Zheng, Z; Zhang, D; Chan, L C; Wong, Y C

    1999-08-01

    The exact pathogenesis for prostate cancer is not known. Progress made in prostate cancer research has been slow, largely due to the lack of suitable in vitro models. Here, we report our work on the immortalization of a human prostate epithelial cell line and show that it can be used as a model to study prostate tumorigenesis. Replication-defective retrovirus harboring the human papillomavirus (HPV) type 16 E6 and E7 open reading frames was used to infect primary human prostate epithelial cells. Polymerase chain reaction, followed by Southern hybridization for the HPV 16 E6/E7, Western blot for prostatic acid phosphatase, telomeric repeat amplification protocol assay for telomerase activity, two-dimensional gels for cytokeratins, and cytogenetic analysis were undertaken to characterized the infected cells. The retrovirus-infected cell line, HPr-1, continued to grow in culture for more than 80 successive passages. Normal primary cells failed to proliferate after passage 6. HPr-1 cells bore close resemblance to normal primary prostate epithelial cells, both morphologically and biochemically. However, they possessed telomerase activity and proliferated indefinitely. Cytogenetic analysis of HPr-1 cells revealed a human male karyotype with clonal abnormalities and the appearance of multiple double minutes. The HPr-1 cells expressed prostatic acid phosphatase and cytokeratins K8 and K18, proving that they were prostate epithelial cells. They were benign in nude mice tumor formation and soft agar colony formation assay. The HPr-1 cell line is an in vitro representation of early prostate neoplastic progression. Copyright 1999 Wiley-Liss, Inc.

  6. ALK gene copy number gain and immunohistochemical expression status using three antibodies in neuroblastoma.

    PubMed

    Kim, Eun Kyung; Kim, Sewha

    2016-03-17

    Anaplastic lymphoma kinase (ALK) gene aberrations-such as mutations, amplifications, and copy number gains-represent a major genetic predisposition to neuroblastoma (NB). This study aimed to evaluate the correlation between ALK gene copy number status, ALK protein expression, and clinicopathological parameters. We retrospectively retrieved 30 cases of poorly differentiated NB and constructed tissue microarrays (TMAs). ALK copy number changes were assessed by fluorescence in situ hybridization (FISH) assays, and ALK immunohistochemistry (IHC) testing was performed using three different antibodies (ALK1, D5F3, and 5A4 clones). ALK amplification and copy number gain were observed in 10% (3/30) and 53.3% (16/30) of the cohort, respectively. There were positive correlations between ALK copy number and IHC positive rate in ALK1 and 5A4 antibodies (p= < 0.001 and 0.019, respectively). ALK1, D5F3, and 5A4 antibodies equally showed 100% sensitivity in detecting ALK amplification. However, the sensitivity for detecting copy number gain differed among the three antibodies, with 75% sensitivity in D5F3 and 0% sensitivity in ALK1. ALK-amplified NBs were correlated with synchronous MYCN amplification and chromosome 1p deletion. ALK IHC positivity was frequently observed in INSS stage IV and high-risk group patients. In conclusion, this study identified that an increase in the ALK copy number is a frequent genetic alteration in poorly differentiated NB. ALK-amplified NBs showed consistent ALK IHC positivity with all kinds of antibodies. In contrast, the detection performance of ALK copy number gain was antibody dependent, with the D5F3 antibody showing the best sensitivity.

  7. ALK Gene Copy Number Gain and Immunohistochemical Expression Status Using Three Antibodies in Neuroblastoma.

    PubMed

    Kim, Eun Kyung; Kim, Sewha

    2017-01-01

    Anaplastic lymphoma kinase ( ALK) gene aberrations-such as mutations, amplifications, and copy number gains-represent a major genetic predisposition to neuroblastoma (NB). This study aimed to evaluate the correlation between ALK gene copy number status, ALK protein expression, and clinicopathological parameters. We retrospectively retrieved 30 cases of poorly differentiated NB and constructed tissue microarrays (TMAs). ALK copy number changes were assessed by fluorescence in situ hybridization (FISH) assays, and ALK immunohistochemistry (IHC) testing was performed using three different antibodies (ALK1, D5F3, and 5A4 clones). ALK amplification and copy number gain were observed in 10% (3/30) and 53.3% (16/30) of the cohort, respectively. There were positive correlations between ALK copy number and IHC-positive rate in ALK1 and 5A4 antibodies ( P < 0.001 and P = 0.019, respectively). ALK1, D5F3, and 5A4 antibodies equally showed 100% sensitivity in detecting ALK amplification. However, the sensitivity for detecting copy number gain differed among the three antibodies, with 75% sensitivity in D5F3 and 0% sensitivity in ALK1. ALK-amplified NBs were correlated with synchronous MYCN amplification and chromosome 1p deletion. ALK IHC positivity was frequently observed in INSS stage IV and high-risk group patients. In conclusion, this study identified that an increase in the ALK copy number is a frequent genetic alteration in poorly differentiated NB. ALK-amplified NBs showed consistent ALK IHC positivity with all kinds of antibodies. In contrast, the detection performance of ALK copy number gain was antibody dependent, with the D5F3 antibody showing the best sensitivity.

  8. HPV16 and 18 genome amplification show different E4-dependence, with 16E4 enhancing E1 nuclear accumulation and replicative efficiency via its cell cycle arrest and kinase activation functions

    PubMed Central

    Jackson, Deborah; Mahmood, Radma

    2017-01-01

    To clarify E1^E4’s role during high-risk HPV infection, the E4 proteins of HPV16 and 18 were compared side by side using an isogenic keratinocyte differentiation model. While no effect on cell proliferation or viral genome copy number was observed during the early phase of either virus life cycle, time-course experiments showed that viral genome amplification and L1 expression were differently affected upon differentiation, with HPV16 showing a much clearer E4 dependency. Although E4 loss never completely abolished genome amplification, its more obvious contribution in HPV16 focused our efforts on 16E4. As previously suggested, in the context of the virus life cycle, 16E4s G2-arrest capability was found to contribute to both genome amplification success and L1 accumulation. Loss of 16E4 also lead to a reduced maintenance of ERK, JNK and p38MAPK activity throughout the genome amplifying cell layers, with 16E4 (but not 18E4) co-localizing precisely with activated cytoplasmic JNK in both wild type raft tissue, and HPV16-induced patient biopsy tissue. When 16E1 was co-expressed with E4, as occurs during genome amplification in vivo, the E1 replication helicase accumulated preferentially in the nucleus, and in transient replication assays, E4 stimulated viral genome amplification. Interestingly, a 16E1 mutant deficient in its regulatory phosphorylation sites no longer accumulated in the nucleus following E4 co-expression. E4-mediated stabilisation of 16E2 was also apparent, with E2 levels declining in organotypic raft culture when 16E4 was absent. These results suggest that 16E4-mediated enhancement of genome amplification involves its cell cycle inhibition and cellular kinase activation functions, with E4 modifying the activity and function of viral replication proteins including E1. These activities of 16E4, and the different kinase patterns seen here with HPV18, 31 and 45, may reflect natural differences in the biology and tropisms of these viruses, as well as

  9. A comparative genomic hybridization approach to study gene copy number variations among Chinese hamster cell lines.

    PubMed

    Vishwanathan, Nandita; Bandyopadhyay, Arpan; Fu, Hsu-Yuan; Johnson, Kathryn C; Springer, Nathan M; Hu, Wei-Shou

    2017-08-01

    Chinese Hamster Ovary (CHO) cells are aneuploid in nature. The genome of recombinant protein producing CHO cell lines continuously undergoes changes in its structure and organization. We analyzed nine cell lines, including parental cell lines, using a comparative genomic hybridization (CGH) array focused on gene-containing regions. The comparison of CGH with copy-number estimates from sequencing data showed good correlation. Hierarchical clustering of the gene copy number variation data from CGH data revealed the lineage relationships between the cell lines. On analyzing the clones of a clonal population, some regions with altered genomic copy number status were identified indicating genomic changes during passaging. A CGH array is thus an effective tool in quantifying genomic alterations in industrial cell lines and can provide insights into the changes in the genomic structure during cell line derivation and long term culture. Biotechnol. Bioeng. 2017;114: 1903-1908. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  10. Engineered promoters enable constant gene expression at any copy number in bacteria.

    PubMed

    Segall-Shapiro, Thomas H; Sontag, Eduardo D; Voigt, Christopher A

    2018-04-01

    The internal environment of growing cells is variable and dynamic, making it difficult to introduce reliable parts, such as promoters, for genetic engineering. Here, we applied control-theoretic ideas to design promoters that maintained constant levels of expression at any copy number. Theory predicts that independence to copy number can be achieved by using an incoherent feedforward loop (iFFL) if the negative regulation is perfectly non-cooperative. We engineered iFFLs into Escherichia coli promoters using transcription-activator-like effectors (TALEs). These promoters had near-identical expression in different genome locations and plasmids, even when their copy number was perturbed by genomic mutations or changes in growth medium composition. We applied the stabilized promoters to show that a three-gene metabolic pathway to produce deoxychromoviridans could retain function without re-tuning when the stabilized-promoter-driven genes were moved from a plasmid into the genome.

  11. Copy number analysis reveals a novel multiexon deletion of the COLQ gene in congenital myasthenia.

    PubMed

    Wang, Wei; Wu, Yanhong; Wang, Chen; Jiao, Jinsong; Klein, Christopher J

    2016-12-01

    Congenital myasthenic syndrome (CMS) is genetically and clinically heterogeneous. 1 Despite a considerable number of causal genes discovered, many patients are left without a specific diagnosis after genetic testing. The presumption is that novel genes yet to be discovered will account for the majority of such patients. However, it is also possible that we are neglecting a type of genetic variation: copy number changes (>50 bp) as causal for some of these patients. Next-generation sequencing (NGS) can simultaneously screen all known causal genes 2 and is increasingly being validated to have a potential to identify copy number changes. 3 We present a CMS case who did not receive a genetic diagnosis from previous Sanger sequencing, but through a novel copy number analysis algorithm integrated into our targeted NGS panel, we discovered a novel copy number mutation in the COLQ gene and made a genetic diagnosis. This discovery expands the genotype-phenotype correlation of CMS, leads to improved genetic counsel, and allows for specific pharmacologic treatment. 1 .

  12. Diversity in copy number and structure of a silkworm morphogenetic gene as a result of domestication.

    PubMed

    Sakudoh, Takashi; Nakashima, Takeharu; Kuroki, Yoko; Fujiyama, Asao; Kohara, Yuji; Honda, Naoko; Fujimoto, Hirofumi; Shimada, Toru; Nakagaki, Masao; Banno, Yutaka; Tsuchida, Kozo

    2011-03-01

    The carotenoid-binding protein (CBP) of the domesticated silkworm, Bombyx mori, a major determinant of cocoon color, is likely to have been substantially influenced by domestication of this species. We analyzed the structure of the CBP gene in multiple strains of B. mori, in multiple individuals of the wild silkworm, B. mandarina (the putative wild ancestor of B. mori), and in a number of other lepidopterans. We found the CBP gene copy number in genomic DNA to vary widely among B. mori strains, ranging from 1 to 20. The copies of CBP are of several types, based on the presence of a retrotransposon or partial deletion of the coding sequence. In contrast to B. mori, B. mandarina was found to possess a single copy of CBP without the retrotransposon insertion, regardless of habitat. Several other lepidopterans were found to contain sequences homologous to CBP, revealing that this gene is evolutionarily conserved in the lepidopteran lineage. Thus, domestication can generate significant diversity of gene copy number and structure over a relatively short evolutionary time. © 2011 by the Genetics Society of America

  13. Diversity in Copy Number and Structure of a Silkworm Morphogenetic Gene as a Result of Domestication

    PubMed Central

    Sakudoh, Takashi; Nakashima, Takeharu; Kuroki, Yoko; Fujiyama, Asao; Kohara, Yuji; Honda, Naoko; Fujimoto, Hirofumi; Shimada, Toru; Nakagaki, Masao; Banno, Yutaka; Tsuchida, Kozo

    2011-01-01

    The carotenoid-binding protein (CBP) of the domesticated silkworm, Bombyx mori, a major determinant of cocoon color, is likely to have been substantially influenced by domestication of this species. We analyzed the structure of the CBP gene in multiple strains of B. mori, in multiple individuals of the wild silkworm, B. mandarina (the putative wild ancestor of B. mori), and in a number of other lepidopterans. We found the CBP gene copy number in genomic DNA to vary widely among B. mori strains, ranging from 1 to 20. The copies of CBP are of several types, based on the presence of a retrotransposon or partial deletion of the coding sequence. In contrast to B. mori, B. mandarina was found to possess a single copy of CBP without the retrotransposon insertion, regardless of habitat. Several other lepidopterans were found to contain sequences homologous to CBP, revealing that this gene is evolutionarily conserved in the lepidopteran lineage. Thus, domestication can generate significant diversity of gene copy number and structure over a relatively short evolutionary time. PMID:21242537

  14. Models for loosely linked gene duplicates suggest lengthy persistence of both copies.

    PubMed

    O'Hely, Martin; Wockner, Leesa

    2007-06-21

    Consider the appearance of a duplicate copy of a gene at a locus linked loosely, if at all, to the locus at which the gene is usually found. If all copies of the gene are subject to non-functionalizing mutations, then two fates are possible: loss of functional copies at the duplicate locus (loss of duplicate expression), or loss of functional copies at the original locus (map change). This paper proposes a simple model to address the probability of map change, the time taken for a map change and/or loss of duplicate expression, and considers where in the spectrum between loss of duplicate expression and map change such a duplicate complex is likely to be found. The findings are: the probability of map change is always half the reciprocal of the population size N, the time for a map change to occur is order NlogN generations, and that there is a marked tendency for duplicates to remain near equi-frequency with the gene at the original locus for a large portion of that time. This is in excellent agreement with simulations.

  15. [Copy number variation of trinucleotide repeat in dynamic mutation sites of autosomal dominant cerebellar ataxias related genes].

    PubMed

    Chen, Pu; Ma, Mingyi; Shang, Huifang; Su, Dan; Zhang, Sizhong; Yang, Yuan

    2009-12-01

    To standardize the experimental procedure of the gene test for autosomal dominant cerebellar ataxias (ADCA), and provide the basis for quantitative criteria of the dynamic mutation of spinocerebellar ataxia (SCA) genes in Chinese population. Genotyping of the dynamic mutation loci of the SCA1, SCA2, SCA3, SCA6 and SCA7 genes was performed, using florescence PCR-capillary electrophoresis followed by DNA sequencing, to investigate the variation range of copy number of CAG tandem repeat of the genes in 263 probands of ADCA pedigrees and 261 non-related normal controls. Based on the sequencing result, the bias of the CAG copy number estimation using capillary electrophoresis with different DNA controls was compared to analyze the technical detailes of the electrophresis method in testing the dynamic mutation sites. PCR products containing dynamic mutation loci of the SCA genes showed significantly higher mobility than that of molecular weigh marker with relatively balanced GC content. This was particularly obvious in the SCA2, SCA 6 and SCA7 genes whereas the deviation of copy number could be corrected to +/-1 when known CAG copy number fragments were used as controls. The mobility of PCR products was primarily related to the copy number of CAG repeat when the fragments contained normal CAG repeat. In the 263 ADCA pedigrees, 6 (2.28%) carried SCA1 gene mutation, 8 (3.04%) had SCA2 mutation and 81 (30.80%) harbored SCA3 mutation. The gene mutation of SCA6 and SCA7 was not found. The normal variation range of the CAG repeat was 17-36 copies in SCA1 gene, 13-30 copies in SCA2, 14-39 copies in SCA3, 6-16 copies in SCA6 and 6-13 copies in SCA7. The heterozygosity was 76.1%, 17.7%, 74.4%, 72.1% and 41.3%, respectively. The mutation range of the CAG repeat was 49-56 copies in SCA1 gene, 36-41 copies in SCA2, 59-81 copies in SCA3. Neither homozygous mutation of an SCA gene nor double heterozygous mutation of the SCA genes was observed in the study. The copy number of the CAG

  16. TTT and PIKK Complex Genes Reverted to Single Copy Following Polyploidization and Retain Function Despite Massive Retrotransposition in Maize.

    PubMed

    Garcia, Nelson; Messing, Joachim

    2017-01-01

    The TEL2, TTI1, and TTI2 proteins are co-chaperones for heat shock protein 90 (HSP90) to regulate the protein folding and maturation of phosphatidylinositol 3-kinase-related kinases (PIKKs). Referred to as the TTT complex, the genes that encode them are highly conserved from man to maize. TTT complex and PIKK genes exist mostly as single copy genes in organisms where they have been characterized. Members of this interacting protein network in maize were identified and synteny analyses were performed to study their evolution. Similar to other species, there is only one copy of each of these genes in maize which was due to a loss of the duplicated copy created by ancient allotetraploidy. Moreover, the retained copies of the TTT complex and the PIKK genes tolerated extensive retrotransposon insertion in their introns that resulted in increased gene lengths and gene body methylation, without apparent effect in normal gene expression and function. The results raise an interesting question on whether the reversion to single copy was due to selection against deleterious unbalanced gene duplications between members of the complex as predicted by the gene balance hypothesis, or due to neutral loss of extra copies. Uneven alteration of dosage either by adding extra copies or modulating gene expression of complex members is being proposed as a means to investigate whether the data supports the gene balance hypothesis or not.

  17. Dietary Variation and Evolution of Gene Copy Number among Dog Breeds

    PubMed Central

    Reiter, Taylor; Jagoda, Evelyn; Capellini, Terence D.

    2016-01-01

    Prolonged human interactions and artificial selection have influenced the genotypic and phenotypic diversity among dog breeds. Because humans and dogs occupy diverse habitats, ecological contexts have likely contributed to breed-specific positive selection. Prior to the advent of modern dog-feeding practices, there was likely substantial variation in dietary landscapes among disparate dog breeds. As such, we investigated one type of genetic variant, copy number variation, in three metabolic genes: glucokinase regulatory protein (GCKR), phytanol-CoA 2-hydroxylase (PHYH), and pancreatic α-amylase 2B (AMY2B). These genes code for proteins that are responsible for metabolizing dietary products that originate from distinctly different food types: sugar, meat, and starch, respectively. After surveying copy number variation among dogs with diverse dietary histories, we found no correlation between diet and positive selection in either GCKR or PHYH. Although it has been previously demonstrated that dogs experienced a copy number increase in AMY2B relative to wolves during or after the dog domestication process, we demonstrate that positive selection continued to act on amylase copy number in dog breeds that consumed starch-rich diets in time periods after domestication. Furthermore, we found that introgression with wolves is not responsible for deterioration of positive selection on AMY2B among diverse dog breeds. Together, this supports the hypothesis that the amylase copy number expansion is found universally in dogs. PMID:26863414

  18. Dietary Variation and Evolution of Gene Copy Number among Dog Breeds.

    PubMed

    Reiter, Taylor; Jagoda, Evelyn; Capellini, Terence D

    2016-01-01

    Prolonged human interactions and artificial selection have influenced the genotypic and phenotypic diversity among dog breeds. Because humans and dogs occupy diverse habitats, ecological contexts have likely contributed to breed-specific positive selection. Prior to the advent of modern dog-feeding practices, there was likely substantial variation in dietary landscapes among disparate dog breeds. As such, we investigated one type of genetic variant, copy number variation, in three metabolic genes: glucokinase regulatory protein (GCKR), phytanol-CoA 2-hydroxylase (PHYH), and pancreatic α-amylase 2B (AMY2B). These genes code for proteins that are responsible for metabolizing dietary products that originate from distinctly different food types: sugar, meat, and starch, respectively. After surveying copy number variation among dogs with diverse dietary histories, we found no correlation between diet and positive selection in either GCKR or PHYH. Although it has been previously demonstrated that dogs experienced a copy number increase in AMY2B relative to wolves during or after the dog domestication process, we demonstrate that positive selection continued to act on amylase copy number in dog breeds that consumed starch-rich diets in time periods after domestication. Furthermore, we found that introgression with wolves is not responsible for deterioration of positive selection on AMY2B among diverse dog breeds. Together, this supports the hypothesis that the amylase copy number expansion is found universally in dogs.

  19. Long-term persistence of systemic and mucosal immune response to HPV-16/18 AS04-adjuvanted vaccine in preteen/adolescent girls and young women.

    PubMed

    Petäjä, Tiina; Pedersen, Court; Poder, Airi; Strauss, Gitte; Catteau, Gregory; Thomas, Florence; Lehtinen, Matti; Descamps, Dominique

    2011-11-01

    Vaccination against oncogenic human papillomavirus (HPV) types is one key intervention for cervical cancer prevention. This follow-up study assessed the persistence of the systemic and mucosal immune responses together with the safety profile of the HPV-16/18 AS04-adjuvanted vaccine administered to young women aged 10-25 years. Serum and cervicovaginal secretion (CVS) samples were collected at prespecified time-points during the 48-month follow-up period. Anti-HPV-16/18 antibody levels in serum and CVS were measured by enzyme-linked immunosorbent assay (ELISA). At Month 48, all subjects remained seropositive for serum anti-HPV-16 and -18 antibodies. As previously observed, anti-HPV-16 and -18 antibodies levels (ELISA Units/mL) were higher in subjects vaccinated at the age of 10-14 years (2862.2 and 940.8) compared to subjects vaccinated at the age of 15-25 years (1186.2 and 469.8). Moreover, anti-HPV-16 and -18 antibodies in CVS were still detectable for subjects aged 15-25 years (84.1% and 69.7%, respectively). There was a strong correlation between serum and CVS anti-HPV-16 and -18 antibodies levels (correlation coefficients = 0.84 and 0.90 at Month 48, respectively) supporting the hypothesis of transudation or exudation of serum immunoglobulin G antibodies through the cervical epithelium. The HPV-16/18 AS04-adjuvanted vaccine had a clinically acceptable safety profile. In conclusion, this follow-up study shows that the HPV-16/18 AS04-adjuvanted vaccine administered to preteen/adolescents girls and young women induces long-term systemic and mucosal immune response and has a clinically acceptable safety profile up to 4 years after the first vaccine dose. Copyright © 2010 UICC.

  20. Identification of human papillomavirus (HPV) 16 DNA integration and the ensuing patterns of methylation in HPV-associated head and neck squamous cell carcinoma cell lines.

    PubMed

    Hatano, Takashi; Sano, Daisuke; Takahashi, Hideaki; Hyakusoku, Hiroshi; Isono, Yasuhiro; Shimada, Shoko; Sawakuma, Kae; Takada, Kentaro; Oikawa, Ritsuko; Watanabe, Yoshiyuki; Yamamoto, Hiroyuki; Itoh, Fumio; Myers, Jeffrey N; Oridate, Nobuhiko

    2017-04-01

    Recent studies showed that human papillomavirus (HPV) integration contributes to the genomic instability seen in HPV-associated head and neck squamous cell carcinoma (HPV-HNSCC). However, the epigenetic alterations induced after HPV integration remains unclear. To identify the molecular details of HPV16 DNA integration and the ensuing patterns of methylation in HNSCC, we performed next-generation sequencing using a target-enrichment method for the effective identification of HPV16 integration breakpoints as well as the characterization of genomic sequences adjacent to HPV16 integration breakpoints with three HPV16-related HNSCC cell lines. The DNA methylation levels of the integrated HPV16 genome and that of the adjacent human genome were also analyzed by bisulfite pyrosequencing. We found various integration loci, including novel integration sites. Integration loci were located predominantly in the intergenic region, with a significant enrichment of the microhomologous sequences between the human and HPV16 genomes at the integration breakpoints. Furthermore, various levels of methylation within both the human genome and the integrated HPV genome at the integration breakpoints in each integrant were observed. Allele-specific methylation analysis suggested that the HPV16 integrants remained hypomethylated when the flanking host genome was hypomethylated. After integration into highly methylated human genome regions, however, the HPV16 DNA became methylated. In conclusion, we found novel integration sites and methylation patterns in HPV-HNSCC using our unique method. These findings may provide insights into understanding of viral integration mechanism and virus-associated carcinogenesis of HPV-HNSCC. © 2016 UICC.

  1. Multi-gene fluorescence in situ hybridization to detect cell cycle gene copy number aberrations in young breast cancer patients

    PubMed Central

    Li, Chunyan; Bai, Jingchao; Hao, Xiaomeng; Zhang, Sheng; Hu, Yunhui; Zhang, Xiaobei; Yuan, Weiping; Hu, Linping; Cheng, Tao; Zetterberg, Anders; Lee, Mong-Hong; Zhang, J

    2014-01-01

    Breast cancer is a disease of cell cycle, and the dysfunction of cell cycle checkpoints plays a vital role in the occurrence and development of breast cancer. We employed multi-gene fluorescence in situ hybridization (M-FISH) to investigate gene copy number aberrations (CNAs) of 4 genes (Rb1, CHEK2, c-Myc, CCND1) that are involved in the regulation of cell cycle, in order to analyze the impact of gene aberrations on prognosis in the young breast cancer patients. Gene copy number aberrations of these 4 genes were more frequently observed in young breast cancer patients when compared with the older group. Further, these CNAs were more frequently seen in Luminal B type, Her2 overexpression, and tiple-negative breast cancer (TNBC) type in young breast cancer patients. The variations of CCND1, Rb1, and CHEK2 were significantly correlated with poor survival in the young breast cancer patient group, while the amplification of c-Myc was not obviously correlated with poor survival in young breast cancer patients. Thus, gene copy number aberrations (CNAs) of cell cycle-regulated genes can serve as an important tool for prognosis in young breast cancer patients. PMID:24621502

  2. Gene and Chromosomal Copy Number Variations as an Adaptive Mechanism Towards a Parasitic Lifestyle in Trypanosomatids.

    PubMed

    Reis-Cunha, João Luís; Valdivia, Hugo O; Bartholomeu, Daniella Castanheira

    2018-02-01

    Trypanosomatids are a group of kinetoplastid parasites including some of great public health importance, causing debilitating and life-long lasting diseases that affect more than 24 million people worldwide. Among the trypanosomatids, Trypanosoma cruzi, Trypanosoma brucei and species from the Leishmania genus are the most well studied parasites, due to their high prevalence in human infections. These parasites have an extreme genomic and phenotypic variability, with a massive expansion in the copy number of species-specific multigene families enrolled in host-parasite interactions that mediate cellular invasion and immune evasion processes. As most trypanosomatids are heteroxenous, and therefore their lifecycles involve the transition between different hosts, these parasites have developed several strategies to ensure a rapid adaptation to changing environments. Among these strategies, a rapid shift in the repertoire of expressed genes, genetic variability and genome plasticity are key mechanisms. Trypanosomatid genomes are organized into large directional gene clusters that are transcribed polycistronically, where genes derived from the same polycistron may have very distinct mRNA levels. This particular mode of transcription implies that the control of gene expression operates mainly at post-transcriptional level. In this sense, gene duplications/losses were already associated with changes in mRNA levels in these parasites. Gene duplications also allow the generation of sequence variability, as the newly formed copy can diverge without loss of function of the original copy. Recently, aneuploidies have been shown to occur in several Leishmania species and T. cruzi strains. Although aneuploidies are usually associated with debilitating phenotypes in superior eukaryotes, recent data shows that it could also provide increased fitness in stress conditions and generate drug resistance in unicellular eukaryotes. In this review, we will focus on gene and chromosomal copy

  3. [Detection of the exogenous gene copy number of the transgenic tomato anti-caries vaccine].

    PubMed

    Bai, Guo-hui; Liu, Jian-guo; Tian, Yuan; Chen, Zhu; Bai, Peng-yuan; Han, Qi; Gu, Yu; Guan, Xiao-yan; Wang, Hai-hui

    2013-12-01

    To detect the exogenous gene copy number of the transgenic tomato anti-caries vaccine by using the SYBR Green real-time PCR. Recombinant plasmid pEAC10 and pEPC10 were used as standard to detect genome samples of exogenous gene pacA-ctxB and pacP-ctxB by SYBR green fluorescent quantitation, then the average value was calculated as gene copy number. The copy number of the transgenic tomato carrying pacA-ctxB was 1.3 and the pacP-ctxB was 3.2. The transgenic tomato plants which have high stability are low-copy transgenic plants. Supported by National Natural Science Foundation of China (30160086, 81260164), Science and Technical Fund of Guizhou Province (LKZ[2011]41), Project of Technology Innovation Team in Guizhou Province, Leading Academic Discipline Construction Project in Guizhou Province and Excellent Scientific Research Team Cultivation Project in Zunyi Medical College ([2012]12).

  4. [Gene copy number, mRNA transcription and protein expression of PD-1 gene in primary hepatocarcinoma patients].

    PubMed

    Fan, Hui-Min; Wu, Ling-Jie; Hu, Feng-Yu; Yang, Zhan

    2012-08-01

    To study the gene copy number, mRNA transcription and protien expression of programmed cell death 1 (PD-1) gene in primary hepatocellular carcinoma (PHC) patients and normal control individuals (NC) who are anti-HBs positive, and to investigate the variations in PD-1 gene copy numbers and its relationship with PHC. Real-time PCR was adopted to detect the PD-1 gene copy numbers and their mRNA expressions in peripheral blood mononuclear cells (PBMCs) from 24 samples of PHC patients and 26 of NC. Protein expression level of PD-1 on CD8+ T was analyzed by flow cytometry. In terms of number of PD-1 gene copy numbers, the percentage of cases of haploid (single) was 34.62% and 4.17% in PHC group and control group respectively while the percentage of cases of diploid (double) was 61.54% and 95.83% respectively. The difference between the two was statistically significant (chi2 = 7.639, P = 0.006). The rate of cases with double PD-1 gene copy numbers was found to be higher in patients with PHC than in control group. It was also found that the average expression of PD-1 mRNA was 2.35E-03 in control group and 1.23E-03 in PHC group. The expression level was significant lower in PHC group than that in control group when compared by using Mann-whitey technic (U = 153, P = 0.009). Furthermore, the frequency of PD-1 protein expression on CD8+ T cells was 3.72 +/- 0.32 in control group and 16.13 +/- 1.68 in PHC group. The level of PD-1 mRNA expression was higher in PHC and significant differences was shown between two groups (t = -7.073, P = 0.000). Our study suggests that the variation in PD-1 gene copy number may trigger primary hepatocellular carcinoma to HBV carriers. The relationship between the variation of PD-1 gene copy numbers and its association with primary hepatocellular carcinoma is worth further focus.

  5. The Orphan Gene dauerless Regulates Dauer Development and Intraspecific Competition in Nematodes by Copy Number Variation

    PubMed Central

    Mayer, Melanie G.; Rödelsperger, Christian; Witte, Hanh; Riebesell, Metta; Sommer, Ralf J.

    2015-01-01

    Many nematodes form dauer larvae when exposed to unfavorable conditions, representing an example of phenotypic plasticity and a major survival and dispersal strategy. In Caenorhabditis elegans, the regulation of dauer induction is a model for pheromone, insulin, and steroid-hormone signaling. Recent studies in Pristionchus pacificus revealed substantial natural variation in various aspects of dauer development, i.e. pheromone production and sensing and dauer longevity and fitness. One intriguing example is a strain from Ohio, having extremely long-lived dauers associated with very high fitness and often forming the most dauers in response to other strains´ pheromones, including the reference strain from California. While such examples have been suggested to represent intraspecific competition among strains, the molecular mechanisms underlying these dauer-associated patterns are currently unknown. We generated recombinant-inbred-lines between the Californian and Ohioan strains and used quantitative-trait-loci analysis to investigate the molecular mechanism determining natural variation in dauer development. Surprisingly, we discovered that the orphan gene dauerless controls dauer formation by copy number variation. The Ohioan strain has one dauerless copy causing high dauer formation, whereas the Californian strain has two copies, resulting in strongly reduced dauer formation. Transgenic animals expressing multiple copies do not form dauers. dauerless is exclusively expressed in CAN neurons, and both CAN ablation and dauerless mutations increase dauer formation. Strikingly, dauerless underwent several duplications and acts in parallel or downstream of steroid-hormone signaling but upstream of the nuclear-hormone-receptor daf-12. We identified the novel or fast-evolving gene dauerless as inhibitor of dauer development. Our findings reveal the importance of gene duplications and copy number variations for orphan gene function and suggest daf-12 as major target for

  6. The Orphan Gene dauerless Regulates Dauer Development and Intraspecific Competition in Nematodes by Copy Number Variation.

    PubMed

    Mayer, Melanie G; Rödelsperger, Christian; Witte, Hanh; Riebesell, Metta; Sommer, Ralf J

    2015-06-01

    Many nematodes form dauer larvae when exposed to unfavorable conditions, representing an example of phenotypic plasticity and a major survival and dispersal strategy. In Caenorhabditis elegans, the regulation of dauer induction is a model for pheromone, insulin, and steroid-hormone signaling. Recent studies in Pristionchus pacificus revealed substantial natural variation in various aspects of dauer development, i.e. pheromone production and sensing and dauer longevity and fitness. One intriguing example is a strain from Ohio, having extremely long-lived dauers associated with very high fitness and often forming the most dauers in response to other strains' pheromones, including the reference strain from California. While such examples have been suggested to represent intraspecific competition among strains, the molecular mechanisms underlying these dauer-associated patterns are currently unknown. We generated recombinant-inbred-lines between the Californian and Ohioan strains and used quantitative-trait-loci analysis to investigate the molecular mechanism determining natural variation in dauer development. Surprisingly, we discovered that the orphan gene dauerless controls dauer formation by copy number variation. The Ohioan strain has one dauerless copy causing high dauer formation, whereas the Californian strain has two copies, resulting in strongly reduced dauer formation. Transgenic animals expressing multiple copies do not form dauers. dauerless is exclusively expressed in CAN neurons, and both CAN ablation and dauerless mutations increase dauer formation. Strikingly, dauerless underwent several duplications and acts in parallel or downstream of steroid-hormone signaling but upstream of the nuclear-hormone-receptor daf-12. We identified the novel or fast-evolving gene dauerless as inhibitor of dauer development. Our findings reveal the importance of gene duplications and copy number variations for orphan gene function and suggest daf-12 as major target for

  7. Molecular Inversion Probe Analysis of Gene Copy Alterations Reveals Distinct Categories of Colorectal Carcinoma

    PubMed Central

    Ji, Hanlee; Kumm, Jochen; Zhang, Michael; Farnam, Kyle; Salari, Keyan; Faham, Malek; Ford, James M.; Davis, Ronald W.

    2006-01-01

    Genomic instability is a major feature of neoplastic development in colorectal carcinoma and other cancers. Specific genomic instability events, such as deletions in chromosomes and other alterations in gene copy number, have potential utility as biologically relevant prognostic biomarkers. For example, genomic deletions on chromosome arm 18q are an indicator of colorectal carcinoma behavior and potentially useful as a prognostic indicator. Adapting a novel genomic technology called molecular inversion probes which can determine gene copy alterations, such as genomic deletions, we designed a set of probes to interrogate several hundred individual exons of >200 cancer genes with an overall distribution covering all chromosome arms. In addition, >100 probes were designed in close proximity of microsatellite markers on chromosome arm 18q. We analyzed a set of colorectal carcinoma cell lines and primary colorectal tumor samples for gene copy alterations and deletion mutations in exons. Based on clustering analysis, we distinguished the different categories of genomic instability among the colorectal cancer cell lines. Our analysis of primary tumors uncovered several distinct categories of colorectal carcinoma, each with specific patterns of 18q deletions and deletion mutations in specific genes. This finding has potential clinical ramifications given the application of 18q loss of heterozygosity events as a potential indicator for adjuvant treatment in stage II colorectal carcinoma. PMID:16912164

  8. Copy number variations in the amylase gene (AMY2B) in Japanese native dog breeds.

    PubMed

    Tonoike, A; Hori, Y; Inoue-Murayama, M; Konno, A; Fujita, K; Miyado, M; Fukami, M; Nagasawa, M; Mogi, K; Kikusui, T

    2015-10-01

    A recent study suggested that increased copy numbers of the AMY2B gene might be a crucial genetic change that occurred during the domestication of dogs. To investigate AMY2B expansion in ancient breeds, which are highly divergent from modern breeds of presumed European origins, we analysed copy numbers in native Japanese dog breeds. Copy numbers in the Akita and Shiba, two ancient breeds in Japan, were higher than those in wolves. However, compared to a group of various modern breeds, Akitas had fewer copy numbers, whereas Shibas exhibited the same level of expansion as modern breeds. Interestingly, average AMY2B copy numbers in the Jomon-Shiba, a unique line of the Shiba that has been bred to maintain their appearance resembling ancestors of native Japanese dogs and that originated in the same region as the Akita, were lower than those in the Shiba. These differences may have arisen from the earlier introduction of rice farming to the region in which the Shiba originated compared to the region in which the Akita and the Jomon-Shiba originated. Thus, our data provide insights into the relationship between the introduction of agriculture and AMY2B expansion in dogs. © 2015 Stichting International Foundation for Animal Genetics.

  9. Clinical omics analysis of colorectal cancer incorporating copy number aberrations and gene expression data.

    PubMed

    Yoshida, Tsuyoshi; Kobayashi, Takumi; Itoda, Masaya; Muto, Taika; Miyaguchi, Ken; Mogushi, Kaoru; Shoji, Satoshi; Shimokawa, Kazuro; Iida, Satoru; Uetake, Hiroyuki; Ishikawa, Toshiaki; Sugihara, Kenichi; Mizushima, Hiroshi; Tanaka, Hiroshi

    2010-07-29

    Colorectal cancer (CRC) is one of the most frequently occurring cancers in Japan, and thus a wide range of methods have been deployed to study the molecular mechanisms of CRC. In this study, we performed a comprehensive analysis of CRC, incorporating copy number aberration (CRC) and gene expression data. For the last four years, we have been collecting data from CRC cases and organizing the information as an "omics" study by integrating many kinds of analysis into a single comprehensive investigation. In our previous studies, we had experienced difficulty in finding genes related to CRC, as we observed higher noise levels in the expression data than in the data for other cancers. Because chromosomal aberrations are often observed in CRC, here, we have performed a combination of CNA analysis and expression analysis in order to identify some new genes responsible for CRC. This study was performed as part of the Clinical Omics Database Project at Tokyo Medical and Dental University. The purpose of this study was to investigate the mechanism of genetic instability in CRC by this combination of expression analysis and CNA, and to establish a new method for the diagnosis and treatment of CRC. Comprehensive gene expression analysis was performed on 79 CRC cases using an Affymetrix Gene Chip, and comprehensive CNA analysis was performed using an Affymetrix DNA Sty array. To avoid the contamination of cancer tissue with normal cells, laser micro-dissection was performed before DNA/RNA extraction. Data analysis was performed using original software written in the R language. We observed a high percentage of CNA in colorectal cancer, including copy number gains at 7, 8q, 13 and 20q, and copy number losses at 8p, 17p and 18. Gene expression analysis provided many candidates for CRC-related genes, but their association with CRC did not reach the level of statistical significance. The combination of CNA and gene expression analysis, together with the clinical information

  10. Reduction in HPV 16/18 prevalence in sexually active young women following the introduction of HPV immunisation in England.

    PubMed

    Mesher, D; Soldan, K; Howell-Jones, R; Panwar, K; Manyenga, P; Jit, M; Beddows, S; Gill, O N

    2013-12-17

    Reduction in the prevalence of vaccine type HPV infection in young women is an early indication of the impact of the HPV immunisation programme and a necessary outcome if the subsequent impact on cervical cancer is to be realised. Residual vulva-vaginal swab (VVS) specimens from young women aged 16-24 years undergoing chlamydia screening in community sexual health services (formerly known as family planning clinics), general practice (GP), and youth clinics in 2010-2012 were submitted from 10 laboratories in seven regions around England. These specimens were linked to demographic and sexual behaviour data reported with the chlamydia test, anonymised, and tested for type-specific HPV DNA using a multiplex PCR and Luminex-based genotyping test. Estimated immunisation coverage was calculated and findings were compared to a baseline survey conducted prior to the introduction of HPV immunisation in 2008. A total of 4664 eligible specimens were collected and 4178 had a valid test result. The post-immunisation prevalence of HPV 16/18 infection was lowest in this youngest age group (16-18 years) and increased with age. This increase with age was a reversal of the pattern seen prior to immunisation and was inversely associated with estimates of age-specific immunisation coverage (65% for 16-18 year olds). The prevalence of HPV 16/18 infection in the post-immunisation survey was 6.5% amongst 16-18 year olds, compared to 19.1% in the similar survey conducted prior to the introduction of HPV immunisation. These findings are the first indication that the national HPV immunisation programme is successfully preventing HPV 16/18 infection in sexually active young women in England. The reductions seen suggest, for the estimated coverage, high vaccine effectiveness and some herd-protection benefits. Continued surveillance is needed to determine the effects of immunisation on non-vaccine HPV types. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  11. Impact of duplicate gene copies on phylogenetic analysis and divergence time estimates in butterflies.

    PubMed

    Pohl, Nélida; Sison-Mangus, Marilou P; Yee, Emily N; Liswi, Saif W; Briscoe, Adriana D

    2009-05-13

    The increase in availability of genomic sequences for a wide range of organisms has revealed gene duplication to be a relatively common event. Encounters with duplicate gene copies have consequently become almost inevitable in the context of collecting gene sequences for inferring species trees. Here we examine the effect of incorporating duplicate gene copies evolving at different rates on tree reconstruction and time estimation of recent and deep divergences in butterflies. Sequences from ultraviolet-sensitive (UVRh), blue-sensitive (BRh), and long-wavelength sensitive (LWRh) opsins,EF-1 and COI were obtained from 27 taxa representing the five major butterfly families (5535 bp total). Both BRh and LWRh are present in multiple copies in some butterfly lineages and the different copies evolve at different rates. Regardless of the phylogenetic reconstruction method used, we found that analyses of combined data sets using either slower or faster evolving copies of duplicate genes resulted in a single topology in agreement with our current understanding of butterfly family relationships based on morphology and molecules. Interestingly, individual analyses of BRh and LWRh sequences also recovered these family-level relationships. Two different relaxed clock methods resulted in similar divergence time estimates at the shallower nodes in the tree, regardless of whether faster or slower evolving copies were used, with larger discrepancies observed at deeper nodes in the phylogeny. The time of divergence between the monarch butterfly Danaus plexippus and the queen D. gilippus (15.3-35.6 Mya) was found to be much older than the time of divergence between monarch co-mimic Limenitis archippus and red-spotted purple L. arthemis (4.7-13.6 Mya), and overlapping with the time of divergence of the co-mimetic passionflower butterflies Heliconius erato and H. melpomene (13.5-26.1 Mya). Our family-level results are congruent with recent estimates found in the literature and indicate

  12. Impact of duplicate gene copies on phylogenetic analysis and divergence time estimates in butterflies

    PubMed Central

    Pohl, Nélida; Sison-Mangus, Marilou P; Yee, Emily N; Liswi, Saif W; Briscoe, Adriana D

    2009-01-01

    Background The increase in availability of genomic sequences for a wide range of organisms has revealed gene duplication to be a relatively common event. Encounters with duplicate gene copies have consequently become almost inevitable in the context of collecting gene sequences for inferring species trees. Here we examine the effect of incorporating duplicate gene copies evolving at different rates on tree reconstruction and time estimation of recent and deep divergences in butterflies. Results Sequences from ultraviolet-sensitive (UVRh), blue-sensitive (BRh), and long-wavelength sensitive (LWRh) opsins,EF-1α and COI were obtained from 27 taxa representing the five major butterfly families (5535 bp total). Both BRh and LWRh are present in multiple copies in some butterfly lineages and the different copies evolve at different rates. Regardless of the phylogenetic reconstruction method used, we found that analyses of combined data sets using either slower or faster evolving copies of duplicate genes resulted in a single topology in agreement with our current understanding of butterfly family relationships based on morphology and molecules. Interestingly, individual analyses of BRh and LWRh sequences also recovered these family-level relationships. Two different relaxed clock methods resulted in similar divergence time estimates at the shallower nodes in the tree, regardless of whether faster or slower evolving copies were used, with larger discrepancies observed at deeper nodes in the phylogeny. The time of divergence between the monarch butterfly Danaus plexippus and the queen D. gilippus (15.3–35.6 Mya) was found to be much older than the time of divergence between monarch co-mimic Limenitis archippus and red-spotted purple L. arthemis (4.7–13.6 Mya), and overlapping with the time of divergence of the co-mimetic passionflower butterflies Heliconius erato and H. melpomene (13.5–26.1 Mya). Our family-level results are congruent with recent estimates found in

  13. Epidemiology of HPV 16 and Cervical Cancer in Finland and the Potential Impact of Vaccination: Mathematical Modelling Analyses

    PubMed Central

    Barnabas, Ruanne V; Laukkanen, Päivi; Koskela, Pentti; Kontula, Osmo; Lehtinen, Matti; Garnett, Geoff P

    2006-01-01

    Background Candidate human papillomavirus (HPV) vaccines have demonstrated almost 90%-100% efficacy in preventing persistent, type-specific HPV infection over 18 mo in clinical trials. If these vaccines go on to demonstrate prevention of precancerous lesions in phase III clinical trials, they will be licensed for public use in the near future. How these vaccines will be used in countries with national cervical cancer screening programmes is an important question. Methods and Findings We developed a transmission model of HPV 16 infection and progression to cervical cancer and calibrated it to Finnish HPV 16 seroprevalence over time. The model was used to estimate the transmission probability of the virus, to look at the effect of changes in patterns of sexual behaviour and smoking on age-specific trends in cancer incidence, and to explore the impact of HPV 16 vaccination. We estimated a high per-partnership transmission probability of HPV 16, of 0.6. The modelling analyses showed that changes in sexual behaviour and smoking accounted, in part, for the increase seen in cervical cancer incidence in 35- to 39-y-old women from 1990 to 1999. At both low (10% in opportunistic immunisation) and high (90% in a national immunisation programme) coverage of the adolescent population, vaccinating women and men had little benefit over vaccinating women alone. We estimate that vaccinating 90% of young women before sexual debut has the potential to decrease HPV type-specific (e.g., type 16) cervical cancer incidence by 91%. If older women are more likely to have persistent infections and progress to cancer, then vaccination with a duration of protection of less than 15 y could result in an older susceptible cohort and no decrease in cancer incidence. While vaccination has the potential to significantly reduce type-specific cancer incidence, its combination with screening further improves cancer prevention. Conclusions HPV vaccination has the potential to significantly decrease HPV

  14. Unique LCR variations among lineages of HPV16, 18 and 45 isolates from women with normal cervical cytology in Ghana.

    PubMed

    Awua, Adolf K; Adanu, Richard M K; Wiredu, Edwin K; Afari, Edwin A; Zubuch, Vanessa A; Asmah, Richard H; Severini, Alberto

    2017-04-21

    In addition to being useful for classification, sequence variations of human Papillomavirus (HPV) genotypes have been implicated in differential oncogenic potential and a differential association with the different histological forms of invasive cervical cancer. These associations have also been indicated for HPV genotype lineages and sub-lineages. In order to better understand the potential implications of lineage variation in the occurrence of cervical cancers in Ghana, we studied the lineages of the three most prevalent HPV genotypes among women with normal cytology as baseline to further studies. Of previously collected self- and health personnel-collected cervical specimen, 54, which were positive for HPV16, 18 and 45, were selected and the long control region (LCR) of each HPV genotype was separately amplified by a nested PCR. DNA sequences of 41 isolates obtained with the forward and reverse primers by Sanger sequencing were analysed. Nucleotide sequence variations of the HPV16 genotypes were observed at 30 positions within the LCR (7460 - 7840). Of these, 19 were the known variations for the lineages B and C (African lineages), while the other 11 positions had variations unique to the HPV16 isolates of this study. For the HPV18 isolates, the variations were at 35 positions, 22 of which were known variations of Africa lineages and the other 13 were unique variations observed for the isolates obtained in this study (at positions 7799 and 7813). HPV45 isolates had variations at 35 positions and 2 (positions 7114 and 97) were unique to the isolates of this study. This study provides the first data on the lineages of HPV 16, 18 and 45 isolates from Ghana. Although the study did not obtain full genome sequence data for a comprehensive comparison with known lineages, these genotypes were predominately of the Africa lineages and had some unique sequence variations at positions that suggest potential oncogenic implications. These data will be useful for comparison

  15. Inferring species trees from incongruent multi-copy gene trees using the Robinson-Foulds distance

    PubMed Central

    2013-01-01

    Background Constructing species trees from multi-copy gene trees remains a challenging problem in phylogenetics. One difficulty is that the underlying genes can be incongruent due to evolutionary processes such as gene duplication and loss, deep coalescence, or lateral gene transfer. Gene tree estimation errors may further exacerbate the difficulties of species tree estimation. Results We present a new approach for inferring species trees from incongruent multi-copy gene trees that is based on a generalization of the Robinson-Foulds (RF) distance measure to multi-labeled trees (mul-trees). We prove that it is NP-hard to compute the RF distance between two mul-trees; however, it is easy to calculate this distance between a mul-tree and a singly-labeled species tree. Motivated by this, we formulate the RF problem for mul-trees (MulRF) as follows: Given a collection of multi-copy gene trees, find a singly-labeled species tree that minimizes the total RF distance from the input mul-trees. We develop and implement a fast SPR-based heuristic algorithm for the NP-hard MulRF problem. We compare the performance of the MulRF method (available at http://genome.cs.iastate.edu/CBL/MulRF/) with several gene tree parsimony approaches using gene tree simulations that incorporate gene tree error, gene duplications and losses, and/or lateral transfer. The MulRF method produces more accurate species trees than gene tree parsimony approaches. We also demonstrate that the MulRF method infers in minutes a credible plant species tree from a collection of nearly 2,000 gene trees. Conclusions Our new phylogenetic inference method, based on a generalized RF distance, makes it possible to quickly estimate species trees from large genomic data sets. Since the MulRF method, unlike gene tree parsimony, is based on a generic tree distance measure, it is appealing for analyses of genomic data sets, in which many processes such as deep coalescence, recombination, gene duplication and losses as

  16. Integrative analysis of copy number and gene expression data suggests novel pathogenetic mechanisms in primary myelofibrosis.

