(KIFAP3) Has No Effect on Survival in a Population-Based Cohort of ALS Patients

Kinesin-associated protein 3 (KIFAP3) has no effect on survival in a population-based cohort of ALS patients

Bryan J. Traynora,1, Michael Nallsb, Shiao-Lin Laia,c,d, Raphael J. Gibbsb,c, Jennifer C. Schymicka, Sampath Arepallib, Dena Hernandezb,c, Marcel P. van der Bruge,f, Janel O. Johnsona, Allissa Dillmane, Mark Cooksone,CristinaMogliag, Andrea Calvog, Gabriella Restagnoh,2, Gabriele Morai,2, and Adriano Chiòg,1,2

aNeuromuscular Diseases Research Group, bMolecular Genetics Section, and eCell Biology and Gene Expression Unit, Laboratory of Neurogenetics, National Institute on Aging, Bethesda, MD 20892; cDepartment of Molecular Neuroscience and Reta Lila Weston Institute of Neurological Studies, Institute of Neurology, London WC1 3BG, United Kingdom; dChang Gung University Hospital and College of Medicine, Chiayi, Taiwan; fDepartment of Neuroscience, The Scripps Research Institute, Jupiter, FL 33458; gDepartment of Neuroscience, University of Turin, 10126 Turin, Italy; hMolecular Genetics Unit, Department of Clinical Pathology, Azienda Ospedialiera Ospedale Infantile Regina Margherita-S.Anna, 10126 Turin, Italy; and iFondazione Salvatore Maugeri, 20138 Milan, Italy

