Guy Van Camp
email: guy.vancamp@ua.ac.be

Lut Van Laer

Jeroen R Huyghe

Samuli Hannula

Els Van Eyken

Dietrich A Stephan

Elina Mäki-Torkko

Pekka Aikio

Alana Lysholm-Bernacchi

Martti Sorri

Erik Fransen

Matthew J Huentelman

Dr Guy Van Camp

A genome-wide association study for age-related hearing impairment in the Saami

Keywords:

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Age-related hearing impairment (ARHI), or presbycusis, is the most frequent sensory disability among the elderly. [START_REF] Davis | Prevalence of Hearing Impairment[END_REF][START_REF] Gates | Presbycusis[END_REF] The involvement of genetic factors in the development of this multifactorial disorder was demonstrated by several heritability studies. [START_REF] Christensen | Genetic and environmental influences on self-reported reduced hearing in the old and oldest old[END_REF][START_REF] Gates | Genetic associations in age-related hearing thresholds[END_REF][START_REF] Karlsson | Description and primary results from an audiometric study of male twins[END_REF] Only recently the first studies aiming at the identification of these genetic factors have been carried out.

Hitherto, three linkage studies have been performed, [START_REF] Destefano | Genomewide linkage analysis to presbycusis in the Framingham Heart Study[END_REF][START_REF] Garringer | Hearing impairment susceptibility in elderly men and the DFNA18 locus[END_REF][START_REF] Huyghe | Genome-wide SNP-based linkage scan identifies a locus on 8q24 for an age-related hearing impairment trait[END_REF] but only one region on chromosome 8q24.13-q24.22 reached genome-wide significance. [START_REF] Huyghe | Genome-wide SNP-based linkage scan identifies a locus on 8q24 for an age-related hearing impairment trait[END_REF] In addition, several association studies based on a candidate gene approach and two genome-wide association studies (GWAS) [START_REF] Huyghe | Genome-wide SNP-based linkage scan identifies a locus on 8q24 for an age-related hearing impairment trait[END_REF][START_REF] Friedman | GRM7 variants confer susceptibility to age-related hearing impairment[END_REF] have been conducted. Early candidate gene-based association studies are reviewed in Van Eyken et al. [START_REF] Van Eyken | The complexity of age-related hearing impairment: contributing environmental and genetic factors[END_REF] Recently, based on a candidate gene approach, we reported associations between ARHI and variants in GRHL2, [START_REF] Van Laer | The grainyhead like 2 gene (GRHL2), alias TFCP2L3, is associated with age-related hearing impairment[END_REF] encoding a transcription factor that is also involved in monogenic hearing loss. In addition, based on a pooling GWAS, we reported association with variants in GRM7, [START_REF] Friedman | GRM7 variants confer susceptibility to age-related hearing impairment[END_REF] encoding a metabotropic glutamate receptor assumed to modulate hair cell excitability and synaptic efficacy. Acquired mitochondrial mutations have also been proposed as a cause of ARHI in humans. These include both the "common" 4977 bp mitochondrial DNA deletion associated with ageing [START_REF] Bai | Mitochondrial DNA deletions associated with aging and possibly presbycusis: a human archival temporal bone study[END_REF] and an accumulation of many different mitochondrial mutations. [START_REF] Fischel-Ghodsian | Temporal bone analysis of patients with presbycusis reveals high frequency of mitochondrial mutations[END_REF] Despite these first series of studies, the genetic etiology of ARHI remains elusive. In this paper we present the results of a GWAS for ARHI in the Finnish Saami. The Saami, previously known as Lapps, live across Northern Scandinavia and the Kola Peninsula.

Traditionally, Saami have been fishers, hunters and reindeer herders. A minority of the Saami continues to be engaged in these traditional economic activities, while the majority has adopted other occupations and a more westernised lifestyle. Among European populations, the Saami are considered a population genetic "outlier". Despite the fact that both the Saami languages and Finnish belong to the Uralic language group, there is strong genetic differentiation between Saami and Finns. [START_REF] Cavalli-Sforza | Human genomic diversity in Europe: a summary of recent research and prospects for the future[END_REF] Several genetic studies have been conducted to shed light on the genetic origin of the Saami. [START_REF] Lahermo | The genetic relationship between the Finns and the Finnish Saami (Lapps): analysis of nuclear DNA and mtDNA[END_REF][START_REF] Lahermo | Y chromosomal polymorphisms reveal founding lineages in the Finns and the Saami[END_REF][START_REF] Sajantila | Genes and languages in Europe: an analysis of mitochondrial lineages[END_REF][START_REF] Tambets | The western and eastern roots of the Saami--the story of genetic "outliers" told by mitochondrial DNA and Y chromosomes[END_REF] These studies suggested that the Saami are descendants of Europeans that, several thousand years ago, reached northern Fennoscandia from both a western and an eastern route and subsequently got admixed. They also found a small eastern Asian contribution to the gene pool. [START_REF] Tambets | The western and eastern roots of the Saami--the story of genetic "outliers" told by mitochondrial DNA and Y chromosomes[END_REF][START_REF] Ingman | A recent genetic link between Saami and the Volga-Ural region of Russia[END_REF] Several genetic studies further have suggested that the extent of linkage disequilibrium (LD) in the Saami is dramatically higher compared to outbred populations. [START_REF] Johansson | Evaluation of the SNP tagging approach in an independent population sample--array-based SNP discovery in Saami[END_REF][START_REF] Johansson | Linkage disequilibrium between microsatellite markers in the Swedish Saami relative to a worldwide selection of populations[END_REF][START_REF] Kaessmann | Extensive linkage disequilibrium in small human populations in Eurasia[END_REF][START_REF] Laan | Demographic history and linkage disequilibrium in human populations[END_REF] Higher levels of LD are observed in other population isolates too. [START_REF] Bonnen | Evaluating potential for whole-genome studies in Kosrae, an isolated population in Micronesia[END_REF][START_REF] Service | Magnitude and distribution of linkage disequilibrium in population isolates and implications for genome-wide association studies[END_REF] Most of the genetic studies on genetically isolated populations were conducted on young founder populations, in which the high extent of LD can be attributed to a founder effect. In ancient small genetically isolated populations without evidence of demographic expansion, however, high levels of LD are believed to have been generated by genetic drift. [START_REF] Slatkin | Linkage disequilibrium in growing and stable populations[END_REF][START_REF] Terwilliger | Mapping genes through the use of linkage disequilibrium generated by genetic drift: 'drift mapping' in small populations with no demographic expansion[END_REF] The Saami represent such a population. Therefore, the choice of this study population was based on its anticipated statistical power advantage for association mapping, offering a better genomic coverage for a given set of markers, compared to outbred populations.

Due to the expected higher levels of LD in the Saami population and based on what was known about the structure of the humane genome at the time this study was designed, we anticipated that the Affymetrix 100K array pair would entail sufficient genomic coverage for the identification of putative ARHI susceptibility genes.

Materials and methods

Subjects Saami subjects, aged between 50 and 75 years, were enrolled through the public population register from four municipalities in northern Finland (Ivalo, Utsjoki, Vuotso and Enontekiö) in three stages. In a first stage, geographical criteria were applied; only subjects from an area with a high probability of including Saami were selected. In a second stage, putative study subjects were invited based on an evaluation made by an expert in Saami Communities. Finally, the Saami identity of the subject in question was confirmed in a discussion taking place between the subject and the researcher. Except for the age range that was used, ARHI selection criteria were as described previously. [START_REF] Van Eyken | KCNQ4: a gene for age-related hearing impairment?[END_REF] Briefly, all volunteers completed an extended questionnaire detailing medical history and exposure to environmental factors putatively influencing hearing. Subjects suffering from one or more medical pathologies from a standardized list of conditions that could potentially affect hearing ability were excluded. All subjects who passed this initial screening underwent an otoscopic investigation before pure tone audiometry. Air conduction thresholds were measured at 0.125, 0.25, 0.5, 1, 2, 3, 4, 6 and 8 kHz, and bone conduction at 0.5, 1, 2 and 4 kHz for all participating volunteers. Audiological exclusion criteria were an air-bone gap of more than 15 dB averaged over 0.5, 1 and 2 kHz in at least one ear or asymmetrical hearing impairment with a difference in air conduction thresholds exceeding 20 dB in at least 2 frequencies between 0.5, 1 and 2 kHz. Written informed consent was obtained from all volunteers. The study was approved by the Finnish National Advisory Board on Health Care Ethics and by the ethical committees or the appropriate local institutional review boards at all participating institutions.

Description of the phenotype

For each subject, only hearing thresholds from the best ear, defined as the ear with the lowest average threshold over all measured frequencies, were used in the analyses. Supplementary Figure 1 shows that hearing thresholds increase with age and that, for the higher frequencies, thresholds for males are higher and display greater variability (i.e. there is heteroscedasticity) than for females. We therefore adjusted hearing thresholds for age and sex. The effect of age was removed by regressing the log-transformed threshold values (log 10 (20+threshold)) for each of the measured frequencies on age, age squared and age cubed. To resolve the heteroscedasticity problem and to correct for sex, this adjustment for age was carried out in males and females separately, after which the residuals of each regression were scaled and subsequently combined. Note that adding the age and sex covariates in the model for association testing would not deal with this problem satisfactorily.

To deal with the multivariate audiometric phenotype, we applied principal component analysis as a data reduction technique. Performing association tests for each frequency separately would have resulted in a large number of tests, worsening the multiple testing problem. In addition, such analysis would completely have ignored the correlation structure of the audiometric data, i.e. the shape of the audiogram. The latter would also apply when simple pure-tone averages (PTAs), which are more widely used in hearing research, would have been analysed. Our first principal component (PC1) was highly correlated with the PTA over all frequencies, adjusted for sex, age, age square and age cubed (Spearman correlation = 0.96).

Spearman correlations between PC1 and sex and age adjusted PTAs for the high frequencies (2 kHz to 8 kHz) and low frequencies (0.125 kHz to 1 kHz) were 0.88 and 0.61, respectively.

