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Genetic modifiers in carriers of repeat expansions in the C9ORF72 gene van Blitterswijk, Marka; Mullen, Bianca; Wojtas, Aleksandra; Heckman, Michael G; Diehl, Nancy N; Baker, Matthew C; DeJesus-Hernandez, Mariely; Brown, Patricia H; Murray, Melissa E; Hsiung, Ging-Yuek R; Stewart, Heather; Karydas, Anna M; Finger, Elizabeth; Kertesz, Andrew; Bigio, Eileen H; Weintraub, Sandra; Mesulam, Marsel; Hatanpaa, Kimmo J; White, Charles L; Neumann, Manuela; Strong, Michael J; Beach, Thomas G; Wszolek, Zbigniew K; Lippa, Carol; Caselli, Richard; Petrucelli, Leonard; Josephs, Keith A; Parisi, Joseph E; Knopman, David S; Petersen, Ronald C; Mackenzie, Ian R; Seeley, William W; Grinberg, Lea T; Miller, Bruce L; Boylan, Kevin B; Graff-Radford, Neill R; Boeve, Bradley F; Dickson, Dennis W; Rademakers, Rosa Sep 20, 2014

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RESEARCH ARTICLEes1drConclusions: Variants identified through this study were previously reported to be involved in FTD and/or MND,but we are the first to describe their effects as potential disease modifiers in the presence of a clear pathogenicvan Blitterswijk et al. Molecular Neurodegeneration 2014, 9:38http://www.molecularneurodegeneration.com/content/9/1/38in carriers of these expansions, including heterogeneityin age at onset and disease duration [10]. While recentJacksonville, FL 32224, USAFull list of author information is available at the end of the articlemutation (i.e. C9ORF72 repeat expansion). Although validation of our findings is necessary, these variantshighlight the importance of protein degradation, antioxidant defense and RNA-processing pathways, andadditionally, they are promising targets for the development of therapeutic strategies and prognostic tests.Keywords: C9ORF72, Frontotemporal dementia, Motor neuron disease, Genetic modifier, Repeat expansionBackgroundTwo fatal neurodegenerative diseases, frontotemporaldementia (FTD) and motor neuron disease (MND),demonstrate clinical, pathological and genetic overlap.In up to 50% of FTD patients, for instance, signs ofmotor neuron dysfunction are present and an equal per-centage of MND patients can show cognitive symptoms offrontal lobe impairment [1-4]. Moreover, inclusions oftransactive response DNA-binding protein 43 (TDP-43)are the most common subtype of FTD and are also apathological hallmark of MND [5,6]. Interestingly,hexanucleotide repeat expansions in the chromosome 9open reading frame 72 (C9ORF72) gene have beenidentified in FTD and MND [7,8], representing themost frequent genetic cause of both diseases [9]. Con-siderable clinical variability, however, has been detected* Correspondence: Rademakers.Rosa@mayo.edu1Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road,Marsel Mesulam6, Kimmo J Hatanpaa7, Charles L White III7, Manuela Neumann8, Michael J Strong9,Thomas G Beach10, Zbigniew K Wszolek11, Carol Lippa12, Richard Caselli13, Leonard Petrucelli1, Keith A Josephs14,Joseph E Parisi14, David S Knopman14, Ronald C Petersen14, Ian R Mackenzie15, William W Seeley4, Lea T Grinberg4,Bruce L Miller4, Kevin B Boylan11, Neill R Graff-Radford11, Bradley F Boeve14, Dennis W Dickson1 andRosa Rademakers1*AbstractBackground: Hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9ORF72) are causativefor frontotemporal dementia (FTD) and motor neuron disease (MND). Substantial phenotypic heterogeneity hasbeen described in patients with these expansions. We set out to identify genetic modifiers of disease risk, age atonset, and survival after onset that may contribute to this clinical variability.Results: We examined a cohort of 330 C9ORF72 expansion carriers and 374 controls. In these individuals, we assessedvariants previously implicated in FTD and/or MND; 36 variants were included in our analysis. After adjustment formultiple testing, our analysis revealed three variants significantly associated with age at onset (rs7018487 [UBAP1;p-value = 0.003], rs6052771 [PRNP; p-value = 0.003], and rs7403881 [MT-Ie; p-value = 0.003]), and six variants significantlyassociated with survival after onset (rs5848 [GRN; p-value = 0.001], rs7403881 [MT-Ie; p-value = 0.001], rs13268953 [ELP3;p-value = 0.003], the epsilon 4 allele [APOE; p-value = 0.004], rs12608932 [UNC13A; p-value = 0.003], and rs1800435[ALAD; p-value = 0.003]).Genetic modifiers in carriin the C9ORF72 geneMarka van Blitterswijk1, Bianca Mullen1, Aleksandra WojtaMatthew C Baker1, Mariely DeJesus-Hernandez1, Patricia