DETECCIÓN MOLECULAR DE REGIONES ONCOGÉNICAS E6 Y E7 DE VIRUS DEL PAPILOMA HUMANO MEDIANTE PCR EN PACIENTES PAPANICOLAOU NEGATIVO DEL INSTITUTO REGIONAL DE ENFERMEDADES NEOPLÁSICAS DE LA LIBERTAD
Resumen
La presente investigación tuvo como objetivo detectar regiones oncogénicas E6 y E7 de Virus del Papiloma Humano mediante PCR en pacientes Papanicolaou negativo del Instituto Regional de Enfermedades Neoplásicas de La Libertad. Se seleccionaron 20 muestras Papanicolaou negativo y se procedió a extracción de ADN utilizando el kit “High Pure PCR Template Preparation Kit” de Roche. La amplificación de genes L1 y L2 se realizó utilizando el set de primers genéricos MY09/11 y GP5+/6+, seguido la amplificación de oncogenes E6 y E7 utilizando primers específicos para genotipos de alto riesgo (VPH 16, 18, 31, 33 y 52) y bajo riesgo (VPH 6/11). Se obtuvo como resultado que el 75 % de pacientes presentaron oncogenes E6 y E7 de VHP 16, el 15 % de VPH 52, el 10 % de VPH 18, el 5 % VPH 33, 0 % de VPH 31 y el 20 % de VPH 6/11. También se observó que un 50 % presentaron infecciones simples por VPH 16 y 20 % por VPH 6/11, mientras que un 25 % presentaron infecciones múltiples por VPH 16, 18, 33 y 52. Se concluyó que se detectó regiones oncogénicas E6 y E7 de VPH 16, 18, 33, 52 y 6/11 mediante PCR en pacientes Papanicolaou negativo, con alta incidencia de oncogenes de VPH 16, y en menor grado VPH 6/11, 18, 33, y 52.
Palabras clave: Regiones oncogénicas E6 y E7, Virus del Papiloma Humano, PCR.
ABSTRACT
This study aimed to detect oncogenic regions E6 and E7 of Human papillomavirus by PCR in patients with negative Pap diagnosis of the “Instituto Regional de Enfermedades Neoplásicas” of La Libertad. 20 negative Pap samples were selected and DNA extraction was performed using "High Pure PCR Template Preparation Kit" of Roche. L1 and L2 genes amplification was performed using the generic primers sets MY09/11 and GP5+/6+, followed by E6 and E7 oncogenes amplification using specific primers for high risk genotypes (HPV 16, 18, 31, 33 and 52) and low risk (HPV 6/11). The result was that 75 % of patients had E6 and E7 oncogenes of HPV 16, 15 % of HPV 52, 10 % of HPV 18, 5 % of HPV 33, 0 % of HPV 31 and 20 % of HPV 6/11. Furthermore, it was observed that 50 % had HPV simple infections HPV 16 and 20 % of HPV 6/11, while 25 % had multiple infections by HPV 16, 18, 33 and 52. We concluded that the oncogenic regions E6 and E7 of HPV 6/11, 16, 18, 33 and 52 were detected by PCR in patients with negative Pap diagnosis, showing high incidence of oncogenes of HPV 16 and lesser extent HPV 6/11, 18, 33 and 52.
Key words: Oncogenic regions E6 and E7, Human Papillomavirus, PCR.
Recibido: 13 Diciembre de 2014
Aceptado: 8 de Junio de 2015
Citas
Donoso E. Cuello M. Villarroel L. Reducción de la mortalidad por cáncer cervicouterino en Chile, 1990-2003. Rev Chil Obstet y Ginecol. 2006; 71(5):307-312.
International Agency for Research of Cancer. Estimated cancer, Incidence, mortality and prevalence worldwide. GLOBOCAN [Internet].
[Citado 10 Nov 2014]. Disponible en: http://globocan.iarc.fr/Pages/Map.aspx.
Franco L, Villa L, Sobrinho P. Epidemiology of acquisition and clearance of cervical Human Papillomavirus infection in women from a high-risk area for cervical cancer. J. Infect Dis. 1999; 180(5):1415-1423.
