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CAT53 and HFE alleles in Alzheimer's disease: A putative protective role of the C282Y HFE mutation
Neuroscience Letters 457 (2009) 129–132
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
CAT53 and HFE alleles in Alzheimer’s disease: A putative protective role of the C282Y HFE mutation Ana Paula Correia a,b , Jorge P. Pinto c , Vera Dias c , Cláudia Mascarenhas c , Susana Almeida c , Grac¸a Porto a,c,d,∗ a
HGSA – Centro Hospitalar do Porto, Hospital Santo António, Porto, Portugal HML – Hospital Magalhães Lemos, Porto, Portugal IBMC – Institute for Molecular and Cellular Biology, Porto, Portugal d ICBAS – Abel Salazar Institute for the Biomedical Sciences, Porto, Portugal b c
a r t i c l e
i n f o
Article history: Received 26 February 2009 Received in revised form 25 March 2009 Accepted 27 March 2009 Keywords: CAT53 HFE C282Y MHC Alzheimer’s disease
a b s t r a c t Alzheimer’s disease (AD) is a complex disorder, resulting from an interaction between environmental and genetic factors. Several studies addressed the association of AD with MHC class-I polymorphisms without definite conclusions. Considering the remarkable linkage disequilibrium at the MHC region, it is not possible to assume if the reported associations result from a direct effect of the respective genes or result from associations with other closely linked genes transmitted in an extended conserved haplotype. Recent evidence pointed to CAT53, a newly described gene located at the MHC class-I region in the vicinity of HLA-C, as a candidate modifier gene in AD. CAT53 encodes a phosphatase 1 nuclear inhibitor protein and is strongly expressed in brain regions involved in memory and AD. Here we tested the potential association of CAT53 with the risk of developing AD and searched for potential haplotypic associations of CAT53 with two common mutations (H63D, C282Y) in the HFE gene, also located at chromosome 6p21.3. The allele frequencies of these mutations in AD patients were compared to the expected frequencies previously established in the normal Portuguese population. We detected only one polymorphism (G>A) in CAT53, at position 8232, in intron 17. Screening of this polymorphism in 113 AD patients and 82 controls did not show any evidence of association, therefore excluding the hypothetical role of the CAT53 polymorphism as modifier in AD. In contrast, we found a significant negative association of the C282Y HFE mutation with AD, thus supporting a putative protective role of this protein variant in neurodegeneration. © 2009 Elsevier Ireland Ltd. All rights reserved.
Alzheimer’s disease (AD) is the most common cause of dementia in the elderly. It is a progressive neurodegenerative disorder pathologically characterized by the presence of extracellular amyloid -peptide aggregated in soluble A oligomers and insoluble deposits as senile plaques and intracellular neurofibrillar tangles [18,20]. AD is a complex, multifactorial disorder. Although the genetics of early onset, autosomal dominant AD is relatively well characterized, the susceptibility to the most common late-onset forms of the disease is still debated. This form results most probably from an interaction between environmental and genetic factors. To date, only the 4 allele of apolipoprotein E is generally recognized as a susceptibility genotype for late-onset AD [9,27,28] but the fact that it is neither necessary nor sufficient to cause the disease, indicates that other genes may be involved in AD susceptibility.
∗ Corresponding author at: IRIS, IBMC – Instituto de Biologia Molecular e Celular, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. Tel.: +351 226074956; fax: +351 226098480. E-mail address: [email protected] (G. Porto). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.03.088
MHC class-I gene polymorphisms have been the focus of a large number of AD association studies, with weak associations identified for several of these [3]. However, conflicting data have not allowed so far a definite exclusion or confirmation of their real impact in AD. The presence of the human leukocyte antigen (HLA) A2, as well as variations in the regulatory regions of the tumor necrosis factor alpha (TNF-␣) and in the HLA-B-associated transcript 1 (BAT1) gene, all have been reported to modify the risk of developing AD [10,12,16,30]. In addition, several reports have implicated the H63D variant of the HFE gene as a modifier of the age of onset in AD, and as an interacting factor with the APOE 4 allele [6,21,26]. HFE is a non-classical MHC class-I gene that has a role in the regulation of iron metabolism. Although the role of the H63D variant in iron homeostasis is still not completely understood, another common mutation in this gene, the C282Y mutation, is strongly associated with iron overload, both in human and in animal models, being the major genetic variant found in patients with hereditary hemochromatosis [23]. While no direct role of MHC class I proteins has been demonstrated so far in the susceptibility to AD, it is still possible that the
130
A.P. Correia et al. / Neuroscience Letters 457 (2009) 129–132 Table 2 CAT53 genotype frequencies (and %) in AD patients according to the HFE genotypes.
previously reported associations may result from another closely linked gene transmitted in an extended conserved haplotype, given the remarkably high linkage disequilibrium in this region. Recently, a newly characterized gene located on chromosome 6p21.3, in the vicinity of HLA-C, previously identified and named FB19 [29] and now designated as CAT53 [24] was suggested as a candidate gene involved in the pathogenesis and progression of AD [24]. High levels of expression of CAT53 were found in human and mouse brain and a very high expression was found in the neurofibrillary tangles of AD brain [24]. SMART analysis of CAT53 domains was supportive of a nuclear regulatory role on PP1, a protein involved in several brain functions including learning and memory [24]. To assess the potential impact of CAT53 on the risk of developing AD we searched for polymorphic associations in a case control study involving 113 Portuguese AD patients (age 78.2 ± 7.2 years, age of onset 73.5 ± 7.7 years, 24% males) and 82 unrelated apparently healthy normal subjects (age 60.0 ± 18.0 years, range 19–100 years, 46% males). All patients and controls were Caucasian born in the north and central regions of Portugal. The same physician at the Neurology Department of Hospital Santo António and Psychogeriatric Department of Hospital Magalhães Lemos, both in Porto, performed the clinical characterization and selection of patients. The diagnosis of probable AD was established according to criteria defined by the Diagnostic and Statistical Manual, revision 4 (DSM-IV) [1] and the guidelines of the National Institute of Neurological Disorders and Stroke, and the Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) [17]. An early age at onset was defined as ≤65 years. Controls were defined as subjects without DMS-IV dementia criteria and with integrity of daily living functions. In order to test for potential haplotype associations, all AD patients studied were HFE genotyped for the C282Y and H63D mutations by a PCR amplification method with selective hybridization to allele-specific oligonucleotide probes, using commercially available Kits (Gene Mutation Assay I and II, Vienna-Lab, Vienna, Austria). Written informed consent was obtained from all recruited subjects, or their legal representatives, according to the 1975 Declaration of Helsinki and the study was approved by the ethical committee of Hospital Santo António and Hospital Magalhães Lemos, Porto. Comparisons between allele or genotype frequencies were tested by the Chi-square and the Fisher’s exact tests, and significant results considered for P-values lower than 0.05. All 18 exons, exon/intron boundaries, the 5 UTR and the first 1000 base pairs of the proximal promoter of the CAT53 gene were first sequenced double-strand in 12 controls for the detection of common DNA sequence variants. Briefly, genomic DNA was extracted from peripheral blood, using the “salting out” method, and quantified. Each amplicon was amplified by PCR using a specific primer pair and PCR products were analyzed by agarose gel electrophoresis and documented by photography. DNA was excised from the gel and purified using the QIAquick Gel Extraction Kit (Qiagen). Sequencing was performed with the BigDye® Terminator v1.1 Cycle Sequencing Kit, in an ABI Prism Avant DNA sequencer. A striking high degree of conservation was found in the sequences of all individuals analyzed and only one polymorphism (G>A) was found at position 8232, in intron 17 of the CAT53 gene. We next
HFE genotype
CAT53 genotype
C282Y/wt (n = 3) H63D/wt (n = 28) H63D/H63D (n = 6) wt/wt (n = 76)
GG
GA
AA
2 (66.7%) 16 (57.1%) 6 (100%) 55 (72.4%)
1 (33.3%) 11 (39.3%) – 20 (26.3%)
0 (–) 1 (3.6%) – 1 (1.3%)
The P-value for the Chi-square test of independence was 0.51.