HHeather Stewart3, Anna M Karydas4, Elizabeth Finger5, An© 2014 van Blitterswijk et al.; licensee BioMedCreative Commons Attribution License (http:/distribution, and reproduction in any mediumDomain Dedication waiver (http://creativecomarticle, unless otherwise stated.Open Accessrs of repeat expansions, Michael G Heckman2, Nancy N Diehl2,Brown1, Melissa E Murray1, Ging-Yuek R Hsiung3,ew Kertesz5, Eileen H Bigio6, Sandra Weintraub6,Central Ltd. This is an Open Access article distributed under the terms of the/creativecommons.org/licenses/by/4.0), which permits unrestricted use,, provided the original work is properly credited. The Creative Commons Publicmons.org/publicdomain/zero/1.0/) applies to the data made available in thisstudies implicated variants in transmembrane protein106 B (TMEM106B), intermediate repeats in ataxin-2(ATXN2), C9ORF72 expansion size, and the presence ofdouble mutations as genetic modifiers of the clinicalpresentation in C9ORF72 expansion carriers [11-15], itremains largely unknown why some individuals developdisease symptoms in their 40s whereas others remainunaffected until old age.In C9ORF72 expansion carriers, FTD and/or MND-was 4.42 years later in probands with two copies of theminor allele, than in probands with at least one copy ofthe major allele (p-value = 0.003; recessive genotypicmodel). Probands carrying at least one copy of the minorallele in rs7403881 (metallothionein 1 E [MT-Ie] haplo-block), demonstrated a delay of 3.95 years in mean age atonset as compared to probands homozygous for the majorallele (p-value = 0.003; dominant genotypic model). Wedid not detect significant associations for any of the dis-A655665van Blitterswijk et al. Molecular Neurodegeneration 2014, 9:38 Page 2 of 10http://www.molecularneurodegeneration.com/content/9/1/38associated variants that modify disease risk, age at onset orsurvival after onset have not been studied systematically.For this reason, we conducted a thorough literature searchand included 36 known variants in our study. Thesevariants were investigated in a cohort of 330 C9ORF72expansion carriers and 374 controls; importantly, we iden-tified eight potential disease modifiers that may aid inexplaining the reported phenotypic heterogeneity.ResultsWe investigated a cohort of 330 C9ORF72 expansioncarriers and 374 controls for 36 variants known to mod-ify disease risk, age at onset or survival after onset inFTD and/or MND (Table 1; Additional file 1: Table S1).For simplicity, we have included an overview of signifi-cant associations, displaying only the genotypic modelfor which evidence of association was strongest (Table 2);results of all genotypic models for analyses that con-tained significant associations are shown in the supple-ment (Additional file 1: Table S2 [age at onset] andAdditional file 1: Table S3 [survival after onset]).Our primary analysis focused on the 265 probandscarrying C9ORF72 repeat expansions with FTD, FTD/MND, or MND. Under a false discovery rate (FDR) of10%, none of the variants studied was significantly associ-ated with disease risk, neither in our overall group nor inany of our disease subgroups. Age at onset analysis, how-ever, revealed three significant associations in our overallgroup (Table 2; Figure 1). Each additional minor allele ofrs7018487 (ubiquitin-associated protein 1 [UBAP1]) wasassociated with a decrease in mean age at onset of2.62 years (p-value = 0.003; additive genotypic model). Forrs6052771 (prion protein [PRNP]), the mean age at onsetTable 1 Subject characteristicsGroup N Female genderControls 374 172 (46.0%)All repeat expansion carriers 330 149 (45.2%)FTD, FTD/MND, and MND probands 265 115 (43.4%)FTD probands 74 29 (39.2%)FTD/MND probands 71 25 (35.2%)MND probands 120 61 (50.8%)Continuous variables are summarized with the sample mean ± standard deviation (clinically diagnosed patients, and age at death in pathologically diagnosed patientsease subgroups.In the 221 FTD, FTD/MND, and MND probands withinformation available regarding survival after onset,median follow-up length after onset was three years (range:4 months – 24 years [FTD: 1 year – 24 years, FTD/MND:10 months – 24 years, MND: 4 months – 9 years]). Thesurvival after onset analysis resulted in significant associa-tions with six variants (Table 2). Of those associations, onewas present in our overall group, three were present in ourFTD subgroup, and two were present in our MND sub-group. When concentrating on our overall group (Table 2;Figure 2), we