The association testing results for PC1, therefore, would not differ much from those that would be obtained when PTAs would have been analysed. Table 1 lists the eigenvalues and eigenvectors for the first three principal components (PCs), PC1 to PC3. Results of calculations on males and females separately yielded similar values, justifying data-pooling (results not shown). Each of the PCs provides independent bits of information. The first three PCs were retained for the analysis as they captured approximately 80% of the total variation. The coefficients of the eigenvectors (loadings) are directly proportional to the correlations between the transformed hearing thresholds corrected for age and gender, and each of the PCs (see e.g. Johnson and Wichern [START_REF] Johnson | Applied Multivariate Statistical Analysis[END_REF] ). PC1 is clearly a 'size' variable, providing an overall measure of a subject's hearing ability. PC2 and PC3 are 'shape' variables. PC2 contrasts low with high frequencies and is a measure for the slope of the audiogram. PC3 contrasts the middle frequencies with the lower and higher frequencies and can be considered a measure for the concavity of an audiogram (Table 1 andFigure 1). These phenotypes are biologically meaningful [START_REF] Nelson | Presbycusis: a human temporal bone study of individuals with downward sloping audiometric patterns of hearing loss and review of the literature[END_REF][START_REF] Schuknecht | Cochlear pathology in presbycusis[END_REF] and we have previously shown that they have significant heritabilities. [START_REF] Huyghe | Genome-wide SNP-based linkage scan identifies a locus on 8q24 for an age-related hearing impairment trait[END_REF] Genotyping and Quality Control of the data Genomic DNA was extracted using standard protocols and diluted to 50 ng/µl. The quality of the genomic DNA was assessed by spectrophotometric analysis and gel electrophoresis. Each sample was genotyped on the GeneChip ® Human Mapping 100K array pair from Affymetrix (Santa Clara, CA, USA) as described in the GeneChip ® Human Mapping 100K Assay Manual (Affymetrix, Santa Clara, CA, USA). Genotype calling was performed by the BRLMM algorithm of the Affymetrix GeneChip Genotyping Analysis Software (GTYPE) version 4.1. Quality control of the data was performed based on the following criteria: a sample call rate of > 94%, a marker call rate of > 90%, a Hardy-Weinberg equilibrium P-value of > 0.001 and a minor allele frequency of > 5%. In total 352 Saami samples were genotyped with the Affymetrix 100K array. One subject was excluded because of a low sample call rate. The total genotyping rate in the remaining samples was 0.9931. One additional sample was removed due to an unintentional duplication event, and three further subjects were excluded due to missing phenotypic information. After data quality control, 347 subjects and 83,381 SNPs were taken forward for statistical analysis. Genotyping for SNP rs11928865 was performed using a Taqman assay (Applied Biosystems, Foster City, CA, USA) on a Lightcycler480 realtime PCR machine (Roche, Basel, Switzerland).

Association testing Estimates of genome-wide pairwise identity-by-descent sharing, provided by PLINK, [START_REF] Purcell | PLINK: a tool set for whole-genome association and population-based linkage analyses[END_REF] revealed a substantial degree of undocumented relatedness among subjects. This was also reflected in an excess of SNPs that were not in Hardy-Weinberg equilibrium. The results of a linear regression for each of the three quantitative traits would, therefore, not be trustworthy as dependent data lead to underestimation of the standard errors for the regression coefficients, and hence, P-values that are too small. In addition, population stratification may confound the results of the analysis. To account for the dependency in the data as well as population stratification, we analysed the data using a mixed model. [START_REF] Yu | A unified mixed-model method for association mapping that accounts for multiple levels of relatedness[END_REF] With this approach the covariance matrix of the phenotype is modelled as: e are the genetic and the residual variance respectively. This was implemented using the MIXED procedure of SAS 9.1.3 within a SAS macro, which was based on that used by Yu et al. [START_REF] Yu | A unified mixed-model method for association mapping that accounts for multiple levels of relatedness[END_REF] The 2K matrix with the proportion of alleles shared identical-by-descent, was estimated using PLINK. [START_REF] Purcell | PLINK: a tool set for whole-genome association and population-based linkage analyses[END_REF] The model also corrected for origin. As samples originated from 4 municipalities, 3 dummy variables were included as covariates in the model. An additive genetic model was assumed for each SNP. As it was computationally infeasible to run this analysis for the entire set of SNPs, for each trait only the top 4000 SNPs (approximately 5%

of the total number of SNPs passing quality control) resulting from an initial screening based on a linear regression analysis (naive analysis) went to the mixed model analysis. To verify that no true association signals would be lost by using this analytical strategy, we first examined how well the P-values of the naive analysis correlated with those of the mixed model analysis. For this purpose, we analysed 8000 randomly selected SNPs meeting quality control standards, representing approximately 10% of the SNPs that passed quality control, with the mixed model. Comparison of P-values between naive analysis and mixed model results showed that no SNPs with highly significant P-values in the mixed model analysis showed a non-significant P-value in the naive analysis. A reciprocal relationship would be attributed to inflated test statistics. Therefore, for the data at hand, by analysing the top 5%

(4000 SNPs) of a naive linear regression analysis, the chance of missing any SNPs that would not be significant in the naive analysis but highly significant in the mixed model analysis seems very low (Supplementary Figure 2). Quantile-quantile plots in Supplementary Figure 2 show that residual population stratification or cryptic relatedness are of minor concern.

Genomic control inflation factors varied from 1.02 to 1.08. The graph at the right of each panel in Supplementary Figure 2 further shows that P-values obtained from the naive analysis tend to be too low. For example, for PC1, the most extreme -log 10 (P-value) from the naive analysis is 4, while this is 3.5 for the mixed model.

Results

After quality control, 347 subjects and 83,381 SNPs were taken forward for association testing. The quantitative traits tested were the first three principal components (PCs) calculated from the log-transformed hearing threshold values, adjusted for age and sex. To account for the multiple levels of relatedness present in our data and potential population stratification, associations between SNPs and the traits were tested using a mixed model approach, based upon genome-wide kinship estimates. Due to computational constraints, only the top 4000 SNPs (approximately 5% of the total number of SNPs passing quality control)

resulting from an initial screening based on a linear regression analysis went to the mixed model analysis. In the methods section we have outlined that, using this strategy, it is unlikely that strong association signals will be lost. For each of the three PCs, the top 25 SNPs resulting from this analysis are listed in Tables 2 to 4.

If, in agreement with other GWAS, [START_REF]Wellcome Trust Case Control Consortium: Genome-wide association study of 14,000 cases of seven common diseases and 3000 shared controls[END_REF] we used a P-value cutoff of 5 × 10 -7 to declare genomewide significance, the top-ranked SNP for PC3 (rs457717; Table 4) survived this significance threshold (P-value = 3.55 x 10 -7 ). This SNP is located in an intron of the IQ motif-containing GTPase-activating-like protein (IQGAP2). The second ranked SNP (rs1697845) is also positioned in IQGAP2, and is highly correlated with the first (D´= 1; r 2 = 0.965), indicating that this signal is not a genotyping artefact.

For the other two traits, PC1 and PC2, none of the SNPs reached this level of significance (Table 2 and3). Intriguingly, however, the eight-ranked SNP for PC1 (rs161927; Table 2; Pvalue 0.000149) was positioned 40 kb downstream of the metabotropic glutamate receptor 7 gene (GRM7; Supplementary Figure 3). We have previously reported an association of a common variant in the GRM7 gene, rs11928865, with hearing ability based on a poolingbased GWAS in a European sample set. [START_REF] Friedman | GRM7 variants confer susceptibility to age-related hearing impairment[END_REF] Therefore, we also genotyped this SNP in the Saami and analysed this data using the mixed model approach. This resulted in a P-value of 0.045.

However, the A-allele was associated with an elevated hearing threshold in the Saami sample set, while in the European sample set the associated allele was T. [START_REF] Friedman | GRM7 variants confer susceptibility to age-related hearing impairment[END_REF] One of the two top-scoring GRM7 SNPs resulting from a GWAS in a Finnish sample set, 9 rs779701, was present on the 100K Affymetrix array. In the Saami sample set this SNP did, however, not result in a significant association (P-value of 0.735). The LD-pattern within the GRM7 region deduced for the Saami (Supplementary Figure 3D) was similar to that observed in the HapMap for the European population (Supplementary Figure 3C). No obvious biological meaning related to the inner ear could be deduced for the gene regions of the seven higher-ranked SNPs for PC1.

For PC2 no strong biological candidates were present among the top 25 ranking SNPs (Table 3). The presence of six SNPs from the same gene (KH domain-containing RNA-binding signal transduction-associated protein 2; KHDRBS2) within Table 3 is due to the fact that all of these SNPs reside in the same region of high LD.

Discussion

We conducted a genome-wide association scan for ARHI traits derived from audiometric data in the genetically isolated Finnish Saami population. We summarised the information contained in each audiogram by the first three components obtained with a principal component analysis. Previously we have shown that these traits have significant heritabilities. [START_REF] Huyghe | Genome-wide SNP-based linkage scan identifies a locus on 8q24 for an age-related hearing impairment trait[END_REF] By using these audiometric measures we retain biologically important information on the shape of the audiogram that would be lost when simple pure-tone averages or individual frequencies would be analysed. Indeed, premortem-derived audiometric patterns have been correlated to pathophysiological findings in human temporal bones. [START_REF] Nelson | Presbycusis: a human temporal bone study of individuals with downward sloping audiometric patterns of hearing loss and review of the literature[END_REF][START_REF] Schuknecht | Cochlear pathology in presbycusis[END_REF] Nelson et al. [START_REF] Nelson | Presbycusis: a human temporal bone study of individuals with flat audiometric patterns of hearing loss using a new method to quantify stria vascularis volume[END_REF] studied individuals with downward sloping audiograms and reported that the severity of hearing loss, based on audiometric thresholds, was highly associated with a decrease in stria vascularis volume and the degeneration of outer and inner hair cell and ganglion cell populations. The slope of the audiogram was significantly associated with the extent of ganglion cell degeneration.

Our association scan resulted in two SNPs in or near genes that are reasonable biological candidates and that are candidates for further research. The top-ranked SNP, rs457717 (Pvalue 3.55x10 -7 ), that reached genome-wide significance, was associated with PC3 and was localised in an intron of the IQ motif-containing GTPase-activating-like protein (IQGAP2).

