Novel loss-of-function PRRT2mutation causes paroxysmal kinesigenic dyskinesia in a Han Chinese family

Abstract

BackgroundMutations in proline-rich transmembrane protein 2 (PRRT2) are a cause of paroxysmal kinesigenic dyskinesia (PKD). In this study, we investigated the PRRT2 gene mutation in a Chinese Han family with PKD and study the pathogenesis of the mutation with PRRT2 gene.MethodsPeripheral venous blood was taken from the family members. Sanger sequencing was used for novel mutation sequencing. For the pathogenesis with the novel mutation was analyzed by bioinformatics, real-time PCR, subcellular localization and Western blot.ResultsThe Sanger sequencing showed a novel mutation, c.186-187delGC, a deletion mutation, in exon 2 of the PRRT2 gene, the frameshift mutation generated a truncated protein that was stably expressed in transfected Human embryonic kidney (HEK) 293 cells. A subcellular localization assay in COS-7 cells with GFP-tagged protein showed nuclear localization for the mutant protein while the wild-type protein was localized in membranes. Co-transfection of HEK293 cells with wild-type and mutant expression plasmids cells did not influence mRNA or protein expression from the wild-type plasmid.ConclusionsOur findings demonstrated that the c.186-187delGC mutation resulted in a truncated protein from the PRRT2 gene to involve in PKD pathogenesis with haploinsufficiency. The results extend the mutation spectrum of the PRRT2 gene and provide a new example for studying the pathogenesis of the mutated PRRT2 gene.

PRRT2 mutations lead to neuronal dysfunction and neurodevelopmental defects

“…Consistent with this result, a recent in vitro study also showed a decrease in synapses in cultured hippocampal neurons when Prrt2 was knocked down [32]. Delayed neuronal migration and abnormal spine density are frequently associated with various cognitive, learning and memory deficits [28, 34–36]. Our results indicated that these defects may contribute to the severe encephalopathy caused by the rare homozygous or biallelic PRRT2 mutations [10, 15, 16, 37].…”

“…Reduced expression has been reported in cells expressing other PRRT2 mutations [19, 33, 34] and could be a common functional loss of PKD-related mutations. It remains unclear whether the decreased protein level in cells was caused by decreased production, protein instability, or both.…”

Functional study and pathogenicity classification of PRRT2 missense variants in PRRT2‐related disorders

“…Among them, c.649dupC (p.R217Pfs*8) is the most frequent variant, accounting for 78.5% of mutation carriers . Truncated variants leading to conspicuous reduced protein level have been reported in various in vitro studies . Besides the truncated variants, about 29 missense variants of PRRT2 are documented in PRRT2 ‐related disorders.…”

Activity‐dependent cleavage of dyskinesia‐related proline‐rich transmembrane protein 2 (PRRT2) by calpain in mouse primary cortical neurons

“…Amino acids used in calpain-resistant substitution mutants are highlighted with yellow. C, Lysates of Neuro2A cells transfected with PRRT2wt-EGFP cDNA (lanes 1-3), PRRT2 LSRHP243_247FIDDD-EGFP cDNA (244-uncleaved mutant; lanes 4-6), PRRT2 HSPP201_204IDDD-EGFP cDNA (201-mut; lanes 7-9), PRRT2 LAGPG251_255FIDDD-EGFP cDNA (252-mut; lanes 10-12) or PRRT2 LQQLV219_223FIDDD-EGFP cDNA (220-mut; lanes 13-15) were treated with 4 mM CaCl 2 and 16.8 ng/µL native calpain-1 at 30°C for 0 hr (lanes 1, 4, 7, 10, 13, 16), 0.5 hr (lanes 2, 5, 8, 11, 14, 17), or 2 hr (lanes 3,6,9,12,15), and then immunoblotted with anti-GFP antibody. A black arrowhead and arrow indicate the full-length PRRT2-EGFP (lanes 1, 4, 7, 10, 13) and 12K-CTF-EGFP (lanes 2, 3, 8, 9, 11, 12), respectively.…”

“…4 PRRT2 is composed of 340 amino acids, including a large intracellular domain (aa 1-268) harboring a proline-rich motif (aa 131-216), followed by an intramembrane region (aa 269-289) and a transmembrane domain (aa 318-338) at the carboxy (C)-terminal region (see Figure 1C). 5 Many of the PRRT2 mutations found in PKD patients result in truncation of PRRT2 protein lacking transmembrane regions, causing a rapid degradation or subcellular mislocalization, 1,6,7 | 181 HATTA eT Al suggesting that loss-of-function of PRRT2 might cause PKD. This hypothesis is supported by the observation that Prrt2 knockout mice show PKD-like phenotypes, 8 although the possibility remains that the truncated mutants might be slightly expressed and cause gain-of-abnormal-function.…”