focal cervical dystonia cases were recruited from 2 movement disorders clinics in uk : ( 1 ) national hospital for neurology and neurosurgery , london ; and ( 2 ) salford royal foundation trust , manchester . only cases that remained focal involvement were recruited and most of them were followed up in botulinum toxin clinic .
cases were reviewed by movement disorder specialists , and only presumptive primary cases were recruited , with secondary causes like wilson s disease or other neurodegenerative disease ruled out where clinically appropriate .
because of small sample size , subclassification according to family history of movement disorders and age of onset was not attempted
all patients gave informed consent to the study , approved by the respective local ethics committee .
dna was extracted locally from blood and genotyped in the university college london genomics microarray centre , using illumina human 610-quad beadchip ( illumina , san diego , ca , usa ) .
control data of european descent was drawn from the wellcome trust case control consortium ( wtccc ) data set as previously reported .
a total of 2930 samples from 1958 birth cohort and 2737 samples from national blood services were genotyped in illumina 1.2 m duo array by the wellcome trust sanger institute .
quality control measure was performed both before and after merging with the cases as detailed in the supporting materials .
genotyped data from cervical dystonia cases was assembled in genomestudio ( v2011.1 ) per the manufacturer s suggestion ( illumina ) .
postassembly quality control was performed in genomestudio and plink ( version 1.07 ) . in brief , genotyping quality of the sample and single - nucleotide polymorphisms ( snps ) were controlled for in genomestudio ( illumina ) .
further checking for gender mismatch , sample relatedness ( identity - by - descent , excluding pihat > 0.125 ) ( pi - hat  a parameter in plink using estimates of pairwise ibd to find pairs of individuals who are possibly related . ) , hardy - weinberg equilibrium ( excluding p < 13 10 ) , allele frequency ( excluding minor allele frequency [ maf ] < 0.01 ) , nonrandom missingness ( missingness - by - haplotype , excluding p < 1  10 ; missingness - by - genotype , excluding p < 1  10 ) and population substructure ( excluding 6 sd from combined mean of northern and western european ancestry ( ceu ) and toscani in italia ( tsi ) in multidimensional scaling [ mds ] component 1 or 2 ) were performed in plink ( details in supporting materials ) .
the dystonia sample data was merged with the wellcome trust case control consortium ( wtccc ) , and association analysis was performed using logistic regression in the plink package .
three covariates were used in the regression : gender and the first 2 components of plink mds analysis to adjust for gender and genetic variation .
because of our relatively small sample size , we did not perform separate analysis that looked specifically at known loci .
imputation for autosomal chromosome was performed with mach and minimac , in chunks of 10 megabases with 1 megabase overlapping at both ends .
postimputation association was done in mach2dat excluding imputed snps with r squared ( rsqr ) < 0.3 as suggested .
a higher cutoff was chosen as the small sample size in this study contributed to false - positive association in snps with low maf .
reference for imputation was taken from the 1000 genomes project 2010 august release and composed of 283 individuals from the european continental group .
a total of 233 cases were genotyped and 212 cases ( 66 m , 146 f , mean age 60.6 years , sd 10.9 years ) remained after quality control and 494 k snps had maf > 0.01 ( exclusion breakdown in supporting tables 1 and 2 ) .
the cases were clustered around the ceu - tsi controls in the mds plot confirming european descent ( supporting fig .
these were compared with 5173 controls ( 2609 m , 2564 f ) from wtccc .
quantile - quantile ( qq ) plot did not deviate from the expected ( supporting fig .
no single snp had reached a genomewide significant association ( defined as p < 5  10 ) .
the best signal was rs9416795 ( p = 2.00  10 ) , located in an intergenic region on chromosome 10 .
this was followed by rs1338041 on chromosome 13 , intron region of nalcn , coding for sodium leak channel , nonselective ( fig .
1a , supporting table 3 ) . after imputation and quality controls ( rsqr > 0.3 and maf 0.03 )
these had satisfactory genomewide coverage , except a few regions , mainly telomeric and centromeric regions ( manhattan plot , fig .
the regions not well covered with gwas and imputation are listed in supporting table 7 .
there were no snps with genomewide significance ( defined as p < 5  10 ) .
a few clusters of possible associations ( defined as p < 5  10 ) were found and shown in the manhattan plot ( fig .
1b , table 1 ) . with imputation , the best signals found clustered around nalcn with best p value of 9.8  10 in rs61973742 and 5 more snps in the same gene just short of the best p value .
the majority of the associated snps were found in the first intron between the first and second exon .
the remaining were within a few kilobases of the 5 region of exon 1 and 5 untranslated region ( utr ) ( fig .
the second cluster with peak p at 3.1  10 was found in rs67863238 , chromosome 11 base position 48,267,856 ( hg19 ) .
this cluster codes for a number of olfactory receptors ( or4x1 , or4x2 , or4s1 , and or4b1 ) ( fig .
