paroxysmal kinesigenic dyskinesia ( pkd ) is an episodic movement disorder characterized by recurrent and brief attacks of involuntary movements or dystonia , without alteration of consciousness .
attacks are usually triggered by sudden movement or change in velocity , presenting with choreoathetosis , dystonia , or ballism .
the episodes of pkd usually start in childhood or early adolescence and tend to remit with age .
pkd cases usually have a good response to antiepileptic drugs , such as carbamazepine and phenytoin .
sporadic pkd cases are also reported , which are considered to be attributed to incomplete penetrance or de novo mutations .
prrt2 encoding proline - rich transmembrane protein 2 has been identified as a causative gene for pkd by several independent groups .
a hotspot mutation c.649dupc has been found in pkd cases from different ethnic origins . however , prrt2 mutations do not account for all pkd cases , especially for those sporadic ones .
the low frequency of prrt2 mutations in sporadic pkd suggested an existence of additional gene mutation or possible misdiagnosis due to overlapping clinical manifestations .
of note , prrt2 mutations were not only implicated in pkd but also identified in other paroxysmal disorders , such as benign familial infantile seizures , paroxysmal nonkinesigenic dyskinesia ( pnkd ) , and paroxysmal exertion - induced dyskinesia ( ped ) .
however , it is still elusive whether the causative genes of other paroxysmal disorders are involved in the etiology of prrt2-negatives poradic pkd . in this study , we collected a cohort of 28 chinese patients who were clinically diagnosed with sporadic pkd and were excluded prrt2 mutations .
we investigated the genetic variants of mr-1 , slc2a1 , and clcn1 , which are the causative genes of pnkd , ped , and myotonia congenita ( mc ) , respectively , in these cases .
this study was approved by the medical ethics committee of huashan hospital , fudan university .
twenty - eight chinese patients with a presumptive diagnosis of sporadic pkd were recruited in this study [ table 1 ] .
all patients were primary pkd and met the diagnostic criteria proposed by bruno et al . among these pkd cases ,
the other nine patients ( patient 2028 ) were collected between august 2012 and december 2012 .
the mean age at onset was 12.3  3.6 years ( range 618 years ) .
none of them was accompanied by infantile febrile , hemiplegic migraine , migraine or episodic ataxia .
in addition , 200 healthy individuals of chinese ancestry were recruited as control subjects . clinical features and genetic investigations of 28 presumptively sporadic pkd patients
sm : sudden movement ; s : shifting position ; fa : fatigue ; co : cold ; ch : choreoathetosis ; d : dystonia ; st : stiffness ; w : weakness ; pkd : paroxysmal kinesigenic dyskinesia .
genomic dna of 28 subjects was extracted from 3 ml peripheral blood by a standard protocol using qiaamp dna blood mini kit ( qiagen , hilden , germany ) .
all exons of the targeted genes ( mr-1 , slc2a1 , and clcn1 ) and their respective intron - exon boundaries were amplified by the polymerase chain reaction ( pcr ) .
pcrs were carried out in a final volume of 20 l , containing 40 ng of dna and 12.5 pmol of both forward and reverse primers .
the purified pcr products of 28 samples were sequenced using an abi 3730 automated dna sequencer ( applied biosystems , foster city , ca , usa ) .
the sequencing results were aligned to the ncbi human reference dna sequence of mr-1 , slc2a1 , and clcn1 , respectively ( ensembl gene i d : ensg00000153029 , ensg00000117394 , and ensg00000188037 ) .
amino acid and nucleotide changes were identified and numbered corresponding to their position within the prrt2 mrna .
primers and annealing temperatures for mr-1 f : forward ; r : reverse . primers and
this study was approved by the medical ethics committee of huashan hospital , fudan university .
twenty - eight chinese patients with a presumptive diagnosis of sporadic pkd were recruited in this study [ table 1 ] .
all patients were primary pkd and met the diagnostic criteria proposed by bruno et al . among these pkd cases ,
the other nine patients ( patient 2028 ) were collected between august 2012 and december 2012 .
the mean age at onset was 12.3  3.6 years ( range 618 years ) .
none of them was accompanied by infantile febrile , hemiplegic migraine , migraine or episodic ataxia .
in addition , 200 healthy individuals of chinese ancestry were recruited as control subjects . clinical features and genetic investigations of 28 presumptively sporadic pkd patients
sm : sudden movement ; s : shifting position ; fa : fatigue ; co : cold ; ch : choreoathetosis ; d : dystonia ; st : stiffness ; w : weakness ; pkd : paroxysmal kinesigenic dyskinesia .