IQGAP2 is a member of the Ras superfamily of GTPases. This class of proteins regulates a wide variety of cellular signalling pathways. IQGAP2 is expressed inside the cochlea [START_REF] Robertson | Isolation of novel and known genes from a human fetal cochlear cDNA library using subtractive hybridization and differential screening[END_REF] and has been implicated in cadherin-mediated cell adhesion. [START_REF] Yamashiro | IQGAP2 is required for the cadherin-mediated cellto-cell adhesion in Xenopus laevis embryos[END_REF] As cadherin 23 (CDH23) plays an important role in the sensory hair cells [START_REF] Kazmierczak | Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells[END_REF] and mutations in CDH23 are responsible for syndromic and nonsyndromic autosomal recessive deafness in humans [START_REF] Bolz | Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D[END_REF][START_REF] Bork | Usher syndrome 1D and nonsyndromic autosomal recessive deafness DFNB12 are caused by allelic mutations of the novel cadherin-like gene CDH23[END_REF] and age-related hearing loss in inbred mouse strains, [START_REF] Noben-Trauth | Association of cadherin 23 with polygenic inheritance and genetic modification of sensorineural hearing loss[END_REF] this warrants further research. Intriguingly, SNP rs161927 (P-value 0.000149) that ranked eight for PC1, was positioned immediately downstream of the metabotropic glutamate receptor 7 gene (GRM7). A previous GWAS in a

European and a Finnish sample set that was based on an age and gender-corrected Z-score highly correlated with our PC1, [START_REF] Friedman | GRM7 variants confer susceptibility to age-related hearing impairment[END_REF][START_REF] Van Laer | The grainyhead like 2 gene (GRHL2), alias TFCP2L3, is associated with age-related hearing impairment[END_REF][START_REF] Van Eyken | KCNQ4: a gene for age-related hearing impairment?[END_REF][START_REF] Van Eyken | The N-acetyltransferase 2 polymorphism NAT2*6A is a causative factor for Age Related Hearing Impairment[END_REF] already suggested a role for GRM7 in ARHI. 9 GRM7 encodes a metabotropic glutamate receptor which can be activated by L-glutamate, the primary excitatory neurotransmitter in the cochlear hair cell. GRM7 expression was observed in inner and outer hair cells and in the spiral ganglion nerve cell bodies of the inner ear. [START_REF] Friedman | GRM7 variants confer susceptibility to age-related hearing impairment[END_REF] We have hypothesized that certain GRM7 variants may cause an increased susceptibility to glutamate excitotoxicity, which may consequently lead to the development of ARHI.

However, variants that were significant in our previous study did not replicate in our current study. The LD structure within the GRM7 region indicates that these are independent signals.

These results, therefore, are consistent with allelic heterogeneity which is a plausible scenario given the likely absence of any selective pressure for this disorder. Independent studies are needed to investigate the role of common variants in GRM7 in ARHI.

This study was carried out in the genetically isolated Saami population. A number of studies provide evidence that the extent of LD is much higher in the Saami compared to other European populations. [START_REF] Johansson | Evaluation of the SNP tagging approach in an independent population sample--array-based SNP discovery in Saami[END_REF][START_REF] Johansson | Linkage disequilibrium between microsatellite markers in the Swedish Saami relative to a worldwide selection of populations[END_REF][START_REF] Kaessmann | Extensive linkage disequilibrium in small human populations in Eurasia[END_REF][START_REF] Laan | Demographic history and linkage disequilibrium in human populations[END_REF] This would provide an important power advantage, as higher LD leads to better genomic coverage. It has been proposed that association studies based on a map of modest marker density within a population isolate such as the Saami, would provide a first rough identification of the region associated with a particular complex disease. This first demarcation of the region should then preferably be refined by association studies in an outbred population using a denser map. [START_REF] Kaessmann | Extensive linkage disequilibrium in small human populations in Eurasia[END_REF] Elsewhere, we will publish the results of an elaborate genome-wide evaluation of the relative power for GWAS in the Saami.

A possible limitation of this study is the limited sample size. Recent studies indicate that several thousand subjects need to be recruited in order to obtain sufficient power to detect the effect sizes typically expected for complex phenotypes (with each contributing variant explaining as little as 0.1 to 1.3% of the variation), [START_REF] Purcell | Genetic Power Calculator: design of linkage and association genetic mapping studies of complex traits[END_REF][START_REF] Weedon | Genome-wide association analysis identifies 20 loci that influence adult height[END_REF] although successful GWAS have been conducted using only 96 cases and 50 controls. [START_REF] Klein | Complement factor H polymorphism in age-related macular degeneration[END_REF] Obviously, large sample sizes can never be recruited in small isolated populations such as the Saami. A specific difficulty of working with isolated populations became apparent in the current study. Due to the fact that isolated populations are often derived from a limited number of founders and that inbreeding is sometimes inevitable, an increased relatedness is observed within such populations. As a consequence, classical statistical methods that assume unrelatedness, cannot be applied to analyse an association study performed in a population isolate. In our exploratory phase we investigated several approaches including a naive linear regression analysis and a principal components-based correction (EIGENSTRAT [START_REF] Price | Principal components analysis corrects for stratification in genome-wide association studies[END_REF] ). However, for the latter approach the basic assumptions were violated and the inbreeding and relatedness present in our Saami sample created spurious axes of variation. [START_REF] Patterson | Population structure and eigenanalysis[END_REF] Mixed models offered a solution. In this approach we estimated genetic relationships between study subjects based on genome-wide data and subsequently accounted for them in the analysis. [START_REF] Yu | A unified mixed-model method for association mapping that accounts for multiple levels of relatedness[END_REF] In conclusion, we present the results of a first GWAS performed in the genetically isolated Saami population. We identified IQGAP2 as a candidate ARHI gene. Independent replication is needed to confirm this lead. In addition, we found further evidence implicating GRM7 in ARHI. Eigenvalue for the PC divided by the sum of all eigenvalues which corresponds to the total number of variables in the original dataset (9). b The correlations between the PC and the age and sex corrected transformed hearing thresholds for each frequency can be obtained by multiplying each coefficient by the square root of its corresponding eigenvalue. The number of homozygotes for the minor allele/ the number of heterozygotes/ the number of homozygotes for the major allele. b The Hardy-Weinberg equilibrium P-value should be larger than 0.001 for the SNP genotyping to be valid. c "/" indicates that this SNP was not located within a gene region. d "upstream" or "downstream" was based on the Affymetrix annotation. Chr.: chromosome; MAF: Minor Allele Frequency; P HWE: P-value resulting after Hardy-Weinberg equilibrium testing; P Mixed: P-value resulting after analysis with the mixed model (indicated in bold); Pos. Rel. Gene: Position of the SNP relative to the gene listed in the previous column The number of homozygotes for the minor allele/ the number of heterozygotes/ the number of homozygotes for the major allele. b The Hardy-Weinberg equilibrium P-value should be larger than 0.001 for the SNP genotyping to be valid. c "/" indicates that this SNP was not located within a gene region. d "upstream" or "downstream" was based on the Affymetrix annotation. P-values are indicated in bold. Chr.: chromosome; MAF: Minor Allele Frequency; P HWE: P-value resulting after Hardy-Weinberg equilibrium testing; P Mixed: P-value resulting after analysis with the mixed model (indicated in bold); Pos. Rel. Gene: Position of the SNP relative to the gene listed in the previous column; Prot: protein; Prec: Precursor

2 g and σ 2

 22 the column vector with the phenotypes of the 347 subjects, K is a matrix with kinship coefficients estimated based on the genome-wide data, I is an identity matrix and σ

Table 1

 1 Eigenvectors and eigenvalues for the first three principal components calculated from age and sex corrected hearing thresholds

	Principal Component (PC)	PC1	PC2	PC3
	Eigenvalue	4.421	1.790	0.904
	% of variation explained a	0.491	0.199	0.100
	Coefficients eigenvectors
	(loadings) b
	res125	0.208	0.460	0.527
	res250	0.288	0.470	0.287
	res500	0.333	0.382	-0.186
	res1000	0.348	0.186	-0.481
	res2000	0.373	0.020	-0.408
	res3000	0.393	-0.237	-0.109
	res4000	0.358	-0.324	0.088
	res6000	0.356	-0.328	0.276
	res8000	0.305	-0.345	0.335
	a

Table 2

 2 Top 25 SNPs resulting from the mixed model analysis for Principal Component 1

				Minor
	SNP	Chr.	Position Alleles	allele	MAF	Genotypes a P HWE b P Mixed Gene Region c

Table 3

 3 Top 25 SNPs resulting from the mixed model analysis for Principal Component 2The number of homozygotes for the minor allele/ the number of heterozygotes/ the number of homozygotes for the major allele. b The Hardy-Weinberg equilibrium P-value should be larger than 0.001 for the SNP genotyping to be valid.

			Minor
	SNP	Chr. Position Alleles	allele	MAF	Genotypes a P HWE b P mixed Gene Region c

a c "/" indicates that this SNP was not located within a gene region. d "upstream" or "downstream" was based on the Affymetrix annotation.

Table 4

 4 Top 25 SNPs resulting from the mixed model analysis for Principal Component 3

			Minor
	SNP	Chr. Position Alleles	allele	MAF	Genotypes a P HWE b P Mixed Gene Region c

Acknowledgements

The authors would like to express their most sincere gratitude to all the Saami volunteers who have participated in this study. J.R.H. wishes to thank Gael Pressoir and Jianming Yu from the Ed Buckler Lab for kindly providing help with their SAS macro. This work was funded by the European Community (5th Framework project QLRT-2001-00331), by the University of Antwerp (TOP project), by the Research Foundation -Flanders (FWO grant G.0163.09) and by the State of Arizona. J.R.H. is a fellow of the Research Foundation -Flanders (FWO).

Supplementary information is available at the European Journal of Human Genetics' website.

Titles and Legends to Figures

Legends to Supplementary Figures

Supplementary Figure 1 Boxplots showing the effects of age and sex on hearing thresholds. This figure illustrates that hearing deteriorates with age and that this deterioration is most pronounced for the higher frequencies and in males. Note also that for the higher frequencies, within a certain age class, the hearing thresholds tend to be more variable for males compared to females. This is most apparent for 3 kHz and 4 kHz. Supplementary Figure 2 Evaluation of the mixed model versus the naive analysis. Eight thousand randomly selected SNPs (approximately 10% of the SNPs meeting quality control standards) were analysed with both methods and the results were compared for the three principal components under study (PC1, PC2 and PC3 in respectively A, B and C). The extreme left graph of each panel shows a quantile-quantile plot for the test statistics from the mixed model analysis together with the genomic control inflation factor λ. The blue dashed line goes through the first and third quartile. There is little evidence for residual confounding.

Scatter plots, together with Pearson correlation coefficients, are given in the middle graph of each panel. The extreme right graph of each panel shows that the P-values from the naive analysis tend to be too low relative to the more correct mixed model analysis.

