Investigation of Copy Number Changes in CYP21A2 Gene By Using MLPA Technique in Patients with Congenital Adrenal Hyperplasia

Öz

Congenital adrenal hyperplasia (CAH) belongs to the group of familial diseases with autosomal recessive inheritance, which is caused by a disorder of adrenal steroid production. It is caused by the deficiency of any of the five enzymes that provide cortisol synthesis from cholesterol in the adrenal cortex. Characteristic of CAH is ambiguous genital structure and impaired sexual development. The most common cause of CAH is 21-hydroxylase enzyme deficiency. The CAH, which is caused by 21-hydroxylase deficiency, has 2 types as 'classic' and 'non-classical'. The differences in the clinical picture depend on the structural and functional changes caused by mutations in the 21-hydroxylase gene.
The two steroid 21-hydroxylase genes (CYP21A2 active gene, CYP21P pseudogene) are located on the short arm of chromosome 6. The mutations that cause defect of the active gene are due to the fact that active and pseudogene are very close and quite homologous. Deletions, duplications, gene conversions and point mutations occur as a result of intergenic recombination of the DNA sequence. Due to the presence of CYP21A2 pseudogene; there are often problems in diagnosing CAH, which develops due to 21-hydroxylase deficiency only with sequence analysis studies for the CYP21A2 gene.
In this study; patients who were directed to Department of Medical Genetics in Cerrahpasa Medical Faculty for CYP21A2 gene mutation analysis were reevaluated with clinical and laboratory findings. It was aimed to determine the number of copy changes that could not be detected in the sequence analysis. Therefore; ensuring the treatment of the patients without delay in their diagnosis; In addition, it is aimed to demonstrate that MLPA method is an easy, relatively cheap, fast and reliable method in mutation analysis for the CYP21A2 gene.
With the MLPA method, 28 patients were included in the study, homozygous deletion covering the majority of the gene in 1 patient, heterozygous deletion in 2 patients and heterozygous duplication in 1 patient were detected.

Anahtar Kelimeler

• Congenital adrenal hyperplasia,CYP21A2,CYP21P,MLPA

Konjenital Adrenal Hiperplazi Vakalarında CYP21A2 Geni Kopya Sayısı Değişimlerinin MLPA Tekniği İle İncelenmesi

Öz

Konjenital adrenal hiperplazi (KAH), adrenal steroid yapımının bozukluğu ile oluşan ve otozomal resesif kalıtım gösteren ailesel hastalıklar grubundandır. Adrenal kortekste kolesterolden kortizol sentezini sağlayan beş enzimden herhangi birinin eksikliği sonucu oluşmaktadır. KAH’ın karakteristik özelliği, kuşkulu genital yapı ve bozulmuş cinsel gelişimdir. KAH’ın en sık nedeni ise 21-hidroksilaz enzim eksikliğidir. 21-hidroksilaz enzim eksikliğine bağlı KAH’ın, ‘klasik’ ve ‘klasik olmayan’ olarak 2 tipi vardır. Klinik tablodaki farklılıklar 21-hidroksilaz genindeki mutasyonların oluşturduğu yapısal ve fonksiyonel değişikliklere bağlıdır.
İki steroid 21-hidroksilaz geni (CYP21A2 aktif gen, CYP21P psödogen) 6. kromozomun kısa kolu üzerinde bulunur. Aktif genin defektine neden olan mutasyonlar aktif gen ile psödogenin birbirine çok yakın ve oldukça homolog olmalarından kaynaklanmaktadır. DNA dizisinin intergenik rekombinasyonları sonucunda delesyonlar, duplikasyonlar, gen konversiyonları ve nokta mutasyonlar meydana gelir. CYP21A2’nin psödogeninin bulunması nedeniyle 21-hidroksilaz enzim eksikliğine bağlı gelişen KAH’a sadece CYP21A2 genine yönelik dizi analizi çalışmalarıyla doğru tanı konulmasında çoğu kez aksaklıklar yaşanmaktadır.
Çalışmamızda; Cerrahpaşa Tıp Fakültesi Tıbbi Genetik Anabilim Dalı’na CYP21A2 geni mutasyon analizi için yönlendirilen hastalar klinik ve laboratuvar bulgularıyla tekrar değerlendirilmeye alındı ve dizi analizinde saptanması mümkün olmayan kopya sayısı değişikliklerinin belirlenmesi hedeflendi. Böylece; hastaların tanısını kesinleştirerek, tedavilerinin gecikmeden yapılmasının sağlanması ile birlikte CYP21A2 genine yönelik yapılan mutasyon analizlerinde MLPA yönteminin kolay, nispeten ucuz, hızlı ve güvenilir bir yöntem olduğunun gösterilmesi amaçlanmıştır.
Çalışmaya dahil ettiğimiz 28 hastada uygulamış olduğumuz MLPA yöntemiyle, 1 hastada genin büyük kısmını kapsayan homozigot, 2 hastada heterozigot delesyon ve 1 hastada ise heterozigot duplikasyon saptanmıştır.

Anahtar Kelimeler

• Konjenital adrenal hiperplazi,CYP21A2,CYP21P,MLPA

Kaynakça

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