Edited* by Don W. Cleveland, University of California, La Jolla, CA, and approved May 10, 2010 (received for review December 4, 2009) It was recently reported that rs1541160 on chromosome 1q24.2 has population structure (component vectors 1 and 2 from multidi- a marked effect on survival of amyotrophic lateral sclerosis (ALS) mensional scaling) as covariates, was also not significant (β co- patients by influencing KIFAP3 expression. The cohorts used in that efficient = 0.024, P = 0.89). Survival analysis limited to the cohort study were collected from ALS specialty clinics. We attempted to of patients attending a specialized ALS clinic was similarly nega- replicate these findings in a population-based cohort of 504 Italian tive [n = 89 patients (Fig. S1)]. None of the 139 other SNPs within ALS patients. None of 140 SNPs genotyped within the KIFAP3 locus the 1-Mb region surrounding KIFAP3 significantly influenced (including rs1541160) had an effect on survival (log-rank P value for survival of the Italian ALS cohort (Table S1). rs1541160 = 0.47) or on gene expression in that region. These data Expression quantitative trait loci (eQTL) mapping of the illustrate the complexities associated with analyzing ALS pheno- chromosome 1q24 region demonstrated that rs1541160 had no types for association. effect on KIFAP3 expression within the frontal cortex, temporal cortex, pons, or cerebellum of ∼144 neurologically normal brains amyotrophic lateral sclerosis | Italian | survival analysis | genome-wide [r2 in cerebellum = 0.01, P = 0.232; r2 in frontal cortex = 7.0 × − − association study | gene expression quantitative trait loci mapping 10 4, P =0.76;r2 in pons = 2.1 × 10 5, P =0.96;r2 in temporal cortex = 0.035, P = 0.03 (Fig. 2)]. Furthermore, none of the other myotrophic lateral sclerosis (ALS) is a fatal neurodegenera- SNPs within the locus influenced KIFAP3 expression or expres- Ative disease characterized by rapidly progressive paralysis sion of any nearby gene (Fig. 3). To confirm that this discrepancy leading to death from respiratory failure. Population-based epi- with the work of Landers et al. (5) was not due to methodological demiological studies indicate that the median survival of ALS differences, we quantified expression of KIFAP3 in our 143 fron- patients is 20–36 mo (1–3). Demographic and clinical features, as tal cortex samples using the same Taqman gene expression assays well as riluzole and certain symptomatic therapies, are known to used in their work. These experiments confirmed the lack of ef- modestly modify survival, but the influence of genetics on the rate fect of rs1541160 on KIFAP3 expression levels [hs00183973, r2 = of disease progression and survival is not known (4). Landers et al. 0.003, P = 0.86; hs00946074, r2 = 0.018, P = 0.38 (Fig. S2)]. We (5) recently reported that the CC genotype of SNP rs1541160 also attempted to replicate the semiquantitative Western blot within the kinesin-associated protein 3 (KIFAP3)geneonchro- analysis of KIFAP3 published by Landers et al. (5) using frontal mosome 1q24.2 conferred a 14-mo survival advantage on ALS cortex samples from neurologically normal individuals who were patients. Furthermore, expression data based on 26 occipital lobe homozygous for either the minor or major allele of rs1541160. brain samples suggested that the CC genotype of this SNP re- These experiments used the same Santa Cruz Biotechnology duced KIFAP3 expression by 41% compared with the TT geno- KIFAP3 antibody as in the original work, and a second KIFAP3 type (5). To confirm the accuracy of this report, we attempted to antibody from BD Biosciences. Neither antibody demonstrated replicate this finding in a population-based cohort of 504 Italian a significant effect of SNP genotype on protein level [Santa Cruz ALS patients collected using the Piemonte and Valle d’Aosta Biotechnology antibody sc-55598, P = 0.88; BD Biosciences an- Registry for ALS (PARALS). We also evaluated the effect of the tibody 610637, P = 0.57 (Fig. S3)]. identified SNP, and all SNPs within the 1-Mb surrounding region, on the expression of KIFAP3 in four brain regions obtained from 144 neurologically normal individuals. Author contributions: B.J.T., M.N., R.J.G., J.C.S., M.P.v.d.B., M.C., G.R., G.M., and A. Chiò designed research; B.J.T., S.-L.L., J.C.S., S.A., D.H., M.P.v.d.B., J.O.J., A.D., M.C., C.M., Results A. Calvo, G.R., G.M., and A. Chiò performed research; B.J.T., M.N., R.J.G., S.A., D.H., M.P.v.d.B., J.O.J., A.D., M.C., C.M., A. Calvo, G.R., G.M., and A. Chiò analyzed data; and B.J.T., Rs1541160 had no effect on survival in our population-based co- M.N., S.-L.L., R.J.G., J.C.S., S.A., D.H., M.P.v.d.B., J.O.J., A.D., M.C., C.M., A. Calvo, G.R., G.M., and hort of 504 Italian ALS cases either under the genotypic model A. Chiò wrote the paper. NEUROSCIENCE [median survival for CC genotype = 4.09 y; CT genotype = 3.48 y; The authors declare no conflict of interest. TT genotype = 3.58 y; log-rank P = 0.48, Peto P = 0.60 (Fig. 1A)] *This Direct Submission article had a prearranged editor. or under the allelic model [log-rank P =0.33,PetoP =0.53(Fig. Freely available online through the PNAS open access option. B 1 )]. Similarly, survival analysis limited to the 318 deceased ALS Data deposition: The data have been deposited in the dbGaP database, phs000101.v2.p1. patients failed to demonstrate an effect of rs1541160 on survival 1To whom correspondence may be addressed. E-mail: [email protected] or achio@ either under the genotypic model [log-rank P = 0.55, Peto P = usa.net. 0.92 (Fig. 1C)] or under the allelic model [log-rank P = 0.42, Peto 2G.R., G.M., and A. Chiò contributed equally to this work. P D =0.71(Fig.1 )]. Linear regression modeling of the deceased- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. only cohort, incorporating age, gender, site of symptom onset, and 1073/pnas.0914079107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0914079107 PNAS | July 6, 2010 | vol. 107 | no. 27 | 12335–12338 Downloaded by guest on September 26, 2021 Fig. 1. Survival from time of symptom onset in a population- based cohort of Italian ALS patients according to rs1541160 status. (A) Kaplan–Meier curves for all 504 patients according to rs1541160 genotype status. (B) Kaplan–Meier curves for all 504 patients according to rs1541160 C allele carrier status. (C) Kaplan–Meier curves restricted to 318 deceased patients according to rs1541160 genotype status. (D) Kaplan–Meier curves for 318 deceased patients according to rs1541160 C allele carrier status. +, censored events.