screened the whole group of controls and AD patients for the presence of the 8232 G>A polymorphism by Denaturing High Performance Liquid Chromatography (DHPLC) using a specific primer pair. Allele and genotype distributions of the 8232 polymorphisms were compared between AD patients and controls (Table 1), with no significant differences found between groups. When analysis was performed including only the selected group of late-onset AD patients, the same results were obtained (see also Table 1). Searching for eventual haplotypic associations between the CAT53 8232 polymorphisms and HFE mutations was done by comparing the CAT53 genotype frequencies in AD patients grouped according to their HFE genotype (Table 2). No significant association was found between variables. Allele frequencies of the HFE mutations in the AD patient groups are shown in Table 3, in comparison with the expected frequencies previously determined in a representative, geographically matched, Portuguese sample described by Cardoso et al. (n = 259) [5]. It should be noted that, due to the demonstrated differences in expected C282Y allele frequencies in the Portuguese population according to the region of origin [5], only the data from subjects originating from the north or central region (n = 259) were used in the present study. In accordance with previous studies [6,26] the H63D mutation was associated with the age of onset in lateonset AD, with a significantly lower frequency found in patients presenting with more than 75 years (a.f. = 0.115 vs. 0.203 in controls; Chi-square test, P = 0.012; Fisher’s exact test, P = 0.0125) but not in patients presenting with 66–75 years (a.f. = 0.206). In addition, a global highly significant negative association was found in AD patients for the presence of the C282Y mutation (a.f. = 0.013 vs. 0.058 in controls; Chi-square test, P = 0.0022; Fisher’s exact test, P = 0.00197). This result was confirmed when the analysis was restricted to the group of late-onset AD patients (a.f. = 0.010 vs. 0.058 in controls; Chi-square test, P = 0.0025; Fisher’s exact test, P = 0.0002). MHC class I variation is a key determinant of susceptibility and resistance to a large number of diseases but the identification of the MHC variants conferring susceptibility to disease is problematic due to high levels of variation and linkage disequilibrium. With this in mind, we focused our interest in testing CAT53 as a candidate gene in AD in the context of its putative linkage to other genes on 6p21.3, namely with HFE mutations. Only one single nucleotide polymorphism was found in the CAT53 gene and no association was found of this polymorphism with the presence of AD. This does not exclude a putative role of the CAT53 gene product in the pathogenesis of
Table 1 Allele and genotype frequencies and distributions (n) of the CAT 53 8232 polymorphisms in AD patients and controls. 8232 variant
AD patients Late-onset AD patientsa Controls
N
116 99 82
Allele frequencies (n)
Genotype frequencies (n)
G
A
GG
GA
AA
0.841 (190) 0.843 (167) 0.848 (139)
0.159 (36) 0.157 (31) 0.152 (25)
0.699 (79) 0.707 (70) 0.707 (58)
0.283 (32) 0.272 (27) 0.280 (23)
0.018 (2) 0.020 (2) 0.012 (1)
N = number of subjects in samples. No statistical significant differences were found for allele or genotype frequencies between groups. a Subgroup of AD patients with >65 years.
A.P. Correia et al. / Neuroscience Letters 457 (2009) 129–132
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Table 3 Allele frequencies of the HFE mutations in AD patients, according to age group, in comparison to expected frequencies in a control populationa . H63D allele frequencies
b
C282Y allele frequencies
b
All patients (N = 113) Late-onset AD patientsc (N = 99) Age group 66–75 years (N = 51) Age group >75 years (N = 48)
0.172 0.162 0.206 0.115
n.s. P = 0.0504 n.s. P = 0.0125
0.013 0.010
P = 0.00197 P = 0.0002
Controls (N = 259)
0.203
0.058
N, number of subjects in samples; n.s., not significant. a From Ref. [5]. b Significance of the differences between each AD group and controls (Fisher’s exact test). c Subgroup of AD patients with >65 years.
