Prognostic value of Dna and Mrna E6/e7 of human papillomavirus in the evolution of cervical intraepithelial neoplasia grade 2

Objective: This study aimed at evaluating whether human papillomavirus (HPV) groups and E6/E7 mRNA of HPV 16, 18, 31, 33, and 45 are prognostic of cervical intraepithelial neoplasia (CIN) 2 outcome in women with a cervical smear showing a low-grade squamous intraepithelial lesion (LSIL).Methods: This cohort study included women with biopsy-confirmed CIN 2 who were followed up for 12 months, with cervical smear and colposcopy performed every three months.Results: Women with a negative or low-risk HPV status showed 100% CIN 2 regression. The CIN 2 regression rates at the 12-month follow-up were 69.4% for women with alpha-9 HPV versus 91.7% for other HPV species or HPV-negative status (P < 0.05). For women with HPV 16, the CIN 2 regression rate at the 12-month follow-up was 61.4% versus 89.5% for other HPV types or HPV-negative status (P < 0.05). The CIN 2 regression rate was 68.3% for women who tested positive for HPV E6/E7 mRNA versus 82.0% for the negative results, but this difference was not statistically significant.Conclusions: The expectant management for women with biopsy-confirmed CIN 2 and previous cytological tests showing LSIL exhibited a very high rate of spontaneous regression. HPV 16 is associated with a higher CIN 2 progression rate than other HPV infections. HPV E6/E7 mRNA is not a prognostic marker of the CIN 2 clinical outcome, although this analysis cannot be considered conclusive. Given the small sample size, this study could be considered a pilot for future larger studies on the role of predictive markers of CIN 2 evolution. © the authors.This study aimed at evaluating whether human papillomavirus (HPV) groups and E6/E7 mRNA of HPV 16, 18, 31, 33, and 45 are prognostic of cervical intraepithelial neoplasia (CIN) 2 outcome in women with a cervical smear showing a low-grade squamous intraepithelial lesion (LSIL).Methods: This cohort study included women with biopsy-confirmed CIN 2 who were followed up for 12 months, with cervical smear and colposcopy performed every three months.Results: Women with a negative or low-risk HPV status showed 100% CIN 2 regression. The CIN 2 regression rates at the 12-month follow-up were 69.4% for women with alpha-9 HPV versus 91.7% for other HPV species or HPV-negative status (P < 0.05). For women with HPV 16, the CIN 2 regression rate at the 12-month follow-up was 61.4% versus 89.5% for other HPV types or HPV-negative status (P < 0.05). The CIN 2 regression rate was 68.3% for women who tested positive for HPV E6/E7 mRNA versus 82.0% for the negative results, but this difference was not statistically significant.Conclusions: The expectant management for women with biopsy-confirmed CIN 2 and previous cytological tests showing LSIL exhibited a very high rate of spontaneous regression. HPV 16 is associated with a higher CIN 2 progression rate than other HPV infections. HPV E6/E7 mRNA is not a prognostic marker of the CIN 2 clinical outcome, although this analysis cannot be considered conclusive. Given the small sample size, this study could be considered a pilot for future larger studies on the role of predictive markers of CIN 2 evolution9152

Identification And Complete Sequencing Of Novel Human Transcripts Through The Use Of Mouse Orthologs And Testis Cdna Sequences