Supplementary Figure 3 Results of the statistical analysis for PC1 in the GRM7 region. A)

The GRM7 genomic region. The positions of the exons are indicated with numbered vertical bars. The positions of the significant SNPs from our previous study [START_REF] Friedman | GRM7 variants confer susceptibility to age-related hearing impairment[END_REF]


Vittoria Petruzzella
email: v.petruzzella@biochem.uniba.it

Luigi Bisceglia

Stefano Zoccolella

Alessandra Torraco

Maria Rosaria Piemontese

Angela Amati

Rosa Dell'aglio

Patrizia De Bonis

Lucia Artuso

Filippo M Santorelli

Massimiliano Copetti

Maria Filippo

Santorelli

Luigi Serlenga

Leopoldo Zelante

Enrico Bertini

A new locus on 3p23-p25 for an autosomal dominant limb-girdle muscular dystrophy, LGMD1H

Keywords: Autosomal Dominant Limb-Girdle Muscular Dystrophy, multiple mtDNA deletions, linkage analysis, chromosome 3

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

Introduction

The limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of hereditary neuromuscular disorders with predominant or selective weakness in proximal limb and axial muscles having an estimated incidence of 1:100.000. The clinical phenotype is characterized by a great variability, ranging from early onset, with a severe and rapidly progressive clinical progression to milder forms, with a later onset and a slower progression. [START_REF] Beckmann | 66th/67th ENMC sponsored international workshop: the limb-girdle muscular dystrophies, 26-28 March 1999, Narden, the Nederlands[END_REF] To date, seven autosomal dominant (AD) and 12 autosomal recessive (AR) forms of LGMD have been described, with AD families representing less than 10% of the whole group of LGMDs. Among the AD forms, LGMD1A caused by mutations in the TTID gene encoding myotilin on chromosome 5q31, is characterized by the presence of dysarthric speech which probably relates to palatal weakness in about 50% of the patients. [START_REF] Hauser | Myotilin is mutated in girdle muscular dystrophy 1A[END_REF] LGMD1B, due to mutations in LMNA encoding lamin A/C on chromosome 1q21, and LGMD1E on 6q23 are also associated with heart conduction system abnormalities, including atrio-ventricular blocks, arrhythmia, and sudden death. [START_REF] Van Der Kooy | Genetic localization of a newly recognized autosomal dominant limb-girdle muscular dystrophy with cardiac involvement (LGMD1B) to chromosome 1q 11-21[END_REF][START_REF] Muchir | Identification of mutation in the gene encoding lamins A/C in autosomal dominant limb-girdle muscular dystrophy with atrioventricular conduction disturbances (LGMD1B)[END_REF][START_REF] Messina | Linkage of familial dilated cardiomyopathy with conduction defect and muscular dystrophy to chromosome 6q23[END_REF] Mutations in CAV3 on chromosome 3p25 (LGMD1D) encoding caveolin, are associated with high CK levels with normal strength and distal myopathy, and usually occur in early childhood. [START_REF] Minetti | Mutations in the caveolin-3 gene cause autsomal dominant limb-girdle muscular dystrophy[END_REF] Both the LGMD1D [START_REF] Speer | Identification of a new autosomal dominant limb-girdle muscular dystrophy locus on chromosome 7q[END_REF] and the

LGMD1F [START_REF] Gamez | Autosomal dominant limb-girdle dystrophy: a large kindred with evidence of anticipation[END_REF][START_REF] Palenzuela | A novel autosomal dominant limb-girdle muscular dystrophy (LGMD1F) maps to 7q32.1-32.2[END_REF] loci map to chromosome 7q and they do not present distinctive clinical features. The LGMD1G form, associated with progressive limitation of fingers and toes flexion, has been positionally cloned on chromosome 4p21 in a Brasilian-Caucasian family. [START_REF] Starling | A new form of autosomal dominant limb-girdle muscular dystrophy (LGMD1G) with progressive fingers and toes flexion limitation maps to chromosome 4p21[END_REF] Fewer than ten AD-LGMD families show allelism with Bethlem myopathy and, less frequently, with facioscapulohumeral dystrophy (FSHD), two neuromuscular disorders which are mapped respectively to chromosomes 2q37, 21q22.3, and 4q35

(www.neuro.wustl.edu/neuromuscular/musdist/lg.html). Pedigrees not linked to any of the listed loci warrant further genetic heterogeneity.

We describe the clinical, pathological, and genetic features of a large Italian family with a novel form of AD-LGMD characterized by slowly progressive clinical features and mapped to chromosome 3p23-p25 upon whole genome scan.

Subjects and methods

Clinical and morphological studies

A total of 36 individuals (17 men and 19 women) spanning four generations in a pedigree from southern Italy were investigated. Figure 1 Having obtained the patients' informed consent, we purified genomic DNA from blood of 19 members of the family and from 6 spouses and from skeletal muscles of II-5 and II-9, using a standard phenol-chlorophorm procedure. Approximately 6 µg of total DNA from muscle samples were cleaved with the endonuclease PvuII to linearize mtDNA molecules, electrophoresed through a 0.8% agarose gel, and blotted onto a nylon membrane (N+ Hybond, Amersham Biosciences, UK). The entire mtDNA was PCR-amplified using four overlapping primer pairs (primers and PCR conditions are available upon request), labelled with alpha-32 P-dATP by random priming (Random Primed DNA Labelling Kit, Roche Diagnostic Corp., Germany), and used as a probe.

Signal intensities were analyzed with a Molecular Imager FX (BioRad, Hercules, CA).

Linkage analysis

A genome wide scan was carried out by using the ABI PRISM Linkage Mapping Set LMS-MD10 (Applied Biosystems, Foster City, CA) which includes 382 highly polymorphic microsatellite markers from the 22 autosomes with an average intermarker distance of 10 cM. Additional, more densely-spaced markers were used to refine chromosomal regions on 3p, 4q, 7q, and 9p according to available genetic maps.

Genotyping and fragments analysis was performed as previously described. [START_REF] Bisceglia | Linkage analysis in keratoconus: replication of locus 5q21.2 and identification of other suggestive loci[END_REF] Statistical analyses were carried out assuming an autosomal dominant mode of inheritance with a disease allele frequency of 0.001 and assuming equal frequencies and recombination rates between men and women for all markers analyzed. Physical and genetic distances between them were based upon publically available electronic databases from the University of California Santa Cruz (www.genome.ucsc.edu) and the Marshfield genetic map (www.marshfieldclinic.org). Two age-dependent liability classes were considered for at-risk individuals: 0.80 in < 35 years and 0.95 in >35 years subjects. In addition, because of the presence of two different groups of patientsthe first made of definitively affected subjects and the second consisting of individuals with younger age at onset or milder clinical presentation, or bothwe also conducted data analysis under a more stringent scenario where patients belonging to the second group were considered as having an -unknown phenotype‖. Data were simulated using SLINK [START_REF] Ott | Computer-simulation methods in human linkage analysis[END_REF][START_REF] Weeks | SLINK: a general simulation program for linkage analysis[END_REF] in order to determine the power to detect linkage. Parametric two-point linkage analysis was performed using MLINK from the Linkage Analysis Package II version

Sequencing of candidate genes

The entire coding sequence of CAV3 was amplified and directly sequenced in subjects II-1 and II-5 using previously published primer pairs and PCR conditions. [START_REF] Minetti | Mutations in the caveolin-3 gene cause autsomal dominant limb-girdle muscular dystrophy[END_REF] The CMYA1, CAPN7 and MGC15763 genes were screened for mutations in the same individuals. PCR primers were designed with the Primer 3 software [START_REF] Rozen | Primer3 on the WWW for general users and for biologist programmers[END_REF] (sequences are available upon request). All PCR fragments were agarose gel-purified and directly sequenced on both strands using the Big Dye Terminator chemistry on an ABI 3100 DNA sequencer (Applied Biosystem, Foster City, CA, USA).

Results

Table 1 summarizes clinical, morphological, and laboratory data in the examined family members. ID numbers refer to the pedigree represented in Figure 1. A subset of severely affected members developed a slowly progressive proximal muscle weakness in both upper and lower limbs; among these, five subjects (II-1, II-3, II-5, II-9, II-13)

were considered as affected fulfilling the criteria for a diagnosis of LGMD. [START_REF] Bushby | Limb girdle muscular dystrophy[END_REF] We also identified a mildly affected group of patients (II:7, III:3, III:4, III:8, IV:1, IV:2) who had calf hypertrophy, and occasional raise of CK and lactate levels. These patients were considered as possibly affected, though to a lesser extent. Histochemistry performed on muscle biopsies in patients II-1, II-5, II-9 and II-13

showed abnormal fiber size and shape variation, increased endo-and perimysial connective tissue on hematoxylin-eosin staining. Central nuclei were occasionally present. Ragged red fibers (RRFs) were observed in patient II-1 and subsarcolemmal accumulation of mitochondria upon succinate dehydrogenase (SDH) staining as well as fibers negative at cytochrome c oxidase staining were evidenced in patients II-5 and II-9. Immunohistochemical stains for dystrophin, sarcoglycan, caveolin and δ-sarcoglican were normal. In addition to the wild-type mtDNA, Southern blotting in skeletal muscle biopsies from patients II-5 and II-9 showed multiple bands of lower size corresponding to deleted mtDNA molecules (Figure 2).

Following the initial 10 cM genome-wide screening, multipoint analysis showed four chromosomal regions (3p23, 4q13.2, 7qter, and 9p13.3) with a maximum location score >2. Using more densely-spaced markers within each identified region we increased the resolution map to 1.5 cM, and conclusively excluded linkage to the 4q and 7q regions. A maximum location score of 2.36 was obtained for chromosome 9p but the highest value of 3.236 was reached at marker D3S1286, located on chromosome 3p23-p25. A value>3.0 was observed, in the ~16 cM interval between markers D3S3610 and D3S1266 (Figure 3). Two point analysis showed a MLS=3.23 (θ=0.0) at markers D3S3613 and D3S1286 (Table 2). Non parametric multipoint analysis supported this conclusion at both markers, yielding a STAT D score of 2.26 (p=0.0004) and a STAT E score of 15.4358 (p=0.0004), where -statistic D‖ is the extent of allele sharing among all affected pairs as measured by their IBD kinship coefficient [START_REF] Sobel | Descent graphs in pedigree analysis: applications to haplotyping, location scores, and marker sharing statistics[END_REF] and -statistic E‖ is the NPL_all statistic as implemented in GeneHunter 17 (Table 2). When we adopted a more conservative analysis, which considers as -unknown‖ cases belonging to the group of mildly affected patients, the highest value of LOD score (MLS>1.50) was reached in the interval between markers D3S3610-D3S3726. Taken together, our results made chromosome 3p23-25p the most likely region where the new LGMD1H locus is potentially located.

Haplotype reconstruction confirmed the results obtained from linkage analysis data (Figure 1). The telomeric boundary of the candidate interval was defined by an obligate recombination event observed between D3S1263 and D3S1259 in subject III-4, whereas the centromeric flanking marker is defined by another recombination event between markers D3S3521 and D3S3685 in subject III-3. All the affected individuals shared the same disease haplotype. Two healthy individuals (III-2 and III-6) shared part of the same haplotype between markers D3S1619 and D3S1277, thus further refining the

LGMD1H locus to a 25 cM region.

