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Analisis Polimorfisme GABRA1 rs2279020 dan GABRG2 rs211037 dan penanda Inflamasi pada Pasien Status Epileptikus Konvulsif yang Mendapatkan Diazepam = An Analysis of GABRA1 rs2279020 DAN GABRG2 rs211037 Gene Polymorphism and Inflammatory Biomarkers of patients with Convulsive Status Epilepticus treated with Diazepam.

Latar belakang: Status epileptikus (SE) merupakan kedaruratan neurologis yang membutuhkan penanganan yang cepat dan tepat untuk mengurangi morbiditas dan mortalitas. Sepertiga kasus SE tidak bisa dikendalikan secara adekuat oleh benzodiazepine (termasuk diazepam [DZP]) yang merupakan obat lini pertama. Beberapa hal yang bisa mempengaruhi resistensi DZP pada SE antara lain internalisasi reseptor GABAA (GABAAR) sinaptik, perubahan komposisi subunit GABAAR fungsional, dan beratnya respons inflamasi yang terjadi pada SE. Pada penelitian ini, kami menganalisis hubungan polimorfisme gen GABAA (GABRA1 dan GABRG2), berbagai penanda inflamasi dari darah dengan respons terhadap DZP. Metode: Sembilan-puluh-dua pasien SE dewasa yang dirawat di IGD atau ruang perawatan RS Cipto Mangunkusumo Jakarta, dan mendapatkan terapi DZP sejak Mei 2022 hingga Oktober 2023 dimasukkan ke dalam studi ini. Kami mengumpulkan data klinis dan demografik dari masing-masing subyek. Setelah mendapatkan persetujuan (informed consent), dilakukan pengambilan sampel darah dalam waktu 72 jam dari awitan bangkitan. Setelah itu dilakukan ekstraksi DNA, isolasi PBMC dan serum. Hasil isolasi disimpan pada suhu -80 °C hingga digunakan selanjutnya. Serum digunakan untuk pemeriksaan kadar Human mobility group box-1 (HMGB1), soluble Toll-like receptors-4 (sTLR4), interleukin: IL-6 dan IL-10, dan Glial fibrillary acidic protein (GFAP) dengan metode ELISA; hasil DNA dilanjutkan dengan PCR dan DNA sequencing untuk melihat polimorfisme GABRA1 rs2279020 dan GABRG2 rs211037; dan sebagian hasil isolasi PBMC (N=18) dilakukan kultur dengan/tanpa lipopolisakarida (LPS), kemudian di-harvest, dilakukan ekstraksi mRNA, sintesis cDNA, dilanjutkan PCR kuantitatif untuk melihat ekspresi gen HMGB1, TLR4, IL- 6, IL-10, NF-κB dan GFAP. Hasil: Kadar GFAP dan sTLR4 serum berbeda signifikan antara kelompok resisten (DRT) dan responsif DZP (DRV). Tidak ditemukan perbedaan frekuensi genotip dan alotip GABRA1 rs2279020 dan GABRG2 rs211037 pada kelompok DRT dan DRV. Ditemukan hubungan antara kadar GFAP dan sTLR4 serum dengan durasi bangkitan pada kelompok DRT dan DRV. Ditemukan perbedaan signifikan ekspresi mRNA HMGB1, IL-6 dan rasio IL-6/IL-10 pada kultur PBMC dengan stimulasi LPS dibandingkan kontrol pada kelompok DRV. Kesimpulan: Kadar GFAP serum yang tinggi di kelompok DRT dapat menjadi penanda beratnya neuroinflamasi yang terjadi pada SE dengan resistensi DZP. Perlu dikembangkan studi lanjutan untuk menekankan pentingnya peranan GFAP lebih lanjut pada SE terutama dengan resistensi obat.
Kata kunci: Diazepam, HMGB1, GFAP, Interleukin, NF-κB, Polimorfisme GABA, Status epilepticus, TLR4


Introduction: Status epilepticus (SE) is a neurological emergency that requires prompt and appropriate treatment to reduce morbidity and mortality. It has been observed that one-third of SE cases cannot be effectively managed with benzodiazepines, such as diazepam (DZP), which are first-line agents. Several factors have been identified that may contribute to DZP resistance in SE, including internalization of synaptic GABAA receptors (GABAARs), changes in functional GABAAR subunit composition, and the severity of the inflammatory response that occurs in SE. This study investigated the potential relationship between GABAA gene polymorphisms (specifically GABRA1 and GABRG2), various inflammatory markers in the blood, and the response to DZP. Method: This study involved 92 adult SE patients who were treated with DZP therapy at Cipto Mangunkusumo Hospital Jakarta from May 2022 to October 2023. Clinical and demographic information was collected from After obtaining informed consent, blood samples were collected within 72 hours of seizure onset. DNA extraction, isolation of peripheral blood mononuclear cells (PBMC), and serum isolation were carried out. The samples were stored at -80°C until further analysis. Serum was used to determine the levels of human mobility group box-1 (HMGB1), soluble toll-like receptor-4 (sTLR4), interleukin-6 (IL-6), IL-10 and glial fibrillary acidic protein (GFAP) by ELISA; DNA results were followed by PCR and DNA sequencing to determine the polymorphism of GABRA1 rs2279020 and GABRG2 rs211037; Eighteen PBMC isolations were cultured with and without lipopolysaccharides (LPS), then harvested for mRNA extraction and cDNA synthesis. Quantitative PCR was performed to measure the expression of HMGB1, TLR4, IL-6, IL-10, NF-κB, and GFAP genes. Results: The levels of GFAP and sTLR4 in serum were significantly different between the resistant group (DRT) and the responsive group (DRV). However, no differences were detected in the genotype and allele frequencies of GABRA1 rs2279020 and GABRG2 rs211037 in either group. It was observed that there is a correlation between the duration of seizures and the levels of serum GFAP and sLTR4 in both the DRT and DRV groups. In the DRV group, some differences were observed in mRNA expression of HMGB1, IL-6, and IL-6/IL-10 ratio in PBMC culture with LPS stimulation compared to the control. Conclusion: The high levels of GFAP in the serum of the DRT group may be a marker of the severity of the neuroinflammation that occurs in SE with DZP resistance. Further research is needed to explore the role of GFAP in SE, especially in cases of drug resistance.
Keywords: Diazepam resistance, HMGB1, GFAP, Interleukin, NF-κB, GABA Polymorphism, Status epilepticus, TLR4