Truncating mutations in NRXN2 and NRXN1 in autism spectrum disorders and schizophrenia

Abstract

Growing genetic evidence is converging in favor of common pathogenic mechanisms for autism spectrum disorders (ASD), intellectual disability (ID or mental retardation) and schizophrenia (SCZ), three neurodevelopmental disorders affecting cognition and behavior. Copy number variations and deleterious mutations in synaptic organizing proteins including NRXN1 have been associated with these neurodevelopmental disorders, but no such associations have been reported for NRXN2 or NRXN3. From resequencing the three neurexin genes in individuals affected by ASD (n = 142), SCZ (n = 143) or non-syndromic ID (n = 94), we identified a truncating mutation in NRXN2 in a patient with ASD inherited from a father with severe language delay and family history of SCZ. We also identified a de novo truncating mutation in NRXN1 in a patient with SCZ, and other potential pathogenic ASD mutations. These truncating mutations result in proteins that fail to promote synaptic differentiation in neuron coculture and fail to bind either of the established postsynaptic binding partners LRRTM2 or NLGN2 in cell binding assays. Our findings link NRXN2 disruption to the pathogenesis of ASD for the first time and further strengthen the involvement of NRXN1 in SCZ, supporting the notion of a common genetic mechanism in these disorders.

Fig. 1

a Schematic structure of the two major isoforms (alpha and beta) of NRXN1 and NRXN2 proteins (LNS 1–6, laminin neurexin sex hormone binding domains; EGF, epidermal growth factor-like domains; SP, signal peptide; TM, transmembrane region; 4.1, band 4.1 N binding region; purple line, PDZ domain binding site). Arrows indicate non-synonymous variants found during the screening of our diseases (top) and control (bottom) cohorts. b Pedigrees of families showing putative causative variations in NRXN1 or NRXN2 that have been tested functionally

Fig. 2

NRXN1 c.4205insACGG results in functional deficiency in surface trafficking and binding to LRRTM2 and NLGN2 in cell binding assays. a COS-7 cells were co-transfected with Flag-neurexin1α human cDNA expression constructs and CFP to mark transfected cells. CFP-positive COS cells cotransfected for wild-type neurexin1α showed variable, often bright, surface Flag immunofluorescence by live cell primary antibody staining. In contrast, little or no surface Flag immunofluorescence was detectable for the insACGG neurexin1α mutant, fluorescence was similar to that of untransfected cells (*p < 0.005 t test, n = 20 CFP-positive cells each group from two experiments). The mutant construct was expressed as shown by bright intracellular Flag staining of permeabilized cells. b Defective surface trafficking of Flag-neurexin1α insACGG mutant was confirmed in primary cultured hippocampal neurons. Whereas neurons expressing wild-type Flag neurexin1α showed bright staining for Flag on the surface including along the axon length, surface Flag staining was not detectable for neurons expressing the insACGG mutant (the small localized signal may represent non-specific permeabilization). Neurons were subsequently stained with phalloidin for F-actin. c Western blot analysis revealed a similar amount of mutant insACGG Flag-neurexin1α as wild type in transfected cell lysate and none in media, indicating that the mutant protein is trapped in the cell. d Insertion of ACGG at the equivalent position in mouse NRXN1 results in aberrant translation of a homologous sequence (bold) and truncation (*) at the equivalent residue. Thus, for further analysis we used a rodent cDNA. e Live cell surface HA immunofluorescence was bright in cells expressing wild-type HA-neurexin1α, but reduced to background level for the insACGG mutant. Permeabilized cell HA immunofluorescence confirmed expression of both constructs. For quantitative assays comparing wild type and insACGG HA-neurexin1α-CFP, we used cotransfected CFP as a marker of expressing cells (since the frameshift resulted in lack of expression of the downstream CFP from the mutant construct, and all other fluorescence channels were needed for antibody staining in the coculture assays); we confirmed that the majority of CFP-positive cells co-labeled for permeabilized HA, equally for wild type (88 ± 2%) and insACGG mutant (86 ± 3%). f Surface expression was strong in CFP-positive cells cotransfected for HA-neurexin1α-CFP wild type but reduced to background in cells cotransfected for the insACGG mutant (**p < 0.0001 t test, n = 20 cells each). The R813C missense variant, and also the H8P and E715K variants (not shown), exhibited surface accumulation similar to wild type (ANOVA p = 0.043, each missense p > 0.05 compared with wild type by Tukey–Kramer post hoc test, n = 40 cells each; also t test p > 0.1 for R813C vs. wild type). g LRRTM2-AP binding was strong in CFP-positive cells cotransfected for HA-neurexin1α-CFP wild type, but reduced to background in cells cotransfected for the insACGG mutant (**p < 0.0001 t test, n = 25 cells each). The R813C missense variant, and also the H8P and E715K variants (not shown), exhibited normal binding of LRRTM2-AP undistinguishable from wild type (ANOVA p > 0.1, n = 30 cells each; also t test p > 0.1 for R813C vs. wild type). HA-CD8 is shown here as a negative control. h NLGN2-Fc binding was strong in CFP-positive cells cotransfected for HA-neurexin1α-CFP wild type but reduced to background in CFP-positive cells cotransfected for the insACGG mutant (**p < 0.0001 t test, n = 15 cells each). The R813C missense variant, and also the H8P and E715K variants (not shown), exhibited normal binding of NLGN2-Fc undistinguishable from wild type (ANOVA p > 0.1, n = 40 cells each; also t test p > 0.1 for R813C vs. wild type). Scale bars, 10 μm

