Javier Simón-Sánchez
email: j.simonsanchez@vumc.nl

CA Jacobus J Van Hilten

Bart C P Van De Warrenburg

Henk W Berendse

Sampath Arepalli

Dena G Hernandez

Rob M A De Bie

Daan Velseboer

Hans Scheffer

Bastiaan Bloem

Bart Post

Karin D Van Dijk

Fernando Rivadeneira

Albert Hofman

André G Uitterlinden

Patrizia Rizzu

Zoltan Bochdanovits

Andrew B Singleton

Peter Heutink

Genome-wide association study confirms extant PD risk loci among the Dutch

Keywords: Parkinsons disease, SNCA, BST1, GAK/DGKQ, HLA, MAPT, GWAS

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INTRODUCTION

A genetic contribution to Parkinson's disease (PD) is well recognized [START_REF] Burn | Parkinson's disease in twins studied with 18F-dopa and positron emission tomography[END_REF][START_REF] Piccini | Dopaminergic function in familial Parkinson's disease: a clinical and 18F-dopa positron emission tomography study[END_REF][START_REF] Piccini | The role of inheritance in sporadic Parkinson's disease: evidence from a longitudinal study of dopaminergic function in twins[END_REF] with ~10% of the cases carrying mutations that lead to rare Mendelian forms of the disease [START_REF] Pirkevi | From genes to proteins in mendelian Parkinson's disease: an overview[END_REF] .

Recently, independent genome-wide association studies (GWAS) established an unequivocal role for common genetic variants in the etiology of PD [START_REF] Pankratz | Genomewide association study for susceptibility genes contributing to familial Parkinson disease[END_REF][START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF][START_REF]Dissection of the genetics of Parkinson's disease identifies an additional association 5' of SNCA and multiple associated haplotypes at 17q21[END_REF][START_REF] Edwards | Genome-wide association study confirms SNPs in SNCA and the MAPT region as common risk factors for Parkinson disease[END_REF][START_REF] Hamza | Common genetic variation in the HLA region is associated with late-onset sporadic Parkinson's disease[END_REF][START_REF] Saad | Genome-wide association study confirms BST1 and suggests a locus on 12q24 as risk loci for Parkinson's disease in the European population[END_REF] and suggested a population-specific genetic heterogeneity, with SNCA, PARK16, BST1 and LRRK2 as shared risk loci for PD [START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF][START_REF]Dissection of the genetics of Parkinson's disease identifies an additional association 5' of SNCA and multiple associated haplotypes at 17q21[END_REF][START_REF] Hamza | Common genetic variation in the HLA region is associated with late-onset sporadic Parkinson's disease[END_REF][START_REF] Saad | Genome-wide association study confirms BST1 and suggests a locus on 12q24 as risk loci for Parkinson's disease in the European population[END_REF] , and MAPT as an European-specific risk locus [START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] . Besides, variation in the GAK/DGKQ [START_REF] Pankratz | Genomewide association study for susceptibility genes contributing to familial Parkinson disease[END_REF] , the HLA region [START_REF] Hamza | Common genetic variation in the HLA region is associated with late-onset sporadic Parkinson's disease[END_REF] and a locus in chromosome 12q24 [START_REF] Saad | Genome-wide association study confirms BST1 and suggests a locus on 12q24 as risk loci for Parkinson's disease in the European population[END_REF] have been proposed to exert a risk for PD in different European populations.

In order to determine the role of these loci in the Dutch population and to find new genetic factors exerting a risk for PD, we performed what is, to our knowledge, the first GWAS for PD in the Dutch population.

MATERIALS AND METHODS

Subjects

As a product of a national collaborative venture, a total of 841 PD patients were recruited from four different centers within the Netherlands (Scales for Outcomes in Parkinson's disease, SCOPA, http://www.scopa-propark.eu; the Academic Medical Center Amsterdam, AMC, http://www.amc.uva.nl); the Parkinson Centrum Nijmegen, ParC, http://www.umcn.nl; and the VU University medical centre, VUmc, http://www.vumc.nl). All patients were self-reported Caucasian individuals from the Netherlands. The assessed samples consisted of 533 males and 308 females with a mean age at onset ranging from 16 to 84 years (mean = 57.5 years; standard deviation = 12). For more information about these samples, please visit the websites listed above.

Genome-wide genotyping data from 2,082 control participants from the Rotterdam study III [START_REF] Hofman | Determinants of disease and disability in the elderly: the Rotterdam Elderly Study[END_REF][START_REF] Hofman | The Rotterdam Study: objectives and design update[END_REF][START_REF] Hofman | The Rotterdam Study: 2010 objectives and design update[END_REF] (ERGO Young) genotyped with Human610K beadchips from Illumina (www.illumina.com) were used as our control population. Of these, 912 were males and 1,116 females. The mean age was 53.75 years with a range of 45-95.

Genotyping

All 841 PD cases were genotyped at 592,839 unique positions with Human660W-Quad beadchips from Illumina, a powerful tool for GWAS. For more information about this genotyping platform, please visit www.illumina.com.

Quality control (QC) procedures

After extensive QC approaches (see supplementary material for details), the final number of fully genotyped samples from the Netherlands was 2,796 including 772 cases and 2,024 controls. Each of these was genotyped in a total of 514,799 unique autosomal SNPs.

Statistical analyses

Quanto software was used to estimate power. Odds ratios (OR) considering a p value of 9.71 x 10 -8 (genome-wide significance level after Bonferroni correction) and different allele frequencies were calculated (supplementary figure 1).

For association analyses, all estimates and tests were performed with PLINK [START_REF] Purcell | PLINK: a tool set for whole-genome association and population-based linkage analyses[END_REF] (http://pngu.mgh.harvard.edu/~purcell/plink/).

For each SNP that successfully passed our QC process a multi-covariate logistic regression analysis following an additive model was applied. Covariates used in our model were gender, age at onset and age at ascertainment (for cases and controls respectively) and the first two components of the MDS values after calculation of pairwise IBS. Although the genomic inflation factor (based on median chi-squared) was low (λ = 1.06, supplementary figure 3) we chose to adjust for genomic control to correct for possible population substructure.

In a first approach aiming to determine (in the Dutch) the role of previously-identified PD genetic risk factors, we decided to look closely at the results for SNCA (chromosome 4q21) [START_REF] Pankratz | Genomewide association study for susceptibility genes contributing to familial Parkinson disease[END_REF][START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] , MAPT locus (chromosome 17q21.1) [START_REF] Pankratz | Genomewide association study for susceptibility genes contributing to familial Parkinson disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] , LRRK2 (chromosome 12q12) [START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] , PARK16 [START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] (chromosome 1q32), BST1 (chromosome 4p15) [START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF] , the GAK/DGKQ locus (chromosome 4p16) [START_REF] Pankratz | Genomewide association study for susceptibility genes contributing to familial Parkinson disease[END_REF] , the HLA region (chromosome 6p21.3) 10 and chromosome 12q24 locus [START_REF] Saad | Genome-wide association study confirms BST1 and suggests a locus on 12q24 as risk loci for Parkinson's disease in the European population[END_REF] . A total of 30 SNPs from these loci were selected for closer scrutiny.

The LD structure of the associated loci was analyzed using Haploview 4.1 [START_REF] Barrett | Haploview: analysis and visualization of LD and haplotype maps[END_REF] and blocks delimited using the D'-based confidence interval method developed by Gabriel et al. [START_REF] Gabriel | The structure of haplotype blocks in the human genome[END_REF] as implemented in Haploview. Risk haplotypes were counted and OR values plotted with R v.2.7.2 (http://cran.r-project.org/).

A logistic regression analysis of the most associated SNP in each block conditioned to variation at the most associated SNP in the other block (if applicable) was performed to test for statistical independence of the signals detected.

For SNCA, the population attributable risk (PAR) was calculated using the formula "PAR = (p[RR-1])/(p[RR-1]+1) × 100", where p is the prevalence of the risk allele in the population and RR is the relative risk. As we showed that association from the 3' and 5' LD block is independent (see results), the combined PAR was calculated with the formula "cPAR = 1 -(1 -PAR 3' block ) × (1 -PAR 5' block )".

For MAPT locus, the presence of alleles in the H1 and H2 haplotypes was accomplished using rs1981997 as a haplotype tag SNP because the major (G) and minor (A) alleles of this SNP are fixed in the H1 and H2 haplotypes respectively [START_REF] Stefansson | A common inversion under selection in Europeans[END_REF][START_REF] Edwards | Genome-wide association study confirms SNPs in SNCA and the MAPT region as common risk factors for Parkinson disease[END_REF] . To determine which of the associated alleles in the MAPT locus were present in H1, (previously associated with PD [START_REF] Golbe | The tau A0 allele in Parkinson's disease[END_REF][START_REF] Maraganore | Case-Control study of the extended tau gene haplotype in Parkinson's disease[END_REF][START_REF] Skipper | Linkage disequilibrium and association of MAPT H1 in Parkinson disease[END_REF][START_REF] Zhang | The tau gene haplotype h1 confers a susceptibility to Parkinson's disease[END_REF][START_REF] Zabetian | Association analysis of MAPT H1 haplotype and subhaplotypes in Parkinson's disease[END_REF][START_REF] Tobin | Haplotypes and gene expression implicate the MAPT region for Parkinson disease: the GenePD Study[END_REF] ) a 2-locus haplotype association analysis of rs1981997 and SNPs in the MAPT region was performed using PLINK [START_REF] Purcell | PLINK: a tool set for whole-genome association and population-based linkage analyses[END_REF] .