noted a significant association only forrs5848 (granulin precursor [GRN]; relative risk [RR] = 1.64;p-value = 0.001; additive genotypic model). However, wealso performed an additional analysis to evaluate the com-bined effect of two other variants, rs13268953 andrs6985069 (elongator acetyltransferase complex subunit 3[ELP3]; not in linkage disequilibrium [LD]), on survivalafter onset, especially because these variants both showednon-significant trends towards an association and werelocated near the same gene. When combining these vari-ants, we did detect a significant association with survivalafter onset (p-value = 0.001; Additional file 1: Table S4).In our disease subgroups (Table 2; Figure 2), we observedsignificant associations in our FTD probands for rs7403881(MT-Ie; RR = 3.81; p-value = 0.001; recessive genotypicmodel), rs13268953 (ELP3; RR = 3.65; p-value = 0.003; re-cessive genotypic model), and the epsilon 4 allele (apolipo-protein E [APOE]; rs429358 and rs7412; RR = 3.13; p-value= 0.004; dominant genotypic model). In our MND pro-bands, significant associations were found for rs12608932(unc-13 homolog A, C. elegans [UNC13A]; RR = 5.65; p-value = 0.003; recessive genotypic model) and rs1800435ge Age at onset Pathological diagnosis1.2 ± 10.2 (35–90) N/A N/A9.4 ± 10.0 (35–90) 56.5 ± 9.1 (34–83) 123 (37.3%)9.6 ± 10.0 (35–90) 56.8 ± 9.1 (34–83) 112 (42.3%)3.1 ± 12.2 (35–90) 57.7 ± 9.8 (34–79) 45 (60.8%)0.6 ± 8.5 (39–80) 56.2 ± 9.0 (34–74) 51 (71.8%)6.9 ± 8.6 (37–83) 56.5 ± 8.7 (36–83) 16 (13.3%)range). The age provided is age at blood draw in controls, age at onset in. Information was unavailable for age (n = 41) and age at onset (n = 59).Table 2 Variants significantly associated with age at onset or survival after onsetVariant (gene/disease group) Number of patients with each genotypea Model Association measure (95% CI) P-valueAge at onset (overall) Regression coefficientrs7018487 (UBAP1) 122 / 96 / 23 Additive −2.62 (−4.36, −0.89) 0.003rs6052771 (PRNP) 92 / 104 / 46 Recessive 4.42 (1.51, 7.32) 0.003rs7403881 (MT-Ie) 65 / 118 / 60 Dominant 3.95 (1.36, 6.54) 0.003Survival after onset (overall) Relative riskrs5848 (GRN) 116 / 86 / 19 Additive 1.64 (1.22, 2.22) 0.001Survival after onset (disease subgroups) Relative riskrs7403881 (MT-Ie): FTD 13 / 31 / 14 Recessiveb 3.81 (1.71, 8.46) 0.001rs13268953 (ELP3): FTD 16 / 31 / 11 Recessive 3.65 (1.56, 8.55) 0.003Epsilon 4 (APOE): FTD 42 / 13 / 2 Dominant 3.13 (1.45, 6.74) 0.004rs12608932 (UNC13A): MND 44 / 40 / 23 Recessiveb 5.65 (1.82, 17.58) 0.003rs1800435 (ALAD): MND 88 / 18 / 1 Dominant N/Ac 0.003Association measure = regression coefficient (age at onset analysis) and relative risk (survival after onset analysis); CI = confidence interval. Additive models, dominantmodels, and recessive models were utilized. We adjusted for multiple testing using a false discovery rate (FDR) of 10%. aOrder of genotypes: major-major/major-minor/minor-minor. bIndicates that the variant was also significantly associated with the given outcome under an additive model. cFor rs1800435, none of the 19 MND patients(0.0%) who carried the minor allele died as compared to 14 of 78 MND patients (15.9%) who did not carry the minor allele; the p-value of 0.003 results from a log-ranktest. The strongest association with the given outcome is displayed in this table; other associations are shown in Additional file 1: Table S2 (age at onset) and Additionalfile 1: Table S3 (survival after onset).Figure 1 Associations with age at onset in the overall group of FTD, FTD/MND, and MND probands. Three variants are shown thatdemonstrate a significant association with age at onset in C9ORF72 expansion carriers (rs7018487:T > G [UBAP1; panel A], rs6052771:A > G [PRNP;panel B], and rs7403881:G > C [MT-Ie; panel C]). In each panel, the mean in the given group is denoted by a solid horizontal line; associations arespecified in Table 2 and genotype frequencies in Additional file 1: Table S1.van Blitterswijk et al. Molecular Neurodegeneration 2014, 9:38 Page 3 of 10http://www.molecularneurodegeneration.com/content/9/1/38Figure 2 Variants significantly associated with survival after onset. Six significant associations with survival after onset are presented (rs5848:G > A [GRN; panel A], rs7403881:G > C [MT-Ie; panel B], rs13268953:A > G [ELP3; panel C], the epsilon 4 allele:E4- > E4+ [APOE; panel D], rs12608932:A > C [UNC13A; panel E], and rs1800435G > C [ALAD; panel F]). When three curves are shown (rs5848), zero copies of the