AD but excludes a strong impact of common gene polymorphisms in this condition. We are aware that the limited number of subjects tested for the screening of polymorphisms impairs the detection of variants of low frequencies and therefore their putative association to AD is neglected. However, and assuming a high frequency of AD in the general population, we did not intend to search for associations with rare polymorphisms. Although the results do not support a role for CAT53 as a susceptibility gene in AD and do not suggest its possible linkage to HFE in a conserved haplotype, our data support, however, a possible role of the HFE genotype as a genetic modifier in this clinical condition. While the H63D mutation was associated to an earlier progression of AD, the C282Y mutation showed a negative association with the disease, thus appearing as a putative protective genotype. Interestingly, similarly low frequencies (between 0.008 and 0.026) were previously reported in AD patients from other Northern Italian, Canadian and Portuguese populations [2,4,11,26]. A higher C282Y allele frequency (0.078) was found in a selected AD population from the Oxford region [25]. In this particular case, however, the presence of the C282Y mutation was clearly and strongly associated with the co-inheritance of the transferrin gene allele C2 [25]. The mechanism how HFE could influence the clinical expression of AD is still elusive. It is relevant to note, however, the recent study by Lee et al. [15] where it was shown that the two different HFE mutations have unique effects on human neuroblastoma stable cell lines over-expressing HFE. While the presence of the C282Y mutation was associated with an up-regulation of the signal transduction pathway in response to stress, H63D-expressing cells had higher levels of lipid peroxidation, protein oxidation and lower mitochondrial membrane potential, suggesting a higher baseline stress, and were also more vulnerable to exposure to oxidative stress agents [15]. That was the first study providing insights into how the different HFE mutations could have different clinical consequences. More recently, an association of the H63D mutation with increased tau phosphorylation and up-regulated protein kinase activity provided more evidence that this allelic variant is associated with an altered intracellular environment, consistent with its putative role as a risk factor for neurodegenerative disorders (Hall et al., personal communication). On the other side, another recent report of gene expression in the brain from Hfe knockout mice showed a remarkable decrease in the expression of several genes involved in the pathogenesis of AD namely the genes encoding the amyloid precursor protein (APP) and the presenilin 1 gene (PSEN1), thus challenging again the notion of the role of HFE as predisposing or protective in AD (Johnstone et al., personal communication). The mechanism how the HFE C282Y mutation could confer protection to neurodegeneration is still speculative. In hereditary hemochromatosis patients it has been shown that monocyte chemoattractant protein-1 (MCP-1) serum levels are differentially associated with the HFE mutations, being increased in patients with the H63D mutation but significantly decreased in those carrying the C282Y mutation [14], the authors suggesting that HFE could have a potential role in neurodegeneration via its effect
on MCP-1 serum expression [14]. Importantly, in a more recent study in human neuroblastoma cell lines expressing different variants of HFE it was shown that the HFE polymorphisms differentially influenced the synthesis and release of MCP-1 [19]. The mechanism of action in this case involved cellular iron status but it appears there could be additional influences such as ER stress [19]. It has been recently described that the presence of the mutant C282Y protein stimulates an unfolded protein response (UPR) with consequent increased expression of other chaperone proteins implicated both in the response to cellular iron overload and in protection from oxidative stress [13,7,8]. One of these proteins, calreticulin, was shown to have a protective role in the clinical expression of hereditary hemochromatosis [22]. It is plausible to consider that other proteins may be also involved in the HFE-induced UPR that could explain the protective response associated with an up-regulation of the signal transduction pathway in response to stress [15]. In conclusion, the present study does not support the putative role of CAT53 polymorphisms in AD but, instead, it stresses the interest to further study the function of the variants of the HFE protein as modifiers in neurodegeneration. Larger studies in other populations will be important to support the present findings of a putative protective role of the C282Y polymorphism in AD. Acknowledgements We gratefully acknowledge all the nurse and medical staff at the Neurology Department of Centro Hospitalar do Porto, Hospital Santo António and at the Psychogeriatric Department of Hospital Magalhães Lemos, and especially to nurse Grac¸a Melo who took the responsibility of handling the blood samples. We are also grateful to Ruma Raha-Chowdury for inspiring this work. This work was partially supported by the research grant 53/2007 of the “Comissão de Fomento da Investigac¸ão em Cuidados de Saúde” awarded by the Portuguese Ministry of Health and partially funded by a research grant from Lundbeck Portugal, Lda. References [1] American Psychiatric Association, Diagnostic and Statistical Manual of Mental Disorders, 4th revised edition (DSM IV), American Psychiatric Press, Washington, DC, 1994. [2] D. Berlin, G. Chong, H. Chertkow, H. Bergman, N.A. Phillips, H.M. Schipper, Evaluation of HFE (hemochromatosis) mutations as genetic modifiers in sporadic AD and MCI, Neurobiol. Aging 25 (2004) 465–474. [3] G. Candore, C.R. Balistreri, G. Colonna-Romano, D. Lio, C. Caruso, Major histocompatibility complex and sporadic Alzheimer’s disease: a critical reappraisal, Exp. Gerontol. 39 (2004) 645–652. [4] G. Candore, F. Licastro, M. Chiappelli, C. Franceschi, D. Lio, C.R. Balistreri, G. Piazza, G. Colonna-Romano, L.M. Grimaldi, C. Caruso, Association between the HFE mutations and unsuccessful ageing: a study in Alzheimer’s disease patients from Northern Italy, Mech. Ageing Dev. 124 (2003) 525–528. [5] C. Cardoso, P. Oliveira, C. Oberkanis, M. Mascarenhas, P. Rodrigues, C. Sá Miranda, F. Kury, M. de Sousa, G. Porto, Comparative study of the two more frequent HFE mutations (C282Y and H63D): significant different allelic frequencies between the North and South of Portugal, Eur. J. Hum. Genet. 9 (2001) 843–848. [6] O. Combarros, M. García-Román, A. Fontalba, J.L. Fernández-Luna, J. Llorca, J. Infante, J. Berciano, Interaction of the H63D mutation in the hemochromato-
132
[7] [8]
[9] [10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