    PubMed

    Salati, Simona; Zini, Roberta; Nuzzo, Simona; Guglielmelli, Paola; Pennucci, Valentina; Prudente, Zelia; Ruberti, Samantha; Rontauroli, Sebastiano; Norfo, Ruggiero; Bianchi, Elisa; Bogani, Costanza; Rotunno, Giada; Fanelli, Tiziana; Mannarelli, Carmela; Rosti, Vittorio; Salmoiraghi, Silvia; Pietra, Daniela; Ferrari, Sergio; Barosi, Giovanni; Rambaldi, Alessandro; Cazzola, Mario; Bicciato, Silvio; Tagliafico, Enrico; Vannucchi, Alessandro M; Manfredini, Rossella

    2016-04-01

    Primary myelofibrosis (PMF) is a Myeloproliferative Neoplasm (MPN) characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and transformation to Acute Myeloid Leukemia (AML). A number of phenotypic driver (JAK2, CALR, MPL) and additional subclonal mutations have been described in PMF, pointing to a complex genomic landscape. To discover novel genomic lesions that can contribute to disease phenotype and/or development, gene expression and copy number signals were integrated and several genomic abnormalities leading to a concordant alteration in gene expression levels were identified. In particular, copy number gain in the polyamine oxidase (PAOX) gene locus was accompanied by a coordinated transcriptional up-regulation in PMF patients. PAOX inhibition resulted in rapid cell death of PMF progenitor cells, while sparing normal cells, suggesting that PAOX inhibition could represent a therapeutic strategy to selectively target PMF cells without affecting normal hematopoietic cells' survival. Moreover, copy number loss in the chromatin modifier HMGXB4 gene correlates with a concomitant transcriptional down-regulation in PMF patients. Interestingly, silencing of HMGXB4 induces megakaryocyte differentiation, while inhibiting erythroid development, in human hematopoietic stem/progenitor cells. These results highlight a previously un-reported, yet potentially interesting role of HMGXB4 in the hematopoietic system and suggest that genomic and transcriptional imbalances of HMGXB4 could contribute to the aberrant expansion of the megakaryocytic lineage that characterizes PMF patients. © 2015 UICC.

  17. An HPV 16 L1-based chimeric human papilloma virus-like particles containing a string of epitopes produced in plants is able to elicit humoral and cytotoxic T-cell activity in mice

    PubMed Central

    De la Rosa, Georgina Paz; Monroy-García, Alberto; Mora-García, María de Lourdes; Peña, Cristina Gehibie Reynaga; Hernández-Montes, Jorge; Weiss-Steider, Benny; Lim, Miguel Angel Gómez

    2009-01-01

    Background Even though two prophylactic vaccines against HPV are currently licensed, infections by the virus continue to be a major health problem mainly in developing countries. The cost of the vaccines limits wide-scale application in poor countries. A promising strategy for producing affordable and efficient vaccines involves the expression of recombinant immunogens in plants. Several HPV genes have been expressed in plants, including L1, which can self-assemble into virus-like particles. A plant-based, dual prophylactic/therapeutic vaccine remains an attractive possibility. Results We sought to express in tomato plants chimeric HPV 16 VLPs containing L1 fused to a string of epitopes from HPV 16 E6 and E7 proteins. The L1 employed had been modified to eliminate a strong inhibitory region at the 5' end of the molecule to increase expression levels. Several tomato lines were obtained expressing either L1 alone or L1-E6/E7 from 0.05% to 0.1% of total soluble protein. Stable integration of the transgenes was verified by Southern blot. Northern and western blot revealed successful expression of the transgenes at the mRNA and protein level. The chimeric VLPs were able to assemble adequately in tomato cells. Intraperitoneal administration in mice was able to elicit both neutralizing antibodies against the viral particle and cytotoxic T-lymphocytes activity against the epitopes. Conclusion In this work, we report for the first time the expression in plants of a chimeric particle containing the HPV 16 L1 sequence and a string of T-cell epitopes from HPV 16 E6 and E7 fused to the C-terminus. The particles were able to induce a significant antibody and cytotoxic T-lymphocytes response. Experiments in vivo are in progress to determine whether the chimeric particles are able to induce regression of disease and resolution of viral infection in mice. Chimeric particles of the type described in this work may potentially be the basis for developing prophylactic

  18. An HPV 16 L1-based chimeric human papilloma virus-like particles containing a string of epitopes produced in plants is able to elicit humoral and cytotoxic T-cell activity in mice.

    PubMed

    Paz De la Rosa, Georgina; Monroy-García, Alberto; Mora-García, María de Lourdes; Peña, Cristina Gehibie Reynaga; Hernández-Montes, Jorge; Weiss-Steider, Benny; Gómez-Lim, Miguel Angel

    2009-01-06

    Even though two prophylactic vaccines against HPV are currently licensed, infections by the virus continue to be a major health problem mainly in developing countries. The cost of the vaccines limits wide-scale application in poor countries. A promising strategy for producing affordable and efficient vaccines involves the expression of recombinant immunogens in plants. Several HPV genes have been expressed in plants, including L1, which can self-assemble into virus-like particles. A plant-based, dual prophylactic/therapeutic vaccine remains an attractive possibility. We sought to express in tomato plants chimeric HPV 16 VLPs containing L1 fused to a string of epitopes from HPV 16 E6 and E7 proteins. The L1 employed had been modified to eliminate a strong inhibitory region at the 5' end of the molecule to increase expression levels. Several tomato lines were obtained expressing either L1 alone or L1-E6/E7 from 0.05% to 0.1% of total soluble protein. Stable integration of the transgenes was verified by Southern blot. Northern and western blot revealed successful expression of the transgenes at the mRNA and protein level. The chimeric VLPs were able to assemble adequately in tomato cells. Intraperitoneal administration in mice was able to elicit both neutralizing antibodies against the viral particle and cytotoxic T-lymphocytes activity against the epitopes. In this work, we report for the first time the expression in plants of a chimeric particle containing the HPV 16 L1 sequence and a string of T-cell epitopes from HPV 16 E6 and E7 fused to the C-terminus. The particles were able to induce a significant antibody and cytotoxic T-lymphocytes response. Experiments in vivo are in progress to determine whether the chimeric particles are able to induce regression of disease and resolution of viral infection in mice. Chimeric particles of the type described in this work may potentially be the basis for developing prophylactic/therapeutic vaccines. The fact that they are

  19. iGC-an integrated analysis package of gene expression and copy number alteration.

    PubMed

    Lai, Yi-Pin; Wang, Liang-Bo; Wang, Wei-An; Lai, Liang-Chuan; Tsai, Mong-Hsun; Lu, Tzu-Pin; Chuang, Eric Y

    2017-01-14

    With the advancement in high-throughput technologies, researchers can simultaneously investigate gene expression and copy number alteration (CNA) data from individual patients at a lower cost. Traditional analysis methods analyze each type of data individually and integrate their results using Venn diagrams. Challenges arise, however, when the results are irreproducible and inconsistent across multiple platforms. To address these issues, one possible approach is to concurrently analyze both gene expression profiling and CNAs in the same individual. We have developed an open-source R/Bioconductor package (iGC). Multiple input formats are supported and users can define their own criteria for identifying differentially expressed genes driven by CNAs. The analysis of two real microarray datasets demonstrated that the CNA-driven genes identified by the iGC package showed significantly higher Pearson correlation coefficients with their gene expression levels and copy numbers than those genes located in a genomic region with CNA. Compared with the Venn diagram approach, the iGC package showed better performance. The iGC package is effective and useful for identifying CNA-driven genes. By simultaneously considering both comparative genomic and transcriptomic data, it can provide better understanding of biological and medical questions. The iGC package's source code and manual are freely available at https://www.bioconductor.org/packages/release/bioc/html/iGC.html .

  20. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number

    PubMed Central

    Brockman, Alan; McGready, Rose; Ashley, Elizabeth; Phaipun, Lucy; Patel, Rina; Laing, Kenneth; Looareesuwan, Sornchai; White, Nicholas J; Nosten, François; Krishna, Sanjeev

    2015-01-01

    Summary Background The borders of Thailand harbour the world’s most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known. Methods The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism. Findings Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6·3 (95% CI 2·9–13·8, p<0·001) after mefloquine monotherapy and 5·4 (2·0-14·6, p=0·001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo. Interpretation Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria. Relevance to practice Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a

  1. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number.

    PubMed

    Price, Ric N; Uhlemann, Anne-Catrin; Brockman, Alan; McGready, Rose; Ashley, Elizabeth; Phaipun, Lucy; Patel, Rina; Laing, Kenneth; Looareesuwan, Sornchai; White, Nicholas J; Nosten, François; Krishna, Sanjeev

    The borders of Thailand harbour the world's most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known. The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism. Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6.3 (95% CI 2.9-13.8, p<0.001) after mefloquine monotherapy and 5.4 (2.0-14.6, p=0.001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo. Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria. Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with

  2. Copy number variation of the APC gene is associated with regulation of bone mineral density☆

    PubMed Central

    Chew, Shelby; Dastani, Zari; Brown, Suzanne J.; Lewis, Joshua R.; Dudbridge, Frank; Soranzo, Nicole; Surdulescu, Gabriela L.; Richards, J. Brent; Spector, Tim D.; Wilson, Scott G.

    2012-01-01

    Introduction Genetic studies of osteoporosis have commonly examined SNPs in candidate genes or whole genome analyses, but insertions and deletions of DNA, collectively called copy number variations (CNVs), also comprise a large amount of the genetic variability between individuals. Previously, SNPs in the APC gene have been strongly associated with femoral neck and lumbar spine volumetric bone mineral density in older men. In addition, familial adenomatous polyposis patients carrying heterozygous mutations in the APC gene have been shown to have significantly higher mean bone mineral density than age- and sex-matched controls suggesting the importance of this gene in regulating bone mineral density. We examined CNV within the APC gene region to test for association with bone mineral density. Methods DNA was extracted from venous blood, genotyped using the Human Hap610 arrays and CNV determined from the fluorescence intensity data in 2070 Caucasian men and women aged 47.0 ± 13.0 (mean ± SD) years, to assess the effects of the CNV on bone mineral density at the forearm, spine and total hip sites. Results Data for covariate adjusted bone mineral density from subjects grouped by APC CNV genotype showed significant difference (P = 0.02–0.002). Subjects with a single copy loss of APC had a 7.95%, 13.10% and 13.36% increase in bone mineral density at the forearm, spine and total hip sites respectively, compared to subjects with two copies of the APC gene. Conclusions These data support previous findings of APC regulating bone mineral density and demonstrate that a novel CNV of the APC gene is significantly associated with bone mineral density in Caucasian men and women. PMID:22884971

  3. Divergent gene copies in the asexual class Bdelloidea (Rotifera) separated before the bdelloid radiation or within bdelloid families.

    PubMed

    Mark Welch, David B; Cummings, Michael P; Hillis, David M; Meselson, Matthew

    2004-02-10

    Rotifers of the asexual class Bdelloidea are unusual in possessing two or more divergent copies of every gene that has been examined. Phylogenetic analysis of the heat-shock gene hsp82 and the TATA-box-binding protein gene tbp in multiple bdelloid species suggested that for each gene, each copy belonged to one of two lineages that began to diverge before the bdelloid radiation. Such gene trees are consistent with the two lineages having descended from former alleles that began to diverge after meiotic segregation ceased or from subgenomes of an alloploid ancestor of the bdelloids. However, the original analyses of bdelloid gene-copy divergence used only a single outgroup species and were based on parsimony and neighbor joining. We have now used maximum likelihood and Bayesian inference methods and, for hsp82, multiple outgroups in an attempt to produce more robust gene trees. Here we report that the available data do not unambiguously discriminate between gene trees that root the origin of hsp82 and tbp copy divergence before the bdelloid radiation and those which indicate that the gene copies began to diverge within bdelloid families. The remarkable presence of multiple diverged gene copies in individual genomes is nevertheless consistent with the loss of sex in an ancient ancestor of bdelloids.

  4. Integrative analysis of copy number alteration and gene expression profiling in ovarian clear cell adenocarcinoma.

    PubMed

    Sung, Chang Ohk; Choi, Chel Hun; Ko, Young-Hyeh; Ju, Hyunjeong; Choi, Yoon-La; Kim, Nyunsu; Kang, So Young; Ha, Sang Yun; Choi, Kyusam; Bae, Duk-Soo; Lee, Jeong-Won; Kim, Tae-Joong; Song, Sang Yong; Kim, Byoung-Gie

    2013-05-01

    Ovarian clear cell adenocarcinoma (Ov-CCA) is a distinctive subtype of ovarian epithelial carcinoma. In this study, we performed array comparative genomic hybridization (aCGH) and paired gene expression microarray of 19 fresh-frozen samples and conducted integrative analysis. For the copy number alterations, significantly amplified regions (false discovery rate [FDR] q <0.05) were 1q21.3 and 8q24.3, and significantly deleted regions were 3p21.31, 4q12, 5q13.2, 5q23.2, 5q31.1, 7p22.1, 7q11.23, 8p12, 9p22.1, 11p15.1, 12p13.31, 15q11.2, 15q21.2, 18p11.31, and 22q11.21 using the Genomic Identification of Significant Targets in Cancer (GISTIC) analysis. Integrative analysis revealed 94 genes demonstrating frequent copy number alterations (>25% of samples) that correlated with gene expression (FDR <0.05). These genes were mainly located on 8p11.21, 8p21.2-p21.3, 8q22.1, 8q24.3, 17q23.2-q23.3, 19p13.3, and 19p13.11. Among the regions, 8q24.3 was found to contain the most genes (30 of 94 genes) including PTK2. The 8q24.3 region was indicated as the most significant region, as supported by copy number, GISTIC, and integrative analysis. Pathway analysis using differentially expressed genes on 8q24.3 revealed several major nodes, including PTK2. In conclusion, we identified a set of 94 candidate genes with frequent copy number alterations that correlated with gene expression. Specific chromosomal alterations, such as the 8q24.3 gain containing PTK2, could be a therapeutic target in a subset of Ov-CCAs. Copyright © 2013. Published by Elsevier Inc.

  5. Increased Growth of a Newly Established Mouse Epithelial Cell Line Transformed with HPV-16 E7 in Diabetic Mice.

    PubMed

    He, Lan; Law, Priscilla T Y; Boon, Siaw Shi; Zhang, Chuqing; Ho, Wendy C S; Banks, Lawrence; Wong, C K; Chan, Juliana C N; Chan, Paul K S

    2016-01-01

    Epidemiological evidence supports that infection with high-risk types of human papillomavirus (HPV) can interact with host and environmental risk factors to contribute to the development of cervical, oropharyngeal, and other anogenital cancers. In this study, we established a mouse epithelial cancer cell line, designated as Chinese University Papillomavirus-1 (CUP-1), from C57BL/KsJ mice through persistent expression of HPV-16 E7 oncogene. After continuous culturing of up to 200 days with over 60 passages, we showed that CUP-1 became an immortalized and transformed epithelial cell line with continuous E7 expression and persistent reduction of retinoblastoma protein (a known target of E7). This model allowed in-vivo study of interaction between HPV and co-factors of tumorigenesis in syngeneic mice. Diabetes has been shown to increase HPV pathogenicity in different pathological context. Herein, with this newly-established cell line, we uncovered that diabetes promoted CUP-1 xenograft growth in syngeneic db/db mice. In sum, we successfully established a HPV-16 E7 transformed mouse epithelial cell line, which allowed subsequent studies of co-factors in multistep HPV carcinogenesis in an immunocompetent host. More importantly, this study is the very first to demonstrate the promoting effect of diabetes on HPV-associated carcinogenesis in vivo, implicating the importance of cancer surveillance in diabetic environment.

  6. A Non-oncogenic HPV 16 E6/E7 Vaccine Enhances Treatment of HPV Expressing Tumors

    PubMed Central

    Wieking, Bryant G.; Vermeer, Daniel W.; Spanos, William C.; Lee, Kimberly M.; Vermeer, Paola; Lee, Walter T.; Xu, Younong; Gabitzsch, Elizabeth S.; Balcaitis, Stephanie; Balint, Joseph P.; Jones, Frank R.; Lee, John H.

    2012-01-01

    Human papillomaviruses (HPVs) are the causative factor for greater than 90% of cervical cancers and 25% of head and neck cancers. The incidence of HPV positive (+) head and neck squamous cell carcinomas (HNSCCs) has greatly increased in the last 30 years. E6 and E7 are the two key viral oncoproteins that induce and propagate cellular transformation. An immune response generated during cisplatin/radiation therapy improves tumor clearance of HPV(+) cancers. Augmenting this induced response during therapy with an adenoviral HPV16 E6/E7 vaccine improves long term survival in preclinical models. Here we describe the generation of an HPV16 E6/E7 construct, which contains mutations that render E6/E7 non-oncogenic, while preserving antigenicity. These mutations do not allow E6/E7 to degrade p53, pRb, PTPN13, or activate telomerase. Non-oncogenic E6/E7 (E6Δ/E7Δ) expressed as a stable integrant, or in the [E1-, E2b-] adenovirus, lacks the ability to transform human cells while retaining the ability to induce an HPV specific immune response. Moreover, E6Δ/E7Δ plus chemotherapy/radiation statistically enhances clearance of established HPV(+) cancer in vivo. PMID:22918471

  7. HPV16 E7 protein associates with the protein kinase p33CDK2 and cyclin A.

    PubMed

    Tommasino, M; Adamczewski, J P; Carlotti, F; Barth, C F; Manetti, R; Contorni, M; Cavalieri, F; Hunt, T; Crawford, L

    1993-01-01

    E7 is the major transforming protein of human papillomavirus type 16 (HPV16). It has been found to associate with the retinoblastoma protein Rb1. We investigated whether HPV16 E7 protein was associated with other cellular proteins, in particular with those involved in cell cycle control. Immunoprecipitates from CaSki cell extracts with an anti E7 monoclonal antibody contained a histone H1 kinase. Recombinant E7, synthesized in yeast, when mixed with protein extracts from epithelial cells bound histone H1 kinase activity in vitro. The in vivo and the in vitro-formed E7-kinase complex had the same periodicity of activity during the cell cycle, being most active in S and G2/M. Immunoblotting of E7 immunoprecipitates with an antibody raised against the p33CDK2, revealed a 33 kDa protein band not detected by an anti-p34cdc2 antibody, suggesting that the E7-associated kinase activity is due to the p33CDK2. The interaction appears to be via cyclin A, since probing of similar immunoblots showed a 50 kDa band corresponding to cyclin A. The association of E7 with cyclin A appeared to be direct, not involving Rb 1 or other proteins.

  8. Detection of MET Gene Copy Number in Cancer Samples Using the Droplet Digital PCR Method.

    PubMed

    Zhang, Yanni; Tang, En-Tzu; Du, Zhiqiang

    2016-01-01

    The analysis of MET gene copy number (CN) has been considered to be a potential biomarker to predict the response to MET-targeted therapies in various cancers. However, the current standard methods to determine MET CN are SNP 6.0 in the genomic DNA of cancer cell lines and fluorescence in situ hybridization (FISH) in tumor models, respectively, which are costly and require advanced technical skills and result in relatively subjective judgments. Therefore, we employed a novel method, droplet digital PCR (ddPCR), to determine the MET gene copy number with high accuracy and precision. The genomic DNA of cancer cell lines or tumor models were tested and compared with the MET gene CN and MET/CEN-7 ratio determined by SNP 6.0 and FISH, respectively. In cell lines, the linear association of the MET CN detected by ddPCR and SNP 6.0 is strong (Pearson correlation = 0.867). In tumor models, the MET CN detected by ddPCR was significantly different between the MET gene amplification and non-amplification groups according to FISH (mean: 15.4 vs 2.1; P = 0.044). Given that MET gene amplification is defined as MET CN >5.5 by ddPCR, the concordance rate between ddPCR and FISH was 98.0%, and Cohen's kappa coefficient was 0.760 (95% CI, 0.498-1.000; P <0.001). The results demonstrated that the ddPCR method has the potential to quantify the MET gene copy number with high precision and accuracy as compared with the results from SNP 6.0 and FISH in cancer cell lines and tumor samples, respectively.

  9. Efficacy of fewer than three doses of an HPV-16/18 AS04-adjuvanted vaccine: combined analysis of data from the Costa Rica Vaccine and PATRICIA Trials.

    PubMed

    Kreimer, Aimée R; Struyf, Frank; Del Rosario-Raymundo, Maria Rowena; Hildesheim, Allan; Skinner, S Rachel; Wacholder, Sholom; Garland, Suzanne M; Herrero, Rolando; David, Marie-Pierre; Wheeler, Cosette M; González, Paula; Jiménez, Silvia; Lowy, Douglas R; Pinto, Ligia A; Porras, Caroline; Rodriguez, Ana Cecilia; Safaeian, Mahboobeh; Schiffman, Mark; Schiller, John T; Schussler, John; Sherman, Mark E; Bosch, F Xavier; Castellsague, Xavier; Chatterjee, Archana; Chow, Song-Nan; Descamps, Dominique; Diaz-Mitoma, Francisco; Dubin, Gary; Germar, Maria Julieta; Harper, Diane M; Lewis, David J M; Limson, Genara; Naud, Paulo; Peters, Klaus; Poppe, Willy A J; Ramjattan, Brian; Romanowski, Barbara; Salmeron, Jorge; Schwarz, Tino F; Teixeira, Julio C; Tjalma, Wiebren A A

    2015-07-01

    There is some evidence to suggest that one or two doses of the HPV vaccine provides similar protection to the three-dose regimen. The main aim of the study was to ascertain HPV-16/18 vaccine efficacy in both full and naive cohorts and to explore protection conferred against non-vaccine HPV types, by number of doses received. Summary data from the Costa Rica Vaccine Trial (CVT; NCT00128661) and ~the PATRICIA trial (NCT001226810), two phase 3, double-blind, randomised controlled clinical trials of the HPV-16/18 AS04-adjuvanted vaccine in young women, were combined in a post-hoc analysis (GlaxoSmithKline [GSK] e-track number 202142) to investigate the efficacy of fewer than three doses of the HPV-16/18 vaccine after 4 years of follow-up. Women were randomly assigned to receive three doses of the HPV-16/18 vaccine or to a control vaccine; yet, some received fewer doses. After exclusion of women with less than 12 months of follow-up or those who were HPV-16/18 DNA-positive at enrolment (for the HPV-16/18 endpoint), we calculated vaccine efficacy against one-time detection of incident HPV infections after three, two, and one dose(s). The primary study endpoint was one-time detection of first incident HPV-16/18 infections accumulated during the follow-up phase. We assessed vaccine efficacy against incident HPV-16/18 infection in the modified total vaccinated cohort (22 327 received three doses, 1185 two doses, 543 one dose). Vaccine efficacy against incident HPV-16/18 infections for three doses was 77·0% (95% CI 74·7-79·1), two doses was 76·0% (62·0-85·3), and one dose was 85·7% (70·7-93·7). Vaccine efficacy against incident HPV-31/33/45 infections for three doses was 59·7% (56·0-63·0), two doses was 37·7% (12·4-55·9), and one dose was 36·6% (-5·4 to 62·2). Vaccine efficacy against incident HPV-16/18 infection for two-dose women who received their second dose at 1 month was 75·3% (54·2-87·5) and 82·6% (42·3-96·1) for those who received the second

  10. Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using the Pichia pastoris PGK1 Promoter.

    PubMed

    Mariz, F C; Coimbra, E C; Jesus, A L S; Nascimento, L M; Torres, F A G; Freitas, A C

    2015-01-01

    The human papillomavirus (HPV) L1 major capsid protein, which forms the basis of the currently available vaccines against cervical cancer, self-assembles into virus-like particles (VLPs) when expressed heterologously. We report the development of a biotechnology platform for HPV16 L1 protein expression based on the constitutive PGK1 promoter (PPGK1) from the methylotrophic yeast Pichia pastoris. The L1 gene was cloned under regulation of PPGK1 into pPGKΔ3 expression vector to achieve intracellular expression. In parallel, secretion of the L1 protein was obtained through the use of an alternative vector called pPGKΔ3α, in which a codon optimized α-factor signal sequence was inserted. We devised a work-flow based on the detection of the L1 protein by dot blot, colony blot, and western blot to classify the positive clones. Finally, intracellular HPV VLPs assembly was demonstrated for the first time in yeast cells. This study opens up perspectives for the establishment of an innovative platform for the production of HPV VLPs or other viral antigens for vaccination purposes, based on constitutive expression in P. pastoris.

  11. HPV-16 E7 expression up-regulates phospholipase D activity and promotes rapamycin resistance in a pRB-dependent manner.

    PubMed

    Rabachini, Tatiana; Boccardo, Enrique; Andrade, Rubiana; Perez, Katia Regina; Nonogaki, Suely; Cuccovia, Iolanda Midea; Villa, Luisa Lina

    2018-04-27

    Human Papillomavirus (HPV) infection is the main risk factor for the development and progression of cervical cancer. HPV-16 E6 and E7 expression is essential for induction and maintenance of the transformed phenotype. These oncoproteins interfere with the function of several intracellular proteins, including those controlling the PI3K/AKT/mTOR pathway in which Phospolipase D (PLD) and Phosphatidic acid (PA) play a critical role. PLD activity was measured in primary human keratinocytes transduced with retroviruses expressing HPV-16 E6, E7 or E7 mutants. The cytostatic effect of rapamycin, a well-known mTOR inhibitor with potential clinical applications, was evaluated in monolayer and organotypic cultures. HPV-16 E7 expression in primary human keratinocytes leads to an increase in PLD expression and activity. Moreover, this activation is dependent on the ability of HPV-16 E7 to induce retinoblastoma protein (pRb) degradation. We also show that cells expressing HPV-16 E7 or silenced for pRb acquire resistance to the antiproliferative effect of rapamycin. This is the first indication that HPV oncoproteins can affect PLD activity. Since PA can interfere with the ability of rapamycin to bind mTOR, the use of combined strategies to target mTOR and PLD activity might be considered to treat HPV-related malignancies.

  12. Cellular immune responses to HPV-18, -31, and -53 in healthy volunteers immunized with recombinant HPV-16 L1 virus-like particles

    SciTech Connect

    Pinto, Ligia A.; Viscidi, Raphael; Harro, Clayton D.

    Human papillomavirus-like particles (HPV VLP) are candidate vaccines that have shown to be efficacious in reducing infection and inducing robust antiviral immunity. Neutralizing antibodies generated by vaccination are largely type-specific, but little is known about the type-specificity of cellular immune responses to VLP vaccination. To determine whether vaccination with HPV-16 L1VLP induces cellular immunity to heterologous HPV types (HPV-18, HPV-31, and HPV-53), we examined proliferative and cytokine responses in vaccine (n = 11) and placebo (n = 5) recipients. Increased proliferative and cytokine responses to heterologous types were observed postvaccination in some individuals. The proportion of women responding to heterologousmore » types postvaccination (36%-55%) was lower than that observed in response to HPV-16 (73%). Response to HPV-16 VLP predicted response to other types. The strongest correlations in response were observed between HPV-16 and HPV-31, consistent with their phylogenetic relatedness. In summary, PBMC from HPV-16 VLP vaccine recipients can respond to L1VLP from heterologous HPV types, suggesting the presence of conserved T cell epitopes.« less

  13. Prevention of persistent human papillomavirus infection by an HPV16/18 vaccine: a community-based randomized clinical trial in Guanacaste, Costa Rica.

    PubMed

    Herrero, Rolando; Wacholder, Sholom; Rodríguez, Ana C; Solomon, Diane; González, Paula; Kreimer, Aimee R; Porras, Carolina; Schussler, John; Jiménez, Silvia; Sherman, Mark E; Quint, Wim; Schiller, John T; Lowy, Douglas R; Schiffman, Mark; Hildesheim, Allan

    2011-10-01

    Target groups for human papillomavirus (HPV) vaccination are controversial. We evaluated vaccine efficacy (VE) against 1-year persistent infection, stratified by age and sexual behavior, among young women in Costa Rica. We randomized 7,466 healthy women 18 to 25 years of age to HPV16/18 or hepatitis A vaccine (follow-up, 50.4 months). According-to-protocol (ATP) cohorts included compliant HPV-negative women; intention-to-treat (ITT) included all randomized women. ATP VE was 90.9% (95% CI, 82.0-95.9) against HPV16/18 infections, 44.5% against HPV31/33/45 (95% CI, 17.5-63.1), and 12.4% (95% CI, -3.2 to 25.6) against any oncogenic infection. Overall ITT VE against HPV16/18 infections was 49.0%, but ATP and ITT VE almost reached 100% in year 4 of follow-up. ATP efficacy against HPV16/18 was similar by age, but ITT VE was greatest among youngest women (68.9% among those 18-19 years of age; 21.8% among those 24-25 years of age) and 79.8% among virgins. Among previously unexposed women, vaccination is highly efficacious against HPV16/18 and partially against HPV31/33/45. Vaccination is most effective in women and girls before they initiate sexual activity, with programmatic and individual decision implications.

  14. Regression of Established Human Papillomavirus Type 16 (HPV-16) Immortalized Tumors In Vivo by Vaccinia Viruses Expressing Different Forms of HPV-16 E7 Correlates with Enhanced CD8+ T-Cell Responses That Home to the Tumor Site

    PubMed Central

    Lamikanra, Abigail; Pan, Zhen-Kun; Isaacs, Stuart N.; Wu, Tzyy-Choou; Paterson, Yvonne

    2001-01-01

    Using vaccinia virus as a live vector, we show that the expression of human papillomavirus type 16 (HPV-16) E7 fused to a nonhemolytic portion of the Listeria monocytogenes virulence factor, listeriolysin O (LLO), induces an immune response that causes the regression of established HPV-16 immortalized tumors in C57BL/6 mice. The vaccinia virus construct expressing LLO fused to E7 (VacLLOE7) was compared with two previously described vaccinia virus constructs: one that expresses unmodified E7 (VacE7) and another that expresses E7 in a form designed to direct it to intracellular lysosomal compartments and improve major histocompatibility complex class II-restricted responses (VacSigE7LAMP-1). C57BL/6 mice bearing established HPV-16 immortalized tumors of 5 or 8 mm were treated with each of these vaccines. Fifty percent of the mice treated with VacLLOE7 remained tumor free 2 months after tumor inoculation, whereas 12 to 25% of the mice were tumor free after treatment with VacSigE7LAMP-1 (depending on the size of the tumor). No mice were tumor free in the group given VacE7. Compared to VacE7, VacSigE7LAMP-1 and VacLLOE7 resulted in increased numbers of H2-Db-specific tetramer-positive CD8+ T cells in mouse spleens that produced gamma interferon and tumor necrosis factor alpha upon stimulation with RAHYNIVTF peptide. In addition, the highest frequency of tetramer-positive T cells was seen in the tumor sites of mice treated with VacLLOE7. An increased efficiency of E7-specific lysis by splenocytes from mice immunized with VacLLOE7 was also observed. These results indicate that the fusion of E7 with LLO not only enhances antitumor therapy by improving the tumoricidal function of E7-specific CD8+ T cells but may also increase the number of antigen-specific CD8+ T cells in the tumor, the principle site of antigen expression. PMID:11559797

  15. The positioning logic and copy number control of genes in bacteria under stress

    NASA Astrophysics Data System (ADS)

    Zhang, Qiucen; Austin, Robert; Vyawahare, Saurabh; Lau, Alexandra

    2013-03-01

    Escherichia coli (E. coli) cells when challenged with sublethal concentrations of the genotoxic antibiotic ciprofloxacin cease to divide and form long filaments which contain multiple bacterial chromosomes. These filaments are individual mesoscopic environmental niches which provide protection for a community of chromosomes (as opposed to cells) under mutagenic stress and can provide an evolutionary fitness advantage within the niche. We use comparative genomic hybridization to show that the mesoscopic niche evolves within 20 minutes of ciprofloxacin exposure via replication of multiple copies of genes expressing ATP dependent transporters. We show that this rapid genomic amplification is done in a time efficient manner via placement of the genes encoding the pumps near the origin of replication on the bacterial chromosome. The de-amplification of multiple copies back to the wild type number is a function of the duration is a function of the ciprofloxacin exposure duration: the longer the exposure, the slower the removal of the multiple copies. The project described was supported by the National Science Foundation and the National Cancer Institute

  16. Increased copy number of the DLX4 homeobox gene in breast axillary lymph node metastasis

    PubMed Central

    Torresan, Clarissa; Oliveira, Márcia M.C.; Pereira, Silma R.F.; Ribeiro, Enilze M.S.F.; Marian, Catalin; Gusev, Yuriy; Lima, Rubens S.; Urban, Cicero A.; Berg, Patricia E.; Haddad, Bassem R.; Cavalli, Iglenir J.; Cavalli, Luciane R.

    2017-01-01

    DLX4 is a homeobox gene strongly implicated in breast tumor progression and invasion. Our main objective was to determine the DLX4 copy number status in sentinel lymph node (SLN) metastasis to assess its involvement in the initial stages of the axillary metastatic process. A total of 37 paired samples of SLN metastasis and primary breast tumors (PBT) were evaluated by fluorescence in situ hybridization, quantitative polymerase chain reaction and array comparative genomic hybridization assays. DLX4 increased copy number was observed in 21.6% of the PBT and 24.3% of the SLN metastasis; regression analysis demonstrated that the DLX4 alterations observed in the SLN metastasis were dependent on the ones in the PBT, indicating that they occur in the primary tumor cell populations and are maintained in the early axillary metastatic site. In addition, regression analysis demonstrated that DLX4 alterations (and other DLX and HOXB family members) occurred independently of the ones in the HER2/NEU gene, the main amplification driver on the 17q region. Additional studies evaluating DLX4 copy number in non-SLN axillary lymph nodes and/or distant breast cancer metastasis are necessary to determine if these alterations are carried on and maintained during more advanced stages of tumor progression and if could be used as a predictive marker for axillary involvement. PMID:24947980

  17. Origin of a function by tandem gene duplication limits the evolutionary capability of its sister copy.

    PubMed

    Hasselmann, Martin; Lechner, Sarah; Schulte, Christina; Beye, Martin

    2010-07-27

    The most remarkable outcome of a gene duplication event is the evolution of a novel function. Little information exists on how the rise of a novel function affects the evolution of its paralogous sister gene copy, however. We studied the evolution of the feminizer (fem) gene from which the gene complementary sex determiner (csd) recently derived by tandem duplication within the honey bee (Apis) lineage. Previous studies showed that fem retained its sex determination function, whereas the rise of csd established a new primary signal of sex determination. We observed a specific reduction of nonsynonymous to synonymous substitution ratios in Apis to non-Apis fem. We found a contrasting pattern at two other genetically linked genes, suggesting that hitchhiking effects to csd, the locus under balancing selection, is not the cause of this evolutionary pattern. We also excluded higher synonymous substitution rates by relative rate testing. These results imply that stronger purifying selection is operating at the fem gene in the presence of csd. We propose that csd's new function interferes with the function of Fem protein, resulting in molecular constraints and limited evolvability of fem in the Apis lineage. Elevated silent nucleotide polymorphism in fem relative to the genome-wide average suggests that genetic linkage to the csd gene maintained more nucleotide variation in today's population. Our findings provide evidence that csd functionally and genetically interferes with fem, suggesting that a newly evolved gene and its functions can limit the evolutionary capability of other genes in the genome.

  18. From DNA Copy Number to Gene Expression: Local aberrations, Trisomies and Monosomies

    NASA Astrophysics Data System (ADS)

    Shay, Tal

    The goal of my PhD research was to study the effect of DNA copy number changes on gene expression. DNA copy number aberrations may be local, encompassing several genes, or on the level of an entire chromosome, such as trisomy and monosomy. The main dataset I studied was of Glioblastoma, obtained in the framework of a collaboration, but I worked also with public datasets of cancer and Down's Syndrome. The molecular basis of expression changes in Glioblastoma. Glioblastoma is the most common and aggressive type of primary brain tumors in adults. In collaboration with Prof. Hegi (CHUV, Switzerland), we analyzed a rich Glioblastoma dataset including clinical information, DNA copy number (array CGH) and expression profiles. We explored the correlation between DNA copy number and gene expression at the level of chromosomal arms and local genomic aberrations. We detected known amplification and over expression of oncogenes, as well as deletion and down-regulation of tumor suppressor genes. We exploited that information to map alterations of pathways that are known to be disrupted in Glioblastoma, and tried to characterize samples that have no known alteration in any of the studied pathways. Identifying local DNA aberrations of biological significance. Many types of tumors exhibit chromosomal losses or gains and local amplifications and deletions. A region that is aberrant in many tumors, or whose copy number change is stronger, is more likely to be clinically relevant, and not just a by-product of genetic instability. We developed a novel method that defines and prioritizes aberrations by formalizing these intuitions. The method scores each aberration by the fraction of patients harboring it, its length and its amplitude, and assesses the significance of the score by comparing it to a null distribution obtained by permutations. This approach detects genetic locations that are significantly aberrant, generating a 'genomic aberration profile' for each sample. The 'genomic

  19. Diversity and population-genetic properties of copy number variations and multicopy genes in cattle

    PubMed Central

    Bickhart, Derek M.; Xu, Lingyang; Hutchison, Jana L.; Cole, John B.; Null, Daniel J.; Schroeder, Steven G.; Song, Jiuzhou; Garcia, Jose Fernando; Sonstegard, Tad S.; Van Tassell, Curtis P.; Schnabel, Robert D.; Taylor, Jeremy F.; Lewin, Harris A.; Liu, George E.

    2016-01-01

    The diversity and population genetics of copy number variation (CNV) in domesticated animals are not well understood. In this study, we analysed 75 genomes of major taurine and indicine cattle breeds (including Angus, Brahman, Gir, Holstein, Jersey, Limousin, Nelore, and Romagnola), sequenced to 11-fold coverage to identify 1,853 non-redundant CNV regions. Supported by high validation rates in array comparative genomic hybridization (CGH) and qPCR experiments, these CNV regions accounted for 3.1% (87.5 Mb) of the cattle reference genome, representing a significant increase over previous estimates of the area of the genome that is copy number variable (∼2%). Further population genetics and evolutionary genomics analyses based on these CNVs revealed the population structures of the cattle taurine and indicine breeds and uncovered potential diversely selected CNVs near important functional genes, including AOX1, ASZ1, GAT, GLYAT, and KRTAP9-1. Additionally, 121 CNV gene regions were found to be either breed specific or differentially variable across breeds, such as RICTOR in dairy breeds and PNPLA3 in beef breeds. In contrast, clusters of the PRP and PAG genes were found to be duplicated in all sequenced animals, suggesting that subfunctionalization, neofunctionalization, or overdominance play roles in diversifying those fertility-related genes. These CNV results provide a new glimpse into the diverse selection histories of cattle breeds and a basis for correlating structural variation with complex traits in the future. PMID:27085184

  20. The nuclear retention signal of HPV16 L2 protein is essential for incoming viral genome to transverse the trans-Golgi network

    PubMed Central

    DiGiuseppe, Stephen; Bienkowska-Haba, Malgorzata; Hilbig, Lydia; Sapp, Martin

    2014-01-01

    The Human papillomavirus (HPV) capsid is composed of the major and minor capsid proteins, L1 and L2, respectively. Infectious entry requires a complex series of conformational changes in both proteins that lead to uptake and allow uncoating to occur. During entry, the capsid is disassembled and host cyclophilins dissociate L1 protein from the L2/DNA complex. Herein, we describe a mutant HPV16 L2 protein (HPV16 L2-R302/5A) that traffics pseudogenome to the trans-Golgi network (TGN) but fails to egress. Our data provide further evidence that HPV16 traffics through the TGN and demonstrates that L2 is essential for TGN egress. Furthermore, we show that cyclophilin activity is required for the L2/DNA complex to be transported to the TGN which is accompanied by a reduced L1 protein levels. PMID:24928042

  1. [Preinvasive vulvar and cervical cancer in a 32-year-old woman, DNA HPV 16 positive with mtDNA mutation--case study].

    PubMed

    Kedzia, Witold; Malkowska-Walczak, Blanka; Józefiak, Agata; Wadowicka, Alicja; Guglas, Bogna; Pruski, Dominik; Kedzia, Helena; Spaczyński, Marek

    2009-07-01

    Coincidence of preinvasive vulvar and cervical cancer in young women is very rare. Lesions like VIN 3/preinvasive vulvar cancer and CIN 3/preinvasive cervical cancer are strictly connected with viral infection and are multilocular. In the presented case the following tests have been performed: HPV DNA test for the presence of 13 oncogenic HPV types, mRNA HPV test for the presence of transcripts for HPV 16, 18, 31, 33, 45 and the analysis of mtDNA D-Loop region. In the examined patient HPV 16 infection, as well as the presence of transcripts for HPV 16 E6/7 were diagnosed. The analysis of mtDNA D-Loop region showed nucleotide lesions like: T>C 16.192, T>C 16.223, T>C 16.292, C>T 16.325, C>T 16.579.

  2. Systematic Prioritization and Integrative Analysis of Copy Number Variations in Schizophrenia Reveal Key Schizophrenia Susceptibility Genes

    PubMed Central

    Luo, Xiongjian; Huang, Liang; Han, Leng; Luo, Zhenwu; Hu, Fang; Tieu, Roger; Gan, Lin

    2014-01-01

    Schizophrenia is a common mental disorder with high heritability and strong genetic heterogeneity. Common disease-common variants hypothesis predicts that schizophrenia is attributable in part to common genetic variants. However, recent studies have clearly demonstrated that copy number variations (CNVs) also play pivotal roles in schizophrenia susceptibility and explain a proportion of missing heritability. Though numerous CNVs have been identified, many of the regions affected by CNVs show poor overlapping among different studies, and it is not known whether the genes disrupted by CNVs contribute to the risk of schizophrenia. By using cumulative scoring, we systematically prioritized the genes affected by CNVs in schizophrenia. We identified 8 top genes that are frequently disrupted by CNVs, including NRXN1, CHRNA7, BCL9, CYFIP1, GJA8, NDE1, SNAP29, and GJA5. Integration of genes affected by CNVs with known schizophrenia susceptibility genes (from previous genetic linkage and association studies) reveals that many genes disrupted by CNVs are also associated with schizophrenia. Further protein-protein interaction (PPI) analysis indicates that protein products of genes affected by CNVs frequently interact with known schizophrenia-associated proteins. Finally, systematic integration of CNVs prioritization data with genetic association and PPI data identifies key schizophrenia candidate genes. Our results provide a global overview of genes impacted by CNVs in schizophrenia and reveal a densely interconnected molecular network of de novo CNVs in schizophrenia. Though the prioritized top genes represent promising schizophrenia risk genes, further work with different prioritization methods and independent samples is needed to confirm these findings. Nevertheless, the identified key candidate genes may have important roles in the pathogenesis of schizophrenia, and further functional characterization of these genes may provide pivotal targets for future therapeutics and

  3. Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting.

    PubMed

    Aguirre, Andrew J; Meyers, Robin M; Weir, Barbara A; Vazquez, Francisca; Zhang, Cheng-Zhong; Ben-David, Uri; Cook, April; Ha, Gavin; Harrington, William F; Doshi, Mihir B; Kost-Alimova, Maria; Gill, Stanley; Xu, Han; Ali, Levi D; Jiang, Guozhi; Pantel, Sasha; Lee, Yenarae; Goodale, Amy; Cherniack, Andrew D; Oh, Coyin; Kryukov, Gregory; Cowley, Glenn S; Garraway, Levi A; Stegmaier, Kimberly; Roberts, Charles W; Golub, Todd R; Meyerson, Matthew; Root, David E; Tsherniak, Aviad; Hahn, William C

    2016-08-01

    The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest. By examining single-guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR/Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR/Cas9 elicits a gene-independent antiproliferative cell response. This effect has important practical implications for the interpretation of CRISPR/Cas9 screening data and confounds the use of this technology for the identification of essential genes in amplified regions. We found that the number of CRISPR/Cas9-induced DNA breaks dictates a gene-independent antiproliferative response in cells. These observations have practical implications for using CRISPR/Cas9 to interrogate cancer gene function and illustrate that cancer cells are highly sensitive to site-specific DNA damage, which may provide a path to novel therapeutic strategies. Cancer Discov; 6(8); 914-29. ©2016 AACR.See related commentary by Sheel and Xue, p. 824See related article by Munoz et al., p. 900This article is highlighted in the In This Issue feature, p. 803. 2016 American Association for Cancer Research.

  4. Proof-of-principle evaluation of the efficacy of fewer than three doses of a bivalent HPV16/18 vaccine.

    PubMed

    Kreimer, Aimée R; Rodriguez, Ana Cecilia; Hildesheim, Allan; Herrero, Rolando; Porras, Carolina; Schiffman, Mark; González, Paula; Solomon, Diane; Jiménez, Silvia; Schiller, John T; Lowy, Douglas R; Quint, Wim; Sherman, Mark E; Schussler, John; Wacholder, Sholom

    2011-10-05

    Three-dose regimens for human papillomavirus (HPV) vaccines are expensive and difficult to complete, especially in settings where the need for cervical cancer prevention is greatest. We evaluated the vaccine efficacy of fewer than three doses of the HPV16/18 vaccine Cervarix in our Costa Rica Vaccine Trial. Women were randomly assigned to receive three doses of the HPV16/18 vaccine or to a control vaccine and were followed for incident HPV16 or HPV18 infection that persisted in visits that were 10 or more months apart (median follow-up 4.2 years). After excluding women who had no follow-up or who were HPV16 and HPV18 DNA positive at enrollment, 5967 women received three vaccine doses (2957 HPV vaccine vs 3010 control vaccine), 802 received two doses (422 HPV vs. 380 control), and 384 received one dose (196 HPV vs. 188 control). Reasons for receiving fewer doses and other pre- and post-randomization characteristics were balanced within each dosage group between women receiving the HPV and control vaccines. Incident HPV16 or HPV18 infections that persisted for 1 year were unrelated to dosage of the control vaccine. Vaccine efficacy was 80.9% for three doses of the HPV vaccine (95% confidence interval [CI] = 71.1% to 87.7%; 25 and 133 events in the HPV and control arms, respectively), 84.1% for two doses (95% CI = 50.2% to 96.3%; 3 and 17 events), and 100% for one dose (95% CI = 66.5% to 100%; 0 and 10 events). Four years after vaccination of women who appeared to be uninfected, this nonrandomized analysis suggests that two doses of the HPV16/18 vaccine, and maybe even one dose, are as protective as three doses.

  5. Comparison of cytology, HPV DNA testing and HPV 16/18 genotyping alone or combined targeting to the more balanced methodology for cervical cancer screening.

    PubMed

    Chatzistamatiou, Kimon; Moysiadis, Theodoros; Moschaki, Viktoria; Panteleris, Nikolaos; Agorastos, Theodoros

    2016-07-01

    The objective of the present study was to identify the most effective cervical cancer screening algorithm incorporating different combinations of cytology, HPV testing and genotyping. Women 25-55years old recruited for the "HERMES" (HEllenic Real life Multicentric cErvical Screening) study were screened in terms of cytology and high-risk (hr) HPV testing with HPV 16/18 genotyping. Women positive for cytology or/and hrHPV were referred for colposcopy, biopsy and treatment. Ten screening algorithms based on different combinations of cytology, HPV testing and HPV 16/18 genotyping were investigated in terms of diagnostic accuracy. Three clusters of algorithms were formed according to the balance between effectiveness and harm caused by screening. The cluster showing the best balance included two algorithms based on co-testing and two based on HPV primary screening with HPV 16/18 genotyping. Among these, hrHPV testing with HPV 16/18 genotyping and reflex cytology (atypical squamous cells of undetermined significance - ASCUS threshold) presented the optimal combination of sensitivity (82.9%) and specificity relative to cytology alone (0.99) with 1.26 false positive rate relative to cytology alone. HPV testing with HPV 16/18 genotyping, referring HPV 16/18 positive women directly to colposcopy, and hrHPV (non 16/18) positive women to reflex cytology (ASCUS threshold), as a triage method to colposcopy, reflects the best equilibrium between screening effectiveness and harm. Algorithms, based on cytology as initial screening method, on co-testing or HPV primary without genotyping, and on HPV primary with genotyping but without cytology triage, are not supported according to the present analysis. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Human papillomavirus (HPV) types 16, 18, 31, 45 DNA loads and HPV-16 integration in persistent and transient infections in young women

    PubMed Central

    2010-01-01

    Background HPV burden is a predictor for high-grade cervical intraepithelial neoplasia and cancer. The natural history of HPV load in young women being recently exposed to HPV is described in this paper. Methods A total of 636 female university students were followed for 2 years. Cervical specimens with HPV-16, -18, -31, or -45 DNA by consensus PCR were further evaluated with type-specific and β-globin real-time PCR assays. Proportional hazards regression was used to estimate hazard ratios (HR) of infection clearance. Generalized estimating equations assessed whether HPV loads was predictive of HPV infection at the subsequent visit. Results HPV loads were consistently higher among women <25 years old, and those who had multiple sex partners, multiple HPV type infections and smokers. HPV-16 integration was encountered only in one sample. Infection clearance was faster among women at lower tertiles of HPV-16 (HR = 2.8, 95%CI: 1.0-8.1), HPV-18 (HR = 3.5, 95%CI: 1.1-11.2) or combined (HR = 2.4, 95%CI: 1.8-6.2) DNA loads. The relationship between HPV-16 and HPV-18 DNA loads and infection clearance followed a clear dose-response pattern, after adjusting for age and number of sexual partners. GEE Odds Ratios for HPV persistence of the middle and upper tertiles relative to the lower tertile were 2.7 and 3.0 for HPV-16 and 3.8 and 39.1 for HPV-18, respectively. There was no association between HPV-31 or -45 DNA loads and persistence. Conclusions The association between HPV load and persistence is not uniform across high-risk genital genotypes. HPV-16 integration was only rarely demonstrated in young women. PMID:21070660

  7. Multiple copies of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708.

    PubMed Central

    Gopal-Srivastava, R; Mallonee, D H; White, W B; Hylemon, P B

    1990-01-01

    Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium which possesses inducible bile acid 7-dehydroxylation activity. Several new polypeptides are produced in this strain following induction with cholic acid. Genes coding for two copies of a bile acid-inducible 27,000-dalton polypeptide (baiA1 and baiA2) have been previously cloned and sequenced. We now report on a gene coding for a third copy of this 27,000-dalton polypeptide (baiA3). The baiA3 gene has been cloned in lambda DASH on an 11.2-kilobase DNA fragment from a partial Sau3A digest of the Eubacterium DNA. DNA sequence analysis of the baiA3 gene revealed 100% homology with the baiA1 gene within the coding region of the 27,000-dalton polypeptides. The baiA2 gene shares 81% sequence identity with the other two genes at the nucleotide level. The flanking nucleotide sequences associated with the baiA1 and baiA3 genes are identical for 930 bases in the 5' direction from the initiation codon and for at least 325 bases in the 3' direction from the stop codon, including the putative promoter regions for the genes. An additional open reading frame (occupying from 621 to 648 bases, depending on the correct start codon) was found in the identical 5' regions associated with the baiA1 and baiA3 clones. The 5' sequence 930 bases upstream from the baiA1 and baiA3 genes was totally divergent. The baiA2 gene, which is part of a large bile acid-inducible operon, showed no homology with the other two genes either in the 5' or 3' direction from the polypeptide coding region, except for a 15-base-pair presumed ribosome-binding site in the 5' region. These studies strongly suggest that a gene duplication (baiA1 and baiA3) has occurred and is stably maintained in this bacterium. Images PMID:2376563

  8. Phylogenetic Resolution of Deep Eukaryotic and Fungal Relationships Using Highly Conserved Low-Copy Nuclear Genes

    PubMed Central

    Ren, Ren; Sun, Yazhou; Zhao, Yue; Geiser, David

    2016-01-01

    Abstract A comprehensive and reliable eukaryotic tree of life is important for many aspects of biological studies from comparative developmental and physiological analyses to translational medicine and agriculture. Both gene-rich and taxon-rich approaches are effective strategies to improve phylogenetic accuracy and are greatly facilitated by marker genes that are universally distributed, well conserved, and orthologous among divergent eukaryotes. In this article, we report the identification of 943 low-copy eukaryotic genes and we show that many of these genes are promising tools in resolving eukaryotic phylogenies, despite the challenges of determining deep eukaryotic relationships. As a case study, we demonstrate that smaller subsets of ∼20 and 52 genes could resolve controversial relationships among widely divergent taxa and provide strong support for deep relationships such as the monophyly and branching order of several eukaryotic supergroups. In addition, the use of these genes resulted in fungal phylogenies that are congruent with previous phylogenomic studies that used much larger datasets, and successfully resolved several difficult relationships (e.g., forming a highly supported clade with Microsporidia, Mitosporidium and Rozella sister to other fungi). We propose that these genes are excellent for both gene-rich and taxon-rich analyses and can be applied at multiple taxonomic levels and facilitate a more complete understanding of the eukaryotic tree of life. PMID:27604879

  9. Evaluation of HPV-16 and HPV-18 specific antibody measurements in saliva collected in oral rinses and merocel® sponges.

