the hereditary ataxias are a clinically and genetically heterogeneous group of inherited neurodegenerative disorders with ataxia as the predominant symptom . the hereditary ataxias include the autosomal recessive ataxias , the dominantly inherited spinocerebellar ataxias ( sca ) and episodic ataxias ( ea ) and the x - linked ataxias . although the causative mutations have been identified in a number of ataxia subtypes , the underlying genetic defects for many forms of hereditary ataxia remain unknown . the transient receptor potential ( trp ) channel family member trpc3 is a non - selective cation channel , which is highly enriched in the purkinje cells of the cerebellum [ 2 , 8 ] . trpc3 is a key mediator of metabotropic glutamate receptor ( mglur)-dependent synaptic transmission in the purkinje cells   and has also been implicated in cerebellar development [ 2 , 10 ] . interestingly , trpc3 is associated with several proteins that are linked to human cerebellar ataxia , including mglur1 , the ip3 receptor and protein kinase c  ( pkc ) [ 8 , 14 ] . in vitro studies have shown that trpc3 is a substrate of pkc , and that mutants of pkc-causing sca14 fail to phosphorylate trpc3 , resulting in dysfunctional calcium homeostasis . furthermore , in a mouse model of sca1 , trpc3 is downregulated before the onset of degeneration . notably , genetic mouse models of trpc3 dysfunction exhibit cerebellar ataxia [ 2 , 8 ] . collectively , these studies point to an important role of trpc3 in the pathways leading to cerebellar ataxia in mice and humans and suggest trpc3 as a promising candidate gene for hereditary forms of human cerebellar ataxia . here , we report a mutation screen of the human trpc3 gene in a cohort of 108 patients with suspected but , as yet , undetermined genetic forms of ataxia . ninety - eight samples were from patients with late - onset cerebellar ataxia seen at a large tertiary referral centre . patients had sporadic onset or did not have a clearly defined family history of ataxia . evaluation was performed for acquired and hereditary causes of late - onset cerebellar ataxia according to established clinical protocols , and patients did not have a defined genetic diagnosis at the time of screening . prior genetic analysis was performed individually based on phenotype but , in general , minimal analysis included screening for sca1 , sca2 , sca3 , sca6 , sca7 and fxn repeat expansions . patient consent was obtained and dna was extracted from peripheral blood leukocytes using the gentra puregene blood kit ( qiagen ) . all human subject research protocols were approved by the ucla institutional review board and comply with the current laws of the united states . dna samples from ten individuals with clinical features consistent with episodic ataxia type 2 ( ea2 ) were obtained with informed consent . these patients had been previously sequenced for mutations in the coding and flanking intronic regions of cacna1a and no disease - causing mutations were identified . dna from 96 healthy adults ( european collection of cell cultures ) were screened as controls . patient genomic dna extracted from blood was amplified using standard pcr techniques with primers for all coding regions of trpc3 ( exons 111 of the trpc3 - 001 transcript according to the ensembl genome browser 58 ) and an average of 80 nucleotides of flanking intronic regions . all amplicons were sequenced using big dye 3.1 dideoxy terminator methods ( applied biosystems ) using the amplification primers . to confirm sequence variants , exonic splicing enhancer ( ese ) motifs were predicted using esefinder 3.0 ( cold spring harbor laboratory ) and rescue - ese . ninety - eight samples were from patients with late - onset cerebellar ataxia seen at a large tertiary referral centre . patients had sporadic onset or did not have a clearly defined family history of ataxia . evaluation was performed for acquired and hereditary causes of late - onset cerebellar ataxia according to established clinical protocols , and patients did not have a defined genetic diagnosis at the time of screening . prior genetic analysis was performed individually based on phenotype but , in general , minimal analysis included screening for sca1 , sca2 , sca3 , sca6 , sca7 and fxn repeat expansions . patient consent was obtained and dna was extracted from peripheral blood leukocytes using the gentra puregene blood kit ( qiagen ) . all human subject research protocols were approved by the ucla institutional review board and comply with the current laws of the united states . dna samples from ten individuals with clinical features consistent with episodic ataxia type 2 ( ea2 ) were obtained with informed consent . these patients had been previously sequenced for mutations in the coding and flanking intronic regions of cacna1a and no disease - causing mutations were identified . dna from 96 healthy adults ( european collection of cell cultures ) were screened as controls . patient genomic dna extracted from blood was amplified using standard pcr techniques with primers for all coding regions of trpc3 ( exons 111 of the trpc3 - 001 transcript according to the ensembl genome browser 58 ) and an average of 80 nucleotides of flanking intronic regions . all amplicons were sequenced using big dye 3.1 dideoxy terminator methods ( applied biosystems ) using the amplification primers . to confirm sequence variants , exonic splicing enhancer ( ese ) motifs were predicted using esefinder 3.0 ( cold spring harbor laboratory ) and rescue - ese . to identify possible mutations in trpc3 , we screened all 11 coding exons and flanking exon / intron boundaries of this gene in 98 patients with undiagnosed late - onset cerebellar ataxia . previous work in mice had suggested that trpc3 mutations cause a dominant pure cerebellar phenotype , therefore patients with similar phenotypes were selected from a population of undiagnosed ataxia cases at a large tertiary referral centre . late - onset cases ( onset greater than age 25  years ) were chosen as this is the most common presentation of a dominantly inherited cerebellar ataxia . table  1characteristics of the patient populations studiednumberage at exam ( years)female ( % ) cerebellar only ( % ) sca - like ( % ) spasticity ( % ) episodic ( % ) msa - like ( % ) 9855.5    15.857.128.637.812.28.213.31025    1433.30001000ninety - eight patients presented with late - onset gait and appendicular ataxia . additional phenotypic features include involvement of spinocerebellar pathways and/or peripheral neuropathy ( sca - like ) , increased lower and/or upper extremity tone ( spasticity ) , fluctuating periods of ataxia ( episodic ) or autonomic neuropathy and/or parkinsonism not diagnostic for multiple system atrophy ( msa - like ) . a further panel of ten patients presented with episodic ataxia clinically similar to ea2 characteristics of the patient populations studied ninety - eight patients presented with late - onset gait and appendicular ataxia . additional phenotypic features include involvement of spinocerebellar pathways and/or peripheral neuropathy ( sca - like ) , increased lower and/or upper extremity tone ( spasticity ) , fluctuating periods of ataxia ( episodic ) or autonomic neuropathy and/or parkinsonism not diagnostic for multiple system atrophy ( msa - like ) . a further panel of ten patients presented with episodic ataxia clinically similar to ea2 as trpc3 is an ion channel , it is possible that the human phenotype may demonstrate a paroxysmal course . we included an additional ten samples from patients with a dominant history of episodic ataxia whose clinical features were most consistent with ea2 in terms of attack precipitants and duration , interictal signs and response to acetazolamide . all ten individuals were initially sequenced for mutations in the cacna1a gene and found to be negative . three known single nucleotide polymorphisms ( snps ) were found in the patient cohort at similar frequencies as published for the hapmap - ceu control population ( table  2 ) . in addition , we identified two novel variants ( c.585g > a , c.2271a > g ) , each in a single patient ( table  2 ) . both of these snps result in synonymous changes at the protein level ( p.lys195lys , p.ser757ser ) . interestingly , the c.2271a > g variant in exon 9 , which was found in a patient with a complex phenotype of intermittent ataxia and episodic hemiplegia , is predicted to weaken an ese motif according to bioinformatics calculation . the mutation was also detected in the proband s mother , who had hemiplegic migraine . in an in vitro splicing assay , this putative ese motif did not have an effect on the splicing of an artificial minigene construct ( data not shown ) . however , this does not rule out the potential importance of this ese motif for splicing of the full trpc3 gene in vivo in the patient s nervous system . unfortunately , this patient was lost to follow - up and material was not available for further splicing analysis . 
 table  2identified genetic variants in trpc3refsnpvariationexonaa changegenotype frequencygenotype frequency in populationrs13121031c.78c > g1p.ala26ala0.148 ( g / c)0.183 ( g / c)c.585g > a1p.lys195lys0.009 ( c / t)n / ars11732666c.2199g > a8p.arg733arg0.352 ( c / t)0.5 ( c / t)0.111 ( t / t)0.1 ( t / t)c.2271a > g9p.ser757ser0.009 ( t / c)n / ars61741700c.2451a > g10p.glu817glu0.019 ( c / t)n / anomenclature is based on the national center for biotechnology information ( ncbi ) reference sequences nm_003305.2 ( mrna ) and np_003296.1 ( protein ) . snp reference numbers , genetic variants and population frequency ( hapmap - ceu ) are listed according to the ncbi dbsnp database . newly identified variants are indicated in boldn / a not applicable identified genetic variants in trpc3 nomenclature is based on the national center for biotechnology information ( ncbi ) reference sequences nm_003305.2 ( mrna ) and np_003296.1 ( protein ) . snp reference numbers , genetic variants and population frequency ( hapmap - ceu ) are listed according to the ncbi dbsnp database . genetic mouse models of loss of trpc3 or dysfunctional trpc3 result in an ataxic phenotype [ 2 , 8 ] . furthermore , several studies have linked trpc3 to key signalling pathways that are affected in human cerebellar ataxia [ 1 , 14 ] . trp family member genes have recently been implicated in human neurological diseases [ 4 , 5 ] . we , therefore , hypothesized that mutations in the trpc3 gene might underlie previously undiagnosed late - onset forms of human cerebellar ataxia or episodic ataxia . our findings suggest that mutations in trpc3 are not a common cause of late - onset or episodic cerebellar ataxia . we did identify a novel variant in one patient with episodic ataxia that is predicted to alter splicing of the trpc3 gene . protein interaction motifs in the intracellular c - terminus , including the crib and cam binding domains . as a consequence , this shorter form of trpc3 could act as a dominant negative and impair proper calcium handling in purkinje cells of the cerebellum . despite the absence of trpc3 mutations in our group of patients , trpc3 mutations may still contribute to human disease , as it is well - known that genetic mutations do not always produce equivalent phenotypes in mice and humans . therefore , future genetic studies in larger patient cohorts and/or different ethnic / geographic populations , as well as in patients with different subtypes of cerebellar ataxia , are necessary to fully elucidate the role trpc3 may play in human cerebellar ataxia .
the hereditary cerebellar ataxias are a diverse group of neurodegenerative disorders primarily characterised by loss of balance and coordination due to dysfunction of the cerebellum and its associated pathways . 
 although many genetic mutations causing inherited cerebellar ataxia have been identified , a significant percentage of patients remain whose cause is unknown . 
 the transient receptor potential ( trp ) family member trpc3 is a non - selective cation channel linked to key signalling pathways that are affected in cerebellar ataxia . 
 furthermore , genetic mouse models of trpc3 dysfunction display cerebellar ataxia , making the trpc3 gene an excellent candidate for screening ataxic patients with unknown genetic aetiology . here , we report a genetic screen for trpc3 mutations in a cohort of 98 patients with genetically undefined late - onset cerebellar ataxia and further ten patients with undefined episodic ataxia . 
 we identified a number of variants but no causative mutations in trpc3 . 
 our findings suggest that mutations in trpc3 do not significantly contribute to the cause of late - onset and episodic human cerebellar ataxias .