Giuliano R, Harris R, Sedjo L. Incidence, prevalence and clearance of type specific Human Papillomavirus infections. The Young Women’s Health Study. J. Infect Dis. 2002; 186(4):462-469.
Strauss J, Bunting H, Melnick L. Virus-like particles and inclusion bodies in skin papillomas, J Invest Dermato.1950; 15:433-444.
Nobbenhuis A, Helmerhorst J, Van den Brule J, Rozendaal L, Voorhorst J, Bezemer D, et al. Cytological regression and clearance of high-risk Human Papillomavirus in women with an abnormal cervical smear. Lancet, 2001; 358:1782-1783.
De Villiers M, Ruhland A. Do specific Human Papillomavirus types causepsoriasis. Arch. Dermatol. 2001; 137(3):384-394.
De Villiers M, Fauquet C, Broker R, Bernard U, Zur Hausen H. Classification of Papillomaviruses, Virology. 2004; 324(1):17-27.
Broken R. Structure and genetic expression of Papillomaviruses. Obstet Gynecol Pathol. 2000; 19:16-28.
Yee C, Krishnan-Hewlen I, Berjer C, Schlegel R, Howley M. Presence and expression of Human Papillomavirus sequences in human cervical carcionoma cell lines. AmJpathol. 1985; 119:361-366.
Álvarez M, López E. Regulación genética de los Papilomavirus humanos genitales. Salud Pública de México. 1995; 37:240-247.
Jayshree S, Sreenivas A, Tessy M, Krishna S. Cell intrinsic y extrinsic factors in cervical carcinogenesis. J Med Res. 2009; 13:286-295.
Majewski S, Jablonska S. Human papillomavirus-associated tumors of the skin and mucosa. J Am Acad Dermatol. 1997; 36:659-685.
Ziegert C, Wentzensen N, Vinokurova S, Kisseljov F, Einenkel J, et al. A comprehensive analysis of HPV integration loci in anogenital lesions combining transcript and genome-based amplification techniques. Oncogene. 2003; 22:3977-3984.
Zur Hausen H. Papillomaviruses and cancer from basic studies to clinical application. Nat Rev Cancer. 2002; 2:342-350.
Winer L, Kiviat B, Hughes P, Adam E, Lee K, Kuypers M, et al. Development and duration of Human papillomavirus lesions, after initial infection. J Infect Dia. 2005; 191(5):731-738.
Dobbs P, Asmussen T, Nunns D, Hollingworth J, Brown R, Ireland D. Does histological incomplete excision of cervical intraepithelial neoplasia following large loop excision of transformation zone increase recurrence rates a six year cytological following up. Br J Obstet Gynaecol. 2000; 107:1025-1030.
Hahn C, Weinberg A. Modelling the molecular circuitry of cancer. Nat Rev Cancer. 2002; 2:331-341.
Huibregtse M, Scheffner M, Howley M. Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the Human Papillomavirus E6 oncoprotein with p53. Mol Cell Biol. 1993; 13:775-784.
Be X, Hong Y, Wei J, Androphy J, Chen J, Baleja D. Solution structure determination and mutational analysis of the papillomavirus E6 interacting peptide of E6AP. Biochemistry. 2001; 40:1293-1299.
Chen J, Hong Y, Rustamzadeh E, Baleja D, Androphy J. Identification of an alpha helical motif sufficient for association with Papillomavirus E6. J Biol Chem. 1998; 273:13537-13544.
Solomon D, Davey D, Kurman R, Moriarty A, O'Connor D, Prey M, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA. 2002; 287(16):2114-2119.
Phelps W, Yee C, Munger K, Howley P. The Human Papilomavirus type 16 E7 gene encodes transactivation and transformation funtions similar to those of adenovirus E1A. cell. 1988; 53:539-546.
Moody A, Laimins A. Human Papillomavirus oncoproteins pathways to transformation. Nature reviews. 2010; 550-560.
Pim D, Banks L. Interaction of viral oncoproteins with cellular target molecules infection with high-risk vs low-risk Human Papillomaviruses. APMIS. 2010; 118:471-493.20
Werness B, Levine A, Howley B. Association of Human Papillomavirus types 16 and 18 E6 proteins with p53. Science. 1990; 248:76-79.
