Biallelic ADAM22 pathogenic variants cause progressive encephalopathy and infantile-onset refractory epilepsy

Abstract

Pathogenic variants in A Disintegrin And Metalloproteinase (ADAM) 22, the postsynaptic cell membrane receptor for the glycoprotein leucine-rich repeat glioma-inactivated protein 1 (LGI1), have been recently associated with recessive developmental and epileptic encephalopathy. However, so far, only two affected individuals have been described and many features of this disorder are unknown. We refine the phenotype and report 19 additional individuals harbouring compound heterozygous or homozygous inactivating ADAM22 variants, of whom 18 had clinical data available. Additionally, we provide follow-up data from two previously reported cases. All affected individuals exhibited infantile-onset, treatment-resistant epilepsy. Additional clinical features included moderate to profound global developmental delay/intellectual disability (20/20), hypotonia (12/20) and delayed motor development (19/20). Brain MRI findings included cerebral atrophy (13/20), supported by post-mortem histological examination in patient-derived brain tissue, cerebellar vermis atrophy (5/20), and callosal hypoplasia (4/20). Functional studies in transfected cell lines confirmed the deleteriousness of all identified variants and indicated at least three distinct pathological mechanisms: (i) defective cell membrane expression; (ii) impaired LGI1-binding; and/or (iii) impaired interaction with the postsynaptic density protein PSD-95. We reveal novel clinical and molecular hallmarks of ADAM22 deficiency and provide knowledge that might inform clinical management and early diagnostics.

Keywords: ADAM22; LGI1; developmental and epileptic encephalopathy; refractory seizures.

Figure 1

Brain MRI and histology. (A and B) Relevant neuroimaging features associated with ADAM22 variants, including cerebral atrophy with enlargement of the CSF spaces (thin arrows) and lateral ventricles (asterisks), cerebellar atrophy with prevalent vermian involvement (empty arrows), corpus callosum hypoplasia/thinning (thick arrows) and anterior commissure hypoplasia (arrowheads). Additional diffuse hyperintensity of the supratentorial white matter with bilateral pulvinar involvement (dotted arrows) was noted in one subject on FLAIR images (A) from Patient P4 and (B) from Patient P5. (C–F) Post-mortem examination of brain tissue obtained from Patient P10 (deceased at the age of 28 years). (C) Haematoxylin and eosin-staining (×200 magnification) of the visual cortex, which showed profound atrophy and neuronal depletion with only some pyramidal cells in layers V–VI. (D) Haematoxylin and eosin-staining (×200 magnification) of the medial thalamus which was extremely atrophic and gliotic. (E) PAS staining (×100 magnification) of the frontal cortex which was very atrophic with a vast number of corpora amylacea. (F) Neurofilament SMI32 staining by immunohistochemistry (×40 magnification), showing the pronounced loss of neurons at the sulcal region.

Figure 2

Structural mapping and cell-surface expression of ADAM22 variants. (A) ADAM22 gene structure and protein domain overview. The immature form of ADAM22 contains the N-terminal prosequence (Pro). The mature ADAM22 consists of the metalloprotease-like, disintegrin, cysteine-rich, EGF-like, transmembrane (TM) and cytoplasmic domains. The major ADAM22 isoform has a PDZ-binding motif at its C-terminus. The positions of ADAM22 variants are indicated. Missense variants are all conserved across various species and in ADAM22 family proteins (ADAM11 and ADAM23). The RefSeq ID NM_021723.3 (a long spliced form of ADAM22) is used to indicate all variants. p.C401Y, p.S799IfsTer96 and p.R896Ter are reported variants. (B) Maturation and expression levels of ADAM22 variants. COS7 cells were transfected with the indicated ADAM22 variants. Cell lysates were subjected to western blotting (WB) with anti-ADAM22 antibody. An arrow and an arrowhead indicate the positions of immature and mature forms of full-length ADAM22. Asterisks indicate the immature form of frame-shifted ADAM22. An open arrowhead indicates the mature form of E859DfsTer2 (indicated as E859Dfs*2). Maturation (%) was calculated by the ratio of the band intensity of the mature form to the total band intensity (mature plus immature forms). The data shown are representative of two independent experiments. (C) Indicated cDNAs of ADAM22 variants were transfected into COS7 cells. Cell-surface expressed ADAM22 was live-labelled by an antibody against the extracellular domain of ADAM22. To see the intracellular pool of ADAM22 expressed (total), ADAM22 was labelled with different fluorescence after the fixation and permeabilization of cells. Nuclear DNA was stained by Hoechst 33342 to distinguish transfected from untransfected cells. (D) P438T, G448D and S799IfsTer96 variants were predominantly localized in the endoplasmic reticulum labelled by the anti-KDEL antibody. Regions outlined with squares are magnified (large insets). Scale bars = 20 μm (C and D). Please note that the provided immunoblots have been cropped; full images are provided in Supplementary Fig. 6.

Figure 3

LGI1- and PSD-95-binding activities of ADAM22 variants. (A) The interaction of ADAM22 variants with LGI1-FLAG was examined by immunoprecipitation with FLAG antibody in lysates derived from COS7 cells transiently co-transfected with wild-type or indicated variant ADAM22 and LGI1-FLAG. ADAM22 variants besides E859Dfs*2 showed reduced or no binding to LGI1. Immature ADAM22 (arrow and asterisks) was often observed when overexpressed in cells and seemed to be non-specifically precipitated under the conditions. In the rodent brain lysate, immature forms are hardly detected. (B) LGI1-FLAG and ADAM22 variants were co-expressed and cell-surface bound LGI1 through ADAM22 was live-labelled by anti-FLAG antibody. After fixation and permeabilization of cells, protein expression of ADAM22 (total) and LGI1 (in insets; total) was validated. (C) The interaction of ADAM22 variants with PSD-95 was investigated as in A. E859DfsTer2 selectively lost the binding to PSD-95. Extracellular missense variants showed various levels of PSD-95 binding, according to their expression levels. Fold changes in LGI1 (A) or PSD-95 (C) binding of variants relative to the wild-type are shown. The data shown are representative of two independent experiments. (D) Mapping of eight ADAM22 variants on the LGI1 EPTP-ADAM22 structure. The corresponding amino-acid residues are shown. (E–G) Close-up views of G448 (E), T578 (F) and C694 (G). The G448D mutation causes a steric hindrance to C447 (E, right) and impairs the disulphide bond formation between C447 and C477, which supports the Ca²⁺ coordination (E, left). The T578M mutation impairs the hydrogen bond formation between T578 and E576 (F). The C694L mutation disrupts the disulphide bond formation between C679 and C694 (G). Note that provided immunoblots have been cropped; full images are provided in Supplementary Fig. 6. ECD = extracellular domain of ADAM22.