Exome sequencing identifies truncating mutations in PRRT2 that cause paroxysmal kinesigenic dyskinesia

Paroxysmal kinesigenic dyskinesia is the most common type of paroxysmal dyskinesia. Approximately half of the cases of paroxysmal kinesigenic dyskinesia worldwide are attributable to proline-rich transmembrane protein 2 mutations. The objective of this study was to investigate potential causative genes and clinical characteristics in proline-rich transmembrane protein 2-negative patients with paroxysmal kinesigenic dyskinesia. We analyzed clinical manifestations and performed exome sequencing in a cohort of 163 proline-rich transmembrane protein 2-negative probands, followed by filtering data with a paroxysmal movement disorders gene panel. Sanger sequencing, segregation analysis, and phenotypic reevaluation were used to substantiate the findings. The clinical characteristics of the enrolled 163 probands were summarized. A total of 39 heterozygous variants were identified, of which 33 were classified as benign, likely benign, and uncertain significance. The remaining 6 variants (3 novel, 3 documented) were pathogenic and likely pathogenic. Of these, 3 were de novo (potassium calcium-activated channel subfamily M alpha 1, c.1534A>G; solute carrier family 2 member 1, c.418G>A; sodium voltage-gated channel alpha subunit 8, c.3640G>A) in 3 sporadic individuals, respectively. The other 3 (paroxysmal nonkinesiogenic dyskinesia protein, c.956dupA; potassium voltage-gated channel subfamily A member 1, c.765C>A; Dishevelled, Egl-10, and Pleckstrin domain containing 5, c.3311C>T) cosegregated in 3 families. All 6 cases presented with typical paroxysmal kinesigenic dyskinesia characteristics, except for the Dishevelled, Egl-10, and Pleckstrin domain containing 5 family, where the proband's mother had abnormal discharges in her temporal lobes in addition to paroxysmal kinesigenic dyskinesia episodes. Our findings extend the genotypic spectrum of paroxysmal kinesigenic dyskinesia and establish the associations between paroxysmal kinesigenic dyskinesia and genes classically related to other paroxysmal movement disorders. De novo variants might be a cause of sporadic paroxysmal kinesigenic dyskinesia. © 2018 International Parkinson and Movement Disorder Society.

Paroxysmal kinesigenic dyskinesia (PKD) is characterized by paroxysms of dystonic, choreic, ballistic, or athetoid movements. Attacks usually commence during childhood or early adulthood, typically lasting a few seconds to a few minutes, and they can occur up to 100 times daily. Attacks usually respond to low-dose carbamazepine [1]. Mutations in PRRT2 have been identified as a cause of autosomal dominant PKD [2] and replicated in other studies [3-7]. It remains important to report the clinical characteristics of genetically defined families in order to fully describe the clinical syndrome, including the presence of additional associated features beyond the movement disorder. In a large Caucasian family with PKD (Fig. 1), a detailed neurological history and clinical examination were undertaken. Sanger sequencing of PRRT2 was performed. Three individuals have PKD alone, and three individuals have PKD and infantile convulsions (IC). Two individuals had isolated IC, whilst two other individuals had at least one adolescent seizure but no movement disorder. The age of onset for the movement disorder ranged between 3 and 30 years. Infantile convulsions occurred between the age of 6 and 12 months. In all cases, PKD attacks were choreic or dystonic in nature, lasting less than a minute, and responded well to carbamazepine. Attacks were provoked by sudden movements in all, with stress in II:10 and III:9. Four individuals were treated with low-dose carbamazepine and/or phenytoin for PKD/IC, which abolished symptoms of the movement disorder. Neurological examination was normal (Table 1). Fig. 1 Pedigree of Family 1. All individuals with symptoms of PKD, PKD and IC, or isolated IC were found to have a frameshift mutation in PRRT2 (c.649_650InsC p.P217fsX7) indicated by a plus symbol; individuals found not to have the mutation are indicated by ... Table 1 Clinical descriptions and genotyping results for family members Sanger sequencing of PRRT2 revealed the recognized pathogenic (c.649_650InsC p.P217fsX7) heterozygous mutation [2-7] in all eight clinically affected individuals as well as in three of the seven clinically unaffected individuals (Fig. 1). PRRT2 mutations are unlikely to be the cause of adolescent-onset seizures in this family, as individual III:12 did not carry the mutation. Non-penetrance was observed in three individuals. Migraine with aura, as classified using the International Headache Society diagnostic criteria [8], co-segregated in all but one individual with the p.P217fsX7 mutation, including in three individuals who do not have symptoms of IC/PKD, but was not observed in individuals who did not carry the p.P217fsX7 mutation. Visual aura (positive visual phenomena) was most frequently reported, other types of aura including expressive dysphasia, alexia and alien hand syndrome were also described. Migraine frequency ranged from twice a year to three times a month. Reported triggers included stress, sleep deprivation and exercise. Two individuals were treated with low-dose propranolol for several months, which reduced the frequency and severity of migraine attacks, but did not improve symptoms of the movement disorder. Previously reported linkage studies for this kindred (family 3, Spacey et al.) [9] generated LOD scores that were not significant for the analysis of PKD alone or PKD ± IC. We repeated linkage analysis using Merlin [10], assuming an autosomal dominant mode of inheritance with 80 % penetrance. LOD scores for the PRRT2 mutation and PKD/IC and the PRRT2 mutation and migraine with aura are 1.65 and 2.7, respectively. Several kindreds with hemiplegic migraine and PRRT2 mutations have recently been reported [11]. Migraine (with and without aura) is over-represented in individuals with PRRT2 mutations and clustering of migraine has been identified in small kindred’s harboring PRRT2 mutations [12] In this large family, migraine with aura segregates in a Mendelian fashion in nine of 10 individuals with the PRRT2 mutation, the remaining individual is still in adolescence and could still go onto develop migraine. Furthermore, it highlights that in some families, migraine with aura may be the predominant phenotype associated with PRRT2 mutations, which is important to bear in mind when determining whether a patient with PKD/IC has a positive family history, to enable appropriate genetic counseling and testing. The association of migraine with PRRT2 mutations raises the question of whether genetic variation in PRRT2 might play a role in susceptibility to ‘common’ migraine with aura.