2b , supporting table 5 ) . remaining imputed snps that passed the possible associations were found on chromosome ( chr ) 1 rgl1 , chr 2 intergenic 3 of kiaa1715 , chr 10 intergenic , and chr 11 col4a1 .
the snps and local plots of these regions are shown in supporting table 4 and supporting figures 4 and 5 .
in this cervical dystonia gwas , no loci reached a statistically significant association with dystonia .
the most crucial limitation was the small sample size , which was underpowered to detect loci with a smaller disease effect .
assuming maf of 0.4 , odds ratio 1.3 , additive model , and prevalence of 430 per million in dystonia , it would need more than 1800 cases to achieve an 80% power .
if the odds ratio was as high as with the complement factor h in macular degeneration , the cohort of 212 cases with same assumptions would have a power of almost 100% to detect the snp .
hence , we conclude that a common snp with large effect - size is unlikely to be present in idiopathic cervical dystonia , at least in chr 1 to 22 as shown in manhattan plot ( fig . 1 ) and within the regions that were usually genotyped by microarray ( supporting table 7 for uncovered regions ) .
our group has also recently identified mutations in anoctamin ( ano3-dyt23 ) , a calcium - gated chloride channel gene , leading to autosomal dominant craniocervical dystonia .
nalcn protein is a member of 4  6 transmembrane voltage - independent , nonselective , noninactivating ion channel found in all animals studied and universally expressed in mouse brain and spinal cord .
our uk brain expression consortium data shows nalcn is universal expressed in brain ( supporting fig .
3 ) . in schizophrenia , the associated snp in nalcn was rs2044117 , located at the last intron .
the c - terminal end of the protein is the important site in coupling with unc79 and unc80 proteins . this complex senses calcium level and results in alteration of leaking current and neuronal excitability , comparable to ano3 as a calcium - gated chloride channel .
mice with exon 1 knocked out die from disrupted respiratory neuronal firing in the brainstem , suggesting regions other than the c - terminal are also critical . our snps were clustered around the exon 1 both at 5 and within the first intron .
there are a few synonymous and non - synonymous snps found in the exon 1 ( rs144447052 , rs145910377 , rs74707055 , rs76774740 , rs75606652 , rs77203309 , rs188237867 , rs79047578 , and rs9557636 ) .
these snps are very rare , with maf well below 0.01 and not included in the imputation and analysis
. these may be pathogenic but unidentified rare pathogenic variants tagged to our snps are also possible .
the best snp from imputation ( rs61973742 ) has a low r with the best snp rs1338041 in gwas .
there is a great discrepancy of the maf , 0.062 in rs61973742 and 0.34 in rs1338041 .
a discrepancy in maf can lead to low r ( vanliere and rosenberg and wray ) .
these 2 snps have a |d| 5 0.88 despite its low r ( 0.05 ) in 1000 genomes 378 european population .
it was not found in the initial gwas , but multiple snps with borderline p value were identified from imputation ( supporting table 5 ) . if replicated , this represents the power of finding new associations that are not well tagged by common snps and are identified through haplotypes inferred during imputation .
this group of associations , namely or4x1 , or4x2 , or4s1 , and or4b1 , is interesting ( fig .
they belong to the olfactory receptor , family 4 , a type of g - protein - coupled receptors ( gpcrs ) . olfactory function may seem unrelated to dystonia .
recently fuchs et al . reported the association of gnal mutation with primary torsion dystonia , and the predominant clinical feature in these patients is cervical dystonia .
gnal , coding an olfactory g protein [ g(olf ) ] is found highly expressed in striatum and coupled with the expression of dopamine d1 receptor ( drd1 ) .
the 4 olfactory genes ( or4x1 , or4x2 , or4s1 , and or4b1 ) are universally expressed in brain and orb1 is highly expressed in striatum as well ( allen brain atlas ) .
this may lead to slightly different p values in gwas and imputation for the same snp .
the 3 other hits found in gwas were located at intergenic at chr 6 92 mb , chr 10 28 mb , and chr 19 29 mb .
the local plots of postimputation snps in these regions are shown in supporting figure 4 .
the sparse imputed snps around chr 10 suggested that might be a false - positive result , as were the imputation finding at chr 1 183 mb and chr 2 176 mb ( supporting figure 4 ) .
the extra snps found with imputation at chr 6 92 mb and chr 19 29 mb suggested potential association .
the p value was lower than the best 2 clusters in nalcn and or4x1 and the functional role within an intergenic region is difficult to predict .
given the recent findings from encode , intergenic region may still play a significant function in transcription . in summary , we found a plausible association , though not statistically confirmed , of cervical dystonia with snps in the nalcn region .
replication on another cohort of cervical dystonia cases would be essential to confirm the association .
as dystonia gwas is relatively understudied , we make all these data publicly available to encourage further analyses of the problem .
 dystonia is a common movement disorder . 
 a number of monogenic causes have been identified . 
 however , the majority of dystonia cases are not explained by single gene defects . 
 cervical dystonia is one of the commonest forms without genetic causes identified . 
 this pilot study aimed to identify large effect - size risk loci in cervical dystonia . a genomewide association study ( gwas ) was performed . 
 british resident cervical dystonia patients of european descent were genotyped using the illumina-610-quad . 
 comparison was made with controls of european descent from the wellcome trust case control consortium using logistic regression algorithm from plink . 
 snps not genotyped by the array were imputed with 1000 genomes project data using the mach algorithm and minimac . 
 postimputation analysis was done with the mach2dat algorithm using a logistic regression model . 
 after quality control measures , 212 cases were compared with 5173 controls . 
 no single snp passed the genome - wide significant level of 5  108 in the analysis of genotyped snp in plink . 
 postimputation , there were 5 clusters of snps that had p value < 5  106 , and the best cluster of snps was found near exon 1 of nalcn , ( sodium leak channel ) with p = 9.76  107 . 
 several potential regions were found in the gwas and imputation analysis . 
 the lowest p value was found in nalcn . 
 dysfunction of this ion channel is a plausible cause for dystonia . 
 further replication in another cohort is needed to confirm this finding . 
 we make this data publicly available to encourage further analyses of this disorder . 