Neuroscience Letters 344 (2003) 99–102 www.elsevier.com/locate/neulet Simultaneous analysis of five genetic risk factors in Polish patients with Alzheimer’s disease Maria Styczynskaa,*, Dorota Religaa,b, Anna Pfeffera, Elzbieta Luczywekc, Boguslaw Wasiaka, Grzegorz Styczynskid, Beata Peplonskaa, Tomasz Gabryelewicza, Marek Golebiowskie, Malgorzata Kobrysa, Maria Barcikowskaa a Department of Neurodegenerative Disorders, Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego Str., 02-106 Warsaw, Poland b Department of Neurotec, Section of Experimental Geriatrics, Karolinska Institutet, Stockholm, Sweden c Department of Neurosurgery, Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland d Department of Internal Medicine and Hypertension, Medical University of Warsaw, Warsaw, Poland e Department of Radiology, Medical University of Warsaw, Warsaw, Poland Received 17 February 2003; received in revised form 3 April 2003; accepted 7 April 2003 Abstract As Alzheimer’s disease (AD) is a complex disease, we decided to estimate how previously reported genetic polymorphisms interact to increase the risk for the disease. Five candidate genes were chosen: apolipoprotein E (APOE), a2-macroglobulin, cathepsin D, myeloperoxidase and nitric oxide synthase. Genotyping was performed in 100 cases of late-onset AD and 100 healthy controls. We found a highly significant difference in APOE 14 distribution between groups (P , 0:005). However, no evidence of association for other studied loci was found. Cumulative analysis of five genetic polymorphisms was performed, but it also failed to reveal any synergistic effect of candidate genes greater than that caused by APOE itself. Our results suggest that the APOE 14 allele is the only known genetic risk factor for late-onset, sporadic AD. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; Polymorphism; Apolipoprotein E; a2-Macroglobulin; Cathepsin D; Myeloperoxidase; Nitric oxide synthase Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with complex pathogenesis. Among genetic factors that contribute to the development of the late-onset form of AD, apolipoprotein E (APOE) polymorphism is the most important one. The association between the disease and the APOE 14 allele was first reported in 1993 and strongly supported later [21]. The presence of the APOE 14 allele increases the risk of AD in a dose-related fashion and is associated with about 40 – 70% of late-onset cases. Nevertheless, APOE genotyping does not provide sufficient sensitivity or specificity to be used alone as a diagnostic test for AD. This explains the attempts to identify other genetic factors associated with AD. In 1997 the results of linkage studies suggested the existence of an AD susceptibility locus on chromosome 12, * Corresponding author. Tel.: þ 48-22-608-65-38; fax: þ 48-22-668-55- 32. E-mail address: desperat@cmdik.pan.pl (M. Styczynska). in a region encompassing the a2-macroglobulin (a2M) [18]. The a2M polymorphism was considered as a candidate risk factor for AD for several reasons. In vitro studies have suggested that this serum protease inhibitor might be involved in the binding, degradation and clearance of the b-amyloid protein and that it might be a major ligand of the lipoprotein receptor-related protein [13]. Two main polymorphisms in the a2M gene were found: a deletion polymorphism in exon 18 (a2M-I/D) and a missense mutation polymorphism in exon 24 (a2M Ile1000Val). Some authors suggested that the carriers of the a2M-D allele genotype are at an increased risk of AD [3]. Others reported overrepresentation of the a2M-Val/Val genotype in AD cases compared to controls [16]. Three other candidate genes were found in 1999. Papassotiropoulos et al. hypothesized that cathepsin D (CatD) polymorphism at exon 2 (CatD*C ! T:Ala224Val) is associated with an increased risk of developing AD [17]. CatD is an intracellular protease with in vitro b-secretase- 0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00438-5 100 M. Styczynska et al. / Neuroscience Letters 344 (2003) 99–102 like features [6]. It has been suggested that this polymorphism has functional consequences in terms of enhancing enzyme activity, which results in an increased amount of the b-amyloid peptide. The authors found significant overrepresentation of the CatD *T (Val) allele in demented patients compared to controls. Reynolds et al. [20] have found an association between myeloperoxidase (MPO) promoter polymorphism and a gender specific risk for AD. This polymorphism is a 2 463 G/A nucleotide exchange, which results in increased gene expression in the subjects