    PubMed

    Parker, Katherine H; Kemp, Troy J; Pan, Yuanji; Yang, Zhen; Giuliano, Anna R; Pinto, Ligia A

    2018-05-03

    Current Human papillomavirus (HPV) L1 VLP vaccines protect against HPV-16 and HPV-18-associated cancers, in females and males. Although correlates of protection have not been identified, HPV-specific antibodies at sites of infection are thought to be the main mechanism of protection afforded by vaccination. Oral sampling has gained increased attention as a potential alternative to serum in monitoring immunity to vaccination and understanding local immunity in oral cancers. Serum was collected via venipuncture, and saliva was collected via oral rinses and Merocel® sponges from healthy volunteers: 16 unvaccinated females, 6 females (ages 24-41) and 6 mid-adult aged males (ages 27-45) recipients of three doses of the HPV-16/18/6/11 vaccine (Gardasil®). Mid-adult male vaccine trial participants were compared to female participants. Samples were tested for anti-HPV-16 and anti-HPV-18 immunoglobulin G levels by an L1 virus-like particle-based enzyme-linked immunosorbent assay (ELISA). All vaccinated participants had detectable serum anti-HPV-16 and anti-HPV-18 antibodies. Optimal standard concentration range and sample serial dilutions for oral rinses were determined. The standard curve was not affected by the type of solution examined. Reproducibility of HPV-16 and HPV-18 antibody titers in mouthwash (overall CV < 10%) or in Merocel® extraction buffer was robust (CV < 13%). Excellent assay linearity (R 2  > 0.9) was observed for sera spiked controls in both solutions. HPV-16 and HPV-18 specific antibodies were detectable in saliva from vaccine recipients, both in mouthwash and in Merocel® sponges but levels were several logs lower than those in serum. This study confirms the application of HPV-16 and HPV-18 ELISAs currently used in sero-epidemiological studies of immunogenicity of HPV vaccines for use with oral samples. Oral samples may be a useful resource for the detection of HPV-16 and HPV-18-specific antibodies in saliva following vaccination

  10. Population structuring of multi-copy, antigen-encoding genes in Plasmodium falciparum

    PubMed Central

    Artzy-Randrup, Yael; Rorick, Mary M; Day, Karen; Chen, Donald; Dobson, Andrew P; Pascual, Mercedes

    2012-01-01

    The coexistence of multiple independently circulating strains in pathogen populations that undergo sexual recombination is a central question of epidemiology with profound implications for control. An agent-based model is developed that extends earlier ‘strain theory’ by addressing the var gene family of Plasmodium falciparum. The model explicitly considers the extensive diversity of multi-copy genes that undergo antigenic variation via sequential, mutually exclusive expression. It tracks the dynamics of all unique var repertoires in a population of hosts, and shows that even under high levels of sexual recombination, strain competition mediated through cross-immunity structures the parasite population into a subset of coexisting dominant repertoires of var genes whose degree of antigenic overlap depends on transmission intensity. Empirical comparison of patterns of genetic variation at antigenic and neutral sites supports this role for immune selection in structuring parasite diversity. DOI: http://dx.doi.org/10.7554/eLife.00093.001 PMID:23251784

  11. Mapping of single-copy genes by TSA-FISH in the codling moth, Cydia pomonella.

    PubMed

    Carabajal Paladino, Leonela Z; Nguyen, Petr; Síchová, Jindra; Marec, František

    2014-01-01

    We work on the development of transgenic sexing strains in the codling moth, Cydia pomonella (Tortricidae), which would enable to produce male-only progeny for the population control of this pest using sterile insect technique (SIT). To facilitate this research, we have developed a number of cytogenetic and molecular tools, including a physical map of the codling moth Z chromosome using BAC-FISH (fluorescence in situ hybridization with bacterial artificial chromosome probes). However, chromosomal localization of unique, single-copy sequences such as a transgene cassette by conventional FISH remains challenging. In this study, we adapted a FISH protocol with tyramide signal amplification (TSA-FISH) for detection of single-copy genes in Lepidoptera. We tested the protocol with probes prepared from partial sequences of Z-linked genes in the codling moth. Using a modified TSA-FISH protocol we successfully mapped a partial sequence of the Acetylcholinesterase 1 (Ace-1) gene to the Z chromosome and confirmed thus its Z-linkage. A subsequent combination of BAC-FISH with BAC probes containing anticipated neighbouring Z-linked genes and TSA-FISH with the Ace-1 probe allowed the integration of Ace-1 in the physical map of the codling moth Z chromosome. We also developed a two-colour TSA-FISH protocol which enabled us simultaneous localization of two Z-linked genes, Ace-1 and Notch, to the expected regions of the Z chromosome. We showed that TSA-FISH represents a reliable technique for physical mapping of genes on chromosomes of moths and butterflies. Our results suggest that this technique can be combined with BAC-FISH and in the future used for physical localization of transgene cassettes on chromosomes of transgenic lines in the codling moth or other lepidopteran species. Furthermore, the developed protocol for two-colour TSA-FISH might become a powerful tool for synteny mapping in non-model organisms.

  12. Mapping of single-copy genes by TSA-FISH in the codling moth, Cydia pomonella

    PubMed Central

    2014-01-01

    Background We work on the development of transgenic sexing strains in the codling moth, Cydia pomonella (Tortricidae), which would enable to produce male-only progeny for the population control of this pest using sterile insect technique (SIT). To facilitate this research, we have developed a number of cytogenetic and molecular tools, including a physical map of the codling moth Z chromosome using BAC-FISH (fluorescence in situ hybridization with bacterial artificial chromosome probes). However, chromosomal localization of unique, single-copy sequences such as a transgene cassette by conventional FISH remains challenging. In this study, we adapted a FISH protocol with tyramide signal amplification (TSA-FISH) for detection of single-copy genes in Lepidoptera. We tested the protocol with probes prepared from partial sequences of Z-linked genes in the codling moth. Results Using a modified TSA-FISH protocol we successfully mapped a partial sequence of the Acetylcholinesterase 1 (Ace-1) gene to the Z chromosome and confirmed thus its Z-linkage. A subsequent combination of BAC-FISH with BAC probes containing anticipated neighbouring Z-linked genes and TSA-FISH with the Ace-1 probe allowed the integration of Ace-1 in the physical map of the codling moth Z chromosome. We also developed a two-colour TSA-FISH protocol which enabled us simultaneous localization of two Z-linked genes, Ace-1 and Notch, to the expected regions of the Z chromosome. Conclusions We showed that TSA-FISH represents a reliable technique for physical mapping of genes on chromosomes of moths and butterflies. Our results suggest that this technique can be combined with BAC-FISH and in the future used for physical localization of transgene cassettes on chromosomes of transgenic lines in the codling moth or other lepidopteran species. Furthermore, the developed protocol for two-colour TSA-FISH might become a powerful tool for synteny mapping in non-model organisms. PMID:25471491

  13. Development of universal genetic markers based on single-copy orthologous (COSII) genes in Poaceae.

    PubMed

    Liu, Hailan; Guo, Xiaoqin; Wu, Jiasheng; Chen, Guo-Bo; Ying, Yeqing

    2013-03-01

    KEY MESSAGE : We develop a set of universal genetic markers based on single-copy orthologous (COSII) genes in Poaceae. Being evolutionary conserved, single-copy orthologous (COSII) genes are particularly useful in comparative mapping and phylogenetic investigation among species. In this study, we identified 2,684 COSII genes based on five sequenced Poaceae genomes including rice, maize, sorghum, foxtail millet, and brachypodium, and then developed 1,072 COSII markers whose transferability and polymorphism among five bamboo species were further evaluated with 46 pairs of randomly selected primers. 91.3 % of the 46 primers obtained clear amplification in at least one bamboo species, and 65.2 % of them produced polymorphism in more than one species. We also used 42 of them to construct the phylogeny for the five bamboo species, and it might reflect more precise evolutionary relationship than the one based on the vegetative morphology. The results indicated a promising prospect of applying these markers to the investigation of genetic diversity and the classification of Poaceae. To ease and facilitate access of the information of common interest to readers, a web-based database of the COSII markers is provided ( http://www.sicau.edu.cn/web/yms/PCOSWeb/PCOS.html ).

  14. Prevalence of HPV 16 and 18 and attitudes toward HPV vaccination trials in patients with cervical cancer in Mali.

    PubMed

    Téguété, Ibrahima; Dolo, Amadou; Sangare, Kotou; Sissoko, Abdoulaye; Rochas, Mali; Beseme, Sarah; Tounkara, Karamoko; Yekta, Shahla; De Groot, Anne S; Koita, Ousmane A

    2017-01-01

    Cervical cancer is one of the most common and lethal cancers in West Africa. Even though vaccines that protect against the most common Human papillomavirus (HPV) strains, 16 and 18, are currently in use in developed countries, the implementation of these vaccines in developing countries has been painfully slow, considering the pre-eminence of HPV-associated cervical cancer among women in those countries. We performed serological and PCR-based assessment of blood and tissue specimens obtained from women undergoing cervical cancer-related surgery at a major urban hospital in Bamako. Since several therapeutic HPV vaccines are currently in clinical trials, we also assessed willingness to participate in HPV cancer vaccine trials. Blood and biopsy samples of 240 women were evaluated for HPV types 16 and 18 by serology and PCR. Knowledge regarding the HPV vaccine and autonomy to decide to vaccinate their own child was assessed with a standardized questionnaire. HPV 16 and 18 were identified in 137/166 (82.5%) cervical cancer biopsy samples by PCR. Co-infection with both HPV 16 and 18 was significantly more frequent in women over 50 years of age than in younger women (63.0% vs. 37.0%). 44% of study participants said they would be willing to vaccinate their child with HPV vaccine. Only 39% of women participating in this study reported that they would be able to make an autonomous decision to receive HPV vaccination. Permission from a male spouse or head of household was identified as important for participation by 59% of the women. This study provides strong support for the introduction of currently available HPV vaccines in Mali, and also provides key information about conditions for obtaining informed consent for HPV vaccine trials and HPV vaccination in Mali.

  15. Prevalence of HPV 16 and 18 and attitudes toward HPV vaccination trials in patients with cervical cancer in Mali

    PubMed Central

    Téguété, Ibrahima; Dolo, Amadou; Sangare, Kotou; Sissoko, Abdoulaye; Rochas, Mali; Beseme, Sarah; Tounkara, Karamoko; Yekta, Shahla; De Groot, Anne S.; Koita, Ousmane A.

    2017-01-01

    Background Cervical cancer is one of the most common and lethal cancers in West Africa. Even though vaccines that protect against the most common Human papillomavirus (HPV) strains, 16 and 18, are currently in use in developed countries, the implementation of these vaccines in developing countries has been painfully slow, considering the pre-eminence of HPV-associated cervical cancer among women in those countries. Aim We performed serological and PCR-based assessment of blood and tissue specimens obtained from women undergoing cervical cancer-related surgery at a major urban hospital in Bamako. Since several therapeutic HPV vaccines are currently in clinical trials, we also assessed willingness to participate in HPV cancer vaccine trials. Methods Blood and biopsy samples of 240 women were evaluated for HPV types 16 and 18 by serology and PCR. Knowledge regarding the HPV vaccine and autonomy to decide to vaccinate their own child was assessed with a standardized questionnaire. Results HPV 16 and 18 were identified in 137/166 (82.5%) cervical cancer biopsy samples by PCR. Co-infection with both HPV 16 and 18 was significantly more frequent in women over 50 years of age than in younger women (63.0% vs. 37.0%). 44% of study participants said they would be willing to vaccinate their child with HPV vaccine. Only 39% of women participating in this study reported that they would be able to make an autonomous decision to receive HPV vaccination. Permission from a male spouse or head of household was identified as important for participation by 59% of the women. Conclusion This study provides strong support for the introduction of currently available HPV vaccines in Mali, and also provides key information about conditions for obtaining informed consent for HPV vaccine trials and HPV vaccination in Mali. PMID:28231334

  16. Development of a simple and quick immunochromatography method for detection of anti-HPV-16/-18 antibodies

    PubMed Central

    Endo, Fumiko; Tabata, Tsutomu; Sadato, Daichi; Kawamura, Machiko; Ando, Noriyuki; Ukaji, Masako; Kobayashi, Kaoru; Kobayashi, Yukuharu; Ikeda, Tomoaki; Shibasaki, Futoshi

    2017-01-01

    Immunochromatography (IC) is widely used to detect target molecules in biological fluids. Since this method can be performed without a special technique or device, IC is a convenient way to assess the existence of antibodies or pathogens such as viruses and bacteria, simply and quickly. In this study, we established an IC method to detect serum antibodies against oncogenic human papillomavirus (HPV)-16 and HPV-18 L1 proteins using recombinant L1 proteins produced by silkworms as antigens. Infection of oncogenic HPVs is a major risk factor of cervical cancer, which is one of the most common cancers in women worldwide. We first measured blood sera of two groups by magnetic beads enzyme-linked immunosorbent assay (MB-ELISA). For the first group, sera were collected prospectively from young women who planned to receive HPV vaccination. The second group consisted of children under 20 years of age, non-vaccinated healthy women, vaccinated healthy women, dysplasia, cervical intraepithelial neoplasia III, and cervical cancer patients. We confirmed that standard vaccination doses significantly increased serum HPV antibody concentrations, and the level was sustained at least more than 30 months after vaccination. In contrast, an increase in antibody concentration was not observed in patients with precancerous cervical changes and cervical cancer. We next measured the samples in both groups using the IC method we originally developed, and found that the measurement values of IC highly correlated with those of MB-ELISA. The simple and quick IC method would be a useful tool for rapid monitoring of L1 specific antibody levels in a non-laboratory environment. With less than one drop of serum, our IC can easily detect serum HPV-16/-18 antibodies within 15 minutes, without the need for electronic devices or techniques. PMID:28158224

  17. 8q24 allelic imbalance and MYC gene copy number in primary prostate cancer.

    PubMed

    Chen, H; Liu, W; Roberts, W; Hooker, S; Fedor, H; DeMarzo, A; Isaacs, W; Kittles, R A

    2010-09-01

    Four independent regions within 8q24 near the MYC gene are associated with risk for prostate cancer (Pca). Here, we investigated allelic imbalance (AI) at 8q24 risk variants and MYC gene DNA copy number (CN) in 27 primary Pcas. Heterozygotes were observed in 24 of 27 patients at one or more 8q24 markers and 27% of the loci exhibited AI in tumor DNA. The 8q24 risk alleles were preferentially favored in the tumors. Increased MYC gene CN was observed in 33% of tumors, and the co-existence of increased MYC gene CN with AI at risk loci was observed in 86% (P<0.004 exact binomial test) of the informative tumors. No AI was observed in tumors, which did not reveal increased MYC gene CN. Higher Gleason score was associated with tumors exhibiting AI (P=0.04) and also with increased MYC gene CN (P=0.02). Our results suggest that AI at 8q24 and increased MYC gene CN may both be related to high Gleason score in Pca. Our findings also suggest that these two somatic alterations may be due to the same preferential chromosomal duplication event during prostate tumorigenesis.

  18. Epidermal growth factor receptor and AKT1 gene copy numbers by multi-gene fluorescence in situ hybridization impact on prognosis in breast cancer.

    PubMed

    Li, Jiao; Su, Wei; Zhang, Sheng; Hu, Yunhui; Liu, Jingjing; Zhang, Xiaobei; Bai, Jingchao; Yuan, Weiping; Hu, Linping; Cheng, Tao; Zetterberg, Anders; Lei, Zhenmin; Zhang, Jin

    2015-05-01

    The epidermal growth factor receptor (EGFR)/PI3K/AKT signaling pathway aberrations play significant roles in breast cancer occurrence and development. However, the status of EGFR and AKT1 gene copy numbers remains unclear. In this study, we showed that the rates of EGFR and AKT1 gene copy number alterations were associated with the prognosis of breast cancer. Among 205 patients, high EGFR and AKT1 gene copy numbers were observed in 34.6% and 27.8% of cases by multi-gene fluorescence in situ hybridization, respectively. Co-heightened EGFR/AKT1 gene copy numbers were identified in 11.7% cases. No changes were found in 49.3% of patients. Although changes in EGFR and AKT1 gene copy numbers had no correlation with patients' age, tumor stage, histological grade and the expression status of other molecular makers, high EGFR (P = 0.0002) but not AKT1 (P = 0.1177) gene copy numbers correlated with poor 5-year overall survival. The patients with co-heightened EGFR/AKT1 gene copy numbers displayed a poorer prognosis than those with tumors with only high EGFR gene copy numbers (P = 0.0383). Both Univariate (U) and COX multivariate (C) analyses revealed that high EGFR and AKT1 gene copy numbers (P = 0.000 [U], P = 0.0001 [C]), similar to histological grade (P = 0.001 [U], P = 0.012 [C]) and lymph node metastasis (P = 0.046 [U], P = 0.158 [C]), were independent prognostic indicators of 5-year overall survival. These results indicate that high EGFR and AKT1 gene copy numbers were relatively frequent in breast cancer. Co-heightened EGFR/AKT1 gene copy numbers had a worse outcome than those with only high EGFR gene copy numbers, suggesting that evaluation of these two genes together may be useful for selecting patients for anti-EGFR-targeted therapy or anti-EGFR/AKT1-targeted therapy and for predicting outcomes. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  19. Copy number variation analysis implicates the cell polarity gene glypican 5 as a human spina bifida candidate gene

    PubMed Central

    Bassuk, Alexander G.; Muthuswamy, Lakshmi B.; Boland, Riley; Smith, Tiffany L.; Hulstrand, Alissa M.; Northrup, Hope; Hakeman, Matthew; Dierdorff, Jason M.; Yung, Christina K.; Long, Abby; Brouillette, Rachel B.; Au, Kit Sing; Gurnett, Christina; Houston, Douglas W.; Cornell, Robert A.; Manak, J. Robert

    2013-01-01

    Neural tube defects (NTDs) are common birth defects of complex etiology. Family and population-based studies have confirmed a genetic component to NTDs. However, despite more than three decades of research, the genes involved in human NTDs remain largely unknown. We tested the hypothesis that rare copy number variants (CNVs), especially de novo germline CNVs, are a significant risk factor for NTDs. We used array-based comparative genomic hybridization (aCGH) to identify rare CNVs in 128 Caucasian and 61 Hispanic patients with non-syndromic lumbar-sacral myelomeningocele. We also performed aCGH analysis on the parents of affected individuals with rare CNVs where parental DNA was available (42 sets). Among the eight de novo CNVs that we identified, three generated copy number changes of entire genes. One large heterozygous deletion removed 27 genes, including PAX3, a known spina bifida-associated gene. A second CNV altered genes (PGPD8, ZC3H6) for which little is known regarding function or expression. A third heterozygous deletion removed GPC5 and part of GPC6, genes encoding glypicans. Glypicans are proteoglycans that modulate the activity of morphogens such as Sonic Hedgehog (SHH) and bone morphogenetic proteins (BMPs), both of which have been implicated in NTDs. Additionally, glypicans function in the planar cell polarity (PCP) pathway, and several PCP genes have been associated with NTDs. Here, we show that GPC5 orthologs are expressed in the neural tube, and that inhibiting their expression in frog and fish embryos results in NTDs. These results implicate GPC5 as a gene required for normal neural tube development. PMID:23223018

  20. Frequent loss of lineages and deficient duplications accounted for low copy number of disease resistance genes in Cucurbitaceae

    PubMed Central

    2013-01-01

    Background The sequenced genomes of cucumber, melon and watermelon have relatively few R-genes, with 70, 75 and 55 copies only, respectively. The mechanism for low copy number of R-genes in Cucurbitaceae genomes remains unknown. Results Manual annotation of R-genes in the sequenced genomes of Cucurbitaceae species showed that approximately half of them are pseudogenes. Comparative analysis of R-genes showed frequent loss of R-gene loci in different Cucurbitaceae species. Phylogenetic analysis, data mining and PCR cloning using degenerate primers indicated that Cucurbitaceae has limited number of R-gene lineages (subfamilies). Comparison between R-genes from Cucurbitaceae and those from poplar and soybean suggested frequent loss of R-gene lineages in Cucurbitaceae. Furthermore, the average number of R-genes per lineage in Cucurbitaceae species is approximately 1/3 that in soybean or poplar. Therefore, both loss of lineages and deficient duplications in extant lineages accounted for the low copy number of R-genes in Cucurbitaceae. No extensive chimeras of R-genes were found in any of the sequenced Cucurbitaceae genomes. Nevertheless, one lineage of R-genes from Trichosanthes kirilowii, a wild Cucurbitaceae species, exhibits chimeric structures caused by gene conversions, and may contain a large number of distinct R-genes in natural populations. Conclusions Cucurbitaceae species have limited number of R-gene lineages and each genome harbors relatively few R-genes. The scarcity of R-genes in Cucurbitaceae species was due to frequent loss of R-gene lineages and infrequent duplications in extant lineages. The evolutionary mechanisms for large variation of copy number of R-genes in different plant species were discussed. PMID:23682795

  1. Identification of copy number variation-driven genes for liver cancer via bioinformatics analysis.

    PubMed

    Lu, Xiaojie; Ye, Kun; Zou, Kailin; Chen, Jinlian

    2014-11-01

    To screen out copy number variation (CNV)-driven differentially expressed genes (DEGs) in liver cancer and advance our understanding of the pathogenesis, an integrated analysis of liver cancer-related CNV data from The Cancer Genome Atlas (TCGA) and gene expression data from EBI Array Express database were performed. The DEGs were identified by package limma based on the cut-off of |log2 (fold-change)|>0.585 and adjusted p-value<0.05. Using hg19 annotation information provided by UCSC, liver cancer-related CNVs were then screened out. TF-target gene interactions were also predicted with information from UCSC using DAVID online tools. As a result, 25 CNV-driven genes were obtained, including tripartite motif containing 28 (TRIM28) and RanBP-type and C3HC4-type zinc finger containing 1 (RBCK1). In the transcriptional regulatory network, 8 known cancer-related transcription factors (TFs) interacted with 21 CNV-driven genes, suggesting that the other 8 TFs may be involved in liver cancer. These genes may be potential biomarkers for early detection and prevention of liver cancer. These findings may improve our knowledge of the pathogenesis of liver cancer. Nevertheless, further experiments are still needed to confirm our findings.

  2. ROS1 gene rearrangement and copy number gain in non-small cell lung cancer.

    PubMed

    Jin, Yan; Sun, Ping-Li; Kim, Hyojin; Park, Eunhyang; Shim, Hyo Sup; Jheon, Sanghoon; Kim, Kwhanmien; Lee, Choon-Taek; Chung, Jin-Haeng

    2015-01-01

    ROS1 has attracted much attention as a possible oncogenic driver and ROS1-rearranged tumors show sensitivity to most ALK inhibitors. We aimed to clarify the prevalence of ROS1 gene rearrangement and investigate the clinical implications of ROS1 gene copy number gain (CNG) in non-small cell lung cancer (NSCLC) patients. We carried out fluorescent in situ hybridization with ROS1 and centromere enumeration 6 probes and immunohistochemistry for ROS1 protein expression. ROS1 rearrangement was detected in 3 of 375 samples (0.8 %); all of whom were female, never-smokers, and harbored an adenocarcinoma component. ROS1 gene CNG was found in 18 cases (4.8 %). ROS1 gene CNG was significantly associated with shorter disease-free survival (DFS, 12 vs. 58 months; p = 0.003) and shorter overall survival (OS, 40 vs. 67 months; p <0.001) than the group without CNG. Multivariate analysis confirmed that ROS1 gene CNG was significantly associated with poorer DFS (hazard ratio [HR]=2.16, 95 % confidence interval [CI] = 1.22-3.81, p = 0.008), and OS ([HR] = 2.53, 95 % [CI] = 1.31-4.89, p = 0.006). ROS1 protein overexpression was observed in 5.0 % (18 out of 357), of which 2 cases harbored ROS1 gene rearrangement. There was no statistically significant correlation between ROS1 gene CNG and protein overexpression. This study demonstrated ROS1 gene rearrangement was detected in 0.8 % of surgically resected NSCLC; and ROS1 gene CNG is an independent poor prognostic factor. This survival analyses may contribute to future studies on the utility of ROS1-targeted therapy for patients.

  3. Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy.

    PubMed

    Lin, Huan-Ting; Okumura, Takashi; Yatsuda, Yukinori; Ito, Satoru; Nakauchi, Hiromitsu; Otsu, Makoto

    2016-10-01

    Stable gene transfer into target cell populations via integrating viral vectors is widely used in stem cell gene therapy (SCGT). Accurate vector copy number (VCN) estimation has become increasingly important. However, existing methods of estimation such as real-time quantitative PCR are more restricted in practicality, especially during clinical trials, given the limited availability of sample materials from patients. This study demonstrates the application of an emerging technology called droplet digital PCR (ddPCR) in estimating VCN states in the context of SCGT. Induced pluripotent stem cells (iPSCs) derived from a patient with X-linked chronic granulomatous disease were used as clonable target cells for transduction with alpharetroviral vectors harboring codon-optimized CYBB cDNA. Precise primer-probe design followed by multiplex analysis conferred assay specificity. Accurate estimation of per-cell VCN values was possible without reliance on a reference standard curve. Sensitivity was high and the dynamic range of detection was wide. Assay reliability was validated by observation of consistent, reproducible, and distinct VCN clustering patterns for clones of transduced iPSCs with varying numbers of transgene copies. Taken together, use of ddPCR appears to offer a practical and robust approach to VCN estimation with a wide range of clinical and research applications.

  4. Association between salivary amylase (AMY1) gene copy numbers and insulin resistance in asymptomatic Korean men.

    PubMed

    Choi, Y-J; Nam, Y-S; Yun, J M; Park, J H; Cho, B L; Son, H-Y; Kim, J I; Yun, J W

    2015-12-01

    Salivary amylase gene (AMY1) copy number variations (CNVs) correlate directly with salivary amylase activity and serum amylase levels. Previously, individuals with high AMY1 CNVs exhibited low postprandial glucose levels and postprandial early insulin surge, suggesting that high AMY1 gene copy numbers may play a role in lowering the risk of insulin resistance. We verified the relationship between AMY1 CNVs and homeostatic model assessment-insulin resistance (HOMA-IR) in a cohort of 1257 Korean men aged 20-65 years who visited two medical centres for regular health check-ups, and in subgroups of current smokers and regular alcohol drinkers. Individuals with fasting plasma glucose levels > 10.0 mmol/l, HbA1c ≥ 64 mmol/mol (8.0%) or who used oral hypoglycaemic agents or insulin were excluded. AMY1 CNVs correlated negatively with HOMA-IR even after adjusting for covariates (e.g. BMI, systolic blood pressure, triacylglycerol, alcohol consumption, smoking and physical activity). When the participants were divided according to current smoking and alcohol consumption habits, negative correlations between AMY1 CNVs and HOMA-IR were more evident among non-smokers and regular drinkers and were non-significant among smokers and non-regular drinkers. Low AMY1 CNVs correlated with high insulin resistance in asymptomatic Korean men, and such a relationship presented differently according to the status of smoking and alcohol consumption. © 2015 The Authors. Diabetic Medicine © 2015 Diabetes UK.

  5. Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy

    PubMed Central

    Lin, Huan-Ting; Okumura, Takashi; Yatsuda, Yukinori; Ito, Satoru; Nakauchi, Hiromitsu; Otsu, Makoto

    2016-01-01

    Stable gene transfer into target cell populations via integrating viral vectors is widely used in stem cell gene therapy (SCGT). Accurate vector copy number (VCN) estimation has become increasingly important. However, existing methods of estimation such as real-time quantitative PCR are more restricted in practicality, especially during clinical trials, given the limited availability of sample materials from patients. This study demonstrates the application of an emerging technology called droplet digital PCR (ddPCR) in estimating VCN states in the context of SCGT. Induced pluripotent stem cells (iPSCs) derived from a patient with X-linked chronic granulomatous disease were used as clonable target cells for transduction with alpharetroviral vectors harboring codon-optimized CYBB cDNA. Precise primer–probe design followed by multiplex analysis conferred assay specificity. Accurate estimation of per-cell VCN values was possible without reliance on a reference standard curve. Sensitivity was high and the dynamic range of detection was wide. Assay reliability was validated by observation of consistent, reproducible, and distinct VCN clustering patterns for clones of transduced iPSCs with varying numbers of transgene copies. Taken together, use of ddPCR appears to offer a practical and robust approach to VCN estimation with a wide range of clinical and research applications. PMID:27763786

  6. The E6 and E7 genes of human papillomavirus type 6 have weak immortalizing activity in human epithelial cells.

    PubMed Central

    Halbert, C L; Demers, G W; Galloway, D A

    1992-01-01

    Previous studies have shown that the E7 gene of human papillomavirus (HPV) type 16 or 18 alone was sufficient for immortalization of human foreskin epithelial cells (HFE) and that the efficiency was increased in cooperation with the respective E6 gene, whereas the HPV6 E6 or E7 gene was not active in HFE. To detect weak immortalizing activities of the HPV6 genes, cells were infected with recombinant retroviruses containing HPV genes, alone and in homologous and heterologous combinations. The HPV6 genes, alone or together (HPV6 E6 plus HPV6 E7), were not able to immortalize cells. However the HPV6 E6 gene, in concert with HPV16 E7, increased the frequency of immortalization threefold over that obtained with HPV16 E7 alone. Interestingly, 6 of 20 clones containing the HPV16 E6 gene and the HPV6 E7 gene were immortalized, whereas neither gene alone was sufficient. Thus, the HPV6 E6 and E7 genes have weak immortalizing activities which can be detected in cooperation with the more active transforming genes of HPV16. Acute expression of the HPV6 and HPV16 E6 and E7 genes revealed that only HPV16 E7 was able to stimulate the proliferation of cells in organotypic culture, resulting in increased expression of the proliferative cell nuclear antigen and the formation of a disorganized epithelial layer. Additionally, combinations of genes that immortalized HFE cells (HPV16 E6 plus HPV16 E7, HPV16 E6 plus HPV6 E7, and HPV6 E6 plus HPV16 E7) also stimulated proliferation. Images PMID:1312623

  7. Beneficial effect of a high number of copies of salivary amylase AMY1 gene on obesity risk in Mexican children.

    PubMed

    Mejía-Benítez, María A; Bonnefond, Amélie; Yengo, Loïc; Huyvaert, Marlène; Dechaume, Aurélie; Peralta-Romero, Jesús; Klünder-Klünder, Miguel; García Mena, Jaime; El-Sayed Moustafa, Julia S; Falchi, Mario; Cruz, Miguel; Froguel, Philippe

    2015-02-01

    Childhood obesity is a major public health problem in Mexico, affecting one in every three children. Genome-wide association studies identified genetic variants associated with childhood obesity, but a large missing heritability remains to be elucidated. We have recently shown a strong association between a highly polymorphic copy number variant encompassing the salivary amylase gene (AMY1 also known as AMY1A) and obesity in European and Asian adults. In the present study, we aimed to evaluate the association between AMY1 copy number and obesity in Mexican children. We evaluated the number of AMY1 copies in 597 Mexican children (293 obese children and 304 normal weight controls) through highly sensitive digital PCR. The effect of AMY1 copy number on obesity status was assessed using a logistic regression model adjusted for age and sex. We identified a marked effect of AMY1 copy number on reduced risk of obesity (OR per estimated copy 0.84, with the number of copies ranging from one to 16 in this population; p = 4.25 × 10(-6)). The global association between AMY1 copy number and reduced risk of obesity seemed to be mostly driven by the contribution of the highest AMY1 copy number. Strikingly, all children with >10 AMY1 copies were normal weight controls. Salivary amylase initiates the digestion of dietary starch, which is highly consumed in Mexico. Our current study suggests putative benefits of high number of AMY1 copies (and related production of salivary amylase) on energy metabolism in Mexican children.

  8. Dietary starch intake modifies the relation between copy number variation in the salivary amylase gene and BMI.

    PubMed

    Rukh, Gull; Ericson, Ulrika; Andersson-Assarsson, Johanna; Orho-Melander, Marju; Sonestedt, Emily

    2017-07-01

    Background: Studies have shown conflicting associations between the salivary amylase gene ( AMY1 ) copy number and obesity. Salivary amylase initiates starch digestion in the oral cavity; starch is a major source of energy in the diet. Objective: We investigated the association between AMY1 copy number and obesity traits, and the effect of the interaction between AMY1 copy number and starch intake on these obesity traits. Design: We first assessed the association between AMY1 copy number (genotyped by digital droplet polymerase chain reaction) and obesity traits in 4800 individuals without diabetes (mean age: 57 y; 60% female) from the Malmö Diet and Cancer Cohort. Then we analyzed interactions between AMY1 copy number and energy-adjusted starch intake (obtained by a modified diet history method) on body mass index (BMI) and body fat percentage. Results: AMY1 copy number was not associated with BMI ( P = 0.80) or body fat percentage ( P = 0.38). We observed a significant effect of the interaction between AMY1 copy number and starch intake on BMI ( P -interaction = 0.007) and body fat percentage ( P -interaction = 0.03). Upon stratification by dietary starch intake, BMI tended to decrease with increasing AMY1 copy numbers in the low-starch intake group ( P = 0.07) and tended to increase with increasing AMY1 copy numbers in the high-starch intake group ( P = 0.08). The lowest mean BMI was observed in the group of participants with a low AMY1 copy number and a high dietary intake of starch. Conclusions: Our findings suggest an effect of the interaction between starch intake and AMY1 copy number on obesity. Individuals with high starch intake but low genetic capacity to digest starch had the lowest BMI, potentially because larger amounts of undigested starch are transported through the gastrointestinal tract, contributing to fewer calories extracted from ingested starch. © 2017 American Society for Nutrition.

  9. ALK gene copy number gain and its clinical significance in hepatocellular carcinoma.

    PubMed

    Jia, Shou-Wei; Fu, Sha; Wang, Fang; Shao, Qiong; Huang, Hong-Bing; Shao, Jian-Yong

    2014-01-07

    To examine the status and clinical significance of anaplastic lymphoma kinase (ALK) gene alterations in hepatocellular carcinoma (HCC) patients. A total of 213 cases of HCC were examined by fluorescent in situ hybridization using dual color break-apart ALK probes for the detection of chromosomal translocation and gene copy number gain. HCC tissue microarrays were constructed, and the correlation between the ALK status and clinicopathological variables was assessed by χ(2) test or Fisher's exact test. Survival analysis was estimated using the Kaplan-Meier approach with a Log-rank test. Univariate and multivariate analyses of clinical variables were performed using the Cox proportional hazards regression model. ALK gene translocation was not observed in any of the HCC cases included in the present study. ALK gene copy number gain (ALK/CNG) (≥ 4 copies/cell) was detected in 28 (13.15%) of the 213 HCC patients. The 3-year progression-free-survival (PFS) rate for ALK/CNG-positive HCC patients was significantly poorer than ALK/CNG-negative patients (27.3% vs 42.5%, P = 0.048), especially for patients with advanced stage III/IV (0% vs 33.5%, P = 0.007), and patients with grade III disease (24.8% vs 49.9%, P = 0.023). ALK/CNG-positive HCC patients had a significantly poorer prognosis than ALK/CNG-negative patients in the subgroup that was negative for serum hepatitis B virus DNA, with significantly different 3-year overall survival rates (18.2% vs 63.6%, P = 0.021) and PFS rates (18.2% vs 46.9%, P = 0.019). Multivariate Cox proportional hazards regression analysis suggested that ALK/CNG prevalence can predict death in HCC (HR = 1.596; 95%CI: 1.008-2.526, P = 0.046). ALK/CNG, but not translocation of ALK, is present in HCC and may be an unfavorable prognostic predictor.

  10. ALK gene copy number gain and its clinical significance in hepatocellular carcinoma

    PubMed Central

    Jia, Shou-Wei; Fu, Sha; Wang, Fang; Shao, Qiong; Huang, Hong-Bing; Shao, Jian-Yong

    2014-01-01

    AIM: To examine the status and clinical significance of anaplastic lymphoma kinase (ALK) gene alterations in hepatocellular carcinoma (HCC) patients. METHODS: A total of 213 cases of HCC were examined by fluorescent in situ hybridization using dual color break-apart ALK probes for the detection of chromosomal translocation and gene copy number gain. HCC tissue microarrays were constructed, and the correlation between the ALK status and clinicopathological variables was assessed by χ2 test or Fisher’s exact test. Survival analysis was estimated using the Kaplan-Meier approach with a Log-rank test. Univariate and multivariate analyses of clinical variables were performed using the Cox proportional hazards regression model. RESULTS: ALK gene translocation was not observed in any of the HCC cases included in the present study. ALK gene copy number gain (ALK/CNG) (≥ 4 copies/cell) was detected in 28 (13.15%) of the 213 HCC patients. The 3-year progression-free-survival (PFS) rate for ALK/CNG-positive HCC patients was significantly poorer than ALK/CNG-negative patients (27.3% vs 42.5%, P = 0.048), especially for patients with advanced stage III/IV (0% vs 33.5%, P = 0.007), and patients with grade III disease (24.8% vs 49.9%, P = 0.023). ALK/CNG-positive HCC patients had a significantly poorer prognosis than ALK/CNG-negative patients in the subgroup that was negative for serum hepatitis B virus DNA, with significantly different 3-year overall survival rates (18.2% vs 63.6%, P = 0.021) and PFS rates (18.2% vs 46.9%, P = 0.019). Multivariate Cox proportional hazards regression analysis suggested that ALK/CNG prevalence can predict death in HCC (HR = 1.596; 95%CI: 1.008-2.526, P = 0.046). CONCLUSION: ALK/CNG, but not translocation of ALK, is present in HCC and may be an unfavorable prognostic predictor. PMID:24415871

  11. Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting | Office of Cancer Genomics

    Cancer.gov

    The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest.

  12. Differentially expressed microRNAs in lung adenocarcinoma invert effects of copy number aberrations of prognostic genes

    PubMed Central

    Tokar, Tomas; Pastrello, Chiara; Ramnarine, Varune R.; Zhu, Chang-Qi; Craddock, Kenneth J.; Pikor, Larrisa A.; Vucic, Emily A.; Vary, Simon; Shepherd, Frances A.; Tsao, Ming-Sound; Lam, Wan L.; Jurisica, Igor

    2018-01-01

    In many cancers, significantly down- or upregulated genes are found within chromosomal regions with DNA copy number alteration opposite to the expression changes. Generally, this paradox has been overlooked as noise, but can potentially be a consequence of interference of epigenetic regulatory mechanisms, including microRNA-mediated control of mRNA levels. To explore potential associations between microRNAs and paradoxes in non-small-cell lung cancer (NSCLC) we curated and analyzed lung adenocarcinoma (LUAD) data, comprising gene expressions, copy number aberrations (CNAs) and microRNA expressions. We integrated data from 1,062 tumor samples and 241 normal lung samples, including newly-generated array comparative genomic hybridization (aCGH) data from 63 LUAD samples. We identified 85 “paradoxical” genes whose differential expression consistently contrasted with aberrations of their copy numbers. Paradoxical status of 70 out of 85 genes was validated on sample-wise basis using The Cancer Genome Atlas (TCGA) LUAD data. Of these, 41 genes are prognostic and form a clinically relevant signature, which we validated on three independent datasets. By meta-analysis of results from 9 LUAD microRNA expression studies we identified 24 consistently-deregulated microRNAs. Using TCGA-LUAD data we showed that deregulation of 19 of these microRNAs explains differential expression of the paradoxical genes. Our results show that deregulation of paradoxical genes is crucial in LUAD and their expression pattern is maintained epigenetically, defying gene copy number status. PMID:29507679

  13. Clinical features associated with copy number variations of the 14q32 imprinted gene cluster.

    PubMed

    Rosenfeld, Jill A; Fox, Joyce E; Descartes, Maria; Brewer, Fallon; Stroud, Tracy; Gorski, Jerome L; Upton, Sheila J; Moeschler, John B; Monteleone, Berrin; Neill, Nicholas J; Lamb, Allen N; Ballif, Blake C; Shaffer, Lisa G; Ravnan, J Britt

    2015-02-01

    Uniparental disomy (UPD) for imprinted chromosomes can cause abnormal phenotypes due to absent or overexpression of imprinted genes. UPD(14)pat causes a unique constellation of features including thoracic skeletal anomalies, polyhydramnios, placentomegaly, and limited survival; its hypothesized cause is overexpression of paternally expressed RTL1, due to absent regulatory effects of maternally expressed RTL1as. UPD(14)mat causes a milder condition with hypotonia, growth failure, and precocious puberty; its hypothesized cause is absence of paternally expressed DLK1. To more clearly establish how gains and losses of imprinted genes can cause disease, we report six individuals with copy number variations of the imprinted 14q32 region identified through clinical microarray-based comparative genomic hybridization. Three individuals presented with UPD(14)mat-like phenotypes (Temple syndrome) and had apparently de novo deletions spanning the imprinted region, including DLK1. One of these deletions was shown to be on the paternal chromosome. Two individuals with UPD(14)pat-like phenotypes had 122-154kb deletions on their maternal chromosomes that included RTL1as but not the differentially methylated regions that regulate imprinted gene expression, providing further support for RTL1 overexpression as a cause for the UPD(14)pat phenotype. The sixth individual is tetrasomic for a 1.7Mb segment, including the imprinted region, and presents with intellectual disability and seizures but lacks significant phenotypic overlap with either UPD(14) syndrome. Therefore, the 14q32 imprinted region is dosage sensitive, with deletions of different critical regions causing UPD(14)mat- and UPD(14)pat-like phenotypes, while copy gains are likely insufficient to recapitulate these phenotypes.

  14. Phylogeny and Divergence Times of Gymnosperms Inferred from Single-Copy Nuclear Genes

    PubMed Central

    Guo, Dong-Mei; Yang, Zu-Yu; Wang, Xiao-Quan

    2014-01-01

    Phylogenetic reconstruction is fundamental to study evolutionary biology and historical biogeography. However, there was not a molecular phylogeny of gymnosperms represented by extensive sampling at the genus level, and most published phylogenies of this group were constructed based on cytoplasmic DNA markers and/or the multi-copy nuclear ribosomal DNA. In this study, we use LFY and NLY, two single-copy nuclear genes that originated from an ancient gene duplication in the ancestor of seed plants, to reconstruct the phylogeny and estimate divergence times of gymnosperms based on a complete sampling of extant genera. The results indicate that the combined LFY and NLY coding sequences can resolve interfamilial relationships of gymnosperms and intergeneric relationships of most families. Moreover, the addition of intron sequences can improve the resolution in Podocarpaceae but not in cycads, although divergence times of the cycad genera are similar to or longer than those of the Podocarpaceae genera. Our study strongly supports cycads as the basal-most lineage of gymnosperms rather than sister to Ginkgoaceae, and a sister relationship between Podocarpaceae and Araucariaceae and between Cephalotaxaceae-Taxaceae and Cupressaceae. In addition, intergeneric relationships of some families that were controversial, and the relationships between Taxaceae and Cephalotaxaceae and between conifers and Gnetales are discussed based on the nuclear gene evidence. The molecular dating analysis suggests that drastic extinctions occurred in the early evolution of gymnosperms, and extant coniferous genera in the Northern Hemisphere are older than those in the Southern Hemisphere on average. This study provides an evolutionary framework for future studies on gymnosperms. PMID:25222863

  15. Target genes discovery through copy number alteration analysis in human hepatocellular carcinoma.

    PubMed

    Gu, De-Leung; Chen, Yen-Hsieh; Shih, Jou-Ho; Lin, Chi-Hung; Jou, Yuh-Shan; Chen, Chian-Feng

    2013-12-21

    High-throughput short-read sequencing of exomes and whole cancer genomes in multiple human hepatocellular carcinoma (HCC) cohorts confirmed previously identified frequently mutated somatic genes, such as TP53, CTNNB1 and AXIN1, and identified several novel genes with moderate mutation frequencies, including ARID1A, ARID2, MLL, MLL2, MLL3, MLL4, IRF2, ATM, CDKN2A, FGF19, PIK3CA, RPS6KA3, JAK1, KEAP1, NFE2L2, C16orf62, LEPR, RAC2, and IL6ST. Functional classification of these mutated genes suggested that alterations in pathways participating in chromatin remodeling, Wnt/β-catenin signaling, JAK/STAT signaling, and oxidative stress play critical roles in HCC tumorigenesis. Nevertheless, because there are few druggable genes used in HCC therapy, the identification of new therapeutic targets through integrated genomic approaches remains an important task. Because a large amount of HCC genomic data genotyped by high density single nucleotide polymorphism arrays is deposited in the public domain, copy number alteration (CNA) analyses of these arrays is a cost-effective way to reveal target genes through profiling of recurrent and overlapping amplicons, homozygous deletions and potentially unbalanced chromosomal translocations accumulated during HCC progression. Moreover, integration of CNAs with other high-throughput genomic data, such as aberrantly coding transcriptomes and non-coding gene expression in human HCC tissues and rodent HCC models, provides lines of evidence that can be used to facilitate the identification of novel HCC target genes with the potential of improving the survival of HCC patients.

  16. Sex bias in copy number variation of olfactory receptor gene family depends on ethnicity.

    PubMed

    Shadravan, Farideh

    2013-01-01

    Gender plays a pivotal role in the human genetic identity and is also manifested in many genetic disorders particularly mental retardation. In this study its effect on copy number variation (CNV), known to cause genetic disorders was explored. As the olfactory receptor (OR) repertoire comprises the largest human gene family, it was selected for this study, which was carried out within and between three populations, derived from 150 individuals from the 1000 Genome Project. Analysis of 3872 CNVs detected among 791 OR loci, in which 307 loci showed CNV, revealed the following novel findings: Sex bias in CNV was significantly more prevalent in uncommon than common CNV variants of OR pseudogenes, in which the male genome showed more CNVs; and in one-copy number loss compared to complete deletion of OR pseudogenes; both findings implying a more recent evolutionary role for gender. Sex bias in copy number gain was also detected. Another novel finding was that the observed sex bias was largely dependent on ethnicity and was in general absent in East Asians. Using a CNV public database for sick children (International Standard Cytogenomic Array Consortium) the application of these findings for improving clinical molecular diagnostics is discussed by showing an example of sex bias in CNV among kids with autism. Additional clinical relevance is discussed, as the most polymorphic CNV-enriched OR cluster in the human genome, located on chr 15q11.2, is found near the Prader-Willi syndrome/Angelman syndrome bi-directionally imprinted region associated with two well-known mental retardation syndromes. As olfaction represents the primitive cognition in most mammals, arguably in competition with the development of a larger brain, the extensive retention of OR pseudogenes in females of this study, might point to a parent-of-origin indirect regulatory role for OR pseudogenes in the embryonic development of human brain. Thus any perturbation in the temporal regulation of olfactory

  17. Copy number of the Adenomatous Polyposis Coli gene is not always neutral in sporadic colorectal cancers with loss of heterozygosity for the gene.

    PubMed

    Zauber, Peter; Marotta, Stephen; Sabbath-Solitare, Marlene

    2016-03-12

    Changes in the number of alleles of a chromosome may have an impact upon gene expression. Loss of heterozygosity (LOH) indicates that one allele of a gene has been lost, and knowing the exact copy number of the gene would indicate whether duplication of the remaining allele has occurred. We were interested to determine the copy number of the Adenomatous Polyposis Coli (APC) gene in sporadic colorectal cancers with LOH. We selected 38 carcinomas with LOH for the APC gene region of chromosome 5, as determined by amplification of the CA repeat region within the D5S346 loci. The copy number status of APC was ascertained using the SALSA® MLPA® P043-B1 APC Kit. LOH for the DCC gene, KRAS gene mutation, and microsatellite instability were also evaluated for each tumor, utilizing standard polymerase chain reaction methods. No tumor demonstrated microsatellite instability. LOH of the DCC gene was also present in 33 of 36 (91.7%) informative tumors. A KRAS gene mutation was present in 16 of the 38 (42.1%) tumors. Twenty-four (63.2%) of the tumors were copy number neutral, 10 (26.3%) tumors demonstrated major loss, while two (5.3%) showed partial loss. Two tumors (5.3%) had copy number gain. Results of APC and DCC LOH, KRAS and microsatellite instability indicate our colorectal cancer cases were typical of sporadic cancers following the 'chromosomal instability' pathway. The majority of our colorectal carcinomas with LOH for APC gene are copy number neutral. However, one-third of our cases showed copy number loss, suggesting that duplication of the remaining allele is not required for the development of a colorectal carcinoma.

