Fig 2: Identification of a p.R356Q mutation in ARHGEF9 in family D228. (A) Pedigree of the D228 family. Open symbols represent normal individuals, filled squares represent affected males. Individuals who are deceased have a slash through the symbol. Individuals tested for the nucleotide substitution in each family are indicated with either a T (mutant allele) or a C (normal allele). (B–D) DNA sequence electropherograms for the chrX:62875607C>T, p.R356Q variant reported in this study, showing wild-type, hemizygous and heterozygous states. (E) Schematic of the human collybistin protein with a regulatory SH3 domain, a catalytic RhoGEF domain and a pleckstrin homology (PH) domain. The relative locations of known missense and nonsense variants in ARHGEF9 are shown. (F) Sequence alignments of collybistin proteins from various species showing the high conservation of R356 in the PH domain. R356 is one of two predicted PI3P binding residues (R356 and R357, green highlighting).

Fig 3: Collybistin phosphoinositide binding mutants R356Q and R356N/R357N alter gephyrin cluster number and size in cultured hippocampal neurones. Triple-label immunofluorescence of transfected hippocampal neurons with mouse anti-myc (green), rabbit anti-gephyrin (red) and sheep anti-GAD (blue). (A–C) Transfection with myc-CB3SH3- (wild-type, WT). (D–F) Transfection with myc-CB3SH3-R356Q (R356Q). (G–I) Transfection with myc-CB3SH3-R356N/R357N (R356N/R357N). The two panels on the right side of each row correspond to the boxed areas in (A,D,G), respectively. Arrows point to GABAergic synapses. Arrowheads point to aggregates of gephyrin that form in transfections with collybistin R356Q or R356N/R357N. Note that there is colocalization of mutant collybistin and gephyrin in these aggregates. Wild-type collybistin does not induce the formation of aggregates but accumulates at GABAergic synapses and colocalizes with gephyrin (arrows, panel C). The R356Q and the R356N/R357N mutants do not accumulate at synapses (arrows, panels F,I). The R356N/R357N mutant (panels H,I) shows a clear decrease in the overall number of gephyrin clusters, including synaptic gephyrin clusters. In neurons overexpressing the R356N/R357N mutant, GAD positive puncta often make contacts without apposed gephyrin clusters (crossed arrows). Scale bar = 26 µm for (A,D,G) and 10 µm for (B,C,E,F,H,I).