Although we are aware that the sample size of this cohort has a limited power and a GWAS would probably fail to find any associated locus after correcting for 514,799 independent tests, we decided to perform this analysis to look for specific PD risk loci in the Dutch population. For this purpose, each genotyped SNP was tested for association using the multi-covariate logistic regression explained above.

RESULTS

After QC, a total of 772 Dutch PD cases and 2,024 controls from the Rotterdam study [START_REF] Hofman | Determinants of disease and disability in the elderly: the Rotterdam Elderly Study[END_REF][START_REF] Hofman | The Rotterdam Study: objectives and design update[END_REF][START_REF] Hofman | The Rotterdam Study: 2010 objectives and design update[END_REF] genotyped in a total of 514,799 unique autosomal SNPs, were included in our analyses (table 1). To assess the homogeneity of our cohort, identity by state (IBS) distances were calculated. This analysis revealed that both cases and controls share common ancestry (supplementary figure 1A), consistent with that seen in CEU (CEPH Europeans from Utah) (supplementary figure 1B,C). Power calculations showed that our sample has adequate power to detect variants conferring a risk with an OR of 1.4-1.5 depending on the minor allele frequency of such variant (supplementary figure 2).

Because we are aware that our population is of limited size to detect risk loci with the effect size previously described for PD in Europeans at the significance level requested by GWAS, the first objective of this project was to determine the role, in the Dutch population, of previously-identified genetic risk factors. Thus, association results from 30 SNPs in SNCA [START_REF] Pankratz | Genomewide association study for susceptibility genes contributing to familial Parkinson disease[END_REF][START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] , MAPT locus [START_REF] Pankratz | Genomewide association study for susceptibility genes contributing to familial Parkinson disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] , LRRK2 6,7 , PARK16 6 , BST1 [START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF] and GAK/DGKQ [START_REF] Pankratz | Genomewide association study for susceptibility genes contributing to familial Parkinson disease[END_REF][START_REF] Hamza | Common genetic variation in the HLA region is associated with late-onset sporadic Parkinson's disease[END_REF] , HLA [START_REF] Hamza | Common genetic variation in the HLA region is associated with late-onset sporadic Parkinson's disease[END_REF] and chromosome 12q24 loci 11 , were selected for close scrutiny. Two SNPs in SNCA and BST1 were significantly associated with PD in our population after correcting for 30 independent tests (p < 0.00166 in both instances, table 2). Although no association was found in any of the other SNPs, a trend towards an association in the DGKQ/GAK and MAPT loci was detected (table 2). Post-hoc analysis of results at these two locations and the HLA region, provided evidence of a role of these three loci in the pathogenesis of PD in the Dutch. No association was found in LRRK2, PARK16 or the chromosome 12q24 locus. A more detailed description of the results obtained at each locus is given below.

SNCA

Although association of SNCA with PD is well established, it remains unclear whether it is derived from the 3' or the 5' end of the gene [START_REF] Mueller | Multiple regions of alpha-synuclein are associated with Parkinson's disease[END_REF][START_REF] Maraganore | Collaborative analysis of alpha-synuclein gene promoter variability and Parkinson disease[END_REF][START_REF] Mizuta | Multiple candidate gene analysis identifies alpha-synuclein as a susceptibility gene for sporadic Parkinson's disease[END_REF][START_REF] Winkler | alpha-Synuclein and Parkinson disease susceptibility[END_REF][START_REF] Kay | Genetic association between alphasynuclein and idiopathic Parkinson's disease[END_REF][START_REF] Myhre | Multiple alpha-synuclein gene polymorphisms are associated with Parkinson's disease in a Norwegian population[END_REF][START_REF] Rajput | Alpha-synuclein polymorphisms are associated with Parkinson's disease in a Saskatchewan population[END_REF][START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF][START_REF] Sotiriou | A single nucleotide polymorphism in the 3'UTR of the SNCA gene encoding alpha-synuclein is a new potential susceptibility locus for Parkinson disease[END_REF][START_REF]Dissection of the genetics of Parkinson's disease identifies an additional association 5' of SNCA and multiple associated haplotypes at 17q21[END_REF] . In an effort to delineate the signal at this locus, we examined the LD structure across this region. This analysis revealed two LD blocks delimited at the intron 4 of SNCA as previously described [START_REF] Mueller | Multiple regions of alpha-synuclein are associated with Parkinson's disease[END_REF][START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF][START_REF]Dissection of the genetics of Parkinson's disease identifies an additional association 5' of SNCA and multiple associated haplotypes at 17q21[END_REF] (supplementary figure 4). Although the strongest signal detected in SNCA was located at the 3' block (rs2736990, p = 1.63 x 10 -5 ), a closer examination of the association signals revealed that SNPs both in the 3' (lowest p = 1.63 x 10 -5 , rs2736990) and the 5' blocks (lowest p = 1.78 x 10 -3 , rs356188) of the gene appeared to be associated with PD in our population (supplementary table 1). A logistic regression analysis using the allele dosage of the most associated SNP in each block as a covariate, did not change the results observed at the most associated signal in the other, indicating that the two signals are independent (supplementary table 2) and being in disagreement with previous results [START_REF] Mueller | Multiple regions of alpha-synuclein are associated with Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF][START_REF] Sotiriou | A single nucleotide polymorphism in the 3'UTR of the SNCA gene encoding alpha-synuclein is a new potential susceptibility locus for Parkinson disease[END_REF] . LD analysis between these two SNPs revealed a r 2 value of 0.028 and a D' value of 0.388, further supporting this hypothesis.

Examination of the risk conferred by each of the haplotypes contained in these blocks confirmed these results and showed that a single haplotype contained in the 3' block and two other in the 5' block exerted the largest risk for PD in our population with ORs of 1.42, 1.41 and 1.48 respectively (supplementary figure 5).

BST1

Rs12502586 showed significant association with PD in our population, with a p value of 1.63 x 10 -3 and an OR of 1.337. A closer look into the association results at this locus revealed that three SNPs presented with p values ~ 10 -3 (range 1.63 x 10 -3 -3.33 x 10 -3 , supplementary table 3). Haplotype analysis of this region showed the existence of two LD blocks containing haplotypes highly associated with PD (p values of 6 x 10 -4 and 4 x 10 -4 respectively). These blocks span from LOC285550 to intron 3 of BST1 and from intron 4 of BST1 to intron 8 of this same gene. The associated haplotypes exert a risk for PD with an OR of 1.24 and 1.36 respectively (supplementary figure 6). A logistic regression analysis of the most associated SNP in each block (rs4583752 and rs12502586 respectively) conditioned to variation at the most associated SNP in the other, yielded a prominent reduction of the detected association, indicating that both risk haplotypes are tagging the same risk variant (supplementary table 4). The linkage disequilibrium between these two had r 2 value of 0.280 and a D' of 0.938, further supporting the previous results.

GAK/DGKQ

Only two out of the three SNPs in the GAK/DGKQ locus reported to be associated with PD by Pankratz and collaborators [START_REF] Pankratz | Genomewide association study for susceptibility genes contributing to familial Parkinson disease[END_REF] were genotyped in our cohort. One of these (rs1564282), which is the most associated in Pankratz's study, was associated with PD in our population (table 1). Although this association did not surpass our conservative Bonferroni correction for 30 independent tests, post-hoc analysis revealed the existence of 11 SNPs with p values < 0.05 spanning 260.5 kb (lowest p = 1.22 x 10 -4 , rs2242235) (supplementary table 5, supplementary figure 7). Because of this, and because Bonferroni correction is unnecessarily punitive when replicating genes that are known to be associated with a certain trait, we consider these results as positive.

The two most associated SNPs in this region (rs2242235 and rs4690296 with p values of 1.22 x 10 -4 and 2.02 x 10 -4 respectively) are located in PCGF3, a gene 78 kb away from GAK and 188 kb away from DGKQ, which encodes a protein with unknown function containing a C3HC4 type RING finger for protein-protein interaction. These two SNPs are contained in two different haplotype blocks spanning from intron 1 to intron 8 of PCGF3 and intron 8 to intron 9 of this same gene respectively. In addition, a predicted transcript, LOC100118084 is contained in the first block. Each of the two blocks contains a single haplotype associated with PD in our population (p = 2 x 10 -4 and 7.38 x 10 -5 respectively) with an OR of 1.47 and 1.52 respectively (supplementary figure 8). The most associated SNPs in each of these haplotype blocks are in strong LD with each other (r 2 = 0.818, D' = 0.916) indicating that they are, most likely, tagging the same risk variant in the region. A logistic regression analysis using the allele dosage at the other marker as a covariate confirmed this hypothesis (supplementary table 6).

HLA region

In the GWAS performed by Hamza and collaborators [START_REF] Hamza | Common genetic variation in the HLA region is associated with late-onset sporadic Parkinson's disease[END_REF] , only one SNP (rs3129882) in this region exceeded genome-wide significance level. After in silico replication and meta-analysis of the most significant SNPs, another six SNPs in this region were associated with PD in their population [START_REF] Hamza | Common genetic variation in the HLA region is associated with late-onset sporadic Parkinson's disease[END_REF] . Of these, only rs3129882 was present in our genotyping platform. Although this SNP was not associated with PD in our population (p = 0.13), post-hoc analysis of this region revealed the existence of 36 SNPs with p values below 0.05 (lowest p = 4.39 x 10 -5 , rs4248166) spanning a total of 440.1 kb (supplementary table 7, supplementary figure 9).

