Investigating the Molecular and Cellular Effects of Pathogenic Variants of GNAI1 in Developmental and Epileptic Encephalopathy
Abstract number :
1.142
Submission category :
2. Translational Research / 2E. Other
Year :
2022
Submission ID :
2204020
Source :
www.aesnet.org
Presentation date :
12/3/2022 12:00:00 PM
Published date :
Nov 22, 2022, 05:23 AM
Authors :
Esmat Fathi, PhD – St. Jude Children's Research Hospital; Helen Chen, PhD – Lead Researcher, Ctr-Ped Neurological Dis Res, St. Jude Children's Research Hospital; Edith Almanza Fuerte, BS – Sr Researcher, Ctr-Ped Neurological Dis Res, St. Jude Children's Research Hospital; Heather Mefford, MD, PhD – Faculty, Member, Ctr-Ped Neurological Dis Res, St. Jude Children's Research Hospital
Rationale: Developmental and epileptic encephalopathies (DEE) are characterized by intellectual disability, significant developmental delay, intractable seizures, and autistic features. Recently our group identified 16 unique de novo mutations in GNAI1 (G protein subunit alpha I1) in 24 individuals with DEE. GNAI1 encodes Gαi1, a G-protein subunit that binds to and hydrolyzes GTP and interacts with a variety of downstream effectors in signaling cascades inside the cell. However, the effect of the identified variants on Gαi1 functions such as GDP/GTP binding, GTP hydrolysis, and affecting downstream signaling pathways is not known. Although GNAI1 is predicted to be intolerant to loss-of-function variants, only a single disease-causing variant has been studied to date. Therefore, there is an urgent need to determine the molecular and cellular consequences of disease-causing variants and develop a cellular model in which to study disease and test potential therapies.
Methods: To investigate the molecular and cellular function of GNAI1 disease-causing variants, we selected four pathogenic mutations, each found in two or more patients, including three missense variants in different positions of the GDP binding domain (Gly40Cys, Thr48Lys, Lys270Arg), and one single amino acid deletion just outside of the GDP binding domain (Gln172del). We designed a series of GNAI1 mutations in pcDNA3.1 vector with a flag tag using site-directed mutagenesis. We also designed one early termination (Thr48Ter) as a control for haploinsufficiency and loss of function effects. In addition, we are generating CRISPR-engineered cells with the same selected variants and have patient cells for three variants. We are investigating the effect of disease-related G⍺i1 variants on protein localization, protein-protein interactions, GTP binding and downstream pathways.
Results: We transiently transfected HEK-293T cells with each mutant construct (Gly40Cys, Thr48Lys, Lys270Arg, Gln172del, Thr48Ter). We showed that the protein expression of all five GNAI1 mutant constructs is significantly decreased compared with wild type (WT) control in HEK-293T cells. In addition, preliminary results suggest that the Lys270Arg and Gln172del variants disrupt protein localization compared with control.
Conclusions: Our engineered and patient-derived cells represent a valuable tool for studying the role of GNAI1 mutations in the pathogenesis of G⍺i1 mutant proteins. We will validate our preliminary results from in vitro studies in 3D brain organoids from patient cells, which will serve as a beneficial disease model to investigate molecular and cellular function of G⍺i1 variants and to test promising therapeutic strategies for GNAI1-related DEE.
Funding: St. Jude Children's Research Hospital
Translational Research