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Istanbul Üniversitesi Sağlik Bilimleri Enstitüsü T.C. İSTANBUL ÜNİVERSİTESİ SAĞLIK BİLİMLERİ ENSTİTÜSÜ ( YÜKSEK LİSANS TEZİ ) MKA/MR OLGULARINDA GENETİK ETİYOLOJİNİN ARRAY-CGH TEKNİĞİ İLE ARAŞTIRILMASI MARYAM BARGHI MASTAN ABAD PROF. DR. BEYHAN TÜYSÜZ GENETİK ANABİLİM DALI GENETİK PROGRAMI İSTANBUL-2018 BEYAN Bu tez çalışmasının kendi çalışmam olduğunu, tezin planlanmasından yazımına kadar bütün safhalarda etik dışı davranışımın olmadığını, bu tezdeki bütün bilgileri akademik ve etik kurallar içinde elde ettiğimi, bu tez çalışmayla elde edilmeyen bütün bilgi ve yorumlara kaynak gösterdiğimi ve bu kaynakları da kaynaklar listesine aldığımı, yine bu tezin çalışılması ve yazımı sırasında patent ve telif haklarını ihlal edici bir davranışımın olmadığı beyan ederim. MARYAM BARGHI MASTAN ABAD ii İTHAF Sevgili Aileme… iii TEŞEKKÜR Yüksek lisans eğitimim boyunca engin bilgi ve tecrübelerinden yararlandığım, desteğini her zaman yanımda hissettiğim, tez çalışmamın her satırında emeği olan sevgili danışman hocam sayın Prof. Dr. Beyhan TÜYSÜZ’e; Yol gösterici ve anlayışlı tavrıyla her zaman destek olan, bilgi ve tecrübelerini esirgemeyen sevgili hocam sayın Doç. Dr. Birsen KARAMAN’a; Tez çalışmalarım boyunca birlikte çalışma fırsatı yakaladığım, bilgi ve tecrübelerinden yararlandığım, desteğini her zaman hissettiğim sayın Dr. Dilek ULUDAĞ’a; Eğitimim boyunca hiçbir konuda yardımlarını esirgemeyen, her zaman destekleyen, her birinin ayrı ayrı emeği olan tüm Çocuk Genetik Anabilim Dalı çalışanlarına ve arkadaşlarıma, Desteklerini hiçbir zaman esirgemeyen ve her an yanımda olan sevgili aileme sonsuz teşekkür ederim. iv İÇİNDEKİLER TEZ ONAYI……………………………………………………………………………….... İ BEYAN ………...................................................................................................................... İİ İTHAF..................................................................................................................................... İİİ TEŞEKKÜR............................................................................................................................ İV İÇİNDEKİLER..........................................................................................................................V TABLOLAR LİSTESİ......................................................................................................... Vİİİ ŞEKİLLER LİSTESİ.............................................................................................................. İX SEMBOLLER / KISALTMALAR LİSTESİ.......................................................................... Xİ ÖZET.................................................................................................................................... Xİİİ ABSTRACT......................................................................................................................... XİV 1. GİRİŞ VE AMAÇ..................................................................................................................1 2. GENEL BİLGİLER............................................................................................................... 3 2.1 Mental RetardasyonTanımı ve Epidemiyolojisi…………………….