Disertasi

Analisis Molekular Mutasi Glycogen Branching Enzyme 1 (GBE1) via Protein Expression Study pada Penderita Glycogen Storage Disease IV dan Patogenisitasnya = Molecular Analysis of Glycogen Branching Enzyme 1 (GBE1) Mutation via Protein Expression Study in Glycogen Storage Disease IV Patients and its Pathogenicity.

Latar Belakang: Kelainan kongenital seperti Glycogen Storage Disease (GSD) IV memiliki prevalensi rendah namun berdampak besar pada kesehatan dan ekonomi. Diagnostik multiomik, yang melibatkan analisis fenotipe, genotipe, dan protein, dapat meningkatkan akurasi diagnosis dan klasifikasi patogenisitas varian mutasi baru. Penelitian ini bertujuan membuat model alur diagnostik multiomik untuk menegakkan patogenisitas varian baru GBE1 dan dampaknya terhadap ekspresi glycogen branching enzyme (GBE). Penelitian ini membuat library analisis fenotipe (IDeRare-pheno) dan pipeline fenotipe-genotipe terintegrasi (IDeRare) baru. Metode: Penelitian ini menggunakan pendekatan multiomik in silico dan pemodelan in vitro pada sel HEK293T untuk mengklasifikasikan patogenisitas berdasarkan kriteria ACMG. Ekspresi protein varian baru GBE1 dibandingkan dengan kontrol normal GBE1 wild type (WT), kontrol patogenik L224P, dan Y329S. Analisis in silico mencakup fenotipe, genotipe, dan protein, termasuk prediksi struktur dan degradasi protein. Studi in vitro melibatkan transfeksi plasmid, pembentukan galur sel transgenik, dan analisis ekspresi protein melalui immunoblotting. Hasil: Penelitian ini berhasil memodelkan alur diagnosis multiomik penyakit langka dengan pemodelan penemuan varian baru GBE1 R198T. Varian GBE1 R198T semula diklasifikasikan sebagai VUS secara in silico, dapat diklasifikasikan menjadi likely pathogenic paska studi fungsional in vitro. Eksperimen multiomik in silico mendukung perubahan struktur primer, sekunder, tersier, ikatan ligan-protein, dan degradasi protein GBE R198T. Studi in vitro menunjukkan penurunan signifikan intensitas sinyal GBE R198T pada kondisi basal dibandingkan GBE wild type. Alur multiomik ini disusun dengan memodifikasi alur penelitian sebelumnya, mempertimbangkan ketersediaan modalitas pemeriksaan di Indonesia dan efisiensi biaya. Kesimpulan: Penelitian ini berhasil membuktikan kaitan patomekanisme perubahan struktural protein GBE R198T secara in silico, yang didukung oleh penurunan ekspresi protein in vitro, dan menyusun alur diagnosis multiomik untuk penyakit langka di Indonesia.
Kata Kunci: kelainan bawaan, whole exome sequencing, analisis trio, undiagnosed case, mutasi baru


Introduction: Congenital anomalies like Glycogen Storage Disease (GSD) IV are rare but have significant health and economic impacts. Multiomic diagnostics, which analyze phenotype, genotype, and protein, can improve the accuracy of diagnosing new mutation variants. This study aims to develop a multiomic diagnostic model to establish the pathogenicity of novel GBE1 variant and its impact toward glycogen branching enzyme (GBE) expression. The study create novel phenotype analysis library (IDeRare-pheno) and an integrated phenotypegenotype pipeline (IDeRare). Method: An in silico multiomic approach and in vitro modeling in HEK293T cells were used to classify novel variant pathogenicity according to ACMG criteria. The novel GBE1 variant protein expression was compared with normal GBE1 wild type, and pathogenic control L224P and Y329S variants. In silico analysis included phenotype, genotype, and protein structure predictions. In vitro studies involved plasmid transfection, creation of transgenic mammalian cell lines, and protein expression analysis through immunoblotting. Results: The study successfully modeled a multiomic diagnostic pathway by discovering novel GBE1 R198T variant. GBE1 R198T variant, previously classifed as VUS, was able to be reclassified as likely pathogenic after in vitro functional study. In silico multiomic experiments supported structural changes, ligand-protein binding, and degradation of GBE R198T. In vitro studies showed a significant decrease in GBE R198T signal intensity under basal conditions compared to GBE wild type. The multiomic pathway was adapted from previous research, considering the availability of diagnostic modalities in Indonesia and cost efficiency. Conclusion: This study demonstrated the pathomechanism of structural changes in GBE R198T in silico, supported by decreased protein expression in vitro, and established a multiomic diagnostic pathway for rare diseases in Indonesia.
Keywords: congenital anomalies, whole exome sequencing, trio analysis, undiagnosed case, novel mutation