The four most associated SNPs in this region (p values ranging from 4.39 x 10 -5 to 7.98 x 10 -5 ) are located in two consecutive LD blocks. Each of these blocks contains a haplotype associated with PD in our population (p values of 6.90 x 10 -5 and 6.63 x 10 -5 respectively) with ORs of 1.32 and 1.28 respectively (supplementary figure 10). The most associated SNPs in each of these haplotype blocks are in strong LD with each other (r 2 = 0.758, D' = 0.984) indicating that they are tagging the same risk variant. A logistic regression analysis using the allele dosage at the other marker as a covariate confirmed this hypothesis (supplementary table 8).

The next association signal in this locus lies on rs17533090. Although this signal is 223.46 Kb apart from the one explained in the previous paragraph, it is in relatively strong LD with it (r 2 = 0.376, D' = 0.616), indicating that they are, most likely, tagging the same risk variant. A logistic regression analysis using the allele dosage at rs4248166 as a covariate confirmed this hypothesis (supplementary table 9).

MAPT locus

Again, Bonferroni correction is unnecessarily conservative when replicating genes known to be associated with PD. A total of 20 SNPs in this locus were nominally associated with PD in our population (lowest p = 1.9 x 10 -3 at rs3785880) (supplementary table 10). Genotypes at rs1981997 allowed us to differentiate between H1 and H2 haplotypes at this locus as the major (G) and the minor (A) alleles of this SNP are fixed in the H1 and H2 haplotypes respectively [START_REF] Stefansson | A common inversion under selection in Europeans[END_REF][START_REF] Edwards | Genome-wide association study confirms SNPs in SNCA and the MAPT region as common risk factors for Parkinson disease[END_REF] . This analysis showed that the H1 haplotype frequency in the Dutch population is 77.64%. Two-locus haplotype analysis of this SNP and the most associated SNPs in our dataset (p < 0.01) revealed medium to high r 2 values in all SNP pairs (supplementary figure 11). A two-locus association analysis showed that, in all instances, positive effect sizes were found in haplotypes in which the G allele of rs1981997 was present (supplementary table 11).

Genome-wide analysis

As expected, none of the SNPs tested reached genome-wide significance (figure 1).

The most significant signal was found on rs7995973, with a p value of 5.41 x 10 -6 and an OR of 0.72. This SNP is part of an associated locus containing 39 SNPs nominally associated with PD in our population of which 8 had a p value below 10 -3 (supplementary table 1). This locus is on chromosome 13q31 and contains LOC729479 and SPRY2 (supplementary figure 12).

In an attempt to replicate these results, logistic regression models were applied to these approach failed to find any association after correcting for 39 independent tests (supplementary table 12).

DISCUSSION

Here, we present the results of the first GWAS of PD in the Dutch population. Given the population-specific heterogeneity in reported genetic risk factors for PD [START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] , the first objective of this project was to determine the role, in our population, of the genetic risk factors identified in previous GWAS in European and Japanese populations [START_REF] Pankratz | Genomewide association study for susceptibility genes contributing to familial Parkinson disease[END_REF][START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF][START_REF] Edwards | Genome-wide association study confirms SNPs in SNCA and the MAPT region as common risk factors for Parkinson disease[END_REF] . This approach confirmed SNCA and BST1 as PD risk loci. Association of the tested SNPs in GAK/DGKQ, HLA and MAPT loci did not surpass our stringent Bonferroni correction for 30 independent tests. However, given the strong a priori for each of these loci, correcting across all the tests performed is unnecessarily conservative. For this reason and because post-hoc analyses revealed the existence of 11, 36 and 20 SNPs nominally associated with PD at these loci (GAK/DGKQ, HLA and MAPT respectively), we are confident that the signals detected represent a true association with PD.

Although a role of SNCA in the risk for PD is well established, it is still unclear whether this association is derived from the 3' or the 5' end of the gene [START_REF] Mueller | Multiple regions of alpha-synuclein are associated with Parkinson's disease[END_REF][START_REF] Maraganore | Collaborative analysis of alpha-synuclein gene promoter variability and Parkinson disease[END_REF][START_REF] Mizuta | Multiple candidate gene analysis identifies alpha-synuclein as a susceptibility gene for sporadic Parkinson's disease[END_REF][START_REF] Winkler | alpha-Synuclein and Parkinson disease susceptibility[END_REF][START_REF] Kay | Genetic association between alphasynuclein and idiopathic Parkinson's disease[END_REF][START_REF] Myhre | Multiple alpha-synuclein gene polymorphisms are associated with Parkinson's disease in a Norwegian population[END_REF][START_REF] Rajput | Alpha-synuclein polymorphisms are associated with Parkinson's disease in a Saskatchewan population[END_REF][START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF][START_REF] Sotiriou | A single nucleotide polymorphism in the 3'UTR of the SNCA gene encoding alpha-synuclein is a new potential susceptibility locus for Parkinson disease[END_REF][START_REF]Dissection of the genetics of Parkinson's disease identifies an additional association 5' of SNCA and multiple associated haplotypes at 17q21[END_REF] . The data presented here supports the hypothesis that two different signals in each block are present in the Dutch population, which is in controversy with previous results [START_REF] Winkler | alpha-Synuclein and Parkinson disease susceptibility[END_REF][START_REF] Myhre | Multiple alpha-synuclein gene polymorphisms are associated with Parkinson's disease in a Norwegian population[END_REF][START_REF] Rajput | Alpha-synuclein polymorphisms are associated with Parkinson's disease in a Saskatchewan population[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF][START_REF] Sotiriou | A single nucleotide polymorphism in the 3'UTR of the SNCA gene encoding alpha-synuclein is a new potential susceptibility locus for Parkinson disease[END_REF] .

In a report by Mueller and colleagues [START_REF] Mueller | Multiple regions of alpha-synuclein are associated with Parkinson's disease[END_REF] , the authors also detected two association signals in the 3' and 5' blocks of SNCA respectively. Interestingly, when stratifying their cohort into males and females as well young and old PD groups (according to the median age of 57 years), additional significant association in the intron 4 region of the 5' block was found for the female and the young patients subgroups. Stratifying our data based on gender and median age at onset/ascertainment did not replicate such differences between either of the subgroups (data not shown). These results do not support gender and age heterogeneity for PD at this locus in the Dutch population and support that two different variants are exerting a risk for PD in our population.

Collectively, these results indicate that the population attributable risk (PAR) by variation at the SNCA locus in the Dutch is larger than previously described in other European populations [START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] as it should be calculated by adding the effect of variation from each independent LD block. When the most associated SNPs at each block are considered, the combined PAR at the SNCA locus would be 17.56% (6.39% from the 3' and 11.97% from the 5' block).

Several groups have focused their association studies on polymorphisms in the promoter region of SNCA, especially a microsatellite marker ~ 10Kb upstream of the gene (REP1, D4S3481). Although there are some conflicting results regarding the association of this marker with PD [START_REF] Izumi | Genetic studies in Parkinson's disease with an alpha-synuclein/NACP gene polymorphism in Japan[END_REF][START_REF] Khan | Parkinson's disease is not associated with the combined alpha-synuclein/apolipoprotein E susceptibility genotype[END_REF][START_REF] Holzmann | Polymorphisms of the alphasynuclein promoter: expression analyses and association studies in Parkinson's disease[END_REF][START_REF] Spadafora | NACP-REP1 polymorphism is not involved in Parkinson's disease: a case-control study in a population sample from southern Italy[END_REF][START_REF] Tan | Alpha-synuclein haplotypes implicated in risk of Parkinson's disease[END_REF] , most reports consistently describe a positive association between the longer forms of REP1 and PD [START_REF] Farrer | alpha-Synuclein gene haplotypes are associated with Parkinson's disease[END_REF][START_REF] Pals | alpha-Synuclein promoter confers susceptibility to Parkinson's disease[END_REF][START_REF] Maraganore | Collaborative analysis of alpha-synuclein gene promoter variability and Parkinson disease[END_REF][START_REF] Winkler | alpha-Synuclein and Parkinson disease susceptibility[END_REF] . Gene expression differences caused by this repeat have also been described both in vitro [START_REF] Chiba-Falek | Effect of allelic variation at the NACP-Rep1 repeat upstream of the alpha-synuclein gene (SNCA) on transcription in a cell culture luciferase reporter system[END_REF][START_REF] Holzmann | Polymorphisms of the alphasynuclein promoter: expression analyses and association studies in Parkinson's disease[END_REF] and in vivo [START_REF] Cronin | Expansion of the Parkinson diseaseassociated SNCA-Rep1 allele upregulates human alpha-synuclein in transgenic mouse brain[END_REF][START_REF] Linnertz | Genetic regulation of alpha-synuclein mRNA expression in various human brain tissues[END_REF] . We agree that examining this microsatellite in our population would help understanding whether the effect detected on the 5' block is a residual effect of variation in REP1 caused by LD between the associated alleles. However, these experiments could not be performed because only genotypic information was available for the control population used in this study.

Results derived from the analyses performed herein, successfully replicate the association at BST1. Although association at this locus was originally presented as specific for the Japanese population [START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] , it has recently been replicated in British [START_REF]Dissection of the genetics of Parkinson's disease identifies an additional association 5' of SNCA and multiple associated haplotypes at 17q21[END_REF] and French 11 populations. Thus, this is the third study implicating BST1 on PD risk in a European population.

