Loss of SYNJ1 dual phosphatase activity leads to early onset refractory seizures and progressive neurological decline

Abstract

SYNJ1 encodes a polyphosphoinositide phosphatase, synaptojanin 1, which contains two consecutive phosphatase domains and plays a prominent role in synaptic vesicle dynamics. Autosomal recessive inherited variants in SYNJ1 have previously been associated with two different neurological diseases: a recurrent homozygous missense variant (p.Arg258Gln) that abolishes Sac1 phosphatase activity was identified in three independent families with early onset parkinsonism, whereas a homozygous nonsense variant (p.Arg136*) causing a severe decrease of mRNA transcript was found in a single patient with intractable epilepsy and tau pathology. We performed whole exome or genome sequencing in three independent sib pairs with early onset refractory seizures and progressive neurological decline, and identified novel segregating recessive SYNJ1 defects. A homozygous missense variant resulting in an amino acid substitution (p.Tyr888Cys) was found to impair, but not abolish, the dual phosphatase activity of SYNJ1, whereas three premature stop variants (homozygote p.Trp843* and compound heterozygote p.Gln647Argfs*6/p.Ser1122Thrfs*3) almost completely abolished mRNA transcript production. A genetic follow-up screening in a large cohort of 543 patients with a wide phenotypical range of epilepsies and intellectual disability revealed no additional pathogenic variants, showing that SYNJ1 deficiency is rare and probably linked to a specific phenotype. While variants leading to early onset parkinsonism selectively abolish Sac1 function, our results provide evidence that a critical reduction of the dual phosphatase activity of SYNJ1 underlies a severe disorder with neonatal refractory epilepsy and a neurodegenerative disease course. These findings further expand the clinical spectrum of synaptic dysregulation in patients with severe epilepsy, and emphasize the importance of this biological pathway in seizure pathophysiology.

Keywords: SYNJ1; SYNJ1 dual phosphatase activity; early onset epilepsy; neurodegenerative disorder; recessive disorder.

SYNJ1 encodes a polyphosphoinositide phosphatase (Synaptojanin 1) with a prominent role in synaptic vesicle dynamics. Hardies et al. report three families (six patients) with autosomal recessive SYNJ1 variants, who display early-onset refractory seizures and progressive neurological decline. The pathogenic variants entail loss of the dual phosphatase activity of Synaptojanin 1.

Figure 1

Families with recessive inherited SYNJ1 variants reported here. (A) Pedigrees of Families A–C with an indication of the segregating SYNJ1 variants (NM_203446.2; NP_982271.2). All parents are healthy, affected children are indicated by filled symbols. Double horizontal lines indicate consanguinity between the parents and arrows point towards the patients whose fibroblasts were used in the mRNA expression studies. (B) Visualization of sequencing reads produced by direct Sanger sequencing during variant validation.

Figure 2

Phosphatase activity assay of the identified SYNJ1 missense variants. The effect on enzymatic activity of SYNJ1 was tested for mutants resulting from missense variants. Wild-type or mutant FLAG-tagged SYNJ1 (NM_203446.2) was expressed in Expi293F cells, purified and tested for enzymatic activity against short chain phosphoinositides using a malachite-based assay (Maehama et al., 2000). The dephosphorylation of domain specific substrates was investigated: PI(4)P for the Sac1 domain, and PI(4,5)P₂ and PI(3,4,5)P₃ for the 5’PP domain. Because dephosphorylation of PI(4,5)P₂ by the 5’PP domain results in PI4P, which can then be dephosphorylated by the Sac1 domain, the phosphate released when using PI(4,5)P₂ as substrate reflects the action of both phosphatase domains. When PI(3,4,5)P₃ was used as a substrate, selective action of the 5’PP domain could be assessed. Graphics depict mean values of released inorganic free phosphate from three independent experiments (bars reflect standard error of mean). A clear reduction, but not a complete loss, of phosphate activity towards all tested substrates was found for the p.Tyr888Cys substitution identified in Family A. Compared to wild-type expressing cells, no differences in enzymatic activity was found for cells expressing the p.Met1020Ile/p.Tyr1057Ser substitutions. Unpaired t-test was used to analyse the difference between wild-type and mutants (***P = 0.0002–0.0003, ****P < 0.0001). ns = non-significant.

Figure 3

Protein structure of human SYNJ1. Domains of the protein are shown in different colours. The positions of newly identified (above) and previously reported (below) pathogenic variants are indicated (NP_982271.2). Variants identified in patients with early onset refractory epilepsy and progressive neurological decline are marked in red, the recurrent substitution identified in three families with early onset parkinsonism in blue, and the compound heterozygous substitutions that are likely to be polymorphisms (p.Met1020Ile/p.Tyr1057Ser), in green. Sac = Sac1 domain; 5PPtase = 5-phosphatase domain; RRM = RNA recognition motif; PRD = proline rich domain.

Figure 4

Levels of SYNJ1 mRNA in patient and control fibroblasts. The effect of the premature stop variants identified in Families B and C was evaluated by the amount of mutant mRNA transcript in patient fibroblasts. Messenger RNA was extracted from human fibroblasts expressing either homozygous wild-type (control) or mutant SYNJ1: patient Family B homozygous for p.Trp843* and patient Family C compound heterozygous for p.Gln647Argfs*6/p.Ser1122Thrfs*3. Quantitative PCR was performed on the converted cDNA. Graphics depict mean values of normalized ΔCt, relatively compared to wild-type mRNA levels, from at least two independent experiments (bars reflect standard error of mean). P-values were determined by comparing the normalized ΔCt (unpaired t-test, ****P < 0.0001). A significant reduction in SYNJ1 transcript expression was found in both patients’ fibroblasts compared to control cells.