An association study between granulin gene polymorphisms and Alzheimer's disease in Finnish population.код для вставкиСкачать
BRIEF RESEARCH COMMUNICATION Neuropsychiatric Genetics An Association Study Between Granulin Gene Polymorphisms and Alzheimer’s Disease in Finnish Population Jayashree Viswanathan, Petra M€akinen, Seppo Helisalmi, Annakaisa Haapasalo, Hilkka Soininen, and Mikko Hiltunen* Department of Neurology and Brain Research Unit, Clinical Research Centre/Mediteknia, University Hospital and University of Kuopio, Kuopio, Finland Received 30 April 2008; Accepted 7 October 2008 Granulin protein plays an important role in neurite outgrowth and neuronal survival. Recently, it was shown that mutations in granulin (GRN) gene cause tau-negative frontotemporal dementia supporting the idea that granulin is involved in neurodegeneration. Here we have investigated whether genetic variability in the GRN gene influences also the risk of developing Alzheimer’s disease (AD). Genotyping of six single nucleotide polymorphisms (SNPs) in the GRN gene among 512 AD patients and 649 control subjects originating from Finland did not show significant association with AD. However, stratification according to gender revealed a significant male-specific allele, genotype and haplotype association between AD and GRN SNPs rs4792939, rs850713, and rs5848. These data suggest that genetic variability in the GRN gene may also increase the risk for developing AD in a gender-specific manner. 2008 Wiley-Liss, Inc. Key words: Alzheimer’s disease; polymorphisms; risk gene; association; neurodegeneration Alzheimer’s disease (AD) is the most common cause of progressive neurological disorder leading to dementia. Several attempts have been made to find novel AD risk genes using candidate genebased association studies in both case–control and family-based settings (see details http://www.alzgene.org). Recently, mutations in the granulin (GRN, also known as progranulin) gene leading to haploinsufficiency of the granulin protein were found to cause taunegative frontotemporal dementia (FTD) linked to chromosome 17q21 [Baker et al., 2006; Cruts et al., 2006]. Although the precise biological functions of granulin have not been determined yet, the initial findings suggest that granulin is crucial for neuronal survival and that the down regulation of this protein leads to neurodegeneration [He and Bateman, 2003; Baker et al., 2006; Cruts et al., 2006]. Consistent with this idea, it was shown that granulin levels were reduced in the cerebrospinal fluid of the FTD patients carrying a GRN mutation [Van Damme et al., 2008]. On the other hand, granulin expression was increased in activated microglial cells obtained from amyotrophic lateral sclerosis patients [Malaspina et al., 2001]. Here we have investigated whether genetic variability in the GRN gene influences also the risk of developing AD using a 2008 Wiley-Liss, Inc. How to Cite this Article: Viswanathan J, M€akinen P, Helisalmi S, Haapasalo A, Soininen H, Hiltunen M. 2009. An Association Study Between Granulin Gene Polymorphisms and Alzheimer’s Disease in Finnish Population. Am J Med Genet Part A 150B:747–750. clinic-based series of AD patients and control subjects originating from Finland. All patients fulfilled the NINCDS-ADRDA criteria for probable AD [McKhann et al., 1984]. The study group consisted of 512 patients with AD (mean age at onset of AD 71 7 years; range 43–89 years; 68.8% women) and 649 age-matched healthy control subjects (mean age at time of neuropsychological examination 69 6 years; range 37–87 years; 59.6% women). Control subjects had no signs of dementia according to interview and neuropsychological testing. There were 87 AD patients and 151 controls with an onset age or age at examination of 65 years. These early onset AD patients did not show conclusive evidence of autosomal dominant transmission [Lehtovirta et al., 1996] and there were no APP or PSEN1/2 mutations. The Ethics Committee of Kuopio University Hospital and Kuopio University has approved the study. We assessed the statistical power for detecting significant differences between AD and control cohorts and