minor allele aredisplayed in black, one copy of the minor allele is displayed in blue, and two copies of the minor allele are displayed in red. If two curves arepresent (other variants), then the common genotype is shown in black and the rare genotype is shown in blue.van Blitterswijk et al. Molecular Neurodegeneration 2014, 9:38 Page 4 of 10http://www.molecularneurodegeneration.com/content/9/1/38van Blitterswijk et al. Molecular Neurodegeneration 2014, 9:38 Page 5 of 10http://www.molecularneurodegeneration.com/content/9/1/38(delta-aminolevulinate dehydratase [ALAD]; 0.0% deathin carriers of minor allele versus 15.9% in non-carriers;p-value = 0.003; dominant genotypic model).Of note, all results of statistical analyses involving dis-ease risk, age at onset and survival after onset were verysimilar when including individuals who were family mem-bers or who had received another diagnosis, and alsowhen additionally adjusting models for age in the diseaserisk analysis (data not shown).DiscussionThis study was designed to help elucidate the clinical vari-ability observed in C9ORF72 expansion carriers. Weinvestigated variants previously implicated in FTD and/orMND, and determined their effects in a unique cohort ofsubjects with known pathogenic expansions in C9ORF72.Excitingly, we discovered eight variants that may assist inexplaining the reported phenotypic variability, especiallywith regard to age at onset and survival after onset(Table 2). Although it should be stressed that replicationis needed, our results represent a major step forward inthe search for genetic modifiers, and they provide direc-tions for future validation and meta-analytical studies.We identified one single nucleotide polymorphism(SNP) located near UBAP1 (rs7018487) that was associ-ated with age at onset in our overall group of C9ORF72expansion carriers (p-value = 0.003). UBAP1 functions inubiquitin-dependent sorting at the multivesicular body(MVB), and depletion of UBAP1 severely disrupts thiscomplex process [16,17]. Variants in UBAP1 have alreadybeen linked to FTD risk, and colocalization of UBAP1 andTDP-43 in neuronal cytoplasmic inclusions has been dem-onstrated [18]. Our results also revealed an associationbetween PRNP (rs6052771; in LD with rs1799990) andage at onset in our overall group (p-value = 0.003). Thecontribution of PRNP to the pathogenesis of FTD and/orMND has not been studied thoroughly [19,20], and conse-quently, little is known about its effects on these diseases.One study, however, reported an association of PRNP withage at onset in a small number of FTD patients harboringGRN mutations [21], supporting the premise of a commonunderlying mechanism.Moreover, we discovered a variant in metallothionein(rs7403881) that is associated with a delayed age at onsetin our overall group (p-value = 0.003). In addition to thisdelay, we detected a decrease in survival after onset in ourFTD subgroup (p-value = 0.001). Currently, only a fewstudies investigating FTD and/or MND have focused onthe metallothionein family, which is involved in antioxi-dant defense [22]. One of these studies suggested thatrs7403881 increases MND risk [23]. A recent study insuperoxide dismutase-1 (Sod1) mice, revealed that overex-pression of metallothioneins slows disease progressionand extends lifespan [24]. Further evidence for a potentialrole of oxidative stress is provided by the association be-tween survival after onset and a coding SNP in ALAD(rs1800435; p-value = 0.003). The ALAD enzyme influ-ences susceptibility to lead exposure, which may contrib-ute to MND risk; although studies published thus far areinsufficient for a definitive conclusion [25-27].Interestingly, we also found a significant association be-tween a functional SNP in GRN (rs5848; 3’-untranslatedregion [UTR]) and survival after onset in our overall group.It has already been reported that carriers homozygous forthe minor allele of rs5848 demonstrate an increased FTDrisk as compared to homozygous major allele carriers [28],but no associations with either FTD risk or age at onsetwere observed in other studies [21,29-32]. Thus, althoughthe contribution of GRN SNPs to neurodegenerativediseases has not been elucidated, our present finding sug-gests that GRN is associated with survival after onset incarriers of C9ORF72 repeat expansions (p-value = 0.001).We also examined two variants near ELP3 (rs13268953and rs6985069; not in LD). ELP3 is a component of theRNA polymerase II complex, and as such, is involved inthe