Mutations in TNK2 in severe autosomal recessive infantile onset epilepsy

Abstract

We identified a small family with autosomal recessive, infantile onset epilepsy and intellectual disability. Exome sequencing identified a homozygous missense variant in the gene TNK2, encoding a brain-expressed tyrosine kinase. Sequencing of the coding region of TNK2 in 110 patients with a similar phenotype failed to detect further homozygote or compound heterozygote mutations. Pathogenicity of the variant is supported by the results of our functional studies, which demonstrated that the variant abolishes NEDD4 binding to TNK2, preventing its degradation after epidermal growth factor stimulation. Definitive proof of pathogenicity will require confirmation in unrelated patients.

FIGURE 1

Family pedigree and genotypes. The indicated by an arrow. Wt 5 wild type (chr3:197079609_hg18); Mut 5 mutated (chr3:197079609_T).

FIGURE 2

Family pedigrees and genotypes for detected heterozygous variants in TNK2. wt5 wild type.

FIGURE 3

Loss of binding between TNK2-716Met and neural precursor cell expressed developmentally down-regulated 4-1(NEDD4-1) or NEDD4-2. cDNA of each TNK2 allele and NEDD4-1 or NEDD4-2 was cotransfected into COS-7 cells, and binding was detected by immunoprecipitation (IP) using an anti-TNK2 monoclonal antibody after 48 hours of transfection. The top row of both parts shows binding of wild-type TNK2 (716V) to NEDD4-1 and NEDD4-2, whereas binding of variant TNK2 (716M) is abolished. GAPDH 5 glyceraldehyde-3-phosphate dehydrogenase.

FIGURE 4

Loss of TNK2-716Met degradation after epidermal growth factor (EGF) treatment. cDNA of each TNK2 allele was transfected into COS-7 cells and 10lg=ml of recombinant human EGF was added after 48 hours of transfection. (A) After 1, 2, and 4 hours of incubation, expression of TNK2 was detected by Western blotting. Degradation of wild-type TNK2 (716V) but not of variant TNK2 (716M) is observed. (B) Plotted data represent averages and standard error of triplicated assays. *p < 0.01 (Student t test). GAPDH 5 glyceraldehyde-3-phosphate dehydrogenase. [Color figure can be viewed in the online issue, which is

FIGURE 5

Postulated functional effects of TNK2 wild-type and Val716Met variant. (A) Binding of wild-type TNK2 (716V) to neural precursor cell expressed developmentally down-regulated 4-1 (NEDD4-1) and NEDD4-2 results in TNK2 degradation in response to epidermal growth factor (EGF) stimulation. (B) The 716M variant abolishes TNK2 binding with NEDD4-1 and NEDD4-2 and inhibits TNK2 degradation in response to EGF stimulation. Increased expression of TNK2 and EGF receptor induces epilepsy through enhanced extracellular signal-regulated kinase (ERK) activity. EGFR 5 epidermal growth factor receptor. [Color figure can be viewed in the online issue, which is available at www.annalsofneurology.org.]available at www.annalsofneurology.org.]