The Sac1 domain of SYNJ1 identified mutated in a family with early-onset progressive Parkinsonism with generalized seizures

Abstract

This study aimed to elucidate the genetic causes underlying early-onset Parkinsonism (EOP) in a consanguineous Iranian family. To attain this, homozygosity mapping and whole-exome sequencing were performed. As a result, a homozygous mutation (c.773G>A; p.Arg258Gln) lying within the NH2 -terminal Sac1-like inositol phosphatase domain of polyphosphoinositide phosphatase synaptojanin 1 (SYNJ1), which has been implicated in the regulation of endocytic traffic at synapses, was identified as the disease-segregating mutation. This mutation impaired the phosphatase activity of SYNJ1 against its Sac1 domain substrates in vitro. We concluded that the SYNJ1 mutation identified here is responsible for the EOP phenotype seen in our patients probably due to deficiencies in its phosphatase activity and consequent impairment of its synaptic functions. Our finding not only opens new avenues of investigation in the synaptic dysfunction mechanisms associated with Parkinsonism, but also suggests phosphoinositide metabolism as a novel therapeutic target for Parkinsonism.

Keywords: SYNJ1; autosomal recessive Parkinsonism; homozygosity mapping; whole-exome sequencing.

Figure 1

Left side Upper panel: B allele frequency plots from Genome studio software (Illumina) showing the loss of heterozygosity area (LOH (blank areas); 21q21.1-22.2) shared by both affected siblings; the location of SYNJ1 within the disease-associated LOH area is highlighted in red. Middle panel: The pedigree structure of the Iranian family with EOP is shown in the left side while the Sanger chromatograms of the human reference sequence (bottom) as well as both heterozygous (middle) and homozygous (top) mutant sequences are shown in the right side; a black arrow highlights the pathogenic mutation. The p.Arg258Gln mutation is represented as p.R258Q. Bottom panel shows the conservation of R258 amino-acid (highlighted in red) within different orthologs in both SYNJ1 and SYNJ2 proteins. HS: Homo sapiens; BT: Bos Taurus; CL: Canis lupus; MM: Mus musculus; PT: Pan troglodytes; RN: Rattus norvegicus. Right side: Brain FLAIR magnetic resonance images (MRIs) of patient 1 showing bilateral periventricular (a) and subcortical (b and c) white matter hyperintense signals predominantly in parietal-occipital regions. Axial (e & e) and sagittal (f) post-gadolinium brain MR images of patient 2 show an intensely enhancing meningioma with compressing the left pons and lower midbrain. There was no evidence of obstructive hydrocephalus.

Figure 2

2a: Domain organization of SYNJ1-145 (3) and SYNJ1-170 (4) isoforms (Haffner, et al., 1997; Perera, et al., 2006). SYNJ1-145 is highly expressed in the nervous system, specifically in mature brain and synapses, while the alternatively spliced isoform SYNJ1-170, which has a longer C-terminal, is ubiquitously expressed but at lower levels. The domain organization was drawn based on published reports and SMART predictions (http://smart.embl-heidelberg.de/) (Guo, et al., 1999; Jha, et al., 2004) and includes the two polyphosphoinositide phosphatase regions and the C-terminal region, which is responsible for protein-protein interactions. The extended C-terminal tail of SYNJ1-170 contains binding sites for proteins of the endocytic machinery. Both p.Arg219Gln (R219Q) and p.Cys383Ser (C383S) mutations lying in the Sac1-like region are shown. 2b: Anti-Flag Western-Blot showing equal amounts of affinity purified Flag-tagged wild-type and mutant SYNJ1. 2c: Wild-type and mutants SYNJ1 were expressed in HEK 293T cells and purified by immunoprecipitation. The enzymatic activity was measured by malachite-green based assays. Error bars are SEM. Graphics represent means and SEM of at least nine independent experiments. (***) indicates p values of p≤0.001.