Investigation of Unknown Neurogenetic Variants: Integrative Computational Analysis Reveals Critical Variants of GABRB3 Gene Associated with Epilepsy

Yıl 2025, Cilt: 25 Sayı: 6, 1239 - 1260

Derya Türkmen Delioğlu , Ayla Arslan

Öz

Alterations of GABA (A) receptors are linked to various disorders including epilepsy, which is diagnosed through a comprehensive approach including genetic screening. The functional consequence of many genetic variants in the β3 subunit of the GABA(A) receptor remain unknown. This presents a challenge for genetic testing and precision medicine. Addressing this obstacle, in the present study, we analyzed the 141 missense variants with unknown function in the GABRB3 gene using a comprehensive in silico approach. Algorithmic computing based on the sequence homology and other features including the functional and disease-related analysis of missense variants revealed the prediction of the most pathogenic variants, mapped onto the different domains of the β3 subunit, with Y48C, D49E, D73H, M80R, D94E, H132Y, R142C, P169L, C175W and Y182H being located in the N terminus extracellular domain, S264F in the first transmembrane domain, G279R, T281A the second transmembrane region, R294Q is at the end of the second transmembrane domain, P298S in the linker between the second and the third transmembrane domains, and Y467S/H in the fourth transmembrane domains. These variants were generally associated with childhood absence epilepsy. Our results provide guidance for the laboratory research aiming for the identification of new pathogenic epilepsy mutations.

Anahtar Kelimeler

Epilepsy , GABA (A) receptor , GABRB3 , Variants of uncertain significance

Etik Beyan

This study is based on the master's thesis entitled “Virtual functional profiling of variants of unknown significance in the GABRB3 gene associated with epilepsy’’ (Thesis number: 10612321), completed on January 19, 2024, under the supervision of Asst. Prof. Dr. Ayla Arslan. It is hereby declared that the preparation of this study adhered to scientific and ethical standards, and that all sources consulted have been duly cited in the bibliography.

Bilinmeyen Nörogenetik Varyantların Araştırılması: Epilepsi ile İlişkili GABRB3 Geninin Kritik Varyantlarının Belirlenmesi

Öz

GABA (A) reseptörlerindeki mutasyonlar, genetik testler de dahil olmak üzere bütünleştirici bir yaklaşımla teşhis edilen epilepsi gibi çeşitli hastalıklarla bağlantılıdır. Öte yandan, β3 alt ünitesini kodlayan GABRB3 geninin varyantları da dahil olmak üzere GABA (A) reseptör alt ünitesi gen varyantlarının büyük bir kısmının fonksiyonel sonucu bilinmemektedir ve bu durum genetik testler ve hassas tıp için bir zorluk teşkil etmektedir. Mevcut çalışmada, kapsamlı in siliko analizi kullanılarak GABRB3 geninin işlevi bilinmeyen 141 varyantı analiz edildi. Sekans homolojisine ve varyantların fonksiyonel ve hastalıkla ilgili analizi de dahil çeşitli özelliklere dayanan algoritmik hesaplamalar, β3 alt ünitesinin farklı protein alanlarında bulunan en patojenik varyantları ortaya çıkardı. Y48C, D49E, D73H, M80R, D94E, H132Y, R142C, P169L, C175W, ve Y182H, N -terminal hücre dışı alanda, S264F birinci transmembran alanında, G279R ve T281A ikinci transmembran alanında, R294Q, ikinci transmembran bölgesinin sonunda, P298S ikinci ve üçüncü transmembran alanları arasındaki bölgede ve Y467S/H dördüncü transmembran alanda bulunan patojenik varyantlar olarak tespit edildi. Bu varyantlar, çocukluk çağı absans epilepsisi ile ilişkili bulunmuştur. Sonuçlarımız, yeni patojenik epilepsi mutasyonlarının tanımlanmasına yönelik laboratuvar araştırmalarına rehberlik sağlamaktadır.