To describe the phenotypes and penetrance of paroxysmal kinesigenic dyskinesia (PKD), a movement disorder characterized by attacks of involuntary movements occurring after sudden movements, infantile convulsion and choreoathetosis (ICCA) syndrome, and benign familial infantile convulsions (BFIC), caused by PRRT2 mutations. We performed clinical and genetic studies in 3 large families with ICCA, 2 smaller families with PKD, and 4 individuals with sporadic PKD. Migraine was also present in several individuals. We detected 3 different PRRT2 heterozygous mutations: the recurrent p.Arg217Profs*8 mutation, previously reported, was identified in 2 families with ICCA, 2 families with PKD, and one individual with sporadic PKD; one novel missense mutation (p.Ser275Phe) was detected in the remaining family with ICCA; and one novel truncating mutation (p.Arg217*) was found in one individual with sporadic PKD. In the 2 remaining individuals with sporadic PKD, PRRT2 mutations were not detected. Importantly, PRRT2 mutations did not cosegregate with febrile convulsions or with migraine. The estimated penetrance of PRRT2 mutations was 61%, if only the PKD phenotype was considered; however, if infantile convulsions were also taken into account, the penetrance was nearly complete. Considering our findings and those reported in literature, 23 PRRT2 mutations explain ∼56% of the families analyzed. PRRT2 mutations are the major cause of PKD or ICCA, but they do not seem to be involved in the etiology of febrile convulsions and migraine. The identification of PRRT2 as a major gene for the PKD-ICCA-BFIC spectrum allows better disease classification, molecular confirmation of the clinical diagnosis, and genetic testing and counseling.

Characteristics of patients with benign partial epilepsy in infancy without PRRT2 mutations

Paroxysmal kinesigenic dyskinesia (PKD) is the most prevalent kind type of paroxysmal Dyskinesia, characterized by recurrent and transient episodes of involuntary movements. Most PKD cases were attributed to the proline-rich transmembrane protein 2 (PRRT2) gene, in which the c.649 region is a hotspot for known mutations. Even though some patients with PKD have been genetically diagnosed using whole-exome sequencing (WES) and Sanger sequencing, there are still cases of missed diagnoses due to the limitations of sequencing technology and analytic methods on throughput. Patients meeting the diagnosis criteria of PKD with negative results of PRRT2-Sanger sequencing and WES were included in this study. Mutation screening and targeted high-throughput sequencing were performed to analyze and verify the sequencing results of the potential mutations. Six patients with PKD with high mutation ratios of c.649dupC were screened using our targeted high-throughput sequencing from 26 PKD patients with negative results of PRRT2-Sanger sequencing and WES (frequency = 23.1%), which compensated for the comparatively shallow sequencing depth and statistical flaws in this region. Compared with the local normal population and other patients with PKD, the mutation ratios of c.649dupC of these six patients with PKD were much higher and also had truncated protein structures and differentially altered mRNA expression. Based on the above studies, we emphasize the routine targeted high-throughput sequencing of the c.649 site in the PRRT2 gene in so-called genetic-testing-negative patients with PKD, and manually calculate the deletion and duplication mutations depth and ratios to lower the rate of clinical misdiagnosis.