A.P. Correia et al. / Neuroscience Letters 457 (2009) 129–132 sis gene with the apolipoprotein E epsilon 4 allele modulates age at onset of Alzheimer’s disease, Dement. Geriatr. Cogn. Disord. 15 (2003) 151–154. S.F. De Almeida, M. de Sousa, The unfolded protein response in hereditary hemochromatosis, J. Cell Mol. Med. 12 (2008) 421–434. S.F. De Almeida, J.V. Fleming, J.E. Azevedo, M. Carmo-Fonseca, M. de Sousa, Stimulation of an unfolded protein response impairs MHC class I expression, J. Immunol. 178 (2007) 3612–3619. M.R. Farlow, Alzheimer’s disease: clinical implications of the apolipoprotein E genotype, Neurology 48 (1997) S30–S34. A. Gnjec, K.J. D’Costa, S.M. Laws, R. Hedley, K. Balakrishnan, K. Taddei, G. Martins, A. Paton, G. Verdile, S.E. Gandy, G.A. Broe, W.S. Brooks, H. Bennett, O. Piguet, P. Price, J. Miklossy, J. Hallmayer, P.L. McGeer, R.N. Martins, Association of alleles carried at TNFA-850 and BAT1-22 with Alzheimer’s disease, J. Neuroinflamm. 5 (2008) 36. R.J. Guerreiro, J.M. Bras, I. Santana, C. Januario, B. Santiago, A.S. Morgadinho, M.H. Ribeiro, J. Hardy, A. Singleton, C. Oliveira, Association of HFE common mutations with Parkinson’s disease, Alzheimer’s disease and mild cognitive impairment in a Portuguese cohort, BMC Neurol. 6 (2006) 24. J.M. Harris, A.M. Cumming, N. Craddock, D.S. Clair, C.L. Lendon, Human leucocyte antigen-A2 increases risk of Alzheimer’s disease but does not affect age of onset in a Scottish population, Neurosci. Lett. 294 (2000) 37–40. M.W. Lawless, A.K. Mankan, M. White, M.J. O’Dwyer, S. Norris, Expression of hereditary hemochromatosis C282Y HFE protein in HEK293 cells activates specific endoplasmic reticulum stress responses, BMC Cell Biol. 8 (2007) 30. M.W. Lawless, M. White, A.K. Mankan, M.J. O’Dwyer, S. Norris, Elevated MCP1 serum levels are associated with the H63D mutation and not the C282Y mutation in hereditary hemochromatosis, Tissue Antigens 70 (2007) 294–300. S.Y. Lee, S.M. Patton, R.J. Henderson, J.R. Connor, Consequences of expressing mutants of the hemochromatosis gene (HFE) into a human neuronal cell line lacking endogenous HFE, FASEB J. 21 (2007) 564–576. F. Listì, G. Candore, C.R. Balistreri, M.P. Grimaldi, V. Orlando, S. Vasto, G. ColonnaRomano, D. Lio, F. Licastro, C. Franceschi, C. Caruso, Association between the HLA-A2 allele and Alzheimer disease, Rejuvenation Res. 9 (2006) 99–101. G. McKhann, D. Drachman, M. Folstein, R. Katzman, D. Price, E.M. Stadlan, Clinical diagnosis of Alzheimer’s disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer’s Disease, Neurology 34 (1984) 939–944. S.S. Mirra, A. Heyman, D. McKeel, S.M. Sumi, B.J. Crain, L.M. Brownlee, F.S. Vogel, J.P. Hughes, G. van Belle, L. Berg, Participating CERAD neuropathologists, The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer’s disease, Neurology 41 (1991) 479–486. R.M. Mitchell, S.Y. Lee, W.T. Randazzo, Z. Simmons, J.R. Connor, Influence of HFE variants and cellular iron on monocyte chemoattractant protein-1, J. Neuroinflamm. 6 (2009) 6.
[20] National Institute on Aging, and Reagan Institute Working Group on Diagnostic Criteria for Neuropathological Assessment of Alzheimer’s disease, Consensus recommendations for the postmortem diagnosis of Alzheimer’s disease, Neurobiol. Aging 18 (1997) S1–S2. [21] M. Percy, S. Moalem, A. Garcia, M.J. Somerville, M. Hicks, D. Andrews, A. Azad, P. Schwarz, R.B. Zavareh, R. Birkan, C. Choo, V. Chow, S. Dhaliwal, V. Duda, A.L. Kupferschmidt, K. Lam, D. Lightman, K. Machalek, W. Mar, F. Nguyen, P.J. Rytwinski, E. Svara, M. Tran, L. Yeung, K. Zanibbi, R. Zener, M. Ziraldo, M. Freedman, Involvement of ApoE E4 and H63D in sporadic Alzheimer’s disease in a folate-supplemented Ontario population, J. Alzheimers Dis. 14 (2008) 69–84. [22] J.P. Pinto, P. Ramos, S.F. de Almeida, S. Oliveira, L. Breda, M. Michalak, G. Porto, S. Rivella, M. de Sousa, Protective role of calreticulin in HFE hemochromatosis, Free Radic. Biol. Med. 44 (2008) 99–108. [23] G. Porto, C.S. Cardoso, F. Macedo, E. Cruz, Hereditary hemochromatosis type I: genetic, clinical and immunological aspects, in: H. Fuchs (Ed.), Iron Metabolism and Disease, Research Signpost, Kerala, 2008, pp. 435–460. [24] R. Raha-Chowdhury, S.R. Andrews, J.R. Gruen, CAT 53: a protein phosphatase 1 targeting subunit encoded in the MHC class I region strongly expressed in regions of the brain involved in memory, learning, and Alzheimer’s disease, Mol. Brain Res. 138 (2005) 70–83. [25] K.J. Robson, D.J. Lehmann, V.L. Wimhurst, K.J. Livesey, M. Combrinck, A.T. Merryweather-Clarke, D.R. Warden, A.D. Smith, Synergy between the C2 allele of transferrin and the C282Y allele of the haemochromatosis gene (HFE) as risk factors for developing Alzheimer’s disease, J. Med. Genet. 41 (2004) 261– 265. [26] M. Sampietro, L. Caputo, A. Casatta, M. Meregalli, A. Pellagatti, J. Tagliabue, G. Annoni, C. Vergani, The hemochromatosis gene affects the age of onset of sporadic Alzheimer’s disease, Neurobiol. Aging 22 (2001) 563– 568. [27] A.M. Saunders, W.J. Strittmatter, D. Schmechel, P.H. George-Hyslop, M.A. Pericak-Vance, S.H. Joo, B.L. Rosi, J.F. Gusella, D.R. Crapper-MacLachlan, M.J. Alberts, C. Hulette, B. Crain, D. Goldgaber, A.D. Roses, Association of apolipoprotein E allele -C4 with late-onset familial and sporadic Alzheimer’s disease, Neurology 43 (1993) 1467–1472. [28] W.J. Strittmatter, A.M. Saunders, D. Schemechel, M. Pericak-Vance, J. Enghild, G.S. Salvesen, A.D. Roses, Apolipoprotein E: high-avidity binding to betaamyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 1977–1981. [29] A. Totaro, A. Grifa, M. Carella, J.M. Rommens, M.A. Valentino, A. Roetto, L. Zelante, P. Gasparini, Cloning of a new gene (FB19) within HLA class I region, Biochem. Biophys. Res. Commun. 250 (1998) 555–557. [30] S. Zareparsi, D.M. James, J.A. Kaye, T.D. Bird, G.D. Schellenberg, H. Payami, HLAA2 homozygosity but not heterozygosity is associated with Alzheimer disease, Neurology 58 (2002) 973–975.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
CAT53 and HFE alleles in Alzheimer’s disease: A putative protective role of the C282Y HFE mutation Ana Paula Correia a,b , Jorge P. Pinto c , Vera Dias c , Cláudia Mascarenhas c , Susana Almeida c , Grac¸a Porto a,c,d,∗ a
HGSA – Centro Hospitalar do Porto, Hospital Santo António, Porto, Portugal HML – Hospital Magalhães Lemos, Porto, Portugal IBMC – Institute for Molecular and Cellular Biology, Porto, Portugal d ICBAS – Abel Salazar Institute for the Biomedical Sciences, Porto, Portugal b c
a r t i c l e
i n f o
Article history: Received 26 February 2009 Received in revised form 25 March 2009 Accepted 27 March 2009 Keywords: CAT53 HFE C282Y MHC Alzheimer’s disease
a b s t r a c t Alzheimer’s disease (AD) is a complex disorder, resulting from an interaction between environmental and genetic factors. Several studies addressed the association of AD with MHC class-I polymorphisms without definite conclusions. Considering the remarkable linkage disequilibrium at the MHC region, it is not possible to assume if the reported associations result from a direct effect of the respective genes or result from associations with other closely linked genes transmitted in an extended conserved haplotype. Recent evidence pointed to CAT53, a newly described gene located at the MHC class-I region in the vicinity of HLA-C, as a candidate modifier gene in AD. CAT53 encodes a phosphatase 1 nuclear inhibitor protein and is strongly expressed in brain regions involved in memory and AD. Here we tested the potential association of CAT53 with the risk of developing AD and searched for potential haplotypic associations of CAT53 with two common mutations (H63D, C282Y) in the HFE gene, also located at chromosome 6p21.3. The allele frequencies of these mutations in AD patients were compared to the expected frequencies previously established in the normal Portuguese population. We detected only one polymorphism (G>A) in CAT53, at position 8232, in intron 17. Screening of this polymorphism in 113 AD patients and 82 controls did not show any evidence of association, therefore excluding the hypothetical role of the CAT53 polymorphism as modifier in AD. In contrast, we found a significant negative association of the C282Y HFE mutation with AD, thus supporting a putative protective role of this protein variant in neurodegeneration. © 2009 Elsevier Ireland Ltd. All rights reserved.