acetylation of histones H3 and H4 to make DNA ac-cessible for transcription [33,34]. Importantly, anothertype of histone modification has already been implicatedin C9ORF72 expansion carriers: a recent report demon-strated that trimethylation of lysine residues within his-tones H3 and H4 might reduce C9ORF72 expression inexpansion carriers [35]. An association study and muta-genesis screen have also exposed associations betweenELP3 and MND susceptibility [36], representing one ofmany FTD and/or MND-associated genes that function inRNA-processing pathways [37]. Our present findings arein agreement with these studies, as shown by thecombined effects of these ELP3 SNPs in our overall group(p-value = 0.001), and one ELP3 SNP (rs13268953) in ourFTD subgroup (p-value = 0.003).In addition, we assessed APOE, a gene that has beencarefully investigated, particularly in patients withdementia. A recent meta-analysis included 28 case–con-trol studies, and demonstrated that the epsilon 4 alleleincreases susceptibility to FTD [38]. Interestingly, wediscovered that the APOE epsilon 4 allele was associatedwith a decline in survival after onset in our FTD sub-group (p-value = 0.004).Our last potential modifier (rs12608932), an intronicSNP in UNC13A, has been identified through a genome-wide association study in MND patients [39]. This findingwas strengthened by an analysis of expression quantitativetrait loci (eQTLs) that demonstrated genome-wide signifi-cance for UNC13A [40]. UNC13A is involved in neuro-transmitter release [41], a tightly regulated process that isthought to be disrupted in MND patients. Our resultsshow that variants in UNC13A are also associated with sur-vival after onset in the presence of a C9ORF72 repeatvan Blitterswijk et al. Molecular Neurodegeneration 2014, 9:38 Page 6 of 10http://www.molecularneurodegeneration.com/content/9/1/38expansion: we detected an association in our MND sub-group (p-value = 0.003).We would like to reiterate that we performed a system-atic study of variants reported in the literature. For manyvariants, however, previous findings were inconclusive andbased on our current discoveries we speculate that someof the seemingly conflicting results are due to differencesin the composition (and size) of study cohorts, most im-portantly: (1) the number of patients with predominantFTD, predominant MND or a mixture of both diseases,(2) the percentage of subjects with a pathologically con-firmed diagnosis, and (3) the subset of individuals withpathogenic mutations in particular FTD and/or MND-associated genes (such as C9ORF72). Hence, because ofour present findings and results of aforementioned stud-ies, reinvestigation of previously published data after ex-clusion of certain subgroups seems warranted, and newwell-sized studies should be performed concentrating onthese subgroups, in order to determine the specificity ofresults.In our study, we used an FDR rather than a family-wise error rate (FWER)-controlling procedure formultiple testing adjustments. The FDR procedure is rela-tively new, and controlling the FDR is a valid method toadjust for multiple comparisons [42]. An FDR correc-tion, however, is less conservative than an FWER correc-tion and its interpretation is different (Methods). Weused an FDR of 10%, which means that for each groupof statistically significant associations we would expectthe vast majority (90%) to be real (i.e. for each grouponly 1 out of 10 significant findings is expected to befalse). Naturally, there is always a balance between thetwo different types of statistical error that can occur forany given conclusion – a type I error (i.e. a false-positiveassociation) and a type II error (i.e. a false-negative asso-ciation), both of which are undesirable. Because the bal-ance tips more in the direction of type I error for theFDR than for the FWER procedures, it is important tohighlight that our results, though promising, do requirevalidation.Additionally, it should be noted that we focused ourarticle on those associations that remained significantafter adjustment for multiple testing. Future studiescould investigate nominally significant associations(Additional file 1: Table S2 and Additional file 1: TableS3) in larger cohorts and/or meta-analyses, to determinewhether any of these potential associations contribute tothe pleiotropy detected in C9ORF72 expansion carriers.Other studies could also concentrate on variants not in-cluded in our present study (i.e. recently published vari-ants); especially