Two presently unaffected individuals (III-1 and III-11) shared the disease haplotypes. Subject III-1 is a 30-year-old man with normal serum CK levels. Subject III-11 is a 14-year-old girl with a history of recurrent migraine who has never had muscle complains nor high CK. However, she is younger than the median age at onset of her affected relatives. Neurological examinations in both individuals was normal.

The region refined by linkage analysis contains more than 150 genes. Four possible candidates were prioritised for mutational screening because of their function or position: CAV3, CAPN7, MGC15763 and CMYA1. Although located well outside the interval refined in linkage with this family, the caveolin-3 gene (CAV3) was analyzed to firmly exclude allelism with LGMD1C. CAPN7 encodes an 814-residues protein which contains a calpain-like cysteine protease domain and a C-terminal PBH domain.

Expressed mostly in the pancreas and only moderately in skeletal muscle and liver, [START_REF] Futai | Molecular cloning of PalBH, a mammalian homologue of the Aspergillus atypical calpain PalB[END_REF] CAPN7 shares sequence homologies with CAPN3, the LGMD2A causative gene. The MGC15763 gene encodes for a 312 amino acid long protein which is predicted to be exported to mitochondria (MitoProt II 1.0α4 probability is 0.8599; predicted cleavage site at amino acid 44). By primary structure analysis, the gene product has a NADH binding domain but no strong homology with any known protein. CMYA1 encodes the human orthologue of mXinα   a protein believed to play a regulatory role in cardiac morphogenesis, differentiation, and localized to the adherence junctions of intercalated discs. [START_REF] Lin | Structure, Expression, and Function of a Novel Intercalated Disc Protein, Xin[END_REF] The entire coding regions of CAV3, CAPN7, MGC15763 and CMYA1 and their flanking intronic sequences were analyzed but no pathogenic mutations were found.

Discussion

We identified a large pedigree from southern Italy segregating a novel AD form of muscular dystrophy that fulfilled the diagnostic criteria for LGMDs. LGMDs, we propose to define this form of muscular dystrophy as LGMD1H.

Evidence of histochemical features suggestive of a defect of oxidative phosphorylation and multiple mtDNA deletions in skeletal muscle from two patients, appeared to suggest a mitochondrial dysfunction. A similar feature was observed in one patient from a single LGMD1F family thus far reported. [START_REF] Gamez | Autosomal dominant limb-girdle dystrophy: a large kindred with evidence of anticipation[END_REF] Multiple mtDNA rearrangements usually result from mutations in genes involved in mtDNA replication and maintenance. [START_REF] Mauro | Mitochondrial respiratory-chain diseases[END_REF] At least seven genes have been identified in this subset of mitochondrial disorders (reviewed in Zeviani et al [START_REF] Zeviani | Nuclear genes in mitochondrial disorders[END_REF] ), but none locate in the 3p23-p25 interval defined in our LGMD1H family. Low levels of multiple mtDNA deletions can also be a secondary event, such as it happens in sporadic and familial Inclusion Body Myosites [START_REF] Jansson | 23-28. mitochondrial DNA point mutations and deletions cause focal impairment of mitochondrial function[END_REF] and in the ageing processes. 24 In our patients, mtDNA deletions are likely to be considered as by-products of mitochondrial dysfunction of still unknown significance.

Whole genome scan for the LGMD1H locus showed data compatible with linkage to a 31 cM region on chromosome 3p23-p25. Haplotype reconstruction allowed the refinement of the candidate region to 25 cM between markers D3S1263 and D3S1277.

In this interval, in silico analyses revealed the presence of more than 150 genes (www.ensembl.org). However, no evident disease-causative gene emerged, and two of the potentially candidates (CAPN7 and MGC15763) were ruled out by direct sequencing. The function of the human CAPN7 gene product is presently unknown but the mouse orthologue is thought to be an enzyme calcium-independent with protease activity. The human protein seems to have a nuclear localization and is the most divergent element in the calpain team, sharing only 26-35% of its sequence with the other members of this family. [START_REF] Futai | Molecular cloning of PalBH, a mammalian homologue of the Aspergillus atypical calpain PalB[END_REF] The MGC15763 gene encodes the hypothetical protein BC008322 which contains a region of NADH -cytochrome b-reductase involved in coenzyme transport and metabolism, energy production and conversion, and it is predicted to have a mitochondrial targeting.

In summary, we have presented evidence for a novel form of AD-LGMD mapping on chromosome 3p23-p25.1 characterized by a slowly progression of proximal muscle weakness in both upper and lower limbs. The transmission model of the disease is consistent with an AD pattern of inheritance with variable expressivity and incomplete penetrance. In fact, the symptoms vary in terms of age at onset and clinical severity. The identification of additional LGMD1H families, further refinement of the genetic interval in our kindred or both are needed before considering screening for additional candidate genes. LGMD and microsatellite markers on chromosome 3p23-p25.

Titles and legends to figures

  shows a partial pedigree of this kindred with at least two instances of a father-to-son transmission and refers to the branches directly examined in the present study. According to established guideline criteria,[START_REF] Bushby | Limb girdle muscular dystrophy[END_REF] index subjects were considered affected by LGMD when neurological examination revealed a typical pattern of muscle weakness affecting the upper and lower girdles. However, we recognized a second group of relatives with younger age at onset or milder clinical presentation, or both, and considered them as affected for genome-wide purposes. Muscle strength was appraised using the MRC (British Medical Research Council) Scale. Age at onset was determined at the time when muscle complains first became manifested. Nineteen subjects (6 men and 13 women) were clinically assessed by two neurologists skilled in the evaluation of neuromuscular disorders. The clinical features of the remaining individuals were obtained by either family recall or consultation of available clinical notes. Routine blood tests were obtained to investigate metabolic causes of the disease. Serum creatin kinase (CK) and lactate levels were determined in all subjects. Electromyography (EMG) was performed in five patients. Four patients (II-1, II-5, II-9 and II-13) underwent a skeletal muscle biopsy. The specimens from the deltoid or vastus lateralis were oriented, snap-frozen in liquid nitrogen-chilled isopentane and the cryostat-cut sections were stained using standard histochemical methods. Immunohistochemistry was performed using the following antibodies: desmin and vimentin (Biogenex, CA), ubiquitin (DAKO, DK), Tau protein and Beta-amyloid (Sigma, MO), sarcoglycans, dystrophin, betadystroglican (YLEM s.r.l. Becton Dickinson Company), caveolin-3 (Transduction Laboratories), and the amino terminal of utrophin (DRP2, Novocastra Laboratories, Newcastle upon Tyne, UK).

Figure 1 .

 1 Figure 1. Pedigree of the family affected by the novel AD LGMD form and haplotype analysis of microsatellite markers from chromosome 3p23-p25. The haplotype segregating with the disease is "boxed". Black filled symbols indicate affected subjects; clear symbols indicate the unaffected subjects.

Figure 2 .

 2 Figure 2. Southern blot performed on DNA extracted from skeletal muscle biopsies.

Figure 3 .

 3 Figure 3. Diagrammatic representation of the region 3p22-p25.3. (a) Schematic map of the short arm of the chromosome 3 showing markers and genes of interest located between 11 and 42 Mb. (b) Multipoint LOD scores between the disease and markers on chromosome 3p.

  [START_REF] Bushby | Limb girdle muscular dystrophy[END_REF][START_REF] Guglieri | Limb-girdle muscular dystrophies[END_REF] Variable expressivity was observed in term of age at onset and muscular symptoms. With few exceptions, the adult patients showed the -canonical‖ clinical features of LGMD, characterised by slowly progressive proximal muscle weakness in upper and lower limbs with onset during the fifth decade and a relatively benign course. Neither ocular-An earlier onset of the disease was found in a group of patients complaining muscle weakness or calf hypertrophy and occasional raise of CK and lactate levels.Based on the clinical history of the five adult members, this juvenile group is likely to develop later in life the typical pattern of LGMD. Two unaffected subjects (III:1 and III:11) carried the disease haplotype, suggesting an incomplete penetrance for the disease. However, the follow up for the subject III:11 remains unpredictable since she is younger than the mean age of the second generation relatives.

	facial-bulbar muscles, nor cardiac muscle involvement, nor early joint contractures were
	observed. A single individual (I:1) had a likely hypertensive cardiomyopathy, while
	echocardiography performed in three subjects affected by LGMD (II:1, II:3, II:13) was
	normal.

Consistently, immunohistochemistry and linkage analysis excluded any other known dominant form of LGMD. In keeping with the current classification of inherited

Table 1 .

 1 Principal clinical and laboratory data in family members.

Table 2 .

