Disertasi

Analisis Mutasi Gen Globin-β Penyebab Talasemia Mayor dan Pengujian Awal Short Tandem Repeat dengan Memanfaatkan Cell Free-DNA pada Spent Culture Media = Analysis of β-globin Gene in Thalassemia Major and Pilot Study of Short Tandem Repeat Testing on cell free-DNA from Spent Culture Media.

Talasemia adalah salah satu penyakit hemoglobinopati monogenic disorder autosomal resesiftersering di dunia sebagai salah satu dari akibat adanya perkawinan konsanguinitas yang dimulai di daerah Mediterrania. Mengacu pada data tahun 2021, penyandang talasemia mayor di Indonesia terdaftar berobat rutin sebanyak 10.973. Berbagai upaya guna mencegah penyakit talasemia telah banyak dilakukan. Namun demikian, upaya mencegah kelahiran bayi sehat dari orangtua penyandang talasemia belum pernah dilakukan sebelumnya. Penelitian ini bertujuan memetakan mutasi gen globin-β penyebab talasemia mayor pada populasi Indonesia, mengevaluasi penggunaan short tandem repeat (STR) sebagai marker segregasi keluarga, serta menguji kemunculan STR dalam cfDNA dari SCM sebagai metode skrining non-invasif. Penelitian terdiri atas dua tahap. Tahap pertama melibatkan sekuensing Sanger pada subjek dan orangtua, serta analisis segregasi STR (D11S2362 dan D11S4181). Tahap kedua mengevaluasi identifikasi STR pada cfDNA dari media kultur embrio sehat. Hasil dari penelitian ini adalah ditemukannya varian mutasi baru (novel) yakni IVS1nt.42 (c.92+42G > C) dan IVS2nt.16 (c.315+16G > C) serta mutasi umum IVS1nt.5, IVS1nt.1, dan Cd26 di populasi Indonesia. Marker STR terbukti mampu diturunkan secara stabil dalam keluarga dengan marker STR D11S2362 sebesar 96% fully informative dan marker STR D11S4181 sebesar 70.67 fully informative. Marker STR ini juga berhasil terdeteksi dalam cfDNA dari SCM, dimana marker STR D11S2362 sebesar 97.91% terdeteksi dan D11S4181 sebesar 96.87% terdeteksi. Kesimpulan dari penelitian ini adalah STR dapat dimanfaatkan sebagai penanda linkage untuk deteksi mutasi gen globin-β dalam pendekatan preimplantation genetic testing non-invasif di Indonesia.
Kata Kunci: Talasemia mayor, short tandem repeat, cell free-DNA, globin-β gene


Thalassemia is one of the most common autosomal recessive monogenic hemoglobinopathy disorders in the world as one of the consequences of consanguineous marriages that began in the Mediterranean region. Referring to 2021 data, 10.973 people with thalassemia major in Indonesia are registered for routine treatment. Various efforts to prevent thalassemia have been made. However, study to prevent the birth of healthy babies from parents with thalassemia have never been done before. This study aims to map β-globin gene mutations causing thalassemia major in Indonesian subjects, assess short tandem repeat (STR) markers for familial segregation analysis, and evaluate STR detectability in cfDNA from SCM as a potential non-invasive genetic screening tool. The study was conducted in two phases. Phase one involved Sanger sequencing of thalassemia subjects and parents, followed by STR segregation analysis (D11S2362 and D11S4181). Phase two tested the presence of STRs in cfDNA isolated from culture media of healthy IVF embryos. Result of this study shown there were 2 novel variants identified (IVS1 IVS1nt.42 (c.92+42G > C) and IVS2nt.16 (c.315+16G > C)) and common mutations were identified in the Indonesian cohort (the IVS1nt.5, IVS1nt.1, dan Cd26). STR markers shwon reliable familial inheritance patterns (STR D11S2362 with fully informative rate 96% and STR D114181 with fully informative rate 70.67%) and were successfully detected in cfDNA from SCM (STR D11S2362 detection percentage rate is 97.91% and STR D11S4181 detection percentage rate is 96.87%). To conclude, STR analysis using cfDNA from SCM holds promise as a non-invasive tool for preimplantation genetic testing of β-globin mutations in thalassemia-prone populations.
Keywords: Major thalassemia, short tandem repeat, cell free-DNA, globin-β gene