Alzheimer’s disease (AD) is the most common cause of dementia in the elderly. It is a progressive neurodegenerative disorder pathologically characterized by the presence of extracellular amyloid -peptide aggregated in soluble A oligomers and insoluble deposits as senile plaques and intracellular neurofibrillar tangles [18,20]. AD is a complex, multifactorial disorder. Although the genetics of early onset, autosomal dominant AD is relatively well characterized, the susceptibility to the most common late-onset forms of the disease is still debated. This form results most probably from an interaction between environmental and genetic factors. To date, only the 4 allele of apolipoprotein E is generally recognized as a susceptibility genotype for late-onset AD [9,27,28] but the fact that it is neither necessary nor sufficient to cause the disease, indicates that other genes may be involved in AD susceptibility.
∗ Corresponding author at: IRIS, IBMC – Instituto de Biologia Molecular e Celular, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. Tel.: +351 226074956; fax: +351 226098480. E-mail address: [email protected] (G. Porto). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.03.088
MHC class-I gene polymorphisms have been the focus of a large number of AD association studies, with weak associations identified for several of these [3]. However, conflicting data have not allowed so far a definite exclusion or confirmation of their real impact in AD. The presence of the human leukocyte antigen (HLA) A2, as well as variations in the regulatory regions of the tumor necrosis factor alpha (TNF-␣) and in the HLA-B-associated transcript 1 (BAT1) gene, all have been reported to modify the risk of developing AD [10,12,16,30]. In addition, several reports have implicated the H63D variant of the HFE gene as a modifier of the age of onset in AD, and as an interacting factor with the APOE 4 allele [6,21,26]. HFE is a non-classical MHC class-I gene that has a role in the regulation of iron metabolism. Although the role of the H63D variant in iron homeostasis is still not completely understood, another common mutation in this gene, the C282Y mutation, is strongly associated with iron overload, both in human and in animal models, being the major genetic variant found in patients with hereditary hemochromatosis [23]. While no direct role of MHC class I proteins has been demonstrated so far in the susceptibility to AD, it is still possible that the
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A.P. Correia et al. / Neuroscience Letters 457 (2009) 129–132 Table 2 CAT53 genotype frequencies (and %) in AD patients according to the HFE genotypes.
previously reported associations may result from another closely linked gene transmitted in an extended conserved haplotype, given the remarkably high linkage disequilibrium in this region. Recently, a newly characterized gene located on chromosome 6p21.3, in the vicinity of HLA-C, previously identified and named FB19 [29] and now designated as CAT53 [24] was suggested as a candidate gene involved in the pathogenesis and progression of AD [24]. High levels of expression of CAT53 were found in human and mouse brain and a very high expression was found in the neurofibrillary tangles of AD brain [24]. SMART analysis of CAT53 domains was supportive of a nuclear regulatory role on PP1, a protein involved in several brain functions including learning and memory [24]. To assess the potential impact of CAT53 on the risk of developing AD we searched for polymorphic associations in a case control study involving 113 Portuguese AD patients (age 78.2 ± 7.2 years, age of onset 73.5 ± 7.7 years, 24% males) and 82 unrelated apparently healthy normal subjects (age 60.0 ± 18.0 years, range 19–100 years, 46% males). All patients and controls were Caucasian born in the north and central regions of Portugal. The same physician at the Neurology Department of Hospital Santo António and Psychogeriatric Department of Hospital Magalhães Lemos, both in Porto, performed the clinical characterization and selection of patients. The diagnosis of probable AD was established according to criteria defined by the Diagnostic and Statistical Manual, revision 4 (DSM-IV) [1] and the guidelines of the National Institute of Neurological Disorders and Stroke, and the Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) [17]. An early age at onset was defined as ≤65 years. Controls were defined as subjects without DMS-IV dementia criteria and with integrity of daily living functions. In order to test for potential haplotype associations, all AD patients studied were HFE genotyped for the C282Y and H63D mutations by a PCR amplification method with selective hybridization to allele-specific oligonucleotide probes, using commercially available Kits (Gene Mutation Assay I and II, Vienna-Lab, Vienna, Austria). Written informed consent was obtained from all recruited subjects, or their legal representatives, according to the 1975 Declaration of Helsinki and the study was approved by the ethical committee of Hospital Santo António and Hospital Magalhães Lemos, Porto. Comparisons between allele or genotype frequencies were tested by the Chi-square and the Fisher’s exact tests, and significant results considered for P-values lower than 0.05. All 18 exons, exon/intron boundaries, the 5 UTR and the first 1000 base pairs of the proximal promoter of the CAT53 gene were first sequenced double-strand in 12 controls for the detection of common DNA sequence variants. Briefly, genomic DNA was extracted from peripheral blood, using the “salting out” method, and quantified. Each amplicon was amplified by PCR using a specific primer pair and PCR products were analyzed by agarose gel electrophoresis and documented by photography. DNA was excised from the gel and purified using the QIAquick Gel Extraction Kit (Qiagen). Sequencing was performed with the BigDye® Terminator v1.1 Cycle Sequencing Kit, in an ABI Prism Avant DNA sequencer. A striking high degree of conservation was found in the sequences of all individuals analyzed and only one polymorphism (G>A) was found at position 8232, in intron 17 of the CAT53 gene. We next
HFE genotype
CAT53 genotype
C282Y/wt (n = 3) H63D/wt (n = 28) H63D/H63D (n = 6) wt/wt (n = 76)
GG
GA
AA
2 (66.7%) 16 (57.1%) 6 (100%) 55 (72.4%)
1 (33.3%) 11 (39.3%) – 20 (26.3%)
0 (–) 1 (3.6%) – 1 (1.3%)
The P-value for the Chi-square test of independence was 0.51.