Although association at this locus was not detected in the GWAS performed in a combined cohort of German and American samples [START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] , separating the dataset by country of origin shows that rs12502586 is significantly associated with PD in the Germans but not in North Americans (p values of 4.82 x 10 -3 and 0.179 respectively) (supplementary table 13).

These results together point to the hypothesis that variation in BST1 represents a risk factor for PD in Japanese [START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF] and European populations [START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF][START_REF]Dissection of the genetics of Parkinson's disease identifies an additional association 5' of SNCA and multiple associated haplotypes at 17q21[END_REF][START_REF] Saad | Genome-wide association study confirms BST1 and suggests a locus on 12q24 as risk loci for Parkinson's disease in the European population[END_REF] (British, French, German and Dutch populations) and that this effect has been diluted in the North America [START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] . A recent report by Hamza and collaborators in which a larger cohort of North American samples have been assayed (2,000 North American PD cases versus 971 typed in the previous German/North American study) [START_REF] Hamza | Common genetic variation in the HLA region is associated with late-onset sporadic Parkinson's disease[END_REF] , identified two of the six reported SNPs at this locus to be nominally associated with PD. These results point to the hypothesis that the association of BST1 is, indeed, diluted in North America and that large datasets are needed to detect the effect of variation at this locus. Given the known European population genetic substructure (specially between northern and southern European populations groups) [START_REF] Seldin | European population substructure: clustering of northern and southern populations[END_REF][START_REF] Moskvina | Genetic Differences between Five European Populations[END_REF] and that American population represents an admixture of individuals with northern and southern European ancestry [START_REF] Price | Discerning the ancestry of European Americans in genetic association studies[END_REF][START_REF] Hamza | Common genetic variation in the HLA region is associated with late-onset sporadic Parkinson's disease[END_REF] one would expect allele frequency differences between North American and European populations at the most heterogeneous loci. In case of rs12502586, the allele frequency of the associated allele has been diluted from 11.24% and 10.41% in the Dutch and German controls respectively, to 9.3% in the North American controls (supplementary table 14). Because of the small size effect of the associated allele, this minor allele frequency changes can lead to differences of statistical power to detect association. Thus, it is not surprising that that there is a diminished power to detect association at BST1 locus in the North American population.

The events that trigger neuronal degeneration in PD remain unknown. Some authors argue that PD may result from inflammatory processes within the nervous system [START_REF] Mcgeer | Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains[END_REF][START_REF] Hirsch | Neuroinflammation in Parkinson's disease: a target for neuroprotection?[END_REF][START_REF] Rugbjerg | Autoimmune disease and risk for Parkinson disease: a population-based case-control study[END_REF] . From this perspective, it is interesting that BST1 encodes a molecule that facilitates pre-B-cell growth [START_REF] Kaisho | BST-1, a surface molecule of bone marrow stromal cell lines that facilitates pre-B-cell growth[END_REF] and has an aminoacid sequence that is 33% similar to CD38, a key player in inflammatory processes. Although no autoimmune disease has been found to be a convincing risk factor for PD, a possible pathogenic link has been proposed between rheumatoid arthritis and PD [START_REF] Hrycaj | Severe Parkinson's disease in rheumatoid arthritis patient treated with infliximab[END_REF][START_REF] Melikoglu | Rheumatoid-like hand deformities in Parkinson disease[END_REF][START_REF] Kogure | Rheumatoid arthritis accompanied by Parkinson disease[END_REF] . It is noteworthy that BST1 expression is enhanced in bone marrow stromal cell lines derived from patients with rheumatoid arthritis [START_REF] Kaisho | BST-1, a surface molecule of bone marrow stromal cell lines that facilitates pre-B-cell growth[END_REF] . Interestingly, another region with genes implicated in neuroinflammation (HLA region) has recently been associated with PD [START_REF] Hamza | Common genetic variation in the HLA region is associated with late-onset sporadic Parkinson's disease[END_REF] . In this report, the authors identified rs3129882 to be associated with PD in their population and chose to name this locus PARK18. Although rs3129882 is not associated with PD in our population, post-hoc analysis revealed 36 SNPs nominally associated with PD in the Dutch (lowest p = 4.39

x 10 -5 ), confirming, for the first time, the role of this locus in PD etiology.

Association between certain HLA antigens and PD has largely been suggested since postmortem analysis of the substantia nigra (SN) in PD patients revealed the presence of activated microglia [START_REF] Mcgeer | Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains[END_REF] . Additionally, the frequency of various HLA antigens was increased in patients with PD and in patients dying from post-encephalitic PD [START_REF] Emile | Association of Parkinson's disease with HLA-B17 and B18 antigens][END_REF][START_REF] Elizan | HLA-B14 antigen and postencephalitic Parkinson's disease. Their association in an American-Jewish ethnic group[END_REF][START_REF] Lampe | HLA typing and Parkinson's disease[END_REF] .

Although PD is clearly not an autoimmune disorder, the occurrence of a localized attack of microglia during the disease course has been demonstrated with epidemiological [START_REF] Chen | Nonsteroidal anti-inflammatory drugs and the risk of Parkinson disease[END_REF][START_REF] Esposito | Non-steroidal anti-inflammatory drugs in Parkinson's disease[END_REF] , animal model [START_REF] Mcgeer | Presence of reactive microglia in monkey substantia nigra years after 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine administration[END_REF][START_REF] Barcia | Evidence of active microglia in substantia nigra pars compacta of parkinsonian monkeys 1 year after MPTP exposure[END_REF] and cell culture [START_REF] Gao | Distinct role for microglia in rotenoneinduced degeneration of dopaminergic neurons[END_REF][START_REF] Lee | Thrombin-activated microglia contribute to death of dopaminergic neurons in rat mesencephalic cultures: dual roles of mitogen-activated protein kinase signaling pathways[END_REF][START_REF] Wang | Parkinson disease IgG and C5ainduced synergistic dopaminergic neurotoxicity: role of microglia[END_REF] approaches. Zhang et al.

reported that aggregated α-synuclein activates microglia which, as a consequence, are toxic towards cultured dopaminergic neurons [START_REF] Zhang | Aggregated alpha-synuclein activates microglia: a process leading to disease progression in Parkinson's disease[END_REF] . Thus, is it hypothesized that in response to something related to α-synuclein accumulation, microglial cells are activated to undergo a neuroinflammation process that, ultimately, kills the dopaminergic neurons of the SN.

In a recent GWAS by Pankratz and colleagues, the authors performed a GWAS focusing only on familial PD cases. With this approach they detected evidence of association to several chromosomal regions including a locus containing GAK, TMEM175 and DGKQ; of which GAK is of special interest because it was one of the 137 genes shown to be differentially expressed in PD cases in a gene expression profiling approach of parkinsonian SN [START_REF] Grunblatt | Gene expression profiling of parkinsonian substantia nigra pars compacta; alterations in ubiquitinproteasome, heat shock protein, iron and oxidative stress regulated proteins, cell adhesion/cellular matrix and vesicle trafficking genes[END_REF] . In our cohort, only two out of the three SNPs reported to be associated with PD by Pankratz et al. [START_REF] Pankratz | Genomewide association study for susceptibility genes contributing to familial Parkinson disease[END_REF] , were successfully genotyped, one of them (rs1564282) being associated with PD in our population.

Although this association did not surpass our conservative Bonferroni correction, the a priori for this hypothesis is strong, as it has been replicated in three independent studies [START_REF]Dissection of the genetics of Parkinson's disease identifies an additional association 5' of SNCA and multiple associated haplotypes at 17q21[END_REF][START_REF] Hamza | Common genetic variation in the HLA region is associated with late-onset sporadic Parkinson's disease[END_REF][START_REF] Saad | Genome-wide association study confirms BST1 and suggests a locus on 12q24 as risk loci for Parkinson's disease in the European population[END_REF] . Thus, we would at most correct for the SNPs tested within this locus and both of them would be significantly associated with PD in our population (p < 0.025 in both instances, table 2).

Post-hoc analysis revealed the existence of 11 SNPs nominally associated with PD at this locus. Interestingly, the two most associated ones are located in PCGF3, which is 78 kb away from GAK and 188 kb away from DGKQ and encodes a protein with unknown function containing a C3HC4 type RING finger for protein-protein interaction. Although the function of this transcript is still unknown, it is expressed in brain. Haplotype analyses also point to this gene as the one carrying the risk variants for the development of PD in the Dutch population, pointing to population-specific heterogeneity for PD in this genomic region with different genes exerting risk in different Caucasian populations. Further genotyping of cases and controls from different populations will help understanding the role of this locus in PD.

Although no significant association was detected in the MAPT locus after multiple tests correction, this is probably due to the relatively small size of the sample used for these experiments, as this locus is a well-known PD risk locus amongst Europeans 65, [START_REF] Zabetian | Association analysis of MAPT H1 haplotype and subhaplotypes in Parkinson's disease[END_REF][START_REF] Pankratz | Genomewide association study for susceptibility genes contributing to familial Parkinson disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF]66,[START_REF] Edwards | Genome-wide association study confirms SNPs in SNCA and the MAPT region as common risk factors for Parkinson disease[END_REF] . If the same hypothesis as that presented for GAK/DGKQ is applied, 5 out of the 9 tested SNPs would be borderline significant (in the range of 10 -3 ) supporting a role of this locus in the development of PD among the Dutch.

In our population, associated alleles at this locus occur in the H1 haplotype.

Genotyping of a larger cohort of Dutch PD cases and controls will help confirming these results and defining what H1 subhaplotypes are those exerting the risk for PD in the Dutch.