PRRT2 encodes for proline-rich transmembrane protein 2 involved in synaptic vesicle fusion and presynaptic neurotransmitter release. Mutations in human PRRT2 have been related to paroxysmal kinesigenic dyskinesia (PKD), infantile convulsions with choreoathetosis, benign familial infantile epilepsies, and hemiplegic migraine. PRRT2 mutations cause neuronal hyperexcitability, which could be related to basal ganglia or cortical circuits dysfunction, leading to paroxysmal disorders. PRRT2 is expressed in the cerebral cortex, basal ganglia, and cerebellum. Approximately, 90% of pathogenic variants are inherited and 10% are de novo. Paroxysmal attacks in PKD are characterized by dystonia, choreoathetosis, and ballismus. In the benign familial infantile epilepsy (BFIE), seizures are usually focal with or without generalization, usually begin between 3 and 12 months of age and remit by 2 years of age. In 30% of cases of PRRT2-associated PKD, there is an association with BFIE, and this entity is referred to as PKD with infantile convulsions (PKD/IC). PRRT2 mutations are the cause of benign family childhood epilepsy and PKD/IC. On the other hand, PRRT2 mutations do not seem to correlate with other types of epilepsy. The increasing incidence of hemiplegic migraine in families with PRRT2-associated PKD or PKD/IC suggests a common disease pathway, and it is possible to assert that BFIE, paroxysmal kinesigenic dyskinesia, and PKD with IC belong to a continuous disease spectrum of PRRT2-associated diseases.

Paroxysmal kinesigenic dyskinesia (PKD) is the most common type of paroxysmal dyskinesias. Only one-third of PKD patients are attributed to proline-rich transmembrane protein 2 (PRRT2) mutations. We aimed to explore the potential causative gene for PKD. A cohort of 196 PRRT2-negative PKD probands were enrolled for whole-exome sequencing (WES). Gene Ranking, Identification and Prediction Tool, a method of case-control analysis, was applied to identify the candidate genes. Another 325 PRRT2-negative PKD probands were subsequently screened with Sanger sequencing. Transmembrane Protein 151 (TMEM151A) variants were mainly clustered in PKD patients compared with the control groups. 24 heterozygous variants were detected in 25 of 521 probands (frequency=4.80%), including 18 missense and 6 nonsense mutations. In 29 patients with TMEM151A variants, the ratio of male to female was 2.63:1 and the mean age of onset was 12.93 ± 3.15 years. Compared with PRRT2 mutation carriers, TMEM151A-related PKD were more common in sporadic PKD patients with pure phenotype. There was no significant difference in types of attack and treatment outcome between TMEM151A-positive and PRRT2-positive groups. We consolidated mutations in TMEM151A causing PKD with the aid of case-control analysis of a large-scale WES data, which broadens the genotypic spectrum of PKD. TMEM151A-related PKD were more common in sporadic cases and tended to present as pure phenotype with a late onset. Extensive functional studies are needed to enhance our understanding of the pathogenesis of TMEM151A-related PKD. © 2021 International Parkinson and Movement Disorder Society.

The proline-rich transmembrane protein (PRRT2) gene was recently identified using exome sequencing as the cause of autosomal dominant paroxysmal kinesigenic dyskinesia (PKD) with or without infantile convulsions (IC) (PKD/IC syndrome). Episodic neurologic disorders, such as epilepsy, migraine, and paroxysmal movement disorders, often coexist and are thought to have a shared channel-related etiology. To investigate further the frequency, spectrum, and phenotype of PRRT2 mutations, we analyzed this gene in 3 large series of episodic neurologic disorders with PKD/IC, episodic ataxia (EA), and hemiplegic migraine (HM). The PRRT2 gene was sequenced in 58 family probands/sporadic individuals with PKD/IC, 182 with EA, 128 with HM, and 475 UK and 96 Asian controls. PRRT2 genetic mutations were identified in 28 out of 58 individuals with PKD/IC (48%), 1/182 individuals with EA, and 1/128 individuals with HM. A number of loss-of-function and coding missense mutations were identified; the most common mutation found was the p.R217Pfs*8 insertion. Males were more frequently affected than females (ratio 52:32). There was a high proportion of PRRT2 mutations found in families and sporadic cases with PKD associated with migraine or HM (10 out of 28). One family had EA with HM and another large family had typical HM alone. This work expands the phenotype of mutations in the PRRT2 gene to include the frequent occurrence of migraine and HM with PKD/IC, and the association of mutations with EA and HM and with familial HM alone. We have also extended the PRRT2 mutation type and frequency in PKD and other episodic neurologic disorders.