screened the whole group of controls and AD patients for the presence of the 8232 G>A polymorphism by Denaturing High Performance Liquid Chromatography (DHPLC) using a specific primer pair. Allele and genotype distributions of the 8232 polymorphisms were compared between AD patients and controls (Table 1), with no significant differences found between groups. When analysis was performed including only the selected group of late-onset AD patients, the same results were obtained (see also Table 1). Searching for eventual haplotypic associations between the CAT53 8232 polymorphisms and HFE mutations was done by comparing the CAT53 genotype frequencies in AD patients grouped according to their HFE genotype (Table 2). No significant association was found between variables. Allele frequencies of the HFE mutations in the AD patient groups are shown in Table 3, in comparison with the expected frequencies previously determined in a representative, geographically matched, Portuguese sample described by Cardoso et al. (n = 259) [5]. It should be noted that, due to the demonstrated differences in expected C282Y allele frequencies in the Portuguese population according to the region of origin [5], only the data from subjects originating from the north or central region (n = 259) were used in the present study. In accordance with previous studies [6,26] the H63D mutation was associated with the age of onset in lateonset AD, with a significantly lower frequency found in patients presenting with more than 75 years (a.f. = 0.115 vs. 0.203 in controls; Chi-square test, P = 0.012; Fisher’s exact test, P = 0.0125) but not in patients presenting with 66–75 years (a.f. = 0.206). In addition, a global highly significant negative association was found in AD patients for the presence of the C282Y mutation (a.f. = 0.013 vs. 0.058 in controls; Chi-square test, P = 0.0022; Fisher’s exact test, P = 0.00197). This result was confirmed when the analysis was restricted to the group of late-onset AD patients (a.f. = 0.010 vs. 0.058 in controls; Chi-square test, P = 0.0025; Fisher’s exact test, P = 0.0002). MHC class I variation is a key determinant of susceptibility and resistance to a large number of diseases but the identification of the MHC variants conferring susceptibility to disease is problematic due to high levels of variation and linkage disequilibrium. With this in mind, we focused our interest in testing CAT53 as a candidate gene in AD in the context of its putative linkage to other genes on 6p21.3, namely with HFE mutations. Only one single nucleotide polymorphism was found in the CAT53 gene and no association was found of this polymorphism with the presence of AD. This does not exclude a putative role of the CAT53 gene product in the pathogenesis of
Table 1 Allele and genotype frequencies and distributions (n) of the CAT 53 8232 polymorphisms in AD patients and controls. 8232 variant
AD patients Late-onset AD patientsa Controls
N
116 99 82
Allele frequencies (n)
Genotype frequencies (n)
G
A
GG
GA
AA
0.841 (190) 0.843 (167) 0.848 (139)
0.159 (36) 0.157 (31) 0.152 (25)
0.699 (79) 0.707 (70) 0.707 (58)
0.283 (32) 0.272 (27) 0.280 (23)
0.018 (2) 0.020 (2) 0.012 (1)
N = number of subjects in samples. No statistical significant differences were found for allele or genotype frequencies between groups. a Subgroup of AD patients with >65 years.
A.P. Correia et al. / Neuroscience Letters 457 (2009) 129–132
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Table 3 Allele frequencies of the HFE mutations in AD patients, according to age group, in comparison to expected frequencies in a control populationa . H63D allele frequencies
b
C282Y allele frequencies
b
All patients (N = 113) Late-onset AD patientsc (N = 99) Age group 66–75 years (N = 51) Age group >75 years (N = 48)
0.172 0.162 0.206 0.115
n.s. P = 0.0504 n.s. P = 0.0125
0.013 0.010
P = 0.00197 P = 0.0002
Controls (N = 259)
0.203
0.058
N, number of subjects in samples; n.s., not significant. a From Ref. [5]. b Significance of the differences between each AD group and controls (Fisher’s exact test). c Subgroup of AD patients with >65 years.