Last, we failed to find any new PD risk loci in the Dutch population. The most associated locus in our cohort was located in chromosome 13q31 and contained LOC729479 and SPRY2. Because the signal detected was not significant after multiple tests correction and because we failed to replicate it in two other Caucasian GWAS datasets [START_REF] Pankratz | Genomewide association study for susceptibility genes contributing to familial Parkinson disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF] , we refrained from drawing any further conclusions. For the same reason, further conclusions cannot be drawn for other clusters of nominally associated SNPs in regions with interesting biological candidates for PD that did not reach genome-wide significance level. A table showing the top 100 results is displayed in the supplementary material (supplementary table 10).

In summary, we presented the results of the first GWAS in PD in the Dutch population. These data directly confirmed SNCA and BST1 as PD genes in the Dutch population. Besides, post-hoc analysis confirmed a role for DGKQ/GAK, HLA and MAPT loci in modulating the risk for PD in this population.

ACKNOWLEDGEMENTS

We thank the subjects involved in this study for making this work possible.

We also want to thank the Hersenstichting Nederland (http://www.hersenstichting.nl), the Neuroscience Campus Amsterdam and the section of Medical genomics (MGA) and the Intramural Research Program of the National Institute on Aging (National Institutes of Health, Department of Health and Human services; project number Z01 AG000949-04) for supporting in part the work presented here.

Last, we would like to thank the Prinses Beatrix Fonds (http://www.prinsesbeatrixfonds.nl) for sponsoring this work.

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no competing financial interests.

TABLES

Table 1: population characteristics of the Dutch cohort used for these experiments.

Country of origin Number Male/Female ratio

Age at onset [Mean (range)]

Age at sampling [Mean (range)]

Cases

The Thirty SNPs significantly associated with PD in 4 GWAS performed in European [START_REF] Pankratz | Genomewide association study for susceptibility genes contributing to familial Parkinson disease[END_REF][START_REF] Simon-Sanchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF][START_REF] Hamza | Common genetic variation in the HLA region is associated with late-onset sporadic Parkinson's disease[END_REF][START_REF] Saad | Genome-wide association study confirms BST1 and suggests a locus on 12q24 as risk loci for Parkinson's disease in the European population[END_REF] and Japanese 6 populations were selected for closer scrutiny. P values and odds ratios for these SNPs are compared with the results at those same positions in the Dutch cohort. To note, although variation in rs3129882 was not associated with PD in our population, post-hoc analysis revealed 36 SNPs nominally associated with PD in this locus (see HLA region results section).


Dr Sabine Hoffjan
email: sabine.hoffjan@rub.de

Stephan Waldmüller

Wulf Blankenfeldt

Judith Kötting

Petra Gehle

Priska Binner

Joerg T Epplen

Thomas Scheffold

Three novel mutations in the ACTA2 gene in German patients with thoracic aortic aneurysms and dissections

Keywords: ACTA2, Marfan syndrome, TAAD, aneurysm, dissection, aorta, actin

Mutations in the gene encoding smooth muscle cell alpha actin (ACTA2) have recently been shown to cause familial thoracic aortic aneurysms leading to type A dissections (TAAD) and predispose to premature stroke and coronary artery disease. In order to further explore the role of ACTA2 variations in the pathogenesis of TAAD, we sequenced the coding regions of this gene in 40 unrelated German patients with TAAD (with [n=21] or without [n=19] clinical features suggestive of Marfan syndrome). All patients had previously tested negative for mutations in the FBN1 and TGFBR2 genes. We identified three novel ACTA2 mutations and mapped them on a 3-dimensional model of actin.

Two mutations affect residues within (M49V) or adjacent to (R39C) the DNAse-I-binding loop within sub-domain 2 of alpha actin. They were observed in families with recurrent aortic aneurysm (R39C) or aortic dissection (M49V). The third mutation causes an exchange in the vicinity of the ATP-binding site (G304R) in a patient thought to have isolated TAAD. None of the affected individuals had clinical features typical for Marfan syndrome, and no case of premature stroke or coronary artery disease was reported from the affected families.

In conclusion, we underscore the role of ACTA2 mutations in non-syndromic TAAD and suggest that ACTA2 should be included in the genes routinely investigated for syndromic and non-syndromic TAAD. Detailed clinical investigations of additional families are warranted to further explore the full range of phenotypic signs associated with the three novel mutations described here.

Introduction

Thoracic aortic aneurysms leading to type A dissections (TAAD) can result from inherited connective tissue disorders such as Marfan syndrome (MFS), Ehlers-Danlos syndrome or Loeys-Dietz syndrome caused by mutations in the FBN1, COL3A1, TGFBR2 and TGFBR1 genes, respectively. In addition, familial aggregation studies revealed that a significant proportion of non-syndromic TAAD also has a familial background and appears to be inherited in an autosomal dominant manner 1 . Recently, mutations in two genes encoding smooth muscle cell (SMC) contractile proteins were identified in families with TAAD: the gene encoding SMC α-actin (ACTA2) on chromosome 10q22-24 2 and the gene encoding the β-myosin heavy chain (MYH11) on chromosome 16p13 [START_REF] Zhu | Mutations in myosin heavy chain 11 cause a syndrome associating thoracic aortic aneurysm/aortic dissection and patent ductus arteriosus[END_REF] . The encoded proteins are involved in SMC contraction through ATPfuelled cyclic interaction of the myosin motor with actin filaments [START_REF] Dillon | Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle[END_REF] . Similar to the situation found in hypertrophic and dilated cardiomyopathy, mutations of the vascular isoforms of actin and myosin are thought to disturb SMC contractile function, thereby triggering profound remodelling of the vessel wall, followed by an increase in aortic stiffness and, ultimately, aortic dilation and dissection 2, [START_REF] Zhu | Mutations in myosin heavy chain 11 cause a syndrome associating thoracic aortic aneurysm/aortic dissection and patent ductus arteriosus[END_REF][START_REF] Pannu | MYH11 mutations result in a distinct vascular pathology driven by insulin-like growth factor 1 and angiotensin II[END_REF][START_REF] Guo | Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease[END_REF] .

It is worth noting that MYH11 mutations appear to be rare (only five mutations reported so far), and have been exclusively found in patients displaying a syndrome comprising of TAAD and patent ductus arteriosus, a common congenital cardiovascular malformation [START_REF] Hoffman | The incidence of congenital heart disease[END_REF] . In contrast, ACTA2 mutations were found to be responsible for approximately 14% of familial TAAD and are believed to interfere with the normal assembly of actin filaments 2 . The resulting vascular phenotype includes SMC hyperplasia and disarray and is therefore reminiscent of the histopathological picture seen in carriers of mutations in the MYH11 gene [START_REF] Pannu | MYH11 mutations result in a distinct vascular pathology driven by insulin-like growth factor 1 and angiotensin II[END_REF] . It has been claimed that in addition to TAAD, ACTA2 mutations predispose patients to a variety of vascular diseases including premature coronary artery disease (CAD) and premature ischemic strokes [START_REF] Guo | Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease[END_REF] .

In order to further explore the role of ACTA2 variations in the pathogenesis of TAAD, we sequenced the coding regions of this gene in 40 unrelated German patients with TAAD who had previously tested negative for mutations in the FBN1 and TGFBR2 genes.

Material and methods

Subjects

Mutation detection

6

DNA was extracted from peripheral blood according to a standard method [START_REF] Miller | A simple salting out procedure for extracting DNA from human nucleated cells[END_REF] . In all 40 patients, the 9 exons of the ACTA2 gene were amplified by PCR using primers specific to exon-flanking intronic sequences and both strands of the DNA were sequenced using the Beckman platform as previously described 9 .

The exact PCR and sequencing conditions as well as the primer sequences are available upon request. The mutation nomenclature used follows the recommendations of the Human Genome Variation Society (HGVS) and refers to the GenBank reference sequences NM_001613.2 (mRNA) and NP_001604.1 (protein). Exons affected by possibly deleterious mutations were sequenced in 96 healthy control individuals, in order to confirm that these variants are not frequent polymorphisms. Online amino acid substitution prediction programs used included PolyPhen and Panther.

Protein modelling

The protein model of rabbit α-actin (1J6Z; [START_REF] Otterbein | The crystal structure of uncomplexed actin in the ADP state[END_REF] ) was derived from the RCBS Protein Data Bank (PDB; [START_REF] Berman | The Protein Data Bank[END_REF]

Results

Detection of three novel mutations in the ACTA2 gene

We identified novel ACTA2 mutations in 3 of the 40 analysed patients with TAAD. Two mutations were found in families with recurrent aortic aneurysm (family 1) or aortic dissection (family 2). The third mutation was found in a patient thought to have isolated TAAD (family 3; see below for a detailed description of the clinical findings in these families). None of the 21 individuals with clinical features suggestive of MFS was found to carry a possibly diseasecausing mutation in ACTA2. Among the remaining 19 patients, no significant clinical differences were observed between the 3 individuals carrying ACTA2 mutations and the non-mutated patients. The three mutations c.115C>T (p.R39C) in exon 2, c.145A>G (p.M49V) in exon 3 and c.910G>C (p.G304R) in exon 8 of the ACTA2 gene all are heterozygous missense mutations that affect evolutionarily conserved amino acids (Figure 1), one of which, Arg 39, has already been implicated in TAAD (Table 1). It is worth noting that the second mutation, M49V, has previously been described for the skeletal isoform of αactin (encoded by the gene ACTA1) in patients with nemaline or cap-type congenital myopathy [START_REF] Laing | Mutations and polymorphisms of the skeletal muscle alpha-actin gene (ACTA1)[END_REF][START_REF] Hung | Cap myopathy caused by a mutation of the skeletal alpha-actin gene ACTA1[END_REF] . All three mutations were absent from 192 control chromosomes and were not listed in the GenBank dbSNP library. The mutation R39C was predicted to have benign consequences by the PolyPhen program but it was called "deleterious" by the program PANTHER. Both programs predicted a damaging effect of the two other mutations, M49V and G304R.

