Büket KOSOVA, Z. EROĞLU, B. YILMAZ, C. GÜNDÜZ, R. ÖZEL, E. SAYIN, Ö. ÇOĞULU, F. ÖZKINAY, C. ÖZKINAY, N. TOPÇUOĞLU

ΔF508, ΔI507 ve F508C kistik fibroz mutasyonlarının gerçek-zamanlı multipleks PCR ile hızlı analizleri

Amaç: Kistik fibroz genellikle çocukluk yaşlarında ortaya çıkan ve tüm ekzokrin bezlerin fonksiyon bozukluğu ile seyreden kalıtsal bir hastalıktır. Avrupa' da yaygın olarak görülen bu hastalık kişilerin yaşam kalitesini etkilemekte ve tekrarlayan ağır solunum yolu enfeksiyonlarının açtıkları komplikasyonlar nedeniyle, erken yaşta ölüme yol açmaktadır. Kistik fibroz' daki en yaygın mutasyon ΔF508' dir. Bununla birlikte, binden fazla kistik fibroz gen (CFTR) mutasyonu tanımlanmıştır; ΔI507 ve F508C gibi. F508C ve ΔF508 mutasyonları ayrıca konjenital vas deferens agenezin gelişiminde de rol oynarlar. Çalışmadaki amacımız, aynı CFTR gen bölgesine düşen bu üç mutasyonu gerçek-zamanlı multipleks PCR yöntemi ile hızlı analizlerini gerçekleştirmekti. Gereç ve Yöntem: Ege Bölgesinde yaşayan kistik fibrozlu veya konjenital unilateral vas deferens agenezisi bulunan toplam 116 olgunun DNA örnekleri, tek bir çalışmada üç CFTR mutasyonun ayırıcı tanılarına gidilecek şekilde tasarlanmış olan gerçek zamanlı bir multipleks PCR yöntemiyle çalışıldı. Kullanılan yöntem ayrıca jel elekroforezi ve dHPLC yöntemleriyle de karşılaştırıldı. Bulgular: Çalışmanın sonunda, ΔI507 ve F508C mutasyonları için taşıyıcı olgu saptanmamasına karşılık, olguların % 6.0' sı ΔF508 aleli için heterozigot taşıyıcı ve % 1.8' i de homozigot hasta bulundu. ΔF508 mutasyonunun görülme sıklığı % 4.7 olarak belirlendi. Gerçek-zamanlı multipleks PCR yönteminin jel elektroforezi ve dHPLC' den daha hızlı sonuç verdiği görüldü. Sonuç: Kistik fibrozlu veya konjenital unilateral agenezisi bulunan olgularda CFTR ΔF508, ΔI507 ve F508C gen mutasyonlarının hızlı tayininde gerçek-zamanlı multipleks PCR ilk tercih edilen yöntem olmalıdır.

Rapid analysis of the cystic fibrosis ΔF508, ΔI507 and ΔF508C mutations by real-time multiplex PCR

Purpose: Cystic fibrosis, usually seen in childhood, is a hereditary disease that proceeds with the dysfunction of all exocrine glands. The disease is widely spread in Europe, affects the life quality of the affected individuals and causes their early death because of complications resulting from repeatedly serious respiratory tract infections. The most common mutation in cystic fibrosis is ΔF508. But so far, more than a thousand of other mutations have been discovered in the cystic fibrosis gene (CFTR); like ΔI507 and F508C. F508C and ΔF508 mutations are also implicated in the development of congenital vas deferens aplasia. The aim of this study was the rapid analyses of these three mutations that reside in the same CFTR gene region with a real-time multiplex PCR method. Methods: A total of 116 DNA samples of cases coming from the Aegean Region with cystic fibrosis or unilateral vas deferens aplasia were analyzed by a specifically designed real-time multiplex PCR method that detects all three CFTR mutations in one-step. The applied method was also compared with gel electrophoresis and dHPLC methods. Results: Although, we could not detect any carrier for the ΔI507 and F508C mutations at the end of our study; 6.0 % of the cases were heterozygous carriers for the ΔF508 allele and 1.8 % homozygous ill. The frequency of the ΔF508 mutation was defined as 4.7 %. The applied PCR method was also found to be faster in obtaining results compared to gel electrophoresis and dHPLC. Conclusion: The employed real-time multiplex PCR method should be the preferential method for the rapid analysis of the CFTR ΔF508, ΔI507 and F508C gene mutations in cases with cystic fibrosis or congenital unilateral vas deferens aplasia.

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