  18. Screen for mitochondrial DNA copy number maintenance genes reveals essential role for ATP synthase

    PubMed Central

    Fukuoh, Atsushi; Cannino, Giuseppe; Gerards, Mike; Buckley, Suzanne; Kazancioglu, Selena; Scialo, Filippo; Lihavainen, Eero; Ribeiro, Andre; Dufour, Eric; Jacobs, Howard T

    2014-01-01

    The machinery of mitochondrial DNA (mtDNA) maintenance is only partially characterized and is of wide interest due to its involvement in disease. To identify novel components of this machinery, plus other cellular pathways required for mtDNA viability, we implemented a genome-wide RNAi screen in Drosophila S2 cells, assaying for loss of fluorescence of mtDNA nucleoids stained with the DNA-intercalating agent PicoGreen. In addition to previously characterized components of the mtDNA replication and transcription machineries, positives included many proteins of the cytosolic proteasome and ribosome (but not the mitoribosome), three proteins involved in vesicle transport, some other factors involved in mitochondrial biogenesis or nuclear gene expression, > 30 mainly uncharacterized proteins and most subunits of ATP synthase (but no other OXPHOS complex). ATP synthase knockdown precipitated a burst of mitochondrial ROS production, followed by copy number depletion involving increased mitochondrial turnover, not dependent on the canonical autophagy machinery. Our findings will inform future studies of the apparatus and regulation of mtDNA maintenance, and the role of mitochondrial bioenergetics and signaling in modulating mtDNA copy number. PMID:24952591

  19. Extensive Copy Number Variation in Fermentation-Related Genes Among Saccharomyces cerevisiae Wine Strains.

    PubMed

    Steenwyk, Jacob; Rokas, Antonis

    2017-05-05

    Due to the importance of Saccharomyces cerevisiae in wine-making, the genomic variation of wine yeast strains has been extensively studied. One of the major insights stemming from these studies is that wine yeast strains harbor low levels of genetic diversity in the form of single nucleotide polymorphisms (SNPs). Genomic structural variants, such as copy number (CN) variants, are another major type of variation segregating in natural populations. To test whether genetic diversity in CN variation is also low across wine yeast strains, we examined genome-wide levels of CN variation in 132 whole-genome sequences of S. cerevisiae wine strains. We found an average of 97.8 CN variable regions (CNVRs) affecting ∼4% of the genome per strain. Using two different measures of CN diversity, we found that gene families involved in fermentation-related processes such as copper resistance ( CUP ), flocculation ( FLO ), and glucose metabolism ( HXT ), as well as the SNO gene family whose members are expressed before or during the diauxic shift, showed substantial CN diversity across the 132 strains examined. Importantly, these same gene families have been shown, through comparative transcriptomic and functional assays, to be associated with adaptation to the wine fermentation environment. Our results suggest that CN variation is a substantial contributor to the genomic diversity of wine yeast strains, and identify several candidate loci whose levels of CN variation may affect the adaptation and performance of wine yeast strains during fermentation. Copyright © 2017 Steenwyk and Rokas.

  20. MulRF: a software package for phylogenetic analysis using multi-copy gene trees.

    PubMed

    Chaudhary, Ruchi; Fernández-Baca, David; Burleigh, John Gordon

    2015-02-01

    MulRF is a platform-independent software package for phylogenetic analysis using multi-copy gene trees. It seeks the species tree that minimizes the Robinson-Foulds (RF) distance to the input trees using a generalization of the RF distance to multi-labeled trees. The underlying generic tree distance measure and fast running time make MulRF useful for inferring phylogenies from large collections of gene trees, in which multiple evolutionary processes as well as phylogenetic error may contribute to gene tree discord. MulRF implements several features for customizing the species tree search and assessing the results, and it provides a user-friendly graphical user interface (GUI) with tree visualization. The species tree search is implemented in C++ and the GUI in Java Swing. MulRF's executable as well as sample datasets and manual are available at http://genome.cs.iastate.edu/CBL/MulRF/, and the source code is available at https://github.com/ruchiherself/MulRFRepo. ruchic@ufl.edu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma

    PubMed Central

    Yan, Deng; Yi, Song; Chiu, Wang Chi; Qin, Liu Gui; Kin, Wong Hoi; Kwok Hung, Chung Tony; Linxiao, Han; Wai, Choy Kwong; Yi, Sui; Tao, Yang; Tao, Tang

    2017-01-01

    Objective This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. Methods Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). Results Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification (P=0.009) or deletion (P=0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly (P=1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. Conclusion Chromosomal CNVs may contribute to their transcript expression in cervical cancer. PMID:29312578

  2. Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma.

    PubMed

    Yan, Deng; Yi, Song; Chiu, Wang Chi; Qin, Liu Gui; Kin, Wong Hoi; Kwok Hung, Chung Tony; Linxiao, Han; Wai, Choy Kwong; Yi, Sui; Tao, Yang; Tao, Tang

    2017-12-12

    This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification ( P =0.009) or deletion ( P =0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly ( P =1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. Chromosomal CNVs may contribute to their transcript expression in cervical cancer.

  3. Effect of HPV16 L1 virus-like particles on the aggregation of non-functionalized gold nanoparticles.

    PubMed

    Palomino-Vizcaino, Giovanni; Valencia Reséndiz, Diana Gabriela; Benítez-Hess, María Luisa; Martínez-Acuña, Natalia; Tapia-Vieyra, Juana Virginia; Bahena, Daniel; Díaz-Sánchez, Mauricio; García-González, Octavio Patricio; Alvarez-Sandoval, Brenda Arizaí; Alvarez-Salas, Luis Marat

    2018-02-15

    Colorimetric assays based on gold nanoparticles (GNPs) are of considerable interest for diagnostics because of their simplicity and low-cost. Nevertheless, a deep understanding of the interaction between the GNPs and the intended molecular target is critical for the development of reliable detection technologies. The present report describes the spontaneous interaction between HPV16 L1 virus-like particles (VLPs) and non-functionalized GNPs (nfGNPs) resulting in the inhibition of nfGNPs salt-induced aggregation and the stabilization of purified VLPs. Ionic-competition experiments suggested that the nature of nfGNPs-VLPs interaction is non-covalent. Adsorption of an RNA aptamer on nfGNPs surface showed an additive aggregation-inhibitory effect. The use of mutant VLPs confirmed that the interaction nfGNPs-VLPs is not mediated by the opposing superficial electrostatic charges, suggesting that non-electrostatic forces participate in the arrangement of nfGNPs on the VLPs surface. Competition experiments using increasing ethanol concentrations on nfGNPs-VLPs complexes suggested hydrophobic interactions as the main stabilizing force. Therefore, the nfGNPs-VLPs interaction described here should facilitate the development of adsorption assays based on nfGNPs for HPV detection and cervical cancer prevention. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Induction of MAGE-A3 and HPV-16 immunity by Trojan vaccines in patients with head and neck carcinoma

    PubMed Central

    Voskens, Caroline J.; Sewell, Duane; Hertzano, Ronna; DeSanto, Jennifer; Rollins, Sandra; Lee, Myounghee; Taylor, Rodney; Wolf, Jeffrey; Suntharalingam, Mohan; Gastman, Brian; Papadimitriou, John C.; Lu, Changwan; Tan, Ming; Morales, Robert; Cullen, Kevin; Celis, Esteban; Mann, Dean; Strome, Scott E.

    2013-01-01

    Background We performed a pilot study using Trojan vaccines in patients with advanced squamous cell carcinoma of the head and neck (SCCHN). These vaccines are composed of HLA-I and HLA-II restricted melanoma antigen E (MAGE)-A3 or human papillomavirus (HPV)-16 derived peptides, joined by furin-cleavable linkers, and linked to a “penetrin” peptide sequence derived from HIV-TAT. Thirty-one patients with SCCHN were screened for the trial and 5 were enrolled. Methods Enrolled patients were treated with 300 lg of Trojan peptide supplemented with Montanide and granulocyte-macrophage colony-stimulating factor (GM-CSF) at 4-week intervals for up to 4 injections. Results Following vaccination, peripheral blood mononuclear cells (PBMCs) from 4 of 5 patients recognized both the full Trojan constructs and constituent HLA-II peptides, whereas responses to HLA-I restricted peptides were less pronounced. Conclusion This treatment regimen seems to have acceptable toxicity and elicits measurable systemic immune responses against HLA-II restricted epitopes in a subset of patients with advanced SCCHN. PMID:22287423

  5. Induction of MAGE-A3 and HPV-16 immunity by Trojan vaccines in patients with head and neck carcinoma.

    PubMed

    Voskens, Caroline J; Sewell, Duane; Hertzano, Ronna; DeSanto, Jennifer; Rollins, Sandra; Lee, Myounghee; Taylor, Rodney; Wolf, Jeffrey; Suntharalingam, Mohan; Gastman, Brian; Papadimitriou, John C; Lu, Changwan; Tan, Ming; Morales, Robert; Cullen, Kevin; Celis, Esteban; Mann, Dean; Strome, Scott E

    2012-12-01

    We performed a pilot study using Trojan vaccines in patients with advanced squamous cell carcinoma of the head and neck (SCCHN). These vaccines are composed of HLA-I and HLA-II restricted melanoma antigen E (MAGE)-A3 or human papillomavirus (HPV)-16 derived peptides, joined by furin-cleavable linkers, and linked to a "penetrin" peptide sequence derived from HIV-TAT. Thirty-one patients with SCCHN were screened for the trial and 5 were enrolled. Enrolled patients were treated with 300 μg of Trojan peptide supplemented with Montanide and granulocyte-macrophage colony-stimulating factor (GM-CSF) at 4-week intervals for up to 4 injections. Following vaccination, peripheral blood mononuclear cells (PBMCs) from 4 of 5 patients recognized both the full Trojan constructs and constituent HLA-II peptides, whereas responses to HLA-I restricted peptides were less pronounced. This treatment regimen seems to have acceptable toxicity and elicits measurable systemic immune responses against HLA-II restricted epitopes in a subset of patients with advanced SCCHN. Copyright © 2012 Wiley Periodicals, Inc.

  6. Optimization of supercoiled HPV-16 E6/E7 plasmid DNA purification with arginine monolith using design of experiments.

    PubMed

    Almeida, A M; Queiroz, J A; Sousa, F; Sousa, A

    2015-01-26

    The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA. Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. The supercoiled HPV-16 E6/E7 plasmid-based vaccine was recently purified with the arginine monolith, with 100% of purity, but only 39% of recovery was achieved. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree. In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. The Composite Central Face design was validated and the recovery of the target molecule was successfully improved from 39% to 83.5%, with an outstanding increase of more than double, while maintaining 100% of purity. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. MenACWY-TT is immunogenic when co-administered with Tdap and AS04-HPV16/18 in girls and young women: Results from a phase III randomized trial.

    PubMed

    Rivera, Luis; Chanthavanich, Pornthep; Põder, Airi; Suryakiran, P V; Jastorff, Archana; Van der Wielen, Marie

    2018-06-22

    Co-administration of vaccines in adolescents may improve coverage. We assessed co-administration of quadrivalent meningococcal serogroups A, C, W and Y tetanus toxoid-conjugate vaccine (MenACWY-TT), human papillomavirus 16/18 AS04-adjuvanted vaccine (AS04-HPV16/18) and tetanus-diphtheria-acellular pertussis vaccine (Tdap) in girls and young women. In this phase IIIb study (NCT01755689), 1300 healthy 9-25-year-old females were randomized (1:1:1:1:1) to receive: MenACWY-TT at month (M) 0 and AS04-HPV16/18 at M1, M2, M7; MenACWY-TT and AS04-HPV16/18 at M0 and AS04-HPV16/18 at M1, M6; AS04-HPV16/18 at M0, M1, M6; MenACWY-TT, Tdap and AS04-HPV16/18 at M0 and AS04-HPV16/18 at M1, M6; Tdap and AS04-HPV16/18 at M0 and AS04-HPV16/18 at M1, M6. Immunogenicity, safety and reactogenicity were evaluated. Immunogenicity of MenACWY-TT and AS04-HPV16/18 when co-administered was non-inferior to that of the 2 vaccines given separately. Co-administration of MenACWY-TT, AS04-HPV16/18 and Tdap was non-inferior to MenACWY-TT administered alone or to Tdap co-administered with AS04-HPV16/18 in terms of immunogenicity for all vaccine components, except pertussis antigens. Post-vaccination, ≥89.5% of participants reached antibody levels above the pre-specified threshold for all antigens. No safety concerns were identified. Our data support co-administration of MenACWY-TT with Tdap and AS04-HPV16/18 vaccines in adolescents. Copyright © 2018 GlaxoSmithKline Biologicals SA. Published by Elsevier Ltd.. All rights reserved.

  8. Immunogenicity and safety of the HPV-16/18 AS04-adjuvanted vaccine in healthy Chinese girls and women aged 9 to 45 years

    PubMed Central

    Zhu, Fengcai; Li, Juan; Hu, Yuemei; Zhang, Xiang; Yang, Xiaoping; Zhao, Hui; Wang, Junzhi; Yang, Jianguo; Xia, Guodong; Dai, Qinyong; Tang, Haiwen; V Suryakiran, Pemmaraju; Datta, Sanjoy K; Descamps, Dominique; Bi, Dan; Struyf, Frank

    2014-01-01

    Immunogenicity and safety of the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine were evaluated in healthy Chinese females aged 9–45 years in 2 phase IIIB, randomized, controlled trials. Girls aged 9–17 years (ClinicalTrials.gov, NCT00996125) received vaccine (n = 374) or control (n = 376) and women aged 26–45 years (NCT01277042) received vaccine (n = 606) or control (n = 606) at months 0, 1, and 6. The primary objective was to show non-inferiority of anti-HPV-16 and -18 immune responses in initially seronegative subjects at month 7, compared with Chinese women aged 18–25 years enrolled in a separate phase II/III trial (NCT00779766). Secondary objectives were to describe the anti-HPV-16 and -18 immune response, reactogenicity and safety. At month 7, immune responses were non-inferior for girls (9–17 years) vs. young women (18–25 years): the upper limit of the 95% confidence interval (CI) for the geometric mean titer (GMT) ratio (women/girls) was below the limit of 2 for both anti-HPV-16 (0.37 [95% CI: 0.32, 0.43]) and anti-HPV-18 (0.42 [0.36, 0.49]). Immune responses at month 7 were also non-inferior for 26–45 year-old women vs. 18–25 year-old women: the upper limit of the 95% CI for the difference in seroconversion (18–25 minus 26–45) was below the limit of 5% for both anti-HPV-16 (0.00% [–1.53, 1.10]) and anti-HPV-18 (0.21% [–1.36, 1.68]). GMTs were 2- to 3-fold higher in girls (9–17 years) as compared with young women (18–25 years). The HPV-16/18 AS04-adjuvanted vaccine had an acceptable safety profile when administered to healthy Chinese females aged 9–45 years. PMID:25424785

  9. Immunogenicity and safety of the HPV-16/18 AS04-adjuvanted vaccine in healthy Chinese girls and women aged 9 to 45 years.

    PubMed

    Zhu, Fengcai; Li, Juan; Hu, Yuemei; Zhang, Xiang; Yang, Xiaoping; Zhao, Hui; Wang, Junzhi; Yang, Jianguo; Xia, Guodong; Dai, Qinyong; Tang, Haiwen; Suryakiran, Pemmaraju; Datta, Sanjoy K; Descamps, Dominique; Bi, Dan; Struyf, Frank

    2014-01-01

    Immunogenicity and safety of the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine were evaluated in healthy Chinese females aged 9-45 years in 2 phase IIIB, randomized, controlled trials. Girls aged 9-17 years (ClinicalTrials.gov, NCT00996125) received vaccine (n = 374) or control (n = 376) and women aged 26-45 years (NCT01277042) received vaccine (n = 606) or control (n = 606) at months 0, 1, and 6. The primary objective was to show non-inferiority of anti-HPV-16 and -18 immune responses in initially seronegative subjects at month 7, compared with Chinese women aged 18-25 years enrolled in a separate phase II/III trial (NCT00779766). Secondary objectives were to describe the anti-HPV-16 and -18 immune response, reactogenicity and safety. At month 7, immune responses were non-inferior for girls (9-17 years) vs. young women (18-25 years): the upper limit of the 95% confidence interval (CI) for the geometric mean titer (GMT) ratio (women/girls) was below the limit of 2 for both anti-HPV-16 (0.37 [95% CI: 0.32, 0.43]) and anti-HPV-18 (0.42 [0.36, 0.49]). Immune responses at month 7 were also non-inferior for 26-45 year-old women vs. 18-25 year-old women: the upper limit of the 95% CI for the difference in seroconversion (18-25 minus 26-45) was below the limit of 5% for both anti-HPV-16 (0.00% [-1.53, 1.10]) and anti-HPV-18 (0.21% [-1.36, 1.68]). GMTs were 2- to 3-fold higher in girls (9-17 years) as compared with young women (18-25 years). The HPV-16/18 AS04-adjuvanted vaccine had an acceptable safety profile when administered to healthy Chinese females aged 9-45 years.

  10. Heritable heading time variation in wheat lines with the same number of Ppd-B1 gene copies.

    PubMed

    Ivaničová, Zuzana; Valárik, Miroslav; Pánková, Kateřina; Trávníčková, Martina; Doležel, Jaroslav; Šafář, Jan; Milec, Zbyněk

    2017-01-01

    The ability of plants to identify an optimal flowering time is critical for ensuring the production of viable seeds. The main environmental factors that influence the flowering time include the ambient temperature and day length. In wheat, the ability to assess the day length is controlled by photoperiod (Ppd) genes. Due to its allohexaploid nature, bread wheat carries the following three Ppd-1 genes: Ppd-A1, Ppd-B1 and Ppd-D1. While photoperiod (in)sensitivity controlled by Ppd-A1 and Ppd-D1 is mainly determined by sequence changes in the promoter region, the impact of the Ppd-B1 alleles on the heading time has been linked to changes in the copy numbers (and possibly their methylation status) and sequence changes in the promoter region. Here, we report that plants with the same number of Ppd-B1 copies may have different heading times. Differences were observed among F7 lines derived from crossing two spring hexaploid wheat varieties. Several lines carrying three copies of Ppd-B1 headed 16 days later than other plants in the population with the same number of gene copies. This effect was associated with changes in the gene expression level and methylation of the Ppd-B1 gene.

  11. Heritable heading time variation in wheat lines with the same number of Ppd-B1 gene copies

    PubMed Central

    Ivaničová, Zuzana; Valárik, Miroslav; Pánková, Kateřina; Trávníčková, Martina; Doležel, Jaroslav; Šafář, Jan

    2017-01-01

    The ability of plants to identify an optimal flowering time is critical for ensuring the production of viable seeds. The main environmental factors that influence the flowering time include the ambient temperature and day length. In wheat, the ability to assess the day length is controlled by photoperiod (Ppd) genes. Due to its allohexaploid nature, bread wheat carries the following three Ppd-1 genes: Ppd-A1, Ppd-B1 and Ppd-D1. While photoperiod (in)sensitivity controlled by Ppd-A1 and Ppd-D1 is mainly determined by sequence changes in the promoter region, the impact of the Ppd-B1 alleles on the heading time has been linked to changes in the copy numbers (and possibly their methylation status) and sequence changes in the promoter region. Here, we report that plants with the same number of Ppd-B1 copies may have different heading times. Differences were observed among F7 lines derived from crossing two spring hexaploid wheat varieties. Several lines carrying three copies of Ppd-B1 headed 16 days later than other plants in the population with the same number of gene copies. This effect was associated with changes in the gene expression level and methylation of the Ppd-B1 gene. PMID:28846721

  12. FAS Gene Copy Numbers are Associated with Susceptibility to Behçet Disease and VKH Syndrome in Han Chinese.

    PubMed

    Yu, Hongsong; Luo, Le; Wu, Lili; Zheng, Minming; Zhang, Lijun; Liu, Yunjia; Li, Hua; Cao, Qingfeng; Kijlstra, Aize; Yang, Peizeng

    2015-11-01

    Previous studies have identified that disturbed apoptosis was involved in the pathogenesis of Behçet disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome. This study aims to investigate whether copy number variations of apoptosis-related genes, including FAS, CASPASE8, CASPASE3, and BCL2, are associated with BD and VKH syndrome in Han Chinese. A two-stage association study was performed in 1,014 BD patients, 1,051 VKH syndrome patients, and 2,076 healthy controls. TaqMan(®) Copy Number Assays and real-time PCR were performed. The first-stage study showed that increased frequency of high FAS copy number (>2) was found in BD (P = 1.05 × 10(-3) ) and VKH syndrome (P = 2.56 × 10(-3) ). Replication and combined study confirmed the association of high copy number (>2) of FAS with BD (P = 3.35 × 10(-8) ) and VKH syndrome (P = 9.77 × 10(-8) ). A significant upregulated mRNA expression of FAS was observed in anti-CD3/CD28 antibodies-stimulated CD4(+) T cells from individuals carrying a high gene copy number (>2) as compared to normal diploid 2 copy number carriers (P = 0.004). Moreover, the mRNA expression of FAS both in active patients with BD and VKH syndrome was significantly higher than that in controls (P = 0.001 and P = 0.007, respectively). Our findings suggest that a high copy number of FAS gene confers risk for BD and VKH syndrome. © 2015 WILEY PERIODICALS, INC.

  13. Copy Number Variations in the Survival Motor Neuron Genes: Implications for Spinal Muscular Atrophy and Other Neurodegenerative Diseases

    PubMed Central

    Butchbach, Matthew E. R.

    2016-01-01

    Proximal spinal muscular atrophy (SMA), a leading genetic cause of infant death worldwide, is an early-onset, autosomal recessive neurodegenerative disease characterized by the loss of spinal α-motor neurons. This loss of α-motor neurons is associated with muscle weakness and atrophy. SMA can be classified into five clinical grades based on age of onset and severity of the disease. Regardless of clinical grade, proximal SMA results from the loss or mutation of SMN1 (survival motor neuron 1) on chromosome 5q13. In humans a large tandem chromosomal duplication has lead to a second copy of the SMN gene locus known as SMN2. SMN2 is distinguishable from SMN1 by a single nucleotide difference that disrupts an exonic splice enhancer in exon 7. As a result, most of SMN2 mRNAs lack exon 7 (SMNΔ7) and produce a protein that is both unstable and less than fully functional. Although only 10–20% of the SMN2 gene product is fully functional, increased genomic copies of SMN2 inversely correlates with disease severity among individuals with SMA. Because SMN2 copy number influences disease severity in SMA, there is prognostic value in accurate measurement of SMN2 copy number from patients being evaluated for SMA. This prognostic value is especially important given that SMN2 copy number is now being used as an inclusion criterion for SMA clinical trials. In addition to SMA, copy number variations (CNVs) in the SMN genes can affect the clinical severity of other neurological disorders including amyotrophic lateral sclerosis (ALS) and progressive muscular atrophy (PMA). This review will discuss how SMN1 and SMN2 CNVs are detected and why accurate measurement of SMN1 and SMN2 copy numbers is relevant for SMA and other neurodegenerative diseases. PMID:27014701

  14. Copy-number variations are enriched for neurodevelopmental genes in children with developmental coordination disorder.

    PubMed

    Mosca, Stephen J; Langevin, Lisa Marie; Dewey, Deborah; Innes, A Micheil; Lionel, Anath C; Marshall, Christian C; Scherer, Stephen W; Parboosingh, Jillian S; Bernier, Francois P

    2016-12-01

    Developmental coordination disorder is a common neurodevelopment disorder that frequently co-occurs with other neurodevelopmental disorders including attention-deficit hyperactivity disorder (ADHD). Copy-number variations (CNVs) have been implicated in a number of neurodevelopmental and psychiatric disorders; however, the proportion of heritability in developmental coordination disorder (DCD) attributed to CNVs has not been explored. This study aims to investigate how CNVs may contribute to the genetic architecture of DCD. CNV analysis was performed on 82 extensively phenotyped Canadian children with DCD, with or without co-occurring ADHD and/or reading disorder, and 2988 healthy European controls using identical genome-wide SNP microarrays and CNV calling algorithms. An increased rate of large and rare genic CNVs (p=0.009) was detected, and there was an enrichment of duplications spanning brain-expressed genes (p=0.039) and genes previously implicated in other neurodevelopmental disorders (p=0.043). Genes and loci of particular interest in this group included: GAP43, RBFOX1, PTPRN2, SHANK3, 16p11.2 and distal 22q11.2. Although no recurrent CNVs were identified, 26% of DCD cases, where sample availability permitted segregation analysis, were found to have a de novo rare CNV. Of the inherited CNVs, 64% were from a parent who also had a neurodevelopmental disorder. These findings suggest that there may be shared susceptibility genes for DCD and other neurodevelopmental disorders and highlight the need for thorough phenotyping when investigating the genetics of neurodevelopmental disorders. Furthermore, these data provide compelling evidence supporting a genetic basis for DCD, and further implicate rare CNVs in the aetiology of neurodevelopmental disorders. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  15. Integrative analysis of gene expression and copy number alterations using canonical correlation analysis.

    PubMed

    Soneson, Charlotte; Lilljebjörn, Henrik; Fioretos, Thoas; Fontes, Magnus

    2010-04-15

    With the rapid development of new genetic measurement methods, several types of genetic alterations can be quantified in a high-throughput manner. While the initial focus has been on investigating each data set separately, there is an increasing interest in studying the correlation structure between two or more data sets. Multivariate methods based on Canonical Correlation Analysis (CCA) have been proposed for integrating paired genetic data sets. The high dimensionality of microarray data imposes computational difficulties, which have been addressed for instance by studying the covariance structure of the data, or by reducing the number of variables prior to applying the CCA. In this work, we propose a new method for analyzing high-dimensional paired genetic data sets, which mainly emphasizes the correlation structure and still permits efficient application to very large data sets. The method is implemented by translating a regularized CCA to its dual form, where the computational complexity depends mainly on the number of samples instead of the number of variables. The optimal regularization parameters are chosen by cross-validation. We apply the regularized dual CCA, as well as a classical CCA preceded by a dimension-reducing Principal Components Analysis (PCA), to a paired data set of gene expression changes and copy number alterations in leukemia. Using the correlation-maximizing methods, regularized dual CCA and PCA+CCA, we show that without pre-selection of known disease-relevant genes, and without using information about clinical class membership, an exploratory analysis singles out two patient groups, corresponding to well-known leukemia subtypes. Furthermore, the variables showing the highest relevance to the extracted features agree with previous biological knowledge concerning copy number alterations and gene expression changes in these subtypes. Finally, the correlation-maximizing methods are shown to yield results which are more biologically

  16. Long-term immunogenicity and safety of the HPV-16/18 AS04-adjuvanted vaccine in 10- to 14-year-old girls: open 6-year follow-up of an initial observer-blinded, randomized trial.

    PubMed

    Schwarz, Tino F; Huang, Li-Min; Lin, Tzou-Yien; Wittermann, Christoph; Panzer, Falko; Valencia, Alejandra; Suryakiran, Pemmaraju V; Lin, Lan; Descamps, Dominique

    2014-12-01

    Immunogenicity and safety of the HPV-16/18 AS04-adjuvanted vaccine were evaluated up to 6 years postvaccination (month 72) in preteen/adolescent girls. Participants, who had received 3 HPV-16/18 AS04-adjuvanted vaccine doses at 10-14 years of age in an initial controlled, observer-blinded, randomized study (NCT00196924) and participated in the open 3-year follow-up (NCT00316706), were invited to continue the follow-up for up to 10 years postvaccination (NCT00877877). Anti-HPV-16 and -18 antibody titers were measured by enzyme-linked immunosorbent assays at yearly visits and were used to fit the modified power-law and piecewise models, predicting long-term immunogenicity. Serious adverse events (SAEs) and pregnancy information were recorded. In the according-to-protocol immunogenicity cohort, all participants (N = 505) with data available remained seropositive for anti-HPV-16 and -18 antibodies at month 72. In initially seronegative participants, anti-HPV-16 and -18 antibody geometric mean titers were 65.8- and 33.0-fold higher than those associated with natural infection (NCT00122681) and 5.0- and 2.5-fold higher than those measured at month 69-74 in a study demonstrating vaccine efficacy in women aged 15-25 years (NCT00120848). Exploratory antibody modeling, based on the 6-year data, predicted that vaccine-induced population anti-HPV-16 and -18 antibody geometric mean titers would remain above those associated with natural infection for at least 20 years postvaccination. The HPV-16/18 AS04-adjuvanted vaccine safety profile was clinically acceptable. In preteen/adolescent girls, the HPV-16/18 AS04-adjuvanted vaccine induced high anti-HPV-16 and -18 antibody levels up to 6 years postvaccination, which were predicted to remain above those induced by natural infection for at least 20 years.

  17. Associations of GBP2 gene copy number variations with growth traits and transcriptional expression in Chinese cattle.

    PubMed

    Zhang, Gui-Min; Zheng, Li; He, Hua; Song, Cheng-Chuang; Zhang, Zi-Jing; Cao, Xiu-Kai; Lei, Chu-Zhao; Lan, Xian-Yong; Qi, Xing-Lei; Chen, Hong; Huang, Yong-Zhen

    2018-03-20

    Copy number variations (CNVs) recently have been recognized as another important genetic variability followed single nucleotide polymorphisms (SNPs). The guanylate binding protein 2 (GBP2) gene plays an important role in cell proliferation. This study was performed to determine the presence of GBP2 CNV (relative to Angus cattle) in 466 individuals representing six main cattle breeds from China, identify its relationship with growth, and explore the biological effects of gene expression. There were two CNV regions in the GBP2 gene, for three types, CNV1 loss type (relative to Angus cattle) was more frequent in XN than other breeds, and CNV2 loss type (relative to Angus cattle) was more frequent in XN and CDM than other breeds. Though the GBP2 gene copy number presented no correlation with the transcriptional expression of JX (P > .05), but the transcriptional expression in heart is higher than other tissues, and the copy number in muscles and fat of JX is higher than others breeds. Statistical analysis revealed that the GBP2 gene CNV1 and CNV2 were significantly associated with growth traits (P < .05). In conclusion, this research established the correlations between CNVs of GBP2 gene and growth traits in different cattle breeds, and our results suggested that the CNVs in GBP2 gene may be considered markers for the molecular breeding of Chinese beef cattle. Copyright © 2018. Published by Elsevier B.V.

  18. Introduction and sustained high coverage of the HPV bivalent vaccine leads to a reduction in prevalence of HPV 16/18 and closely related HPV types.

    PubMed

    Kavanagh, K; Pollock, K G J; Potts, A; Love, J; Cuschieri, K; Cubie, H; Robertson, C; Donaghy, M

    2014-05-27

    In 2008, a national human papillomavirus (HPV) immunisation programme began in Scotland for 12-13 year old females with a three-year catch-up campaign for those under the age of 18. Since 2008, three-dose uptake of bivalent vaccine in the routine cohort aged 12-13 has exceeded 90% annually, while in the catch-up cohort overall uptake is 66%. To monitor the impact of HPV immunisation, a programme of national surveillance was established (pre and post introduction) which included yearly sampling and HPV genotyping of women attending for cervical screening at age 20. By linking individual vaccination, screening and HPV testing records, we aim to determine the impact of the immunisation programme on circulating type-specific HPV infection particularly for four outcomes: (i) the vaccine types HPV 16 or 18 (ii) types considered to be associated with cross-protection: HPV 31, 33 or 45; (iii) all other high-risk types and (iv) any HPV. From a total of 4679 samples tested, we demonstrate that three doses (n=1100) of bivalent vaccine are associated with a significant reduction in prevalence of HPV 16 and 18 from 29.8% (95% confidence interval 28.3, 31.3%) to 13.6% (95% confidence interval 11.7, 15.8%). The data also suggest cross-protection against HPV 31, 33 and 45. HPV 51 and 56 emerged as the most prevalent (10.5% and 9.6%, respectively) non-vaccine high-risk types in those vaccinated, but at lower rates than HPV 16 (25.9%) in those unvaccinated. This data demonstrate the positive impact of bivalent vaccination on the prevalence of HPV 16, 18, 31, 33 and 45 in the target population and is encouraging for countries which have achieved high-vaccine uptake.

  19. Comparison of the sensitivity and specificity of real-time PCR and in situ hybridization in HPV16 and 18 detection in archival cervical cancer specimens.

    PubMed

    Biesaga, Beata; Szostek, Sława; Klimek, Małgorzata; Jakubowicz, Jerzy; Wysocka, Joanna

    2012-07-04

    The aim of this study was to analyze the correlation between real-time PCR (RT-PCR) treated as a reference method and in situ hybridization with tyramide amplification system (ISH-TSA) in the detection of HPV16 and 18 infection and the assessment of viral genome status. The study was performed on cervical cancer biopsies fixed in 10% neutral buffered formalin and embedded in paraffin obtained from 85 women. TaqMan-based 5'exonuclease RT-PCR with type-specific primers was used to assess HPV16 and 18 infections and genome status. Viral infection and genome status was also assessed by ISH-TSA. RT-PCR revealed 76 (89.4%), and ISH-TSA 81 (95.3%) cancers with HPV16 and 18 infections. The ISH-TSA sensitivity and specificity were: 96.1% and 11.1% compared to RT-PCR. The difference between these techniques in HPV detection was significant (p = 0.000). Among 76 HPV16/18 positive cancers in RT-PCR, there were 30 (39.5%) with integrated and 46 (60.5%) with mixed viral genome form. According to ISH-TSA, there were 39 (51.3%) samples with integrated and 37 with mixed form (48.7%). The sensitivity and specificity of ISH-TSA in genome status assessment were 70.0% and 60.9%, respectively. The difference between RT-PCR and ISH-TSA in genome state detection was not statistically significant (p = 0.391). These results suggest that ISH-TSA shows insufficient specificity in HPV detection for use in clinical practice. However, this assay could be applied for viral genome status assessment.

  20. Phylogeny of the cycads based on multiple single copy nuclear genes: congruence of concatenation and species tree inference methods

    USDA-ARS?s Scientific Manuscript database

    Despite a recent new classification, a stable tree of life for the cycads has been elusive, particularly regarding resolution of Bowenia, Stangeria and Dioon. In this study we apply five single copy nuclear genes (SCNGs) to the phylogeny of the order Cycadales. We specifically aim to evaluate seve...

  1. The study of immunogenicity of the antigenic protein of high risk oncogenic type of the human papillomavirus HPV16 L1 produced in the plant expression system on the base of transgenic tomato.

    PubMed

    Salyaev, R K; Rekoslavskaya, N I; Stolbikov, A S; Tretyakova, A V

    2017-05-01

    The immunogenicity of plant-made peroral vaccine against cervical cancer was studied in mice during 342 days after vaccination with the material of tomato fruits genetically transformed with HPV16 L1 Ώ 5'UTR TMV. The immune response was found on day 4 after vaccination in blood serum of vaccinated mice. On days 90-100, the rise to maximum value of the level of antibodies to HPV16 L1 was in the range of 500 ng of the standard antibodies to HPV16 L1 (Santa Cruz Biotechnology, United States). This level of antibodies was retained until the end of the study.

  2. Safety and immunogenicity of the HPV-16/18 AS04-adjuvanted vaccine in HIV-positive women in South Africa: a partially-blind randomised placebo-controlled study.

    PubMed

    Denny, Lynette; Hendricks, Bronwyn; Gordon, Chivaugn; Thomas, Florence; Hezareh, Marjan; Dobbelaere, Kurt; Durand, Christelle; Hervé, Caroline; Descamps, Dominique

    2013-11-19

    In developing countries, risk of human papillomavirus (HPV) infection may be increased by the high prevalence of human immunodeficiency virus (HIV) infection. We evaluated the safety and immunogenicity of the HPV-16/18 AS04-adjuvanted vaccine in HIV-infected women in South Africa. Asymptomatic HIV-positive women aged 18-25 years (N=120) were stratified by CD4⁺ T-cell count and randomised (1:1) to receive HPV-16/18 vaccine (Cervarix®; GlaxoSmithKline Vaccines) or placebo (Al[OH]3) at 0, 1 and 6 months (double-blind). HIV-negative women (N=30) received HPV-16/18 vaccine (open label). Anti-HPV-16/18 antibody and CD4⁺ T-cell responses, CD4⁺ T-cell count, HIV viral load, HIV clinical stage and safety were evaluated for 12 months. The safety and reactogenicity profile of the HPV-16/18 vaccine was comparable in HIV-positive and HIV-negative women. Irrespective of baseline HPV status, all HIV-positive and HIV-negative women who received the HPV-16/18 vaccine were seropositive for both HPV-16 and HPV-18 after the second vaccine dose (month 2) and remained seropositive for both antigens at month 12. Anti-HPV-16/18 antibody titres at month 12 remained substantially above levels associated with natural infection. The HPV-16/18 vaccine induced sustained anti-HPV-16/18 CD4⁺ T-cell responses in both HIV-positive and HIV-negative women. No impact of baseline CD4⁺ T-cell count or HIV viral load was observed on the magnitude of the immune response in HIV-positive women. In HIV-positive women, CD4⁺ T-cell count, HIV viral load and HIV clinical stage were unaffected by HPV-16/18 vaccine administration. In conclusion, the HPV-16/18 AS04-adjuvanted vaccine appears immunogenic and well-tolerated in women with HIV infection. Study ID: 107863/NCT00586339. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  3. Impact of human papillomavirus (HPV) 16 and 18 vaccination on prevalent infections and rates of cervical lesions after excisional treatment

    PubMed Central

    Hildesheim, Allan; Gonzalez, Paula; Kreimer, Aimee R.; Wacholder, Sholom; Schussler, John; Rodriguez, Ana C.; Porras, Carolina; Schiffman, Mark; Sidawy, Mary; Schiller, John T.; Lowy, Douglas R.; Herrero, Rolando

    2016-01-01

    BACKGROUND Human papillomavirus (HPV) vaccines prevent HPV infection and cervical precancers. The impact of vaccinating women with a current infection or after treatment for an HPV-associated lesion is not fully understood. OBJECTIVES To determine whether HPV-16/18 vaccination influences the outcome of infections present at vaccination and the rate of infection and disease after treatment of lesions. STUDY DESIGN We included 1711 women (18–25 years) with carcinogenic human papillomavirus infection and 311 women of similar age who underwent treatment for cervical precancer and who participated in a community-based trial of the AS04-adjuvanted HPV-16/18 virus-like particle vaccine. Participants were randomized (human papillomavirus or hepatitis A vaccine) and offered 3 vaccinations over 6 months. Follow-up included annual visits (more frequently if clinically indicated), referral to colposcopy of high-grade and persistent low-grade lesions, treatment by loop electrosurgical excisional procedure when clinically indicated, and cytologic and virologic follow-up after treatment. Among women with human papillomavirus infection at the time of vaccination, we considered type-specific viral clearance, and development of cytologic (squamous intraepithelial lesions) and histologic (cervical intraepithelial neoplasia) lesions. Among treated women, we considered single-time and persistent human papillomavirus infection, squamous intraepithelial lesions, and cervical intraepithelial neoplasia 2+. Outcomes associated with infections absent before treatment also were evaluated. Infection-level analyses were performed and vaccine efficacy estimated. RESULTS Median follow-up was 56.7 months (women with human papillomavirus infection) and 27.3 months (treated women). There was no evidence of vaccine efficacy to increase clearance of human papillomavirus infections or decrease incidence of cytologic/histologic abnormalities associated with human papillomavirus types present at

  4. HPV16-E2 induces prophase arrest and activates the cellular DNA damage response in vitro and in precursor lesions of cervical carcinoma.

    PubMed

    Xue, Yuezhen; Toh, Shen Yon; He, Pingping; Lim, Thimothy; Lim, Diana; Pang, Chai Ling; Abastado, Jean-Pierre; Thierry, Françoise

    2015-10-27

    Cervical intraepithelial neoplasia (CIN) is caused by human papillomavirus (HPV) infection and is the precursor to cervical carcinoma. The completion of the HPV productive life cycle depends on the expression of viral proteins which further determines the severity of the cervical neoplasia. Initiation of the viral productive replication requires expression of the E2 viral protein that cooperates with the E1 viral DNA helicase. A decrease in the viral DNA replication ability and increase in the severity of cervical neoplasia is accompanied by simultaneous elevated expression of E6 and E7 oncoproteins. Here we reveal a novel and important role for the HPV16-E2 protein in controlling host cell cycle during malignant transformation. We showed that cells expressing HPV16-E2 in vitro are arrested in prophase alongside activation of a sustained DDR signal. We uncovered evidence that HPV16-E2 protein is present in vivo in cells that express both mitotic and DDR signals specifically in CIN3 lesions, immediate precursors of cancer, suggesting that E2 may be one of the drivers of genomic instability and carcinogenesis in vivo.

  5. HPV-16 virions can remain infectious for 2 weeks on senescent cells but require cell cycle re-activation to allow virus entry.

    PubMed

    Broniarczyk, Justyna; Ring, Nadja; Massimi, Paola; Giacca, Mauro; Banks, Lawrence

    2018-01-16

    Successful infection with Human Papillomaviruses requires mitosis, when incoming viral genomes gain access to nuclear components. However, very little is known about how long HPV particles can remain infectious in non-dividing cells or in which cellular compartments these viruses may reside. To investigate these questions we have used BJ cells as a reversible model of senescence and show that HPV-16 can only infect early-passage proliferating cells. Late-passage senescent cells are resistant to HPV infection, but this can be reversed by inducing cell cycle re-entry with a p53 siRNA. In senescent cells we find that efficient virus entry can be attained upon cell cycle re-entry 16 days after infection, demonstrating that HPV can persist for 2 weeks prior to induction of mitosis. However, exposing cells to anti-HPV-16 L1 neutralising antibody blocks infection at these late time points, suggesting that the virions reside near the cell surface. Indeed, immunofluorescence analysis shows that virions accumulate on the cell surface of senescent cells and only enter endocytic vesicles upon stimulation with p53 siRNA. These results demonstrate that HPV-16 virions can remain viable on a non-dividing cell for extended periods of time, but are nonetheless vulnerable to antibody-induced neutralisation throughout.

  6. Flavonol and imidazole derivatives block HPV16 E6 activities and reactivate apoptotic pathways in HPV+ cells

    PubMed Central

    Yuan, C-H; Filippova, M; Krstenansky, J L; Duerksen-Hughes, P J

    2016-01-01

    High-risk human papillomaviruses (HR-HPVs) cause nearly all cases of cervical cancer, as well as approximately 30% of head and neck cancers. HPV 16 E6, one of two major viral oncogenes, protects cells from apoptosis by binding to and accelerating the degradation of several proteins important in apoptotic signaling, including caspase 8 and p53. We proposed that blocking the interactions between HPV E6 and its partners using small molecules had the potential to re-sensitize HPV+ cells to apoptosis. To test this idea, we screened libraries of small molecules for candidates that could block E6/caspase 8 binding and identified several candidates from different chemical classes. We tested hits for dose-dependency and specificity in vitro and for toxicity in a cell-based assay and then used this information to select the two best candidates for further testing: myricetin, a flavonol, and spinacine, an imidazole amino-acid derivative of histidine. Both compounds clearly inhibited the ability of E6 to bind in vitro to both caspase 8 and E6AP, the protein that mediates p53 degradation. In addition, both compounds were able to increase the level of caspase 8 and p53 in SiHa cervical cancer cells, resulting in an increase of caspase 3/7 activity. Finally, both myricetin and spinacine sensitized HPV+ cervical and oral cancer cells, but not HPV− cervical and oral cancer cells, to apoptosis induced by the cancer-specific ligand TRAIL, as well as the chemotherapeutic agents doxorubicin and cisplatin. New therapies based on this work may improve treatment for HPV+ cancer patients. PMID:26794656

  7. Rare copy number variations in congenital heart disease patients identify unique genes in left-right patterning

    PubMed Central

    Fakhro, Khalid A.; Choi, Murim; Ware, Stephanie M.; Belmont, John W.; Towbin, Jeffrey A.; Lifton, Richard P.; Khokha, Mustafa K.; Brueckner, Martina

    2011-01-01

    Dominant human genetic diseases that impair reproductive fitness and have high locus heterogeneity constitute a problem for gene discovery because the usual criterion of finding more mutations in specific genes than expected by chance may require extremely large populations. Heterotaxy (Htx), a congenital heart disease resulting from abnormalities in left-right (LR) body patterning, has features suggesting that many cases fall into this category. In this setting, appropriate model systems may provide a means to support implication of specific genes. By high-resolution genotyping of 262 Htx subjects and 991 controls, we identify a twofold excess of subjects with rare genic copy number variations in Htx (14.5% vs. 7.4%, P = 1.5 × 10−4). Although 7 of 45 Htx copy number variations were large chromosomal abnormalities, 38 smaller copy number variations altered a total of 61 genes, 22 of which had Xenopus orthologs. In situ hybridization identified 7 of these 22 genes with expression in the ciliated LR organizer (gastrocoel roof plate), a marked enrichment compared with 40 of 845 previously studied genes (sevenfold enrichment, P < 10−6). Morpholino knockdown in Xenopus of Htx candidates demonstrated that five (NEK2, ROCK2, TGFBR2, GALNT11, and NUP188) strongly disrupted both morphological LR development and expression of pitx2, a molecular marker of LR patterning. These effects were specific, because 0 of 13 control genes from rare Htx or control copy number variations produced significant LR abnormalities (P = 0.001). These findings identify genes not previously implicated in LR patterning. PMID:21282601

  8. Rare copy number variations in congenital heart disease patients identify unique genes in left-right patterning.

    PubMed

    Fakhro, Khalid A; Choi, Murim; Ware, Stephanie M; Belmont, John W; Towbin, Jeffrey A; Lifton, Richard P; Khokha, Mustafa K; Brueckner, Martina

    2011-02-15

    Dominant human genetic diseases that impair reproductive fitness and have high locus heterogeneity constitute a problem for gene discovery because the usual criterion of finding more mutations in specific genes than expected by chance may require extremely large populations. Heterotaxy (Htx), a congenital heart disease resulting from abnormalities in left-right (LR) body patterning, has features suggesting that many cases fall into this category. In this setting, appropriate model systems may provide a means to support implication of specific genes. By high-resolution genotyping of 262 Htx subjects and 991 controls, we identify a twofold excess of subjects with rare genic copy number variations in Htx (14.5% vs. 7.4%, P = 1.5 × 10(-4)). Although 7 of 45 Htx copy number variations were large chromosomal abnormalities, 38 smaller copy number variations altered a total of 61 genes, 22 of which had Xenopus orthologs. In situ hybridization identified 7 of these 22 genes with expression in the ciliated LR organizer (gastrocoel roof plate), a marked enrichment compared with 40 of 845 previously studied genes (sevenfold enrichment, P < 10(-6)). Morpholino knockdown in Xenopus of Htx candidates demonstrated that five (NEK2, ROCK2, TGFBR2, GALNT11, and NUP188) strongly disrupted both morphological LR development and expression of pitx2, a molecular marker of LR patterning. These effects were specific, because 0 of 13 control genes from rare Htx or control copy number variations produced significant LR abnormalities (P = 0.001). These findings identify genes not previously implicated in LR patterning.

  9. Comparison of cyanobacterial microcystin synthetase (mcy) E gene transcript levels, mcy E gene copies, and biomass as indicators of microcystin risk under laboratory and field conditions.

    PubMed

    Ngwa, Felexce F; Madramootoo, Chandra A; Jabaji, Suha

    2014-08-01

    Increased incidences of mixed assemblages of microcystin-producing and nonproducing cyanobacterial strains in freshwater bodies necessitate development of reliable proxies for cyanotoxin risk assessment. Detection of microcystin biosynthetic genes in water blooms of cyanobacteria is generally indicative of the presence of potentially toxic cyanobacterial strains. Although much effort has been devoted toward elucidating the microcystin biosynthesis mechanisms in many cyanobacteria genera, little is known about the impacts of co-occurring cyanobacteria on cellular growth, mcy gene expression, or mcy gene copy distribution. The present study utilized conventional microscopy, qPCR assays, and enzyme-linked immunosorbent assay to study how competition between microcystin-producing Microcystis aeruginosa CPCC 299 and Planktothrix agardhii NIVA-CYA 126 impacts mcyE gene expression, mcyE gene copies, and microcystin concentration under controlled laboratory conditions. Furthermore, analyses of environmental water samples from the Missisquoi Bay, Quebec, enabled us to determine how the various potential toxigenic cyanobacterial biomass proxies correlated with cellular microcystin concentrations in a freshwater lake. Results from our laboratory study indicated significant downregulation of mcyE gene expression in mixed cultures of M. aeruginosa plus P. agardhii on most sampling days in agreement with depressed growth recorded in the mixed cultures, suggesting that interaction between the two species probably resulted in suppressed growth and mcyE gene expression in the mixed cultures. Furthermore, although mcyE gene copies and McyE transcripts were detected in all laboratory and field samples with measureable microcystin levels, only mcyE gene copies showed significant positive correlations (R(2) > 0.7) with microcystin concentrations, while McyE transcript levels did not. These results suggest that mcyE gene copies are better indicators of potential risks from microcystins

  10. Analysis of Copy Number Variation in the Abp Gene Regions of Two House Mouse Subspecies Suggests Divergence during the Gene Family Expansions

    PubMed Central

    Pezer, Željka; Chung, Amanda G.; Karn, Robert C.

    2017-01-01

    Abstract The Androgen-binding protein (Abp) gene region of the mouse genome contains 64 genes, some encoding pheromones that influence assortative mating between mice from different subspecies. Using CNVnator and quantitative PCR, we explored copy number variation in this gene family in natural populations of Mus musculus domesticus (Mmd) and Mus musculus musculus (Mmm), two subspecies of house mice that form a narrow hybrid zone in Central Europe. We found that copy number variation in the center of the Abp gene region is very common in wild Mmd, primarily representing the presence/absence of the final duplications described for the mouse genome. Clustering of Mmd individuals based on this variation did not reflect their geographical origin, suggesting no population divergence in the Abp gene cluster. However, copy number variation patterns differ substantially between Mmd and other mouse taxa. Large blocks of Abp genes are absent in Mmm, Mus musculus castaneus and an outgroup, Mus spretus, although with differences in variation and breakpoint locations. Our analysis calls into question the reliance on a reference genome for interpreting the detailed organization of genes in taxa more distant from the Mmd reference genome. The polymorphic nature of the gene family expansion in all four taxa suggests that the number of Abp genes, especially in the central gene region, is not critical to the survival and reproduction of the mouse. However, Abp haplotypes of variable length may serve as a source of raw genetic material for new signals influencing reproductive communication and thus speciation of mice. PMID:28575204

  11. Three copies of a single protein II-encoding sequence in the genome of Neisseria gonorrhoeae JS3: evidence for gene conversion and gene duplication.