Objective To explore the electroencephalography(EEG)features of paroxysmal kinesigenic dyskinesia(PKD)and analyze the influence of proline-rich transmembrane protein 2(PRRT2)mutations in EEG features. Methods Sixty patients with PKD were collected in our hospital from January 2006 to December 2014. The patients were classified into different subtypes according to the paroxysmal symptoms: 27 with pure PKD, 2 with PKD plus benign familial infantile convulsion(ICCA), 4 with PKD plus febrile seizure(FS), and 27 with PKD plus unclassified epilepsy. Video EEG monitoring was performed in all patients. The features of EEG in different subtypes of PKD were analyzed. PRRT2 gene mutations were screened in 39 patients. The EEG features were compared between the patients with and without PRRT2 gene mutations. Results Interictal EEG abnormalities were presented in 31 patients(51.7%), including 12(44.4%)with pure PKD, one(50%)with ICCA, 18(66.7%)with PKD plus unclassified epilepsy. Interictal EEG abnormalities were presented as diffused slowing wave(n=9, 29%), focal slowing wave(n=11, 35.5%), and focal epileptiform discharges(n=11, 35.5%). There were no significant differences in the percentage of diffuse slowing discharges(14.8% vs. 18.5% , P=0.500), focal slowing discharges(14.8% vs. 25.9% , P=0.311), and focal epileptiform discharges(14.8% vs. 25.9%, P=0.311)between patients with pure PKD and PKD plus unclassified epilepsy. PRRT2 gene mutations were identified in 21 patients(53.8%). The ratio of EEG abnormalities between groups with and without PRRT2 gene mutations was not significantly different(57.1% vs. 44.4%, P>0.05). Conclusions Interictal EEG abnormalities can be presented in patients with pure PKD or PKD plus epilepsy. PRRT2 gene mutations could not influence EEG features in PKD. Key words: Paroxysmal kinesigenic dyskinesia; Electroencephalography; Proline-rich transmembrane protein 2; Epilepsy

Paroxysmal kinesigenic dyskinesia (PKD) is an autosomal dominant disorder and PRRT2 is the causative gene of PKD. The aim of this study was to investigate PRRT2 mutations in patients who were clinically diagnosed with PKD. Nine PKD cases, including four familial cases and five sporadic cases, were selected. Peripheral blood was drawn after obtaining informed consent, and genomic DNA was extracted by a standard protocol. Sanger sequencing was performed for the screening of PRRT2 mutations. A total of five cases were detected to harbor PRRT2 mutations. Four familial cases carried a c.649dupC (p.Arg217Profs*8) mutation, while one sporadic case and his asymptomatic father carried a c.133-136delCCAG (p.Pro45Argfs*44) mutation. PRRT2 mutations were not identified in the remaining cases. The study further confirmed that PRRT2 was a causative gene of PKD and implied that PRRT2 mutation has incomplete penetrance.

To investigate the clinical features and proline-rich transmembrane protein 2 (PRRT2) gene mutation in patients with paroxysmal kinesigenic dyskinesia (PKD). Clinical information was collected at Peking University First Hospital from January 2004 to July 2014. In total, 10 patients with PKD were recruited, and all were males. Among them, four patients were the probands from four PKD families and the other six patients were sporadic cases. Clinical information was analyzed. Peripheral blood samples for DNA study were collected from PKD patients and their family members. Genomic DNA was extracted using standard procedures. Mutation analysis of PRRT2 was performed by Sanger sequencing after PCR. Of the 10 patients, the median age of dyskinesias onset was 10 years, ranging from 4 to 13 years. The description of their attacks were abnormal involuntary movements provoked by sudden movements, without loss of consciousness. Five patients exhibited dystonia, two patients exhibited choreoathetosis, and three patients had mixed (dystonia and choreoathetosis) dyskinesias. The duration of the attacks lasted for 3 to 30 seconds. The frequency ranged from once per month to twenty times per day. PRRT2 mutations, c. 649_650insC (p. R217PfsX8), were found in all the four PKD families. Mutation c. 649_650insC was also detected in two of the six sporadic PKD cases, inheriting from their asymptomatic mother. The onset age of PKD could be in the early childhood. The clinical features of the familial cases and sporadic cases showed no difference. The attacks manifested as dystonia, choreathetosis, or mixed. PRR2 mutations could be identified in familial or sporadic cases with PKD. Mutation c. 649_650insC is the hotspot mutation of PRRT2 gene.