Discussion surprising because Landers et al. (5) reported that the survival Our data clearly show that SNP rs1541160 does not modify advantage associated with this SNP on chromosome 1q24 was survival within our cohort of 504 Italian ALS cases. This result is large enough (14.9 mo representing a 44.5% increase in median

Fig. 2. Boxplots illustrating the dose relationship between allele load at rs1541160 and expression of KIFAP3 in human cerebellar tissue samples (n = 143), frontal cortex samples (n = 143), pons samples (n = 141), and temporal cortex samples (n = 144). Binary logarithm of residual expression is shown on the y axis, and rs1541160 genotypes are listed along the x axis. Notably, rs1541160 has no effect on KIFAP3 expression in any of the tested tissue types.

12336 | www.pnas.org/cgi/doi/10.1073/pnas.0914079107 Traynor et al. Downloaded by guest on September 26, 2021 allele frequency of rs1541160 in the Italian population (C allele frequency = 30.8%) was almost identical to those reported for the European and US populations in the original work (28.6–30.2%, respectively) and observed within the CEPH HapMap population (26.7%, release 23, 60 founders). It is also possible that the spe- cificSNPthatinfluences survival within the chromosome 1 locus varies from population to population. However, our data effec- tively exclude this scenario because none of the 1,004 SNPs (140 genotyped SNPs plus 864 imputed SNPs) within the 1 Mb surrounding the KIFAP3 locus had a significant effect on survival within the Italian cohort. Our eQTL mapping data and Taqman gene expression assays failed to demonstrate an effect of rs1541160 or any SNP within the locus on KIFAP3 expression within ∼144 samples from four dis- tinct brain regions. This observation agrees with three previously published eQTL datasets, including a study involving 427 liver samples (11), a previous study from our laboratory that analyzed 279 additional frontal cortex samples (12), and an analysis of data derived from 210 lymphoblast cell lines (Fig. S4) (13). In contrast, Landers et al. (5) found that the CC genotype of rs1541160 de- creased expression of KIFAP3 by 41% based on 26 brain samples. Population differences cannot account for these contrasting results, as all 144 brains used to generate our expression data were non-Hispanic Europeans drawn from a similarly representative US population sample as used in the work of Landers et al. (5). Instead, the larger number of samples included in our expression analysis resulted in increased power to detect true quantitative Fig. 3. Expression quantitative trait loci (eQTL) across the KIFAP3 locus on chromosome 1q24 measured in human cerebellar tissue samples (n = 143), trait loci, and to exclude false-positive associations. frontal cortex samples (n = 143), pons samples (n = 141), and temporal cortex In conclusion, our data did not replicate the reported effect of samples (n = 144). In this analysis the allelic load at each of the 1,004 poly- rs1541160 on ALS survival or on KIFAP3 expression in a pop- morphisms across the locus is tested for association with transcript levels of ulation-based Italian sample. To date, none of the reported asso- each gene within the locus. The results of the analysis are shown as log ciations for ALS have been successfully replicated, suggesting that transformed P values (based on Cochran–Armitage test for trend) that are ALS may be a more genetically heterogeneous disease than pre- color-coded to match the transcript of interest. Notably, genotypes across viously recognized, and that results of genome-wide studies should this locus are not associated with KIFAP3 (blue), SCYL3 (green), or SCYL1BP1 be interpreted with caution. This may be especially true in the (red) expression levels. Only transcripts expressed in each tissue are dis- played. SCYL3 expression was not detected in cerebellar tissue. The dashed analysis of phenotype aspects of ALS (e.g., survival, age at