Anahtar Kelimeler

Epilepsi , GABA (A) reseptörü , Belirsiz Anlamlı Varyant (VUS)

Etik Beyan

Bu çalışma, 19 Ocak 2024 tarihinde Dr. Öğretim Üyesi Ayla Arslan danışmanlığında tamamlanan “Virtual functional profiling of variants of unknown significance in the GABRB3 gene associated with epilepsy” başlıklı yüksek lisans tezine (Tez No: 10612321) dayanmaktadır. Bu çalışmanın hazırlanmasında bilimsel ve etik ilkelere uyulduğu ve başvurulan tüm kaynakların kaynakçada eksiksiz biçimde belirtildiği beyan edilmektedir.

Kaynakça

• Ahn, H.J., Ahn, Y., Kurade, M.B., Patil, S.M., Ha, G.S., Bankole, P.O., Khan, M.A., Chang, S.W., Abdellattif, M.H., Yadav, K.K. ve Jeon, B.H., 2022. The comprehensive effects of aluminum oxide nanoparticles on the physiology of freshwater microalga Scenedesmus obliquus and it’s phycoremediation performance for the removal of sulfacetamide. Environmental Research, 215, 114314. https://doi.org/10.1016/J.ENVRES.2022.114314
• Aksu, M., Has, M., Tanattı, N.P., Erden, B., Sınmaz, G. K., Boysan, F. ve Şengil, A., 2022. Assessment of photocatalytic n-TiO2 /UV and n-TiO2 /H2 O2 /UV methods to treat DB 86, RY 145 and AV 90 dye mix containing wastewater. Desalination and Water Treatment, 266, 226–235. https://doi.org/10.5004/DWT.2022.28656
• Angelini, F., Bellini, E., Marchetti, A., Salvatori, G., Villano, M., Pontiggia, D. ve Ferrari, S., 2024. Efficient utilization of monosaccharides from agri-food byproducts supports Chlorella vulgaris biomass production under mixotrophic conditions. Algal Research, 77, 103358. https://doi.org/10.1016/J.ALGAL.2023.103358
• APHA, 2017. Standard Methods for the Examination of Water and Wastewater. Standard Methods, 541. https://doi.org/ISBN 9780875532356
• Barari, F., Gabrabad, M.E. ve Bonyadi, Z., 2024. Recent progress on the toxic effects of microplastics on Chlorella sp. in aquatic environments, Heliyon, 10,32881. https://doi.org/10.1016/j.heliyon.2024.e32881
• Barbero, F., Mayall, C., Drobne, D., Saiz-Poseu, J., Bastús, N. G. ve Puntes, V., 2021. Formation and evolution of the nanoparticle environmental corona: The case of Au and humic acid. Science of The Total Environment, 768, 144792. https://doi.org/10.1016/J.SCITOTENV.2020.144792
• Beyer, W.F. ve Fridovich, I., 1987. Assaying for superoxide dismutase activity: Some large consequences of minor changes in conditions. Analytical Biochemistry, 161(2), 559–566. https://doi.org/10.1016/0003-2697(87)90489-1
• Canli, E. G. ve Canli, M., 2020. Effects of aluminum, copper and titanium nanoparticles on the liver antioxidant enzymes of the Nile fish (Oreochromis niloticus). Energy Reports, 6, 62–67. https://doi.org/10.1016/J.EGYR.2020.10.047
• Chen, L., Zhou, L., Liu, Y., Deng, S., Wu, H. ve Wang, G., 2012. Toxicological effects of nanometer titanium dioxide (nano-TiO 2) on Chlamydomonas reinhardtii. Ecotoxicology and Environmental Safety, 84, 155–162. https://doi.org/10.1016/j.ecoenv.2012.07.019
• Chen, W., Liu, J., Chu, G., Wang, Q., Zhang, Y., Gao, C. ve Gao, M.,2023. Comparative evaluation of four Chlorella species treating mariculture wastewater under different photoperiods: Nitrogen removal performance, enzyme activity, and antioxidant response. Bioresource Technology, 386 (2023) 129511. https://doi.org/10.1016/j.biortech.2023.129511