PRRT2 mutations in Japanese patients with benign infantile epilepsy and paroxysmal kinesigenic dyskinesia

Paroxysmal kinesigenic dyskinesias is a paroxysmal movement disorder characterized by recurrent, brief attacks of abnormal involuntary movements induced by sudden voluntary movements. Although several loci, including the pericentromeric region of chromosome 16, have been linked to paroxysmal kinesigenic dyskinesias, the causative gene has not yet been identified. Here, we identified proline-rich transmembrane protein 2 (PRRT2) as a causative gene of paroxysmal kinesigenic dyskinesias by using a combination of exome sequencing and linkage analysis. Genetic linkage mapping with 11 markers that encompassed the pericentromeric of chromosome 16 was performed in 27 members of two families with autosomal dominant paroxysmal kinesigenic dyskinesias. Then, the whole-exome sequencing was performed in three patients from these two families. By combining the defined linkage region (16p12.1–q12.1) and the results of exome sequencing, we identified an insertion mutation c.649_650InsC (p.P217fsX7) in one family and a nonsense mutation c.487C>T (p.Q163X) in another family. To confirm our findings, we sequenced the exons and flanking introns of PRRT2 in another three families with paroxysmal kinesigenic dyskinesias. The c.649_650InsC (p.P217fsX7) mutation was identified in two of these families, whereas a missense mutation, c.796C>T (R266W), was identified in another family with paroxysmal kinesigenic dyskinesias. All of these mutations completely co-segregated with the phenotype in each family. None of these mutations was identified in 500 normal unaffected individuals of matched geographical ancestry. Thus, we have identified PRRT2 as the first causative gene of paroxysmal kinesigenic dyskinesias, warranting further investigations to understand the pathogenesis of this disorder.

Mutations in the gene PRRT2 encoding proline-rich transmembrane protein 2 have recently been identified as the cause of three clinical entities: benign familial infantile epilepsy (BFIE), infantile convulsions with choreoathetosis...

Paroxysmal kinesigenic dyskinesia (PKD) is a rare neurological disorder, characterized by attacks of involuntary movements triggered by sudden action. Variants in proline-rich transmembrane protein 2 (PRRT2) are the most common genetic cause of PKD. The objective was to investigate the clinical and genetic characteristics of PKD and to establish genotype-phenotype correlations. We enrolled 219 PKD patients, documented their clinical information and performed PRRT2 screening using Sanger sequencing. Whole exome sequencing was performed on 49 PKD probands without PRRT2 variants. Genotype-phenotype correlation analyses were conducted on the probands. Among 219 PKD patients (99 cases from 39 families and 120 sporadic cases), 16 PRRT2 variants were identified. Nine variants (c.879+4A>G, c.879+5G>A, c.856G>A, c.955G>T, c.884G>C, c.649C>T, c.649dupC, c.649delC and c.696_697delCA) were previously known, while seven were novel (c.367_403del, c.347_348delAA, c.835C>T, c.116dupC, c.837_838insC, c.916_937del and c.902G>A). The mean interval from onset to diagnosis was 7.94 years. Compared to patients without PRRT2 variants, patients with the variants were more likely to have a positive family history, an earlier age of onset and a higher prevalence of falls during pre-treatment attacks (27.14% versus 8.99%, respectively). Patients with truncated PRRT2 variants tend to have bilateral attacks. We identified two transmembrane protein 151A (TMEM151A) variants including a novel variant (c.368G>C) and a reported variant (c.203C>T) in two PRRT2-negative probands with PKD. These findings provide insights on the clinical characteristics, diagnostic timeline and treatment response of PKD patients. PKD patients with truncated PRRT2 variants may tend to have more severe paroxysmal symptoms. This study expands the spectrum of PRRT2 and TMEM151A variants. Carbamazepine and oxcarbazepine are both used as a first-line treatment choice for PKD patients.