Taken together, the findings suggest that these mutations are the primary cause of TAAD in the investigated families.

Clinical findings

Family 1 (p.R39C). The mutation was initially identified in a 38-year-old female proband (patient III.5, Figure 2) suffering from mild aortic dilation and insufficiency. She had undergone two uneventful pregnancies and deliveries and appeared physically stable. One of her brothers (patient III.2) had developed an aneurysm of the ascending aorta (that extended to the abdominal aorta) at the age of 25 years and had died at the age of 46 years in the course of a consecutive thoracic operation. No DNA sample was available from this individual to confirm the suspected presence of the ACTA2 mutation. The second brother (patient III.1) was diagnosed with a thoracic aortic aneurysm at the age of 40 years (aortic root diameter 57 mm), and the 44-year-old sister (patient III.4) also showed mild aortic dilation and insufficiency (aortic root diameter 36 mm). In these two siblings, the R39C mutation was confirmed, as well as in her father who was diagnosed with a thoracic aortic aneurysm at the age of 69 years (patient II.1). The grandfather (patient I.1) was reported to have died suddenly at an early age, but no further information was available. The 47year-old sister of the index patient (patient III. Family 3 (p.G304R). The mutation was identified in a male patient without syndromic features who developed a thoracic aortic aneurysm at the age of 41 years (patient II.1, Figure 2). His six sisters were reported to have no known cardiovascular disease and were not interested in genetic counselling or testing.

His parents died early, presumably due to chronic abuse of alcohol. Additional information was not available from this family.

Positions of the mutations in a protein model of α-actin

In order to find clues to the primary molecular defect, the three mutations were mapped onto a 3-dimensional structure of rabbit α-actin. Two of the three novel mutations were found to affect residues within (M49V) or adjacent to (R39C) the DNAse-I-binding loop (D-loop) within subdomain 2 (SD2) of α-actin (Figure 3), which, upon cleavage of ATP, undergoes conformational changes that are believed to be critical for actin monomer interaction [START_REF] Otterbein | The crystal structure of uncomplexed actin in the ADP state[END_REF][START_REF] Holmes | 16 Mannherz HG: Crystallization of actin in complex with actin-binding proteins[END_REF] . Another missense mutation at position 39 has already been described in the literature (R39H, table 1) and has been associated with TAAD 2 as well as with premature stroke and CAD 2 . The third mutation affects residue Gly 304 which is buried in a hydrophobic pocket of the inter-domain cleft known to bind ATP 16 . Collectively, the positional data suggests that all three mutations could interfere with normal formation of actin filaments. Furthermore, the finding that residues Arg 39 and Met 49 map to an exposed loop of SD2 indicates that the mutations R39C and M49V may directly impact on the interaction with myosin or regulatory actinbinding proteins. These two mutations may therefore reduce the motile force generated by SMCs more directly than via a paucity of actin filaments.

Discussion

Genetic epidemiology of TAAD linked to ACTA2

Since the first description of ACTA2 mutations involved in familial TAAD appeared in 2007 2 , a total of 19 mutations in this gene have been reported (see table 1). ACTA2 mutations were found in 14-21% of familial cases, but only 2.5-3.8% of sporadic TAAD 2,17 . In the present analysis, we detected mutations in ACTA2 in 3 out of 40 German TAAD patients (7.5%). This detection rate (likely) reflects the mean detection rate in an unselected TAAD cohort, after exclusion of mutations in the FBN1 and TGFBR2 genes.

We identified 3 missense mutations in ACTA2 residing in highly conserved regions. This goes along with the fact that the mutations described so far were also almost exclusively missense mutations, except for one splice site mutation resulting in skipping of exon 6 2,6,17 . The three mutations presented in this work add to the single mutations previously reported for exons 2, 3 and 8 (Table 1) and therefore underpin the role of the N-and C-terminal portions of actin.

Collectively, the genetic findings in the published cohorts suggest that routine genetic testing in TAAD should address all coding exons of ACTA2.

Possible pathogenetic mechanisms

The data presented in this work strongly suggest that the newly identified mutations may be pathogenetically relevant. Although functional data on the mutant proteins is not yet available, some conclusions on possible molecular consequences can be drawn based on the position of the affected amino acids in a structural model. For example, G304R replaces an unpolar by a positively charged amino acid residue in the hydrophobic pocket that binds to the adenine moiety of ATP. Moreover, the substitution of glycine by arginine introduces a side chain that is believed to sterically hinder actin-ATP interaction. Therefore, G304R, like R185Q described by Guo and colleagues [START_REF] Guo | Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease[END_REF] , most likely perturbs ATP binding and/or hydrolysis. Since the conformation of actin depends on the state of the bound nucleotide [START_REF] Page | Domain motions in actin[END_REF] , such an alteration of ATP-binding likely translates into structural changes that may impair both fibrous-actin (F-actin) stability and actin-myosin interaction. Altered assembly and/or stability of actin filaments are also likely consequences of the two other mutations reported here.

M49V affects the tip of the D-loop. By adopting an extended conformation, the D-loop contributes to the flattening of the molecule which is thought to be essential for the formation of stable helical F-actin 19 . A critical role may also be postulated for the arginine residue affected by the mutation R39C, which colocates with Met 49 on the surface of SD2 (Fig. 3). A perturbation of the F-actin stability is presumed to lead to a paucity of actin filaments, such as that observed in SMCs from carriers of the R118Q and T353N mutations 2 . Diminished steady-state levels of actin could also be due to efficient degradation of mutant globular actin by cellular surveillance systems, such as the ubiquitin proteasome system (UPS), leading to a functional null-allele and presumably, haplo-insufficiency. In this context it is worth noting that Guo and colleagues suggested that ACTA2 mutations usually have a dominant negative effect and that mutations resulting in null-alleles may be inherited recessively 2 .

Clearly, both scenarios are likely to diminish the contractile power of the SMCs, which is thought to trigger compensatory remodelling of the tunica media, resulting in a stiff aorta prone to dilation and dissection. This remodelling process involves dysregulated SMC proliferation similar to the situation observed in TAAD patients carrying MYH11 mutations [START_REF] Pannu | MYH11 mutations result in a distinct vascular pathology driven by insulin-like growth factor 1 and angiotensin II[END_REF] . In smaller vessels (e.g. coronary or cerebral arteries), focal SMC hyperplasia may lead to stenosis which predisposes patients to occlusive conditions [START_REF] Guo | Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease[END_REF] .

Clinical implications

The finding of R39C, in addition to R39H [START_REF] Guo | Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease[END_REF] , further underpins the role of subdomain 2 of the actin protein. In family 1, the mutation was linked to a variable vascular phenotype, ranging from mild aortic dilation and insufficiency in a woman aged 44 years to overt aortic aneurysm requiring surgical repair in a 25-year-old male patient (suspected to carry R39C). While R39H was found to be associated with type A dissections in two families and with type B dissections in one family, dissections were not observed in the R39C family reported here. Collectively, the data available so far indicate that the outcome of a substitution at Arg 39 is highly variable, ranging from no symptoms in the 7 th decade of life to sudden death before the age of 25 years (Table 1).

In addition to increased risk of aortic aneurysms and dissections, ACTA2 mutations have also been implicated in the development of premature CAD and stroke [START_REF] Guo | Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease[END_REF] and have been associated in some families with livedo reticularis, a skin discoloration due to constriction or occlusion of deep dermal capillaries 2 . Yet, in the three patients harbouring ACTA2 mutations in the present study as well as in their family members, we did not find any case of premature CAD, stroke or livedo reticularis. Guo et al. suggested that the different vascular diseases, e.g. TAAD vs. CAD or stroke, may be associated with specific ACTA2 missense mutations: mutations affecting residue 258 were primarily associated with stroke, while p.R118Q and p.R149C lead primarily to CAD [START_REF] Guo | Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease[END_REF] . Interestingly, the R39H mutation has also been implicated in premature stroke (before the age of 20) [START_REF] Guo | Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease[END_REF] . In the present work, a different amino acid exchange in this latter residue (R39C) was observed in several family members affected by aortic aneurysm, but not by stroke or CAD. The number of mutation carriers investigated so far is certainly far too small to conclude that R39C, unlike R39H, exclusively predisposes to TAAD and not to CAD or stroke. The results by Guo

  40 unrelated German patients with TAAD were recruited through the Department of Human Genetics, Ruhr-University Bochum, and the Institute for Heart and Circulation Research, University of Witten/Herdecke. All probands had been enrolled initially with suspected MFS due to thoracic aortic disease at young age (mostly under the age of 50 years). In detail, 21 patients showed additional clinical features suggestive of MFS but did not fulfil the clinical criteria for this hereditary connective tissue disease, while the remaining 19 patients were thought to have TAAD without additional MFS features. Molecular genetic analysis of the FBN1 and TGFBR2 genes had been previously performed and did not reveal a causative mutation in any of the probands. All participants were Germans of European origin. Written informed consent was obtained from all subjects. The study was approved by the Ethics Committees of the Universities of Bochum and Witten/Herdecke, and the Declaration of Helsinki protocols were followed.