genomic dna of 28 subjects was extracted from 3 ml peripheral blood by a standard protocol using qiaamp dna blood mini kit ( qiagen , hilden , germany ) .
all exons of the targeted genes ( mr-1 , slc2a1 , and clcn1 ) and their respective intron - exon boundaries were amplified by the polymerase chain reaction ( pcr ) .
pcrs were carried out in a final volume of 20 l , containing 40 ng of dna and 12.5 pmol of both forward and reverse primers .
the purified pcr products of 28 samples were sequenced using an abi 3730 automated dna sequencer ( applied biosystems , foster city , ca , usa ) .
the sequencing results were aligned to the ncbi human reference dna sequence of mr-1 , slc2a1 , and clcn1 , respectively ( ensembl gene i d : ensg00000153029 , ensg00000117394 , and ensg00000188037 ) .
amino acid and nucleotide changes were identified and numbered corresponding to their position within the prrt2 mrna .
primers and annealing temperatures for mr-1 f : forward ; r : reverse . primers and
after sequencing in the 28 patients with presumptive diagnosis of sporadic pkd , we identified 16 genetic variants , including 4 in mr-1 gene , 8 in slc2a1 gene , and 4 in clcn1 gene [ table 2 ] . among these variants , 7 slc2a1 variants ( c.45c > t , c.399c > t , c.588g > a , c.987g > a , c.1011c > t , c.1065a > g , and c.1372c > a ) and 3 clcn1 variants ( c.261c > t , c.2154c > t , and c.2180c > t ) were proved to be nonpathogenic variants . however , we detected 2 heterozygous missense mutations , including slc2a1 c.363g > a ( p. met121ile ) [ figure 1a ] in one patient and clcn1 c.1205c > t ( p. ala402val ) [ figure 1b ] in two other cases .
the slc2a1 c.363g > a was absent in 1000 genomes database , exac database , and our in - house 200 controls . it was predicted to be deleterious by sift and polyphen2 . the allele frequency of clcn1 c.1205c > t was reported at 0.03% in 1000 genomes database .
sequencing of clcn1 in our 200 controls also did not reveal the presence of clcn1 c.1205c > t .
moreover , it was identified as a causative mutation in a sporadic mc case by fialho et al .
sixteen variants identified in the coding regions and intronexon boundaries of mr-1 , slc2a1 , and clcn1 gene chromatogram of c.363g > t mutation in slc2a1 and c.1205c > t mutation in clcn1 . direct sequencing revealed slc2a1 c.363g > t mutation ( a ) in one case and clcn1 c.1205c > t mutation ( b ) in two cases with presumptive paroxysmal kinesigenic dyskinesia .
the upper panel for each chromatogram depicts the wild - type sequence whereas the lower panel represents heterozygous mutated sequence .
the patient ( patient 17 ) with slc2a1 c.363g > a mutation was a 15-year - old girl , who had 8 years history of dystonia triggered by sudden movement . at the age of 7
each episode lasted 30 s to 1 min , but occasionally up to 35 min .
the attacks initially occurred once or twice per week , but increased to 210 times / day when she was 12 years old .
brain computed tomography and magnetic resonance imaging ( mri ) scanning were uninformative . at the onset , she was diagnosed with epilepsy in local hospital and sodium valproate ( 100 mg / d ) was prescribed . however , her symptoms were not improved .
she then sought her medical care in our hospital when she was 14 years old .
a diagnosis of pkd was rendered , and carbamazepine was prescribed at a dose of 100 mg daily . the frequency of attacks was dramatically reduced from 210 times / day to 12 times / week . a 14-year - old boy ( patient 8) with the clcn1 c.1205c > t mutation developed paroxysmal dystonia in lower limbs when he was 12 years old .
the episodes usually occurred at the moment he began to walk , shift position , or ascend the stairs .
the attacks occurred several times a month , and each spell lasted 1020 s. consciousness remained undisturbed for the duration of attacks . during the intermission of attacks , he was totally normal . over the next 1 year , the frequency of attacks increased to 510 times / day .
in addition , he complained that the attacks occurred more frequently and longer in winter or cold weather .
moreover , the attacks seemed to be more frequent in cold weather . during the follow - up period
, we made a cold water test which showed muscle relaxation delayed in this case .