• Dalai, S., Pakrashi, S., Bhuvaneshwari, M., Iswarya, V., Chandrasekaran, N. ve Mukherjee, A., 2014. Toxic effect of Cr(VI) in presence of n-TiO2 and n-Al2O3 particles towards freshwater microalgae. Aquatic Toxicology, 146, 28–37. https://doi.org/10.1016/j.aquatox.2013.10.029
• Deng, X.Y., Cheng, J., Hu, X.L., Wang, L., Li, D. ve Gao, K., 2017. Biological effects of TiO2 and CeO2 nanoparticles on the growth, photosynthetic activity, and cellular components of a marine diatom Phaeodactylum tricornutum. Science of the Total Environment, 575, 87–96. https://doi.org/10.1016/j.scitotenv.2016.10.003
• Federici, G., Shaw, B. J. ve Handy, R.D., 2007, Toxicity of titanium dioxide nanoparticles to rainbow trout (Oncorhynchus mykiss): gill injury, oxidative stress, and other physiological effects. Aquatic Toxicology, 84 (4), 415–430.
• Gao, K., Xue, C., Yang, M., Li, L., Qian, P., Gao, Z., Gao, Z. ve Deng, X., Optimization of light intensity and photoperiod for growing Chlorella sorokiniana on cooking cocoon wastewater in a bubble-column bioreactor. Algal Research, 62,102612. https://doi.org/10.1016/j.algal.2021.102612
• Gauthier, M.R., Senhorinho, G.N.A. ve Scott, J.A., 2020. Microalgae under environmental stress as a source of antioxidants. Algal Research, 52, 102104.
• Gong, N., Shao, K., Che, C. ve Sun, Y., 2019. Stability of nickel oxide nanoparticles and its influence on toxicity to marine algae Chlorella vulgaris. Marine Pollution Bulletin, 149,110532. https://doi.org/10.1016/j.marpolbul.2019.110532
• Gunawan, C., Sirimanoonphan, A., Teoh, W.Y., Marquis, C.P. ve Amal, R., 2013. Submicron and nano formulations of titanium dioxide and zinc oxide stimulate unique cellular toxicological responses in the green microalga Chlamydomonas reinhardtii. Journal of Hazardous Materials, 260, 984–992. https://doi.org/10.1016/J.JHAZMAT.2013.06.067
• Hasanuzzaman, M., Bhuyan, M.H.M.B., Parvin, K., Bhuiyan, T.F., Anee, T.I., Nahar, K., Hossen, M.S., Zulfiqar, F., Alam, M.M. ve Fujita, M., 2020. Regulation of ROS metabolism in plants under environmental stress: a review of recent experimental evidence. International Journal of Molecular Science, 21 (22), 8695.
• Hartmann, N.B., Von der Kammer, F., Hofmann, T., Baalousha, M., Ottofuelling, S. ve Baun, A. 2010. Algal testing of titanium dioxide nanoparticles – testing considerations, inhibitory effects and modification of cadmium bioavailability. Toxicology, 269 (2–3), 190–197.
• He, Q., Yang, H., Wu, L. ve Hu, C., 2015. Effect of light intensity on physiological changes, carbon allocation and neutral lipid accumulation in oleaginous microalgae. Bioresource Technology, 191, 219–228.
• Hund-Rinke, K. ve Simon, M., 2006. Ecotoxic effect of photocatalytic active nanoparticles (TiO2) on algae and daphnids. Environmental Science and Pollution Research, 13 (4), 225–232.
• Iannelli, M.A., Bellini, A., Venditti, I., Casentini, B., Battocchio, C., Scalici, M.ve Ceschin, S., 2022. Differential phytotoxic effect of silver nitrate (AgNO3) and bifunctionalized silver nanoparticles (AgNPs-Cit-L-Cys) on Lemna plants (duckweeds). Aquatic Toxicology, 250, 106260. https://doi.org/10.1016/j.aquatox.2022.106260
