A Recurrent Missense Variant in AP2M1 Impairs Clathrin-Mediated Endocytosis and Causes Developmental and Epileptic Encephalopathy

Abstract

The developmental and epileptic encephalopathies (DEEs) are heterogeneous disorders with a strong genetic contribution, but the underlying genetic etiology remains unknown in a significant proportion of individuals. To explore whether statistical support for genetic etiologies can be generated on the basis of phenotypic features, we analyzed whole-exome sequencing data and phenotypic similarities by using Human Phenotype Ontology (HPO) in 314 individuals with DEEs. We identified a de novo c.508C>T (p.Arg170Trp) variant in AP2M1 in two individuals with a phenotypic similarity that was higher than expected by chance (p = 0.003) and a phenotype related to epilepsy with myoclonic-atonic seizures. We subsequently found the same de novo variant in two individuals with neurodevelopmental disorders and generalized epilepsy in a cohort of 2,310 individuals who underwent diagnostic whole-exome sequencing. AP2M1 encodes the μ-subunit of the adaptor protein complex 2 (AP-2), which is involved in clathrin-mediated endocytosis (CME) and synaptic vesicle recycling. Modeling of protein dynamics indicated that the p.Arg170Trp variant impairs the conformational activation and thermodynamic entropy of the AP-2 complex. Functional complementation of both the μ-subunit carrying the p.Arg170Trp variant in human cells and astrocytes derived from AP-2μ conditional knockout mice revealed a significant impairment of CME of transferrin. In contrast, stability, expression levels, membrane recruitment, and localization were not impaired, suggesting a functional alteration of the AP-2 complex as the underlying disease mechanism. We establish a recurrent pathogenic variant in AP2M1 as a cause of DEEs with distinct phenotypic features, and we implicate dysfunction of the early steps of endocytosis as a disease mechanism in epilepsy.

Keywords: Human Phenotype Ontology; clathrin-mediated endocytosis; computational phenotypes; developmental and epileptic encephalopathy; neurodevelopmental disorders; synaptic transmission.

Figure 1

HPO-Based Analysis Demonstrating Phenotypic Similarity in Individuals with De Novo Variants (A) An illustration of the similarity score calculation made with the Most Informative Common Ancestor (MICA) approach. (B) A distribution, using 100,000 permutations, of phenotypic similarities between n = 2 individuals in the cohort of 314 individuals. The vertical line indicates the observed value for two individuals with de novo variants in AP2M1. The observed value of 43.20 is in the top 0.3 percentile of the distribution, translating into an exact p value of 0.00307. (C) A dendrogram of 314 individuals clustered by phenotypic similarity bya ward.D2 algorithm for the clustering of the similarity matrix. The gene labels refer to individuals with de novo variants in genes with two or more de novo variants in the entire cohort. (D) Exact p values for observed versus predicted phenotypic similarity for all 11 genes shared by two or more individuals with de novo variants in the cohort of 314 individuals. P values are uncorrected and refer to the distribution of expected similarities for each number of individuals.

Figure 2

Effect of the AP2M1 p.Arg170Trp Variant on the Thermodynamic Entropy of the AP-2 Complex (A) Both the entire structure and a simplified cartoon model of the AP-2 complex are shown in the inactive closed state. The color-coding is a follows: AP-2α (blue), AP-2β (green), AP-2μ (magenta), AP-2 σ (orange), and the AP-2-bound cargo peptide P (yellow). The p.Arg170Trp variant is depicted as a red sphere. Further pathogenic variants in AP2S1 (AP-2σ) and rare population variants in AP2M1 (AP-2μ) variants are shown as golden spheres. (B) Differences in entropy (entropic change; ΔG) between the wild-type (WT) AP-2 complex and the AP-2 complex containing the AP2M1 p.Arg170Trp variant are graphically depicted as ΔG for each residue across the entire AP-2 complex for the inactive closed state. The inlay shows an enlarged y axis to emphasize the differences in ΔG for each subunit. (C) The structure of the AP-2 complex and a simplified cartoon model including the bound cargo peptide P (yellow) are shown in the active state, and the AP2S1 and AP2M1 variants are labelled. (D) The difference in entropy between the WT AP-2 complex and the AP-2 complex containing the AP2M1 p.Arg170Trp variant for the active open state.

Figure 3

Defective Clathrin-Mediated Endocytosis in Cells Expressing the AP2M1 p.Arg170Trp Variant (A) Representative images of HeLa cells depleted of endogenous AP-2μ; the cells were rescued by the re-expression of siRNA-resistant mCherry-AP-2μ wild-type (WT) or p.Arg170Trp mutant and allowed to internalize AlexaFluor⁶⁴⁷-labeled transferrin (Tf) for 10 min at 37°C. Cells were fixed and immunostained for endogenous AP-2α and RFP. RFP was labeled to amplify the signal for mCherry and to identify transfected cells. The scale bars represent 20 μm. (B) A zoom of the marked area in (A) illustrates reduced Tf endocytosis in cells expressing the p.Arg170Trp variant. The scale bars represent 5 μm. Note that the punctate distribution of WT or Arg170Trp mutant mCherry-AP-2μ is consistent with its proper targeting to endocytic pits (see also Figure S4). (C) A quantification of data shown in (A). The data represent mean ± SEM, N = 3 independent experiments (wherein n = 198 for AP-2μ WT and n = 172 for AP-2μ p.Arg170Trp total cells analyzed). ^∗p < 0.05 from a paired two-tailed t test. (D) Representative images of primary astrocytes from WT or AP-2μ knockout (KO) mice; the cells were rescued by re-expression of untagged AP-2μ WT or p.Arg170Trp together with soluble RFP and allowed to internalize AlexaFluor⁶⁴⁷-labeled transferrin (Tf) for 5 min at 37°C. Cells were fixed and immunostained for endogenous AP-2α and cytoplasmic RFP. RFP expressed from the same construct after an internal ribosomal entry site (IRES) was labeled to identify transfected cells. The scale bars represent 20 μm. (E) A zoom of the marked area in (D) shows less Tf in astrocytes expressing the mutant variant of AP-2μ. The scale bars represent 10 μm. (F) A quantification of data shown in (D). The data represent mean ± SEM, N = 5 independent experiments (wherein n = 74 for AP-2μ WT and n = 72 for AP-2μ p.Arg170Trp total cells analyzed). ^∗p < 0.05 from an unpaired t test.

Figure 4

Intact Localization of an AP-2 Complex Carrying the p.Arg170Trp Variant (A) Representative confocal images (maximum intensity projections) of HeLa cells depleted of endogenous AP-2μ; the cells were rescued by re-expression of siRNA-resistant mCherry-AP-2μ WT or p.Arg170Trp mutant and immunostained with clathrin heavy chain (CHC) and RFP antibodies. RFP was labeled to amplify the signal for mCherry-tagged variants and to identify transfected cells. The scale bars represent 20 μm. (B) Merged magnified views of the boxed area in (A). The scale bars represent 5 μm. (C) Pearson’s correlation coefficient for the co-localization of AP-2μ WT or p.Arg170Trp with clathrin heavy chain (CHC). The data represent mean ± SEM and N = 5 independent experiments. Statistical analysis was done with a paired t test.