    PubMed

    van der Ley, P

    1988-11-01

    Gonococci express a family of related outer membrane proteins designated protein II (P.II). These surface proteins are subject to both phase variation and antigenic variation. The P.II gene repertoire of Neisseria gonorrhoeae strain JS3 was found to consist of at least ten genes, eight of which were cloned. Sequence analysis and DNA hybridization studies revealed that one particular P.II-encoding sequence is present in three distinct, but almost identical, copies in the JS3 genome. These genes encode the P.II protein that was previously identified as P.IIc. Comparison of their sequences shows that the multiple copies of this P.IIc-encoding gene might have been generated by both gene conversion and gene duplication.

  12. Copy number variation and microdeletions of the Y chromosome linked genes and loci across different categories of Indian infertile males.

    PubMed

    Kumari, Anju; Yadav, Sandeep Kumar; Misro, Man Mohan; Ahmad, Jamal; Ali, Sher

    2015-12-07

    We analyzed 34 azoospermic (AZ), 43 oligospermic (OS), and 40 infertile males with normal spermiogram (INS) together with 55 normal fertile males (NFM) from the Indian population. AZ showed more microdeletions in the AZFa and AZFb regions whereas oligospermic ones showed more microdeletions in the AZFc region. Frequency of the AZF partial deletions was higher in males with spermatogenic impairments than in INS. Significantly, SRY, DAZ and BPY2 genes showed copy number variation across different categories of the patients and much reduced copies of the DYZ1 repeat arrays compared to that in normal fertile males. Likewise, INS showed microdeletions, sequence and copy number variation of several Y linked genes and loci. In the context of infertility, STS deletions and copy number variations both were statistically significant (p = 0.001). Thus, semen samples used during in vitro fertilization (IVF) and assisted reproductive technology (ART) must be assessed for the microdeletions of AZFa, b and c regions in addition to the affected genes reported herein. Present study is envisaged to be useful for DNA based diagnosis of different categories of the infertile males lending support to genetic counseling to the couples aspiring to avail assisted reproductive technologies.

  13. Use of next-generation sequencing to detect LDLR gene copy number variation in familial hypercholesterolemia.

    PubMed

    Iacocca, Michael A; Wang, Jian; Dron, Jacqueline S; Robinson, John F; McIntyre, Adam D; Cao, Henian; Hegele, Robert A

    2017-11-01

    Familial hypercholesterolemia (FH) is a heritable condition of severely elevated LDL cholesterol, caused predominantly by autosomal codominant mutations in the LDL receptor gene ( LDLR ). In providing a molecular diagnosis for FH, the current procedure often includes targeted next-generation sequencing (NGS) panels for the detection of small-scale DNA variants, followed by multiplex ligation-dependent probe amplification (MLPA) in LDLR for the detection of whole-exon copy number variants (CNVs). The latter is essential because ∼10% of FH cases are attributed to CNVs in LDLR ; accounting for them decreases false negative findings. Here, we determined the potential of replacing MLPA with bioinformatic analysis applied to NGS data, which uses depth-of-coverage analysis as its principal method to identify whole-exon CNV events. In analysis of 388 FH patient samples, there was 100% concordance in LDLR CNV detection between these two methods: 38 reported CNVs identified by MLPA were also successfully detected by our NGS method, while 350 samples negative for CNVs by MLPA were also negative by NGS. This result suggests that MLPA can be removed from the routine diagnostic screening for FH, significantly reducing associated costs, resources, and analysis time, while promoting more widespread assessment of this important class of mutations across diagnostic laboratories. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  14. Human papillomavirus 16 (HPV16) and HPV52 E6-specific immunity in HIV-infected adults on combination antiretroviral therapy.

    PubMed

    Leng, C Y; Low, H C; Chua, L L; Chong, M L; Sulaiman, H; Azwa, I; Roberts, J M; Kamarulzaman, A; Rajasuriar, R; Woo, Y L

    2017-05-01

    Human papillomavirus (HPV)-associated cancers disproportionately affect those infected with HIV despite effective combination antiretroviral therapy (cART). The primary aim of this study was to quantify HPV16 and HPV52 E6-specific interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) T-cell responses, a correlate of protective immunity, in the first year following cART initiation and subsequently in those patients with suboptimal (sIR) and optimal (oIR) immune reconstitution. Ninety-four HIV-infected patients were recruited to the study; a longitudinal cohort of patients recruited just prior to commencing cART and followed up for 48 weeks (n = 27), and a cross-sectional cohort (n = 67) consisting of patients with sIR (CD4 T-cell count < 350 cells/μL) and oIR (CD4 T-cell count > 500 cells/μL) after a minimum of 2 years on cART. Controls (n = 29) consisted of HIV-negative individuals. IFN-γ ELISPOT responses against HPV16 and HPV52 E6 were correlated to clinical characteristics, anal and oral HPV carriage, T-cell maturational subsets, markers of activation, senescence and T-regulatory cells. HPV16 and HPV52 E6-specific T-cell responses were detected in only one of 27 patients (3.7%) during the initial phase of immune recovery. After at least 2 years of cART, those who achieved oIR had significantly higher E6-specific responses (9 of 34; 26.5%) compared with those with sIR (2 of 32; 6.3%) (P = 0.029). Apart from higher CD4 T-cell counts and lower CD4 T-cell activation, no other immunological correlates were associated with the detection of HPV16 and HPV52 E6-specific responses. HPV16 and HPV52 E6-specific IFN-γ T-cell responses, a correlate of protective immunity, were detected more frequently among HIV-infected patients who achieved optimal immune recovery on cART (26.5%) compared with those with suboptimal recovery (6.3%). © 2016 British HIV Association.

  15. [Abnormality of TOP2A expression and its gene copy number variations in neuroblastic tumors].

    PubMed

    Chen, J M; Zhou, C J; Ma, X L; Guan, D D; Yang, L Y; Yue, P; Gong, L P

    2016-11-08

    Objective: To detect TOP2A protein expression and gene copy number alterations, and to analyze related clinical and pathological implications in pediatric neuroblastic tumors (NT). Methods: Immunohistochemistry was used to detect TOP2A protein expression. Fluorescence in situ hybridization (FISH) was used to detect numerical aberrations of TOP2A. Results: TOP2A protein was expressed in 59.1%(52/88) of cases, which was associated with differentiation ( P =0.006), Ki-67 index ( P <0.01) and MKI ( P =0.001). Twenty-eight cases (35.0%, 28/88) showed TOP2A gene amplification, which was correlated with the age ( P <0.01), clinical stage ( P =0.028), high risk group ( P =0.001), Ki-67 index ( P =0.040) and differentiation ( P =0.014). Survival analysis showed that TOP2A expression was related to survival rate. Multivariate analyses showed that TOP2A expression was an independent predictor for poor prognosis ( P =0.010). Conclusions: More than half of the cases show TOP2A expression, which is more likely associated with NB, high Ki-67 index and high MKI. Cases with TOP2A expression have shorter survivals and poorer prognosis. TOP2A amplification is seen in 35% and likely occurs in patients older than 18 months and at advanced INSS stages (Ⅲ and Ⅳ). As a target of the anthracycline-based adjuvant drugs, TOP2A test can be used to select patient with NT for the therapy.

  16. Aluminum tolerance is associated with higher MATE1 gene copy-number in maize

    USDA-ARS?s Scientific Manuscript database

    Genome structure variation, including copy-number (CNV) and presence/absence variation (PAV), comprise a large extent of maize genetic diversity but their effect on phenotypes remains largely unexplored. Here we describe how copy-number variation in a major aluminum (Al) tolerance locus contributes ...

  17. Industrial fuel ethanol yeasts contain adaptive copy number changes in genes involved in vitamin B1 and B6 biosynthesis.

    PubMed

    Stambuk, Boris U; Dunn, Barbara; Alves, Sergio L; Duval, Eduarda H; Sherlock, Gavin

    2009-12-01

    Fuel ethanol is now a global energy commodity that is competitive with gasoline. Using microarray-based comparative genome hybridization (aCGH), we have determined gene copy number variations (CNVs) common to five industrially important fuel ethanol Saccharomyces cerevisiae strains responsible for the production of billions of gallons of fuel ethanol per year from sugarcane. These strains have significant amplifications of the telomeric SNO and SNZ genes, which are involved in the biosynthesis of vitamins B6 (pyridoxine) and B1 (thiamin). We show that increased copy number of these genes confers the ability to grow more efficiently under the repressing effects of thiamin, especially in medium lacking pyridoxine and with high sugar concentrations. These genetic changes have likely been adaptive and selected for in the industrial environment, and may be required for the efficient utilization of biomass-derived sugars from other renewable feedstocks.

  18. Industrial fuel ethanol yeasts contain adaptive copy number changes in genes involved in vitamin B1 and B6 biosynthesis

    PubMed Central

    Stambuk, Boris U.; Dunn, Barbara; Alves, Sergio L.; Duval, Eduarda H.; Sherlock, Gavin

    2009-01-01

    Fuel ethanol is now a global energy commodity that is competitive with gasoline. Using microarray-based comparative genome hybridization (aCGH), we have determined gene copy number variations (CNVs) common to five industrially important fuel ethanol Saccharomyces cerevisiae strains responsible for the production of billions of gallons of fuel ethanol per year from sugarcane. These strains have significant amplifications of the telomeric SNO and SNZ genes, which are involved in the biosynthesis of vitamins B6 (pyridoxine) and B1 (thiamin). We show that increased copy number of these genes confers the ability to grow more efficiently under the repressing effects of thiamin, especially in medium lacking pyridoxine and with high sugar concentrations. These genetic changes have likely been adaptive and selected for in the industrial environment, and may be required for the efficient utilization of biomass-derived sugars from other renewable feedstocks. PMID:19897511

  19. Exploratory factor analysis of pathway copy number data with an application towards the integration with gene expression data.

    PubMed

    van Wieringen, Wessel N; van de Wiel, Mark A

    2011-05-01

    Realizing that genes often operate together, studies into the molecular biology of cancer shift focus from individual genes to pathways. In order to understand the regulatory mechanisms of a pathway, one must study its genes at all molecular levels. To facilitate such study at the genomic level, we developed exploratory factor analysis for the characterization of the variability of a pathway's copy number data. A latent variable model that describes the call probability data of a pathway is introduced and fitted with an EM algorithm. In two breast cancer data sets, it is shown that the first two latent variables of GO nodes, which inherit a clear interpretation from the call probabilities, are often related to the proportion of aberrations and a contrast of the probabilities of a loss and of a gain. Linking the latent variables to the node's gene expression data suggests that they capture the "global" effect of genomic aberrations on these transcript levels. In all, the proposed method provides an possibly insightful characterization of pathway copy number data, which may be fruitfully exploited to study the interaction between the pathway's DNA copy number aberrations and data from other molecular levels like gene expression.

  20. Suppression of HPV-16 late L1 5′-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs

    PubMed Central

    Li, Xiaoze; Johansson, Cecilia; Glahder, Jacob; Mossberg, Ann-Kristin; Schwartz, Stefan

    2013-01-01

    Human papillomavirus type 16 (HPV-16) 5′-splice site SD3632 is used exclusively to produce late L1 mRNAs. We identified a 34-nt splicing inhibitory element located immediately upstream of HPV-16 late 5′-splice site SD3632. Two AUAGUA motifs located in these 34 nt inhibited SD3632. Two nucleotide substitutions in each of the HPV-16 specific AUAGUA motifs alleviated splicing inhibition and induced late L1 mRNA production from episomal forms of the HPV-16 genome in primary human keratinocytes. The AUAGUA motifs bind specifically not only to the heterogeneous nuclear RNP (hnRNP) D family of RNA-binding proteins including hnRNP D/AUF, hnRNP DL and hnRNP AB but also to hnRNP A2/B1. Knock-down of these proteins induced HPV-16 late L1 mRNA expression, and overexpression of hnRNP A2/B1, hnRNP AB, hnRNP DL and the two hnRNP D isoforms hnRNP D37 and hnRNP D40 further suppressed L1 mRNA expression. This inhibition may allow HPV-16 to hide from the immune system and establish long-term persistent infections with enhanced risk at progressing to cancer. There is an inverse correlation between expression of hnRNP D proteins and hnRNP A2/B1 and HPV-16 L1 production in the cervical epithelium, as well as in cervical cancer, supporting the conclusion that hnRNP D proteins and A2/B1 inhibit HPV-16 L1 mRNA production. PMID:24013563

  1. Cost-Effectiveness of Cervical Cancer Screening With Human Papillomavirus DNA Testing and HPV-16,18 Vaccination

    PubMed Central

    Goldhaber-Fiebert, Jeremy D.; Stout, Natasha K.; Salomon, Joshua A.; Kuntz, Karen M.; Goldie, Sue J.

    2011-01-01

    Background The availability of human papillomavirus (HPV) DNA testing and vaccination against HPV types 16 and 18 (HPV-16,18) motivates questions about the cost-effectiveness of cervical cancer prevention in the United States for unvaccinated older women and for girls eligible for vaccination. Methods An empirically calibrated model was used to assess the quality-adjusted life years (QALYs), lifetime costs, and incremental cost-effectiveness ratios (2004 US dollars per QALY) of screening, vaccination of preadolescent girls, and vaccination combined with screening. Screening varied by initiation age (18, 21, or 25 years), interval (every 1, 2, 3, or 5 years), and test (HPV DNA testing of cervical specimens or cytologic evaluation of cervical cells with a Pap test). Testing strategies included: 1) cytology followed by HPV DNA testing for equivocal cytologic results (cytology with HPV test triage); 2) HPV DNA testing followed by cytology for positive HPV DNA results (HPV test with cytology triage); and 3) combined HPV DNA testing and cytology. Strategies were permitted to switch once at age 25, 30, or 35 years. Results For unvaccinated women, triennial cytology with HPV test triage, beginning by age 21 years and switching to HPV testing with cytology triage at age 30 years, cost $78 000 per QALY compared with the next best strategy. For girls vaccinated before age 12 years, this same strategy, beginning at age 25 years and switching at age 35 years, cost $41 000 per QALY with screening every 5 years and $188 000 per QALY screening triennially, each compared with the next best strategy. These strategies were more effective and cost-effective than screening women of all ages with cytology alone or cytology with HPV triage annually or biennially. Conclusions For both vaccinated and unvaccinated women, age-based screening by use of HPV DNA testing as a triage test for equivocal results in younger women and as a primary screening test in older women is expected to be more

  2. Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes.

    PubMed

    Kawakami, Shuji; Hasegawa, Takuya; Imachi, Hiroyuki; Yamaguchi, Takashi; Harada, Hideki; Ohashi, Akiyoshi; Kubota, Kengo

    2012-02-01

    In situ detection of functional genes with single-cell resolution is currently of interest to microbiologists. Here, we developed a two-pass tyramide signal amplification (TSA)-fluorescence in situ hybridization (FISH) protocol with PCR-derived polynucleotide probes for the detection of single-copy genes in prokaryotic cells. The mcrA gene and the apsA gene in methanogens and sulfate-reducing bacteria, respectively, were targeted. The protocol showed bright fluorescence with a good signal-to-noise ratio and achieved a high efficiency of detection (>98%). The discrimination threshold was approximately 82-89% sequence identity. Microorganisms possessing the mcrA or apsA gene in anaerobic sludge samples were successfully detected by two-pass TSA-FISH with polynucleotide probes. The developed protocol is useful for identifying single microbial cells based on functional gene sequences. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. A Meta-Analysis of Multiple Matched Copy Number and Transcriptomics Data Sets for Inferring Gene Regulatory Relationships

    PubMed Central

    Newton, Richard; Wernisch, Lorenz

    2014-01-01

    Inferring gene regulatory relationships from observational data is challenging. Manipulation and intervention is often required to unravel causal relationships unambiguously. However, gene copy number changes, as they frequently occur in cancer cells, might be considered natural manipulation experiments on gene expression. An increasing number of data sets on matched array comparative genomic hybridisation and transcriptomics experiments from a variety of cancer pathologies are becoming publicly available. Here we explore the potential of a meta-analysis of thirty such data sets. The aim of our analysis was to assess the potential of in silico inference of trans-acting gene regulatory relationships from this type of data. We found sufficient correlation signal in the data to infer gene regulatory relationships, with interesting similarities between data sets. A number of genes had highly correlated copy number and expression changes in many of the data sets and we present predicted potential trans-acted regulatory relationships for each of these genes. The study also investigates to what extent heterogeneity between cell types and between pathologies determines the number of statistically significant predictions available from a meta-analysis of experiments. PMID:25148247

  4. A network of epigenetic modifiers and DNA repair genes controls tissue-specific copy number alteration preference.

    PubMed

    Cramer, Dina; Serrano, Luis; Schaefer, Martin H

    2016-11-10

    Copy number alterations (CNAs) in cancer patients show a large variability in their number, length and position, but the sources of this variability are not known. CNA number and length are linked to patient survival, suggesting clinical relevance. We have identified genes that tend to be mutated in samples that have few or many CNAs, which we term CONIM genes (COpy Number Instability Modulators). CONIM proteins cluster into a densely connected subnetwork of physical interactions and many of them are epigenetic modifiers. Therefore, we investigated how the epigenome of the tissue-of-origin influences the position of CNA breakpoints and the properties of the resulting CNAs. We found that the presence of heterochromatin in the tissue-of-origin contributes to the recurrence and length of CNAs in the respective cancer type.

  5. Stratification of clear cell renal cell carcinoma (ccRCC) genomes by gene-directed copy number alteration (CNA) analysis

    PubMed Central

    Thiesen, H.-J.; Steinbeck, F.; Maruschke, M.; Koczan, D.; Ziems, B.; Hakenberg, O. W.

    2017-01-01

    Tumorigenic processes are understood to be driven by epi-/genetic and genomic alterations from single point mutations to chromosomal alterations such as insertions and deletions of nucleotides up to gains and losses of large chromosomal fragments including products of chromosomal rearrangements e.g. fusion genes and proteins. Overall comparisons of copy number alterations (CNAs) presented in 48 clear cell renal cell carcinoma (ccRCC) genomes resulted in ratios of gene losses versus gene gains between 26 ccRCC Fuhrman malignancy grades G1 (ratio 1.25) and 20 G3 (ratio 0.58). Gene losses and gains of 15762 CNA genes were mapped to 795 chromosomal cytoband loci including 280 KEGG pathways. CNAs were classified according to their contribution to Fuhrman tumour gradings G1 and G3. Gene gains and losses turned out to be highly structured processes in ccRCC genomes enabling the subclassification and stratification of ccRCC tumours in a genome-wide manner. CNAs of ccRCC seem to start with common tumour related gene losses flanked by CNAs specifying Fuhrman grade G1 losses and CNA gains favouring grade G3 tumours. The appearance of recurrent CNA signatures implies the presence of causal mechanisms most likely implicated in the pathogenesis and disease-outcome of ccRCC tumours distinguishing lower from higher malignant tumours. The diagnostic quality of initial 201 genes (108 genes supporting G1 and 93 genes G3 phenotypes) has been successfully validated on published Swiss data (GSE19949) leading to a restricted CNA gene set of 171 CNA genes of which 85 genes favour Fuhrman grade G1 and 86 genes Fuhrman grade G3. Regarding these gene sets overall survival decreased with the number of G3 related gene losses plus G3 related gene gains. CNA gene sets presented define an entry to a gene-directed and pathway-related functional understanding of ongoing copy number alterations within and between individual ccRCC tumours leading to CNA genes of prognostic and predictive value. PMID

  6. Stratification of clear cell renal cell carcinoma (ccRCC) genomes by gene-directed copy number alteration (CNA) analysis.

    PubMed

    Thiesen, H-J; Steinbeck, F; Maruschke, M; Koczan, D; Ziems, B; Hakenberg, O W

    2017-01-01

    Tumorigenic processes are understood to be driven by epi-/genetic and genomic alterations from single point mutations to chromosomal alterations such as insertions and deletions of nucleotides up to gains and losses of large chromosomal fragments including products of chromosomal rearrangements e.g. fusion genes and proteins. Overall comparisons of copy number alterations (CNAs) presented in 48 clear cell renal cell carcinoma (ccRCC) genomes resulted in ratios of gene losses versus gene gains between 26 ccRCC Fuhrman malignancy grades G1 (ratio 1.25) and 20 G3 (ratio 0.58). Gene losses and gains of 15762 CNA genes were mapped to 795 chromosomal cytoband loci including 280 KEGG pathways. CNAs were classified according to their contribution to Fuhrman tumour gradings G1 and G3. Gene gains and losses turned out to be highly structured processes in ccRCC genomes enabling the subclassification and stratification of ccRCC tumours in a genome-wide manner. CNAs of ccRCC seem to start with common tumour related gene losses flanked by CNAs specifying Fuhrman grade G1 losses and CNA gains favouring grade G3 tumours. The appearance of recurrent CNA signatures implies the presence of causal mechanisms most likely implicated in the pathogenesis and disease-outcome of ccRCC tumours distinguishing lower from higher malignant tumours. The diagnostic quality of initial 201 genes (108 genes supporting G1 and 93 genes G3 phenotypes) has been successfully validated on published Swiss data (GSE19949) leading to a restricted CNA gene set of 171 CNA genes of which 85 genes favour Fuhrman grade G1 and 86 genes Fuhrman grade G3. Regarding these gene sets overall survival decreased with the number of G3 related gene losses plus G3 related gene gains. CNA gene sets presented define an entry to a gene-directed and pathway-related functional understanding of ongoing copy number alterations within and between individual ccRCC tumours leading to CNA genes of prognostic and predictive value.

  7. Penicillin production in industrial strain Penicillium chrysogenum P2niaD18 is not dependent on the copy number of biosynthesis genes.

    PubMed

    Ziemons, Sandra; Koutsantas, Katerina; Becker, Kordula; Dahlmann, Tim; Kück, Ulrich

    2017-02-16

    Multi-copy gene integration into microbial genomes is a conventional tool for obtaining improved gene expression. For Penicillium chrysogenum, the fungal producer of the beta-lactam antibiotic penicillin, many production strains carry multiple copies of the penicillin biosynthesis gene cluster. This discovery led to the generally accepted view that high penicillin titers are the result of multiple copies of penicillin genes. Here we investigated strain P2niaD18, a production line that carries only two copies of the penicillin gene cluster. We performed pulsed-field gel electrophoresis (PFGE), quantitative qRT-PCR, and penicillin bioassays to investigate production, deletion and overexpression strains generated in the P. chrysogenum P2niaD18 background, in order to determine the copy number of the penicillin biosynthesis gene cluster, and study the expression of one penicillin biosynthesis gene, and the penicillin titer. Analysis of production and recombinant strain showed that the enhanced penicillin titer did not depend on the copy number of the penicillin gene cluster. Our assumption was strengthened by results with a penicillin null strain lacking pcbC encoding isopenicillin N synthase. Reintroduction of one or two copies of the cluster into the pcbC deletion strain restored transcriptional high expression of the pcbC gene, but recombinant strains showed no significantly different penicillin titer compared to parental strains. Here we present a molecular genetic analysis of production and recombinant strains in the P2niaD18 background carrying different copy numbers of the penicillin biosynthesis gene cluster. Our analysis shows that the enhanced penicillin titer does not strictly depend on the copy number of the cluster. Based on these overall findings, we hypothesize that instead, complex regulatory mechanisms are prominently implicated in increased penicillin biosynthesis in production strains.

  8. MET gene copy number gain is an independent poor prognostic marker in Korean stage I lung adenocarcinomas.

    PubMed

    Jin, Yan; Sun, Ping-Li; Kim, Hyojin; Seo, An Na; Jheon, Sanghoon; Lee, Choon-Taek; Chung, Jin-Haeng

    2014-02-01

    MET gene copy number gain (CNG) and protein overexpression have been reported in lung cancer, but the clinical implications in early stage adenocarcinoma remain unclear. We investigated MET gene copy number and protein expression in 141 cases of surgically resected stage I pulmonary adenocarcinoma. MET gene CNG was determined by silver in situ hybridization, and MET protein expression was assessed by immunohistochemistry. The correlation between MET gene CNG/protein expression and clinicopathologic parameters and prognostic significance was analyzed. MET gene CNG was found in 24.1% (34 of 141) of the cases and was associated with larger tumor size, pleural invasion, and lymphatic vessel invasion. MET gene CNG was inversely correlated with the presence of lepidic subtype (r = -0.17, p = 0.045) and was not associated with EGFR, KRAS mutation, or ALK gene rearrangement. In addition, MET gene CNG was significantly associated with shorter disease-free survival (DFS) (49 vs. 75 months; p < 0.001) and shorter overall survival (OS) (65 vs. 78 months; p = 0.01). Multivariate analysis confirmed that MET gene CNG was significantly associated with poorer DFS [p < 0.001; hazard ratio (HR) 5.5; 95% confidence interval (CI) 2.2-13.9] but was not significantly associated with OS. MET overexpression was observed in 71.3% of cases (97 of 136), but it was not correlated with gene CNG. MET gene CNG is an independent poor prognostic factor in patients with stage I lung adenocarcinoma. It is associated with aggressive pathologic features and is inversely correlated with the presence of lepidic subtype.

  9. Clinical significance of ESR1 gene copy number changes in breast cancer as measured by fluorescence in situ hybridisation.

    PubMed

    Lin, Ching-Hung; Liu, Jacqueline M; Lu, Yen-Shen; Lan, Chieh; Lee, Wei-Chung; Kuo, Kuan-Ting; Wang, Chung-Chieh; Chang, Dwan-Ying; Huang, Chiun-Sheng; Cheng, Ann-Lii

    2013-02-01

    The ESR1 gene encodes for oestrogen receptor (ER) α, which plays a crucial role in mammary carcinogenesis and clinical outcome in patients with breast cancer. However, the clinical significance of the ESR1 gene copy number change for breast cancer has not been clarified. ESR1 gene copy number was determined by fluorescence in situ hybridisation (FISH) on tissue sections. A minimum of 20 tumour cells were counted per section, and a FISH ratio of ESR1 gene to CEP6 ≥ 2.0 was considered ESR1 amplification. A ratio >1.2 but <2.0 was considered ESR1 gain. The ESR1 copy number was further measured by quantitative real-time PCR (Q-PCR) with ASXL2 as a reference. FISH revealed ESR1 amplification in six cases (4.0%) and ESR1 gain in 13 cases (8.7%) from a total of 150 cases. ESR1 gain and amplification were more common in older patients (p<0.001), and correlated well with ER protein expression (p=0.03) measured by immunohistochemistry, and ESR1 copy number (p<0.001) measured by Q-PCR. Furthermore, the multivariate analysis revealed that ESR1 amplification was associated with a shorter disease-free survival (HR=5.56, p=0.03) and a shorter overall survival (HR=5.11, p=0.04). In general, the frequency of ESR1 amplification in breast cancer is low when measured by FISH in large sections. ESR1 gain and amplification in breast cancer may be associated with older age and poorer outcomes.

  10. Increased TERC gene copy number and cells in senescence in primary sclerosing cholangitis compared to colitis and control patients.

    PubMed

    Laish, Ido; Katz, Hila; Sulayev, Yael; Liberman, Meytal; Naftali, Timna; Benjaminov, Fabiana; Stein, Assaf; Kitay-Cohen, Yona; Biron-Shental, Tal; Konikoff, Fred; Amiel, Aliza

    2013-10-25

    Primary sclerosing cholangitis (PSC) is a chronic cholestatic disorder that involves inflammatory and fibrotic changes in the bile ducts. Up to 80% of patients have concomitant inflammatory bowel disease (IBD) with colitis. PSC patients are predisposed to develop hepatobiliary, colonic and other extrahepatic malignancies, probably related to inflammatory processes that might promote carcinogenesis. Telomerase is an enzyme complex that lengthens telomeres and has enhanced expression in numerous malignancies. In this study, we evaluated the TERC gene copy number, the proportion of cells in senescence and the amount of fragmentation in the senescent state. Fluorescence in situ hybridization (FISH) for the TERC gene was applied to lymphocytes retrieved from PSC (N=19), colitis (N=20) and healthy control patients (N=20) to determine the TERC copy number. On the same FISH slides, cells stained with DAPI were also analyzed for senescence-associated heterochromatin foci (SAHF) status, including the number of cells with fragments and the number of SAHF fragments in each cell. A higher TERC gene copy number was observed in cells from PSC patients compared to colitis and control group patients. It was also higher in the colitis than in the control group. Significantly more cells in the senescent state and more fragmentation in each cell were observed in the PSC group compared to colitis and control groups. The TERC gene copy number and the number of cells in the senescent state were increased in PSC patients compared to the colitis and control groups. These findings are probably related to the genetic instability parameters that reflect the higher tendency of this patient group to develop malignancies. © 2013.

  11. Detection of the E7 transform gene of human papilloma virus type 16 in human oral squamous cell carcinoma.

    PubMed

    Wang, J; Li, J; Huang, H; Fu, Y

    1998-12-01

    To determine, with the use of polymerase chain reaction, the prevalence of human papillomavirus (HPV) 16 in 30 patients with primary oral squamous cell carcinoma (OSCC) and 30 healthy control patients. DNA was extracted from freshly frozen tumor tissues of 30 patients with primary oral squamous cell carcinoma and from the oral mucosa of 30 controls. A pair of specific primers of the E7 early gene of HPV 16 were designed. PCR products were run by 1.5% agarose gel and the results of electrophoresis were photographed. HPV 16 was detected in 36.7% (11/30) of oral squamous cell carcinoma patients and 11.1% (4/30) of controls. HPV 16 has a significant association with oral squamous cell carcinoma. However, the role HPV 16 plays in the tumorigenesis of oral cancer and its clinical significance remain to be investigated.

  12. Copy number variation of lipocalin family genes for male-specific proteins in tilapia and its association with gender.

    PubMed

    Shirak, A; Golik, M; Lee, B-Y; Howe, A E; Kocher, T D; Hulata, G; Ron, M; Seroussi, E

    2008-11-01

    Lipocalins are involved in the binding of small molecules like sex steroids. We show here that the previously reported tilapia male-specific protein (MSP) is a lipocalin encoded by a variety of paralogous and homologous genes in different tilapia species. Exon-intron boundaries of MSP genes were typical of the six-exon genomic structure of lipocalins, and the transcripts were capable of encoding 200 amino-acid polypeptides that consisted of a putative signal peptide and a lipocalin domain. Cysteine residues are conserved in positions analogous to those forming the three disulfide bonds characteristic of the ligand pocket. The calculated molecular mass of the secreted MSP (20.4 kDa) was less than half of that observed, suggesting that it is highly glycosylated like its homologue tributyltin-binding protein. Analysis of sequence variations revealed three types of paralogs MSPA, MSPB and MSPC. Expression of both MSPA and MSPB was detected in testis. In haploid Oreochromis niloticus embryos, each of these types consisted of two closely related paralogs, and asymmetry between MSP copy numbers on the maternal (six copies) and the paternal (three copies) chromosomes was observed. Using this polymorphism we mapped MSPA and MSPC to linkage group 12 of an F(2) mapping family derived from a cross between O. niloticus and Oreochromis aureus. Females with high MSP copy number were more frequent by more than twofold than males. Gender-MSPC combinations showed significant deviation from expected Mendelian segregation (P=0.009) suggesting elimination of males with MSPC copies. We discuss different hypotheses to explain this elimination, including possibility for allelic conflict resulted by the hybridization.

  13. Topoisomerase-1 and -2A gene copy numbers are elevated in mismatch repair-proficient colorectal cancers.

    PubMed

    Sønderstrup, Ida Marie Heeholm; Nygård, Sune Boris; Poulsen, Tim Svenstrup; Linnemann, Dorte; Stenvang, Jan; Nielsen, Hans Jørgen; Bartek, Jiri; Brünner, Nils; Nørgaard, Peter; Riis, Lene

    2015-06-01

    Topoisomerase 1 (TOP1) and 2A (TOP2A) are potential predictive biomarkers for irinotecan and anthracycline treatment, respectively, in colorectal cancer (CRC), and we have recently reported a high frequency of gene gain of the TOP1 and TOP2A genes in CRC. Furthermore, Mismatch Repair (MMR) subtypes of CRC have been associated with benefit from adjuvant chemotherapy of primary CRC. Given the involvement of the topoisomerase enzymes in DNA replication and repair, we raised the hypothesis that an association may exist between TOP gene copy numbers and MMR proficiency/deficiency in CRC. Test cohort: FISH analysis with an in-house TOP1/CEN20 probe mix and a commercially available TOP2A/CEN17 (Dako, Glostrup, Denmark) probe mix was performed on archival formalin fixed paraffin embedded (FFPE) tissue samples from 18 patients with proficient MMR (pMMR) CRC and 18 patients with deficient MMR (dMMR) CRC. TOP1 and TOP2A gene copy numbers and their ratios per nucleus were correlated with MMR status using the Mann-Whitney test. Validation cohort: FFPE samples from 154 patients with primary stage III CRC (originally included in the RANX05 study) were classified according to MMR status by immunohistochemical analysis using validated antibodies for MLH1, MLH2, MSH6 and PMS2, and information on TOP1, CEN20, TOP2A and CEN17 status was previously published for this cohort. The observed TOP1 gene copy numbers in the 36 CRC test cohort were significantly greater (p < 0.01) in the pMMR subgroup (mean: 3.84, SD: 2.03) than in the dMMR subgroup (mean: 1.50, SD: 0.12). Similarly, the TOP2A copy numbers were significantly greater (p < 0.01) in the pMMR subgroup (mean: 1.99, SD: 0.52) than in the dMMR subgroup (mean: 1.52, SD: 0.10). These findings were confirmed in the validation cohort, where in the pMMR subgroup 51% had ≥2 extra TOP1 copies per cell, while all tumors classified as dMMR had diploid TOP1 status and mean TOP2A copy numbers were 2.30 (SD: 1.36) and 1.80 (SD: 0.31) (p = 0

  14. Construction of a novel gene bank of Bacillus subtilis using a low copy number vector in Escherichia coli.

    PubMed

    Hasnain, S; Thomas, C M

    1986-07-01

    Low copy number vector plasmid pCT571 was constructed to clone Bacillus subtilis genomic fragments in Escherichia coli. pCT571 confers KmR, TcR and CmR in E. coli and CmR in B. subtilis. It has unique restriction sites within the KmR and TcR markers to allow screening for recombinant plasmids by insertional inactivation of these genes. It contains the pSC101 replicon and replicates normally at six to eight copies per chromosome equivalent in E. coli. It also contains oriVRK2, which when supplied with the product of the trfA gene of RK2 in trans, allows pCT571 to replicate at 35-40 copies per chromosome equivalent. A B. subtilis gene bank was created by cloning partially Sau3A-digested and size-fractionated fragments of B. subtilis chromosomal DNA into the BamHI site of pCT571. DNA from 1097 KmR TcS transformants was extracted and analysed electrophoretically as supercoiled DNA and after digesting with EcoRI or EcoRI and SalI. Approximately 1000 hybrid plasmids were found with reasonably sized B. subtilis fragments. The mean size of the inserts in pCT571 is 8 kb, ranging from 4 to 20 kb in different plasmids. The gene bank covers most of the B. subtilis chromosome, as demonstrated by the results of screening the gene bank for selectable nutritional markers in E. coli and B. subtilis. Hybrid plasmids which complement E. coli mutants for arg, his, lys, met, pdx, pyr and thr markers were identified from the gene bank. In B. subtilis the presence of argC, cysA, dal, hisA, ilvA, leuA, lys, metB, metC, phe, purA, purB, thr and trpC was established by transformation experiments. The effects of copy number on cloning and long-term maintenance in the bacterial strains were also investigated. At high copy number some hybrid plasmids cannot be maintained at all, while others show an increased rate of structural deletions and rearrangements.

  15. Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression

    PubMed Central

    Ajiro, Masahiko; Jia, Rong; Zhang, Lifang; Liu, Xuefeng; Zheng, Zhi-Ming

    2012-01-01

    HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5′ splice sites (5′ ss) and three 3′ splice sites (3′ ss) normally used in HPV16+ cervical cancer and its derived cell lines. The choice of two novel alternative 5′ ss (nt 221 5′ ss and nt 191 5′ ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5′ ss and nt 409 3′ ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3′ ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3′ ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of 91QYNK94 to 91PSFW94 displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression. PMID:23056301

  16. Cervarix, the GSK HPV-16/HPV-18 AS04-adjuvanted cervical cancer vaccine, demonstrates stability upon long-term storage and under simulated cold chain break conditions.

    PubMed

    Le Tallec, David; Doucet, Diane; Elouahabi, Abdelatif; Harvengt, Pol; Deschuyteneer, Michel; Deschamps, Marguerite

    2009-07-01

    Cervarix is a recombinant human papillomavirus (HPV)-16 and -18 L1 virus-like-particle (VLP) AS04-adjuvanted vaccine designed to protect against cervical intraepithelial neoplasia and cervical cancer caused by the HPV types 16 and 18. Assessment of the stability of the vaccine during long-term storage and after transient exposure to temperatures out of normal storage range is an integrated part of vaccine quality evaluation. This assessment was done with vaccine samples stored at 2-8 degrees C for up to 36 months, with or without simulated cold chain break (either one week at 37 degrees C, or two or four weeks at 25 degrees C). Among the stability-indicating parameters, antigenicity and immunogenicity were evaluated along with L1 antigen integrity and adsorption to aluminum. Differential scanning calorimetry (DSC) was used to investigate the structural stability of the VLPs before and after vaccine formulation and over time. Cervarix was stable at 2-8 degrees C for at least three years, and the occurrence of cold chain break had no impact, as shown by unchanged product characteristics during the full storage period. DSC analysis demonstrated that the structure of the HPV-16 and -18 L1 proteins and their corresponding VLPs was not affected throughout the manufacturing process. Moreover, the structure of aluminum-adsorbed HPV-16 and -18 L1 VLPs was robust over a 14-month test period. In conclusion, Cervarix was very stable upon long-term storage at 2-8 degrees C with or without transient exposure to higher temperatures (up to 37 degrees C). The observed robust structure of the L1 VLPs contributes to the excellent stability of Cervarix.

  17. The expression of miR-21 and miR-143 is deregulated by the HPV16 E7 oncoprotein and 17β-estradiol.

    PubMed

    Gómez-Gómez, Yazmín; Organista-Nava, Jorge; Ocadiz-Delgado, Rodolfo; García-Villa, Enrique; Leyva-Vazquez, Marco Antonio; Illades-Aguiar, Berenice; Lambert, Paul F; García-Carrancá, Alejandro; Gariglio, Patricio

    2016-08-01

    MicroRNAs (miRNAs) are a class of non-coding RNAs that negatively regulate their target mRNAs at a posttranscriptional level, thereby affecting crucial processes in cancer development. However, little is known about the molecular events that control expression of miRNAs in cervical cancer (CC). HPV16 E7 oncoprotein in conjunction with estrogen are sufficient to produce high grade cervical dysplasia and invasive cervical malignancies in a mouse model. In the present study, we determined the potential role that the E7 oncoprotein and 17β-estradiol (E2) play in the deregulation of miR-21 and miR-143 expression levels by these two risk factors. We found that, while the expression of miR-21 was upregulated and the expression of miR-143 was downregulated by the HPV16 E7 oncoprotein in vivo, and in vitro and that E2 treatment is also implicated in the deregulation of these important miRNAs in vivo. Sustained upregulation of miR-21 resulted in suppression of PTEN expression, and repression of miR-143 increased the mRNA and protein levels from Bcl-2. These results suggested that HPV type 16 E7 oncoprotein and E2 play an important role in regulating miR-21 and miR-143 expression. We have observed similar results in CC patients containing HPV16 sequences, suggesting that these miRNAs could serve as diagnostic biomarkers in CC. The present study highlights the roles of miRNAs in cervical tissue and implicates these important molecules in cervical carcinogenesis.

  18. An evaluation of clinical performance of FTA cards for HPV 16/18 detection using cobas 4800 HPV Test compared to dry swab and liquid medium.

    PubMed

    Dong, Li; Lin, Chunqing; Li, Li; Wang, Margaret; Cui, Jianfeng; Feng, Ruimei; Liu, Bin; Wu, Zeni; Lian, Jia; Liao, Guangdong; Chen, Wen; Qiao, Youlin

    2017-09-01

    Effective dry storage and transport media as an alternative to conventional liquid-based medium would facilitate the accessibility of women in the low-resource settings to human papillomavirus (HPV)- based cervical cancer screening. To evaluate analytical and clinical performance of indicating FTA™ Elute Cartridge (FTA card) for the detection of HPV16/18 and cervical precancerous lesions and cancer compared to dry swab and liquid medium. Ninety patients with abnormal cytology and/or HPV infection were included for analysis. Three specimens of cervical exfoliated cells from each woman were randomly collected by FTA card, dry swab or liquid-based medium prior to colposcopy examination. The subsequent HPV DNA tests were performed on cobas 4800 HPV platform. High-risk HPV (hrHPV) positivity rate was 63.3%, 62.2% and 65.6% for samples collected by FTA card, dry swab and liquid medium, respectively. The overall agreements and kappa values for the detection of hrHPV, HPV 16 and HPV 18 between FTA card and liquid-based medium were 88.9% (κ=0.76), 97.8% (κ=0.94) and 100% (κ=1.0),respectively; between FTA card and dry swab were 92.1% (κ=0.83), 94.5% (κ=0.87) and 100% (κ=1.0), respectively. The performances of hrHPV tested by FTA card, dry swab, and liquid-based medium for detecting CIN2+ were comparable in terms of the sensitivity and specificity. The specificity of detection of CIN2+ by HPV16/18 increased by approximately 40% compared to hrHPV for any medium albeit at cost of a moderate loss of sensitivity. Dry medium might offer an alternative to conventional liquid-based medium in the HPV-based cervical cancer screening program especially in low-resource settings but still needs further evaluation. Copyright © 2017. Published by Elsevier B.V.

  19. Clinical Significance of an HPV DNA Chip Test with Emphasis on HPV-16 and/or HPV-18 Detection in Korean Gynecological Patients.

    PubMed

    Yeo, Min-Kyung; Lee, Ahwon; Hur, Soo Young; Park, Jong Sup

    2016-07-01

    Human papillomavirus (HPV) is a major risk factor for cervical cancer. We evaluated the clinical significance of the HPV DNA chip genotyping assay (MyHPV chip, Mygene Co.) compared with the Hybrid Capture 2 (HC2) chemiluminescent nucleic acid hybridization kit (Digene Corp.) in 867 patients. The concordance rate between the MyHPV chip and HC2 was 79.4% (kappa coefficient, κ = 0.55). The sensitivity and specificity of both HPV tests were very similar (approximately 85% and 50%, respectively). The addition of HPV result (either MyHPV chip or HC2) to cytology improved the sensitivity (95%, each) but reduced the specificity (approximately 30%, each) compared with the HPV test or cytology alone. Based on the MyHPV chip results, the odds ratio (OR) for ≥ high-grade squamous intraepithelial lesions (HSILs) was 9.9 in the HPV-16/18 (+) group and 3.7 in the non-16/18 high-risk (HR)-HPV (+) group. Based on the HC2 results, the OR for ≥ HSILs was 5.9 in the HR-HPV (+) group. When considering only patients with cytological diagnoses of "negative for intraepithelial lesion or malignancy" and "atypical squamous cell or atypical glandular cell," based on the MyHPV chip results, the ORs for ≥ HSILs were 6.8 and 11.7, respectively, in the HPV-16/18 (+) group. The sensitivity and specificity of the MyHPV chip test are similar to the HC2. Detecting HPV-16/18 with an HPV DNA chip test, which is commonly used in many Asian countries, is useful in assessing the risk of high-grade cervical lesions.

  20. Expression profile of microRNA-146a along HPV-induced multistep carcinogenesis: a study in HPV16 transgenic mice.

    PubMed

    Araújo, Rita; Santos, Joana M O; Fernandes, Mara; Dias, Francisca; Sousa, Hugo; Ribeiro, Joana; Bastos, Margarida M S M; Oliveira, Paula A; Carmo, Diogo; Casaca, Fátima; Silva, Sandra; Medeiros, Rui; Gil da Costa, Rui M

    2018-02-01

    Persistent human papillomavirus (HPV) infection is associated with the development of certain types of cancer and the dysregulation of microRNAs has been implicated in HPV-associated carcinogenesis. This is the case of microRNA-146a (miR-146a), which is thought to regulate tumor-associated inflammation. We sought to investigate the expression levels of miR-146a during HPV16-mediated carcinogenesis using skin samples from K14-HPV16 transgenic mice which develop the consecutive phases of the carcinogenesis process. Female transgenic (HPV +/- ) and wild-type (HPV -/- ) mice were sacrificed at 24-26 weeks-old or 28-30 weeks-old. Chest and ear skin samples from HPV +/- and HPV -/- mice were histologically classified and used for microRNA extraction and quantification by qPCR. Chest skin samples from 24 to 26 weeks-old HPV +/- mice presented diffuse epidermal hyperplasia and only 22.5% showed multifocal dysplasia, while at 28-30 weeks-old all (100.0%) HPV +/- animals showed epidermal dysplasia. All HPV +/- ear skin samples showed carcinoma in situ (CIS). MiR-146a expression levels were higher in HPV +/- compared to HPV -/- mice (p = 0.006). There was also an increase in miR-146a expression in dysplastic skin lesions compared with hyperplasic lesions (p = 0.011). Samples showing CIS had a significant decrease in miR-146a expression when compared to samples showing epidermal hyperplasia (p = 0.018) and epidermal dysplasia (p = 0.009). These results suggest that HPV16 induces the overexpression of miR-146a in the initial stages of carcinogenesis (hyperplasia and dysplasia), whereas decreases its expression at later stages (CIS). Taken together, these data implicate and suggest different roles of miR-146a in HPV-mediated carcinogenesis.

  1. Differences in AMY1 Gene Copy Numbers Derived from Blood, Buccal Cells and Saliva Using Quantitative and Droplet Digital PCR Methods: Flagging the Pitfall.

    PubMed

    Ooi, Delicia Shu Qin; Tan, Verena Ming Hui; Ong, Siong Gim; Chan, Yiong Huak; Heng, Chew Kiat; Lee, Yung Seng

    2017-01-01

    The human salivary (AMY1) gene, encoding salivary α-amylase, has variable copy number variants (CNVs) in the human genome. We aimed to determine if real-time quantitative polymerase chain reaction (qPCR) and the more recently available Droplet Digital PCR (ddPCR) can provide a precise quantification of the AMY1 gene copy number in blood, buccal cells and saliva samples derived from the same individual. Seven participants were recruited and DNA was extracted from the blood, buccal cells and saliva samples provided by each participant. Taqman assay real-time qPCR and ddPCR were conducted to quantify AMY1 gene copy numbers. Statistical analysis was carried out to determine the difference in AMY1 gene copy number between the different biological specimens and different assay methods. We found significant within-individual difference (p<0.01) in AMY1 gene copy number between different biological samples as determined by qPCR. However, there was no significant within-individual difference in AMY1 gene copy number between different biological samples as determined by ddPCR. We also found that AMY1 gene copy number of blood samples were comparable between qPCR and ddPCR, while there is a significant difference (p<0.01) between AMY1 gene copy numbers measured by qPCR and ddPCR for both buccal swab and saliva samples. Despite buccal cells and saliva samples being possible sources of DNA, it is pertinent that ddPCR or a single biological sample, preferably blood sample, be used for determining highly polymorphic gene copy numbers like AMY1, due to the large within-individual variability between different biological samples if real time qPCR is employed.

  2. Primary screening for cervical cancer based on high-risk human papillomavirus (HPV) detection and HPV 16 and HPV 18 genotyping, in comparison to cytology.

    PubMed

    Agorastos, Theodoros; Chatzistamatiou, Kimon; Katsamagkas, Taxiarchis; Koliopoulos, George; Daponte, Alexandros; Constantinidis, Theocharis; Constantinidis, Theodoros C

    2015-01-01

    The objective of the present study is to assess the performance of a high-risk human papillomavirus (HR-HPV) DNA test with individual HPV-16/HPV-18 genotyping as a method for primary cervical cancer screening compared with liquid-based cytology (LBC) in a population of Greek women taking part in routine cervical cancer screening. The study, conducted by the "HEllenic Real life Multicentric cErvical Screening" (HERMES) study group, involved the recruitment of 4,009 women, aged 25-55, who took part in routine cervical screening at nine Gynecology Departments in Greece. At first visit cervical specimens were collected for LBC and HPV testing using the Roche Cobas 4800 system. Women found positive for either cytology or HPV were referred for colposcopy, whereas women negative for both tests will be retested after three years. The study is ongoing and the results of the first screening round are reported herein. Valid results for cytology and HPV testing were obtained for 3,993 women. The overall prevalence of HR-HPV was 12.7%, of HPV-16 2.7% and of HPV-18 1.4%. Of those referred for colposcopy, cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was detected in 41 women (1.07%). At the threshold of CIN2+, cytology [atypical squamous cells of undetermined significance (ASC-US) or worse] and HPV testing showed a sensitivity of 53.7% and 100% respectively, without change between age groups. Cytology and HPV testing showed specificity of 96.8% and 90.3% respectively, which was increased in older women (≥30) in comparison to younger ones (25-29). Genotyping for HPV16/18 had similar accuracy to cytology for the detection of CIN2+ (sensitivity: 58.5%; specificity 97.5%) as well as for triage to colposcopy (sensitivity: 58.5% vs 53.7% for cytology). HPV testing has much better sensitivity than cytology to identify high-grade cervical lesions with slightly lower specificity. HPV testing with individual HPV-16/HPV-18 genotyping could represent a more accurate

  3. Primary Screening for Cervical Cancer Based on High-Risk Human Papillomavirus (HPV) Detection and HPV 16 and HPV 18 Genotyping, in Comparison to Cytology

    PubMed Central

    Constantinidis, Theocharis; Constantinidis, Theodoros C.