carrying the G/G genotype. MPO is a common enzyme that generates hypochlorus acid and other reactive radicals and is believed to be involved in the pathogenesis of AD. Many studies showed an increased bamyloid deposition under oxidative conditions. Another candidate gene encodes nitric oxide synthase (NOS3 gene). Dahiyat et al. reported that the possession of a G/G genotype of the common 2 894G/T polymorphism is associated with the increased risk for AD [11]. These functionally important variants may influence susceptibility to degenerative changes caused by reactive radicals by altering the amount of NO generated by the endothelium. There are numerous reports of positive association findings for the genes mentioned above, but most of them analyzed the influence of a single candidate gene polymorphism and have not been replicated later on other populations. As AD is a complex disease, we decided to estimate how previously reported polymorphisms interact to increase the risk for the disease. Five genes of interest, APOE, a2M, CatD, MPO and NOS3, were chosen based on the previously published reports of significant association in AD. The dominant approach has been to employ polymorphisms within candidate genes from biological pathways assumed to be involved in the pathogenesis of AD. Simultaneous investigation of these five factors in one population has to our knowledge not been conducted as yet. The AD group consisted of 100 patients (66 females, 34 males, mean age 76.4 ^ 4.7 years, mean age of onset 71.0 ^ 4.6 years) recruited from the Department of Neurodegenerative Disorders at the Medical Research Centre in Warsaw. In all AD patients the disease was diagnosed as probable according to the NINCDS-ADRDA criteria. All of them were examined by a neurologist, a neuropsychologist (evaluation in MMSE, Global Deterioration Scale, Alzheimer’s Disease Assessment Scalecognitive subscale [ADAS-cog] and Blessed Dementia Rating Scale) and a psychiatrist. In addition, a CT scan with assessment of hippocampal fissure was obtained for each patient. The control group consisted of 100 nondemented people (58 females, 42 males, mean age 74.24 ^ 6.6 years). Blood samples were taken after obtaining written informed consent from the patient or his representative. DNA was isolated from leukocytes using standard protocols. The APOE, a2M, CatD, MPO and NOS3 polymorphisms were determined as described previously [5,16,17,20,11]. The allele distributions were compared between patients and controls by using the x 2 test for independence. Additionally, in order to check if different combinations of alleles of genes such as APOE, a2M, CatD, MPO and NOS3 influence the risk of AD, the regression analysis was performed. P , 0:05 was considered as significant. Statistica 6.0 was used for the statistical analysis. Table 1 shows frequencies of APOE alleles and genotypes. The frequencies of APOE 14, 13 and 12 alleles were 0.325, 0.645 and 0.030 in the AD group and 0.11, 0.845 and 0.045 in the control group, respectively. There was a highly significant overrepresentation of the APOE 14 allele in the AD patients compared with the controls (x2 ¼ 8:5, P , 0:005). We did not find a significant difference in APOE 12 allele frequency between the groups. Our study revealed an increased frequency of the 14/4 genotype in AD patients (7% vs. 1%). However, because of the absolute small number of APOE 14/4 carriers in both groups, it was impossible to demonstrate the significance of that difference. Also the APOE 13/4 genotype frequency was increased in the AD group (x2 ¼ 9:51, P , 0:005). Genotypes APOE 13/3 and APOE 12/3 were found more often in the control group compared to the AD one. The results of a2M, CatD, MPO and NOS3 genotyping are presented in Table 2. We found no statistically significant difference between the AD and control groups either in the frequencies of the alleles or in the distribution of the subsequent genotypes of those four genes. Cumulative analysis of five genetic polymorphisms was performed, but it also failed to reveal any synergistic effect of candidate genes greater than that caused by APOE itself. Many studies confirmed that the inheritance of different combinations of APOE alleles can either increase or decrease the risk of AD. The presence of the APOE 14 allele is the most important genetic risk factor for late-onset AD [21]. Its frequency in AD patients ranges between 0.32 and 0.42, while in normal individuals this frequency is estimated as between 0.13 and 0.17. However, some ethnic variations in genotypic risk are reported. Results of our study were