since it seems plausible that morevariants (either known or unknown) modify the pheno-type of C9ORF72 expansion carriers. Furthermore, ourstudy was designed to investigate associations withdisease risk (i.e. by comparing patients and controls) andto identify factors that could modify age at onset or sur-vival after onset. Interestingly, some of the associationswe observed were only significant in the phenotypic sub-group for which the risk variant was originally reported;for example, APOE genotypes only affected survival afteronset in our subgroup of C9ORF72 expansion carrierswith FTD, whereas the UNC13A variant only affectedsurvival after onset in our MND subgroup. To furtherinvestigate the clinical phenotype, a larger number ofexpansion carriers with either FTD or MND is needed(e.g. international genome-wide association study), sothat direct comparisons of expansion carriers with FTDand MND could be performed.ConclusionsOur present study reveals eight variants that may accountfor the phenotypic variability reported in C9ORF72 expan-sion carriers. These variants strongly emphasize the import-ance of proper protein degradation, antioxidant defense,and processing of RNA. Although identified genes (andtheir corresponding pathways) have already been linked toFTD and/or MND, it was unclear whether they were ableto act as disease modifiers on the background of aC9ORF72 repeat expansion. Our findings, thus, underscorethe complex interplay between many factors that influencethe occurrence and prognosis of these destructive diseases,particularly in C9ORF72 expansion carriers. Though largefor a study of C9ORF72 expansion carriers, our findings re-sult from a relatively small sample size, and therefore, re-peated replication and meta-analyses will be necessary toincrease our understanding of these potential genetic dis-ease modifiers. With that said, the factors identified in thisstudy may represent excellent targets for novel treatments,including preventative treatment strategies, and for thedevelopment of predictive tests aiming at the continuum ofFTD and MND.MethodsSubjectsWe collected DNA from a cohort of 330 C9ORF72 ex-pansion carriers, obtained at the Mayo Clinic (n = 121),Coriell Research Institute (n = 71), University of BritishColumbia, Canada (n = 58), University of California,San Francisco (n = 38), Robarts Research Institute (n =11), Northwestern University Feinberg School of Medicine(n = 9), Drexel University College of Medicine (n = 7),University of Western Ontario, Canada (n = 7), BannerSun Health Research Institute (n = 5), and University ofTübingen (n = 3). Based on available clinical and/orpathological data, these subjects were diagnosed with FTD(n = 91), FTD/MND (n = 78) or MND (n = 127), withanother diagnosis (n = 7; e.g. dementia due to Alzheimer’sdisease, alcohol abuse or behavioral impairment), or theyvan Blitterswijk et al. Molecular Neurodegeneration 2014, 9:38 Page 7 of 10http://www.molecularneurodegeneration.com/content/9/1/38were asymptomatic at time of last evaluation (n = 27; meanage at evaluation: 43.6 ± 12.7 standard deviation [SD]). Ofthose expansion carriers 45.2% (n = 149) were female, theirmean age was 59.4 ± 10.0 years, their main age at onsetwas 56.5 ± 9.1 years, and 37.3% (n = 123) had received aneuropathological diagnosis (Table 1). Age at onset was es-timated based on the appearance of the first disease symp-toms, namely progressive cognitive dysfunction injudgment, language, or memory; or changes in behavior orpersonality (FTD patients); or fasciculations, muscle weak-ness, falls, dysarthria, and dysphagia (MND patients).When symptoms of both FTD and MND were noted, theearliest observation of decline was recorded for age at on-set. Survival after onset was defined as the interval betweenage at onset of disease symptoms and the age at death fordeceased patients, and as the interval between age at onsetand present age for other patients (when follow-up datawas available).We also included neurologically normal controls (n =374), of whom 46.0% (n = 172) were female, and whosemean age was 61.2 ± 10.2 years. All subjects agreed to be inthe study, and biological samples were obtained after in-formed consent with ethical committee approval from therespective institutions. Approval for the genetic analyseswas performed in agreement with ethical committee ap-proval at Mayo ClinicGenotypingC9ORF72 expansion carriers were identified using ourpreviously published 2-step PCR protocol [7]; Southernblotting