    2015-01-01

    Objectives The objective of the present study is to assess the performance of a high-risk human papillomavirus (HR-HPV) DNA test with individual HPV-16/HPV-18 genotyping as a method for primary cervical cancer screening compared with liquid-based cytology (LBC) in a population of Greek women taking part in routine cervical cancer screening. Methods The study, conducted by the “HEllenic Real life Multicentric cErvical Screening” (HERMES) study group, involved the recruitment of 4,009 women, aged 25–55, who took part in routine cervical screening at nine Gynecology Departments in Greece. At first visit cervical specimens were collected for LBC and HPV testing using the Roche Cobas 4800 system. Women found positive for either cytology or HPV were referred for colposcopy, whereas women negative for both tests will be retested after three years. The study is ongoing and the results of the first screening round are reported herein. Results Valid results for cytology and HPV testing were obtained for 3,993 women. The overall prevalence of HR-HPV was 12.7%, of HPV-16 2.7% and of HPV-18 1.4%. Of those referred for colposcopy, cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was detected in 41 women (1.07%). At the threshold of CIN2+, cytology [atypical squamous cells of undetermined significance (ASC-US) or worse] and HPV testing showed a sensitivity of 53.7% and 100% respectively, without change between age groups. Cytology and HPV testing showed specificity of 96.8% and 90.3% respectively, which was increased in older women (≥30) in comparison to younger ones (25–29). Genotyping for HPV16/18 had similar accuracy to cytology for the detection of CIN2+ (sensitivity: 58.5%; specificity 97.5%) as well as for triage to colposcopy (sensitivity: 58.5% vs 53.7% for cytology). Conclusion HPV testing has much better sensitivity than cytology to identify high-grade cervical lesions with slightly lower specificity. HPV testing with individual HPV-16/HPV-18

  4. Phylogeny reconstruction in the Caesalpinieae grade (Leguminosae) based on duplicated copies of the sucrose synthase gene and plastid markers.

    PubMed

    Manzanilla, Vincent; Bruneau, Anne

    2012-10-01

    The Caesalpinieae grade (Leguminosae) forms a morphologically and ecologically diverse group of mostly tropical tree species with a complex evolutionary history. This grade comprises several distinct lineages, but the exact delimitation of the group relative to subfamily Mimosoideae and other members of subfamily Caesalpinioideae, as well as phylogenetic relationships among the lineages are uncertain. With the aim of better resolving phylogenetic relationships within the Caesalpinieae grade, we investigated the utility of several nuclear markers developed from genomic studies in the Papilionoideae. We cloned and sequenced the low copy nuclear gene sucrose synthase (SUSY) and combined the data with plastid trnL and matK sequences. SUSY has two paralogs in the Caesalpinieae grade and in the Mimosoideae, but occurs as a single copy in all other legumes tested. Bayesian and maximum likelihood phylogenetic analyses suggest the two nuclear markers are congruent with plastid DNA data. The Caesalpinieae grade is divided into four well-supported clades (Cassia, Caesalpinia, Tachigali and Peltophorum clades), a poorly supported clade of Dimorphandra Group genera, and two paraphyletic groups, one with other Dimorphandra Group genera and the other comprising genera previously recognized as the Umtiza clade. A selection analysis of the paralogs, using selection models from PAML, suggests that SUSY genes are subjected to a purifying selection. One of the SUSY paralogs, under slightly stronger positive selection, may be undergoing subfunctionalization. The low copy SUSY gene is useful for phylogeny reconstruction in the Caesalpinieae despite the presence of duplicate copies. This study confirms that the Caesalpinieae grade is an artificial group, and highlights the need for further analyses of lineages at the base of the Mimosoideae. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Analysis of Copy Number Variation in the Abp Gene Regions of Two House Mouse Subspecies Suggests Divergence during the Gene Family Expansions.

    PubMed

    Pezer, Željka; Chung, Amanda G; Karn, Robert C; Laukaitis, Christina M

    2017-06-01

    The Androgen-binding protein ( Abp ) gene region of the mouse genome contains 64 genes, some encoding pheromones that influence assortative mating between mice from different subspecies. Using CNVnator and quantitative PCR, we explored copy number variation in this gene family in natural populations of Mus musculus domesticus ( Mmd ) and Mus musculus musculus ( Mmm ), two subspecies of house mice that form a narrow hybrid zone in Central Europe. We found that copy number variation in the center of the Abp gene region is very common in wild Mmd , primarily representing the presence/absence of the final duplications described for the mouse genome. Clustering of Mmd individuals based on this variation did not reflect their geographical origin, suggesting no population divergence in the Abp gene cluster. However, copy number variation patterns differ substantially between Mmd and other mouse taxa. Large blocks of Abp genes are absent in Mmm , Mus musculus castaneus and an outgroup, Mus spretus , although with differences in variation and breakpoint locations. Our analysis calls into question the reliance on a reference genome for interpreting the detailed organization of genes in taxa more distant from the Mmd reference genome. The polymorphic nature of the gene family expansion in all four taxa suggests that the number of Abp genes, especially in the central gene region, is not critical to the survival and reproduction of the mouse. However, Abp haplotypes of variable length may serve as a source of raw genetic material for new signals influencing reproductive communication and thus speciation of mice. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  6. Variable Copy Number, Intra-Genomic Heterogeneities and Lateral Transfers of the 16S rRNA Gene in Pseudomonas

    PubMed Central

    Bodilis, Josselin; Nsigue-Meilo, Sandrine; Besaury, Ludovic; Quillet, Laurent

    2012-01-01

    Even though the 16S rRNA gene is the most commonly used taxonomic marker in microbial ecology, its poor resolution is still not fully understood at the intra-genus level. In this work, the number of rRNA gene operons, intra-genomic heterogeneities and lateral transfers were investigated at a fine-scale resolution, throughout the Pseudomonas genus. In addition to nineteen sequenced Pseudomonas strains, we determined the 16S rRNA copy number in four other Pseudomonas strains by Southern hybridization and Pulsed-Field Gel Electrophoresis, and studied the intra-genomic heterogeneities by Denaturing Gradient Gel Electrophoresis and sequencing. Although the variable copy number (from four to seven) seems to be correlated with the evolutionary distance, some close strains in the P. fluorescens lineage showed a different number of 16S rRNA genes, whereas all the strains in the P. aeruginosa lineage displayed the same number of genes (four copies). Further study of the intra-genomic heterogeneities revealed that most of the Pseudomonas strains (15 out of 19 strains) had at least two different 16S rRNA alleles. A great difference (5 or 19 nucleotides, essentially grouped near the V1 hypervariable region) was observed only in two sequenced strains. In one of our strains studied (MFY30 strain), we found a difference of 12 nucleotides (grouped in the V3 hypervariable region) between copies of the 16S rRNA gene. Finally, occurrence of partial lateral transfers of the 16S rRNA gene was further investigated in 1803 full-length sequences of Pseudomonas available in the databases. Remarkably, we found that the two most variable regions (the V1 and V3 hypervariable regions) had probably been laterally transferred from another evolutionary distant Pseudomonas strain for at least 48.3 and 41.6% of the 16S rRNA sequences, respectively. In conclusion, we strongly recommend removing these regions of the 16S rRNA gene during the intra-genus diversity studies. PMID:22545126

  7. Autistic-like behavioral phenotypes in a mouse model with copy number variation of the CAPS2/CADPS2 gene.

    PubMed

    Sadakata, Tetsushi; Shinoda, Yo; Oka, Megumi; Sekine, Yukiko; Furuichi, Teiichi

    2013-01-04

    Ca²⁺-dependent activator protein for secretion 2 (CAPS2 or CADPS2) facilitates secretion and trafficking of dense-core vesicles. Recent genome-wide association studies of autism have identified several microdeletions due to copy number variation (CNV) in one of the chromosome 7q31.32 alleles on which the locus for CAPS2 is located in autistic patients. To evaluate the biological significance of reducing CAPS2 copy number, we analyzed CAPS2 heterozygous mice. Our present findings suggest that adequate levels of CAPS2 protein are critical for normal brain development and behavior, and that allelic changes due to CNV may contribute to autistic symptoms in combination with deficits in other autism-associated genes. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  8. Pervasive gene content variation and copy number variation in maize and its undomesticated progenitor

    USDA-ARS?s Scientific Manuscript database

    Different individuals of the same species are generally thought to have very similar genomes. However, there is growing evidence that structural variation in the form of copy number variation (CNV) and presence-absence variation (PAV) can lead to variation in the genome content of individuals withi...

  9. Diversity and population-genetic properties of copy number variations and multicopy genes in cattle

    USDA-ARS?s Scientific Manuscript database

    The diversity and population-genetics of copy number variation (CNV) in domesticated animals are not well understood. In this study, we analyzed 75 genomes of major taurine and indicine cattle breeds (including Angus, Brahman, Gir, Holstein, Jersey, Limousin, Nelore, Romagnola), sequenced to 11-fold...

  10. [Diagnostic value of MYB protein expression in adenoid cystic carcinoma and status of MYB gene copy number].

    PubMed

    Huo, Zhen; Zeng, Xuan; Wu, Shafei; Wu, Huanwen; Meng, Yunxiao; Liu, Yuanyuan; Luo, Yufeng; Cao, Jinling; Liang, Zhiyong

    2015-08-01

    To explore the diagnostic value of MYB protein expression for adenoid cystic carcinoma and its differential diagnosis from other salivary gland tumors, and to further investigate the status of MYB gene copy number. MYB expression was studied by immunohistochemistry in 34 adenoid cystic carcinomas, 55 non-adenoid cystic carcinomas (other salivary gland tumors) including 10 pleomorphic adenomas, 10 basal cell adenomas, 10 epithelial-myoepithelial carcinomas, 9 basal cell adenocarcinomas, 8 mucoepidermoid carcinomas, 4 carcinoma in pleomorphic adenomas, and 4 polymorphous low-grade adenocarcinoma. MYB gene copy number status was detected by FISH in MYB protein-positive cases. 82.4% (28/34) of adenoid cystic carcinomas were MYB protein-positive, compared with 9.1% (5/55) of non-adenoid cystic carcinomas, and the difference between the two groups was statistically significant (P < 0.01). 2/18 of adenoid cystic carcinomas had duplication of MYB gene by FISH, and all non-adenoid cystic carcinomas were negative although the difference was not statistically significant (P = 0.435). MYB protein expression is a useful diagnostic marker for adenoid cystic carcinomas in its separation from other salivary gland tumors. In addition, duplication of MYB gene is no a major mechanism for the MYB protein overexpression.

  11. Expression of unique chimeric human papilloma virus type 16 (HPV-16) L1-L2 proteins in Pichia pastoris and Hansenula polymorpha.

    PubMed

    Bredell, Helba; Smith, Jacques J; Görgens, Johann F; van Zyl, Willem H

    2018-04-30

    Cervical cancer is ranked the fourth most common cancer in women worldwide. Despite two commercially available prophylactic vaccines, it is unaffordable for most women in developing countries. We compared the optimized expression of monomers of the unique HPV type 16 L1-L2 chimeric protein (SAF) in two yeast strains of Pichia pastoris, KM71 (Mut s ) and GS115 (Mut + ), with Hansenula polymorpha NCYC 495 to determine the preferred host in bioreactors. SAF was uniquely created by replacing the h4 helix of the HPV-16 capsid L1 protein with a L2 peptide. Two different feeding strategies in fed-batch cultures of P. pastoris Mut s were evaluated: a predetermined feed rate versus feeding based on the oxygen consumption by maintaining constant dissolved oxygen levels (DO stat). All cultures showed a significant increase in biomass when methanol was fed using the DO stat method. In P. pastoris the SAF concentrations were higher in the Mut s strains than in the Mut + strains. However, H. polymorpha produced the highest level of SAF at 132.10 mg.L -1 culture while P. pastoris Mut s only produced 23.61 mg.L -1 . H. polymorpha showed greater potential for the expression of HPV-16 L1/L2 chimeric proteins despite the track record of P. pastoris as a high level producer of heterologous proteins. This article is protected by copyright. All rights reserved.

  12. Focal epithelial hyperplasia by human papillomavirus (HPV)-32 misdiagnosed as HPV-16 and treated with combination of retinoids, imiquimod and quadrivalent HPV vaccine.

    PubMed

    Gemigniani, Franco; Hernández-Losa, Javier; Ferrer, Berta; García-Patos, Vicente

    2015-12-01

    Focal epithelial hyperplasia (FEH) or Heck's disease is a rare, benign and asymptomatic mucosal proliferation associated with human papillomavirus (HPV) infection, mainly with genotypes 13 and 32. We report a florid case of FEH in an 11-year-old Haitian girl with systemic lupus erythematosus receiving immunosuppressive therapy. Cryotherapy was previously performed on numerous occasions with no results. We decided to prescribe a non-invasive and more comfortable treatment. A combination of topical retinoid and imiquimod cream was well tolerated and led to an important improvement. The evidence of infection by HPV-16 detected by polymerase chain reaction (PCR) technique, prompted us to prescribe the quadrivalent HPV vaccine (types 6, 11,16 and 18). Subsequent PCR sequencing with generic primers GP5-GP6 and further BLAST comparative analysis confirmed that genomic viral sequence in our case truly corresponded with HPV-32. This molecular misdiagnosis can be explained by the similarity between genomic sequences of both HPV-16 and -32 genotypes. At the 1-year follow up, we observed total clinical improvement and no recurrences of the disease. Complete healing in this case may correspond to a potential action of topical retinoid, imiquimod and the cross-protection mechanism of the quadrivalent HPV vaccine. © 2015 Japanese Dermatological Association.

  13. Evaluation of the Cow Rumen Metagenome: Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

    SciTech Connect

    Sczyrba, Alex

    2011-10-13

    DOE JGI's Alex Sczyrba on "Evaluation of the Cow Rumen Metagenome" and "Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

  14. Evaluation of the Cow Rumen Metagenome: Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

    ScienceCinema

    Sczyrba, Alex

    2018-02-13

    DOE JGI's Alex Sczyrba on "Evaluation of the Cow Rumen Metagenome" and "Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

  15. Ten-year immune persistence and safety of the HPV-16/18 AS04-adjuvanted vaccine in females vaccinated at 15-55 years of age.

    PubMed

    Schwarz, Tino F; Galaj, Andrzej; Spaczynski, Marek; Wysocki, Jacek; Kaufmann, Andreas M; Poncelet, Sylviane; Suryakiran, Pemmaraju V; Folschweiller, Nicolas; Thomas, Florence; Lin, Lan; Struyf, Frank

    2017-11-01

    Women remain at risk of human papillomavirus (HPV) infection for most of their lives. The duration of protection against HPV-16/18 from prophylactic vaccination remains unknown. We investigated the 10-year immune response and long-term safety profile of the HPV-16/18 AS04-adjuvanted vaccine (AS04-HPV-16/18 vaccine) in females aged between 15 and 55 years at first vaccination. Females who received primary vaccination with three doses of AS04-HPV-16/18 vaccine in the primary phase-III study (NCT00196937) were invited to attend annual evaluations for long-term immunogenicity and safety. Anti-HPV-16/18 antibodies in serum and cervico-vaginal secretions (CVS) were measured using enzyme-linked immunosorbent assay (ELISA). Serious adverse events (SAEs) were recorded throughout the follow-up period. Seropositivity rates for anti-HPV-16 remained high (≥96.3%) in all age groups 10 years after first vaccination. It was found that 99.2% of 15-25-year olds remained seropositive for anti-HPV-18 compared to 93.7% and 83.8% of 26-45-year olds and 45-55-year olds, respectively. Geometric mean titers (GMT) remained above natural infection levels in all age groups. Anti-HPV-16 and anti-HPV-18 titers were at least 5.3-fold and 3.1-fold higher than titers observed after natural infection, respectively, and were predicted to persist above natural infection levels for ≥30 years in all age groups. At Year 10, anti-HPV-16/18 antibody titers in subjects aged 15-25 years remained above plateau levels observed in previous studies. Correlation coefficients for antibody titers in serum and CVS were 0.64 (anti-HPV-16) and 0.38 (anti-HPV-18). This study concluded that vaccinated females aged 15-55 years elicited sustained immunogenicity with an acceptable safety profile up to 10 years after primary vaccination, suggesting long-term protection against HPV. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  16. Copy number variations of six and seven α-globin genes in a family with intermedia and major thalassemia phenotypes.

    PubMed

    Farashi, Samaneh; Vakili, Shadi; Faramarzi Garous, Negin; Ashki, Mehri; Imanian, Hashem; Azarkeivan, Azita; Najmabadi, Hossein

    2015-10-01

    Copy number variations in α-globin genes are results of unequal crossover between homologous segments in the α-globin gene cluster that misalign during the meiosis phase of the gametogenesis process. Reduction or augmentation of α-globin genes leads to imbalance of α/β chains in hemoglobin tetramer and consequently attenuate or worsen the β-thal clinical symptoms, respectively. Multiplications in α-globin genes have been found in some populations, justifying unexpected severe phenotype of β-thal carriers. Unexpected severe phenotype in the family members may result from coexistence of extra α-globin genes, which is an important factor in the causation of thalassemia intermedia and major in heterozygous β-thalassemia. We described different multiplications in α-globin locus in an Iranian family with one, two or three extra α-globin genes (ααα/αα, αααα/αα and αααα/ααα). The excess α-globin gene/genes cause increment in β/α chain imbalance and leads to worsening pathophysiology and clinical severity of β-thalassemia carriers.

  17. Copy number gain of MYCN gene is a recurrent genetic aberration and favorable prognostic factor in Chinese pediatric neuroblastoma patients

    PubMed Central

    2013-01-01

    Background Amplification of MYCN oncogene is an established marker indicating aggressive tumor progression of neuroblastoma (NBL). But copy number analyses of MYCN gene in ganglioneuroblastoma (GNBL) and ganglioneuroma(GN) is poorly described in the literature. In the study, we evaluated the copy number aberrations of MYCN gene in clinical samples of NBLs, GNBLs and GNs and analyzed their association with clinical outcome of the patients. Methods In this study, we analyzed MYCN gene and chromosome 2 aneusomy by using fluorescence in situ hybridization (FISH) method in a total of 220 patients with NBL, GNBL and GN cases. Kaplan-Meier curves were generated by using SPSS 12.0 software. Results Of 220 patients, 178 (81.0%) were NBLs, 32 (14.5%) were GNBLs and 10 (4.5%) were GNs. MYCN gain is a recurrent genetic aberration of neuroblastic tumors (71.8%, 158/220), which was found in 129 NBLs (58.6%, 129/220), 25 GNBLs (11.4%, 25/220) and 4 GN cases (1.8%, 4/220). However, MYCN amplification was only present in 24 NBL tumors (13.5%, 24/178) and 1 GNBL case (3.1%, 1/32). Kaplan-Meier survival analysis indicated that MYCN amplification is significantly correlated with decreased overall survival in NBLs (P=0.017). Furthermore, a better prognosis trend was observed in patients with MYCN gain tumors compared with those with MYCN gene normal copy number tumors and MYCN amplification tumors (P=0.012). Conclusions In summary, the frequency of MYCN amplification in NBLs is high and is rarely observed in GNBLs and GNs, which suggest MYCN plays an important role in neuroblastic tumors differentiation. MYCN gain appeared to define a subgroup of NBLs with much better outcome and classification of MYCN gene copy number alteration as three groups (amplification, gain and normal) can provide a powerful prognostic indicator in NBLs. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/6417541528559124 PMID:23320395

  18. Accurately Assessing the Risk of Schizophrenia Conferred by Rare Copy-Number Variation Affecting Genes with Brain Function

    PubMed Central

    Raychaudhuri, Soumya; Korn, Joshua M.; McCarroll, Steven A.; Altshuler, David; Sklar, Pamela; Purcell, Shaun; Daly, Mark J.

    2010-01-01

    Investigators have linked rare copy number variation (CNVs) to neuropsychiatric diseases, such as schizophrenia. One hypothesis is that CNV events cause disease by affecting genes with specific brain functions. Under these circumstances, we expect that CNV events in cases should impact brain-function genes more frequently than those events in controls. Previous publications have applied “pathway” analyses to genes within neuropsychiatric case CNVs to show enrichment for brain-functions. While such analyses have been suggestive, they often have not rigorously compared the rates of CNVs impacting genes with brain function in cases to controls, and therefore do not address important confounders such as the large size of brain genes and overall differences in rates and sizes of CNVs. To demonstrate the potential impact of confounders, we genotyped rare CNV events in 2,415 unaffected controls with Affymetrix 6.0; we then applied standard pathway analyses using four sets of brain-function genes and observed an apparently highly significant enrichment for each set. The enrichment is simply driven by the large size of brain-function genes. Instead, we propose a case-control statistical test, cnv-enrichment-test, to compare the rate of CNVs impacting specific gene sets in cases versus controls. With simulations, we demonstrate that cnv-enrichment-test is robust to case-control differences in CNV size, CNV rate, and systematic differences in gene size. Finally, we apply cnv-enrichment-test to rare CNV events published by the International Schizophrenia Consortium (ISC). This approach reveals nominal evidence of case-association in neuronal-activity and the learning gene sets, but not the other two examined gene sets. The neuronal-activity genes have been associated in a separate set of schizophrenia cases and controls; however, testing in independent samples is necessary to definitively confirm this association. Our method is implemented in the PLINK software package

  19. Normal exon copy number of the GLI2 and GLI3 genes in patients with esophageal atresia.

    PubMed

    Bednarczyk, D; Smigiel, R; Patkowski, D; Laczmanska, I; Lebioda, A; Laczmanski, L; Sasiadek, M M

    2013-01-01

    Esophageal atresia (EA) is a congenital developmental defect of the alimentary tract concerning the interruption of the esophagus with or without connection to the trachea. The incidence of EA is 1 in 3000-3500 of live-born infants, and occurs in both isolated and syndromic (in combination with abnormalities in other organ systems) forms. The molecular mechanisms underlying the development of EA are poorly understood. Knockout studies in mice indicate that genes like Sonic hedgehog, Gli2, and Gli3 play a role in the etiology of EA. These facts led us to hypothesize that Sonic hedgehog-GLI gene rearrangements are associated with EA in humans. To test this hypothesis, we screened patients with isolated and syndromic EA for GLI2 and/or GLI3 microrearrangements using methods to estimate the copy number (Multiplex Ligation-dependent Probe Amplification, real-time polymerase chain reaction). To our best knowledge this is the first study assessing copy number of GLI2 and GLI3 genes in patients with EA. © 2013 Wiley Periodicals, Inc. and the International Society for Diseases of the Esophagus.

  20. Integrated Analysis of Genome-wide Copy Number Alterations and Gene Expression in MSS, CIMP-negative Colon Cancer

    PubMed Central

    Loo, Lenora WM; Tiirikainen, Maarit; Cheng, Iona; Lum-Jones, Annette; Seifried, Ann; Church, James M; Gryfe, Robert; Weisenberger, Daniel J; Lindor, Noralane M; Gallinger, Steven; Haile, Robert W; Duggan, David J; Thibodeau, Stephen N; Casey, Graham; Le Marchand, Loïc

    2014-01-01

    Microsatellite stable (MSS), CpG island methylator phenotype (CIMP)-negative colorectal tumors, the most prevalent molecular subtype of colorectal cancer, are associated with extensive copy number alteration (CNA) events and aneuploidy. We report on the identification of characteristic recurrent CNA (with frequency >25%) events and associated gene expression profiles for a total of 40 paired tumor and adjacent normal colon tissues using genome-wide microarrays. We observed recurrent CNAs, namely gains at 1q, 7p, 7q, 8p12-11, 8q, 12p13, 13q, 20p, 20q, Xp, and Xq and losses at 1p36, 1p31, 1p21, 4p15-12, 4q12-35, 5q21-22, 6q26, 8p, 14q, 15q11-12, 17p, 18p, 18q, 21q21-22, and 22q. Within these genomic regions we identified 356 genes with significant differential expression (P<0.0001 and ±1.5 fold change) in the tumor compared to adjacent normal tissue. Gene ontology and pathway analyses indicated that many of these genes were involved in functional mechanisms that regulate cell cycle, cell death, and metabolism. An amplicon present in >70% of the tumor samples at 20q11-20q13 contained several cancer-related genes (AHCY, POFUT1, RPN2, TH1L and PRPF6) that were up-regulated and demonstrated a significant linear correlation (P<0.05) for gene dosage and gene expression. Copy number loss at 8p, a CNA associated with adenocarcinoma and poor prognosis, was observed in >50% of the tumor samples and demonstrated a significant linear correlation for gene dosage and gene expression for two potential tumor suppressor genes, MTUS1 (8p22) and PPP2CB (8p12). The results from our integration analysis illustrate the complex relationship between genomic alterations and gene expression in colon cancer. PMID:23341073

  1. The Symbiotic Performance of Chickpea Rhizobia Can Be Improved by Additional Copies of the clpB Chaperone Gene.

    PubMed

    Paço, Ana; Brígido, Clarisse; Alexandre, Ana; Mateos, Pedro F; Oliveira, Solange

    2016-01-01

    The ClpB chaperone is known to be involved in bacterial stress response. Moreover, recent studies suggest that this protein has also a role in the chickpea-rhizobia symbiosis. In order to improve both stress tolerance and symbiotic performance of a chickpea microsymbiont, the Mesorhizobium mediterraneum UPM-Ca36T strain was genetically transformed with pPHU231 containing an extra-copy of the clpB gene. To investigate if the clpB-transformed strain displays an improved stress tolerance, bacterial growth was evaluated under heat and acid stress conditions. In addition, the effect of the extra-copies of the clpB gene in the symbiotic performance was evaluated using plant growth assays (hydroponic and pot trials). The clpB-transformed strain is more tolerant to heat shock than the strain transformed with pPHU231, supporting the involvement of ClpB in rhizobia heat shock tolerance. Both plant growth assays showed that ClpB has an important role in chickpea-rhizobia symbiosis. The nodulation kinetics analysis showed a higher rate of nodule appearance with the clpB-transformed strain. This strain also induced a greater number of nodules and, more notably, its symbiotic effectiveness increased ~60% at pH5 and 83% at pH7, compared to the wild-type strain. Furthermore, a higher frequency of root hair curling was also observed in plants inoculated with the clpB-transformed strain, compared to the wild-type strain. The superior root hair curling induction, nodulation ability and symbiotic effectiveness of the clpB-transformed strain may be explained by an increased expression of symbiosis genes. Indeed, higher transcript levels of the nodulation genes nodA and nodC (~3 folds) were detected in the clpB-transformed strain. The improvement of rhizobia by addition of extra-copies of the clpB gene may be a promising strategy to obtain strains with enhanced stress tolerance and symbiotic effectiveness, thus contributing to their success as crop inoculants, particularly under

  2. The Symbiotic Performance of Chickpea Rhizobia Can Be Improved by Additional Copies of the clpB Chaperone Gene

    PubMed Central

    Paço, Ana; Brígido, Clarisse; Alexandre, Ana; Mateos, Pedro F.; Oliveira, Solange

    2016-01-01

    The ClpB chaperone is known to be involved in bacterial stress response. Moreover, recent studies suggest that this protein has also a role in the chickpea-rhizobia symbiosis. In order to improve both stress tolerance and symbiotic performance of a chickpea microsymbiont, the Mesorhizobium mediterraneum UPM-Ca36T strain was genetically transformed with pPHU231 containing an extra-copy of the clpB gene. To investigate if the clpB-transformed strain displays an improved stress tolerance, bacterial growth was evaluated under heat and acid stress conditions. In addition, the effect of the extra-copies of the clpB gene in the symbiotic performance was evaluated using plant growth assays (hydroponic and pot trials). The clpB-transformed strain is more tolerant to heat shock than the strain transformed with pPHU231, supporting the involvement of ClpB in rhizobia heat shock tolerance. Both plant growth assays showed that ClpB has an important role in chickpea-rhizobia symbiosis. The nodulation kinetics analysis showed a higher rate of nodule appearance with the clpB-transformed strain. This strain also induced a greater number of nodules and, more notably, its symbiotic effectiveness increased ~60% at pH5 and 83% at pH7, compared to the wild-type strain. Furthermore, a higher frequency of root hair curling was also observed in plants inoculated with the clpB-transformed strain, compared to the wild-type strain. The superior root hair curling induction, nodulation ability and symbiotic effectiveness of the clpB-transformed strain may be explained by an increased expression of symbiosis genes. Indeed, higher transcript levels of the nodulation genes nodA and nodC (~3 folds) were detected in the clpB-transformed strain. The improvement of rhizobia by addition of extra-copies of the clpB gene may be a promising strategy to obtain strains with enhanced stress tolerance and symbiotic effectiveness, thus contributing to their success as crop inoculants, particularly under

  3. Anaplastic Lymphoma Kinase Gene Copy Number Gain in Inflammatory Breast Cancer (IBC): Prevalence, Clinicopathologic Features and Prognostic Implication

    PubMed Central

    Kim, Min Hwan; Lee, Soohyeon; Koo, Ja Seung; Jung, Kyung Hae; Park, In Hae; Jeong, Joon; Kim, Seung Il; Park, Seho; Park, Hyung Seok; Park, Byeong-Woo; Kim, Joo-Hang; Sohn, Joohyuk

    2015-01-01

    Background Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer, and its molecular pathogenesis still remains to be elucidated. This study aimed to evaluate the prevalence and implication of anaplastic lymphoma kinase (ALK) copy number change in IBC patients. Methods We retrospectively collected formalin-fixed, paraffin-embedded tumor tissues and medical records of IBC patients from several institutes in Korea. ALK gene copy number change and rearrangement were assessed by fluorescence in situ hybridization (FISH) assay, and ALK expression status was evaluated by immunohistochemical (IHC) staining. Results Thirty-six IBC patients including those with HER2 (+) breast cancer (16/36, 44.4%) and triple-negative breast cancer (13/36, 36.1%) were enrolled in this study. ALK copy number gain (CNG) was observed in 47.2% (17/36) of patients, including one patient who harbored ALK gene amplification. ALK CNG (+) patients showed significantly worse overall survival compared to ALK CNG (-) patients in univariate analysis (24.9 months vs. 38.1 months, p = 0.033). Recurrence free survival (RFS) after curative mastectomy was also significantly shorter in ALK CNG (+) patients than in ALK CNG (-) patients (n = 22, 12.7 months vs. 43.3 months, p = 0.016). Multivariate Cox regression analysis with adjustment for HER2 and ER statuses showed significantly poorer RFS for ALK CNG (+) patients (HR 5.63, 95% CI 1.11–28.44, p = 0.037). Conclusion This study shows a significant presence of ALK CNG in IBC patients, and ALK CNG was associated with significantly poorer RFS. PMID:25803816

  4. Anaplastic lymphoma kinase gene copy number gain in inflammatory breast cancer (IBC): prevalence, clinicopathologic features and prognostic implication.

    PubMed

    Kim, Min Hwan; Lee, Soohyeon; Koo, Ja Seung; Jung, Kyung Hae; Park, In Hae; Jeong, Joon; Kim, Seung Il; Park, Seho; Park, Hyung Seok; Park, Byeong-Woo; Kim, Joo-Hang; Sohn, Joohyuk

    2015-01-01

    Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer, and its molecular pathogenesis still remains to be elucidated. This study aimed to evaluate the prevalence and implication of anaplastic lymphoma kinase (ALK) copy number change in IBC patients. We retrospectively collected formalin-fixed, paraffin-embedded tumor tissues and medical records of IBC patients from several institutes in Korea. ALK gene copy number change and rearrangement were assessed by fluorescence in situ hybridization (FISH) assay, and ALK expression status was evaluated by immunohistochemical (IHC) staining. Thirty-six IBC patients including those with HER2 (+) breast cancer (16/36, 44.4%) and triple-negative breast cancer (13/36, 36.1%) were enrolled in this study. ALK copy number gain (CNG) was observed in 47.2% (17/36) of patients, including one patient who harbored ALK gene amplification. ALK CNG (+) patients showed significantly worse overall survival compared to ALK CNG (-) patients in univariate analysis (24.9 months vs. 38.1 months, p = 0.033). Recurrence free survival (RFS) after curative mastectomy was also significantly shorter in ALK CNG (+) patients than in ALK CNG (-) patients (n = 22, 12.7 months vs. 43.3 months, p = 0.016). Multivariate Cox regression analysis with adjustment for HER2 and ER statuses showed significantly poorer RFS for ALK CNG (+) patients (HR 5.63, 95% CI 1.11-28.44, p = 0.037). This study shows a significant presence of ALK CNG in IBC patients, and ALK CNG was associated with significantly poorer RFS.

  5. MYC and Human Telomerase Gene (TERC) Copy Number Gain in Early-stage Non–small Cell Lung Cancer

    PubMed Central

    Flacco, Antonella; Ludovini, Vienna; Bianconi, Fortunato; Ragusa, Mark; Bellezza, Guido; Tofanetti, Francesca R.; Pistola, Lorenza; Siggillino, Annamaria; Vannucci, Jacopo; Cagini, Lucio; Sidoni, Angelo; Puma, Francesco; Varella-Garcia, Marileila; Crinò, Lucio

    2015-01-01

    Objectives We investigated the frequency of MYC and TERC increased gene copy number (GCN) in early-stage non–small cell lung cancer (NSCLC) and evaluated the correlation of these genomic imbalances with clinicopathologic parameters and outcome. Materials and Methods Tumor tissues were obtained from 113 resected NSCLCs. MYC and TERC GCNs were tested by fluorescence in situ hybridization (FISH) according to the University of Colorado Cancer Center (UCCC) criteria and based on the receiver operating characteristic (ROC) classification. Results When UCCC criteria were applied, 41 (36%) cases for MYC and 41 (36%) cases for TERC were considered FISH-positive. MYC and TERC concurrent FISH-positive was observed in 12 cases (11%): 2 (17%) cases with gene amplification and 10 (83%) with high polysomy. By using the ROC analysis, high MYC (mean ≥2.83 copies/cell) and TERC (mean ≥2.65 copies/cell) GCNs were observed in 60 (53.1%) cases and 58 (51.3%) cases, respectively. High TERC GCN was associated with squamous cell carcinoma (SCC) histology (P = 0.001). In univariate analysis, increased MYC GCN was associated with shorter overall survival (P = 0.032 [UCCC criteria] or P = 0.02 [ROC classification]), whereas high TERC GCN showed no association. In multivariate analysis including stage and age, high MYC GCN remained significantly associated with worse overall survival using both the UCCC criteria (P = 0.02) and the ROC classification (P = 0.008). Conclusions Our results confirm MYC as frequently amplified in early-stage NSCLC and increased MYC GCN as a strong predictor of worse survival. Increased TERC GCN does not have prognostic impact but has strong association with squamous histology. PMID:25806711

  6. Comparative analyses of gene copy number and mRNA expression in GBM tumors and GBM xenografts

    SciTech Connect

    Hodgson, J. Graeme; Yeh, Ru-Fang; Ray, Amrita

    2009-04-03

    Development of model systems that recapitulate the molecular heterogeneity observed among glioblastoma multiforme (GBM) tumors will expedite the testing of targeted molecular therapeutic strategies for GBM treatment. In this study, we profiled DNA copy number and mRNA expression in 21 independent GBM tumor lines maintained as subcutaneous xenografts (GBMX), and compared GBMX molecular signatures to those observed in GBM clinical specimens derived from the Cancer Genome Atlas (TCGA). The predominant copy number signature in both tumor groups was defined by chromosome-7 gain/chromosome-10 loss, a poor-prognosis genetic signature. We also observed, at frequencies similar to that detected in TCGA GBM tumors,more » genomic amplification and overexpression of known GBM oncogenes, such as EGFR, MDM2, CDK6, and MYCN, and novel genes, including NUP107, SLC35E3, MMP1, MMP13, and DDX1. The transcriptional signature of GBMX tumors, which was stable over multiple subcutaneous passages, was defined by overexpression of genes involved in M phase, DNA replication, and chromosome organization (MRC) and was highly similar to the poor-prognosis mitosis and cell-cycle module (MCM) in GBM. Assessment of gene expression in TCGA-derived GBMs revealed overexpression of MRC cancer genes AURKB, BIRC5, CCNB1, CCNB2, CDC2, CDK2, and FOXM1, which form a transcriptional network important for G2/M progression and/or checkpoint activation. Our study supports propagation of GBM tumors as subcutaneous xenografts as a useful approach for sustaining key molecular characteristics of patient tumors, and highlights therapeutic opportunities conferred by this GBMX tumor panel for testing targeted therapeutic strategies for GBM treatment.« less

  7. Physical Mapping of Amplified Copies of the 5-Enolpyruvylshikimate-3-Phosphate Synthase Gene in Glyphosate-Resistant Amaranthus tuberculatus1[OPEN

    PubMed Central

    Dillon, Andrew; Varanasi, Vijay K.; Koo, Dal-Hoe; Nakka, Sridevi; Peterson, Dallas E.; Friebe, Bernd

    2017-01-01

    Recent and rapid evolution of resistance to glyphosate, the most widely used herbicides, in several weed species, including common waterhemp (Amaranthus tuberculatus), poses a serious threat to sustained crop production. We report that glyphosate resistance in A. tuberculatus was due to amplification of the 5-enolpyruvylshikimate-3-P synthase (EPSPS) gene, which encodes the molecular target of glyphosate. There was a positive correlation between EPSPS gene copies and its transcript expression. We analyzed the distribution of EPSPS copies in the genome of A. tuberculatus using fluorescence in situ hybridization on mitotic metaphase chromosomes and interphase nuclei. Fluorescence in situ hybridization analysis mapped the EPSPS gene to pericentromeric regions of two homologous chromosomes in glyphosate sensitive A. tuberculatus. In glyphosate-resistant plants, a cluster of EPSPS genes on the pericentromeric region on one pair of homologous chromosomes was detected. Intriguingly, two highly glyphosate-resistant plants harbored an additional chromosome with several EPSPS copies besides the native chromosome pair with EPSPS copies. These results suggest that the initial event of EPSPS gene duplication may have occurred because of unequal recombination mediated by repetitive DNA. Subsequently, gene amplification may have resulted via several other mechanisms, such as chromosomal rearrangements, deletion/insertion, transposon-mediated dispersion, or possibly by interspecific hybridization. This report illustrates the physical mapping of amplified EPSPS copies in A. tuberculatus. PMID:27956489

  8. Genome-wide copy number variation study associates metabotropic glutamate receptor gene networks with attention deficit hyperactivity disorder

    PubMed Central

    Elia, Josephine; Glessner, Joseph T; Wang, Kai; Takahashi, Nagahide; Shtir, Corina J; Hadley, Dexter; Sleiman, Patrick M A; Zhang, Haitao; Kim, Cecilia E; Robison, Reid; Lyon, Gholson J; Flory, James H; Bradfield, Jonathan P; Imielinski, Marcin; Hou, Cuiping; Frackelton, Edward C; Chiavacci, Rosetta M; Sakurai, Takeshi; Rabin, Cara; Middleton, Frank A; Thomas, Kelly A; Garris, Maria; Mentch, Frank; Freitag, Christine M; Steinhausen, Hans-Christoph; Todorov, Alexandre A; Reif, Andreas; Rothenberger, Aribert; Franke, Barbara; Mick, Eric O; Roeyers, Herbert; Buitelaar, Jan; Lesch, Klaus-Peter; Banaschewski, Tobias; Ebstein, Richard P; Mulas, Fernando; Oades, Robert D; Sergeant, Joseph; Sonuga-Barke, Edmund; Renner, Tobias J; Romanos, Marcel; Romanos, Jasmin; Warnke, Andreas; Walitza, Susanne; Meyer, Jobst; Pálmason, Haukur; Seitz, Christiane; Loo, Sandra K; Smalley, Susan L; Biederman, Joseph; Kent, Lindsey; Asherson, Philip; Anney, Richard J L; Gaynor, J William; Shaw, Philip; Devoto, Marcella; White, Peter S; Grant, Struan F A; Buxbaum, Joseph D; Rapoport, Judith L; Williams, Nigel M; Nelson, Stanley F; Faraone, Stephen V; Hakonarson, Hakon

    2014-01-01

    Attention deficit hyperactivity disorder (ADHD) is a common, heritable neuropsychiatric disorder of unknown etiology. We performed a whole-genome copy number variation (CNV) study on 1,013 cases with ADHD and 4,105 healthy children of European ancestry using 550,000 SNPs. We evaluated statistically significant findings in multiple independent cohorts, with a total of 2,493 cases with ADHD and 9,222 controls of European ancestry, using matched platforms. CNVs affecting metabotropic glutamate receptor genes were enriched across all cohorts (P = 2.1 × 10−9). We saw GRM5 (encoding glutamate receptor, metabotropic 5) deletions in ten cases and one control (P = 1.36 × 10−6). We saw GRM7 deletions in six cases, and we saw GRM8 deletions in eight cases and no controls. GRM1 was duplicated in eight cases. We experimentally validated the observed variants using quantitative RT-PCR. A gene network analysis showed that genes interacting with the genes in the GRM family are enriched for CNVs in ~10% of the cases (P = 4.38 × 10−10) after correction for occurrence in the controls. We identified rare recurrent CNVs affecting glutamatergic neurotransmission genes that were overrepresented in multiple ADHD cohorts. PMID:22138692

  9. Rationale and design of a community-based double-blind randomized clinical trial of an HPV 16 and 18 vaccine in Guanacaste, Costa Rica

    PubMed Central

    Herrero, Rolando; Hildesheim, Allan; Rodríguez, Ana C; Wacholder, Sholom; Bratti, Concepción; Solomon, Diane; González, Paula; Porras, Carolina; Jiménez, Silvia; Guillen, Diego; Morales, Jorge; Alfaro, Mario; Cyr, Jean; Morrisey, Kerrygrace; Estrada, Yenory; Cortés, Bernal; Morera, Lidia Ana; Freer, Enrique; Schussler, John; Schiller, John; Lowy, Douglas; Schiffman, Mark

    2008-01-01

    We report the rationale, design, methods and details of participation of a community-based, double blind, randomized clinical trial of an HPV 16 and 18 vaccine conducted in two provinces of Costa Rica to investigate the efficacy and population impact of the vaccine in the prevention of cervical cancer precursors. More than 24,000 women between 18 and 25 years of age were invited to participate and pre-screened for eligibility, with recruitment of 7,466 women (30% of those prescreened, 59% of those eligible) who were randomized to receive 3 doses of the HPV vaccine or hepatitis A vaccine as control. A complex protocol of data and specimen collection was applied, including an interview, pelvic exam for sexually active women, blood for serology and cell-mediated immunity, cervical secretions for local immunity and cells for HPV, Chlamydia trachomatis and Gonorrhea testing. Eighty percent of the women received 3 doses, 12.4% two doses and 7.4% one dose. At visits, compliance with data and specimen collection was close to 100%. Baseline characteristics and age-specific prevalence of HPV and cervical neoplasia are reported. Overall prevalence of HPV was high (50%), with 8.3% of women having HPV 16 and 3.2% HPV 18. LSIL was detected in 12.7% of women at baseline and HSIL in 1.9%. Prevalence of Chlamydia was 14.2%. There was very good agreement in HPV detection between clinician-collected and self-collected specimens (89.4% agreement for all types, kappa 0.59). Follow up will continue with yearly or more frequent examinations for at least 4 years for each participant. PMID:18640170

  10. Efficacy of the HPV-16/18 AS04-Adjuvanted Vaccine Against Low-Risk HPV Types (PATRICIA Randomized Trial): An Unexpected Observation

    PubMed Central

    Szarewski, Anne; Skinner, S. Rachel; Garland, Suzanne M.; Romanowski, Barbara; Schwarz, Tino F.; Apter, Dan; Chow, Song-Nan; Paavonen, Jorma; Del Rosario-Raymundo, M. Rowena; Teixeira, Julio C.; De Carvalho, Newton S.; Castro-Sanchez, Maria; Castellsagué, Xavier; Poppe, Willy A. J.; De Sutter, Philippe; Huh, Warner; Chatterjee, Archana; Tjalma, Wiebren A.; Ackerman, Ronald T.; Martens, Mark; Papp, Kim A.; Bajo-Arenas, Jose; Harper, Diane M.; Torné, Aureli; David, Marie-Pierre; Struyf, Frank; Lehtinen, Matti; Dubin, Gary

    2013-01-01

    Background. Public Health England has reported a decrease of up to 20.8% in new diagnoses of external genital warts (GWs) among women aged <19 years since the national vaccination program with the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine began in 2008. A post hoc analysis of the phase III PATRICIA (PApilloma TRIal against Cancer In young Adults) trial (NCT00122681) was performed to ascertain whether protection against low-risk HPV types was apparent. Methods. Vaccine efficacy (VE) at 48 months was assessed against 6-month persistent infection (6MPI) with low-risk HPV types in the total vaccinated cohort (TVC) and in the TVC naive (for 25 HPV types tested) populations. Results. In the TVC naive cohort, VE against 6MPI (95% confidence interval) was 34.5% (11.3 to 51.8) for HPV-6/11, 34.9% (9.1 to 53.7) for HPV-6, 30.3% (−45.0 to 67.5) for HPV-11, and 49.5% (21.0 to 68.3) for HPV-74. Conclusions. The HPV-16/18 AS04-adjuvanted vaccine appears to have moderate efficacy against persistent infections with a number of low-risk HPV types (HPV-6/11/74), which are responsible for the majority of external GWs, and recently, antibody and cell-mediated immune response to HPV-6/11 have been observed. These findings may help to explain the decrease in external GW diagnoses seen in England. PMID:24092907

  11. Rationale and design of a long term follow-up study of women who did and did not receive HPV 16/18 vaccination in Guanacaste, Costa Rica.

    PubMed

    Gonzalez, Paula; Hildesheim, Allan; Herrero, Rolando; Katki, Hormuzd; Wacholder, Sholom; Porras, Carolina; Safaeian, Mahboobeh; Jimenez, Silvia; Darragh, Teresa M; Cortes, Bernal; Befano, Brian; Schiffman, Mark; Carvajal, Loreto; Palefsky, Joel; Schiller, John; Ocampo, Rebeca; Schussler, John; Lowy, Douglas; Guillen, Diego; Stoler, Mark H; Quint, Wim; Morales, Jorge; Avila, Carlos; Rodriguez, Ana Cecilia; Kreimer, Aimée R

    2015-04-27

    The Costa Rica Vaccine Trial (CVT) was a randomized clinical trial conducted between 2004 and 2010, which randomized 7466 women aged 18 to 25 to receive the bivalent HPV-16/18 vaccine or control Hepatitis-A vaccine. Participants were followed for 4 years with cross-over vaccination at the study end. In 2010 the long term follow-up (LTFU) study was initiated to evaluate the 10-year impact of HPV-16/18 vaccination, determinants of the immune response, and HPV natural history in a vaccinated population. Herein, the rationale, design and methods of the LTFU study are described, which actively follows CVT participants in the HPV-arm 6 additional years at biennial intervals (3 additional study visits for 10 years of total follow-up), or more often if clinically indicated. According to the initial commitment, women in the Hepatitis-A arm were offered HPV vaccination at cross-over; they were followed 2 additional years and exited from the study. 92% of eligible CVT women accepted participation in LTFU. To provide underlying rates of HPV acquisition and cervical disease among unvaccinated women to compare with the HPV-arm during LTFU, a new unvaccinated control group (UCG) of women who are beyond the age generally recommended for routine vaccination was enrolled, and will be followed by cervical cancer screening over 6 years. To form the UCG, 5000 women were selected from a local census, of whom 2836 women (61% of eligible women) agreed to participate. Over 90% of participants complied with an interview, blood and cervical specimen collection. Evaluation of comparability between the original (Hepatitis-A arm of CVT) and new (UCG) control groups showed that women's characteristics, as well as their predicted future risk for cervical HPV acquisition, were similar, thus validating use of the UCG. LTFU is poised to comprehensively address many important questions related to long-term effects of prophylactic HPV vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. DNA Copy Number Aberrations, and Human Papillomavirus Status in Penile Carcinoma. Clinico-Pathological Correlations and Potential Driver Genes.

    PubMed

    La-Touche, Susannah; Lemetre, Christophe; Lambros, Maryou; Stankiewicz, Elzbieta; Ng, Charlotte K Y; Weigelt, Britta; Rajab, Ramzi; Tinwell, Brendan; Corbishley, Cathy; Watkin, Nick; Berney, Dan; Reis-Filho, Jorge S

    2016-01-01

    Penile squamous cell carcinoma is a rare disease, in which somatic genetic aberrations have yet to be characterized. We hypothesized that gene copy aberrations might correlate with human papillomavirus status and clinico-pathological features. We sought to determine the spectrum of gene copy number aberrations in a large series of PSCCs and to define their correlations with human papillomavirus, histopathological subtype, and tumor grade, stage and lymph node status. Seventy formalin-fixed, paraffin embedded penile squamous cell carcinomas were centrally reviewed by expert uropathologists. DNA was extracted from micro-dissected samples, subjected to PCR-based human papillomavirus assessment and genotyping (INNO-LiPA human papillomavirus Genotyping Extra Assay) and microarray-based comparative genomic hybridization using a 32K Bacterial Artificial Chromosome array platform. Sixty-four samples yielded interpretable results. Recurrent gains were observed in chromosomes 1p13.3-q44 (88%), 3p12.3-q29 (86%), 5p15.33-p11 (67%) and 8p12-q24.3 (84%). Amplifications of 5p15.33-p11 and 11p14.1-p12 were found in seven (11%) and four (6%) cases, respectively. Losses were observed in chromosomes 2q33-q37.3 (86%), 3p26.3-q11.1 (83%) and 11q12.2-q25 (81%). Although many losses and gains were similar throughout the cohort, there were small significant differences observed at specific loci, between human papillomavirus positive and negative tumors, between tumor types, and tumor grade and nodal status. These results demonstrate that despite the diversity of genetic aberrations in penile squamous cell carcinomas, there are significant correlations between the clinico-pathological data and the genetic changes that may play a role in disease natural history and progression and highlight potential driver genes, which may feature in molecular pathways for existing therapeutic agents.

  13. DNA Copy Number Aberrations, and Human Papillomavirus Status in Penile Carcinoma. Clinico-Pathological Correlations and Potential Driver Genes

    PubMed Central

    Lambros, Maryou; Stankiewicz, Elzbieta; Ng, Charlotte K. Y.; Weigelt, Britta; Rajab, Ramzi; Tinwell, Brendan; Corbishley, Cathy; Watkin, Nick; Berney, Dan; Reis-Filho, Jorge S.