The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. In silico expression analysis suggests that some of these transcripts are expressed at low levels and in a restricted set of tissues.34493511Adams, M.D., Kelley, J.M., Gocayne, J.D., Dubnick, M., Polymeropoulos, M.H., Xiao, H., Merril, C.R., Moreno, R.F., Complementary DNA sequencing: Expressed sequence tags and human genome project (1991) Science, 252, pp. 1651-1656Blanco, E., Parra, G., Guigó, R., Finding genes (2002) Current Protocols in Bioinformatics, , (Baxevanis, A., ed.). John Wiley & Sons Ltd., New York (in press)Bonaldo, M.F., Lennon, G., Soares, M.B., Normalization and subtraction: Two approaches to facilitate gene discovery (1996) Genome Res., 6, pp. 791-806Boon, K., Osorio, E.C., Greenhut, S.F., Schaefer, C.F., Shoemaker, J., Polyak, K., Morin, P.J., Riggins, G.J., An anatomy of normal and malignant gene expression (2002) Proc. Natl. Acad. Sci. USA, 99, pp. 11287-11292Brentani, H., Caballero, O.L., Camargo, A.A., Da Silva, A.M., Da Silva Jr., W.A., Dias Neto, E., Grivet, M., Luna, A.M., The generation and utilization of a cancer-oriented representation of the human transcriptome by using expressed sequence tags (2003) Proc. Natl. Acad. Sci. USA, 100, pp. 13418-13423Burge, C., Karlin, S., Prediction of complete gene structures in human genomic DNA (1997) J. Mol. Biol., 268, pp. 78-94Burset, M., Guigó, R., Evaluation of gene structure prediction programs (1996) Genomics, 34, pp. 353-367Camargo, A.A., Samaia, H.P., Dias-Neto, E., Simao, D.F., Migotto, I.A., Briones, M.R., Costa, F.F., Simpson, A.J., The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome (2001) Proc. Natl. Acad. Sci. USA, 98, pp. 12103-12108Carninci, P., Shibata, Y., Hayatsu, N., Sugahara, Y., Shibata, K., Itoh, M., Konno, H., Hayashizaki, Y., Normalization and subtraction of cap-trapper-selected cDNA molecules to prepare full-length cDNA libraries for rapid discovery of new genes (2000) Genome Res., 10, pp. 1617-1630Carninci, P., Shibata, Y., Hayatsu, N., Itoh, M., Shiraki, T., Hirozane, T., Watahiki, A., Hayashizaki, Y., Balanced-size and long-size cloning of full-length, cap-trapped cDNAs into vectors of the novel lambda-FLC family allows enhanced gene discovery rate and functional analysis (2001) Genomics, 77, pp. 79-90Chirgwin, J.M., Przybyla, A.E., McDonald, R.J., Rutter, W.J., Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease (1979) Biochemistry, 18, pp. 5294-5299De Souza, S.J., Camargo, A.A., Briones, M.R., Costa, F.F., Nagai, M.A., Verjovski-Almeida, S., Zago, M.A., Simpson, A.J., Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags (2000) Proc. Natl. Acad. Sci. USA, 97, pp. 12690-12693Dias-Neto, E., Correa, R.G., Verjovski-Almeida, S., Briones, M.R., Nagai, M.A., Da Silva Jr., W., Zago, M.A., Simpson, A.J., Shotgun sequencing of the human transcriptome with ORF expressed sequence tags (2000) Proc. Natl. Acad. Sci. USA, 97, pp. 3491-3496Guigó, R., Knudsen, S., Drake, N., Smith, T., Prediction of gene structure (1992) J. Mol. Biol., 226, pp. 141-157Henke, W., Herdel, K., Jung, K., Schoor, D., Lorning, S.A., Betaine improves the PCR amplification of GC-rich DNA sequences (1997) Nucleic Acids Res., 25, pp. 3957-3958Hickox, D.M., Gibbs, G., Morrison, J.R., Sebire, K., Edgar, K., Keah, H.H., Alter, K., O'Bryan, M.K., Identification of a novel testis-specific member of the phosphatidylethanolamine binding protein family, pebp-2 (2002) Biol. Reprod., 67, pp. 917-927Houlgatte, R., Mariage-Samson, R., Duprat, S., Tessier, A., Bentolila, S., Lamy, B., Auffray, C., The Genexpress Index: A resource for gene discovery and the genic map of the human genome (1995) Genome Res., 5, pp. 272-304Huang, X., Madan, A., CAP3: A DNA sequence assembly program (1999) Genome Res., 9, pp. 868-877Kent, W.J., Sugnet, C.W., Furey, T.S., Roskin, K.M., Pringle, T.H., Zahler, A.M., Haussler, D., The Human Genome Browser at UCSC (2002) Genome Res., 12, pp. 996-1006Kikuno, R., Nagase, T., Waki, M., Ohara, O., HUGE: A database for human large proteins identified in the Kazusa cDNA sequencing project (2002) Nucleic Acids Res., 30, pp. 166-168Lander, E.S., Linton, L.M., Birren, B., Nusbaum, C., Zody, M.C., Baldwin, J., Devon, K., Uberbacher, E., Initial sequencing and analysis of the human genome (2001) Nature, 409, pp. 