consistent with those performed on a Caucasian population and revealed a statistically significant increase of APOE 14 frequency in the AD group compared to the controls. In 1998 Blacker et al. [3] and Liao et al. [16] reported a genetic association between two a2M polymorphisms and AD. Liao et al. found a significant increase of the a2M-Val/Val genotype in AD patients. Results of our study do not confirm this observation – there was no significant difference in a2M allele and genotype distribution between the AD group and the controls. This is in accordance with the results of another study performed in 1999 [10], which failed to reveal any significant increase in a2M Val/Val genotype in the AD patients. Also, studies on a2M-I/D polymorphism performed on various ethnic groups failed to confirm the importance of this pentanucleo- 101 M. Styczynska et al. / Neuroscience Letters 344 (2003) 99–102 Table 1 Frequencies of APOE alleles and genotypes APOE Allele frequency n AD Controls 100 100 Genotype, n (%) 12 13 14 12/2 12/3 12/4 13/3 13/4 14/4 0.030 0.045 0.645 0.845 0.325* 0.11 0 (0) 0 (0) 3 (3) 8 (8) 3 (3) 1 (1) 39 (39) 71 (71)** 48 (48)* 19 (19) 7 (7) 1 (1) *P , 0:005; **P , 0:05. tide mutation in exon 18 [10,12]. Koster et al. performed meta-analysis using all published findings to evaluate the effect of these two polymorphisms [14]. The authors concluded that a2M is not genetically associated with AD. Since 1999, when Papassotiropoulos reported strong association between CatD polymorphism and the risk of AD [17], the results of at least two other studies have been published. Crawford et al. found only a small tendency towards an increase in CatD*T-carrying genotypes in Caucasian AD patients [8]. In contrast, the results of the studies performed by Bhojak et al. indicated no significant association between CatD polymorphism and the risk of AD [2]. The results of our study are consistent with those findings – we found no significant difference in CatD genotypes distribution in AD patients and controls. Reynolds et al. [20] found in one study conducted in California that the MPO G/G genotype is overrepresented in women with AD. Later on the same author failed to replicate these findings in a Finnish population whose MPO A allele was associated with an increased risk for AD in men [19]. Crawford et al. also found a significant association between MPO G/G genotype and AD in Caucasians but not in a Spanish sample from Florida [9]. Combarros et al. [7] also failed to detect a significant difference in genotypic frequencies of MPO in a Spanish population. This indicates that ethnic variation in the genetic risk for AD is relatively strong. In the presented study we found no association Table 2 a2M, CatD, MPO and NOS3 allele and genotype distribution in AD patients and controls n a2M AD Controls CatD AD Controls MPO AD Controls NOS3 AD Controls 100 100 100 100 100 100 100 100 Allele frequency Genotype, n (%) Ile 0.675 0.605 C 0.935 0.955 G 0.76 0.805 G 0.79 0.765 Ile/Ile 49 (49) 40 (40) C/C 88 (88) 91 (91) G/G 71 (71) 72 (72) G/G 65 (65) 57 (57) Val 0.325 0.395 T 0.065 0.045 A 0.24 0.195 T 0.21 0.235 Ile/Val 37 (37) 41 (41) C/T 11 (11) 9 (9) G/A 10 (10) 17 (17) G/T 28 (28) 39 (39) Val/Val 14 (14) 19 (19) T/T 1 (1) 0 (0) A/A 19 (19) 11 (11) T/T 7 (7) 4 (4) between MPO polymorphism and AD in a Polish population. The initial observation on the association between the NOS3 gene polymorphism by Dahiyat et al. [11] has not been replicated later by other groups [15]. Our results also indicate a lack of such an association. In conclusion, we confirmed that of the five candidate polymorphisms, only the APOE 14 allele appears to be a reliable risk marker in AD. The absence of a statistically significant increase in frequency of previously reported a2M, CatD, MPO and NOS3 variants in the AD group indicates that the latter four genes are less reliable for diagnostic purposes. We suggest that if an association between a2M, CatD, MPO and NOS3 and the risk of AD exists, it is likely to be smaller than previously reported. Recent studies have indicated the existence of additional AD genes on chromosome 10 [1]. The insulin degrading enzyme (IDE) gene located near the linkage region was investigated, but none of the examined single nucleotide polymorphisms were associated with AD [4]. Further studies of this region are being performed and they probably will lead to identification of the new candidate genes. Acknowledgements This study was supported by the State Committee for Scientific Research grant no. 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