found that the population size Grant sponsor: Health Research Council of the Academy of Finland; Grant sponsor: Kuopio University Hospital; Grant Number: 5772708; Grant sponsor: Nordic Centre of Excellence of Neurodegeneration; Grant sponsor: Marie Curie Early Stage Researcher Training Program, BiND. *Correspondence to: Mikko Hiltunen, Ph.D., Department of Neurology, University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland. E-mail: firstname.lastname@example.org Published online 14 November 2008 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/ajmg.b.30889 747 748 AMERICAN JOURNAL OF MEDICAL GENETICS PART B is large enough to detect 1.5-fold risk effect at 80% probability, if the allele frequency difference between cohorts is 0.05. Single nucleotide polymorphisms (SNPs) rs850714, rs3785817, rs4792939, rs850713, rs5848, and rs850737 in the GRN gene were genotyped with TaqMan Assays using TaqMan Master Mix (Applied Biosystems, Foster City, CA). As a quality control measure, 10% of the samples were genotyped in duplicate and the accuracy for genotype calling was 100%. Overall genotyping efficiency for GRN SNPs varied from 94.5% (rs3785817) to 99.5% (rs5848). Apolipoprotein E (APOE) genotyping was performed as previously described [Lehtovirta et al., 1996]. Single locus allele and genotype analyses were performed using SPSS 14.0 software. Pairwise linkage disequilibrium, Hardy–Weinberg equilibrium, haplotype block, haplotype estimation and association, as well as simulation analyses were performed using Haploview 4.1 program (http://www. broad.mit.edu/mpg/haploview/). Sample size and power determinations were performed using nQuery Advisor Release 4.0 software (www.statsolusa.com). Statistical significance was set at P < 0.05 and the P-values for allele and genotype data were corrected for multiple comparisons using Bonferroni correction. The APOE e4 allele was significantly overrepresented among 512 AD patients when compared to 649 control subjects (P < 0.001; OR ¼ 4.5 (95% CI 3.7–5.4)). Six non-coding SNPs around GRN gene were chosen for genotyping based on the allele frequency, linkage disequilibrium (LD) and haplotype block structure information obtained from the population of Western European ancestry (CEU) (Genotype data retrieved from HapMap, http:// www.hapmap.org/) (Table I). These tagged SNPs covered the entire GRN gene including also a significant portion of the 50 and 30 regions flanking UTRs (Fig. 1). Genotyping of Finnish case–control cohort revealed two haplotype blocks at 50 (Block 1; SNPs rs850714 and rs3785817) and 30 (Block 2; SNPs rs850713, rs5848, and rs850737) ends of the GRN gene. Observed haplotype block structure among the whole case–control cohort was similar to that observed initially in the CEU population. Comparison of the genotype and allele distribution of GRN SNPs between AD and control cohorts did not reveal significant allele or genotype association in the whole case–control cohort (Table I). Also, individual haplotype association analysis with SNPs in block 1 (rs850714-rs3785817) or block 2 (rs850713-rs5848-rs850737) did not reveal any significant association. However, stratification according to age (>65 years as a cut-off age), gender and APOE status (APOE e4þ and e4 subgroups) showed a nominally significant allele and genotype association among the subgroup of men with SNPs rs4792939, rs850713, and rs5848 (Table II). SNPs rs850713 (TT þ TC vs. CC, OR ¼ 2.18, 95% CI (1.37–3.48)) and rs5848 (AA þ AG vs. GG, OR ¼ 1.84, 95% CI (1.15–2.96)) conferred an approximately twofold increased risk for AD in logistic regression TABLE I. Allele and Genotype Frequencies of GRN SNPs Among Finnish AD and Control Subjects Allele frequency SNP NCBI rs-number and location (kb)a rs850714 (0) Allele C T Controls n ¼ 1286 0.313 0.687 AD n ¼ 988 0.322 0.678 T C n ¼ 1266 0.648 0.352 A G Genotype frequency P-value* Genotype 0.64 CC TC TT n ¼ 932 0.651 0.349 0.86 TT TC CC n ¼ 1282 0.808 0.192 n ¼ 1004 0.837 0.163 0.08 AA AG GG T C n ¼ 1280 0.295 0.705 n ¼ 1010 0.305 0.695 0.59 TT TC CC G A n ¼ 1298 0.642 0.358 n ¼ 1012 0.622 0.378 0.32 GG AG AA A G n ¼ 1292 0.553 0.447 n ¼ 994 