    2016-01-01

    Penile squamous cell carcinoma is a rare disease, in which somatic genetic aberrations have yet to be characterized. We hypothesized that gene copy aberrations might correlate with human papillomavirus status and clinico-pathological features. We sought to determine the spectrum of gene copy number aberrations in a large series of PSCCs and to define their correlations with human papillomavirus, histopathological subtype, and tumor grade, stage and lymph node status. Seventy formalin-fixed, paraffin embedded penile squamous cell carcinomas were centrally reviewed by expert uropathologists. DNA was extracted from micro-dissected samples, subjected to PCR-based human papillomavirus assessment and genotyping (INNO-LiPA human papillomavirus Genotyping Extra Assay) and microarray-based comparative genomic hybridization using a 32K Bacterial Artificial Chromosome array platform. Sixty-four samples yielded interpretable results. Recurrent gains were observed in chromosomes 1p13.3-q44 (88%), 3p12.3-q29 (86%), 5p15.33-p11 (67%) and 8p12-q24.3 (84%). Amplifications of 5p15.33-p11 and 11p14.1-p12 were found in seven (11%) and four (6%) cases, respectively. Losses were observed in chromosomes 2q33-q37.3 (86%), 3p26.3-q11.1 (83%) and 11q12.2-q25 (81%). Although many losses and gains were similar throughout the cohort, there were small significant differences observed at specific loci, between human papillomavirus positive and negative tumors, between tumor types, and tumor grade and nodal status. These results demonstrate that despite the diversity of genetic aberrations in penile squamous cell carcinomas, there are significant correlations between the clinico-pathological data and the genetic changes that may play a role in disease natural history and progression and highlight potential driver genes, which may feature in molecular pathways for existing therapeutic agents. PMID:26901676

  14. Copy number variation and missense mutations of the agouti signaling protein (ASIP) gene in goat breeds with different coat colors.

    PubMed

    Fontanesi, L; Beretti, F; Riggio, V; Gómez González, E; Dall'Olio, S; Davoli, R; Russo, V; Portolano, B

    2009-01-01

    In goats, classical genetic studies reported a large number of alleles at the Agouti locus with effects on coat color and pattern distribution. From these early studies, the dominant A(Wt) (white/tan) allele was suggested to cause the white color of the Saanen breed. Here, we sequenced the coding region of the goat ASIP gene in 6 goat breeds (Girgentana, Maltese, Derivata di Siria, Murciano-Granadina, Camosciata delle Alpi, and Saanen), with different coat colors and patterns. Five single nucleotide polymorphisms (SNPs) were identified, 3 of which caused missense mutations in conserved positions of the cysteine-rich carboxy-terminal domain of the protein (p.Ala96Gly, p.Cys126Gly, and p.Val128Gly). Allele and genotype frequencies suggested that these mutations are not associated or not completely associated with coat color in the investigated goat breeds. Moreover, genotyping and sequencing results, deviation from Hardy-Weinberg equilibrium, as well as allele copy number evaluation from semiquantitative fluorescent multiplex PCR, indicated the presence of copy number variation (CNV) in all investigated breeds. To confirm the presence of CNV and evaluate its extension, we applied a bovine-goat cross-species array comparative genome hybridization (aCGH) experiment using a custom tiling array based on bovine chromosome 13. aCGH results obtained for 8 goat DNA samples confirmed the presence of CNV affecting a region of less that 100 kb including the ASIP and AHCY genes. In Girgentana and Saanen breeds, this CNV might cause the A(Wt) allele, as already suggested for a similar structural mutation in sheep affecting the ASIP and AHCY genes, providing evidence for a recurrent interspecies CNV. However, other mechanisms may also be involved in determining coat color in these 2 breeds. Copyright 2009 S. Karger AG, Basel.

  15. Potential use of low-copy nuclear genes in DNA barcoding: a comparison with plastid genes in two Hawaiian plant radiations

    PubMed Central

    2013-01-01

    Background DNA barcoding of land plants has relied traditionally on a small number of markers from the plastid genome. In contrast, low-copy nuclear genes have received little attention as DNA barcodes because of the absence of universal primers for PCR amplification. Results From pooled-species 454 transcriptome data we identified two variable intron-less nuclear loci for each of two species-rich genera of the Hawaiian flora: Clermontia (Campanulaceae) and Cyrtandra (Gesneriaceae) and compared their utility as DNA barcodes with that of plastid genes. We found that nuclear genes showed an overall greater variability, but also displayed a high level of heterozygosity, intraspecific variation, and retention of ancient alleles. Thus, nuclear genes displayed fewer species-diagnostic haplotypes compared to plastid genes and no interspecies gaps. Conclusions The apparently greater coalescence times of nuclear genes are likely to limit their utility as barcodes, as only a small proportion of their alleles were fixed and unique to individual species. In both groups, species-diagnostic markers from either genome were scarce on the youngest island; a minimum age of ca. two million years may be needed for a species flock to be barcoded. For young plant groups, nuclear genes may not be a superior alternative to slowly evolving plastid genes. PMID:23394592

  16. Comparative humoral and cellular immunogenicity and safety of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine and HPV-6/11/16/18 vaccine in healthy women aged 18-45 years: follow-up through Month 48 in a Phase III randomized study.

    PubMed

    Einstein, Mark H; Levin, Myron J; Chatterjee, Archana; Chakhtoura, Nahida; Takacs, Peter; Catteau, Grégory; Dessy, Francis J; Moris, Philippe; Lin, Lan; Struyf, Frank; Dubin, Gary

    2014-01-01

    We previously reported higher anti-HPV-16 and -18 immune responses induced by HPV-16/18 vaccine compared with HPV-6/11/16/18 vaccine at Month 7 (one month after completion of full vaccination series) in women aged 18-45 y in an observer-blind study NCT00423046; the differences of immune response magnitudes were maintained up to Month 24. Here we report follow-up data through Month 48. At Month 48, in according-to-protocol cohort for immunogenicity (seronegative and DNA-negative for HPV type analyzed at baseline), geometric mean titers of serum neutralizing antibodies were 2.0- to 5.2-fold higher (HPV-16) and 8.6- to 12.8-fold higher (HPV-18) in HPV-16/18 vaccine group than in HPV-6/11/16/18 vaccine group. The majority of women in both vaccine groups remained seropositive for HPV-16. The same trend was observed for HPV-18 in HPV-16/18 vaccine group; however, seropositivity rates in HPV-6/11/16/18 vaccine group decreased considerably, particularly in the older age groups. In the total vaccinated cohort (regardless of baseline serological and HPV-DNA status), anti-HPV-16 and -18 neutralizing antibody levels induced by HPV-16/18 vaccine were higher than those induced by HPV-6/11/16/18 vaccine. CD4+ T-cell response for HPV-16 and HPV-18 was higher in HPV-16/18 vaccine group than in HPV-6/11/16/18 vaccine group. Memory B-cell responses appeared similar between vaccine groups. Both vaccines were generally well tolerated. Overall, the higher immune response observed with the HPV-16/18 vaccine was maintained up to Month 48. A head-to-head study incorporating clinical endpoints would be required to confirm whether the observed differences in immune response between the vaccines influence the duration of protection they provided.

  17. Type 2 diabetes mellitus disease risk genes identified by genome wide copy number variation scan in normal populations.

    PubMed

    Prabhanjan, Manasa; Suresh, Raviraj V; Murthy, Megha N; Ramachandra, Nallur B

    2016-03-01

    To identify the role of copy number variations (CNVs) on disease risk genes and its effect on disease phenotypes in type 2 diabetes mellitus (T2DM) in 12 random populations using high throughput arrays. CNV analysis was carried out on a total of 1715 individuals from 12 populations, from ArrayExpress Archive of the European Bioinformatics Institute along with our subjects using Affymetrix Genome Wide SNP 6.0 array. CNV effect on T2DM genes were analyzed using several bioinformatics tools and a molecular protein interaction network was constructed to identify the disease mechanism altered by the CNVs. Analysis showed 34.4% of the total population to be under CNV burden for T2DM, with 83 disease causal and associated genes being under CNV influence. Hotspots were identified on chromosomes 22, 12, 6, 19 and 11.Overlap studies with case cohorts revealed significant disease risk genes such as EGFR, E2F1, PPP1R3A, HLA and TSPAN8. CNVs play a significant role in predisposing T2DM in normal cohorts and contribute to the phenotypic effects. Thus, CNVs should be considered as one of the major contributors in predisposition of the disease. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. Assessing the impact of copy number variants on miRNA genes in autism by Monte Carlo simulation.

    PubMed

    Marrale, Maurizio; Albanese, Nadia Ninfa; Calì, Francesco; Romano, Valentino

    2014-01-01

    Autism Spectrum Disorders (ASDs) are childhood neurodevelopmental disorders with complex genetic origins. Previous studies have investigated the role of de novo Copy Number Variants (CNVs) and microRNAs as important but distinct etiological factors in ASD. We developed a novel computational procedure to assess the potential pathogenic role of microRNA genes overlapping de novo CNVs in ASD patients. Here we show that for chromosomes # 1, 2 and 22 the actual number of miRNA loci affected by de novo CNVs in patients was found significantly higher than that estimated by Monte Carlo simulation of random CNV events. Out of 24 miRNA genes over-represented in CNVs from these three chromosomes only hsa-mir-4436b-1 and hsa-mir-4436b-2 have not been detected in CNVs from non-autistic subjects as reported in the Database of Genomic Variants. Altogether the results reported in this study represent a first step towards a full understanding of how a dysregulated expression of the 24 miRNAs genes affect neurodevelopment in autism. We also propose that the procedure used in this study can be effectively applied to CNVs/miRNA genes association data in other genomic disorders beyond autism.

  19. Genome-wide copy number analysis reveals candidate gene loci that confer susceptibility to high-grade prostate cancer.

    PubMed

    Poniah, Prevathe; Mohd Zain, Shamsul; Abdul Razack, Azad Hassan; Kuppusamy, Shanggar; Karuppayah, Shankar; Sian Eng, Hooi; Mohamed, Zahurin

    2017-09-01

    Two key issues in prostate cancer (PCa) that demand attention currently are the need for a more precise and minimally invasive screening test owing to the inaccuracy of prostate-specific antigen and differential diagnosis to distinguish advanced vs. indolent cancers. This continues to pose a tremendous challenge in diagnosis and prognosis of PCa and could potentially lead to overdiagnosis and overtreatment complications. Copy number variations (CNVs) in the human genome have been linked to various carcinomas including PCa. Detection of these variants may improve clinical treatment as well as an understanding of the pathobiology underlying this complex disease. To this end, we undertook a pilot genome-wide CNV analysis approach in 36 subjects (18 patients with high-grade PCa and 18 controls that were matched by age and ethnicity) in search of more accurate biomarkers that could potentially explain susceptibility toward high-grade PCa. We conducted this study using the array comparative genomic hybridization technique. Array results were validated in 92 independent samples (46 high-grade PCa, 23 benign prostatic hyperplasia, and 23 healthy controls) using polymerase chain reaction-based copy number counting method. A total of 314 CNV regions were found to be unique to PCa subjects in this cohort (P<0.05). A log 2 ratio-based copy number analysis revealed 5 putative rare or novel CNV loci or both associated with susceptibility to PCa. The CNV gain regions were 1q21.3, 15q15, 7p12.1, and a novel CNV in PCa 12q23.1, harboring ARNT, THBS1, SLC5A8, and DDC genes that are crucial in the p53 and cancer pathways. A CNV loss and deletion event was observed at 8p11.21, which contains the SFRP1 gene from the Wnt signaling pathway. Cross-comparison analysis with genes associated to PCa revealed significant CNVs involved in biological processes that elicit cancer pathogenesis via cytokine production and endothelial cell proliferation. In conclusion, we postulated that the CNVs

  20. Copy Number Variations in Candidate Genes and Intergenic Regions Affect Body Mass Index and Abdominal Obesity in Mexican Children

    PubMed Central

    Burguete-García, Ana Isabel; Bonnefond, Amélie; Peralta-Romero, Jesús; Froguel, Philippe

    2017-01-01

    Introduction. Increase in body weight is a gradual process that usually begins in childhood and in adolescence as a result of multiple interactions among environmental and genetic factors. This study aimed to analyze the relationship between copy number variants (CNVs) in five genes and four intergenic regions with obesity in Mexican children. Methods. We studied 1423 children aged 6–12 years. Anthropometric measurements and blood levels of biochemical parameters were obtained. Identification of CNVs was performed by real-time PCR. The effect of CNVs on obesity or body composition was assessed using regression models adjusted for age, gender, and family history of obesity. Results. Gains in copy numbers of LEPR and NEGR1 were associated with decreased body mass index (BMI), waist circumference (WC), and risk of abdominal obesity, whereas gain in ARHGEF4 and CPXCR1 and the intergenic regions 12q15c, 15q21.1a, and 22q11.21d and losses in INS were associated with increased BMI and WC. Conclusion. Our results indicate a possible contribution of CNVs in LEPR, NEGR1, ARHGEF4, and CPXCR1 and the intergenic regions 12q15c, 15q21.1a, and 22q11.21d to the development of obesity, particularly abdominal obesity in Mexican children. PMID:28428959

  1. Sequence polymorphisms at the growth hormone GH1/GH2-N and GH2-Z gene copies and their relationship with dairy traits in domestic sheep (Ovis aries).

    PubMed

    Vacca, G M; Dettori, M L; Balia, F; Luridiana, S; Mura, M C; Carcangiu, V; Pazzola, M

    2013-09-01

    The purpose was to analyze the growth hormone GH1/GH2-N and GH2-Z gene copies and to assess their possible association with milk traits in Sarda sheep. Two hundred multiparous lactating ewes were monitored. The two gene copies were amplified separately and each was used as template for a nested PCR, to investigate single strand conformation polymorphism (SSCP) of the 5'UTR, exon-1, exon-5 and 3'UTR DNA regions. SSCP analysis revealed marked differences in the number of polymorphic patterns between the two genes. Sequencing revealed five nucleotide changes at the GH1/GH2-N gene. Five nucleotide changes occurred at the GH2-Z gene: one was located in exon-5 (c.556G > A) and resulted in a putative amino acid substitution G186S. All the nucleotide changes were copy-specific, except c.*30delT, which was common to both GH1/GH2-N and GH2-Z. Variability in the promoter regions of each gene might have consequences on the expression level, due to the involvement in potential transcription factor binding sites. Both gene copies influenced milk yield. A correlation with milk protein and casein content was also evidenced. These results may have implications that make them useful for future breeding strategies in dairy sheep breeding.

  2. Conserved Organisation of 45S rDNA Sites and rDNA Gene Copy Number among Major Clades of Early Land Plants

    PubMed Central

    Rosato, Marcela; Kovařík, Aleš; Garilleti, Ricardo; Rosselló, Josep A.

    2016-01-01

    Genes encoding ribosomal RNA (rDNA) are universal key constituents of eukaryotic genomes, and the nuclear genome harbours hundreds to several thousand copies of each species. Knowledge about the number of rDNA loci and gene copy number provides information for comparative studies of organismal and molecular evolution at various phylogenetic levels. With the exception of seed plants, the range of 45S rDNA locus (encoding 18S, 5.8S and 26S rRNA) and gene copy number variation within key evolutionary plant groups is largely unknown. This is especially true for the three earliest land plant lineages Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts). In this work, we report the extent of rDNA variation in early land plants, assessing the number of 45S rDNA loci and gene copy number in 106 species and 25 species, respectively, of mosses, liverworts and hornworts. Unexpectedly, the results show a narrow range of ribosomal locus variation (one or two 45S rDNA loci) and gene copies not present in vascular plant lineages, where a wide spectrum is recorded. Mutation analysis of whole genomic reads showed higher (3-fold) intragenomic heterogeneity of Marchantia polymorpha (Marchantiophyta) rDNA compared to Physcomitrella patens (Bryophyta) and two angiosperms (Arabidopsis thaliana and Nicotiana tomentosifomis) suggesting the presence of rDNA pseudogenes in its genome. No association between phylogenetic position, taxonomic adscription and the number of rDNA loci and gene copy number was found. Our results suggest a likely evolutionary rDNA stasis during land colonisation and diversification across 480 myr of bryophyte evolution. We hypothesise that strong selection forces may be acting against ribosomal gene locus amplification. Despite showing a predominant haploid phase and infrequent meiosis, overall rDNA homogeneity is not severely compromised in bryophytes. PMID:27622766

  3. RefCNV: Identification of Gene-Based Copy Number Variants Using Whole Exome Sequencing.

    PubMed

    Chang, Lun-Ching; Das, Biswajit; Lih, Chih-Jian; Si, Han; Camalier, Corinne E; McGregor, Paul M; Polley, Eric

    2016-01-01

    With rapid advances in DNA sequencing technologies, whole exome sequencing (WES) has become a popular approach for detecting somatic mutations in oncology studies. The initial intent of WES was to characterize single nucleotide variants, but it was observed that the number of sequencing reads that mapped to a genomic region correlated with the DNA copy number variants (CNVs). We propose a method RefCNV that uses a reference set to estimate the distribution of the coverage for each exon. The construction of the reference set includes an evaluation of the sources of variability in the coverage distribution. We observed that the processing steps had an impact on the coverage distribution. For each exon, we compared the observed coverage with the expected normal coverage. Thresholds for determining CNVs were selected to control the false-positive error rate. RefCNV prediction correlated significantly (r = 0.96-0.86) with CNV measured by digital polymerase chain reaction for MET (7q31), EGFR (7p12), or ERBB2 (17q12) in 13 tumor cell lines. The genome-wide CNV analysis showed a good overall correlation (Spearman's coefficient = 0.82) between RefCNV estimation and publicly available CNV data in Cancer Cell Line Encyclopedia. RefCNV also showed better performance than three other CNV estimation methods in genome-wide CNV analysis.

  4. The role of HPV RNA transcription, immune response-related gene expression and disruptive TP53 mutations in diagnostic and prognostic profiling of head and neck cancer.

    PubMed

    Wichmann, Gunnar; Rosolowski, Maciej; Krohn, Knut; Kreuz, Markus; Boehm, Andreas; Reiche, Anett; Scharrer, Ulrike; Halama, Dirk; Bertolini, Julia; Bauer, Ulrike; Holzinger, Dana; Pawlita, Michael; Hess, Jochen; Engel, Christoph; Hasenclever, Dirk; Scholz, Markus; Ahnert, Peter; Kirsten, Holger; Hemprich, Alexander; Wittekind, Christian; Herbarth, Olf; Horn, Friedemann; Dietz, Andreas; Loeffler, Markus

    2015-12-15

    Stratification of head and neck squamous cell carcinomas (HNSCC) based on HPV16 DNA and RNA status, gene expression patterns, and mutated candidate genes may facilitate patient treatment decision. We characterize head and neck squamous cell carcinomas (HNSCC) with different HPV16 DNA and RNA (E6*I) status from 290 consecutively recruited patients by gene expression profiling and targeted sequencing of 50 genes. We show that tumors with transcriptionally inactive HPV16 (DNA+ RNA-) are similar to HPV-negative (DNA-) tumors regarding gene expression and frequency of TP53 mutations (47%, 8/17 and 43%, 72/167, respectively). We also find that an immune response-related gene expression cluster is associated with lymph node metastasis, independent of HPV16 status and that disruptive TP53 mutations are associated with lymph node metastasis in HPV16 DNA- tumors. We validate each of these associations in another large data set. Four gene expression clusters which we identify differ moderately but significantly in overall survival. Our findings underscore the importance of measuring the HPV16 RNA (E6*I) and TP53-mutation status for patient stratification and identify associations of an immune response-related gene expression cluster and TP53 mutations with lymph node metastasis in HNSCC. © 2015 UICC.

  5. Apparent polyploidization after gamma irradiation: pitfalls in the use of quantitative polymerase chain reaction (qPCR) for the estimation of mitochondrial and nuclear DNA gene copy numbers.

    PubMed

    Kam, Winnie W Y; Lake, Vanessa; Banos, Connie; Davies, Justin; Banati, Richard

    2013-05-30

    Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization.

  6. Study of MET protein levels and MET gene copy number in 72 sinonasal intestinal-type adenocarcinomas.

    PubMed

    Projetti, Fabrice; Mesturoux, Laura; Coulibaly, Béma; Durand, Karine; Chaunavel, Alain; Léobon, Sophie; Gadeaud, Emilie; Caire, François; Bessède, Jean-Pierre; Labrousse, François

    2015-11-01

    Sinonasal intestinal-type adenocarcinomas (ITACs) have a poor prognosis, and are defined on the basis of their morphological similarities to colorectal adenocarcinomas. MET signaling pathway is involved in oncogenesis in various cancers. Nothing is currently known about the role of MET in ITACs. In a series of 72 ITACs, we investigated MET protein levels by immunohistochemistry (IHC) and gene copy number by in situ hybridization. These findings were analyzed as a function of clinical data, histological typing, and patient outcome. MET protein was overproduced in 64% of cases and chromosome 7 polysomy was observed in 52% of cases. No tumor displayed MET amplification. The presence of mucinous or solid histological components, T3/T4 tumors, and incomplete resection were associated with a poor outcome. MET is overproduced in about two third of ITACs, suggesting a role for the MET signaling pathway in the oncogenesis of these tumors. © 2014 Wiley Periodicals, Inc.

  7. Exploration of the gene fusion landscape of glioblastoma using transcriptome sequencing and copy number data.

    PubMed

    Shah, Nameeta; Lankerovich, Michael; Lee, Hwahyung; Yoon, Jae-Geun; Schroeder, Brett; Foltz, Greg

    2013-11-22

    RNA-seq has spurred important gene fusion discoveries in a number of different cancers, including lung, prostate, breast, brain, thyroid and bladder carcinomas. Gene fusion discovery can potentially lead to the development of novel treatments that target the underlying genetic abnormalities. In this study, we provide comprehensive view of gene fusion landscape in 185 glioblastoma multiforme patients from two independent cohorts. Fusions occur in approximately 30-50% of GBM patient samples. In the Ivy Center cohort of 24 patients, 33% of samples harbored fusions that were validated by qPCR and Sanger sequencing. We were able to identify high-confidence gene fusions from RNA-seq data in 53% of the samples in a TCGA cohort of 161 patients. We identified 13 cases (8%) with fusions retaining a tyrosine kinase domain in the TCGA cohort and one case in the Ivy Center cohort. Ours is the first study to describe recurrent fusions involving non-coding genes. Genomic locations 7p11 and 12q14-15 harbor majority of the fusions. Fusions on 7p11 are formed in focally amplified EGFR locus whereas 12q14-15 fusions are formed by complex genomic rearrangements. All the fusions detected in this study can be further visualized and analyzed using our website: http://ivygap.swedish.org/fusions. Our study highlights the prevalence of gene fusions as one of the major genomic abnormalities in GBM. The majority of the fusions are private fusions, and a minority of these recur with low frequency. A small subset of patients with fusions of receptor tyrosine kinases can benefit from existing FDA approved drugs and drugs available in various clinical trials. Due to the low frequency and rarity of clinically relevant fusions, RNA-seq of GBM patient samples will be a vital tool for the identification of patient-specific fusions that can drive personalized therapy.

  8. Modeling the impact of the difference in cross-protection data between a human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine and a human papillomavirus (HPV)-6/11/16/18 vaccine in Canada.

    PubMed

    Kohli, Michele; Lawrence, Donna; Haig, Jennifer; Anonychuk, Andrea; Demarteau, Nadia

    2012-10-13

    In Canada, two vaccines that have demonstrated high efficacy against infection with human papillomavirus (HPV) types -16 and -18 are available. The HPV-6/11/16/18 vaccine provides protection against genital warts (GW) while the HPV-16/18 vaccine may provide better protection against other oncogenic HPV types. In this analysis, the estimated clinical and economic benefit of each of these vaccines was compared in the Canadian setting. A Markov model of the natural history of HPV infection among women, cervical cancer (CC) and GW was used to estimate the impact of vaccinating a cohort of 100,000 12-year-old females on lifetime outcomes and healthcare system costs (no indirect benefit in males included). A budget impact model was used to estimate the impact of each vaccine by province. In the base case, vaccination with the HPV-16/18 vaccine was predicted to prevent 48 additional CC cases, and 16 additional CC deaths, while vaccination with the HPV-6/11/16/18 vaccine was predicted to prevent 6,933 additional GW cases. Vaccination with the HPV-16/18 vaccine was estimated to save 1 additional discounted quality adjusted life year (QALY) at an overall lower lifetime cost to the healthcare system compared to the HPV-6/11/16/18 vaccine (assuming vaccine price parity). In sensitivity analyses, the HPV-6/11/16/18 vaccine was associated with greater QALYs saved when the cross-protection efficacy of the HPV-16/18 vaccine was reduced, or the burden of GW due to HPV-6/11 was increased. In most scenarios with price parity, the lifetime healthcare cost of the strategy with the HPV-16/18 vaccine was predicted to be lower than the HPV-6/11/16/18 vaccine. In the probabilistic sensitivity analyses, the HPV-16/18 vaccine provided more QALY benefit than the HPV-6/11/16/18 vaccine in 49.2% of scenarios, with lower relative lifetime costs in 83.5% of scenarios. Overall, the predicted lifetime healthcare costs and QALYs saved by implementing each of the vaccines are similar. Vaccination

  9. Modeling the impact of the difference in cross-protection data between a human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine and a human papillomavirus (HPV)-6/11/16/18 vaccine in Canada

    PubMed Central

    2012-01-01

    Background In Canada, two vaccines that have demonstrated high efficacy against infection with human papillomavirus (HPV) types −16 and −18 are available. The HPV-6/11/16/18 vaccine provides protection against genital warts (GW) while the HPV-16/18 vaccine may provide better protection against other oncogenic HPV types. In this analysis, the estimated clinical and economic benefit of each of these vaccines was compared in the Canadian setting. Methods A Markov model of the natural history of HPV infection among women, cervical cancer (CC) and GW was used to estimate the impact of vaccinating a cohort of 100,000 12-year-old females on lifetime outcomes and healthcare system costs (no indirect benefit in males included). A budget impact model was used to estimate the impact of each vaccine by province. Results In the base case, vaccination with the HPV-16/18 vaccine was predicted to prevent 48 additional CC cases, and 16 additional CC deaths, while vaccination with the HPV-6/11/16/18 vaccine was predicted to prevent 6,933 additional GW cases. Vaccination with the HPV-16/18 vaccine was estimated to save 1 additional discounted quality adjusted life year (QALY) at an overall lower lifetime cost to the healthcare system compared to the HPV-6/11/16/18 vaccine (assuming vaccine price parity). In sensitivity analyses, the HPV-6/11/16/18 vaccine was associated with greater QALYs saved when the cross-protection efficacy of the HPV-16/18 vaccine was reduced, or the burden of GW due to HPV-6/11 was increased. In most scenarios with price parity, the lifetime healthcare cost of the strategy with the HPV-16/18 vaccine was predicted to be lower than the HPV-6/11/16/18 vaccine. In the probabilistic sensitivity analyses, the HPV-16/18 vaccine provided more QALY benefit than the HPV-6/11/16/18 vaccine in 49.2% of scenarios, with lower relative lifetime costs in 83.5% of scenarios. Conclusions Overall, the predicted lifetime healthcare costs and QALYs saved by implementing each

  10. Sustained efficacy and immunogenicity of the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine: analysis of a randomised placebo-controlled trial up to 6.4 years.

    PubMed

    Romanowski, B; de Borba, P Colares; Naud, P S; Roteli-Martins, C M; De Carvalho, N S; Teixeira, J C; Aoki, F; Ramjattan, B; Shier, R M; Somani, R; Barbier, S; Blatter, M M; Chambers, C; Ferris, D; Gall, S A; Guerra, F A; Harper, D M; Hedrick, J A; Henry, D C; Korn, A P; Kroll, R; Moscicki, A-B; Rosenfeld, W D; Sullivan, B J; Thoming, C S; Tyring, S K; Wheeler, C M; Dubin, G; Schuind, A; Zahaf, T; Greenacre, Mary; Sgriobhadair, An

    2009-12-12

    Prophylactic human papillomavirus (HPV) vaccines have to provide sustained protection. We assessed efficacy, immunogenicity, and safety of the HPV-16/18 AS04-adjuvanted vaccine up to 6.4 years. Women aged 15-25 years, with normal cervical cytology, who were HPV-16/18 seronegative and oncogenic HPV DNA-negative (14 types) at screening participated in a double-blind, randomised, placebo-controlled initial study (n=1113; 560 vaccine group vs 553 placebo group) and follow-up study (n=776; 393 vs 383). 27 sites in three countries participated in the follow-up study. Cervical samples were tested every 6 months for HPV DNA. Management of abnormal cytologies was prespecified, and HPV-16/18 antibody titres were assessed. The primary objective was to assess long-term vaccine efficacy in the prevention of incident cervical infection with HPV 16 or HPV 18, or both. We report the analyses up to 6.4 years of this follow-up study and combined with the initial study. For the primary endpoint, the efficacy analysis was done in the according-to-protocol (ATP) cohort; the analysis of cervical intraepithelial neoplasia grade 2 and above (CIN2+) was done in the total vaccinated cohort (TVC). The study is registered with ClinicalTrials.gov, number NCT00120848. For the combined analysis of the initial and follow-up studies, the ATP efficacy cohort included 465 women in the vaccine group and 454 in the placebo group; the TVC included 560 women in the vaccine group and 553 in the placebo group. Vaccine efficacy against incident infection with HPV 16/18 was 95.3% (95% CI 87.4-98.7) and against 12-month persistent infection was 100% (81.8-100). Vaccine efficacy against CIN2+ was 100% (51.3-100) for lesions associated with HPV-16/18 and 71.9% (20.6-91.9) for lesions independent of HPV DNA. Antibody concentrations by ELISA remained 12-fold or more higher than after natural infection (both antigens). Safety outcomes were similar between groups: during the follow-up study, 30 (8%) participants

  11. MG132 plus apoptosis antigen-1 (APO-1) antibody cooperate to restore p53 activity inducing autophagy and p53-dependent apoptosis in HPV16 E6-expressing keratinocytes.

    PubMed

    Lagunas-Martínez, Alfredo; García-Villa, Enrique; Arellano-Gaytán, Magaly; Contreras-Ochoa, Carla O; Dimas-González, Jisela; López-Arellano, María E; Madrid-Marina, Vicente; Gariglio, Patricio

    2017-01-01

    The E6 oncoprotein can interfere with the ability of infected cells to undergo programmed cell death through the proteolytic degradation of proapoptotic proteins such as p53, employing the proteasome pathway. Therefore, inactivation of the proteasome through MG132 should restore the activity of several proapoptotic proteins. We investigated whether in HPV16 E6-expressing keratinocytes (KE6 cells), the restoration of p53 levels mediated by MG132 and/or activation of the CD95 pathway through apoptosis antigen-1 (APO-1) antibody are responsible for the induction of apoptosis. We found that KE6 cells underwent apoptosis mainly after incubation for 24 h with MG132 alone or APO-1 plus MG132. Both treatments activated the extrinsic and intrinsic apoptosis pathways. Autophagy was also activated, principally by APO-1 plus MG132. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in the elevation of p53 protein levels and its phosphorylation in Ser46 and Ser20; the p53 protein was localized mainly at nucleus after treatment with MG132 or APO-1 plus MG132. In addition, induction of its transcriptional target genes such as p21, Bax and TP53INP was observed 3 and 6 h after treatment. Also, LC3 mRNA was induced after 3 and 6 h, which correlates with lipidation of LC3B protein and induction of autophagy. Finally, using pifithrin alpha we observed a decrease in apoptosis induced by MG132, and by APO-1 plus MG132, suggesting that restoration of APO-1 sensitivity occurs in part through an increase in both the levels and the activity of p53. The use of small molecules to inhibit the proteasome pathway might permit the activation of cell death, providing new opportunities for CC treatment.

  12. Obesity, starch digestion and amylase: association between copy number variants at human salivary (AMY1) and pancreatic (AMY2) amylase genes

    PubMed Central

    Carpenter, Danielle; Dhar, Sugandha; Mitchell, Laura M.; Fu, Beiyuan; Tyson, Jess; Shwan, Nzar A.A.; Yang, Fengtang; Thomas, Mark G.; Armour, John A.L.

    2015-01-01

    The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in association with body mass index. In this work, we use paralogue ratio tests, microsatellite analysis, read depth and fibre-FISH to demonstrate that human amylase CNV is not a smooth continuum, but is instead partitioned into distinct haplotype classes. There is a fundamental structural distinction between haplotypes containing odd or even numbers of AMY1 gene units, in turn coupled to CNV in pancreatic amylase genes AMY2A and AMY2B. Most haplotypes have one copy each of AMY2A and AMY2B and contain an odd number of copies of AMY1; consequently, most individuals have an even total number of AMY1. In contrast, haplotypes carrying an even number of AMY1 genes have rearrangements leading to CNVs of AMY2A/AMY2B. Read-depth and experimental data show that different populations harbour different proportions of these basic haplotype classes. In Europeans, the copy numbers of AMY1 and AMY2A are correlated, so that phenotypic associations caused by variation in pancreatic amylase copy number could be detected indirectly as weak association with AMY1 copy number. We show that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures we use and that qPCR data in other studies have been further compromised by systematic miscalibration. Our results uncover new patterns in human amylase variation and imply a potential role for AMY2 CNV in functional associations. PMID:25788522

  13. Obesity, starch digestion and amylase: association between copy number variants at human salivary (AMY1) and pancreatic (AMY2) amylase genes.

    PubMed

    Carpenter, Danielle; Dhar, Sugandha; Mitchell, Laura M; Fu, Beiyuan; Tyson, Jess; Shwan, Nzar A A; Yang, Fengtang; Thomas, Mark G; Armour, John A L

    2015-06-15

    The human salivary amylase genes display extensive copy number variation (CNV), and recent work has implicated this variation in adaptation to starch-rich diets, and in association with body mass index. In this work, we use paralogue ratio tests, microsatellite analysis, read depth and fibre-FISH to demonstrate that human amylase CNV is not a smooth continuum, but is instead partitioned into distinct haplotype classes. There is a fundamental structural distinction between haplotypes containing odd or even numbers of AMY1 gene units, in turn coupled to CNV in pancreatic amylase genes AMY2A and AMY2B. Most haplotypes have one copy each of AMY2A and AMY2B and contain an odd number of copies of AMY1; consequently, most individuals have an even total number of AMY1. In contrast, haplotypes carrying an even number of AMY1 genes have rearrangements leading to CNVs of AMY2A/AMY2B. Read-depth and experimental data show that different populations harbour different proportions of these basic haplotype classes. In Europeans, the copy numbers of AMY1 and AMY2A are correlated, so that phenotypic associations caused by variation in pancreatic amylase copy number could be detected indirectly as weak association with AMY1 copy number. We show that the quantitative polymerase chain reaction (qPCR) assay previously applied to the high-throughput measurement of AMY1 copy number is less accurate than the measures we use and that qPCR data in other studies have been further compromised by systematic miscalibration. Our results uncover new patterns in human amylase variation and imply a potential role for AMY2 CNV in functional associations. © The Author 2015. Published by Oxford University Press.

  14. Single-Copy Nuclear Genes Place Haustorial Hydnoraceae within Piperales and Reveal a Cretaceous Origin of Multiple Parasitic Angiosperm Lineages

    PubMed Central

    Naumann, Julia; Salomo, Karsten; Der, Joshua P.; Wafula, Eric K.; Bolin, Jay F.; Maass, Erika; Frenzke, Lena; Samain, Marie-Stéphanie; Neinhuis, Christoph

    2013-01-01

    Extreme haustorial parasites have long captured the interest of naturalists and scientists with their greatly reduced and highly specialized morphology. Along with the reduction or loss of photosynthesis, the plastid genome often decays as photosynthetic genes are released from selective constraint. This makes it challenging to use traditional plastid genes for parasitic plant phylogenetics, and has driven the search for alternative phylogenetic and molecular evolutionary markers. Thus, evolutionary studies, such as molecular clock-based age estimates, are not yet available for all parasitic lineages. In the present study, we extracted 14 nuclear single copy genes (nSCG) from Illumina transcriptome data from one of the “strangest plants in the world”, Hydnora visseri (Hydnoraceae). A ∼15,000 character molecular dataset, based on all three genomic compartments, shows the utility of nSCG for reconstructing phylogenetic relationships in parasitic lineages. A relaxed molecular clock approach with the same multi-locus dataset, revealed an ancient age of ∼91 MYA for Hydnoraceae. We then estimated the stem ages of all independently originated parasitic angiosperm lineages using a published dataset, which also revealed a Cretaceous origin for Balanophoraceae, Cynomoriaceae and Apodanthaceae. With the exception of Santalales, older parasite lineages tend to be more specialized with respect to trophic level and have lower species diversity. We thus propose the “temporal specialization hypothesis” (TSH) implementing multiple independent specialization processes over time during parasitic angiosperm evolution. PMID:24265760

  15. Deciphering the associations between gene expression and copy number alteration using a sparse double Laplacian shrinkage approach

    PubMed Central

    Shi, Xingjie; Zhao, Qing; Huang, Jian; Xie, Yang; Ma, Shuangge

    2015-01-01

    Motivation: Both gene expression levels (GEs) and copy number alterations (CNAs) have important biological implications. GEs are partly regulated by CNAs, and much effort has been devoted to understanding their relations. The regulation analysis is challenging with one gene expression possibly regulated by multiple CNAs and one CNA potentially regulating the expressions of multiple genes. The correlations among GEs and among CNAs make the analysis even more complicated. The existing methods have limitations and cannot comprehensively describe the regulation. Results: A sparse double Laplacian shrinkage method is developed. It jointly models the effects of multiple CNAs on multiple GEs. Penalization is adopted to achieve sparsity and identify the regulation relationships. Network adjacency is computed to describe the interconnections among GEs and among CNAs. Two Laplacian shrinkage penalties are imposed to accommodate the network adjacency measures. Simulation shows that the proposed method outperforms the competing alternatives with more accurate marker identification. The Cancer Genome Atlas data are analysed to further demonstrate advantages of the proposed method. Availability and implementation: R code is available at http://works.bepress.com/shuangge/49/ Contact: shuangge.ma@yale.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26342102

  16. Copy number variations of genes involved in stress responses reflect the redox state and DNA damage in brewing yeasts.

    PubMed

    Adamczyk, Jagoda; Deregowska, Anna; Skoneczny, Marek; Skoneczna, Adrianna; Natkanska, Urszula; Kwiatkowska, Aleksandra; Rawska, Ewa; Potocki, Leszek; Kuna, Ewelina; Panek, Anita; Lewinska, Anna; Wnuk, Maciej

    2016-09-01

    The yeast strains of the Saccharomyces sensu stricto complex involved in beer production are a heterogeneous group whose genetic and genomic features are not adequately determined. Thus, the aim of the present study was to provide a genetic characterization of selected group of commercially available brewing yeasts both ale top-fermenting and lager bottom-fermenting strains. Molecular karyotyping revealed that the diversity of chromosome patterns and four strains with the most accented genetic variabilities were selected and subjected to genome-wide array-based comparative genomic hybridization (array-CGH) analysis. The differences in the gene copy number were found in five functional gene categories: (1) maltose metabolism and transport, (2) response to toxin, (3) siderophore transport, (4) cellular aldehyde metabolic process, and (5) L-iditol 2-dehydrogenase activity (p < 0.05). In the Saflager W-34/70 strain (Fermentis) with the most affected array-CGH profile, loss of aryl-alcohol dehydrogenase (AAD) gene dosage correlated with an imbalanced redox state, oxidative DNA damage and breaks, lower levels of nucleolar proteins Nop1 and Fob1, and diminished tolerance to fermentation-associated stress stimuli compared to other strains. We suggest that compromised stress response may not only promote oxidant-based changes in the nucleolus state that may affect fermentation performance but also provide novel directions for future strain improvement.

  17. UGT2B17 and SULT1A1 gene copy number variation (CNV) detection by LabChip microfluidic technology.

    PubMed

    Gaedigk, Andrea; Gaedigk, Roger; Leeder, J Steven

    2010-05-01

    Gene copy number variations (CNVs) are increasingly recognized to play important roles in the expression of genes and hence on their respective enzymatic activities. This has been demonstrated for a number of drug metabolizing genes, such as UDP-glucuronosyltransferases 2B17 (UGT2B17) and sulfotransferase 1A1 (SULT1A1), which are subject to genetic heterogeneity, including CNV. Quantitative assays to assess gene copy number are therefore becoming an integral part of accurate genotype assessment and phenotype prediction. In this study, we evaluated a microfluidics-based system, the Bio-Rad Experion system, to determine the power and utility of this platform to detect UGT2B17 and SULT1A1 CNV in DNA samples derived from blood and tissue. UGT2B17 is known to present with 0, 1 or 2 and SULT1A1 with up to 5 gene copies. Distinct clustering (p<0.001) into copy number groups was achieved for both genes. DNA samples derived from blood exhibited less inter-run variability compared to DNA samples obtained from liver tissue. This variability may be caused by tissue-specific PCR inhibitors as it could be overcome by using DNA from another tissue, or after the DNA had undergone whole genome amplification. This method produced results comparable to those reported for other quantitative test platforms.

  18. Elevated PDGFRB gene copy number gain (CNG) is prognostic for improved survival outcomes in resected malignant pleural mesothelioma

    PubMed Central

    Tsao, Anne S.; Harun, Nusrat; Fujimoto, Junya; Devito, Vikki; Lee, J. Jack; Kuhn, Elisabetta; Mehran, Reza; Rice, David; Moran, Cesar; Hong, Waun Ki; Shen, Li; Suraokar, Milind; Wistuba, Ignacio

    2014-01-01

    Background PDGF/PDGFR pathway has been implicated in malignant pleural mesothelioma (MPM) carcinogenesis and evidence suggests autocrine mechanisms of proliferation. We sought to evaluate the incidence of PDGFRB gene copy number gain (CNG) by fluorescence in situ hybridization (FISH) and PDGFR pathway protein expression by immunohistochemistry (IHC) and correlate it to patient clinical outcome. Methods 88 archived tumor blocks from resected MPM with full clinical information were used to perform IHC biomarkers (PDGFRα, PDGFRβ, p-PDGFRβ) and FISH analysis of PDGFRB gene CNG. Spearman's rank correlation, Wilcoxon rank-sum test, Kruskal-Wallis test, BLiP plots, and Kaplan-Meier method were used to analyze the biomarkers and correlation to clinical outcome. Results Several correlations between the IHC biomarkers were seen; however, none correlated to clinically relevant patient demographics or histology. In the CNG analysis, PDGFRB gene CNG in > 10% of tumor cells had lower cytoplasmic p-PDGFRβ (p=0.029), while PDGFRB gene CNG in > 40% of tumor cells had a higher cytoplasmic PDGFRβ (p=0.04). PDGFRB gene CNG status did not associate with patient demographics or tumor characteristics. PDGFR pathway IHC biomarkers did not associate with survival outcomes. However, patients with PDGFRB CNG > 40% of tumor cells had improved relapse-free survival [HR 0.25 (95% CI 0.09, 0.72), p=0.0096] and improved overall survival [HR 0.32 (95% CI 0.11, 0.89), p=0.029]. Conclusions PDGFRB CNG > 40% of MPM tumor cells is a potential prognostic biomarker for surgery and may identify a unique population of mesothelioma patients. Future validation of this biomarker in prospective trials is needed. PMID:24747001

  19. Disruption of the psbA gene by the copy correction mechanism reveals that the expression of plastid-encoded genes is regulated by photosynthesis activity.

    PubMed

    Khan, Muhammad Sarwar; Hameed, Waqar; Nozoe, Mikio; Shiina, Takashi

    2007-05-01

    The functional analysis of genes encoded by the chloroplast genome of tobacco by reverse genetics is routine. Nevertheless, for a small number of genes their deletion generates heteroplasmic genotypes, complicating their analysis. There is thus the need for additional strategies to develop deletion mutants for these genes. We have developed a homologous copy correction-based strategy for deleting/mutating genes encoded on the chloroplast genome. This system was used to produce psbA knockouts. The resulting plants are homoplasmic and lack photosystem II (PSII) activity. Further, the deletion mutants exhibit a distinct phenotype; young leaves are green, whereas older leaves are bleached, irrespective of light conditions. This suggests that senescence is promoted by the absence of psbA. Analysis of the transcript levels indicates that NEP (nuclear-encoded plastid RNA polymerase)-dependent plastid genes are up regulated in the psbA deletion mutants, whereas the bleached leaves retain plastid-encoded plastid RNA polymerase activity. Hence, the expression of NEP-dependent plastid genes may be regulated by photosynthesis, either directly or indirectly.

  20. Novel genes involved in severe early-onset obesity revealed by rare copy number and sequence variants.

    PubMed

    Serra-Juhé, Clara; Martos-Moreno, Gabriel Á; Bou de Pieri, Francesc; Flores, Raquel; González, Juan R; Rodríguez-Santiago, Benjamín; Argente, Jesús; Pérez-Jurado, Luis A

    2017-05-01

    Obesity is a multifactorial disorder with high heritability (50-75%), which is probably higher in early-onset and severe cases. Although rare monogenic forms and several genes and regions of susceptibility, including copy number variants (CNVs), have been described, the genetic causes underlying the disease still remain largely unknown. We searched for rare CNVs (>100kb in size, altering genes and present in <1/2000 population controls) in 157 Spanish children with non-syndromic early-onset obesity (EOO: body mass index >3 standard deviations above the mean at <3 years of age) using SNP array molecular karyotypes. We then performed case control studies (480 EOO cases/480 non-obese controls) with the validated CNVs and rare sequence variants (RSVs) detected by targeted resequencing of selected CNV genes (n = 14), and also studied the inheritance patterns in available first-degree relatives. A higher burden of gain-type CNVs was detected in EOO cases versus controls (OR = 1.71, p-value = 0.0358). In addition to a gain of the NPY gene in a familial case with EOO and attention deficit hyperactivity disorder, likely pathogenic CNVs included gains of glutamate receptors (GRIK1, GRM7) and the X-linked gastrin-peptide receptor (GRPR), all inherited from obese parents. Putatively functional RSVs absent in controls were also identified in EOO cases at NPY, GRIK1 and GRPR. A patient with a heterozygous deletion disrupting two contiguous and related genes, SLCO4C1 and SLCO6A1, also had a missense RSV at SLCO4C1 on the other allele, suggestive of a recessive model. The genes identified showed a clear enrichment of shared co-expression partners with known genes strongly related to obesity, reinforcing their role in the pathophysiology of the disease. Our data reveal a higher burden of rare CNVs and RSVs in several related genes in patients with EOO compared to controls, and implicate NPY, GRPR, two glutamate receptors and SLCO4C1 in highly penetrant forms of familial obesity.

  1. Novel genes involved in severe early-onset obesity revealed by rare copy number and sequence variants

    PubMed Central

    Flores, Raquel; González, Juan R.; Argente, Jesús; Pérez-Jurado, Luis A.

    2017-01-01

    Obesity is a multifactorial disorder with high heritability (50–75%), which is probably higher in early-onset and severe cases. Although rare monogenic forms and several genes and regions of susceptibility, including copy number variants (CNVs), have been described, the genetic causes underlying the disease still remain largely unknown. We searched for rare CNVs (>100kb in size, altering genes and present in <1/2000 population controls) in 157 Spanish children with non-syndromic early-onset obesity (EOO: body mass index >3 standard deviations above the mean at <3 years of age) using SNP array molecular karyotypes. We then performed case control studies (480 EOO cases/480 non-obese controls) with the validated CNVs and rare sequence variants (RSVs) detected by targeted resequencing of selected CNV genes (n = 14), and also studied the inheritance patterns in available first-degree relatives. A higher burden of gain-type CNVs was detected in EOO cases versus controls (OR = 1.71, p-value = 0.0358). In addition to a gain of the NPY gene in a familial case with EOO and attention deficit hyperactivity disorder, likely pathogenic CNVs included gains of glutamate receptors (GRIK1, GRM7) and the X-linked gastrin-peptide receptor (GRPR), all inherited from obese parents. Putatively functional RSVs absent in controls were also identified in EOO cases at NPY, GRIK1 and GRPR. A patient with a heterozygous deletion disrupting two contiguous and related genes, SLCO4C1 and SLCO6A1, also had a missense RSV at SLCO4C1 on the other allele, suggestive of a recessive model. The genes identified showed a clear enrichment of shared co-expression partners with known genes strongly related to obesity, reinforcing their role in the pathophysiology of the disease. Our data reveal a higher burden of rare CNVs and RSVs in several related genes in patients with EOO compared to controls, and implicate NPY, GRPR, two glutamate receptors and SLCO4C1 in highly penetrant forms of familial obesity

  2. Threshold cost-effectiveness analysis for a therapeutic vaccine against HPV-16/18-positive cervical intraepithelial neoplasia in the Netherlands.

    PubMed

    Luttjeboer, Jos; Setiawan, Didik; Cao, Qi; Cahh Daemen, Toos; Postma, Maarten J

    2016-12-07

    In this study, the potential price for a therapeutic vaccine against Human Papilloma Virus (HPV)-16 & 18 (pre)-malignant cervical lesions is examined. A decision tree model was built in the context of the new Dutch cervical cancer-screening program and includes a primary test for the presence of HPV. Based on data of cervical cancer screening and HPV prevalence in the Netherlands, cohorts were created with HPV-16 or 18 positive women with cervical intraepithelial neoplasia (CIN) 2 or 3 or cervical cancer stage 1A (FIGO 1A). In the base case, the vaccine price was based on equal numbers of effective treatments in the vaccine branch and the current treatments branch of the model, and parity in cost, i.e. total cost in both branches are the same. The vaccine price is calculated by subtracting the cost of the vaccine branch from cost in the standard treatment branch and divided by the total number of women in the cohort, thereby equalizing costs in both strategies. Scenario analyses were performed taking quality adjusted life years (QALYs) into account with €20,000/QALY, €50,000/QALY and €80,000/QALY as corresponding thresholds. Sensitivity analyses were specifically targeted at the characteristics of the type-specific HPV test in the screening practice and vaccine efficacy. A probabilistic sensitivity analysis (PSA) was performed to quantify the level of uncertainty of the results found in the base case. In the base case, break-even vaccine prices of €381, €568 and €1697 were found for CIN 2, CIN 3 and FIGO 1A, respectively. The PSA showed vaccine pricing below €310, €490 and €1660 will be cost saving with a likelihood of 95% for CIN 2, CIN 3 and FIGO 1A, respectively. The vaccine price proved to be very sensitive for inclusion of QALY gains, including the HPV-type specific test into the Dutch screening practice and vaccine efficacy. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Efficacy of the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical intraepithelial neoplasia and cervical infection in young Japanese women

    PubMed Central

    Konno, Ryo; Yoshikawa, Hiroyuki; Okutani, Marie; Quint, Wim; V Suryakiran, Pemmaraju; Lin, Lan; Struyf, Frank

    2014-01-01

    In this open, extended follow-up study (NCT00929526, Clinicaltrials.gov), we evaluated the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine efficacy, immunogenicity and safety up to 4 years after first vaccination in Japanese women aged 20–25 years. In the initial randomized, double-blind study (NCT00316693), 1040 women received the study vaccine or hepatitis A control vaccine; 752 women were included in the follow-up study. In women from the according-to-protocol efficacy cohort (ATP-E), who were initially seronegative for the HPV type analyzed, no cervical intraepithelial neoplasia (CIN) grade 1 or greater (CIN1+) cases associated with HPV-16/18 were reported in the HPV group, while in the control group, 5 cases were identified in extended follow-up analyses (vaccine efficacy [VE] 100% [95% CI: −3.7–100]) and 8 cases in combined initial and follow-up studies analyses (VE 100% [42.2–100]). In the ATP-E, VE against CIN1+ and CIN2+ associated with high-risk HPV types reached 66.4% (21.6–87.1) and 83.0% (22.1–98.2) in extended follow-up analyses, and 63.4% (28.8–82.3) and 77.3% (30.4–94.4) in analyses of combined studies, respectively. During the 4-year period, protection against CIN1+ and CIN2+, irrespective of the HPV type, was 56.7% (32.8–72.6) and 54.9% (20.5–75.3) in women receiving ≥1 vaccine dose, regardless of baseline serostatus (total vaccinated cohort [TVC]) and 61.0% (11.8–84.2) and 73.9% (1.1–95.3) in women naïve to HPV infection at baseline (TVC-naïve), respectively. The high VE observed in Japanese women, accompanied by a sustained immune response and a clinically acceptable safety profile, support findings of large, international trials. PMID:25424783

  4. Rapid evolution and copy number variation of primate RHOXF2, an X-linked homeobox gene involved in male reproduction and possibly brain function.