• Kalitsov, A., Zermatten, P.J., Bonell, F., Mitra, A., Mohapatra, J. ve Aslam, M., 2024. Magnetic and electronic properties of anisotropic magnetite nanoparticles. Materials Research Express, 11(2), 022002. https://doi.org/10.1088/2053-1591/AD2A84
• Katırcıoğlu Sınmaz, G., Erden, B. Ve Şengil, İ.A,. 2023. Cultivation of Chlorella vulgaris in alkaline condition for biodiesel feedstock after biological treatment of poultry slaughterhouse wastewater. International Journal of Environmental Science and Technology, 20(3), 3237–3246. https://doi.org/10.1007/s13762-022-04137-4
• Li, F., Liang, Z., Zheng, X., Zhao, W., Wu, M. ve Wang, Z., 2015. Toxicity of nano-TiO2on algae and the site of reactive oxygen speciesproduction. Aquatic Toxicology, 158, 1–13. http://dx.doi.org/10.1016/j.aquatox.2014.10.014
• Li, J., a, Sun, Z.Z., Chen, Y.P. ve Jing Wang, J., 2024. Behavior and toxicity of silver nanoparticles to Chlorella vulgaris: A new perspective based on surface charges. Journal of Environmental Chemical Engineering, 12, 111673. https://doi.org/10.1016/j.jece.2023.111673
• Liu, S., Han, J., Ma, X., Zhu, X., Qu, H., Xin, G. ve Huang, X., 2024.Repeated release of cerium oxide nanoparticles altered algal responses: Growth, photosynthesis, and photosynthetic gene expression. Eco-Environment & Health, 3, 290-299. https://doi.org/10.1016/j.eehl.2024.04.002
• Liu, X., Wang, X., Zhang, F., Yao, X., Qiao, Z., Deng, J., Jiao, Q., Gong, L. ve Jiang, X., 2022. Toxic effects of fludioxonil on the growth, photosynthetic activity, oxidative stress, cell morphology, apoptosis, and metabolism of Chlorella vulgaris. Science of The Total Environment, 838, 156069. https://doi.org/10.1016/J.SCITOTENV.2022.156069
• Mahana, A., Guliy, O.I. ve Mehta, S.K., 2021. Accumulation and cellular toxicity of engineered metallic nanoparticle in freshwater microalgae: Current status and future challenges. Ecotoxicology and Environmental Safety, 208, 111662, https://doi.org/10.1016/j.ecoenv.2020.111662
• Malik, S., Muhammad, K. ve Waheed, Y., 2023. Nanotechnology: A Revolution in Modern Industry. Molecules 2023, Vol. 28, Page 661, 28(2), 661. https://doi.org/10.3390/MOLECULES28020661
• Markou, G. ve Nerantzis, E., 2013. Microalgae for high-value compounds and biofuels production: A review with focus on cultivation under stress conditions. Biotechnology Advances, 31(8), 1532–1542. https://doi.org/10.1016/j.biotechadv.2013.07.011
• Matouke, M.M., Elewa, D.T. ve Abdullahi, K., 2018. Binary effect of titanium dioxide nanoparticles (nTiO2) and phosphorus on microalgae (Chlorella ‘Ellipsoides Gerneck, 1907). Aquatic Toxicology, 198, 40–48. https://doi.org/10.1016/j.aquatox.2018.02.009
• Narayanan, M., Srinivasan, S., Gnanasekaran, C., Ramachandran, G., Chelliah, C.K., Rajivgandhi, G., Maruthupandy, M., Quero, F., Li W.J., Hayder, G., Khaled, J.M., Arunachalam, A. Ve Manoharan, N., 2024. Synthesis and characterization of marine seagrass (Cymodocea serrulata) mediated titanium dioxide nanoparticles for antibacterial, antibiofilm and antioxidant properties. Microbial Pathogenesis, 189, 106595, https://doi.org/10.1016/j.micpath.2024.106595