techniques were employed to confirm the pres-ence of the repeat expansion when sufficient high qualityDNA was available (>25% of expansion carriers) [14]. Toselect candidates that could potentially act as diseasemodifiers in carriers of C9ORF72 repeat expansions, weperformed a literature search on PubMed (August 2012)that revealed all publications on a combination of FTDand/or MND with SNPs. Subsequently, we selected one ortwo variants per gene possibly associated with these dis-eases (top SNPs were preferred), which were suitable forthe Sequenom MassArray iPLEX platform (San Diego,CA, USA) and could be incorporated in Sequenom panels;these variants were analyzed with Typer 4.0 software.Sequenom genotype data was supplemented with five Taq-man SNP genotyping assays (C_3084793_20, C_1085600_10, C_2070266_20, C_8921964_20, and C_7563736_10;Invitrogen, Carlsbad, CA, USA) performed on a 7900HTFast Real Time PCR system; genotype calls were madeusing SDS 2.4 software (Applied Biosystems, Foster City,CA, USA). After genotyping, we excluded SNPs with asignificant deviation from the Hardy-Weinberg equilib-rium (HWE) in our control cohort (rs45559331 andrs6903982), rare SNPs with a minor allele frequency(MAF) of less than 1% in expansion carriers and controls(rs121909536, rs75654767, rs121909541, rs140547520,rs80265967, rs80356715, and rs35070491), and SNPs witha call rate below 95% (rs4680, rs4859146, rs854560,rs7277748, rs4880, and rs2275294). In total, 36 variantswere included in our analysis (Additional file 1: Table S1);the call rate of these variants was greater than 99% andnone of these variants was in LD. All genetic analyses wereperformed at the Mayo Clinic, and genotypes were assignedusing all of the data from the study simultaneously.Statistical analysisIn order to satisfy the statistical assumption of independ-ent measurements, our primary analysis focused on a sub-set of C9ORF72 expansion carriers: 265 unrelatedprobands with FTD (n = 74), FTD/MND (n = 71), orMND (n = 120). We performed secondary analyses, how-ever, that included the remaining expansion carriers toexamine the sensitivity of our results. The entire cohort ofC9ORF72 expansion carriers was assessed, and also dis-ease subgroups separately (FTD, FTD/MND and MND).First, we used logistic regression models adjusted for gen-der to evaluate associations of each of the 36 variants withdisease risk; odds ratios (ORs) and 95% confidence inter-vals (CIs) were estimated. In addition, we examined asso-ciations of each of these variants with age at onset usinglinear regression models adjusted for gender and diseasesubgroup; while associations with survival after onset wereassessed using Cox proportional hazards regressionmodels adjusted for age at onset, gender, and disease sub-group. Regression coefficients (interpreted as changes inmean age at onset) and 95% CIs were estimated in the ageat onset linear regression analysis; whereas in Cox regres-sion analysis, RRs and 95% CIs were estimated, and datawas censored at last follow-up. Each variant was investi-gated under an additive genotypic model (effect of eachadditional minor allele), a dominant genotypic model(presence versus absence of the minor allele), and a reces-sive genotypic model (presence versus absence of two cop-ies of the minor allele). Models were not adjusted forC9ORF72 expansion size, since expansion sizes were onlyavailable for a subset of samples (>25%) and they were es-timated in DNA obtained from various tissues, whichhampers analyses [14]. In order to reduce the chance ofspurious findings and non-informative tests, associationanalyses were not performed for variants with fewer thanten carriers of the minor allele in the given group, orunder an additive or recessive genotypic model whenfewer than ten rare homozygotes were present in the givengroup.To account for multiple testing, we made an adjust-ment separately for each disease group and separatelyfor each outcome measure (disease risk, age at onset,and survival after onset). Given the relatively small sam-ple size of this study, controlling the FWER (i.e. theUSA. 