- Email: [email protected]
No association between high temperature requirement 1 (HTRA1) gene polymorphisms and Alzheimer's disease
Neurobiology of Aging 32 (2011) 547.e7–547.e9
Negative results
No association between high temperature requirement 1 (HTRA1) gene polymorphisms and Alzheimer’s disease Mari Turunen 1 , Saila Vepsäläinen 1 , Petra Mäkinen, Seppo Helisalmi, Annakaisa Haapasalo, Hilkka Soininen, Mikko Hiltunen ∗ Department of Neurology and Brain Research Unit, Clinical Research Centre/Mediteknia, University Hospital and University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland Received 7 November 2008; received in revised form 20 March 2009; accepted 24 August 2009 Available online 23 September 2009
Abstract High temperature requirement 1 (HTRA1) gene is a plausible risk factor in Alzheimer’s disease (AD) as it encodes a protease known to degrade amyloid- peptide. Here we have studied whether single nucleotide polymorphisms (SNPs) in the HTRA1 gene or its nearby regions associated with AD in a large clinic-based case–control cohort originating from Finland. We did not observe significant association of the HTRA1 SNPs with AD among the whole case–control cohort or age-at-onset risk effect among AD patients. © 2009 Elsevier Inc. All rights reserved. Keywords: Alzheimer’s disease; HTRA1; Risk gene; SNP
Alzheimer’s disease (AD) is characterized by typical neuropathological changes including extracellular amyloid plaques consisting of amyloid- (A) peptide and intracellular neurofibrillary tangles. Several enzymes, such as insulin degrading enzyme (IDE) and neprilysin (NEP), are capable of degrading A (Miners et al., 2008). Interestingly, high temperature requirement (HTRA) proteases have also been proposed to play a role in APP metabolism in addition to apoptosis and aging (Clausen et al., 2002; Huttunen et al., 2007). HTRA1 has been shown to degrade A and C99, the -secretase cleavage product of APP, and it also colocalizes with amyloid deposits in human brain (Grau et al., 2005). Importantly, HTRA1 gene locates in chromosome 10 (10q25–q26), close to the region 10q21–10q25, which has been suggested to influence AD risk and plasma A42 levels (Bertram et al., 2000; Ertekin-Taner et al., 2000; Myers et al., 2000). Moreover, HTRA1 resides in a region that has been reported to control the age-at-onset in AD (Li et al., 2002). In the light of the above-mentioned ∗ 1
Corresponding author. Tel.: +358 40 355 2014; fax: +358 17 162048. E-mail address: [email protected] (M. Hiltunen). These authors contributed equally to this work.
0197-4580/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2009.08.010
biochemical and genetic findings, we set our goal to investigate whether single nucleotide polymorphisms (SNP) in the HTRA1 gene or its nearby regions are associated with AD among clinic-based case–control cohort originating from eastern Finland. The APOE 4 allele was significantly overrepresented among the 464 AD patients as compared to 558 controls (OR 4.8; 95% CI: 3.9–5.9; p < 0.001). Five non-coding SNPs from the HTRA1 gene and one SNP in a nearby gene encoding agerelated maculopathy susceptibility 2 (ARMS2) were selected for genotyping using TaqMan chemistry (Table 1). Allele and genotype frequencies of the studied SNPs did not differ significantly between the whole AD and control cohorts (Table 1). SNPs rs2268345, rs2239586, and rs714816 in the HTRA1 gene were in strong linkage disequilibrium (LD) (D values 0.96–1.0) and they were part of the same haplotype block. LD and haplotype block structure were similar to that observed among the Central European population (CEU) in HapMap (http://www.hapmap.org). Using these SNPs, four haplotypes were identified and individual haplotype association analysis did not reveal significant association with AD (Table 1). Stratification according to age (65 years as a cut-off age), gender and APOE status (APOE 4+ and 4−
547.e8
M. Turunen et al. / Neurobiology of Aging 32 (2011) 547.e7–547.e9
Table 1 Allele, genotype, and haplotype distributions of HTRA1 and ARMS2 SNPs among AD patients and controls. SNP
Allele
Allele frequency
p-value
AD (n = 928)
Ctrl (n = 1116)
Genotype
Genotype frequency
p-value
AD (n = 464)
Ctrl (n = 558)
ARMS2 rs10490924
G T
0.79 0.21
0.78 0.22
0.408
GG GT TT
0.63 0.33 0.05
0.62 0.31 0.07
0.230
HTRA1 rs2268345
G T
0.82 0.18
0.82 0.18
0.819
GG GT TT
0.67 0.30 0.03
0.66 0.32 0.03
0.757
HTRA1 rs2239586
C T
0.92 0.08
0.93 0.07
0.437
CC CT TT
0.85 0.14 0.01
0.86 0.14 0.002
0.194
HTRA1 rs714816
C T
0.69 0.31
0.68 0.32
0.800
CC CT TT
0.46 0.45 0.09
0.47 0.43 0.11
0.579
HTRA1 rs2268356
C T
0.49 0.51
0.50 0.50
0.903
CC CT TT
0.24 0.52 0.25
0.25 0.49 0.26
0.765
HTRA1 rs2293871
C T
0.89 0.11
0.90 0.10
0.748
CC CT TT
0.80 0.19 0.01
0.81 0.19 0.01
0.709
Haplotypesa
AD
Ctrl
p-value
H1 (GCC) H2 (GCT) H3 (TCC) H4 (TTC)
0.50 0.32 0.10 0.08
0.50 0.32 0.11 0.07
0.826 0.800 0.524 0.676
Overall
0.910
a
Haplotypes consist of SNPs rs2268345, rs2239586, and rs714816. SNPs were in Hardy–Weinberg equilibrium (HWE) in both cases and controls. HWE p-value for rs10490924 was p = 0.03, while the genotype distribution was the same as in the CEU population based on the NCBI SNP database (http://www.ncbi.nlm.nih.gov/SNP/).