• Narayanan, M., Rajagopal, D.B., Krishnamoorthi, V. Gnanasekaran, C., Palanisamy, B., Manoharan, N., Ramachandran, G., Rajivgandhi, G., Bhaviripudi, V.R. ve Quero, F., 2025. Synthesis of TiO2 nanoparticles using endophytic Streptomyces zaomyceticus MNDV: Characterization and evaluation of antibacterial, antioxidant, antidiabetic and photocatalytic properties. Inorganic Chemistry Communications, 171, 113560. https://doi.org/10.1016/j.inoche.2024.113560
• Navarro, E., Baun, A., Behra, R., Hartmann, N.B., Filser, J., Miao, A.J., Quigg, A., Santschi, P.H. ve Sigg, L., 2008. Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology, 17 (5), 372–386.
• Noori, A., Hasanuzzaman, M., Roychowdhury, R., Sarraf, M., Afzal, S., Das, S. ve Rastogi, A., 2024. Silver nanoparticles in plant health: Physiological response to phytotoxicity and oxidative stress. Plant Physiology and Biochemistry, 209, 108538. https://doi.org/10.1016/J.PLAPHY.2024.108538
• Nurkiewicz, T.R., Porter, D.W., Hubbs, A.F., Cumpston, J.L., Chen, B.T., Frazer, D.G. ve Castranova,V., 2008. Nanoparticle inhalation augments particle-dependent systemic microvascular dysfunction. Particle and Fibre Toxicology, 5, 1–12. https://doi.org/10.1186/1743-8977-5-1
• Porra, R.J., 2002. The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynthesis Research, 73(1), 149–156. https://doi.org/10.1023/A:1020470224740
• Rezayian, M., Niknam, V. ve Ebrahimzadeh, H., 2019. Oxidative damage and antioxidative system in algae. Toxicology Reports, 6, 1309–1313. https://doi.org/10.1016/J.TOXREP.2019.10.001
• Sachdev, S. ve Ahmad, S., 2021. Role of nanomaterials in regulating oxidative stress in plants. In Nanobiotechnology: Mitigation of Abiotic Stress in Plants (pp. 305–326). Springer International Publishing. https://doi.org/10.1007/978-3-030-73606-4_13
• Service, R.F., 2005. Calls rise for more research on toxicology of nanomaterials. Science, 310 (5754), 1609. https://doi.org/10.1126/science.310.5754.1609
• Sezer, M., Tanattı, N.P. ve İ.A.,c, Interaction of TiO2 nanoparticles with the C. vulgaris: oxidative stress, lipid peroxidation and lipid amount. Water Science & Technology, 86 - 8, 2020 https://doi.org/10.2166/wst.2022.335
• Shi, H., Magaye, R., Castranova, V. ve Zhao, J. 2013. Titanium dioxide nanoparticles: a review of current toxicological data. Particle and FibreToxicology, 10 (1), 15.
• Simeonidis, K. ve Mourdikoudis, S., 2023. Nanoparticles as Sustainable Environmental Remediation Agents. In Nanoparticles as Sustainable Environmental Remediation Agents. https://doi.org/10.1039/9781837670215
• Sizochenko, N., Mikolajczyk, A., Syzochenko, M., Puzyn, T. ve Leszczynski, J., 2021. Zeta potentials (ζ) of metal oxide nanoparticles: A meta-analysis of experimental data and a predictive neural networks modeling. NanoImpact, 22, 100317. https://doi.org/10.1016/J.IMPACT.2021.100317