electromyography ( emg ) examination was refused because the patient believed his symptoms were tolerable .
the other patient ( patient 10 ) with the clcn1 c.1205c > t mutation was a 12-year - old boy , who began to experience intermittent rigidity of his left leg at the age of 8 .
he sought his medical care in our hospital 3 years ago and was diagnosed with pkd .
carbamazepine was prescribed 100 mg twice daily , which showed mild alleviation of his symptoms .
after sequencing in the 28 patients with presumptive diagnosis of sporadic pkd , we identified 16 genetic variants , including 4 in mr-1 gene , 8 in slc2a1 gene , and 4 in clcn1 gene [ table 2 ] . among these variants , 7 slc2a1 variants ( c.45c > t , c.399c > t , c.588g > a , c.987g > a , c.1011c > t , c.1065a > g , and c.1372c > a ) and 3 clcn1 variants ( c.261c > t , c.2154c > t , and c.2180c > t ) were proved to be nonpathogenic variants . however , we detected 2 heterozygous missense mutations , including slc2a1 c.363g > a ( p. met121ile ) [ figure 1a ] in one patient and clcn1 c.1205c > t ( p. ala402val ) [ figure 1b ] in two other cases .
the slc2a1 c.363g > a was absent in 1000 genomes database , exac database , and our in - house 200 controls . it was predicted to be deleterious by sift and polyphen2 . the allele frequency of clcn1 c.1205c > t was reported at 0.03% in 1000 genomes database .
sequencing of clcn1 in our 200 controls also did not reveal the presence of clcn1 c.1205c > t .
moreover , it was identified as a causative mutation in a sporadic mc case by fialho et al .
sixteen variants identified in the coding regions and intronexon boundaries of mr-1 , slc2a1 , and clcn1 gene chromatogram of c.363g > t mutation in slc2a1 and c.1205c > t mutation in clcn1 . direct sequencing revealed slc2a1 c.363g > t mutation ( a ) in one case and clcn1 c.1205c > t mutation ( b ) in two cases with presumptive paroxysmal kinesigenic dyskinesia .
the upper panel for each chromatogram depicts the wild - type sequence whereas the lower panel represents heterozygous mutated sequence .
the patient ( patient 17 ) with slc2a1 c.363g > a mutation was a 15-year - old girl , who had 8 years history of dystonia triggered by sudden movement . at the age of 7
each episode lasted 30 s to 1 min , but occasionally up to 35 min .
the attacks initially occurred once or twice per week , but increased to 210 times / day when she was 12 years old .
brain computed tomography and magnetic resonance imaging ( mri ) scanning were uninformative . at the onset , she was diagnosed with epilepsy in local hospital and sodium valproate ( 100 mg / d ) was prescribed . however , her symptoms were not improved .
she then sought her medical care in our hospital when she was 14 years old .
a diagnosis of pkd was rendered , and carbamazepine was prescribed at a dose of 100 mg daily . the frequency of attacks was dramatically reduced from 210 times / day to 12 times / week .
a 14-year - old boy ( patient 8) with the clcn1 c.1205c > t mutation developed paroxysmal dystonia in lower limbs when he was 12 years old .
the episodes usually occurred at the moment he began to walk , shift position , or ascend the stairs .
the attacks occurred several times a month , and each spell lasted 1020 s. consciousness remained undisturbed for the duration of attacks . during the intermission of attacks , he was totally normal . over the next 1 year , the frequency of attacks increased to 510 times / day .
in addition , he complained that the attacks occurred more frequently and longer in winter or cold weather .
moreover , the attacks seemed to be more frequent in cold weather . during the follow - up period
, we made a cold water test which showed muscle relaxation delayed in this case .
electromyography ( emg ) examination was refused because the patient believed his symptoms were tolerable .
the other patient ( patient 10 ) with the clcn1 c.1205c > t mutation was a 12-year - old boy , who began to experience intermittent rigidity of his left leg at the age of 8 .
he sought his medical care in our hospital 3 years ago and was diagnosed with pkd .
carbamazepine was prescribed 100 mg twice daily , which showed mild alleviation of his symptoms .
in the present study , we screened mr-1 , slc2a1 , and clcn1 genes in 28 patients who were diagnosed with sporadic pkd but not carrying prrt2 mutations . among these cases , 26 of them are males and the remaining are females , which is consistent with the male predominance of pkd .
we identified slc2a1 c.363g > a in one case and clcn1 c.1205c > t in two cases .