…………………... 3 2.2 MR’nin Sınıflandırılması……………………………………………………………….. 3 2.3 Multipl Konjenital Anomali (MKA)Tanımı ve Epidemiyolojisi……………..………… 4 2.4 MKA/MR’nin Etiyolojisi………………………………………………………………. 5 2.4.1 MKA/MR'ye Sebep Olan Genetik Nedenler……………………….…………………. 8 2.4.1.1 MKA/MR Etiyolojisinde Kromozom Anomalileri…………………………………. 9 2.4.1.1.2 Sayısal Kromozom Anomalileri…………………………………………………. 10 2.4.1.1.3 Yapısal Kromozom Anomalileri………………………………………………… 10 2.4.1.1.3.1 Dengeli Yapısal Kromozom Anomalileri……………………………………… 10 2.4.1.1.3.2 Dengesiz Yapısal kromozom Anomalileri…………………………………….. 12 2.4.1.2 Tek Gen Hastalıkları………………………………………………………………. 14 Otozomal Dominant Geҫişli Olan MR……………………………………………………… 14 Otozomal Resesif Geҫişli Olan MR……………..………………………………………….. 15 X’e Bağlı MR……………………………………………………………………………..… 15 v 2.5 MKA/MR Tanısında Kullanılan Teknikler…………………………………………...... 16 2.5.1 Klasik Sitogenetik Teknikleri………………………………………………………… 16 Kromozom Elde Edilmesi…………………………………………………………………... 16 Giemsa Bandlama Yöntemi (G-Banding) ………………………………………………...... 17 Yüksek Rezollüsyonlu Bantlama Tekniği (HRBT)…………………………………………. 18 2.5.2 Moleküler Sitogenetik Teknikler……………………………………………………... 19 2.5.2.1 Floresan İn situ Hibridizasyon (FISH)……………………………………………... 19 2.5.2.2 CGH (Comperative Genomic Hybridisation) ……………………………………… 22 2.5.2.3 Mikroarray Analizleri………………………………...…………………………….. 23 2.5.2.4 Array-CGH…………………………………………………………………………. 23 2.4.2.5 SNP-Array………………………………………………………………………...... 28 2.4.2.6 CGH Array'ler ile SNP Array'lerin Karşılaştırılması………………………………. 29 3. GEREÇ-YÖNTEM………………………………………………………………………. 31 3.2 Gereçler………………………………………………………………………………… 31 3.2.1 Kullanılan Taşınabilir Cihazlar………………………………………………………. 31 3.2.2. Kullanılan Kimyasal Malzemeler……………………………………………………. 32 3.3 Kullanılan Teknikler…………………………………………………………………… 33 3.3.2 Array-CGH Çalışması……………………………………………………………...… 34 3.3.2.1 Hasta ve Referans DNA’nın İşaretlenerek Hibridizasyonu…..……………………. 34 3.3.2.2 Yıkama Aşaması…………………………………………………………………… 36 3.3.2.3 Tarama Aşaması……………………………………………………………………. 36 3.3.2.4 Analiz Aşaması……………………………………………………….……………. 37 4. BULGULAR……………………………………………………………………….…….. 39 4.1 a-CGHʼde Patolojik Varyant Saptanan 4 Olgunun Sunumu…………………………… 40 5. TARTIŞMA……………………………………………………………………………… 58 KAYNAKLAR…………………………………………………………………………...…. 67 FORMLAR………………………………………………………………………………….. 72 vi ETİK KURUL KARARI……………………………………………………………….…… 73 ÖZGEÇMİŞ………………………………………………………………………………… 74 vii TABLOLAR LİSTESİ Tablo2-1: IQ test puanlamasına göre MR sınıflandırılması ....................................................... 4 Tablo 2-2: Fetal hayatın farklı evrelerinde ve postnatal dönemde kromozom anomalilerinin sıklığı…………………………………………………………………………………………. 9 Tablo 2-3: Sık görülen bazı mikrodelesyon sendromları ve lokalizasyonları………………. 13 Tablo 4-1: Çalışmaya Dahil Edilen Bireyler ve Bilgileri…………………………………… 39 Tablo 4-2: a-CGHʼde patolojik varyant saptanan 4 olgunun özeti…………………………. 