Μεταλλαγές και έκφραση των ογκογονιδίων RAS και BRAF καθώς και αναζήτηση DNA ιών σε ρινικούς πολύποδες του ανθρώπου
Authors: Ζαραβίνος Απόστολος 
Published: January 2014

Although it is well established that ras genes contribute to tumourigenesis either through the accumulation of mutations or by aberrant expression in a wide range of human cancers, little is known regarding their involvement in human nasal polyps (NPs). Moreover, little is known about the implication of BRAF and RKIP expression, or about the incidence of BRAF mutations, as well as about the presence of viral DNA in the formation of nasal polyps. In the present study, the occurrence of mutations in codons 11, 12 and 13 of the ras family genes was examined by PCR/RFLP and direct sequencing in 23 human NPs and their adjacent turbinates [Control Inferior Turbinates (CIT) and Control Middle Turbinates (CMT)], as well as in turbinates from 13 control subjects (CIT and CMT). Moreover, the expression pattern of ras mRNA levels was assessed in NP specimens and compared to adjacent and control tissues. Regarding the second part of this study, we determined the expression levels of the genes BRAF and RKIP, and inspected the frequency of BRAF mutations in exons 11, 14 and 15 in the nasal polyps and their adjacent and control turbinates. We analysed 24 NPs and their adjacent turbinates (CIT and CMT), as well as 14 CITs and 14 CMTs. The expression pattern of BRAF and RKIP was assessed with real-time RT-PCR. A real-time allele-specific PCR method, in combination with direct sequencing, was performed in order to inspect the frequency of the V600E mutation in exon 15, and to examine mutation status within exons 11 and 14. The presence of amplifiable DNA was confirmed with β2-microglobulin primers, and the HPV, EBV and HSV viral DNA detection was performed with specific primer sets, using PCR. K-ras codon 11 and 12 mutations were detected in 17 and 35% of NPs, respectively, and were found in the adjacent inferior turbinate (AIT) (22 and 16%, respectively) and adjacent middle turbinates (AMT) (16 and 26%, respectively). K- and H-ras expression levels were elevated, whereas N-ras mRNA levels were lower in NPs and adjacent turbinates as compared to the control tissues. K-ras mRNA levels were up-regulated in advanced-stage polyps (P=0.037), while N-ras levels were found elevated in small polyps (P=0.046). Statistically significant negative correlations between K- and N-ras expression profiles arose in NPs and AITs (P=0.009 and 0.003, respectively). Regarding the second part of our study, our results were as follows: The control mucosae presented significantly higher mRNA levels for both genes, compared to the NP and the AIT–AMT. Moreover, in NP, AIT and AMT, RKIP was found to xi xii present higher mRNA levels, in relation to the equivalent values of the BRAF gene (P = 0.003 in NP; P < 0.001 both in AIT and AMT). No mutation was detected in exon 14, whereas a silent mutation (A1380G, G460G) was noted for one NP sample in exon 11. Another NP sample was found to carry two mutations, one T1799A (V600E) and one A1801G (K601E). A significant co-expression of the two genes was noted in NP (P = 0.012) and AIT (P = 0.019). Regarding the viral DNA detection, three NP samples (3/23, 13%) and one sample from each adjacent turbinate [AIT, 123 (4%); AMT, 1/12 (4%)] were found to be positive for the HPV virus, yet no oncogenic type (HPV-11, 16, -18, -33) was recognized. Eight NP samples (8/23, 35%) were EBV positive, and two NP samples (2/23, 8%) were HSV positive, respectively. No adjacent turbinate was positive for the presence of neither of these two herpes viruses. Moreover, al the control turbinates were also found to be negative for the presence of the HPV, EBV and HSV viruses. This, to our knowledge, is the first report on ras mutations and expression analysis in NPs. Our findings suggest a potential key role for activated members of ras family genes in terms of their contribution to the development of NPs as well as to the hypertrophy of adjacent turbinates. The results of the expression levels of RKIP and BRAF, reflect the strong connection between the two genes. RKIP could play an important role in the down-regulation of wild-type BRAF, serving thus as an endogenous inhibitor of the MAPK pathway in nasal polyps and their adjacent turbinate mucosa. The results upon the presence of viral DNA in the nasal polyps, confirm the hypothesis that HPV is not frequently associated with sinunasal polyposis, and it is correlated to a lesser extent to infectious mucosal lesions, than to proliferative lesions. Regarding EBV, the percentage of infection shows that nasal mucosa could be one of the sites of persistence of the virus, and there’s a possibility that it is responsible for presence of viral-associated nasal tumors, whereas, HSV does not seem to be implicated in the development of nasal polyps