Publication: Konjenital adrenal hiperplazili bireylerde, 21 hidroksilaz gen kusurlarının sıklığı, fenotipik etkileri ve diğer moleküler mekanizmaları
Abstract
Konjenital Adrenal Hiperplazi (KAH), kolesterolden kortizol sentezini sağlayan enzimlerin eksikliği sonucu oluşan, genetik, endokrin ve metabolik bir hastalıktır. KAH vakalarının % 90’ından fazlasında 21-hidroksilaz enzim eksikliği bulunmaktadır. 21-hidroksilaz enzim eksikliğine neden olan CYP21A2 genindeki mutasyonları belirlemek için bugüne kadar çeşitli yöntemlerle mutasyon taramaları yapılmasına rağmen, ülkemizde bu konu ile ilgili yapılan çalışmalar yetersizdir. Bu nedenle çalışmamızda, ülkemizdeki KAH’li bireylerde 21-hidroksilaz enzim eksikliğine neden olan mutasyonların dizi analizi yöntemi ile belirlenmesi ve bu mutasyonların hangi tip (klasik ya da kasik olmayan tip) KAH hastalığına neden olduğunun araştırılması planlanmıştır. Çalışmamıza 2007-2009 tarihleri arasında, Marmara Üniversitesi Pediatrik Endokrin Bilim Dalına ve Bakırköy Ana ve Çocuk Sağlığı Eğitim Merkezine değişik şikayetlerle başvuran ve 21-hidroksilaz eksikliği tanısı alan 100 hasta dahil edilmiştir. Kontrol grubuna ise sağlıklı 80 gönüllü vaka alınmıştır. Her iki gruptaki bireylerden periferik kan örnekleri toplanmıştır. Öncelikle periferik kan örneklerinden DNA izolasyonu yapılmış, CYP21A2 genine uygun primerler kullanılarak polimeraz zincir reaksiyonu (PZR) yöntemi ile CYP21A2 geni promotor bölgesinden 4. ekzonun sonuna kadar amplifiye edilmiş ve amplifiye edilen PZR ürünleri saflaştırma işleminden geçirilmiştir. Daha sonra dizi analizi yöntemi ile mutasyon taraması yapılmıştır. Elde edilen bulgular istatistiksel analizler sonucunda değerlendirilmiştir. Çalışmamız sonucunda 10’u yeni olmak üzere toplam 11 farklı mutasyon bulunmuştur. Bu mutasyonlardan 2. intronda yer alan 711. pozisyondaki GA ve 777. pozisyondaki A/ CG mutasyonlarının istatistiksel analizler sonucunda kontrole göre anlamlı olduğu ve genellikle tuz kaybedici tipte gözlenen mutasyonlar olduğu tespit edilmiştir. Bu mutasyonlar dışında 16 farklı polimorfizm belirlenmiştir. CYP21A2, KAH, 21-hidroksilaz eksikliği, mutasyon, steroid hormonlar
Congenital adrenal hyperplasia (CAH) is a genetic, endocrine and metabolic disorder, caused by enzyme deficiencies in the convertion of cholesterol to cortisol. More than 90 % of cases are caused by a deficiency of the 21-hydroxylase enzyme. Although several methods have been used to determine the CYP21A2 gene mutations that cause 21-hydroxylase deficiency, studies about this disease are insufficient in our country. So in our study, it is planned to detect the mutations that cause 21-hydroxylase deficieny in CAH patients by using direct sequencing method and to investigate the type of CAH (classic or non-classic type) that these mutations cause. In our study, 100 patients who were attended with several complaints to Marmara University Pediatric Endocrinology Department and Bakırköy Mother and Child Health Education Center and were diagnosed as 21-hydroxylase deficiency, between 2007 and 2009 were included. 80 healthy volunteers were studied as control group. Peripheric blood samples were collected from each of the groups. Initially DNA isolation from peripheric blood was performed, and then CYP21A2 gene regions starting from promoter to the end of exon 4 were amplified by PCR using appropriate primers. Then, amplified PCR products were purificated and mutations were detected by using direct sequencing. Results were evaluated statistically. At the end of our study, a total number of 11 mutations were found, in which one of them is novel. As a result, G®A mutation at position 711 and A/ C®G mutation at position 777 which were detected in intron 2, were statistically significant when compared to controls. Especially, these mutations were detected in salt-wasting type. Except to these mutations, 16 different polymorphisms were also detected. Key words: CYP21A2, CAH, 21-hydroxylase deficiency, mutation, steroid hormones
Congenital adrenal hyperplasia (CAH) is a genetic, endocrine and metabolic disorder, caused by enzyme deficiencies in the convertion of cholesterol to cortisol. More than 90 % of cases are caused by a deficiency of the 21-hydroxylase enzyme. Although several methods have been used to determine the CYP21A2 gene mutations that cause 21-hydroxylase deficiency, studies about this disease are insufficient in our country. So in our study, it is planned to detect the mutations that cause 21-hydroxylase deficieny in CAH patients by using direct sequencing method and to investigate the type of CAH (classic or non-classic type) that these mutations cause. In our study, 100 patients who were attended with several complaints to Marmara University Pediatric Endocrinology Department and Bakırköy Mother and Child Health Education Center and were diagnosed as 21-hydroxylase deficiency, between 2007 and 2009 were included. 80 healthy volunteers were studied as control group. Peripheric blood samples were collected from each of the groups. Initially DNA isolation from peripheric blood was performed, and then CYP21A2 gene regions starting from promoter to the end of exon 4 were amplified by PCR using appropriate primers. Then, amplified PCR products were purificated and mutations were detected by using direct sequencing. Results were evaluated statistically. At the end of our study, a total number of 11 mutations were found, in which one of them is novel. As a result, G®A mutation at position 711 and A/ C®G mutation at position 777 which were detected in intron 2, were statistically significant when compared to controls. Especially, these mutations were detected in salt-wasting type. Except to these mutations, 16 different polymorphisms were also detected. Key words: CYP21A2, CAH, 21-hydroxylase deficiency, mutation, steroid hormones