Kolon kanseri tanılı olgularda PCR-RFLP metodu ile p53 gen mutasyonlarının saptanması

Abstract

Kolon kanseri sporadik veya kal-tsal olabilen yayg-n bir hastal-kt-r. Kanserolu.umunda etkili olan en önemli tümör süpressör gen p53'dür. Normal p53replikasyon s-ras-nda DNA tamirini kolayla.t-rarak hücre siklusunu G1'de durmas-naveya apoptosise (programlanm-. hücre ölümü) neden olmas-nda rol oynar. Arg72Propolimorfizm kolorektal kanserin erken evrelerinde ve belki cinsiyete ve bölgeye ba=l-olarak invasiv hastal-=a ilerlemede rol oynayabilir. p53 intron 6'daki nükleotitde=i.imleri tümör geli.iminde ve p53 fonksiyonun düzensizli=iyle ili.kili oldu=urapor edilmi.tir. G13964C baz de=i.imi missense, nonsense ve splice bölgemutasyonu benzer dominant mutasyon olarak görev yapar.Çal-.mam-zda 35 (20 kad-n ve 15 erkek) kolon kanser te.hisi konmu. kolon kanserdoku örneklerinden DNA izole edildi. Dokular- ya.lar- 40-90 aras-nda kad-n (ya.ortalamas- 65.95±2.76) ve ya.lar- 40-79 aras-ndaki erkek olmak üzere (61.67±3.07)Ikolon kanserli 35 olgudan topland-. Parafine gömülü dokudan, EZNA Doku DNAKiti kullanarak, genomik DNA elde edildi. G13964C (intron 6) varyant-n- belirlemekiçin PCR-RFLP metodunu kullan-ld-. p53 4. eksonu PCR metodu ile amplifiye ettikve BstFNI restriksiyon enzimi kullanarak 4. ekson için p53 mutasyon analizibelirlendi. p53 6. varyant-n- PCR metoduyla amplifiye ettik ve BstHHI enziminikullanarak 6. intron için p53 mutasyonunu saptand-. Arg72Pro polimorfizmi için 2(%7.4) olguda Pro/Pro, 14 (%51.9) olguda Arg/Arg ve 11 (% 40.7) olguda Arg/Progenotiplerini bulundu. G13964C polimorfizmi için 7 (%23.3) olguda GG, 21(%70.0) GC ve 2 (%6.7) olguda CC bulundu.2006, 126 sayfaAnahtar Kelimeler: Kolon kanseri, p53 geni, Ar72Pro, G13964C, PCR, RFLP,mutasyon, polimorfizm.II

Colon cancer is a common disease that can be sporadic or inherited. p53 is the mostimportant tumor suppressor gene that is effective in cancer formation. Normal p53acts by causing G1 cell-cycle arrest to facilitate DNA repair during replication or toinduce apoptosis (programmed cell death). Arg72Pro may play a role in the earlystages of colorectal neoplasia and possibly in progression to invasive disease,depending on site and sex. Nucleotide alterations in p53 intron 6 have been reportedto be associated with the dysregulation of p53 function and tumor development.G13964C base change functioned as dominant mutation similar to the more commonmissense, nonsense and splice-site mutations.IIn this study, DNA was isolated from colon cancer tissue samples of (20 females and15 males) 35 cases diagnosed to be colon carcinoma. Tissues were collected from 35cases with colon cancer ages of female 40-90 (mean age 65.95±2.76) and ages ofmale 40-79 (mean age 61.67±3.07). Genomic DNA was extracted from paraffinembedded tissues using EZNA Tissue DNA Kit (Omega) following the protocol. Todetect the Arg72Pro region (exon 4) and G13964C variant (intron 6) PCR-RFLPassay was used. After then 4th exon of p53 were amplified by PCR method and p53mutation analyses was performed by using BstFNI restriction enzyme determined for4th exon. After then 6th variant of p53 were amplified by PCR method and p53mutation analyses was performed by using BstHHI restriction enzyme determined for6th intron. In 2 (%7.4) cases Pro/Pro, in 14 (%51.9) cases Arg/Arg and in 11 (% 40.7)Arg/Pro were found for Arg72Pro polymorphism. In 7 (%23.3) cases G/G, in 21(%70.0) cases G/C and in 2 (%6.7) C/C were found for G13964C polymorphism2006, 126 pageKey Words: Colon Cancer, p53 gene, Ar72Pro, G13964C, PCR, RFLP, mutation,polymorphism.II