Fig. 3

NRXN1 c.4205insACGG results in functional deficiency in synaptogenic activity in neuron coculture assays. a, b COS cells were transfected with HA-neurexin1α-CFP vectors and CFP to mark the transfected cells and cocultured with rat hippocampal neurons transfected with YFP-LRRTM2. CFP-positive cells cotransfected for HA-neurexin1α-CFP wild type a induced clustering of YFP-LRRTM2 and endogenous PSD-95 at contact sites with transfected neuron dendrites (arrowheads; 87 of 92 contacts positive). Only synapsin-negative clusters were counted as induced (arrowheads); synapsin-positive clusters were considered to represent endogenous synapses (arrows). In contrast, in spite of equal contact of the CFP-positive cotransfected cells with MAP2-positive dendrites, the insACGG mutant b failed to induce clustering of either glutamatergic postsynaptic component (0 of 75 contacts positive). Only synapsin-positive clusters of YFP-LRRTM2 and PSD-95 (arrows) were seen corresponding to endogenous synapses. c CFP-positive cells cotransfected for HA-neurexin1α-CFP wild type and cocultured with hippocampal neurons induced clustering of endogenous NLGN2 and gephyrin at contact sites with neuron processes (arrowheads; 15 positives scored). d In contrast, the insACGG mutant failed to induce clustering of either GABAergic postsynaptic component (0 positives). Only VGAT-positive clusters of NLGN2 and gephyrin (arrows) were seen corresponding to endogenous synapses. Scale bar, 10 μm

Fig. 4

NRXN2 c.2733delT (delA in mouse) results in functional deficiency in binding to LRRTM2 and NLGN2 in cell binding assays, and deficiency in synaptogenic activity in neuron coculture assays. a Compared with delT in human, delA at the equivalent position in mouse NRXN2 results in aberrant translation of a homologous sequence (bold) and truncation (*) at the equivalent residue. Thus for further analysis we used a mouse cDNA. b LRRTM2-AP binding was strong in CFP-positive cells cotransfected for neurexin2α-CFP wild type, but reduced to background in cells cotransfected for the delA mutant (**p < 0.0001 t test, n = 15 cells each). c NLGN2-Fc binding was strong in CFP-positive cells cotransfected for neurexin2α-CFP wild type, but reduced to background in cells cotransfected for the delA mutant (**p < 0.0001 t test, n = 15 cells each). d, e COS cells were transfected with neurexin2α-CFP vectors and CFP to mark the transfected cells and cocultured with rat hippocampal neurons transfected with YFP-LRRTM2. CFP-positive cells cotransfected for neurexin2α-CFP wild type d induced clustering of YFP-LRRTM2 and endogenous PSD-95 at contact sites with transfected neuron dendrites (arrowheads; 65 of 71 contacts positive). In contrast, despite equal contact with MAP2-positive dendrites, the delA mutant e essentially failed to induce clustering of either glutamatergic postsynaptic component (2 of 88 contacts positive). Only synapsin-positive clusters of YFP-LRRTM2 and PSD-95 (arrows) were seen corresponding to endogenous synapses. f, g CFP-positive cells cotransfected for neurexin2α-CFP wild type f and cocultured with hippocampal neurons induced clustering of endogenous NLGN2 and gephyrin at contact sites with neuron processes (arrowheads; 13 positives scored). In contrast, the delA mutant g failed to induce clustering of either GABAergic postsynaptic component (0 positive). Only VGAT-positive clusters of NLGN2 and gephyrin (arrows) were seen corresponding to endogenous synapses. Scale bars, 10 μm