  Figure2). She had previously had two uneventful pregnancies and deliveries

  et al. rather suggest that the substitution of Arg 39 leads to a general risk of occlusive arterial disease that is not secondary to aortic dissection or aortic surgical repair. Therefore, all mutation carriers and their offspring should be closely monitored by clinical means and predictive genetic testing may be discussed for asymptomatic family members. Regarding the possible clinical consequences of the M49V mutation, our data suggest a risk of aortic dissection within the first four decades of life. Taking into account that Met 49 locates in the vicinity of Arg 39, one might argue that M49V can predispose to Oda T, Iwasa M, Aihara T, Maeda Y, Narita A: The nature of the globular-to fibrous-actin transition. Nature 2009; 457: 441-445. 20 Akutsu K, Morisaki H, Okajima T et al: Genetic analysis of young adult patients with aortic disease not fulfilling the diagnostic criteria for Marfan syndrome. Circ J 2010; 74: 990-997.

Figure 1 .

 1 Figure 1. Three novel mutations in ACTA2 affecting evolutionarily conserved

Figure 2 .

 2 Figure 2. Family trees showing the segregation and clinical manifestations of

Figure 3 .

 3 Figure 3. Localization of mutated amino acids in a protein model of actin. The mutations were plotted onto a model of rabbit α-actin (PDB code 1J6Z, 10 ). The

Figure 3 ,

 3 Figure 1, Hoffjan et al., 2010

Table1. Location of all reported ACTA2 mutations and vascular phenotypes observed in heterozygous mutation carriers, summarized from the studies by Guo et al.[START_REF] Guo | Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease[END_REF] , Morisaki et al.[START_REF] Morisaki | Mutation of ACTA2 gene as an important cause of familial and nonfamilial nonsyndromatic thoracic aortic aneurysm and/or dissection (TAAD)[END_REF] and the present analysis.

Acknowledgements

We would like to thank Joanne Davies, Janine Haremza, Melanie Müller and Kirsten Rackebrandt for technical assistance and the patients for participating in this study.

affects a protein region that has been linked to a high frequency of CAD [START_REF] Guo | Mutations in smooth muscle alpha-actin (ACTA2) cause coronary artery disease, stroke, and Moyamoya disease, along with thoracic aortic disease[END_REF] .

Additional studies which include a higher number of mutation carriers are warranted to further elucidate the full phenotypic spectrum associated with these mutations.

In conclusion, we confirm the role of ACTA2 mutations in non-syndromic TAAD.

Particularly in young patients with aortic disease that do not fulfil the diagnostic criteria for MFS, ACTA2 analysis is likely to have an important place in routine diagnostics 20 . ACTA2 should therefore definitely be included in the genes investigated by future resequencing chips for syndromic and non-syndromic TAAD.

Conflict of interest.

The authors declare no conflict of interest.


Janneke H M Schuurs-Hoeijmakers

Bert Ba De Vries

Jane Y Hehir-Kwa

Bregje Rolph Pfundt

Wm Van Bon

Nicole De Leeuw

Tjitske Kleefstra

Ad Michel Aap Willemsen

Geurts Van Kessel

Han G Brunner

Jayne Y Hehir-Kwa

Bregje W M Van Bon

Ad Michèl A Willemsen

Joris A Veltman

Hans Van Bokhoven

Arjan P M De Brouwer

Bert B A De Vries
email: b.devries@antrg.umcn.nl

Homozygosity mapping in outbred families with mental retardation

Keywords: intellectual disability, mental retardation, autosomal recessive, homozygosity mapping, outbred

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Mental Retardation (MR), also referred to as Intellectual Disability (ID), is a common neurodevelopmental disorder affecting approximately 1-3 % of the general population [START_REF] Leonard | The epidemiology of mental retardation: challenges and opportunities in the new millennium[END_REF] .

Clinically, DSM-IV defines MR as significant subaverage intellectual functioning -Intelligence Quotients (IQ) below 70-with an onset before the age of 18 years and impairment in adaptive functioning such as self care, social and interpersonal skills and work [Am. Psych.

Ass. 1994. DMS-IV.]. MR can be subdivided into syndromic and non-syndromic forms based upon the presence or absence of additional features, although this distinction is clinically not always obvious. The etiology of MR is heterogeneous and despite recent improvement of cytogenetic and molecular technologies, less than 50% of patients have an etiological diagnosis in clinical practice, hampering medical care and prognosis of the patient and genetic counseling of the families [START_REF] Stevenson | Genetic syndromes among individuals with mental retardation[END_REF][START_REF] Rauch | Diagnostic yield of various genetic approaches in patients with unexplained developmental delay or mental retardation[END_REF] . Genetic causes contribute significantly to MR and among these autosomal recessive inheritance may account for a substantial part of this disorder. Although no recent estimations have been made, autosomal recessive genes have previously been estimated to account for up to 25% of unexplained MR [START_REF] Bartley | Mental retardation and multiple congenital anomalies of unknown etiology: frequency of occurrence in similarly affected sibs of the proband. Birth Defects Orig[END_REF][START_REF] Chelly | Genetics and pathophysiology of mental retardation[END_REF][START_REF] Priest | An Approach to Genetic-Factors in Mental-Retardation -Studies of Families Containing at Least 2 Siblings Admitted to A State Institution for the Retarded[END_REF][START_REF] Wright | Investigation of Families with 2 Or More Mentally Defective Siblings -Clinical Observations[END_REF] . This is more than two times as frequent as the contribution of single X-chromosomal genes to MR, which is estimated to explain MR in 10% of affected males [START_REF] De Brouwer | Mutation frequencies of X-linked mental retardation genes in families from the EuroMRX consortium[END_REF][START_REF] Mandel | Monogenic X-linked mental retardation: Is it as frequent as currently estimated? The paradox of the ARX (Aristaless X) mutations[END_REF][START_REF] Ropers | X-linked mental retardation[END_REF] . For practical reasons, over the last decades the focus in genetic MR research has been on X-linked MR, leading to the identification of 90 disease genes on the Xchromosome of which 38 (42%) lead to non-syndromic MR [START_REF] Gecz | The genetic landscape of intellectual disability arising from chromosome X[END_REF] . In contrast, of the 348 genes contributing to AR-MR phenotypes, only six genes (1.6%) have been identified giving rise to non-syndromic AR-MR (OMIM: http://www.ncbi.nlm.nih.gov/omim). Based on the number of MR genes on the X chromosome -11% of X-chromosome protein coding genes are implicated in X-linked MR-we estimate that there are about 2,000 AR-MR genes (11% of all [START_REF] El-Aziz | EYS, encoding an ortholog of Drosophila spacemaker, is mutated in autosomal recessive retinitis pigmentosa[END_REF][START_REF] Collin | Identification of a 2 Mb human ortholog of Drosophila eyes shut/spacemaker that is mutated in patients with retinitis pigmentosa[END_REF]14 . The percentage of homozygous mutations in these cohorts might be as high as 70% 14 . (Remark 1) Here, we report the first study of homozygosity mapping in outbred MR families.

Patients, Materials & Methods

Families

Ten families with brother-sister pairs with MR were included in this study (in total 22 individuals). Eight families consisted of two affected individuals. In two families, three individuals were analyzed: in family ARMR1 a brother-sister pair with an unaffected brother and in family ARMR10 monozygotic twin brothers and their sister (Remark 6). Patients were clinically evaluated by a clinical geneticist in the human genetics department of the Radboud University Nijmegen Medical Centre in Nijmegen, the Netherlands (supplementary table 1).

The study was approved by the Medical Ethics Committee of the Radboud University Nijmegen Medical Centre.

Homozygosity mapping

Patient DNA was isolated from lymphocytes as described by Miller et al. [START_REF] Miller | A simple salting out procedure for extracting DNA from human nucleated cells[END_REF] . Samples were hybridized on an Affymetrix NspI 250K SNP array. SNP array experiments were performed according to the manufacturer's protocols (Affymetrix, Santa Clara, CA, USA).

Copy number estimates were determined using the CNAG software package (v2.0) to exclude causative copy number aberrations [START_REF] Nannya | A robust algorithm for copy number detection using high-density oligonucleotide single nucleotide polymorphism genotyping arrays[END_REF] . Genotypes were called by Affymetrix Genotype Console Software v2.1. All hybridizations had >85% successful genotype calls.

To systematically identify runs of homozygous called SNPs in the 22 siblings, 19 MR patients from consanguineous parents, 817 MR patients from non-consanguineous parents and 159 healthy controls from an outbred population, PLINK v1.06 was used [START_REF] Purcell | PLINK: A tool set for whole-genome association and population-based linkage analyses[END_REF] . In each window of 50 SNPs, up to five SNPs with a missing call and a maximum of two heterozygous called SNPs were allowed. We determined i) regions of at least 1 Mb that contained a minimum of 50 contiguous, genotyped homozygous SNPs, ii) regions ≥ 1.5 Mb and containing a minimum 75 contiguous SNPs, iii) regions ≥ 2 Mb and containing a minimum of 100 SNPs and iv) regions ≥ 5 Mb and containing a minimum of 250 SNPs. For allelic matching, segments were compared pairwise, and samples were grouped into the same haplotype group if at least 0.95 of jointly non-missing, jointly homozygous SNPs were identical (Remark 2). Regions that were homozygous in both sibs, but having different haplotypes, were excluded.

Results and Discussion

Clinical data

We studied ten families with mild to severe MR (families ARMR1-10, supplementary table 1).