8Department of Neuropathology, University of Tübingen and Germanlobar degeneration: consensus recommendations. Acta Neuropathol 2009,van Blitterswijk et al. Molecular Neurodegeneration 2014, 9:38 Page 8 of 10http://www.molecularneurodegeneration.com/content/9/1/38probability of any false-positive finding among the entiregroup of tests) at 5% using a procedure such as a single-step minP permutation correction [43] would result in verylow power to detect associations. We, therefore, opted foran alternative approach and utilized an FDR correction[44]. This increasingly used method has a different inter-pretation than FWER-controlling procedures; an FDR pro-cedure attempts to control the expected proportion offalse-positive findings among those associations consideredsignificant. Note that due to this difference in interpret-ation, the FDR does not necessarily need to be controlledat 5%, only at a reasonable level to allow for high confi-dence in results, which was deemed at 10% for our study[42]. All statistical tests were two-sided, and were per-formed using SAS (version 9.2; SAS Institute, Inc., Cary,NC, USA) and R Statistical Software (version 2.14.0; RFoundation for Statistical Computing, Vienna, Austria).Additional fileAdditional file 1: Genotype counts and frequencies (Table S1),Associations with age at onset under additive, dominant andrecessive models in the overall group of FTD, FTD/MND, and MNDprobands (n = 243; Table S2), Associations with survival after onsetunder additive, dominant and recessive models in the overall groupof FTD, FTD/MND, and MND probands (n = 221; Table S3a),Associations with survival after onset under additive, dominant andrecessive models in FTD probands (n = 58; Table S3b), Associationswith survival after onset under additive, dominant and recessivemodels in MND probands (n = 107; Table S3c), Combinations ofELP3 variants rs13268953 and rs6985069 in relation to survival afteronset in the overall group (Table S4).AbbreviationsFTD: Frontotemporal dementia; MND: Motor neuron disease;TDP-43: transactive response DNA-binding protein 43;C9ORF72: Chromosome 9 open reading frame 72;TMEM106B: Transmembrane protein 106 B; ATXN2: Ataxin-2; FDR: Falsediscovery rate; UBAP1: Ubiquitin-associated protein 1; PRNP: Prion protein;MT-Ie: Metallothionein 1 E; GRN: Granulin precursor; RR: Relative risk;ELP3: Elongator acetyltransferase complex subunit 3; LD: Linkagedisequilibrium; APOE: Apolipoprotein E; UNC13A: unc-13 homolog A, C.elegans; ALAD: Delta-aminolevulinate dehydratase; SNP: Single nucleotidepolymorphism; MVB: Multivesicular body; Sod1: Superoxide dismutase-1;UTR: Untranslated region; eQTLs: Expression quantitative trait loci;FWER: Family-wise error rate; SD: Standard deviation; HWE: Hardy-Weinberg equilibrium; MAF: Minor allele frequency; OR: Odds ratio;CI: Confidence interval.Competing interestsMariely DeJesus-Hernandez and Rosa Rademakers hold a patent on methodsto screen for the hexanucleotide repeat expansion in the C9ORF72 gene.None of the other authors declares that they have competing interests.Authors' contributionsMvB participated in the study concept and design, carried out moleculargenetic studies, interpreted data, and additonally, drafted and revised themanuscript. BM, AW, MCB, MD-H, PHB, and MEM made substantialcontributions to the acquisition of data, and the analysis or interpretation ofdata. MGH and NND performed statistical analyses and revised themanuscript. G-YRH, HS, AMK, EF, AK, EHB, SW, MM, KJH, CLWIII, MN, MJS, TGB,ZKW, CL, RC, LP, KAJ, JEP, DSK, WWS, and LTG contributed vital reagents/tools/patients and revised the manuscript. RCP, IRM, BLM, KBB, NRG-R,BFB, and DWD obtained study funding, contributed vital reagents/tools/117:15–18.7. DeJesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M,Rutherford NJ, Nicholson AM, Finch NA, Flynn H, Adamson J, Kouri N,Wojtas A, Sengdy P, Hsiung GY, Karydas A, Seeley WW, Josephs KA, CoppolaG, Geschwind DH, Wszolek ZK, Feldman H, Knopman DS, Petersen RC, MillerCenter for Neurodegenerative Diseases, Calwerstr. 3, Tübingen 72076,Germany. 9Molecular Brain Research Group, Robarts Research Institute, 100Perth Drive, London, ON N6A 5 K8, Canada. 10Banner Sun Health ResearchInstitute, 10515 W Santa Fe Dr, Sun City, AZ 85351, USA. 11Department ofNeurology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224, USA.12Department of Neurology, Drexel University College of Medicine, 2900 WQueen Ln, Philadelphia, PA 19129, USA. 13Department of Neurology, MayoClinic, 5777 E Mayo Blvd, Phoenix, AZ 85054, USA. 14Department ofNeurology, Mayo Clinic, 1216 2nd St SW, Rochester, MN 55902, USA.15Department of Pathology and Laboratory Medicine, University of BritishColumbia, 2329W Mall, Vancouver, BC V6T 1Z4, Canada.Received: 7 August 2014 Accepted: 29 August 2014Published: 20 September 2014References1. 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