subgroups) revealed a nominally significant allele and genotype association with SNP rs2239586 among the subgroup of early onset AD patients (≤65 years of age) (Table S1). According to logistic regression analysis, gender- and APOEadjusted odds ratio for T-allele carriers of the rs2239586 was 2.5 (95% CI: 1.01–6.13; p = 0.048). Assessment of individual haplotype distributions among the early onset AD patients showed a nominally significant association of the TTC haplotype with AD (Table S1). No significant differences were found among the late-onset AD group. To investigate whether the T-allele affects the age-at-onset of AD, we combined rs2239586 CT+TT genotypes and performed Kaplan–Meier survival analysis, which consequently did not reveal a statistically significant age-at-onset risk effect among AD patients ((p = 0.58, Figure S1). Collectively, we did not find association of HTRA1 gene polymorphisms with AD or a significant age-at-onset risk effect among AD patients originating from eastern Finland. Stratification according to age showed a nominally significant association among early onset AD group (≤65 years of age) with the HTRA1 SNP rs2239586 in allele, genotype, and haplotype analyses. After correction for multiple testing, however, these p-values did not maintain statistical signifi-
cance, which in turn could be related to reduced power due to the small number of patients and controls among this subgroup. Alternatively, stratification of the case–control cohort by age may have led to type I error. HTRA1 is a plausible candidate gene in AD as it locates in chromosome 10, close to a region that has been implicated to influence AD risk and plasma A levels (Bertram et al., 2000; Ertekin-Taner et al., 2000; Myers et al., 2000). HTRA1 also resides inside a region that showed linkage with age-at-onset in AD (Li et al., 2002). In addition, HTRA1 degrades A and C99, the secretase cleavage product of APP (Grau et al., 2005). Based on these observations, it is tempting to speculate that alterations in the HTRA1 gene may impair proteolytic function of this protease and consequently affect, e.g. accumulation and deposition of A. However, our present genetic study together with the recent studies from other ethnic populations (see details http://www.AlzGene.org), do not support a significant risk effect for HTRA1 gene alterations in AD.
Conflict of interest statement None.
M. Turunen et al. / Neurobiology of Aging 32 (2011) 547.e7–547.e9
547.e9
Acknowledgements
References
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 University of Kuopio Graduate School of Molecular Medicine.
Clausen, T., Southan, C., Ehrmann, M., 2002. The HtrA family of proteases: Implications for protein composition and cell fate. Mol. Cell 10, 443–455. Grau, S., Baldi, A., Bussani, R., Tian, X., Stefanescu, R., Przybylski, M., Richards, P., Jones, S.A., Shridhar, V., Clausen, T., Ehrmann, M., 2005. Implications of the serine protease HtrA1 in amyloid precursor protein processing. Proc. Natl. Acad. Sci. U.S.A. 102, 6021–6026. Li, Y.J., Scott, W.K., Hedges, D.J., Zhang, F., Gaskell, P.C., Nance, M.A., Watts, R.L., Hubble, J.P., Koller, W.C., Pahwa, R., Stern, M.B., Hiner, B.C., Jankovic, J., Allen Jr., F.A., Goetz, C.G., Mastaglia, F., Stajich, J.M., Gibson, R.A., Middleton, L.T., Saunders, A.M., Scott, B.L., Small, G.W., Nicodemus, K.K., Reed, A.D., Schmechel, D.E., Welsh-Bohmer, K.A., Conneally, P.M., Roses, A.D., Gilbert, J.R., Vance, J.M., Haines, J.L., Pericak-Vance, M.A., 2002. Age at onset in two common neurodegenerative diseases is genetically controlled. Am. J. Hum. Genet. 70, 985–993.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neurobiolaging. 2009.08.010.
