Recessive loss-of-function mutations in AP4S1 cause mild fever-sensitive seizures, developmental delay and spastic paraplegia through loss of AP-4 complex assembly

Abstract

We report two siblings with infantile onset seizures, severe developmental delay and spastic paraplegia, in whom whole-genome sequencing revealed compound heterozygous mutations in the AP4S1 gene, encoding the σ subunit of the adaptor protein complex 4 (AP-4). The effect of the predicted loss-of-function variants (p.Gln46Profs*9 and p.Arg97*) was further investigated in a patient's fibroblast cell line. We show that the premature stop mutations in AP4S1 result in a reduction of all AP-4 subunits and loss of AP-4 complex assembly. Recruitment of the AP-4 accessory protein tepsin, to the membrane was also abolished. In retrospect, the clinical phenotype in the family is consistent with previous reports of the AP-4 deficiency syndrome. Our study reports the second family with mutations in AP4S1 and describes the first two patients with loss of AP4S1 and seizures. We further discuss seizure phenotypes in reported patients, highlighting that seizures are part of the clinical manifestation of the AP-4 deficiency syndrome. We also hypothesize that endosomal trafficking is a common theme between heritable spastic paraplegia and some inherited epilepsies.

Figure 1.

Genetic findings from the family reported here. (A) Pedigree of the family with segregation of the identified AP4S1 mutations. Filled symbols indicate affected individuals. The deletion of four base pairs is noted with a dash (-). (B and C) Pictures of the oldest and youngest sibling to illustrate the facial hypotonia and dysmorphic features consisting of a broad nasal bridge, hypertelorism with telecantus (most prominent in the youngest), arched eyebrows (most prominent in the oldest), bulbous nose, short philtrum, wide mouth, full lips and high palate. (D) Sequence NM_007077.4 was used as reference. Variant c.289C>T is a nonsense mutation (p.Arg97*) inherited from the father. Variant c.137_140delAAGT is a frameshift mutation (p.Gln46Profs*9) inherited from the mother. Parents are homozygous reference for the other variant. The patients carry both recessive variants, each on a different allele and are thus compound heterozygous.

Figure 2.

Molecular characterization of the AP4S1 mutations described here. (A) Western blot of whole cell lysates show loss of σ4 and reduced levels of the other subunits in human fibroblasts from a patient with loss-of-function mutations in the AP4S1 gene (AP4S1*) versus a control sample. CHC was used as a loading control and did not show any differences. Bands corresponding to the different subunits are indicated with arrows, whereas cross-reaction bands are noted with a black dot. (B) Native immunoprecipitations (IPs) of control and patient cell lines were performed with antibodies against the β4 and ε subunit. Note the reduction of β4 and ε in their own IP, as well as the loss or reduction of all subunits, suggesting that the assembly of AP-4 is impaired. The interaction with the accessory protein tepsin is also clearly reduced in the AP-4-deficient patient. IgG bands confirm the use of equal amount of antibody in each IP. (C) Supernatant of the first IP (B) was used for a second IP with an antibody against AP-1γ. This shows no reduction of AP-1 complex assembly and is a control for equal amounts of starting material in the primary IP. (D and E) Results of immunofluorescence using an antibody against AP-4ε only (D) and double staining against tepsin and AP-1γ (E) on control- and patient AP4S1* patient-derived cell lines. The control line shows the punctuated perinuclear pattern typical for AP-4 localization. Whereas the patient line shows loss of AP-4 labeling (D) and loss of AP-4-associated tepsin (E). In contrast, the localization of AP-1γ is not affected in AP4S1* patient fibroblasts compared with the control.

Figure 3.

Overview of reported AP-4 subunit mutations. In blue the ε subunit (NM_007347.4), in green the β4 subunit (NM_006594.3), in yellow the µ4 subunit (NM_004722.3) and in red the σ4 subunit (NM_007077.4). The scale bar on top indicates the size of the different proteins. Black arrows pinpoint mutation locations. Family IDs and mutations correspond to Table 1. Family 15, underlined, is the family described here; the patients carry both mutations in a compound heterozygous state. All other mutations (Family 1–14) were found homozygous in the patients and heterozygous in the respective parents.