AD but excludes a strong impact of common gene polymorphisms in this condition. We are aware that the limited number of subjects tested for the screening of polymorphisms impairs the detection of variants of low frequencies and therefore their putative association to AD is neglected. However, and assuming a high frequency of AD in the general population, we did not intend to search for associations with rare polymorphisms. Although the results do not support a role for CAT53 as a susceptibility gene in AD and do not suggest its possible linkage to HFE in a conserved haplotype, our data support, however, a possible role of the HFE genotype as a genetic modifier in this clinical condition. While the H63D mutation was associated to an earlier progression of AD, the C282Y mutation showed a negative association with the disease, thus appearing as a putative protective genotype. Interestingly, similarly low frequencies (between 0.008 and 0.026) were previously reported in AD patients from other Northern Italian, Canadian and Portuguese populations [2,4,11,26]. A higher C282Y allele frequency (0.078) was found in a selected AD population from the Oxford region [25]. In this particular case, however, the presence of the C282Y mutation was clearly and strongly associated with the co-inheritance of the transferrin gene allele C2 [25]. The mechanism how HFE could influence the clinical expression of AD is still elusive. It is relevant to note, however, the recent study by Lee et al. [15] where it was shown that the two different HFE mutations have unique effects on human neuroblastoma stable cell lines over-expressing HFE. While the presence of the C282Y mutation was associated with an up-regulation of the signal transduction pathway in response to stress, H63D-expressing cells had higher levels of lipid peroxidation, protein oxidation and lower mitochondrial membrane potential, suggesting a higher baseline stress, and were also more vulnerable to exposure to oxidative stress agents [15]. That was the first study providing insights into how the different HFE mutations could have different clinical consequences. More recently, an association of the H63D mutation with increased tau phosphorylation and up-regulated protein kinase activity provided more evidence that this allelic variant is associated with an altered intracellular environment, consistent with its putative role as a risk factor for neurodegenerative disorders (Hall et al., personal communication). On the other side, another recent report of gene expression in the brain from Hfe knockout mice showed a remarkable decrease in the expression of several genes involved in the pathogenesis of AD namely the genes encoding the amyloid precursor protein (APP) and the presenilin 1 gene (PSEN1), thus challenging again the notion of the role of HFE as predisposing or protective in AD (Johnstone et al., personal communication). The mechanism how the HFE C282Y mutation could confer protection to neurodegeneration is still speculative. In hereditary hemochromatosis patients it has been shown that monocyte chemoattractant protein-1 (MCP-1) serum levels are differentially associated with the HFE mutations, being increased in patients with the H63D mutation but significantly decreased in those carrying the C282Y mutation [14], the authors suggesting that HFE could have a potential role in neurodegeneration via its effect
on MCP-1 serum expression [14]. Importantly, in a more recent study in human neuroblastoma cell lines expressing different variants of HFE it was shown that the HFE polymorphisms differentially influenced the synthesis and release of MCP-1 [19]. The mechanism of action in this case involved cellular iron status but it appears there could be additional influences such as ER stress [19]. It has been recently described that the presence of the mutant C282Y protein stimulates an unfolded protein response (UPR) with consequent increased expression of other chaperone proteins implicated both in the response to cellular iron overload and in protection from oxidative stress [13,7,8]. One of these proteins, calreticulin, was shown to have a protective role in the clinical expression of hereditary hemochromatosis [22]. It is plausible to consider that other proteins may be also involved in the HFE-induced UPR that could explain the protective response associated with an up-regulation of the signal transduction pathway in response to stress [15]. In conclusion, the present study does not support the putative role of CAT53 polymorphisms in AD but, instead, it stresses the interest to further study the function of the variants of the HFE protein as modifiers in neurodegeneration. Larger studies in other populations will be important to support the present findings of a putative protective role of the C282Y polymorphism in AD. Acknowledgements We gratefully acknowledge all the nurse and medical staff at the Neurology Department of Centro Hospitalar do Porto, Hospital Santo António and at the Psychogeriatric Department of Hospital Magalhães Lemos, and especially to nurse Grac¸a Melo who took the responsibility of handling the blood samples. We are also grateful to Ruma Raha-Chowdury for inspiring this work. This work was partially supported by the research grant 53/2007 of the “Comissão de Fomento da Investigac¸ão em Cuidados de Saúde” awarded by the Portuguese Ministry of Health and partially funded by a research grant from Lundbeck Portugal, Lda. References [1] American Psychiatric Association, Diagnostic and Statistical Manual of Mental Disorders, 4th revised edition (DSM IV), American Psychiatric Press, Washington, DC, 1994. [2] D. Berlin, G. Chong, H. Chertkow, H. Bergman, N.A. Phillips, H.M. Schipper, Evaluation of HFE (hemochromatosis) mutations as genetic modifiers in sporadic AD and MCI, Neurobiol. Aging 25 (2004) 465–474. [3] G. Candore, C.R. Balistreri, G. Colonna-Romano, D. Lio, C. Caruso, Major histocompatibility complex and sporadic Alzheimer’s disease: a critical reappraisal, Exp. Gerontol. 39 (2004) 645–652. [4] G. Candore, F. Licastro, M. Chiappelli, C. Franceschi, D. Lio, C.R. Balistreri, G. Piazza, G. Colonna-Romano, L.M. Grimaldi, C. Caruso, Association between the HFE mutations and unsuccessful ageing: a study in Alzheimer’s disease patients from Northern Italy, Mech. Ageing Dev. 124 (2003) 525–528. [5] C. Cardoso, P. Oliveira, C. Oberkanis, M. Mascarenhas, P. Rodrigues, C. Sá Miranda, F. Kury, M. de Sousa, G. Porto, Comparative study of the two more frequent HFE mutations (C282Y and H63D): significant different allelic frequencies between the North and South of Portugal, Eur. J. Hum. Genet. 9 (2001) 843–848. [6] O. Combarros, M. García-Román, A. Fontalba, J.L. Fernández-Luna, J. Llorca, J. Infante, J. Berciano, Interaction of the H63D mutation in the hemochromato-