 2 Two point LOD score and multipoint non parametric LOD scores between

11.4 12.9 15.2 15.7 16.2 16.8

		Mb		8.7 27.9	3 1 4 5 42.4	D3S1286 D3S2338
	Chromosomal bands	3p25.3		3p25.2	3p25.1	3p24.3 3p24.1 3p22	3 3 1 2 3 3	D3S3726 D3S3700 D3S3659
											5 6	D3S2336
											1 1	D3S1266
											8 5 8 5	D3S3727 D3S3727
											1 4	D3S3567
		Tel								1 1	D3S1619
											2 4	D3S1277
											3 3	D3S3521
		4									3 3	D3S3685
		3
		2
	re LOD sco	II 2 II:2 1 3 7 7 3 1 1 1 1 -1 0 1 -2 9 9 4 7 1 4 6 4 2 2 2 2 -3 -4 2 2 3 3 5 7 2 4 3 3 2 2 4 2 4 4 1 1	-50 50	-30 30	-10 10	II 4 II:4 30 30 Genetic distance (cM) 10 II 3 II:3 50 70 10 50 70	II 5 II:5 3 6 3 2 5 5 2 1 6 2 4 3 2 4 3 3 1 1 90 90 1 3 1 3 1 3 5 4 3 1 1 4 1 1 1 5 2 3 5 6	II 6 II:6 2 3 1 1 5 5 3 2 Figure 2 2 3 2 4 4 2 1 2 2 1 110 1 110 1 7 3 7 3 4 4 2 3 3 3 4 3 2 2 1 2 5 4 6 7	II 8 II:8 II:5 II:9 II 7 II:7 3 4 3 8 5 1 2 1 6 8 4 1 2 5 3 3 1 1 1 3 1 3 1 3 5 2 3 1 1 1 1 1 1 8 2 3 5 5	II 10 II:10 2 1 6 6 4 3 4 2 8 1 6 7 1 3 3 4 5 1 3 6 3 6 1 1 9 1 3 3 4 4 2 4 8 5 Wt mtDNA 16.6 kb II 9 II:9 3 4 3 8 5 1 2 1 6 8 4 1 2 5 3 3 1 2 1 3 1 3 1 3 5 4 Deleted mtDNA 3 1 1 1 1 1 1 8 3 3 5 6 2 3 5 6	II 11 II:11 1 6 4 2 1 5 2 1 4 2 2 3 4 4 4 3 2 2 6 3 6 3 3 3 4 4 1 1 4 4 2 1 1 5 1 3 4 6	II 12 II:12 4 7 5 6 2 5 2 2 5 4 6 6 1 8 1 4 3 2 5 3 5 3 3 4 4 6 1 2 2 2 1 3 5 4 4 1 5 5	II 13 II:13 3 6 3 2 5 5 2 1 6 2 4 3 2 4 3 3 1 2 1 3 1 3 1 3 5 4 3 1 1 4 1 5 1 1 2 3 5 6
	III:2			III:3		III:4	III:5		III:6		III:7	III:8	III:9	III:10
	4 3			3 1		1 6	6 2		6 2		4 1	3 2	1 7	6 5
	8 7			3 7		3 4	3 3		2 1		8 2	3 6	4 6	2 4
	1 1			5 3		5 5	3 5		5 5		1 5	5 4	1 5	5 3
	1 1			2 1		2 1	2 4		1 3		1 2	2 4	2 2	1 2
	8 9			6 9		6 7	4 10		2 2		8 1	6 8	4 4	2 6
	1 7			4 4		4 8	9 1		3 2		1 6	4 6	2 6	3 7
	5 4			2 1		2 4	6 5		4 4		5 7	2 3	4 8	4 4
	3 4			3 6		3 5	4 4		3 1		3 4	3 4	4 4	3 3
	1 2			1 2		1 1	1 4		1 2		1 2	1 1	2 3	2 2
	3 2			2 2		2 3	3 4		3 5		3 4	2 3	1 4	3 2
	5 3			5 3		5 5	1 1		6 6		5 5	5 6	4 5	6 1
	1 1			1 1		1 3	3 4		1 2		1 3	1 4	2 1	1 1
	5 7			1 5		1 5	6 8		5 1		8 7	1 5	1 5	5 3
	4 4 4 4			1 4 1 4		1 4 1 4	4 4 4 4		4 4 4 4		1 3 1 3	1 4 1 4	4 2 4 2	4 4 4 4
	1 2			3 2		3 3	4 2		1 3		1 1	3 3	1 1	1 4
	5 2			5 2		5 7	5 5		5 2		2 4	5 1	4 4	4 8
	1 3			1 3		1 1	4 3		1 4		3 2	1 1	3 3	3 4
	1 4			3 4		1 5	8 5		1 7		3 5	1 6	6 5	3 7
						IV:1	IV:2
						1 2	1 2
						3 3	3 3
						5 5	5 5
						2 4	2 4
						6 10	6 10
						4 1	4 1
						2 5	2 5
						3 4	3 4
						1 4	1 4
						2 4 2 4	2 4 2 4
						5 1	5 1
						1 4	1 4
						1 8	1 8
						1 4	1 4
						3 2	3 2
						5 5	5 5
						1 3	1 3

Table 1 .

 1 Principal clinical and laboratory data in family members. : skeletal muscle biopsy showing dystrophic pattern; na: not available; a clinical information based on anamnestic recall; b consultation of clinical notes;

			Patient	Age	Age	Sex	Biopsy c	EMG	CK	Lactate	Neuromuscular examination	Other symptoms
				at	at				level	level
				diagnosis	onset				(U/L)	(mg/dl)
			I:1 a	dead at 77	-	m	na	na	na	na	calf hypertrophy	Headache, heart problems
				years
			II:1	60	50	m	++	myopathic	2295	13.2	muscle weakness, calf	dyspnea , scoliosis
	Severely affected		II:3 b II:5 II:9	59 57 49	49 50 45	f f f	na ++ ++	pattern myopathic pattern myopathic pattern pattern myopathic	1551 997 2139	11.0 8.7 6	hypertrophy muscle weakness muscle weakness hypertrophy muscle weakness, calf	scoliosis
			II:13	42	39	m	++	normal	1600	10.7	muscle weakness
			III:3	24	20	f	na	na	2300	11.9	muscle weakness
			II:7	50	46	m	na	na	153	20.5	calf hypertrophy
	Mildly	affected	III:4 III:8 IV:1	37 22 17	34 22 17	f f f	na na na	na na na	160 155 67	18.2 38.7 11.6	calf hypertrophy calf hypertrophy calf hypertrophy	headache
			IV:2	16	16	f	na	na	98	11.3	calf hypertrophy
			II:11	45	-	m	na	na	62	8,2	normal
			III:1	30	-	m	na	na	185	10,9	normal
	Unaffected		III:2 III:6 III:7 III:9	27 10 10 24	----	f f f f	na na na na	na na na na	110 97 183 78	12,3 11,1 9,9 11,7	normal normal normal normal
			III:10	17	-	m	na	na	162	8,1	normal
			III:11	14	-	f	na	na	130	11,3	normal	recurrent migraine without aura

++c description of bioptical findings in the text.

Table 2 .

 2 Two point LOD score and multipoint non parametric LOD scores between LGMD and microsatellite markers on chromosome 3p23-p25.

	Markers	Position		Two point LOD score at θ (m=f) =			Multipoint NPL
		cM	Mb	0.0	0.01	0.05	0.1	0.2	0.3	0.4	STAT D	p-value STAT E	p-value
	D3S1263	36.10 11.492	-2.52	0.90	1.47	1.57 1.39 0.97 0.44	1.3998	0.2250	0.1383	0.1527
	D3S1259	36.65 12.073	2.32	2.30	2.21	2.06 1.66 1.14 0.53	1.8495	0.1087	8.1269	0.0746
	D3S3610	37.20 12.980	0.66	0.66	0.63	0.59 0.46 0.31 0.15	2.2600	0.0010 15.4095	0.0008
	D3S1554	39.52 14.342	0.88	0.90	0.92	0.86 0.71 0.49 0.24	2.2591	0.0005 15.3722	0.0004
	D3S3613	41.56 15.336	3.23	3.19	3.05	2.83 2.27 1.58 0.76	2.2614	0.0005 15.4394	0.0005
	D3S1286	41.56 15.794	3.23	3.19	3.05	2.83 2.27 1.58 0.76	2.2615	0.0004 15.4396	0.0004
	D3S2338	42.10 16.824	2.75	2.72	2.57	2.35 1.81 1.16 0.48	2.2615	0.0005 15.4397	0.0004
	D3S3726	42.64 19.509	2.21	2.18	2.02	1.81 1.37 0.88 0.36	2.2611	0.0005 15.4268	0.0004
	D3S3659	47.44 22.913	1.23	1.21	1.13	1.01 0.73 0.42 0.12	2.2610	0.0002 15.4261	0.0002
	D3S3700	48.09 23.885	2.75	2.72	2.57	2.35 1.80 1.15 0.46	2.2624	0.0002 15.4443	0.0002
	D3S2336	49.18 24.889	1.75	1.73	1.63	1.44 1.15 0.74 0.29	2.2614	0.0004 15.4163	0.0004
	D3S1266	52.60 27.932	2.21	2.18	2.02	1.81 1.37 0.88 0.36	2.2608	0.0007 15.4012	0.0005
	D3S3727	56.07 30.652	1.21	1.24	1.28	1.26 1.06 0.74 0.37	2.2622	0.0008 15.4436	0.0007
	D3S3567	56.69 30.713	1.20	1.18	1.10	0.97 0.66 0.32 0.07	2.2622	0.0007 15.4436	0.0007
	D3S1619	60.98 34.087	2.62	2.60	2.49	2.32 1.87 1.29 0.60	2.3114	0.0005 15.7125	0.0004
	D3S1277	61.52 34.627	1.83	1.84	1.84	1.79 1.54 1.11 0.53	2.3241	0.0004 15.7821	0.0003
	D3S3521	63.12 38.830	1.23	1.24	1.29	1.28 1.13 0.82 0.37	2.3136	0.0005 15.3653	0.0004
	D3S3685	67.94 42.429	-7.75	-1.46	-0.10	0.40 0.70 0.61 0.29	1.7626	0.0321	0.6551	0.0331

Acknowledgements: This work was supported in part by grants from the Italian Ministry of Health -Ricerca Corrente 2009-2011‖, MIUR-PRIN -Molecular mechanisms, physiology and pathology of membrane bioenergetics system -, Project No. 2005052128_001 and Research Foundation Cassa di Risparmio di Puglia.


MSc Natalie L Marchant
email: n.l.marchant@sussex.com.ac.uk

PhD Sarah L King

MD Naji Tabet

PhD Jennifer M Rusted

Marchant

Positive effects of

Keywords: Apolipoprotein E, Nicotine, Prospective Memory, Cognitive Enhancement, Cholinergic System, Cognition

come

Introduction

The recent proliferation of research concerning "smart drug" use in normally functioning young adults has identified a number of compounds capable of improving cognitive performance [START_REF] Farah | Neurocognitive enhancement: what can we do and what should we do?[END_REF]. A call has been made for increased research into the factors that may influence the potential for compounds to boost cognition in healthy adults given that usage of these compounds may now be surpassing associated knowledge [START_REF] Greely | Towards responsible use of cognitive-enhancing drugs by the healthy[END_REF]. Thus far compounds that affect different neurotransmitter systems in the brain, different cognitive domains, and different baseline profiles have been explored, but no clear cut indices of efficacy have been achieved for any of these factors [START_REF] Lanni | Cognition enhancers between treating and doping the mind[END_REF].