0.554 0.446 0.94 GG GA AA rs3785817 intron 1 (6.1) rs4792939 intron 1 (7.7) rs850713 intron 5 (10.1) rs5848 UTR 30 (12.5) rs850737 (26.0) Controls n ¼ 643 0.092 0.442 0.467 n ¼ 633 0.409 0.477 0.114 n ¼ 641 0.654 0.309 0.037 n ¼ 640 0.086 0.417 0.497 n ¼ 649 0.411 0.461 0.128 n ¼ 646 0.206 0.483 0.311 AD n ¼ 494 0.115 0.413 0.472 n ¼ 466 0.406 0.491 0.103 n ¼ 502 0.701 0.271 0.028 n ¼ 505 0.093 0.424 0.483 n ¼ 506 0.381 0.480 0.138 n ¼ 497 0.191 0.509 0.300 a Location of SNPs are indicated in 50 –30 orientation with respect to SNP rs850714. *Allele and genotype frequencies were compared using two-sided Pearson’s c2 test. All the studied SNPs were in Hardy–Weinberg equilibrium in both cases and controls (P > 0.05). P-value* 0.35 0.82 0.21 0.87 0.58 0.67 VISWANATHAN ET AL. 749 analysis adjusted for age and APOE genotype. Power calculations showed that the case–control cohort has a power of 80% (Corrected a level ¼ 0.05) for detecting an association with SNPs rs850713 and rs5848 in men. Five haplotypes were identified for SNPs rs850713, rs5848, and rs850737 in the AD and controls cohorts among the subgroup of men (Table III). Association analysis showed that the haplotype T-A-G was significantly overrepresented among the male AD patients (OR ¼ 1.52, 95% CI (1.11–2.06)). Granulin protein is a plausible candidate in AD based on its neurotrophic properties and involvement in neuronal degeneration [He and Bateman, 2003; Baker et al., 2006; Cruts et al., 2006; Van Damme et al., 2008]. Reinforced by these facts, we wanted to elucidate whether genetic variability in GRN gene affects the risk of AD in Finnish population. Since mutations in GRN gene have been shown to cause FTD linked to chromosome 17 [Baker et al., 2006; Cruts et al., 2006], it is important to assess whether the other sequence alterations, which may affect, for example, transcription, splicing, or stability of GRN, could conversely increase the susceptibility for AD. Although we did not find any of the studied GRN SNPs to be associated with AD in the whole case–control cohort, the fact that SNPs rs4792939, rs850713, and rs5848 were nominally associated in men suggests that genetic variability in the GRN gene may influence the risk of AD in a gender-specific manner. Consistent with our haplotype-based association analysis showing that the haplotype T-A-G (SNPs rs850713-rs5848-rs850737) is nominally overrepresented among the male AD patients, it was recently shown that haplotype HapE in block 2 of GRN gene (consisting of allele T FIG. 1. A schematic view of the GRN gene showing the location of SNPs used in the present study. Haplotype blocks and LD values (D0 -values) were obtained using the whole case–control cohort. UTRs and coding exons (Ex) in GRN gene are indicated as gray and white boxes, respectively. 50 and 30 end regions of the GRN gene are not in scale (dashed line). TABLE II. Allele and Genotype Frequencies of GRN SNPs in Men Allele frequency SNP rs850714 Allele T C Controls n ¼ 522 0.308 0.692 AD n ¼ 316 0.332 0.668 T C n ¼ 514 0.654 0.346 A G Genotype frequency P-value* Genotype 0.47 CC TC TT n ¼ 286 0.661 0.339 0.84 TT TC CC n ¼ 518 0.801 0.199 n ¼ 318 0.865 0.135 0.02 (0.12) AA AG GG T C n ¼ 520 0.267 0.733 n ¼ 314 0.354 0.646 0.009 (0.05) TT TC CC G A n ¼ 524 0.672 0.328 n ¼ 314 0.580 0.420 0.007 (0.04) GG AG AA G A n ¼ 520 0.425 0.575 n ¼ 314 0.481 0.519 0.12 GG GA AA rs3785817 rs4792939 rs850713 rs5848 rs850737 Controls n ¼ 261 0.088 0.441 0.471 n ¼ 257 0.416 0.475 0.109 n ¼ 259 0.641 0.320 0.039 n ¼ 260 0.088 0.358 0.554 n ¼ 262 0.443 0.458 0.099 n ¼ 260 0.173 0.504 0.323 AD n ¼ 158 0.108 0.449 0.443 n ¼ 143 0.420 0.483 0.098 n ¼ 159 0.755 0.220 0.025 n ¼ 157 0.115 0.478 0.408 n ¼ 157 0.331 0.497 0.172 n ¼ 157 0.223 0.516 0.261 *Allele and genotype frequencies were compared using two-sided Pearson’s c2 test. P-values were corrected for multiple testing using Bonferroni correction (in parenthesis). P-value* 0.75 0.94 0.05 (0.30) 0.02 (0.12) 0.02 (0.12) 0.28 750 AMERICAN JOURNAL OF MEDICAL GENETICS PART B REFERENCES TABLE III. Haplotype Frequencies of GRN SNPs rs850713, rs5848 and rs850737 in Men Haplotype (rs850713-rs5848-rs850737) C-G-A