ADAM22 ethnic-specific variant reducing binding of membrane-associated guanylate kinases causes focal epilepsy and behavioural disorder

Abstract

Pathogenic variants of ADAM22 affecting either its biosynthesis and/or its interactions with either LGI1 and/or PSD-95 have been recently identified in individuals with developmental and epileptic encephalopathy. Here, we describe a girl with seizures, delayed psychomotor development, and behavioural disorder, carrying a homozygous variant in ADAM22 (NM_021723.5:c.2714C > T). The variant has a surprisingly high frequency in the Roma population of the Czech and Slovak Republic, with 11 of 213 (∼5.2%) healthy Roma individuals identified as heterozygous carriers. Structural in silico characterization revealed that the genetic variant encodes the missense variant p.S905F, which localizes to the PDZ-binding motif of ADAM22. Studies in transiently transfected mammalian cells revealed that the variant has no effect on biosynthesis and stability of ADAM22. Rather, protein-protein interaction studies showed that the p.S905F variant specifically impairs ADAM22 binding to PSD-95 and other proteins from a family of membrane-associated guanylate kinases, while it has only minor effect on ADAM22-LGI1 interaction. Our study indicates that a significant proportion of epilepsy in patients of Roma ancestry may be caused by homozygous c.2714C > T variants in ADAM22. The study of this ADAM22 variant highlights a novel pathogenic mechanism of ADAM22 dysfunction and reconfirms an essential role of interaction of ADAM22 with membrane-associated guanylate kinases in seizure protection in humans.

Keywords: ethnic-specific variant; focal epilepsy; insufficient ADAM22–MAGUK interaction.

Graphical Abstract

Figure 1

Genetic study and structural correlates of the ADAM22 c.2714C > T variant. (A) Pedigree of the family and segregation analysis of the ADAM22 c.2714C > T variant. (B) Domain organization of ADAM22 and location of all reported pathogenic ADAM22 variants. The p.S905F variant is located in the PDZ-binding motif at the C-terminus (magenta stick). Pro, prodomain; Cys, cysteine-rich domain; EGF, EGF-like domain; TM, transmembrane domain. Note that the ADAM22 has no metalloprotease activity. (C) Cross-species sequence alignment of the PDZ-binding motif of ADAM22. hs, Homo sapiens; mm, Mus musculus; xl, Xenopus laevis; dr, Danio rerio. The PDZ-biding motifs of neuroligin 1, SynGAP1 and CRIPT that bind to the 3rd PDZ domain of PSD-95 (PDZ3) are aligned. (D and E) Structure of the complex of the ADAM22 PDZ-binding motif (green) and PSD-95-PDZ3 (purple) (D). Close-up view of the interface between the ADAM22 C-terminal tail and PSD-95-PDZ3 (E). The magenta indicates the position of the Ser905 residue. The yellow dotted lines represent hydrogen bonds between ADAM22 Ser905 residue and PSD-95-PDZ3 domain. PDB accession code, 7CQF.

Figure 2

Functional studies of the ADAM22 protein with S905F variant. (A and B) The interaction of ADAM22 S905F with PSD-95 is significantly reduced. Indicated cDNAs for ADAM22 variants and PSD-95–FLAG were co-transfected into HEK293T cells and PSD-95–FLAG was immunoprecipitated (IP). ADAM22 and PSD-95 were detected by western blotting (WB). ADAM22ΔC5, lacking the C-terminal PDZ-binding motif, is used as a control. Arrows and arrowheads indicate the positions of immature and mature forms of ADAM22, respectively. The immature form of ADAM22 is often observed in overexpressed cells. P values were determined by Kruskal–Wallis with post hoc Steel test. *P < 0.05; n.s., not significant; n = 3 experiments. Results are shown as means ± SE. WT, wild-type. See Supplementary material for uncropped blots for A. (C) The S905F variant reduces the binding of ADAM22 to MAGUK proteins including PSD-95, PSD-93 and SAP102 in HEK293T cells. Conserved Asn and Lys residues are shown in blue. (D and E) ADAM22 S905F binds to LGI1. ADAM22 C401Y is used as a control, which shows the reduced binding to LGI1.^,  P values are determined by Kruskal–Wallis with post hoc Steel test. *P < 0.05; n = 3 experiments. Results are shown as means ± SE. Immature ADAM22 (arrows) seems to non-specifically bind to LGI1 under the overexpressed conditions. In the brain, we do not detect such an immature form of ADAM22. See Supplementary material for uncropped blots for C and D. (F and G) ADAM22 S905F is expressed at the cell surface and binds to LGI1. Cell-surface-expressed ADAM22 (F, magenta) and cell-surface-bound LGI1 (G, magenta) were live-labelled. After fixation and permeabilization of the cells, expressed ADAM22 (total) was stained (green). Nuclear DNA was stained by Hoechst 33342 (F and upper panel in G, blue). ADAM22 S905F is expressed on the cell surface and binds to LGI1 as the wild-type. When ADAM22 C401Y is expressed, no cell-surface-bound LGI1 is detected. Regions outlined with white squares are total expression of ADAM22 (F) and LGI1–FLAG (lower panel in G, blue). Bars: 20 μm.