TEK GEN HASTALIĞI VE HLA UYUMUNDA PREİMPLANTASYON GENETİK TANI: TEK MERKEZ DENEYİMİ Single Gene Disorder and HLA Matched Preimplantation Genetic Diagnosis: Single Center Experience

OZET Amac: Preimplantasyon Genetik Tani (PGT)’da tek gen hastaliklari taramasi ile kombine HLA doku tiplemesi tayini Talasemi Major gibi yasami tehdit eden ve kordon kani ve /veya kemik iligi nakli ile tam olarak tedavi edilebilen genetik hastaliklara sahip cocuklar icin etkin bir yontemdir. Bu calismada, Saglik Bilimleri Universitesi Diskapi Yildirim Beyazit Egitim Arastirma Hastanesi Genetik Tani Merkezi Preimplantasyon Genetik Tani Laboratuvarinda 2014-2017 Mayis doneminde calisilan vakalarin verileri sunulmustur. Gerec ve Yontem: Laboratuvarimiza Saglik Bilimleri Universitesi Etlik Zubeyde Hanim Kadin Hastaliklari Tup Bebek Merkezinden gelen 6-8 blastomer asamasindaki 3. Gun embriyolarindan alinan biyopsi orneklerinde preimplantasyon genetik tani uygulamalari yapilan 91 vakanin genetik analiz sonuclari, mutasyon tipleri, implantasyon ve gebelik basarisi ile canli dogum oranlari degerlendirildi. Biyopsi orneklerine uygulanan lizis isleminin ardindan REPLI-g Advanced DNA Single Cell Kit (Qiagen, USA) ile Whole Genom Amplification (Biorad T100, USA) gerceklestirildikten sonra mutasyonu tasiyan DNA fragmentlerinin ve HBB geni ile iliskili 11 markirin belirlenebilir seviyeye kadar multiplex Polimeraz Zincir Reaksiyonu (PZR) ile cogaltilmasi yapildi. Amplifikasyondan sonra DNA, normal DNA fragmentlerini mutasyonu tasiyan fragmentlerden ayirt etmeye olanak saglayan mini-sekanslama teknigi ile kapiller elektroforez kullanilarak analiz edildi. Secilmis Linked Markerlar (STR: Short Tandem Repeat/ Kisa tekrar dizileri) mutasyonun tanisi icin bir destek olusturma ve bazi DNA kontaminasyonlarinin belirlenmesi amaciyla kullanildi. HLA tipleme analizlerinde uyumlu HLA genotiplerinin belirlenmesi icin HLA kompleksi ile iliskilendirilen 32 markir, multipleks PZR ile calisilarak DNA fragmentleri kapiller elektroforez kullanilarak analiz edildi Bulgular: 86 vaka Beta Talasemi (HBB geni) , bir vaka Orak Hucreli Anemi (HBB geni), bir vaka Blackfan Diamond Anemisi (RPS19 geni), bir vaka akut lenfoblastik losemi, bir vaka Agir Konjental Notropeni (HAX1 geni) ve bir vaka Fankoni Aplastik Anemisi (FANCA geni) olmak uzere toplamda 91 vaka calisildi. Toplam calisilan blastomer sayisi 328, transferi yapilan vaka sayisi 41, gebelik gerceklesen vaka sayisi 12, gerceklesen canli dogum sayisi 8 ve kemik iligi nakil islemi yapilan vaka sayisi 6 olarak saptandi. Canli dogumlarin tumunde gerceklestirilen ikinci genetik analizde saglikli/tasiyici gen ve HLA tipinde hasta kardes ile tam uyum goruldu. Sonuc: Sonuclarimiz kemik iligi nakli ile tam kur saglanan genetik hastaliklarin tedavisinde ailede saglikli ve HLA uyumlu cocuk varliginin onemi acisindan anlamlidir. Diger preimplantasyon genetik test uygulanan hastaliklarla karsilastirildiginda transfer edilebilir blastomer bulma oraninin dusuk oldugu gorulmus ve bunun blastomerde iki farkli genetik secilim yapilmasi gerekliliginden kaynaklandigi dusunulmustur. Anahtar Kelimeler: Preimplantasyon genetik tani; HLA uyumu; Tek gen hastaligi; Multipleks polimeraz zincir reaksiyonu; Kisa tekrar dizileri markiri; DNA sekans analizi ABSTRACT Purpose: Preimplantation Genetic Diagnosis (PGD) of single gene disorders combined with HLA typing is an effective method for life-threatening genetic diseases such as Beta thalassemia major which can be treated with bone marrow transplantation. The aim of this study is to present the data of the cases evaluated between May 2014-May 2017 in the PGD Laboratory of Genetic Diagnosis Center, Diskapi Yildirim Beyazit and Research Hospital, Health Sciences Faculty. Material and Methods: We evaluated the results of genetic analysis, the success of implantation and pregnancy, and the rate of live births of 91 cases which was biopsied from blastomeres of 6-8 stage of the 3th day embryos. After the Whole Genome Amplification with REPLI-g Advanced DNA Single Cell Kit (Qiagen, USA) preceded by the lysis of biopsy samples, DNA fragments and HBB related 11 markers were amplified until the predicted level with polymerase chain reaction (PCR). After the amplification, DNA was analyzed with mini-sequencing technique using capillary electrophoresis in order to distinguish normal DNA fragments from the mutated ones. Selected linked markers were used to support the diagnosis of mutation and determinate DNA contaminants. HLA complex related 32 markers were studied with multiplex PCR and DNA fragments were analyzed using capillary electrophoresis in order to determinate compatible HLA genotypes in HLA typing analyses. Results: We studied a total of 91 cases consisting of 86 cases of beta thalassemia (HBB gene), one case of sickle cell disease (HBB gene), one case of acute lymphoblastic lymphoma, one case of severe congenital neutropenia (HAX1 gene) and one case of Fanconi aplastic anemia (FANCA gene). The total number of blastomeres studied were 328, 41cases were transferred, and there were 12 successful pregnancies with 8 live births. There were also 6 cases which were treated with bone marrow transplantation. The second genetic analysis of all live births revealed that healthy/carrier gene and HLA typing of affected sibling was full matched. Conclusion: Our results have significant importance for the treatment of genetic disease which cure is possible with bone marrow transplantation in the presence of HLA matched sibling. The rate of transferable blastomere were lower than other genetically tested diseases; however, the reason of this difference was probably the necessity of making two different genetic selections. Key words: Preimplantation Genetic Diagnosis, HLA Matching, Single Gene Disorder, Multiplex polimeraz chain reaction, Short Tandem Repeat Marker, Sequence analysis DNA