Negative results
No association between high temperature requirement 1 (HTRA1) gene polymorphisms and Alzheimer’s disease Mari Turunen 1 , Saila Vepsäläinen 1 , Petra Mäkinen, Seppo Helisalmi, Annakaisa Haapasalo, Hilkka Soininen, Mikko Hiltunen ∗ Department of Neurology and Brain Research Unit, Clinical Research Centre/Mediteknia, University Hospital and University of Kuopio, P.O. Box 1627, 70211 Kuopio, Finland Received 7 November 2008; received in revised form 20 March 2009; accepted 24 August 2009 Available online 23 September 2009
Abstract High temperature requirement 1 (HTRA1) gene is a plausible risk factor in Alzheimer’s disease (AD) as it encodes a protease known to degrade amyloid- peptide. Here we have studied whether single nucleotide polymorphisms (SNPs) in the HTRA1 gene or its nearby regions associated with AD in a large clinic-based case–control cohort originating from Finland. We did not observe significant association of the HTRA1 SNPs with AD among the whole case–control cohort or age-at-onset risk effect among AD patients. © 2009 Elsevier Inc. All rights reserved. Keywords: Alzheimer’s disease; HTRA1; Risk gene; SNP
Alzheimer’s disease (AD) is characterized by typical neuropathological changes including extracellular amyloid plaques consisting of amyloid- (A) peptide and intracellular neurofibrillary tangles. Several enzymes, such as insulin degrading enzyme (IDE) and neprilysin (NEP), are capable of degrading A (Miners et al., 2008). Interestingly, high temperature requirement (HTRA) proteases have also been proposed to play a role in APP metabolism in addition to apoptosis and aging (Clausen et al., 2002; Huttunen et al., 2007). HTRA1 has been shown to degrade A and C99, the -secretase cleavage product of APP, and it also colocalizes with amyloid deposits in human brain (Grau et al., 2005). Importantly, HTRA1 gene locates in chromosome 10 (10q25–q26), close to the region 10q21–10q25, which has been suggested to influence AD risk and plasma A42 levels (Bertram et al., 2000; Ertekin-Taner et al., 2000; Myers et al., 2000). Moreover, HTRA1 resides in a region that has been reported to control the age-at-onset in AD (Li et al., 2002). In the light of the above-mentioned ∗ 1
Corresponding author. Tel.: +358 40 355 2014; fax: +358 17 162048. E-mail address: [email protected] (M. Hiltunen). These authors contributed equally to this work.
0197-4580/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2009.08.010
biochemical and genetic findings, we set our goal to investigate whether single nucleotide polymorphisms (SNP) in the HTRA1 gene or its nearby regions are associated with AD among clinic-based case–control cohort originating from eastern Finland. The APOE 4 allele was significantly overrepresented among the 464 AD patients as compared to 558 controls (OR 4.8; 95% CI: 3.9–5.9; p < 0.001). Five non-coding SNPs from the HTRA1 gene and one SNP in a nearby gene encoding agerelated maculopathy susceptibility 2 (ARMS2) were selected for genotyping using TaqMan chemistry (Table 1). Allele and genotype frequencies of the studied SNPs did not differ significantly between the whole AD and control cohorts (Table 1). SNPs rs2268345, rs2239586, and rs714816 in the HTRA1 gene were in strong linkage disequilibrium (LD) (D values 0.96–1.0) and they were part of the same haplotype block. LD and haplotype block structure were similar to that observed among the Central European population (CEU) in HapMap (http://www.hapmap.org). Using these SNPs, four haplotypes were identified and individual haplotype association analysis did not reveal significant association with AD (Table 1). Stratification according to age (65 years as a cut-off age), gender and APOE status (APOE 4+ and 4−
547.e8
M. Turunen et al. / Neurobiology of Aging 32 (2011) 547.e7–547.e9
Table 1 Allele, genotype, and haplotype distributions of HTRA1 and ARMS2 SNPs among AD patients and controls. SNP
Allele
Allele frequency
p-value
AD (n = 928)
Ctrl (n = 1116)
Genotype
Genotype frequency
p-value
AD (n = 464)
Ctrl (n = 558)
ARMS2 rs10490924
G T
0.79 0.21
0.78 0.22
0.408
GG GT TT
0.63 0.33 0.05
0.62 0.31 0.07
0.230
HTRA1 rs2268345
G T
0.82 0.18
0.82 0.18
0.819
GG GT TT
0.67 0.30 0.03
0.66 0.32 0.03
0.757
HTRA1 rs2239586
C T
0.92 0.08
0.93 0.07
0.437
CC CT TT
0.85 0.14 0.01
0.86 0.14 0.002
0.194
HTRA1 rs714816
C T
0.69 0.31
0.68 0.32
0.800
CC CT TT
0.46 0.45 0.09
0.47 0.43 0.11
0.579
HTRA1 rs2268356
C T
0.49 0.51
0.50 0.50
0.903
CC CT TT
0.24 0.52 0.25
0.25 0.49 0.26
0.765
HTRA1 rs2293871
C T
0.89 0.11
0.90 0.10
0.748
CC CT TT
0.80 0.19 0.01
0.81 0.19 0.01
0.709
Haplotypesa
AD
Ctrl
p-value
H1 (GCC) H2 (GCT) H3 (TCC) H4 (TTC)
0.50 0.32 0.10 0.08
0.50 0.32 0.11 0.07
0.826 0.800 0.524 0.676
Overall
0.910
a
Haplotypes consist of SNPs rs2268345, rs2239586, and rs714816. SNPs were in Hardy–Weinberg equilibrium (HWE) in both cases and controls. HWE p-value for rs10490924 was p = 0.03, while the genotype distribution was the same as in the CEU population based on the NCBI SNP database (http://www.ncbi.nlm.nih.gov/SNP/).