the slc2a1 c.363g > a mutation was absent in 1000 genomes database and was predicted to be deleterious by sift and polyphen2 .
it has not been reported so far and is therefore identified as a novel mutation .
the clcn1 c.1205c > t was previously reported as a causative mutation in a sporadic mc patient . the absence of 200 controls and the predicted deleteriousness demonstrated that clcn1 c.1205c > t mutation was responsible for this case .
typically , pkd was characterized by a kinesigenic trigger , sudden attacks of involuntary movements , preservation of consciousness , remission with age , and good response to antiepileptic drug .
also , a variety of diseases can mimic the phenotype of pkd , such as ped , tics , seizures , pseudoseizures , and functional movement disorders , increasing the diagnostic challenge in clinical practice . both pkd and ped
the clinical overlap between pkd and ped sometimes make it difficult to distinguish these two diseases . wang et al .
described a family with a phenotype overlapping pkd , pnkd , and ped , and with a prrt2 c.649c > t mutation . in this study , the patient with slc2a1 c.363g > a mutation met the diagnostic criteria proposed by bruno et al
however , an alternative trigger of prolonged exercise and duration of up to 5-min attacks were not typical for pkd and prone to ped .
therefore , a diagnosis of pxds or slc2a1-related disease might be more appropriate for this case .
in addition , we identified clcn1 c.1205c > t mutation in two cases , both of whom exhibited paroxysmal stiffness of limbs .
since both of them had a precipitating factor of sudden movement , childhood onset , and preserved consciousness during attacks , we diagnosed them with pkd .
however , the 14-year - old boy 's symptoms were worsened in cold weather whereas the 12-year - old boy had a mild response to carbamazepine . these were not typical clinical features of pkd but mimicked the manifestations of mc .
regrettably , we did not perform the emg test for these two cases , which is the limitation of our study .
we did not identify any mr-1 exon mutation in our cohort of 28 sporadic pkd cases .
this is consistent with another study , in which mr-1 mutations were not identified in 57 no - prrt2 associated pkd cases .
therefore , these reports demonstrate that there are much overlapping between pkd and other paroxysmal disorders . in summary , we identified slc2a1 and clcn1 mutations in three cases who were diagnosed with pkd .
our results suggested that the phenotypic overlap may lead to the difficulty in distinguishing pkd from pnkd and mc .
also , our report revealed that misdiagnosis is a rational explanation for the low frequency of prrt2 mutation in sporadic pkd .
for these patients who had atypical phenotype of pkd or negative prrt2 mutation , screening of slc2a1 or clcn1 may be helpful .
supplementary information is linked to the online version of the paper on the chinese medical journal website .
this work was supported by grants from the national natural science foundation of china to zhi - ying wu .
this work was supported by grants from the national natural science foundation of china to zhi - ying wu .
 background : paroxysmal kinesigenic dyskinesia ( pkd ) is the most common subtype of paroxysmal dyskinesias and is caused by mutations in prrt2 gene . 
 the majority of familial pkd was identified to harbor prrt2 mutations . however , over two - third of sporadic pkd patients did not carry any prrt2 mutation , suggesting an existence of additional genetic mutations or possible misdiagnosis due to clinical overlap.methods:a cohort of 28 chinese patients clinically diagnosed with sporadic pkd and excluded prrt2 mutations were recruited . 
 clinical features were evaluated , and all subjects were screened for mr-1 , slc2a1 , and clcn1 genes , which are the causative genes of paroxysmal nonkinesigenic dyskinesia ( pnkd ) , paroxysmal exertion - induced dyskinesia , and myotonia congenita ( mc ) , respectively . 
 in addition , 200 genetically matched healthy individuals were recruited as controls.results:a total of 16 genetic variants including 4 in mr-1 gene , 8 in slc2a1 gene , and 4 in clcn1 gene were detected . among them , slc2a1 
 c.363g > a mutation was detected in one case , and clcn1 c.1205c > t mutation was detected in other two cases . 
 neither of them was found in 200 controls as well as 1000 genomes database and exac database . 
 both mutations were predicted to be pathogenic by sift and polyphen2 . 
 the slc2a1 c.363g > a mutation was novel.conclusions:the phenotypic overlap may lead to the difficulty in distinguishing pkd from pnkd and mc . for those prrt2- negative pkd cases , screening of slc2a1 and clcn1 genes are useful in confirming the diagnosis . 
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