onset, horizontal line represents the threshold for genome-wide significance (8.85 rate of progression) based on patients attending specialty ALS × 10−8), and the dotted horizontal line represents the threshold for signifi- clinics, because the demographics of such cohorts may be influ- cance correcting for 1,004 SNPs within the locus [0.05/(1,004 × 4 tissues) = enced by referral bias. − 1.25 × 10 5]. The vertical black arrow indicates the position of rs1541160. Material and Methods Samples. Included in the study were 504 cases diagnosed with probable or survival of patients) to be consistently observed among the dif- definite ALS according to the El Escorial criteria (14). These cases were ferent, similar sized cohorts included in their analysis. One pos- ascertained through PARALS, an ongoing population-based epidemiological sible explanation for this discrepancy may arise from the manner study of ALS based in two regions of northwestern Italy (population = in which the different cohorts were ascertained; the Italian cohort 4,332,842 in 2001) (6). The cohort consisted of 270 (53.6%) men and 234 was collected in a population-based manner (6), whereas the (46.4%) women. 367 (72.8%) described limb-onset symptoms, whereas the remaining 137 (27.2%) presented with bulbar-onset disease. Mean age at cohorts in the original work were mostly drawn from patients at- onset was 61.5 y (SD = 11.1, range = 20.5–87.3), and 318 (63.1%) patients tending ALS clinics (i.e., clinic series) (5). Survival statistics based were deceased or had undergone tracheostomy at the time of last follow- on such clinic cohorts are known to consistently underestimate up. Of the entire cohort, 449 (89.1%) were taking riluzole medication; of ALS mortality by up to one-third because of referral bias (4, 7). deceased patients, 283 (89.0%) were taking riluzole. Median survival of the Longer living patients are more likely to attend an ALS specialty entire cohort (n = 504) was 3.54 y (95% CI, 3.33–3.95 y), whereas median clinic, which in turn means they are more likely to access the survival of the deceased-only cohort (n = 318) was 2.58 y (95% CI, 2.42–2.8 y). disease-modifying medication riluzole and to have access to Median survival and riluzole use by rs1541160 genotype status is listed in Table S2. Written informed consent for genetic analysis was obtained from symptomatic therapies that further prolong their survival (8, 9). In fl each individual, and appropriate institutional review board approval was contrast, the population-based cohorts are more re ective of the obtained concerning human subjects. true survival pattern within the general ALS population. Another possible explanation for our observations is that the original as- Genotyping. Two hundred sixty-eight samples were genotyped on Infinium NEUROSCIENCE sociation of KIFAP3 with ALS survival was a false-positive finding HumanHap550 beadchips (Illumina) (15), and 236 samples were genotyped arising by chance from the several hundred thousand tests per- on Infinium HumanHap610-Quad beadchips (Illumina) according to the formed as part of any GWAS (10). manufacturer’s specifications; 535,468 SNPs were common across both Population-specific genetic variability within the chromosome 1 platforms. These included rs1541160 and 140 other SNPs extending from rs10800456 (167.7 Mb) to rs1928716 (168.7 Mb, genome build 36.3) on region is unlikely to account for the discrepancy between our data chromosome 1q24.2. and the original Landers et al. report (5). It is unlikely that fl rs1541160 would in uence survival within the four European-an- Statistical Analysis. Kaplan–Meier survival modeling (16) and differences in cestry populations reported in the original work and have no ef- survival were measured by the logrank sum test (all cases weighted equally) and fect within a similarly sized Italian cohort. Furthermore, the minor the Peto & Peto Generalized Wilcoxon test (earlier events weighted more