subgroups) revealed a nominally significant allele and genotype association with SNP rs2239586 among the subgroup of early onset AD patients (≤65 years of age) (Table S1). According to logistic regression analysis, gender- and APOEadjusted odds ratio for T-allele carriers of the rs2239586 was 2.5 (95% CI: 1.01–6.13; p = 0.048). Assessment of individual haplotype distributions among the early onset AD patients showed a nominally significant association of the TTC haplotype with AD (Table S1). No significant differences were found among the late-onset AD group. To investigate whether the T-allele affects the age-at-onset of AD, we combined rs2239586 CT+TT genotypes and performed Kaplan–Meier survival analysis, which consequently did not reveal a statistically significant age-at-onset risk effect among AD patients ((p = 0.58, Figure S1). Collectively, we did not find association of HTRA1 gene polymorphisms with AD or a significant age-at-onset risk effect among AD patients originating from eastern Finland. Stratification according to age showed a nominally significant association among early onset AD group (≤65 years of age) with the HTRA1 SNP rs2239586 in allele, genotype, and haplotype analyses. After correction for multiple testing, however, these p-values did not maintain statistical signifi-
cance, which in turn could be related to reduced power due to the small number of patients and controls among this subgroup. Alternatively, stratification of the case–control cohort by age may have led to type I error. HTRA1 is a plausible candidate gene in AD as it locates in chromosome 10, close to a region that has been implicated to influence AD risk and plasma A levels (Bertram et al., 2000; Ertekin-Taner et al., 2000; Myers et al., 2000). HTRA1 also resides inside a region that showed linkage with age-at-onset in AD (Li et al., 2002). In addition, HTRA1 degrades A and C99, the secretase cleavage product of APP (Grau et al., 2005). Based on these observations, it is tempting to speculate that alterations in the HTRA1 gene may impair proteolytic function of this protease and consequently affect, e.g. accumulation and deposition of A. However, our present genetic study together with the recent studies from other ethnic populations (see details http://www.AlzGene.org), do not support a significant risk effect for HTRA1 gene alterations in AD.
Conflict of interest statement None.
M. Turunen et al. / Neurobiology of Aging 32 (2011) 547.e7–547.e9
547.e9
Acknowledgements
References
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 University of Kuopio Graduate School of Molecular Medicine.
Clausen, T., Southan, C., Ehrmann, M., 2002. The HtrA family of proteases: Implications for protein composition and cell fate. Mol. Cell 10, 443–455. Grau, S., Baldi, A., Bussani, R., Tian, X., Stefanescu, R., Przybylski, M., Richards, P., Jones, S.A., Shridhar, V., Clausen, T., Ehrmann, M., 2005. Implications of the serine protease HtrA1 in amyloid precursor protein processing. Proc. Natl. Acad. Sci. U.S.A. 102, 6021–6026. Li, Y.J., Scott, W.K., Hedges, D.J., Zhang, F., Gaskell, P.C., Nance, M.A., Watts, R.L., Hubble, J.P., Koller, W.C., Pahwa, R., Stern, M.B., Hiner, B.C., Jankovic, J., Allen Jr., F.A., Goetz, C.G., Mastaglia, F., Stajich, J.M., Gibson, R.A., Middleton, L.T., Saunders, A.M., Scott, B.L., Small, G.W., Nicodemus, K.K., Reed, A.D., Schmechel, D.E., Welsh-Bohmer, K.A., Conneally, P.M., Roses, A.D., Gilbert, J.R., Vance, J.M., Haines, J.L., Pericak-Vance, M.A., 2002. Age at onset in two common neurodegenerative diseases is genetically controlled. Am. J. Hum. Genet. 70, 985–993.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neurobiolaging. 2009.08.010.