Abstracts

Rationale: a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPARs) play important roles in nervous system development and various pathological conditions, Here, we report 12 rare variants in multiple AMPAR subunits gene GRIA in children with epilepsy and/or developmental/intellectual disabilities by next-generation sequencing. The patients’ phenotypes are summarized and functional properties of the AMPA receptors with these variants are evaluated along with 11 missense variants reported on ClinVar database in addition to wild type (WT) human AMPA receptors. Methods: Variants were introduced into human cDNAs encoding GluA receptors by the QuikChange protocol. cRNA were synthesized from cDNA and injected into Xenopus laevis oocytes, and two-electrode voltage-clamp recordings were used to evaluate agonist potency. Results: We studied 23 patients with GRIA variants, which included 4 GRIA1 variants, 5 GRIA2 variants, 14 GRIA3 variants.  Of these 23 patients, 70% (16/23) show epilepsy and developmental delay/intellectual disability, and 13% (3/23) present only with developmental delay/intellectual disability without seizures. 13% (3/23) of the variants are located in the amino terminal domain (ATD), 13% (3/23) are in the agonist binding domain (ABD), 30% (7/23) are in transmembrane domains (TMs), 35% (8/23) of variants are in linker regions, and 4 % (1/23) are in the C-terminus (CTD). Functional evaluation using voltage clamp current recordings revealed 5 gain-of-function variants that enhanced glutamate and kainate potency by 2.5~53-fold, compared to the corresponding WT receptors.  We also found 8 loss-of-function variants that reduced glutamate and kainate potency by 4.5~10-fold, compared to the WT receptors. All of the gain-of-function variants and 7 of the 8 loss-of-function variants are located in TMs or the linker regions. Notably, similar diseases were associated with both gain- and loss-of-function variants in the same gene. Conclusions: Overall, our results suggest that AMPAR variants are highly relevant with epilepsy and developmental/intellectual disabilities in children, and the transmembrane domain or the linker regions between transmembrane domains were particularly intolerant to functional variation. Funding: This work was supported by CSC scholarship to WX, by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) of the National Institutes of Health (NIH) under award number R01HD082373 to H.Y., by the National Institute of Neurological Disorders and Stroke (NINDS) of the NIH under award numbers NIH-NINDS R01NS036654, R01NS065371, and R24NS092989 to S.F.T., and by the Beijing Municipal Natural Science Key Project under award numbers 15G10050 to YJ.