Cox T, Cuzick J. “HPV DNA testing in cervical cáncer screening: From evidence to policies”. Gynecol Oncol 2006; 103:8-11.
Boulet G, Horvath C, Berghmans S, Bogers J. Human Papillomavirus in cervical cancer Screening: Important role as biomarker. Cancer Epidemiol Biomarkers Prev. 2008; 17(4):810-817.
Morris BJ. Cervical Human Papillomavirus screening by PCR: Advantages of targeting the E6/E7 region. Clin Chem Lab Med. 2005; 43(11):1171-1177.
Lorincz T, Reid R, Jenson B, Greenberg D, Lancaster W, Kurman J. Human Papillomavirus infection of the cervix: Relative risk associations of 15 common anogenital types. Obstet Gynecol 1992; 79:328-337.
Muñoz N, Bosch X, de Sanjose S, Herrero R, Castellsague X, Shah V. Epidemiologic classification of Human Papillomavirus types associated with cervical cancer. N Engl J Med. 2003; 348:518-527.
Koutsky A, Holmes K; Critchlow W, et al. A cohort study of the risk of cervical intraepitelial neoplasia grade 2 or 3 in relation to papilomavirus infection. N Engl J Med 1992; 327:1972-1976.
Roda Husman M, Walboomers M, van den Brule J, Meijer J, Snijders J. The use of general primers GP5 and GP6 elongated at their 3' ends with adjacent highly conserved sequences improves Human Papillomavirus detection by PCR. J Gen Virol. 1995; 76(4):1057-1062.
Gravitt E, Peyton Q, Alessi M, Wheeler F, Coutlee A. Hildesheim H, et al. Improved amplification of genital Human Papillomaviruses. J. Clin. Microbiol. 2000; 38:357-361.
Sasagawa T, Minemoto Y, Basha W, Yamazaki H, Nakamura M, Yoshimoto H, et al. A new PCR-based assay amplifies the E6-E7 genes of most mucosal Human Papillomaviruses (HPV). Virus Res.2000; 67:127-139.
Melo A, García P, Capurro I, Guzmán P, Brebi P, Ili C, et al. Genotipificación del virus papiloma humano en mujeres con adenocarcinoma cervical de la Región de La Araucanía-Chile. Rev Chil Infect. 2010; 27(4):297-301.
Zehbe I, Wilander E. Two consensus primer systems and nested polymerase chain reaction for Human Papillomavirus detection in cervical biopsies: A study of sensitivity. Hum Pathol. 1996; 27(8):812-815.
Iwasawa A, Nieminen P, Lehtinen M, Paavonen J. Human Papillomavirus DNA in uterine cervix squamous cell carcinoma and adenocarcinoma detected by polymerase chain reaction. Cancer. 1996; 77(11):2275-2279.
Andersson S, Rylander E, Larson B, Sigurdardottir S, Backlund I, Sallstrom J, et al. Types of Human Papillomavirus revealed in cervical adenocarcinomas after DNA sequencing. Oncol Rep 2003; 10(1):175-179.
Weimin U, Jiang G, Cruz Y, Chang J, Ho Y, Klein S, et al. PCR detection of Human Papillomavirus: comparison between MY09/MY11 and GP5+/GP6+ primer systems. J Clin Microbiol. 1997; 35(6):1304-1310.
Sotlar K, Diemer D, Dethleffs A, Hack Y, Stubner A, Vollmer N, et al. Detection and Typing of Human Papillomavirus by E6 Nested Multiplex PCR. J Clin Microbiol. Jul 2004; 42(7):3176-3184.
Mejlhede N, Bonde J, Fomsgaard A. High frequency of multiple HPV types in cervical specimens from danish women. Apmis. 2009; 117:108-114.
Wentzensen N, Schiffman M, Dunn T, Zuna E, Gold A, Allen A, et al. Multiple Human Papillomavirus genotype infections in cervical cáncer progression in the study to understand cervical cancer early endpoints and determinants. Int J Cancer 2009; 125:2151-2158.
Rousseau C, Abrahamowicz M, Villa L, Costa C, Rohan E, et al. Predictors of cervical coinfection with multiple Human Papillomavirus types. Cancer Epidemiol Biomarkers Prev. 2003; 12:1029-1037.