Traynor et al. PNAS | July 6, 2010 | vol. 107 | no. 27 | 12337 Downloaded by guest on September 26, 2021 heavily) using the survival package within R statistical software (version 2.9.0). (SuperscriptIII; Invitrogen). After generating cDNA, quantitative RT-PCR was Calculations were performed using the date of onset as day 0, and the primary performed with four replicates per sample using Taqman gene expression endpoint was tracheostomy-free survival. Date of last follow up of patients was primers for KIFAP3 (Applied Biosystems; hs00183973 for human KIFAP3, fi rst August 2009. Multivariate linear regression models examining minor allele hs00946074 for human KIFAP3, and 4326317E for human GAPDH). dosage associations with survival duration were adjusted for age at onset, gender,siteofonset,andthefirst two components vectors generated from Semiquantitative Western Blot Analysis of KIFAP3. Brain lysates were prepared multidimensional scaling. Identical covariates were used in subsequent iter- by adding 500 mL of RIPA buffer with protease inhibitor (Protease Inhibitor ations of the time-dependent models. A cohort of 504 patients has 99.99% Mixture Tablets; Roche Applied Science) to ≈50 mg of tissue followed by potential power to detect a 41% difference in survival assuming a 70% 5-y mortality rate in controls and a minor allele frequency of 0.308 (see Fig. S5 for dounce homogenization. The lysate was then sonicated by twenty 1-s pulses power curves). and kept on ice for 30 min. After centrifugation (5,000 × g for 10 min at 4 °C), the supernatant was stored in aliquots at −80 °C. The concentration of each Expression Analysis. As part of a larger project in our laboratory, frozen tissue sample was determined by a BCA assay. Twenty micrograms of lysate was samples of the frontal cortex, temporal cortex, pons, and cerebellum were separated by SDS/PAGE and blotted onto PVDF membrane. The membranes obtained from 144 neurologically normal, American subjects of European an- were hybridized with a monoclonal antibody direct against KIFAP3 (sc-55598; cestry. One hundred- to 200-mg aliquots of frozen tissue were sub-dissected Santa Cruz Biotechnology) at a 1:100 dilution. The BD Biosciences KIFAP3 from each of the samples and used for genotyping and expression assays. Geno- antibody (#610637) was used at 1:500. Rabbit polyclonal antibody directed fi typing was performed using In nium HumanHap550 beadchips (Illumina) fol- against β-Actin (A1978; Sigma-Aldrich) was used at a 1:2,000 dilution. De- lowed by imputation to ∼1.6 million SNPs after data cleaning. Profiling of tection was performed using the ECL Plus Western Blotting Detection System 22,000 mRNA transcripts was performed using HumanRef-8 Expression bead- (GE Healthcare) in conjunction with a Storm 860 Imaging System (Molecular chips (Illumina). A regression analysis was performed on the expression in- fi tensities generated for mRNA. Gender, age at symptom onset, postmortem Dynamics). Quanti cation of band intensity was performed using Image- interval, tissue source, and hybridization batch were included as covariates. Quant software (GE Healthcare). Residuals from the regression analysis for each probe were then used as the quantitative trait for that probe in genome-wide association analysis looking ACKNOWLEDGMENTS. This work was supported by Ministero della Salute, for eQTLs, performed using the assoc function within PLINK, which correlates Ricerca Sanitaria Finalizzata 2007 (to A. Chiò, G.R., and G.M.), Fondazione allele dosage with change in the trait (17, 18). Vialli e Mauro for ALS, Torino (to A. Chiò and G.M.), and Regione Pie- monte, Progetti Finalizzati (to G.R.), and the Intramural Research Program of the National Institutes of Health, National Institute on Aging (Z01- Taqman Expression Assays of KIFAP3. Total RNA was isolated from human AG000949-02), National Institute of Neurological Disorders and Stroke, frontal cortex samples using TRIzol (Invitrogen). RT-PCR was performed from 0.5 the Packard Center for ALS Research at Hopkins (to B.J.T.), and the ALS μg of RNA using oligo-dT primers according to the manufacturer’sinstructions Association (to B.J.T.).

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