T-A-G C-A-G C-G-G T-G-A Control 0.550 0.238 0.090 0.095 0.027 AD 0.489 0.323 0.096 0.060 0.031 P-value* 0.09 0.007 (0.05) 0.77 0.08 0.74 *Simulated P-value (1,000 simulations) is shown in parenthesis. for rs850713 and allele A for rs5848) was also significantly overrepresented among Belgian AD patients [Brouwers et al., 2008]. These findings collectively suggest that genetic variation(s) at the 30 end of the GRN gene may increase the risk of developing AD. However, we found the association with AD only after stratification with gender, which may have increased the likelihood of false positive result owing to multiple testing and reduced number of AD patients and control subjects. Also, it is difficult to explain how variation(s) in GRN gene could affect the risk of AD only in men. It is possible that the observed gender-specific effect is linked to neurotrophic functions of granulin in conjunction with sex hormones, which could explain the observed interaction. Consistent with this idea, it has been shown that granulin plays a role in the development of male-specific differentiation of hypothalamus [Suzuki and Nishiahara, 2002]. Therefore, replication of the present findings in different populations, identifying the novel sequence alteration(s) affecting the risk as well as comprehensive functional studies are required before making firm conclusions concerning the role of GRN in AD. ACKNOWLEDGMENTS Financial support for this project was provided by the Health Research Council of the Academy of Finland, EVO grant 5772708 of Kuopio University Hospital, the Nordic Centre of Excellence of Neurodegeneration, and the Marie Curie Early Stage Researcher Training Program, BiND. Baker M, Mackenzie IR, Pickering-Brown SM, Gass J, Rademakers R, Lindholm C, Snowden J, Adamson J, Sadovnick AD, Rollinson S, Cannon A, Dwosh E, Neary D, Melquist S, Richardson A, Dickson D, Berger Z, Eriksen J, Robinson T, Zehr C, Dickey CA, Crook R, McGowan E, Mann D, Boeve B, Feldman H, Hutton M. 2006. Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17. Nature 442:916–919. Brouwers N, Sleegers K, Engelborghs S, Maurer-Stroh S, Gijselinck I, van der Zee J, Pickut BA, Van den Broeck M, Mattheijssens M, Peeters K, Schymkowitz J, Rousseau F, Martin JJ, Cruts M, De Deyn PP, Van Broeckhoven C. 2008. Genetic variability in progranulin contributes to risk for clinically diagnosed Alzheimer disease. Neurology 71:656– 664. Cruts M, Gijselinck I, van der Zee J, Engelborghs S, Wils H, Pirici D, Rademakers R, Vandenberghe R, Dermaut B, Martin JJ, van Duijn C, Peeters K, Sciot R, Santens P, De Pooter T, Mattheijssens M, Van den Broeck M, Cuijt I, Vennekens K, De Deyn PP, Kumar-Singh S, Van Broeckhoven C. 2006. Null mutations in progranulin cause ubiquitinpositive frontotemporal dementia linked to chromosome 17q21. Nature 442:920–924. He Z, Bateman A. 2003. Progranulin (granulin-epithelin precursor, PC-cell -derived growth factor, acrogranin) mediates tissue repair and tumorigenesis. J Mol Med 81:600–612. Lehtovirta M, Soininen H, Helisalmi S, Mannermaa A, Helkala EL, Hartikainen P, Hanninen T, Ryynanen M, Riekkinen PJ. 1996. Clinical and neuropsychological characteristics in familial and sporadic Alzheimer’s disease: Relation to apolipoprotein E polymorphism. Neurology 46:413–419. Malaspina A, Kaushik N, de Belleroche J. 2001. Differential expression of 14 genes in amyotrophic lateral sclerosis spinal cord detected using gridded cDNA arrays. J Neurochem 77:132–145. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. 1984. Clinical diagnosis of Alzheimer’s disease: Report of the NINCDSADRDA work group under the auspices of department of health and human services task force on Alzheimer’s disease. Neurology 34:939–944. Suzuki M, Nishiahara M. 2002. Granulin precursor gene: A sex steroidinducible gene involved in sexual differentiation of the rat brain. Mol Genet Metab 75:31–37. Van Damme P, Van Hoecke A, Lambrechts D, Vanacker P, Bogaert E, van Swieten J, Carmeliet P, Van Den Bosch L, Robberecht W. 2008. Progranulin functions as a neurotrophic factor to regulate neurite outgrowth and enhance neuronal survival. J Cell Biol 181:37–41.