| Paroxysmal kinesigenic dyskinesia （PKD） is the most common subgroup of paroxysmal dyskinesia, manifesting as episodic chorea, athetosis, dystonia, or ballism, with a brief duration and trigger of sudden movement. Generally, familial PKD is transmitted in an autosomal dominant inheritance, while sporadic cases are also reported. More males are affected than females with the male:female ratio from 3:1 to 4:1. PKD usually commences in childhood or early adolescence but remits spontaneously in their third or forth decade years old. The attacks of PKD are usually frequent; most patients have several or dozens of attacks per day, whereas some patients may have more than 100 attacks per day.We previously found that PRRT2 was the causative gene of PKD, which was soon confirmed by several independent groups. However, there are still some unsolved issues concerning PRRT2 and PKD. First, we do not know if the sporadic PKD was also caused by PRRT2 mutations. Second, did PRRT2 mutations correlate with the phenotype of PKD? At last, what are the potential mechanisms of PKD? Our study is aimed to investigate these issues.Section 1:The characteristics of PRRT2 mutations in sporadic PKD casesObjective:We aimed to investigate the characteristics of PRRT2 mutations in sporadic PKD cases.Methods:A total of 41 sporadic PKD cases were collected. Peripheral blood （3 mL） was collected, followed by DNA extraction. Direct sequencing of PRRT2 was performed. MR1, SLC2A1, and CLCN1 gene were further investigated in the cases without PRRT2 mutation. Haplotype analysis using 18 SNPs was conducted to confirm the biological relationship.Results:Seven of the 41 cases were identified to harbor PRRT2 mutations, including the c.649dupC in 5 cases, c.649delC in one case and c.133136delCCAG in another one case. In addition,5 parents of the 7 mutation carriers were also identified to carry the same PRRT2 mutations, while the remaining two did not harbor any PRRT2 mutation. Among the 34 non-PRRT2 mutations carriers, one case carried SLC2A1 mutation c.363G>A, two cases carried CLCN1 c.1205C>T mutation. Haplotype analysis confirmed the biological relationship of the family.Conclusions:PRRT2 mutations account for a small number of sporadic PKD cases, are not completely penetranced, and can derive from de novo mutagenesis. Particular attentions should be paid to distinguish PKD from PED and MC.Section 2:The correlation of PRRT2 mutations with clinical presentations of PKD and responses to treatmentObjective:We aimed to investigate the correlations of PRRT2 mutations with clinical phenotypes of PKD and differential responses to carbamazepine.Methods:Eighty-one PKD patients including 44 familial cases in 14 pedigrees and 37 sporadic cases were enrolled in two stages. Direct sequencing of PRRT2 was performed. Detailed clinical phenotypes were evaluated. Carbamazepine was prescribed in a subset of PKD patients, and drug response was followed up. Differences in characteristics between PRRT2 mutations carriers and non-carriers were analyzed using t-test or chi-square test.Results:Four different PRRT2 mutations including c.514517delTCTG, c.649dupC, c.649delC, c.972delA were identified in 44 familial cases and 4 sporadic cases. All 48 PRRT2 mutation carriers expressed the choreoathetosis phenotype, while 33 non-PRRT2 mutation carriers expressed either the dystonia phenotype （26 cases） or the choreoathetosis phenotype （7 cases; P= 3.8×10-15）. Compared to the non-PRRT2 mutation carriers, the PRRT2 mutation carriers were significantly younger at onset （7.63±3.72 vs 13.52±2.69 years old, P=2.2×10-11） and had a longer duration of attack episodes （P=2.2×10-4）. All PRRT2 mutation carriers responded completely to low-dose of carbamazepine （50 mg/d）. However,94%of the non-PRRT2 mutation carriers did not have a full response to carbamazepine, even after the dose was increased （P=1.1×10-20）. Interestingly, one of the PRRT2 c.649dupC mutation carrier was also identified to harbor the CLCN1 mutation c.1723C>T and c.2492A>G, indicating that the case also combined with myotonia congenita.Conclusions:Compared to non-PRRT2 mutation carriers, the mutation carriers had younger age at onset, more severe phenotype, but complete response to low dose of carbamazepine. Our study also revealed that PRRT2 mutation carrier without a complete response to carbamazepine should be investigated other gene mutation.Section 3:The potential altered brain region of PKD investigated by fMRIObjective:We aimed to investigate the potential altered brain region of PKD using fMRI scanningMethods:Thirty PKD patients including 15 PRRT2 mutation carriers and 15 non-PRRT2 mutatio carriers, as well as 14 control individuals were collected. Sanger sequencing was performed to investigate the PRRT2 sequence in all the subjects. All participants were scanned with a Siemens Tim Trio 3.0 scanner.Results:Compared with healthy subjects, increased function connectivity networks and decreased structure connectivity networks were found in PKD cases. In the brain functional network of PKDm patient, the left superior parietal gyrus, inferior temporal gyrus and bilateral postcentral gyrus became the hubs while supplementary motor area and dorsal cingulate cortex degraded to non-hubs. In PKDnm group, the bilateral precuneus was changed from hubs to non-hubs and the superior frontal gyrus emerged as the hub region. Structural network analysis revealed that the dorsal cingulate cortex changed into a non-hub node and calcarine changed into a hub node in PKD groups. Superior frontal gyrus was changed from non-hubs to hubs in PKDm group, while the right caudate and left insula was removed from the hub club in PKDnm group.Conclusions:The function connectivity network was increasd but the structure connectivity network was decreased in PKD cases. The sensory and sensorimotor integration networks are indispensable components for the pathogenesis of PKD.Section 4:The potential mechanisms of PKDObjective:We aimed to investigate the potential mechanisms of PKDMethods:We constructed several recombinant lentivirus vectors encoding wild PRRT2 （WT-PRRT2） and mutant PRRT2 （including c.514517delTCTQ c.649dupC, c.796C>T, c.913G>A, and c.972delA）. After transferring SH-SY5Y cell, we exerted the total RNA from each cell line. RT-PCR and Real Time PCR were performed to analyze the PRRT2 mRNA. Lactacystin was dissolved in DMSO and added to the SH-SY5Y at a dose 10 μmol/L. Western Blot was performed to investigate the change of PRRT2 protein. Urine epithelial cell collected from PKD patient and control subject was cultured in conditional medium. Recombinant adenovirus with six factors （Oct4, Sox2, cMyc, Klf4, Nanog, and Lin28） was genetrated to introduce reprogramming. After infecting the urine cell, induced pluripotent stem （iPS） was obtained. Alkaline phosphatase activity assay and teratoma were used to evaluate the iPS.Results:We generated WT and Mutant PRRT2 recombinant lentivirus vectors, which were confirmed by direct sequencing. RT-PCR and Real Time PCR results revealed that the PRRT2 mRNA was not different among the cell line transferred with the WT or various mutants PRRT2. After lactacystin intervention, the WT-PRRT2 protein changed slightly, while the mutant-PRRT2 protein increased significantly. Thirty days after infecting, we obtained iPS cell which exhibited the morphology and growth properties of ES cells. Alkaline phosphatase activity assay revealed that the iPS did not differentiate, and the teratoma demonstrated that the iPS could form three germs.Conclusions:Mutant PRRT2 lost its function by increasing the degradation of PRRT2 protein rather than the mRNA decay. It is a feasible approach to establish iPS from urine cell, and the obtained iPS has the ability of multipotential differentiation. |
