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An association study between granulin gene polymorphisms and Alzheimer's disease in Finnish population.

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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: mikko.hiltunen@uku.fi
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.
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