• Tekbaba, A., Özpınar, S.Ç., Tunca, H., Sevindik, T.O., Doğru, A., Günsel, A., Bilgiçli, A.T. ve Yarasir, M.N,. 2021. Synthesis, characterization and investigation of algal oxidative effects of water-soluble copper phthalocyanine containing sulfonate groups. Journal of Biological Inorganic Chemistry, 26(2–3), 355–365. https://doi.org/10.1007/s00775-021-01860-0
• Tunca, H., Dogru, A., Kockar, F., Onem, B. ve Sevindik, T.O., 2020. Evaluation of Azadirachtin on Arthrospira plantensis Gomont growth parameters and antioxidant enzymes. Annales de Limnologie - International Journal of Limnology, 56, 8. https://doi.org/10.1051/LIMN/2020008
• Türkmen, E.U., Arslan, P., Erkoç, F., Günal, A. Ç. ve Duran, H., 2024. The cerium oxide nanoparticles toxicity induced physiological, histological and biochemical alterations in freshwater mussels, Unio crassus. Journal of Trace Elements in Medicine and Biology, 83, 127371. https://doi.org/10.1016/J.JTEMB.2023.127371
• Van Nerom, S., Buyse, K., Van Immerseel, F., Robbens, J. ve Delezie, E., 2024. Pulsed electric field (PEF) processing of microalga Chlorella vulgaris and its digestibility in broiler feed. Poultry Science, 103(6), 103721. https://doi.org/10.1016/J.PSJ.2024.103721
• Vazquez-Muñoz, R., Meza-Villezcas, A., Fournier, P.G.J., Soria-Castro, E., Juarez-Moreno, K., Gallego-Hernandez, A.L., Bogdanchikova, N., Vazquez-Duhalt, R. ve Huerta-Saquero, A., 2019. Enhancement of antibiotics antimicrobial activity due to the silver nanoparticles impact on the cell membrane, Plos One, 8, https://doi.org/10.1371/journal.pone.0224904
• Wang, S.Y., Jiao, H.J. ve Faust, M., 1991. Changes in ascorbate, glutathione, and related enzyme activities during thidiazuron-induced bud break of apple. Physiologia Plantarum, 82(2), 231–236. https://doi.org/10.1111/J.1399-3054.1991.TB00086.X
• Wang, Y.L., Lee, Y.H., Chou, C.L., Chang, Y.S., Liu, W.C. ve Chiu, H.W., 2024. Oxidative stress and potential effects of metal nanoparticles: A review of biocompatibility and toxicity concerns. Environmental Pollution, 346, 123617. https://doi.org/10.1016/J.ENVPOL.2024.123617
• Xiong, D., Fang, T., Yu, L., Sima, X. ve Zhu,W. 2011 Effects of nano-scale TiO2, ZnO and their bulk counter parts on zebrafish: acutetoxicity, oxidative stress and oxidative damage. Science of the Total Environment, 409, 1444–1452.
• Yahyaee, A., Vatankhah, P. ve Sørensen, H., 2024. Effects of nanoparticle size on surface dynamics and thermal performance in film boiling of Al2O3 water-based nanofluids. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 134267. https://doi.org/10.1016/J.COLSURFA.2024.134267
• Yang, W., Zhang, H., Yang, S., Xiao, Y., Ye, K., He, R., Liu, Y., Hu, Z., Guo, W., Zhang, Q., Qu, H. ve Mao, Y., 2024. Combined effects of microplastics and pharmaceutical and personal care products on algae: A critical review. Environmental Pollution, 358, 124478. https://doi.org/10.1016/j.envpol.2024.124478
• Zhang, J., Xie, X., Li, Q., Wang, J. ve Zhang, S.,2023. Combined toxic effects of TiO2 nanoparticles and organochlorines on Chlorella pyrenoidosa in karst area natural waters, Aquatic Toxicology, 257, 106442. https://doi.org/10.1016/j.aquatox.2023.106442
• Zhu, X., Chang, Y. ve Chen, Y., 2010. Toxicity and bioaccumulation of TiO2 nanoparticle aggregates in Daphnia magna. Chemosphere, 78 (3), 209–215