All families had one affected male and one affected female, except for ARMR10. This family had an affected female and two affected males, the latter two being monozygotic twins.

Additional clinical features were observed in eight families, ranging from minor clinical features such as pectus carinatum in ARMR4 to major clinical features such as hypokinetic rigid syndrome with severe autism in AMR10.

Homozygosity mapping

Individual regions of homozygosity (ROHs)

Population studies often use a size cut-off value for the detection of regions of homozygosity (ROHs) of 1 Mb [START_REF] Curtis | Study of regions of extended homozygosity provides a powerful method to explore haplotype structure of human populations[END_REF][START_REF] Gibson | Extended tracts of homozygosity in outbred human populations[END_REF][START_REF] Nalls | Measures of Autozygosity in Decline: Globalization, Urbanization, and Its Implications for Medical Genetics[END_REF][START_REF] Lencz | Runs of homozygosity reveal highly penetrant recessice loci in schizophrenia[END_REF] . Homozygous mutations in outbred families have been identified in regions as small as 2.1 Mb and mutations in consanguineous families are usually found in large linkage intervals encompassing several to tenths of megabases 14,[START_REF] Najmabadi | Homozygosity mapping in consanguineous families reveals extreme heterogeneity of non-syndromic autosomal recessive mental retardation and identifies 8 novel gene loci[END_REF] . Therefore showing on average 31 ROHs in healthy individuals (range 0-115 in 2,429 individuals) [START_REF] Curtis | Study of regions of extended homozygosity provides a powerful method to explore haplotype structure of human populations[END_REF][START_REF] Gibson | Extended tracts of homozygosity in outbred human populations[END_REF][START_REF] Nalls | Measures of Autozygosity in Decline: Globalization, Urbanization, and Its Implications for Medical Genetics[END_REF] .

To test whether the siblings were truly from outbred families, we compared the total amount of genomic homozygosity (all regions > 1 Mb) of the siblings with (i) 19 2). The size of these homozygous segments varied from 950 kb to 1.9 Mb, except for the segment in overlap with MRT11 which was 11.0 Mb in size (figure 2). Families ARMR2, 3 and 6 shared the same haplotype in the MRT8 locus (1.1 Mb overlap: rs16929951; rs16930750, 15 genes) as did ARMR1, 6, 7, 8 and 10 for the MRT10 locus. Since more than 2000 genes might contribute to AR-MR and the mutation frequency of each single gene is presumably below 0.1%, each family in this study will most probably have a unique genetic defect, giving rise to the MR. Therefore, the overlap of several families with part of the MRT7, MRT8 and MRT10 loci, is unlikely to contain the causative genetic defect in all families, but likely to reflect common regions of homozygosity as reported by Lencz et al. [START_REF] Lencz | Runs of homozygosity reveal highly penetrant recessice loci in schizophrenia[END_REF] # ARMR10: data of the female patient and one of the monozygotic twin brothers is shown.

Titles and legends to figures

  we choose to analyze the ten outbred families with four different ROH size cut-off values, namely 1 Mb, 1.5 Mb, 2 Mb and 5 Mb (Remark 4). The number of ROHs over 1 Mb in size varied from 20 in the male patient of ARMR3 to 74 in the male patient of ARMR1, and was on average 57 per individual (table 1). We observed on average 14 ROHs over 1.5 Mb (range 2-22) and 4 ROHs over 2 Mb (range 0-9) in size (Remark 4). ROHs over 5 Mb in size were observed in three families (ARMR1, 7 and 8). Notably the female patient of ARMR7 has seven ROHs considerably larger than 5 Mb (Range: 6.4 to 27.1 Mb) indicating that in this family the parents are likely related, although her affected brother has only one homozygous stretch of 6 Mb. The average of 57 ROHs per individual is in line with previous reports

Figure 1 .

 1 Figure 1. This graph shows the number of shared regions of homozygosity between siblings

Figure 2 .

 2 Figure 2. SROHs showing overlap with MRT7-10 (http://genome.ucsc.edu/, hg18). A)

  MR patients from consanguineous parents, (ii) 817 MR patients from non-consanguineous parents and (iii) 159 healthy controls from an outbred population. With Student's t-test, the siblings having on average 178 Mb (SD: 75 Mb) of the genome homozygous, differed significantly (p=8.34 E-08) from the consanguineous MR patients, who had on average 358 Mb (SD: 98 Mb) of their genome homozygous. There was no significant difference between the siblings and the non-consanguineous MR patients (p=0.441, average homozygosity 191 Mb, SD: 49 Based on Mendelian inheritance, siblings are expected to share 25% of their individual ROHs. Therefore, the 40% overlap of ROHs longer than 1Mb observed between sibs is most likely partly due to false positive ROHs. However, the rate of false positive ROHs drops as the size of the ROHs increases, as we observed for the 1.5 and 2

	Shared regions of homozygosity (SROHs) and known AR-MR loci
	None of the reported SROHs encompassed one of the six reported non-syndromic
	AR-MR genes or encompassed syndromic AR-MR genes that could explain the phenotype in
	Shared regions of homozygosity (SROHs) We observed shared regions of homozygosity (SROHs) in the siblings in all 10 families and categorized these SROHs according to sizes longer than 1Mb, 1.5 Mb, 2 Mb and 5 Mb, respectively (figure 1). On average, siblings shared 21 regions longer than 1 Mb (range: 8-36) which represents almost 40% of the individual ROHs per sib (table 1). Siblings shared on average four regions over 1.5 Mb in size (average: 27% of individual ROHs) and one region over 2 Mb in size (average: 25% of individual ROHs). Two families, ARMR1 and 8, showed one single SROH longer than 5 Mb; 11.0 Mb on chromosome 19q13 and 8.4 Mb on In ARMR1, consisting of four siblings (an affected brother and sister and two healthy brothers), we also genotyped one of the healthy brothers. In this family, 18 ROHs longer the number of candidate regions by 55% (Remark 7). Notably, the longest SROH of 11.0 Mb on chromosome 19q13 was heterozygous in the healthy brother. minimal overlap of 2 Mb. Three SROHs had a unique haplotype (ARMR1, chr16: 78.198.864-80.847.224; ARMR8, chr6: 130.485.817-138.854.223 and chr9: 131.487.840-133.745.038, http://genome.ucsc.edu/, hg18), among which the second longest SROH of 8.4 Mb on chromosome 6 in ARMR8. The 11.0 Mb SROH of family ARMR1 (chr19: 38.737.515-48.316.888) had a 2.3 Mb overlap with one patient (chr19: 387.37.515-41.057.851), the haplotype of the remaining 8.7 Mb of this 11.0 Mb SROH showed no overlap. Another SROH of 2.6 Mb on chromosome 6 in ARMR9 had an overlap with three patients (chr6: 62.030.184-64.647.424). Six SROHs had a more common haplotype occurring in 2.6 to 14.4 % of the 976 studied individuals (supplementary table 3). (Remark 2) these families. Overlap of SROHs with known AR-MR loci was observed for MRT7 in ARMR1 and 2, for MRT8 in ARMR2, 3, 6 and 7, for MRT9 in ARMR7, for MRT10 in ARMR1, 6, 7, 8 and 10, and for MRT11 in ARMR1 (OMIM: #611093, %611094, %611095, %611096, %611097) chromosome 6q23, respectively. Mb regions with 27% and 25% homozygous regions shared between siblings, respectively. than 1 Mb were shared between the patients but not with the healthy sib, thereby reducing (figure 2, supplementary table

Mb) or the healthy controls (p=0.337, average homozygosity 194 Mb, SD: 74 Mb). This shows that the total amount of genomic homozygosity of the siblings, in the current study, is not different from that observed in an outbred population.

For all SROHs over 2 Mb in size (12 in total), we compared haplotypes of the ARMR families with haplotypes of the 817 non-consanguineous MR patients and the 159 healthy controls to see whether these haplotypes were unique or part of a frequently occurring haplotype. We considered a haplotype as shared between individuals when there was a

Table 1 .

 1 Summary of the number of reported homozygous regions in 10 AR-MR families with a size cut-off value of 1 Mb (Remark 4) for homozygous regions. Shown are the individual numbers of homozygous runs (ROH), the shared homozygous runs between siblings of each family (SROH), and the shortest and longest SROH for each family, with two families having a relatively long SROH of over 5Mb in size.

	. In their study, 9% and 15% of 144 healthy individuals were homozygous for
	MRT8 and MRT10, respectively. Of more interest is the overlap of the single families with the
	MRT9 and MRT11 loci. Especially the latter, as this overlap of 2.9 Mb with MRT11 is part of
	an 11.0 Mb homozygous region (388 SNPs: rs1864132; rs16959168) that is shared between
	the affected siblings of family 1 and is heterozygous in the unaffected brother. Besides an
	individual 5.4 Mb ROH in the female patient of this family, this 11.0 Mb ROH is the only
	homozygous stretch exceeding 5 Mb in both siblings. The MRT11 locus (MIM: %611097),
	reported by Najmabadi et al. 22 , was mapped in a consanguineous Iranian family with four

* Homozygosity mapping of an unaffected brother reduced the amount of SROHs in the patients of ARMR1 from 33 to 18 regions.

Acknowledgements: We are grateful to the patients and their families for their support and cooperation. This work has been supported by grants from the Dutch Organisation for Health Research and Development (ZON-MW) (917-86-319 to B.B.A.d.V.), Hersenstichting Nederland (B.B.A.d.V.).

Competing interest: the authors declare no conflict of interest.