40 Tablo 5-1: 6qterminal delesyonuyla ilşkili en önemli genler, fonksiyonları ve yol aҫtıkları hastalıklar…………………………………………………………………………………… 60 Tablo 5-2: 22q11.2 delesyonuyla ilşkili en önemli genler, fonksiyonları ve yol aҫtıkları hastalıklar…………………………………………………………………………………… 61 Tablo 5-3: Xq27.3q28 duplikasyonu ile ilşkili en önemli genler, fonksiyonları ve yol aҫtıkları hastalıklar…………………………………………………………………………………… 62 Tablo 5-4: Xp22.33 delesyonuyla ilşkili en önemli genler, fonksiyonları ve yol aҫtıkları hastalıklar…………………………………………………………………………………… 64 Tablo 5-5: 16p11.2 mikrodelesyon ile ilşkili en önemli genler, fonksiyonları ve yol aҫtıkları hastalıklar…………………………………………………………………………………… 65 viii ŞEKİLLER LİSTESİ Şekil 2-1: MKA/MR’ye sebep olan genetik nedenler………………………………………... 9 Şekil 2-2: 3.kromozomda perisentrik inversiyon örneği……………………………………. 12 Şekil 2-3: MR ile ilişkili bazı otozomal genler ve kromozomlar üzerindeki yerleri………... 14 Şekil 2-4: OMIM'de X'e bağlı MR sorumlu olarak rapor edilen genlerin kromozomal lokalizasyonu ve ilişkili oldukları hastalıklar………………………………………………... 16 Şekil 2-5: CTF’de yapılan G-bantlama örneği……………………………………………… 18 Şekil 2-6: FISH çalışma prensibi…………………………………………………………… 20 Şekil 2-7: CGH yönteminin akış şeması……………………………………………………. 22 Şekil 2-8: Database of Genomic Variants’da arşivlenmiş CNV’lerin büyüklük dağılımları…………………………………………………………………………………... 28 Şekil 2-9: CNV’lerin tek ve çift vuruş fenotip etkisi……………………………………….. 25 Şekil 2-10: a-CGH tekniğinin uygulama aşamaları………………………………………… 26 Şekil 2-11: Kromozom analizi normal sonuҫlanan olgulardaki a-CGH incelemelerinde akış şeması……………………………………………………………………………………….. 28 Şekil 2-13: CGH arrayʼler ile SNP arrayʼlerin karşılaştırılması……………………………. 30 Şekil 4-1: olgu 17ʼye ait aile ağacı………………………………………………………….. 40 Şekil 4-2: Olgu 17’e ait 5.5 yaş fotoğrafları………………………………………………… 41 Şekil 4-3: Olgu 17'e ait karyotip görüntüsü………………………………………………… 42 Şekil 4-4: Olgu 17’ye ait a-CGH (Agilent platformu ile) görüntüsü……………………….. 42 Şekil 4-5: Olgu 17’e ait 6. kromozomundaki delesyon bölgesinin a-CGH görüntüsü……… 43 Şekil 4-6: Olgu 18ʼe ait aile ağacı…………………………………………………………... 44 Şekil 4-7: Olgu 18’e ait 16 yaş fotoğrafları…………………………………………………. 44 Şekil 4-8: Olgu 18'e ait karyotip görüntüsü………………………………………………… 45 Şekil 4-9: Olgu 18’e ait a-CGH (Agilent platformu ile) görüntüsü………………………… 46 Şekil 4-10: Olgu 18’e ait 22. kromozomundaki delesyon bölgesinin a-CGH görüntüsü…… 46 Şekil 4-11: Olgu 18'e ait FISH görüntüsü…………………………………………………... 47 ix Şekil 4-12: Olgu 19ʼa ait aile ağacı…………………………………………………………. 48 Şekil 4-13: Olgu 19’a ait 4.5 yaş fotoğrafları……………………………………………….. 49 Şekil 4-14: Olgu 19'a ait karyotip görüntüs………………………………………………… 49 Şekil 4-15: Olgu 19’a ait a-CGH (Agilent platformu ile) görüntüsü……………………….. 50 Şekil 4-16: Olgu 19’a ait X. kromozomundaki delesyon/duplikasyon bölgelerinin a-CGH görüntüsü……………………………………………………………………………………. 51 Şekil 4-17: olgu 19'a ait FISH görüntüsü…………………………………………………… 52 Şekil 4-18: olgu 19'un annesine ait FISH görütüsü…………………………………………. 53 Şekil 4-19: Olgu 20ʼye ait aile ağacı………………………………………………………... 53 Şekil 4-20: Olgu 20’ye ait 5.5 yaş fotoğrafları……………………………………………… 55 Şekil 4-21: Olgu 20'ye ait karyotip görüntüsü……………………………………………… 55 Şekil 4-22: Olgu 20’ye ait a-CGH (Agilent platformu ile) görüntüsü…………………….... 56 Şekil 4-23: Olgu 20’ye ait 16. kromozomundaki delesyon bölgelerinin a-CGH görüntüsü…56 x
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