With this in mind we sought to investigate whether genetic differences might interact with the potential for neuromodulation of cognition by putative cognitive enhancers. The most widely studied genetic variation is that associated with a common polymorphism of the apolipoprotein E (APOE) gene, which results in three alleles (ε2, ε3, ε4). Emerging studies now indicate that young adult ε4 carriers may comprise a cognitive supergroup [START_REF] Han | Revision of the apolipoprotein E compensatory mechanism recruitment hypothesis[END_REF], and in fact that the well-documented negative consequences of possessing an ε4 allele (eg greater risk for developing dementia, poorer cognitive aging [START_REF] Corder | Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families[END_REF][START_REF] Saunders | Association of apolipoprotein E allele epsilon 4 with late-onset familial and sporadic Alzheimer's disease[END_REF]) may not emerge until after the fifth decade of life [START_REF] Savitz | Apolipoprotein E variants and cognition in healthy individuals: a critical opinion[END_REF]. Specifically, recent behavioural studies report that ε4 carriers in younger life have higher IQs [START_REF] Yu | Intelligence and event-related potentials for young female human volunteer apolipoprotein E epsilon4 and non-epsilon4 carriers[END_REF], higher educational achievement [START_REF] Hubacek | A possible role of apolipoprotein E polymorphism in predisposition to higher education[END_REF], and better performance on certain cognitive tasks [START_REF] Mondadori | Better memory and neural efficiency in young apolipoprotein E epsilon4 carriers[END_REF] compared to their non-ε4 peers. Therefore we asked the question: does the ε4 advantage in healthy young adults reduce or increase the potential for pharmacologic cognitive enhancement?

One such cognitive enhancer, nicotine, acts via cholinergic stimulation. It improves attention and memory performance in younger and older adults without a history of nicotine use [START_REF] Levin | Nicotinic effects on cognitive function: behavioral characterization, pharmacological specification, and anatomic localization[END_REF][START_REF] Robbins | The 5-choice serial reaction time task: behavioural pharmacology and functional neurochemistry[END_REF]. Additionally, it improves prospective memory (PM) in normally functioning nicotine-naïve young adults [START_REF] Marchant | Prospective memory or prospective attention: physiological and pharmacological support for an attentional model[END_REF][START_REF] Rusted | Comparable effects of nicotine in smokers and nonsmokers on a prospective memory task[END_REF][START_REF] Rusted | Nicotine improves memory for delayed intentions[END_REF]. PM is a type of memory particularly relevant to everyday living and as such is a valuable indicator of real world memory capability against which to test cognitive enhancers. It is engaged when we interrupt an ongoing activity to retrieve and act upon a previously formed intention, and allows us to multi-task successfully through the day.

Cognitively healthy older adult ε4 carriers show poorer PM performance than non-ε4 peers [START_REF] Driscoll | Apolipoprotein E and prospective memory in normally aging adults[END_REF], suggesting that PM performance is a sensitive index of genetic as well as pharmacologic manipulation. Therefore we chose this task as the primary focus of a study that co-varied genotype (ε3/ε3 vs ε4 carriers) and nicotine administration in normally functioning young-adults.

Methods and Materials

Participants

One-hundred and fifty-six volunteers were recruited from Sussex University. All volunteers met strict criteria for physical and psychological health, were non-smokers (with >5 years abstinence), had a body mass index within the normal range (18-30), and were aged 18-30. APOE genotype was determined by a blood sample or buccal swab. Twenty-seven ε4 (1 ε2/ε4, 2 ε4/ε4, 24 ε3/ε4; furthermore referred to as ε4 carriers) and 29 ε3/ε3 participants (furthermore referred to as the ε3 controls) returned for the full study (ε3/ε3's were chosen as it is normal practice to provide a homogenous control group representing the genotype most frequent in the general population [START_REF] Rebeck | Apolipoprotein E in sporadic Alzheimer's disease: allelic variation and receptor interactions[END_REF]). This e4 allelic frequency is aligned with population percentages and from other studies (eg [START_REF] Corder | Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families[END_REF]. Forty-four participants were Caucasian, 7 Asian, 1 Black, and 4 participants described themselves as 'mixed ethnicity'. The study was approved by the National Health Service (NHS) -National Research Ethics Service.

Written informed consent was obtained, and all participants were reimbursed for their time.

Participants in each group (ε3/placebo, ε3/nicotine, ε4/placebo, ε4/nicotine) were matched on age, gender, IQ, and appropriate behavioral and physiological measures (see table 1). Alcohol consumption was measured using the Alcohol Usage Quotient (AUQ; [START_REF] Mehrabian | A questionnaire measure of habitual alcohol use[END_REF] , a questionnaire that provides a composite measure based on units of alcohol consumed per week, drinking rate, number of times the participant was drunk in the last six months, and percentage of times the participant gets drunk. Exercise was determined by self-reported number of times the participant exercised per week (Sedentary: <1 day of exercise, Moderate: 1-3 days, Active: 4-5 days, Athletic: >5 days).

Neuropsychological Assessment

Spatial Working Memory (Mechaeil, unpublished thesis) was assessed during the familiarization session. Participants were instructed to remember the locations of seven rectangles presented simultaneously for 1 second. After an 8 second delay, one rectangle appeared and participants decided whether the solitary rectangle was in the same location as one of the initial seven rectangles by circling either 'yes' (the rectangle is in the same location as one of the seven presented earlier) or 'no' (the rectangle is not in the same location as one of the seven presented earlier) on a trial-by-trial template. They were allowed 10 seconds to make their decision before the program progressed to the next stimuli. There were 2 practice trials and 16 experimental trials.

The National Adult Reading Test (NART; [START_REF] Nelson | National Adult Reading Test (NART), Second edn[END_REF] provides an estimate of IQ and was administered as part of the baseline battery of tests. Participants are required to read 50 phonetically irregular words aloud and their responses are individually scored as correct or incorrect based on their pronunciation. The total numbers of errors is well correlated with the Wechsler Adult Intelligence Scale-R [START_REF] Nelson | National Adult Reading Test (NART), Second edn[END_REF].

Immediate Verbal Free Recall was measured during the familiarization session and again at the end of the experimental session. Participants were instructed to complete written recall of a 20-word list with each word presented on screen for 2 s with an inter-stimulus interval (ISI) of 1 s. Different lists were used at the different time points, matched on frequency of occurrence, imageability and number of syllables [START_REF] Rusted | Dissociative effects of scopolamine on working memory in healthy young volunteers[END_REF].

A verbal fluency task, in which participants generate as many words as possible that begin with a certain letter within 60 seconds, was included to measure 'executive functioning'. 1 They were given the letters "F", "A", and "S", and the number of unique words for each of these letters were summed and reported in 15-second segments.

Cognitive failures questionnaire (CFQ; [START_REF] Broadbent | The Cognitive Failures Questionnaire (CFQ) and its correlates[END_REF] is self-report questionnaire that assesses four different appraisals of cognitive functioning: memory, distractibility, blunders, and naming. Participants are asked to respond to the questions using a 5-point scale (very often, quite often, occasionally, very rarely, never).

Sustained attention was measured using the Rapid Visual Information Processing (RVIP) task [START_REF] Wesnes | Effects of smoking on rapid information processing performance[END_REF]. Single digit numbers appeared in the middle of the computer screen and participants were instructed to press 'spacebar' each time they saw either three odd numbers or three even numbers in a row. Digits were presented for 750 ms with no interstimulus interval. The task lasted continuously for five minutes with five target sequences occurring per minute. Target hits, misses, false alarms, and correct rejections for each minute were entered into a d' transformation. The d' transformation minimizes the potential influence of response bias by subtracting the z-transformed false alarm rate from the hit rate; therefore a higher d' value indicates better performance.

Decision making ability was measured using the ongoing task of the PM paradigm.

Participants first completed this task prior to drug administration as a measure of 'decision making' ability. They were instructed to sort a deck of 52 playing cards according to suit [START_REF] Rusted | Positive effects of nicotine on cognition: the deployment of attention for prospective memory[END_REF]. Each card image remained on the computer screen for 750 ms with an ISI of 1000 ms (where the back of a playing card was displayed). Therefore participants were allowed 1750 ms from stimulus onset to respond. This task was completed prior to drug administration.

Prospective Memory was measured in the context of the ongoing decision-making task.

In addition to completing the decision-making task, participants were told to withhold their suitsorting responses and press the spacebar key if they saw a number '7' card -the PM target. There were two decks of cards comprising 104 trials in total; with 8 target cards. Therefore, PM targets constituted roughly 4% of trials. Participants received these instructions prior to drug administration and completed the task approximately 15 minutes after using the nasal spray.

Design and Procedure

Participants visited the laboratory 1-7 days prior to the experimental session to become familiar with the nasal spray, complete the spatial working memory task, immediate verbal free recall task, and practice the RVIP task.

Baseline physiological and mood measures were taken at the beginning of the experimental session, followed by instructions and practice of the ongoing decision-making task.

On completion, participants received the added PM instructions. They then self-administered the nasal spray and immediately performed the verbal fluency task, NART, and CFQ. Completion of these tasks required 12-15 minutes, the time necessary for nicotine to reach peak plasma concentration [START_REF] Schneider | Clinical pharmacokinetics of nasal nicotine delivery. A review and comparison to other nicotine systems[END_REF]. Physiological and mood measures were again taken. They then completed the ongoing/PM task and then restated the ongoing/PM instructions to confirm compliance. Four participants incorrectly reported these instructions and were removed from analyses of this task. A five-minute version of the RVIP task was then performed, followed by a second immediate verbal free recall task. Final physiological and mood measures were taken.

Participants also reported whether they believed they had received nicotine or not, and were subsequently debriefed and discharged from the lab.

APOE Genotyping and Data Analysis

APOE genotype was determined following a standardized protocol using a polymerase chain reaction (PCR)-based assay that employs the Afl III and HaeII restriction enzymes to distinguish among the alleles [START_REF] Ossendorf | Rapid and easy apolipoprotein E genotyping using an improved PCR-RFLP technique[END_REF].

Statistical Analyses

Statistical analyses were conducted using Statistical Package of Social Sciences (SPSS 14) software and an alpha level of .05 was used to interpret significant differences in the results.

In all cases genotype and drug represented between subjects factors, and time represented the within subject factor. Pre-nicotine analyses were conducted using independent t-tests (genotype) or a mixed design analysis of variance (ANOVA; genotype x time). Post-nicotine analyses were conducted using between subjects ANOVAs (genotype x drug) or mixed design ANOVAs (genotype x drug x time). Post hoc analyses were performed using independent t-tests with Bonferroni corrections reported when appropriate. Means with ± 1 standard error of the mean (SEM) are reported.

All of the analyses examining the cognitive measures were run using only Caucasian participants (see supplementary table), and also comparing only ε3/ε3 and ε3/ε4 participants.

The same pattern of results emerged for these smaller groups as those reported in the results section which used the entire sample.

Results

Pre-nicotine Assessments

ε4 carriers were faster at the 'decision making' task (measured in milliseconds, ms; 472 ± 8) than ε3 controls (499 ± 8; t (54) = 2.38, p = .02), while maintaining equal levels of accuracy (t (34.52) = 1.51, P = .14, equal variances not assumed; figure 1a).