860-921Liang, F., Holt, I., Pertea, G., Karamycheva, S., Salzberg, S.L., Quackenbush, J., Gene index analysis of the human genome estimates approximately 120,000 genes (2000) Nature, 25, pp. 239-240Makalowski, W., Zhang, J., Boguski, M.S., Comparative analysis of 1196 orthologous mouse and human full-length mRNA and protein sequences (1996) Genome Res., 6, pp. 846-857Modrek, B., Lee, C., A genomic view of alternative splicing (2002) Nat. Genet., 30, pp. 13-19Nakajima, D., Okazaki, N., Yamakawa, H., Kikuno, R., Ohara, O., Nagase, T., Construction of expression-ready cDNA clones for KIAA genes: Manual curation of 330 KIAA cDNA clones (2002) DNA Res., 9, pp. 99-106Rogic, S., Mackworth, A.K., Ouellette, F.B., Evaluation of gene-finding programs on mammalian sequences (2001) Genome Res., 11, pp. 817-832Sakharkar, M.K., Kangueane, P., Genome SEGE: A database for 'intronless' genes in eukaryotic genomes (2004) BMC Bioinformatics, 5, p. 67Sakharkar, M.K., Kangueane, P., Petrov, D.A., Kolaskar, A.S., Subbiah, S., SEGE: A database on 'intron less/single exonic' genes from eukaryotes (2002) Bioinformatics, 18, pp. 1266-1267Sambrook, J., Fritsch, E., Maniatis, T., (1989) Molecular Cloning, , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USAShabalina, S.A., Ogurtsov, A.Y., Rogozin, I.B., Koonin, E.V., Lipman, D.J., Comparative analysis of orthologous eukaryotic mRNAs: Potential hidden functional signals (2004) Nucleic Acids Res., 32, pp. 1774-1782Shendure, J., Church, G.M., Computational discovery of sense-antisense transcription in the human and mouse genomes (2002) Genome Biol., 3, pp. 1-14Sogayar, M.C., Camargo, A.A., Bettoni, F., Carraro, D.M., Pires, L.C., Parmigiani, R.B., Ferreira, E.N., Zanette, D.L., A transcript finishing initiative for closing gaps in the human transcriptome (2004) Genome Res., 14, pp. 1413-1423Solovyev, V., Statistical approaches in eukaryotic gene prediction (2001) Handbook of Statistical Genetics, pp. 83-127. , (Balding, D.J., Bishop, M. and Cannings, C., eds.). John Wiley & Sons Ltd., UKSorek, R., Safer, H.M., A novel algorithm for computational identification of contaminated EST libraries (2003) Nucleic Acids Res., 31, pp. 1067-1074Strausberg, R.L., Feingold, E.A., Klausner, R.D., Collins, F.S., The mammalian gene collection (1999) Science, 286, pp. 455-457Strausberg, R.L., Feingold, E.A., Grouse, L.H., Derge, J.G., Klausner, R.D., Collins, F.S., Wagner, L., Marra, M.A., Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences (2002) Proc. Natl. Acad. Sci. USA, 99, pp. 16899-16903Genome sequence of the nematode C. elegans: A platform for investigating biology (1998) Science, 282, pp. 2012-2018Velculescu, V.E., Zhang, L., Vogelstein, B., Kinzler, K.W., Serial analysis of gene expression (1995) Science, 270, pp. 484-487Venter, J.C., Adams, M.D., Myers, E.W., Li, P.W., Mural, R.J., Sutton, G.G., Smith, H.O., Xiao, C., The Sequence of the Human Genome (2001) Science, 291, pp. 1304-1351Vettore, A.L., Da Silva, F.R., Kemper, E.L., Arruda, P., The libraries that made SUCEST (2001) Gen. Mol. Biol., 24, pp. 1-7Warrington, J.A., Nair, A., Mahadevappa, M., Tsyganskaya, M., Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes (2000) Physiol. Genomics, 2, pp. 143-147Wiemann, S., Weil, B., Wellenreuther, R., Gassenhuber, J., Glassl, S., Ansorge, W., Bocher, M., Poustka, A., Toward a catalog of human genes and proteins: Sequencing and analysis of 500 novel complete protein coding human cDNAs (2001) Genome Res., 11, pp. 422-435Yao, J., Chiba, T., Sakai, J., Hirose, K., Yamamoto, M., Hada, A., Kuramoto, K., Mori, M., Mouse testis transcriptome revealed using serial analysis of gene expression (2004) Mamm. Genome, 15, pp. 433-451Yelin, R., Dahary, D., Sorek, R., Levanon, E.Y., Goldstein, O., Shoshan, A., Diber, A., Rotman, G., Widespread occurrence of antisense transcription in the human genome (2003) Nat. Biotechnol., 21, pp. 379-38