    PubMed

    Niu, Ao-lei; Wang, Yin-qiu; Zhang, Hui; Liao, Cheng-hong; Wang, Jin-kai; Zhang, Rui; Che, Jun; Su, Bing

    2011-10-12

    Homeobox genes are the key regulators during development, and they are in general highly conserved with only a few reported cases of rapid evolution. RHOXF2 is an X-linked homeobox gene in primates. It is highly expressed in the testicle and may play an important role in spermatogenesis. As male reproductive system is often the target of natural and/or sexual selection during evolution, in this study, we aim to dissect the pattern of molecular evolution of RHOXF2 in primates and its potential functional consequence. We studied sequences and copy number variation of RHOXF2 in humans and 16 nonhuman primate species as well as the expression patterns in human, chimpanzee, white-browed gibbon and rhesus macaque. The gene copy number analysis showed that there had been parallel gene duplications/losses in multiple primate lineages. Our evidence suggests that 11 nonhuman primate species have one RHOXF2 copy, and two copies are present in humans and four Old World monkey species, and at least 6 copies in chimpanzees. Further analysis indicated that the gene duplications in primates had likely been mediated by endogenous retrovirus (ERV) sequences flanking the gene regions. In striking contrast to non-human primates, humans appear to have homogenized their two RHOXF2 copies by the ERV-mediated non-allelic recombination mechanism. Coding sequence and phylogenetic analysis suggested multi-lineage strong positive selection on RHOXF2 during primate evolution, especially during the origins of humans and chimpanzees. All the 8 coding region polymorphic sites in human populations are non-synonymous, implying on-going selection. Gene expression analysis demonstrated that besides the preferential expression in the reproductive system, RHOXF2 is also expressed in the brain. The quantitative data suggests expression pattern divergence among primate species. RHOXF2 is a fast-evolving homeobox gene in primates. The rapid evolution and copy number changes of RHOXF2 had been driven by

  5. Contribution of copy number variants involving nonsense-mediated mRNA decay pathway genes to neuro-developmental disorders.

    PubMed

    Nguyen, Lam S; Kim, Hyung-Goo; Rosenfeld, Jill A; Shen, Yiping; Gusella, James F; Lacassie, Yves; Layman, Lawrence C; Shaffer, Lisa G; Gécz, Jozef

    2013-05-01

    The nonsense-mediated mRNA decay (NMD) pathway functions not only to degrade transcripts containing premature termination codons (PTC), but also to regulate the transcriptome. UPF3B and RBM8A, important components of NMD, have been implicated in various forms of intellectual disability (ID) and Thrombocytopenia with Absent Radius (TAR) syndrome, which is also associated with ID. To gauge the contribution of other NMD factors to ID, we performed a comprehensive search for copy number variants (CNVs) of 18 NMD genes among individuals with ID and/or congenital anomalies. We identified 11 cases with heterozygous deletions of the genomic region encompassing UPF2, which encodes for a direct interacting protein of UPF3B. Using RNA-Seq, we showed that the genome-wide consequence of reduced expression of UPF2 is similar to that seen in patients with UPF3B mutations. Out of the 1009 genes found deregulated in patients with UPF2 deletions by at least 2-fold, majority (95%) were deregulated similarly in patients with UPF3B mutations. This supports the major role of deletion of UPF2 in ID. Furthermore, we found that four other NMD genes, UPF3A, SMG6, EIF4A3 and RNPS1 are frequently deleted and/or duplicated in the patients. We postulate that dosage imbalances of these NMD genes are likely to be the causes or act as predisposing factors for neuro-developmental disorders. Our findings further emphasize the importance of NMD pathway(s) in learning and memory.

  6. A Recombinant Rabies Virus Encoding Two Copies of the Glycoprotein Gene Confers Protection in Dogs against a Virulent Challenge

    PubMed Central

    Sun, Zhaojin; Chen, Jing; Ai, Jun; Dun, Can; Fu, Zhen F.; Niu, Xuefeng; Guo, Xiaofeng

    2014-01-01

    The rabies virus (RABV) glycoprotein (G) is the principal antigen responsible for the induction of virus neutralizing antibodies (VNA) and is the major modality of protective immunity in animals. A recombinant RABV HEP-Flury strain was generated by reverse genetics to encode two copies of the G-gene (referred to as HEP-dG). The biological properties of HEP-dG were compared to those of the parental virus (HEP-Flury strain). The HEP-dG recombinant virus grew 100 times more efficiently in BHK-21 cell than the parental virus, yet the virulence of the dG recombinant virus in suckling mice was lower than the parental virus. The HEP-dG virus can improve the expression of G-gene mRNA and the G protein and produce more offspring viruses in cells. The amount of G protein revealed a positive relationship with immunogenicity in mice and dogs. The inactivated HEP-dG recombinant virus induced higher levels of VNA and conferred better protection against virulent RABV in mice and dogs than the inactivated parental virus and a commercial vaccine. The protective antibody persisted for at least 12 months. These data demonstrate that the HEP-dG is stable, induces a strong VNA response and confers protective immunity more effectively than the RABV HEP-Flury strain. HEP-dG could be a potential candidate in the development of novel inactivated rabies vaccines PMID:24498294

  7. Individual Differences in AMY1 Gene Copy Number, Salivary α-Amylase Levels, and the Perception of Oral Starch

    PubMed Central

    Mandel, Abigail L.; Peyrot des Gachons, Catherine; Plank, Kimberly L.; Alarcon, Suzanne; Breslin, Paul A. S.

    2010-01-01

    Background The digestion of dietary starch in humans is initiated by salivary α-amylase, an endo-enzyme that hydrolyzes starch into maltose, maltotriose and larger oligosaccharides. Salivary amylase accounts for 40 to 50% of protein in human saliva and rapidly alters the physical properties of starch. Importantly, the quantity and enzymatic activity of salivary amylase show significant individual variation. However, linking variation in salivary amylase levels with the oral perception of starch has proven difficult. Furthermore, the relationship between copy number variations (CNVs) in the AMY1 gene, which influence salivary amylase levels, and starch viscosity perception has not been explored. Principal Findings Here we demonstrate that saliva containing high levels of amylase has sufficient activity to rapidly hydrolyze a viscous starch solution in vitro. Furthermore, we show with time-intensity ratings, which track the digestion of starch during oral manipulation, that individuals with high amylase levels report faster and more significant decreases in perceived starch viscosity than people with low salivary amylase levels. Finally, we demonstrate that AMY1 CNVs predict an individual's amount and activity of salivary amylase and thereby, ultimately determine their perceived rate of oral starch viscosity thinning. Conclusions By linking genetic variation and its consequent salivary enzymatic differences to the perceptual sequellae of these variations, we show that AMY1 copy number relates to salivary amylase concentration and enzymatic activity level, which, in turn, account for individual variation in the oral perception of starch viscosity. The profound individual differences in salivary amylase levels and salivary activity may contribute significantly to individual differences in dietary starch intake and, consequently, to overall nutritional status. PMID:20967220

  8. Vitamin K3 induces antiproliferative effect in cervical epithelial cells transformed by HPV 16 (SiHa cells) through the increase in reactive oxygen species production.

    PubMed

    de Carvalho Scharf Santana, Natália; Lima, Natália Alves; Desoti, Vânia Cristina; Bidóia, Danielle Lazarin; de Souza Bonfim Mendonça, Patrícia; Ratti, Bianca Altrão; Nakamura, Tânia Ueda; Nakamura, Celso Vataru; Consolaro, Marcia Edilaine Lopes; Ximenes, Valdecir Farias; de Oliveira Silva, Sueli

    2016-10-01

    Cervical cancer is characterized as an important public health problem. According to latest estimates, cancer of the cervix is the fourth most common cancer among women. Due to its high prevalence, the search for new and efficient drugs to treat this infection is continuous. The progression of HPV-associated cervical cancer involves the expression of two viral proteins, E6 and E7, which are rapidly degraded by the ubiquitin-proteasome system through the increase in reactive oxygen species generation. Vitamins are essential to human substances, participate in the regulation of metabolism, and facilitate the process of energy transfer. Some early studies have indicated that vitamin K3 exerts antitumor activity by inducing cell death by apoptosis through an increase in the generation of reactive oxygen species. Thus, we evaluated the antiproliferative effect and a likely mechanism of action of vitamin K3 against cervical epithelial cells transformed by HPV 16 (SiHa cells) assessing the production of total ROS, the mitochondrial membrane potential, the cell morphology, the cell volume, and the cell membrane integrity. Our results show that vitamin K3 induces an increase in ROS production in SiHa cells, triggering biochemical and morphological events, such as depolarization of mitochondrial membrane potential and decreasing cell volume. Our data showed that vitamin K3 generates an oxidative imbalance in SiHa cells, leading to mechanisms that induce cell death by apoptosis.

  9. No evidence for cross-protection of the HPV-16/18 vaccine against HPV-6/11 positivity in female STI clinic visitors.

    PubMed

    Woestenberg, Petra J; King, Audrey J; van der Sande, Marianne A B; Donken, Robine; Leussink, Suzan; van der Klis, Fiona R M; Hoebe, Christian J P A; Bogaards, Johannes A; van Benthem, Birgit H B

    2017-04-01

    Data from a vaccine trial and from post-vaccine surveillance in the United Kingdom have suggested that the bivalent HPV-16/18 vaccine offers cross-protection against HPV-6/11 and protection against anogenital warts (AGW). We studied the effect of the bivalent vaccine on genital HPV-6/11 positivity and AGW in the Netherlands. We included all vaccine-eligible women from the PASSYON study, a biennial cross-sectional study among 16- to 24-year-old sexually transmitted infection (STI) clinic attendants. Vaginal self-swabs were analyzed for type specific HPV and AGW were diagnosed at the STI-clinic. Prevalence of HPV-6 and/or HPV-11 and AGW were compared between self-reported vaccinated and unvaccinated women by log-binomial regression analysis, adjusted for demographics and risk behavior. Of the 1198 women included, 56% reported to be vaccinated at least once. Relative to unvaccinated women, the adjusted prevalence ratio (PR) for HPV-6/11 was 1.03 (95% confidence interval [CI] 0.74-1.43) for women vaccinated at least once. The crude PR for AGW was 0.67 (95% CI 0.22-2.07) for women vaccinated at least once. Adjustment did not change these results. We observed no cross-protective effect of the bivalent vaccine on genital HPV-6/11 positivity and a non-significant partially protective effect on AGW. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Solution structure analysis of the HPV16 E6 oncoprotein reveals a self-association mechanism required for E6-mediated degradation of p53

    PubMed Central

    Zanier, Katia; Sidi, Abdellahi ould M’hamed ould; Boulade-Ladame, Charlotte; Rybin, Vladimir; Chappelle, Anne; Atkinson, Andrew; Kieffer, Bruno; Travé, Gilles

    2012-01-01

    The viral oncoprotein E6 is an essential factor for cervical cancers induced by “high-risk” mucosal HPV. Among other oncogenic activities, E6 recruits the ubiquitin ligase E6AP to promote the ubiquitination and subsequent proteasomal degradation of p53. E6 is prone to self-association, which long precluded its structural analysis. Here we found that E6 specifically dimerizes through its N-terminal domain and that disruption of the dimer interface strongly increases E6 solubility. This allowed us to raise the first structural data covering the entire HPV16 E6 protein, including the high-resolution NMR structures of the two zinc-binding domains of E6 and a robust data-driven model structure of the N-terminal domain homodimer. Interestingly, homodimer interface mutations that disrupt E6 self-association also inactivate E6-mediated p53 degradation. These data suggest that E6 needs to self-associate via its N-terminal domain to promote the poly-ubiquitination of p53 by E6AP. PMID:22483108

  11. Copy Number Variation Affecting the Photoperiod-B1 and Vernalization-A1 Genes Is Associated with Altered Flowering Time in Wheat (Triticum aestivum)

    PubMed Central

    Isaac, Peter; Laurie, David A.

    2012-01-01

    The timing of flowering during the year is an important adaptive character affecting reproductive success in plants and is critical to crop yield. Flowering time has been extensively manipulated in crops such as wheat (Triticum aestivum L.) during domestication, and this enables them to grow productively in a wide range of environments. Several major genes controlling flowering time have been identified in wheat with mutant alleles having sequence changes such as insertions, deletions or point mutations. We investigated genetic variants in commercial varieties of wheat that regulate flowering by altering photoperiod response (Ppd-B1 alleles) or vernalization requirement (Vrn-A1 alleles) and for which no candidate mutation was found within the gene sequence. Genetic and genomic approaches showed that in both cases alleles conferring altered flowering time had an increased copy number of the gene and altered gene expression. Alleles with an increased copy number of Ppd-B1 confer an early flowering day neutral phenotype and have arisen independently at least twice. Plants with an increased copy number of Vrn-A1 have an increased requirement for vernalization so that longer periods of cold are required to potentiate flowering. The results suggest that copy number variation (CNV) plays a significant role in wheat adaptation. PMID:22457747

  12. Exome sequencing and arrayCGH detection of gene sequence and copy number variation between ILS and ISS mouse strains.

    PubMed

    Dumas, Laura; Dickens, C Michael; Anderson, Nathan; Davis, Jonathan; Bennett, Beth; Radcliffe, Richard A; Sikela, James M

    2014-06-01

    It has been well documented that genetic factors can influence predisposition to develop alcoholism. While the underlying genomic changes may be of several types, two of the most common and disease associated are copy number variations (CNVs) and sequence alterations of protein coding regions. The goal of this study was to identify CNVs and single-nucleotide polymorphisms that occur in gene coding regions that may play a role in influencing the risk of an individual developing alcoholism. Toward this end, two mouse strains were used that have been selectively bred based on their differential sensitivity to alcohol: the Inbred long sleep (ILS) and Inbred short sleep (ISS) mouse strains. Differences in initial response to alcohol have been linked to risk for alcoholism, and the ILS/ISS strains are used to investigate the genetics of initial sensitivity to alcohol. Array comparative genomic hybridization (arrayCGH) and exome sequencing were conducted to identify CNVs and gene coding sequence differences, respectively, between ILS and ISS mice. Mouse arrayCGH was performed using catalog Agilent 1 × 244 k mouse arrays. Subsequently, exome sequencing was carried out using an Illumina HiSeq 2000 instrument. ArrayCGH detected 74 CNVs that were strain-specific (38 ILS/36 ISS), including several ISS-specific deletions that contained genes implicated in brain function and neurotransmitter release. Among several interesting coding variations detected by exome sequencing was the gain of a premature stop codon in the alpha-amylase 2B (AMY2B) gene specifically in the ILS strain. In total, exome sequencing detected 2,597 and 1,768 strain-specific exonic gene variants in the ILS and ISS mice, respectively. This study represents the most comprehensive and detailed genomic comparison of ILS and ISS mouse strains to date. The two complementary genome-wide approaches identified strain-specific CNVs and gene coding sequence variations that should provide strong candidates to

  13. Copy Number Variation of TLR-7 Gene and its Association with the Development of Systemic Lupus Erythematosus in Female Patients from Yucatan Mexico

    PubMed Central

    Pacheco, Guillermo Valencia; Cruz, Darig Cámara; González Herrera, Lizbeth J; Pérez Mendoza, Gerardo J; Adrián Amaro, Guadalupe I; Nakazawa Ueji, Yumi E; Angulo Ramírez, Angélica V

    2014-01-01

    Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of autoantibodies against self-antigens, which occurs most often in women between 15 and 40 years of age. The innate immunity is involved in the pathogenesis of SLE through TLR- 7. Genetic factors such as copy number variation (CNV) of target genes may contribute to disease development, but this possible risk has not yet been studied in SLE patients from Yucatan, Mexico. The CNV of TLR-7 gene was determined by quantitative polymerase chain reaction assay using TaqMan probes in 80 SLE women and 150 control subjects. The results showed that 10% of SLE patients exhibited more than two copies of TLR-7 gene, whereas no mRNA overexpression was detected. These data suggested that increased CNV of the TLR-7 gene in Yucatan SLE women can be a risk factor for this disease. PMID:25512712

  14. Construction of a restriction map and gene map of the lettuce chloroplast small single-copy region using Southern cross-hybridization.

    PubMed

    Mitchelson, K R

    1996-01-01

    The small single-copy region (SSCR) of the chloroplast genome of many higher plants typically contain ndh genes encoding proteins that share homology with subunits of the respiratory-chain reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase complex of mitochondria. A map of the lettuce chloroplast SSCR has been determined by Southern cross-hybridization, taking advantage of the high degree of homology between a tobacco small single-copy fragment and a corresponding lettuce chloroplast fragment. The gene order of the SSCR of lettuce and tobacco chloroplasts is similar. The cross-hybridization method can rapidly create a primary gene map of unknown chloroplast fragments, thus providing detailed information of the localization and arrangement of genes and conserved open reading frame regions.

  15. Copy number variations in "classical" obesity candidate genes are not frequently associated with severe early-onset obesity in children.

    PubMed

    Windholz, Jan; Kovacs, Peter; Schlicke, Marina; Franke, Christin; Mahajan, Anubha; Morris, Andrew P; Lemke, Johannes R; Klammt, Jürgen; Kiess, Wieland; Schöneberg, Torsten; Pfäffle, Roland; Körner, Antje

    2017-05-01

    Obesity is genetically heterogeneous and highly heritable, although polymorphisms explain the phenotype in only a small proportion of obese children. We investigated the presence of copy number variations (CNVs) in "classical" genes known to be associated with (monogenic) early-onset obesity in children. In 194 obese Caucasian children selected for early-onset and severe obesity from our obesity cohort we screened for deletions and/or duplications by multiplex ligation-dependent probe amplification reaction (MLPA). As we found one MLPA probe to interfere with a polymorphism in SIM1 we investigated its association with obesity and other phenotypic traits in our extended cohort of 2305 children. In the selected subset of most severely obese children, we did not find CNV with MLPA in POMC, LEP, LEPR, MC4R, MC3R or MC2R genes. However, one SIM1 probe located at exon 9 gave signals suggestive for SIM1 insufficiency in 52 patients. Polymerase chain reaction (PCR) analysis identified this as a false positive result due to interference with single nucleotide polymorphism (SNP) rs3734354/rs3734355. We, therefore, investigated for associations of this polymorphism with obesity and metabolic traits in our extended cohort. We found rs3734354/rs3734355 to be associated with body mass index-standard deviation score (BMI-SDS) (p = 0.003), but not with parameters of insulin metabolism, blood pressure or food intake. In our modest sample of severely obese children, we were unable to find CNVs in well-established monogenic obesity genes. Nevertheless, we found an association of rs3734354 in SIM1 with obesity of early-onset type in children, although not with obesity-related traits.

  16. Low gene copy number shows that arbuscular mycorrhizal fungi inherit genetically different nuclei.

    PubMed

    Hijri, Mohamed; Sanders, Ian R

    2005-01-13

    Arbuscular mycorrhizal fungi (AMF) are ancient asexually reproducing organisms that form symbioses with the majority of plant species, improving plant nutrition and promoting plant diversity. Little is known about the evolution or organization of the genomes of any eukaryotic symbiont or ancient asexual organism. Direct evidence shows that one AMF species is heterokaryotic; that is, containing populations of genetically different nuclei. It has been suggested, however, that the genetic variation passed from generation to generation in AMF is simply due to multiple chromosome sets (that is, high ploidy). Here we show that previously documented genetic variation in Pol-like sequences, which are passed from generation to generation, cannot be due to either high ploidy or repeated gene duplications. Our results provide the clearest evidence so far for substantial genetic differences among nuclei in AMF. We also show that even AMF with a very large nuclear DNA content are haploid. An underlying principle of evolutionary theory is that an individual passes on one or half of its genome to each of its progeny. The coexistence of a population of many genomes in AMF and their transfer to subsequent generations, therefore, has far-reaching consequences for understanding genome evolution.

  17. Safety and Immunogenicity of the HPV-16/18 AS04-adjuvanted Vaccine in 4-6-year-old Girls: Results to Month 12 From a Randomized Trial.

    PubMed

    Lin, Lan; Parra, Mercedes Macias; Sierra, Victor Y; Cespedes, Albino Salas; Granados, Maria Angelica; Luque, Adriana; Damaso, Silvia; Castrejon Alba, Maria Mercedes; Romano-Mazzotti, Luis; Struyf, Frank

    2018-04-01

    The burden of cervical cancer caused by human papillomavirus (HPV) is high in Latin America. The suboptimal HPV vaccination coverage in adolescents could be improved by pediatric immunization. HPV vaccination has not yet been reported in girls <9 years of age. This ongoing phase III, controlled, randomized, single-blind, multicenter study conducted in Colombia, Mexico and Panama (NCT01627561) evaluated the safety and immunogenicity of AS04-HPV-16/18 vaccine in 4-6-year-old girls. Healthy girls (randomized 1:1) received either 2 doses of AS04-HPV-16/18 vaccine (HPV group, N=74) or 1 dose of each measles-mumps-rubella and diphtheria-tetanus-acellular-pertussis vaccines (control group, N=74) 6 months apart. We report the safety and serum anti-HPV-16 and anti-HPV-18 antibodies (measured by enzyme-linked immunosorbent assay) up to 6 months postvaccination, that is, month (M) 12. Injection site pain was the most frequently reported solicited local symptom in HPV vaccinees. The incidence of other solicited and unsolicited symptoms after each vaccination was similar between the HPV and control group. Until M12, 1 girl in the HPV group and 2 in the control group reported serious adverse events; all serious adverse events were assessed as unrelated to study vaccines. No potential immune-mediated diseases were identified. All girls seroconverted for both antigens after 2 doses of AS04-HPV-16/18. In initially seronegative girls, anti-HPV-16 geometric mean concentrations were 20080.0 enzyme-linked immunosorbent assay units (EU)/mL at M7 and 3246.5 EU/mL at M12; anti-HPV-18 geometric mean concentrations were 10621.8 EU/mL at M7 and 1216.6 EU/mL at M12. Two-dose vaccination with AS04-HPV-16/18 was well tolerated and induced adequate antibody responses in 4-6-year-old girls.

  18. Analysis of the L1 gene product of human papillomavirus type 16 by expression in a vaccinia virus recombinant.

    PubMed

    Browne, H M; Churcher, M J; Stanley, M A; Smith, G L; Minson, A C

    1988-06-01

    The L1 open reading frame of human papillomavirus type 16 (HPV16) has been expressed in vaccinia virus under the control of both the 7.5K early and late promoter, and the 4b major late promoter. Antibodies to a beta-galactosidase fusion protein containing a C-terminal portion of the HPV16 L1 gene product were used to compare the levels of L1 expression in the two recombinants, and showed that greater levels of expression were obtained when the gene was placed under the control of the 4b late promoter. Immunofluorescence studies revealed a nuclear location of the L1 gene product when expressed in vaccinia virus. Antibodies to the beta-galactosidase fusion protein detected a major polypeptide species of 57K and a minor species of 64K in Western blots of recombinant-infected cell lysates. The 64K species was not detected when cells were infected in the presence of tunicamycin, indicating that the primary translation product of the HPV16 L1 open reading frame is modified by N-linked glycosylation when expressed in vaccinia virus. Whereas antibodies to HPV16 L1 fusion proteins and to a peptide containing amino acids from the C terminus of HPV16 L1 reacted well in Western blots with the HPV16 L1 target expressed in vaccinia virus, no reactivity was observed with antibodies to bovine papillomavirus type 1 particles or to a HPV6b fusion protein.

  19. Elevated PDGFRB gene copy number gain is prognostic for improved survival outcomes in resected malignant pleural mesothelioma.

    PubMed

    Tsao, Anne S; Harun, Nusrat; Fujimoto, Junya; Devito, Vikki; Lee, J Jack; Kuhn, Elisabetta; Mehran, Reza; Rice, David; Moran, Cesar; Hong, Waun Ki; Shen, Li; Suraokar, Milind; Wistuba, Ignacio

    2014-06-01

    PDGF/PDGFR pathway has been implicated in malignant pleural mesothelioma (MPM) carcinogenesis, and evidence suggests autocrine mechanisms of proliferation. We sought to evaluate the incidence of PDGFRB gene copy number gain (CNG) by fluorescence in situ hybridization and PDGFR pathway protein expression by immunohistochemistry (IHC) and correlate it to patient clinical outcome. Eighty-eight archived tumor blocks from resected MPM with full clinical information were used to perform IHC biomarkers (PDGFRα, PDGFRβ, p-PDGFRβ) and fluorescence in situ hybridization analysis of PDGFRB gene CNG. Spearman rank correlation, Wilcoxon rank-sum test, Kruskal-Wallis test, BLiP plots, and Kaplan-Meier method were used to analyze the biomarkers and correlation to clinical outcome. Several correlations between the IHC biomarkers were seen; however, none correlated to clinically relevant patient demographics or histology. In the CNG analysis, PDGFRB gene CNG in >10% of tumor cells had lower cytoplasmic p-PDGFRβ (P=.029), while PDGFRB gene CNG in >40% of tumor cells had a higher cytoplasmic PDGFRβ (P=.04). PDGFRB gene CNG status did not associate with patient demographics or tumor characteristics. PDGFR pathway IHC biomarkers did not associate with survival outcomes. However, patients with PDGFRB CNG >40% of tumor cells had improved relapse-free survival (HR 0.25 [95% CI 0.09-0.72], P=.0096) and improved overall survival (HR 0.32 [95% CI 0.11-0.89], P=.029). PDGFRB CNG >40% of MPM tumor cells is a potential prognostic biomarker for surgery and may identify a unique population of mesothelioma patients. Future validation of this biomarker in prospective trials is needed. From a retrospective review of archived tissue specimens from patients with resected malignant pleural mesothelioma tumors, we show that patients with PDGFRB CNG >40% of tumor cells had improved relapse-free survival (HR 0.25 [95% CI 0.09-0.72], P=.0096) and improved overall survival (HR 0.32 [95% CI 0

  20. Use of next-generation sequencing to detect LDLR gene copy number variation in familial hypercholesterolemia[S

    PubMed Central

    Iacocca, Michael A.; Wang, Jian; Dron, Jacqueline S.; Robinson, John F.; McIntyre, Adam D.; Cao, Henian

    2017-01-01

    Familial hypercholesterolemia (FH) is a heritable condition of severely elevated LDL cholesterol, caused predominantly by autosomal codominant mutations in the LDL receptor gene (LDLR). In providing a molecular diagnosis for FH, the current procedure often includes targeted next-generation sequencing (NGS) panels for the detection of small-scale DNA variants, followed by multiplex ligation-dependent probe amplification (MLPA) in LDLR for the detection of whole-exon copy number variants (CNVs). The latter is essential because ∼10% of FH cases are attributed to CNVs in LDLR; accounting for them decreases false negative findings. Here, we determined the potential of replacing MLPA with bioinformatic analysis applied to NGS data, which uses depth-of-coverage analysis as its principal method to identify whole-exon CNV events. In analysis of 388 FH patient samples, there was 100% concordance in LDLR CNV detection between these two methods: 38 reported CNVs identified by MLPA were also successfully detected by our NGS method, while 350 samples negative for CNVs by MLPA were also negative by NGS. This result suggests that MLPA can be removed from the routine diagnostic screening for FH, significantly reducing associated costs, resources, and analysis time, while promoting more widespread assessment of this important class of mutations across diagnostic laboratories. PMID:28874442

  1. EGFR gene copy number alterations are not a useful screening tool for predicting EGFR mutation status in lung adenocarcinoma.

    PubMed

    Russell, Prudence A; Yu, Yong; Do, Hongdo; Clay, Timothy D; Moore, Melissa M; Wright, Gavin M; Conron, Matthew; Wainer, Zoe; Dobrovic, Alexander; McLachlan, Sue-Anne

    2014-01-01

    We investigated if gene copy number (GCN) alterations of the epidermal growth factor receptor (EGFR), as detected by silver enhanced in situ hybridisation (SISH), could be used to select patients for EGFR mutation testing. Resected lung adenocarcinoma specimens with adequate tumour were identified. EGFR SISH was performed using the Ventana Benchmark Ultra platform. EGFR GCN was classified according to the Colorado Classification System. EGFR mutations were scanned by high resolution melting and confirmed by Sanger sequencing. Thirty-four of 96 tumours were EGFR SISH positive (35%), and 31 of 96 tumours harboured one or more EGFR mutations (32%). Of 31 EGFR-mutant tumours, 18 were EGFR SISH positive (58%). There was a statistically significant relationship between the presence of an EGFR mutation and EGFR GCN (p = 0.003). Thirteen of 31 EGFR-mutant tumours were EGFR SISH negative (42%), and 16 of 65 EGFR-wild type tumours were EGFR SISH positive (24%). The sensitivity, specificity, positive predictive value and negative predictive value were 58%, 75%, 52.9% and 79%, respectively. Despite a significant relationship between EGFR GCN alterations and EGFR mutations, our results indicate that EGFR GCN as detected by SISH is not a suitable way to select patients for EGFR mutation testing.

  2. Identification and qualification of 500 nuclear, single-copy, orthologous genes for the Eupulmonata (Gastropoda) using transcriptome sequencing and exon capture.

    PubMed

    Teasdale, Luisa C; Köhler, Frank; Murray, Kevin D; O'Hara, Tim; Moussalli, Adnan

    2016-09-01

    The qualification of orthology is a significant challenge when developing large, multiloci phylogenetic data sets from assembled transcripts. Transcriptome assemblies have various attributes, such as fragmentation, frameshifts and mis-indexing, which pose problems to automated methods of orthology assessment. Here, we identify a set of orthologous single-copy genes from transcriptome assemblies for the land snails and slugs (Eupulmonata) using a thorough approach to orthology determination involving manual alignment curation, gene tree assessment and sequencing from genomic DNA. We qualified the orthology of 500 nuclear, protein-coding genes from the transcriptome assemblies of 21 eupulmonate species to produce the most complete phylogenetic data matrix for a major molluscan lineage to date, both in terms of taxon and character completeness. Exon capture targeting 490 of the 500 genes (those with at least one exon >120 bp) from 22 species of Australian Camaenidae successfully captured sequences of 2825 exons (representing all targeted genes), with only a 3.7% reduction in the data matrix due to the presence of putative paralogs or pseudogenes. The automated pipeline Agalma retrieved the majority of the manually qualified 500 single-copy gene set and identified a further 375 putative single-copy genes, although it failed to account for fragmented transcripts resulting in lower data matrix completeness when considering the original 500 genes. This could potentially explain the minor inconsistencies we observed in the supported topologies for the 21 eupulmonate species between the manually curated and 'Agalma-equivalent' data set (sharing 458 genes). Overall, our study confirms the utility of the 500 gene set to resolve phylogenetic relationships at a range of evolutionary depths and highlights the importance of addressing fragmentation at the homolog alignment stage for probe design. © 2016 John Wiley & Sons Ltd.

  3. A common copy number variation polymorphism in the CNTNAP2 gene: sexual dimorphism in association with healthy aging and disease.

    PubMed

    Iakoubov, Leonid; Mossakowska, Malgorzata; Szwed, Malgorzata; Puzianowska-Kuznicka, Monika

    2015-01-01

    New therapeutic targets are needed to fight aging-related diseases and increase life span. A new female-specific association with diseases and limited survival past 80 years was recently reported for a copy number variation (CNV) in the CNTNAP4 gene from the neurexin superfamily. We asked whether there are CNVs that are associated with aging phenotypes within other genes from the neurexin superfamily and whether this association is sex specific. Select CNV polymorphisms were genotyped with proprietary TaqMan qPCR assays. A case/control study, in which a group of 81- to 90-year-old community-dwelling Caucasians with no chronic diseases (case) was compared to a similar control group of 65- to 75-year-olds, revealed a negative association with healthy aging for the ins allele of common esv11910 CNV in the CNTNAP2 gene (n = 388; OR = 0.29, 95% CI: 0.14-0.59, p = 0.0004 for males, and OR = 0.82, 95% CI: 0.42-1.57, p = 0.625 for females). This male-specific association was validated in a study of an independent group of 76- to 80-year-olds. To look for a corresponding positive association of the allele with aging-related diseases, two case subgroups of 81- to 90-year-olds, one composed of individuals with cognitive impairment and the other with various diseases not directly related to the nervous system, such as cardiovascular diseases, etc., were compared to a healthy control subgroup of the same age. A positive male-specific association was found for both cases (OR = 2.75, p = 0.008 for association with cognitive impairment, and OR = 3.18, p = 0.002 for other diseases combined). A new male-specific association with aging is reported for a CNV in the CNTNAP2 gene. The polymorphism might be useful for diagnosing individual genetic predispositions to healthy aging versus aging complicated by chronic diseases. © 2014 S. Karger AG, Basel.

  4. A functional promoter shift of a chloroplast gene: a transcriptional fusion between a novel psbA gene copy and the trnK (UUU) gene in Pinus contorta.

    PubMed

    Lidholm, J; Gustafsson, P

    1992-11-01

    A comparative transcription analysis of the chloroplast trnK-psbA-trnH region of the two pine species Pinus contorta and Pinus sylvestris is reported. The chloroplast genome of P. contorta has previously been shown to contain a duplicated psbA gene copy integrated closely upstream of the split trnK gene. This rearrangement has resulted in the gene order psbAI-trnK-psbAII-trnH, where psbAII is the ancestral psbA gene copy. In P. sylvestris, a species which lacks the psbA duplication, transcription of the trnK gene originates from a position 291 bp upstream of the trnK 5' exon, adjacent to a canonical promoter structure. In P. contorta, the corresponding promoter structure has been separated from the trnK gene by the insertion of psbAI, and has, in addition, been partially deleted. Analysis of the transcriptional organization of the trnK-psbA-trnH region of the two pine species revealed that the trnK gene in P. contorta is transcriptionally fused to the inserted psbAI gene copy. As a result, trnK is under the control of the psbA promoter in this species and has therefore acquired psbA-like expression characteristics. In P. sylvestris, accumulation of trnK transcripts is not significantly higher in light-grown than in dark-grown seedlings. In contrast, the level of trnK transcripts in P. contorta is approximately 12-fold higher in the light than in the dark. When light-grown seedlings of the two pine species were compared, an approximately 20-fold higher level of trnK RNAs was found in P. contorta. In both pine species, evidence was obtained for trnK-psbA and psbA-trnH co-transcription.

  5. Establishing a novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure for the direct detection of gene doping.

    PubMed

    Beiter, Thomas; Zimmermann, Martina; Fragasso, Annunziata; Armeanu, Sorin; Lauer, Ulrich M; Bitzer, Michael; Su, Hua; Young, William L; Niess, Andreas M; Simon, Perikles

    2008-01-01

    So far, the abuse of gene transfer technology in sport, so-called gene doping, is undetectable. However, recent studies in somatic gene therapy indicate that long-term presence of transgenic DNA (tDNA) following various gene transfer protocols can be found in DNA isolated from whole blood using conventional PCR protocols. Application of these protocols for the direct detection of gene doping would require almost complete knowledge about the sequence of the genetic information that has been transferred. Here, we develop and describe the novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure that overcomes this difficulty. Apart from the interesting perspectives that this spiPCR procedure offers in the fight against gene doping, this technology could also be of interest in biodistribution and biosafety studies for gene therapeutic applications.

  6. Zinc-dependent global transcriptional control, transcriptional deregulation, and higher gene copy number for genes in metal homeostasis of the hyperaccumulator Arabidopsis halleri.

    PubMed

    Talke, Ina N; Hanikenne, Marc; Krämer, Ute

    2006-09-01

    The metal hyperaccumulator Arabidopsis halleri exhibits naturally selected zinc (Zn) and cadmium (Cd) hypertolerance and accumulates extraordinarily high Zn concentrations in its leaves. With these extreme physiological traits, A. halleri phylogenetically belongs to the sister clade of Arabidopsis thaliana. Using a combination of genome-wide cross species microarray analysis and real-time reverse transcription-PCR, a set of candidate genes is identified for Zn hyperaccumulation, Zn and Cd hypertolerance, and the adjustment of micronutrient homeostasis in A. halleri. Eighteen putative metal homeostasis genes are newly identified to be more highly expressed in A. halleri than in A. thaliana, and 11 previously identified candidate genes are confirmed. The encoded proteins include HMA4, known to contribute to root-shoot transport of Zn in A. thaliana. Expression of either AtHMA4 or AhHMA4 confers cellular Zn and Cd tolerance to yeast (Saccharomyces cerevisiae). Among further newly implicated proteins are IRT3 and ZIP10, which have been proposed to contribute to cytoplasmic Zn influx, and FRD3 required for iron partitioning in A. thaliana. In A. halleri, the presence of more than a single genomic copy is a hallmark of several highly expressed candidate genes with possible roles in metal hyperaccumulation and metal hypertolerance. Both A. halleri and A. thaliana exert tight regulatory control over Zn homeostasis at the transcript level. Zn hyperaccumulation in A. halleri involves enhanced partitioning of Zn from roots into shoots. The transcriptional regulation of marker genes suggests that in the steady state, A. halleri roots, but not the shoots, act as physiologically Zn deficient under conditions of moderate Zn supply.

  7. Correlation of P16 (Ink4a) and CK17 to HPV (16E6+18E6) in Premalignant and Malignant Lesions of Uterine Cervix: A Clinicopathologic Study

    PubMed Central

    Chaloob, Mohammed K.; Hussein, Alaa G.; Qasim, Ban J.

    2016-01-01

    Background: This research was accomplished to evaluate the IHC expression of p16 (ink4a) and CK17 in low grade cervical intraepithelial lesions (LSIL), high grade cervical intraepithelial lesions (HSIL) and invasive cervical carcinomas and to assess their correlation to HPV (16E6+18E6). Methods: The study included (127) formalin-fixed paraffin-embedded cervical biopsies; of which 22 cases were chronic cervicitis, 24 cases were LSIL, 28 cases were HSIL and 53 cases were invasive cervical carcinomas. Sections were immunohistochemically stained for p16 (ink4a), CK17 and HPV (16E6+18E6). Results: The study established a highly significant increase in IHC of expression of p16 (ink4a), CK17 and HPV (16E6+18E6) from LSIL through HSIL to invasive carcinomas (P-value˂0.001). There was non-significant association between IHC expression of all makers with age of patients; types, grade and stage of cervical carcinomas (P-value˃0.05). HPV (16E6+18E6) revealed a significantly positive correlation with p16 (ink4a) (P-value˂0.05) and a non- significant correlation with CK17 (P-value˃0.05); in LSIL, HSIL and invasive carcinoma cases. Conclusion: p16 (ink4a) expression directly reflects infection with high risk HPV in cervical lesions and can add a significant diagnostic accuracy in the evaluation of CIN. CK 17 is a good marker of malignant transformation, with increasing in its expression according to the severity of cervical lesions; however, it is not related to HPV infection. Both markers are not related to prognostic variables of patients with cervical carcinoma. PMID:28855930

  8. Decreased Migration of Langerhans Precursor-Like Cells in Response to Human Keratinocytes Expressing HPV-16 E6/E7 is Related to Reduced Macrophage Inflammatory Protein-3Alpha Production

    DTIC Science & Technology

    2005-01-01

    high-risk human papillomavirus ( HPV ) types, particularly type 16 and 18, contributes to 90% of cervical cancer cases. HPV infects cutaneous or mucosal...been implicated in cervical/ anogenital cancer and oral squamous cell carcinomas (41). The mucosal lesions caused by HPVs often resolve over time, and a...Decreased Migration of Langerhans Precursor-Like Cells in Response to Human Keratinocytes Expressing HPV -16 E6/E7 is Related to Reduced Macrophage

  9. Long-Read Single Molecule Sequencing to Resolve Tandem Gene Copies: The Mst77Y Region on the Drosophila melanogaster Y Chromosome

    PubMed Central

    Krsticevic, Flavia J.; Schrago, Carlos G.; Carvalho, A. Bernardo

    2015-01-01

    The autosomal gene Mst77F of Drosophila melanogaster is essential for male fertility. In 2010, Krsticevic et al. (Genetics 184: 295−307) found 18 Y-linked copies of Mst77F (“Mst77Y”), which collectively account for 20% of the functional Mst77F-like mRNA. The Mst77Y genes were severely misassembled in the then-available genome assembly and were identified by cloning and sequencing polymerase chain reaction products. The genomic structure of the Mst77Y region and the possible existence of additional copies remained unknown. The recent publication of two long-read assemblies of D. melanogaster prompted us to reinvestigate this challenging region of the Y chromosome. We found that the Illumina Synthetic Long Reads assembly failed in the Mst77Y region, most likely because of its tandem duplication structure. The PacBio MHAP assembly of the Mst77Y region seems to be very accurate, as revealed by comparisons with the previously found Mst77Y genes, a bacterial artificial chromosome sequence, and Illumina reads of the same strain. We found that the Mst77Y region spans 96 kb and originated from a 3.4-kb transposition from chromosome 3L to the Y chromosome, followed by tandem duplications inside the Y chromosome and invasion of transposable elements, which account for 48% of its length. Twelve of the 18 Mst77Y genes found in 2010 were confirmed in the PacBio assembly, the remaining six being polymerase chain reaction−induced artifacts. There are several identical copies of some Mst77Y genes, coincidentally bringing the total copy number to 18. Besides providing a detailed picture of the Mst77Y region, our results highlight the utility of PacBio technology in assembling difficult genomic regions such as tandemly repeated genes. PMID:25858959

  10. Two Functional Copies of the DGCR6 Gene Are Present on Human Chromosome 22q11 Due to a Duplication of an Ancestral Locus

    PubMed Central

    Edelmann, Lisa; Stankiewicz, Pavel; Spiteri, Elizabeth; Pandita, Raj K.; Shaffer, Lisa; Lupski, James; Morrow, Bernice E.

    2001-01-01

    The DGCR6 (DiGeorge critical region) gene encodes a putative protein with sequence similarity to gonadal (gdl), a Drosophila melanogaster gene of unknown function. We mapped the DGCR6 gene to chromosome 22q11 within a low copy repeat, termed sc11.1a, and identified a second copy of the gene, DGCR6L, within the duplicate locus, termed sc11.1b. Both sc11.1 repeats are deleted in most persons with velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS), and they map immediately adjacent and internal to the low copy repeats, termed LCR22, that mediate the deletions associated with VCFS/DGS. We sequenced genomic clones from both loci and determined that the putative initiator methionine is located further upstream than originally described, but in a position similar to the mouse and chicken orthologs. DGCR6L encodes a highly homologous, functional copy of DGCR6, with some base changes rendering amino acid differences. Expression studies of the two genes indicate that both genes are widely expressed in fetal and adult tissues. Evolutionary studies using FISH mapping in several different species of ape combined with sequence analysis of DGCR6 in a number of different primate species indicate that the duplication is at least 12 million years old and may date back to before the divergence of Catarrhines from Platyrrhines, 35 mya. These data suggest that there has been selective evolutionary pressure toward the functional maintenance of both paralogs. Interestingly, a full-length HERV-K provirus integrated into the sc11.1a locus after the divergence of chimpanzees and humans. PMID:11157784

  11. Comparative immunogenicity and safety of human papillomavirus (HPV)-16/18 vaccine and HPV-6/11/16/18 vaccine: follow-up from months 12-24 in a Phase III randomized study of healthy women aged 18-45 years.

    PubMed

    Einstein, Mark H; Baron, Mira; Levin, Myron J; Chatterjee, Archana; Fox, Bradley; Scholar, Sofia; Rosen, Jeffrey; Chakhtoura, Nahida; Meric, Dorothée; Dessy, Francis J; Datta, Sanjoy K; Descamps, Dominique; Dubin, Gary

    2011-12-01

    In this observer-blind study (NCT00423046), women (N=1,106), stratified by age (18-26, 27-35, 36-45 y), were randomized (1:1) to receive the HPV-16/18 vaccine (Cervarix®, GlaxoSmithKline Biologicals, Months 0, 1, 6) or the HPV-6/11/16/18 vaccine (Gardasil® Merck & Co., Inc., Months 0, 2, 6). Month 7 results were previously reported; we now report Month 24 results. In the according-to-protocol cohort for immunogenicity (seronegative and DNA-negative at baseline for HPV type analyzed), seropositivity rates of neutralizing antibodies (nAbs) [pseudovirion-based neutralization assay] were, across all age strata, 100% (HPV-16/18 vaccine) and 97.5-100% (HPV-6/11/16/18 vaccine) for HPV-16, and 99.0-100% (HPV-16/18 vaccine) and 72.3-84.4% (HPV-6/11/16/18 vaccine) for HPV-18. Corresponding geometric mean titers (GMTs) were 2.4-5.8-fold higher for HPV-16 and 7.7-9.4-fold higher for HPV-18 with the HPV-16/18 vaccine versus the HPV-6/11/16/18 vaccine; HPV-16 and HPV-18 GMTs were significantly higher with the HPV-16/18 vaccine than the HPV-6/11/16/18 vaccine (p< 0.0001) in the total vaccinated cohort (received ≥1 vaccine dose, irrespective of baseline sero/DNA-status). Similar results were obtained using enzyme-linked immunosorbent assay (ELISA). Positivity rates and GMTs of antigen-specific IgG antibodies in cervicovaginal secretions (ELISA) were not significantly different between vaccines. At Month 24, CD4⁺ T-cell responses for HPV-16 and HPV-18 were higher with the HPV-16/18 vaccine; memory B-cell response was higher for HPV-18 with the HPV-16/18 vaccine and similar between vaccines for HPV-16. Both vaccines were generally well tolerated. Although an immunological correlate of protection has not been defined, differences in the magnitude of immune response between vaccines may represent determinants of duration of protection.

  12. C33-A cells transfected with E6*I or E6*II the short forms of HPV-16 E6, displayed opposite effects on cisplatin-induced apoptosis.

    PubMed

    Vaisman, Carolina E; Del Moral-Hernandez, Oscar; Moreno-Campuzano, Samadhi; Aréchaga-Ocampo, Elena; Bonilla-Moreno, Raul; Garcia-Aguiar, Israel; Cedillo-Barron, Leticia; Berumen, Jaime; Nava, Porfirio; Villegas-Sepúlveda, Nicolas

    2018-03-02

    The HPV-16 E6/E7 bicistronic immature transcript produces 4 mature RNAs: the unspliced HPV-16 E6/E7 pre-mRNA product and 3 alternatively spliced mRNAs. The 3 spliced mRNAs encode short forms of the E6 oncoprotein, namely E6*I, E6*II and E6^E7. In this study we showed that transfection of C-33A cells with monocistronic constructs of these cDNAs fused to GFP, produced different effects on apoptosis, after the treatment with cisplatin. Transfection of C-33A cells with the full-length E6-GFP oncoprotein resulted in a 50% decrease in cell death, while the transfection with the E6*I-GFP construct showed only a 25% of diminution of cell death, compared to the control cells. Transfection with the E6^E7-GFP or E7-GFP construct had no effect on the number of the apoptotic cells, compared with control cells. Conversely, transfection with the E6*II construct resulted in higher cell death than the control cells. Taken together, these results suggested that E6*I or E6*II, the short forms of HPV-16 E6, displayed opposite effects on cisplatin-induced apoptosis, when transfected in C-33A cells. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. In situ hybridization detection methods for HPV16 E6/E7 mRNA in identifying transcriptionally active HPV infection of oropharyngeal carcinoma: an updating.

    PubMed

    Volpi, Chiara C; Ciniselli, Chiara M; Gualeni, Ambra V; Plebani, Maddalena; Alfieri, Salvatore; Verderio, Paolo; Locati, Laura; Perrone, Federica; Quattrone, Pasquale; Carbone, Antonino; Pilotti, Silvana; Gloghini, Annunziata

    2018-04-01

    The aim of this study is to compare 2 in situ hybridization (ISH) detection methods for human papilloma virus (HPV) 16 E6/E7 mRNA, that is, the RNAscope 2.0 High Definition (HD) and the upgraded RNAscope 2.5 HD version. The RNAscope 2.5 HD has recently replaced the RNAscope 2.0 HD detection kit. Therefore, this investigation starts from the need to analytically validate the new mRNA ISH assay and, possibly, to refine the current algorithm for HPV detection in oropharyngeal squamous cell carcinoma with the final goal of applying it to daily laboratory practice. The study was based on HPV status and on generated data, interpreted by a scoring algorithm. The results highlighted that the compared RNAscope HPV tests had a good level of interchangeability and enabled to identify oropharyngeal squamous cell carcinoma that are truly driven by high-risk HPV infection. This was also supported by the comparison of the RNAscope HPV test with HPV E6/E7 mRNA real-time reverse-transcription polymerase chain reaction in a fraction of cases where material for HPV E6/E7 mRNA real-time reverse-transcription polymerase chain reaction was available. Furthermore, the algorithm that associates p16 immunohistochemistry with the identification of HPV mRNA by RNAscope was more effective than the one that associated p16 immunohistochemistry with the identification of HPV DNA by ISH. Copyright © 2017 Elsevier Inc. All rights reserved.