Examination of the verbal fluency task revealed a main effect of time, with participants producing fewer words over the course of the task (F (3,63) = 103.44, p < .001). There was no main effect of genotype for overall performance on the 60 second task (t (22) = 1.72, p = .1), but there was a time x genotype interaction (F (3,63) = 3.18, p = .03). ε4 carriers (19.2 ± 1.72) produced more words in the first quarter of the task than ε3 controls (14.85 ± .77; t (21) = 2.5, p = .02), and while they continued to produce more words in each subsequent quarter the differences were not significant (figure 1b).

For the other cognitive tasks administered prior to nicotine administration, no differences were found between gene groups: spatial working memory (t (53) = .19, p = .85); immediate verbal recall (t (54) = .71, p = .48). Additionally, no differences emerged on any of the factors from the CFQ: memory (t (54) = 1.15, p = .25), distractibility (t (54) = .4, p = .69), blunders (t (54) = .31, p = .76), and naming (t (54) = .89, p = .38).

Following nasal spray administration, participants completed the ongoing task of sorting the playing cards by suit but this time with the embedded PM component. The ε4 carriers were again faster at the ongoing task than ε3 controls (F (1,48) = 5.11, p = .03), with no main effect of drug or interaction (figure 2a). There was a near significant main effect of genotype on levels of accuracy for the ongoing task (F (1,48) = 3.87, p = .055), with an additional near significant interaction with drug (F (1,48) = 3.41, p = .07; figure 2b). Given the investigative nature of the study, post hoc analyses were conducted. The ε4/nicotine group (95.24 ± .84) was significantly more accurate than the ε4/placebo group (89.21 ± 1.7; t (21) = 3.1, p = .005), the ε3/nicotine group (86.73 ± 2.87; t (23) = 2.55, p = .02), and ε3/placebo group (88.94 ± 2.29; t (17.55) = 2.59, p = .02, equal variances not assumed). There was no difference between the ε3/placebo and ε3/nicotine groups, or ε4/placebo and ε3/placebo groups. After employing the Bonferroni correction (p < .01) the only difference to remain significant was between ε4/nicotine and ε4/placebo groups.

PM was measured by the number of times the volunteer pressed the spacebar to register the detection of the PM target, regardless of whether they had first made a response to the ongoing task (maximum = 8). No main effects or interactions were found between groups when analyzing reaction time. A main effect of gene was found for PM target percent accuracy (F (1,48) = 5.78, p = .02), with ε4 carriers (71.2 ± 4.8) surpassing ε3 controls (53.45 ± 5.39). Post hoc analyses indicated that the ε4/nicotine group were significantly more accurate than the ε3/nicotine group (t (23) = 2.77, p = .01), and the ε3/placebo group (t (23.79) = 2.25, p = .03; equal variances not assumed), but not the ε4/placebo group (t (21) = 1.39, p = .18; figure 2c). There was no difference between the ε3/placebo group and the ε3/nicotine group. After employing the Bonferroni correction (p < .01) the only difference to remain significant was between ε4/nicotine and ε3/nicotine groups. Thus not only did ε4 carriers show superior decision-making and PM performance, this advantage was augmented by the cognitive enhancer, nicotine. In fact, nicotine appeared to benefit only ε4 carriers.

Sustained attention was measured using the Rapid Visual Information Processing (RVIP) task. A d' transformation revealed a main effect of time on task (F (4,208) = 17.44, p < .001), which was qualified by a three way (time x gene x drug) interaction (F (4,208) = 3.95, p = .004). Post hoc analyses revealed that the ε4/placebo group were more accurate (3.77 ± .11) than the ε3/placebo group (3.0 ±.28) in minute 1, (t (17.83) = 2.58, p = .02; equal variances not assumed), and in minute 2, (t (25) = 2.4, p = .02). When corrected for multiple comparisons using the Bonferroni post hoc analysis (p < .01), these differences did not remain significant. A Chi square analysis was conducted to identify the number of people who were unable to correctly identify 50% of the targets. Significantly more ε3 controls recorded 50% or less hits (11 out of 29) than ε4 carriers (2 out of 27; χ 2

(1) = 7.31, p = .007). There was no differential effect of nicotine on this measure.

Response times (ms) to correctly identified targets were not differentiated by gene or drug. The results from this task indicate that ε4 carriers were better able to sustain their attention, and showed an early advantage in accuracy.

Physiological measures and subjective assessments of mood were collected at baseline, following nicotine absorption, and upon completion of the experimental tasks. Mean arterial blood pressure (MAP) increased in a linear fashion over the course of the experiment (F (2,104) = 4.85, p = .01). There were no main effects of drug or gene, and no interactions. Heart rate showed no main effects or interactions. Mood was assessed using two standardized measures:

the Profile of Mood States (McNair et al 1971) and Thayer's measures of alert and tense arousal [START_REF] Thayer | Toward a psychological theory of multidimensional activation (arousal)[END_REF]. Both measures registered a decline in arousal/alertness across the time course of the study, independent of gene type or drug condition (p's < .02). There were no other main effects or interactions. These findings are consistent with previous reports using nicotine nasal sprays [START_REF] Marchant | Prospective memory or prospective attention: physiological and pharmacological support for an attentional model[END_REF].

Discussion

The potential of putative cognitive-enhancers and of select genetic polymorphisms to boost performance in normally functioning adults is an area of growing interest. We sought to investigate the extent of the ε4 advantage in young adults across a broad coverage of cognitive domains, and also, using a double-blind placebo-controlled study, to explore the interactive effect of APOE genotype and the cognitive enhancer, nicotine. The results from these cognitive tasks indicate that possession of an ε4 allele produces domain-specific advantages rather than a global cognitive advantage. Specifically, ε4 carriers excelled in decision-making, PM performance and verbal fluency, all tasks that require frontal lobe involvement, but not on spatial memory or word recall. Thus we argue that, early in life, frontally-mediated tasks appear to benefit from possession of an ε4 allele. Indeed these findings support emerging literature showing cognitive superiority of young ε4 carriers [START_REF] Alexander | The contribution of apolipoprotein E alleles on cognitive performance and dynamic neural activity over six decades[END_REF][START_REF] Han | Revision of the apolipoprotein E compensatory mechanism recruitment hypothesis[END_REF][START_REF] Mondadori | Better memory and neural efficiency in young apolipoprotein E epsilon4 carriers[END_REF]. In our study, this cognitive superiority was not explained by an alternate hypothesis, namely that ε4 carriers were consciously compensating for poorer memory skills with metacognitive strategies. ε4 and ε3 participants demonstrated similar self-reported abilities in cognitive functioning as measured by the CFQ.

In addition, the manipulation of performance by the introduction of a cognitive enhancer revealed that even though ε4 carriers already showed a cognitive advantage over their e3 counterparts, nicotine potentiated this advantage on measures of decision-making and PM performance. These results indicate the capacity for cognitive performance in young healthy volunteers to be modified both by pharmacologic and genetic factors, and, critically, for those effects to be cumulative. The result is in contrast with recent suggestions [START_REF] Muller | Effects of modafinil on working memory processes in humans[END_REF][START_REF] Randall | Cognitive effects of modafinil in student volunteers may depend on IQ[END_REF] that cognitive enhancement in younger adults is most likely to be achieved when baseline performance is below average.

Our results, then, add further support for the emerging view that while the ε4 allele produces detrimental consequences in later life, it in fact confers a cognitive advantage in younger adulthood. These findings support the recent proposition of APOE ε4 antagonistic pleitropy [START_REF] Han | Revision of the apolipoprotein E compensatory mechanism recruitment hypothesis[END_REF][START_REF] Wright | Apolipoprotein E genotype predicts 24-month bayley scales infant development score[END_REF], whereby the ε4 variant might have contrasting effects across the lifespan. Our results suggest that this labile cognitive profile of ε4 carriers may be related to and influenced by the integrity of the cholinergic system. Older carriers of the ε4 allele have significantly less choline (the precursor to acetylcholine) and less choline acetyltransferase (the enzyme involved in converting choline to acetylcholine) activity than matched non-ε4 carriers [START_REF] Poirier | Apolipoprotein E4 allele as a predictor of cholinergic deficits and treatment outcome in Alzheimer disease[END_REF]. These deficiencies certainly contribute to the more rapid cognitive decline in older ε4 carriers and may explain why, in our study, ε4 carriers were more receptive to the cholinergic agonist, nicotine. Our findings offer new implications for the antagonistic pleitropy theory and suggest the possible molecular basis or locus for the opposing effects of APOE across the lifespan. A similar advantage for carriers of the APOE polymorphism from other putative cognitive enhancers would implicate a noncholinergic explanation, however.

Knowing that possession of an ε4 allele increases susceptibility to dementia and cognitive deterioration in later life, our results raise the question of how long this advantage in younger life might last, and whether cholinergic stimulation in earlier life might delay or hasten this decline.

These results thus have implications for the development of cognitive enhancing drugs for healthy individuals and treatment approaches for dementia. Specifically, individual differences may play a significant role in the efficacy of these compounds across the lifespan. This study provides the first study to cross the ε4 polymorphism with a pharmacological manipulation. The sample size, while typical of pharmacological manipulation studies, may be considered small in relation to genetic studies. As such, it may be viewed as a preliminary step in the field of gene x drug interaction. It argues firmly, however, for the value of further research into the individual factors that may moderate the effects of putative cognitive modulators across the lifespan.

Disclosure/Conflict of Interest

Naji Tabet has received research grants (unrelated to this study) from Pfizer and Novartis. He also received consultation and/or speaker fees from Pfizer, Eli Lilly, AstraZenca, Shire, Novartis and Lundbeck. Natalie Marchant, Sarah King, and Jennifer Rusted have no potential conflicts of interest to disclose.

Footnotes

1 Only data from participants whose first language was English and who were in the placebo condition are reported because this task was conducted during the nicotine absorption phase (making any possible effects of nicotine ambiguous).

Title and legend to table.

Table 1. Mean baseline scores for volunteers on measured participant characteristics (standard deviations below).*Based on data from participants for whom English is their first language. ○Exercise Classifications per week: S=Sedentary (<1 day of exercise), M=Moderate (1-3 days), Ac=Active (4-5 days), At=Athletic (>5 days). Titles and legends to figures.

Figure 1 .

 1 Figure1. Genotype differences on cognitive measures that require frontal cortex involvement.

Figure 2 .

 2 Figure 2. Cognitive measures showing different effects of nicotine on performance by ε4 carriers

Acknowledgements

This work was supported by a Sussex University D.Phil. studentship and an Overseas Research Scheme Award to the first author. We thank McNeil AB, Helsingborg, Sweden for providing the nasal sprays.