132
[7] [8]
[9] [10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
A.P. Correia et al. / Neuroscience Letters 457 (2009) 129–132 sis gene with the apolipoprotein E epsilon 4 allele modulates age at onset of Alzheimer’s disease, Dement. Geriatr. Cogn. Disord. 15 (2003) 151–154. S.F. De Almeida, M. de Sousa, The unfolded protein response in hereditary hemochromatosis, J. Cell Mol. Med. 12 (2008) 421–434. S.F. De Almeida, J.V. Fleming, J.E. Azevedo, M. Carmo-Fonseca, M. de Sousa, Stimulation of an unfolded protein response impairs MHC class I expression, J. Immunol. 178 (2007) 3612–3619. M.R. Farlow, Alzheimer’s disease: clinical implications of the apolipoprotein E genotype, Neurology 48 (1997) S30–S34. A. Gnjec, K.J. D’Costa, S.M. Laws, R. Hedley, K. Balakrishnan, K. Taddei, G. Martins, A. Paton, G. Verdile, S.E. Gandy, G.A. Broe, W.S. Brooks, H. Bennett, O. Piguet, P. Price, J. Miklossy, J. Hallmayer, P.L. McGeer, R.N. Martins, Association of alleles carried at TNFA-850 and BAT1-22 with Alzheimer’s disease, J. Neuroinflamm. 5 (2008) 36. R.J. Guerreiro, J.M. Bras, I. Santana, C. Januario, B. Santiago, A.S. Morgadinho, M.H. Ribeiro, J. Hardy, A. Singleton, C. Oliveira, Association of HFE common mutations with Parkinson’s disease, Alzheimer’s disease and mild cognitive impairment in a Portuguese cohort, BMC Neurol. 6 (2006) 24. J.M. Harris, A.M. Cumming, N. Craddock, D.S. Clair, C.L. Lendon, Human leucocyte antigen-A2 increases risk of Alzheimer’s disease but does not affect age of onset in a Scottish population, Neurosci. Lett. 294 (2000) 37–40. M.W. Lawless, A.K. Mankan, M. White, M.J. O’Dwyer, S. Norris, Expression of hereditary hemochromatosis C282Y HFE protein in HEK293 cells activates specific endoplasmic reticulum stress responses, BMC Cell Biol. 8 (2007) 30. M.W. Lawless, M. White, A.K. Mankan, M.J. O’Dwyer, S. Norris, Elevated MCP1 serum levels are associated with the H63D mutation and not the C282Y mutation in hereditary hemochromatosis, Tissue Antigens 70 (2007) 294–300. S.Y. Lee, S.M. Patton, R.J. Henderson, J.R. Connor, Consequences of expressing mutants of the hemochromatosis gene (HFE) into a human neuronal cell line lacking endogenous HFE, FASEB J. 21 (2007) 564–576. F. Listì, G. Candore, C.R. Balistreri, M.P. Grimaldi, V. Orlando, S. Vasto, G. ColonnaRomano, D. Lio, F. Licastro, C. Franceschi, C. Caruso, Association between the HLA-A2 allele and Alzheimer disease, Rejuvenation Res. 9 (2006) 99–101. G. McKhann, D. Drachman, M. Folstein, R. Katzman, D. Price, E.M. Stadlan, Clinical diagnosis of Alzheimer’s disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer’s Disease, Neurology 34 (1984) 939–944. S.S. Mirra, A. Heyman, D. McKeel, S.M. Sumi, B.J. Crain, L.M. Brownlee, F.S. Vogel, J.P. Hughes, G. van Belle, L. Berg, Participating CERAD neuropathologists, The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer’s disease, Neurology 41 (1991) 479–486. R.M. Mitchell, S.Y. Lee, W.T. Randazzo, Z. Simmons, J.R. Connor, Influence of HFE variants and cellular iron on monocyte chemoattractant protein-1, J. Neuroinflamm. 6 (2009) 6.
[20] National Institute on Aging, and Reagan Institute Working Group on Diagnostic Criteria for Neuropathological Assessment of Alzheimer’s disease, Consensus recommendations for the postmortem diagnosis of Alzheimer’s disease, Neurobiol. Aging 18 (1997) S1–S2. [21] M. Percy, S. Moalem, A. Garcia, M.J. Somerville, M. Hicks, D. Andrews, A. Azad, P. Schwarz, R.B. Zavareh, R. Birkan, C. Choo, V. Chow, S. Dhaliwal, V. Duda, A.L. Kupferschmidt, K. Lam, D. Lightman, K. Machalek, W. Mar, F. Nguyen, P.J. Rytwinski, E. Svara, M. Tran, L. Yeung, K. Zanibbi, R. Zener, M. Ziraldo, M. Freedman, Involvement of ApoE E4 and H63D in sporadic Alzheimer’s disease in a folate-supplemented Ontario population, J. Alzheimers Dis. 14 (2008) 69–84. [22] J.P. Pinto, P. Ramos, S.F. de Almeida, S. Oliveira, L. Breda, M. Michalak, G. Porto, S. Rivella, M. de Sousa, Protective role of calreticulin in HFE hemochromatosis, Free Radic. Biol. Med. 44 (2008) 99–108. [23] G. Porto, C.S. Cardoso, F. Macedo, E. Cruz, Hereditary hemochromatosis type I: genetic, clinical and immunological aspects, in: H. Fuchs (Ed.), Iron Metabolism and Disease, Research Signpost, Kerala, 2008, pp. 435–460. [24] R. Raha-Chowdhury, S.R. Andrews, J.R. Gruen, CAT 53: a protein phosphatase 1 targeting subunit encoded in the MHC class I region strongly expressed in regions of the brain involved in memory, learning, and Alzheimer’s disease, Mol. Brain Res. 138 (2005) 70–83. [25] K.J. Robson, D.J. Lehmann, V.L. Wimhurst, K.J. Livesey, M. Combrinck, A.T. Merryweather-Clarke, D.R. Warden, A.D. Smith, Synergy between the C2 allele of transferrin and the C282Y allele of the haemochromatosis gene (HFE) as risk factors for developing Alzheimer’s disease, J. Med. Genet. 41 (2004) 261– 265. [26] M. Sampietro, L. Caputo, A. Casatta, M. Meregalli, A. Pellagatti, J. Tagliabue, G. Annoni, C. Vergani, The hemochromatosis gene affects the age of onset of sporadic Alzheimer’s disease, Neurobiol. Aging 22 (2001) 563– 568. [27] A.M. Saunders, W.J. Strittmatter, D. Schmechel, P.H. George-Hyslop, M.A. Pericak-Vance, S.H. Joo, B.L. Rosi, J.F. Gusella, D.R. Crapper-MacLachlan, M.J. Alberts, C. Hulette, B. Crain, D. Goldgaber, A.D. Roses, Association of apolipoprotein E allele -C4 with late-onset familial and sporadic Alzheimer’s disease, Neurology 43 (1993) 1467–1472. [28] W.J. Strittmatter, A.M. Saunders, D. Schemechel, M. Pericak-Vance, J. Enghild, G.S. Salvesen, A.D. Roses, Apolipoprotein E: high-avidity binding to betaamyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 1977–1981. [29] A. Totaro, A. Grifa, M. Carella, J.M. Rommens, M.A. Valentino, A. Roetto, L. Zelante, P. Gasparini, Cloning of a new gene (FB19) within HLA class I region, Biochem. Biophys. Res. Commun. 250 (1998) 555–557. [30] S. Zareparsi, D.M. James, J.A. Kaye, T.D. Bird, G.D. Schellenberg, H. Payami, HLAA2 homozygosity but not heterozygosity is associated with Alzheimer disease, Neurology 58 (2002) 973–975.